Lathyrus Lathyrism Newsletter Vol 4 · G. B.Polignano, P. Uggenti, G. Olita, V. Bisignano, V. Alba and P. Perrino- Italy 15 Model plant type in Khesari (Lathyrus sativus L.) suitable

Lathyrus Lathyrism Newsletter 4 (2005)

Contents

Page

Editor's Comment 1 Colin Hanbury -Australia

Articles 2 Fatty acid composition of grass pea (Lathyrus sativus L.)

seeds Gurusamy Chinnasamy, Arya Kumar Bal, and David Bruce McKenzie- Canada

5 Performance of grass pea (Lathyrus sativus L.) somaclones at Adet, northwest Ethiopia

D. Tsegaye, W. Tadesse and M. Bayable- Ethiopia

7 Search for resistance to crenate broomrape (Orobanche crenata) in Lathyrus

J.C. Sillero, J.I. Cubero, M. Fernández-Aparicio and D. Rubiales- Spain

10 Characterization of grass pea (Lathyrus sativus L.) entries by means of agronomically useful traits

G. B. Polignano, P. Uggenti, G. Olita, V. Bisignano, V. Alba and P. Perrino- Italy

15 Model plant type in Khesari (Lathyrus sativus L.) suitable for hill farming

Vedna Kumari and Rajendra Prasad- India

18 Resilience of South Asian disabling conditions: a glimpse of lathyrism among comparative histories

M. Miles- UK

22 Considerations on the reintroduction of grass pea in China Hui-Min Yang and Xiao-Yan Zhang- China

27 Effects of drought on stomatal character, photosynthetic character and seed chemical composition in grass pea, and their relationships

Hui-Min Yang, Xiao-Yan Zhang and Gen-Xuan Wang- China

28 Scope of growing lathyrus and lentil in relay cropping systems after rice in West Bengal, India

S. Gupta and M.K. Bhowmick- India

34 The same goal, a different approach: a new Belgian-Ethiopian project

Fernand Lambein and Seid Ahmed- Belgium/Ethiopia

THIS VOLUME IS STILL BEING COMPILED- THERE WILL BE MORE ARTICLES TO FOLLOW

ISSN 1832-8431 (Print) and ISSN 1832-844X (Online)

The Lathyrus Lathyrism Newsletter can be obtained on-line at

http://go.to/lathyrus OR

http://www.clima.uwa.edu.au/lathyrus

All research articles are provided there in PDF format.


Jointly supported by:

Third World Medical Research Foundation (TWMRF), PO Box 9171, Portland, Oregon 97207, USA

http://www.twmrf.org and

Centre for Legumes in Mediterranean Agriculture (CLIMA), University of Western Australia, 35 Stirling Highway

Crawley 6009, Australia http://www.clima.uwa.edu.au


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Editor’s comment

Dear Readers Welcome to the Lathyrus Lathyrism Newsletter Vol. 4 and thank you all for your support of the newsletter. You will see from the variety of articles that there is enthusiasm for Lathyrus related research in many countries, this bodes well for the increased knowledge and cultivation of these useful legumes. Recently, there has been a change in the editing process. In order to give authors an advantage of on-line publication from now onward articles will be placed on the web page as soon as the editing process is finalised. This will enable the maximum quick exposure of information. Then at the end of the year the volume will be closed off and compiled, for those who require the printed version this is when it will be completed. Hence as you revisit the web page through the year you will notice more articles appearing. Any suggestions for further improvements are welcome. The Lathyrus Lathyrism Newsletter has been assigned ISSN 1832-8431 (Print) and ISSN 1832-844X (Online) and has been placed on a number of directories of open access journals, ensuring wider exposure and listings in library resources internationally. Citations of articles in the newsletter have been steadily increasing. Thanks to the Third World Medical Research Foundation (TWMRF) and the Centre for Legumes in Mediterranean Agriculture (CLIMA) for supporting the newsletter from 2000 until now. Please consider summarising your recent research for the newsletter. Your contribution will be useful not only as science but also toward the greater stability of cropping systems in regions with increasing pressure on natural resources. Most research submissions should be approximately 1500 words and can include a small number of tables or figures, with electronic copies preferred. Introduction, Methods, Results followed by Discussion is the preferred layout for research summaries, although this can be altered as necessary. Abstracts of complete work are also welcome, if they have been published elsewhere then full acknowledgment will be made. I hope you find the articles contained interesting and useful to your work. Colin Hanbury Editor contact details: Dr Colin Hanbury Department of Agriculture, Western Australia 3 Baron-Hay Court South Perth 6151 Australia E-mail: [email protected]


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Fatty acid composition of grass pea (Lathyrus sativus L.) seeds

Gurusamy Chinnasamy1*, Arya Kumar Bal1, and David Bruce McKenzie2

1. Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada A1B 3X9.

2. Atlantic Cool Climate Crop Research Centre, Agriculture and Agri-Food Canada, 308 Brookfield Road, St. John’s, Newfoundland and Labrador, Canada A1E 5Y7.

*Author for correspondence. Present address: Imaging Program, Lawson Health Research Institute, St.

Joseph’s Health Care, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2 Email: [email protected] OR [email protected]

Introduction Grass pea or chickling vetch (Lathyrus sativus L.) is a well-established, commercially available, tropical semi-arid crop. Pods of this crop are flat, dorsally broad with two ridges, short and 3-5 cm in length. Each pod contains 2-7 seeds. Mature seeds are rhomboid or triangular in shape, dull whitish grey brown and variously mottled (4). Grass pea is a relatively productive crop compared to other pulses in regions characterized by poor soil (7). It is very well adapted to adverse climatic conditions and requires very little management for crop production. Moreover, its deep taproot and nitrogen-fixing ability make this crop an ideal choice for sustainable agriculture. Grass pea seeds are a major source of protein for large sections of the population in Bangladesh, China, Ethiopia and India (13, 15). It is also grown to a lesser extent in the Middle East, southern Europe and some parts of South America. In India grass pea is considered one of the most economical pulses for fodder and green manure in rice fields during the cool winter period (1). Lathyrus species contain very high protein, but a neurotoxin, 3-(-N-oxalyl)-L-2,3-diamino propionic acid (ODAP), is present in wild and most cultivated forms that if consumed in sufficient amounts can cause the irreversible crippling disease known as lathyrism (10, 17). This toxin to a considerable extent has been bred out of some cultivars although lathyrism in Asia from consuming grass pea is common. Because of its drought tolerance, grass pea has been judged to have good potential as a future new pulse crop for low rainfall areas of the Canadian prairies (12). It acts as a ground cover alternative to summer fallow, helping to prevent wind and water erosion, as well as adding nitrogen to the soil (11). The nutritional health and well being of humans are entirely dependent on plant foods. Plants are critical components of the dietary food chain in that they provide almost all essential minerals and organic nutrients to humans either directly, or indirectly when plants are consumed by animals, which are then consumed by humans (9). Grass pea seeds may

represent a potential source of several important nutrients for human and animal nutrition. Therefore, it is necessary to analyze grass pea seeds for their nutrient composition. The present investigation proposes to determine the fatty acid composition of different lipid classes in mature seeds of Indian grass pea. Material and Methods Seed materials Mature seeds of grass pea were procured from a local market in Kolkatta (Calcutta), West Bengal, India. Extraction and estimation of total lipids Total lipids were determined by the gravimetric method (2). One gram of dried grass pea seeds in each of three replicates was powdered using a 700S Waring blender (Waring Products Co., USA) and homogenized in 10 ml of 50 mM Tris-HCl buffer containing 0.5 M NaCl at pH 7.2. The homogenate was combined with a mixture of chloroform and methanol in a ratio of 1.25:2.25 (v/v) to extract lipids. The chloroform layer (supernatant) was separated by centrifugation at 5000 g for 20 min and allowed to stand overnight after combining with a mixture of chloroform and distilled water in a ratio of 1:1 (v/v). The chloroform layer was collected in a pre-weighed vial for evaporation under nitrogen gas. The vial with the lipid residue was weighed again to estimate the amount of total lipids. Separation of lipids and analysis of fatty acid composition Total lipids were fractionated into 5 lipid classes [phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FFA) and triglycerides (TG)] by thin layer chromatography and the constituent fatty acids in each lipid class were separated and estimated using gas chromatography as described in Chinnasamy et al. (6). The measurement of each fatty acid was calculated as a relative weight percentage to 10 selected fatty acids [C14:0 (myristic acid), C14:1 (myristoleic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:3 (linolenic


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acid), C18:4 and C20:4 (arachidonic acid)]. The double bond index (DBI) was calculated (14) using the formula: DBI=Σ (% of fatty acid content × no. of double bonds) 100 Among 10 major fatty acids, sum of all saturated fatty acids, unsaturated fatty acids with one double bond and unsaturated fatty acids with more than one double bond gave total saturated fatty acids (TSFA), total monounsaturated fatty acids (TMUFA) and total polyunsaturated fatty acids (TPUFA) respectively. Unsaturated to saturated ratio (USR) was calculated by dividing total unsaturated fatty acids by total saturated fatty acids. Statistical analysis For all sets of data, one way analysis of variance was performed using the SPSS computer package (16). Means were compared by Duncan’s multiple comparison test at P = 0.05. For the purpose of statistical analysis, data in percentage were transformed to arcsine values (18). Results and Discussion Total lipid content of mature grass pea seeds was 20.93 ± 0.27 mg/g dry weight. The nutritive value of seeds is determined by not only quantity but also by quality of lipids they contain. Thus, fatty acids present in lipids are playing important role in deciding shelf life, nutrition and flavor of food products (8). Chavan et al. (3) reported the presence of 20 fatty acids varying

from C8 to C22 in total lipids of Canadian grass pea seeds. The relative weight percentages of 10 major fatty acids in 5 lipid classes isolated from mature seeds of Indian grass pea are summarized in Table 1. C18:2, C18:1, C18:0 and C16:0 were the major fatty acids present in PL, MG, FFA and TG separated from grass pea seeds. DG contained higher quantities of C18:0, C16:0 and C18:2 compared to other fatty acids. PL and TG showed higher overall DBI than FFA, MG and DG. The content of TSFA was higher in DG than in other lipid classes. High amounts of TMUFA were observed in FFA and TG. PL registered high quantity of TPUFA. TG, PL and FFA showed higher USR compared to MG and DG. Although lipids constitute a minor portion of many leguminous seeds, their profiles indicate the desirable nature of fatty acid constituents present (3). In the present study, grass pea seeds exhibited a high amount of total unsaturated fatty acids (56.37% – 59.98%) and a low amount of total saturated fatty acids (40.01 – 43.65%) in all lipid classes except MG and DG, which contained 31.49 – 47.29 % total unsaturated fatty acids and 52.72 – 68.53% total saturated fatty acids. Overall, in the present work, Indian grass pea seeds showed higher total unsaturated fatty acids than total saturated fatty acids that are in agreement with fatty acid composition of beach pea and Canadian grass pea seeds (3-5). Therefore, grass pea seeds may be important for nutritional health and may serve as a valuable nutritional source. Further in depth study is necessary to elucidate the nutritional quality and importance of grass pea.

Table 1. Composition of major fatty acids (relative weight percentage1) in phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FFA) and triglycerides (TG) isolated from total lipids of mature seeds of Indian grass pea. Values are means (± SE) of three replications. Fatty acids PL MG DG FFA TG C14:0 0.65 ± 0.82b 4.51 ± 2.78b 2.01 ± 0.24d 2.09 ± 1.61c 1.06 ± 0.85c C14:1 0.66 ± 0.53b 0.77 ± 1.41b 0.91 ± 1.63de 0.49 ± 0.09c 0.82 ± 1.03c C16:0 24.88 ± 1.67a 26.62 ± 2.89a 31.21 ± 0.75a 26.07 ± 3.60ab 24.57 ± 5.25ab C16:1 0.57 ± 0.40b 2.77 ± 2.53b 1.10 ± 0.48de 2.66 ± 2.61c 2.26 ± 1.17c C18:0 16.24 ± 4.90a 21.59 ± 1.82a 35.31 ± 0.92a 15.49 ± 1.15b 14.38 ± 1.96b C18:1 26.20 ± 6.64a 23.44 ± 2.18a 9.30 ± 0.12c 35.99 ± 8.32a 33.91 ± 6.64a C18:2 28.65 ± 11.13a 17.84 ± 8.39a 17.55 ± 1.86b 14.31 ± 2.85b 19.17 ± 3.73ab C18:3 1.16 ± 0.61b 1.57 ± 2.27b 0.75 ± 0.69de 0.81 ± 0.26c 2.55 ± 1.90c C18:4 0.64 ± 0.91b 0.23 ± 0.19b 0.73 ± 0.99e 1.93 ± 1.61c 0.75 ± 1.35c C20:4 0.36 ± 1.32b 0.67 ± 0.70b 1.15 ± 0.89de 0.18 ± 0.04c 0.52 ± 1.03c DBI2 0.92 ± 0.18 0.71 ± 0.20 0.56 ± 0.08 0.79 ± 0.02 0.88 ± 0.04 TSFA3 41.77 52.72 68.53 43.65 40.01 TMUFA4 27.43 26.98 11.31 39.14 36.99 TPUFA5 30.81 20.31 20.18 17.23 22.99 USR6 1.39 0.90 0.46 1.29 1.50 1The value of each fatty acid was calculated as a relative weight percentage to 10 selected fatty acids. 2 Double bond index. 3 Total saturated fatty acids. 4 Total monounsaturated fatty acids. 5 Total polyunsaturated fatty acids. 6 Unsaturated/saturated ratio.

a-e Means in the same column followed by different letters are significantly different using Duncan’s multiple comparison test at P = 0.05.


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Acknowledgements We wish to thank Dr. P. J. Davis, Rhonda and Dona for the help in fatty acid analysis. This work was supported by the Dean of Science Grant to A. K. Bal.

References 1. Allen ON, Allen EK. 1981. The leguminosae.

University of Wisconsin Press, Madison, WI. 2. Bligh EG, Dyer WJ. 1959. A rapid method of

total lipid extraction and purification. Can J Biochem Physiol 37, 911-917.

3. Chavan UD, Shahidi F, Bal AK, McKenzie DB. 1999. Physico-chemical properties and nutrient composition of beach pea (Lathyrus maritimus L.). Food Chem 66, 43-50.

4. Chavan UD. 1998. Chemical and biochemical components of beach pea (Lathyrus maritimus L.). Ph.D. Thesis. Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada.

5. Chinnasamy G, Bal AK, McKenzie DB. 2004. Fatty acid and elemental composition of mature seeds of beach pea [Lathyrus maritimus (L.) Bigel.]. Can J Plant Sci 84, 65-69.

6. Chinnasamy G, Davis PJ, Bal AK. 2003. Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea. J Plant Physiol 160, 355-365.

7. Duke JA, Reed CF, Weder JKP. 1981. Lathyrus sativus L. In Handbook of legumes of world economic importance. Duke JA (Ed.). Plenum Press, New York, NY. pp. 107-110 and 345-349.

8. Gaydou EM, Rasoarahona J, Bianchini JP. 1983. A micromethod for the estimation of oil content and fatty acid composition in seeds with special reference to cyclopropenoic acids. J Sci Food Agric 34, 1130-1136.

9. Grusak MA, DellaPenna D. 1999. Improving the nutrient composition of plants to enhance human nutrition and health. Ann Rev Plant Physiol Plant Mol Biol 50, 133-161.

10. Hanbury C, White C, Mullan BP, Siddique KHM. 2000. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Lathyrus Lathyrism Newsletter 1, 34.

11. Henkes R. 1995. The remaking of grasspea. The Furrow 100, 25-26.

12. Kiehn FA, Reimer M. 1992. Alternative crops for the prairies. Agriculture Canada Publication. 1887/E, Ottawa, Ontario, Canada.

13. Kuo YH, Khan JK, Lambein F. 1994. Biosynthesis of the neurotoxin β-ODAP in developing seeds of Lathyrus sativus. Phytochem 35, 911-913.

14. Skoczowski A, Filek M, Dubert F. 1994. The long-term effect of cold on the metabolism of winter wheat seedlings. II. Composition of fatty acids of phospholipids. J Therm Biol 19, 171-176.

15. Spencer PS, Roy DN, Ludolph A, Dwivedi MP, Roy DN, Hugon J, Schaumburg HH. 1986. Lathyrism: evidence for the role of the neuroexcitatory amino acid BOAA. Lancet 2, 1066-1067.

16. SPSS Inc. 1990. SPSS/PC + StatisticsTM 4.0 for the IBM PC/XT/AT and PS/2. SPSS Inc., Chicago, IL.

17. White C, Hanbury C, Siddique KHM. 2001. The nutritional value of Lathyrus cicera and Lupinus angustifolius grain for sheep. Lathyrus Lathyrism Newsletter 2, 49-50.

18. Zar JH. 1996. Biostatistical analysis. Third edition. Prentice Hall, New Jersey, NJ.


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Performance of grass pea (Lathyrus sativus L.) somaclones at Adet, northwest Ethiopia

D. Tsegaye*, W. Tadesse and M. Bayable

Adet Agricultural Research Center, P.O Box 08, Bahir Dar, Ethiopia.

*Email: [email protected]

Introduction Grass pea (Lathyrus sativus L.) is one of the important crops of economic significance in Ethiopia. It is the fifth most important pulse crop in Ethiopia after faba bean, field pea, chickpea and haricot bean. It is the cheapest source of protein in the diets of most people. Estimates of the total cultivated area and production of grass pea in Ethiopia was reported to be 83,522 hectares and 92,339 tonnes, respectively. Of this total sum, northwest Ethiopia exceeds other regions, having 39,983 hectares (47.9 %) and 40,840 tonnes of production (44.2 %) (3). Grass pea is a highly popular food and feed legume in the farming system due to tolerance of drought, flooding and disease and its importance in ameliorating soil fertility (2). It is commonly grown as a double crop after the cereals tef or barley. Despite its importance, the presence of the neurotoxin β-N-oxalyl L-a, β-diaminopropanoic acid (ODAP) is a discouraging factor for grass pea production. Irreversible crippling can occur, if the seeds are consumed as a major part of the diet for an extended period (1). To date no single improved variety has been developed and released in Ethiopia due to the inconsistency of ODAP content of the promising grass pea genotypes across environments. The development of biotechnology and its application in grass pea has resulted in somaclones with neurotoxin ODAP content of less than 0.1% (100 mg ODAP\100gm seed) in India (5). As a result the government of India released one of the somaclones, Bio L212 (Ratan), for cultivation (8). Low ODAP lines are also available at the International Center for Agricultural Research in Dry land Areas (ICARDA) (6). A study was carried out to test the agronomic performances and the neurotoxin ODAP content of those lines developed by somacloning technique at ICARDA.

Material and Methods Eleven low neurotoxin grass pea somaclones introduced from ICARDA and a local check were grown for two consecutive years (2000/01 and 2001/02) at Adet Agricultural Research Center, which is located at 37029’E and 11016’N latitude, with an altitude of 2240 m above sea level in the Amhara National Regional State, Ethiopia. The soil is vertisol with pH 6.0. The weather variables during the testing periods were generally conducive for normal growth of grass pea. The genotypes were planted in a randomized complete block design with two replications at a seeding rate of 40 kg ha-1. A spacing of 60 cm between plots and 20 cm between rows were used, with a plot size of 0.8 m x 4 m. The materials were evaluated for stand percent, days to flowering, days to maturity, plant height (cm), number of pods per plant, number of seeds per pod, 100 seed weight (g) and grain yield (kg ha-1). ODAP content analysis on seed was done at Addis Ababa University, Department of Chemistry, using Rao Method for the 2001/02 cropping season only. Analysis of variance was computed for each season using an MSTAT computer program (7). Results and Discussion The result of agronomic performance of the tested genotypes is presented in Table 1. Genotypes showed significant difference (P<0.05) in days to maturity, 100 seed weight and grain yield. The grain yield level ranged from 3705 to 5233 kg ha-1. ILAT-LS-K-290 was the best yielder (5233 kg ha-1) and early in maturity; whereas the local check was later to mature, lower in 100 seed weight and grain yield (3705 kg ha-

1) than the other tested lines. ILAT-LS-K-289 and ILAT-LS-K-444 were promising, both with 0.104 % ODAP (Table 1). ILAT-LS-K-288 and ILAT-LS-K-33 were also promising with low ODAP contents of 0.119 and 0.125 % seed. The local variety scored the


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highest ODAP content, 0.252 %. The results clearly indicate that the somaclone technique is promising in developing grass pea varieties with ODAP content presumed at a safe level for human consumption (< 0.2 %) (4) in the future. Therefore, the promising lines

will be included in the regional as well as the national breeding programme for testing under different environmental conditions and for possible release.

Table 1. Agronomic performance of grass pea somaclones at Adet, northwest Ethiopia (2000/01 and 2001/02).

Variety Stand %

Days to flowering

Days to maturity

Plant height (cm)

Pods/ plant

Seeds/pod

100 seed weight (g)

Seed yield (kg/ha)

Seed ODAP (%)*

ILAT –LS-K-290 81 64 142 115 71 3 7.3 5233 0.168 ILAT –LS-K-30 81 66 143 111 59 4 8.7 4317 0.216 ILAT –LS-K-104 83 66 145 121 69 4 10.2 5064 0.140 ILAT –LS-K-33 85 64 143 119 63 3 8.9 4261 0.125 ILAT –LS-K-299 84 67 147 112 56 3 7.2 4300 0.206 ILAT –LS-K-289 82 65 143 121 72 4 7.5 4807 0.104 ILAT –LS-K-444 82 64 144 114 64 4 7.6 4464 0.104 ILAT –LS-K-387 78 64 145 122 74 3 7.9 4405 0.211 ILAT –LS-K-190 78 64 142 112 64 4 7.6 4468 0.202 ILAT –LS-K-288 82 66 145 104 56 4 8.5 4209 0.119 ILAT –LS-K-390 85 57 143 104 56 4 7.4 4206 0.168 Local variety 86 54 149 126 67 4 6.5 3705 0.259 Mean 82 64 114 115 64 4 7.9 4462 - S.E. 6.69 1.44 1.18 8.38 18.2 0.32 0.53 504 - LSD (5%) 19.6 4.2 3.4 24.6 53.4 0.95 1.55 1478 - CV (%) 11.5 3.2 1.2 10.3 40.3 12.9 9.4 16.0 -

*2001/02 only References1. Campbell CG. 1997. Grass pea (Lathyrus sativus

L.). Promoting the Conservation and use of underutilized and neglected crops. 18. Institute of Plant Genetic and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institutes, Rome, Italy.

2. Campbell CG, Tiwari KR. 1997. Breeding grass pea for reduced seed levels of neurotoxin (ODAP). In: R. Teklehaimanot and F. Lambein (eds) Lathyrus and Lathyrism: A Decade of Progress. University of Ghent, pp. 85-86

3. Dahiya BS. 1976. Seed morphology as an indicator for low neurotoxin in Lathyrus sativus. Qual Plant-Pl Fds Hum Nut 25, 391-394.

4. Central Statistics Authority, Ethiopia (CSA). 2001/02. Agricultural Sample Survey. Addis Ababa, pp. 27.

5. Mehta S.L. 1997. Plant biotechnology for removal of ODAP from Lathyrus. In: R. Teklehaimanot and F. Lambein (eds) Lathyrus and Lathyrism: A Decade of Progress. University of Ghent, pp 103.

6. Moneim, AM. Saxena MC, El-Saleh A, Nakkoul H. 1997. The status of breeding grass pea (Lathyrus sativus) for improved yield and quality at ICARDA. In: R. Teklehaimanot and F. Lambein (eds) Lathyrus and Lathyrism: A Decade of Progress. University of Ghent, pp. 81-82.

7. MSTATC. 1989. A micro-computer statistical program for experimental design, data management and data analysis. Michigan State University. Crop and Soil Science, Agricultural Economics and Institute of International Agriculture, Michigan, USA.

8. Santha IM, Mehta SL. 2001. Development of low ODAP somaclones of Lathyrus sativus. Lathyrus Lathyrism Newsletter 2, 42-45.


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Search for resistance to crenate broomrape (Orobanche crenata) in Lathyrus

J.C. Sillero1*, J.I. Cubero2, M. Fernández-Aparicio3 and D. Rubiales3

1. CIFA, Dep. Mejora y Agronomía, Apdo. 3092, E-14080 Córdoba, Spain. 2. ETSIAM-UCO, Dep. Genética, Apdo. 3048, E-14080 Córdoba, Spain.

3. CSIC, Instituto de Agricultura Sostenible. Apdo.4084, E-14080 Córdoba, Spain.

* Email: [email protected]

Updated from original publication: Sillero JC, Cubero JI and Rubiales D. 2001. Resistance to broomrape (Orobanche crenata) in Lathyrus.

7th International Parasitic Weed Symposium, (eds. A. Fer et al.), pp. 224-227, Faculté des Sciences,Nantes, France.

Introduction Grass pea has been widely cultivated in South Asia and the Mediterranean area since antiquity for food and feed uses(4). Its cultivation was in continuous recession in last decades, but there is an increasing interest in its reintroduction in the cropping systems in marginal areas due to its adaptability to unfavourable environments (4) and its beneficial effects on subsequent crops. Recently, plant breeders have been working to improve its utilities (2) as well as to reduce anti-nutritional factors responsible for lathyrism (1). An additional priority for breeding L. sativus and L. cicera is resistance to crenate broomrape (Orobanche crenata) (4). O. crenata is a holoparasitic weed that seriously attacks legume crops, such as faba beans, lentils, peas, chickpeas, grass peas and vetches (5,6,9). Different control methods have been proposed to avoid this serious problem, but none has been completely effective and the development of resistant cultivars is a major need (8,9). Breeders are actively working on this matter and resistance to O. crenata has been found both in cultivated and wild legume species (7,9). The genus Lathyrus can be satisfactorily grown in marginal areas, so it is necessary to develop varieties resistant to broomrape to prevent this parasitic weed problem in infested areas. The purpose of this study was to search for sources of resistance to O. crenata in different species of the genus Lathyrus.

Material and Methods Fourteen accessions belonging to 10 different species of the genus Lathyrus (Table 1) were screened for resistance to broomrape under field conditions in 1995/96 in at Córdoba (Spain). Accessions were kindly provided by IPK (Germany) and USDA (USA). Each accession was sown in a 1m row, surrounded by four rows of a faba bean susceptible check (cv. Prothabon). The sowing took place on 26 November 1995, in a field heavily infested with O. crenata seeds. Hand weeding was done when required, but no herbicides were applied. The intensity of broomrape attack was evaluated at crop maturity (from early June), by counting the final number of emerged and non-emerged broomrape shoots per plant. Data were expressed as a percentage of the mean of its four surrounding rows of Prothabon (=100%). The most resistant lines were studied in a second field season, with field design and evaluation criteria similar to those described above. The resistance found in two of the most resistant accessions was confirmed in pots and petri dishes experiments. The faba bean cv. Prothabon and a pea variety (cv. Messire) were included as susceptible checks. For the pot experiment five day old plants were transplanted into 1 litre plastic pots filled with vermiculite, previously mixed with 25 mg of broomrape seeds (about 8000 seeds). Each genotype was represented by 12 plants, 1 plant/pot. When the plants were mature, 90 to 120 days after sowing, the plants were extracted, the roots were washed in water and the number of broomrape tubercles was counted. The petri dish experiment was carried out using the procedure described by Sauerborn (10). One month after transplanting the seedlings, 500 seeds per dish were studied and classified to determine the percentages of germination and the number of tubercles per plant.


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Table 1. Emerged and non-emerged broomrape attacks in selected accessions of Lathyrus, under field conditions, during the growing seasons 1995/96 and 1996/97 in Córdoba (Spain)1.

1995/96 season 1996/97 season Accession

Code

Species

Emerged shoots (%)

Non-emerged shoots (%)

Emerged shoots (%)

Non-emerged shoots (%)

Lat-340 PI 255365 Lathyrus annuus 246 380 Lat-323 LAT 150/88 Lathyrus aphaca 73 91 Lat-341 PI 227529 Lathyrus aphaca 145 727 Lat-321 LAT 201/89 Lathyrus cicera 66 205 Lat-343 PI 208307 Lathyrus cicera 90 141 Lat-322 LAT 165/86 Lathyrus clymenum 0 0 0 0 Lat-344 PI 283488 Lathyrus clymenum 0 0 0 11 Lat-342 PI 229794 Lathyrus choranthus 0 123 16 34 Lat-345 PI 358859 Lathyrus gorgoni 34 129 Lat-348 PI 358864 Lathyrus inconspicuus 74 175 Lat-320 LAT 340/92 Lathyrus ochrus 0 0 0 0 Lat-352 PI 271361 Lathyrus ochrus 0 0 0 0 Lat-354 PI 165528 Lathyrus sativus 130 746 Lat-356 PI 269921 Lathyrus szowitsii 160 139 1 Emerged and non-emerged broomrapes referred to the susceptible faba bean check, cv. Prothabon (=100%).

Table 2. Established broomrape in pot and petri dishes and germination of broomrape seeds in petri dishes in two selected accessions of Lathyrus and two susceptible checks.

Pot experiment Petri dishes experiment

Line

Species

No. of established broomrapes/ plant

% germination

No. of tubercles/ plant

Messire Pisum sativum 9.4 a 53.2 a 26.4 a Protabon Vicia faba 7.6 a 37.6 b 13.3 b Lat-320 Lathyrus ochrus 0.0 b 0.2 c 0.0 c Lat-322 Lathyrus clymenum 0.0 b 0.1 c 0.0 c Data with the same letter per column are not significantly different (P<0.05, Duncan test).

Results In none of the accessions of L. clymenum and L. ochrus studied was there any broomrape emergence (Table 1), although in one accession of L. clymenum a few non-emerged tubercles were found in the second field season. Low broomrape emergence but high levels of non-emerged tubercles were recorded in both seasons for the L. choranthus accession studied. All the accessions of the species L. annuus, L. aphaca, L. cicera, L. gorgoni, L. inconspicuus, L. sativus and L. szowitsii were moderately to highly susceptible, all with more than 34% emerged broomrape shoots.

Results of in vitro experiments showed that no broomrape was installed in any of the accessions, in neither pots nor in petri dishes (Table 2), which suggests a barrier to the broomrape establishment. However, when the germination of the broomrape seeds was recorded, almost no germination was found in any of the accessions, so the low germination of the broomrape seeds seems to be the main barrier to the broomrape attack.


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Discussion High levels of resistance to O. crenata have been found in the genus Lathyrus, especially in the species L. ochrus and L. clymenum. In both species the main mechanism of resistance seems to be an early barrier to the establishment of the broomrape, as none or few broomrape tubercles were recorded under field conditions. This low establishment is due to the low induction of germination of the broomrape seeds. Low levels of induction of germination have been described in other resistant legume species such as chickpea (7). We cannot exclude the presence of additional mechanisms of resistance preventing attachment and/or development of tubercles, but the low germination precludes its study and quantification. Such a mechanism preventing development of tubercles is suggested in L. chloranthus by the fact that tubercle formation was allowed, but they did not emerge. Both mechanisms have been previously detected in the genus Cicer (7). Interspecific variation in resistance to broomrape has been described in the genus Lathyrus (3) and has been confirmed in this study. All the accessions of the same species were susceptible (i.e. L. annuus, L. aphaca, L. cicera, L. gorgoni, L. inconspicuus, L. sativus and L. szowitsii) or resistant (i.e. L. clymenum and L. ochrus) to the broomrape attack. L. clymenum and L. ochrus have been used as human food since antiquity (1) and can also be cultivated for forages. Farmers can grow these two species in heavily infested fields and avoid broomrape attack. L. choranthus (at least the line we studied) could be used as a ‘trap crop’ to reduce the broomrape seed bank in the soil; as it permitted very low emergence of broomrape shoots but allowed a relatively high establishment of the parasite, which imply that the stimulation and germination of the broomrape seeds occur. Several legume species have been successfully used with this aim (3,11).

References 1. Enneking D (2000) The riddle of lathyrism.

Lathyrus Lathyrism Newsletter 1, 6. 2. Lazányi J. 2000. Grass pea and green manure

efffects in the Great Hungarian Plain. Lathyrus Lathyrism Newsletter 1, 28-30.

3. Linke KH, Abd El-Monein AM and Saxena MC. 1993. Variation in resistance of some forage legumes species to Orobanche crenata Forsk. Field Crops Research 32, 277-285.

4. Robertson LD and El-Moneim AMA. 1997. Status of Lathyrus germplasm held at ICARDA and its use in breeding programmes. In: Lathyrus Genetic Resources Network: Proceedigns of a IPGRI-ICARDA-ICAR Regional Working Group Meeting (eds. Mathur PN, RAmanatha Rao V and Arora RK), pp 30-41, IPGRI New Delhi, India

5. Rubiales D. 2001. Parasitic plants: an increasing threat. Grain Legumes 33, 10-11.

6. Rubiales D. 2003. Parasitic plants, wild relatives and the nature of resistance. New Phytologist 160, 459-461.

7. Rubiales D, Alcántara C and Sillero JC. 2004. Variation in resistance to crenate broomrape (Orobanche crenata) in species of Cicer. Weed Research 44, 27-32.

8. Rubiales D, Pérez-de-Luque A, Cubero JI and Sillero JC. 2003. Crenate broomrape (Orobanche crenata) infection in field pea cultivars. Crop Protection 22, 865-872.

9. Rubiales D, Sillero JC, Román MB, Moreno MT, Fondevilla S, Pérez-de-Luque A, Cubero JI, Zermane N, Kharrat M and Khalil S. 2002. Management of broomrape in Mediterranean agriculture. In: Legumed: Grain Legumes in the Mediterranean Agriculture (ed. European Association for Grain Legume Research), pp 67-73, Rabat, Morocco.

10. Sauerborn J, Masri H, Saxena MC and Erskine W. 1987. A rapid test to screen lentil under laboratory conditions to susceptibility to Orobanche. Lens Newsletter 14, 15-16.

11. Saxena MC, Linke KH and Sauerborn J. 1994. Integrated control of Orobanche in cool-season food legumes. In: Biology and management of Orobanche (eds AH Pieterse, JAC Verkleij and SJ Ter Borg), pp 419-431. The Netherlands.


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Characterization of grass pea (Lathyrus sativus L.) entries by means of agronomically useful traits

G. B. Polignano1 1*, P. Uggenti1, G. Olita1, V. Bisignano1, V. Alba2 and P. Perrino1

1. Istituto di Genetica Vegetale, C.N.R., Via Amendola 165/A, 70126 Bari, Italia

2. Dipartimento di Biologia Difesa e Biotecnologie Agro Forestali, Università della Basilicata, Potenza

*E-mail: [email protected]

Introduction Grass pea or chickling pea (Lathyrus sativus L.) is a diploid (2n=14), self-pollinated annual with branched, straggling, or climbing habit, blue (sometimes violet or white) flowers and characteristic smooth seed with pressed sides (16). The center of origin and diversification of the Lathyrus gene pool is in the Mediterranean region (15). The earliest archaeological remains of Lathyrus appear in the Neolithic in the Balkans and Near East of Bulgaria, Cyprus, Iraq, Iran and Turkey (4). According to Kupicha (8), the genus Lathyrus contains about 150 species but only L. sativus is widely cultivated for human consumption, particularly in Bangladesh, China, Ethiopia, India, Nepal and Pakistan (10). In Italy, this crop has mostly disappeared and today it is no longer seen as the ‘food of the poor’ as it was in the past. Fortunately, grass pea is still used by local populations in marginal areas, and sold in some marketplaces. Emerging global and national strategies on sustainable farming systems, sustainable development and the preservation of biological diversity reflect concern at adequate quantification of local biodiversity. Consequently, researchers, farmers and policy makers have focused their attention on the neglected and/or underutilised crops to improve the food security, nutrition and economic welfare of humans all around the world (5). In Italy, among these species the grain legume grass pea has received renewed attention as a local and typical product, it is becoming an exclusive and fashionable food for which discerning consumers are prepared to pay a higher price than for other pulse products. In addition, the most interesting agronomical feature of the species are drought tolerance, resistance to pests and diseases, adaptability to different types of soil as well as to adverse climatic conditions (9). Despite these and other advantages, L. sativus is inadequately exploited and studied. In fact, it is well known that this crop is grown mainly as landraces; their genetic diversity is used and maintained largely by a small number of farmers in very limited areas of central southern Italy. In other words, valuable genetic resources of L. sativus are exposed to the threat of genetic erosion and disappearance. Therefore collection and storage of germplasm and deeper knowledge of the nature, entity, and geographical

distribution of the residual genetic variation is recommended for the benefit of both direct users and crop improvement programmes (12). In this regard it is important to underline that the consumption of L. sativus seeds by humans and animals has been limited by presence of a neurotoxin known as β-N-oxalyl-L-α,β-diaminoproprionic acid (β-ODAP) in the seeds, which when taken in large quantity can lead to “lathyrism” a disease causing paralysis of the limbs (3). With that premise, breeding programmes evolving genotypes combining high yield with high protein content and low neurotoxin (ODAP) are in progress all over the world (7). At the same time it was felt that it was necessary to evaluate and describe the genetic diversity available in the grass pea collections (2,6,10,11,12). In other words there is a need to survey, collect, conserve and characterise the valuable resources of the Lathyrus species germplasm for the benefit of both users and crop improvement programmes. The main objective of the present research was to study the variation in a collection of grass pea entries with respect to yield capacity and other important agronomic traits (such as biomass) with the aim of a direct utilisation of the most promising material and their use in cross combinations for breeding purposes. Material and Methods Seventy-six grass pea entries of different geographical origin were used. These were subset of the whole collection including entries characterized by desirable traits: erect plants, high podded node, early flowering, high seed yield, big and light seeds, high biomass, low ODAP content and high protein content. All entries were grown in 2002-2003 winter season on clay-sand soil at the experimental farm “Pantanello”, belonging to the Basilicata region, at Metaponto (Matera) in southern Italy. Generally the climate in the Metaponto area (0-300m a.s.l.) is a strong Mediterranean type with an annual rainfall less than 600 mm and an annual temperature trend consisting of mild or absent winters and hot summers. Sowing was done in mid November after a deep summer plowing and two secondary tillages. During summer tillage 120 kg/ha


11

of P2O5 was applied. Harvest occurred at the end of June at full maturity stage. Five randomly chosen plants from each entry from a single row plot were scored for the 18 quantitative and qualitative descriptors reported in Table 1. Frequency distributions for the qualitative descriptors flower and seed colour were also determined. Multivariate data analysis followed three steps: a) estimation of standardized entry means of 16 quantitative traits; b) derivation of orthogonal, uncorrelated traits for each entry using Principal Component Analysis (PCA); c) clustering entries into similarity groups using uncorrelated traits (PCA coefficients). The SAS procedure PRINCOMP computed the correlation matrix and determined the principal components (SAS Institute, 1987). The sum of the first eight PC axes, representing 88% of total variation were used in subsequent analyses. Entries were clustered using Ward’s minimum-variance method (SAS Institute, 1987). The cluster routine was stopped to form five discrete clusters looking for a consensus among the four statistics R2 (RSQ), cubic clustering criterion (CCC), pseudo-F (PSF) and pseudo-t2 (PST2). Results of this clustering were combined with results of the PCA analysis as a visual aid in discerning clusters. Results and Discussion Means, minimum and maximum values, coefficients of variation for 16 quantitative traits and the frequency distributions of the flower colour and seed colour are reported in Table 1. Entries showed a wide range of variation as evidenced by coefficients of variation. The most variable traits were seed yield, biomass, leaf width and seeds/pod; and the lowest values of variation were estimated for time to flowering and time of emergence; all the other traits showed intermediate values. The variability of means for yield components was lower than variability for seed yield. Extreme values for seed yield and biomass were 7.0-214.0 and 13.0-481.0g respectively. The distribution in frequency classes for flower colour showed that nearly 51% of entries were characterized by violet flowers; while the prevalent seed colour was beige (68.4%). The eigenvalues representing the variance of the principal components, and the cumulative percent of the eigenvalues indicating percentage contribution to the total variance attributable to each principal component are given in Table 2. Eigenvectors indicating the degree of association among original data and each principal components are also reported. The first two PC axes accounted for >48% of the multivariate variation among entries and the first eight axes >88% of variation, indicating a moderate degree

of correlation among traits for these entries. The first principal component accounted for 31% of variation reflected mostly influence on pod and seed traits. The second component accounts for 17% of the variance and thus is comparable in importance to the first. The traits with the largest coefficients and which contribute to it are the length of longest stem, biomass, seed yield and pedicel length. Time to flowering, leaf length, seed thickness, length of internode, leaf width, time to emergence and height of first podded node have some importance in the other components. Although there is no clear demarcation between important and unimportant principal components, it is interesting to note that yield and some yield components appear strongly in the first two components. Clustering entries based on similarity of the first eight principal components identified five large groups accounting for a 31% share of variance. Cluster memberships are reported in Table 3. Cluster I included the highest number of Italian entries; while a large number of Cyprus entries were in cluster II. Entries from the other less represented origins spread over all five groups. Table 1. Means, minimum and maximum values, and coefficient of variation (C.V.) for 16 quantitative descriptors and frequency distributions for 2 qualitative descriptors observed in 76 grass pea landraces. Descriptora Mean Min Max C.V.

Time to emergence (d) 23.9 21.0 33.0 5.8 Time to flowering (d) 79.5 74.0 90.0 3.1 Length of longest stem (cm) 74.0 25.0 98.0 18.9 Height first podded node (cm) 22.2 8.0 48.0 24.1 Length of internode (cm) 3.9 2,0 9.0 24.6 Leaf lenght (cm) 7.9 0.7 9.9 23.9 Leaf width (cm) 0.7 0.2 1.5 34.3 Pod lengthb (cm) 3.8 2.7 5.2 12.4 Pod widthb (cm) 1.3 0.8 2.0 17.7 Pedicel lengthb (cm) 5.0 1.7 8.5 23.0 Seeds/podb (no.) 2.6 1.0 5.0 33.8 Seed lengthc (cm) 0.8 0.4 1.5 25.0 Seed widthc (cm) 0.8 0.4 1.3 23.0 Seed thicknessc (cm) 0.5 0.2 1.0 16.0 Seed yield (g) 82.4 7.0 214.0 48.7 Biomass (g) 201.7 13.0 481.0 44.0 Colour Flower colour White Violet Pink - Frequency (%) 37.6 51.1 11.3 - Seed colour White Beige Brown Green

-grey Frequency (%) 3.9 68.4 5.5 7.9 aData collected on single plant; b Average of 5 dry pods/plant; c Average of 5 seeds/ plant.


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Table 2. Principal component analysis (PCA) of descriptors associated with 76 grass pea landraces showing eigenvalues and proportion of variation associated with the first eight axes and eigenvectors of descriptors. PC axis 1 2 3 4 5 6 7 8 Eigenvalues 5.01 2.61 1.67 1.38 1.06 0.98 0.71 0.62 Variation (%) com. 31 48 58 67 73 79 84 88 Descriptora Eigenvectors Time to emergence (d) -0.12 0.15 0.40 0.27 0.17 0.44 -0.45 0.35 Time to flowering (d) -0.11 0.04 0.61 0.04 -0.07 0.09 0.31 0.05 Length of longest stem (cm) 0.17 0.45 -0.18 0.00 0.11 0.00 -0.03 -0.40 Height of 1° podded node (cm) 0.25 0.13 0.18 0.36 0.28 0.20 -0.16 -0.49 Length of internode (cm) 0.11 0.21 -0.23 0.23 0.64 -0.14 0.26 0.20 Leaf lenght (cm) 0.08 0.11 0.21 -0.52 0.20 0.42 0.52 -0.13 Leaf width (cm) 0.06 -0.05 -0.41 -0.34 -0.01 0.60 -0.31 0.03 Pod lengthb (cm) 0.38 -0.04 0.13 -0.15 -0.06 -0.10 -0.10 0.14 Pod widthb (cm) 0.42 -0.09 0.07 -0.06 0.00 -0.07 -0.05 0.01 Pedicel lengthb (cm) -0.03 0.42 -0.01 -0.31 0.24 -0.19 -0.11 0.50 Seeds/podb (n.) -0.33 0.31 -0.07 0.02 -0.08 0.08 0.04 -0.08 Seed lengthc (cm) 0.42 -0.11 0.06 -0.01 0.01 -0.01 -0.04 0.11 Seed widthc (cm) 0.41 -0.14 0.07 -0.00 0.04 0.02 0.03 0.13 Seed thicknessc (cm) 0.12 -0.05 -0.31 0.46 -0.21 0.38 0.44 0.32 Seed yield (g) 0.18 0.43 -0.02 0.12 -0.40 -0.01 0.11 0.07 Biomass (g) 0.20 0.44 0.06 -0.05 -0.40 -0.02 -0.08 0.01 aData collected on single plant; b Average of 5 dry pods/plant; c Average of 5 seeds/ plant. Table 3. Cluster memberships: entry number and geographical origin of 76 grass pea landraces.

Cluster I (n=19) Entry1 Origin2 Entry Origin Entry Origin

100263 ITA 112411 CYP 115243 ITA 100288 ESP 106531 AUS 113090 ITA 115099 ITA 112252 ITA 100290 ITA 100291 MAR 103641 ITA 112390 CYP 115242 ITA 103203 ITA 113874 ITA 115795 BGR 100041 Unk.3 100042 Unk.3 100287 AUS

Cluster II (n=16) 106529 AUS 111982 ITA 112401 CYP 112414 CYP 112418 CYP 116171 ALB 112403 CYP 112399 CYP 112407 CYP 112415 CYP 112417 CYP 112419 CYP 112408 CYP 112412 CYP 116170 ALB 112410 CYP

Cluster III (n=15) 110434 ITA 110435 ITA 110437 ITA 110492 ITA 115833 HUN 110955 ITA 109680 ESP 110262 ITA 111986 ITA 100289 RUS 111985 ITA 115097 ITA 115834 HUN 100043 Unk.3 103585 ETH

Cluster IV (n=17) 112400 CYP 112416 CYP 113873 ITA 115653 ITA 103244 ITA 103376 ITA 103468 ETH 103579 ETH 113949 HUN 115093 ITA 115094 ITA 100293 Unk.3 103212 ITA 110957 ITA 112251 ITA 116250 ALB 113089 ITA

Cluster V (n=9) 100044 Unk. 112413 CYP 115096 ITA 100292 FRA 103237 ITA 106385 AUS 106434 ITA 106530 AUS 115241 ITA 1Mediterranean Germplasm number (MG); 2ISO Country code ; 3Unknown

Table 4. Cluster means of 16 quantitative descriptors observed in 76 grass pea landraces. Descriptor Cluster I II III IV V Time to emergence (days) 24 24 23 24 24 Time to flowering (days) 80 99 79 79 79 Length of longest stem (cm) 67 74 75 79 76 Height of 1st podded node (cm) 20.7 19.2 21.8 25.0 25.0Length of internode (cm) 3.7 3.9 3.7 3.9 4.5 Leaf length (cm) 7.3 8.3 11.0 8.0 7.5 Leaf width (cm) 0.7 0.7 0.8 0.8 0.6 Pod length (cm) 4.0 3.5 3.9 4.0 4.0 Pod width (cm) 1.3 1.0 0.8 1.2 1.2 Pedicel length (cm) 4.8 5.4 5.1 4.9 5.0 Seeds/pod (n.) 2 3 2 3 2 Seed length (cm) 0.9 0.6 0.9 0.9 0.9 Seed width (cm) 0.9 0.6 0.8 0.8 0.9 Seed thickness (cm) 0.5 0.5 0.4 0.5 0.4 Seed yield (g) 83.5 81.9 73.1 94.9 70.0Biomass (g) 195 197 206 228 163 The mean values of the original traits for each cluster are listed in Table 4. For some traits it was impossible to clearly differentiate the phenotypic diversity among clusters, such as the time to emergence and seed thickness; while, by the other traits the clusters were better differentiated. In particular, means indicate shorter plants with larger pods and seeds in cluster I. Entries in cluster II with smaller seeds and shorter pods flower nearly three weeks later. Highest yield and biomass means characterize entries in cluster IV, which also showed taller plants and higher first podded node. On the contrary, cluster V grouped entries with longer internode and lower mean values for yield and biomass. Entries in cluster III had larger leaves; indicated by leaf length and width.


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Fig 1. Plot of principal component analysis and clusters using the first two axes PRIN1 and PRIN2. Each cluster is represented by a different symbol. A combined spatial distribution of entries and clusters can be represented in two-dimensional scatter diagrams as shown in Figure 1. Conclusions The phenotypic diversity among grass pea entries was well defined by both Principal Component and Cluster analyses. Considering the different morpho-bio-agronomic descriptors, it has been possible to observe a remarkable inter and intra-group diversity. The covariation structure in the material studied revealed a different association between traits. The traits with dominant roles in the first two components are closely related to yield and yield components; while, vegetative traits like flowering time, height of first podded node and leaf traits were separately linked to other components. This suggests the possibility of obtaining, though selection, suitable genotypes combining high yield with desirable traits for direct release as cultivars in marginal areas of southern Italy. Cluster analysis also helped us to differentiate entries on the ground of their different levels of similarity. Five groups were identified with clear-cut differences according to the first principal component, which mostly accounted for yield and yield components. Smaller differences among groups were seen according to the second principal component, which accounted for vegetative traits. Compared with other groups, group IV show highest mean values for yield, biomass, seed size and height of first podded node, which are useful agronomic traits to breed new grass pea varieties. With the exception of group V, which included the less productive entries, groups I, II and III were moderately similar. Among the entries tested the analysis provides useful information in order to utilise directly the most promising materials for

production (for example MG 113089, MG 112416) or to use them in future selection programs. As reported in previous experiences (1,14) grouping germplasm entries into morphologically similar and presumably genetically similar groups is useful when little is known about the crop history and the population structure; as is the case for the grass pea collection. It is evident that entries clustered together are more alike than entries from other clusters. However, the data must be considered with caution because they are an expression of linked genetic and environmental effects. So, it is important to emphasise that the groups were defined on the basis of results from in a single location of southern Italy. The clusters could be different if the examination took place elsewhere. There seems to be no significant differences in relation to the origin of entries, most of which were from Italy and distributed in all groups. However, the entries from Cyprus with some exception were found to form a distinct group (II), which was characterized by entries showing smaller pods and seeds, longer pedicels and later flowering time. A more detailed geographical differentiation was impossible with many origins underrepresented, with the exception of Italy and Cyprus. In fact, at the level of five clusters, the proportion of variance accounted for by the clusters is 31%. This is a low percentage for the variance explained by the identified groups. Thus the differentiation according to these clusters can only be considered as a preliminary approach until more detailed analysis and information is available. Finally, the observed wide diversity in the Italian grass pea entries distributed in all groups suggests the use of this adapted material to breed new improved grass pea varieties.

Plot of PCAs and clusters using the first two axes, PRIN1 and PRIN2

0 1 2 3 4-1-2-3

0

1

2

3

-1

-2

-3

Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5


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References 1. Alba E, Polignano GB, Notarnicola L. 1997.

Diversity analysis in some Amaranthus entries. Agr. Med. 127, 198-204.

2. Bisignano V, Della Gatta C, Polignano GB. 2002. Variation for protein content and seed weight in grass pea (Lathyrus spp.) germplasm. Plant Genetic Resources Newsletter 132, 30-34.

3. Eyzaguirre PB, Paludosi S, Hodgkin T. 1999. IPGRI’s strategy for neglected and underutilized species and the human dimension of agrobiodiversity. In: Priority-setting for underutilized and neglected plant species of the Mediterranean region (Paludosi S, ed.). Report of the IPGRI Conference, 9-11 February 1998, ICARDA, Aleppo, Syria. IPGRI, Rome, Italy. Pp 1-20.

4. Enneking D, M. Wink. 2000. Towards the elimination of anti-nutritional factors in grain legumes. In: Linking Research and Marketing Opportunities for Pulses in the 21st Century (Knight R, ed.), Kluwer. Pp 671-681.

5. Erskine W, Smartt J, Muehlbauer FJ. 1994. Mimicry of lentil and the domestication of common vetch and grass pea. Economic Botany 48, 326-332.

6. Granati E, Bisignano V, Chiaretti D, Polignano GB, Crinò P. 2002. Characterization of Italian and exotic Lathyrus germplasm for quality traits. Genetic Resources and Crop Evolution 50, 273-280.

7. Kupicha FK, 1983. The infrageneric structure of Lathyrus. Notes from the Royal Botanic Garden Edinburgh 41, 287-326.

8. Hanbury CD, White CL, Mullan BP, Siddique KHM. 2000. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Animal Feed Science and Technology 87, 1-27.

9. Noto F, Poma I, Gristina L, Venezia G, Ferrotti F. 2001. Bioagronomic and qualitative characteristics in Lathyrus sativus lines. In: Proceedings 4th European Conference on Grain Legumes (eds. AEP), 8-12 July 2001, Cracow, Poland. P 183.

10. Polignano GB, Uggenti P, Perrino P. 2001. Phenotypic diversity in Bari grass pea (Lathyrus spp.) collection. In: Proceedings 4th European Conference on Grain Legumes (eds. AEP), 8-12 July 2001, Cracow, Poland. Pp 184-185.

11. Polignano GB, Uggenti P, Bisignano V, Alba E. 2003. Patterns of variation in Lathyrus sativus and some related species. Agr. Med. 133, 1-9

12. Sarker A, Robertson D, Campbell CG. 2000. Lathyrus spp.: Conserved Resources, Priorities for collection and future prospects. In: Linking Research and Marketing Opportunities for Pulses in the 21st Century (R. Knight, ed.), Kluwer. Pp 645-654.

13. SAS/STAT Guide for Personal Computers. Version 6. SAS Institute Inc., Cary, NC USA, 1987.

14. Souza E, Sorrells ME. 1991. Relationships among 70 North American Oat Germplasm: I. Cluster analysis using quantitative characters. Crop Sci 31, 599-605.

15. Zeven AC, Zhukovsky PM. 1975. Dictionary of cultivated plants and their centres of diversity. Centre for Agricultural Publication And Documentation, Wageningen, The Netherlands.

16. Zohary D, Hopf M. 1988. Domestication of Plants in the Old World. Clarendon, Oxford.


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Model plant type in Khesari (Lathyrus sativus L.) suitable for hill farming

Vedna Kumari and Rajendra Prasad

CSK, Himachal Pradesh Krishi Vishvavidyalaya, Oilseeds Research Station, Kangra, Himachal Pradesh 176001, India

Introduction Khesari is grown over large areas in Bangladesh, China, India, Burma, Nepal and Pakistan, as well as being cultivated in southern Europe and parts of Africa and South America. In India, it is widely grown in Madhya Pradesh, West Bengal, Maharastra and parts of eastern Uttar Pradesh despite the Government of India’s ban on its cultivation and sale. The crop has adaptive advantages, such as tolerance to moisture stress, requirement of minimum management, possessing good quality of amino acid composition in its protein and resistance to many biotic stresses. In spite of these attributes, the excessive consumption of khesari dal causes irreversible paralysis of lower limbs (neurolathyrism) due to the presence of a neurotoxin called (β-N-oxalyl-L-αβ- diaminopropionic acid or ODAP (3). This disorder is not confined to India alone, but has been present in other countries including Germany, Greece, Italy, Algeria, Bangladesh and Iran. The natural germplasm of grasspea in parts of the north-western hills of India has shown large variation in seed ODAP content. The landraces grown by farmers have poor plant type, low yield potential and high neurotoxin content (0.2 to >0.7%) that shows instability over locations and seasons. The basic cause of low yields in pulse crops, including grasspea is attributed to changes in plant type, which mainly includes morphological changes, many of which may be related to physiological functions as well. Material and Methods The experimental material for the present investigation comprised of 24 diverse and determinate landraces/germplasm lines collected from farmers fields from Kangra district as well as from the Division of Genetics, Indian Agricultural Research Institute, New Delhi. All the lines were grown in randomised complete block design with 3 replications at the experimental farm area of Himachal Pradesh Krishi Vishvavidyalaya, Palampur (32°6’N, altitude 1290 m). Each plot was 1.5 x 1.4 m with inter and intra row spacings of 30 and 15 cm, respectively. Locally recommended practices were used to raise the crop. Observations on various morphological characters and yield components were

recorded at appropriate stages; these included plant height, number of branches, days to flower, days to podding, days to maturity, pods per plant, seeds per pod, 100 seed weight and seed yield per plot. The seeds were biochemically analysed for ODAP content as per standard procedure. Mean values of 5 random plants per replication and seed yield from each plot were used for computing correlations. Path-coefficient analysis was performed (1). Results and Discussion The analysis of variance (not shown) for all characters except for days to maturity, showed the presence of substantial genetic variability. Seed yield was positively and significantly associated with plant height, days to flowering, days to podding, pods per plant and seeds per pod (Table 1). Seed weight was negatively but non-significantly correlated with seed yield. Plant height was had a significant positive correlation with days to flower. Further, days to flower and days to podding were strongly positively correlated. Seed ODAP content was significantly and positively correlated with number of branches, days to flowering, days to podding, pods per plant, seeds per pod and seed yield. Path-coefficient analysis (Table 2) showed that the seed ODAP content, pods per plant, plant height, days to podding and seeds per pod had direct positive effects on seed yield. The contribution of the remaining characters to seed yield was also mainly through these characters, their direct effect being negative. Seed ODAP content had the highest contribution to seed yield, followed by pods per plant and plant height. The effect of number of branches and seed weight was via pods per plant and plant height respectively. A comparison of correlations and path analysis revealed that plant height, days to podding, pods per plant and seeds per pod were the principal yield components. The character days to flower also contributed to seed yield but mainly via pods per


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plant. Path coefficient analysis revealed that pods per plant, followed by plant height contributed the most toward seed yield, while correlations also showed similar associations. A strong positive correlation of plant height, pods per plant and seeds per pod have previously been reported (2,4). An alternative approach to identify model plant characteristics is by comparing highest and lowest yielding lines in all morphological features to identify the character responsible for difference in yield potential (Table 3). There were significantly large differences in pods per plant, days to podding, plant height and seeds per pod which could account for differences in yield potential of these lines. These characters are incidentally the same ones indicated by correlations and path analysis. Further, a comparison of the pattern of seed yield in lines with high vis-à-vis low level of expression of

seed yield components would help to obtain their relative importance and consequently model plant architecture. This comparison also suggested large differences in yield potential due to the differences in pods per plant, followed by seeds per pod, days to podding, days to flowering, number of branches and plant height. Greater seed weight, though desirable in many pulse crops, was found to be a less important yield component in grasspea. The plant type suggested by correlation, path-coefficient analysis and from a comparison of plant morphology of high and low yielding line, as well as the seed yield pattern of lines with high vis-à-vis low level of expression of seed yield components agree to a large extent (5). Accordingly, such a model plant was characterised by higher numbers of days to flowering and podding, pods per plant, seed per pod and was definitely a taller plant.

Table 1. Correlation coefficients among different pairs of characters in grasspea.

Branches per plant

Days to flower

Days to podding

Pods per plant

Seeds per pod

100 seed weight

Seed yield per plot

Seed ODAP

Plant height -0.03 0.44* 0.36 0.32 0.35 0.29 0.47** 0.32 Branches per plant 0.08 0.01 0.36 0.19 0.06 0.22 0.41* Days to flower 0.75** 0.34 0.28 0.10 0.45* 0.44* Days to podding 0.24 0.30 0.12 0.41* 0.40* Pods per plant 0.31 -0.11 0.59** 0.45* Seeds per pod 0.05 0.45* 0.47* 100 seed weight -0.14 -0.23 Seed yield per plot 0.66**

* significant P<0.05, ** significant P<0.01 Table 2. Path-coefficients, direct (diagonal in bold) and indirect (off-diagonal) effects of characters on yield.

Plant height

Branches per plant

Days to flower

Days to podding

Pods per plant

Seeds per pod

100 seed weight

Seed ODAP

Correlation with yield

Plant height 0.212 0.002 0.004 0.033 0.097 0.031 -0.025 0.119 0.47** Branches per plant -0.006 -0.057 0.001 0.001 0.112 0.017 -0.005 0.154 0.22 Days to flower 0.093 -0.004 0.009 0.068 0.106 0.024 -0.009 0.165 0.45* Days to podding 0.076 -0.001 0.007 0.091 0.075 0.027 -0.010 0.149 0.41* Pods per plant 0.067 -0.021 0.003 0.022 0.309 0.027 0.010 0.170 0.59** Seeds per pod 0.075 -0.011 0.002 0.028 0.094 0.087 -0.004 0.176 0.45* 100 seed weight 0.060 -0.004 0.001 0.011 -0.034 0.004 -0.087 -0.087 -0.14 Seed ODAP 0.067 -0.023 0.004 0.036 0.139 0.041 0.020 0.376 0.66**

* significant P<0.05, ** significant P<0.01, Residual effect = 0.39


17

Table 3. Comparison of the highest and lowest yielding lines in grasspea. Character Highest Lowest Difference Seed yield (g/plot) 206.7 81.0 125.7* Plant height 56.3 52.4 3.9** Branches per plant 15.4 13.5 1.9 Days to flower 133.1 131.4 1.7 Days to podding 159.4 145.6 13.8** Pods per plant 50.0 33.8 16.2 Seeds per pod 3.0 2.6 0.5 100 seed weight 6.2 7.4 -1.2 * significant P<0.05, ** significant P<0.01

Table 4. Pattern of seed yield (g/plot) in lines with high and low levels of expression of quantitative traits in grasspea. Character Highest Lowest Difference Plant height 123.1 15.1 108.0** Branches per plant 124.5 15.1 109.4** Days to flower 179.0 31.0 148.0** Days to podding 184.4 35.2 149.2** Pods per plant 249.0 15.1 233.9** Seeds per pod 206.7 15.1 191.6** 100 seed weight 60.9 15.1 45.8 * significant P<0.05, ** significant P<0.01

References 1. Dewey DR, Lu KH. 1959. A correlation and path-

coefficient analysis of components of crested wheat grass seed production. Agron J 51, 515-518

2. Kumar S, Dubey DK. 2001. Variability, heritability and correlation studies in grasspea (Lathyrus sativus L.). Lathyrus Lathyrism Newsletter 2, 79-81.

3. Roy DN, Nagarajan V, Gopalan C. 1963. Production of neurolathyrism in chicks by injection of Lathyrus sativus concentrates. Current Sci 32, 116-118.

4. Sharma RN, Chitale MW, Ganvir GB, Geda AK, Pandey RL. 2000. Observations on the development of selection criterion for high yield and low neurotoxin in grasspea based on genetic resources. Lathyrus Lathyrism Newsletter 1, 15-16.

5. Solanki IS, Singh VP, Waldia RS. 1992. Model plant type in lentil (Lens culinaris Medik.). Legume Research 15, 1-6.


18

Resilience of South Asian disabling conditions: a glimpse of lathyrism among comparative histories

M. Miles

West Midlands, UK

E-mail: [email protected]

Some disabling conditions in South Asia, such as lathyrism, iodine deficiency disorders (IDD), cataract, poliomyelitis, epilepsy and leprosy, have mechanisms and impairment effects that differ substantially. Yet they share socio-cultural features that have promoted their strong resilience in face of efforts to eliminate them as public health problems. For each condition a 'magic bullet' or much-improved technical fix has been applied, often with increasing vigour over decades. Successes are reported in some regions during some periods (so far as genuine data can be distinguished from data adjusted to fit externally-prescribed 'targets'). Yet the longer view is that 'vertical' applications of a seemingly effective fix are not enough, without concurrent broad and long-term measures for poverty alleviation, community health education, and local self-help. The widespread resurgence of malaria and tuberculosis warns that none of these diseases or conditions is beaten. None can be relegated to low-profile 'mopping up' work. The eradication of smallpox may have been seriously misleading. Such a hypothesis was examined recently with focus on leprosy, comparing evidence from the public careers of other major disabling conditions including lathyrism (15). The idea was that the stigma and separateness of the 'leprosy world' has been self-reinforcing, and obscured some common factors. People involved with each disability-related condition have something to learn from history and from one another, though the precise biomedical focus may be dissimilar. There is nothing new in taking a broader comparative view. In the 1920s, disabling ailments such as goitre, rickets and lathyrism were mapped together, across South Asia, for epidemiological purposes (13). The historical background of lathyrism in South Asia, and socio-cultural features

foreshadowed there, are reproduced here to contribute to an appraisal of the broader picture. Evidence of Lathyrus sativus (grass pea or chickling vetch) cultivation has been found in Indian archaeological sites from the second millennium BC (17). A physical impairment attributed to eating khesari dal (the Indian food name of this pulse) was described in the period 200 BC - 200 CE, in Susruta's Nidanasthana: "When there is trembling in taking the first few steps with limping and when organisation of the joint gets loose, it is known as Kalayakhanja [Footnote: Kalaya -- Khesari pulse.]", though some translators are cautious about identifying lathyrism here (20,23). The condition is mentioned in South Asian writing in the late 16th century, in the Bhavaprakasa of Bhavamisra, and in Abul Fazal's Ain-i-Akbari (1,23). The latter notes that, "Kisari is the name of a pulse, resembling peas, eaten by the poor, but is unwholesome", with footnote on Kisari as Lathyrus sativus. Symptoms were more precisely described by the physician and surveyor Francis Buchanan reporting on Bihar and Patna in 1811-1812: "It seems to consist in a weakness and irregular motion of the muscles moving the knees, which are bent and moved with a tremulous irregular motion, somewhat as in the chorea, but not so violent. When the disease has lasted some time, and has become confirmed, the legs suffer emaciation. It is not accompanied by fever, but in the commencement is often, though not always, attended with pain" (4). The first institutional service for lathyrism sufferers was probably the Mejah Cripples' Asylum (Allahabad, India), maintained "by the charity of the local rajas and land-holders under the supervision of the Tahsildar" (21). Some blind inmates and some with leprosy were also listed, but this asylum arose mainly


19

to care for sufferers from lathyrism, affecting an estimated 4% of the local population in 1861. Between 1859 and 1868, the Indian Annals of Medical Science carried four detailed papers by James Irving, Civil Surgeon, Allahabad, reporting this cumulative disaster. He found it "remarkable that thousands of people, who know that a particular grain may render them lame, yet continue to use it for food. Is this because they must either eat the poison or starve? Will no other grain grow and be productive in the affected [areas]? If not at present - will drainage or other means not render the soil capable of bearing other and less deleterious crops? Are there no means, in fact, of inducing the people to give up the use of the poisonous food?" (10). Forty years later, Irving's questions were still unanswered. The government commissioned an enquiry into lathyrism, by Major Andrew Buchanan (6). He duly reported, and the report was approved and filed away. A few years later it was practically unknown and unobtainable (22). Sixty years after Irving's questions, the social problems were underlined by a senior pathologist, Major Hugh Acton. He went upcountry from Calcutta to examine 204 people with lathyrism, who were breaking limestone at a kiln. Acton estimated that 60,000 people had lathyrism in North Rewah alone, many of whom "migrate to the larger cities, Patna, Benares, Bombay and Calcutta, and form a large percentage of the beggar population" (2). Acton was a hard-boiled military scientist, sceptical of anything not viewable on a microscope slide; yet he concluded that the solution to lathyrism must be "a sociological one", starting with abolition of the rural debt-slavery that forced workers to accept risky food in lieu of wages. Sixty more years down the line, Dr Gopalan of the Nutrition Foundation of India noted that the Indian government in the 1950s had tried to ban the payment of Lathyrus sativus to agricultural workers in lieu of cash, and to dissuade rural folk from excessive consumption of kesari dal. These efforts "had no impact whatever" (7). Gopalan was publishing on lathyrism as early as 1950. In a remarkable turn, he noted in 1999 that lathyrism had now practically disappeared from some Indian regions where it was long endemic -- not because of any direct technical advance, but because "Green Revolution" investment in wheat and rice has reduced their price, while that of Lathyrus sativus (still widely

grown) has risen sharply. "Evidently, the poor landless labourers were being 'saved' from the poisonous seed not because of the researches and educational programme of the last two decades, but solely due to the intervention of market forces. The very greed and profit motive of the landed gentry, which for centuries was responsible for the perpetuation of neurolathyrism among the poor of Rewa, has apparently helped to redeem the poor by putting Lathyrus sativus out of their economic reach" (8). During the 1990s, researchers made laudable technical progress in breeding and testing types of Lathyrus sativus with the neurotoxin significantly reduced, while retaining the plant's remarkable capacity to flourish in barren conditions. Yet there remain complex tasks of promoting the new breeds, re-orienting the centuries-long folk awareness of lathyrism dangers, organising distribution networks so that the new seeds are used by hundreds of thousands of subsistence farmers scattered across South and West Asia and the Horn of Africa, and monitoring the feeding outcomes with humans and livestock. Technically this process could perhaps succeed in less than ten years; yet agricultural realities, and the imminent prospect of water catastrophes in the region, suggest that some decades may pass before the benefits of current research reach those needing them. Availability of research funding for technical advances may depend more on the commercial potential of the grass pea as a strong, high-protein crop, than the protection of subsistence farmers from paralysis. Health and nutrition development for rural people tends to be long, slow and of doubtful outcome, unless people see a sure and tangible gain for themselves, without drawbacks. The uncertainty factors can be verified from experiences in the cataract surgery field. The offer of 'eyesight regained' should be overwhelmingly attractive; yet hundreds of thousands of South Asians, within reach of low-cost cataract surgery, do not avail themselves of it. There are deep-rooted folk memories of 'development' and 'new methods' which turned out to have unexpected costs and pains. So what could be the drawbacks of 'new Lathyrus sativus'? One predictable irony of history could occur if, in 30 years time, lathyrism has been eliminated and a million Asian farmers have lost their smallholdings to agrobusinesses that mass-produce the toxin-free crop with minimal need of


20

human labour. There is more than one way to cripple a community, even with the most benevolent intentions. For further comparison, Gopalan notes "unforeseen factors" introduced by technological intervention, which are moving IDD nearer to the children of urban planners. Iodine deficiency, once considered a hill country problem, has been increasing in the densely populated plains of India. This may arise from intensive irrigation and multiple cropping, resulting in the depletion of soil micronutrients, plus food additives and contaminants that boost goitrogen or reduce body utilisation of iodine (8). Since no microbe works in a vacuum, no body lives solely in a laboratory, efforts are needed to 'foresee the unforeseen', without resort to astrology. Meanwhile, some Afghans still suffer lathyrism, unseen by modern health services. Lathyrus sativus has been cultivated in many parts of Afghanistan (5), and lathyrism seems to have been noticed in 1839, when Indian soldiers on a British military expedition to Kabul (probably via the Bolan Pass, Kandahar and Ghazni) suffered physical harm after being reduced to minimal food rations. To survive, they had supplemented their diet with khesari dal, though they knew the risks (10). Modern reports of lathyrism in Afghanistan appeared in 1953 and 1988, at Kabul (16,17). In 1999, a medical team visiting Afghanistan's north-eastern "Wakhan corridor", reported lathyrism endemic in several villages (19). The corridor runs north of Chitral and Gilgit in Pakistan, where in 1908 McCarrison found ten cases of lathyrism, all male,

among a village population of 101, and Mackenzie reported further cases (11,12). The timescale for rural change is usually long, but not all the inertia and delay can be blamed on villagers. International action in translating scientific discovery to practical effect, e.g. on iodinisation, smallpox vaccination, polio immunisation, or detoxifying lathyrus, is also painfully slow-moving. Coindet published the effective use of iodine against goitre in 1820, and by 1825 iodine was being applied successfully by David Scott, District Commissioner and amateur physician, in a remote area of Bengal (18). Yet prophylactic iodisation of salt took another 160 years to start being taken seriously in South Asia, and is still pursued in a half-hearted way. In the specific region where Scott made the first move, goitre remains endemic. Nearly half the Bangladesh population reportedly had goitres in 1993, though by 2000 this dropped below 20% (9,14). Major Acton in 1922 proposed an evidence-based plan of action against lathyrism, but lamented that "In India one publishes results and waits patiently for years to see them carried out into practice." (2). His comment could draw a grimace of recognition from scientists and development agents in any country at any time during the following eighty years. The historical lesson is that technical fixes alone are seldom sufficient. Concerted and sustained action is needed on many fronts and at many levels to address issues of poverty and exploitation, and to enlighten planners as well as the masses.

References 1. Abul Fazal. Ain-i-Akbari, transl. HS Jarrett, 1891.

Calcutta: Asiatic Society of Bengal, vol. II, 151. 2. Acton HW. 1922. An investigation into the

causation of lathyrism in man. Ind Med Gaz 57, 241-247.

3. Arya LS, Qureshi MA, Jabor A, Singh M. 1988. Lathyrism in Afghanistan. Ind J Pediatr 55, 440-442.

4. Buchanan F (n.d.) An Account of the Districts of Bihar and Patna in 1811-1812. Patna: Bihar & Orissa Research Society, p. 274.

5. Chrtkova-Zertova A, van der Maesen LJG, Rechinger KH. 1979. Papilionaceae I - Vicieae. Flora Iranica No. 140. Graz: Akademische Druck, pp. 78-79.

6. Editorial. 1903. The inquiry into lathyrism. Ind Med Gaz 38, 61-62.

7. Gopalan C. 1983. Recent studies and efforts. In: The Lathyrism Problem: current status and new dimensions. Nutrition Foundation of India.

8. Gopalan C. 1999. The changing epidemiology of malnutrition in a developing society - The effect of unforeseen factors. Bull. Nutrition Foundation of India 20, 1-5.

9. IDD Prevalence and Control Program Data: Bangladesh. At: www.people.virginia.edu/~jtd/ iccidd/mi/idd_014.htm

10. Irving J. 1860. Report on a species of palsy prevalent in Pergunnah Khyraghur. Ind Ann Med Sci 7 (No. 13), 127-137.

11. Mackenzie LHL. 1927. Lathyrism in the Gilgit Agency. Ind Med Gaz 62, 201-202.

12. McCarrison R. 1926. A note on lathyrism in the Gilgit Agency. Ind J Med Res 14, 379-381.


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13. Megaw JWD, Gupta JC. 1927. The geographical distribution of some of the diseases of India. Ind Med Gaz 62, 299-313.

14. Miles M. 1998. Goitre, cretinism and iodine in South Asia: historical perspectives on a continuing scourge. Med Hist 42, 47-67.

15. Miles M. 2003. Knowledge and management of disabling conditions in South Asian histories: implications for leprosy futures. Ind J Lepr 75, 153-167. Revised version at: www.disabilityworld.org/ 09-10_03/news/southasia.shtml .

16. Rouault De La Vigne A, Ahmad A. 1953. Lathyrisme en Afghanistan. Revue Médicale du Moyen-Orient 10, 325-336.

17. Saraswat KS. 1980. The ancient remains of the crop plants at Atranjikhera (c. 2000 - 1500 B.C.). J Ind Botan Soc 59, 306-319.

18. Scott D. 1825. Extract from a letter from D. Scott, Esq. Commissioner, Rungpore district, communicated by G. Swinton, etc. Trans Med Phys Soc Calcutta, 1, 367.

19. Simpson RA. 2002. North West Frontier mission in Afghanistan. Med J Australia 177 (11), 633-637.

20. Singhal GD, Singh LM, Singh KP. 1972. Diagnostic Considerations in Ancient Indian Surgery. Allahabad: Singhal, pp. 77-78.

21. Steel CD. 1884. Statistical, Descriptive, and Historical Account. Vol VIII, Pt II - Allahabad. NW Provinces & Oudh Govt Press, Allahabad, pp. 131-132, 203.

22. Stockman R. 1917. Lathyrism. Edinburgh Med J 19 (n.s.), 277-296.

23. Wujastyk D. 1998. The Roots of Ayurveda. New Delhi: Penguin, pp. 15, 169.


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Considerations on the reintroduction of grass pea in China

Hui-Min Yang* and Xiao-Yan Zhang

College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China

*E-mail: [email protected]

Introduction Grass pea (Lathyrus sativus L.) is probably the most drought tolerant legume crop and it is also resistant to moderate salinity. In drought prone areas the plant is considered an insurance crop for subsistence farmers. During drought-triggered famines, grass pea can be the only food available and a lifesaver. In addition, grass pea is valuable for its abundant nutrients, especially protein and lysine. Historically, grass pea has been a daily food for millions in Asia and Africa, and is now reintroduced into popular use in different parts of the world as a fine green manure, forage, and food. For better and safer use of grass pea, there have been a number of efforts of different research groups from different disciplines. As a developing country in which agriculture plays a very important role in people’s daily lives, China has to further develop its food productivity with high nutritional quality, and meanwhile protect the environment for coming generations. Grass pea, which has been utilized and then banned several decades ago, should be reintroduced into China and utilized again in various ways. Below we summarize the history of grass pea in China and discuss various considerations. This will contribute to the understanding of grass pea in China by the international community of researchers. The new challenges for China: food, environment, and health Food availability and human health China produced 25% of the global food yield and fed 22% of the world population on only 9% of the world farmland. However, yields decreased from 1996 and people suffered, especially in the areas of the north and west of China. For the purpose of national “food security”, it was necessary to provide sufficient and safe food as well as healthy nutrition (7), and efforts have been made to develop better crop varieties to improve food quality. In addition, it was also important to modulate food composition, i.e. the ratio of plant food to meat, in order to provide more nutrients from meat (which contains more proteins, lipids, etc.). A resolution to develop stockbreeding in some regions also implied the development of forage.

Undoubtedly a highly efficient crop plant species, which can be used as food and forage, would be an excellent candidate for this effort. Environment protection and ecosystem health The development of human activities has heavily influenced the environment and the global climate, and desertification of the land and the degradation of farmland has been a very serious problem for years in the north and west of China. The global changes in climate have also affected the environment, especially that of the rangelands. In the north and west of China, where rangelands are dominant, low precipitation, salinity, high temperature and high irradiation all lead to more severe drought stress. Water deficit results in the degradation of the rangeland and soil infertility. The deterioration of the environment has seriously affected the food yield and people’s food security, consequently the protection of environment has become urgent. In order to develop stockbreeding and also protect the environment, forage species with the potential to be used as food for the domestic animals and as ground cover in these regions should have high priority. The reintroduction of a highly resilient species is a very urgent requirement. The chosen species should be tolerant to the environmental stresses and should contribute to the protection of the environment. The nature and character of grass pea: the scope of its reintroduction High nutrient value Besides containing high amounts of starch, grass pea contains 26.3%-34.3% protein in the seed and 13.8%-20.1% protein in the stem and leaf, which is higher than those in Pisum sativum, Vicia faba or Medicago sativa. The protein contains 17 amino acids in sufficient quantities, especially lysine at levels higher than that in other legumes or in cereal crops (8). High tolerance and resistance to environmental stresses Grass pea plants can grow well under drought, cold and moderately saline conditions. They fix and use atmospheric nitrogen, require neither fertile soil nor


23

costly inputs, and can be the main food source when other crops fail due to drought stress. In addition, the plant can resist many pests and pathogens, and control some weeds (5). Multi-use for daily life Grass pea is a good green manure, forage, and food crop. As a legume plant, grass pea can fix atmospheric nitrogen and use it, and so is a good green manure for improvement of soil quality (5). It covers the ground well, acting to reduce evaporation and conserve soil water. In spite of the neurotoxin ODAP (3-(-N-oxalyl)-L-2,3-diamino propionic acid), the stem, leaf, and seed of grass pea can be used as fine forage and is suitable for feeding cattle, sheep, etc. In addition, as a food source it can also serve people in seriously stressed areas due to high protein content and other nutrients, as well as retaining productivity under stress. Studies on grass pea in China: the theoretical and technological basis for reintroduction The brief utilization of grass pea in China Grass pea-like species have been reported in China in antiquity (10). Although it might not be the same species as that today, it indicated the existence of grass pea. There are at least 43 varieties of L. sativus stored in the Institute of Crop Germplasm Resources of China, however most varieties planted at present were introduced from abroad. Before the 1960s, grass pea was widely planted in northwest China, such as Shanxi, Sichuan, Yunnan and Gansu provinces. It was reported that the planting area exceeded 20,000 ha at that time in Gansu province alone (15), the crop was mainly used as green manure and forage. In the 1970s, a severe drought occurred and caused a terrible famine in this area. Since grass pea could cope with the severe drought, poor soil fertility, and cost little, it was widely planted and its seed was used as a main food source. The continuous consumption of grass pea seeds as almost the only food available caused an epidemic of “lathyrism” in this region during the period of famine. The lathyrism epidemic led to the banning of the utilization and development of grass pea in China, but studies continued. These researches ranged from the investigation of clinical symptoms, the toxicity mechanism, breeding to produce low-toxicity varieties, and to the development of detoxification methods. Clinical investigations The clinical effects of the consumption of grass pea were studied in various kinds of experimental animals to understand how grass pea affects human health (3, 6,

14, 36). This contributed to understanding the mechanisms of lathyrism and its clinical symptoms,

and confirmed that ODAP caused lathyrism and even determined the lethal dose of ODAP. However there was still no effective way to cure lathyrism patients. Analysis of ODAP Since its discovery in 1964 (16, 20), multidisciplinary research in many groups has focused on ODAP. All these efforts heavily depended on the analytical methods available for quantification of ODAP, over time researchers have developed different methods. A paper-based chromatographic method was established to detect the presence of ODAP (6). The research group in Lanzhou University established a paper chromatography-based colorimetric analytical method to quantitatively measure ODAP for the first time in China (6). This method could quantitatively measure ODAP content, but was not very accurate and could not separate the isomers, a modified method was then developed (3, 11). The method established by Rao (19) proved to be a good and cheap one to measure ODAP, it had been used widely by researchers with some modifications (3, 12, 13), since it could not separate the two isomers, was not reproducible and time-consuming, its use is therefore limited. The accurate measurement of α- and β-ODAP separately seemed especially necessary, since the former isomer was proven to be non-toxic. Zhao et al. (38, 39) developed a capillary zone electrophoresis (CZE) method to measure ODAP, with the successful separation of the two isomers and another non-protein amino acid. This method still had the drawback that the high working pH (9.2) easily caused the hydrolysis of ODAP to DAP. A high performance liquid chromatographic (HPLC) method has been developed and improved, proving efficient and based on widely used technology. The HPLC method was further developed using off-line pre-column derivatization with 6-aminoquinolyl-N-hydroxy-succinimyl carbamate (AQC) (2). Other improvements were pre-column derivatization with 1-fluoro-2, 4-dinitrobenzene(23), or dansyl chloride (25, 26). These modified methods could accurately separate the two isomers and developed into a classic routine method to measure ODAP. However, the above methods still have some problems, such as experimental inconvenience, time-consuming derivatization steps, and especially, the easy decomposition of the derivatives. Further optimization of ODAP analysis is expected. Efforts on detoxification For the safe use of grass pea, much effort on crop improvement has been addressed, including direct seed treatment, mutagenesis and mutant breeding. All these approaches are described below.


24

It was found that water soaking of grass pea seed could lower the ODAP content but not sufficiently for continuous safe human consumption (6). Further treatment of the seed into some products could almost remove all toxicity and be safe (6), but these methods were too complicated to be widely used and reduced the nutritional components in the seed, such as water-soluble vitamins and minerals. Physical and chemical treatments have also been used in the detoxification of grass pea. Treatment of the seeds with highly charged C6+ ions could induce some mutants (22). Some mutants were also obtained using the 60Co γ-ray irradiation combined with ethyl methane sulfonate (EMS) treatment (18). These treatments could lower ODAP content to some extent in the seedlings but not in the F1 seeds. The mutants were not widely used as their characters were unstable or ODAP content was not sufficiently low. Further efforts on this aspect could still give results but have not been pursued. Mutation would be a good choice for further improvements if combined with genetic engineering methods. Agronomists have also paid much attention to the breeding selection of low toxicity varieties. Two low toxin varieties were found among 14 local strains (4), further tests were performed by the addition of phosphorus to the soil in an attempt to select a stable and low toxin variety (3). Yu et al. (36) studied some local and introduced varieties to investigate the relationships between location, species, genus and ODAP content. Bao et al. (1) screened 65 varieties looking for low toxin lines. These comparative studies afforded some data on the development of low toxin varieties. Physiological and eco-physiological studies Since some low toxin varieties have been obtained, some researchers turned their eyes to the physiology and eco-physiology of grass pea. Increases in ODAP content were observed under prolonged water stress or osmotic stress (27-30). Polyamines in the seedlings were closely related to ODAP content under water stress induced by PEG treatment (29). It was found that ABA (28) and H2O2 (30) accumulation affected ODAP content when the plant suffered drought stress. During seed germination, ODAP content in the radicles increased as water deficit extended, while ODAP decreased in the cotyledons (31, 32). Zhou et al. (40) concluded that ODAP could scavenge the hydroxyl radical. Extensive experimentation showed that ODAP could protect the activity of glycolate oxidase under stress conditions

(37). In addition, oxalate influenced ODAP content and its distribution in the plant(24). Physiological characteristics of grass pea during treatment with Eu3+, Ca2+ and PEG were studied under laboratory conditions (21). The results showed effects on the moisture levels, root activity, Na+-K+ ATPase, antioxidases, hydroxyl radicals and MDA content, polyamines, chlorophyll and carotenoid content, proline and other free amino acids. This represents a detailed study of this aspect of grass pea and implies that some mechanisms exist in this species to respond and cope with environmental stress. Our group has made some efforts to study the eco-physiology of grass pea under water deficit conditions (34, 35). Drought may affect stomata, photosynthesis, and yield of grass pea plant as well as ODAP content (35). We found a significant relationship between stomata and photosynthesis and yield, and drought may enhance the correlations (34), more extensive research is proceeding. Concluding remarks Grass pea is a fine green manure, forage, and food species. Its multiple beneficial properties and various uses make it suitable for reintroduction into China, completed studies show good groundwork for these purposes (33). Reintroduction of grass pea will help improve food availability and environment protection, not only in China, but also in other parts of the world with similar environmental conditions. The studies on grass pea are being carried out in a multidisciplinary approach by researchers in China, in some instances ahead of other international efforts. Development of low toxin varieties could be further advanced. A lot of work has been carried out in breeding, selection, detoxification (9) and the metabolic pathway of ODAP (17) in the past. Now attention should be turned to the ecological, physiological and biochemical properties of grass pea. In addition, further work should be devoted to incorporation of molecular and gene engineering technology. Acknowledgements This work was supported by the International Cooperation Project between China and Australia (2005DFA30030-6) and the Cuiying Programme of Lanzhou University, Lanzhou, China (to H-MY).


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References 1. Bao XG, Lv FH, Liu SZ, Shu QP. 1995. Sifting

and cultivating utilization of low toxin varieties of Lathyrus. Pratacultural Science 12, 48-54.

2. Chen X, Wang F, Chen Q, Qin XC, Li ZX. 2000. Analysis of neurotoxin 3-N-oxalyl-L-2,3-diaminopropionic acid and its α-isomer in Lathyrus sativus by high-performance liquid chromatography with 6-aninoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Journal of Agriculture and Food Chemistry 48, 3383-3386.

3. Chen YZ, Li ZX, Lv FH, Bao XG, Liu SZ, Liu XC, Zhang GW, Li YR. 1992. Studies on the screening of low toxic species of Lathyrus, analysis of toxins and toxicology. Journal of Lanzhou University (Natural Sciences) 28, 93-98.

4. Chen YZ, Wang XG, Ma ZL, Cai WT. 1980. Studies on Lathyrus sativus L. Journal of Lanzhou University (Natural Sciences) 16, 114-115.

5. Das NR. 2000. Lathyrus sativus in rainfed multiple cropping systems in West Bengal, India-a review. Lathyrus Lathyrism Newsletter 1, 25-27.

6. Department of Chemistry, Lanzhou University. 1975. Analysis on the toxin and studies on the removal of the toxin in Lathyrus sativus. Journal of Lanzhou University (Natural Sciences) 11(2), 45-65.

7. Food and Agriculture Organization of the United Nation. 2004. The state of world food security. In: Scanes CG, Miranowski JA. Perspectives in World Food and Agriculture. Iowa, USA: Iowa State Press. 3-29.

8. Hanbury CD, White CL, Mullan BP, Siddique KHM. 2000. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Animal Feed Science and Technology 87, 1-27.

9. Jiao CJ, Wang CY, Li ZX, Wang YF. 2005. Advances in both toxic biochemistry and detoxification of Lathyrus sativus. Acta Prataculturae Sinica 14(1), 100-105.

10. Li SZ. 1578. Ben Cao Gang Mu. Vol. 16, issue 24 for grain, 160.

11. Li ZX, Cai WT, Chen YZ. 1986. Paper chromatography with scanner for determination of BOAA of Lathyrus sativus. Journal of Lanzhou University (Natural Sciences) 22(2), 76-80.

12. Li ZX, Cai WT, Xu ZG. 1987. The determination of ODAP in Lathyrus sativus using OPT colorimetry. Journal of Lanzhou University (Natural Sciences) 23(2), 130-132.

13. Li ZX, Meng YF, Zhang L, Chen YZ. 1992. Determination of α- and β-oxalyldiaminopropionic acid in Lathyrus sativus

by electrophoresis. Journal of Lanzhou University (Natural Sciences) 28(3), 89-92.

14. Liu XC, Zhang GW, Li YR, Wang JX, Liang ZN. 1989. Toxicological study on grass pea vine (Lathyrus sativus L.) and its toxic-component BOAA. Scientia Agricultura Sinica 22(5), 86-93.

15. Lv FH, Bao XG, Liu SZ. 1990. A study of vetchling (Lathyrus L.) germplasm resources. Crop Genetic Resources (China) 33(3), 17-19.

16. Murti VVS, Seshadri TR, Venkitasubramanian TA. 1964. Neurotoxic compound of the seeds of Lathyrus sativus. Phytochemistry 3, 73-78.

17. Qin XC, Li ZX, Wang YF. 2000. Studies of Lathyrus sativus and its neurotoxin (ODAP). Chinese Bulletin of Life Sciences 12, 52-56.

18. Qin XC, Wang F, Wang XJ, Zhou GK, Li ZX. 2000. Effect of combined treatment of 60Coγ-ray and EMS on antioxidase activity and ODAP content in Lathyrus sativus. Chinese Journal of Applied Ecology 11(6), 957-958.

19. Rao SLN. 1978. A sensitive and specific colormetric method for determination of α, β-diaminopropionic acid and Lathyrus sativus neurotoxin. Analytical Biochemistry 86, 386-395.

20. Rao SLN, Adiga PR, Saima PS. 1964. The isolation and characterization of β-N-oxalyl-α,β-diaminopropionic acid: a neurotoxin from the seeds of Lathyrus sativus. Biochemistry 3, 432-436.

21. Tian H. 2003. Study of Stress Resistance on Different Environment in Lathyrus sativus L. Ph D thesis. Lanzhou University, Lanzhou, China.

22. Wang CY, Yang HM, Wang YF. 1993. Mutation effect of C6+ heavy ion on seed of Lathyrus sativus L. Hereditas (Beijing) 15(1), 28-31.

23. Wang F, Chen X, Chen Q, Qin XC, Li ZX. 2000. Determination of neurotoxin 3-N-oxalyl-L-2,3-diaminopropionic acid and non-protein amino acids in Lathyrus sativus by precolumn derivatization with 1-fluoro-2, 4-dinitrobenzene. Journal of Chromatography A 883, 113-118.

24. Xing GM. 2002. Molecular and Physiological Responses to Stress in the Wheat Line with Resistance Rust and Lathyrus sativus L. Ph D thesis. Lanzhou University, Lanzhou, China.

25. Xing GM, Chen X, Li ZX. 2001. Quantitative high-performance liquid chromatography determination of neurotoxin β-ODAP and α-isomer in grass pea (Lathyrus sativus). Chemical Research in Chinese Universities 17, 224-225.

26. Xing GM, Wang F, Cui KR, Li ZX. 2001. Assay of neurotoxin β-ODAP and non-protein amino acids in Lathyrus sativus by high-performance liquid chromatography with dansylation. Analytical Letters 34(15), 2649-2657.

27. Xing GS, Cui KR, Li J, Wang YF, Li ZX. 2001. Water stress and accumulation of β-N-oxalyl-L-α, β-diaminopropionic acid in grass pea


26

(Lathyrus sativus). Journal of Agriculture and Food Chemistry 49, 216-220.

28. Xing GS, Zhou GK, Li ZX. 2000. Accumulation of ABA and ODAP in Lathyrus sativus under water stress. Chinese Journal of Applied Ecology 11(5), 693-698.

29. Xing GS, Zhou GK, Li ZX, Cui KR. 2000. Studies of polyamine metabolism and β-N-oxalyl-L-α,β-diaminopropionic acid accumulation in grass pea (Lathyrus sativus) under water stress. Acta Botanica Sinica 42(10), 1039-1044.

30. Xing GS, Zhou GK, Li ZX, Cui KR. 2001. Effect of osmotic stress on accumulation of H2O2 and ODAP in grass pea (Lathyrus sativus). Acta Phytophysiologica Sinica 27(1), 5-8.

31. Xue S, Zhang LS, Cao R, Wang MH, Wang PH. 2001. Effect of drought on β-ODAP and amino acids in the process of Lathyrus seed germination. Acta Botanica Boreali-Occidentalia Sinica 21(4), 620-624.

32. Xue S, Zhang LS, Cao R, Wang MH, Wang PH. 2001. Effects of water stress on the proteins and free amino acid of the Lathyrus sativus seed during the germination. Journal of Northwest Sci-Tech University of Agriculture and Forestry (Natural Science Edition) 29(5), 79-83.

33. Yan ZY, Xing GM, Wang CY, Wang YF, Li ZX. 2004. Recent advance in Lathyrus sativus and toxin ODAP. Acta Botanica Boreali-Occidentalia Sinica 24(5), 911-920.

34. Yang HM, Zhang XY, Wang GX. 2004. Relationships between stomatal character, photosynthetic character and seed chemical composition in grass pea at different water

availabilities. Journal of Agricultural Science 142, 675-681.

35. Yang HM, Zhang XY, Wang GX, Wang YF, Qiao LX. 2004. Stomatal characteristics and the contents of seed ODAP, protein and starch in two varieties of grass pea under stress condition. Journal of Lanzhou University (Natural Sciences) 40, 64-67.

36. Yu JZ, Wang HQ, Yu ZC, Chen YZ, Ma ZL, Cai WT. 1994. Researches of biological laws of lower noxious Lathyrus sativus and lower poison varieties selecting. Acta Botanica Boreali-Occidentalia Sinica 3, 94-98.

37. Zhang J, Xing GM, Yan ZY, Li ZX. 2003. β-N-oxalyl-L-α,β-diaminopropionic acid protects the activity of glycolate oxidase in Lathyrus sativus seedlings under high light. Russian Journal of Plant Physiology 50(5), 618-622.

38. Zhao L, Chen XG, Hu ZD, Li Q, Chen Q, Li ZX. 1999a. Analysis of β-N-oxalyl-L-α,β-diaminopropionic acid and homoarginine in Lathyrus sativus by capillary zone electrophoresis. Journal of Chromatography A 857, 295-302.

39. Zhao L, Li ZX, Li GB, Chen XG, Hu ZD. 1999. Kinetics studies on thermal isomerization of β-N-oxalyl-L-α,β-diaminopropionic acid by capillary zone electrophoresis. Physical Chemistry Chemical Physics 1, 3771-3773.

40. Zhou GK, Kong YZ, Cui KR, Li ZX, Wang YF. 2001. Hydroxy radical scavenging activity of β-N-oxalyl-L-α,β-diaminopropionic acid. Phytochemistry 58, 759-762.


27

Effects of drought on stomatal character, photosynthetic character and seed chemical composition in grass pea, and their relationships

Hui-Min Yang1*, Xiao-Yan Zhang1 and Gen-Xuan Wang2

1. College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China.

2. College of Life Sciences, Zhejiang University, Hangzhou 310027, China *Email: [email protected]

Full article published in Journal of Agricultural Science (2004) 142: 675-681.

Summary The neurotoxin β-N-oxalyl-L-α, β-diaminopropionic

acid (β-ODAP) in grass pea (Lathyrus sativus L.) can

cause the disease “lathyrism”. Much work has been

done on lowering toxicity and on selection of low

toxicity varieties, while research on the eco-

physiological characteristics of grass pea is very rare.

Stomatal character (stomatal density and aperture),

photosynthetic character (Pn and E) and seed chemical

composition (ODAP, protein and starch) were

measured in four varieties of L. sativus at different

water availabilities. Under drought conditions,

stomatal aperture, Pn and E were lower than those

under the control, while the other parameters were

higher. A significant positive correlation was observed

between stomatal density and water use efficiency

(WUE), while negative correlations were found

between stomatal density and the remaining

parameters. Obvious positive correlations were also

observed between stomatal aperture and Pn, E, the

concentrations of seed ODAP, protein and starch,

while a negative correlation appeared between

stomatal aperture and WUE. Under drought

conditions, R2 values were more comparable with the

control. Intriguingly, the R2 values of stomatal

aperture were higher than of stomatal density,

especially under drought conditions. These results

indicate that stomatal aperture may be more closely

related to photosynthetic character and seed chemical

composition in grass pea, and drought may enhance

the correlations.

Grass pea is a potentially valuable feed and food crop

in semi-arid regions. Our group is continuing research

on the regulation of water and nutrient utilization

efficiency in grass pea. Further researches in grass pea

are waiting for multidisciplinary efforts.


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Scope of growing lathyrus and lentil in relay cropping systems after rice in West Bengal, India

S. Gupta* and M.K. Bhowmick

Pulses and Oilseeds Research Station (PORS)

Berhampore-742-101, West Bengal, India.

* Email: [email protected] OR [email protected]

Introduction The State of West Bengal in the eastern part of India, bordering Bangladesh, is a unique example where rice is cultivated in all the three seasons viz., summer, autumn and winter. The State has to feed almost 70 million people with the support of only 5.8 million hectares of cultivable land. Since independence the state has, therefore, had to resort to more areas under rice than for other crops, especially pulses, the productivity of which are comparatively low. At present the area under rice occupies about 66 percent of the total gross cropped area, which is about 9.24 million hectares with an average cropping intensity of 171 percent (9). The total area under pulses has diminished gradually every year from 582,000 ha during 1957-58 to 242,000 ha during 2002-03 (11). It is a general practice of the farmers of this region to sow various winter pulse crops like lentil (Lens culinaris L.), lathyrus (Lathyrus sativus L.), chickpea (Cicer arietinum L.) and pea (Pisum sativum) in the standing rice crop field, just before the harvest to ensure germination using the residual moisture and to avoid tillage operations during pulse growing. Such a relay cropping operation (known by the terms utera or paira) is very popular for growing lathyrus. Subsequently due to high ODAP content of local land races and also with the advent of irrigation facilities, the farmers tended to shift from relay cropping of lathyrus to more remunerative crops like rapeseed, mustard, potato, other vegetables and winter rice which require more water. Thus the area under lathyrus, in particular, diminished drastically from 54,000 ha during 1981-82 to 40,000 ha during 2000-01 (10). With the backdrop of the aforesaid facts, efforts were concentrated to develop high yielding lathyrus varieties with ODAP content less than a critical limit (0.20 %). A variety Nirmal (B-1) was developed at the Pulses and Oilseeds Research Station (PORS). Other varieties (BioL 212, BioR 202, LSD-3 and P-24) are also of late recommended by the Indian Council of Agricultural Research for cultivation in India. Therefore, the PORS conducted a series of experiments to establish the suitability for growing pulses in the utera cropping

system; especially in the mono-cropped areas of the state situated in the Coastal, Old Alluvial and Terai (Foot Hills) Zones of West Bengal with total area under cultivation comprising nearly 1.36 million hectares. Recently efforts are also going on to grow black gram (Vigna mungo) and green gram (Vigna radiata) under utera condition in wheat fallows with the objective to grow an early duration pulse as a catch crop and to sustain soil health in wheat-rice rotations.

Experimental Findings and Discussion The PORS, Berhampore, West Bengal, India has engaged in various field experiments since 1970-71 to identify the cultivars suitable for the utera system, to standardize the improved package of practices. As such, a trial was undertaken at the Block Seed Farm, Kandi, Murshidabad, West Bengal in 1970-71 with different varieties of lentil and lathyrus. Seeds of lentil and lathyrus were broadcast on standing aman (wet season) paddy field (variety Bhasamanik) @ 50 kg per hectare on 13th and 14th November 1970, respectively, 15 days before rice harvest. The variety yields are presented in Table 1. Table 1. Yield of different varieties of lentil and lathyrus grown as utera at Block Seed Farm, Kandi, Murshidabad, India in 1970-71. Lentil Yield

(kg/ha) Lathyrus Yield

(kg/ha) L6-42 578 Kh-3 3878 L6-8 1022 Kh-4 3306 L6-85 1056 Kh-18 2817 LL6-4 1089 Kh-17 2672 L6-77 1111 B-77 1133 L6-21 1478 L6-36 1500 L6-20 1556 L6-23 2256 Source: Chakraborty et al. (13)


29

It is evident from Table 1 that both lentil and lathyrus could be successfully grown under muddy land conditions in the standing rice crop 15 days before its maturity, provided that there is no standing water in the field. The yield ranged from 578 to 2256 kg/ha in case of lentil and 2672 to 3878 kg/ha in case of lathyrus, compared to the respective average yield of 485 kg/ha and 511 kg/ha for these two crops in the state (13). During 1999-2000 and 2000-01 different genotypes of lathyrus were tested for their suitability and competitiveness for growing under utera conditions in muddy land of standing rice fields in mono-cropped

coastal saline tracts of West Bengal. The trials were laid out in two Government Farms viz., Sub-Divisional Adaptive Research Farm, Baruipur and Block Seed Farm, Mathurapur. The yield data obtained are in Table 2. The variety Nirmal developed at PORS is the most adapted (Table 2) in the rice-utera system in the Coastal Zone of the state. The field experiments were conducted under rainfed conditions on a clayey soil where salinity develops gradually after cessation of rainfall in the dry winter months, when the crop was grown.

Table 2. Seed yield of lathyrus after rice in utera conditions at 2 sites (Mathurapur and Baruipur) in the coastal zone of West Bengal.

Varieties 1999-2000 2000-2001 Mean yield Mathurapur Baruipur Baruipur (kg/ha) BioL 212 2083 1104 775 1321 BioR 202 1875 1251 650 1259 LSD 3 1782 923 670 1125 P-24 1771 855 570 1065 P-505 1375 1004 670 1016 Nirmal 1729 1529 1295 1494 LSD (P<0.05) 268 162 104 - CV (%) 10.1 9.7 10.2 -

Source: Annual Reports (4,5)

Table 3. Performance of lentil genotypes in rice-utera conditions.

Variety Seed yield (kg/ha) 2001-02 2003-04 L 4661 - 1233 WBL-75 - 1642 ILL 8006 - 666 B-77 903 1366 WBL-58 1158 1708 PL-639 - 908 IPL-304 - 592 K-75 841 - LSD(P <0.05) 26.8 -

Source: Annual Reports (6,8) Table 4. Time to flowering and maturity and 1000 grain weight of 7 varieties of lentil grown in rice-utera conditions.

Variety Days to 50% flowering

Days to maturity

1000 grain wt. (g)

L-4661 77 122 22.33 WBL-75 84 127 18.29 ILL 8006 66 114 20.32 B-77 69 120 19.98 WBL-58 68 126 21.48 PL-639 70 133 15.62 IPL-304 73 139 28.76

Source: Annual Report (8)

Likewise, variety suitability and competitiveness of lentil genotypes under rice-utera system were tested for two years at PORS. Altogether 8 cultivars were included in the experimentation during 2003-04, whereas only 3 varieties were tested during 2001-02. The yield data of individual lentil genotype tested for the two years are presented in Table 3. The duration and other characters of individual variety recorded during 2003-04 are in Table 4. The rice variety cultivated in the experiment was MTU 7029, which matured in 140-145 DAS. The maturity period of all lentil varieties (Table 4) were delayed for at least 10 days when grown under utera cropping system. Genotypes with characteristics of early vigour and comparatively tall stature could perform well under utera conditions. Similar observations were made of lathyrus cultivars when grown under utera conditions. Some experiments were conducted to optimise sowing times and other agronomic management in the utera system of sowing of pulses in muddy fields of standing rice, utilizing residual moisture and fertilizer. One such trial was conducted in 1994-95 with lathyrus under rice-utera conditions and the results are given in Table 5.


30

Table 5. Effect of sowing time and weed management on lathyrus yield under rice-utera conditions during 1994-95 (rice variety IR-36 and lathyrus variety Nirmal). Treatment Seed yield

(kg/ha) Sowing time (days before rice harvest) 10 743 20 973 30 824 LSD (P<0.05) 100 Weed Management No hand weeding 500 One hand weeding at 45 DAS 932 Two hand weedings at 45 and 60 DAS 1100 LSD (P<0.05) 100 CV (%) 14.04 LSD(P<0.05) for sowing time x weeding interaction

NS

Source: Annual Report (3)

The yield of lathyrus variety Nirmal was maximum when sown 20 days before harvest of rice (Table 5). Keeping the plot weed free after rice harvest was found to be significantly superior to the existing farmers’ practice of not weeding the plot. Another experiment was conducted during 1999-2000 and 2001-2002 to verify the sowing time results of 1994-95 and to examine whether basal fertilizer by means of placement of fertilizer in between the rows of standing rice crop had any effect on yield of utera crop of lathyrus. The results are given in Table 6. The results in the Table 6 clearly corroborated the results of the previous experiment of 1994-95. Sowing at 15-20 days before rice harvest gave a better result in respect of yield, probably due to the fact that earlier sowing might have more effective utilization of residual moisture in the rice field. Duary et al. (14) were of the same opinion. The utera crop might need some initial nutrition as a starter dose of NPK, possibly to enhance symbiotic activities with the Rhizobium bacteria.

Table 6. Effect of sowing time and fertilizer management on seed yield of lathyrus under rice-utera conditions (rice variety MTU 7029 and lathyrus variety Nirmal)

Treatment Seed yield (kg/ha) 1999-2000 2001-2002 Mean Sowing time (days before rice harvest) 0 - 889 889 10 1150 912 1031 15 1225 993 1109 20 1525 - 1525 LSD (P<0.05) 69 - - CV (%) 6 - - Fertilizer(N:P205:K20 in kg/ha) No fertilizer (Control) 1033 799 916 10 : 25 : 20 (Basal) 1300 890 1095 20 : 50 : 20 (Basal) 1317 1022 1169 10 : 25 : 20 (Basal) + DAP spray (2% solution) at flowering

1550 1013 1282

LSD (P<0.05) 88 - - CV (%) 8 5 - LSD (P<0.05) for sowing time x fertilizer interaction

153 71 -

Source: Annual Report (4,6)


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Table 7. Effect of seed soaking and foliar nutrition on lathyrus under rice-utera conditions during 2003-04 (rice variety IR-36 and lathyrus variety BioL 212).

Treatment Mature plant

height (cm)

Branches/ plant

Pods/ plant

Seeds/pod 1000 seed weight (g)

Seed yield (kg/ha)

Seed priming No soaking 45 3.69 8.94 2.22 63.6 731 Soaking in water for 6 h 50 3.87 9.48 2.54 66.6 802 Soaking in 2% KH2PO4 solution for 6 h 52 3.92 10.70 2.76 70.4 910 Sowing of sprouted seeds 54 4.26 11.55 3.00 71.8 938 LSD (P<0.05) 3.0 0.23 0.90 0.18 1.7 53 Foliar nutrition Control 46 3.68 9.18 2.44 66.4 675 Water spray 48 3.79 9.62 2.54 67.7 798 Urea spray (2% solution) 54 4.29 11.25 2.87 69.5 963 DAP spray (2% solution) 51 4.11 10.84 2.69 68.7 912 KCl spray (2% solution) 51 3.81 9.95 2.63 68.1 878 LSD (P<0.05) C.V.(%)

3.4 8.2

0.26 8.1

1.01 12.1

0.20 9.33

1.9 3.5

58.7 8.4

Source: Annual Report (8) Table 8. Effect of sowing time and seed priming on yield and other characters of lentil sown under rice-utera conditions during 2003-04 (lentil variety WBL-58 and rice variety MTU-7029).

Treatment Plant population (‘000/ha)

Mature plant height

(cm)

Pods/plant 1000 seed weight (g)

Seed yield (kg/ha)

Sowing time 14 Nov 2003 842 36 64 18.3 1162 21 Nov 2003 940 38 77 19.7 1232 LSD (P<0.05) 85 NS 4.5 NS 41 Seed priming No soaking 773 34 62 17.7 1018 Soaking in water for 6 h 855 35 70 18.5 1174 Soaking in 2% KH2PO4 solution for 6 h 928 36 74 19.0 1266 Sowing of sprouted seeds 1007 42 77 20.7 1330 LSD (P<0.05) 120 3.2 6.4 NS 58 LSD (P<0.05) for sowing time x priming interaction

NS NS NS NS NS

C.V. (%) 10.9 7.0 7.3 10.4 3.89 Source: Annual Report (8)


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Table 9. Effect of weed management and seed rate on seed yield of lentil under rice-utera conditions.

Treatment Seed yield (kg/ha) 1991-92 1992-93 Mean Weed management Control 599 741 670 Weed free (Hand weeding at 30, 45 and 60 DAS)

815 831 823

One hand weeding (30 DAS) 730 824 777 One hand weeding (45 DAS) - 931 931 Two hand weedings (30 and 50 DAS) 746 918 832 Oxyfluorfen @ 50 g/ha as post- emergence (30 DAS)

704 927 816

LSD (P<0.05) 79 54 - CV (%) 10.2 6.0 - Seed rate(kg/ha) 60 676 755 716 80 762 969 866 LSD (P<0.05) 57 27 - CV (%) 11.7 5.0 - LSD (P<0.05) for weeding x seed rate interaction

NS 65 -

Source: Annual Report (1,2) As no tillage operation is done for sowing pulses as a relay (paira) crop, it is difficult to apply fertilizer either through placement or through top dressing and consequently, no fertilization is made by the farmers to the succeeding pulse crops (12). Therefore, the scope of fertilization becomes confined to foliar spray or top dressing. With this view, an experiment was conducted to study the effect of pre-sowing seed soaking and foliar nutrition on yield improvement in lathyrus. This was done in 2003-04 at PORS Sub-Station at Beldanga and the results are given in Table 7. Sowing of sprouted seeds significantly increased yield. Spraying of 2% urea solution at 10 days after rice harvest had the greatest beneficial effect and it was followed by 2% solution of di-ammonium phosphate. The effect of seed soaking of lentil genotypes under rice-utera conditions was also tested in the same year at PORS Sub-Station, Beldanga. The results are given in

Table 8. The sowing of sprouted lentil seeds increased the plant stand and yield quite significantly. This was also true in the case of lathyrus (Table 7). The significantly increased yields are possibly due to increased germination and crop emergence. An experiment was conducted to ascertain the effect of weeding and also to determine the actual seed rate of lentil. It was felt that seed rate should be higher when either lentil or lathyrus were sown under utera conditions in comparison to being sown as a sole crop. The results of two consecutive year- trials (1991-92 and 1992-93) are shown in Table 9. The higher seed rate (80 kg/ha) was effective in significantly increasing both the plant stand and yield. Thus, it is recommended that seed rate should be more than double of the normal rate (30-35 kg/ha ) when lentil is sown under rice-utera conditions, presumably indicating that lathyrus will respond similarly.


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Future strategies To achieve higher productivity of the pulses under utera conditions, the following points should be considered: 1. Generation and/or selection of efficient genotypes: Location specific high yielding genotypes with early vigour, earliness, close canopy, synchronous maturity, resistance to key diseases and insect-pests and tolerance to moisture stress need to be developed for various agro-climatic zones of West Bengal. 2. Development of an efficient fertilizer use schedule: A. Identification and use of efficient strains of

Rhizobium and phosphate solubilizing bacteria (PSB) for seed inoculation

B. Developing an integrated nutrient management schedule for the system as a whole.

C. Exploring the possibilities of foliar nutrition. D. Maintaining proper plant population: E. Using quality seeds of small-seeded varieties. F. Timely sowing to exploit the residual soil moisture. G. Finding the optimum seed rate. H. Seed treatment with effective fungicide to combat

seedling mortality and seed borne diseases. I. Following specific technique for pre-sowing seed

soaking (seed priming) to enhance drought tolerance and seedling vigour.

4. Weed management: A. Choosing genotypes with higher weed suppression

ability. B. Higher seed rate to increase smothering effect. C. Identification of post-emergence herbicides and

efficient application techniques. D. Utilizing residual efficacy of herbicides by applying

the same in preceding crops. E. Timely intercultural operation. Epilogue The above observations indicate some trend in maximizing the productivity of lathyrus and lentil under utera conditions in rice. Similar progress can be made for other utera crops grown in rotation with wheat, such as green gram (Vigna radiata) and black gram (Vigna mungo). Further experiments on utera cropping need to be conducted in order to make recommendations to the farmers. However, such research is being continued on this unique system of growing pulses under residual moisture and fertility, paving the way towards increasing cropping intensity in mono-cropped areas. This will practically maintain soil health, productivity and production of high protein legume crops in developing countries, where many people currently suffer from malnutrition. In conclusion, paira (utera) cropping with winter pulses in low land rice-fallows holds great promise, which would not only step up the pulse production but also help in protecting the environment from the risks of high input agriculture.

References 1. Annual Report (1991-92). Pulses and Oilseeds

Research Station (PORS), Berhampore-742101, Govt. of West Bengal, India.

2. Annual Report (1992-93). PORS, Berhampore-742101, Govt. of West Bengal, India.







9. Anonymous (2000). Status of Agriculture 2000. Dept. of Agriculture, Govt. of West Bengal, India.

10. Anonymous (2002-03). Area, Production and Productivity of Some Principal Crops in West Bengal (2002-03). Socio-Economic and Development Branch, Dept. of Agriculture, Govt. of West Bengal, India.

11. Anonymous (2003-04). Area, Production and Productivity of Some Principal Crops in West Bengal (2003-04). Socio-Economic and Development Branch, Dept. of Agriculture, Govt. of West Bengal, India.

12. Bhowmick MK, Aich A, Aich SS, Shrivastava MP, Gupta S, Man GC. 2005. Crop diversification through paira (utera) cropping with rabi pulses. SATSA Mukhapatra-Annual Technical Issue 9, 43-60.

13. Chakraborty LN, Sen SN, Mandal SK, Sengupta K and Mukherjee D. 1973. Possibility of utilizing rice fallow in West Bengal. Proc. Sem. on possibility of growing a second crop after rice in West Bengal (Das Gupta DK, ed.), Sept. 15, 1973, Calcutta: 71-76.

14. Duary B, Hazra D, Ghosh AK. 2004. Response of lathyrus (Lathyrus sativus L.) to different dates of sowing and fertilizer application under paira cropping in rice. Indian Agriculturist 48, 157-159.


34

The same goal, a different approach: a new Belgian-Ethiopian project

Fernand Lambein1* and Seid Ahmed 2

1. Ghent University, Institute for Plant Biotechnology for Developing Countries (IPBO), Belgium http://www.ipbo.ugent.be

2. Ethiopian Institute for Agricultural Research (EIAR) http://www.eiar.gov.et

*Email: [email protected]

Ever since the discovery of the structure and the neuro-excitatory activity of β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid) in grass pea seeds, research has focused on the reduction/removal of this secondary metabolite from the plant on the one hand, and the better understanding of its physiological activity in the brain on the other hand. Forty years later, the plant still produces the toxic metabolite albeit in a lesser amount, while the understanding of the brain's physiology and the action of β-ODAP on motoneurons has made great strides. But still no prevention has reached the people at risk, nor has a cure reached the victims of neurolathyrism. Only a few years ago an epidemic of neurolathyrism did occur in Ethiopia1. Has something been overlooked? Or is there another road?

In many respectable publications, the nutritional quality of grass pea is praised as being rich in high quality protein, a statement that is being repeated again and again. When calculating the amino acid score of grass pea seed, the low score is stunning: only 20 % of the WHO/FAO proposed standard in which all essential amino acids are present in well-balanced optimal ratios. This is the lowest amino acid score of all commercial legumes. This means that even β-ODAP-free grass pea is not a healthy staple food and needs to be mixed with ingredients richer in those amino acids that are low in grass pea protein. This has been explained before and documented with historical accounts2. The ancient Aztecs of Central America were smart enough to mix cereals and beans, which together form a much better complement of essential amino acids. Essential amino acids that are present at insufficient level in grass pea and are the limiting factor for the low amino acid score are the sulphur containing amino acids cysteine and methionine, needed for our protection against oxidative stress. Oxidative stress is involved in neuronal cell death that occurs in the upper motoneurons in neurolathyrism3.

Lathyrism has often been described as a disease of poverty, ignorance and drought-triggered famine.

Improving the commercial value of grass pea can therefore solve one of the problems underlying the occurrence of lathyrism. Understanding the physiology of drought and salt tolerance and the role of β-ODAP can also be important for agronomic planning for a better quality product from the best suitable soil. Ignorance is a socio-political issue that is out of the reach of science. However, when we find a statistically significant link between the incidence of neurolathyrism and illiteracy, we may find out what information or habit is protecting literate people from developing this irreversibly crippling neurolathyrism4. What condiments are consumed together with grass pea that may protect the consumer from neurolathyrism, and what nutrients are present in those protective condiments?

An alternative road to improve grass pea and prevent neurolathyrism may then be to develop varieties of grass pea that are richer in those nutrients, together with the reduction of antinutritional factors such as β-ODAP. A project funded by the Flemish Inter-university Council (VLIR) will explore this alternative road. This project: "Improving the Nutritional Quality of Grass Pea (Lathyrus sativus)" is a collaborative effort between the Ethiopian Institute for Agricultural Research (EIAR) (formerly the Ethiopian Agricultural Research Organization, EARO) and Ghent University in Belgium. It will be carried out by the Crops Research Department of EIAR and the Institute for Plant Biotechnology for Developing Countries (IPBO) in Ghent. The project started on May 1st, 2004 and will run for four years. Focal points of the project are: -Training Ethiopian researchers in plant biotechnology. -Dissemination of information to the populations at risk, concerning the prevention of neurolathyrism. -Selection of both mutants and somaclones for low β-ODAP and improved amino acid composition.


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-Study stability of the low-β-ODAP trait under abiotic stress. -Examining the potential for applying genetic transformations to grass pea. -Studies on the effect of essential amino acids as food/feed supplements on the nutritional quality of grass pea seed.

A mid-term evaluation will be done by an external expert. The project also calls for an open scientific conference to be organized near the end of the project in April 2008.

References 1. Getahun H, Mekonnen A, Teklehaimanot R,

Lambein F. 1999. Epidemic of neurolathyrism in Ethiopia. The Lancet 354, 306-307.

2. Lambein F, Diasolua Ngudi D, Kuo YH. 2001. Vapniarca revisited: Lessons from an inhuman human experience. Lathyrus Lathyrism Newsletter 2, 5-7.

3. Rao AV, Balachandran B. 2002. Role of oxidative stress and antioxidants in neurodegenerative diseases. Nutritional Neurosciences. 5, 291-309.

4. Getahun H, Lambein F, Vanhoorne M, Van der Stuyft P. 2002. Pattern and associated factors of the neurolathyrism epidemic in Ethiopia. Tropical Medicine and International Health 7, 118-124.

Documents

Lathyrus Lathyrism Newsletter Vol 4 · G. B.Polignano, P. Uggenti, G. Olita, V. Bisignano, V. Alba and P. Perrino- Italy 15 Model plant type in Khesari (Lathyrus sativus L.) suitable