Indian Journal of Experimental Biology Vol. 38, October 2000, pp. 1054-1057

Assessment of androc1onal variation in an indica rice PTB 28

Asit B. Mandai, T E Sheeja & Bidhan Roy

Biotechnology Section, Central Agricultural Research Institute, Port Blair 74410 I, India

Received 10 April 2000; revised 25 July 2000

Anther culture (AC) derived 160 doubled haploid (DH) families of an indica rice PTB 28 were field evaluated at Port Blair (Jat llu.4I ' 13.04/1N ; long 92°43'30.16/1E), to assess the extent and range of induced variability among the androclones ' at segregating A2 generation and identify promising selectants for genetic improvement. Though the coefficient of variation (CV) for plant height was least (2.65%), majority of the androclones were shorter (81.7%). CV was high in case of panicle number per plant (24.59%) and tiller number per plant (28.28%), while it was maximum in respect of yield per plant (37.0%). Tiller and panicle number per plant registered maximum reduction compared to parent, however, displayed larger variations in double haploid (DH) lines. Out of 160 DH families, only 10 (6.3%) lines out- yielded the parent. Extreme types with wide variations were encountered which reaffirms the possibility of employing anther culture in rice improvement by restoring yield advantage in advanced generations or by tailoring them through recombination breeding.

Induction of in vitro culture associated variation (SV) in the regenerants is a frequently occurring phenomenon in somatic tissue cul ture1.2. Variations do occur in plants developed through anther culture (AC)3 also, which are known as androclonal variations (AV). SV has been exploited extensively in rice improvement4

-9

• Unlike somaclonal variation (SV), utilization of A V in rice improvement is very less. Upon culturing anthers from an inbred line or from FI recombinants, one might generate androclones with altered traits different from the donor parent due to culture induced AV. However, relatively mild in vitro culture condition does not drastically alter the genetic background of the regenerants/variants and thus the agronomic values of the regenerants are generally not much affected. Hence one might expect variationls in near-isogenic background especially if the variability is associated with single gene mutation with large recognizable effects. This kind of variation results in development of variants almost identical to the mother except for the altered trait. However, occurrence of such variations largely depends upon the biological and chemical environments and duration of in vitro culture_ It further differs from crop to crop, even among different genotypes within the same crop. Following a few cycles of field evaluation the stability of the new traitls and mode of inheritance could easily be predicted. The present study attempts to assess the range and magnitude of variation in pollen plants in respect of yield and yield components of an AC responsive indica variety PTB 28 as a

model system and to recover elite androclones for rice genetic improvement.

Materials and Methods One in vitro AC responsive indica rice variety PTB

28 (IRGC 19321) obtained from International Rice Research Institute, Philippines was employed as a model system in this study. Donor plants were grown and managed in experimental net house and anthers were cultured 10. Healthy tillers with panicles enclosed within the leaf sheath (selected based on distance of 4-5 cm between the auricle of the flag leaf and the next subtending leaf) to approximate mid-uninucleate and early bi-nucleate stages, which are considered to be the most responsive to AC were collected between 8.30-9.00 a.m. They were thoroughly washed in tap water. After clipping off the flag leaves, boots were sprayed with 70% ethyl alcohol, wrapped in moist paper towels, kept in polyethylene bags and incubated at 80 C for 8 days. Light green spikelets with anthers having reached up to 50 % of the total length were chosen. Selected portions were covered in sterile gauze cloth and immersed in freshly prepared 0.1 % HgClz solution for 5 min. They were washed 3 times in sterile distilled water and bottles were kept upside down on sterile tissue paper for 5-10 minutes to drain out excess water. About 50-60 spikelets were cut at the base (to free the anthers from their filaments) individually at a time on sterile petri dish. Approximately 90-100 anthers were implanted onto 7-8 rnl well dried semi-solid N6 mediumll with 1.0, 2.0, 3.0 and 4.0 mg rl 2,4-D by tapping them on the

MANDALetal.: ANDROCLONAL VARIATION IN RICE 1055

rim of radio sterile polystyrene petri dishes (Tars on make, 60 mm dia.). Dishes were sealed with parafilm and kept in dark for callus induction. Small calli started developing from cultured anthers, which were transferred (average calli diameter 2-3 mm) after 30 days with the help of a flattened spatula onto regeneration medium (RM) containing MS with 1.0, 2.0 and 3.0 mg r l of kinetin and 0.5 mg rl NAA in combination in culture tubes with cotton plugs. The cultures were kept under 16/8 h light/dark cycle (light intensity -130 !lE m·2 S·I) at 25±2° C. Regenerated plants (AI generation) were counted at the end of 60 days. Plants with vigorous roots were directly transferred to soil (otherwise maintained for a few days in Hoagland solution) in small plastic pots and covered with polyethylene bags for 4-5 days. Plants of approximately 8-10 cm height were transferred to big cement pots (12Ix12Ix18") and grown to maturity. Panicles of individual plants at flowering were covered with butter paper bags to prevent any cross-

. pollination. Seeds were harvested at maturity, kept in separate packets, threshed, cleaned and stored. Plant to row progeny trial was performed at A2 generation under rainfed condition under the humid tropics of Port Blair. Plants were grown at 15x20 cm spacing. Standard fertilizer schedule and crop protection measures were adopted 12 to ensure good crop growth. Major agronomic characters viz. plant height, tiller number per plant, panicle number per plant, panicle length, yield per plant and spikelet sterility at maturity were recorded and employed for statistical analyses to assess the extent of androclonal variation

Results and Discussion Maximum (16.7%) anthers produced profuse calli

(8.9 calli/anther) on N6 medium supplemented with 2.0 mg rl 2,4 -D. Calli were initiated within 26 days after plating. When calli of 2-3 mm diameter were shifted onto RM, maximum (88.4%) plant regeneration was observed on MS with 2.0 mg rl

kinetin and 0.5 mg rl NAA. Of the total regenerants 57.3% were green, rest were albinos. About 12% of the total green plants were found to be sterile haploids in the field (data not presented).

A wide range of variation was observed for major agronomic characters in 160 DH families of PTB 28 (Table 1). Phenotypically variant plants of rice derived through AC were first described l3 and it was suggested that variation in regenerants from haploid somatic tissue is greater than any other chronic mutagenic treatments. Commonly occurring phenotypic variations were reported in number of tillers per plant, fertile tillers per plant, average panicle length, numb<rr of fertile seeds, plant height and flag leaf length in the primary regenerants of rice I4.16. Wide variations In several agronomic traits in A2 from haploid somaclones were reported 17, which is corollary to our observations. Overall mean values of all the characters were reduced except panicle length (23.7 cm), which was little higher than the parent (21.5 cm). Reduced plant height was observed in the present set of androclones to the higher extent of 81.7%. SD of mean, a reliable parameter to measure uniformity of a character was found to be more uniform for plant height and panicle length across the androclone families in comparison to other characters as evident from their low SD (2.76 and 0.99), low range (2.65) and very low CV (4.17 %) values. Though overall tiller and panicle number per plant were reduced, higher SD and per cent CV revealed that these two characters varied largely among the DH lines. Mean yield of 160 DH androclones (13.6 g/plant) was less compared to the mean yield of parent (14.8 glplant) with high SD (5.04), wide range (0.3-28.3) and maximum CV (37.0%). Frequency polygon and histogram of yield variations among andro<;:lones (Fig. I) clearly elucidate that yield improvement due to A V was very less (5-6%). Leptokurtic frequency distribution curve indicated that mean yield of majority of the androclones was either less or equivalent

Table 1 - Androclonal variation for major agronomic characters in PTB 28

Character PTB 28 Androclone families (parent) Range Mean SD CV

Mean

Plant height (cm.) 107.5 96.7- 113.4 104.1 2.76 2.65 Panicle number per plant 13.0 5.0 - 18.6 9.3 2.28 24.59 Tiller number per plant 14.0 5.5 - 27.0 13.9 3.94 28.28 Panicle length (cm.) 21.5 20.2 - 25.9 23.7 0.99 4.17 Yield (glpl.) 14.8 0.3 - 28.3 13.6 5.04 37.00 Spikelet sterility (%) 29.6 1.9 - 90.9 52.8 19.03 36.04

1056 INDIAN J EXP BIOL, OCTOBER 2000

to the parent. Yield improvement was observed in 10 amongst 160 DH families, however, the increment was marginal (4.0-22.86%) except A3-34. This indicates the less applicability of AC in improvising polygenic traits like seed yield. Alike SV when A V arises due to single gene mutation, chance for improving a polygenic trait becomes remote. However, the substantial yield increase in a very few androclones (de novo variation) seems to be promising in rice improvement. Sterility of the androclones was also very high (52.80%) and varied markedly as evident from the maximum SD (19.03), wide range (1.89-90.01) and high CV (36.04%). The average spikelet sterility of the androclones was high (52.50%) and was exceptionally high as 70% in A5-48 , probably due to in vitro culture induced alteration in chromosome number and structure, which in turn affected the fel1ility status 18. The frequency of variation for different characters distributed widely among the androclones is inflicted from their segregating nature (Table 2). Of the 160 DH families , 81.7% were shorter, while the rest were taller (5.7%) than the parent. Near normal distribution was observed in case of number of panicles per plant among the DH lines. About 25-30% of the

androclones had almost equal number of panicles and tiller per plant alike the parent. Number of tillers per plant was reduced in most of the androclones (70.6%) while panicle number per plant was reduced in 42.5% only. A limited number of androclones had more tillers than the parent (4.4%) while 28.7% had more panicles per plant (about 50% of parental mean). Though 65 androclones yielded almost equal to the parent, majority (53.4%) yielded less (-50% of parental mean). However, only 10 androclones (6.25%) out-yielded the parent of which one DH line produced as high as 48.57% over the parental mean. Thus, a wide variation was observed in the DH families for various characters. These variations are presumably induced by key determinants like physical factors, chemical constituents of culture medium, biological condition of the explants and departure from duration of unorganized calli from organized growth. Gene amplification, diminution, gene conversion, somatic recombination, movement of transposable elements and position effect mutations arising from translocation and other chromosomal rearrangements may be the other possible sources of AV in DH plants l8

. Alteration in the number of the copies of DNA in tobacco by A V is already

Table 2 - Distribution of androclones based on major agronomic traits in comparison to parental mean

Variation

50-26% less than the parental mean 25-1 % less than the parental mean Equal to parental mean 1-25% more than the parental mean 26-S0% more than the parental mean

Plant height (cm.)

No. %

131 81.7 20 12.6

9 5.7

Tiller number

No. %

21 \3.1 92 57.5 40 25 .0

7 4.4

Frequency distribution Character

Panicle number Panicle length (cm)

No. % No. %

20 12.5 48 30.0 140 87.5 46 28.7 14 2.5 32 20.0 16 10.0 14 8.8

Table 3 - Agronomic performance of selected elite androclones of PTB 28

Androclone Plant height Panicle number Panicle length Yield designation (cm) per plant (cm) (g/pl)

A5-3 102.0 (-7 .27) 12.0 (-7 .69) 23.4 (+1.74) 20.0 (+ 14.28) A5-7 104.0 (-5.45) 16.0 (+23.08) 25.4 (+10.44) 208 (+15.86) AS-I 106.0 (-3.64) 12.0 (-7.69) 22.4 (-2.61 ) 21.0 (+20.00) A3-23 109.0 (-0.90) 20.0 (+53.85) 24.6 (+6.95) 26.0 (+48.S7) Al-S 106.0 (-3.64) 19.0 (+46.15) 23.0 (0.00) 20.S (+1 7.\5) A5-8 108.0 (-1.82) 15.0 (+15.38) 24.0 (+4.3S) 18.7 (+6.86) A3-25 108.0 (-1.82) 16.0 (+23.08) 23.7 (+3.04) 21.0 (+20.00) A3-1 10"7.0 (-2.73) 17.0 (+30.77) 22.8 (-0.87) 18.2 (+4.00) A2-1 108.0 (-1.82) 16.0 (+23.08) 23.7 (+3.04) 21.S (+22.86) AS-15 109.0 (-0.90) 16.0 (+23.08) 24.6 (+6.96) 19.5 (+11.43) PTB 28 (parent) 109.99 12.99 23.00 17.5 C.D. (P=O.05) 1.09 I.S9 1.37 3.S9

*Values in parentheses indicate per cent increase (+) or decrease (-) over parent

Yield (g/pl)

No. %

30 18.7 55 34.4 65 40.6 9 5.6

·0.7

MANDAL et al.: ANDROCLONAL VARIATION IN RICE lOS7

70

65

60

55

50

45

40

35

20

15

10

" III

VARIA liON IN YIELD

Leg~nds :

~ 26% LESS THAN PARENT MEAN 11 25 - 1% LESS THAN PARENT MEAN III. Ea.UAL TO PARENT MEAN IV. 1 - 25% MORE THAN PARENT MEAN V. 26 - 50% MORE THAN PARENT MEAN

IV V

Fig. 1 - Frequency polygon and histogram of yield variation among androclones of PTB 28

reported l9, which substantiates this study too. Further

more, AC allows the early expression of recessive genes, attributing to the major variations.

Single gene androclonal variants, especially the genes with large diagnosable effects20 have enormous practical applications though the chance of improvement of polygenically controlled characters is less. Based on yield performance, 10 semi-tall DH lines were selected with high yield and improved yield attributing characters (Table 3). All selectants were short (0.9-7.3%) and majority had more number (1S.38 - S3.8S%) of longer (1.74 - 10.44%) panicles in comparison to parent. Shortest plant was AS-3 with a height of 102.0 cm. Maximum panicle number per plant (20.0) and panicle length (2S.4 cm.) was met in A3-23 and AS-7, respectively. Amongst all, A3-23 out-yielded the parent (26.0 glpl.) to the extent of 48.S7%. Those few selected lines, which displayed significant differences from control may be exploited in future in rice improvement especially for varietal improvement if yield advantages are restored in advanced generations or by tailoring extreme types through recombination breeding.

Acknowledgement

The authors thank the Director for his keen interest.

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