International Rice Research Notes Vol.21 No.2

International Rice Research Notes The International Rice Research Notes (IRRN) expedites communication among scientists concerned with the development of improved technology for rice and rice- based systems.

other informed of current rice research findings. The concise scientific notes are meant to encourage rice scientists to communicate with one another to obtain details on the research reported.

The IRRN is published three times a year in April, August, and December by the International Rice Research Institute.

The IRRN is a mechanism to help scientists keep each

Focus on rice genetics The specter of food shortages is looming once again, with the annual rate of increase of rice production slowing to where it is lower than the rate of increase of rice consumers.

Recent advances in cellular and molecular genetics of rice have come perhaps in the nick of time to provide us with new tools to develop rice varieties for the future. Only 10 years ago, the status of rice genetics was considered far behind that of other food crops, such as maize and wheat. The past decade, however, has seen an explosion of knowledge in this arena. Rice is now considered a model plant for such research on cereal crops.

In October 1995, IRRI hosted the Third International Rice Genetics Symposium. More than 500 scientists from 31 countries attended. Along with a dramatic increase in the attendance over the years has come a major shift in the complexion of the program. During the first symposium in 1985, around 90% of the papers were on classical genetics; at this symposium, about 80% of the papers addressed topics on cellular and molecular genetics.

IRRI book. The posters displayed at the symposium appear as notes (in a modified format) throughout this double issue of IRRN, and will also be featured in the next issue. They are denoted by the

We hope you find these notes to be a valuable source of information.

The key papers presented have been published as an

IRRN production team . . . . . . . . . . . . . . .

Editors: Carolyn Dedolph and Theresa A. Castillo Assistant editor: Teresita Rola Layout and design: Erlie Putungan Artwork: Doris Rifareal

2 IRRN 21:2-3 (August-December 1996)

Contents August-December 1996

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Genetic resources Distribution of rice land races in China 5 Some suggestions for in situ conservation of wild rices 6 Panicle culture and karyotype analysis from callus cells of a diploid wild

A study of Neolithic carbonized rice grains excavated from Hemudu,

Classification of A genome species in the genus Oryza using nuclear

Detection of D genome chromosomes bygenomic in situ

Fingerprinting rice germplasm using ALP and PCR-based RFLP 10 Distribution and evolutionary differentiation of mitochondrial plasmid-

Genetic diversity of Chinese wild rice populations 13

Genetics Molecular analysis of introgression in Oryza sativa/O. brachyantha and

Acrotrisomics in rice 15 Molecular mapping of fertility-restoring gene Rf3 in rice 16 Characterization of common cis -regulatory elements responsible for

endosperm-specific expression of rice glutelin gene 17 A fertility-restoring revertant, controlled by a single gene induced from a

rice cytoplasmic male sterile line 18 Effects of Se1 gene on basic vegetative growth of rice 19 Genetic and cytochemical analysis of high 57-kD polypeptide mutants in

Molecular mapping of genes for F 1 pollen sterility in rice 20 Chloroplast ribosomal protein genes encoded in rice nuclear

Stability of mitochondrial plasmidlike DNAs of rice 22 Sensitivity of plant height genes to gibberellic acid and their regulation

Genetic diversity estimated among rice cultivars using restriction

Character expression of a rice dwarf mutant with lax panicle 25 Genetic differentiation between Japanese lowland and upland

Evolutionary variations in the Gramineae: rearrangements of DNA

rice, Oryza meyeriana 7

China 8

DNA markers 8

hybridization 10

like DNAs in the genus Oryza 12

O. sativa/O. granulata derivatives 14

rice 19

genome 21

by endogenous plant hormones in rice 22

landmark genomic scanning method 24

rices 26

fragments transferred from chloroplast genomes to mitochondrial genomes 27

Identifying transposonlike element Tnr2 in rice 29

Breeding methods New cytoplasmic male sterile lines developed in Andhra Pradesh,

Maintainers and restorers identified in some rice cultivars of

Development of rice cytoplasmic male sterile line 47456 A in Kala Shah

India 30

Pakistan 31

Kaku, Pakistan 31

Germplasm improvement

symbol

Comparison of promoters and selectable marker genes for indica RCPL-3-2 and RCPL-3-6: two promising rice lines for the mid-hills of

Transfer of cytoplasmic male sterility in indica rice through Toag92: a short-duration rice cultivar for Turkey 59

Characters of plants regenerated from protoplasts of photoperiod- Papua New Guinea 60

Attempted hybridization between Oryza sativa L. and Porteresia India 60

DNA content in rice hybrids and heterosis 36 lntegrated germplasm improvement-rainfed lowland Induction of Agrobacterium tumefaciens vir genes by rice 36 Shakuntla: a rice variety for rainfed lowlands in Bihar, India 61 Isolation of a tapetum-specific gene and promoter from rice 37 CMS lines for shallow rainfed lowland situation 62 Production and characterization of Oryza sativa L./ O. minuta Presl.

Histological observation of callus morphology in rice 39 Majhera 7, a direct seeded rainfed upland spring rice for Uttar Pradesh, Genetic analysis of epicuticular structure involving a dripping-wet India 62

Embryogenic cell suspensions established from a high-protein MTU9993, a promising rainfed upland rice for Andhra Pradesh,

rice transformation 32 Sikkim, India 59

protoplast fusion 33 KK15-36-C: a modern high-yielding rice variety for irrigated lowlands in

sensitive genic male sterile rice 34 Birsa Dhan 201 and Birsa Dhan 202: early-maturing varieties for Bihar,

coarctata T. 35

hybrids and backcross progenies 38 lntegrated germplasm improvement—upland

leaf mutant of rice 39

purple black rice 40 India 64

Improved upland rice for the hillsides of Colombia 63

Factors affecting pollen embryogenesis of rice anther culture 41 Genetic studies on purple pigmentation in rice and its use in

breeding two-line rice hybrids 42

Physiology and plant nutrition

The processes of pollination and fertilization in rice 65 Grain quality Analysis of growth duration and heat units of different rice genotypes 65 Calorific values of 60 rice varieties 43 Cibodas, a high-yielding variety with good grain quality 43 Studies on intercellular space of embryo and seedling tissues in rice Improving provitamin A (carotenoid) content of rice endosperm 43 plants 66 Phytoene-forming activities in wild-type and transformed rice

endosperm 44 Fertilizer management

Pest resistance—diseases Effects of time of split application of K on soil N forms and lowland rice

yield 67 Screening of Basmati rice genotypes against blast 45 Resistance spectrum, race specificity, and expression of the Xa21

A bacterial blight-resistant, wide-compatible indica line 46 RFLP mapping of blast resistance gene Pi-k m in rice 47 Indica/japonica doubled haploid population as a model for mapping

Characterization of a pathogenesis-related gene family induced in

Genes controlling field resistance to blast in Japanese upland rice

gene family 45

rice yellow mottle virus and blast resistance genes 47

rice infected with Magnapor the grisea 49

Effect of Biozyme and NPK on rice yield 68 Effects of fertilizer and green manure on survival and productivity of

detached deepwater rice plants 69 lntegrated N management with Gliricidia maculata and Sesbania

aculeata for transplanted rice 70

Fertilizer management—inorganic sources Response of late-transplanted rice to NPK under irrigated

conditions 70

detected using RFLP markers 50 Fertilizer management—organic sources Genetic analysis for true resistance to blast in rice varieties 51 Evaluating stem-nodulating green manure crops for lowland rice 71

Managing crop residue in rice 71 Pest resistance-insects Evaluation of green manures and grain legumes 72 Preliminary evidence of aphid resistance in transgenic plants 52 Effect of soil amendments on rice yield 72 Role of host plant resistance in successful control of brown

planthopper in Central Luzon, Philippines 53

rice leafhopper 53

in rice 54

Graphical genotypes of rice parental lines for resistance to green

RFLP mapping of brown planthopper resistance gene Bph1

Stress tolerance—salinity

Crop management Use of DWR varietal mixtures to minimize hydrological risk in basin lands

of Bihar, India 73 Effect of transplanting times on hybrid rice in Haryana, lndia 74 Effect of solar radiation and temperature on rice yields in different

planting dates 75

Principal component analysis and variety classification in relation to Integrated pest management—diseases rice seedling salinity tolerance 55 Effects of neem derivatives on sheath rot in scented rice 76

Seedborne nature and seed transmission of rice sheath blight 76 Serological evidence for inducing resistance to rice tungro viruses using

lntegrated germplasm improvement—irrigated Variability, heritability, correlation, path analysis, and genetic

divergence studies in M 2 generation of gamma-irradiated upland antiviral principles 77

rice 56

IRRN 21:2-3 (August-December 1996) 3

Crop and resource management

Integrated pest management—insects Hibernation sites of the rice stalk stink bug Tibraca limbativentris in

the central region of Rio Grande do SUI, Brazil 78

Integrated pest management—weeds Predicting the effects of Echinochloa crus-galli on rice ( Oryza sativa L.)

Weed control in direct seeded puddled rice 79 using the ecophysiological model INTERCOM 78

Integrated pest management—other pests Effect of soil solarization of rice nursery beds to suppress plant

parasitic nematodes 80

Water management Supplemental irrigation for dry seeded upland rice 81 Rice yield under sprinkler irrigation 81

Farming systems Effect of different rice cultures on crops yield in rice-based

systems 82

Farm machinery A low-cost, in-store dryer for small-scale farmers 83

A simple and rapid method for isolation of bacterial genomic DNA 84 A simulation model of rice sheath blight epidemics (I) Structure and

model development 85 A simulation model of rice sheath blight epidemics (II) Model

performance derived from sensitivity analysis 86 Use of CERES-Rice model to assess potential yield 87

Effect of contour hedgerow on runoff, soils loss, and upland rice production 87

Recommendations of the 3rd International Symposium on Hybrid Rice 88

Note to IRRN readers We sincerely apologize for the delay in producing this special combined issue. Regular production of the IRRN will resume in 1997. Thank you for your continued support.


Research methodology

Environment

Recommendations

Erratum

rmguevarra

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Germplasm improvement

Genetic resources

Distribution of rice land races in China

Cao Yongsheng and Zhang Xianzhen, Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Cultivated rice (Oryza sativa L.) is widely distributed in China (see figure and table). These germplasm resources are highly diverse and extremely localized because of the country's vast territory, the complicated terrain, the continental and oceanic monsoon climates, and various other ecological factors. These factors make the

distribution of rice land races in China regional and noncontinuous.

The summer monsoon brings warm temperatures and abundant precipitation to southeastern China, making for a long rice- growing season and a concentration of ricefields. The landscape is very hilly, which causes significant differences in the climate. The rice germplasm resources in the region are rich.

The majority (93%) of the rice land races in China are distributed in the area south of the Qing Mountains and Huaihe River, where the annual precipitation is more than 800 mm and the annual mean temperature is above 15°C. The Changjiang Delta, the Zhujiang Delta, the central Anhui Plain, and the Chendu Plain are the main regions of

rice land race distribution; the plains in Yunnan and Guizhou provinces and the coastal plains in Zhejiang, Fujian, and Hainan provinces are also important areas.

The rice-growing season is short in the area to the north of the Huaihe River in eastern China because of the high latitude and low mean temperature. Rice land races are less common (only 6% of the total accessions) than in southern China because most of the area's annual precipitation (400- 800 mm) occurs in the summer and is significantly less than that in southern China. Additionally, rivers and lakes in the area are few.

In the southern part of the Huanghuai Plain, the abundant rainfall at the end of spring and the beginning of summer

~~


Rice land races from several provinces in China.

Province

Anhui 725 Beijing 9 Fujian 1894

No.

Gansu Guangdong Guangxi Guizhou Hainan Hebei Heilongjiang Henan Hubei Hunan Jiangsu Jiangxi Jilin Liaoning Neimenggu Ningxia Shaanxi Shandong Shanghai Shanxi Sichuan Taiwan TianJin Xinjiang Xizang Yunnan Zhejiang

7 4863 8967 4279 668 325 113 365

1417 5001 2320 2869

68 78 10 18

642 128 304 169

3255 1190

27 16 38

5532 1585

Total 46,882

negatively affects rice growth. The rice- growing area and the number of land races there are much less than those in areas to the south of the Chanjiang River. In the Huanghuai Plain, the land races are concentrated in the lower valley of the Huaihe River and the coastal regions where water resources are abundant.

The area near the Bohai Sea in Hebei Province, the lower valley of the Liaohe River in northeastern China, and the Songhuajiang River Valley are the main areas for rice land races in regions at high latitudes.

The climate in northwestern China is continental and dry with a short wet season. Rice land races are mainly—but sparsely— distributed over river valleys. They constitute only about 1% of all the land races in China.

Some suggestions for in situ conservation of wild rices

S. N. Singh and M. Gadgil, Centre for Ecological Sciences (CES) , Indian Institute of Science, Bangalore 560012, India

India has seven species of wild rices. Four of these, Oryza nivara, O. rufipogon, O. sativa f. spontaea, and Posteresia coarctata, occur in the coastal and hilly districts of Uttara Kannada (13º 55' N to 15° 32' N latitude and 74° 5' E to 75° 5' E longitude) and Shimoga in Karnataka.

rices to determine their habitat require- We examined 125 populations of wild

ments and how ongoing ecological changes are affecting them. From 22 Oct 1992 to 6 Oct 1993, we collected seed and plant specimens from 43 locations and deposited them in a herbarium at the CES (see table).

Karnataka had not previously been explored for wild rices, so the intraspecific variation that remains can still be captured. Moreover, large patches of wild rices still exist that can be easily conserved in situ. These wild rices are locally known as Nyarai, Nyarai batta, Kadu batta, Kadu hullu, Uddinakardi battu, Uddirige Urbu, Chungu Nyari, and Navane.

A few plants of P. coarctata that were seen on 22 Oct 1992 at Kumta-Vannalli

Collection date, locality, and other information for some wild rice samples. Karnataka, India.

Collection Locality Taluk a Latitude Longitude Altitude Habitat Relative Species d

date (m) type b abundance c

22-10-92 Kumta-Vannalli K 14°25.25 74°24' 3.5 E E K 14°25.5' 74°24'

P 28-10-92 Dhareshwar 3 S F s/n 06-10-93 Dhareshwar K 14°25.5' 74°24' 3 S R s/n 23-10-92 Handigon K 14°23.5' 74°24.5' 4 F A s/n 23-10-92 Handigon K 4°23.5' 74°24.5' 4 F A s/n06-10-93 Handigon K 14°23.5' 74°24.5' 4 F E s/n 28-10-92 Handigon K 14°23.5' 74°24.5' 4 F F s/n 23-10-92 Handigon K 14°23.5' 74°24.5' 4 F A s/n 30-10-92 Manki K 14°26.75' 74°26' 5 C R s/n 23-10-92 Manki K 14°26.75' 74°26' 5 F A s/n 06-10-93 Manki K 14°26.75' 74°26' 5 F O s/n 05-11-92 Gudwi So 14°27' 75°01' 550 P A n/r 06-11-92 Gudnapur So 14°33' 74°59' 570 T A n/r 05-11-92 Yelasi So 14°22' 75°03' 580 T A n/r 04-11-92 Kondli Si 14°21' 74°54.5' 590 T A n/r 22-10-92 Sashitlu-Kumta K 14°25.5' 74°24' 3.25 F F S 03-11-92 Mattigar Si 14°18' 74°52.5' 580 F A S 03-11-92 Menasi Si 14°17' 74°49' 580 F A S 03-11-92 Menasi Si 14°17' 74°49' 580 F A S 03-11-92 Mattigar Si 14°18' 74°52.5' 580 F A S 05-11-92 Tyavgod So 14°25' 75°03' 585 T A r 04-11-92 Balekoppa Si 14°21' 74°54' 590 T F r 04-11-92 Avragoppa Si 14°22' 74°52' 585 T A r 04-11-92 Aigod Si 14°24' 74°56' 570 T A r 06-11-92 Kantraji So 14°22' 74°59' 555 T A 05-11-92 Konan mane So 14°22' 75°01' 560

r T A r

04-11-92 Andawalli So 14°22' 74°57' 570 T A r 04-11-92 Andawalli So 14°22' 74°57' 570 T A r 03-11-92 Akkunji Si 14°18' 74°53.5' 570 T A r 05-11-92 Kallambi So 14°26' 75°02' 570 T A r 04-11-92 Andawalli So 14°22' 74°57' 570 T A r 05-11-92 Sirlige Si 14°21' 74°56.25' 575 T A r 05-11-92 Sirlige Si 14°21' 74°56.25' 575 T A r 05-11-92 Hale Sorab So 14°23' 75°05' 580 T A r 03-11-92 Kawachur Si 14°16' 74°54' 580 T A r 05-11-92 Hale Sorab So 14°23' 75°05' 580 T A r 03-11-92 Nagarbawi Si 14°19' 74°51.8' 590 T A r 03-11-92 Hosur Si 14°20' 74°53' 595 T R r 03-11-92 Hosur Si 14°20' 74°53' 595 T A r 03-11-92 Hasvante Si 14°14' 74°54' 595 T A r 05-11-92 Gundasettikoppa So 14°22' 75°04' 595 T A r 03-11-92 Talguppa Sa 14°13' 74°54.25' 615 T A r

estuarine rice cultivation, F = farmer's field, P = pond, S = seasonal stream, T = tank. c A = abundant, F = frequent, a Taluk is an administrative unit below the district level; K = Kumta, Si = Siddapur, So = Sorab, Sa = Sagar. b E =

O = occasional, R = rare, E = locally extinct. d n = Oryza nivara, r = O. rufipogon, s = O. sativa f. spontanea. p = Porteresia coarctata.


have already become locally extinct when a tidal wetland area was converted into prawn culture. Other wild rice populations are threatened by the encroachment of small irrigation tanks, freshwater aquaculture, and a change in the cultivation system from broadcasting to transplanting.

In situ conservation of these important genetic resources must be initiated and complement ex situ conservation efforts. These measures could include

• Integration into conservation programs of tidal and freshwater swamps. For example, the Karnataka State Forest Department has a program to conserve mangrove

Panicle culture and karyotype analysis from callus cells of a diploid wild rice, Oryza meyeriana

Xiao-Ling Wang, Li-Hui Shu, Wen-Jing Yuan, and Lan-Jie Liao, School of Life Science, Wuhan University, Wuhan, Hubei 430072, China

The genus Oryza, to which the cultivated rice (O. sativa 2n=24) belongs, has 20 wild species with 2n=24 or 48 chromosomes. Oryza meyeriana (2n=24) is highly resistant to bacterial blight. The karyotype of this species has not been well documented and only a few tissue culture studies have been made. We report the results of in vitro culture of young panicles and karyotype analysis of this species.

Embryogenic calli with high plant regeneration ability. We obtained embryogenic calli with high plant regeneration ability using N6 and MS

swamps at Kundapur in Dakshina Kannada and a freshwater wetland at Gudwi Pakshidham in Shimoga. Other wetlands, such as those in Bharatpur in the state of Rajasthan. are being protected under the Ramsar Convention. Special attention should be paid to in situ conservation. including deliberately introducing into the wetlands appropriate indigenous wild rice species.

• Integration into ecotourism-based conservation programs. One of the study sites, Gudwi. is a bird sanctuary close to the tourist attraction of the Gerusoppa Waterfalls. Conservation

culture media alternately. The N6 medium contained 2 mg 2.4-D L -1 and 4.5% sucrose while the MS medium was supplemented with 2 mg kinetin L -1 , 0.5 mg NAA L -1 , and 3.0% sucrose. Green plant regeneration frequency of the selected calli was up to 76%, but that of the unselected calli was only 33% after the 10th culture. The free amino acids in the calli were determined. The methionine glutamic acid and the total amino acid contents in the selected calli were lower than those in the control, while the alanine and phenylalanine contents in the selected calli were higher than those in the control. The main components of the free amino acid pool in the selected calli were alinine. glutamic acid, and phenylalanine, which together constituted 65.8% of the total amino acids.

It appears that alanine. glutamic acid, methionine, and phenylalanine occupy a key position in amino acid metabolism and influence the regeneration capacity of calli.

of wild rices, along with environmental awareness programs, could be effectively integrated with the tourist attraction of aquatic birds.

• Use of indigenous knowledge. Some local indigenous communities, such as the Mushahars of eastern Uttar Pradesh and Bihar, depend on wild rice grain and other wild foods for survival. These people have an intimate knowledge of the distribution and habitat requirements of wild rices. It would be highly useful to involve these communities in in situ conservation programs.

High IAA oxydase activity was helpful in regenerating the calli.

The regenerated plants were tested for reaction to three bacterial blight strains: PXO61, T7174, and Jiang Ling 691. Fifty regenerated plants were inoculated with the three strains on different tillers of the same plant. The regenerated plants were resistant to the strains, but the degree of resistance differed among the plants.

Karyotype analysis. O. meyeriana has strong seed shattering and low seed fertility, making karyotype analysis using seed difficult. So instead, we used the method of Kurata et a1 to conduct karyotype analysis using calli.

The O. meyeriana had 2n=24 (see figure). Based on the arm ratio. the karyotype of O. meyeriana consisted of 4 pairs of metacentrics. 7 pairs of submetacentrics, and a pair of satellite- submetacentric chromosomes (see table).

a) The somatic metaphase showing 24 chromosomes in O. meyeriana; b) the karyotype of O. meyeriana (x7000).


Relative length, arm ratio, and classification of the chromosomes of Oryza meyeriana.

Chromosome number

1 2 3 4 5 6 7 8

10 9

11 12

Relative length (%)

11.80 11.48

9.87 8.80 8.69 8.31 8.11

6.97 7.26

6.79 6.17 5.74 b

Arm ratio (short/long)

0.82 0.90 0.67 0.70 0.73 0.69 0.73 0.63 0.93 0.88 0.69 0.37

Classification a

M M SM SM SM SM SM SM M M SM SAT

a M = metacentric, SM = submetacentric, SAT = satellite. Arm ratio: M = (1.0-0.76), SM = (0.75-0.25). b The length of the satellite is not inluded in the length of the chromosome.

IRRN reminder

only one report for a single Multiple submissions. Normally,

experiment will be accepted. Two or more items about the same work submitted at the same time will be returned for merging. Submitting at different times multiple notes from the same experiment is highly inappropriate. Detection will result in the rejection of all submissions on that research.

A study of Neolithic carbon-

from Hemudu, China ized rice grains excavated

Shengxiang Tang and Hanyong Yu, China National Rice Research Institute, Hangzhou 310006, China

Rice grains excavated from Hemudu, China (6950±130 BC) are known to be some of the world’s oldest remains of rice cultivation. We studied the variation in

these grains to improve our understanding that the Hemudu rice population had large of the origin and domestication of variation in grain shape and awn cultivated rice in China.

The length and width of 105 of the characteristics.

Our earlier study found a few wild rice grains were measured using an enlarged photograph. About half of the grains were excavated at Hemudu.

grains ( O. rufipogon ) among the rice grains

awnless. The grain length ranged from 5.2 We therefore infer that about 7,000 yr to 8.6 mm, with an average of 7.1 mm, and ago, people in Hemudu did primitive rice the grain width ranged from 2.1 to 3.2 mm, cultivation, and that the middle and lower with an average of 2.8 mm. The grain length-width ratio (L:W) was 1.7-3.2. with cultivated rice in China.

basin of Yangtze River is a homeland of

an average of 2.6. These findings suggest

Length, width, and shape distribution of 105 rice grains excavated from Hemudu, China.

Grain trait Distribution

Length (mm) (no.) (%)

(no.) (%)

Width (mm)

L-W ratio (no.) (%)

<5.4 1 0.9

2.1 2 1.9

1.7

0.9 1

5.6 2 1.9

2.2 3 2.9

1.8 0 0

5.9 3 2.9

6.2 6 5.7

2.3 3 2.9

2.4

0.9 1

1.9

0.9 1

2.0 2 1.9

6.5

6.7 7

2.5 5 4.8

2.1 5 4.8

21 6.8

20.0

2.6 9 8.6

2.2

6.6 7

29 7.9

27.6 11

7.4

10.5

7.7 9 8.6

11 8.0

10.5

20 2.7

19.0 24

2.8

22.9 18

2.9

17.1 15

3.0

14.3

2.3 5 4.8

12 2.4

11.4 12

2.5

11.4 16 2.6

15.2

8.3 4 3.8

3.1 4 3.8

13 2.7

12.4

8.6

0.9 1

3.2

0.9 1

2.8 5 4.8

16 2.9

15.2

3.0 4 3.8

>3.1 6 5.7

Classification of A genome species in the genus Oryza using nuclear DNA markers

K. Doi, A. Yoshimura, M. Nakano, and

of Agriculture, Kyushu University, Fukuoka N. Iwata, Plant Breeding Laboratory, Faculty

812-81, Japan; and D. A. Vaughan, National Institute of Agrobiological Resources, Tsukuba 305, Japan

We did a new analysis of the restriction

of 67 accessions of A genome species in the fragment lcngth polymorphisms (RFLPs)


genus Oryza, which contains O. rufipogon, O. nivara, O. glaberrima, O. barthii, O. longistaminata, O. glumaepatula, and O. meridionalis, and of the materials previously analyzed by Nakano et al (1992) (see figure). A total of 192 accessions of the A genome species were analyzed.

RFLPs were detected for combinations of Dra I-digested total DNA and 21 single copy genomic clones. Genetic distances between accessions were quantified as

D = -1n [2Mxy/(Mx+My)]

where Mx and My were the total fragments in accessions X and Y, respectively, and Mxy were the common fragments observed between accessions X and Y. A dendrognm was then constructed by the UPGMA method using 64 of the 67 new accessions and 12 previously analyzed accessions as a reference (see figure).

five major groups: Asian ( O. sativa, A genome species were classified into

O. rufipogon, and O. nivara ), O. glumaepatula, O. glaberrima- O. barthii, O. longistaminata, and O. meridionalis. O. glumaepatula had

rmguevarra

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RFLP-derived dendrogram of 76 accessions. Sixty-four out of 67 accessions provided by IRRI, and 12 previously analyzed accessions of O. sativa, O. rufipogon, and O. glaberrima (Nakano et al 1992), were included for reference; three O. rufipogon accessions from Papua New Guinea were excluded.


closer affinity to the O. glaberrima- O. barthii group than to the Asian O. rufipogon group, although it had been considered a subtype of O. rufipogon.

The Asian group comprised four or five loosely knit groups, each corresponding to the indica type (including some O. rufipogon ), japonica type (including O. rufipogon from China), O. rufipogon (consisting of several subgroups), and O. nivara (see figure). Some O. rufipogon- O. nivara showed close affinity to cultivated rice, as suggested by previous studies (Wang et al 1992, Nakano et al 1992).

Three accessions of perennial O. rufipogon from Papua New Guinea (excluded from the figure) carried both fragments of O. rufipogon and O. meridionalis. Although no O. meridionalis has been reported from Papua New Guinea so far, it is possible that some exist in the country and that the natural hybrids between O. rufipogon and O. meridionalis could have survived until now.

The classification among species matched well with the findings of previous studies (Morishima 1969, Second 1985, Wang et al 1992). Further analysis is needed to reveal the relationships among Asian subgroups.

Cited references Morishima H. 1969. Phenetic similarity and

phylogenetic relationships among strains or Oryza perennis, estimated by methods of numerical taxonomy. Evolution 23:429-443.

Phylogenetic study of cultivated rice and its wild relatives by RFLP. Rice Genet. Newsl. 9:132- 134.

Second G. 1985. Evolutionary relationships in the sativa group of Oryza based on isozyme data. Genet. Sel. Evol. 17:89-114.

Wang ZY, Second G, Tanksley SD. 1992. Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by nuclear RFLPs. Theor. Appl. Genet. 83:565-581.

Nakano M, YoshimuraY, Iwata N. 1992.

Detection of D genome chromosomes by genomic in situ hybridization

R. Shishido and T. Kinoshita, Hokkaido University, Sapporo 060, Japan; N. Ohmido and K. Fukui, Hokuriku National Agricultural Experiment Station, Joetsu 943-01, Japan

Based on chromosome pairing at meiosis, it is known that the genus Oryza has five genomes (A, B, C, E, and F) in the diploid species. Tetraploid species with BC or CD genomes have also been found, although a diploid species with the D genome has not yet been discovered. The D genome is only found in tetraploids ( O. alta, O. grandi- glumis, and O. latifolia from Central and South America) in combination with the C genome.

chromosomes on a glass slide in a CD genome tetraploid species. We used a chromosome painting method in which we applied the genomic in situ hybridization (GISH) method, using the total genomic DNA isolated from a C genome diploid species, O. officinalis (2n=24), labeled with biotin as the probe. O. latifolia was used to prepare chromosomes.

Chromosome samples were prepared by an enzymatic maceratiod/air drying method. The chromosome samples were treated with an enzymatic mixture (2% cellulase onozuka RS, 1.5% macerozyme R-200,0.3% Pectolyase Y-23), proteinase K (1 mg ml -1 ), 4.5% acetic acid, and RNase A (1 mg ml -1 ) prior to in situ hybridization to get clear genome-specific fluorescent signals on small chromosomes.

In this study, we visualized D genome

The post-treatment removed cytoplasmic debris, cellular protein, and RNAs that cause nonspecific signals. In addition, a modified thermal cycler with a flat aluminum plate was developed and used to precisely control temperature. The results of GISH were analyzed using CHIAS 2, an image-analyzing system that provides clear fluorescent signal reproduction. This system can capture the fluorescent signals of GISH within a second, enhance weak signals, and even superimpose the signal images onto the original chromosome

chromosomes, detected by the fluorescent probe of O. officinalis, from a mixture of 48 O. latifolia chromosomes belonging to the C and D genomes. All 24 D genome chromosomes were clearly identified using the imaging method, as were 24 C genome chromosomes from the 48 chromosomes with B and C genomes.

strength of fluorescent signals between C and D genomes, and between B and C genomes. The differences in the fluorescent signal strength between B and C genome chromosomes were more distinct than those between C and D genome chromosomes, despite applying the same probe. These differences reflect the sequence homology between the C and D or B genomes. The D genome is considered to be more similar to the C genome than to the B genome.

We will continue to use the GISH

We identified 24 C genome

We also found differences in the relative

method to provide new information about the relationships among the six genomes in Oryza.

Fingerprinting rice germplasm using ALP and PCR-based RFLP

B. Ghareyazie, Faculty of Agriculture, University of Guilan, Rasht, The Islamic Republic of Iran; N. Huang, G. Second, J. Bennett, and G. S. Khush, IRRI

Morphological and isozyme markers provide a simple method for classifying rice collections. However, the markers available and their level of polymorphism are inconveniently low. DNA markers, on the

other hand, are abundant, high in their level of polymorphism, and known to be useful for germplasm surveys.

We evaluated the usefulness of convert- ing mapped restriction fragment length polymorphism (RFLP) markers to polymerase chain reaction (PCR)-based markers, such as amplicon length polymorphism (ALP), and PCR-based RFLP (Ghareyazie et al 199.5). This is the first report on applying ALP and or PCR to classify rice and on classifying Iranian rice varieties at the DNA level.


We used a set of 35 Iranian varieties, along with three indica and two japonica varieties as references (Fig. 1). A set of 15 pairs of primers were used to amplify genomic DNA isolated from the 40 varieties. To examine ALPS, PCR products were separated in agarose gels containing ethidium bromide. The products were then digested with nine different restriction endonucleases (recognizing 4-5 bases) and separated on either agarose or polyacrylamide gels. Amplified PCR products and patterns generated upon their digestion were scored for the presence or absence of bands in each variety. These data were used to construct a resemblance matrix according to Dice distance coefficients. We used the NTSYS program to perform cluster analysis and to build the dendrogram according to the UPGMA clustering method.

Agarose and or polyacrylamide gel analysis of the digested PCR products showed a variety of banding patterns.

Diversity at loci RG118, RG13, and RG235 was exhibited prior to and/or after digestion of PCR products (Fig. 2). Six of the 15 pairs ofprimers used showed ALPS (Fig.2c, e). The others generated monomorphic PCR products across 40 varieties (Fig. 2a). These monomorphic banding patterns were converted to polymorphisms by digesting PCR products with Mval (Fig, 2b).

Using the formula

where n is the total number of alleles in a given locus, and X i and X j are the number of varieties carrying the ith and jth alleles. respectively, we calculated the number of polymorphic pairs (NP) as a measure of the abundance of ALP (13%) and PCR (28%) in rice.

distances’ matrix (Fig. 1). A clear distinc- tion exists between the two major rice subspecies, indica and japonica. Three groups were distinguished among Iranian

A dendrogram was built from the genetic

varieties, (indicas and japonicas, and varieties that are genetically distinct from both indica and japonica types). The varieties are traditionally divided into the major groups of Sadri, Champa, and Gerdeh.

One major group included all of the Sadri-type varieties from northern Iran (Guilan and Mazandaran provinces), where rice is the major crop. This group was clustered with two japonica standards. The second group included only six varaties (entries 5, 9, 14, 15, 21, and 16, Fig. 1). It was not clustered with any of the standard varieties, indicating the varieties probably evolved independently within the country.

included only three Champa varieties from Iranian germplasm and was clustered with the three indica standard varieties.

Sadri-type varieties, which are strongly scented and well known for their cooking quality, are genetically closer to japonicas

The third group. the smallest one,

1. Dendrogram based on Dice's genetic distances demonstrating relationships among 40 rice varieties. Genetic distance was calculated according to Dice's method based on the pooled ALP and P-BR data of 13 loci. The NTSYS program was used to build the dendrogram according to the UPGMA clustering method.


n–1 n NP = S (X i × S X j )

i = 1 j = i + 1

2. Ethidium bromide staining of PCR products for RG118 (A), RG13 (C) and RG235 (E) loci amplified from total genomic DNA of Iranian varieties and restriction fragments generated from the products digested with Mva l (B) and Hin Fl (D). The numbers for each lane are the entry numbers used in Figure 1. The approximate size of bands is shown in the base pair.

than to indicas. Despite their very typical indica morphology (long and cylindrical seed, tall stature, narrow leaves), these varieties, as well as some of the Champa- type varieties, are clustered with standard japonicas. This suggests that the morphological characteristics are not representative of the genetic status of a given variety, and as revealed by Glaszmann (1987), these varieties may belong to isozyme group V

and are therefore neither pure indica nor Cited references pure japonica. Ghareyazie B, Huang N, Second G, Bennett J,

We found PCR-based RFLP to be faster, Khush GS. 1995. Classification of rice easier, and relatively cheaper than South- germplasm. I. Analysis using ALP and PCR- ern-based RFLP. PCR markers are now based RFLP. Theor. Appl. Genet. 91:218-

abundant, with known map locations. Their application is highly reproducible and more informative than randomly amplified

227. Glaszmann JC. 1987. Isozymes and Asian rice

varieties. Theor. Appl. Genet. 74:21-30.

polymorphic DNA.

Distribution and evolutionary differentiation of mitochondrial plasmidlike DNAs in the genus Oryza

S. Miyata, A. Kanazawa, N. Tsutsumi, Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan; Y. Sano, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan; and A. Hirai, Faculty of Agriculture, The University of Tokyo

We investigated the distribution of plasmidlike DNAs homologous to those of O. sativa in the genus Oryza and found some evolutionary differentiations of plasmidlike DNAs. Four kinds of circular mitochondrial plasmid-like DNAs, designated B1, B2, B3, and B4, have been detected in many Oryza sativa strains and analyzed in DNA sequences.

We first analyzed the distribution of plasmidlike DNAs homologous to those of O. sativa in 40 strains of the genus Oryza with AA, BB, BBCC, CC, CCDD, and EE genomes (Miyata et al 1995). Using the B1, B2, B3, and B4 clones as probes, plasmidlike DNAs were observed only in varieties with AA, CC, and CCDD genomes. The distribution patterns of strains with the AA genome were highly polymorphic. This suggests that the disappearance of plasmidlike DNAs occurred during the diversification of strains with the AA genome.

from strains with the AA genome by polymerase chain reaction (PCR) and examined restriction fragment length polymorphisms (RFLPs). RFLPs were detected among plasmidlike DNAs amplified from different strains (Miyata et al 1995), indicating some mutations, such as base substitutions.

We then amplified the plasmidlike DNAs

In CC and CCDD genomes, some signals hybridized with the B1 probe showed rather different restriction patterns, indicating that these signals are new plasmidlike DNAs homologous to B1. We cloned them and named them M1, M2, and M3. Their entire nucleotide sequences were determined; M1 is 2,430 bp in size, M2 is 2,034 bp, and M3 is 2,096 bp. We compared each of these sequences and found that M1, M2, and M3 share high homology to B1. B1, M2, and M3 each lack about 300 bp of a region that is present in M1, and small repeats were found at the sites of deleted sequences (see figure).

the B1 family differentiated from a common ancient molecule that was similar to M1 through slipped mispairing during DNA replication at several stages in the evolution in the genus Oryza.

We therefore propose the hypothesis that



Genetic diversity of Chinese wild rice populations

Cai Hong-wei, National Institute of Genetics, Mishima, 411 Japan; Wang Xiang-kun, Beijing Agricultural University, Beijing, 100094, China; and H. Morishima, National Institute of Genetics, Mishima, 411 Japan

The genetic diversity of Chinese rice Oryza rufipogon and its relationships with the strains distributed in tropical Asia have not been fully investigated.

We examined three samples from populations collected in southern China from Dongxiang, Jiangxi (DX, the northern- most site of this species), Yuangjing, Yunnan (YJ), and Guigang, Guangxi (GG) for

various phenotypic characters, isozymes, and nuclear genome restriction fragment length polymorphism (RFLP). Four populations collected from Thailand, India, and Indonesia were used as the controls. A total of 292 plants were examined.

O. rufipogon is differentiated into two types: perennial and annual (often referred to as O. nivara). We have found Chinese wild rices that are perennial, but they tend to differ from the typical perennial type from tropical countries in terms of root and shoot regeneration pattern from basal nodes, dry weight proportion of newly emerged tillers after heading, and panicle number per plant.

The result of factor analysis based on 11 characters indicated that the populations examined, though they contained intrapopulational variability, showed inter- populational differentiation into annual and perennial types. Chinese populations were

intermediate between the annual and perennial types.

Polymorphism was found in 29 isozyme loci and 15 probe/restriction enzyme combinations. Differences among populations seemed to be clearer in RFLP than in isozyme variation. Chinese populations share some common markers that are rarely found in other populations, indicating that they differ from one another (Table 1). The result of factor analysis of isozyme data revealed geographic variation, but not ecological variation (perennial vs annual differentiation).

Interestingly, populations of Chinese rice O. rufipogon contain some “japonica- specific alleles,” such as Est-13 (t)-1 and Cat1-2, at higher frequencies than in other populations (Table 1). At other loci, however, they carry some “indica-specific alleles,” as in other populations.

The schematic representation of the explanation for the generation of members of the B1 family of mitochondrial plasmidlike DNAs during the diversification of the genus Oryza a

Shaded portions indicate M1 and the regions a

homologous to M1. Large arrows indicate the differentiation of the B1 family. The dashed lines of A, B, and C indicate the deleted regions of M1 and the corresponding sites in B1, M2, and M3, respectively. White and black arrowheads indicate the small repeats of 3 bp and 15 bp at the deleted sites of A and B, respectlvely.

.

Cited reference Miyata S, Kanazawa A, Tsutsumi N, Sano Y,

Hirai A. 1995. Polymorphic distribution and molecular diversification of mitochondrial plasmid-like DNAs in the genus Oryza, Jpn. J. Genet. (in press.)

Table 1. Allelic frequencies at six isozyme loci and four RFLP markers.

Population

China Thailand Indonesia India

DX YJ GG NE3 NE88 W1981 W120

P a P P A P P P

Locus/allele

Isozyme Sdh1-2 0.90 0.89 0.92 0.00 0.00 0.00 Amp1-3 1.00 1.00 0.50 0.43

0.68 0.96

Acp7 (t)-1 0.00 0.04

1.00 1.00 0.81 0.19 0.33 0.56 0.92 0.47

0.21 Est9-1 Est13 (t)-1

0.29 0.00 0.01 0.00 0.98 1.00

0.07 0.59 0.00 0.00

Cat1-2 0.99 1.00 0.00 0.00

0.04 0.00 0.00 0.00 0.25

RFLP 0.65 1.00 0.10 0.00 0.07 1.00 1.00 0.96 0.65 0.01 1.00 0.65 0.63

0.64 0.00 0.00

0.65 1.00 0.78 0.00 0.01 0.00 0.00 G20-20 G249-1 2

a P = perennial, A = annual.

Table 2. Parameter values showing intrapopulational genetic diversity estimated from data of 29 isozyme loci and 15 RFLP markers.

Population Parameter

DX YJ GG NE3 NE88 W1981 W120

Isozyme (no. of plants examined) 54 14 68 74 46 22 14 Av gene diversity (H) 0.07 0.04 0.20 0.14 0.21 0.22 0.25 Proportion of polymorphic loci 0.55 0.10 0.83 0.41 0.69 0.55 0.59 Alleles/locus (av no.) 1.55 1.10 2.10 1.45 1.97 1.72 1.75 Frequency of heterozygote 0.13 0.14 0.53 0.07 0.46 0.46 0.71

RFLP (no. of plants examined) 46 14 63 74 41 Av gene diversity (H) 0.13 0.19 0.27 0.20 0.12 - Proportion of polymorphic loci 0.31 0.46 0.73 0.53 0.60 Alleles/locus (av no.) 1.38 1.46 1.80 1.53 1.67 - Frequency of heterozygote 0.54 0.00 0.30 0.03 0.27 -

G187-1 2 G282-2 2

- -

- - - - -

- - -

-

-

Several parameters for intrapopulational genetic diversity were estimated for each population using isozyme and RFLP data (Table 2). These parameters showed no correlation between isozyme and RFLP level. The DX and YJ populations, which are completely isolated from cultivated ricefields, showed less polymorphism than the perennial populations from other countries at the isozyme level, but this trend was not found at the RFLP level. The high gene diversity at both the isozyme and DNA levels in the GG population adjacent to cultivated ricefields is probably due to the flow of genes from these neighboring cultivated rices.

Molecular analysis of introgression in Oryza sativa/ O. brachyantha and O. sativa/O. granulata derivatives

R. K. Aggarwal, D. S. Brar, N. Huang, and G. S. Khush, IRRI

The genus Oryza, to which cultivated rice belongs, has 20 wild species that provide an important reservoir of useful genes for rice improvement. O. brachyantha (2n=24, FF) and O. granulata (2n=24), two distantly related species, possess genes for resistance to yellow stem borer, bacterial blight, and brown planthopper. Several backcross progenies of these two species with elite breeding lines of O. sativa have been developed and are being analyzed for

possible introgression at the phenotypic and molecular levels.

The objective of our investigation was to determine the nature and magnitude of introgression from O. brachyantha and O. granulata into rice and to characterize the nature of extra chromosome of wild species in monosomic alien addition lines (MAALs) using molecular markers.

The material comprised parents ( O. sativa cultivar IR56 and an elite breeding line IR31917-45-3-2, O. brachyantha accession 101232, and O. granulata accession 100879), F 1 , BC 1 , and advanced backcross progenies. We analyzed 30 derivatives (12 BC 3 F 3 , 11BC 3 F 5 , 7 MAALs) of O. sativa/O. brachyantha and 40 derivatives (32 BC 2 F 3 , 5 BC 3 F 1 , 3 BC 4 F 1 ) of O. sativa/O. granulata. RFLP analysis was carried out using mapped markers on chromosomes 6, 7, 9, 10, 11, and 12 (Causse et al 1994). Forty-seven markers comprising 27 cDNA and 20 genomic probes were used with seven restriction enzymes ( Eco RI, Eco RV, Dra 1, Hind III. Bam HI, Xba 1, Sca 1). The hybridizations and washings were carried out at 60 °C under high salt conditions.

All of the marker-enzyme combinations, except a few, were polymorphic (see table). The hybridization signal intensities for the marker-specific target DNA sequences. done under conditions permissible to detect 70-75% DNA homology (washing stringency of 2X SSC at 60 °C), were moderate to low for most of the markers in both the wild parents, especially O. granulata (see table). The divergence was pronounced for chromosomes 9 and 12

~

specific-marker sequences in both wild species.

were identified based on phenotypic and cytological observations. were confirmed at the molecular level. In most of the MAALs, the alien chromosome was modified.

Five MAALs of O. brachyantha, which

Despite the high divergence of alien genomes from cultivated rice, intro- gressions were detected for a few of the markers in the derivatives from both of the crosses (Fig. 1a). The analysis of O. granulata derivatives revealed two new MAALs for chromosomes 9 and 11 (Fig. 1b). In general, the frequency of introgression was low. Several markers detected nonparental bands in wide-cross derivatives (Fig. 1b). About half of these involved modifica- tion(s) of Eco RI-specific alleles. Further analysis is under way to determine why such bands occurred.

The results showed that the genomes of O. brachyantha and O. granuluta are highly diverged from that of cultivated rice, with O. granulata being more diverse. Despite high divergence, chromatin material can get introgressed into cultivated rice from distantly related genomes of Oryza. The occurrence of nonparental bands in wide crosses is probably due to genome repatterning resulting from the interaction between unrelated genomes.

Cited reference Causse MA, Fulton TM, Cho YG, Ahn SN,

Chunwongse J, Wu K, Xiao J, Yu Z, Ronald PC, Harrington SE, Second G. McCouch SR. Tanksley SD. 1994. Saturated molecular map

backcross population. Genetics 138:1251- 1274.

of the rice genome based on an interspecific

Polymorphism and hybridization signal intensities for marker-specific target DNA sequences in the wild parents relative to those of O. sativa.

Chromosome Markers Polymorphic Monomorphic marker- Markers with null Hybridization signal a

tested markers enzyme combinations phenotype (%) (no.) (%) O. granulata O. brachyantha O. granulata O. brachyantha O. granulata O. brachyantha

6 9 100 bc 1/63 2/63 2 2 20-50 50-100 7 9 100 2/63 2/63 1 d 1 d 30-50 35-100 9 8 100 0/56 0/56 1 d 0 10-20

10 6 100 1/42 1/42 1 1 30-50 11 8 100 1/56 1/56 1 1 20-50 40-100 12 7 100 0/49 0/49 1 d 0 10-20 20- 30

20- 40 35-100

Total 47 100 5/329 6/329 8 5

a Under relaxed conditions of hybridizations and washings permissible to detect 70.75% DNA homology relative to O. sativa. b For at least five restriction enzymes. c Markers with null phenotype are considered polymorphic. d No discrete bands: signal highly dispersed: O. sativa alleles multicopy.


Genetics

Dra 1 - RFLP patterns of introgression lines ( O. sativa/O. granulata ). Lanes. - molecular weight marker; 1-10 and 14-23 - different derived lines; 11 - O. sativa lR31917-45-3-2; 12- O. granulata (acc. 100879); 13-F 1 hybrid. a) Probe RZ884, chromosome 6. See O. granulata - specific allele in lanes 2 and 3 (marked with arrow). b) Probe R2797, chromosome 11. Nonparental band in lane 1 and O. granulata alleles in derived-MAALs for chromosome 11 (lanes 6 and 7).

Acrotrisomics in rice

K. Ikeda, H. Furuumi, A. Yoshimura, H. Yasui, and N. Iwata, Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka 812-81, Japan

Researchers have extensively used primary trisomics in genetic mapping of rice ( Oryza sativa L.). However, no information

existed previously on the occurrence of acrotrisomics in rice.

We isolated six acrotrisomic plants from selfed progenies of primary trisomics and from the F 1 obtained from pollination of primary trisomics with irradiated pollen. An extra chromosome was identified in these acrotrisomics. One of the acrotrisomics was used in chromosome mapping.

and Triplo 7 were used for crossing as female plants. Among the F 1 plants,

Triplo 4, with liguleless gene ( lg lg lg ),

acrotrisomic-like plants were selected based on the phenomenon of pseudo- dominance (Triplo 4) and abnormal morphological characters (Triplo 7). In the progenies of primary trisomics (Triplo 4, 5, 7, and 11), acrotrisomic-like plants showed abnormal morphological characters spontaneously.

somics were identified by mitotic chromosome analysis (Acro4S 4L -a, Acro4S 4L -b, Acro5, Acro 11) and/or restriction fragment

Among the selected plants, six acrotri-


l

length polymorphism (RFLP) gene dosage analysis (Acro4S 4L -a, Acro7a, Acro7b). To distinguish two Acro4S 4L isolated from different origins, the acrotrisomics were designated as Acro4S 4L -a and Acro4S 4L -b, and Acro7.

Acro4S 4L -a and Acro7a were selected in the F 1 plants; the other acrotrisomics were obtained from the progenies of primary trisomics. Acro4S 4L -a and Acro4S 4L -b each had a complete short arm (4S) and heterochromatin region of the long arm (4L) on chromosome 4 in addition to a normal chromosome complement as identified by mitotic chromosome analysis. RFLP gene dosage analyses were studied based on RFLP map (Saito et a1 1991). RFLP gene dosage effects of Acro4S 4L -a were observed at the loci on XNpb203, 237, and 311 but not at the loci on XNpb49, 114, and 120 on chromosome 4. These plants had narrow grains, as did those of Acro4S 4L -b.

of chromosome 5, and the plants had small grains and short panicles. RFLP gene dosage effects of Acro7a and Acro7b were observed at the locus of XNpb338 but not at the loci XNpb33 to 22 on chromosome 7. These plants had compact panicles and round grains. Acrol1 had a fragment containing heterochromatin. Morpho- logical features of the plants were late heading and short stature.

acrotrisomic was easily transmitted to the

Acro5 had a fragment of about one-third

The acrocentric chromosome of each

Molecular mapping of fertility-restoring gene Rf3 in rice

G. Zhang and Y. Lu, South China Agricultural University, Guangzhou 510642, China; and N. Huang, IRRI

Cytoplasmic male sterility in WA-Zhen- shan 97 A (ZSA), a male sterile line widely used for developing hybrid rice in China, is caused by the interaction between wild abortive cytoplasm and two nuclear genes.

A series of near-isogenic lines (NILS) carrying different genotypes for restoration was developed by nine backcrosses to ZSA using IR24, a restorer line with two fertility- restoring genes, as the donor (Zhang et a1 1994).


Breeding behavior of acrotrisomics upon selfing.

Plants (%) Acrotrisomlc

Disomics Acrotrlsomics Primary trisomics

Acro4S 4L -a 55.2 35.8 9.1 Acro4S 4L -b 61.9 37.1 0.9 Acro5 59.4 38.1 2.5 Acro7a 71.3 27.3 1.5 Acro7b 66.4 30.6 3.0

Av (total) 63.5 33.3 3.2

Plants (no.)

618 854 286 891 265

(2914)

next generation. The average frequency of acrotrisomics in the selfed progenies of five acrotrisomics was 33.3%, ranging from 27.3% in Acro7a to 38.1% in Acro5 (see table).

a suggested that the break point of the acrocentric chromosome was in the region between XNpb311 and 49 (10.5 cM) on chromosome 4, and that the chromosome lacked the region from XNph49 to lg on the long arm. These findings and integrated linkage map (Ideta et a1 1992) revealed the orientation of the marker genes on chromosome 4 (see figure). The arrangement of classical and RFLP marker genes from the long arm are lg-XNph120 -

RFLP gene dosage analysis of Acro4S 4L -

114 - d - 11 - 49- 311 - 237 - 203.

Cited references Ideta O, Yoshimura A, Matsumo T. Tsunematsu

H, Iwata N. 1992. Integration of convention- a1 and RFLP linkage maps in rice. I. Chromo- somes 1, 2, 3, and 4. Rice Genet. Newsl. 9:128-129.

Construction of chromosome 4 map using acrotrisomic-Acro4S 4L -a: classical linkage map (left), RFLP linkage map (center), and ideogram of chromosome 4 (right). a Italicized numbers indicate RFLP markers.

Saito A, Yano M, Kishimoto N, Nakagahra M, Yoshimura A, Saito K, Kuhara S, Ukai Y, Kawase M, Nagamine T, Yoshimura S, Ideta O, Ohsawa R, Hayano Y, Iwata N, Sugiura N. 1991. Linkage map of restriction fragment length polymorphism loci in rice. Jpn. J. Breed. 41:665-670.

To tag the restorer genes, the set of NILS

and the donor IR24 were used for randomly amplified polymorphic DNA (RAPD) analysis. From a survey of 720 random primers, we identified six RAPD markers that were associated with one of the two restorer genes. Three of the six RAPD markers (OPKO5-800,OPU10-1100, and OPWOl-350) were used as probes. They were located on chromosome 1 in a population of doubled haploid lines derived from cross IR64/Azucena.

An F 2 population was developed from the cross between ZSA and ZSRR21. ZSR21 is a NIL carrying two restorer genes. Thirty-six sterile plants in the F 2 segregating population were selected for linkage analysis. The three RAPD markers and three restriction fragment length

polymorphism markers (RG532, RG140, and RG458 located on chromosome 1) were found to be closely linked to the restorer gene. The distances between each of the six markers and the restorer gene were less than 4 cM in this population (see figure).

For fertility restorer genes in rice, Rf1 was the notation given to the gene located on chromosome 10, which restores male fertility in line CMS-B (Shinjyo 1975). Rf2 was the notation given to the gene located on chromosome 2, which restores male fertility in line CMS-L (Shinjyo and Sato 1994). In this study, the restorer gene located on chromosome 2, named Rf2 in a previous paper, is named Rf3 (Zhang et a1 1994).

Location of the restorer gene Rf3 on chromosome 1. The map was developed using the F 2

population of 36 sterile plants derived from the cross Zhenshan 97 A/ZSR21. Molecular markers are shown on the right side of the figure. Map distances (in cM) are based on the Kosambi function.

Cited references Shinjyo C. 1975. Genetical studies of

cytoplasmic male sterility and fertility restoration in rice, Oryza sativa L. Sci. Bull. Coll. Agric. Univ. Ryukyus 22:1-57.

Shinjyo C., Sato S. 1994. Chromosomal location of fertility-restoring gene Rf2. Rice Genet. Newsl. 11:93-95.

Zhang G, Lu Y, Huang N. 1994. Molecular analysis of introgressed chromosomal segments in a set of near-isogenic lines of rice for fertility-restoring genes. Rice Genet. Newsl. 11:147-149.

Characterization of common cis -regulatory elements responsible for endosperm- specific expression of rice glutelin gene

F. Takaiwa, Cell Biology Department, National Institute of Agrobiological Resources (NIAR), Tsukuba 305, Japan; T. Yoshihara, Biotechnology Department, Central Research Institute Electric Power Industry, Abiko 1646, Japan; H. Washida, F. Tanabe, and K. Ozawa, Cell Biological Department, NIAR, Japan; U. Yamanouchi, and K. Yamada, Biology Department, Toyama University, Toyama 930, Japan; and C-Y Wu and S. Toki, Cell Biology Department, NIAR, Japan

Glutelin is the major seed storage protein of rice, accounting for about 80% of total endosperm protein. Since glutelin genes are expressed only in maturing endosperm tissue and are regulated mainly at the transcriptional level, they provide excellent

model systems of regulation of tissue- specific gene expression.

Glutelin is coded for by a small multigene family composed of about 10 copies per haploid genome (Okita et al 1989, Takaiwa and Oono 1991, Takaiwa et al 1991). These genes are clearly classified into two groups—designated as GluA and GluB subfamilies—that share about 60-65% homology between members of each subfamily at the nucleotide sequence level (Takaiwa et al 1991). Comparison of the nucleotide sequence in the 5' flanking regions of six glutelin genes showed that the conserved sequences are restricted to the 13 bp of AACA motif around -70, the GCN4 motif around -160 and -90, and the GCAA motif around -350 from the transcriptional start site. Therefore, these motifs may be candidate for cis -regulatory elements controlling the endosperm- specific expression found in the glutelin genes.

elements responsible for the endosperm- specific expression of glutelin genes, the 5' and 3' nested deletions and internal deletions of the 5' flanking regions of the GluB-1 and GluA-3 genes were transcriptionally fused to either ß- glucuronidase (GUS) or firefly luciferase (LUX) reporter genes. These chimeric genes were transferred into the tobacco genome via Agrobacterium- mediated transformation system or into the rice genome using electroporation method. The GUS or LUX activities were assayed in maturing seeds.

The GUS reporter gene was expressed at high levels in the sub-aleurone layer of the maturing rice seed. Essential cis -regulatory elements governing the spatially and temporally specific expression of the glutelin genes were located within the first 437 bp and 245 bp of the promoter regions of the GluA-3 and GluB-1 genes, respectively (Yoshihara and Takaiwa 1995).

The AACA motif and the GCNA4 motif within the 100 bp of the 5' flanking region, common to all the glutelin penes, were the key elements determining endosperm- specific expression of glutelin genes. Deletion or site-specific mutagenesis of this proximal AACA motif remarkably reduced

To identify the common cis -regulatory ~

~

promoter activities. The GCN4 motif also acted as a major cis-regulatory element determining the quantitative level. Each of these elements is necessary, but not sufficient, for endosperm-specific expression because tissue specificity is retained irrespective of the absence of each element. However, deleting the entire region, which includes both the AACA and GCN4 motifs, abolished seed-specific expression, suggesting that the synergistic interaction between both elements determines the endosperm-specific expression of the glutelin genes.

To confirm the significance of com- bining the AACA and GCN4 motifs to confer the endosperm-specific expression, the regions (-245 to -145, - 113 to - 40), including both motifs, were fused to the truncated (-90 to +9) or core (-46 to +1) CaMV35S promoter/GUS cassette, and then introduced into tobacco and rice. Although neither the AACA motif nor the GCN4 motif—by itself—is capable of enhancing the truncated promoter of CaMV35S promoter, their combination conferred the endosperm-specific expression. However, the combination failed to activate promoter activity of the core CaMV promoter.

of two sets of AACA and GCN4 motifs or the combination of one set of these two motifs and the G-box motif is required for the endosperm-specific expression of the rice glutelin gene.

Cited references Okita TW, Hwang YS, Hnilo J, Kim WT, Aryan

AP, Larson R, Krishnan HB. 1989. Structure and expression of the rice glutelin mutigene family. J. Biol. Chem. 265:12573-12581.

sequences of two new genes for new storage proteinglutelin in rice. Jpn. J. Genet. 66:161- 171.

The results suggest that the combination

Taikawa F, Oono K. 1991. Genomic DNA

Takaiwa et al. 1991, Sequences of three members and expression of a new major subfamily of glutelin gene from rice. Plant Mol. Bid. 17:875-885.

Yoshihara T, Takaiwa F. 1995. cis -regulatory elements responsible for quantitative regulation of rice seed storage protein glutelin GluA-3 gene. (in press)


A fertility-restoring revertant, controlled by a single gene induced from a rice cytoplasmic male sterile line

Shen Yuwei, Institute of Genetics (IG), Fudan University, Shanghai 200433, China: Cai Qihua and Gao Mingwei, Institute of Nuclear- Agricultural Sciences, Zhejiang Agricultural University, Hangzhou 310029, China; and Wang Xunming, IG, Fudan University

Cytoplasmic male sterility is inherited steadily through the maternal parent. However, reversions of cytoplasmic male sterile (CMS) lines to male fertile lines, either by cytoplasmic or nuclear gene mutation, have been documented (Nawa et al 1987, Smith 1987, He et al 1989). We report a fertility-restoring revertant induced from an indica rice CMS line II-32A treated with -rays, which is perhaps the first case in rice.

Dry seeds (13% moisture) of CMS rice line II-32A, which possesses the cytoplasm of an Indonesian rice variety, were irradiated with 60 C- rays at a dose of 290 Gy. Eight plants with an open air seed-setting rate of more than 30% were selected from about 1,000 M 1 II-32A individuals planted in an isolated area. These plants showed segregation in later generations. Among them, line T24 segregated for completely sterile and fully fertile plants. The sterile plants had 100% pollen grains that were shrunken and not stainable with I 2 -KI. The fertile plants had a seed-setting rate of about

80% in the open air and more than 90% dark-stained pollen grains.

In the 1993 early season, revertant T24 line was testcrossed with Zhenshan 97 A and II-32 A for its fertility-restoring ability. The results were grouped into two sets. In set one, both the parental T24 and the F 1 plants were fertile. In set two, the parental T24 plants segregated into a 3 fertile-1 sterile ratio. F 1 plants showed a 1 fertile-1 sterile ratio, indicating that the revertant could restore the fertility of CMS lines and one pair of nuclear genes of T24 might control the restoring ability.

To clarify the above facts, the seed- setting rate distribution in F 2 s of the crosses between II-32 A, Zhenshan 97 A, DShan A, Xieqinzao A, and T24 were investigated in the 1993 late season in Hangzhou and in the 1994 early season in Hainan Island. The F 2 seed-setting rate distributions of all four crosses were clearly divided into sterility (0-40%) and fertility(70- 100%). The supplemental pollen vigor assay with I 2 -KI revealed the pollen grains of the sterile plants in the open air to be completely abortive, the seed setting of which resulted from cross pollination.

The ratio of the fertile plants to sterile ones in F 2 s of the four crosses was 3:1 ( = 1.12,0.20<p>0.30 for II-32 A/T24,

2 = 0.2735, 0.50<p>0.70 for Zhenshan 97 A/T24, =0.003,p>0.99 for DShan A,

2 =0.1077, p=0.30 for Xieqinzao A/T24). The genetic mode of the fertility-restoring ability of T24 was different from that of Minghui 63 and 20964, the leading

restorers in southern China (see table). The bagged seed-setting rate and the seed- setting rate in open air of II-32 A/20964 and II-32A/Minghui 63 were widely distri-

buted. The fertility-restoring ability possessed by 20964 and Minghui 63 was thought to be controlled by two pairs of nuclear genes, based on the proportion of sterile individuals to total segregating F 2 plants.

The fertility-restoring revertant created in this study is of significance because first, the reversion process inferred that rice CMS is not as complicated as previously thought (conditioned by the incompatibility between cytoplasm and nucleus). The revertant, still possessing sterile cytoplasm, provides not only a restoring gene source, but also a restorer selection system. The revertant also provides excellent material for CMS molecular studies.

Cited References He P, Li Z, Li T. 1989. Fertility restoring

mutation in T type wheat cytoplasmic male sterile line irradiated with 60 Co- g rays. Acta Genet. Sin. 16:1-6.

Nawa S, Sano Y, Yamada M, Fuji T. 1987. Cloning the plasmids in cytoplasmic male sterile rice and changes of organization of mitochondrial and nuclear DNA in cytoplasmic reversion. Jpn. J. Genet. 62:301- 3 14.

Smith RL. 1987. Mitochondrial DNA rearrangements in Pennisetum associated with reversion from cytoplasmic male sterility to fertility. Plant Mol. Biol. 9:277- 286.

Distribution of F 2 seed-setting rate for crosses of T24 with indica CMS rice lines.

Seed-setting rate distribution a

Cross 0 10 20 30 40 50 60 70 80 combination b 90 100 (3:1)

llA/T24 16 12 5 1 8 16 25 45 1.1200 0.20-0.30 ZSA/T24 13 11 2 3 2 3 21 42 0.2735 0.50-0.70 DSA/T24 19 3 5 6 7 21 30 0.0003 >0.99 XQZA/T24 18 4 3 3 3 11 18 33 0.1077 0.3 llA/MH63 3 6 6 9 9 10 9 3 1 1 llA/20964 3 6 10 7 7 18 17 7 3

a Data for llA/MH63 and llA/20964 are bagged seed-setting rate while others are seed-setting rate in open air. b ZSA = Zhenshan 97 A, DSA = DShan A, ZQZA = Xieqinzao A, MH63 = Minghui 63. c The number of restoring genes in MH63 and 20964 were estimated with this formula.

P

k=(logN-logM)/0/6021 c


c c 2

c c 2

g

c 2

Y. Takemoto, M. Y. Son, T. Ito, and H. Satoh, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan; T. Kumamaru, Plant Genetics and Breeding Laboratory, Japan Tobacco Inc., lwata, Sizuoka 438, Japan; and M. Ogawa, Faculty of Home Economics, Yamaguchi Women's University, Yamaguchi 753, Japan

High 57-kD polypeptide mutants (57H mutants) were induced by mutagen N-

methyl-N-nitrosourea (MNU) treatment (Kumamaru et al 1988), while the 57H spontaneous mutants were found in northern Asian varieties. Four 57H mutant loci ( esp2, Glup1, glup2, and glup3 ) have been located on chromosomes 11, 9,9, and 4, respectively (Satoh et al 1994).

The 57-kD polypeptides of these mutants reacted with anti-glutelin ß subunit antibody, indicating that 57H mutants accumulate 57 kD glutelin precursors. Glutelins in wild-type Kinmaze were extracted using 1% lactic acid solution (acid solution). The presence of prolamins did not affect extraction of glutelins at all. In

esp2 mutant CM1787, the glutelin precursors could not be extracted without first removing the prolamins. In Glup1 mutant EM61 and glup2 mutant EM305, the glutelin precursors were extracted to a slight extent by acid solution, whereas removing the prolamins promoted extensive extraction of glutelin precursors.

These results indicate that glutelin precursors in these mutants coexist with prolamins. In glup3 mutant HO1274, the glutelin precursors were extracted as easily as the glutelins of Kinmaze, suggesting that the deposition site of the glutelin precursors is different from that of the prolamins.

Effects of Se1 gene on basic vegetative growth of rice

M. Niwa, K. Terakado, and N. Takeda, School of Agriculture, lbaraki University, Ami-machi, Inashiki-gun, Ibaraki, 300-03, Japan

The Se1 gene, located on the sixth chromosome of rice (Oryza sativa), plays an important role in determining the heading time in many rice varieties (Yokoo et al 1980). Through backcrossing, one of the alleles on the Se1 locus, Se1-u, was introgressed from Malaysian rice variety Morak Sepilaito to a genetic background of Fujisaka 5, which has Se1-e (Yokoo and Fujimaki 1971). This occurrence suggested that Se1-u brought late heading by increasing photoperiod sensitivity. Yokoo and Kikuchi (1982) proposed that Se1-u controlled basic vegetative growth.

Our objectives in this study were to clarify the effects of Se1-e on vegetative growth and determine whether those effects varied with photoperiod.

Two near-isogenic lines of rice with the genetic background for Fujisaka 5 and different alleles on the Se1 locus, ER ( Se1- e ) and LR ( Se1-u ), were exposed to various photoperiod regimes in two series of experiments.

and grown in a phytotron at 28°C for 10 d under a 24-h, long-day (LD) photoperiod. Then some of the plants were shifted to a 12-h, short-day (SD) photoperiod at 4-d intervals (LD-SD shifting). In the second series, LD-SD shifting was conducted, using a 10- or 13-h photoperiod as SD and a

In the first series, the plants were seeded

24-h photoperiod as LD in the open air chamber with a mean temperature of 32°C.

Three plants were grown per pot; two pots were used per treatment. White fluorescent light (600 1x at the soil surface) was supplemented with sunlight to prolong photoperiod as needed.

1. Days after sowing to heading of isogenic lines ER and LR, as affected by days after sowing when the plants were alternately shifted from 12 to 24 h photoperiod every 4th day.

2. Days from sowing to heading of isogenic lines

sowing when the plants were shifted from a 10- or 13-h to a 24-h photoperiod.

ER (a) and LR (b) as affected by days from

In the first series, the horizontal parts of the line graphs were determined by averaging days to heading (DTH), which did not differ significantly from those of the control plants grown under 12 h SD from sowing to heading (Fig. 1). Increase in DTH with days from sowing to shifting was approximated by linear regression using significantly larger DTH than those of the control.

Consequently, the turning points of the graphs project estimated days after sowing during which SD did not accelerate DTH. These days are the photoperiod-insensitive phase (PIP) for each isoline. The estimated length of PIP was 24.8 d for ER and 18.0 d for LR.

In the second series. the estimated length of PIP for each isoline and for each photoperiod regime was 16.2 d for ER-10 SD, 18.8 d for ER-13 SD, 12.7 d for LR-10 SD, and 10.7 d for LR-13 SD (Fig.2). Here, ER also exhibited longer PIP than LR. PIP did not differ significantly between regimes within the same isoline.

We concluded that Se1 affected not only photoperiod sensitivity but also duration of PIP, and that duration of PIP did not vary with photoperiod but rather with temperature.

Cited references Yokoo, M, Fujimaki H. 1971. Tight linkage of blast

resistance with late maturity observed in different indica varieties of rice. Jpn. J. Breed. 21:35-39.

Yokoo M, Kukuchi F, Nakane A, Fujumaki H. 1980. Genetical analysis of heading time by aid of close linkage with blast resistance in rice. Bull. Natl. Inst. Agric. Sci. D31:96 - 126.

Yokoo M, Kukuchi F. 1982. Monogenic control of basic vegetative phase and photoperiod- sensitive phase in rice. Jpn. J. Breed. 32: 1-8.


Genetic and cytochemical analysis of high 57-kD polypeptide mutants in rice

1. Electron micrographs of the developing endosperm of a) Kinmaze and b) CM1787 ( esp2 mutant). Arrow denotes new type of protein bodies. Bar = 0.5 µm.

Two types of protein bodies (PB) (PB-I and PB-II) were observed in the endosperm of Kinmaze, as reported by Tanaka et al (1980). In CM1787, although new types of PB and PB-II were observed, PB-I was absent. Many ribosomes were observed on the surface of the new type of PB (Fig. 1 b), suggesting that these PBs, such as PB- 1, were derived from endoplasmic reticulum. Immunogold labeling (Fig. 2) showed that the glutelin precursors of esp2 mutants were deposited in the new type of PB together with prolamin, suggesting that the presence of glutelin precursor in PB-I leads to the formation of the new PB type. In three other types of mutants, both PB-I and PB-II were observed; the new type of PB was not found. The localization of glutelin precursors in these mutants was examined. These observations indicate that the different mutations in the processing pathway of glutelin precursors can cause PB variations among mutants.


2. Electron micrographs of the developing endosperm of esp2 mutant showing the specificities of a) anti- b subunit antibody and b) anti-13b prolamin polypeptide antibody for a new type of protein body (denoted by arrow). Bar = 0.5 µm.

We conclude that these mutations are concerned with the genes controlling the processing of glutelin precursors. The study confirmed that esp2 mutation can result in the deposition of 57-kD glutelin precursors in PB-I, and that Esp2 is a gene controlling the proteins responsible for targeting glutelin precursors toward the protein vacuole.

Cited references Kumamaru T, Satoh H, Iwata N, Omura T,

Ogawa M, Tanaka K. 1988. Mutant of rice storage proteins. 1. Screening of mutants for storage proteins in the starchy endosperm. Theor. Appl. Genet. 76:11-16.

Satoh H, Kumamaru T, Yoshimura S, Ogawa M. 1994. New 57kDa glutelin genes on chromosome 9 in rice. Rice Genet. Newsl. 11:158-161.

Tanaka K, Sugimoto T, Ogawa M, Kasai Z. 1980. Isolation and characterization of two types of protein bodies in the rice endosperm. Agric. Biol. Chem. 44:1633-1639.

Molecular mapping of genes for F 1 pollen sterility in rice

Zhuang Chuxiong, Zhang Guiquan, Mei Mantong, and Lu Yonggen, South China Agricultural University (SCAU), Guangzhou 510642, China

F 1 hybrid sterility, probably caused mainly by pollen sterility constrains the use of heterosis in the subspecific hybrid between indica (Hsein) and japonica (Keng). Zhang and Lu (1993) proposed that at least six loci are involved in controlling F 1 hybrid pollen sterility.

To map S-c, one of the F 1 pollen sterility genes, Taichung 65 (a japonica variety, denoted as E 1 ) and E 5 , its isogenic F 1 pollen sterile line carrying this gene, were used as parents. E 5 was developed using indica variety Pehku as the donor. It was then backcrossed with E 1 for 11 generations. One hundred and four F 2 plants from E 5 /E 1

were used for the segregation analysis. Pollen fertility was 98.5% for E 1 and

98.6% for E 5 ; however, pollen fertility for F 1 plants from the cross E 1 /E 5 was 53.2%. Segregation of fertile and sterile plants in the F 2 population from this cross fit well to a 1-1 ratio ( c 2 =0).

One hundred and seventy restriction fragment length polymorphism (RFLP) markers were used to survey polymorphism between E 1 and E 5 , and only three markers located on the same chromosome detected different RFLP patterns. The three positive markers (RG227, RG166, RG369A) were tightly linked to each other and were mapped on chromosome 3 (Causse et al 1994). Polymorphism was low between E 1 and E 5 , which could be explained by the short length of introgressed segments in the isogenic line, because it was developed through repeated backcrossing.

The polymorphic RFLP markers were then used to survey the filters with DNA from 104 F 2 plants, and cosegregation of fertility and RFLP patterns were analyzed. The genetic distances between the S-c locus and markers RG227 and RG369 were 0.5 and 2.5 cM, respectively (see figure).

functionally very similar to eubacterial ribosomes, most likely reflecting their endosymbiotic origin. Several chloroplast ribosomal protein genes have been cloned and sequenced, mostly in dicotyledonous plants. Publication of complete nucleotide sequences of the rice chloroplast genome (Hiratsukaet al 1989) has allowed scientists

Location of the S-c gene on chromosome 3. Markers are on the right and map distances (in chloroplast ribosomal proteins encoded in cM) are on the left. the nucleus. These nucleus-encoded

chloroplast ribosomal proteins are F 2 Segregation Patterns for RFLP markers in the synthesized on cytoplasmic 80S ribosomes cross between E 1 and E 5 . as precursor polypeptides with a transit Locus j/j j/i i/i c 2 peptide sequence, which is cleaved off

to predict about two-thirds of the 55-60

(1:2:1) during transport into the organelle.

RG227 0 51 53 50.1 RG166 2 52 50 44.3 clones for nuclear-encoded genes rpl13 and RG369 2 52 50 44.3 rpl28, which encode chloroplast ribosomal

proteins L13 and L28, respectively, in rice. Distorted F 2 ratios were observed for the With poly(A) + -RNA isolated from green

three RFLP markers in the cross (see table). leaves of 10-d-old rice seedlings (Oryza The results suggest the use of the one-locus sativa L. cv Nipponbare), cDNA libraries sporo-gametophytic model in explaining F 1 were constructed in ZAPII (Stratagene). pollen sterility in cultivated rice (Zhang and Based on the highly conserved region Lu 1993). among chloroplast ribosomal protein

Cited references tobacco and spinach, the corresponding rice Causse MA, Fulton TM, Cho YG. Ahn SN, DNA fragments were amplified using

Chunwongse J, Wu K, Xiao J, Yu Z, Ronald reverse transcriptase-polymerase chain PC, Harrington SE, Second G. McCouch SR, reaction and used as probes. Nucleotide Tanksley SD. 1994. Saturated molecular map sequences of positive clones were of the rice genome based on an interspecific backcross population. Genetics 138: 1251-

determined using the dideoxy nucleotide

1274. chain termination method on double-

Zhang G, Lu Y. 1993. Genetic studies on the stranded DNA templates.

hybrid sterility in cultivated rice ( Oryza The cDNA clone of rpl13 consists of 1,076 nucleotides, including poly(A) 29 tail, sativa ). II. A genic model for F 1 pollen

sterility. Chin. J. Genet. 20:249-255. and contains an open reading frame of 702 bp. The cDNA clone of rpl28 consists of 781 nucleotides, including poly(A) 20 , tail,

sequences reported in other plants, such as

Chloroplast ribosomal and contains an open reading frame of 411 protein genes encoded in bp. Compared with sequences from rice nuclear genome spinach and tobacco, amino acid sequences

deduced from cDNA sequences of rpl13 N. Tsutsumi, S. Takusagawa, M. Kurashige, and rpl28 are more conserved in the mature Y. Li, and A. Hirai, Laboratory of Radiation peptide region, which is actually assembled Genetics, Faculty of Agriculture, The into chloroplast ribosome. than in the transit University of Tokyo, Yayoi, Bunkyo-ku, Tokyo peptide region. The mature L13 protein has 113, Japan 85 and 58% amino acid sequence identity

with the counterparts of spinach (Phua et al Plant cells contain three distinct types of 1989) and Escherichia coli (Isono et al ribosomes that are found in the cytosol, 1985), respectively, in the homologous mitochondria, and chloroplasts. Chloro- overlapping regions (see figure, region C). plast ribosomes are structurally and The amino acid identity of the mature

A schematic representation of local alignment of amino acid sequences of rp128. Predicted amino acid sequence of rice rp128 cDNA was aligned with the homologues of spinach and E. coli using MACAW algorithm (Lawrence et al 1993). Darker shade indicates sequence blocks with higher homology scores.

protein of L28 is 75 or 32% relative to tobacco (Yokoi and Sugiura 1992) and E. coli (Lee et al 1981). The mature L13 protein of rice contains 24 amino acid residues of N-terminal exlension (see figure, region B), which is 16 residues shorter than that of spinach.

Cited references Isono S, Thamm S, Kitakawa M, Isono K. 1985.

Cloning and nucleotide sequencing of the genes for ribosomal proteins S9 (rpsl) and L13 (rplM) of Escherichia coli. Mol. Gen. Genet. 198:779-282.

Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. 1993. Detecting subtle sequence signals: a gibbs sampling strategy for multiple alignment. Science 262:208-214.

Lee JS, Am G, Friesen JD. Isono K. 1981. Cloning and the nucleotide sequence of the genes for Escherichia coli ribosomal protein L28 (rpmB) and L33 (rpmG). Mol. Gen. Genet. 184:218-223.

Phua SH, Srinivasa BR, Subramanian SR. 1989. Chloroplast ribosomal protein L13 is encoded in the nucleus and is considerably larger than its bacterial homologue. J. Biol. Chem. 264:

Hiratsuka J, Shimada H, Whiottier RF, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji R, Sun CR, Mong BY, Li YQ, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M. 1989. The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct RNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol. Gen. Genet. 217:185- 194.

Yokoi F, Sugiura M. 1992. Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28. FENS Lett. 308:258- 260.

1968-1971.


We isolated and sequence CDNA

Stability of mitochondrial plasmidlike DNAs of rice

A. Kanazawa, N. Tsutsumi, and A. Hirai, Laboratory of Radiation Genetics, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

B1 and B4 are small, circular plasmidlike DNAs found in rice mitochondria. We previously investigated the distribution of four kinds of plasmidlike DNA molecules in 85 Oryza sativa accessions (Kanazawa et al 1992). These molecules were found mostly in indicas, with a few in javanicas, and none in japonicas. Copy number also differed among the four kinds of molecules. Copy number was not conserved among cultivated rice varieties with different cytoplasms. It seems possible that mitochondrial plasmidlike DNAs could be replicated and stably maintained for at least several generations under normal environmental conditions. However, transmission of these DNAs may become irregular as a result of suppressed replication under unstable environmental conditions.

We examined whether temperature influences copy numbers of mitochondrial plasmidlike DNAs and main mitochondrial genomic DNAs, especially during cell proliferation. The observed quantitative fluctuations in levels of plasmidlike DNAs were larger and less closely related to cell proliferation than those of main mitochondrial genomic DNAs on which genes indispensable for mitochondrial biogenesis were located. The copy number of plasmidlike DNAs was reduced significantly in calli cultured at high temperatures (Fig. 1).

We developed an assay system for monitoring DNA synthesis in isolated mitochondria to examine the contribution of mtDNA replication to the quantitative fluctuations. Synthesis of the main mitochondrial genomic DNAs occurred at all the temperatures examined, whereas that of plasmidlike DNAs occurred only over a limited range of temperatures (Fig. 2). These results indicate the instability of plasmidlike DNAs relative to main mitochondrial genomic DNAs.


1. Gel blot analysis of DNAs from calli cultured at various temperatures. Total DNA (10 µg) isolated from calli cultured at 20 °C (lane 1), 25 °C (lane 2), 30 °C (lane 3), and 35 °C (lane 4) was digested with Stul to linearize plasmidlike DNA, B1. After electrophoresis, the gel blot was probed with 32 P-labeled B1 and atpA. Signals corresponding to B1 and atpA are indicated on the right.

2. Gel electrophoresis of products of DNA synthesis in isolated mitochondria. MtDNAs purified from reaction mixtures incubated at 5 °C (lane 1), 13 °C (lane 2), 20 °C (lane 3), 25 °C (lane 4), 30 °C (lane 5), 35 °C (lane 6), 42 °C (lane 7), and 50 °C (lane 8) were separated on an agarose gel. After electrophoresis, the gel was placed onto an imaging plate and radioactivity was visualized and quantified using a bio-imaging analyzer. One signal of low mobility and two signals of higher mobility indicate the incorporation of labeled nucleotide into the main mitochondrial genomic DNAs and the plasmidlike DNAs, respectively.

We have detected sequences homologous to mitochondrial plasmidlike DNAs that are integrated within limited chromosomal regions on the rice nuclear genome (Kanazawa et al 1993). This suggests interorganellar sequence transfer between mitochondria and nucleus. The unstable transmission of plasmidlike DNAs shown in this study implies that plasmidlike DNA sequences have been transferred from the mitochondria to the nucleus during at least the early differentiation phase among rice varieties, before the sequences were lost from the mitochondria of some rice varieties.

Cited references Kanazawa A, Sakamoto W, Nakagahra M,

Kadowaki K, Tsutsumi N, Tano S. 1992. Distribution and quantitative variation of mitochondrial plasmid-like DNAs in cultivated rice (Oryza sativa L.) Jpn. J.

Kanazawa A, Kishimoto N. Sakamoto W, Ohsawa R, Ukai Y, Tsutsumi N, Hirai A, Saito A. 1993. Restriction fragments homologous to mitchondrial plasmid-like DNAs are located within limited chromosomal regional on the rice nuclear genome. Theor. Appl. Genet. 87:3577-586.

Genet. 67:309-319.

Sensitivity of plant height genes to gibberellic acid and their regulation by endogenous plant hormones in rice

He Zuhua and Li Debao, Institute of Biotechnology, Zhejiang Agricultural University, Hangzhou 310029, China

We studied the sensitivities to gibberellic acid (GA 3 ) of genes governing plant height in five rice genotypes and how endogenous hormones regulate them. We also investigated proteins related to plant elongation.

Sensitivity of plant height genes to GA 3 . Five rice genotypes. Er-jiu-feng (EJF. semidwarf, sd1 gene), tall EJF (near- isogenic line [NIL] of EJF, tall Sd1 gene), tall Zhen-shan 97 (TZS, NIL of ZS, tall, eui gene), Chao ai (CA, dwarf, sdss(t) gene), and Kyushu-3 (D53, dwarf, D53 gene) were treated with 20 and 100 mg GA 3 L -1 at seedling, tillering, and heading stages. Plant elongation was measured 8 d after treatment. The sensitivities of rice genes to GA 3

1. Endogenous plant hormone contents in different rice genotypes: (a) GA 3 content (µg g -1 FW), (b) IAA content (nmol g -1 FW), (c) ZR content (pmol g -1 FW), (d) ABA content (nmol g -1 FW). GA contents of dwarf genotypes were 16.9-55.6% lower than those of tall genotypes. IAA contents were lower by 12.0-81.7% in the dwarf genotypes than in the tall genotypes. ZR and ABA contents varied among genotypes during plant growth.

2. Protein patterns in GA3-treated and nontreated NILS. Lanes 1-6 consisted of nontreated seedlings of TEJF, EJF, TZS, ZS, 320T, and CA; lanes 7-12 consisted of TEJF, EJF, TZS, ZS, 320T, and CA seedlings treated with GA 3 for 3 d. Proteins a (about 50 kD), b (about 62 kD), c (about 50 kD), which existed in nontreated EJF, TZS, and CA, are shown with arrows; these proteins appeared in other lines of the NIL pairs after 3 d of GA 3 treatment. Another new protein d (about 52 kD) also appeared in CA. Protein standard molecular weights are shown on the left.

were found to be ui>D53>sd1>Sd1>sds(t) classified as sensitive at all growth stages, at the seedling stage. However, sensitivities D53 as sensitive during seedling and of the genes changed to tillering stages, and Sd1 and sds(t) as D53>sd1>eui>sds(t)>Sd1 during tillering insensitive at all stages. and sd1>eui>D53>Sd1>sds(t) during Using endogenous hormones to regulate heading. The genes eui and Sd1 were plant height genes. Plant hormones were

assayed using the enzyme-linked immunosorbent assay method. More GA 3 was found in the uppermost internodes of heading plants of the tall genotypes (Fig. 1a) than in the other genotypes. Although the eui plants have the highest GA 3 , they were highly sensitive to GA 3 . The sds(t) plants were less sensitive to GA 3 despite their lower GA 3 content. GA 3 and indole acetic acid (IAA) were higher in the tall genotypes than in the dwarf genotypes (Fig. 1b), the highest IAA content was also found in the eui plants. The tall genotypes had higher zatin ribozide (ZR) content than the dwarf genotypes at seedling and tillering stages (Fig. 1c), while at heading, higher ZR contents appeared in the dwarf genotypes.

Except for the tall genotypes Sd1 and eui, in which the abscisic acid (ABA) contents remained stable from the tillering stage (Fig. 1d). increases in ABA of the three dwarf genotypes began during the seedling stage. Dwarf rice plants had more ABA and less GA 3 and IAA at tillering and heading stages. Dwarf seedlings had lower ABA. Seedlings and tillering plants with low ABA were more sensitive to GA 3 than those with higher ABA. The results indicate that genes governing plant height play an important role in regulating plant hormones.

Identifying GA 3 ,-induced proteins. According to sodium dodecylsulfate polyacrylamide gel electrophoresis of seedling proteins, three proteins (Fig. 2) with molecular weights of about 50,62, and 50 kD, existed only in the EJF (sdl) (a), TZS (eui) (b), and CA (sds(t)) (c) lines, respectively. After a 3-d GA 3 treatment, these proteins appeared in other lines of the NIL pairs and a new protein (d) also appeared in the CA plants. The proteins were possibly related to plant height and could have been induced by GA 3 .

Hormone receptors promote the responses of plants to exogenous hormones. Genes governing plant height may take part in regulating the receptor system through some message molecules, or the receptors may induce plant height genes to affect plant elongation. In another study, we found that amounts of peroxidases and esterases differed among seedlings with different genes.


Genetic diversity estimated among rice cultivars using restriction landmark genomic scanning method

M. Kawase, Laboratory of Plant Biotech- nology, Crop Improvement Department, Shikoku National Agricultural Experiment Station, 3-1, Sen'yu-cho 1, Zentsuji, Kagawa 765, Japan

The restriction landmark genomic scanning (RLGS) method is a new technique developed for analyzing genomic DNA of higher organisms (Hatada et al 1991). RLGS, which uses restriction enzyme sites as landmarks, requires direct labeling at the restriction sites and two-dimensional electrophoresis to resolve these landmarks. It gives a two-dimensional pattern with thousands of scattered spots after autoradiography. The RLGS method provides unbiased information on genetic polymorphism throughout the whole

genome, an accurate estimation of genetic similarity, and is applicable to rice cultivars (Kawase 1994).

We extracted DNA from leaves of 14-d- old seedlings of two Japanese improved cultivars (Nipponbare and Koshihikari) and a Chinese land race (Liu’ Zhou’ Bao’ Ya’ Zao)’ (Kawase 1994). RLGS was conducted based on the procedure described by Hatada et al (1991), which consisted of blocking reaction, landmark cleavage, labeling, first fractionation, and second fractionation. Genetic similarities between cultivars were calculated using the measure devised by Dice (1945), which was suggested for DNA polymorphism by Nei and Li (1979):

GS(i,j) = 2N(i,j)/[N(i)+N(j)], where GS(i,j) = the measure of genetic similarity between cultivars i and j; N(i,j) = the total spots common to i and j; and N(i) and N(j) = the number of spots for cultivars i and j, respectively.

More than 3,000 landmarks were detected as scattered spots on the auto-

radiogram (Fig. I). The relative position and the intensity of each spot were highly reproducible. An RLGS profile was unique to each cultivar used.

The RLGS profiles of Nipponbare and Liu’ Zhou’ Bao’ Ya’ Zao showed quite different patterns, indicating a wide genetic difference between the two cultivars. Restriction fragment length polymorphism analysis also revealed that they were distantly related (Kawase et al 1991). A limited area (Fig. 1) could be compared, although the different patterns made it rather difficult to identify each spot. Nipponbare and Liu’ Zhou’ Bao’ Ya’ Zao have 387 and 403 spots in the area, respectively, of which 136 were scored as common or indistinguishable spots. The low GS value, calculated as 0.344, suggests large genetic diversity among rice cultivars.

Nipponbare and Koshihikari showed similar patterns. An area showing the clearest resolution contained 1,156 spots common to both cultivars (Fig. 2).

1. More than 3,000 spots were scattered on the RLGS profile obtained by autoradiography (Kawase 1994). Nipponbare (a) and Liu'Zhou'Bao'Ya'Zao (b) showed quite different patterns. The enclosed area was used for estimating the GS value between the two cultivars.


2. RLGS profiles of Nipponbare and Koshihikari were highly similar. However, the cultivars were distinguished based on the presence and absence of particular spots in the enclosed area (Kawase 1994). Arrow heads indicate the spots unique to each of the two cultivars compared.

Additionally, 24 and 25 spots were found unique to Nipponbare and Koshihikari, respectively. The GS value was calculated as 0.980.

The RLGS method can be a powerful fingerprinting technique for estimating genetic diversity in rice as well as for identifying cultivars.

Cited references Takeda N, Nagamine T, Nakagahra M. 1991. Dice LR. 1945. Measures of the amount of Intraspecific variation and genetic differen-

ecologic association between species. tiation based on restriction fragment length Ecology 26:297-302. polymorphism in Asian cultivated rice,

Hatada I, Hayashizaki Y, Hirotsune S, Komatsu- Oryza sativa L. Rice genetics II. Manila bara H, Mukai T. 1991. A genomic scanning (Philippines): International Rice Research method for higher organisms using

Nei M, Li WH. 1979. Mathematical model for restriction sites as landmarks. Proc. Natl. Institute. p 467-473.

studying genetic variation in terms of Acad. Sci. USA 88:9523-9527. Kawase M, Kishimoto N, Tanaka T, Yoshimura restriction endonucleases. Proc. Natl. Acad.

A, Yoshimura S, Saito K, Saito A, Yano M, Sci. USA 765269-5273.

Character expression of a rice dwarf mutant with lax panicle

Y. Futsuhara, T. Horio, and S. Tanaka, Faculty of Agriculture, Meijo University, Tenpaku, Nagoya 468, Japan; and J. Yonemaru, Tohoku National Agricultural Experiment Station, Morioka, 020-01, Japan

A dwarf mutant with lax panicle was induced from the parent cultivar Fujiminori by 200 Gy of X-ray irradiation. The mutant exhibits leaf rolling during the early vegetative phase until the appearance of the 6th or 7th leaf. Inheritance of this mutant trait

was studied from an F 2 segregation involving a cross between the dwarf mutant and the parent cultivar. The F 1 was normal and the F 2 segregated into 103 normal plants and 39 dwarf plants, giving a good fit to a 3:1 ratio ( =0.460, P=0.498). Pleio- tropic action of the dwarf mutant gene appears to be responsible for the lax panicles and rolled leaves. Panicle density is a complex character, which consists of many component characters such as spikelet number, panicle length, and number of branches (Futsuhara et al 1979). Panicle laxness in this mutant could be attributed to the decrease in total spikelets and branches, especially secondary branches (see table).

Rolled leaf is a marker trait that can be used in selecting dwarf mutant types during juvenile stage.

variations in various agronomic traits, the dwarf mutant and its parent cultivar were tested in 1992 and 1994. The rice plants were raised under two seeding times (early and late seedings) only in 1994.

The dwarf mutant showed different responses to various seeding conditions (see table). The difference in panicle density between parent and mutant grown under late seeding condition was not significant. Seed fertility of the mutant, which was significantly less than that of the parent

To determine the annual and seasonal

IRRN 21:23 (August-December 1996) 25

Related characters of panicle density in a dwarf mutant with lax panicles and its parent cultivar, Fujiminori. a

Cultivar and Culm Panicle Panicle Spikelets Fertility Primary Primary Secondary Rachis length length density b (no.) (cm)

(%) branch branch branches length (cm) (no.) length (cm) (no.) (cm)

Year Seeding time strain

1992 Normal seeding Fujiminori 88.0 24.1 1.58 158.4 96.0 10.8 8.0 Dwarf mutant 74.8** 21.6* 1.11* 58.6** 66.2** 8.3**

32.8 18.7 7.3 16.5** 17.2

1994 Early seeding Fujiminori 95.3 21.8 1.24 111.5 89.5 10.5 6.3 18.3 16.6 Dwarf mutant 84.2** 18.8* 1.01** 68.1** 52.1** 7.2** 6.8 11.3** 12.7**

1994 Late seeding Fujiminori 62.2 14.0 1.10 47.7 87.6 6.0 6.6 5.8 Dwarf mutant 52.6** 12.8 1.09

8.6 39.9 34.5** 6.2 4.7* 5.1 8.7

a * and ** = significant at the 1 and 0.1% level, respectively. b Panicle density = no. of spikelets/total length of rachis and primary branches (in cm) (Futsuhara et al 1979).

cultivar under all three conditions, was further reduced under late seeding condition (during an abnormally hot summer). Premature heading occurred in all the mutant plants grown under late seeding condition. Most of the panicles with premature heading showed malformed floral organs such as degenerated ovary,

Genetic differentiation between Japanese lowland and upland rices

R. Ishikawa, Y. Harata, T. Harada, and M. Niizeki, Plant Breeding Laboratory, Faculty of Agriculture, Hirosaki University, Hirosaki 036, Japan

Upland and lowland varieties are cultivated under different conditions in Japan. Low- land rice with useful agronomic traits is a major crop. In contrast, upland rice. useful as a genetic source of stress resistance to blast, is regarded as a minor crop. Thus, diagnostic genetic markers to evaluate upland rice will be useful to help improve resistance in modern rice varieties.

Isozyme genotypes were surveyed among Japanese lowland and upland traditional cultivars. Seventeen isozyme loci were monitored, and different genotypes were scored. A total of 27 genotypes were recognized, 16 for lowland rice and 13 for upland cultivars. Genotypic diversity for upland cultivars was 0.072, which is surprisingly higher than that of lowland cultivars (0.020). Such high genotypic diversity could not be seen from the data of Glaszmann (1987). Four hundred and fifty lowland and 200 upland cultivars were classified either as japonica or indica using discriminant score based on 11 loci (Ishikawa et al 1991, Sano and Morishima

multi-ovary, and long empty glumes. Late sowing also promoted premature heading. The results indicate that heat stress tolerance was depressed and premature heading was enhanced in the dwarf mutant.

Induced dwarf mutants with simulta- neous changes in many traits are being used

in developmental genetic research to understand pleiotropic gene action.

Cited references Futsuhara Y, Kondo S, Kitano H. 1979. Genetical

studies on dense and lax panicles in rice. I. Character expression and mode of inheritance of lax paniclerice. Jpn. J. Breed. 29:151-158.

1991). Except for five lowland and five upland cultivars classified as indicas, the rest of the cultivars were japonicas (Table 1 ). The 10 indicas were characterized with allele 3 for Pgd1, which is not common among other cultivars in Japan. Charac- teristics of the indica cultivars, such as short apiculus hair (APH) averaging 0.36 ± 0.11 mm, slender hull type represented by hull

Table 1. Genotypic variation for Pgd among Japanese lowland and upland rices.

Varietal type

Lowland

Pgd1-1 Pgd1-2 Pgd1-3

Indica 0 0 5 Japonica 445 0 0

Indica 1 0 4 Japonica 26 169 0

Upland

length-width ratio (L/W; av of 2.86 ± 0.14), and positive phenol reaction (Table 2), however, seemed to point to the cultivars’ common origin.

Allele 2 for Pgd1, which is never observed in lowland rices, is also a remarkable diagnostic allele for specifying upland varietal groups. Upland cultivars can be classified into three groups based on Pgd and other trait combinations.

Group 1 : characterized by the presence of allele 1 and represents longer APH (0.65 ± 0.12 mm) and round hull type (L/W; 2.14 ± 0.16) same as 445 lowland cultivars (APH; 0.72 ± 0.19 mm, L/W; 2.09 ± 0.34). However, only 19% of cultivars in this group showed positive phenol reaction.

of allele 2 and represents slightly shorter Group 2: characterized by the presence

Table 2. Morphological and physiological characteristics of Japanese lowland and upland varietal groups having different alleles for Pgd.

Phenol reaction a

Group Allele Cultivars Apiculus hair length L/W (no.) Mean ± SD (mrn) Mean ± SD (mm) Cultivars (no.)

+ -

Lowland Japonica Pgd1-1 445 0.72 ± 0.19 2.09 ± 0.34 32 413 Indica b Pgd1-3 5 0.37 ± 0.12 2.79 ± 0.16 4 1

Japonica A Pgd1-1 26 0.65 ± 0.12 2.14 ± 0.16 5 21 Japonica B Pgd1-2 169 0.44 ± 0.14 2.38 ± 0.21 131 38 Indica b Pgd1-3 5 0.34 ± 0.13 2.98 ± 0.19 4 1

Upland


a + = hull of cultivars blackened. - = hull did not blacken. b Total of 10 indica cultivars were found. Av APH and L-W ratios were 0.96 ± 0.11 and 2.8 ± 0.14, respectively.

APH (0.44 ± 0.14 mm) and more slender hull type (L/Wp; 2.38 ± 0.21) than the first group.

Group 3: possesses mainly allele 3 belonging to the indica type mentioned earlier.

Pgd1 is located on chromosome 11 along with la, v4, and Adh1 (Ishikawa et al 1991). Several key resistance genes have already been located on this chromosomal area (Goto 1970), which seemed to be a specific region for upland characters. Landmarks on chromosome 11 were used to obtain restriction fragment length polymorphism (RFLP) markers for constructing a detailed map around Pgd1. Sensho and Taichung 65 (Acc. 504) were used to ensure polymorphic regions on chromosome 11. Eleven probes and seven restriction enzymes were used; four of these probes showed polymorphism with some enzymes. Among the probes, adh1, G181, and G189 showed allelic band

Evolutionary variations in the Gramineae: rearrangements of DNA fragments transferred from chloroplast genomes to mitochondrial genomes

A. Kanno, Institute of Genetic Ecology, Tohoku University, Sendai 980-77, Japan; M. Nakazono, N. Watanabe, N. Tsutsumi, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan; T. Kameya, Institute of Genetic Ecology, Tohoku University; and A. Hirai, Faculty of Agriculture, The University of Tokyo, Japan

The transfer of DNA fragments from chloroplast to mitochondrial genomes is considered a general phenomenon in higher plants (Stem and Lonsdale 1982, Stern and Palmer 1984). For rice, Nakazono and Hirai (1993) have determined the entire set of transferred chloroplast sequences present in the mitochondrial genome. Total length of the transferred chloroplast sequences, whose lengths range from 32 bp to 6.8 kb. accounted for about 6% of the rice mitochondrial genome. Two of the 16 identified ct-derived fragments in rice mitochondrial genome, one comprising

trends specific to the upland group. The Cited references

specific than that of Pgd. These RFLP of Asian rice varieties. Theor. Appl. Genet.

chromosome. Recent plant breeding programs have

failed to introduce upland-specific resistance genes into lowland modem varieties. Ishikawa R, Maeda K, Harada T, Niizeki M,

trend of RFLP patterns, however, was less Glaszmann JC. 1987. Isozyme and classification

markers and Pgd are used to arrange several 74:21-30.

agronomic and stress resistance traits on the Goto I. 1970. Genetic studies on the resistance of rice plant to the blast fungus. I. Inheritance of resistance in crosses Sensho x H79 and Imochi-Shiraru X H79. Ann. Phytopathol. Soc. Jpn. 36:304-312.

This implies the presence of linkage blocks Saito K. 1991. Classification of Japanese rice for useful stress resistance traits of upland varieties into Indica and Japonica types by cultivars, and also of existing undesirable using isozyme genotypes. Jpn. J. Breed. agronomic traits in lowland varieties. 41:605-622.

upland-specific markers are located would T, Niizeki M, Saito K. 1991. Linkage increase understanding of the origin of analysis of nine isozyme genes on the

Japanese upland rices. Also. detecting conventional linkage map in rice. Jpn. J.

RFLP between upland and lowland Breed. 41:265-272. Sano R, Morishima H. 1992. Indica-Japonica

cultivars will help rice breeders to introduce differentiation of rice cultivars viewed from only useful genetic resources from upland the variations in key characters and rice into modem varieties. isozymes, with special reference to landraces

from the Himalayan hilly areas. Theor. Appl. Genet. 84:266-274.

Confirming the chromosomal region where Ishikawa R, Morishima H, Kinoshita T, Harada

rps 19 - trnH - rp12 - rp123 and the other comprising rp123-rbcL-atpB-atpE-trnM- trnV, were found to be separated in the chloroplast genome but were joined in the mitochondrial genome, which might be the cause for homologous recombination between rp123 and rp123. Maize mitochondrial genome also contains these gene clusters (Lonsdale et al 1983).

Watanabe et al (1994) compared this region, which is homologous to chloroplast rps l9 in the mitochondrial genomes, among

sorghum, Italian rye grass, and wheat) by Southern blot hybridization, polymerase chain reaction (PCR), and DNA sequencing techniques. In all the mitochondrial DNAs from the five gramineous plants — except for that from wheat — the ct-derived fragments ofchloroplast DNA were found to be maintained and the same junctions of mitochondrion-specific and chloroplast- like sequences were found at one terminus (rps 19 side). Subsequent analysis revealed that the fragments had been variously rearranged among species with respect to the other terminus. For this region, however, rice rp123 includes a 135-bp deletion in both chloroplast and mitochondrial genomes. In contrast, neither gene in maize has such a deletion. This

five gramineous plants (rice, maize,

suggests that this region must have been transferred separately in rice and maize after their divergence from one another, with subsequent homologous recombination between rp123 and Ψrp123 in the mitochondrial genomes of rice and maize occurring independently (Fig. 1). These findings indicate that the transfer of the choloroplast sequence occurred in the distant past during the evolution of gramineous plants.

Maintenance of a common junction on one side (the rps19 side), despite the extensive rearrangements of mitochondrial genome, points to the possibility that these sequences might function in the mitochondria. For the result of reverse transcriptase(RT)-PCR and Northern blot hybridization in this study, the chloroplast- derived trnH gene was expressed in rice mitochondria (Fig. 2). Comparisons of such ct-derived fragments among or within species may provide some interesting information about the evolution of mitochondrial genomes.

Cited references Stem DB, Lonsdale DM. 1992. Mitochondrial

and chloroplast genomes of maize have a 12- kilobase DNA sequence in common. Nature 299:698-702.


y

Stern DB, Palmer JD. 1984. Extensive and widespread homologies between mitochondrial DNA and chloroplast DNA in plants. Proc. Natl. Acad. Sci. USA 81:1946- 1950.

Nakazono M, Hirai A. 1993. Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome on rice. Mol. Gen. Genet. 236:341-346.

Watanabe N, Nakazono M, Kanno A, Tsutsumi N, Hirai A. 1994. Evolutionary variations in DNA sequences transferred from chloroplast genomes to mitochondrial genomes in the Gramineae. Curr. Genet. 26:512-5 18.

Lonsdale DM, Hodge TP, Howe CJ, Stern DB. 1983. Maize mitochondrial of DNA contains a sequence homologous to the ribulose-1, 5- bisphosphate carboxylase large subunit gene ofchloroplast DNA. Cell 34:1007-1014.

1. Schematic representation of possible transfer and homologous recombination of ct-derived DNA fragments. Event I (transfer of DNA fragment from chloroplast to mitochondrial genome) occurred before the divergence of rice and maize. Events II (secondary DNA transfer from chloroplast to mitochondrial genome) and III (homologous recombination) occurred after the divergence of rice and maize.

2. Gene expression of the ct-derived DNA fragment in mitochondrial genome. (a) Positions of primers for PCR experiment are indicated by P1 and P2. (b) RT-PCR experiment. Total RNA was prepared from rice green leaves and cDNA were synthesized (or not synthesized) with reverse transcriptase (RT+/RT-) using P2 primer. M = molecular weight marker of ΦX174 DNA digested with Hae lll. (c) Northern hybridization was carried out using nonradioactively labeled probes, indicated under each Northern hybridization pattern. Oligo-trnfM and B9 Kpnl 1.8 kb are specific probes for mitochondrial and chloroplast tRNA, respectively. Lane 1 = rice mtRNA; lane 2 = total RNA extracted from green leaves; lane 3 q total RNA extracted from etiolated leaves.


Identifying transposonlike element Tnr2 in rice

Y. lida, H. Ohtsubo, and E. Ohtsubo, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan

Tnr2 was found as an insertion sequence within p-SINEl-r4, a member of the retroposon p-SINE1, in rice. This sequence is 157 bp in length and present in multiple copies in the rice genome. Tnr2 has the 56- bp terminal inverted repeat sequence (TIR) and is flanked by an 8-bp direct repeat sequence. Tnr2 shows no homology with known transposable elements, suggesting that Tnr2 is a new transposable element (Fig. 1) (Mochizuki et al 1993, Ohtsubo et al 1993).

To analyze the inner region of Tnr2, we synthesized primers that hybridize the sequences of inverted repeats and carried out polymerase chain reaction (PCR) using genomic DNA of Oryza sativa L. cultivars IR36 (indica) and Nipponbare (japonica) as the template. The PCR-amplified fragments were cloned, and their nucleotide sequences determined. The Tnr2 members contained substitutions and deletions in their sequences. A consensus sequence derived from the nucleotide sequences of these members showed a 20-bp tandem repeat in the region between the TIR sequences (Fig. 2). A few members, however, had only one of the repeating sequences.

We identified three more Tnr2 members with flanking sequences by screening from the genomic library of IR36. Nucleotide sequencing of these members revealed that

1. The stem-loop structure of Tnr2 element, 157 bp in length. Arrows indicate TSD of 8 bp.

2. Nucleotide sequences of Tnr2 members. The TIR sequences (56 bp) are underlined. - - = bases identical to those of consensus sequence of Tnr2, / = bases deleted, * = primer sequences used for PCR. Tandem repeat sequences of 20 bp are double-underlined. Clones pYK2A and pYK4 lack one of the repeating units.


each member had target site duplication (TSD) of 8 bp, confirming that Tnr2 is a transposable element (Fig. 2). PCR using oligonucleotides derived from the Tnr2 flanking sequences as primers revealed that several accessions of AA genome species O. longistaminata and O. glaberrima also had Tnr2 in the corresponding loci. This indicates that Tnr2 was inserted into each locus before the rice varieties with AA genome had differentiated during evolu-

however, showed length polymorphism caused by deletion within the Tnr2 sequence or in the region flanking Tnr2.

study are small and are assumed to be a nonautonomous element derived by deletion from an autonomous one that can transpose by itself. Several hundred copies of Tnr2 exist in the rice genome, one of which may correspond to the autonomous Tnr2.

All theTnr2 members identified in the

Cited references Ohtsubo E, Mochizuki K, Tenzen T, Ohtsubo H.

1993. Gamma Field Symp. 32:71-83. Mochizuki K, Ohtsubo H, Hirani H, Sano Y,

Ohtsubo E. 1993. Classification and relationship of rice strains with AA genome by identification of transposable elements at nine loci. Jpn. J. Genet. 68:205-217.

New cytoplasmic male sterile lines developed in Andhra Pradesh, India

R. V. Kumar, P. V. Satyanarayana, and M. S. Rao, Agricultural Research Station (ARS), Maruteru 534122, Andhra Pradesh, India

Successful use of hybrid vigor in rice largely depends on availability of local cytoplasmic male sterile (CMS) and restorer lines. In India, many IRRI-bred CMS lines from the wild abortive (WA) source are being used to develop rice hybrids, which could lead to genetic vulnerability. Use of local CMS lines will help to alleviate this problem and to develop adaptable, heterotic hybrids. We therefore screened several hundred elite genotypes with diverse genetic backgrounds for their maintaining ability at ARS, Maruteru, India.

cytoplasmic source and one effective maintainer for the ARC cytoplasmic source were identified and successfully converted into local CMS lines through backcrossing. Lines with complete pollen sterility identified from BC 6 generation were

Two effective maintainers for the WA

designated as APMS1 A (IR54755 A/WGL 3010-407 - ARC source), APMS2 A (V20 A/WGL 3010-407 - WA source) and APMS5A(IR58025 A/MTU4870 - WA source) during 1994-95. These lines, along with IRRI-bred CMS line IR58025 A, were evaluated for their agronomic and floral traits and natural outcrossing potential. The experiment was laid out in a randomized block design with five replications during the 1995 wet season. Plants were raised at 20- × 20-cm spacing in a 4-m 2 plot. Five plants from the central row in each replication were observed. To study their natural outcrossing potential, the CMS lines were planted adjacent to the corresponding maintainer lines. Good flowering synchrony was achieved. Seed set was attained without resorting to any supplementary pollination techniques.

Data on six characters were statistically analyzed. The four CMS lines significantly differed in agronomic and floral traits,

table). Early-duration (110-115 d) APMS1 A

(ARC) and APMS2 A (WA) possess field resistance to gall midge and bacterial leaf

blight (BLB). Their anthers are white and shivered with moderately well-exserted stigmas. These lines may be extremely valuable in developing early to super-early rice hybrids adaptable to different cropping systems.

Long-duration (147 d) APMS5 A has sturdy culms and possesses resistance to brown planthopper, BLB, and rice tungro virus. Spikelet opening angle, stigma exsertion, and natural outcrossing potential are comparable with those of popular CMS line IR58025A. APMS5 A is the first long- duration CMS line with desirable floral and agronomic traits developed in India. This line will be useful in developing long- duration rice hybrids suitable for wet- season cultivation in coastal Andhra Pradesh.

We used the three CMS lines for testcrossing with several elite lines, and effective restorers and maintainer were identified. The three CMS lines are potential female parents for developing heterotic rice hybrids adapted to different ecological conditions, including coastal regions of Andhra Pradesh, India.

Agronomic and floral traits of new CMS lines in rice.

Days to Plant Productive Duration of Angle of Seed set Pollen Stigma Anther

flowering (cm) (no.) opening (min) opening (º) outcrossing (%) (%) CMS line 50% height tillers/plant spikelet spikelet on natural sterility exsertion characteristic

APMS1 A 78.0 86.3 10.3 192.5 26.8 10.0 100 Moderately well White and shriveled

APMS2 A 80.0 87.5 10.1 183.5 27.0 10.8 100 Moderately well White and shriveled

APMS5 A 116.7 115.5 9.8 194.0 31.0 12.1 100 Well exserted White and shriveled

exserted

exserted

lR58025 A 90.5 90.8 10.1 222.5 30.0 12.6 100 Well exserted White and shriveled

SEM 0.57 0.63 0.23 4.36 0.21 0.3 CD (0.05) 1.75 1.94 ns 13.39 0.63 0.93 CV (%) 1.40 1.30 5.00 4.90 1.60 6.00


tion. The PCR-amplified fragments,

Breeding methods

except for productive tillers plant -1 (see

- - - - - -

- - -

Maintainers and restorers identified in some rice cultivars of Pakistan

S. S. Ali and M. G. Khan, Rice Research Institute (RRI), Kala Shah Kaku, Lahore, Pakistan

We crossed 38 aromatic and nonaromatic local rice varieties and lines with IR58025 A and IR62829 A wild abortive (WA) cytoplasmic male sterile (CMS) lines from IRRI during 1994 kharif season. Seventy- six F 1 hybrids, their male parents, and two isogenic maintainers were transplanted in rows of 30 plants with 23-cm spacing on each side. The crop received 120 kg N ha -1

and 60 kg P ha -1 . Standard agronomic and plant protection measures were used.

Ten plants from each hybrid were labeled. Three panicles from each of these plants were marked and bagged before flowering. Spikelet fertility was calculated as a percentage of filled grains. Five spikelets from the upper part of each panicle were collected before anthesis and fixed in 70% alcohol. Two to three anthers from each spikelet were placed onto a glass slide, squashed in 1% IKI solution, and screened for sterile and fertile pollens. Fertile pollens, which became deeply stained, were round and fully developed.

Male parents of the hybrids with 100% pollen and spikelet sterility were classified as potential maintainers, those with 80-100% pollen and spikelet fertility as restorers, and the rest as partial restorers.

Frequency of maintainers (63%) was much higher than that of restorers among 76 hybrids tested (see table). 1021-8 and KS282 were the only potential restorers for both CMS lines, whereas Basmati 385 restored fertility only in IR58025 A (>80%), indicating that marked cytoplasmic nuclear interaction exists and expression of restorer genes varies with the genetic background of

Maintainer and restorer lines identified for IRRI CMS lines at Kala Shah Kaku, Pakistan. 1994 95.

Spikelet fertility of hybrids with a

lR58025 A lR62829 A Pollen parent

KS282 R R IR6 B B 49744 PR PR 4048-3 B B Basmati 385 R PR 4029-A PR PR 4029-B B B 4439 PR PR 50189-8-6 PR PR 40555 PR PR GP-6 B B GP-10 PR PR GP-15 B B GP-16 B B GP-43 B B 4029-2 B B 4029-3 PR PR Basmati 5854 PR PR 47456 B B

C622 PR PR Jhona 349 PR PR PK3717-12 B B

DR82 B B Basmati 370 B B 1021-8 R R 4289 B B 4334 B B 4365 B B PK3355-5-1-4 B B PK3303-7-2 B B

PK3732-28 B B

PK3727-2 B B

49818 B B 33608 B B 50020 B B 50021 B B Basmati 198 B B Basmati 6129 PR PR

a R = restorer (80-100% pollen and spikelet fertility). PR = partial restorer (5-79% pollen and spikelet fertility). B = maintainer (100% pollen and spikelet sterility).

female parents. Apparently, restorers among Pakistani rice cultivars are scarce.

Maintainer lines identified are being used in a backcrossing program for inducing CMS in local germplasm. Restorer lines 1021-8, KS282, and Basmati 385 will be used to develop new hybrid combinations.

Development of rice cytoplasmic male sterile line 47456 A in Kala Shah Kaku, Pakistan

S. S. Ali and M. G. Khan, Rice Research Institute, Kala Shah Kaku, Pakistan

Hybrid rice research was initiated at Kala Shah Kaku in 1991. We crossed IRRI cytoplasmic male sterile (CMS) lines IR58025 A and IR62829 A, with wild abortive (WA) cytoplasm, with aromatic rice breeding line 47456 in 1992. 47456 [4048-3/PK4112 (Basmati 370/4439)] has extra-long grains and short stature, and flowers early. Plants in the F 1 were raised in 1993. Crosses of IR58025 A and IR62829 A with 47456 showed complete pollen sterility as well as spikelet sterility under bagged conditions (see table).

Backcrosses were made with recurrent parent 47456. The derived progenies (F1, BC 1 , and BC 2 ) and the parents were transplanted in the field in rows of 30 plants at 23-cm spacing from each side in Jul 1995. The crop received 120 kg N ha -1 and 60 kg P ha -1 . Standard agronomic and plant protection measures were followed.

Ten plants from each generation were selected, with three panicles from each plant bagged before flowering. Spikelet fertility was calculated as the percentage of filled grains. Five spikelets from the upper part of each panicle were collected before anthesis and fixed in 70% alcohol. Two to three anthers from each spikelet were squashed in 1% IKI solution to determine pollen sterility. Complete pollen sterility was observed in the F1 and backcross generations, indicating successful transfer of CMS from IR58025 A and IR62829 A into Basmati line 47456. Restorers for the Basmati CMS line 47456 A are being identified.


Pollen and spikelet sterility of parents, F 1 , BC 1 , and BC 2 generations at Kala Shah Kaku, Pakistan. the hph gene constructs (pEmu-hph and 1995. pUbi1-hph) either alone or combined with

Plant height Days to 50% Pollen sterility Spikelet sterility the gus constructs (p40CSD35SAcl-gus (cm) flowering (%) on bagging (%) and pUbi1-gus). With the cobombarded

Parent calli, about 300-1,500 BEUs were obtained lR58025 A 97.1 lR62829 A 85.5 47456 90.1

F 1 lR58025 A/47456 99.1 lR62829 A/47456 92.2

BC 1 lR58025 A/(47456) a 99.3 IR62829 A/(47456) 94.4

BC 2 lR58025 A/(47456) b 99.5 lR62829 A/(47456) 94.6

a = generation 1. b = generation 2.

Comparison of promoters and selectable marker genes for indica rice transformation

Z. Li, N. M. Upadhyaya, and P. M. Water- house, Commonwealth Scientific and Industrial Research Organization (CSIRO) Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia; and A. J. Gibbs, Research School of Biological Sciences, Australia National University, Canberra, ACT, Australia

Several selectable marker genes (nptII, hptII, dhfr; bar; and csr1) have been used for cereal transformation. However, the bar gene and, to a lesser degree, the hpt gene, have been the most commonly used in recent years. A comparison of the efficacy of these selectable marker genes in indica rice has not been previously reported.

porated protoplasts and bombarded suspension cells. Plasmids with the gus gene under the control of promoters pCaMV35S-gus, pEmu-gus, pRolC-gus, and pActl-gus were electroporated into protoplasts. Similarly, pCaMV35S-gus, pEmu-gus, pActl-gus, and pUbi1-gus were used to bombard suspension cells. Except for the RolC promoter, which directs vascular-specific expression, GUS expression was detectable under the control of all promoters tested. Constitutive promoters Act1 and Ubi1 gave the highest expression levels. The order of promoter

Transient expression of GUS in electro-


84 100 100 83 100 100 85 10 13.5

92 100 100 90 100 100

95 100 100 90 100 100

90 100 100 89 100 100

strength (from strong to weak) in nonorgan- ized cells of indica rice Chinsurah Boro II was Ubi1 > Act1 > Emu > CaMV35S>RolC. The Ubil, Actl, and Emu gave 12-, 8-, or 2-fold higher expression levels, respectively, compared with the CamV35S promoter.

Comparison of hpt and bar genes driven by various promoters for stable transformation. Embryogenic calli were cobombarded with pUbi1-gus and a range of plasmids containing either the hph or bar gene controlled by various promoters. Calli staining for GUS activity 1 d after shooting produced more than 300 blue expression units (BEUs) per petri dish of calli bombarded with each plasmid. Although most of the BEUs diminished or disappeared, a few from the pUbi1-gus-bombarded calli and any of the three hph -containing plasmids gradually increased in size over 2 wk of selection. Four weeks after selection. they formed nodular structures from the surface of the original calli.

Cobombardment of pUbi1-gus and pUbil-hph gave the most large BEUs after 4 wk, pEmu-hph produced a similar number of smaller BEUs, and pCaMV35S- hph gave the fewest and smallest BEUs. Some cultures cobombarded with gus and bar constructs had modest increase in size and number of BEUs. None of the BEUs developed nodular structures.

and regeneration of transgenic plants. Embryogenic calli were bombarded with

Selection of stably transformed cell lines

1 d after shooting. Without staining for GUS activity,

resulting transformed cells or microcalli cannot be identified under the dissecting microscope for at least 1 mo after bombard- ment. However, staining a few plates of target calli for GUS activity after 2 wk of selection allowed us to observe transformed cell clusters. After three to four subcultures on selection media, fast-growing callus lines could be distinguished among the bombarded calli. These callus lines were

selection medium for further growth. From 10 experiments and after 2 mo of

selection on media, 50 transformed callus lines were obtained from 72 bombarded plates (see table). Of these, 16 regenerated into 79 putative transgenic plants (about 30% regeneration frequency) when placed onto the regeneration medium. Of the six putative transgenic plant lines from cotransformation of gus and hph genes, two were GUS-positive when the plants were about 5 cm tall. However, GUS activity of one line ceased as the plants matured. All 79 putative transgenic plants (24 sterile and 55 fertile) were transferred to soil in the glasshouse. Most of the sterile plants were derived from experiment 1, which used foundation callus line 1. About 87% of the transgenic plants derived from foundation callus lines 2 and 3 were fertile. Fertile plants were derived from 12 different transformed callus lines (6 from pUbil-hph and 6 from pEmu-hph). Southern blot analysis showed that at least one plant from each line contained between one and eight copies of the transgene. Ten of the 12 transgenic plant lines contained both selectable and nonselectable genes, which were introduced on separate plasmids. giving an 83% cointegration frequency.

Calli bombarded with bar constructs grew poorly on bialaphos-containing media irrespective of the promoter used to control the bar gene. The few BEUs that expanded over time failed to form nodular cell clusters. This suggests the limited use of the bar gene in indica rice transformation. Rapid growth of hygromycin-resistant calli

individually subcultured onto a new

Summary of stable transformation of indica rice cv Chinsurah Boro II.

Experiment Callus age Plates hph- GUS+ Embryogenic Plants GUS+ Fertile Selectable Nonselectable (mo)/ bombarded resistant callus lines lines (no.) plant lines plants marker gene

callus line (no.) callus lines (no.) (no.) (no.) (no.) gene

1 8.5/1 8 13 10 3 16 1 (1) a 0 pEmu- hph p40CSD35S- gus 2 9/1 4 5 4 0 - pEmu- hph p40CSD35S- gus 3 7.5/2 3 3 1 2 8 0 7 p Ubi1- hph pUbi1- gus 4 8/2 12 5 2 1 2 0 1 p Ubi1- hph pUbi1- gus 5 7.5/2 7 7 3 23 - 22 pEmu- hph RRSV b

6 8/2 7 5 1 4 4 pEmu- hph RRSV 7 8.3/2 8 1 1 1 1 pEmu- hph RRSV 8 8.3/2 9 2 1 2 1 9

pEmu- hph RRSV 5.3/3 7 3 3 17 13 p Ubi1- hph RRSV

10 5.3/3 7 6 1 6 6 p Ubi1- hph RRSV Total 72 50 17 16 79 1 55

a Number in parentheses shows transgenic plant line expressing GUS when about 5 cm tall, but not at 20 cm tall. b Coding region of rice ragged stunt virus segment 5 drlven by various promoters.

and BEU expansion were observed when the hph gene was driven by the Ubi 1 promoter, followed by Emu and then CaMV35S. In all three cases, expanding BEUs formed nodular structures indicative of regeneration. The Ubi 1>Emu>CaMV35S ranking

follows the relative levels of GUS after shooting are required to achieve each expression driven by these promoters in our

data show that about 2,000-4,000 BEUs 1 d enable the most effective selection. The transgene. From this we infer that stronger promoters genic rice plants with 1-8 copies of the

such BEUs regenerate into fertile trans- transient and stable expression studies. hygromycin-resistant callus line and that

Transfer of cytoplasmic male sterility in indica rice through protoplast fusion

H. S. Gupta, B. Bhattacharjee, and A. Pattanayak, Plant Breeding Division, Indian Council for Agricultural Research (ICAR) Research Complex for NEH Region, Umroi Road, Barapani 793103, Meghalaya, India

Cytoplasmic male sterility (CMS) is widely used in hybrid seed production and requires routine transfer of CMS to new genetic backgrounds. Transfer of CMS through conventional breeding requires five to six backcrossings, taking 2-3 yr. However, a one-step transfer of CMS through asymmetric protoplast fusion can be used to shorten this period (Kyozuka et al 1989).

CMS, maintainer, and restorer lines. Embryogenic calli initiated from the scutella obtained from mature embryos of CMS line V20A were repeatedly subcultured to make them friable. Five hundred 1,000-mg calli were used to initiate cell suspension, which was established after 3-4 mo of regular subculturing at a 4-5 d

Plant regeneraion, from protoplastsof

interval. Protoplasts, which were isolated, purified, and cultured as described by Bhattacharjee and Gupta (1995), underwent sustained division into to microcolonies that became macroscopic after 30-40 d. Protocalli differentiation was obtained 20-30 d after transfer to regeneration medium. Plants were transferred to pots where they flowered and produced sterile pollen grains. Likewise, protocols for protoplasts to plant systems were developed for RCPL1-2C, V20 B, and IR36.

protoplasts and asymmetric fusion. Because protoplasts of all four lines were dividing on culture, we standardized the dose for their inactivation to undertake asymmetric fusion. Protoplasts of V20A were irradiated with 30 krad gamma ray (Fig. 1a); those of RCPL1-2C, V20 B, and IR36 were inactivated with 10 mM iodo- acetamide for 15 min at 20°C (Fig. 1b).

Fusion of gamma ray- and iodo- acetamide-inactivated protoplasts of V20A with RCPL1-2C, V20 B, and IR36 was attained with 25 V/1 KHz AC amplitude for 25 s, followed by 900 V DC pulse (2.52 KV/cm field strength) amplitude for 30 µs. Seven 10% fusions were obtained.

Inactivation of donor and recipient

Culture of fusion products and plant regeneration, from putative cybrid calli. Fused protoplasts of V20 A with RCPL1- 2C, V20 B, and IR36, along with inactivated protoplasts of V20 A, V20 B, RCPL1-2C, and lR36, were cultured separately. The inactivated protoplasts served as the control. Only fused protoplasts underwent sustained division and formed microcolonies (Fig. 1c), which became macroscopic after 25-30 d of culture. Macrocolonies of putative cybrids between V20A and IR36 differentiated into plantlets when transferred to regeneration medium (Fig. 1 d). The plantlets transferred to pots (Fig. 1 e) grew to maturity and, as expected. were fertile. Putative cybrid calli between V20 A/RCPL1-2C and V20A/V20 B also differentiated and gave rise to plantlets, which were grown to maturity to check the expression of male sterility. Molecular analyses of mitochondrial DNA of both the sets and parents are in progress.

Cited reference Bhattacharjee B, Gupta HS. 1995. Fertile plants

regenerated from protoplasts of cold-tolerant rice line RCPL1-2C. J. Biochem. Biotechnol. 4:61-65.


- - - - - -

- -

- - - - -

Protoplast fusion between V20 A and IR36 and regeneration of putative cybrid. (a) gamma- irradiated protoplasts of V20 A; (b) iodoaceta- mide-treated protoplasts of IR36; (c) microcolonies resulting from sustained division of the fusion product; (d) plantlets regenerated from putative cybrid calli; and (e) putative cybrid plants.

Characters of plants regene rated from protoplasts of photoperiod-sensitive genic male sterile rice

Qingzhong Xue and K. Etoh, Agronomy Department, Zhejiang Agricultural University, Hangzhou 310029, People's Republic of China; and S. McCouch and E. D. Earle, Plant Breeding and Biometry Department, Cornell University, Ithaca, NY 14853-1902, USA

Identifying photoperiod-sensitive genic male sterility (PGMS) in rice (Shi 1981) makes it possible to produce hybrid rice seed through the two-line system. We studied the response of PGMS protoplast clones to photoperiod and temperature,


variation in ploidy, and agronomic traits. ploids (44.5 cm). The diploid plants had Promising PGMS line Zhenongdal 1s 96.5 spikelets per panicle whereas (X126- 11) has already been developed autotetraploids had few spikelets (19.3). through protoplast culture. Change in fertility. Typical sterile pollen

A cell suspension with small globular was >85% at the sterile stage and ranged calli was established from the scutellum of from 10 to 30% at the fertile stage. How- mature embryos of PGMS cultivar N5047s. ever, the spherical sterile pollen ranged A high density (1.5 × 10 6 ml -1 ) of from 5 to 20% at the two stages. Percentage protoplasts was obtained, and green of staining pollen exhibited one or three regenerates (plantlets) were produced peaks at the fertile stage. Four PGMS following Xue and Earle’s (1995) method. protoplast clones had obvious differences To distinguish autotetraploids from both in duration of sterility and in stability diploids, the DNA content of regenerates before mid-August. The change from was measured using flow cytometry sterility to fertility was clear. Seed set was according to the procedure of Arumuga- more than 30% under shorter daylength in muthan and Earle (1991). Sowing at September. different times under natural conditions at Agronomic traits. Agronomic characters Hangzhou (30° 15' N) promoted changes in significantly differed among the four fertility and agronomic traits of the four diploid protoplast clones and control diploid PGMS protoplast clones and their cultivar N5047s. Spikelets panicle -1 , control cultivar N5047s. panicle number plant -1 , spikelet density, and

Variation in ploidy. Flow cytometric 100-grain weight of X126-11 were analysis indicated that nuclear DNA content significantly higher than those of other in protoplasts of diploid plants ranged protoplast clone and NS047s. between 0.715 and 0.873 pg 2c -1 , and that Photoperiod and temperature from autotetraploids ranged between 1.537 responses. We used computer simulation and 1.786 pg 2c -1 . This accounted for 60%. analysis of PGMS protoplast clones to diploid plants and 40% autotetraploid measure photoperiod-sensitive and plants. Diploid regenerates were taller temperature-sensitive coefficients for (78.0 cm) compared with the autotetra- defining their model of seed set rate. The

Comparison of the anatomical structure of anther connective tissue and thrum at the fertile and sterile stages X126-11. (a) Fertile anther connective tissue, (b) sterile anther connective tissue, (c) fertile thrum, and (d) sterile thrum. NVB = normal vascular bundle. AVB = abnormal vascular bundle. V = vascular bundle. S = sieve tubes.

analysis showed that photoperiod and temperature can affect fertility of PGMS protoplast clones.

and fertile periods in PGMS rice. The pollen sac and thrum (or filament) at the sterile stage were smaller than those at the fertile stage. Poorly developed vessel elements and sieve tubes of the anther vascular bundle and thrum vascular bundle (see figure) are abnormalities that can affect the transport of nutrients to the pollen sac, leading to microspore sterility.

Cited references Arumugamuthan K, Earle ED. 1991. Estimate of

Anatomical differences between sterile

nuclear DNA content of plants by flow cytometry. Plant Mol. Biol. Rep. 9:229-231.

Shi MS. 1981. Preliminary research report on breeding and utilization of natural two-uses line in late Japonica rice. Sci. Agric. Hubei 7: 1-3.

Xue QZ, Earle ED. 1995. Plant regeneration from protoplasts of cytoplasmic male sterile lines of rice ( Oryza sativa L.). Plant Cell Rep. 15:76-81.

Attempted hybridization between Oryza sativa L. and Porteresia coarctata T.

Z. I. Seraj, M. O. Faruque, Biochemistry Department, University of Dhaka (UD), Dhaka 1000, Bangladesh; K. G. Hossain, Institute of Post Graduate Studies in Agriculture, Salna, Gazipur, Bangladesh; R. H. Sarker, Botany Department, UD; T. Devi, Z. Islam, Biochemistry Department, UD; and A. S. Islam, Botany Department, UD

Wild halophytic Porteresia coarctata (2n=4x=48), endemic to the coastal areas in Bangladesh, is a potential source of salt tolerance for Oryza sativa. Crosses between these two genera were unsuccess- ful until Jena (1994) reported a hybrid between IR36 and P. coarctata. This triploid hybrid was much smaller than either parent and was completely male sterile. Sarker et al (1993) found that P. coarctata accepts 0. sativa pollen readily, but not vice versa. Our crossing strategy therefore involved P. coarctata

(4x)/ O. sativa (2n) and tetraploid O. sativa (4x-c)/ P. coarctata.

Hand pollinations were made between the two genera. Gibberellic acid (75 ppm) with 0.1 % Tween was sprayed once 20 min after crossing. Embryos were rescued 10- 12 d after pollination and cultured on 1/4 Murashige and Skoog (MS) medium with 0.45% agarose. Hybrids showing slow growth were kept on 1/4 MS for 8-10 wk. Plants were then transferred to the soil. Rapidly dividing root tips were stained with Feulgen.

Seed set percentage and survival among crosses were recorded (see table) The tetraploid hybrid from the cross BR-7 (4x- c)/ P. coarctata grew vigorously and was taller than the induced tetraploid mother variety BR-7 (4x-c). Leaf texture was rough or intermediate between that of the parents. Leaves of the hybrid taper gradually compared with the abrupt taper of BR-7 (4x-c) leaves but not as gradually as those of P. coarctata. The hybrid did not set any seed on selfing. It has spikelets showing short awn and the curled morphology and asynchronized flowering pattern of P. coarctata. The hybrid from cross IR36/ P. coarctata, which showed parental isozyme bands both of O. sativa and P. coarctata, did not survive. The hybrid from BR-7(4x)/ P. coarctata showed a faint band corresponding to that of P. coarctata.

Both P. coarctata and the tetraploid hybrid had 48 chromosomes. Chromo-

somes in the hybrid, counted from 50 cells, appear to be a mixture of the parents.

We examined 70 cells in triploid hybrids P. coarctata × Rajashail/Binnatoa and found them to have 36 chromosomes. The morphology of the triploid hybrid is close to that of P. coarctata. Three F1s involving P. coarctata with Binnatoa 1, Rajashail 1, and Rajashail 4 showed 24.3, 15.3, and 30.8% seed set, respectively. Seed set in selfed P. coarctata was 94%. Embryos from three seeds obtained after backcrossing P. coarctata /Rajashail 4 with Rajashail pollen were rescued 11-13 d after anthesis and are currently being grown in 1/4 MS medium with 0.45% agarose.

Chromosomes of P. coarctata and O. sativa are small and not morphologically distinct from each other, making studies difficult on the tetraploid hybrid nature through standard karyotype analysis. We have multiplied the hybrid through vegetative propagation. We will examine it for hybridity based on meiotic chromosome and restriction fragment length polymorphism analysis.

Cited references Jena KK. 1994. Production of intergeneric

hybrid between Oryza sativa L. and Porteresia coarctata T. Curr. Sci. 67:744-746.

Sarkar RH, Samad MA, Seraj ZI, Hoque MI, Islam AS. 1993. Pollen tube growth in crosses between Porteresia coarctata and Oryza sativa. Euphytica 67:744-746.

Percent seed set and survival in crosses of O. sativa and P. coarctata.

Cross combination

Seed set Germinated plants Surviving plants (%) a (no.) (no.)

IR36 (4x)/ P. coarctata 1.6 (180) IR64 (4x)/ P. coarctata 1.6 (120) Latisail (4x)/ P. coarctata 0.16 (1250) BR-7 (4x)/ P. coarctata 0.44 (900) P. coarctata /Rajashail (2x) 2.34 (940) P. coarctata /Binnatoa (2x) 4.16 (240) P. coarctata /BR-7 (2x) 2 (1140)

a Values in parentheses indicate total number of florets used in crossing.

3 2 2 4

22 10

6

0 0 0 1 4 4 0


DNA content in rice hybrids and heterosis

X. C. Liu, J. L. Wu, S. Q. Tang, and S. K. Min, China National Rice Research Institute, Hangzhou 310006, Zhejiang, People's Republic of China

Certain metabolic functions in the nucleus are enhanced in heterotic plants compared with those of their parents. However, what happens to the nucleus under heterozygo- sity is still unknown. Nebiolo et al (1983), in a study on RNA metabolism in protoplasts isolated from seedlings of a heterotic maize hybrid and its parents, suggested that a hybrid nucleus may provide advantages in the rate of DNA and RNA synthesis. We studied the nuclear behavior and DNA content in hybrids to improve understanding of the molecular basis of heterosis.

Using a MIPS-1 imaging analytical instrument with analytical software, intact

nuclei were isolated from root tips on slides, stained with Feulgen, and screened for different mitotic stages (prophase, metaphase, anaphase, and telophase). Screened nuclei were synchronically determined based on optic density (OD), integrated optic density (IOD), and size.

lines, six male indica parental lines, and nine hybrids derived from them. We examined 4,401 nuclei from root tips based on OD, IOD, and nucleus size (or an av of 55 nuclei for each mitotic stage for each material). Average OD value of nuclei from the nine hybrids was less than that from the 11 parental lines. However, average IOD value was greater in hybrids than in the parents (see table). Average nucleus size, which significantly expanded during prophase and telophase stages (at the 5% level), was greater in the hybrids. OD, IOD, and nucleus size were compared for hybrids and their parents (see figure). We found that nucleus size is directly proportional to IOD based on the regres-

We selected five female indica parental

sion index of about 0.977. Increases in DNA content and nucleus size and decrease in DNA density occurring in the hybrids indicate that gene expression is probably enhanced. These factors could be important for understanding karyological and molecular bases of rice heterosis. However, we have not yet determined which part of the DNA increases and what its functions are.

Variation in DNA content and nuclear size in hybrids compared with those in their parental lines (represented at zero).

Comparison of nuclear size and DNA content between nine hybrids and their parental lines.

Prophase b Metaphase Anaphase Determinant a

F M H F M H F M H F

Nuclei (no.) 333 374 592 143 218 298 254 313 406 316 OD (arbitrary) 0.158 0.157 0.146 0.300 0.286 0.283 0.240 0.239 0.231 0.176 IOD (arbitrary) 5.69 5.87 6.22 3.89 3.98 4.04 2.19 2.18 2.27 2.5 Size (µm) 2 36.77 38.38 43.41* 13.25 14.09 14.56 9.38 9.29 10.16 14.67

a OD = optic density, IOD = integrated optic density. b F = female line, M = male line, H = hybrld. * = signlficant at the 5% level.

Telophase

M H

416 738 0.176 0.170 2.56 2.73

14.91 17*

Induction of Agrobacterium tumefaciens vir genes by rice

K. Vijayachandra, K. Palanichelvam, and K. Veluthambi, Plant Biotechnology Depart- ment, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India

Transformation of monocotyledonous plants, such as rice, by Agrobacterium has low efficiency and has not yet become a routine laboratory technique. We studied whether rice tissues have limited ability to induce Agrobacterium vir genes and to promote synthesis of T-strands.


We used Agrobacterium tumefaciens strain A348 harboring a cosmid with vir E- lacZ fusion to monitor ability of plant tissues to induce vir genes. Tobacco leaf segments preincubated for 48 h in Mura- shige and Skoog (MS) medium and incubated with Agrobacterium for 24 h induced vir E expression to 4,500 Miller units, which equaled vir induction level obtained using the chemical signal, acetosyringone. Induction of vir E expression by leaf segments of Oryza sativa L. cv Co 43 was low (only about 5% of that achieved by tobacco leaf segments).

We analyzed various parts of rice seedlings of different ages for their ability to induce vir genes. Rice roots, leaf bases,

coleoptiles, and leaf segments induced vir genes to very low levels (see table). Only scutellum from 4-d-old seedlings induced vir E to a high level (about 40% of induction by tobacco). We found that a 72-h preincu- bation is essential for vir gene induction. Most of the vir gene induction was associated with the preincubated scutellum per se. The conditioned medium exhibited very little induction.

Agrobacterium efficiently transformed scutellum-derived calli of japonica rice cultured for 25 d on a 2,4-D-containing medium (Hiei et al 1994). However, scutella excised from 5-d-old seedlings and grown on 2,4-D-containing medium were transformed at very low efficiency.

rmguevarra

Rectangle

rmguevarra

Rectangle

rmguevarra

Rectangle

Induction of A. tumefaciens vir E gene by various parts of rice plants of different ages.

Age of rice Part of rice plant Mean Miller plant (d) preincubated units ± SE

for 72 h

4 Coleoptile 203 ± 4 Scutellum 2919 ± 284

8 Leaf blade 501 ± 47 Leaf base 378 ± 42 Root 362 ± 39

14 Leaf blade 353 ± 17 Leaf base 531 ± 22

20 Leaf blade 528 ± 75 Leaf base 455 ± 15

Control values: MS medium – 175 ± 8 MS medium –5129 ± 90 + 60 µM AS MS medium –6840 ± 235 + tobacco leaf segments

Scutellum-derived calli induced vir E expression to 1,745 Miller units, whereas scutella excised from 5-d-old seedlings and grown on 2,4-D-containing medium

induced vir E only to about 646 Miller units. The scutellum-derived calli that induced vir genes more efficiently exhibited a higher level of susceptibility to Agrobacterium mediated transformation.

However, scutella excised from 4-d-old seedlings and grown on hormone-free medium induced vir gene expression to a level (2,547 Miller units) higher than that achieved with scutellum-derived calli. The results suggest that these scutella may be more susceptible to Agrobacterium than the scutellum-derived calli. We also found that rice scutellum promotes generation of the T-DNA transfer intermediates, T-strands, in Agrobacterium.

Cited references Hiei Y, Ohta S, Komari T, Kumashiro T. 1994.

Efficient transformation office ( Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6:271-282.

each other. A 0.52-kb Eco RI/ Xho I fragment of a cDNA clone (RTS1) was used as a probe for screening the IR54 genomic library. A positive genomic clone (RTS2) was isolated and sequenced. Northern blot analysis (Fig. 1) and in situ hybridization (Fig. 2) showed that the gene was predominantly expressed in the anther's tapetum during vigorous meiosis and disappeared before anthesis. Sequence comparison between the cDNA clone

1. Northern blot of total RNA isolated from leaves, seeds, and panicles at various

Isolation of a tapetum- DNA from rice cultivar IR54. A panicle development stages and probed with putative

specific gene and promoter cDNA library was constructed and anther-specific cDNA. Lanes with RNA are (A)

from rice leaf, (B) panicle, 4 d before C, (C) panicle when

differentially screened for panicle and auricles of first and second leaves are at same anther specificity. We selected two cDNA level, (D) panicle, 4 d after C, (E) panicle, 8 d

J.-Y. Lee, R. R. Aldemita, and T. K. Hodges, clones that could be cross-hybridized with after C, (F) panicle, 12 d after C, and (G) seed. Botany and Plant Pathology Department, Purdue University, West Lafayette, IN 47901, USA

Potential vulnerability of cytoplasmic male sterility (CMS)-derived hybrids calls for developing an alternative method of controlling male fertility/sterility for rice breeding and hybrid development. A tapetal-specific promoter-driving barnase induced sterility in tobacco and oil seed rape plants (Mariani et al 1992). Fertility was restored with the same promoter- driving barstar. Although tapetum-specific cDNA clones have recently been isolated from rice (Tsuchiya et al 1992) no regulating sequences have been reported. Developing a system by which male fert- lity in rice can be controlled requires a promoter that can regulate gene expression. specifically in the male organ, during anther development.

specific cDNA and corresponding genomic We cloned a rice anther (tapetum)-

2. In situ hybridization of transverse sections through rice spikelet, including anthers with denatured cDNA (RTS1) probe. Spikelets were sampled when auricle of first leaf was 2.5 cm above the auricle of the second leaf. (A) and (B) anthers and glumes: (C) lower magnification of A; (D) and (E) RNAse- treated anthers before hybridization; and (F) leaf sheath, a = anther; t = tapetum; lo = locule; g = glume; ep = epidermis; and en = endothecium.


(RTS1) and the corresponding genomic clone (RTS2) and a primer extension assay showed that this gene has no introns and a continuous coding region of 285 base pairs yielding a putative polypeptide of 94 amino acids. The coding region of the gene has a 75.8% GC content (216/285). The nucleotide and deduced amino acid sequences. when compared with those in data banks, did not show any significant homology to known sequences. However, GAATTTGTTA, a sequence in the promoter region, differs only by one or two

Production and characterization of Oryza sativa L./ O. minuta Presl. hybrids and backcross progenies

A. L. Mariam, Biology Department, Faculty of Science and Natural Resources, Universiti Kebangsaan Malaysia, Sabah Campus, Locked Bag No. 62, 88996 Kota Kinabalu, Sabah, Malaysia; A. H. Zakri, M. C. Mahani, Genetics Department, Universiti Kebang- saan Malaysia, 43600 Bangi, Selangor DE, Malaysia: and M. N. Normah, Botany Department, Faculty of Life Sciences, Universiti Kebangsaan Malaysia

We attempted to produce hybrid and backcross progenies between rice and wild species to enable the transfer of some important traits from O. minuta into locally cultivated rice.

Rice varieties Mahsuri, Setanjung, MR84, and MR103 were crossed with one accession of O. minuta (IRRI Acc. 101141), which also served as the male parent. The original F 1 hybrids and two colchicine-treated F 1 s were then backcrossed to the respective O. sativa varieties as the recurrent parent to produce backcross 1 (BC 1 ). Successive backcrosses to the recurrent parents were made to produce BC 2 progenies.

Seeds of the F 1 , BC 1 , and BC 2 with poorly developed endosperm began to degenerate 2 wk after pollination. To ensure survival of the hybrids and BC progenies, the 14-d-old embryos were rescued on 1/4 MS medium following the procedure of Jena and Khush (1984).

nucleotides from one of the conserved Cited references sequence motifs in the promoter region of Mariani C, Gossele V, De Beukeleert M, De two pollen-specific genes of tomato (Twell Block M, Goldberg RB, De Greef M, et al 1991). Leeman J. 1992. Achimaeric ribonuclease-

The promoter function and regulation of inhibitor gene restores fertility to male sterile

RTS2 for tapetum specificity may be limited to rice. Experiments to obtain rice plants transformed with gus A gene- containing constructs under the control of pollen development reveals pollen-specific

plants. Nature 357:348-387.

McCormic S. 1991. Promoter analysis of genes that are coordinately expressed during

Twell D, Yamaguchi J, Wing RA, Ushiba J,

this tapetum-specific promoter are in enhanced sequences and shared regulatory progress. These studies will allow us to elements, Gene Dev. 5:496-507. determine the DNA sequences responsible Tsuchiya T, Toriyama K, Nasrallah ME, Ejiri for controlling tapetum-specific expression. S-I. 1992. Isolation of genes abundantly

expressed in rice anther at the microscope stage. Plant Mol. Biol. 20:1189-1993.

Morphological characteristics, pollen fertility, somatic chromosome number, and meiotic chromosome pairing of the F 1 hybrids and BC progenies were observed.

subjected to starch gel electrophoresis and stained for shikimate dehydrogenase (SDH), phosphogluconate dehydrogenase (PGD), and glutamate oxaloacetate trans- aminase (GOT) activities using the procedure described by Glaszmann et al (1988).

Seed set in O. sativa/O. minuta crosses ranged from 9.5 to 25.1%, depending on the rice variety used (see table). By rescuing 10- to 14-d-old embryos. 414 F 1 hybrids were successfully raised to maturity. The hybrids closely resembled their wild parent, although some of their morphological characteristics were intermediate. The male sterile F 1 plants were vigorous and tillered profusely. All had the expected chromosome number (2n=3x=36) and showed irregular meiotic chromosome pairing, with mostly univalents. Mean chromosome configuration was 29.31 (16-

The hybrids and BC progenies were also

36) Is + 3.32 (0-10)IIs + 0.02 (0-1)IIIs + 0.002(0-1)IVs.

Seed set in the original F 1 backcrossed with O. sativa parents was extremely low (0.03%); however, seed set was slightly higher (0.88%) when backcrosses were done on the colchicine-treated plants (see table). The 17 BC 1 plants obtained after embryo rescue appeared more like the F 1 and were male sterile. These plants, for which chromosome numbers varied from 44 to 48, exhibited irregular meiosis with mostly univalents.

Seed set in BC 2 was about 2.4% (see table). Of the 47 embryos cultured, 18 BC 2 plants grew to maturity and had different morphological characteristics, pollen fertility, and somatic chromosome number. Of these, one plant closely resembled the O. sativa parent and had 24 chromosomes. The partially fertile plant had about 58.8% pollen stainability and 12.47% spikelet fertility and showed 12 bivalents in 60% of the cells and 9-11 bivalents in the other 40% of the cells. The results showed the recovery of O. sativa phenotype in the

Production of Oryza sativa/O. minuta F 1 hybrids and backcross progenies.

Spikelets Seed Embryos Plants Chromosome

(no.) (%) (no.) (no.) (2n) Cross combination a pollinated set cultured obtained number

Mahsuri/ O. minuta (F 1 ) 1,742 15.1 132 108 36 Setanjung/ O. minuta (F 1 ) 1,729 25.1 138 123 36 MR84/ O. minuta (F 1 ) 2,465 12.5 110 89 36 MR103/ O. minuta (F 1 ) 2,346 9.5 112 94 36 (O. sativa / O. minuta) × O. sativa (BC 1 ) 37,080 0.03 10 1 45 (CT F 1 )/ O. sativa (BC 1 ) 3,733 0.88 33 16 44-48 (BC 1 from CT F 1 ) × O. sativa (BC 2 ) 4,452 2.47 110 32 (18) a 24-37

a CT F 1 = colchiclne-treated F 1 (O. sativa/O. minuta). Figure in parentheses shows number of plants with normal growth.


Genetic analysis of epicu- titular structure involving a dripping-wet leaf mutant of rice

K. Nakamura and K. Hattori, Laboratory of Plant Genetics and Breeding, Nagoya University, Chikusa, Nagoya 464-01, Japan

We obtained a dripping-wet leaf mutant among doubled haploid progenies regenerated from rice ( Oryza sativa L. cv Nipponbare) anther culture. This mutant

was derived from callus treated with 20 Gy of gamma rays 2 d after transfer to regeneration medium. Rice leaves, in general, can shed drops of water; however, leaves of the dripping-wet leaf mutant do notbecause of their highly hydrophilic leaf surface. Many of these mutant types, which are controlled by a single recessive gene, have been obtained in rice. Some of these gene loci have already been identified Satoh et al 1983). Although factors causing high leaf wettability in these mutants are not clear, leaf surface structure

second backcross, which probably resulted from the restricted recombination between parental genomes indicated by the low frequency of rod bivalents in the meiosis of the F 1 hybrids. Furthermore, none of the O. minuta allozyme (Sdh1, Pgd2, Got1, and Got3) was detected in the 24-chromosome plant, indicating lack of introgression of chromosome segment from the donor wild species.

Cited references Glaszmann JC, delos Reyes BG, Khush GS.

1988. Electrophoretic variation of isozymes in plumules of rice ( Oryza sativa L.) - a key to the identification of 76 alleles at 24 loci. IRRI Res. Pap. Ser. 134.14 p.

Jena KK, Khush GS. 1984. Embryo rescue of interspecific hybrids and its scope in rice improvement. Rice Genet. Newsl. 1:133- 134.

Histological observation of callus morphology in rice

J. O. Narciso and K. Hattori, Laboratory of Plant Genetics and Breeding, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan

We evaluated callus morphology, empha- sizing cell compositions of rice varieties. through histological observation.

Rc2, Oboshi, 2757, 2764), two japonica (Nipponbare, Tsutsu), and two javanica (Lemonte, Rinatte) varieties was evaluated in two callus induction media. Varieties showing good callus growth 2 and 4 wk after culture were evaluated through histological observations using scanning electron and light microscopes.

Of the varieties tested, IR54, Toboshi, Nipponbare, Tsutsu, and Rinatte exhibited good callus growth. Callus morphology of each subspecies differed from one another. At 2 wk after culture, calli of IR54 and Toboshi were characterized by two callus masses: one was yellow, compact, and smooth-surfaced; the other yellowish with hairy protuberance and green spots. Japonica varieties Nipponbare and Tsutsu and javanica variety Rinatte had almost similar morphology. Calli of both sub-

Callus morphology of five indica (IR54.

1. Dehusked grains of a) indica (IR54), japonica (Nipponbare), and javanica (Rinatte) and b) their corresponding callus types.

species were yellowish, compact, and smooth-surfaced. Slight changes in callus morphology were noted 1 mo after culture. Green spots occupied more than 50% of the whole callus mass in IR54. Calli of Nipponbare and Tsutsu became globular and easily disintegrated into small pieces. Callus of Rinatte became friable. Callus types of the three subspecies represented by IR54, Nipponbare, and Rinatte are shown in Figure lb.

Histological observation through a scanning electron microscope showed that

each callus type had a distinct cell cum- position (Fig. 2a-c). Callus of IR54 showed a large, highly compact cell mass with domelike structures. The surface was rough with corrugations (Fig. 2a). Callus of Nipponbare was made up of compact, globular cell masses of about the same size (Fig. 2b). The cell masses were composed of round cells arranged in layers, like a flower. Callus of Rinatte was composed of small. round, compactly arranged cells (Fig. 2c). Light microscopic observation of the resin sections generally showed darkly stained, meristematic cell clusters.

The results of a study using mungbean suggest that cell types in a callus mass relate to embryogenic potential (Narciso and Hattori 1995). This relationship has not been fully studied in rice. Results of this research could provide the basic information needed to establish this relationship.

Cited reference Narciso JO, Hattori K. 1995. Different cell

compositions in mungbean ( Vigna radiata (L.) Wilczek) cotyledon calli. Breed. Sci. 45(2):173-177.

2. Scanning electron microscopic observation of the cell compositions in the calli of a) IR54, b) Nipponbare, and c) Rinatte. Scale bars = 300 µ.


Scanning electron microscopy observations of the flag leaf blade surface at the abaxial side of a dripping-wet mutant. Nipponbare (wild type) (a and b) dripping-wet leaf mutant (c and d). LP = large papillae, SP = small papillae, S = stomata, P = pili.

is one of the putative causes of wettability. To study the histological basis of leaf wettability, we observed the leaf surface of the newly obtained dripping-wet leaf mutant under a scanning electron microscope.

We observed that the dripping-wet leaf mutant was also an epicuticular waxless mutant. Large and small papillae, stomata, and pili on the mutant leaves were not so different from those on wild types (see figures 1a, 1c). However, the mutant leaf had a plain surface with epicuticular wax less developed than that found in wild types (see figure 1b, 1d). We also investigated the inheritance of epicuticular wax and dripping-wet leaf traits in crosses of mutant type/wild type and wild type/mutant type. The F 1 plants in both crosses were wild types. In the F 2 of both crosses, dripping- wet leaf plants always had the epicuticular waxless trait. The segregation ratio of the wild type to mutant type (dripping-wet leaf and epicuticular waxless) fitted to a 3-1 ratio

in both crosses, indicating that the dripping- wet leaf trait was caused b) reduced leaf epicuticular wax.

Many mutants with less epicuticular wax accumulation were already obtained in Arabidopsis, barley, and wheat (Lemieux et al 1994). Reduced wax on the leaf surface altered the reflection of light, which allowed isolation of the mutants through visual inspection. These mutants were designated as glossy or nonglaucous mutants. The dripping-wet leaf mutant of rice might be one of these mutants with reduced epicuticular wax.

Cited references Lemieux B, Koornneef M. Feldmann KA. 1994.

Epicuticular wax and eceriferum mutants. In: Meyerowitz and Somerville, editors. Arabidopsis. New York: Cold Spring Harbor Laboratory Press. p 1031 - 1047.

Satoh H, Iwata N, Omura T. 1983. Gene analysis of some dripping-wet leaf mutants in rice. Jpn. J. Breed. 33 (Suppl. 2): 243-243.


Embryogenic cell suspensions established from a high-protein purple black rice

Huihua Fu, Tingliang Tian, Biology Department, Central Chinal Normal University, Wuhan 430070, China; and Yingguo Zhu, Biology Department, Wuhan University, Wuhan 430072, China

Establishing embryogenic cell suspensions is important in regenerating plants from rice protoplasts. We studied the effects of explants, media, hormones, and culture procedures on callus induction and cell suspension of indica variety HuaHei 01, a new high-protein purple black rice derived from progenies of Hua 03/Hong Jing 501.

Effects of explants. We cultured explants from young panicles (1-10 mm), anther at mid-uninucleate stage, and mature seeds on Murashige and Skoog (MS) medium supplemented with 2 mg 2,4-D L -1 + 0.2 mg Kin L -1 . The lowest callus induction (0.4%) was obtained from anthers. Young panicles had high callus induction (24.6%), as did mature seeds (21.3%). Calli from anthers and mature seed were compact. globular, and yellow, and could be used to establish embryogenic cell suspensions. Calli from young panicles were scattered and pale yellow.

Effects of media and hormone ratios. We chose MS, N6, and GM supplemented with different ratios of hormones for incubating explants. AA, N6, and MS with 2 mg 2,4-D L -1 + 0.2 mg Kin L -1 was used for the liquid suspension culture. Although the calli induction rate on N6, MS, and GM media did not significantly differ, the quality of calli induced on the various media did. Most of the calli induced on N6 were yellow, compact, and globular, and grew rapidly; those induced on MS and GM grew slowly. Calli in the AA liquid medium remained yellow, while those in the N6 and MS media turned brown in the first month of suspension.

Hormones significantly affected induction frequency and callus quality. Time required for callus induction was shortened, and some calli induced were pale yellow with root hairs when 4 mg NAA L -1 , 1 mg 2,4-D L -1 , and 0.2 mg Kin L -1 were supplemented. However, induction


0.2 0

frequency was slightly higher and calli were fresh yellow, compact, and globular when supplemented with 2 mg 2,4-D L -1

and 0.2 mg Kin L -1 (see table). Effect of culturing procedures. When

calli reached 1-2 mm in diameter, half were transferred into AA, MS, and N6 liquid media supplemented with 2 mg 2,4-D L -1

and 0.2 mg Kin L -1 and shaken at 120 rpm.

The other half was subcultured onto the same induction medium with 500 mg proline L -1 for 10 d to 6 mo (subcultured once a month) and then transferred into liquid suspension culture. Calli turned brown and died in 3-4 d in liquid culture with immediate transfer or subculture for less than 1 mo. On the other hand, calli could propagate quickly when transferred

into liquid media after subculture on solid induction media for 1 mo or longer. At the start of suspension culture, the calli were subcultured every 3 d by replacing all original medium with the same fresh medium. After 4 wk, the aggregates grew well, and cultures were subcultured every 6 d by replacing two-thirds of the original medium with the same fresh medium. Subsequently, the well-grown cultures released many miniclusters into the liquid medium. Thus, through about 3 mo of suspension culture, we obtained embryogenic cell suspensions composed mainly of cell aggregates consisting of 10-60 cells with fresh yellow color and dense cytoplasm.

Factors affecting pollen embryogenesis of rice anther culture

J. K. Sohn, Agronomy Department, Kyung- pook National University, Taegu, Republic of Korea (ROK); G. H. Yi, B. G. Oh, National Youngnam Agricultural Experiment Station, 1085, Milyang, ROK; S. J. Yang, IRRI: and T. S. Kwak, Agronomy Department, Sangi University, Weonju, ROK

Anther culture techniques as a means of breeding are considered effective and time- saving methods for obtaining pure linea in rice and for other major crop improvement.

Pollen embryogenesis is believed to provide new prospects for anther culture because of its stability and enhanced recovery of regenerants.

We occasionally observed pollen embryogenesis during rice anther culture in many cultivars and F 1 plants in our laboratory. Growth regulators. water stress, and media composition affect the induction rate of pollen embryogenesis (Torrizo and Zapata 1986).

Abscisic acid (ABA) in the callus formation medium was most effective for pollen embryogenesis of rice; callus formation from the anthers plated did not increase significantly. However, embryo formations were markedly increased.

Effects of hormones on callus induction from mature seeds of rice. a

Hormone (mg L -1 ) Callus Induction frequency (%) Callus characteristics

NAA (4) + 2,4-D (1) + kinetin (0.2)

2,4-D (2) + kinetin (0.2)

a Basic medium used was Murashige and Skoog (MS).

20.8 Pale yellow; some calli with

24.2 Globular, compact, and yellow root hairs

Table 1. Effects of abscisic acid (ABA) on embryo and callus formation in rice anther cutture.

ABA Responding (mg L -1 ) anthers (%)

0 19.7 ± 2.3 a

0.5 28.8 ± 2.2 1.0 19.7 ± 2.1 5.0 18.1 ± 1.8

10.0 15.1 ± 1.6 20.0 7.8 ± 1.4

a Mean ± SE.

Calli formed (%)

8.4 ± 1.1 9.1 ± 1.1 7.2 ± 1.2 6.6 ± 1.0 7.3 ± 0.9 2.9 ± 0.7

Embryos formed (%)

1.6 ± 0.4 10.8 ± 1.2

7.2 ± 0.8 8.8 ± 0.9 5.5 ± 0.9 3.2 ± 0.5

Table 2. Effects of growth regulators on germination of pollen embryos in rice anther culture.

Growth regulators (mg L -1 ) Plants (%)

Zeatin IAA a Kinetin Green Albino

1 0 0 6.1 ± 2.0 b 5.0 ± 5.0 5 0 0 13.9 ± 3.9 5.0 ± 5.0 1 8.6 ± 1.8 5 0.2 0 9.8 ± 0.2 0 0.5 2 26.7 ± 1.9 0 0 0 8.0 ± 1.0

2.0 ± 2.0 0 0 8.0 ± 5.8

a IAA = indole acetic acid. b Mean ± SE.

Maximum frequency (10.8%) of embryo formation was obtained at 0.5 ppm ABA (Table 1). Also, ABA treatment during the cold pretreatment period (15 °C, 15 d) showed the same tendency as that under in vitro condition.

We tested several combinations of plant growth regulators for germinating pollen embryos. The combination of 0.5 ppm

indole acetic acid and 2.0 ppm kinetin was suitable for germinating embryos (Table 2).

The results indicate that ABA is important in plant regeneration and embryo formation in rice anther culture.

Cited reference Torrizo LB, Zapata FJ. 1986. Anther culture of

rice. IV. The effect of abscisic acid on plant regeneration. Plant Cell Rep. 5:136-139.

Genetic studies on purple pigmentation in rice and its use in breeding two-line rice hybrids

M. Tongmin, L. Chunhai, Y. Guocai, and L. Xinggui, Food Crop Research Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China

Many scientists, particularly those in Japan, have systematically studied how purple color is inherited in rice leaves. Three pairs of basic genes ( C , A, P ) control the inheritance of anthocyanin pigments (Nagao 1951 and Takashi 1957). The C-A- P gene system in japonica rice is suitable for indica rice (Kinoshita 1984). All C, A, P genes have their multiple alleles as well as several main genes with multiple effects. The gene P1, for example, is responsible for giving leaves, nodes, internodes, sheath, and leaf rings their purple color. At least one or two pairs of inhibitor genes influence the expression of purple leaf color.

We studied the inheritance of two newly discovered purple rice lines and the possibility of their use as gene markers in hybrid rice breeding.

OPL and PL 184 with purple leaves were discovered in fields planted to normal green varieties Ketan Nangka and W6184S in 1988 and 1990, respectively. (They were crossed with each other and then crossed with IRRI variety IR1552 in 1991 and 1992.) The resulting F 1 s and F 2 s were purple, and no segregation in F 2 populations was found, inferring that OPL, PL 184, and IRI552 may have allelic genes controlling purple pigmentation of seedlings. How- ever, when OPL and PL 184 were crossed with 23 normal green rice cultivars or lines (12 indica thermosensitive genic male sterile (TGMS) lines, 2 japonica photoperiod-sensitive genic male sterile (PGMS) lines, 2 indica wild abortive (WA) lines, 1 indica BT lines, 4 conventional indica cultivars. and 2 japonica cultivars), all F 1 s had green leaves, indicating that the purple leaf character of OPL and PL 184

Table 1. Segregation and genetic ratio of seedling leaf color in F 2 populations from crosses between purple and green rice varieties.

Cross plants plants plants ratio Total Green Purple Genetic

(no.) (no.) (no.)

W9056 S/PL 184 1,081 827 254 3:1 1.2239 0.50-0.25 W8013 S/PL 184 1,361 1,038 323 3:1 1.0994 0.50-0.25 Shuang-guang S/PL 184 5,360 4,352 1,008 13:3 0.0077 0.90-0.75 Zhen-shan 97 A/PL 184 12,034 9,833 2,201 13:3 1.6425 0.25-0.10 Mi-ai 64 S/PL 184 17,898 14,520 3,378 13:3 0.1711 0.75-0.50 Pei-Ai 64 S/PL 184 6,025 4,852 1,175 13:3 1.9957 0.25-0.10 Yue-tai A/PL 184 2,034 1,655 379 13:3 0.0116 0.90-0.75 W91273 S/PL 184 2,699 2,173 526 13:3 0.9153 0.50-0.25 W6068 S/PL 184 8,127 6,947 1,180 55:9 1.3669 0.25-0.10 An-nong S/OPL 3285 S/PL 184

4,809 4,158 651 55:9 1.0557 0.50-0.25 4,720 4,030 690 55:9 1.1624 0.50-0.25

W91238 S/PL 184 E47 S/PL 184

3,969 3,439 530 55:9 1.5882 0.25-0.10 1,671 1,448 223 55:9 0.6549 0.50-0.25

HN5 S/PL 184 4,143 3,698 445 229:27 0.1454 0.75-0.50

Table 2. Segregation and genetic ratio of seedling leaf color in testcross F 1 and three way cross F 1 .

Generation Total Green Purple Genetic P Cross plants plants plants ratio

(no.) (no.) (no.)

Zhen-shan 97 A/PL 184// TC 148 76 72 1:1 0.0608 0.90-0.75 PL 184 HN5 S/PL 184//PL 184 TC 110 62 48 1:1 1.5364 0.25-0.10 Shuang-guang S//W9056 S/ F 2 8434 7400 1034 113:15 2.3311 0.25-0.10 PL 184

was recessive. This trait can therefore be used as a gene marker in hybrid rice breeding.

We investigated leaf color at the seedling stage (about five leaves) in the F 2 generation of 14 crosses between PL 184 or OPL and green lines (Table 1). Crosses 1 and 2 had a 3:1 genetic ratio for green:purple, indicating that PL 184 and W9056S or W8013S differed in one pair of genes. Crosses 3-8 had a 13:3 genetic ratio for green:purple, indicating differences between parents were based on two pairs of genes, one of which was an inhibitory gene. Crosses 9-13 had a 55:9 genetic ratio, indicating that differences between green and purple parents were based on three pairs of genes, one of which was an inhibitory gene. The F 2 s from HN5S/PL 184 had a 229:27 genetic ratio, indicating that differences between parents were based on four gene pairs, one of which was an inhibitory gene.

Although the green:purple ratios in the F 2 s of Zhen-shan 97 A/PL 184 and HN5S/ PL 184 differed, both backcrosses had a genetic ratio of 1:1 (Table 2), which confirmed an inhibitory gene in the gene system of the purple character in rice. Furthermore, the ratio of green:purple in triple cross Shuang-guang S//W9056S/PL 184 was 113:15, indicating that inhibitory genes in W9056S and Shuang-guang S were nonallelic.

Purple-leafed sterile plants were found in all F 2 s of crosses with TGMS or PGMS lines, indicating that purple genes and TGMS or PGMS can be combined into one line. Several purple-leafed TGMS lines have been developed. However, crossing these lines with normal green rice varieties produced F 1 s with green leaves, and PL 184-5 and IR1552 had better maintainer ability than Zhen-shan 97 A. The results suggest possibility of using purple leaf character as a gene marker in hybrid rice breeding.

Cited references Kinoshita T. 1984. Gene analysis and linkage

map. In: S. Tsund, N Takashi. editors. Biology or rice. Tokyo: JSSP/Elsevier. p 187- 274.

Nagao S. 1951. Gene analysis and linkage relationship of characters in rice. Adv. Genet. 4:181-212.


P c 2

c 2

Improving provitamin A (carotenoid) content of rice endosperm

D. Mozoub, P. D. Matthews, V. N. Upasani, E. T. Wurtzel, Z. H. Li, R. B. Luo, D. Matias,

Biological Sciences Department, Lehman G. Valdez, A. Yoganathan, and J. Yu,

College of The City University of New York, Bronx, New York 10468, USA

Genetic engineering is a feasible approach

Calorific values of 60 rice varieties

to alter or improve carotenoid content of endosperm, an agronomically valuable tissue. However, to plan a strategy for engineering rice endosperm, we must identify the biosynthetic block preventing carotenoid accumulation. Three alternative possibilities are being considered: 1) genes encoding one or more of the biosynthetic

P. V. Nandini, L. Prema and C. E. Ajithkumar,

enzymes may not be expressed in the endosperm, 2) these enzymes are expressed but are not functional, and/or 3) a limitation

College of Agriculture, Thiruvananthapuram, Kerala, India

We determined the calorific values of 60 rice varieties (30 hybrid derivatives, 28 traditional, and 2 improved varieties) in raw and parboiled forms (see table).

Calories significantly differed among the varieties. In raw rice, traditional variety Aryankali had the highest value (371 kcal) and the hybrid derivative Bhadra. the lowest (279 kcal). We observed a significant variation in calories in all rice

varieties after parboiling, with Aryankali (383 kcal) and Bhadra, the lowest (301 again having the highest calorific value kcal).

Calorific values of 60 rice varieties.

Variety

Raw Parboiled Variety rice rice

Raw Parboiled rice rice

(kcal g -1 ) (kcal g -1 ) (kcal g -1 ) (kcal g -1 )

Kavungin-poothala 330 357 Thrissur local 2 312 333 Veluthavattan 317 337 Ponnaryan 332 348 Kattamodan 342 380 Chuvannari Thavalakannan 336 356 Arvan 345 375 Veluthari Thavalakannan 346 359 Vadakken Chitteni 307 358 Thkkencheera

345 353 Cheriya Aryan Thekken

Elappapoochemban 335 372 Sinduram

Chenkayama 342 345 Aruvakkari Chuvannamodan 321 329 Kutticheradi

Navara 341 358 lmproved Thrissur local 1 312 333 CO 25

Mashuri

357 365 316 364 348 354 344 356 316 373

341 358 332 362

Cibodas, a high-yielding variety with good grain quality

A. Partoatmodjo, Allidawati, and Z. Harahap, Research Institute for Food Crop Biotech- nology, JI. Tentara Pelajar 3A, Bogor 16002, Indonesia

B9775b-Mr-8-1-1 (derived from the cross B7004d-Mr-10-1/B6992f-Mr-262) is a

high-yielding line with good grain quality. Released as Cibodas for the irrigated rice ecosystem in Sep 1995, the variety is resistant to brown planthopper biotype 1 and bacterial blight strain III. It yields about 30% more than IR64 and Cisadane at 200- to 500-m above sea level (Table 1). Cibodas seems more suitable for cropping on intermediate elevation (about 400 m).

Table 1. Average yield of Cibodas in seven provinces in Indonesia. 1993-95.

We compared grain quality characteristics of Cibodas with those of IR64 and Cisadane (Table 2). Large grain size (1,000-grain weight = 349) probably contributes to the variety’s high yield potential. Cibodas has intermediate amylose content and tender cooking quality that rice consumers in Java prefer.

Locations Elevation Yleld Increase Province (no.) (m) (t ha -1 ) over check

(%) a

Jakarta West Java West Java

1 3 3

50 200-450 400-600

5.7 8.2 7.0

2 a 30 b 29 a

Central Java 3 2-18 5.8 12 a East Java Lampung Bengkulu

1 1 1

86 750 320

6.2 6.3 6.9

5 a 14 a 12 a

a a = IR64, b = Cisadane.

Table 2. Grain quality characteristics of Cibodas, IR64, and Cisadane.

Characteristic Cibodas IR64 Cisadane

Yield (t ha -1 ) Plant height (cm) Days to maturity 1,000-grain weight (g) Head rice recovery (%) Grain length (mm) Grain length-breadth ratio Amylose content Cooking quality

5.7 - 8.2 115 123

34 80

6.5 2.4

24 Tender

3.8 - 6.2 99

115 25 80

7.0 3.2

22 Tender

4.8 - 7.8 110 135

28 80

6.7 2.6

20 Tender


Grain quality

1. RT-PCR analysis: PSY and PDS transcribed throughout rice endosperm development. a M = mature endosperm. b DAF = days after flowering. c Sh = Sucrose synthetase.

exists in precursors available to the pathway. Rice endosperm lacks both end products of the carotenoid biosynthetic pathway and any intermediates, suggesting that biosynthetic enzymes may not be expressed. Only geranylgeranyl diphosphate (GGPP), a substrate of the first enzyme specific to the carotenoid biosynthetic pathway, is present. GGPP is a precursor common to other pathways, such as the gibberellic acid biosynthetic pathway.

We developed gene and protein probes to test the expression of the PSY and PDS, the first two enzymes specific to the pathway. Genes encoding both enzymes were each mapped to a single genetic locus in rice and in maize and behaved as single

copy genes by Southern blot analysis. Reverse transcriptase-polymerase chain reaction showed transcripts in the leaves and endosperm. In developing rice endosperm. the PSY transcript was constant, whereas the PDS transcript increased over time (Fig. I). Using a polyclonal antiserum raised against maize PSY, Western blot analysis showed PSY to be constantly expressed in developing rice endosperm (Fig. 2). We used a starch gene encoding sucrose synthetase to compare the transcript and protein analyses. Finally, high-pressure liquid chromatography analysis of a rice carotenoid mutant showed phytoene (a carotenoid intermediate) in the leaves and embryos but not in the endosperm.

Phytoene-forming activities in wild-type and transformed rice endosperm

P. Beyer, University of Freiburg, Institute of Biology II, D79104 Freiburg, Germany; P. K. Burkhardt, Swiss Federal Institute of Techno- logy (ETH), Institute for Plant Sciences, CH-8092 Zurich, Switzerland; M. Schledz, S. Al-Babili, M. Bonk, and J. von Lintig, University of Freiburg; G. A. Armstrong and I. Potrykus, ETH

Rice endosperm does not contain carotene, including phytoene, the first carotene in the biosynthetic pathway. To develop a strategy for rice transformation with the aim

of obtaining phytoene synthesis in endosperm (as a first step toward b -carotene accumulation), we investigated in initial experiments the enzymatic prenyl lipid- forming activity of this tissue. We incubated immature endosperm with [1- 14 Clisopentenyl diphosphate and [ 14 C] geranylgeranyldiphosphate (GGPP). After hydrolysis with alkaline phosphatase to convert prenyl phosphates into their corresponding alcohols, followed by extraction, reverse phase-high performance liquid chromatography (RP-HPLC) analysis revealed farnesol, geranylgeraniol, a third unidentified alcohol, and squalene as the main products in incubations with radio-labeled isopentenyl diphosphate.


2. Western blot analysis: PSY protein expressed throughout rice endosperm development.

Several factors likely cause the carotenoid deficiency in rice. Genes encoding PSY and PDS, the first two biosynthetic enzymes, are expressed in developing rice endosperm. However, the absence of intermediates, even in rice mutants blocked in the pathway, suggests that the endosperm PSY does not function or that precursors feeding the pathway are limiting. Also, the level of PDS transcripts was not constant, suggesting the possibility of discordant expression of enzymes at inappropriate levels or times during endosperm development, which would contribute to the general problem of carotenoid deficiency.

Geranyl-geraniol was most important because its diphosphate, besides being a substrate in the formation of various prenyl lipids, is also the first carotenoid-specific precursor—the substrate of the enzyme phytoene synthase. Both phytoene synthase and GGPP are localized in the plastids of plants.

We cloned a cDNA coding for phytoene synthase from the flower Narcissus pseudonarcissus and proved its identity by functional expression (enzymatic activity) in insect cells, using the baculovirus system. To confirm the presence of functional transit sequence, we studied the import of a radio-labeled in vitro translation product into isolated pea chloroplasts.

These experiments showed a high extent of protein import as measured by inaccessi- bility to thermolysin digestion. We conclude that the cDNA from N. pseudonarcissus codes for a functional phytoene synthase and thus is suitable for rice transformation.

The cDNA was subcloned under the control of the CaMV35S promoter and glutelin promoter (Burkhardt et al 1996, Potrykus et al 1996). We examined the resulting transformants for phytoene synthase expression. First, reverse-transcriptase polymerase chain reaction was established to detect mRNA of the introduced transgenes in rice endosperm. The results revealed the presence of specific

mRNA, which was absent in endosperm of control plants. Second, using homologous anti-phytoene synthase antibodies, phytoene synthase was detected in transformed endosperm, whereas no signal was obtained in the control plants. Third, using RP- HPLC, the phytoene content was determined in mature rice seeds deprived of the embryo. Phytoene content was usually below detection limit in the controls: in some cases, however, insignificantly low amounts probably derived from seed coat were present.

In contrast, high amounts of phytoene found in seeds of some transformants allowed clear UV/VIS spectra-proving identity to be obtained. Constructs under glutelin promoter control were successful in

phytoene formation. The best line obtained so far showed a phytoene content of 1 µmol per gram (after quantification by calibrated integration on HPLC and use of a molar extinction coefficient of 68,125 mol -1 cm -1 ). This corresponds to 545 µg phytoene per gram, which is safely within the nutri- tionally useful range (Burkhardt et al 1996).

Cited references Burkhand PK, Beyer P, Terada R, Kloti A, Wünn

J, Lintig JV, Armstrong GA, Potrykus I. 1996. Genetic engineering of provitamin A biosynthesis in rice endosperm. In: Khush GS, ed. Rice genetics III. Manila (Philippines): International Rice Research Institute, p 818-821.

Screening of Basmati rice genotypes against blast

R. Singh and D. S. Dodan, CCS, Haryana Agricultural University, Rice Research Station, Kaul 132021, Haryana, India

Blast caused by Pyricularia grisea Sacc.= Magnaporthe grisea (Hebert) Barr., is a devastating disease of scented, tall varieties in Haryana. We screened 175 rice genotypes in station and coordinated trials at the Rice Research Station in Kaul.

For leaf blast screening in the Uniform Blast Screening Nursery, we alternated each test genotype with susceptible variety Taraori Basmati on all sides during the 1993 kharif (dry season). For neck blast screening, 1-mo-old seedlings of test entries were transplanted in two 5-m-long rows at 20- × 15-cm spacing. One line of Taraori Basmati was planted between each test entry to ensure maximum disease development. We recorded leaf blast incidence during the last week of September when the disease was at its peak. Tillers of 25 randomly selected hills of each test entry were examined for neck blast 1 wk before harvest. We scored the disease severity using the Standard evaluation system for rice (1988). Genotypes showing

Basmati rice genotypes showing resistance to blast. Haryana, India, 1993 and 1994 kharifs.

Leaf blast Neck blast Both leaf and neck blast

HKR86-416 HKR90-403 HKR90-403 HKR90-403 HKR90-404 HKR91-405 HKR90-407 HKR91-405 HKR90-410 HKR91-431 HKR91-401 RP3121-14-10-2 HKR91-402 RP3138-60-91-0-6 HKR91-404 RP3238-38-15-7-1 HKR91-405 RPST328 HKR91-408 NDR637-90 HKR91-413 UPR-BS-92-5 HKR91-424 HKR91-427 HKR91-456 HKR91-459 HKR91-460 HKR91-467 HKR92-405

HKR239 HKR92-409

resistance to leaf or neck blast were retested during 1994 kharif to confirm their reaction to the disease.

We found 19 genotypes resistant to leaf blast and 10 genotypes to neck blast during both years (see table). Only genotypes HKR90-403 and HKR91-405 showed resistance to both leaf and neck blast. Besides blast, three genotypes (HKR86- 4 16, HKR90-404, and HKR92-405) also exhibited resistance to stem rot.

Resistance spectrum, race specificity, and expression of the Xa21 gene family

Guo-Liang Wang, Wen-Yuan Song, Li-Li Chen, R. Ruan, S. Sideris, and P. C. Ronald, Plant Pathology Department, University of California, Davis, CA 95616, USA

The rice gene Xa21 confers resistance to the bacterial pathogen Xanthomonas oryzae ( Xoo ) (Ikeda et al 1990, Ronald et al 1992). The Xa21 locus consists of a small multigene family, and transgenic lines (T 0 ) expressing a single member of the Xa21 gene family confer resistance to Xoo race 6 (Song et al 1996). Unlike all other cloned plant disease resistance genes, the deduced amino acid sequence of Xa21 encodes a glycosylated leucine-rich repeat (LRR) extracellular domain, a single-pass transmembrane domain, and a serine threonine kinase intracellular domain. The sequence of the predicted protein of Xa21 strongly suggests its role in cell-surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response.

To determine the resistance spectrum of cloned resistance gene Xa21. we tested 300 T 1 progeny from the transgenic line 106-17 containing Xa21 for Xoo resistance.


Pest resistance—diseases

Inoculation results indicate that adding a single gene ( Xa21 ) is sufficient to confer resistance to all seven Philippine races of Xoo and 20 isolates from China, Colombia, India, Indonesia, Malaysia, Nepal. and Thailand. Resistance to Xoo strains of Philippine races 1 through 6 cosegregated with polymerase chain reaction (PCR) profile of the Xa21 transgene in a 3:1 ratio. Southern blot analysis confirmed the PCR results, indicating that a single member of the Xa21 multigene family can confer resistance to diverse strains isolated from seven countries.

The Xa21 gene family contains eight members, all located at the Xa21 locus. Each member contains an LRR motif.

A bacterial blight-resistant, wide-compatible indica line

Zhu Lihong and Zhang Hongsheng, Nanjing Agricultural University, Nanjing 210095, China

In China, scientists started to develop inno- vative elite lines with genes for resistance to bacterial blight (BB) Xanthomonas oryzae pv. oryzae ( Xoo ) in the early 1980s. Scientists are now using wide compatibility as an approach to develop indica/japonica hybrid rice through pyramiding genes for wide compatibility, disease resistance, and agronomically important characters.

We determined resistance to BB by evaluating lesion length 20 d after inoculation (9 × 108 cells ml -1 concentration) using the clipping method at booting stage. We used 10 Xoo isolates (provided by the Plant Protection Department, Nanjing Agricultural University) of major

Differences in the LRR domain of the family may play a role in the specificity of pathogen recognition. To test this idea. we generated transgenic plants carrying subclones from other members of the Xa21 gene family. The resistance of these lines to the six Xoo races has been determined. Preliminary screening showed that at least one copy (copy D) confers partial resistance to races 1 and 6 in transgenic plants.

To analyze the expression of the Xa21 gene family, we made a cDNA library from the resistant line infected with Xoo. We identified 17 cDNAs hybridizing with LRR or/and kinase domains. Sequencing results indicate that at least four copies of the gene family are expressed.

Cited references Ikeda R, Khush GS, Tabien RE. 1990. A new

resistance gene to bacterial blight derived from O. longistaminata. Jpn. J. Breed. 40 [suppl.l]:280-281.

Ronald PC, Albani B, Tabien R, Abenes L, Wu KS, McCouch S, Tanksley SD. 1992. Genetic and physical analysis of the rice bacterial blight disease resistance locus, Xa- 21. Mol. Gen. Genet. 236: 113-120.

Song WY, Wang GL, Chen L, Kim KS, Holsten T, Wang B, Zhai Z, Zhu LH, Fauquet C, Ronald PC. 1996. The rice disease resistance gene, Xa-21, encoded a receptor kinase-like protein. Sci. (in press)

KS-6-6 (II) (Jiangsu), HB84-17 (II) characters. 85-21416 was used in 1988 as a (Heibei), OS105 (II) (Guangdong), FJ856 resistance donor in intersubspecific genetic (III) (Fujian), ZHE173 (IV) (Zhejiang). studies. AH023 (IV) (Anfei), GD1358 (V) The shorter lesion lengths of 85-21416 (Guangdong), GX325 (V) (Guangxi), and confirmed its stable BB resistance JS49-6 (VII) (Hunan). (Table 1). A recessive gene allelic to xa5

We evaluated wide compatibility using (data not shown) controls the resistance. spikelet fertility from testcrosses to both 85-21416 may have inherited the resistance typical indica and japonica cultivars. Mean fertility of unbagged F 1 plants was assessed at maturity. Spikelet fertility of up to 70% crossed with various tester varieties. a

was normal and compatible.

Table 2. F 1 spikelet fertilities of 85-21416

85-21416 is a promising indica line Cross combination b Mean Plants (no.)

developed from a 1978 cross between Taichung Native 1, a susceptible and

Balilla/85-21416 87.3 ± 3.58 15 Aikihikari/85-21416 91.7 ± 3.20 14

noncompatible indica cultivar from Taiwan. 85-21416/IR36 87.9 ± 5.04 6

China, and DZ78, a highly resistant, tall indica from Bangladesh. 85-21416 has

Ketan Nangka/ 62.5 ± 1.41 18 02428/85-21416 79.4 ± 5.28 12

85-21416 strong resistance to BB and is extremely 85-21416/CPSL017 68.4 ± 1.73 18

compatible in intersubspecific crosses. We 85-21416 (I) 80.6 ± 5.31 14

grew F 3 lines of this cross combination in Balilla (J) 67.9 ± 4.86 12 Aikihikari (J) 89.3 ± 3.51 10

early spring of 1980. We then selected 02428 (J) 71.4 ± 7.06 10 IR36 (I) 92.9 ± 1.05 15 CPSL017 (I) 82.6 ± 3.00 10 individuals each year through artificial

inoculation evaluation. By 1985,85-21416 pathotypes I through VII from different along with other advanced lines (F 7-8 ) were a Data were collected in 1992, except those for 85-

provinces in China: OS14 (I) (Liaoning), selected for stable resistance and other 21416/IR36 and IR36, which were collected in Sep 1995. b l = indica, J =japonica.

Table 1. Reactions of 85-21416 to bacterial blight isolates compared with Nanjing 11, DV85, and IRBB7.

Isolates Line/variety Gene Evaluation criterion Year

OS14 HB84-17 KS-6-6 OS105 FJ856 ZHE173 AH023 GD1358 GX325 JS94-6

85-21416 xa5(t) 1.7 1.4 1.8 0.8 1.1 1.1 1.5 3.9 1.6 0.6 Lesion length (cm) 1994 DV85 xa5, Xa7 1.2 1.1 1.2 1.5 1.1 1.3 1.3 0.7 1.6 0.9 Lesion length (cm) 1994 IRBB117 Xa7 3.0 3.0 5.0 3.0 Leaf area necrotized (%) 1995 Nanjing 11 None 19.8 26.1 25.8 20.6 13.1 19.8 23.7 9.7 25.6 10.2 Lesion length (cm) 1994


- - - - - -

gene from DZ78, which carries xa5 and xa7.

F 1 hybrids derived from crosses between 85-21416 and 16 japonica cultivarsin 1989 were fertile, with 84.2 ± 2.8% average spikelet fertility. 85-21416 carries a

compatibility gene that appears allelic to compatibility gene carried by 85-21416 S-5 n . Fairly fertile hybrids were obtained could have come from indica DZ78, which from crosses between 85-21416 and produced a compatible intersubspecific cultivars known to have the wide- hybrid when crossed with japonica tester compatibility gene S-5 n (Ketan Nangka, cultivar Akihikari (69.1 ± 1.1% spikelet CPSL017, and 02428) (Table 2). The fertility in 1992).

RFLP mapping of blast resistance gene Pi-k m

in rice

R. Kaji and T. Ogawa, Kyushu National Agricultural Experiment Station, Chikugo, Fukuoka 833, Japan

Blast resistance gene Pi-k is located on chromosome 11 (Goto et al 1981) and has multiple alleles Pi-k h , Pi-k m , and Pi-k p

(Kiyosawa 1978). Although restriction fragment length polymorphism (RFLP) linkage analysis indicated that Pi-k is located on chromosome 11, no RFLP marker closely linked to Pi-k was found at that time. For fine-mapping of Pi-k loci, we conducted RFLP analysis of Pi-k m and

RFLP markers using F 3 lines from the cross between Tohoku IL4 (japonica near- isogenic line with Pi-k m introgressed from Tsuyuake) and CO 39 (susceptible indica variety).

Each F 3 line was inoculated with Japan- ese blast race 003 at 3-4 leaf stage using the spray method. The F 3 lines were classified either as resistant (or segregating) lines or susceptible lines 30 d after inoculation.

DNA was extracted from the leaves of each F 3 line using the cetyltrimethyl ammonium bromide method. Total DNA was digested with restriction enzymes ( Eco RI, Hin dIII, and Dra I) and blotted onto a positively charged nylon membrane filter using capillary transfer after electrophoresis. RFLP landmarkers on chromosome 11 and

Linkage analysis among Pi-k m and RFLP markers.

Gene pair Segregation mode in F 3 Genetic map distance (cM)

A - B AABB AABb AAbb AaBB AaBb Aabb aaBB aaBb aabb

Pi-k m L190 36 61 1 R1506 29 47 3

0 1 37 1.5 ± 1.2

G181 28 62 5 0 2 32 4.3 ± 1.0 0 7 34 8.4 ± 1.9

G1465 31 80 12 L190 R1506 26

1 12 32 15.8 ± 2.4 5 1 1 31 2

G181 26 0 1 27 6.0 ± 2.6

3 1 0 42 5 G1465 20 13 3

0 6 29 7.4 ± 1.8

R1506 G181 23 6 48 8

3 0 1 10 27 19.1 ± 3.0

G1465 18 10 1 3 37 3 0 6 27 7.7 ± 1.8 8 33 8

G181 G1465 19 0 9 26 18.5 ± 1.9

8 1 6 54 9 0 8 31 13.3 ± 2.8

cDNA clone R1506 (provided by NIAR/ STAFF) were used. Linkage analyses of Pi- k m and the RFLP markers are shown in the table. Recombination values calculated using the maximum likelihood method were converted into genetic map distances (cM) using the Kosambi function. All RFLP markers used in this study were linked to Pi-k m , notably L190, which was closely linked to Pi-k m at about 1.5 cM.

RFLP linkage map between L190 and R1546 (see figure). Because L190 is located on the terminal region of chromosome 11 (Nagamura et al 1993), results of our study correspond to those of a classical linkage map. For further fine- mapping of the Pi-k m loci, we will subject more F 3 lines to RFLP analysis.

Pi-k m is located on chromosome 11 in the

Linkage map of Pi-k m and RFLP markers on chromosome 11.

Indica/japonica doubled haploid population as a model for mapping rice yellow mottle virus and blast resistance genes

A. Ghesquiere, M. Lorieux, lnstitut Français de recherche scientifique développement en coopération (ORSTOM), BP 5045, 34032 Montpellier Cedex 1, France; E. Roumen, Centre de coopération internationale en recherche agronomique por le développe- ment (CIRAD), BP 5035, 34032 Montpellier Cedex 1, France; L. Albar, ORSTOM and CIRAD; D. Fargette, ORSTOM; N. Huang, IRRI; and J. L. Notteghem, CIRAD

Rice yellow mottle virus (RYMV) is the most damaging rice pest in irrigated fields of West Africa. Blast is a widespread disease causing severe yield losses. To map several resistance genes, we used two doubled haploid (DH) populations derived from the F 1 hybrids IR64/Azucena and IRAT 177/Apuraj and segregating for many agronomic traits. This analysis relied on core restriction fragment length polymorphism (RFLP) maps developed at IRRI for the IR64/Azucena population (Huang et al 1994) and at Cornell University for the IRAT 177/Apura population (Yu et al 1991).

RYMV scoring. The resistance analysis involved 74 IR64/Azucena DH lines and 48 IRAT 177/Apura DH lines. Nineteen-day- old rice plants were mechanically inoculated with a RYMV isolate collected from Mali. Two weeks after inoculation, newly emerged leaves were harvested for assessment of virus concentration by serological methods involving ACP- or DAS-enzyme-linked immunosorbent assays.

Blast scoring. An initial survey involving the parents and 20 IR64/Azucena DH lines allowed us to select six diverse strains of Magnaporthe grisea apparently inter-


acting with different genes or quantitative trait loci (QTLs) in this population (Roumen et al, 1996, unpubl.data). The 135 mapped DH lines were evaluated for resistance to the six blast strains. Twenty to twenty-five plants from each DH line were grown in the glasshouse and inoculated with spore suspensions by spraying or in- jecting into sheaths of rice plants at the 4-5 leaf stage. Resistance scores ranged from 0 (complete resistance) to 6 (high susceptibility).

Genetic mapping. For chromosome 12, additional RFLP markers were mapped on the available 183 DH lines of IR64/ Azucena progeny to refine QTL/major gene locations. Clones mapped on an interspecific backcross were used as probes (Causse et al 1994), and RFLP was identified using a chemiluminescent kit following CIMMYT protocols (Hoisington et al 1994). In addition, we conducted bulked segregant analysis (BSA) as described by Michelmore et al (1991) to compare the RYMV susceptible/resistant pools with 300 randomly amplified polymorphic DNA (RAPD) primers. Interval mapping identified the loci involved in resistance to RYMV or blast (Lander and Botstein 1989) and confirmed by the Kruskall & Wallis nonparametric test using MapQTL 2.4.

RYMV resistance. For the IR64/ Azucena population, interval mapping revealed only one locus associated with significant effects on RYMV resistance on chromosome 12 (LOD=2.57), close to the RG341 marker. This locus probably corresponds to a QTL with a relatively high percentage of explained trait variance (R 2 =0.30). This localization was found highly consistent with that obtained by BSA. A 800-bp band amplified with Op O10 primer was also detected by BSA and mapped at 2.7 cM from RG341. A QTL was detected at the same location for IRAT 177/Apuraj population (LOD=3.27; R 2 =0.33). Because the two recombinant populations shared the five RFLP markers on chromosome 12, an integrative map of chromosome 12 was established on 2.58 individuals, and the QTL was reinforced (LOD=5.57; R 2 =0.37). This result clearly indicates that the resistance genes from IRAT 177 and Azucena were allelic or closely linked.


Blast resistance. Significant resistance loci observed in the IR64/Azucena DH population are summarized in the table. Some loci were strain-specific while others were common to different strains. For instance, a QTL on chromosome 2 was detected with strains Br26, Ch66, and Ch72. Other strains, such as Br26, could reveal up to four different QTLs. Differen- tial QTLs for strain Ph68 were detected following inoculation by spray or injection. QTLs for blast resistance tend to cluster on rice chromosomes (McCouch et al 1994). Two QTLs or major genes are lying on the opposite sides of RG869 (chromosome 12) in the same pattern as observed by Yu et al (1991) and could correspond to Pi4(t) and Pi6(t) resistance genes. For CD69 strain,

the locus detected on chromosome 12 probably corresponds to a major gene because it explained about 90% of the trait variance. In addition, the results suggest that one or two other different QTLs could exist in the vicinity of RG457. Similarly, loci detected on other chromosomes could correspond to previously located genes (see table).

A cluster of resistance genes or QTLs involving a major QTL for resistance to RYVM and several blast resistance genes or alleles was observed on a relatively small portion of chromosome 12 (see figure). This chromosome segment will need special attention and further evaluation for determining its implications in breeding for resistance in rice.

Loci identified for resistance to 6 blast isolates by a QTL detection method (interval mapping) in IR64/Azucena cross. a

BI isolates b

Chromo- Marker/ Map Br26 Ph68 CD69 Ch66 Ch72 C16 correspondence some interval position spr spr or inj spr inj inj spr with known loci

Possible

(cM)

1 RG173 147.4 2.9 RG532 177.8 24.1 (a) c

RG520 0 5.8 3.0 2.3

2

8 G104 34.6 6.1 Pi11 (t)

12 010 73.5 10.8 Pi4 (t)

(20.5 (i) d 11.6 (i) 9.5 (i)

RZ123 16.6 5.7 RG654 27.4 18.8 (i)

5 RG313 37.7 2.8 ie

14.1 (a) RZ70 132.8 RZ225 152.5 15.2 (a)

RG213 20.7 3.0 Pi2 (t) 6

3.1

13.8 (i) or Pi9 (t)

6 RZ144 33.4 1.9 1.5 RZ667 35.2 7.1 (i) 8.2 (i)

RZ617 41.6 22.2 (i)

RG667 92.8 2.3 RG451 111.1 8.9 (i)

10 RZ500 0.0 2.2 i 2.2 CD093 25.0 8.0 (a) 10.2 (a)

11 RG1094 100.2 4.8 4.2 Pi-a? RG118 111.8 21.6 (i) 20.2 (i)

12 RG457 57.9 100.0 i 010 73.5 34.0 (i)

RG341 76.4 37.0 (i) =Pi62?

12 =Pi-ta?

RG869 76.4 6.1 25.0 Pi6 (t) RZ816 103.2 23.2 (i) 90.0 (i)

a Loci with low significance (1.5 < LOD score < 2.3) are reported if detected with at least two Isolates. Letters in

by injection. c (a) = resistance locus brought by Azucena. d (i) = resistance locus brought by IR64. e i = locus detected regular font = LOD score; in italics = % of explained variance by the locus. b spr = inoculation by spray: inj = inoculation

with injection method only.

2

Characterization of a pathogenesis-related gene family induced in rice infected with Magnaporthe grisea

J. D. McGee, Botany and Plant Pathology Department, Purdue University (PU), West Lafayette, IN 47907, USA; N. J. Talbot, T. Bhargava, and J. E. Hamer, Biology Department, PU; and T. K. Hodges, Botany

Alignment of QTLs/major genes for RYMV and and PIant Pathology Department, PU

blast resistance on rice chromosome 12. Loci

indica/japonica F 1 hybrids (IR64/Azucena and grisea and Rhizoctonia solani can cause IRAT 177/Apura). Vertical dashed line indicates significant yield losses. Increased disease position of RG869 marker, which separates Pi4 resistance can be obtained in plants and Pi6 in other studies. The peaks observed on

transformed with genes encoding known each side of the line could correspond to these antifungal proteins, such as the hydrolytic two genes.

were detected using DH lines derived from two Fungal pathogens, such as Magnaporthe

enzymes chitinase and b -1.3-glucanase

Cited references Causse M, Fulton TM, Cho YG, Ahn SN,

Chungwongse J, Wu K, Xiao J. Yu Z, Ronald PC, Harrington SE, Second G, MacCouch SR, Tanksley SD. 1994. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138:1251-1274.

Hoisington D, Khairallah M, Gonzalez-de-Leon D. 1994. Laboratory protocols: CIMMYT Applied Molecular Genetics Laboratory. Second edition. Mexico D. F.: International Maize and Wheat Improvement Center.

Huang N, McCouch S, Mew T, Parco A, Guiderdoni E. 1994. Development of an RFLP map from a doubled haploid population rice. Rice Genet. Newsl. 11:134- 137.

Lander ES, Botstein D. 1989. Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121:185-199.

McCouch SR. Nelson KJ, Tohme J. Leigler KS. 1993. Mapping of blast resistance genes in rice. In: Rice blast disease. RS Zeigler, SA Leong, PS Teng, editors. London: CABI and Manila: International Rice Research Institute. p 167- 186.

Yu ZH. I991. Molecular mapping of rice ( Oryza sativa L.) genes via linkage to restriction fragment polymorphism, (RFLP) markers. Ph D thesis, Cornell University, Ithaca, N.Y.

Yu ZH, Mackill DJ, Bonmann JM, Tanksley SD. 1991, Tagging genes for blast resistance via linkage to RFLP markers. Theor. Appl. Genet. 81:471-476.

(Jach et al 1995, Zhu et al 1994). We isolated a M. grisea -induced promoter that was later used to drive antifungal genes in

infection. rice to suppress the effects of fungal

Rice cDNAs, w hose genes exhibited elevated expression during rice blast infection. have been isolated through differential screening (Talbot et al 1993).

as a source of the M. grisea -induced promoter. MAG-7 was found to share homology with a known class of PR proteins (PR-10 proteins) found in various plants (van Loon et al 1994).

Southern blot hybridization of rice genomic DNA (varieties Co 39, IR36, and IR54) probed with the MAG-7 cDNA indicated that these varieties contain multiple (2-5) copies of the MAG-7 gene

One of these cDNAs, MAG-7, was chosen

1. Northern blot analyses comparing the presence of different rice mRNA transcripts at various times following Magnaporthe grisea infection. 10-µg samples of total RNA isolated either from uninfected 3- wk-old Co 39 seedlings (0 h) or from 3-wk-old seedlings infected for 12, 18, 24, 36, 48, 72, or 144 h were hybridized to different rice PR-10 probes. (a) Total RNA hybridized with a 814-bp nonspecific probe from a PR-10a cDNA, which shares homology with all three rice PR-10 genes. (b) Total RNA hybridized to a 198-bp probe derived from the 5' untranslated region of the PR-10a gene. (c) Total RNA hybridized to a 230-bp probe derived from the 3' untranslated region of the PR-10b gene. (d) Total RNA hybridized to a 231-bp probe derived from the 5' untranslated region of the PR-10c gene. The intensity of chloroplast 23S rRNA in b, c, and d shows similar amounts of RNA loaded onto gels.


per haploid genome. A 16-kb genomic clone isolated from a Co 39 genomic library was found to contain three copies of the MAG-7 gene (named rice PR-10a, rice PR- 10b, and rice PR-10c). Rice PR-10a, sequenced in its entirety, including about 1.4 kb of the 5' promoter region, shared 100% homology with the MAG-7 cDNA. The rice PR-10b and PR-10c genes have been partially sequenced.

We constructed gene-specific probes to observe transcriptional differences among the three rice PR-10 genes in Northern blots of total RNA isolated from Co 39 seedling leaves at 0, 12, 18, 24, 36, 48, 72, and 144 h after M. grisea infection (Fig. 1). Transcripts of rice PR-10a were induced from a low basal level within 12 h following M. grisea infection, with increasing accumulation overtime. M. grisea also enhanced transcripts of rice PR-10b, for which induction became strongly visible 48 h after inoculation. Unlike rice PR-10a and rice PR-10b, rice PR-10c exhibited no signs of induction by M. grisea in RNA samples isolated from infected rice leaves throughout the 144 h.

Tissue prints of M. grisea -infected Co 39 leaves were made using the PR-10a gene-specific probe (Fig. 2). Hybridization of the rice PR-10a probe occurred only at the infection site, illustrating that M. grisea induces the rice PR-10a transcript in a localized fashion.

Genes controlling field resistance to blast in Japanese upland rice detected using RFLP markers

S. Fukuoka, K. Okuno, National Institute of Agrobiological Resources, Tsukuba, lbaraki 305, Japan; M. Kawase, Shikoku National Agricultural Experiment Station, Zentsuji, Kagawa 765, Japan; K. Miura, Hokkaido National Agricultural Experiment Station, Sapporo, Hokkaido 062, Japan; and S. Kiyosawa, Tsukuba International Agricultural Training Center, Tsukuba, lbaraki 305, Japan

After observing the rapid breakdown of vertical resistance genes to blast, such as Pi-k, rice breeders are now developing


~

Barley genes encoding class II chitinase, class II ß 1,3-gluconase, and Type-I ribosome-inactivating protein (Jach et al 1995) will be introduced into rice via Agrobacterium tumefaciens transformation to study their ability to control fungal diseases. Both the rice PR-10a promoter and the constitutively expressed ubiquitin promoter will be used to drive each of these genes; the effectiveness of both promoters will be compared.

2. Tissue print illustrating the localized transcription of the rice PR-10a gene during Magnaporthe grisea infection. A 2-wk-old (Co 39 rice seedling was inoculated with two adjacent 20-µl drops of M. grisea conidial suspension (0.4% gelatin; 1,000 conidia drop -1 ). Following 72 h of incubation, the seedling was pressed onto a nylon membrane and hybridized to an antisense RNA probe specific for the rice PR- 10a transcript. Inset photo: a 4X magnification of the infection site.

Cited references Jach G, Gornhardt B, Mundy J, Logemann J,

Pinsdorf E, Leah R, Schell J, Maas C. 1995. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J. 8:97-109.

Talbot NJ, Ebbole DJ, Hamer JE. 1993. Identi- fication and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575-1590.

van Loon LC, Pierpoint WS, Boller T, Conjero V. 1994. Recommendations for naming plant pathogenesis-related proteins. Plant Mol. Biol. Rep. 12:245-264.

Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ. 1994. Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Bio/Technology 12:807- 812.

breeding lines with field resistance to blast. Field resistance is defined as the resistance that allows effective control of a parasite under natural field conditions. Japanese upland rice varieties are potential gene donors for field resistance. Studies using linkage analysis with conventional genetic markers have pointed to several different loci controlling field resistance to blast (Goto 1970, Shinoda et al 1971, Higashi and Saito 1985).

We studied the chromosomal location of genes controlling field resistance to blast in Japanese upland rice using restriction fragment length polymorphism (RFLP) markers.

RFLP is frequently detected among Japanese varieties. About 40% of RFLP

markers located on the rice genetic linkage map revealed RFLP between lowland and upland rice varieties using DNA probes polymorphic between lowland and upland varieties.

Field resistance to leaf blast was genetically analyzed. Three hundred twelve F 3 lines from a cross between Nipponbare (moderately susceptible, lowland) and Owarihatamochi (resistant, upland) were field-tested for resistance to blast. Of these, 27 F 3 lines expressed resistance equivalent to that of Owarihata- mochi while 27 lines were susceptible. Total DNA was extracted from leaves of resistant and susceptible F 3 lines and their parents. Using 138 RFLP markers, we determined the relationship between

Location of the regions (loci) with significant correlation between genotype of RFLP markers and blast field resistance in Japanese upland rice.

markers and resistance/susceptibility in the F 3 lines. We found a close relationship between blast resistance and genotypic frequency in eight regions of six chromosomes. One region was detected each on chromosomes 2, 6, 7, and 12; two regions were detected on chromosomes 4 and 11 (see figure). The loci linked to RFLP markers on chromosomes 4 and 11 played a major role in expressing field resistance, confirming the results of an earlier study (Wang et al 1994).

Cited references Goto I. 1970. Genetic studies on the resistance of

rice plant to the blast fungus 1. Inheritance of resistance in cross Sensho x H-79 and Imochi-shirazu x H-79. Ann. Phytopathol. Soc. Jpn. 36:304-3 12.

Higashi T, Saito S. 1985. Linkage group of field resistance genes of upland rice variety “Sensho” to leaf blast caused by Pyricularia oryzae Cav. Jpn. J. Breed. 35:438-448.

Shinoda H, Toriyama K, Yunoki T, Ezuka A, Sakurai T. 197 1. Studies on varietal resistance of rice to blast. 6. Linkage relationship of blast resistance genes. Bull. Chugoku Natl. Exp. Stn. A20: 1-25.

Wang L, MacKill D, Bonman J, McCouch S, Champoux M, Nelson R. 1994. RFLP mapping of genes confering complete and partial resistance to blast in a durably resistant cultivar. Genetics 136:1421-1434.

Genetic analysis for true resistance to blast in rice varieties

M. Yamaguchi, Tohoku National Agricultural Experiment Station, Omagari, Akita 014-01, Japan; H. Saitoh, National Agriculture Research Center (NARC), Tsukuba, lbaraki 305, Japan; H. Yaegashi, National Institute of Agro-Environmental Science, Tsukuba, Ibaraki 305, Japan; R. Ikeda, NARC; and T. Higashi, Tohoku National Agricultural Experiment Station

Analysis of unknown blast resistance

lines fixed for susceptibility to TH68-141 showed resistance to blast isolate Naga66- 16 (race 001, incompatible to Pi-a gene), indicating that IR50 has the Pi-a gene. We selected 29 of the B 1 F 2 lines showing the segregation ratio of 3 resistant: 1 susceptible plants against TH68-141 and inoculated these with seven isolates of different races (Table 2). Of these, eight lines showed susceptibility to isolate TH87-20 (race 007- b+, compatible to Pi-a, Pi-i, and Pi-b ) despite showing segregation against isolate Naga69-50 (race 007, compatible to Pi-a and Pi-i ). This indicates that IR50 also has the Pi-b gene. Based on the response of the

gene(s) of introduced or improved indica varieties showing high blast resistance is useful. We estimated the true resistance genes of indica varieties IR50, Suweon 288 (IR24//IR24/IR747), and Takanari (Mil- yang 42/Milyang 25) using the method of Toriyama et al (1983), in which backcrossed progenies are tested for reactions to blast isolates of different races.

IR50 was crossed with Ouu308, a japonica line having only the Pi-a gene. Resulting F 1 plants were backcrossed with parent Ouu308 to obtain B 1 F 2 lines. We inoculated 141 lines of the B 1 F 2 with blast isolate TH68-141 (race 003, compatible to Pi-a only) to estimate the number of true resistance genes involved. The ratio of the B 1 F 2 lines fixed for susceptibility to the total B 1 F 2 lines tested was 17.0%, indicating that IR50 has two or three true resistance genes, exclusive of Pi-a (Table 1). All 24 B 1 F 2

Table 1. Expected and actual ratios of B1F2 lines fixed for susceptibility to the total B1F2 lines tested for different numbers of true resistance genes involved (except for Pi-a).

Genes involved 2 3 4 5 6 (no.)

Expected ratio 25.0 12.5 6.3 3.1 1.6 Actual ratio

IR50 17.0 Suweon 288 3.0 Takanari 13.0

lines to other isolates, it was difficult to estimate other true resistance genes in IR50 (Table 2). The results suggest that IR50 has the Pi-a, Pi-b, and one or two true resistance genes.

backcrossed with variety Nekken 2, which has the Pi-a and S-5 n (wide-compatibility gene to reduce hybrid sterility). We inoculated 100 lines of these B 1 F 2 with blast isolate TH68-141. The ratio of B 1 F 2 lines fixed for susceptibility to the total lines tested were 3.0 and 13.0%, respectively, indicating that Suweon 288 has four to six genes and Takanari has three genes, exclusive of Pi-a (Table 1). Both varieties were verified to have the Pi-a gene after inoculation with isolate Naga66-16, with 3 and 13 of the B 1 F 2 lines fixed for susceptibility to TH68-141, respectively. When seven isolates (Table 2) were used to check 28 and 50 of the B 1 F 2 lines showing the segregation against TH68- 141 respectively for the two varieties, only Takanari was proven to have the Pi-b gene based on the line’s response to isolates Naga69-150 and TH87-20. The results suggest that Suweon 288 has Pi-a and four to six other genes. Takanari, on the other hand, has Pi-a, Pi-b, and two other genes.

Some of the genes we failed to identify in this study are probably new resistance genes, including recessive ones.

Suweon 288 and Takanari were

Table 2. Blast isolates of different races used and the corresponding true resistance genes.

Isolate (race) 68-141 69-150 -02 N86-95A 67-106 85-101 87-20

TH Naga NAO Ina TH Ina 5A TH

(003) (007) (033) (047) (103) (303) (403) (007-b + )

Compatible gene(s) Pi-a Pi-a Pi-a Pi-a Pi-a Pi-a Pi-a Pi-a for true resistance Pi-i Pi-k Pi-i Pi-ta Pi-ta Pi-z t Pi-i

Pi-k m Pi-z Pi-ta 2 Pi-b


Cited reference Toriyama K, Ezuka A, Asaga K, Yokoo M. 1983.

Note to readers To save on costs, the IRRN is now being published three times a year: April, August, and December. We have also discontinued the

A method of estimating true resistance genes IRRN sections on news about research collaboration and to blast in rice varieties by testing their announcements. The material previously covered in these sections backcrossed progenies for race-specific is now included in the Rice Reporter. reactions. Jpn. J. Breed. 33:448-456.

Preliminary evidence of aphid resistance in transgenic plants

Zhen Zhu, Zhaolan Zhou, Zhaoyang Deng, Yuefeng Gao, Yu Zhu, and Xianghui Li, Institute of Genetics, Academia Sinica, Beijing 100101, China

Sap-sucking insect pests (mainly Homop- tera) can severely damage rice crops and also transmit virus diseases. Promising results have already been achieved with transgenic plants expressing the Bacillus thuringiensis ( Bt ) delta-endotoxin gene and proteinase inhibitor genes, which protect plants from Lepidoptera and Coleoptera insect pests, but are not effective against Homoptera insects. Information from this study. which describes pea lectin (P-lec) transgenic tobacco plants showing apparent aphid resistance, may be valuable for controlling rice Homoptera insect pests.

Plant expression plasmid constructed. We cloned and identified the P-lec gene as described by Liu et al(1995). A Hin dIII/ Sac I fragment from pBS-lec I, including 0.9- kb P-lec gene, was inserted into mini Ti- plasmid pK7-1, regulated by CaMV35S promoter in the 5'-end and rbcS Poly(A) signal in the 3'-end. The recombinant was

then transferred into Agrobacterium tumefaciens LBA4404 by electroporation.

Plant transformation. Leaf discs of tobacco line NC89 were transformed via A. tumefaciens. Transformants were selected using kanamycin, and the transgenic plants were confirmed using Npt-II assay.

Bioassay on transgenic plants.Young transgenic tobacco plants and control tobacco plants (4-5 leaves) were individually infested on the top leaves with 80 peach aphids ( Myzus persicae ). Aphid population density was calculated 4 and 6 wk after infestation and used to assign the resistance score (see table).

We evaluated aphid resistance (divided into four resistance levels) based on density of aphid population on the top leaves. The ratio of plants with high resistance to aphids (scores 3 and 4) and the resistance mean in transgenic plants were higher than in the control (see table), indicating that the transgenic plants had better protection from aphids than the control (see figure).

A dramatic difference exists in aphid resistance between the transgenic tobacco and the control, possibly caused by transgenic plants expressing P-lec. However, evidence indicating the effect of pea lectin against some sap-sucking insect pests fed

Bioassay of transgenic and control tobacco plants.

Weeks after infestation b

Score a 4 6

Plants Control Plants P-lec Plants Control (no.) (%) (no.) (%) (no.) (%)

1 21 61.8 8 2

27.6 27 5

79.4 14.9 4 13.8 5 14.7

3 2 5.9 7 24.1 2 4

5.9 6 17.6 10 54.5 0 0.0

Plants P-lec (no.) (%)

8 27.6 8 27.6 4 13.8 9 31.0

Total 34 29 34 29

between 2 and 5 aphids cm -1 leaf; 2 = denslty is in the range of 1-50; and 1 = denslty of more than 10 aphids cm -2 a 4 = plants where peach aphid population density on top leaves is less than 2 aphids cm 2 leaf: 3 = density is

leaf. b Data represent percentage of particular plants to total plants Infested.

Leaves from transgenic tobacco plant (right) and from control tobacco plant. The photo was taken 6 wk after aphid infestation.

on an artificial diet is not available (Powell et al 1993). Possible explanations for this area) the effects of pea lectin on rice brown planthopper and rice green leafhopper used in Powell’s study differed from the effects on aphids, and b) the pea lectin could have lost its antimetabolic activity on Homoptera insects during the artificial diet preparation.

The P-lec gene expression plasmid suitable for rice transformation has already been constructed, and the transformation of rice is under way.

Cited references Liu CM, Yu ZY, Zhu Z, Zhou ZL, Xiao GF, and

Li XH. 1995. Cloning and sequencing of the pea lectin gene. Acta Genet. Sin. 22(4):302- 306.

Powell KS, Gatehouse AMR, Hilder VA, and Gatehouse JA. 1993. Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cincticeps. Entomol. Exp. Appl. 66:119- 126.


Pest resistance—insects

Role of host plant resistance in successful control of brown planthopper in Central Luzon, Philippines

E. B. Medina, C. C. Bernal, and M. B. Cohen, IRRI

Planting a single rice variety over a large area year after year may promote the breakdown of rice resistance to brown planthopper (BPH) Nilaparvata lugens (Stål). This cropping scenario, when combined with other factors such as overuse of insecticides, may lead to BPH outbreaks. Most farmers in Central Luzon, Philippines, have been planting for almost 10 yr the popular variety IR64. Despite this situation, BPH populations in Central Luzon are extremely low. We conducted life table studies and seedbox resistance tests on four BPH populations from Central Luzon to determine if IR64 is still resistant to BPH in Central Luzon, in the process determining the extent to which host plant resistance is responsible for low and stable BPH populations in that region.

We collected BPH in Oct 1994 from four sites, 5-50 km apart, and took the insects to IRRI to evaluate on four varieties: IR22 (no genes for BPH resistance), IR36 ( bph2 gene), IR64 ( Bph1 gene plus additional

minor gene(s)), and IR72 ( Bph3 gene). Initially, we studied the data by analysis of variance using PROC GLM (general linear model) of the Statistical Analysis Software package, with site as a random effect. Two variables (developmental time and nymphal survival) showed a significant site-by- variety interaction; hence, means were not averaged across sites.

IR22 and IR64 did not differ significantly across populations (see table).

In three populations, survival to the adult stage among BPH reared on IR22 and IR64 either did not differ significantly or survival was higher on IR64. In two populations, the developmental period of female BPH reared on IR64 was significantly longer than those reared on IR22. At three sites, IR64 had a lower damage rating than did IR22 in seedbox tests.

Results indicate that IR64 maintains a slight-to-moderate resistance to BPH populations in Central Luzon. It is possible that the minor gene(s) in IR64 contribute to a greater durability of resistance in this variety. The results also suggest that high levels of host plant resistance are not necessary for successfull BPH management in areas, such as Central Luzon, where insecticide use is low and biological control is not disrupted.

Fecundity among female BPH reared on

Performance of BPH from Central Luzon, Philippines, on four rice varieties. a 1994 wet season.

Population Variety Fecundity b Development time c Nymphal survival d Damage rating e

Graphical genotypes of rice parental lines for resistance to green rice leafhopper

K. Tamura, Y. Fukuta, M. Hirae, K. Fukui, and S. Oya, Hokuriku National Agricultural Experiment Station, Joetsu, Niigata 943-01, Japan

Green rice leafhopper (GRLH), Nephotettix cincticeps, infects rice by transmitting viral diseases such as dwarf disease. Screening for resistance to GRLH has been conducted in Japan since the mid-1960s. The degree of resistance to GRLH was found to differ considerably among several indica varieties, but all Japanese rice varieties were

resistance was introduced from indica donors into japonica backgrounds. Norin PL5 was reported to express GRLH resistance by means of two complementary genes (Imbe and Iwasaki 1987) or by two dominant multiple genes (Takita and Nishiyama 1989). However, scientists have not yet determined chromosomal location of the resistance genes in those lines.

phism (RFLP) linkage map and RFLP markers have been developed in rice

susceptible (Ishii et al 1969). GRLH

Restriction fragment length polymor-

(Kurata et al 1994). Graphical genotype, as designated by Young and Tanksley (1989), is a powerful method for evaluating progeny strains generated from wide crosses.

We studied the resistance to GRLH of Matingkis IR22 623.90 ± 52.24 a 13.34 ± 0.17 a 13.00 ± 0.89 abc 9.0 ± 0 a five parental linesusing thegraphical

IR36 574.20 ± 40.30 a 13.38 ± 0.18 a 13.38 ± 0.18 ab 7.6 ± 0.66 ab genotype method and examined which IR64 477.88 ± 83.81 a 13.16 ± 0.18 a 14.77 ± 0.83 a 7.0 ± 0.66 bc IR72 403.00 ± 52.84 b 13.63 ± 0.24 a 10.00 ± 0.93 c 5.6 ± 0.66 c

regions of the rice chromosomes remain as indica domains of those parental lines.

Silang IR22 467.60 ± 36.19 a 13.06 ± 0.32 b 11.80 ± 1.03 a 9.0 ± 0 a IR36 482.00 ± 56.95 a 13.93 ± 0.35 ab 11.20 ± 0.83 a 6.3 ± 1.76 a

We used five lines (Norin PL2, Kanto

IR64 435.20 ± 56.66 a 14.32 ± 0.45 a 13.90 ± 0.82 a 5.6 ± 1.33 a PL6, Norin PL5, Norin PL6, and Aichi42)

IR72 271.80 ± 59.76 b 14.12 ± 0.16 a 12.20 ± 1.04 a 5.6 ± 1.33 a with resistance genes derived from indica

San Miguel IR22 596.40 ± 55.10 a 13.84 ± 0.16 a 16.20 ± 0.80 a 8.33 ± 0.66 a

varieties Pe-bi-hun, Tadukan, C203-1,

IR36 459.90 ± 60.03 ab 13.09 ± 0.23 b 14.70 ± 0.79 ab 5.00 ± 1.54 b IR64 490.10 ± 59.14 a 13.52 ± 0.23 ab 13.30 ± 0.73 bc 5.00 ± 1.54 b

tively (see table). Lepedumai, and Rantaj-emas 2, respec-

IR72 321.10 ± 75.65 b 13.67 ± 0.24 a 11.30 ± 0.95 c 3.66 ± 0.66 b Total DNAwas extracted from parental

Romero IR22 357.10 ± 50.07 ab 13.12 ± 0.10 b 13.80 ± 1.04 a 9.0 ± 0 a lines, original indica varieties, and three

IR36 398.80 ± 47.04 ab 14.59 ± 0.41 a 12.20 ± 1.04 ab 5.6 ± 0.66 b japonica varieties (Nipponbare, Reiho,

IR72 317.60 ± 66.29 b 14.50 ± 0.14 a 9.80 ± 1.22 b 3.0 ± 0 c IR64 474.90 ± 37.11 a 14.35 ± 0.28 a 12.20 ± 0.90 ab 4.3 ± 0.66 bc

Toyonishiki) using the cetyltrimethyl ammonium bromide method. The extracted

a Mean±SE. Within a column and site, means followed by the same letter are not significantly different (P>0.05) by DNA was digested with four restriction

d Number of nymphs reaching adult stage (out of 20 nymphs female -1 ). n = 10 females. e 0-9 scale of the Standard

enzymes ( Hind III , Bam HI, Eco Rv, and LSD statistical test. b Number of eggs laid per female, n = 10. c Days for completion of female development, n=10.

evaluation system for rice. BgIII) and separated by agarose gel (0.8%) electrophoresis. A nonradioactive method


(ECL system: Amersham) was used for labeling and detection.

Graphical genotypes of the five lines are shown in the figure. Several chromosome domains originating from each indica donor parent were detected in all lines. We found four domains in Norin PL 2 (chromosomes 5, 11, 12); six in Kanto PL6 (chromosomes 1, 2, 3, 4, 8, 12); seven in Norin PL 5 (chromosomes 1, 3, 4, 8, 9,11); two in Norin PL 6 (chromosomes 3, 11); and one in Aichi 42 (chromosome 6). We also detected two domains (on chromosomes 3 and 11) common to both Norin PL 5 and Norin PL 6. Some undetermined domains

Parental lines resistant to green rice leafhopper.

Line Cross combination a

Norin PL 2 Pe-bi-hun /Kanto 98// (Kanto PL 3) 2*Kanto 100

Kanto PL 6 Kanto 107/3/ Tadukan /Kanto 98 / / Kanto 100 / 4 / Nipponbare

Norin PL 5 Reiho/ C203-1 //2*Reiho/3/ (Saikai PL 2) Saikai 137

Norin PL 6 Toyonishiki/ Lepedumai // (Ou PL 1) Ou284/3/2*Toyonishiki

Aichi42 Nipponbare/4/Alchi24/3/Kanto 105//Nipponbare*5/ Rantaj-emas 2

a Indica varietles from which resistance genes were derived are in boldface. Kanto 98, Kanto 100: Kochikaze/ Nipponbare.

did not show polymorphism between indica Takita T, Nishiyama H. 1989. Selection of donors and the three japonica cultivars. To biotypes of green rice leafhopper and genetic determine these domains, other japonica analysis for the resistance in rice. Bull. varieties in those crosses should be tested Kyushu Natl. Agric. Exp. Stn. 25(3):51-259.

further. Chromosomal regions that remained fragment length polymorphism maps and the

indica domains of the five parental lines concept of graphical genotypes. Theor. Appl. Genet. 77:95-101.

were detected in the study. But because GRLH resistance genes may be located on those domains, we will conduct linkage RFLP mapping of brown analysis between resistance to GRLH and planthopper resistance gene RFLP markers in a hybrid population to Bph1 in rice identify the gene loci.

Cited references National Agricultural Experiment Station, Imbe T, Iwasaki M. 1987. Inheritance of resis- Chikugo, Fukuoka 833, Japan

Young ND, Tanksley SD. 1989. Restriction

H. Hirabayashi and T. Ogawa, Kyushu

tance to the green rice leafhopper Nephottettix cincticeps Uhler and dwarf disease in rice Norin PLS (in Japanese, English summary]. Jpn. J. Breed. 37:177-184.

Ishii M, Yasuo S, Yamaguchi T. 1969. Testing methods and analysis of the varietal resistance to rice dwarf disease (in Japanese, English summary]. J. Cent. Agric. Exp. Stn. Jpn. 13:23-44.

Harushima Y, Sue N, Wu J, Antonio BA, Shomura A, Shimizu T, Lin Y-S, Inoue T, Fukuda A, Sinamano T, Kuboki Y, Toyama T, Miyamoto Y, Kirihara T, Hayasaka K, Miyao A, Manna L, Zhong HS, Tamura Y, Wang Z- X, Momma T, Yan M, Sasaki T, Minobe Y. 1994. A 300-kilobase interval genetic map of rice including 883 expressed sequences. Nat. Genet. 8:365.372.

Kurata N, NagamuraY, Yamamoto K.

Graphical genotypes of five rice parental lines. In each of the 12 chromosomes, the 5 strips stand for, from left to right, Norin PL 5, Norin PL 2, Kanto PL 6, Aichi 42, and Norin PL 6. Each bar beside the strips shows the position of 123 RFLP markers used.


The nine brown planthopper (BPH) resistance genes identified so far have not yet been located on the linkage map in detail. In this study, we determined the Bph1 locus using several restriction fragment length polymorphism (RFLP) markers. BPH-resistant indica variety IR28 ( Bph1 ) was crossed with Koshihikari, a susceptible japonica variety. We tested 92 F 3 lines of the cross for resistance in a mass screening using biotype 1 BPH and IR28 and Koshihikari. DNA was extracted from young leaves of the parents and F 3 lines using the cetyltrimethyl ammonium bromide method. The RFLP probes, except XNpb, were provided by NIAR/STAFF.

The F 3 lines from the cross Koshihikari/ IR28 segregated into 68:24 for resistant homozygous or heterozygous (R+H) and homozygous susceptible (S). Although susceptible lines died after infestation, we could not distinguish between resistant homozygous and heterozygous plants. This segregation showed a good fit to the expected 3(R+H):1 (S) ratio ( c 2 = 0.058).

We initially used several markers located on chromosome 4, based on the study of Ikeda and Kaneda (1983). that reported the location of Bph1 on chromosome 4 by trisomic analysis. However, our RFLP analysis showed that Bph1 was not linked to 11 markers on chromosome 4. We then used several RFLP markers located on chromosome 1. The bph2 gene, allelic or closely linked to Bph1, was linked to the d2 gene on chromosome 4 at a 39.4% recombination value (Ikeda 1985). Ideta et al (1984) reported that d2 was located on chromosome 1 using RFLP analysis.

However, Bph1 was not linked to the 21 RFLP markers on chromosome 1. We therefore used several RFLP markers located on the other 10 chromosomes to obtain the marker linked to Bph1. The results showed that Bph1 was linked to G148, C185, XNpb248, and XNpb304-1 on chromosome 12 at recombination values of 18.4, 11.5, 10.7, and 11.9%, respectively (see table). Thus, we conclude that Bph1 is located on chromosome 12. These loci are arranged as G148 - C 185 - Bph1 - XNpb248 - XNpb304- 1 - XNpb319 - XNpb304-2 (see figure).

However, results of earlier studies (Ikeda and Kaneda 1983, Ikeda 1985) were inconsistent with our findings. Mistakes during trisomic analysis for insect and

disease resistance are possible. Weak and scanty trisomic plants that have the resistance gene may appear susceptible under resistance selection pressure. The recombination value of 39.4% between bph2 and d2 is also too distant to confirm a linkage relationship. Previous studies did not use marker genes on chromosome 12. Use of several RFLP markers on all chromosomes enabled us to determine the location of Bph1 on chromosome 12.

Cited references Idetd O, Yoshimura A, Iwata N. 1994. lnte-

gration of convention and RFLP linkage map in rice. V. The locus ebisu dwarf ( d2 ) [in Japanese]. Breed. Sci. 44 (suppl.2): 185.

Arrangement of Bph1 and RFLP markers on chromosome 12. The genetic distances are given in centiMorgan units.

Ikeda R. 1985. Studies on the inheritance of resistance to the rice brown planthopper (Nilaparvata lugens Stål) and the breeding of resistant rice cultivars [in Japanese]. Bull. Natl. Agric. Res. Cent. 3:1-54.

Ikeda R, Kaneda C. 1983. Trisomic analysis of the gene Bphl for resistance to the brown planthopper, Nilaparvata lugens Stål., in rice. Jpn. J. Breed. 33:40-44.

Principal component analysis and variety classification in relation to rice seedling salinity tolerance

L. M. Gonzáles, Soil Science and Agricultural Chemistry Department, Agricultural Re- search Institute Jorge Dimitrov, Bayamo 2360, Cuba

Salinity is one of the major contraints to rice production in Cuba; improved salt-tolerant varieties are essential for achieving economically important gains in yield. We evaluated the salinity tolerance of some varieties important to rice breeding programs in eastern Cuba.

Distilled water (EC 0.02 dS m -1 ) was used as the laboratory medium and as a control. Salinity stress (EC 12 dS m -1 ) was

adjusted by adding NaCl. Twenty seeds were kept in each solution in petri dishes, with five replications. Water volume was maintained by adding solution. Root length, seedling height, and fresh and dry seedling matter were measured after 7 d. Salinity tolerance indexes (STI) were calculated using the equation STI (%) = 100 (IS/IC), where IS is the mean of saline solution indicator and CL is the mean of the control solution indicator.

Principal component analysis showed that more than 83% of the total variability between varieties was accumulated in the first two components (see table). The STI (based on seedling height and seedling dry matter) in the first component and the STI (based on root length) in the second component appear to have the highest value of correlation with the principal axis, indicating the usefulness of these indexes in classifying varietal tolerance.

Based on the first two components, cultivars were placed in four groups (see figure), where the variability in cultivar response to salinity was observed. Group I (Caribe 1, IA Cuba-26,2 196, Ecia 22-8, J-


20 0 41 1 0 24 10.9 (<0.01) 18.4 ± 10.1 19.3 ± 10.3 61 0 5 7 0 17 39.1 (<0.001) 11.5 ± 3.6 11.7 ± 3.6 20 42 6 0 4 20 54.5 (<0.001) 10.7 ± 3.4 10.9 ± 3.4

Bph1 - G148

18 42 6 0 5 19 48.6 (<0.001) 11.9 ± 3.6 12.2 ± 3.6

Bph1 - C185

Bph1 - XNpb304-1 Bph1 - XNpb248

Bph1 - XNpb319 Bph1 - XNpb304-2 Bph1 - XNpb336 Bph1 - XNpb316 Bph1 - G124-1

19 42 6 0 5 19 48.6 (<0.001) 11.9 ± 3.6 12.1 ± 3.6

21 20 21 18

40 38 33 36

Linkage analysis between Bph1 and RFLP markers on chromosome 12.

a Calculated based on the ratio of 3:6:3:1:2:1 (df 2).

0 5 1 6 2 9 2 14 7

14

c 2 a Recombination Genetic map AB A_BB A_Bb A_bb aaBB aaBb aabb value (%) distance (cM)

Segregation mode in F 3

6

Gene pair

19 49.1 (<0.001) 12.1 ± 3.6 12.3 ± 3.6 9 17 33.0 (<0.001) 18.9 ± 4.5 19.9 ± 4.5

13 12.5 (<0.01) 30.3 ± 5.6 35.2 ± 5.6 8 6.6 (<0.01) 32.7 ± 5.9 38.1 ± 6.0

Results of principal component analysis.

Principal components Characteristic a

1 2

STI based on Ht 0.746 0.009 STI based on RL 0.290 0.634 STI based on FM –0.538 0.281 STI based on DM 0.732 0.010

Eigenvectors 2.306 0.934

Contribution to 68.80 14.30 variability (%)

Total variability 68.80 83.60

a STI = salinity tolerance indexes. Ht = seedling height,

seedling dry matter. RL = root length, FM = seedling fresh matter, and DM =

Strees tolerance—salinity

112, 2006, IR42, and IR5931), which gave

matter = 71%), was the most salt-tolerant programs for seedling tolerance because the 79%, root length = 81%, and seedling dry ciency and save on expenses in breeding the highest STI values (seedling height =

Use of this method will improve effi-

mined and used. group. most salt-tolerant cultivars can de deter-

Variability, heritability, correlation,

Analysis of variance showed significant gence studies in M 2 generation of 30-, 40-, and 50-kR gamma rays. path analysis, and genetic diver- eight upland varieties treated with 10-, 20-,

gamma-irradiated upland rice differences among genotypes for all characters (Table 1). Considerable range of

S. S. Mehetre, C. R. Mahajan, P. A. Patil, variation was expressed for percent sterility and P. M. Dhumal, Botany Section, College (43.36), tillers plant -1 (14.91), grain yield of Agriculture, Kolhapur 416004, Maha- plant -1 (14.67), and spikelets panicle -1

rashtra, India (13.51), indicating better scope for selection for genetic improvement. Percent

We studied the genotypic and phenotypic sterility had maximum genotypic coefficients of variation, heritability, coefficient of variation (32.23), followed by genetic advance (GA), coefficients of grain yield plant -1 (29.75), plant height correlation, path analysis, and genetic (26.07), spikelets panicle -1 (19.94), tillers divergence in 75 M 2 families raised from plant -1 (13.85), and days to flowering

(12.26) and maturity (9.33).

Estimates of heritability ranged from 91.20% for plant height to 35.60% for sterility. Although plant height (91.20), and days to flowering (88.00) and maturity (87.00) had high heritability, these characters had low genotypic coefficients of variation (GCV) values. This might be due to the variation of environmental components involved in these traits. Expected GA ranged from 6.92% (panicle length) to 54.91% (grain yield plant -1 ). Grain yield plant -1 , plant height, percent sterility, and spikelets panicle -1 showed high GA with high GCV values. These characters should therefore be considered in efforts to obtain high genetic gains.

Grain yield plant -1 was positively correlated with tillers plant -1 , but was negatively and significantly correlated with days to 50% flowering (–0.409) and maturity (–0.421), plant height (–0.717), panicle length (–0.302), and percent sterility (–0.771). Negative correlation of characters (tillers plant -1 , panicle length, and spikelets panicle -1 ) might be due to the highest correlation ( r 2 = –0.771) with sterility.

Percent sterility was positively and significantly correlated with plant height (0.579) and panicle length (0.575). Panicle length was found significantly and positively correlated with days to 50% flowering (0.382) and maturity (0.370) and plant height (0.68). Both plant height and tillers plant -1 were significantly and positively correlated with days to flowering (0.416 and 0.376) and maturity (0.425 and 0.361). Days to 50% flowering had a highly significant and positive (0.999) correlation with days to maturity.

Correlation and path analysis studies revealed that filled grains panicle -1 , plant height, and panicle length are important yield-contributing characters that must be considered when adopting selection criteria in an upland rice breeding program.

Genetic divergence studies using D2 analysis showed that the 75 M 2 families had formed 14 genetically diverse groups (Table 2). Selection pressure applied on the M2 families is likely to provide targeted yield and desired combinations of yield- contributing traits in future generations.


Integrated germplasm improvement—irrigated

Table 1. Analysis of variance and genetic parameters of variation in genotypic and phenotypic coefficients of correlation and path analysis for 8 quantitative characters in M 2 generation of upland rice.

df Days to Days to Plant Tillers Panicle Spikelets Sterility Grain yield Source 50% maturity height plant -1 length panicle -1 (%) plant -1 (g)

flowering (cm) (no.) (cm) (no.)

Analysis of variance Replication Treatment

CD (5%) Error

1 26.06 36.00 2.94 0.17 0.78 188.25 2.74 9.08 44 313.42** a 303.17** 1382.29** 10.87** 3.55** 1501.70** 10.72* b 45.05** 44 20.03 21.00 63.41 3.99 1.39 280.49 5.09 4.88

8.77 8.98 15.60 3.91 2.31 32.82 4.42 4.33

Genetic parameters of variation c

Mean 98.76 127.31 98.48 13.39 20.51 123.93 5.20 15.06 CV (%) 4.53 3.59 8.08 14.91 5.74 13.51 43.36 14.67 Range (min) 71.00 100.00 69.40 9.00 16.80 78.60 1.49 5.15

(max) 121.00 156.00 161.10 19.40 26.10 259.60 14.58 30.00 Variance (P) 166.73 162.09 122.85 7.44 2.47 891.10 7.91 24.96

(G) 146.69 141.09 659.44 3.44 1.08 610.60 2.81 20.08 Coefficient (GCV) 13.08 10.00 27.30 20.37 7.66 24.08 54.08 33.17 Variance (PCV) 12.26 9.33 26.07 13.85 5.07 19.94 32.23 29.75 Heritability (BS) (%) 88.00 87.00 91.20 46.30 43.90 68.50 35.60 80.40 Genetic advance 23.40 22.81 50.51 2.60 1.42 42.12 2.06 8.27 Expected genetic advance 23.69 17.92 51.28 19.42 6.92 33.99 39.62 54.91

Coefficients of correlation and path analysis d

Days to 50% flowering rp 0.998 0.411 0.419 0.196 0.297* 0.126 –0.379** Days to maturity rg 0.999** 0.466** 0.376** 0.382** 0.288** 0.078 –0.409**

pcg 1.584 –1.878 –0.187 0.001 0.012 –0.017 0.035 – pcp (52.458) (–50.593) (–0.575) (–0.931) (–0.807) (–0.121) (0.161) –

Days to maturity rp 0.420** 0.140 0.186 0.295* 0.134 –0.393** rg 0.425** 0.361** 0.370** 0.281 0.088 –0.421** pcg –1.741 1.581 –0.191 0.001 0.011 –0.017 –0.037 – pcp (–50.634) (52.416) (–0.588) (–0.894) (–0.782) (–0.118) (0.178) –

Plant height (cm) rp –0.288 –0.480** 0.261** 0.365** –0.265** rg –0.284 0.658** 0.265 0.579** –0.717** pcg –0.455 0.651 –0.730 –0.002 0.029 –0.015 –0.102 – pcp (–1.383) (21.815) (–21.523) (0.704) (–1.392) –0.112 (1.173) –

Tillers plant -1 (no.) rp 0.013 –0.136 –0,010 0.118 rg –0.128 –0.142 0.222 0.147 pcg 0.009 0.237 (–0.243) (0.104) 0.001 0.008 –0.003 – pcp (–2.479) (19.705) (–18.260) (0.393) (0.271) (0.060) (0.457) –

rg 0.136 0.575** –0.302** pcg 0.061 0.310 –0.324 –0.219 0.000 –0.012 –0.241 – pcp (–2.114) (20.028) (–18.731) (–0.910) (0.317) (–0.057) (1.165) –

rg pcg –0.058 0.471 –0.513 –0.119 –0.001 0.013 0.007 – pcp (–0.420) (15.105) –14.208 (–0.367) 10.351 (–0.288) (0.303) –

Sterility (%) rp rg pcg –0.280 0.200 –0.233 0.166 –0.000 pcp (–2.027) (4.156) –4.444 (–0.800) (–0.559) (–0.215) (0.063) –

Grain yield/plant (g) rp rg pcg pcp

Panicle length (cm) rp 0.212 0.084 –0.207

Spikelets panicle -1 (no.) rp 0.025 –0.201 –0.150 –0.130

–0.473** –0.771**

0.005 0.001 –

a** = significant at the 1% level. b * = significant at the 5% level. c GCV = genotypic coefficient of variation; PCV = phenotypic coefficient of variation: BS = broad sense. d Figures in parentheses indicate genotypic and underlined figures indicate direct effects; residual effect = 1,354 rg, rp, pcg and pcp = genotypic correlation, phenotypic correlation, genotypic path and phenotypic path coefficient, respectively.


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Table 2. Distribution of 45 M2 families of 8 upland rice varieties treated with different irradiation doses and cluster means for different characters studied.

Clus- Treatments ter (no.) Treatment Days to height plant -1 length panicle -1 (%) plant -1

no.

Plant Tillers Panicle Spikelets Sterility Yield

(cm) (no.) (cm) (no.) (g) Variety Gamma ray 50% Maturity

doses (kR) flowering (I) (II) (III) (IV) (V) (VI) (VII) (VIII) (IX) (X) (XI) (XII) (XIII) (XIV)

I 14 Jaya 10 98.3 123.35 76.34 14.12 20.16 106.65 5.77 17.05 JS180 50 (12.20) R24 30, 50 Kundalika 20, 30, 40, 50 ACK5 10, 20, 30, 40,

50, control II 6 Ghansal 30 89.73 118.67 88.74 11.40 20.04 133.15 5.26 17.62

(13.63) (19.26) R24 20 HS17 30, 40, control Kundalika Control

III 5 Jaya 20, 30, 40, 103.46 131.75 84.56 15.03 20.18 134.77 1.75 21.72 control (27.52) (47.28) (14.07)

Kundalika 10 IV 5 Ghansal 10, control 109.74 147.81 140.86 13.25 21.66 155.48 7.34 7.65

V 3 HS17 10, 20 86.42 115.00 128.83 11.53 22.27 118.47 6.30 15.68 JS180 10, 20, 30, 40 (101.97) (130.12) (121.15) (–15.65)

(117.75) (53.62) (131.35) (155.88) (19.29) Basmati 10

VI 2 Basmati 30, 40 92.00 121.50 115.50 13.15 21.60 106.50 6.91 11.36

VI I 3 Ghansal 40, 50 79.33 108.50 89.47 11.65 19.33 107.20 7.87 10.60 (66.03) (62.28) (99.58) (54.42) (68.06) (23.88)

(80.20) (46.67) (123.03) (148.80) (46.88) (53.76) (15.46) Basmati 50

Vlll 1 Ghansal 20 108.00 136.38 139.70 16.00 20.98 173.95 4.24 9.17 (101.71) (101.10) (108.53) (27.45) (93.86) (47.62) (110.96) (0.00)

(132.60) (207.45) (193.08) (40.96) (222.77) (74.69) (207.52) (93.05) (0.00) IX 1 JS180 Control 123.50 152.50 153.90 11.75 22.50 38.08 28.65 8.76 -

X 1 R24 10 102.67 130.34 84.50 11.47 21.34 210.96 3.68 14.41 -

XI 1 R24 20 99.50 129.00 89.00 13.00 22.10 116.10 2.76 12.46 -

XII 1 R24 Control 95.00 123.50 78.00 20.30 18.40 71.30 17.73 15.39 -

Xlll 1 Basmati 20 95.75 124.00 132.60 30.90 22.69 101.85 6.27 15.06 -

XIV 1 Basmati Control 121.00 150.50 115.60 10.60 17.30 98.80 3.85 7.51 -

(18.75) (46.73) (40.45) (136.58) (120.47) (124.50) (112.18) (103.89) (257.53) (0.00)

(56.86) (41.47) (44.45) (82.27) (107.19) (44.69) (64.66) (38.84) (14.88) (69.50) (0.00)

(29.18) (48.26) (48.53) (109.14) (118.15) (74.11) (74.41) (88.25) (180.88) (65.05) (50.33) (0.00)

(154.12) (134.13) (206.10) (57.07) (142.60) (13.18) (128.98) (64.44) (44.24) (238.28) (118.45) (152.75) (0.00)

(88.45) (134.12) (112.03) (48.35) (183.24) (79.90) (140.27) (75.72) (86.45) (164.27) (61.92) (117.37) (111.74) (0.00)

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RCPL-32 and RCPL-3-6: two promising rice lines for the mid-hills of Sikkim, India

M. Singh, G. Singh, L. S. Srivastava, and R. P. Awasthi, Indian Council for Agricultural Research Complex for NEH Region, Tadong, Gangtok, Sikkim; H. S. Gupta, ICAR Re- search Complex for NEH Region, Meghalaya; and A. C. Sharma, CAR Research Complex for NEH Region, Imphal, Manipur, India

Rice is the second most important crop in Sikkim, India covering about 19,000 ha. Yields average 1.3 t ha -1 . In Sikkim, rice is cultivated on terraces using gravity flow water. Commonly grown local cultivars (Attey, Chirkey, Krishnabhog, Tapre, Kalonunia, Tulasi, Champasare, and Bhuinphool) are late-maturing, tall, and low-tillering. Their yields are poor but they have good quality attributes. Suitable substitutes for these cultivars must be semitall, medium-maturing, nonlodging, have low photoperiod sensitivity, high yield potential, and good quality. After 4 yr of evaluation, lines RCPL-3-2 and RCPL-3-6 outyielded all other lines and checks (Table 1).

RCPL-3-2, a selection from cross Moirangphou/Jaya, is semidwarf (98-105 cm), while RCPL-3-6, a selection from cross CH-988/IR24, is also semidwarf but slightly taller. RCPL-3-2 is nonglutinous, whereas RCPL-3-6 is a semiglutinous rice. Both are medium in maturity and resistant

Tooag92: a shortduration rice cultivar for Turkey

N. Açikgöz and M. N. Gevrek, Field Crops Department, Faculty of Agriculture, Ege University, Bornova, Izmir, Turkey

Toag92, a rice variety derived from the cross Nucleoryza/Labella, was released in 1992. The main aim of the breeding program was to create a variety that does well in the second cropping season in the Aegean and Mediterranean regions of Turkey. Toag92, however, also has high yields in northern Turkey, where it did particularly well when sown late and in areas where existing varieties cannot be grown because of their long vegetative

Table 1. Grain yield (t ha -1 ) of promising lines and checks over years and locations in Sikkim, India.

Location Year RCPL-3-2 RCPL-3-6 DR-92 Attey

Tadong 1990 5.0 4.3 3.8 2.0 Tadong 1991 5.0 4.5 2.9 1.2 Tadong 1992 4.1 4.6 2.5 1.3 Tadong 1993 4.3 4.4 3.0 1.8 Tadong 1994 4.2 4.4 3.6 1.4 Pakyong 1992 4.0 4.6 3.3 1.9 Pakyong 1993 3.9 3.5 2.6 2.2 Dharmdin 1992 6.0 5.5 3.8 2.2 Nazitam 1992 4.9 4.6 3.5 2.1 Nazitam 1993 4.9 4.6 3.2 2.0

Table 2. Important characteristics of RCPL-3-2 and RCPL-3-6.

Characteristic RCPL-3-2

Plant height (cm) 98-105 Days to flowering 60-70 Days to maturity 115-120 Ear-bearing tillers (no.) 9-15 Panicle length (cm) 20-23 Flag leaf Erect Awn type Awnless Husk grain color Straw Kernel color Dull 1,000-grain weight (g) 25.4 Grain length (cm) 0.83 Grain width (cm) 0.30 L-B ratio 2.77 Endosperm type Nonglutinous Grain yield (t ha -1 ) 4.5-5.0 Reaction to leaf blast (0-9 scale) 0-2 Reaction to neck blast (%) 0-1

RCPL-3-6

110-115 67-72

120-125 8-14 25-27 Erect

Awnless Straw Dull 23.5 0.84 0.28 2.94

Semiglutinous 4.2-4.6

0-2 0-1

to lodging, leaf and neck blast. Distin- RCPL-3-2 and RCPL-3-6 are suitable guishing traits for both include panicle for early sowing, and will enable farmers to length, 1,000-grain weight, L-B ratio, adopt more profitable rice-based cropping panicle-bearing tillers. and endosperm type systems such as rice-mustard/pea/barley, (Table 2). that promise to increase cropping intensity

under rainfed conditions.

Table 1. Comparison of some agronomic characters of Toag92 and commercial varieties. Bornova, Turkey (3-yr av) .

Variety Yield Height 1,000- Yield Duration Sterility Leaf blast a

(t ha -1 ) (cm) grain (kg d -1 ) (d) (%) weight (g)

Toag 92 5.3 64 33 5.95 89 16 0 Baldo 4.1 92 38 3.66 112 24 Ribe 4.5 99 33 4.24 106 30 8 Krasnodorsky 424 4.4 101 31 4.40 100 24 4

a Scored using the Standard evaluation system (SES) 0-9 scale where 0 = highly resistant and 9 = highly susceptible.

durations. Toag92 matures 2 wk earlier than Toag92 is of medium height (88 cm), Ribe. 30 cm shorter than Ribe, and is resistant to

Its low sterility under Mediterranean lodging. The yield d -1 of Toag92 is almost conditions makes it promising for the 50% more than that of other improved southeastern Anatolia region, where August cultivars. Toag92 is also resistant to leaf temperatures are quite high. blast (Table 1).


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Toag92 has medium grain and is free of white belly. Quality is good and similar to that of Baldo, Ribe, and Krasnodorsky-424 (Table 2).

Birsa Dhan 201 and Birsa Dhan 202: early-maturing varieties for Bihar, India

M. P. Singh, D. N. Singh, and M. F. Haque, Plant Breeding and Genetics Department, Birsa Agricultural University, Ranchi 834006, India

Birsa Dhan 201 was developed by crossing TN1/Brown Gora 23-19, and Birsa Dhan 202 by crossing Jaya/BR34. Both varieties were released during 1985 and re-released in 1995 for use in the Chotanagpur and Santhal Parganas region of Bihar, India. Birsa Dhan 201 is an early-duration variety that matures in 105-110 d under transplanting. It is nonlodging, nonshattering, and highly responsive to chemical fertilizers, yielding 3.0-3.5 t ha -1 . It has compact, long panicles with bold grains and white kernels. It is moderately resistant to

blast, bacterial leaf blight, stem borer, and gall midge.

Birsa Dhan 202 is intermediate in plant type and provides adequate rice straw for cattle feed, as required by farmers in the region. The variety has very good seedling vigor and produces heavy, drooping panicles. It is stiff-strawed, making it resistant to lodging. The grains are long and bold. producing white kernels. It is moderately

resistant to blast, stem borer, and bacterial leaf blight, but moderately susceptible to gall midge. The variety yields reasonably well, producing 5-6 t ha -1 when 40-60 kg N ha -1 is applied (see table). A lot of this region has been planted to Birsa Dhan 202. Farmers have been sharing its seed with each other, increasing the area planted to it.

Both varieties have acceptable taste. and grain and cooking quality.

Table 2. Quality traits of Toag92. Bornova, Turkey.

Variety Amylose content Protein content KOH reaction White belly a

(%) (%)

Toag92 18.8 13.7 9.8

15.6 7.8 Baldo 16.8 Ribe

Low 5 Low 0

Krasnodorsky 424 7.4 Low 5 Low

a Scored using the SES scale where 0 = no belly. 9 = more than 20% of kernel area.

KK1536-C: a modern high-yielding rice variety for irrigated lowlands in Papua New Guinea

M. S. Sajjad, Agriculture and Livestock Department, Food Management Branch, Erap Research and Development Centre, P. O. Box 1984, Lae, Papua New Guinea

Out of 122 genotypes we evaluated in 1991,

7, Nepalese in origin) performed the best KK15-36-C (IR 5657-33-2/BKNDR1031-

and was selected as one of six genotypes for further evaluation.

We confirmed the high yield potential (10.5 t ha -1 ) of KK15-36-C in local and regional yield trials conducted during 1992- 95. At the OISCA Farm in Kokopo, Rabaul, KK15-36-C and Ayung yielded the most of the varieties tested. At Bubia during 1991 and 1995, the variety was either highest yielding (6-7 t ha -1 during 1994, 9.5 t ha -1

during 1995) or at par with the highest yielding genotypes.

The variety is semidwarf (<110 cm) with medium-tillering ability. It has long (>25 cm), fully filled panicles with more than 100 grains panicle -1 . The 1,000-grain weight is 30 g. Its foliage remains green up to late maturity.

medium bold, awned and translucent. The milling recovery is very good. KK 15-36-C is extremely well suited for general cultivation in the irrigated lowland fields of Papua New Guinea.

The variety’s grains are long and

Performance of Birsa Dhan 202 in the All-India coordinated trials. 1989 and 1990.

Mean of different locations

Variety

Birsa Dhan 202 Vikas Ratna Rasi Local (control)

1989 1990

Yield (t ha -1 ) Days to 50% flowering Yield (t ha -1 ) Days to 50% flowering

6.3 101 3.5 94 6.1 99 6.1 95

2.6 85 6.2 99 3.1 92


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Shakuntla; a rice variety for rainfed lowlands in Bihar, India

V. N. Sahai, B. K. Singh, and S. Saran, Agricultural Research Institute, Mithapur, Patna 800001, Bihar, India; R. C. Chaudhary, IRRI

Shakuntla (RAU73-16-1-40) was released in 1995 for cultivation in rainfed lowland areas of Bihar, India. Rainfed lowland rice is grown on about 40% (2.2 million ha) of the state’s area. The current yield averages less than 1.0 t ha

Developed from the cross Pankaj/Br8,

-1 .

Shakuntla is weakly photoperiod-sensitive, of long duration (140- 145 d), and is medium in height. Its grains are long and slender.

In the All India coordinated yield trials, Shakuntla ranked fifth in 1990 and first in 1991. In state uniform variety trials during 1993 and 1994, Shakuntla yielded 12.3- 50.5% more than check Mahsuri. In the 1994 eastern India rainfed lowland shuttle breeding trial, the variety was tested using normal planting with 30-d-old seedlings and delayed planting with 60-d-old seedlings. The yield advantage was greater under normal than delayed planting (Table 1).

From 1988 to 1990, Shakuntla outperformed check Mahsuri by an average of 20% under direct seeding and transplanting (Table 2).

Table 1. Grain yield of Shakuntla at different sites in India. 1990-94.

Yield (t ha -1 ) Year Site LSD (5%) CV (%)

1990 Pusa 2.2 1.4 0.9 27.1 Sabour 3.1 2.4 1.0 15.0 Chinsurah 2.7 2.6 0.4 10.9 Ghaghraghat 2.2 1.2 0.5 7.1

Shakuntla Mahsuri or Utkal prabha

1991 Ranital 3.4 Canning 2.8 Patna 2.5 Pusa 2.6

1993 Patna 4.1 Pusa 2.8 Sabour 1.7

1994 Patna 4.8 Pusa 2.7 Sabour 1.9 Bikramganj 4.6

1994 Chinsurah Masodha Patna Pusa Raipur Titabar

Chinsurah Masodha Patna Pusa Raipur Titabar

Pooled mean

a ns = not significant.

3.0 6.5 5.2 5.5 2.8 5.5

5.1 2.5 3.6 2.2 4.6 2.6 3.5

2.8 2.1 1.4 2.2

3.4 2.6 1.1

3.9 1.7 0.5 3.3

0.4 8.1 0.5 10.8 0.6 15.3 0.3 7.9

0.1 24.0 ns a

0.8 23.3

0.1 16.3 0.6 16.2 0.8 20.8 0.5 5.7

Normal planting with 30-d-old seedlings 3.4 0.4

Delayed

5.0 6.7 3.0 2.3 3.4

planting 3.3 2.1 2.2 1.3 2.2 2.2 2.6

with 60-d-old

0.9 0.2 0.5 0.7 0.9

0.6 1.0 0.3 0.2 ns a

ns a

seedlings

10.7 9.0

17.4 6.9

27.9 13.5

9.3 27.6

6.4 32.1

Table 2. Grain yield (t ha -1 ) of Shakuntla in farmers' fields under direct seeding and transplanting. ICAR-IRRI collaborative research, 1988-90 wet seasons.

Variety Direct seeding Transplanting Av Increase over check 1988-90 1989-90 (%)

Shakuntla 3.5 3.7 3.6 20 Mahsuri (check) 2.9 3.0 3.0

Integrated germplasm improvement—rainfed lowland

Manuscript preparation. Arrange the note as a brief statement of research objectives, a short description of project design, and a succinct discussion of results. Relate results to the objectives. Do not include abstracts. Do not cite references or include a bibliography. Restrain acknowledgments

Manuscripts must be in English. Limit each note to no more than two pages of double-spaced typewritten text. Submit the original manuscript and a duplicate, each with a clear copy of all tables and figures. Authors should retain a copy of the note and of all tables and figures.


CMS lines for shallow rainfed lowland situation

S. B. Pradhan and P. J. Jachuck, Central Rice Research Institute (CRRI), Cuttack 753006, Orissa, India

We are working to develop parental lines for hybrid rice for the rainfed lowland ecosystem in eastern India. Although hundreds of cytoplasmic male sterile (CMS) lines have been developed for breeding hybrids for the irrigated ecosystem in India, hardly any have been reported so far for the rainfed lowlands.

We therefore converted 13 popular lowland cultivars into CMS lines through repeated backcrossing with three male sterility sources: wild abortive, Kalinga-I, and O. perennis. These sources, in BC 6 generation, were grown in the field along with their maintainers during the 1994 wet season. We recorded pollen sterility, spikelet fertility in bagged panicles, plant height, days to 50% flowering, and panicle exsertion for each of the CMS and maintainer lines.

All 13 CMS lines were completely pollen sterile with white anthers and did not set any seed in bagged panicles (see table). Moti A, Kalashree A, and Padmini A were late-duration varieties while the rest were medium-duration.

in stature, with stiff straw. All were shorter (1-56 cm) than their respective isonuclear

The CMS lines are semidwarf to semitall

Agronomic characters of CMS lines and their maintainers developed at CRRI, Cuttack, Orissa, India. 1994 wet season.

CMS a or Pedigree of A lines Pollen Spikelet Plant Days to Panicle maintainer sterility fertility height 50% exsertion lines (%) (%) (cm) flowering (%)

Moti A V20A/Moti (WA) 100 0 90 127 Moti B

73.1

Kalashree A V20A/Kalashree (WA) 100 0 95 134 68.7 Kalashree B 82.9 113 130 100

Padmini A V20A/Padmini (WA) 100 0 119 119 80.3 Padmini B 89.5 145 118 100

IET11350 A V20A/IET11350 (WA) 100 0 95 97 IET11350 B

80.4

IET10428 A V20A/IET10428 (WA) 100 0 104 98 79.3 IET10428 B 90.0 105 90 100

87.9 120 124 100

82.5 100 95 100

IET11668 A V20A/IET11668 (WA) 100 0 93 96 81.3 IET11668 B 74.4 102 95 100

BKS64 A V20A/BKS 64 (WA) 100 0 85 94 77.8 BKS64 B 86.7 99 95 100

Manipur A Annada A/Manipur (WA) 100 0 82 100 67.3 Manipur B 82.5 92 99 100

Mirai A Mirai B

Blazin A Blazin B

IET10983-I A IET10983 B IET10983-ll

IET10983 B

Krishna A/Mirai (Kalinga-I) 100 0 79 100 70.1 80.6 88 99 100

Krishna A/Blazin (Kalinga-I) 100 0 70 95 66.1 74.8 126 102 100

IR62829A/IET10983 (WA) 100 0 75 100 66.6

lR66707 A/IET10983 100 0 65 98 62.3 (O. perennis)

80.4 82 97 100

81.5 85 97 100

Tharrangsong A lR66707 A/Tharrangsong 100 0 71 88 69.3

Tharrangsong B 78.7 75 85 100

a CMS lines are in the BC 6 generatlon.

(O. perennis)

maintainers. Their plant heights and dura- oping hybrids for the shallow rainfed tions are considered ideal for use in devel- lowland situation in India.

Majhera 7, a direct seeded rainfed upland spring rice for Uttar Pradesh, India

B. V. Singh, A. Prasad, V. P. Singh, and C. S. Prasad, G. B. Pant University of Agriculture and Technology, Research Station, Majhera, Nainital, U. P. 263135, India

Spring rice is a major rainfed crop in the uplands of Uttar Pradesh, India. It is direct dry seeded from mid-March to mid-April and harvested in September-October. Only two spring rice varieties had been released to date: Majhera 3 in the early 1960s and VL 206 in 1983. Varieties grown in this

region must have long duration and drought tolerance, especially during May and June.

To address this need, we developed Majhera 7 from germplasm collected from the Kumaun uplands. After numerous trials (Table 1), Majhera 7 was recommended for release as a commercial variety in the Uttar Pradesh hill zone under direct dry seeded rainfed upland conditions.

In agronomical evaluation trials involving different sowing dates and fertilizer doses, Majhera 7 yielded more than checks Majhera 3 and VL 206. Maximum yield for the variety was obtained using a 7 Apr sowing date. Majhera 7 yielded 23.9% more than Majhera 3 and 6.3% more than

VL 206 when 75 kg N and 11 kg P ha -1 were

Majehra 7 matures in 174-187 d and fits well in common crop rotations: spring rice - wheat - ragi - toria - fallow and spring rice - wheat - barnyard millet - barley. It is 95- 105 cm tall, erect, and with broad, dark- green leaves. It has medium-bold grain, a length-breadth ratio of 2.24. and a 21 g thousand-grain weight. Milling recovery is 80%. Majhera 7 is widely adaptable and shows field resistance to stem borer, leaf- folder, grasshopper, sucking insects, and blast. (See Table 2 for morpho-agronomic characters of Majhera 7, Majhera 3, and VL 206.)

applied.


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Manuscript preparatlon. Arrange the note as a brief statement of research objectives. a short description of project design, and a succinct discussion of results. Relate results to the objectives. Do not include abstracts. Do not cite references or include a bibliography. Restrain acknowledgments.

Manuscripts must be in English. Limit each note to no more than two pages of double-spaced typewritten text. Submit the original manuscript and a duplicate, each with a clear copy of all tables and figures. Authors should retain a copy of the note and of all tables and figures.

Improved upland rice for the hillsides of Colombia

A. M. Moreno-B, Centro Nacional de Investi- gaciones de Café (CENICAFE). A. A. 2427, Manizales, Colombia; E. P. Guimaraes, M. Chatel, and J. Borrero, Centro Inter- national de Agricultura Tropical (CIAT), A. A. 6713, Cali, Colombia

The Andean mountain range runs across Colombia from south to north, rising up to almost 6,000 m above sea level (masl). Coffee, grown by small-scale farmers, is the most important agricultural crop in the middle altitudes. Once the coffee is planted, it takes at least 3 yr to start commercial production. In the meantime, farmers use considerable resources to control weeds and prevent erosion. CENICAFE has been working to develop different cropping alternatives to help farmers have income before coffee starts to produce.

CENICAFE, together with the CIAT Rice Program, worked to identify upland rice germplasm suitable for these hillsides. In 1993, we evaluated 21 upland lines at three locations, each replicated three times. in the heart of the coffee-growing area at 1,300 masl. Average temperature ranges from 20.6 to 23.1 °C. The monthly average maximum (28.5 °C) occurs in February and the minimum (16.9 °C) in September. So germplasm must be cold tolerant. Breeding lines were selected for this trial based on results in Africa, where some of their parents performed well.

Only results of the performance of the top six lines grown in trials in La Catalina, Risaralda state, are reported in the tables. The percentage of empty grains ranged from 12 to almost 100%, indicating that the germplasm presents variability for the trait (data not shown). Selection concentrated on the lines with at least 60% fertility. The average grain yield in those lines was higher than originally expected, ranging from 3.8 to 5.6 t ha -1 (Table 1). Even though the yield was relatively high, little is known about agronomic management under hilly conditions in Colombia. No checks were used because rice was never before planted in this area.

The growth duration of the lines was extended to around 150 d after sowing, compared with 120 d under acid soil

Table 1. Performance of Majhera 7 and checks in various trials. Uttar Pradesh, India. 1991-94.

Mean yield (t in Uttar Pradesh


uplands ha -1 )

over checks Yield increase (% )

VL 206 Majhera 3 Trial/year

Majhera 7 VL 206 (check) Majhera 3 (check)

Standard varietal trials 3.5 2.0 1.0

2.9

0.8 1.8

1.8

1.5 1.8 1.6

1.6

1.1 2.0 2.0

1.7

1.7

22.8

50.0 10.0

48.3

87.5 22.2

1991 1992 1993

Mean

Station trials 1992 1993 1994

Mean

Adaptive trials 1992 1993

Mean -

4.3 2.2 1.5

2.7 2.1 23.8 44.4

2.1 2.0 1.8

1.5 1.5 1.6

40.0 33.3 12.5

40.0 11.1 12.5

1.9

2.5 - - - - 3.0

1.5 26.6 18.7

3.0 - - -

3.0

1.2 Demonstration at research station

66.6 22.2 9.5

22.4

17.6

15.0

81.8 10.0 15.0

29.4

35.3

1992 1993 1994

Mean

2.0 2.2 2.3

2.3

1.8 2.1

1.7

1.7

2.0

Demonstration in farmers' fields 1994

Total mean

2.0

2.3

Table 2. Morphoagronomic character of Majhera 7 and check varieties Majhera 3 and VL 206.

Genotype Days to Days to Plant Panicle Panicles m -2 Grains 1,000- Milling Grain flowering maturity height length (no.) panicle -1 grain recovery color

(cm) (cm) (no.) weight (%) (g)

174 176 175

104 104 103

198.3 227.7 215.8

115.2 116.9 11.4

78.0 80.0 81.0

Brown White White

Majhera 3

VL 206 Majhera 7

139 139 139

21.8 22.1 22.3

19.3 20.9 20.5

rmguevarra

Rectangle

Table 1. Average data from the best lines in the 1993 observational trial conducted in the coffee- growing area of Colombia, Naranjal Experimental Station, Risaralda.

Panicles Tillers Grains Sterile Quality traits a Yield (t ha -1 ) Line (%) (no.) (no.)

Filled Empty GT WB GL Disp (no.) (no.)

CT10037-9-4-M-4-8P-1-M 113 141 376 123 33 I 0.2 L 4 4.4 CT6196-33-11-1-3-M 120 143 348 99 28 I 0.6 L 5 4.0 CT9997-5-3-M-4-M 120 135 345 136 39 IB 0.4 L 3.6 3.8 CT10069-27-3-1-4 132 160 526 66 12 IA 2 CT10037-9-7-M-1-M CT10037-30-3-M-1-2P-2-M 116 124 433 78 18 I 0.2 EL 5 3.8

L 3.4 5.2 120 155 512 176 34 I 0.2 L 5 5.6

a GT = gelatinization temperature, WB = white belly, GL = grain length, and Disp = dispersion.

Table 2. Yield ability, tiller number, and sterility percentage of the six breeding lines evaluated. a

Naranjal Experimental Station, Risaralda, Colombia. 1994.

Line Yield (t ha -1 ) Tillers (no.) Filled grain (%)

3.6 bc 82.53 bc 81.9 a 3.3 c 85.00 ab 81.0 a 3.2 c 84.01 abc 58.0 b 4.3 ab 88.13 ab 87.5 a 4.7 a 90.85 a 83.0 a 3.2 c 76.05 c 82.5 a

CT10037-9-4-M-4-8P-1-M CT6196-33-11-1-3" CT9997-5-3-M-4-M CT10069-27-3-1-4 CT10037-9-7-M-1-M CT10037-30-3-M-1-2P-2-M

a Means in a column followed by the same letter are not significantly different (P = 0.05) by DMRT.

conditions at 700 masl. Rainfall during the March, the end of the growing season. to cropping season ranged from 128 mm in 394 mm in January.

CT10069-27-3-1-4 was selected in 1993 as one of the most promising lines, so agronomic evaluation was done. A simple unreplicated trial was conducted. Row planting produced a higher yield than spaced or hill planting, independent of the N level (0, 60 kg N ha -1 ) and seed density (60, 80, 100 kg ha -1 ). The rice responded to N at all densities, and the ideal seed density was 80 kg ha+ (data not shown).

The best six lines were evaluated again in 1994 in a yield trial in a randomized block design with three replications (Table 2). The yield potential of these lines was confirmed, with the highest yielding line (CT10037-9-7-M-1-M) averaging 4.7 t ha -1 , similar to that of CTl0069-27-3-1-4 and statistically different from the four others.

CIAT upland breeding lines are an alternative for the coffee-growing region of Colombia. Further research will aim to identify the best line for release and suitable agronomic practices.

These preliminary results indicate that

MTU9993, a promising rainfed upland rice for Andhra Pradesh, India

P. S. S. Murthy, S. S. R. Prasad, K. R. K. Murthy, and N. S. R. Reddi, Agricultural

Performance of MTU9993 under rainfed upland conditions at multiple locations during dry seasons 1988-92.

Grain yield (t ha -1 )

1988 1989 1991 1992 1990 Location and variety

Chinthapally Research Station, Maruteru 534122, West Godavari District, Andhra Pradesh, India

MTU9993 3.2 3.5

% increase 10.3 40.0 MTU17 (check) 2.9 2.5

MTU9993 is a high-yielding variety identified from the progeny of Rasi/Fine Gora (white) for the rainfed uplands of coastal Andhra Pradesh. It was released in I993 to replace tall traditional varieties that are low yielding and prone to lodging. The short-duration (105-110 d) variety has straw-colored glumes and white kernels. MTU9993 is superior to the existing rainfed upland rice cultures, such as MTU17 and Mettasannavari, in grain quality and yield, shortness, tolerance for lodging, strong seed dormancy, tolerance for iron deficiency, and resistance to leaf blast.

The variety was tested from 1988 to

Lam, Guntur MTU9993 MS. Vari (check) % increase

Ragolu MTU9993 MTU17 (check) % increase

Jagtial MTU9993 Rasi (check) % increase

V.R. Gudem MTU9993 MTU17 (check) % increase

1.8 1.6

12.5

4.4 3.3

33.3

0.9 0.8

12.5

2.3 1.8

27.5

3.9 3.1

25.8

3.4 2.6

30.8

2.6 2.2

18.2

2.2 1.4

57.1

2.5 1.8

38.8

2.2 2.0

10.0

1.8 1.2

50.0

2.3 1.7 1.5 1.2

53.5 41.7

2.7 2.8 2.2 2.2

22.7 27.3

1.4 1.2

16.7

1992 and yielded 10-57% more than local checks (see table).

Maruteru MTU9993 4.3 2.5 2.2 MTU17 (check)

2.8 2.9 2.0 1.6 2.0

% increase 48.3 25.0 37.5 40.0


- - - - - - - - -

- - -

- - - - - -

- - -

- - -

Crop and resource management

Analysis of growth duration and heat units of different rice genotypes

J. L. Chaudhary, A.S.R.A.S. Sastri, and R. K. Sahu, lndira Gandhi Agricultural Univer- sity, Raipur, Madhya Pradesh 492012, India

In general, higher temperatures accelerate the rate of development at all phenological stages of a crop and thereby reduce the length of a given phenological stage (Oldemann et al 1986).

We analyzed variation in duration of some prominent rice genotypes from sowing to 50% flowering and the corresponding variation in accumulated degree days (DD) (with base temperature of 10 °C) in Raipur, India. Long-term phenological observations and meteorological data from a nearby agrometeoro- logical observatory were used.

Duration from sowing to 50% flowering of 12 selected genotypes varied from 66 d (Poorva) to 111 d (Surekha) [see table). The

DD requirement for the same period was lowest for Poorva (1127 DD) and highest for Surekha (1935 DD). The coefficient of variation for duration varied from 2.6%; for Samridhi to 6.8% for Cauvery. indicating least variability for Samridhi and greatest variability for Cauvery. Variability in DD requirement varied from 4.8% for Samridhi to 6.9% for Phalguna. For Samridhi, this indicates that variability in duration was lower than variation in DD requirement. This is contrary to the general belief that variability in duration of different phenological stages can well be explained by DD.

This trend also suggests that variability in duration of different phenological stages is influenced more by thermal regime within the vicinity of the crop environment. The meteorological observatory was situated in a nearby field and the microclimate there is quite different from the one existing in the ricefields.

Cited reference Oldemann LR, Seshu DV, Cady FB. 1986.

Response of rice to weather variables. Report on the IRRI/WMO Project. Manila (Philippines): International Rice Research Institute.

The processes of pollination and fertilization in rice

K. Hattori and T. Wada, Laboratory of Plant Genetics and Breeding, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 46401, Japan

The processes of pollination and fertilization in higher plants have, to date, been only well studied in Liliaceae species.

In japonica rice variety Nipponbare, we observed conditions of pollen tube elongation on the stigmatic hair and the portion of the style through which the pollen tube passed. Pollen tube behavior in the ovary was also studied. At the flowering stage, every flower was sampled and fixed in formalin-acetic-alcohol after pollination. These samples were examined under both fluorescent and light microscopes after being sectioned using a resin-embedding method.

Light microscopy of a cross-section taken from the top of the stigma to the bottom of the ovule showed that each stigmatic hair had a hole in the center (the intercellular space formed with stigmatic hair cells) (Fig. 1a). These holes are connected to an area (transmitting tissue) made up of cells that are shaped differently than other parenchymatous cells located inside the vascular bundle of the stigma (Fig. 1b). This area continued through the stylar tissue up to the ovary. In the ovary, a narrow space was observed between the outer wall of the ovule and the ovary wall, except at the dorsal portion of the locule (Fig. 1c). This space shows where the pollen tube passed through the micropyle. The longitudinal sections of the ovary reveal that the ovary wall and the outer wall of the ovule fused from the top to near the bottom of the ovule (Fig. 1d).

Analysis of variation in growth duration (from germination to 50% flowering) and degree days of some prominent rice genotypes.

Duration (d from germi- Accumulated nation to 50% flowering) degree days

Variety Observations (no.) Mean SD Mean SD CV Cv

(%) (%)

IR36 31 92 4.95 5.4 1584 100 6.3 Samridhi 6 89 2.33 2.6 1527 73 4.8 Poorva 12 66 4.35 6.6 1127 65 5.8 Tripti 16 76 4.6 6.1 1311 81 6.1 Cauvery 25 75 5.1 6.8 1295 81 6.2 Deepti 13 91 4.6 5.0 1575 89 5.6 Ratna 41 89 4.9 5.5 1539 86 5.6 Madhuri 10 105 5.3 5.0 1794 115 6.3 Anupama 38 88 4.7 6.4 1523 94 6.1 Phalguna 24 104 5.9 5.7 1771 123 6.9 Surekha 9 111 5.6 5.0 1935 113 5.8 Usha 8 102 5.7 5.6 1762 91 5.1


Physiology and plant nutrition

2. Observations of some parts of the rice pistil after pollination using an epifluorescence microscope. a) cross-section of stigmatic hair, b) longitudinal section of stigmatic hair, c) cross-section of the uppermost part of style, and d) cross-section of ovary. Bars show 100 µm.

Fluorescent microscopy showed that pollen germination occurred 1 h after the pollen reached the stigmatic hairs. The pollen tube then elongated, passing through the center hole of the stigmatic hair (Fig. 2a, b). In the stigmatic and stylar tissues. the pollen tube passed through the transmitting tissue located in the space between the center area and the vascular bundle (Fig. 2c). (Among Liliaceae species, the pollen tube is believed to pass through the stylar cavity.)

The cells comprising the transmitting tissue differed from other parenchymatous cells. They appear as a darkened area under the fluorescent microscope. implying that the cells were thin walls. The absence of a narrow space toward the dorsal side (downward) of the ovary explained the observance of pollen tube elongation at both sides of the locule but not in the dorsi- ventral direction of the ovary (Fig. 2d).

The pollen tube reached the micropyle 3 h after pollination. However, time of fertilization could not be determined.

Studies on intercellular space of embryo and seedling tissues in rice plants

T. Wada, R. Goto, S-Y. Kang, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-01, Japan; and K. Hattori, Chungnam Rural Administration, Chungnam 320-860, Korea

The development of intercellular spaces into aerenchyma in rice plants is thought to be important in improving oxygen gas diffusion through the plant body. But details of the developmental process during seedling and early vegetative stages are unclear. To better understand the process, a morphological study of intercellular spaces was conducted using high-resolution microscopy as well as cryo-scanning electron microscopy.

Intercellular spaces form in mature embryos of dry seeds (see figures a, b, c).

The spaces are located mainly in the parenchymatous cell regions including the scutellum, mesocotyl, and coleoptile and radicle, and are evident in the inner regions of these tissues. Intercellular spaces near the apical region of the radicle procortex were found in mature embryos, confirming previous findings (Armstrong 1979) (see figures f, g). However, all these spaces. which appear at the corner of neighboring cells, are very small in dry seeds. They increase in volume during imbibition and germination (see figures a-e).

During the early seedling stages after germination, no obvious changes in intercellular space were observed, but a tubular structure along the longitudinal cell alignment was formed (this occurred after the elongation and development of cortical cells along the shoot-root axis) (see figures e, f, h). In the shoot region, air space was formed in chlorenchymatous cell regions in the coleoptile and young leaves, especially under nonsubmerged culture condition.

The intercellular space near the axis during the early seedling stage seemed to be mostly in the water-rich state as revealed by cryo-scanning electron microscopy. Similar results have been obtained in other plants (Canny and Huang 1993). Seven days after germination, root air space was formed in the basal elongated seminal root region of the nonsubmerged plants. In the submerged condition, inner air space was formed among the elongated coleoptile and young leaves, while seminal root growth was reduced and the development of the cortical air space was delayed.

Cited references Armstrong W. 1979. Aeration in higher plants.

Adv. Bot. Res. 7:225-332. Canny MJ, Huang CX. 1993. What is in the

intercellular spaces of roots? Evidence from the cryo-analytical-scanning electron microscope. Physiol. Plant. 87:561-568.

1. Observations of some parts of the rice pistil using a light microscope. a) cross-section of a stigmatic hair, b) cross-section of a stigma, c) cross-section toward the bottom of an ovary, and d) longitudinal section of the dorsi-ventral direction of an ovary. Bars show 100 µm.


Effects of time of split application

rice yield

D. Muthumanickam, M. Ravichandran, and M. V. Sriramachandrasekaran, Faculty of Agriculture, Annamalai University, 608002, India

An experiment was conducted to study the effect of timing of split application of K on N forms in the soil and on rice yield. The experiment was carried out using clay soil (Typic Chromusters. pH 8.04, electrical conductivity 0.32 dS m -1 , organic carbon 0.41 %. and 294, 10, 290 kg available NPK ha -1 ). The crop received 100-22-41.5 kg NPK ha -1 . Half of the N and K and the entire dose of P were applied basally. The remaining amount of N and K was equally split and topdressed as per treatment

of K on soil N forms and lowland

schedule (see table). The experiment was carried out in two

different seasons (February to June and July to November) using IR20 and a factorial completely randomized block design. Treatments were replicated thrice. Twelve replications were used (three replications at each stage of soil analysis).

Soil samples were taken at a depth of 15 cm during tillering, panicle initiation, flowering, and harvest stages. Wet soil samples were analyzed for NH 4 -N, NO 3 -N, available N, and fixed NH 4 -N. Plants collected at harvest were threshed carefully to separate the grains and straw and weighed separately after drying at 60 °C.

In both seasons, grain and straw yields were highest when K was always applied 7 d earlier than N (see table). Yields were lowest in the control and when K was

Light micrographs of rice embryo tissues, showing intercellular spaces among the cells. A low applied 7 d after N (see table). The higher magnification view of the radicle and mesocotyl regions of a mature embryo (a); higher profiles of the grain and straw yield with early application radicle procortex region (b); inner coleoptile cells (c) in dry seeds; inner scutellar parenchyma cells 48 of K was caused by the greater availability h after imbibition (HAI) (d); meristematic cortical cell region of seminal root (e); and a lower view of of NH 4 -N and NO 3 -N at tillering and

HAI (h). d-h: nonsubmerged condition. Arrows indicate intercellular spaces. Bar = 50 µm. the root (f), 72 HAI; apical region of the root tip, 96 HAI (g); and meristematic cells of the cortex, 120 panicle initiation (see figure),


Fertilizer management

Effect of time of split application of K on grain and straw yield (t ha -1 ) of rice.

Grain yield Treatment a

Straw yield

Feb-Jun Jul-Nov Feb-Jun

Control (T1) 3.4 3.3 Entire basal K (T2)

4.8 4.4 4.4

4.4 6.6

K 7d earlier than N at T and PI (T3) 6.5 6.4 6.4

8.2 K + N at T and PI (T4) 5.1 4.9 7.0 6.7

8.1

K 7d after N at T and PI (T5) 3.8 3.6 5.3 5.0 K 7d earlier than N at T 5.7 5.5 7.2 7.0

K 7d earlier than N at T and K 7d 5.1 4.9 7.6 7.4

K + N at T and K 7d earlier 5.2 5.0 7.3 7.1

K + N at T and K 7d after N at PI (T9) 4.3 4.1 6.6 6.3 K 7d after N at T and K 7d earlier 4.2 4.0 6.5 6.1

K 7d after N at T and K + N at PI (T11) 4.2 4.0 6.7 6.4

JuI-NOV

and K + N at PI (T6)

after N at PI (T7)

than N at PI (T8)

than N at PI (T10)

CD (p = 0.05) 0.544 0.405 0.834

a T = tillering. PI = panicle initiation, K + N = joint application of K and N.

Grain yield was positively correlated with NH 4 -N at maximum tillering in the February-June (Y= 1.51 × -1.85, r = 0.89**) and July-November seasons (Y = 1.49 × -1.91, r = 0.91**). Application of K before N presumably suppressed fixation of NH 4

+ from applied fertilizer

because selective exchange sites in the 2:1 layer clay minerals were occupied by K + . On clay soils with high K + and NH 4

+

fixation potential, rice yields and N recovery efficiency can be increased by applying K fertilizer 1 wk before N topdressing. Changes in ammoniacal (a), nitrate (b), and

available nitrogen (c) due to time of split application of potassium.

Effect of Biozyme and NPK on rice yield

A. Sen and K. Santosh, Agronomy Depart- ment, Institute of Agricultural Sciences, Banaras Hindu University (BHU), Varanasi 221005, Uttar Pradesh, India

Biozyme is a commercial product produced from Ascophyllum nodosum, a seaweed alga known to be rich in cytokinin and auxin precursors, enzymes, and hydrolyzed protein. Farmers apply it in the field to enhance growth and yield of crop by effecting better utilization of nutrients.

We conducted a field trial during the 1993-94 wet season to investigate the effect of Biozyme granules in combination with NPK on yield performance of rice at the BHU research farm. The trial consisted of nine treatments: control (i.e., no Biozyme or NPK) (T1); 15 kg Biozyme ha -1 + 120- 60-60 kg NPK ha -1 (100% fertilizer) (T2); 15 kg Biozyme ha -1 + 50% fertilizer (T3); 20 kg Biozyme ha -1 + 100% fertilizer (T4);

20 kg Biozyme ha -1 + 50% fertilizer (T5); 40 kg Biozyme ha -l + 100% fertilizer (T6); 40 kg Biozyme ha -1 + 50% fertilizer (T7); 50 kg Biozyme ha -1 + 100% fertilizer (T8); and 50 kg Biozyme ha -1 + 50% fertilizer (T9).

The experiment was laid out in a randomized block design with three replications. N, P, and K were applied as urea, diammonium phosphate, and muriate of potash, respectively. All of the P, K, and Biozyme were applied basally, while N was applied in three splits (in 1:2:1 ratio) during transplanting, maximum tillering, and panicle initiation. In treatments 6, 7, 8, and 9, Biozyme was applied in two equal splits, at transplanting and 30 d after transplanting (DAT).

The variety was Early Monsoori, a medium-duration cultivar. Soil was slightly alkaline (pH 7.4), has low available N (210 kg ha -1 ) and P (11 kg ha -1 ) but medium K

The field was kept flooded throughout the growing period.

All treatments recorded significant increases in number of spikelets panicle -1 . panicles m -2 , and grain and straw yield over the control in both years. An increase in yield with an increasing dose of Biozyme + fertilizer was observed up to the 40 kg Biozyme + 100% fertilizer ha -1 treatment, after which a decreasing trend was noted. T6 produced a significantly superior yield compared with T2 and T3; the rest remained statistically on par with each other. It was further observed that the yield gap between 100 and 50% fertilizer doses along with different doses of Biozyme was considerably reduced. A similar trend was also found in all other yield-attributing characters. The stimulatory effect of Biozyme was most probably due to the presence of cytokinin and auxin precursors, which resulted in elevated cellular energy and more effective tillering. The addition of Biozyme at 20 kg ha -1 with 50% of the recommended NPK dose could save as much as Rs 690.35 ha -1 over 100% fertilization.


(179 kg ha -1 ) content, and 0.32% organic C.

0.625

Yield-attributing characters of rice as affected by Biozyme and NPK. Uttar Pradesh, India, 1993-94.

Panicle length (cm) Panicle weight (g) Spikelets panicle -1 (no.) Spikelets m -2 ('000) 1.000-grain weight (g)

1993 1994 1993 1994 1993 1994 1993 1994 1993 1994

16.58

Treatment

Control (no Biozyme 22.60 21.34 3.21 3.83 173.17 190.10 29.13 36.00 16.87 and no NPK)

15 kg Biozyme ha -1 23.34 22.04 3.69 3.90 187.30 210.32 40.44 50.63 16.97

15 kg Biozyme ha -1 + 100% fertilizer a

17.01

23.62 22.22 3.51 3.84 183.40 199.62 38.75 47.34 16.93 16.99 + 50% fertilizer

+ 100% fertilizer 20 kg Biozyme ha -1 22.92 22.66 4.10 4.22 197.23 220.30 45.04 55.41 17.07 17.10

20 kg Biozyme ha -1 22.86 22.26 4.00 4.18 195.13 212.53 42.11 51.25 17.02 17.08 + 50% fertilizer

+ 100% fertilizer

+ 50% fertilizer

+ 100% fertilizer

40 kg Biozyme ha -1 22.85 22.00 4.29 4.47 202.63 225.87 51.37 65.79 17.60 17.85

40 kg Biozyme ha -1 23.74 21.65 4.17 4.37 199.33 215.40 48.17 60.83 17.33 17.58

50 kg Biozyme ha -1 23.74 22.46 3.98 4.11 189.23 207.05 38.72 48.08 17.20 17.33

50 kg Biozyme ha -1 23.11 22.38 3.74 4.00 181.60 203.21 45.07 46.81 17.00 17.23 + 50% fertilizer

LSD (0.05) ns ns ns ns 15.35 17.32 1.58 1.75 ns ns CV (%) 3.27 3.38 11.97 10.29 13.33 13.46 3.35 3.71 3.09 4.58

S d - 0.62 0.61 0.80 0.33 7.23 8.17 0.74 0.83 0.43 0.39

Treatment

Control (no Biozyme and no NPK)

15 kg Biozyme ha -1

+ 100% fertilizer a

15 kg Biozyme ha -1

+ 50% fertilizer 20 kg Biozyme ha -1



Panicles m - 2 (no.)

1993 1994

175.31 200.11

227.33 255.73

218.44 249.42

243.62 261.15

231.23 255.80

265.19 309.43

Filled spikelets (%)

1993 1994

81.12 84.34

82.67 86.83

81.57 86.18

85.03 87.17

83.19 86.99

85.96 89.96

Grain yield (t ha -1 ) Straw yield (t ha -1 )

1993 1994 1993 1994

2.1 2.2 3.8 4.2

2.9 3.2 4.8 6.0

2.8 3.2 4.6 5.8

3.3 3.5 5.4 6.4

3.2 3.4 5.3 6.2

3.5 3.7 5.2 7.0

40 kg Biozyme ha -1 258.92 297.37 85.49 88.89 3.4 3.6 5.6 6.6 + 100% fertilizer

+ 50% fertilizer 50 kg Biozyme ha -1 218.65 247.13 84.96 86.93 3.0 3.3 5.0 5.1 50 kg Biozyme ha -1 264.91 245.56 84.08 86.77 2.9 3.3 5.0 5.9 + 50% fertilizer

18.85 21.22 3.35 3.21 0.3 0.3 0.7 0.5 LSD (0.05) 39.96 44.99 ns ns 0.6 0.5 1.5 1.2

S d -

CV (%) 12.13 13.34 4.87 5.07 11.2 12.0 13.9 11.7

a 120-60-60 kg NPK ha -1 .

Effects of fertilizer and green of stem detachment and improve the

sition of green manure lowered water pH, old) having only nodal roots were floated in plants 9.7 range due to algal growth. Decompo- Detached deepwater rice plants (128 d tivity of detached deepwater rice fertilizers, pH increased to within the 9.3- manure on survival and produc- survival and productivity of deepwater rice. afternoon. After application of NP/NPK

concrete tanks containing 2400 liters of maintaining it between 7.2 and 8.2. M. M. Panda, Regional Research Station water. Treatments consisted of applying Dissolved O2 concentration was generally (RRS), Judia Farm, Keonjhar 758002, various fertilizers and “burying” of higher in the afternoon than in the morning. Orissa, India Sesbania aculeata plants in water (see In the presence of decomposing green

table). The aim was to maintain a nutrient manure, dissolved O 2 was 0.0-0.2 ppm in Deepwater rice plants sometimes get concentration of 5 or 10 ppm N in water. the morning and 1.0-6.6 ppm in the detached from the soil during the vegetative Temperature and dissolved O 2 content of afternoon. stage due to damage by crabs and other the water were measured at 6:20 a.m. and at At crop maturity, four samples were organisms. They then float in water. The 4:00 p.m. Average temperature was 29 °C harvested from each tank. There was a survival of such plants depends on nodal in the morning and 31 °C in the afternoon. positive response to applied nutrients (see roots that draw nutrients from the flood- Water pH was measured at 9:00 a.m. and at table). Application of NP gave NPK gave the water. Nodal roots can appear within 24 h 3:00 p.m. It increased by 0.5-1.0 unit in the best yields and were superior to application


Effect of fertilizers and green manure on yield Effect of green manure and fertilizer N on rice yield and comparative economic data. Maharashtra, of detached deepwater rice plants grown in India. water.

Treatment

No fertilizer (control) 5 ppm N as urea 10 ppm N as urea

Yield (g m -2

tank area)

Grain Straw

14.4 261 25.1 321 27.4 329

5 ppm N through fresh 22.6 326

5 ppm N as urea and 5 ppm 37.5 394

5 ppm N as urea, 5 ppm P 2 O 5 as 38.7 378

Sesbania aculeata plants (3 kg)

P 2 O 5 as single superphosphate

single superphosphate, and 5 ppm K 2 O as muriate of potash

LSD (0.05) 6.9 39.5

of N alone, either through urea or green manure. The detached plants could complete their life cycle to grain filling and ripening without anchorage and nutrition from the soil. However, grain yield was low, with a maximum harvest index of 0.1.

Integrated N management with Gliricidia maculata and Sesbania aculeata for transplanted rice

L. S. Chavan, B. P. PatiI, and T. S. Ingole, Agricultural Research Station, Palghar 401404, Thane District, Maharashtra, India

To save on fertilizer costs, green manure (GM) is used in conjunction with fertilizer N. We evaluated the effect of integrating GM Gliricidia maculata and Sesbania aculeata with different levels of fertilizer N (urea) on grain yield of transplanted rice in the 1989-90 rainy season. Gliricidia seedlings were planted on ricefield bunds at a distance of 0.5-1 m. The plants produced sufficient green loppings after 3 yr with 2.74% N, 0.5% P, and 1.1 % K in the leaves (dry weight basis). Sesbania was grown in situ at a seeding rate of 50 kg ha -1 and incorporated in the soil after 45 d. Sesbania contained 2.9% N, 0.2% P, and 0.98% K. Soil was clay with pH 8.1, 0.6% organic C, and 200-42-346 kg available NPK ha -1 .

The experiment was laid out in a randomized block design with four replications. GM crops were incorporated in the soil at transplanting. On the same day, 30-

Treatment Rice yield (t ha -1 ) Cost of Gross Net B:C a

1989 1990 Pooled (US$ ha -1 ) (US$ ha -1 ) (US$ ha -1 ) cultivation returns returns

mean

100 kg N ha - 1 as urea in 3 splits: 3.6 2.6 3.1 344.99 404.71 59.72 1.17 1/2 at planting + 1/4 at tillering + 1/4 at panicle initiation (recommended)

5 t G. maculata ha -1 (27.4 kg N ha -1 3.1 2.2 2.6 330.10 342.95 12.85 1.04 + 50 kg N ha -1 as urea at planting

7.5 t G. maculata ha -1 (41.1 kg N 3.4 2.5 2.9 334.80 385.00 50.20 1.15 ha -1 ) + 25 kg N ha -1 as urea at planting

5 t S. aculeata ha -1 (29 kg N ha -1 3.2 2.3 2.8 335.70 363.98 28.28 1.08 + 50 kg N ha -1 as urea at planting

7.5 t S. aculeata ha -1 (43.5 kg N 3.4 2.1 2.7 333.35 357.41 24.06 1.07 ha -1 ) + 25 kg N ha -1 as urea at planting

Control (no N) 2.4 1.5 1.9 301.46 253.60 –47.86 0.84

SE± 0.2 0.2 0.1 LSD (0.05) 0.6 0.5 0.2

a Benefit-cost ratio.

to 35-d-old seedlings of 125-d-duration rice variety PLG1 were transplanted. P and K at 50 kg ha -1 each were basally applied. N was applied as per treatment schedule (see table).

Fertilizer application at the recommended dose and integration of fertilizer N with GM proved superior to the control. Basal application of 7.5 t G. maculata with 25 kg urea N ha -1 was on par with 100 kg urea N ha -1 in terms of yield. Costs of cultivation, gross returns, net returns, and benefit-cost ratio of this integrated N management treatment were comparable with those of the recommended dose. With this treatment, a savings of 75 kg urea N ha -1 may be achieved.

~

Response of late-transplanted rice to NPK under irrigated conditions

B. Hasan, A. S. Bali, and K. N. Singh, Agronomy Division, S. K. University of Agricultural Sciences and Technology (SKUAST), P. 0. Box 706, G. P. 0. Srinagar, Kashmir 180001, India

Rice followed by rapeseed - mustard is the most feasible double cropping system in Kashmir Valley (1650 m asl). But aberrant weather conditions (low temperature and cloudiness) delay transplanting. The NPK requirement of a crop under such a situation may differ from that of a timely planted rice crop, which usually requires 80-45-20 kg NPK ha -1 .

We conducted a 2-yr field trial at SKUAST’s Shalimar Campus, on silty clay loam soil (pH 6.8, low available N and P, medium available K) during the 1990-91 wet seasons. The 18 treatments, tested in a randomized block design experiment with three replications, comprised different combinations of NPK levels (see table). Half the N and a full P and K dose were applied basally while the remaining N was applied in two splits: at tillering (18 d after transplanting [DAT]) and at panicle initiation (37 DAT).

Rice variety K39 was transplanted at 15- × 1.5-cm spacing at 3 seedlings per hill by the 1 st week of Jul. During the experiment. temperatures ranged from 23.0 to 31.1 °C (maximum) and from 11.3 to 19.6 °C (minimum). Relative humidity was 45- 76%. Total rainfall averaged 234 mm, spread over 35 d.


Fertilizer management— inorganic sources

Effect of NPK levels on yield and yield-attributing characters of rice. Kashmir, India. 1990-91.

Yield Panicles m -2 Grains 1,000-grain Nutrient level (kg ha -1 ) (t ha -1 ) (no.) panicle -1 weight (g)

(no.)

N 40 4.0 318 57.8 21.25 80 4.5 331 65.8 21.70

LSD (P = 0.05) 0.37 5 1.7 0.50 120 4.7 339 69.0 23.05

P 8.8 3.6 315 58.7 20.90

17.6 4.6 331 64.7 21.50 26.4 5.0 344 69.0 22.15

LSD (P = 0.05) 0.37 5 1.7 0.50

K 16.6 4.9 322 63.0 21.40 33.2 5.5 335 65.4 21.65

LSD (P = 0.05) 0.30 4 1.3 0.40

Results showed that up to 80 kg of N and up to 26.4 kg of P ha -1 significantly increased rice grain yield with concomitant improvement in all yield attributes (see table). The yield levels obtained were, however, lower than those of a timely sown crop (5.5 - 6.0 t ha -1 ). This implies that a combination of 80-26.4-33.2 kg NPK ha -1

will give satisfactory yields under late transplanting conditions in the valley.

Evaluating stem-nodulating green manure crops for lowland rice

D. N. Medhi, Agronomy Department, Assam Agricultural University (AAU), Jorhat 785012, Assam, India; and B. Deka Medhi, Agricul- tural Chemistry Research Unit, AAU

We studied the effect of stem-nodulating green manure (GM) crops Sesbania rostrata, S. speciosa, Aeschynomene afraspera, A. indica, A. schemperi, and A. nilotica on N 2 fixation and biomass production in lowland rice. The experiment was conducted during Aug 1993 and Jul 1994 wet season on a sandy loam acidic soil (pH 5.1) with 205 kg available N ha -1 . S. aculeata was the check.

Treatments were replicated four times in a randomized block design. The GM seeds were broadcast at 50 kg ha -1 in all the 3- × 3- m plots. The GM plants were allowed to grow for 45 d after emergence; they were then cut at ground level from an area of 0.32 m 2 in each plot. N concentration of the whole plants was determined by micro- Kjeldahl method.

Significant differences in dry biomass production, N concentration. and N content of the GM crop were recorded (see table). S. rostrata and A. afraspera were found to be superior to all other species. indicating that these two are the most suitable GM crops for lowland rice soils.

Dry biomass production, N concentration, and N content of different green manure crops. Assam, India, 1993-94.

Managing crop residue in rice

R. D. Misra, V. K. Gupta, and D. S. Pandey, Department of Agronomy, G. E. Pant University of Agriculture and Technology (GBPUAT), Pantnagar, Nainital 263145, India

Burning of crop leads to loss of organic matter and nutrients. Incorporating straw helps in nutrient recycling and maintains soil health. We carried out an experiment on crop residue management at GBPUAT, Pantnagar, during the 1993-94 kharif season.

Soil was loam with pH 7.4,0.084% total N, 1.10% organic C, 17.0 kg available P ha -1 , and 194.0 kg available K ha -1 . The experiment, laid out in randomized block design, had 12 treatments with four replications, (see table).

Green manure (GM) crop Sesbania aculeata was grown and incorporated in the

Dry biomass (t ha -1 ) N concentration (%) N content (kg ha -1 ) Same plot before transplanting rice, This

1993 1994 1993 1994 1993 1994 provided 60 kg excess N ha -1 in 1993 and 57 kg excess N ha -1 in 1994. Berseem

Sesbania aculeata 3.1 3.2 1.4 1.5 41 44 Sesbania rostrata 5.3 6.1 3.0 3.2 142 188

( Trifolium alexandruim L.) was sown in a

Sesbania speciosa 3.1 3.3 2.7 2.9 94 88 standing crop of rice at the milk stage and Aeschynomene afraspera 4.3 5.4 3.6 3.8 139 185 was incorporated in the field 1 mo before

Species

Aeschynomene indica 2.5 3.1 2.7 2.9 65 89 Aeschynomene schemperi 2.3 2.2 2.8 2.8 61 58

transplanting, giving an additional 80 and Aeschvnomene nilotica 2.4 3.5 2.6 2.7 61 84 70 kg N ha -1 in the respective years. Incor-

LSD (0.05) 2.5 2.3 1.8 1.9 30 32 poration of wheat straw. done without a starter dose, provided an additional 74 and 65 kg N ha -1 . With 20 kg of starter dose of N (as urea), straw incorporation provided


Fertilizer management—organic sources

Effect of different crop residue management Evaluation of green manures and practices on rice yield. All of the green manures and grain

grain legumes legumes recorded higher dry matter

Treatment Grain yield production in the February-May season.

(t ha -1 ) S. Porpavai, S. P. Palaniappan, and Sesbania rostrata accumulated more N S. Purushothaman, Centre for Soil and Crop (165 kg ha -1 ) than did the other green Management Studies, Tamil Nadu Agricul- manures.

Rice and what straw removed 5.6 4.6 (control)

tural University (TNAU), Coimbatore 641003, Cowpea ( Vigna unguiculata ) outper- Rice straw burned and wheat 5.6 4.7 India

Only rice straw incorporated 5.7 4.7 Only rice straw incorporated 5.7 4.9 We compared several green manures and After the rice harvest in January, farmers

with 20 kg N ha -1

percentage, and N accumulation at 60 d Only rice straw incorporated and 5.7 4.8 grain legumes in terms of dry weight, N may grow S. aculeata or S. rostrata in situ

excess 20 kg N ha -1 to wheat Rice and wheat straw 5.9 5.0 after sowing in two seasons, Jul-Sep 1991 the next rice crop that is grown during

formed the other grain legumes, producing 10 t green manure on dry-weight basis.

and plow it down during transplanting of

straw removed

incorporated

incorporated with 20 kg N ha -1

and Feb-May 1992. The experiment was kuruvai season (June-September). Rice and wheat straw

Only rice straw incorporated 5.8 5.0 three replications. 6.1 5.1 laid out in a randomized block design with,

with berseem 20 t FYM a before rice 20 t FYM a before wheat

6.7 5.4 Dry matter production and N accumulation in green manures and grain legumes at 60 DAS. a

5.9 5.0 Coimbatore, India. Sesbania aculeata before rice 6.9 5.7 Berseem before wheat 5.8 4.8

LSD (P = 0.05) .15 .23

a FYM = farmyard manure.

90 and 83 kg N ha -1 in 1993 and 1994, respectively. Rice straw was incorporated or burned before the wheat crop.

Twenty-two-day-old seedlings of Pant Dhan 10 was transplanted on 24 Jul 1993 and 7 Jul 1994. The recommended fertilizer dose of 120 kg N ha -1 was applied in all plots and the usual cultural operations were done.

Burning of rice straw was at par with the treatment where straw was removed. Incorporating both rice and wheat straw gave significantly higher yield over rice alone because wheat straw supplied some extra nutrient to the rice crop. Incorporating straw with a starter dose of 20 kg N was slightly better than the treatment without a starter dose because the initial dose helped straw to decompose faster, thereby providing more nutrients to rice.

Green manuring with S. aculeata gave the highest production and was at par with 20 t farmyard manure ha -1 before rice. Use of GM berseem gave a slightly better yield than the control. The higher grain yield obtained from incorporating straw with a starter dose of N, FYM, and GM with S. aculeata was attributed to extra N (more than 120 kg ha -1 ) applied to these treatments. Besides the direct contribution of N, the better soil physical condition must have positively contributed to yield.

Green manure

S. aculeata S. rostrata S. speciosa S. grandiflora Tephrosia purpurea Glycine max Crotalaria juncea Phaseolus trilobus Vigna aureus Vigna unguiculata

Jul-Sep 1991

Dry N (%) N weight (t ha -1 )

accumulation (kg ha -1 )

5.4 1.7 91.8 4.6 2.4 110.4 1.6 1.7 27.2 2.4 1.2 28.8 1.4 1.2 16.2 2.0 1.1 22.0 5.8 1.3 75.4 3.8 1.5 55.1 2.1 1.5 31.1 5.8 1.4 77.6

Feb-May 1992

Dry N (%) N weight accumulation (t ha -1 ) (kg ha -1 )

8.6 1.6 7.5 2.2 6.5 2.1 7.2 1.4 5.0 0.9 8.1 1.0 9.3 1.2 9.5 1.2

137.6 165.0 136.5 100.8 45.0 81.0

107.0 114.0

8.4 1.0 84.0 10.0 1.2 123.0

CD 0.61 0.62 3.8 3.5 1.0 5.2

a DAS = days after sowing.

Effect of soil amendments on rice yield

R. D. Sharma and S. Ali, G. B. Pant University of Agriculture and Technology (GBPUAT), Western Campus, Modipuram Meerut, Uttar Pradesh 250110, India

We conducted a field experiment on a Typic Natrustalf at GBPUAT in the 1987-92 wet seasons (WS) to evaluate the performance of gypsum, pyrite, mud press, and farmyard manure (FYM) as soil amendments. Surface soil (0-15 cm) is silty clay loam (53.5% sand, 25.2% silt, and 21.3% clay), with pH 9.4-9.8, EC 6.9 dS m -1 , 22.6% exchangeable Na, 0.12% organic C, 8.2 kg Olsen P ha -1 , 0.35 c mol exchangeable K kg -1 , and CEC 12.4 c mol kg -1 .

Amendments were applied in Apr 1987 and 35-d-old seedlings (3 hill -1 ) of rice cultivar Saket 4 were transplanted at 15- × 15-cm spacing. A uniform dose of 150 kg N had in three splits, 21.8 kg P ha -1 , 4 1.5 kg K ha -1 , and 25 kg ZnSO 4 ha -1 (basal), was applied. A wheat crop was raised in the rabi (winter) season, with 150 kg N applied in 3 splits, 26.2 kg P, and 41.5 kg K (basal) ha -1 . The same rice - wheat cropping sequence and NPK fertilization were followed during subsequent years on the same layout without additional soil amendments.

The control treatment had an average rice yield of 2.7 t ha -1 (Table 1). The higher yields recorded were with gypsum application at 12 t ha -1 followed by pyrite at 4 t ha -1 . The lowest yield increase over the control was observed with pyrite at 2 t ha -1 , mud press at 6 t ha -1 , and FYM at 20 t ha -1 .


1993 1994

control treatment (Table 1). Maximum

Table 1. Effects of soil amendments on rice yield. Uttar Pradesh, India, 1987-92.

Grain yield (t ha -1 ) Six-year average

Soil amendment 1987 1988 1989 1990 1991 1992

Control 0.9 2.2 2.8 3.1 3.5 3.8 Gypsum (6 t ha -1 ) 1.8 3.1 3.5 3.8 4.1 4.3 Gypsum (9 t ha -1 ) 2.2 3.4 3.8 4.1 4.5 4.8 Gypsum (12 t ha -1 ) 2.6 3.8 4.2 4.6 4.9 5.1 Pyrite (2 t ha -1 ) 1.3 2.5 3.0 3.3 3.7 3.9 Pyrite (3 t ha -1 ) 1.6 2.9 3.1 3.4 3.8 4.0 Pyrite (4 t ha -1 ) 2.5 3.5 3.9 4.3 4.5 4.6 Mud press (6 t ha -1 ) 1.5 2.7 3.1 3.4 3.7 3.8 Mud press (12 t ha -1 ) 1.7 3.1 3.4 3.8 4.1 4.3 FYM a (20 t ha -1 ) 1.4 2.7 2.9 3.3 3.6 3.9 FYM (10 t) + pyrite (2 t ha -1 ) 2.2 3.2 3.6 3.9 4.2 4.4 FYM (10 t )+ mud press 2.4 3.3 3.7 4.1 4.3 4.5

(6 t ha -1 ) SEM (±) 0.12 0.05 0.07 0.06 0.09 0.016 LSD (5%) 0.4 0.2 0.2 0.2 0.3 0.5

a FYM = farmyard manure.

Rice

Yield Increase (t ha -1 ) over control

(%)

2.7 3.4 25.9 3.8 40.7 4.2 55.6 3.0 11.1 3.1 14.8 3.9 44.4 3.0 11.1 3.4 25.9 3.0 11.1 3.6 33.3 3.7 37.0

Wheat

Yield Increase (t ha -1 ) over control

(%)

1.2 2.0 66.7 2.4 100.0 2.9 141.7 1.7 41.7 1.9 58.3 2.5 108.3 1.7 41.7 2.0 66.7 1.5 25.0 2.0 66.7 2.2 83.3

Table 2. Physicochemical properties of surface soil (0-15 cm) a after 6 yr of experimentation. Uttar Pradesh, India, 1987-92.

pH EC (dSm -1 ) CEC Exchangeable cations (c mol (p + ) kg -1 ) Exchangeable Treatment (1:2 soil-water (1:2 soil-water (c mol (p + ) sodium

ratio) ratio) kg -1 ) Na + K Ca ++ Mg ++ percentage

Control 8.2 0.40 12.0 0.58 0.19 5.27 1.27 4.83 Gypsum (6 t ha -1 ) 8.1 0.36 12.1 0.42 0.12 7.00 1.20 3.50 Gypsum (9 t ha -1 ) 7.8 0.33 12.0 0.41 0.11 7.13 1.00 3.45 Gypsum (12 t ha -1 ) 7.3 0.33 12.0 0.40 0.11 7.47 1.13 3.13 Pyrite (2 t ha -1 ) 8.2 0.36 12.1 0.49 0.16 6.80 1.27 3.95 Pyrite (3 t ha -1 ) 8.1 0.34 12.1 0.43 0.14 7.00 1.10 3.58 Pyrite (4 t ha -1 ) 7.8 0.35 12.2 0.40 0.12 7.10 1.00 3.28 Mud press (6 t ha -1 ) 8.1 0.35 12.1 0.40 0.13 6.73 1.00 3.28 Mud press (12 t ha -1 ) 7.8 0.29 11.8 0.39 0.12 7.20 1.27 3.31 FYM (20 t ha -1 ) 7.7 0.30 12.3 0.42 0.19 6.13 0.93 3.44 FYM (10 t) + pyrite (2 t ha -1 ) 8.0 0.33 12.0 0.39 0.19 6.67 1.00 3.22 FYM (10 t) + mud press (6 t ha -1 ) 7.7 0.32 12.1 0.38 0.16 7.47 0.86 3.22

SEM (±) 0.02 0.03 0.03 0.29 0.13 -LSD (5%) ns b 0.09 ns 0.84 ns -

a The Initial values were pH 9.4-9.8, EC 6.9 dS m -1 , CEC 12.4 cmol(p+) kg -1 , and ESP 22.6. b ns = not significant.

Applying FYM at 10 t ha -1 in combination with pyrite at 2 t ha -1 or gypsum at 6 t ha -1

resulted in comparable yields. The amendments were effective in this order: gypsum > pyrite > mud press > FYM.

Wheat yield averaged 1.2 t ha -1 in the

yield increase was observed with gypsum application at 12 t ha -1 and minimum with FYM at 20 t ha -1 . The pattern of effectiveness of soil amendments in the wheat crop was similar to that in the rice crop.

After 6 yr, the pH and exchangeable sodium percentage in the control and treated plots were appreciably reduced, and the level of amendment applied was reflected in this reduction (Table 2). Besides the amendments, the rice - wheat cropping sequence has a profound influence on soil improvement, as reflected in the yield levels from year 1 to year 6. Gypsum alone or gypsum with FYM outperformed pyrite.

Use of deepwater varietal mixtures to minimize hydrological risk in basin lands of Bihar, India

M. M. Sinha, A. Kumar, N. Chaudhary, Rajendra Agricultural University, Bihar Agricultural Research Institute, Mithapur, Patna 800001, India

Chaurs (basin lands) are low-lying fields of variable shape and size having poor or no drainage. Crop production depends mainly on the unpredictable monsoon rainfall. Drought or flooding of varying durations


– –

– – – – – – – –

Crop management

and intensities are the main hydrological constraints to rice productivity.

(DWR) is a prevalent practice in chaurs. The varieties used have almost identical morphological characters and are highly adapted to sowing and transplanting conditions in the area. We studied the submergence tolerance of DWR varieties Darmi and Jagar under different hydrological conditions.

collected randomly from deepwater ricefields (200 cm maximum water depth) cropped with mixtures of Darmi and Jagar during 1990 kharif in chaur. The area was stratified into 50-100, 100-150, and 150- 200 cm water depths. Based on kernel color and panicle characteristics, samples from each water regime were classified into three—Darmi, Jagar, and “others.”

Pregerminated seeds of Darmi and Jagar were sown 5 cm apart in five trays filled with soil on 12 Jun 1991. The trays with 14-d-old seedlings were completely submerged in a pond for 7 d with 30 cm of water above the tip of the seedling. On the 10th day of the recovery period, survival count and phenotypic scoring for submergence tolerance were done. The IRRI standard scoring system was used; a low score indicates good tolerance.

Cultivating mixtures of deepwater rice

Five samples of 100 panicles each were

Effect of transplanting times on hybrid rice in Haryana, India

H. Om, Rice Research Station (RRS), Kaul 132021; S. K. Katyal, CCS Haryana Agricultural University, Hisar 125004; S. D. Dhiman and A. Singh, RRS, Kaul, India

We evaluated the performance of newly developed hybrids in India with respect to staggered planting. The field study involved four transplanting dates (15 Jun, 25 Jun, 5 Jul, and 25 Jul) and four genotypes (hybrids ORI 161, PMS2A/IR31802, and PMS01A/PR106 and check variety HKR126) and was conducted during the 1993-94 wet seasons at RRS, Kaul. Soil was clay loam, with pH 8.2, low available N. but high available P and K.

The experiment was laid out in a split- plot design with four replications; planting dates were main plots and varieties were


Table 1. Number of panicles of Darmi, Jagar, and others per sample of 100 panicles, collected from three water depths. Vaishali, Bihar, India.

Water regimes

100-150 crn

Jagar Darmi Others Jagar Darmi Others Jagar Darmi Others

Sample 50-100 cm 150-200 crn

1 52 45 3 34 56 10 21 2 51 36 41 51

77 13

2

3 40 10 28 8 33

50 61 6

4 49 35 63 9 48 50 2

16 40 5

54 56 40

6 26 66 4

8 30 59 11 43 54 3

Total 248 206 46 173 283 44 181 307 21 49.6 41.2 9.2 34.6 56.6 8.8 36.2 61.4 4.2

Table 2. Submergence tolerance scores of Darmi and Jagar. Bihar, India.

Variety Tray 1 Tray 2 Tray 3 Tray 4 Tray 5 Av

6.8 8.6

Darmi 7 6 8 6 7 Jagar 8 9 9 9 8

Among samples collected from the 50-100 cm water regime, Jagar had more panicles (49.6%) than Darmi (41.2% ), suggesting that Jagar was the better adapted cultivar with a greater number of effective tillers.

Among samples obtained from the lower niches of the chaur. Darmi had higher panicle number (56.6 and 61.4%) in the 100-1.50 and 150-200 cm water regimes compared with Jagar, which had 34.5 and 36.2%, respectively (Table 1). This indi-

cates that Darmi was better adapted to deep water than was Jagar.

Darmi had better submergence tolerance (av 6.8) than Jagar (av 8.6) (Table 2). This suggests that Darmi possesses genes that make it suitable for deepwater areas.

different levels of submergence tolerance but similar agronomic characters could be one way of minimizing hydrological constraints to growing rice in rainfed low-lying areas.

Developing DWR varietal mixtures with

subplots. Plant spacing was 20 × 15 cm. old seedlings were transplanted in all dates The crop was fertilized with 150 kg N.26.4 except in the 25 Jul 1993 planting, where

three equal splits: at transplanting, active Highest grain yields were obtained when tillering, and panicle initiation. Thirty-day- the genotypes were transplanted on 25 Jun

kg P, and 5.75 kg Zn ha -1 , with N applied in 40-d-old seedlings were used.

Effect of transplanting time and genotype on grain yield and harvest index. Kaul, India. 1993-94.

Grain yield Harvest Productivity Spikelet sterility Treatment (t ha -1 ) index (kg d -1 ) (%)

1993 1994 Mean 1993 1994 1993 1994 1993 1994

Time of transplanting 15 Jun 7.2 6.8 7.0 0.51 0.50 46.5 51.0 26.2 27.6 25 Jun 7.9 7.5 7.7 0.54 0.53 51.2 57.1 20.4 23.4 5 Jul 7.6 7.2 7.4 0.53 0.52 50.9 54.6 22.1 25.4 25 Jul 5.5 4.2 4.9 0.46 0.41 41.9 32.1 28.8 37.9

LSD (0.05) 0.4 0.3 - 0.01 0.02 - - 2.1 1.8

Genotypes ORI 161 7.8 7.4 7.6 0.54 0.52 52.9 56.5 19.2 24.4 PMS2 A/IR31802 7.3 6.6 7.0 0.52 0.49 49.1 49.8 25.3 29.7 PMS10 A/PR106 6.3 5.8 6.1 0.48 0.45 42.4 43.7 30.1 33.9 HKR106 6.9 6.1 6.5 0.50 0.49 47.3 46.4 23.5 27.6

LSD (0.05) 0.2 0.2 - 0.01 0.01 - - 2.0 2.0

Av

(7.7 t ha -1 ) and on 5 Jul (7.4 t ha -1 ) in both years (see table). A drastic yield reduction was observed in the 25 Jul planting, but the rate of decrease was slower in 1993 than in 1994. This may be due to the use of aged (40 d) seedlings in 1993. There was 10.1, 4.1, and 57.1% increase in grain yield in the 25 Jun planting over that of 15 Jun, 5 Jul, and 25 July, respectively.

were highest in the 25 Jun planting, followed by the 5 Jul and 15 Jun plantings.

Harvest index and productivity (kg d -1 )

Lowest values of harvest index and produc- HKR126 (6.5 t ha -1 ). Productivity and tivity were registered with the 15 Jun harvest index were highest in ORI 161, planting. Spikelet sterility was lower in followed by PMS2 A/IR3 1802 and 1993. It was similarly lower for 25 Jun and HKR126. Spikelet sterility was minimum 5 Jul than for 15 Jun and 25 Jul. Percentage in ORI 161, followed by HKR126. sterility was relatively higher for the late Planting hybrid OR1 161 resulted in a planting dates in 1994. 17% increase in grain yield over the best

Among genotypes, ORI 161 (PHB71) available pureline genotypes. In produced the highest grain yield (7.6 t ha -1 ). northwestern India, the best time to Hybrids ORI 161 and PMS2 A/IR31802 transplant hybrid rice is between 25 Jun and were found significantly superior to 5 Jul.

Effect of solar radiation and 3-wk intervals were evaluated, the first in temperature on rice yields in 29 Apr and the last in 22 Jul 1993. different planting dates The experiment was laid out in a split-

F. J. Osuna-Canizales, Campo Experimental Zacatepec, Morelos with four replications, Zacatepec, Apartado Postal 12, Zacatepec, with planting dates in the main plot and Morelos 62780, Mexico genotypes in the subplots. Forty-five-day-

old seedlings were planted at 20- × 20-cm Solar radiation and temperature are im- spacing in 4- × 4-cm plots. Water portant climatic factors affecting rice yields management consisted of periodical under irrigated conditions. In an experi- irrigation. Basal fertilization with 90 kg N ment to define optimum planting dates for ha -1 , 40 kg P ha -1 , and 40 kg K ha -1 was the newly released high-yielding variety applied 25 d after transplanting (DAT); Morelos A92 and breeding line CAEZ401, additionally, 45 kg N ha -1 each were applied the effect of these climatic factors on grain at panicle initiation and at booting stage. yield was assessed through regression Grain yield in a 9-m 2 area was measured techniques. and adjusted to 14% moisture on a wet

organic matter, 0.31% total N, and 22 ppm Regression analysis with grain yield as Olsen P. Five planting dates with the dependent variable and global radiation

plot design at the experimental station of

Soil is a Vertisol with pH 7.6,3% basis.

(GR, estimated after sunshine hours) and minimum mean temperature (MMT) as independent variables were performed in three periods: from 20 d before flowering up to flowering (f-20). from flowering to 20 d after flowering (f+20), and from 20 d before to 20 d after flowering (f-20 to f+20).

Growth duration was shortened in the last planting dates because of photoperiod sensitivity of the two genotypes (see table). In the first planting date, Morelos A92 and CAEZ401 were harvested 137 and 125 DAT, respectively, whereas in the last planting date. Morelos A92 and CAEZ401 were harvested 120 DAT. Grain yield declined in Morelos A92 as planting was delayed (see figure), but in CAEZ401, decline was significant only for the late planting date.

Regression equations and parameters to assess the fitness of different models in the three periods. Zacatepec, Morelos, Mexico, 1993.

Model Equation R 2 (%) a MSE b

f-20 c

1 Yield d = 10328 - 4.6 (GR) e 0.16 25.9 2909314 2 Yield = 437393 - 49234 (MMT) e + 1410 (MMT 2 ) 0.07 78.1 3670335 3 Yield = 10599 - 93 (GR/MMT) 0.19 20.2 2776786 4 Yield = 916660 + 980 (GR) - 78858 (MMT) + 1496 (MMT 2 ) - 16751 (GR/MMT) 0.85 2.6 813472

f + 20

1 Yield = 20 (GR) - 2411 0.20 19.9 2768937 2 Yield = 144952 - 17855 (MMT) + 571 (MMT 2 ) 0.81 0.3 3

758453 Yield = 12114 - 137 (GR/MMT)

4 0.02 67.4 3365802

Yield = 163592 + 37 (GR) - 18847 (MMT) + 568 (MMT 2 ) - 652 (GR/MMT) 0.81 4.9 1060182

f - 20 to + 20

1 2

Yield = 28 (GR) - 6705 0.25 14.4 2595018

3 Yield = 47782 (MMT) - 1277 (MMT 2 ) - 437848 0.34 23.2 2594505

4 Yield = 305 (GR/MMT) - 796 0.09 40.2 3138594 Yield = 454149 + 367 (GR) - 44589 (MMT) - 1051 (MMT 2 ) - 6035 (GR/MMT) 0.49 40.9 2799678

a Observed level of significance. b Mean square error. c f - 20 = from 20 d before flowering up to flowering, f + 20 = from flowering to 20 d after flowering. f - 20 to + 20 = from 20 d before to 20 d after flowering. d kg ha -1 . e GR = global radiation. MMT = mean minimum temperature.


Filled grains panicle -1 and grain weight were the most affected by late transplanting. For the two genotypes, filled grains panicle -1 went down from 63 in the first date to 47 in the last date while 1,000 grain weight was reduced from 43.6 to 40.7 g.

The regression equations obtained and the parameters used to evaluate fitness of the models in each period are shown in the table. Within each period, the best fit was obtained when GR and MMT were

Effect of planting date on grain yield of cultivar Morelos A92 and breeding line CAEZ401. Zacatepec, Morelos, Mexico. 1993.

evaluated together (model 4), except in the period f+20, when MMT and MMT2 (model 2) were comparable. The fitness of model 4 was higher for f-20 and f+20 than their combination.

These results show that variation in incident global radiation and mean minimum temperature during the period 20 d before or 20 d after flowering may explain much of the variation in grain yield in the different planting dates for irrigated rice.

Effects of neem derivatives on Comparison of biocides to control sheath rot incidence and grain yield of scented rice JJ92. sheath rot in scented rice Treatment Percent ShR incidence a Graln yield

(t ha -1 ) K. Sivaprakasam and R. Jagannathan, Plant Pathology Department, Agricultural College and Research Institute, Madurai, Tamil Nadu, India

We assessed the efficacy of botanical biocides and pesticides (fungicides carbendazim and mancozeb) for controlling sheath rot (ShR) of scented rice. Leaf extract and fungicides were applied twice as foliar sprays at a 2-wk interval beginning at booting. The field trial was laid out in a randomized block design with three replications using scented hybrid rice JJ92. Plot size was 5 × 2 m.

ShR incidence was measured as the percentage of infected tillers in 25

Seed borne nature and seed transmission of rice sheath blight

S. Acharya and P. K. S. Gupta, Plant

Neem oil (3%) Neem seed kernel extract (5%) Monocrotophos (1250 ml ha -1 ) Carbendazim (500 g ha -1 ) Mancozeb (1 kg ha -1 ) Monocrotophos (1250 ml) + carbendazim (500 g ha -1 ) Monocrotophos (1250 ml) + mancozeb (1 kg ha -1 ) Nochi (10%) ( Vitex negundo ) Untreated check

LSD (P=0.05)

30.22 26.30 25.86 24.27 30.47 27.57 22.87 26.80 37.52 1.10

(33.25) (29.70) (30.48) (29.80) (33.38) (31.61) (28.49) (31.10) (37.67)

5.2 5.0 4.4 4.1 3.4 3.8 4.5 4.2 3.2 .5

a Means of three replications. Data in parentheses are arcsine-transformed values.

randomly selected hills/plot 20 d after the mancozeb (1 kg ha -1 ) effectively reduced last spray. Higher yields were recorded ShR incidence (see table). with neem oil (3%), neem seed kernel Using a spray of neem seed kernel extract (5%), and monocrotophos (1,250 ml extract generated higher grain yield and ha -1 ) + mancozeb (1 kg ha -1 ). Standard effectively reduced disease incidence, fungicides carbendazim (500 g ha -1 ) and although to a lesser degree, than using monocrotophos (1,250 ml ha -1 ), and regular fungicides.

Effect of R. solani infection on rice germination and seed health. West Bengal a , India.

Seeds Germination Seedling Seedling Shoot Shoot Root Root Treatment yielding (%) infection collapse fresh dry fresh dry

R. solani (%) (%) weight weight weight weight (%) (mg) (mg) (mg) (mg)

Pathology Department, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Kalyani

Naturally Infested seeds

741235, Nadia, West Bengal, India Artificially inoculated seeds

Although rice sheath blight (ShB) caused Apparently healthy

by Rhizoctonia solani is documented as a seeds

seedborne disease, evidence of this nature is SE ±

lacking. We investigated spotted and Unsterilized seeds apparently healthy seeds of rice cultivar Surface-sterilized

LSD (0.05)

37.83

56.35

12.17

0.682 1.520

50.92

19.98

59.09

70.66

73.19

0.655 1.459

65.63

69.67

14.88 4.35 279.46 46.83 485.7 64.1

23.58 9.21 361.20 66.05 673.75 120.13

0 0 467.26 94.3 922.4 174.3

0.728 0.127 0.57 0.376 0.575 0.324 1.624 0.284 1.286 0.838 1.281 0.721

8.70 9.09 334.66 60.52 608.66 109

6.93 0 403.94 77.60 779.23 130.02 Swarna Mashuri to find out the mode of seeds

transmission involved. The seeds were collected from a heavily ShB-infected rice Interaction crop from the BCKV Regional Research SE ± 0.9315 0.9266 1.03 0.032 0.81 0.532 0.813 0.459

Farm.

0.545 0.535 0.594 0.018 0.47 0.307 0.469 0.265 LSD (0.05) 1.214 1.19 1.326 0.024 1.050 0.684 1.04 0.590

LSD (0.05) 2.07 2.06 2.29 0.072 1.819 1.85 1.81 1.022

a All percentage data are transformed angular values.


Integrated pest management—diseases

SE ±

Healthy seeds (200 g) were placed in a sterilized erlenmeyer flask and 10 cm of air- dried inoculum of R. solani (grown in sand- maize-meal medium for 15 d at 20 °C) was added to it. The flask was shaken for 5 min.

R. solani was isolated from spotted, artificially inoculated, and apparently healthy seeds. The seeds were placed in 2% sterilized water agar in petri dishes and incubated at 28 °C for 72 h. One lot was seeded without surface sterilization; the other lot was seeded after surface sterilization with NAOCl solution for 1 min.

Serological evidence for inducing resistance to rice tungro viruses using antiviral principles

P. Muthulakshmi, and P. Narayanasamy, Plant Pathology Department, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore, 641003, India; and H. Koganezawa, IRRI

Use of antiviral principles (AVPs) from plant sources is found to reduce rice tungro virus (RTV) infection. We conducted a study to obtain serological evidence for the effectiveness of AVP application in managing rice tungro disease.

AVPs from seed sprouts of pigeonpea (PP-AVP) and mungbean (MB-AVP) were sprayed at 10% concentration on rice variety Co 43. The following day, each plant was inoculated with two viruliferous Nephotettix virescens (Distant). Twenty- five plants were inoculated in each treatment; inoculated plants without AVP application served as the control. Leaf samples were collected 25 d after inoculation and tested using enzyme-linked immunosorbent assay (ELISA) to determine the presence of rice tungro bacilliform (RTBV) and rice tungro spherical virus (RTSV).

The RTV antigen was optimally diluted in carbonate-bicarbonate buffer (pH 9.6), coated at 100 µ1 well -1 of ELISA plate and incubated overnight at 4 °C. The plate was washed three times with PBS Tween 20 (PBS-T). Bovine serum albumin (2%) was added to block nonspecific reaction and the

Among naturally infected seeds (spotted), 40% of unsterilized seeds and 36% of surface-sterilized seeds yielded R. solani (see table). This indicates that in naturally infested seeds, the fungus was both externally and internally seedborne. The data for artificially inoculated and apparently healthy seeds were 100 and 15, and 17 and 0, respectively. Isolation of the fungus from some unsterilized apparently healthy seeds is probably due to surface contamination. None of the apparently healthy seeds showed seedling infection. Seedling infection was higher in artificially

inoculated unsterilized seeds. Surface sterilization resulted in reduced seedling infection, indicating that the fungus in artificially inoculated seeds was externally borne.

Infection of seedlings grown from R. solani -infected seeds had considerable effect on seedling health. This was indicated by the much higher fresh and dry weight of the seedlings grown from healthy seeds as compared with those from naturally infected seeds (both unsterilized and surface-sterilized) and artificially inoculated unsterilized ones.

Detection by indirect ELISA of rice tungro-associated viruses in rice plants following AVP application and RTBV and RTSV inoculation. Coimbatore, India. a

Plants Absorbance at 405 nm infected

Treatment (no.)/plants RTSV RTBV inoculated

(no.) Infected Uninfected Infected Uninfected

RTV-inoculated (control) 24/25 1.111-1.525 0.060 1.200-1.600 –0.025 AVP-treated and RTV-inoculated

10% Pigeonpea AVP 4/25 0.860-0.939 0.069-0.125 0.831-0.945 0.068-0.120 10% Mungbean AVP 5/25 0.550-0.925 0.070-0.090 0.745-0.940 0.070-0.085

Healthy (ELISA check) 0.070-0.152 0.075-0.150

tungro spherical virus, RTBV = rice tungro bacilliform virus. a RTV = rice tungro virus, AVP = antiviral principles, ELISA = enzyme-linked immunosorbent assay, RTSV = rice

plate was washed with PBS-T. The rabbit I g G antiserum specific to RTBV and RTSV was diluted 1:100 with PBS-T buffer and incubated for 90 min at 37 °C, then washed three times with PBS-T. The alkaline phosphatase conjugated goat antirabbit was diluted 1:7,000, placed in the well, and incubated for 1 h at 37 °C. The plate was then washed five times with PBS-T.

Freshly prepared substrate solution containing P-nitrophenyl phosphate (PNP) (1 mg ml -1 in 10% diethanolamine buffer) was added and incubated at room temperature for 1 h. The reaction was stopped with 3 M NaOH. Absorbance at 405 nm was recorded using the ELISA reader. Absorbance values greater than 0.152 (maximum value for healthy plants) indicate a positive reaction. The ELISA test

Application of PP-AVP and MB-AVP was repeated.

reduced virus infection to 16 and 20%,

respectively (untreated control has 96%

detected in plants that did not show any infection). RTBV and RTSV could not be

visible symptoms, indicating their absence in AVP-treated plants. But the presence of RTSV and RTBV was detected by ELISA in all plants showing infection symptoms.

Applying PP-AVP protected the plants from both RTSV and RTBV infection, shown by the reduced percentage of infection in AVP-treated plants. The concentration of both viruses was reduced in AVP-treated plants, even though they are infected by the viruses, indicating that AVPs may interfere with the replication of rice tungro-associated viruses (see table). The AVPs do not appear to have any effect on leafhopper feeding.

Studies on the characterization of AVPs and molecular biology of resistance induced by AVPs in rice are in progress.


- -

Hibernation sites of the rice stalk Average number of bugs on some plant species. stink bug Tibraca limbatiwentris in 94. the central region of Rio Grande do SUI, Brazil

Santa Maria, Rio Grande do SUI, Brazil, 1992- Agudo, and three in Santa Maria. In 1993, 20 fallow fields in Santa Maria were visited.

Species Bushes Bugs Standard Range Fields that were flooded after harvest or (no.) bush -1 deviation were not infested during the growing

D. Link, J. G. Naibo, and J. P. Pelentir, Departamento de Defesa Fitossanitaria, Centro de Ciencias Rurais-UFSM, 97119- 900, Santa Maria-RS, Brazil

We surveyed the preferential sites where the rice stalk stink bug (RSSB) Tibraca limbativentris (Hemiptera: Pentatomidae), an important irrigated rice pest in southern Brazil, hibernates between growing seasons. Visual observation of densely massed plants was made. The insects were counted in each plant located 2.5-100 m away from field borders.

In 1992, we surveyed fallow fields in three counties: five in Sao Sepe, two in

(no.)

1992-93 Eryngium 330 33.99 0.86

Erianthus sp. b 330 14.00 1.57 1-101 Andropogon 330 9.17 0.69 1-24

Paspalum 330 5.73 0.93 1-26

Baccharis 330 2.20 1.20 1-10

Panicum 330 2.14 0.70 1-3

Senecio 330 1.10 1.15 1-5

eburneum a 5-100

lateralis b

urvillei b

trimera c

prionites b

brasiliensis c

1993-94 Andropogon 600 11.58 1.22

Schizachyrium 600 6.89 0.71 1-30

Eryngium 600 5.02 1.01 1-29

Paspalum 600 2.86 0.69 3-23

lateralis b

microstachyum b

eburneum a

urvillei

1-100

a Apiaceae (=Umbelliferae), b Poaceae (=Gramlneae), c Asteraceae (=Compositae).

season were not included. Higher RSSB populations were ob-

served on the bigger and more compact clusters, which provide an ideal refuge for both adult and young forms.

The bugs were near the soil surface. where air moisture is higher. More than 90% of the hibernating forms were located up to 10 m from the field border. The distribution of the hibernating forms of RSSB was observed at 2.5, 5.0, 7.5, 10, 15, 25, 50, and 100 m from the field border. For each site, 10 samples were collected from a 2-m 2 area. More than 40 plant species were found to shelter RSSB, but the last four species listed were preferred by the bugs (see table).

Predicting the effects of Echino- chloa crus-galli on rice ( Oryza sativa L.) using the ecophysiological model INTERCOM

F. J. Abamu, IRRI

Weed research is now focused on finding alternative methods to control weeds as well as on understanding new concepts that underlie weed management. To support this new approach, tools that can be used to understand the complexities of crop-weed relations and to predict weed effects are important.

Between 1994 and 1995, we conducted field experiments and simulation studies at IRRI to evaluate the ability of an ecophysiological crop-weed competition model (INTERCOM) to simulate the effects on rice ( Oryza sativa L.) of competition from different densities of a weed ( Echinochloa crus-galli L. Beauv), emerging at different times, and allowed to grow with the crop for the rest of the season. A split-plot expe-

rimental design was used. Weed density and time of weed emergence/flush were the treatments.

There were three weed density levels — 0, 20, and 40 plants m -2 (designated as weed- free check, low, and high densities, respectively). All weed populations were introduced artificially. To imitate natural conditions in which weeds emerge in different flushes, the weed density treatments were controlled in such a way that weeds emerge at two separate times in the ricefield. These were 2-5 d after rice transplanting (DAT) (early emergence) and 12-15 DAT (late emergence). Nitrogen was applied at a rate of 150 kg ha -1 .

Simulation runs were made with weather data of 6 yr, including the actual year of experimentation.

Results showed that time of weed emergence was a more critical factor than weed density. Competition from weeds that emerged late (both high and low densities) did not have significant (P>0.05) effects on rice. This is because the cultivar used (IR72)


closed its canopy at about 25-30 DAT, thereby increasing the shade over the late- emerging weeds and limiting the light capture ability of this set of weeds for the benefit of the rice crop. It has been reported that the growth, development, and competi- tiveness of the weeds (Echinochloa) are adversely affected by shade.

height did not significantly (P>0.05) re- spond to competition from both high and low weed densities, whereas there were significant (P<0.05) reductions in rice leaf area index (LAI) from maximum tillering. The model comparison for plant height prediction showed that INTERCOM adequately simulated rice height from transplanting to maturity. Simulated and observed heights at maturity did not differ significantly (P>0.05). The pattern of simulated LAI development in time was also similar to that observed, but deviation of the observed from the simulated was significant (P>0.05) around maximum LAI. LAI was underpredicted at this growth stage in most

When the seeds emerged early, rice plant

Integrated pest management—insects

Integrated pest management—weeds

treatments, suggesting a need to further evaluate the model's ability to predict LAI.

Dry matter production and grain yield (Fig. 1) were significantly (P<0.05) reduced by competition from early-emerging weeds. Reduction was greater at high than at low weed density. Simulated and ob-served grain yields across all weed densities and time of weed emergence treatment combinations were highly correlated (R=0.97**)

2. Comparison between simulated and observed dry matter of rice from transplanting to maturity. In the legend, ds and ws = crop seasons (dry and wet), H and L = weed densities (high [40], correlated (R=0.92**) (Fig. 2). and low [20] plants m -2 ) 1 and 2 = time of weed INTERCOM can provide insights into

1. Response of observed and simulated grain emergence (1 = early [2-5 DAT], 2 = late [12-15 the effects of Echinochloa competition on yield of rice to competition from different DAT]). IRRI, 1994-95. rice when the crop receives adequate N, densities of weeds ( Echinochloa crus-galli ) irrespective of time of weed flush or weed emerging at different times. IRRI, 1994-95. density.

Simulated and observed dry matter content from transplanting to maturity were also

Weed control in direct seeded puddled rice

P. S. Bisht, P. C. Pandey, and P. Lal, Agron- omy Department, G. B. Pant University of Agriculture and Technology, Pantnagar, Nainital 263145, India

Weed control in direct seeded rice is a Butachlor, pendimethalin, and thio- serious problem in northern India. Manual bencarb are the recommended herbicides, weeding controls weeds effectively but but at least one additional hand weeding is availability and cost of labor limit its adop- required with these. The use of safener, an tion. There are safe and nonphytotoxic antiphytotoxicity compound that eliminates herbicides, but their effectiveness has not the toxic effect of herbicides on rice yet been evaluated. seedlings, offers opportunities to control

Effect of herbicides and cultural methods on grain yield, panicles m -2 , panicle weight, and weed dry weight in direct-seeded puddled rice. Pantnagar, India, 1993-94.

1993 1994 Time of Dose Concen-

(DAS) a yield dry yield dry Treatment application (kg ai ha -1 ) tration Grain Panicles Weed Grain Panicles Weed

(t ha -1 ) No. m -2 Weight weight (t ha -1 ) No. m -2 Weight weight (g) (g m -2 ) (g) (g m -2 )

Weed-free (check) 5.3 475 1.42 10 5.9 468 1.05 35 Hand weeding twice 20 and 40 - 5.1 357 1.66 91 5.4 486 1.18 175 High plant population 20 (1993) 2.5 432 0.98 247 (125 kg seed ha -1 ) + 1 hand 30 (1994) - 5.4 562 0.93 87

Pretilachlor + safener 3 0.4 30 EC b 3.7 392 1.23 305 3.7 512 0.99 563 Pretilachlor + safener 6 0.4 30 EC 2.6 378 0.93 430 Pretilachlor + safener 3 0.6 30 EC 4.7 400 1.06 225 4.4 463 1.06 304 Pretilachlor + safener 6 0.6 30 EC 4.6 385 1.39 267 Butachlor + safener 3 1.0 50 EC 3.4 322 1.22 279 3.4 484 0.87 678 Butachlor + safener 6 1.0 50 EC 3.0 358 1.08 394 Butachlor + safener 3 1.5 50 EC 3.6 373 1.07 422 4.1 4 98 0.89 357 Butachlor + safener 6 1.5 50 EC 4.0 355 1.16 404 Butachlor 3 1.0 50 EC 3.8 440 0.90 376 Butachlor 3 1.5 50 EC 3.4 389 1.30 305 3.4 425 0.85 444 Butachlor 6 1.5 50 EC 3.0 429 1.13 373 Anilophos 3 0.3 30 EC - 1.5 244 0.69 601 Anilophos 3 0.4 30 EC - 0.9 141 1.31 960 Anilophos 8 0.4 30 EC 3.7 391 1.15 360 3.5 355 1.05 356 Thiobencarb 3 1.0 50 EC 2.2 250 1.15 607 Thiobencarb 3 1.5 50 EC 1.9 202 1.06 678 Weedy (nonweeded control) 1.2 252 0.99 473 0.8 150 0.78 935

LSD (5%) 1.1 97 ns 200 1.5 150 0.34 213

weeding

CV (%) 21.0 18 26.00 46 23.0 28 24.00 31

a DAS = days after seeding. b EC = emulsifiable concentrate.


- - -

- - - - -

- - - -

- - - - - -

- - - - - - - -

- -

- - - -

- - - -

- - - -

- - - -

- - - -

- - - -

and did not differ significantly (P>0.05).

The experiment used a randomized block design and was conducted in the IARI farm during the 1995 summer season. The beds were infested with moderate to heavy population of plant parasitic nematodes. mostly root-knot nematodes Meloidogye spp. Tillage was achieved by digging to a depth of about 10 cm, followed by light irrigation. Mulching was done with 60 µm- thick, transparent, white polyethylene sheet buried on all sides. Daily temperature was recorded for each treatment at 0800 and 1500 h for the entire 3-wk duration of solarization and organic amendment treatments. The organic amendment treatment consisted of incorporating mustard cake (0.5% w/w) into the soil 15 d prior to sowing of rice seeds.

At the end of the solarization treatment, polyethylene mulch was removed and soil samples at 0-15 cm depth were drawn. The modified sieving and decanting method by Cobb was used to extract nematodes from 150 cm 3 of soil sample.

The nursery beds were loosened and irrigated; seeds of rice cultivar Basmati 370 were then sown. Seedlings were harvested 30 d after sowing and data on plant growth

soil were recorded.

weeds in direct seeded rice more effectively.

A field experiment was conducted during the 1993-94 wet season at the Crop Research Centre, Pantnagar (29 °N, 79 °E, 244 m altitude). Soil is silt loam (Aquic Hapludoll) with pH 7.9, 1.1% organic C, 0.1 % total N, and CEC 20 meq 100 g -1 soil. The treatments (see table) were tested in a randomized block design with four replications. Short-duration variety Govind (90 d) was used in 1993 and Saket 4 (110 d) was planted in 1994. Pregeminated seeds were uniformly broadcast in puddled soil at 100 kg dry seeds ha -1 on 10 Jul l993 and 2 Jul 1994. A single dose of 18 kg P ha -1 and 33.2 kg K ha -1 was applied along with three splits of 120 kg N ha -1 (1/2 as basal dressing, 1/4 at tillering, and l/4 at panicle initiation). Soil was kept near saturation during seedling establishment and was later flooded with 0-5 cm water.

Weed samples were taken from a 0.25- m2 area and were dried at 70 ± 5 °C. Dry weights were recorded. The common weeds found were Echinochloa colona, Panicum sp., Digitaria sp., Cyperus iria, Cyperus difformis, Ischaemum rugosum, Commelina henghalensis, Casulia axillaris, and Eclipta prostrata. Grain yield was recorded from a 6-m 2 net plot area. Plant samples from 0.5 m 2 were taken at harvest to evaluate yield components.

Phytotoxicity problems were not observed in treatments with the new herbicide formulation (herbicides with safener). Weeds were controlled effectively in both years. Pretilachlor + safener at 0.6 kg ai ha -1

applied 3 d after sowing was found most effective in controlling weeds. Yield was statistically at par with that of weed-free check and that of the crop that received two hand weedings. Compared with butachlor- and thiobencarb-treated plots, high plant density with one manual weeding at 30 d was found to suppress weeds significantly (see table).


Effect of soil solarization of rice nursery beds to suppress plant parasitic nematodes

A. K. Ganguly, Pankaj, and A. Sirohi, Nematology Division, Indian Agricultural Research Institute (IARI), New Delhi 110012, India

An advantage of solarizing nursery beds, aside from a favorable cost-benefit ratio, is its potential to suppress other soilborne pests besides the target pest. We explored the possibility of using this nonchemical method to suppress plant parasitic nematodes that infect rice seedlings.

Table 1. Effect of solarization and organic amendments on growth characters of rice seedlings. New Delhi, India.

Length (cm) Weight [g)

Shoot Root Shoot Root Treatment

Control 26.20 6.70 0.42 0.165

Solarization 39.30 13.80 1.22 0.631

Organic 38.40 14.80 1.39 0.644 amendment

SE ± 1.31 0.78 0.11 ns

Table 2. Effect of solarization and organic amendment on population of nematodes infecting rice seedlings. New Delhi, India.

Nematode population 250 cm 3 of soil Percentage Nematode species Increase/

Pre-treatment Post-treatment decrease a

Meloidogyne spp. Control 172 250 Solarization 115 15 Organic amendment 116 33

Helicotylenchus spp. Control Solarization Organic amendment

13 33 71 30 41 23

45.3 (+) 87.0 (-) 71.5 (-)

Control Tylenchorhynchus spp.

23 Solarization 61 Organic amendment 33

Pratylenchus spp. Control Solarization Organic amendment

26 37 14

40 25 22

45 18 7

153.8 (-)

43.9 (-) 57.7 (-)

74.0 (+) 59.0 (-) 33.3 (-)

73.0 (+) 51.3 (-) 50.0 (-)

Aphelenchus avenae Control 105 139 32.4 (+) Solarization 56 13 Organic amendment 78 57 27.0 (-)

a + = increase; - = decrease.

76.8 (-)

characters and nematode population in the LSD (5%) 2.75 1.65 0.23 ns

Integrated pest management—other pests

Grain yield of rice as influenced by supplemental irrigation management. a Karnataka, India. 1992 and 1994.

Grain yield (t ha -1 ) % increase Treatment over rainfed

1992 1994 Mean treatment

Rainfed Irrigation at booting Irrigation at flowering Irrigation at grain filling Irrigation at booting and flowering Irrigation at flowering and grain filling Irrigation at booting and grain filling Irrigation at booting, flowering, and grain filling Daily irrigation from booting up to 10 d before harvest

LSD (0.05) 1.3 ns b

3.8 5.4 4.6 5.3 5.4 5.4 4.4 4.9 4.7 3.9 5.6 4.8 5.4 5.7 5.5 4.2 5.9 5.0 5.4 5.0 5.2 5.5 5.2 5.3 5.2 5.3 5.3

- 17

1 3

20 9

16 16 14

a Water depth applied at each irrigation: 5 cm. b ns = not significant.

We investigated rice yield under sprinkler irrigation during three growing seasons 1991-93 in Turkey. The experiment was laid out in a split plot design with three replications. Main plots were two irrigation intervals: 4d (A 1 ) and 8d (A 2 ). The subplots were irrigation depths (or amount): same amount of pan evaporation (B1), 50% more than pan evaporation (B2), and 100% more than pan evaporation (B3). Plots were 12 m 2 .

Seeds of rice variety Ergene (120 d) were drill-seeded in early May at I80 kg ha -1

using 25-cm row spacing. N (150 kg ha -1 ) was applied in two splits—l/2 basal and 1/2 60 d after planting. P (35.2 kg ha -1 ) was applied during soil preparation. Weeds were controlled with two herbicide applications, before drilling and 40 d after drilling. Soil was sandy clay silt with 1.3% organic matter and pH 6.5.

All treatments received similar amounts of irrigation water until seedling emergence to ensure adequate stand establishment. Water use efficiency (WUE) was calculated by dividing grain yield (kg ha -1 ) by total amount of water applied (mm) for the whole crop.

Irrigation interval has no significant effect on yield (see table). No interaction between irrigation interval and irrigation depth was observed. However, irrigation water depth influenced yield significantly.

As the amount of sprinkler irrigation water decreased, the yield decreased. B3 had the highest yield while B2 gave the highest WUE.

Polyethylene mulching increased mean maximum temperature at all observation times (data not shown). An increase of 7- 13 °C in the mulched bed over the non- mulched bed was observed during the 3-wk

With regard to plant growth parameters, no Good control of plant parasitic nema- significant differences were observed be- todes (50-87% reduction) was achieved in tween treatments. The improved growth of the solarized beds (Table 3). The decrease seedlings in the solarized bed could have in root-knot nematode populations was been due to the good control of plant most notable. Solarization controlled

period of solarization. Mulching signifi- parasitic nematodes, other soilborne nematodes better than did organic cantly increased seedling growth (Table 1). pathogens, and weeds. amendment.

Supplemental irrigation for dry seeded upland rice

V. V. Angadi, Agricultural Research Station, Sankeshwar 591314, Belgaum; and P. N. Imapathy, Main Research Station, University of Agricultural Sciences, Dharwad 580005, Karnataka, India

Upland rice in Karnataka is often exposed to moisture stress due to dry spells that coincide with the critical growth stages of the crop. Supplemental irrigation using water from small tanks found all over the rainfed rice tract may increase rice yield.

We studied the effect of supplemental irrigation at various growth stapes on grain yield of rice during the 1992 and 1994 wet seasons. The experiment consisted of rainfed rice and rice grown with supplemental irrigation (5 cm water depth) at booting, flowering, and grain-filling stages and their combination (see table for treatments). Daily irrigation from booting to 10 d before harvest was also one of the treatments. Due to high percolation in the unpuddled soil, the fields were flooded only for several hours after irrigation or heavy rainfall.

The 5.0- × 3.4-m plots were laid out in a randomized complete block design with three replications. Soil was silty clay loam with 0.4% slope. Plots were separated by well-compacted bunds and buffer channels.

Land was plowed and harrowed and direct seeded with rice cultivar Avinash (140 d) at the onset of the rains. A common fertilizer dose (100 kg N, 22 kg P, and 41 kg K ha -1 ) was applied to all plots.

Total rainfall was 1150 in 1992 and 1135 mm in I994 with 75 and 79 rainy days during the respective cropping season. Except for a dry spell of 11 d before booting in 1992, good rainfall was received during other stages in both years.

Irrigation at booting, alone or in combination with other stages, relieved the stress caused by the dry spell in 1992 and significantly increased yields. Irrigation only at flowering and grain-filling stages in 1992 and all irrigation treatments in 1994 did not increase yield. This was attributed to suffi cient rainfall in 1994. During flowering and grain filling in 1992, plants were not stressed, even without irrigation.

Best results are obtained when timely supplemental irrigation is given only when dry spells occur during critical stages (booting, flowering, and grain filling).

Rice yield under sprinkler irrigation

H. Sürek, H. Aydin, Thrace Agricultural Research Institute (TARI), Edirne, Turkey; R. Cakir, H. Karaata, Atatürk Soil and Irri- gation Research Institute (ASIRI), Kirklareli, Turkey; M. Negis and H. Kusku, TARI


Water management

,

Average yield of Ergene under flooded Sprinkler irrigation can be thus used to irrigation conditions (2000 mm water increase WUE in conditions where water is application) was about 6 t ha -1 . With the B3 scarce. Extra investment is, however. treatment (1172 mm water application), needed to install and operate the system. yield was 4.7 t ha -1 .

Yield of rice under sprinkler irrigation. Edirne, Turkey. 1991-93.

Grain yield (t ha -1 )

1991 1992 1993 Mean

A1B1 2.7 d 1.8 b 2.2 c 2.3 c 4 d A1B2 5.5 ab 3.2 abc 3.2 b 3.9 b

A1B3 5.9 a 3.6 ab 4.8 a 4.7 a

Treatment a Water applied (mm)

1991 1992 1993

641 681 804 874 944 1004

1107 1206 1203

Water use efficiency (kg ha -1 mm -1 )

Mean 1991 1992 1993 Mean

709 4.4 2.7 2.7 940

3.2 6.3

1172 3.3 3.1 4.2

5.3 3.0 4.0 4.0

A2B1 3.7 cd 1.6 c 2.4 bc 2.5 c 641 681 804 709 8 d A2B2 4.6 bc 3.5 ab 4.4 a 4.2 ab 874 944 1004

5.7 2.3 2.9 3.6

A2B3 4.8 ab 940

4.4 a 4.8 a 4.7 a 1107 1206 1203 1172 5.3 3.7 4.4 4.4 4.4 3.6 3.9 4.0

F values A 0.87 0.35 14.62 0.43 B 24.36** b 9.28** 49.67** 52.59**

A×B 4.18 0.50 3.77 0.32 CD (0.05) 1.07 1.76 0.82 0.66 CV (%) 12.49 18.06 12.03 16.55

at 1% level. a A: Irrigation interval: A1 = 4 d; A2 = 8 d. B: lrrigation depth (amount): B1 = same as pan evaporation; B2 = 50% of pan evaporation, B3 = 100% of pan evaporation. b ** = significant

Effect of different rice cultures on crop yield in rice-based systems

T. S. Verma and P. K. Sharma, Soil Science Department, Himachal Pradesh Agricultural University (HPAU), Palampur 176062, Himachal Pradesh, India

We investigated the performance of three rice cultures—(i) irrigated lowland (IL, rice seedlings transplanted in puddled soil under irrigated conditions); (ii) rainfed lowland (RL, crop established by dry seeding, followed by wet tillage at the 3-4 leaf stage under rainfed conditions [locally called halod]); and (iii) rainfed upland rice (DSR, rice dry seeded and raised as an upland crop under rainfed conditions)—and their subsequent effects on the yield of the

following upland crops of wheat ( Triticum aestivum ), linseed ( Linum usitatissinum ), and raya ( Brassica juncea ).

We conducted this experiment at the HPAU experimental farm in Palampur (32°6'N, 76°3'E, 1300 m altitude) during the 1993 and 1994 wet seasons. Soil was silty clay loam (Typic Hapludalf). Plots (5 × 3 m) were arranged in randomized complete block design with nine replications for rice and three replications for each upland crop. All crops received fertilizers at the recommended rates.

In both years, rice grain yield followed this trend: IL > RL > DSR. The differences were statistically significant. Grain yield of wheat and raya were highest in DSR and lowest in IL plots; linseed yield was not affected by the land preparation technique

Grain yield of crops (t ha-1) in rice-based cropping systems involving three rice cultures.

Rice Wheat Linseed Raya

1991 1992 1991 1992 1991 1992 1991 1992 Rice culture

Irrigated lowland 4.2 4.5 3.0 3.3 1.2 1.3 0.9 1.0 Rainfed lowland 3.3 3.5 3.6 3.9 1.4 1.4 1.3 1.4 Direct seeded rice 2.9 3.1 3.9 4.1 1.5 1.6 1.6 1.7

LSD (0.05) 0.3 0.3 0.3 0.2 ns ns 0.1 0.2

adopted in the preceding rice crop. The energy required to prepare the land

after rice harvest was significantly affected by the land preparation technique used in rice. Puddling is the most intensive tillage practice in rice, followed by halod and dry tillage. Manual land preparation with the use of spades required about 967 h ha -1 in IL, 633 h ha -1 in RL, and 483 h ha -1 in DSR. Considering that the energy of an adult worker equals 1.96 MJ human h -1 , land preparation after IL, RL, and DSR rice required 1895, 1241, and 947 MJ ha -1 , respectively. Thus, the energy required to prepare the seedbed after RL rice was about 2/3, and the energy needed for the crop after DSR was about 1/2 that of IL rice.

Rice in silty clay loam soil performed best under lowland irrigated conditions. But this rice culture did not favor the following crops of wheat and raya. They performed best after DSR. Linseed was unaffected by rice culture. Because wheat is next to rice in importance, appropriate tillage technology has to be developed to maximize crop yields in rice - wheat cropping systems.


Farming systems

A low-cost, in-store dryer for small- scale farmers

Pham Hieu Hien, Le Van Ban, and Bui Ngoc Hung, University of Agriculture and Forestry (UAF), Ho-Chi-Minh City, Vietnam

We developed a low-cost in-store dryer, called SRR-1, at UAF to meet the needs of small-scale farmers in Vietnam. They require a dryer with adequate capacity, high-quality dried grain. and reasonable investment and drying cost.

The design of the SRR-1 dryer is based on the principle of low-temperature in-store drying. The dryer consists of three components: a two-stage axial fan, an electric heater, and a bamboo mat drying bin (see figure).

The drying bin consists of two concen- tric bamboo mat cylinders, 0.4 and 1.5 m in diameter and 1.1 m high. The bin can contain 1 t of rough rice.

A 0.5-hp electric motor drives the fan. Two 350-mm-diameter rotors are mounted on both ends of the motor shaft inside a steel casing. (Plastic rotors are locally made and readily available in the market.) The fan is positioned on top of the inner bamboo mat cylinder; it can deliver 0.3 m 3 s -1 at 400 Pa static pressure. The heater is a 1000-W resistor from an electric stove; it is mounted beneath the lower rotor. Supplemental heat

Schematic of SRR-1 dryer.

from the resistor is sometimes used at night or during times of continuous rain.

During the day and the first two nights, the fan is turned on to aerate the rice. Sup- plementary heat is provided on the third night. In succeeding nights, the blower is turned off because the grain moisture content is low enough for temporary storage.

(wet basis), drying time is 80-85 h batch -1 , for which total power consumption is 80

At an initial moisture content of 26-28%

kWh batch -1 . The dryer does not require much, and its manufacturing cost is less than US$l00. (SRR stands for “very cheap dryer” in Vietnamese). The aired grain quality from SRR is outstanding. Moisture gradients are less than 1 %. and head rice recovery is increased by at least 2% when compared with sun drying—and the weather risk is eliminated. After drying, the

rice is stored in the drying bin and the fan is moved to the next bin.

Drying costs at UAF were $5 batch -1 , including dryer depreciation. The dryer is designed for farm-level use, mainly for drying and storing rough rice for household con-

wihtin 1 yr of introduction in Vietnam. Many sumption. More than 200 units were sold

farmers in southern Vietnam are enthusias- tically accepting the SRR-1 dryer.


Farm machinery

Research methodology A simple and rapid method for then dissolved in 10 µl of sterile water. To isolation of bacterial genomic DNA check the viability of the ethanol-treated

M. L. C. George, V. Quinto, M. Villamayor, the residual alcohol, and the whole sus- and R. J. Nelson, IRRI pension was plated.

cells, the pelleted cells were resuspended in

One µl of the DNA preparation was used The polymerase chain reaction (PCR) is as template in a 25-µl reaction volume. now an important tool for ecological studies Primers corresponding to conserved because of its simplicity and capacity to sequences in bacterial repetitive elements rapidly screen a large number of samples (REP1R-I, 5'IIIICGICGICATCIGGC 3' with a minimal amount of DNA. It is and REP2-I, 5'ICGICTTATCIGGCCTAC especially useful for studying the popula- 3') were used for all the strains under tion biology of plant pathogens, such as conditions prescribed for rep-PCR.

(which causes bacterial blight of rice), 0.5 µM of the primers JELl where massive sampling is required for (5'CTCAGGTCAGGTCGCC 3') and JEL2 analysis. While it is possible to obtain PCR (5'GCTCTACAATCGTCCGC 3') were fingerprints directly using whole bacterial used with 185 µM each of four dNTPs. cells, more consistent results are obtained approximately 2.5 units of Taq polymerase using good-quality DNA as a template. in an incubation buffer amended with 10% Genomic DNA is isolated in a multistep dimethylsulfoxide (v/v) and 7.5 µl of 1 M process, typically involving sodium Tris, pH 9.5. The reaction mixture was dodecyl sulfate-mediated cell lysis. DNA initially denatured for 1 min at 94 °C and extraction, alcohol precipitation, and then subjected to 30 cycles of PCR (10s ethanol wash. Because it is a lengthy denaturation at 94 °C, 1 min annealing at process, the isolation of genomic DNA is a 62 °C, and 10 min extension at 65 °C) and a bottleneck in large-scale application of final extension for 15 min at 65 °C using a PCR as a fingerprinting tool. Perkin Elmer Cetus DNA thermal cycler.

Here we report a simple alcohol lysis The PCR products were resolved in a gel procedure to isolate PCR-quality genomic containing 0.5% agarose and 0.75% DNA of bacterial cells. In this method, Synergel ™ , visualized by staining with ethanol functions both to lyse the cells and ethidium bromide and photographed using to precipitate the DNA. Cells from Xoo, X. Polaroid 667 film. o. pv. oryzicola, Pseudomonas blumei, Burkholderia glumae, Asorhizobium sp., and Escherichia coli plate cultures were collected using the wide end of a sterile toothpick (Fig. 1) and washed in 400 µl of water to remove the extracellular poly- saccharides. Cells were pelleted in a microcentrifuge by spinning for 2 min (12,400 rpm) and the still turbid supernatant was discarded. The cells were resuspended in the residual liquid, and 200 µl of 95% ethanol was added to a final concentration

Xanthomonas oryzae pv. oryzae (Xoo) Additionally for the Xanthomonas strains,

~

~

1. Cells collected using a sterile toothpick from of approximately 70% alcohol. After 10-30 a 3-d-old plate culture of Xanthomonas oryzae min, the suspension was spun for 30 s to pv. oryzae. DNA isolated from the cells by the pellet the cell debris, and the supernatant

pelleted by spinning for 5 min, dried, and PCR amplification. of water, and 1 µI was used as a template for was transferred to a new tube. DNA was alcohol lysis method was resuspended in 10 µI


With DNA prepared by this method as template for PCR, fingerprint patterns generally comparable with those obtained using DNA prepared by conventional means were obtained from 3-d-old, 1 -wk- old, and 5-wk-old plate cultures, and from 1-d-old broth culture (Fig. 2). Similar patterns were also obtained from cells stored in ethanol for 6 wk prior to DNA isolation.

The alcohol lysis method is simple and fast, requiring about 15 min, and produces enough DNA for several amplification reactions. It also renders cells nonviable and thus suitable for storage and transport and eventual PCR fingerprinting of pathogen sample ex situ.

I

2. PCR fingerprint patterns of Xanthomonas oryzae pv. oryzae strains PX0215 (A), PX0143 (B), PX01 (C) and PX0204 using template DNA prepared by the potassium acetate method (lanes 1, 6, 11, and 16), and by the alcohol lysis method using 3-d old (lanes 2, 7, 12, and 17), 1- wk-old (lanes 3, 8, 13, and 18), and 5-wk-old (lanes 4, 9, 14, and 19) plate cultures, and from 1-d-old broth cultures (lanes 5, 10, 15, and 19). The primers used in the PCR method were JEL1/JEL2. The DNA molecular size markers are on the lanes labeled M on the left (1-kb ladder) and right (BioMarker EXT).

A simulation model of rice sheath blight epidemics (I) Structure and model development

S. Savary, IRRI-Institute Franqais de recher- the scientifique pour le développement en coopération (ORSTOM) Joint Project on Rice Pest Characterization, IRRI: and L. Willocquet, ORSTOM

A systems model was developed to simulate rice sheath blight (ShB) epidemics over time. The model considers the two phases of ShB epidemics: mobilization of soilborne primary inoculum and secondary spread. The mechanism involved in secondary spread is a key feature of ShB epidemiology. Once infected, a tiller may in turn infect its neighbors via direct contact between leaves, which enable mycelial strands of the fungus to spread in the canopy. While the primary phase of epidemics is dependent on soilborne inoculum, the secondary phase involves leafborne inoculum and depends on crowding of canopy and climatic factors such as leaf wetness, light, and relative humidity for its multiplication.

The system under consideration in the model consists of a 1-m 2 rice crop. represented by a growing population of tillers. Tillers may belong to two categories: healthy or diseased. Two independent infection processes may lead to the accumulation of diseased tillers—primary infections generated from primary inoculum and secondary infections resulting from the progress of the pathogen in the crop canopy. The rate of change of infected tillers is thus represented by the sum of a rate of primary infection and of a rate of secondary infection: dNi/dt=(dNi/dt) p + (dNi/dt) s .

The rates of primary and secondary infections are described by a monomole- cular model and a modified logistic model: dNi/dt=r p P(1-(N/(N+Ni))+r s Ni(l-N/ (N+Ni)) a where N is the number of healthy tillers (per m 2 ); Ni is the number of infected tillers (per m 2 ); r p and r s are respectively intrinsic rates of primary and secondary infection; Pis the current amount of primary inoculum (per m 2 ); and a is a parameter for disease aggregation. The structure of the model is described in the flow chart of Figure 1.

1. A systems model for rice ShB epidemics. State variables are indicated by rectangles, flows of individuals by double arrows, parameters (estimated or computed) by circles, and numerical relationships by simple arrows. The intrinsic rate of primary inoculum decay under flooded conditions was derived from Roy (1986).

The term (1-N/(N+Ni)) is commonly the disease develops as a result of the short called correlation factor. It represents the range of dispersal of the pathogen. fractionof healthy tissues (tillers)—i.e.,

tillers available to infection. there is a correction factors for rates of primary andthe progressively declining proportion of materialized by a difference between plication therefore develops. In addition to underlying assumption of the model is additional constraint to disease multi- tissues available for infection. One major

In the course of an epidemic, an

secondary infection. and the introduction of limited number of tillers that actually are a new term, a, that reflects the limited accessible to infection from the diseased accessibility of available (healthy) tissues tillers. The parameter a of the correction for infection in the secondary phase. factor for secondary infection, (1 - (N/

inoculum is considered random due to field alternately, the limited accessibility of plowing, harrowing, and flooding, which tillers available for infection. results in a randomly distributed probability The amount ofprimary inoculum, P, also of each healthy tiller being infected by varies over time, as result of the flooding of soilborne inoculum. This assumption is the soil on which the crop is grown. Varia- translated in the model by the use of a tion of P is assumed to follow an exponen- correction factor (COFR) in this simplest tial decay: dP/dt=k P. form for the rate of primary infection: Some additional features were added to (1 - (N/N+Ni)). the model and are summarized in Figure 1:

When disease becomes established in 1) the size of the tiller population is made the crop and progresses within the canopy. variable over time, reflecting crop growth; a however, the typical aggregated structure of logistic increase (with relative growth rate

The distribution of primary, soilborne N+Ni)) a , represents disease aggregation, or


A series of simulation runs using a sheath blight simulation model, with varying values of three parameters: r p (a), r s (b), and a (c).

[RCRI) was assumed to adequately represent total tiller growth; 2) tillers may recover from infection, and a rate of disease recovery (Recov) was included; 3) severe infection on individual tillers may lead to their death, and a rate of tiller death (Rmort) was included; 4) tiller senescence (Tsen) was also included, and represented as an exponential decay function. This series of equations was linked together and integrated using a 1-d time-step.

Empirical values were derived for the rates of senescence, mortality, and recovery from field experiments at IRRI. An intrinsic rate of primary inoculum decay was derived from published data (k = -0.11; Roy [1986]). Data from field experiments (Gou et al 1983) were then used as a basis for model verification, and to estimate the three parameters of the equation for the rate of infection: r p , r s , and a. This was achieved

A simulation model of rice sheath blight epidemics (II) Model per formance derived from sensitivity analysis

S. Savary, IRRI-ORSTOM Joint Project on Rice Pest Characterization, IRRI; and L. Willocquet, ORSTOM

A preliminary simulation model of rice sheath blight (ShB) epidemics was developed, which incorporates a few important characteristics of the disease: a rate of primary infection (which is proportional to the amount of primary, soilborne inoculum, the amount of healthy tissues [tillers] available for new infection, and an intrinsic rate of primary infection, r p ) and a rate of secondary infection (which is proportional to the amount of infected tissues (tillers) and an intrinsic rate of secondary infection, rs). The rate of secondary infection also depends on the amount of tissues still available for infection and on an aggregation parameter, a.

This model addresses a few research issues, such as 1) the relative effect of variation of parameter values on the behavior of the model; 2) the contribution

, ,

2. Comparison of observed (dots) data and simulated (line) ShB incidence. Observed data were derived from Gou et al (1983).

using disease incidence (i.e.. Ni/(N+Ni) as a synthetic variable representing the processes underlying a ShB epidemic, and the DUD interactive technique of the NLIN procedure of the statistical package SAS. Using this technique, parameter estimates were r p = 0.04, r s = 0.09, and a = 1.77.

The results of the simulation indicate that this system model has the potential of adequately describing ShB epidemics, and may serve as a basis for further improvement (Figure 2). Experiments are currently under way at IRRI to further assess the performance of the model and to better document r s in terms of climatic factors and contact frequency among tillers in a rice crop.

Cited references Gou FS, Li XQ, Xu CL. 1983. Study on the

spatial distribution patterns of the rice sheath blight plant in rice field and its practical amplications. Acta Phytopathol. Sin. 13:77- 34.

Roy AK. 1986. Survival of sclerotia of Rhizoctonia solani F. sp. sasakii in relation to moisture regime of soil. Indian Phytopathol. 39:259-263.

of primary inoculum, relative to that of secondary inoculum, resulting in spread of the disease within the rice crop canopy; and 3) the magnitude of the effect of disease

The figure shows a series of scenarios where the three parameters were independently varied, using optimized parameter values (r p =0.04, r s = 0.09, and a = 1.77) as references. A series of ShB incidence progress curves were generated by increasing or decreasing r p and r s by 25% or 50% of their initial values. Because a cannot, by definition, be smaller than 1, the following values were tested: 1 (random distribution of disease throughout the epidemic), 1.39, 1.77 (optimized value). 2.16, and 2.54.

Variation in r p (Fig. a) has a small effect on the shape of disease epidemics, a decrease of r p resulting primarily in a delay of the epidemics with the same speed. Variation in r s (Fig. b) has strong effects on both the slopes of the curves and the termi-

aggregation on disease spread.

nal incidences. As expected, variation in a (Fig. c) affects the shape of curves in a later stage of disease epidemic—the larger the a value, the stronger the disease aggregation. the lower the accessibility of healthy tillers

~


to diseases, and the slower the epidemic when it enters the polycyclic phase.

This behavior of the model indicates that

variation in r s so as to reflect variation of of a cropping season should prove effective. environment under which ShB epidemics It also indicates a comparatively high develop. This suggests that the polycyclic sensitivity of the model to variation in the

it is sensitive to variation in r s . Future nature of ShB must be considered a key aggregation parameter, and experiments are development of the model must consider characteristic of the disease for its manage- necessary for its estimation. the effect of time-dependent factors on ment, and that measures taken in the course

Use of CERES-Rice model to assess potential yield

G. S. L. H. V. Prasada Rao and N. Subash, Regional Agricultural Research Station (RARS), Pilicode 671353, Kasaragod District, Kerala, India

We conducted a field experiment at RARS, Pilicode (1 2° 12' N; 75° 10' E; 2755 mm rainfall, 4.4 h of bright sunshine d -1 ) to test the CERES-Rice model developed under the IBSNAT Project. Soils were well- drained sandy loam of lateritic origin with pH 5.5. The model was tested for five cropping seasons using different dates of transplanting during 1993 and 1994 kharif and 1992-93, 1993-94, and 1994-95 rabi. Popular varieties Jaya (120- 125 d), Jyothi (110-125d), and Triveni (95-l05d) were used.

The simulated grain yield through the CERES-Rice model was in good agreement with the observed yield during 1993 kharif while simulated grain yield was higher than that of the observed during 1994 kharif on all transplanting dates (Figs. a and b). Heavy rains during I994 kharif brought floods. Incidence of gall midge and rice bug was also high at the time, leading to a high percentage of grain chaffing (see table).

for a floodwater management scheme and takes into account bund height as well as floodwater maintained through irrigation at three different early stages of crop growth. The actual rabi crop at the field site was in a totally different condition from that mentioned previously. However, it should be noted that the CERES-Rice model simulated higher grain when run during rabi in all three test varieties on all transplanting dates.

This study reveals that the CERES-Rice model needs validation for rabi while it could very well be used during kharif to assess potential grain yields based on simple daily weather variables.

The CERES-Rice model during rabi runs

a. Actual and simulated grain yield (t ha -1 ) of rice varieties on different dates of transplanting rice varieties on different dates of transplanting

during 1993 kharif.

b. Actual and simulated grain yield (t ha -1 ) of

during 1994 kharif.

Percentage of grain chaffing of three test varieties during 1994 kharif on different dates of transplanting. Kerala, India, 1992-95.

Jaya Jyothi Triveni Date of transplanting Good grains Chaffing Good grains Chaffing Good grains Chaffing

(%) (%) (%) (%) (%) (%)

8 Jun 68.7 31.3 81.1 18.9 90.3 9.7 15 Jun 82.7 17.3 83.6 16.4 88.7 11.3 22 Jun 75.2 24.8 71.6 28.4 82.8 17.2 29 Jun 67.9 32.1 63.0 37.0 84.0 16.0

6 Jul 58.3 41.7 65.0 35.0 64.2 35.8

Av 70.6 29.4 72.9 27.1 82.0 18.0

Environment Effect of contour hedgerow on Rice (variety 9991 in 1993 and Heera in runoff, soil loss, and upland rice 1994) was dry-seeded in rows with 20-cm production spacing along contours. Hedgerows sepa-

C. R. Subudhi, P. C. Pradhan, and P. C. 1993 and 7 in 1994) arranged in random- Senapati, Dry Land Agricultural Research ized complete block with three replications Project (DLAP), Orissa University of Agri- were the vegetation used to form the

Orissa, India The hedgerows were planted with 1-yr- old grass seedlings (except for stylo where

Most of the hilly areas in Orissa lose a lot of seeds were sown) 1 mo before the experi- soil during the wet season. Soil loss and ment. The distance between two runoff, as affected by different contour hedgerows was 8 m. Plots were 25 m × 3 m. hedgerows, were quantified and their Lengthwise, each plot comprised three effects on upland rice production deter- terraces separated by two hedgerows.

The study was conducted at DLAP sites tislot divisor. Runoff collected by one slot in Bhawanipatna in 1993 and in Phulbani in is fed into in a drum installed at the end of 1994. Soil at both sites is sandy loam with the divisor. A sample of 250 ml was taken about 2% slope. from each drum after thorough stirring and

rated rice terraces. The treatments (9 in

culture and Technology, Phulbani 7602001, hedgerows (see table).

Daily runoff was monitored using a mul-


mined.

Runoff, soil loss, and crop yield during 1993-94 cropping seasons.

1993 Treatment

1994 Mean yield

Runoff Soil loss Crop Runoff Soil loss Crop (t ha -1 ) (mm) (t ha -1 ) yield (mm) (t ha -1 ) yield

(t ha -1 ) (t ha -1 )

T1 No hedgerows 184.1 3.5 1.2 204.0 10.4 1.3 1.3 T2 Cynodon dactylon hedgerow 154.9 3.0 1.0 172.2 5.5 1.7 1.4 T3 Pennisetum purpureum hedgerow 149.9 2.7 1.6 1.6 T4 Vetiveria zizanoidea hedgerow 142.4 2.2 2.1 151.7 4.2 2.0 2.1 T5 Eulaliopsis binata hedgerow 150.7 2.4 1.4 162.4 4.8 1.9 1.7 T6 Cymbopogon flexuosus hedgerow a 151.8 2.6 1.9 1.9 T7 Stylosanthes hamata hedgerow 156.3 2.8 1.3 172.6 5.8 1.8 1.6 T8 Hybrid napier hedgerow b 155.2 3.1 1.6 T9 Fallow land T10 Broadcasting of dry seed, no 235.7 11.1 1.0 1.01

hedgerow c

1.6 201.9 7.9 248.5 20.7

was evaporated to determine soil loss. The vetiver treatment (T4) produced the highest yield, having less runoff and less soil loss. Vetiver, with root penetration up to 1 m and consequently greater chances of survival than other grasses, was able to check the velocity of flow. Highest soil loss was observed in the fallow. Compared with leaving the land fallow (T9), 20% (1993) and 80% (1994) soil loss could be reduced by contour planting using vetiver.

Mean 160.8 3.3 1.5 191.0 8.9 1.5 1.6 LSD (0.01) 0.2 SE (m)± 0.1

a Total rainfall was 859 mm in 1993 and 1023 mm in 1994. b Done only in 1993. c Done only in 1994.

Recommendations Recommendations of the 3rd lnterna- tional Symposium on Hybrid Rice

The global requirement for rice by 2020 is expected to be around 800 million t compared with the current production of 520 million t. With shrinking resources—particularly arable land area, irrigation water, and energy—the only option left is to increase production. Increasing rice production by 300 million t during the next 25 yr will be a very challenging task. Of the possible genetic approaches to meet this challenge, hybrid rice technology is an immediate option since it has been a proven technology over the past 2 decades in China and is now showing commercial prospects in India.

The theme of the recent 3rd International Symposium on Hybrid Rice held in Hyde- rabad, India, 14-16 Nov 1996, was enhancement and sustenance of hybrid rice technology. The symposium was cospon- sored by the Indian Council of Agricultural Research (ICAR), the United Nations Development Programme (UNDP), and IRRI. About 150 Indian delegates and 50 international delegates from 20 countries addressed various aspects and issues for improving the technology and making it available outside of China. These issues were deliberated in six sessions (current scenario, increasing breeding efficiency


and enhancing yield heterosis, sustain- ability of hybrid rice technology, tissue culture and molecular approaches in heterosis breeding, toward true-breeding hybrids, and status of development and adoption of hybrid rice technology in various countries) and a meeting of the International Task Force on Hybrid Rice. During the 3 d of sessions, the following major recommendations emerged.

Technology development • Formation of a research network and

effective collaboration with international agencies have been the main reasons for the remarkable success achieved in India. Use these approaches as models for similar achievements in other countries of South and Southeast Asia.

• Intensify efforts for development of hybrid rice technology in countries, such as India, Philippines, Vietnam, and Indonesia, where there is a demand for hybrid rice seeds and the capability to produce them.

• To meet consumer preference, emphasize improving milling, head rice recovery, and other quality characteristics.

resistance to major pests and diseases in the promising parental lines.

• Especially emphasize incorporating

• To enhance the level of heterosis, the Chinese are successfully using two-line hybrids—photoperiod-sensitive gene male sterility (PGMS) and thermosensitive gene male sterility (TGMS)— and are developing hybrids through intersubspecific crossing of indicas and japonicas. Vigorously pursue these approaches in the tropics using indica/ tropical japonica lines. Countries that

the prospects of temperate japonica/ tropical japonica hybrids.

• For full exploitation of rice hybrids, develop special management practices that promote efficient use of nitrogen, phosphorus, and other nutrients and water.

• Improve parental lines through the use of random mating populations-the private sector, particularly, should pursue this to minimize dependence on the public sector for the supply of improved parental lines.

different ecosystems—especially for the

the irrigated ecosystem.

hybrids for the boro season (India, Bangladesh) and the spring season (Vietnam, Myanmar).

grow japonica rices should be exploring

• Develop rice hybrids adapted to

shallow lowlands, which are similar to

• Intensify research to develop rice

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Technology transfer • To speed up large-scale adoption, create

awareness and the demand for hybrid rice by conducting extensive on-farm trials, front-line demonstrations, and training programs.

• Expeditiously develop mechanisms for registration of parental lines so that they can be shared freely among collaborating countries.

Seed production The higher cost of hybrid seed is a constraint to adoption of the technology. The following measures are recommended to reduce prices to affordable levels. • Intensify studies on proper flowering

synchronization of parental lines to get higher seed yields in the target areas; low seed yield is a major problem faced by seed growers.

• Emphasize producing and supplying parental lines with only the highest purity; identify suitable agencies to perform this task.

• Strengthen the breeding of cytoplasmic male sterile (CMS) lines (possessing higher outcrossing potential) and restorers (providing high pollen load).

seasons that are most favorable for seed production.

(GA 3 ) and simultaneously intensify the search for cheaper alternatives.

• Develop special management practices to obtain higher seed yields.

• Wherever feasible, involve interested nongovernment organizations in producing hybrid rice seed.

• Encourage governments to develop policies that aggressively advocate private sector participation in hybrid rice seed production and research.

on hybrid seed production.

• Identify in each country locations/

• Economize the use of gibberellic acid

• Conduct mass-scale in-country training

Basic studies • Initiate intensive work to identify

molecular markers associated with quantitative trait loci for yield heterosis and subsequently incorporate these heterotic blocks into the parental lines.

• Speed up the work on apomixis as a long-term strategy by utilizing all

possible tools including genetic engineering.

International Task Force on Hybrid Rice (INTAFOHR)

proposed during the 2nd International Symposium on Hybrid Rice at IRRI in 1992, to promote the technology outside of China. Subsequently, this was endorsed by several countries in various international forums organized by FAO and IRRI. In October 1995, IRRI and FAO convened a joint meeting with country representatives and prospective donors (United Nations Development Programme, Asian Develop- ment Bank, and MAHYCO Research Foundation of India). The participants concluded that establishing INTAFOHR involving IRRI-FAO and the national agricultural research systems (NARSs) was an excellent idea and the prospective donors asked IRRI to submit a project profile for their consideration. After this was done, the donors asked IRRI to submit a detailed project proposal for possible funding. During the 3rd symposium. a panel discussion was held on 15 Nov 1996. During this discussion attended by the representatives of 16 countries, consensus was reached on the following points:

The establishment of INTAFOHR was first

Goal Improved food security and sustainable development through increased rice production using hybrid rice.

Objectives • To promote free exchange of registered

germplasm, information, and data from ongoing research and development programs on hybrid rice among interested partners.

• To strengthen national systems’ capabilities for applied and strategic research so that they can develop hybrid rice technology expeditiously.

• To intensify collaborative strategic research on hybrid rice by establishing effective regional/interregional collaboration on hybrid rice research and development.

• To assist member countries in formulation of appropriate policy incentives for hybrid rice development and use.

• To strengthen the hybrid seed industry and the linkage between it and the hybrid rice research centers.

Participation Participation in the Task Force is open to all interested countries.

Strategy General framework. Development of hybrid rice technology in member countries, particularly the charter members, will be expedited by 1) establishing goal- oriented hybrid rice research and development programs aiming to strengthen human resources for research and development of hybrid varieties and hybrid seed production capacity, 2) establishing collaborative research linkages, and 3) freely exchanging breeding materials and information.

Technology transfer will be expedited by strengthening on-farm testing and promo- ting the technology through appropriate channels and identifying policy inter- ventions and by respective governments encouraging investment in hybrid rice research and seed production.

Phasewise development. In recognition of the conditions governing rice production and the situations concerning research, development, and use of hybrid rice in different countries as well as the necessity to obtain time-bound outputs to sustain the activities of the Task Force, the Task Force will initially have three categories of membership: 1) charter (or core) members, 2) observers, and 3) affiliate members. All members will gradually become core members as conditions permit.

have the following features: • Rice is an important crop and planted on

a large area so that an economically viable local hybrid seed industry is possible.

• Hybrid rice research, development, and use have been adopted as national priorities.

• Staff and facilities are adequate to make effective and positive contributions to the Task Force activities, especially in terms of exchange of germplasm, information, and data. Bangladesh, India, Indonesia, Philip- pines, and Vietnam agreed to participate as

The charter member countries should


charter member countries. China and Sri collaboration with selected institutions that Lanka agreed in principle to serve as charter have advance programs concerning member countries but first need to get strategic research for hybrid rice approval from their respective govern- development. ments. Brazil, Colombia, Egypt, and FAO, having expertise in agricultural Thailand agreed to participate as observers. development, will provide guidance and Australia, Japan, and the USA agreed to technical backstopping for seed industry participate as affiliates. development and transfer of hybrid rice

Implementation. IRRI, possessing a technology in member countries. FAO will strong multidisciplinary hybrid rice also facilitate institutional linkage among research program linked with several agencies dealing with hybrid rice research NARSs, will be the executing agency that and seed production within and among coordinates Task Force activities— especially for technical assistance in the Member countries will designate matters of developing hybrid rice respective national coordinators to work technology and establishing necessary collaboratively with IRRI, FAO, and other

member countries.

member countries to implement the work plans as prepared and adopted in annual meetings of the Task Force.

a component of the Council for Collaborative Research in Asia (CORRA) and should be managed on the pattern of the rainfed and upland rice consortia already in operation at IRRI in collaboration with several NARSs.

Management. The Task Force should be

Erratum Replace leaf CHO with stem CHO in Figure 1 in “Leaf carbohydrate analysis—a simple method for integrating daily canopy photosynthesis,” IRRN 21:1, p. 52.


RICE PRODUCTION TRAINING MODULE

Stem borers of rice (RP4-18) INTERNATIONAL RICE RESEARCH INSTITUTE

1988 edition. 80 colored slides, one cassette tape, and two booklets (self-learning and self-testing)10.50x21.30 cm. Paperback. HDC US$254.00, LDC US$67.00 plus airmail (US$17.00) postage.

Stem borers of rice presents an overview of the life cycle of rice stem borers, the damage they cause, and control measures.

The lesson lasts for about 35 min. It will enable the user to

• describe and identify damage to rice caused by stem borers;

• identify and describe the five destructive stem borers of rice, and relate stem borer species with geographic distribution;

• identify and describe the four stages in the life cycle of the major stem borers including their appearance, habitat, behavior, mode of feeding, and duration of each life cycle; and

• describe the most effective methods of controlling the five major stem borers of rice.

Stem borers can damage rice plants from seeding to maturity. A knowledge of their life cycle and damage symptoms is important to implement effective control measures.


Morphology of the rice plant (RP4-01) INTERNATIONAL RICE RESEARCH INSTITUTE

1987 edition. 79 colored slides, one cassette tape, and two booklets (self-learning and self-testing) 10.50x21.30cm. Paperback. HDC US$254.00, LDC US$67.00 plus airmail (US$17.00) postage.

Morphology of the rice plant introduces the terms associated with the different parts of the rice plant.

The lesson lasts for about 20 min. It will enable the user to identify and describe the morphological characters or parts of the following:

• the germinating rice seed and seedling; • a rice tiller — roots, culm, and leaves; • the rice inflorescence or panicle. Understanding of standard terms describing rice

morphology is important to effectively communicate with other scientists about the rice plant.

IRRI INTERNATIONAL RICE RESEARCH INSTITUTE


Growth stages of the rice plant (RP4-02)

INTERNATIONAL RICE RESEARCH INSTITUTE

1988 edition. 80 colored slides, one cassette tape, and two booklets (self-learning and self-testing)10.50x21.30cm. Paperback. HDC US$254.00, LDC US$67.00 plus airmail (US$17.00) postage.

Growth stages of the rice plant relates and describes the various stages of growth of the rice plant from seed to maturity.

The lesson lasts for about 26 min. It will enable the user to

• recognize the three basic growth phases of the rice plant and the stages of development in each phase;

• identify the growth stages of a rice plant according to a 0-9 numerical scale (each number in the scale corresponds to a specific growth stage); and

• explain the specific physical changes in a growing rice plant.

It is essential to know the stages of the growth cycle of rice so that appropriate management practices can be employed at the right time. The effectiveness of management practices, such as fertilization and irrigation, and weed, pathogen, and insect control, largely depends on correct timing.



Documents

International Rice Research Notes Vol.21 No.2