volume 3 (1), december 2003 · 2010. 7. 5. · radovici, societe medicale des hospitaux de...

Volume 3 (1), December 2003

Edited by Colin Hanbury, CLIMA, Australia

Jointly supported by

Lathyrus Lathyrism Newsletter 3 (2003)

Contents

Page

Editor's Comment1 Colin Hanbury -Australia

ArticlesOpinion and Future Directions

2 Dr. Arthur Kessler (1903-2000) David Kessler

Plant Genetic Resources, Evaluation andBreeding

5 Experiences with Lathyrus latifolius in agriculture of highelevation zones of Central America

Raul Alemán, Rinaldo Diaz Votto -Honduras

8 Performance of Rhizobium strains isolated from Lathyrussativus plants growing in southern Chile

L. Barrientos, A. Badilla, M. Mera, A.Montenegro, N. Gaete, N. Espinoza -Chile

10 Seasonal changes in abscisic acid concentration ofperennial root nodules of beach pea (Lathyrus maritimus[L.] Bigel.)

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

13 New green manuring Lathyrus sativus variety ACGreenfix available in USA

David Krause, Ila Krause -USA

15 "Genetic diversity among induced mutants of grasspea(Lathyrus sativus L.)" -Sushil Kumar, D.K. Dubey -India

Sushil Kumar, D.K. Dubey -India

18 Review: A brief history of grasspea and its use in cropimprovement

Jennifer S. McCutchan -Australia

24 Heritability of seed weight in an inbred population oflarge-seeded Lathyrus sativus

M. Mera, A. Montenegro, N. Espinoza, N.Gaete, L. Barrientos -Chile

26 Luanco-INIA, a large-seeded cultivar of Lathyrus sativusreleased in Chile

M. Mera, J. Tay, A. France, A. Montenegro,N. Espinoza, N. Gaete -Chile

27 Mutagenesis as a tool for improvement of traits in grasspea(Lathyrus sativus L.)

Wojciech Rybiński -Poland

32 Stability of grasspea (Lathyrus sativus L.) varieties forODAP content and grain yield in Ethiopia.

Wuletaw Tadesse -Ethiopia

Agronomy 35 Rainfed rice-based 3-crop systems with paira/utera crops

in West Bengal, India.N. R. Das -India

36 Productivity of grasspea (Lathyrus sativus L.) underdifferent levels of phosphorus and foliar spray ofmolybdenum

R.K. Sarkar, B. Biswas, G.C. Malik -India


Animal Feeding 38 Effect of chickling vetch (Lathyrus sativus L.) or alfalfa

(Medicago sativa) hay in gestating ewe dietsC. Poland, T. Faller, L. Tisor -USA

41 Effect of antinutritional factors in khesari seeds (Lathyrussativus) on the biological performance of chicks

Archana Sharma, M. Kalia, S.R. Malhotra -India

44 Lathyrus cicera as quality feed for laying hens Colin Hanbury, Bob Hughes - Australia

Chemistry and Analysis 47 A new amperometric ß-ODAP biosensor Negussie W. Beyene, Helmut Moderegger,

Kurt Kalcher -Austria

50 Items of Interest

The Lathyrus Lathyrism Newsletter can be obtained on-line at

http://go.to/lathyrusOR

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

All research articles are provided there in pdf format.

Jointly supported by:

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

Crawley 6009, Australiahttp://www.clima.uwa.edu.au

and

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

http://www.twmrf.org


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Editor’s comment

Dear Readers

Welcome to the first instalment of the Lathyrus Lathyrism Newsletter Vol.3. Thank you all for your support of thenewsletter. There has been a hiatus in the publishing process, due to alterations in my employment, which should nolonger delay future editions. I can still be contacted via CLIMA, University of Western Australia but am nowemployed at the Department of Agriculture, Western Australia. My old E-mail address will continue to reach me,although I also have a new one (see below). The next edition of the newsletter is due for late in 2004.

Thanks are again due to the Centre for Legumes in Mediterranean Agriculture (CLIMA) and the Third World MedicalResearch Foundation (TWMRF) for supporting the newsletter.

Any suggestions for improving the newsletter in the future are most welcome. Some small changes have beenincluded in this edition of the newsletter. Photographs relevant to articles are particularly welcome, they add interestfor readers and often a feel for what is occurring in areas other than those the reader is familiar with.

Please consider summarising your recent research for the newsletter as contributions from readers are imperative tomaintaining the direction and momentum of the newsletter. Most research submissions should be approximately 1500words and can include a small number of tables or figures. Introduction/Methods/Results and Discussion are thepreferred layout for research summaries, although this can be altered as necessary. Abstracts are also welcome, if theyhave been published elsewhere then full acknowledgment will be made. Electronic copies are preferred, due to thereduction in typing needed, but this is not essential if you do not have access to word processing facilities.

The first article in this edition (Pages 2-4) is an interesting short biography of Dr Arthur Kessler, of interest to thoseaware of his contribution to victims of lathyrism inflicted in a World War II concentration camp.

Colin Hanbury

Editor contact details:

Dr Colin HanburyDepartment of Agriculture, Western Australia3 Baron-Hay CourtSouth Perth 6151Australia

E-mail: [email protected]


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Dr. Arthur Kessler (1903-2000)

David Kessler

Email: [email protected]

NoteDr Arthur Kessler was responsible for an influentialpaper on lathyrism, which concerned an outbreak as aresult of inclusion of high proportions of Lathyrussativus in food consumed in the Vapniarcaconcentration camp during World War II. Dr Kesslerwas himself interned in the camp. The author of thisarticle (David Kessler) is the son of Dr Arthur Kessler.For some more background see Lambein F, Ngudi DDand Kuo Y. (2001). Vapniarca revisited: Lessons froman inhuman experience. Lathyrus LathyrismNewsletter 2, 5-7.

Dr Arthur Kessler in 1973

HistoryArthur Kessler was born in the town of Gewitsch (alsocalled Gewiczor Jevicko) in Moravia (Maehren) onOctober 11, 1903 to Dr. David Kessler (1866-1945)and Anna Gottfried (1875-1947). He was their secondson following Joseph who was born a year earlier.Gewitsch is 56 km north of Brno, now in the CzechRepublic.

In Nov. 1913, the family moved to Czernowitz,Bukovina province, (today in Ukraine). David Kessler

worked as a theology professor and in July 1914 wasappointed rabbi and deputy to chief rabbi Dr.Rosenfeld. However, the First World War started andCzernowitz was too close to the Russians. David andhis family, together with some relatives fromCzernowitz, returned to Gewitsch where they stayedtill the end of the war.

Arthur went to the Gymnasium at Mara Strivova. Thefamily stayed in Gewitsch till April 1918 when theydecided to return to Czernowitz, the war ending inNovember 1918. In Czernowitz, now part of Romania,15 year old Arthur enrolled in State High School L3.The city was quite a diverse one; L1 was the highschool for the Romanians, L2 for the Germans, L3 forthe Jews, L4 for the Ukrainians, and L5 for the Poles.He passed matriculation exams in the classics programwith distinction and graduated in 1923. On 27 Oct.1923, he enrolled at the University of Vienna Facultyof Medicine, and received his diploma in 1929.

Soon after, Dr. Kessler was recruited to the Romanianarmy as a doctor in "Sanitary Company 2" of the 2ndArmy Corps. He was transferred to reserve duty on 1Oct. 1930 and allowed to work in civilian practice. Hethen spent three years in a German hospital south ofZwickau. In 1933, with anti-Semitism increasing inGermany, he could no longer stay there and hereturned to Romania. On 6 May 1937 he marriedChaia Schulsinger in Czernowitz. Their daughter Verawas born in 1940.

When on 28 June 1940 the Russians took overBukovina, the Romanian physicians fled. Arthur thenbecame the hospital manager. On 22 June 1941 theGerman army invaded the Soviet Union with theirRomanian and Hungarian allies and in early July,occupied Czernowitz, reestablishing Romanian rule.Dr. Kessler as a director of a hospital under theSoviets was considered an agent of the Soviet Union.He was accused of "commandeering the CzernowitzHospital" and thrown to jail on 30 Dec 1941. He wasreleased on 7 Feb 1942.

Romania was then under Fascist rule and on 4 Oct.1941, the Czernowitz Military Command instructedthat “All Jews from Bukovina will be sent east of theDniester within 10 days.”


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Dr. Kessler was not transported; he probably stayeddue to the efforts of the mayor of the city, Mr.Popovici, one of the few who tried to circumvent theexpulsion order. This first transport, at the onset of thesevere winter of 1941, was the most horrendous andmurderous.

Later, in Sept. 1942, he was picked up in the street andsent to Vapniarca, a camp run by the Romanians forthe Germans. The camp commander, Lieut. Col. IonC. Murgescu, told the arriving inmates that they “willleave on all fours or on crutches”. The detailedaccount of his experience at the camp is told in his100-page summary titled “Ein Arzt im Lager” (yet tobe published).

The events at this concentration camp were, however,documented in numerous publications. Attached is thededication to Arthur in“L’Epidemie de Lathyrisme duCamp de Concentration Vapniarka” by Dr. A.Radovici, Societe Medicale des Hospitaux deBucarest, Nr. 5-7, Nai-Julliet

“To Dr. Kessler, An example of heroic resistanceagainst the Teutonic fury unleashed against innocentand unarmed population. Deported and imprisoned incamp Vapniarca, he provided moral support to hiscomrades in suffering. He nursed with devotion thesick during the epidemic outburst and used all meansto improve the living conditions by addressing withconsiderable risk repeated protests.

In the midst of the horrible circumstances, in the“house of the dead”, he however conserved thetranquillity of the soul and the purity of the spirit totrace in a scientific manner the progress of paraplegicepidemic called Lathyrismus. He cultivated differentvarieties of the lathyrus and left us a collection ofplants. “

On 1 May 1943 Dr. Kessler and 99 others were movedfrom Vapniarca to the ghetto at Ol'gopol where theyspent almost a year. In 1944, he escaped ,trailing theretreating Germans and arrived ahead of theadvancing Russians back to Romania.

In 1944, Dr. Kessler, with his wife and daughter,immigrated to Israel wherehe started practicing again.His son David was born in 1948. Dr. Kessler becamethe director of the Allergy Department of theZamenhof Clinic in Tel Aviv, and a leading pioneer ofhis field in Israel. His list of publications is attached.In Mar. 1968, he was elected Chairman of the IsraelSociety of Allergology. He engaged in lectures,workshops, and seminars in addition to practicing andpublishing.

He was considered an authority in the area oflathyrism and continued for years to treat the invalidvictims from Vapniarca without pay. Aside from hisoutstanding professional reputation, his empathy andcompassion endeared him to his many patients. Anunusually modest man, he always shunned away fromall trappings of public recognition. He was keenlyinterested in many subjects beside medicine, mostlyhistory and archeology. Arthur Kessler died on August18, 2000.

Publications (not all on Lathyrus related topics)• The struggle for survival and health in the ghettosin the days of the Nazi occupation (Hebrew). DapimRefuim, Volume 6, September 1946 • Poisoning by the Lathyrus sativus beans,(Hebrew) Dr. A Kessler, Harefua, Vol. 31, No.7,October 1946.• Lathyrismus, von A. Kessler, Monthly Review ofPsychiatry and Neurology, Vol. 113 No. 6 (1947).• Survey of Airborne Pollen and Fungus Spores inIsrael, 1952-1953, Arthur Kessler, M.D., Annals ofAllergy, Vol. 11, pages 322-328, May-June, 1953.• Asthma among our Children (Hebrew) , Eitanim,January 1954• Survey of Airborne Pollen and Fungus Spores inIsrael, 1953• Arthur Kessler, M.D., Annals of Allergy, Vol. 12,pages 261-262, May-June, 1954.• On the Problem of Hay-Asthma and Hay-Fever inIsrael (Hebrew) by Dr. A. Kessler. Dapim Refuim,Vol. 13, No. 2, June 1954.• Sensitivity to Penicillin (Hebrew) by Dr. A.Kessler, Dapim Refuim, Vol. 14, No. 1, February1955, Medial Journal, Kupath Cholim, Tel Aviv.• The Indications of Treatment of BronchialAsthma with Cortisone. Dr. A. Kessler, DapimRefuim, vol. 15, No. 5-6, December 1956.• Sensitivity to Olive Pollen (Olea europa) as thecause for allergic diseases, Dr. A. Kessler, DapimRefuim, Vol. 17, No. 2-3, August 1958.• Survey of Airborne Pollen and Fungus Spores inIsrael, 1954-1955• Arthur Kessler, M.D., Annals of Allergy, Vol. 16,pages 445-450, July-August, 1958.• Bronchial Asthma and Heredity, Dr. A. Kessler,Dapim Refuim, Vol. 19, No. 5, November 1960.• La significacion de la herencia en el asmabroncial, por Arthur Kessler, La Semana Medica,Buenos Aires, 30/4/1962• Stings and Bites of Bees, Wasps, Mosquitoes andantes, Dr. A. Kessler, Dapim Refuim, Vol. 21, No. 7,November 1962.• Airborne allergens and clinical response ofasthmatics in Arad, a new town in a desert area inIsrael. by S. Z. Kantor, Arthur Kessler et al, TheJournal of Allergy, vol. 37, No. 2 February 1966.


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• Advances in Diagnosis and Treatment inAllergology (Hebrew) (sometime after 1966)• Bee Stings by Dr. A. Kessler, Harefua, Vol. 73,No. 1, July 2nd 1967, pp28-29.• Allergy of babies, Dr. A. Kessler, Eitanim Vol.21, No. 7, July 1968.• Bronchial Asthma, Hypothyroidism(Myxoedema) and Glaucoma in Homozygous Twins,Dr. A. Kessler, Head of the Allergy Dept., ZamenhoffClinic, Tel Aviv., Presented in honor of Prof.Ostrovsky on the occasion of his 80th birthday. DapimRefuim, Vol. 27, No. 7-8, December 1968. • On Sensitivity to Medications, Dr. A. Kessler,Dapim Refuim, Vol. 28, No. 2-3, Mars 1969.• Panel discussion on Bronchial Asthma, Prof.Rakover, Dr. A. Kessler and 5 others, Dapim Refuim,Vol. 28, No. 2-3, Mars 1969.• The Asthmatic Child from the Point of View ofthe Allergy Expect, Dr. A. Kessler, Eitanim No. 1,January 1970.• Mortality of Asthma Bronchiale and its causes,Dr. A. Kessler, Harefua, Vol. 80, No. 5, March 1971.• House dust, Mites and Bronchial Asthma,Harefua, Vol. 85, No. 6, September 1973.• Short article, same title as above in the 30October 1973 issue of the daily “Davar”• Itching of Dermanyssus Avium (from birds) inIsrael, A. Kessler, Harefua, Vol. 87, No. 9, November1974.• Risks in the Use of Aerosols, Dr. A. Kessler,Harefua, Vol. 89, No. 4, August 1975.• Bees and Wasps Bites, Dr. A. Kessler, Harefua,Vol. 89, No. 12, December 1975.• The Lower Motoric Neuron in ChronicLathyrism, by M. Strifler, A. Arlasarov, A. Kesslerand D. Cohen, Harefua, Vol. 90, No. 10, May 1976.• Bites by Bees, Wasps, Mosquitoes and ants, Dr.A. Kessler, “The Family Physician”. Vol. 7, No. 1-2,December 1977.• The influence of the Israeli climate on Asthmapatients, Dr. A. Kessler, Harefua, Vol. 94, No. 11,June 1978.• Electromyographic observations in chroniclathyrism.in Electroencephalography and ClinicalNeurophysiology. 1967 Dec; 23(6):588 ArlazoroffA, Kessler A, Streifler M.

Unpublished paper• Vascular Diseases by Consumption ofLathyrismus sativus, by Dr. Kessler.

Other publications• Book review: Clinical Allergy by French andHanel, Mosby Co., Harefua, 1953.• Review of the 5th International Conference ofAllergeology in Madrid, by Dr. A. Kessler, DapimRefuim, Vol. 24, No. 3, May 1965.

• Book Review by Dr. A. Kessler: The AllergicChild, by P. Sapir , Dapim Refuim, Vol. 24, No. 7-8,December 1965.• Obituary to Dr. Kissman, 1967. Dapim Refuim.• Obituary to Dr. Ester Lindenbaum, December1967. Dapim Refuim.• Book Review by Dr. A. Kessler : Allergology, byCharpin et al. Harefua, 1973.• Book Review by Dr. A. Kessler : BronchialAsthma, by M.J. Gross, Harper & Row. Harefua,July 1974.• Book Review by Dr. A. Kessler : Asthma inChildren, by R.J. Jones, Edward Arnold Ltd. London.May 1977.• Book Review by Dr. A. Kessler: Asthma, byT.J.H. Clark, Chapman and Hall, London, Harefua,Vol. 94, No. 7-8, April 1978.• Book Review by Dr. A. Kessler: Research andMedical Practice: Their Interaction, Ciba FoundationSymposium 44, Elsevier Experta Medical, NorthHolland, Harefua, Vol. 94, No. 9, May 1978.

Notable citationsDr Kessler’s citation index is quite extensive andincludes many citations in Hebrew papers andprofessional medical magazines. A partial list isshown:• L’Epidemie de Lathyrisme du Camp deConcentration Vapniarka, by Dr. A. Radovici, SocieteMedicale des Hospitaux de Bucarest, Nr. 5-7, Nai-Julliet. 1945. The paper is dedicated to Dr Kessler.• Das motoriche Neuron in chronischenLathyrysmus by D.F. Cohn, M. Striefler andE.Schujman, Nervenarzt 48, 127-129 (1977)• Neurolathyrism, Historical Notes (A Chapterfrom the Holocaust) by Dan F. Cohen and M.Streifler (English and Hebrew) Koroth 7, No. 7-7,(1978).• Guam Amyotrophic lateral Sclerosis-Parkinsonism-Demential Linked to a plant ExitantNeurotoxin , by Peter S, Spencer et al., Science, Vol.237, 465-564 (1987). This extensive paper finds theconnection between the Guam disease and lathyrismuswhere both are initiated by consumption of differentplants but with very similar active ingredients.• Toscanini’s Fumble and other Tales of ClinicalNeurology by H.L. Klawans, CB contemporaryBooks, Chicago NY. ISBN 0-8092-4727-5. The thirdchapter is called “Vapniarka” in which theLathyrismus outbreak is detailed with emphasis on DrKessler’s role.• Barbed Wire on the Dniester, by Ema Talmai,Sifriat Hapoalim (1947). A book in Hebrew,describing from first survivor accounts the events inVapniarka. Dr. Korn is the alias for Dr. Kessler.


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Experiences with Lathyrus latifolius in agriculture of high elevation zones ofCentral America

Raul Alemán1* and Rinaldo Diaz Votto2

1. International Cover Crops Clearinghouse (CIDICCO), P.O. Box 4443, Tegucigalpa MDC, Honduras C.A.2. Honduran Foundation of Agricultural Research (FHIA), Honduras C.A.

*Email: [email protected]

Originally published in Cover Crops News No. 12. http://cidicco.hn/newcidiccoenglish/bulletin12.htm, accessed 11thDecember 2003.

Introduction The utilisation of cover crops and green manures inthe sustainable management of soil fertility in zonesover 1400 meters above sea level (masl) presents achallenge. Species are sought that can be adapted toconditions of temperate climate with temperaturesbelow 10 ºC during the coldest months, in degradedsoils and besides, which could be included in thecropping systems or existing land use in the region.There are few species that have such characteristics,amongst them are the chinapopo beans (Phaseoluscoccineus), an edible species utilised as a cover cropin association with corn; milpero beans (Phaseolusvulgaris) which are similar to chinapopo, faba beans(Vicia faba) that can be associated with corn or potatoproviding in addition to a regular ground cover, foodrich in protein. Dolichos beans (Dolichos lablab) alsogrows well at altitudes of 1700 masl providing a goodbiomass production, although it is affected by freezingtemperatures. Other species with potential for zones ofhigher elevation include the tarwi (Lupinus mutabilis),species utilized in the Andes of Bolivia, severalspecies of the genus Vicia, clovers (Trifolium sp) andalfalfa (Medicago sativa).

Another not so well known but very promising legumeis choreque beans (Lathyrus latifolius). This plantpossesses good tolerance to frost, adaptation to highaltitude, abundant biomass production and above all,rapid growth and easily associated with corn. Thisdocument presents a summary of the utilisation ofchoreque beans by indigenous groups in the higherregion of Chimaltenango, Guatemala and the results oftrials conducted at Santa Catarina ExperimentalStation of the Honduran Foundation of AgriculturalResearch (FHIA) at La Esperanza, Intibuca, Honduras.

Choreque is an annual plant of bushy-vine typegrowth (Fig. 1). At La Esperanza it grows up to 1.4 mhigh. Its flowers are pink in colour and abundant, thepods normally have 4-5 seeds which are brown incolour.

Fig. 1. Choreque (Lathyrus latifolius) in Honduras.

In Honduras, studies on the behaviour of choreque asgreen manure is being conducted in the fields oforganic agriculture of the Honduran Foundation ofAgricultural Research (FHIA) at La Esperanza,Intibuca. The altitude of the region is 1680 masl andhas a mean annual rainfall of 1350 mm. Averageannual temperature of 16 ºC and a minimum of –2 ºCwhich occurs between December and February (theseare commonly frost events) and a maximum of 30 ºCin March. Average relative humidity is 85%. Soils areclay loam with a pH of 4.5-5.0.

http://cidicco.hn/newcidiccoenglish/bulletin12.htm


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In this region, potatoes, vegetables, corn and altitudefruits are extensively grown. Soils are degraded due tointensive use and excessive application of chemicalfertilisers, especially in the case of potatoes.

The idea of testing choreque originated frominformation from Guatemala and there was specialinterest in testing its tolerance to frost. In the highplateau of Chimaltenango, Guatemala, choreque isutilised in association with corn for soil improvementand increasing yields. This region is over 1800 masland has rainfall of 1300mm between May andOctober. Soils are volcanic, clay loam with slopes ofmore than 10%.

Farmers plant in May at the beginning of the rains.Choreque is planted from the middle of July up toSeptember, when the corn has already developed.Planting is done at the time of the second “earth up”of corn. One to two choreque seeds are planted per hillevery 10 cm. Between each hill of corn, which in thisregion, is planted in square at a distance of 1x1 m. Atthis density, 14 kg of choreque seed are needed perhectare.

Apparently, choreque does not limit the growth oryield of the corn, which is harvested in Novemberwhen choreque is 60 cm high. During harvest of corn,choreque is not damaged since it only has between 2-3small branches. From this time on its development isaccelerated. After harvest, choreque remains on theground and tangles on the corn stalks providing acomplete ground coverage. All this material can beincorporated into the soil in the next cropping seasonand some farmers also utilise it as a forage reserve forthe dry season.

The benefits mentioned by the farmers include thefollowing: • Serves as green manure, choreque has been recordedas producing 100 t/ha of green matter in a period of 6months (1)

. All this material greatly improves soils andmake them easier to work with. • Incorporates nitrogen into the soil. Nitrogen fixationin this species is approximately 62 kg/ha (2), but themost important is the nitrogen content in the leaves,which is approximately 4.6% based on dry matter (seeTable 1). • Improves yield of corn. In previous studies it hasbeen concluded that “the yields of grain corn weregreatly increased as a result of the planting andincorporation of choreque. When planting andincorporating choreque, the effects of chemicalfertilisers is not significant for local corn variety usedin this trial" (1).• Protects the soil from rains. The coverage ofchoreque after more than 2 months of growth is 100%protecting the soil from erosion from water drops.

• Control weeds. The coverage of choreque is verythick and does not permit the sunlight ot reach to thesoil and so avoids stimulating the germination ofweeds. Some weeds are eliminated by the greatergrowth of choreque, which covers them with itsfoliage and stems. • Maintains soil moisture. The soil remains soft andeasy to work, although land preparation takes moretime due to the incorporation of choreque residues. • Is tolerant to frost conditions. Frost occurs fromDecember to February and choreque resiststemperatures down to –2 °C without affecting itsgrowth. • Remains green during the dry season. Chorequeremains green a great part of the dry season, coveringthe soil and providing fresh forage for animals. • Is resistant to pests, tolerates drought and is adaptedto poor soils. These characteristics makes this speciesideal for the high elevation conditions where thealternatives are few in comparison to zones under1500 masl.

Material and MethodsChoreque seed was obtained from Chimaltenango,Guatemala and planted in an area of 350 m2 at LaEsperanza in April. Distance between rows was 0.75m and 2 seeds were planted per hill every 0.20 m. Atthis density, ≈1.5 kg of seed was used, 9 kg of 12-24-12 (NPK) fertiliser was utilised at planting (fertiliserwas used for seed multiplication needs).

Germination occurred after 15-20 days. No treatmentwas given to the seed in this trial, however, inGuatemala, the farmers of the high plains ofChimaltenango, recommend sun drying the seed for 4hours before planting to accelerate germination. Initialgrowth of choreque is slow but after 1 ½ months itgrows very fast and covers the soil. Weeding wasdone twice with a hoe, the first one month aftergermination and the second one month later.Additionally, two irrigations were conducted sincenormally there are no rains in the month of April inthis region. Then choreque grew without the need forany additional management and completely coveredthe soil in 2 ½ months.

ResultsTwo biomass measurements were conducted, the firstduring flowering in September. In this case, thecontribution encountered was 95.5 t/ha of greenmaterial (fresh weight), which greatly exceeds themajority of legumes in the high elevation regions andis similar in yield to those reported for choreque inGuatemala (1). The second measurement was done twomonths later obtaining 65 t/ha of green material (freshweight). Foliage analysis based on dry matter is givenin Table 1.


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Seed was harvested in January, 9 ½ months afterplanting. 8.2 kg of seed was obtained from this plot,equivalent to 233 kg/ha. This yield was affected byexcessive rains, especially during the months offlowering, which prevented pod formation.Observations at the garden level indicate that in a less

cold area, choreque produces seeds in a shorter time(3-4 months) and in greater abundance. After harvest,the plants began to dry slowly, but most interestinglymaintained the soil cover during most of the dryseason and the residue protected the soil from the sunand wind.

Table 1: Foliage analysis of choreque (Lathyrus latifolius). Moisture content was 89%.

N P K Ca Mg Fe Mn Cu Zn% DM ppm

4.6 0.42 3.1 1.03 0.24 168 81 10 33Source: Chemical-Agricultural Laboratory FHIA. La Lima. Honduras

Observations on incorporation as green manure Some small plots (6 m x 4 m) were planted on thesame date (April) for observation of choreque as acover crop. In this case only one weeding wasconducted but with two similar irrigations. In thisregion, to incorporate choreque into the soil it is idealto conduct it 4 ½ months after planting, when it is inthe flowering stage. At the time of incorporation thefoliage was slashed with a machete and then the bedsprepared with a hoe incorporating the material, thebeds were left for 2 weeks for decomposition to occur.The vegetables planted in these beds weighed morethan ones planted in the plots without cover crops andwith chemical fertiliser.

Conclusions • Choreque is a viable alternative as a green manure

in rotations in the high elevation zones. It can beplanted after potato or vegetables andincorporated at flowering. This rotationcontributes to restoring the balance in soil fertilityin the soils of this region and to gradually reducethe use of chemical fertilisers.

• In the zone of La Esperanza, it is less likely thatfarmers will be interested in cultivating choreque

with corn, since the legumes in common use aremilpero beans (Phaseolus vulgaris) andchinapopo beans (P. coccineus), obtaining two orthree edible crops in the same plot. Choreque isnot edible.

• It may be useful to plant choreque in fallow landsto accelerate their recuperation and to produceseed.

• Due to the urgent need to improve the soils of thisregion, it is not recommended to use the land withchoreque residues for animal grazing since thecontribution of nutrients will decrease and soilswill be compacted.

References1. Villatoro R. 1977. Evaluation of the effect of

choreque as a green manure and five levels ofchemical fertilization in corn. Thesis Ing.Agrónomo. University of San Carlos, Guatemala,73 pp.

2. Tropical Products Institute. 1979. Daysi E. Kay,ed., Food Legumes, London, pp. 11-123


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Performance of Rhizobium strains isolated from Lathyrus sativus plantsgrowing in southern Chile

L. Barrientos*, A. Badilla, M. Mera, A. Montenegro, N. Gaete and N. Espinoza

INIA-Carillanca, Casilla 58-D, Temuco, Chile


Introduction Grass pea (Lathyrus sativus L.) is grown in southernChile by small farmers (2), in poorly managed soilscharacterised by a high level of erosion and lowfertility (3). As with any other legume, the symbioticnitrogen fixation is a process that must be improved.Grain legume crops are rarely inoculated in Chile andempirical evidence suggests that ineffectiveRhizobium strains may be a significant proportion ofthe nodulating bacteria. This may be particularlyrelevant in the case of grass pea, where areas notpreviously cultivated with this legume are beingutilised. Being a low-cost, environmentally friendly,simple technology, the use of inoculants should bepromoted. However, there is a lack of an inoculantspecific for grass pea in Chile. Consequently, we haveundertaken the task of developing a quality inoculantfor grass pea, starting with the evaluation ofRhizobium strains isolated from well-nodulated grasspea plants sampled in three locations of the LaAraucania region of Chile.

Material and Methods

Locations. Sampling of nodulated plants was done inthree different locations of La Araucania: Lumaco,Selva Oscura and General Lopez. The soils of Lumacoare inceptisols, severely degraded with low fertility.Besides grass peas, other legumes traditionally foundin these soils are peas (Pisum sativum), lentils (Lensculinaris), broad beans (Vicia faba), vetches (Viciaspp.) and chickpeas (Cicer arietinum). The soils ofSelva Oscura are andisols moderately degraded, whichquite often present medium-high levels of aluminiumsaturation. A majority of the soils in Selva Oscura donot have a record of previous grass pea cultivation.Sampling at General Lopez was done in soils of theCarillanca Regional Research Centre, which have ahigh level of fertility, with a long history of severallegume species participating in crop rotations, thoughrather sparsely over time.

Sampling. Twenty small farms were visited in Lumacoand from the nodules collected, 47 strains wereisolated. Three small farms were visited in SelvaOscura, from which eight strains were obtained.

Sampling at Carillanca, in four different fields, yieldedfive additional strains. Therefore, a total of 60 strainswere isolated, presumably corresponding toRhizobium leguminosarum biovar viciae.

Rhizobia strains isolation. Three random, good-looking, pink nodules were excised from each plantand nodule isolates were obtained by the procedure ofVincent (5). Single colonies were picked andmaintained on yeast mannitol agar (YMA) slants at4ºC for further characterisation. Isolates weredesignated by two letters (LU = Lumaco, SO = SelvaOscura, CA = Carillanca) and a correlative number.

Evaluation of symbiotic properties of rhizobialisolates. The isolates were characterised according totheir colony morphology in YMA (5), after 2 and 4days of growth and then evaluated preliminarily inLeonard jars with two replications, under greenhouseconditions. The best 15 strains were selected for asecond evaluation, whose results are reported here.Leonard jars were filled with river sand carefullywashed and watered with a nitrogen-free nutrientsolution (4). Seedlings of grass pea were transferredaseptically to jars (five per jar) and inoculated with 10mL of 2 day-old yeast mannitol broth (YMB) culturesof rhizobia to provide approximately 108 cells seed-1.An non-inoculated control jar with mineral N (CN),supplied as potassium nitrate solution (0.75 g L-1 of Nevery 15 days), was also evaluated. Seedlings werethinned to uniformity to two per jar and covered witha layer of sterilised coarse sand. Jars were arranged ina completely randomised design with three replicatesper treatment. Plants were harvested 45 days afterinoculation and symbiotic effectiveness was estimatedthrough the aerial dry weight according to aneffectiveness criterion (1). A relative production indexwas calculated as RP = I/N, where I is the dry matterproduction by inoculated plants and N is the drymatter production of uninoculated control plants thatreceived fertilizer nitrogen. An RP<1 indicates thatnitrogen fixation was insufficient to cover the plantsrequirement of nitrogen. The size, number and weightof nodules per jar were also recorded (not shown).Data were processed with an analysis of variance andDuncan’s multiple range test using SAS 6.12.


9

Results and Discussion

Table 1. Dry matter production per jar and relativeproduction index of two grass pea plants inoculated withfifteen strains of Rhizobium leguminosarum biovar viciae,isolated from three locations in southern Chile.

Origin Strain Dry matter(g jar-1)

Relativeproduction index

Lumaco LU-29 3.22 a* 1.43Carillanca CA-60 2.68 ab 1.19Selva Oscura SO-51 2.56 ab 1.14Control CN 2.26 bc 1.00Lumaco LU-23 2.07 bcd 0.92Selva Oscura SO-53 1.96 bcde 0.87Lumaco LU-24 1.57 bcdef 0.70Selva Oscura SO-47 1.54 cdef 0.68Selva Oscura SO-48 1.52 cdef 0.67Lumaco LU-19 1.41 def 0.62Lumaco LU-45 1.41 def 0.62Lumaco LU-02 1.40 def 0.62Selva Oscura SO-50 1.20 ef 0.53Lumaco LU-01 1.11 f 0.49Lumaco LU-28 1.06 f 0.47Lumaco LU-27 0.85 f 0.38* Means sharing same letter are not significantlydifferent according to Duncan’s NMRT, P≤0.05.

Strains varied considerably in dry matter production,as shown in Table 1. Only one strain (LU-29) yieldedsignificantly more dry matter than the controlreceiving nitrogen. Seven strains (CA-60, SO-51, LU-23, SO-53, LU-24, SO-47, SO-48) performedsimilarly to the control, and the remaining seven weresignificantly inferior. Twelve strains gave a relativeproduction index lower than 1, suggesting that grasspea roots are being infected by poorly effectivebacteria in terms of nitrogen fixation. In fact, severalstrains gave rise to small, pale nodules (data notshown). At the same time, this result suggests thatmuch can be done regarding grass pea inoculation in

southern Chile. Even when Rhizobium leguminosarumbiovar viciae promiscuously nodulates the Vicieae,which comprises genera Pisum, Lens, Vicia andLathyrus (6), it is widely recognised that maximumbenefit from symbiotic nitrogen fixation can beachieved only when rhizobia and host plant present ahigh affinity. Evaluation of strains LU-29, CA-60 andSO-51 will continue, as they are good candidates toconform a future commercial inoculant, specific forthe grass pea cultivated in the region of La Araucania.Interestingly, these strains come from locations withquite different soil and environmental conditions.

AcknowledgementsFNDR Project BIP 20155696-0 granted by GobiernoRegional de La Araucania.

References1. Bordeleau L, Anton H & Lachance R. 1977.

Effets des souches de Rhizobium meliloti et descoupes successives de la luzerne (Medicagosativa) sur la fixation symbiotique d’azote. Can JPlant Sci 57, 433-439.

2. Mera M, Montenegro A, Espinoza N & Gaete N.2000. Research backs grass pea exports by smallChilean farmers. Lathyrus Lathyrism Newsletter1, 31.

3. Montenegro A. 1991. Diagnóstico preliminar delos tenores de nitrógeno, fósforo, potasio, materiaorgánica y pH de los suelos de la IX Región.Investigación y Progreso Agropecuario Carillanca10(3), 3-11.

4. Norris DO and Date RA. 1976. Legumebacteriology. In: Shaw NH & Bryan W (eds.).Tropical Pastures Research; Principles andMethods. p 134-174. Commonwealth Bureau ofPastures and Field Crops, Hurley, UK.

5. Vincent JM. 1975. Manual Práctico deRhizobiología. Hemisferio Sur, Buenos Aires,Argentina. 200 p.

6. Young JPW. 1996. Phylogeny and taxonomy ofrhizobia. Plant and Soil 186, 45-52.


10

Seasonal changes in abscisic acid concentration of perennial root nodules ofbeach pea (Lathyrus maritimus [L.] Bigel.)

Gurusamy Chinnasamy1*, Arya Kumar Bal1 and David Bruce McKenzie2

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

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

*Author for correspondence. Present address: Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9.

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

Introduction Beach pea (Lathyrus maritimus [L.] Bigel.), apotential cold-climate circumpolar legume crop,naturally grows along the shorelines ofNewfoundland, Canada. It is perennial and persistentfor many years, and is resistant to drought and frost. Itforms large continuous stands by rhizomes and hasprolific seed production. Sometimes, the vegetativeparts of beach pea are used as fodder for cattle (1) andseeds have been used for food or feed purposes duringscarcity of other foods by stranded sailors (11).Vigorous plant growth has occurred in bothgreenhouse and field trials (16). The added advantageof the nutritional value of the seeds and other plantparts (5) suggests that beach pea may be a goodcandidate as a cold-climate crop for food, feed orforage. Having a symbiotic association with thenitrogen-fixing soil bacterium, Rhizobium, makes iteven more attractive as a crop, which allows beachpea to grow well in nutrient-poor areas that can beinhospitable to other plants (2).

The rhizobia-induced nodules of beach pea fixnitrogen at relatively low temperature (1). Thesenodules are perennial and are indeterminate having anapical meristem capable of continuous growth (2), andbecome dormant during the winter when the aerialparts of the plant freeze (12,13). With the advent ofspring, the nodule meristem produces new noduletissues that fix nitrogen (12,13). In our earlier studies, wehave shown seasonal changes in oleosomes (lipidbodies), lipids, carbohydrates, proteins and elementsof perennial root nodules of beach pea (6-8,12,13).

The formation and development of root nodules areregulated by their hormone levels (15). Abscisic acid(ABA), a potent molecule, is present in some plantnodules (4,9,10,19) and it is synthesised both in the leavesand the roots of the plant (17). The concentration ofABA increases as a result of water stress, playing an

important role in the plant response to cold, droughtand salinity; stresses that involve cellular water stress(14,21). In plants, ABA also plays a significant role inregulating gene expression during differentdevelopmental stages and adaptation to various abioticstresses (3). The present investigation was carried outto study seasonal changes in the concentration of ABAin perennial root nodules of beach pea.

Material and MethodsNodules were collected from about 100 beach peaplants growing naturally on the sandy beach ofSalmon Cove, Newfoundland during summer (15June, 1997), early autumn (13 September, 1997) andlate autumn (16 November, 1997) by digging the soiland exposing the root system. Nodule samples werealso collected from beach pea plants grown in potsduring winter (February 20, 1998). For wintersamples, plants with rhizomes and nodules from thesame naturally growing stands were placed in largepots in the same sandy soil and kept outdoors.Sampling was made possible from the above pots afterthawing at 5-10°C in the greenhouse for 18 hr aspreviously reported (13). This was the only waynodules could be harvested from the frozen pots. Noappreciable changes in the native pattern of cellconstituents were apparent from ultra structuralstudies (13).

The method employed for extraction of ABA wasessentially that of Welbaum et al. (20) with minormodifications. One gram of clean fresh nodules each,in three replications, from each season washomogenised with a pestle and mortar and extracted in10 ml of cold extraction solvent (80% methanolcontaining 10 mg/l butylated hydroxytoluene) for 17hr at 4°C in the dark. Homogenates were centrifugedfor 30 min at 10000 g in an ultra centrifugemaintained at 4°C. The pellet was re-extracted with 4ml of extraction solvent for 3 hr at 4°C in the dark and


11

centrifuged as above. Supernatants were combinedand dried using a rotary water bath to removemethanol and toluene. The dried supernatants were re-dissolved in 1 ml of 25mM Tris-buffered saline(25mM Tris, 150mM NaCl and 2mM MgCl2, pH 7.5)and used for the assay.

Abscisic acid concentration in each sample wasdetermined by enzyme-linked immunosorbent assay(ELISA) using the Phytodetek ABA immunoassay kit(Idetek, Inc., Sunnyvale, CA). Samples were assayedto serial dilutions to ensure that they fell within therange of a standard curve. All samples were run intriplicate and ABA standards were included intriplicate in each assay for construction of a standardcurve. The outer rows and columns were not used toimprove uniformity. Colour absorbency, followingreaction with the substrate, was read at 405 nm using amicroplate autoreader EL 311 (Bio-Tek Instruments,Inc., Winooski, VT). For all sets of data, one-wayanalysis of variance was performed using the SPSScomputer package (18) and means were compared byDuncan’s multiple comparison test at P = 0.05 (18). Results and DiscussionThe concentration of ABA significantly increasedfrom early autumn to summer (Table 1). Charbonneauand Newcomb (4) reported a higher concentration ofABA in the nodule meristem of pea (Pisum sativum).In the present study, the highest concentration of ABAin summer nodules may be due to the presence of highamounts of meristematic tissues, which are essentialfor active growth and development of effectivenodules. Dangar and Basu (10) found the highestconcentration of ABA in root nodules of Pterocarpusmursupium in winter. According to Watts et al. (19),ABA concentrations in dormant nodules were up to2.5 times higher than those in actively growing plants.In contrast, winter nodules from lentil (Lens culinaris)showed low concentration of ABA (9). The onset ofnodule senescence has been also associated with anincrease in the concentration of ABA in the nodules(4,9,10).

Table 1. Seasonal changes in abscisic acidconcentration of perennial root nodules of beachpea.

Season Abscisic acid*

(ρmol/g fresh mass)Early autumn 4.217 ± 0.009c

Late autumn 4.457 ± 0.061b

Winter 4.497 ± 0.038ab

Summer 4.600 ± 0.029a

* Values are means (± SE) of three replications.a-c Means (± SE) followed by different letters aresignificantly different using Duncan’s multiple comparisontest at P = 0.05.

In the present study, the high concentrations of ABAin the late autumn and winter may be due to thepresence of large numbers of old/senesced nodulesand dormant nodules, respectively. The highconcentration of ABA in the winter nodules may playan important role in cold tolerance and prolongedwinter dormancy by preventing cellular dehydration.Under hardening conditions in the late autumn, highconcentration of ABA in nodules may protect againstcold stress. The presence of small numbers ofold/senesced nodules, large numbers of active nodulesand less stressful environmental conditions may beresponsible for the low concentration of ABA in theearly autumn.

From this study it is apparent that perennial root nodulesof beach pea reveal changes in the concentration ofABA during over-wintering and growing periods.Further studies of samples taken from plants grown incontrolled growth chambers would provide more precisedata to correlate physiological and biochemical activitieswith climatic changes. It would also be of interest tolocalise seasonal distribution of ABA in different tissuesof perennial root nodules.

AcknowledgementsWe wish to thank Mr. D. A. Martin for the samplecollection and Mrs. Sangeetha for the manuscriptpreparation. This work was supported by the Dean ofScience grant to A. K. Bal.

References1. Bal AK, Barimah-Asare J. 1993. Legume-

Rhizobium symbiosis in the shorelines ofNewfoundland: N2-fixation in beach pea rootnodules. In ‘The scientific challenge of ourchanging environment’. Hall J, Wadleigh M(Eds.). Canadian Global Change ProgrammeIncidental Report Series, Report No. IR93-2. TheRoyal Society of Canada, Ottawa, pp. 58-59.

2. Barimah-Asare J, Bal AK. 1994. Symbioticnitrogen-fixing root nodules of Lathyrusmaritimus (L.) Bigel. (beach pea) fromNewfoundland shorelines with special referenceto oleosomes (lipid bodies). Plant Cell Environ17, 115-119.

3. Chandler PM, Robertson M. 1994. Geneexpression regulated by abscisic acid and itsrelation to stress tolerance. Ann Rev Plant PhysiolPlant Mol Biol 45, 113-141.

4. Charbonneau GA, Newcomb W. 1985. Growthregulators in developing effective root nodules ofthe garden pea (Pisum sativum). Biochem PhysiolPflanzen 180, 667-681.

5. Chavan UD. 1998. Chemical and biochemicalcomponents of beach pea (Lathyrus maritimusL.). PhD Thesis. Memorial Univ ofNewfoundland, St John’s, Newfoundland,Canada.


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6. Chinnasamy G, Bal AK. 2003. Seasonal changesin carbohydrates of perennial root nodules ofbeach pea. J Plant Physiol 160, 1185-1192.

7. Chinnasamy G, Bal AK, McKenzie DB. 2003.Seasonal changes in protein, amino acid andelemental composition of perennial nodules ofbeach pea. Can J Plant Sci 83, 507-514.

8. Chinnasamy G, Davis PJ, Bal AK. 2003. Seasonalchanges in oleosomic lipids and fatty acids ofperennial root nodules of beach pea. J PlantPhysiol 160, 355-365.

9. Dangar TK, Basu PS. 1984. Seasonal changes andmetabolism of plant hormones in root nodules ofLens sp. Biol Plant 26, 253-259.

10. Dangar TK, Basu PS. 1984. Studies on rootnodules of leguminous tress: I. Seasonal variationof plant hormones and IAA metabolism withreference to nitrogen fixation in Pterocarpusmursupium Roxb. Biochem Physiol Pflanzen 179,359-368.

11. Erichson-Brown C. 1979. Use of plants for thepast 500 years. Breezy Creeks Press, Aurora,Ontario.

12. Gurusamy C, Bal AK, McKenzie DB. 1999.Nodulation of beach pea (Lathyrus maritimus [L.]Bigel.) induced by different strains of rhizobia.Can J Plant Sci 79, 239-242.

13. Gurusamy C, Davis PJ, Bal AK. 2000. Seasonalchanges in perennial nodules of beach pea(Lathyrus maritimus [L.] Bigel.) with specialreference to oleosomes. Int J Plant Sci 161, 631-638.

14. Hartung W, Sauter A, Hose E. 2002. Abscisicacid in the xylem: where does it come from,where does it go to? J Exp Bot 53, 27-32.

15. Libbenga KR, Bogers RJ. 1974. Root nodulemorphogenesis. In “The biology of nitrogenfixation”. Quispel A (Ed.). North Holland,Amsterdam. pp. 430-472.

16. McKenzie DB, Donnelly JG, Martin DA. 1997.Beach pea (natural stand) biomass production. Areport submitted to the Parks and Natural AreasDivision of the Department of Tourism, Cultureand Recreation, Government of Newfoundlandand Labrador, St. John’s, Newfoundland, Canada.

17. Sauter A, Davies WJ, Hartung W. 2001. Thelong-distance abscisic acid signal in the droughtedplant: the fate of the hormone on its way fromroot to shoot. J Exp Bot 52, 1991-1997.

18. SPSS Inc. 1990. SPSS/PC + StatisticsTM 4.0 forthe IBM PC/XT/AT and PS/2. SPSS Inc.,Chicago. pp. B24-B34.

19. Watts SH, Wheeler CT, Hillman JR, BerrieAMM, Crozier A, Math VB. 1983. Abscisic acidin the nodulated root system of Alnus glutinosa.New Phytologist 95, 203-208.

20. Welbaum GE, Bian D, Hill DR, Grayson RL,Gunatilaka MK. 1997. Freezing tolerance, proteincomposition, and abscisic acid localization andcontent of pea epicotyl, shoot, and root tissue inresponse to temperature and water stress. J ExpBot 48, 643-654.

21. Wilkinson S, Davies WJ. 2002. ABA-basedchemical signalling: the co-ordination ofresponses to stress in plants. Plant Cell Environ25, 195-210.


13

New green manuring Lathyrus sativus variety AC Greenfix available in USA

David Krause and Ila Krause

Dakota Frontier Seeds, 6520 45th Avenue Flasher, North Dakota 58535, USA


Note: Dakota Frontier Seeds is a commercial company and is the exclusive nationwide distributor of AC Greenfix inthe USA working with seed dealers throughout the USA.

AC Greenfix (Fig. 1), a new variety of grass pea(Lathyrus sativus) has been developed to supply greenmanure nitrogen for both organic and conventionalgrowers, and presents a good new choice for greenmanuring or green fallow.

Partially replacing summer fallow with green manurelegumes not only adds valuable nitrogen, but alsoreduces soil loss from erosion, increasing soil

productivity for subsequent crops. AC Greenfixovercomes the problems of soil moisture depletion andlimited abilities to fix nitrogen that have kept growersfrom using traditional green manure crops in the past.It is a tremendous boon for organic farmers, the fastestgrowing agricultural segment in the US. Its featuresmake it ideal for green fallow rotation, required forcontinued organic certification.

Fig. 1. Lathyrus sativus cv AC Greenfix. From left to right: Seeds, 2-3 weeks growth, flowers and pods.

AC Greenfix was developed in Saskatchewan, Canadaby Dr. V.O. Biederbeck from studies conducted from1984 to 1992. It is useful for farmers wishing toincrease soil fertility, improve yields and conservesoil. AC Greenfix was officially registered under theCanada Seeds Act and became commercially availablein May 1996 from Johnson Seeds, of Arborg,Manitoba, Canada. Its benefits were shown in fouryears of on-farm studies with only 125-150 mm ofrainfall. The studies showed an average productionrate of 90-100 kg N/ha, with more than 200 kgproduced in one research test plot. Nitrogen fixingbegins in the top 30 cm of soil about two weeks afteremergence and reaches maximum activity betweenearly and full bloom. About 80 percent of the nitrogenis contained in aboveground growth. Plant growthusually ranges from 40 to 90 cm high, with plants in

full bloom in about 60 days. AC Greenfix willcontinue to flower and grow as long as there areadequate moisture and temperature.

Early spring planting yields the best results, but isrecommended when the temperature is no longerexpected to fall below -1 °C. Cool spring weatherenhances root development and enables the plant tobenefit from winter moisture.

AC Greenfix also survives well during severe droughtand heat and responds well to irrigation. With itsdrought tolerance, moisture efficiency and resistanceto many insects and diseases, AC Greenfix is suitablefor a variety of climates and soil types, and canenhance the major components of soil quality andboost soil fertility. It has great potential as a soil-


14

improving crop and as an alternative to commercialfertilisers. Seed cost in the US is about US$1 per kgand is available exclusively from Dakota FrontierSeeds for the 2003 growing season.

Proper seed inoculation increases legume growth byabout 100 percent, raises water use efficiency by 130percent and reduces weed population. The inoculantmust be designated for field peas or vetch, rather thanfor alfalfa, sweet clover or soybeans. Canadianresearchers suggest a high-quality, self-sticking, peatpowder-type Rhizobium leguminosarum inoculant,similar to that used for peas and lentils. The seed canbe planted in the top 8 cm of soil, depending on theavailable moisture, soil type and protection frompossible freezing. It should be covered with at least 3cm of soil.

The available moisture, climate and intended use ofthe growing crop determine the plant sowing rate. Arate of 50-70 kg/ha is recommended, andexperimentation will help growers find the rate fortheir situations.

A regular grain drill or air seeder is recommended forplanting AC Greenfix as they will not damage the seedor chip the corners, making them susceptible mouldand other pathogens and reducing germination.

US researchers in North Dakota, Montana andOklahoma have studied the forage quality traits of ACGreenfix against 25 other annual and perenniallegumes. The crude protein normally runs from 22 to26%, with a high ranking for total digestible nutrientsand relative feed value. Cool season hay forage trialswere conducted at North Dakota State University’sCarrington Research Extension Center from 1995-2000. The studies showed that AC Greenfix excelledwhen compared to 15-25 other annual legumes,cereals and cereal/legume mixtures.

For research statistics and other information on ACGreenfix, go to www.acgreenfix.com.


15

Genetic diversity among induced mutants of grasspea (Lathyrus sativus L.)

Sushil Kumar* and D.K. Dubey

Department of Botany, Janata Mahavidyalaya, Ajitmal, Auraiya 206 121 (UP), India

*Present Address: Society for Conservation of Nature (SCON). 576, Karamganj, Punjabi Colony, Etawah 206001 (UP), India

Introduction Knowledge about genetic diversity is a prerequisite ofany breeding programme. Inclusion of diverse parentsin hybridisation programmes serves the purpose ofcombining desirable genes in new recombinations.Mahalanobi’s D2 statistic (7) is a powerful tool inquantifying the degree of divergence at genotypiclevel. Several workers have used this method toquantify the degree of divergence based on phenotypicobservations in different crops (1,2,4,5,6,8,11,12). Thesestudies have shown that accessions from the samegeographical region may differ genetically as well asphenotypically and also in adaptability. In the presentstudy, this analysis was used to determine thedivergence among 81 induced mutants and 6 parentallines of grasspea (Lathyrus sativus L.).

Material and MethodsIn a study on induced mutants in cvs. Nirmal, LSD-3,DL-250, PLK-750, Roma-2 and P-24 of Lathyrussativus using gamma rays, ethyl methyl sulphonate(EMS), diethyl sulphate (DES) and N-nitroso-N-methyl urea (NMU), a number of macromutants wereisolated. Of these, 81 elite mutants were selected forthe present study. M3 populations of these mutantswere grown along with their parental lines (controls)in a randomised block design with five replications.Each replication comprised of three rows 45 cm apart,with plant to plant spacing of 45 cm. Observationswere recorded on five randomly selected plants foreight characters viz. days to first flowering, plantheight, number of primary branches, pods/plant,seeds/pod, seeds/plant, 100 seed weight and seedyield/plant.

Estimates of divergence among the 87 genotypes werebased on multivariate analysis using Mahalanobi’s D2

statistic (7). The formation of clusters was done byToucher’s method (9).

Results and DiscussionAnalysis of variance showed significant differencesamong the genotypes. Based on the degree ofdivergence, 87 genotypes (including parental lines)were grouped into 12 clusters. Cluster V was thelargest one having 17 genotypes (Table 1) and cluster

VIII the smallest with only one genotype. The patternof cluster formation shows that there is a wide geneticdiversity with regard to yield and its components inthe genotypes isolated on the basis of macromutationsfrom the same parent variety. On the other hand it isalso interesting to note that several mutants isolatedfrom genetically diverse varieties fall under the samecluster i.e. they are genetically closer to each other.This is in agreement with findings for pigeonpea (3),where genetic diversity was claimed not be parallelwith geographical diversity and has been supported inother pigeonpea studies (10).

Inter and intra-cluster distances have been given inTable 1. Maximum intra-cluster distance (3.87) wasobserved for cluster XII, whereas the minimumdistance (2.45) was recorded for cluster XI (excludingcluster VIII with only one genotype). Maximum inter-cluster distance was found between cluster VII andVIII (9.57) followed by clusters I and VIII (9.43), VIIIand IX (8.29), III and VIII (8.12), VIII and XI (8.00)and VIII and X (7.62) suggesting wide diversitybetween these clusters. Minimum inter-clusterdistance between IX and X (1.41) indicated closerelationship among genotypes falling in these clusters.

Mean performance of different clusters for variouscharacters is given in Table 2. The table revealsconsiderable differences between the clusters. Earlymaturity was found in clusters IV, VIII and XI, whilecluster I showed delayed maturity over the generalmean. Significant increased plant height was recordedin clusters II, VIII, and IX, while dwarf plant heightwas recorded in clusters I, III, VII and XI. Maximumnumber of primary branches was recorded in clusterII. Maximum numbers of pods, number of seeds andyield per plant were recorded in cluster VI and VIII.Maximum 100 seed weight was recorded in cluster X.Minimum number of seeds/plant, yield/plant andminimum number of pods/plant, 100 seed weight andyield/plant were recorded in clusters I and VII,respectively. Thus, in overall performance, cluster VIand VIII were the best since the mean values for mosttraits were higher. Cluster I showed minimum valuesfor almost all the traits, indicating poorestperformance.


16

Table 1: Number of genotypes in clusters and average intra and inter-cluster distances between clustercentroids in induced mutants of grasspea.

Cluster No. Clustergenotypes I II III IV V VI VII VIII IX X XI XII

I 8 3.05 4.83 2.07 5.47 2.72 6.73 1.78 9.43 3.85 3.87 3.13 4.86II 5 3.81 3.95 3.06 2.91 3.63 4.86 6.22 3.02 3.05 4.31 2.49

III 6 2.94 3.98 1.95 5.52 2.36 8.12 2.96 2.50 1.98 3.50IV 3 3.21 3.71 3.27 5.52 4.62 4.67 3.72 3.65 2.97V 17 2.73 4.34 2.30 7.47 3.19 3.21 2.41 2.19

VI 3 2.77 6.55 3.69 5.67 5.42 5.58 2.88VII 10 2.46 9.57 4.30 4.40 2.52 4.25

VIII 1 0.00 8.29 7.62 8.00 6.13IX 9 2.48 1.41 4.38 3.85X 5 3.07 3.73 3.72

XI 10 2.45 3.42XII 10 3.87

Note: figures in italics denote intra-cluster values.

Table 2: Mean performance of different clusters for various characters in M3 generation of grasspea mutants.

Days to firstflower

Plant height(cm)

No. primarybranches

Pods/ plant Seeds/ pod Seeds/ plant 100 seedweight

Yield/ plant(g)

I 105* 45* 4.8 25.4 1.78 43.4 4.42 2.06*II 96 80* 8.6* 71.4 2.33 164.9 6.46 10.47

III 94 49* 4.6 41.7 2.06 89.2 7.17 5.97IV 74* 70 6.5 103.9 2.08 215.2 6.06 12.66V 101 57 5.6 55.4 2.55 138.3 5.24 7.44

VI 97 71 6.8 125.6* 2.58 325.3 5.92 19.69*VII 102 40* 4.7 21.7* 2.61 54.0 3.93* 2.19*

VIII 76* 84* 7.2 203.8* 1.91 390.5 6.04 23.67*IX 102 85* 6.6 33.7 2.01 66.4 9.97* 6.69X 90 73 6.4 45.4 1.81 81.2 10.23* 7.66

XI 80* 44* 4.7 44.2 2.52 110.0 4.84 5.40XII 94 67 6.4 70.4 3.00* 208.3 6.56 13.23

Mean 93 64 6.1 70.2 2.27 157.2 6.40 9.76CD 5% 12.0 13.6 1.70 41.85 0.57 95.28 2.15 9.19* differs significantly (P<0.05) from the mean.

Of the two best performing clusters, cluster VIcontained three genotypes (AKM-5, AKM-24 andAKM-79), while cluster VIII contained only genotype(AKM-26). These four genotypes hold promise forfurther use in future grasspea improvement programs.

The relative importance of plant height, number ofprimary branches, number of pods and number ofseeds besides seed yield in contributing towardsdivergence was established when inter cluster groupsmeans were compared. This study has clearly broughtout in quantitative terms, the wide divergence induced

in the mutants isolated from the different parentalgenotype and falling under the same cluster throughmutagenic treatments. Thus it could be concluded thatwhile selecting genotypes from a particular cluster,the inter cluster distance, cluster mean and per seperformance should be taken into consideration.

AcknowledgementsThe authors are thankful to S.C.S.T., Lucknow (U.P.)for financial assistance and to Principal J.M.V.Ajitmal for providing facilities.


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References1. Aher RP, Salunke JS, Shinde GC, Kute NS. 1998.

Genetic diversity in early pigeonpea. Ind J PulsesRes 11, 68-71.

2. Balyan HS, Singh S. 1986. Genetic divergence inlentil. LENS Newsletter 13, 3-4.

3. Henry A, Krishna GVSR. 1992. Geneticdivergence in pigeonpea. Madras Agric J 79, 41-43.

4. Khare D, Singh CB. 1992. Divergence analysis inVicia faba L. for nutritional and antinutritionalattributes. Ind J Genetics 51, 58-62.

5. Koul BL, Singh K, Kanwal KS. 1997. Geneticvariation for yield and other quantitative traits inchickpea. Ind J Pulses Res 10, 53-56.

6. Kumar S, Dubey DK. 1996. Divergence amonginduced mutants of grasspea (Lathyrus sativusL.). FABIS Newsletter 38/39, 33-36.

7. Mahalanobis PC. 1928. A statistical study ofChinese head measurement. J Asiatic Soc, Bengal25, 301-307.

8. Mishra AK, Yadav LN, Indrapurkar YM. 1995.Divergence studies in genotypes of mungbean andurdbean. Ind J Pulses Res 8, 175-177.

9. Rao CR. 1952. Advanced statistical methods inbiometrical research. John Willey and Sons Inc.,New York.

10. Sandhu TS, Reddy PK, Gumber RK. 1993.Assessment of genetic divergence in pigeonpeagermplasm. International Pigeonpea Newsletter17, 8-10.

11. Sharma PC, Luthra SK. 1987. Genetic divergencein lentil (Lens culinaris Med.). Genetika Agraria41, 349-359.

12. Vandana, Dubey DK. 1994. Genetic divergenceamong induced mutants of Vicia faba L. J IndianBot Soc 73, 121-123.


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Review: A brief history of grasspea and its use in crop improvement

Jennifer S. McCutchan

ILFR, University of Melbourne, Victoria 3010, Australia.


Introduction Grasspea (Lathyrus sativus L.) is an annual pulse cropbelonging to the family Fabaceae (4) and the tribeVicieae. Grasspea is known by a wide range ofcommon names, include chickling vetch, Indian vetch(UK and USA), khesari or Batura (India) and dhal (44).Along with Pisum, Lens and Vicia, this genus may bea source of new and useful genetic traits for closelyrelated genera for future plant breeding workimproving commercially valuable species.

The Lathyrus genus is believed to have originated insouthwest and central Asia, with a significantsubsequent spread to the east of the Mediterraneanbasin (62, 63). All grasspea lines appear to divide intotwo geographical origins - one group derives from theIndian subcontinent, and another from theMediterranean/European region, which typically hashigher yields and larger seeds (24). Morphologically,this crop resembles field pea, but its leaflets are longand grass-shaped rather than rounded, and it has adeep taproot system (65, 66). This species is now widelydistributed throughout Eurasia, North America,temperate South America and East Africa (44, 62, 63)

with a small amount being cultivated in Australia (58).The most recent studies of grasspea have examinedover 140 accessions in detail (50).

Agronomic and economic importance of grasspeaThere are around 187 species and subspecies in theLathyrus genus. Grasspea is the only member of thisgenus that is widely cultivated as a food crop (44),while L. odoratus (sweet pea) is grown commerciallyfor its flower morphology (51).

Grasspea performs well under adverse agriculturalconditions, and its many cultivars possess differentattributes including the ability to resist both droughtand flooding, high climatic adaptability and the abilityto grow in cool climates and at high altitudes (65, 66) .Grasspea cultures also have the ability to adapt tosaline, alkaline, clay or otherwise poor soils, and arehardy and easy to cultivate (3, 61). In addition tonutritional benefits, grasspea has an important role asa legume crop in crop rotations, reportedly adding

around 67 kg ha–1 of nitrogen to the soil in a singleseason and conferring yield and protein benefits on thesubsequent non-legume crop (44, 68).

Grasspea is grown primarily as a winter pulse crop,and is grown for stockfeed and human consumption inthe Middle East, France, Spain, India, Bangladesh,China, Pakistan and Nepal, Asia and Africa (24, 44, 45, 62),and is known as the ‘poor men’s diet’ in the Centralregion of India (60). This crop is cultivated only as afodder crop in Australia, Europe and North America,and is recommended for low quality soils ofsouthwestern Australia (59). Grasspea is valued as anutritious staple food and fodder crop primarily due toits relatively high protein content 18–34% dry weightin seeds, 17% in mature leaves) and its high lysinecontent (18, 53, 58).

One of the major drawbacks of grasspea is the factthat the seeds contain a major anti-nutritionalcompound β-N-oxalylamino-L-alanine (BOAA) (alsoknown as β-N-oxalyl-L-α, β-diaminopropionic acid orODAP) (6, 61, 68, 70). Following prolonged or excessiveconsumption of grasspea, the neurotoxin causes adrastic paralytic disease known as ‘lathyrism’ or‘neurolathyrism’, manifesting as paralysis of the legmuscles, muscular rigidity and weakness (43, 66). Recentstudies have found that genotype is the maindeterminant of BOAA concentration, with little if anyeffect from environment (24).

Grasspea genetics and applicationsGenetic studies on Lathyrus spp. are rare. Detailedchromosome analysis of grasspea shows that grasspeahas a chromosome number of 2n = 14, with twometacentric and five submetacentric chromosomes (28,

33). Total metaphase chromosome length is estimatedat 40.3 µm (47).

Meiosis and chromosome pairing have been examinedin grasspea, L. odoratus and L. pratensis (29, 30).Variation in genome size (47) or karyotype (28, 31, 32, 33, 34)

in Lathyrus spp. has also been studied. Preliminarystudies on the phylogenetic distance between sevenLathyrus species have been undertaken (1). Studieshave taken place on the inheritance of genetic traits


19

such as BOAA content, flower and seed coat colour(58, 65, 66) and the genetic diversity in germplasmcollections of Lathyrus spp. (52).

Grasspea has been identified as an important source ofnovel genes for use in grain legume breedingprograms, both for abiotic and biotic stress resistance.Compared to other legumes, grasspea is resistant tomany insect pests (66) . Lathyrus spp. is a possiblesource of genes for cold tolerance (52) or downy andpowdery mildew resistance genes (44). In Syria, host-plant screening in Lathyrus spp. has taken place,uncovering potential sources of resistance to powderymildew [induced by Erysiphe pisi Syn. (syn, E.Polgoni D.C.)], botrytis blight (induced by Botrytiscinerea Pers. ex Fr) and ascochyta blight (induced byA. pisi Lib.). These sources of resistance are graduallybeing incorporated into pulse breeding programs overtime (52).

Several studies have demonstrated that many membersof the Lathyrus genus possess resistance to ascochytablight caused by M. pinodes (5, 10, 11, 69). Glasshousetrials conducted at the Victoria Institute for DrylandAgriculture in Victoria, Australia, with controlledinoculation conditions found that 59 accessionsLathyrus spp., representing ten species including L.sativus, exhibited higher levels of resistance toascochyta blight than did P. sativum (13, 48). Attemptswere made to produce field pea–grasspea hybrids byconventional crossing methods followed by embryoculture, but this work was unsuccessful (E. Pang, pers.comm.). Furthermore, preliminary screenings of L.sativus accessions indicated a degree of intraspecificvariation in ascochyta blight resistance (E. Pang, pers.comm.). The inheritance of resistance to this diseasewas studied and resistance was found to be controlledby two major recessive genes (13).

Two potential techniques, more advanced thanconventional crossing, offer possibilities fortransferring any identified resistance gene(s), such asthose from grasspea, across the species barrier. Theseare cloning of a resistance gene from the donorspecies, followed by transformation and regenerationof the transgenic host species directly usingAgrobacterium spp., or through asymmetric somatichybridisation via protoplast fusion (19, 40). Genetictransformation through techniques such as theseprovides a means for the genetic improvement of thegenome of crop species. Judicious application ofbiotechnological tools holds great potential foralleviating some of the major constraints toproductivity of these crops in the agricultural systemsthroughout the world (57).

A transformation/regeneration procedure usingAgrobacterium-mediated gene delivery has been

developed successfully for one agronomicallyimportant crop, field pea (2, 21, 22, 55). This has sincebeen used to transfer the cDNA encoding an α-amylase inhibitor from common bean into field pea,conferring resistance to pea weevil (56), and toherbicides (21, 22). Agrobacterium-mediatedtransformation of field pea has also been shown to beeffective across a range of genotypes (46, 49). However,the use of such a technique to transfer ascochyta blightor other resistance from grasspea into field pea is notyet possible, because the relevant ascochyta blightgenes have not been identified cloned. Once this hasbeen determined, a more rapid means oftransformation of agronomically important crops suchas field pea, using valuable genes from grasspea,should be possible.

Grasspea tissue cultureThe use of wild relatives for introducing resistancegenes originates from the ongoing search for a broadergene pool for continued crop improvement. Resistancemay also be induced by the use of mutagenic agents(39) or tissue culture techniques to produce somaclonalvariants (35, 36). However, the disadvantage of inductionof resistance is the unpredictability of the resultingcombination of other traits in the new plant, orreduced fertility 20). While the intentional introductionof new, known traits is desired, interspecific orintergeneric barriers must be overcome. Manytechniques are available for this task, includingconventional crossing with embryo rescue, somatichybridisation via protoplast fusion, or transformationusing Agrobacterium tumefaciens or A. rhizogenesplasmid vectors. However, each of these techniquesrequires tissue culture protocols to ensure regenerationof mature hybrids. Plant regeneration from in vitrocultures is the major limiting factor in production oftransgenic plants, from either protoplast- orAgrobacterium- derived sources.

Tissue culture is an effective way to study manyaspects of cell development and differentiation (12),and relies on the ability of cells to undergo sustaineddivision on a solid or liquid growth medium. Thebasal medium consists of macro- and micronutrients,trace elements and carbon substrates such as sucroseor glucose, with a pH of around 5.6–6.0 (15). Additivessuch as coconut water, a source of nutritious liquidendosperm (16, 54) have been seen to stimulate cellgrowth (63). The first report of successful isolation andculture of protoplasts in Lathyrus spp. was from callusof L. odoratus (51). There are only two reports ofprotoplast isolation in grasspea; an Australian group(42) were the first to isolate grasspea protoplasts, fromcell suspension cultures, where in the following year aFrench group (17) isolated grasspea protoplasts fromembryonic axis shoots.


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Molecular biology using grasspeaAs genetic transformation of many pulse species hasmet with limited success in the last decade, the searchcontinues for new or improved methods of transferringgenes across the sexual barrier (9). The techniques ofAgrobacterium-mediated gene transfer,electroporation and microprojectile bombardment alldepend on having a knowledge of specific genes to betransferred (7, 8, 14, 25, 26, 27, 38), where protoplastelectrofusion allows large segments of the genome tobe transferred. Electrofusion is an experimental optionfor consideration in cases where the gene(s) of interesthave not yet been characterised or isolated.

Molecular markers are used in plant genome studies toassess genetic diversity, identify plants at cultivarlevel, for construction of genetic maps, as specificprobes for screening of traits in breeding programsand to tag genetic traits (37). For example, RAPDanalysis has been used in cluster analysis studies forclassification of two Lathyrus species, L. odoratus andL. larifolium (23).

One potential application of molecular markers inLathyrus spp. is the development of SCAR primerstightly linked to the gene(s) for ascochyta blightresistance (13). These markers could then be used toselect somatic hybrids with the resistance marker geneincorporated into the genome. This would provide arapid and non-destructive method for screening largenumbers of putative hybrid plants, to identify thosecontaining the introduced resistance gene.

Several studies have developed protoplast sources forgrasspea, through shoot, callus and suspension cellcultures, to develop protoplast isolation andpurification protocols (17, 41. 42). Electrofusion betweenfield pea and grasspea protoplasts has been carried outto introduce useful resistance genes from grasspea intofield pea (17, 41, 42). Analysis of markers linked to thegenes for ascochyta blight resistance in grasspea hasbeen performed (41).

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rDNA loci, and DAPI bands reflect thephylogenetic distance between Lathyrus species.Genome 43, 1027–1032.

2. Bean SJ, Gooding PS, Mullineaux PM, DaviesDR. 1997. A simple system for peatransformation. Plant Cell Reports 16, 513–519.

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4. Biswas SC, Biswas AK. 1997. Inducedtranslocation heterozygosity and sterility inLathyrus sativus L. Bangladesh Journal of Botany26, 131–136.

5. Bretag TW. 1991. Epidemiology and control ofascochyta blight of field peas. PhD thesis, LaTrobe University, Melbourne, Australia.

6. 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 inLathyrus sativus by high-performance liquidchromatography with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)derivatisation. Journal of Agricultural and FoodChemistry 48, 3383–3386.

7. Cho MJ, Jiang W, Lemaux PG. 1999. High-frequency transformation of oat viamicroprojectile bombardment of seed-derivedhighly regenerative cultures. Plant Science 148,9–17.

8. Choi HW, Lemaux PG, Cho MJ. 2000. Highfrequency of cytogenetic aberration in transgenicoat (Avena sativa L.) plants. Plant Science 156,85–94.

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10. Clulow SA. 1989. The resistance of Pisum toMycosphaerella pinodes (Berl and Blox.)Vestergr. PhD thesis, University of East Anglia,UK.

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13. Croft AM. 2000. The development of molecularmarkers for ascochyta blight resistance genes inLathyrus sativus L. PhD Thesis, The Universityof Melbourne, Australia.

14. De Villiers SM, Kamo K, Thomson JA, BornmanCH, Berger DK. 2000. Biolistic transformation ofchincherinchee (Ornithogalum) and regenerationof transgenic plants. Physiologia Plantarum 109,450-455.

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16. Dornelas MC, Vieira MLC. 1994. Tissue culturestudies on species of Passiflora. Plant Cell Tissueand Organ Culture 36, 211–217.

17. Durieu P, Ochatt SJ. 2000. Efficient intergenericfusion of pea (Pisum sativum L.) and grass pea


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(Lathyrus sativus L.) protoplasts. Journal ofExperimental Botany 51, 1237–1242.

18. Gharyal PK, Maheshwari SC. 1980. Plantletformation from callus cultures of a legume,Lathyrus sativus cv. L.S.D.-3. Zeitschrift fürPflanzenphysiologie Bd. 100 S, 359–362.

19. Glimelius K. 1988. Potentials of protoplast fusionin plant breeding programmes. In ‘Progress inplant protoplast research’. (Eds Puite K, Dons,JJM, Herzig, HJ, Kool, AJ, Koorneef, M, Krens,FA. Boston) pp. 159–168. (Kluwer AcademicPublishers: Dordrecht, The Netherlands)

20. Glimelius K, Fahlesson J, Landgren M, Sjödin C,Sundberg E. 1991. Gene transfer via somatichybridization in plants. Tibtech 9, 24–30.

21. Grant JE, Cooper PA McAra AE, Frew TJ. 1995.Transformation of peas (Pisum sativum L.) usingimmature cotyledons. Plant Cell Reports 15, 254–258.

22. Grant JE, Cooper PA, Gilpin BJ, Hoglund SJ,Reader JK, Pither-Joyce MD, Timmerman-Vaughan GM. 1998. Kanamycin is effective forselecting transformed peas. Plant Science 139,159–164.

23. Hanada H, Hirai M. 2000. Classification of sweetpea (Lathyrus odoratus L.) and everlasting pea(Lathyrus latifolium L.) by random amplifiedpolymorphic DNA (RAPD) analysis. Journal ofthe Japanese Society for Horticultural Science 69,758–763.

24. Hanbury CD, Siddique KHM, Galwey NW,Cocks PS. 1999. Genotype-environmentinteraction for seed yield and ODAPconcentration of Lathyrus sativus L. and L. ciceraL. in Mediterranean-type environments.Euphytica 110, 45–60.

25. Huang Y-W, Dennis ES. 1989. Factorsinfluencing stable transformation of maizeprotoplasts by electroporation. Plant Cell, Tissueand Organ Culture 18, 281–296.

26. Ishige T. 1995. Somatic cell fusion betweendiploid potato (Solanum tuberosum) lines usingtransformed antibiotic selection markers. PlantScience 112, 231–238.

27. Jeknic Z, Lee SP, Davis J, Ernst RC, Chen THH.1999. Genetic transformation of Iris germanicamediated by Agrobacterium tumefaciens. Journalof the American Society for HorticulturalScience. 124, 575–580.

28. Kar K, Sen S. 1991. A comparative karyologicalstudy of root and embryo tissue of a few genera ofLeguminosae. Cytologia 56, 403–408.

29. Khawaja HIT, Sybenga J, Ellis JR. 1997.Chromosome pairing and chiasma formation inautopolyploids of different Lathyrus species.Genome 40, 937–944.

30. Khawaja HIT, Sybenga J, Ellis JR. 1998. Meiosisin aneuploids of tetraploid Lathyrus odoratus andL. pratensis. Hereditas 129, 53–57.

31. Klamt A, Schifino-Wittmann MT. 2000.Karyotype morphology and evolution in someLathyrus (Fabaceae) species of southern Brazil.Genetics and Molecular Biology 23, 463–467.

32. Kumar S, Dubey DK. 1996a. Variability andcorrelation studies in grasspea (Lathyrus sativusL.). FABIS Newsletter 38/39, 26–30.

33. Kumar S, Dubey DK. 1996b. Karyotype study inLathyrus sativus L. cv P-505. FABIS Newsletter38/39, 24–26.

34. Kumar S, Dubey DK. 1996c. Divergence amonginduced mutants of grasspea (Lathyrus sativusL.). FABIS Newsletter 38/39, 33–36.

35. Larkin PJ, Scowcroft WR. 1981. Somaclonalvariation- a novel source of variability from cellcultures for plant improvement. Theoretical andApplied Genetics 60, 197–214.

36. Larkin PJ, Banks PM, Bhati R, Brettell RIS,Davies PA, Ryan SA, Scowcroft WR, SpindlerLH, Tanner GJ. 1989. From somatic variation tovariant plants; mechanisms and applications.Genome 31, 705–711.

37. Lawson WR, Henry RJ, Kochman JK, Kong GA.1994. Genetic diversity in sunflower (Helianthusannuus L.) as revealed by random amplifiedpolymorphic DNA analysis. Australian Journal ofAgricultural Research 45, 1319–1327.

38. Li H, Wylie SJ, Jones MGK. 2000. Transgenicyellow lupin (Lupinus luteus). Plant Cell Reports19, 634–637.

39. Maluszynski M, Ahloowalia BS, SigurbjornssonB. 1995. Application of in vivo and in vitromutation techniques for crop improvement.Euphytica 85, 303–315.

40. Marrs KA, Urioste JCC. 1995. Transient geneexpression analysis in electroporated maizeprotoplasts. In ‘Plant cell electroporation andelectrofusion protocols’. (Ed. Nickoloff JA) Vol.55, 133–145 (Humana Press Inc.: Totowa, NJ,USA)

41. McCutchan JS. 2001. Transferring ascochytablight resistance from Lathyrus spp. into field pea(Pisum sativum L.) via protoplast fusion (somatichybridisation). PhD Thesis, The University ofMelbourne, Australia.

42. McCutchan JS, Larkin PJ, Stoutjesdijk PA,Morgan ER, Taylor PWJ. 1999. Establishment ofshoot and suspension cultures for protoplastisolation in Lathyrus sativus L. Journal ofBreeding and Genetics 31, 43–50.

43. Mehta SL, Ali K, Barna KS. 1994. Somaclonalvariation in a food legume - Lathyrus sativus.Journal of Plant Biochemistry and Biotechnology3, 73–77.


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44. Muehlbauer FJ, Tullu A. 1997. NewCROPFactSHEET — Lathyrus sativus L. Internetpublication.http://www.hort.purdue.edu/newcrop/CropFactSheets/grasspea.html. Last updated 24 February1998.

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47. Nandini AV, Murray BG, O’Brien IEW,Hammett KRW. 1997. Intra-and interspecificvariation in genome size in Lathyrus(Leguminosae). Botanical Journal of the LinneanSociety 125, 359–366.

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50. Przybylska J, Zimniak-Przybylska Z, KrajewskiP. 2000. Diversity of seed globulins in Lathyrussativus L. and some related species. GeneticResources and Crop Evolution 47, 239–246.

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52. Robertson LD, Singh KB, Erskine W, Abd elMoneim AM. 1996. Useful genetic diversity ingermplasm collections of food and foragelegumes from West Asia and North Africa.Genetic Resources and Crop Evolution 43, 447–460.

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Heritability of seed weight in an inbred population of large-seeded Lathyrus sativus

M. Mera*, A. Montenegro, N. Espinoza, N. Gaete and L. Barrientos

INIA-Carillanca, Casilla 58-D, Temuco, Chile


Introduction The grass pea (Lathyrus sativus L.) cultivated in Chilehas a large, mostly clear white, grain. It is valued byEuropean communities as the basis for the preparationof traditional dishes. Small Chilean farmers areexporting grass pea to these markets, which pay higherprices for larger seed. However, the size of the graincurrently being produced is quite heterogeneous, andthe export grain is produced after severe sorting of theharvested grain. A short-term project is underway (4),which aims to improve crop management and grainquality, particularly seed diameter. This preliminarywork provides an estimate on how much of theobserved variation for seed weight in a local grass peapopulation has a genetic basis. Seed mass appears tobe a reasonably good estimator of seed diameter (4).


A composite of 1640 seeds was assembled bycombining grass pea samples of equal size from 41farmers of Lumaco, an area located ~150 km NW ofTemuco, southern Chile. The composite was plantedin the winter (August) of 1999 in a sparse stand (6plants.m-2) and 1385 plants were harvestedindividually during the summer (January) of 2000.The grain produced by each single plant was cleaned,counted and weighed, and mean seed weight wascalculated. Ten randomly chosen seeds from each plant wereplanted in hills 0.7 m apart in August 2000, and 1384families were harvested in February 2001. The grainproduced by each family was cleaned and weighed,and mean seed weight was calculated from 200random grains per family.

Heritability was estimated by the standard unit method(1), which uses regression on data coded in standarddeviation units to eliminate the environmental effectexpected when parents are measured in one seasonand their offspring in another. This is equivalent tocalculating the correlation coefficient. Heritabilityestimates were also obtained by using parent-offspringregression (3).

Results and DiscussionThe 1385 plants harvested in 2000 had a seed weightrange of 114-455 mg.seed-1 on a single plant basis,with a mean of 270 mg.seed-1 and a standard deviationof 48.6. Seeds per plant ranged from 10 to 233 with amean of 61 and standard deviation of 30.9. For the1384 families harvested in 2001, seed weight ranged130-442 mg.seed-1, with mean of 279 mg.seed-1 andstandard deviation of 37.8.

Heritability based on the parent-offspring correlationcoefficient calculated from 1384 paired data was 0.58,a value that predicts a good response to selection forseed size (seed mass basis). The correlationcoefficient, b', was highly significant (P<0.0001).

The linear regression coefficient, b, was 0.45, estimatesignificant at P<0.0001. Heritability values for aninbred population of a self-pollinated species baseddirectly on the linear regression coefficient (b=h2) arelargely overestimated, and the correct estimator isb/2rxy=h2, rxy being a measure of the degree of geneticrelationship between the parent and its offspring (5).Therefore, considering for this case a geneticrelationship of unity between the homozygous parentand its self-pollinated offspring, h2=½b, and narrowsense heritability estimate becomes 0.23.

Previous estimates on the heritability of seed weightare scarce. Broad sense heritability values from 0.26to 0.31 were reported for seed weight in segregatingpopulations of Lathyrus sativus derived from crossesbetween a medium-size (132 mg.seed-1) lineoriginating from France and two small-seeded linesfrom Bangladesh and India (7).

According to the data generated from the evaluation ofour families in the 2000-01 cropping season, seedweight and seed yield were positively though weaklycorrelated, with phenotypic correlation coefficient rph= 0.27***. This value agrees with previous findings (7)

and it is higher than rph = 0.15ns reported (2) betweenseed weight and yield per plant in small-seeded grasspeas.


25

The mean seed weights for both seasons are higherthan the 176 mg.seed-1 observed among Chilean grasspea accessions collected in the central-south zone (6).Part of the difference may be due to the higher annualrainfall of the southern zone as compared to thecentral-south, which would provide conditions morefavourable for seed filling.

AcknowledgementsFNDR Project BIP 20155696-0 granted by GobiernoRegional de La Araucania.

References1. Frey KJ and Horner T. 1957. Heritability in

standard units. Agronomy J 49, 59-62.2. Kumar S and Dubey DK. 1996. Variability and

correlation studies in grasspea (Lathyrus sativusL.). FABIS Newsletter 38/39, 26-30.

3. Lush JL. 1940. Intra-sire correlations orregressions of offspring on dam as a method ofestimating heritability of characteristics. Proc AmSoc Anim Prod, 293-301.

4. Mera M, Montenegro A, Espinoza N, Gaete N.2000. Research backs grass pea exports by smallChilean farmers. Lathyrus Lathyrism Newsletter1, 31.

5. Smith JD and Kinman ML. 1965. The use ofparent-offspring regression as an estimator ofheritability. Crop Sci 5, 595-596.

6. Tay J, Valenzuela A and Venegas F. 1999.Collecting and evaluating Chilean germplasm ofgrasspea (Lathyrus sativus L.). FABIS Newsletter42, 3-5.

7. Tiwari KR and Campbell CG. 1996. Inheritanceof seed weight in grasspea (Lathyrus sativus L.).FABIS Newsletter 38/39, 30-33.


26

Luanco-INIA, a large-seeded cultivar of Lathyrus sativus released in Chile

M. Mera1, J. Tay2, A. France2, A. Montenegro1, N. Espinoza1 & N. Gaete1

1. INIA-Carillanca, Casilla 58-D, Temuco, Chile.2. INIA-Quilamapu, Casilla 426, Chillan, Chile.

Emails:

1. [email protected] 2. [email protected]

Grass pea (Lathyrus sativus L.) is a minor crop inChile that has received attention lately because smallsouthern farmers have begun to export this grain toEurope. Grass pea is also locally used for animalfeeding and occasional human consumption. TheChilean grass pea has a large, clear white seed, whichcomplies with the requisites of the European market(1).

A new large-seeded cultivar, named Luanco-INIA,was obtained through pure line selection fromaccession LS-0027, from the Chilean grass peagermplasm collection (2). After preliminary evaluation,it was included in yield trials over three years at fivecentral-south and south locations. Seed yield is notsuperior to the prevalent grass pea landraces, but seedsize is larger. Mean seed weight is usually around 300mg seed-1 though it can reach 350 mg in a favourableenvironment. Generally, mean seed weight of Luanco-INIA is about 50 mg over the prevalent local landrace.A mean seed weight of 300 mg seed-1 matches with aseed calibre (longest seed diameter) of between 9 and10 mm.

The seed coat is clear ivory white with yellowcotyledons. Flowers are white and number of seedsper pod varies from 1 to 3. Foliage is light-green.ODAP content has not been reduced, and it is withinthe range of other Chilean landraces (0.175 - 0.516%). As for most grass pea material evaluated, Luanco-INIA had extremely variable plant height, with stems

being able to reach up to 150 cm in length when theconditions favour soil moisture retention late in theseason. Despite this exuberant vegetative growth, seedyields have also been high in these cases, exceeding4000 kg ha-1. Luanco-INIA is well adapted to winterand spring seeding, either in medium or high rainfallareas in the central-south and south regions.

Seed of this variety is being increased and hopefullywill be distributed soon to farmers, who are expectedto benefit from the better price that should come withimproved grain quality.

Acknowledgements FNDR Project BIP 20155696-0 granted by GobiernoRegional de La Araucania.

References1. Mera M, Montenegro A, Espinoza N & Gaete N.

2000. Research backs grass pea export by smallChilean farmers. Lathyrus Lathyrism Newsletter1, 31-32.

2. Tay J, Valenzuela A, Venegas F. 2000. Collectingand evaluating Chilean germplasm of grasspea(Lathyrus sativus L.). (Abstract). LathyrusLathyrism Newsletter 1, 21.


27

Mutagenesis as a tool for improvement of traits in grasspea (Lathyrus sativus L.)

Wojciech Rybiński

Institute of Plant Genetics, Strzeszyńska 34, 60-479 Poznań, Poland


Introduction Grasspea (Lathyrus sativus L.) is produced as majorcrop in Bangladesh, China, India and Pakistan, and toa lesser extent in many countries of Europe, theMiddle East, northern Africa, as well as in SouthAmerica. It is extensively cultivated and naturalised inthe Middle East countries of Iraq, Iran, Afghanistan,Syria and Lebanon and in Ethiopia, Egypt, Morocco,Algeria and Libya in northern Africa (1). In Europegrasspea was cultivated in the Balkans by around 8000BC

(4). Foods prepared from the seeds have been

popular in Europe and are still being used locally inSpain, France, Germany and Bulgaria.

According to Milczak (5) grasspea was introduced toPoland (Podlasie Region) in the 17th century, probablywith arrival and settling of the Tatars. Now grasspea isgrown on a marginal scale in Eastern Poland, it iscultivated as a vegetable and is locally known as"soczewica podlaska" (Podlaska lentil). The first plantmaterial for breeding purposes in 1991 constituted alocal population from the village of Derewiczna. Asresults of breeding work in the following few years, incooperation with the Breeding Station for Vegetablesin Nochowo, the first Polish cultivars: Derek and Krabwere released (5).

The main advantages of grasspea are: resistance todiseases and environmental stress conditions, highyielding potential (at the level of pea) and favourablenutritional composition of seed. Grasspea is verytolerant to drought, due to a very hardy andpenetrating root system and can be grown on a widerange of soil types, including very poor soils.Undesirable features such as prostrate plant habit,indeterminate growth, late maturity, pod shatteringand neurotoxin content (ODAP) are the mostimportant factors limiting the broader introduction ofgrasspea for growing in different environmentalconditions.

Many crop improvement programs are presentlyaddressing research on different aspects of grasspea,(2,3) and mutagenesis and mutation breeding can be avaluable supplement to conventional breedingmethods. It can be used to create additional geneticvariability that may be utilised by plant breeders in the

development of cultivars for specific purposes or withspecific adaptations. To create new genetic variabilityof traits and mutations of possible economic interestthe seed of two Polish cultivars were treated withchemomutagens.

Material and MethodsA composite of 1640 seeds was assembled by twoPolish cultivars of Lathyrus sativus - Derek and Krabbeing treated with chemical mutagens: N-nitroso-N-methyl urea (NMU) and sodium azide (NaN3).Samples of dry seeds were soaked for 12 h in distilledwater at room temperature (24°C). After soaking theseeds were treated for 3 h with one of eight differentfreshly prepared aqueous solutions of NMU (0.5, 0.8,1.1 and 1.4 mM) or NaN3 (2.0, 4.0, 6.0 and 8.0 mM).The seeds were then washed with running tap-water toremove the mutagens from the seed surface. Onehundred treated seeds of each treatment were sown ina randomised-block design with 3 replications at anexperimental field in Cerekwica with spacing 50 x 20cm, immediately after the treatment. The untreatedseeds were sown as control. Data were recorded onplant height, pod number per plant, pod length, seedsnumber and weight per main stem and seed numberand weight per plant. The obtained data in M1(biological injury level) were expressed stimulation(+) or reduction (-) calculated as a percentage ofcontrol value.

The seeds of M1 plants were harvested and sown infield conditions. In the M2 progeny of the treated andcontrol material was screened for chlorophyll andmorphological mutations. From M2 progeny 82 viablemutants were selected in respect to plant habit,maturity, stem shape, leaf size, flower colour, podsize, seed size and colour according to systemssuggested by Swaminathan (11) and Nerkar (6).

All chosen genotypes and initial cultivars wereanalysed for morphological traits and yieldcomponents. The results allowed estimates of meanvalues and ranges of analysed traits. The plants in M2were grown in conditions of environmental stresscaused by very a strong spring drought.


28

Results and Discussion In the M1 progeny the effect of stimulation orreduction of analysed traits as compared to initialcultivars was observed (Tables 1 and 2). In the case ofNaN3 a small stimulation effect appeared only for lowdose of mutagen and cv. Derek. In other casesdifferent level of reduction was noticed. As comparedto NaN3, NMU showed a markedly stronger influenceon the grade of biological injuries. All doses ofmutagen decreased the plant height, pod length andparameters of yield structure. This reduction was morevisible for NMU (Table 1) than NaN3 (Table 2) forused doses and both cultivars. The level of biologicalinjuries expressed by reduction of traits value increasewith use of higher doses of chemomutagens. Similareffects in Lens culinaris were observed for gammarays, EMS and NMU (8). Particularly useful traits forestimation of susceptibility of plants to thechemomutagen constituted the traits directlyconnected with fertility: number and weight of seedsper pod and plant. The highest dose of NMU (1.4mM) reduced the number and weight of seeds per

plant by about 70% for both cultivars. According toSingh and Chaturvedi (10) who used gamma rays andEMS, seedling height, pollen fertility and plantsurvival were very sensitive indicators for estimationof biological injury level in M1 progeny.Independently of dose the susceptibility of cultivars tomutagens are genetically specific (6). In ourexperiment no distinct differences in susceptibilitybetween both cultivars were observed. It wasparticularly true for higher doses of NMU and NaN3and yield contributing parameters.

Genetically effect of mutagens was analysed in M2 inconditions of strong spring drought (Table 3). A widespectrum of chlorophyll mutations, albina, chlorina,chlorina-virescens, chlorotica, and chlorotica-virescens were observed. There was an increase in thefrequency of mutation with an increase in the dosageof NMU and NaN3. A higher frequency of chlorophyllmutations was noticed for NMU than for NaN3 and incv. Krab compared to Derek (Tables 1 and 2).

Table 1. Stimulation (+) or reduction (-)* of analysed traits of grasspea in M1 progeny and frequency ofchlorophyll mutations in M2 after use of NMU.

NMUdose(mM)

Plantheight

Pods/plant

Podlength

Seeds/pod

Seed weight/pod on mainstem

Seeds/planton main stem

Seed weight/plant

Chlorophyllmutation(%)

Cultivar Derek0.5 +1.0 +18.2 -1.4 -19.5 -6.5 -23.4 -8.8 1.850.8 -0.5 -16.3 -4.8 -38.9 -22.6 -82.4 -50.6 2.741.1 -7.0 -25.3 -11.6 -50.0 -30.7 -76.9 -66.8 3.181.4 -4.8 -68.6 -21.4 -52.8 -50.8 -79.8 -75.3 4.31

Cultivar Krab0.5 -8.2 -19.5 -0.9 -16.1 +2.5 -49.7 -37.9 2.570.8 -19.3 -42.0 -6.0 -32.2 -14.8 -64.5 -53.9 3.011.1 -25.1 -32.5 -11.0 -41.9 -22.4 -67.1 -62.9 4.551.4 -26.2 -46.7 -2.4 -41.9 -25.0 -76.8 -71.3 5.08

*stimulation and reduction level % of control value

Table 2. Stimulation (+) or reduction (-)* of analysed traits of grasspea in M1 progeny and frequency ofchlorophyll mutations in M2 after use of NaN3.

NaN3dose(mM)

Plantheight

Pods/plant

Podlength

Seeds/pod

Seed weight/pod on mainstem

Seeds/planton main stem

Seed weight/plant

Chlorophyllmutation(%)

Cultivar Derek2.0 +17.1 +6.9 +1.2 -20.0 -8.0 -15.1 -3.6 0.904.0 +4.5 +7.6 +1.8 -22.5 -8.8 -24.9 -7.2 1.416.0 -6.8 +0.1 -0.1 -35.0 -14.0 -33.4 -19.2 1.958.0 -2.9 -0.1 -2.8 -27.5 -21.6 -32.0 -35.5 3.27

Cultivar Krab2.0 -6.0 +5.8 -0.3 -3.3 -6.4 +0.4 -0.3 1.084.0 -23.8 -7.0 -5.6 -6.7 -0.1 -20.8 -27.3 1.506.0 -26.8 -13.5 -5.3 -10.0 -2.4 -28.9 -22.6 2.768.0 -28.8 -23.7 -8.4 -19.4 -4.1 -47.8 -38.1 3.77

* stimulation and reduction level was calculated as percent of control value


29

The higher frequency of chlorophyll mutationsobserved for cv. Krab was positively correlated withgenetic variability of visible mutants according to thesystem suggested by Swaminathan (11) (Table 4). FromM2 plants of cv. Krab 68 and cv. Derek 14morphological mutants were selected. The mean valueand range of traits of chosen plants are presented inTable 5. A wide spectrum of viable mutants wereobserved affecting plant habit, branching, maturityand parameters of yield structure. The chemomutagenswere particularly efficient in inducing plant habitvariability as dwarfism, semi-dwarfism, erectness andlow and high branching forms, which confirm dataobtained by Prasad and Das (7). The mean value ofplant height for cv. Krab and Derek was respectively75.3 and 69.2 cm and chosen M2 plants 54.0 and 55.6cm. The range of these traits for M2 plants were

respectively: 24-85 cm and 32-80 cm and for initialforms: 66-85 cm and 61-74 cm. Gamma rays andEMS altered polygenic variability in M2; plant heightranged between 26-120 cm with mean 71.9 cm, andnumber of primary branches per plant: range 2-14with mean 4.95 (13). The number of all branches perM2 plants ranged between 1-44 for cv. Krab and 9-32for cv. Derek. The grasspea showed indeterminategrowth with inclination to produce a high number ofbranches and biomass and in many cases decreasedseed yield, seed size and prolonged maturity period.The semi-dwarf forms with suitable first legumeheight, lower branch number and improved earlinessand stable yield ability seem to be interesting initialmaterial for breeding purposes. Such forms wereselected in M2 progeny.

Table 3. The temperature and rainfall in spring 2000 for region of Wielkopolska (data from Breeding Station Szelejewo).

March April May June

1-10

11-2

0

21-3

1

1-10

11-2

0

21-3

0

1-10

11-2

0

21-3

1

1-10

11-2

0

21-3

0

Temp (°C) 4.1 3.6 5.3 10.3 15.2 20.2 18.3 16.0 15.9 19.3 21.8 19.7Rainfall (mm) 41.7 28.9 34.9 0 17.1 0 0 14.4 36.7 10.2 1.7 7.5

Table 4. Classification of viable mutations found (after Swaminathan, 1965).

Character Visible mutations Macromutations Systematic mutationsPlant habit Erect, spreading, subnormal Small plant type, stunted,

minutum (miniature size)Grassy

Maturity Early flowering synchronous maturity - -Branching Non-branching, shy branching - -Stem - Brittle stem Afila (non-winged stem)Leaf Leaflets-broad, narrow, long, short,

obovate, wiry, three and four leafletsCoriaceata (leatheryleaf), abnormal leaflets,curly leaf, circinnata(rolled leaf)

-

Stipule - - Serrated stipuleFlower Albus (white), roseus (crimson), cyaneus

(light blue)Half-split keel Extra whorl of corolla,

epicalyx, bracteolata,extended peduncle

Pod Large poded, small podded, tereta(cylindrical pod), declinata (curved pod )

- -

Seed Coloured seed coat Small seeded -


30

Particularly wide variability was observed for numberof pods per plant, number and weight of seeds per podand plant as well as for 100 seed weight (Table 5).One of the most important traits influenced yieldingability is pods number per plant (9,12). The value of thistrait for M2 plants Derek und Krab rangedrespectively between 19-168 and 8-222. Studies usinggamma rays and EMS showed this trait in M2 rangedbetween 6-560 (13). In many cases the M2 plantsproduced a high number of pods per plant and werecharacterised by a decrease in 100 seed weight andincreased production of sterile pods. The 100 seedweight in M2 for Krab and Derek ranged respectivelybetween: 4.80-19.41 g and 5.1-10.8 g; for initialcultivars: 12.2- 13.9 and 8.3-9.9 g. After use ofgamma rays and EMS the 100 seed weight in M2range was 6.1-22.4 g (13).

In Table 6 are presented the most promising mutantsfor breeding purposes obtained in M2 progeny. Theanalysis of M3 and M4 may confirm that some mutantshave economic interest with the aim of using this toproduce new Polish cultivars adapted to our climaticconditions.

As mentioned, the M2 progeny were grown inconditions of very strong spring drought (Table 3).This environmental stress had no negative influenceon the growth and development of grasspea, however,for other crops nearby the stress reduced growth. Thisconfirms the high tolerance of this crop to droughtobserved by other investigators (2,3,4,5).

Table 5. Induced polygenic variability in M2.

Traits Cultivar Krab Cultivar DerekInitial form M2 plants Initial form M2 plants

Mean Range Mean Range Mean Range Mean RangePlant height (cm) 75.3 66-85 54.0 24-82 69.2 61-74 55.6 32-80First legume height (cm) 17.2 15-20 11.9 7-25 13.3 10-15 10.8 5-15No. stem/plant 12.6 10-14 17.6 1-44 15.6 13-18 15.7 9-32No. pods/plant 68.1 61-76 63.7 8-222 77.4 64-80 61.5 19-168No. fertile pods/plant 65.0 59-64 55.8 7-196 75.1 74-76 53.9 14-163No. sterile pods/plant 3.1 0-5 7.9 0-36 2.2 0-9 7.7 0-20Pod length (cm) 3.6 3.2-3.9 3.44 2.42-4.17 3.36 3.01-3.54 3.10 1.88-3.67Pod width (cm) 1.28 1.11-1.34 1.25 1.03-1.52 1.22 1.18-1.29 1.21 1.08-1.37No. seeds/pods/main stem 2.9 2.7-3.3 2.68 1.6-3.9 3.20 2.9-3.5 2.91 2.2-3.6Wt. seed/pod/main stem (g) 0.32 0.29-0.34 0.29 0.11-0.49 0.20 0.17-0.24 0.21 0.13-0.25No. seeds/plant 125.2 116-135 125.4 16-361 150.2 141-168 131.3 26-220Wt. seeds/plant (g) 16.9 15.4-17.3 13.55 0.95-43.86 13.5 12.7-13.9 9.85 1.33-33.57100 seed wt. (g) 13.5 12.2-13.9 10.80 4.80-19.41 9.3 8.3-9.9 7.45 51-10.8

Table 6. Mutants means for analyzed traits of chosen forms.

Initialform (initalics)andmutants

Plan

t hei

ght

(cm

)

Firs

t leg

ume

heig

ht (

cm)

No.

stem

s per

plan

t

No.

pod

s per

plan

t

No.

fert

ilepo

ds/p

lant

No.

ster

ilepo

ds/p

latn

t

Pod

leng

th(c

m)

Pod

wid

th(c

m)

Seed

s/po

d on

mai

n st

em

Seed

wt/p

odm

ain

stem

(g)

No.

seed

s per

plan

t

Seed

wt p

erpl

ant (

g)

100

seed

wei

ght (

g)

Day

s to

mat

urity

cv Krab 75.3 17.2 12.6 68.1 65.0 3.1 3.61 1.28 2.9 0.32 125.2 16.91 13.5 11816/K 61.1 12.3 32.0 146.0 108.0 38.0 3.88 1.71 2.3 0.37 195.0 32.12 16.5 11518/K 55.0 13.1 14.0 52.0 36.0 16.0 3.72 1.47 1.9 0.31 43.0 6.23 14.4 11421/K 44.1 7.0 15.0 48.0 48.0 0 3.97 1.37 2.9 0.42 109.0 15.13 13.9 11534/K 56.3 11.4 25.0 61.0 61.0 0 4.09 1.43 3.2 0.46 170.0 24.03 14.1 11153/K 54.4 13.7 22.0 102.0 93.0 9.0 4.05 1.46 3.3 0.40 213.0 26.89 12.6 11855/K 56.4 13.6 20.0 86.0 86.9 0 3.89 1.28 3.2 0.40 210.0 25.62 12.2 11163/K 44.2 11.0 25.0 66.0 61.0 6 2.99 1.35 2.7 0.39 119.0 17.78 14.9 11570/K 45.5 18.1 20.0 65.0 63.0 2 3.22 1.26 2.1 0.28 125.0 16.72 13.4 12072/K 24.1 9.2 9.0 8.0 8.0 0 2.42 1.05 1.8 0.14 18.0 1.45 8.0 109

cv Derek 69.2 13.3 15.6 77.4 75.1 2.2 3.36 1.22 3.2 0.20 150.2 13.51 9.30 12013/D 56.3 11.5 32.0 168.0 163.0 5.0 3.22 1.13 3.0 0.22 415.0 33.57 8.11 12019/D 57.2 9.1 18.0 105.0 92.0 12.0 3.13 1.23 2.5 0.19 177.0 13.81 7.82 11621/D 52.4 18.9 12.0 81.0 71.0 10.0 3.37 1.21 3.6 0.24 220.0 14.34 6.51 112


31

References

1. Campbell CG, Mehra RB, Agraval SK, Chen YZ,Abd El Moneim AM, Khawaja HIT, Yadov CR,Tay JU, Araya WA. 1994. Current status andfuture strategy in breeding of grasspea (Lathyrussativus). Euphytica 73, 167-175.

2. Hanbury CD, Siddique KHM, Galwey NW,Cocks PS. 1999. Genotype-environmentalinteraction for seed yields and ODAPconcentration of Lathyrus sativus L. and L. ciceraL. in Mediterranean-type environments.Euphytica 110, 45-60.

3. Hanbury CD, White CL, Mullan BP, SiddiqueKHM. 2000. A review of potential of Lathyrussativus L. and L. cicera L. grain for use as animalfeed. Animal Feed Science and Technology 82, 1-27.

4. Lambein F. 1997. Lathyrus sativus, a neolithiccrop with a modern future?. An overview of thepresent situation. Int. Scientific Symposium"Lathyrus sativus - cultivation and nutritive valuein animals and humans”, Lublin - Radom, 9-10June 1997, pp. 6-12.

5. Milczk M, Pędziński M, Mnichowska H, Szwed-Urbaś K. 1997. Creative breeding of chicklingvetch (Lathyrus sativus L.) - summation of first-stage of investigations. Int. Scientific Symposium"Lathyrus sativus - cultivation and nutritive valuein animals and humans", Lublin - Radom, 9-10June 1997, pp.13-22.

6. Nerkar YS. 1976. Mutation studies in Lathyrussativus. Indian Journal of Genetics and PlantBreeding 36, 223-229.

7. Prasad AB, Das AK. 1980. Morphologicalvariants in khesari. Indian Journal of Genetics andPlant Breeding 40, 172-175.

8. Sharma RP, Kumar R, Chauhan SVS. 1978.Mutagenic efficiency of gamma rays, EMS andMNU in Lens culinaris. Journal of AgriculturalSciences and Research 18, 37-43.

9. Sharma RN, Chitale MW, Ganvir AK, Geda AK,Pandey RL. 2000. Observation on thedevelopment of selection criterion for high yieldand low neurotoxin in grass pea based on geneticresources. Lathyrus Lathyrism Newsletter 1, 15-16.

10. Singh M, Chaturvedi SN. 1987. Effectiveness andefficiency of mutagen alone or in combinationwith dimethyl sulphoxide in Lathyrus sativusLinn. Indian Journal of Agricultural Sciences 57,503-507.

11. Swaminathan MS. 1965. Report of the meeting ofthe symposium in the use of induced mutations inplant breeding. Rad. Bot. 5, 65-69.

12. Tavoletti S, Capitani E. 2000. Field evaluation ofgrass pea populations collected in the Marcheregion (Italy). Lathyrus Lathyrism Newsletter 1,17-20.

13. Waghmare VN, Mehra RB. 2000. Inducedmutation in grasspea (Lathyrus sativus L.).Lathyrus Lathyrism Newsletter 1, 21-24.


32

Stability of grasspea (Lathyrus sativus L.) varieties for ODAP content and grain yield in Ethiopia

Wuletaw Tadesse

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

Introduction Grasspea (Lathyrus sativus L.) is a popular food andfeed crop in some Asian and African countries, suchas India, Pakistan, Bangladesh and Ethiopia becauseof its resistance to drought, flood and moderatesalinity. Grasspea becomes the only available sourceof food for the poor section of the population andsometimes a means of survival in times of droughtinduced famine. Therefore it is called the “poor mans’insurance crop”.

In Ethiopia, grasspea is an important pulse crop grownin the cambisol and vertisol soil-type areas. It occupies8.7% of the total area and 7.6% of the total productionof food legumes in the country (9). According to therecent Ethiopian Central Statistical Authority report (2)

grasspea is the third most important pulse crop, afterfaba bean and chickpea, with an area of 142 170 haand production of 104 740 tonnes. However, excessiveconsumption of the seeds can cause an upper motorneuron disease called “neurolathyrism” in humans,characterised by paralysis of the lower limbs, due tothe presence of the toxin oxalyl-diamino-propionicacid (ODAP).

The breeding station at Adet Research Center hasidentified some varieties with low ODAP content inmulti-location trials conducted in the potentialgrowing areas of north-west Ethiopia (10). However,genotype, environment and their interactions werefound to be significant for ODAP content and grainyield. The change in ODAP content of the varietiesfrom location to location, and from season to season,creates difficulty in identifying varieties with stableODAP content and fairly good grain yield potential. Insuch cases, stability analysis is useful to identifyvarieties that show minimum interaction withenvironment (1).

Statistical approaches to the study of quality criteriasuch as variance component analyses and jointregression methods of Yates and Cochram have beenfurther developed (4,5) and widely adopted. A stablegenotype is one having a high mean yield, a regressioncoefficient close to 1 and a minimum deviation fromregression (4). This study was made to identify the

most stable varieties for ODAP content and grainyield among the landraces and some Canadianvarieties.

Material and MethodsTwenty grasspea varieties were tested usingrandomised complete block design with 4 replicationsat Adet, Woreta and Bichena in 1994 and 1995. The1995 data at Bichena was not included in this studysince the trial was damaged by excessive flooding.Table 1 shows the soil characteristics of the locations.The plot sizes were 4 m2 (5m length, 4 rows at 20 cmapart) at a seeding rate of 40 kg/ha. The seeds weresown into residual moisture, starting in mid-September, following farmers’ practice and therainfall situation. Each year x location interaction wasconsidered as an individual environment. Ten varietieswith relatively low ODAP content were used forstability analysis, since the objective was to identifyvarieties stable low toxin content.

Table 1. Physiographic features and soilcharacteristics of the test locations.

LocationAdet Woreta Bichena

Alititude (m a.s.l.) 2240 1900 2600Annual rainfall (mm) 1230 1052 1077Soil order Vertisol Vertisol Pellic

vertisolpH (H2O) 6.0 6.0 8.0Organic matter (%) 1.6 1.5 3.3NC (%) 0.17 0.15 0.15P (ppm Bray II) 1.8 5.8 2.6

ODAP content analysis was performed at the AddisAbaba University, Chemistry Department. Samples ofanalysis were taken from seeds from a bulk of allreplicates for reasons of practicality. Analysis ofvariance involving sites and years was done for ODAPcontent and grain yield. Mean comparison method ofstability analysis was followed. Regressioncoefficients (b), deviations from regression (sd) andcoefficients of determination (r2) were used as stabilityindices. The b values were tested for difference fromunity using a t-test.


33

Results and DiscussionThere was a wide range in ODAP content (0.300 to0.529%) and in grain yield (0.32 to 3.0 t/ha) amongthe varieties in different environments (data notshown). This wide range of variation indicates theimportance of stability analysis (7).

According to the combined analysis (Table 2)genotypes and environments have significant effectson both ODAP content and grain yield, even thoughthe relative importance of the environmentalcomponent of variance was larger than the genotypecomponent for both grain yield and ODAP content.This result coincides with the stability result reportedby Dahiya (3). Similar results for most of the qualitytraits of bread wheat have been previously observed(8). Acc. No. 201513 (a landrace) and LS 82 46 (aCanadian variety) had b values for ODAP contentsignificantly different from unity indicating that theyare unstable over a wide range of environments (Table3). These two varieties, however, remain low inODAP content across environments and do not changein ranking, despite season and locational fluctuations.

The regression coefficient (b) is significantly differentfrom unity for grain yield in Acc. No. 46008 and46070. For these 2 varieties the coefficient ofdetermination (r2) is 99%, indicating that the variationis attributed to the linear regression. Varieties with r2

significantly different from unity are not stable (4), andaccordingly such varieties show high yield in suitableenvironments and vice versa. On the other hand, bvalues for ODAP content of these varieties were notsignificantly different from unity implying that theywere stable in this regard. There were positivecorrelations between mean ODAP contents andregression coefficients (r = 0.437) and mean grainyields with regression coefficients (r = 0.323)indicating that the ODAP content and grain yield ofthe varieties increase with favourably goodenvironments. The correlation between standard error

and r2 values were negative (r = -0.0806, and r = -0.859**) for ODAP content and grain yield,respectively, implying stability of the varieties.Similar result for grain yield stability on wheat wasreported by Getenet (6). About 64 to 97% of thevariation in ODAP content and 85 to 99% fo thevariation in grain yield of the individual varieties, inthe case of landraces (acc# 1-5), were explained by thevariation in environmental index (Table 3) whichshowed that the regression coefficients hadconsiderable predictive values for the varieties ofinterest to both ODAP content and grain yield. In thecase of the Canadian varieties (No. 6-10), the valuesof r2 for both ODAP content and grain yield were low,the range being 5.2 to 81.9% and 34.2 to 83.9%,respectively (Table 3). In this case the deviation meansquare (s2d) and the mean values of the charactersconcerned, have the main role in determining stabilitythan environmental index.

The introduced Canadian varieties are not able to keepup their original low ODAP content. LS8246 forexample showed a dramatic increase from season toseason and from location to location. This holds truefor all the Canadian varieties indicating that they areunsuitable.

Table 2. Analysis of variance for ODAP contentand grain yield of 20 grass pea varieties over fiveenvironments (1994-95).

Mean squaresSource ofvariation

df ODAP (%) Grain yield(t/ha)

Genotype (G) 19 0.002* 0.117*Environment (E) 4 0.066* 9.483*G x E 76 0.001** 0.07*CV (%) 8.61 10.3* significant at P<0.05; ** significant at P<0.01.

Table 3. Genotypic means, regression coefficients, deviation from regression and coefficient of determinationfor ODAP content and grain yield of 10 grass pea varieties over 5 environments.

Grain yield (t/ha) ODAP (%)Variety mean b sd r2 mean b sd r2

Acc#201513 0.309 0.497* 0.214 0.642 3.3 1.233 0.208 0.921Acc#46057 0.355 0.921 0.359 0.687 3.3 0.944 0.229 0.850Acc#46008 0.344 0.512 0.142 0.813 3.5 1.267** 0.019 0.999Acc#46070 0.342 0.804 0.078 0.972 3.4 1.790* 0.065 0.991Acc#46053 0.342 1.064 0.114 0.967 3.4 1.060 0.201 0.903LS8246 0.365 -0.212* 0.524 0.052 2.1 1.400 0.371 0.779Nc 8a 7 0.517 0.920 0.170 0.619 1.7 0.900 0.613 0.440Nc 8a 84 0.597 0.640 0.460 0.394 1.7 1.300 0.632 0.595Nc 8a 157 0.494 1.490* 0.406 0.819 1.5 1.200 0.308 0.839Nc 8a 74 0.493 1.18 0.505 0.394 1.8 0.600 0.443 0.342

* significant at P<0.05


34

ConclusionsThis study suggests that environment, genotype andtheir interaction affects the ODAP content and grainyield performance of grass pea varieties. The ODAPcontent is significantly affected by both the genotypeand environment thought the variation by theenvironmental component is high. A stable variety forgrain yield may not be stable for ODAP content andvice versa. As toxicity is the major bottleneck of grasspea production, stability of ODAP content needs moreattention than grain yield. Accordingly, varietiesLS8246 and Acc No. 201513 are low in ODAPcontent across locations and can be used over widerenvironments even though their ODAP value changesin amount.

As such studies had not been previously conducted ongrass pea in Ethiopia, this information may beimportant in evaluation of varieties before release.However, much information should be generated inthe future in more environments since this study wascarried out in limited number of locations in north-west Ethiopia.

References1. Allard RW, Bradshaw AD. 1964. Implications of

genotype environment interactions on appliedplant breeding. Crop Sci 4, 403-507.

2. CSA. 1998. Agricultural sample survey, area andproduction of major crops. Statistical Bulletin189.

3. Dahiya BS. 1986. Genetics and stability analysisin grass pea: Its implications in future breedingprograms. In: Kaul AK, Combes D (Eds).

Lathyrus and Lathyrism. Third World MedicalResearch Foundation, New York, 161-168.

4. Eberhart SA, Russell WA. (1966). Stabilityparameters for comparing varieties. Crop Sci 6,36-40.

5. Finlay RW, Wilkinson GM. 1963. The analysis ofadaptation in plant breeding programs. Aust JAgric Res 14, 742-754.

6. Getenet G. 1988. Grain yield stability of breadwheat cultivars in the highlands of Ethiopia. In:Van Ginkel M, Tanner DG (Eds). Fifth regionalwheat workshop for Eastern, Central andSouthern Africa and the Indian Ocean. CIMMYT,Mexico, 61-65.

7. Kambal AE, Mahmoud MA. 1978. Genotype byenvironment interactions in sorghum variety testsin the Sudan central rainlands. Exp Agric 14, 41-48.

8. Katunzi AL, Maganga TE, Merema A. 1992. Theeffect of genotype, environment and theirinteraction on soft bread wheat quality inTanzania. In: Tanner DG, Mwangi W (Eds).Seventh regional wheat workshop for Eastern,Central and Southern Africa and the IndianOcean. CIMMYT, Mexico.

9. Woldeamlak A, Alelign K. 1990. Status ofgrasspea (Lathyrus sativus) production inEthiopia. Newsletter of Ethiopian AgriculturalResearch 5(1), 4.

10. Tadesse W, Degago Y, Telaye A. 1995. Grass pea(Lathyrus sativus) production and breeding inEthiopia. In: Tekle Haimanot R, Lambein F (Eds).Lathyrus and Lathyrism, a Decade of Progress.University of Ghent, Belgium, 87-90.


35

Rainfed rice-based 3-crop systems with paira/utera crops in West Bengal, India

N.R. Das

Department of Agronomy, Faculty of Agriculture, Bidhan Chandra Agricultural University P.O. Mohanpur – 741 252, Nadia, West Bengal, India


Summary only, full paper published in Advances in Agricultural Research in India (2000) 13, 163-195

SummaryTaking advantage of climatic conditions, landsituation, soil type and different seasons (rabi, pre-kharif and kharif) of West Bengal, differentpaira/utera (no tillage relay) crops were studied underrainfed conditions with other crops in the seasons. Thepurpose was to improve yield production, maintenanceof soil fertility and production economics. Studieswere conducted at University Farms, Bidhan ChandraKrishi Viswavidyalaya (B.C. Agricultural University),Kalyani (22°39'N and 88°54'E) in West Bengal, Indiaand commenced in 1989. Although other systemsmight be utilised according to different situations ofWest Bengal, out of many rainfed rice-based 3-cropsystems studied with paira crops eight have shown tobe important:

(i) Transplanted kharif rice- paira grasspea- jute(ii) Transplanted kharif rice- paira lentil- jute(iii) Transplanted kharif rice- paira oat- mungbean(iv) Transplanted kharif rice- paira grasspea-

mungbean(v) Transplanted kharif rice- paira grasspea-

groundnut(vi) Transplanted kharif rice- paira sunhemp (for

seed)- mungbean(vii) Transplanted kharif rice- paira sunhemp- mesta(viii) Transplanted kharif rice- paira mesta (for seed)-

grasspea

Table 1. Direct, residual and cumulative effect of rainfed rabi/paira (no-tillage relay) crops (grasspea, linseed,mustard, lentil and oat) and tillage number on benefit/cost ratio of each crop in rainfed rice-based 3-cropsystems in West Bengal, India.

Direct effect on rabi/pairacrops

Residual effect on pre-kharif groundnut

Cumulative effect onkharif transplanted rice

Tillage (number ofpasses)



Rabi/paira crops

0 2 4 Mean 0 2 4 Mean 0 2 4 MeanGrasspea 6.5 1.1 0.7 2.8 3.8 2.6 2.6 3.0 0.5 0.1 0.2 0.2Linseed 3.1 0.6 0.1 1.3 3.3 2.3 2.4 2.7 0.2 0.1 0.2 0.2Mustard 4.9 0.0 0.0 1.6 3.7 2.8 2.7 2.9 0.3 0.4 0.1 0.3Lentil 5.6 1.5 1.4 2.6 3.4 2.7 3.1 3.1 0.7 0.4 0.3 0.5Oat 5.1 2.5 1.4 3.0 4.0 2.5 2.8 3.1 0.1 0.1 0.1 0.1Mean 5.0 1.2 0.7 2.3 3.6 2.6 2.7 3.0 0.3 0.2 0.2 0.3

Notes:1. Rabi crops were grown with varying amounts of

tillage from 4 passes to paira (0 passes) crops,whereas in pre-kharif (groundnut) and kharif(transplanted rice) season the land was tilled.

2. In the rabi season, paira crops of grasspea(Lathyrus sativus cv Nirmal 1), linseed (Linumusitattisimum cv Neelam), mustard (Brassicajuncea cv B-85), lentil (Lens culinaris cv B77)and oat (Avena sativa cv Kent) were grown. Inpre-kharif season, groundnut (Arachis hypogea cvICGV 86124) and in kharif season transplantedrice (Oryza sativa cv Khitish) were grown.


36

Productivity of grasspea (Lathyrus sativus L.) under different levels ofphosphorus and foliar spray of molybdenum

R.K. Sarkar1, B. Biswas2 and G.C. Malik1

1. Department of Agronomy, University College of Agriculture, Calcutta University, Calcutta, West Bengal –700 019, India

2. Zonal Adaptive Research Station, Government of West Bengal, Krishnagar, West Bengal – 741 101, India

Introduction Grasspea (Lathyrus sativus L.) is one of the importantfood legumes in many countries of Southern Europe,North Africa and Asia Minor (1). It is grown in dry andwarm regions due to its potential among grainlegumes to tolerate dry and unfavourable conditions(4). Being drought and flood tolerant, with a high seedprotein content and suitability for soil amelioration,grasspea accounts for about 5% of total area in Indiaunder pulses and 4% of production. It is a significantcrop of the Indo-Gangetic plains grown mainly onresidual moisture as a crop succeeding another crop.Adoption of promising varieties and suitable cropmanagement practices are important factors forexploring yield potential of grasspea. Phosphorus isthe most essential nutrient for increasing pulseproductivity (3). Molybdenum is important in thenutrition of legumes as it is essential for the activity ofthe enzyme nitrogenase (5). Hence an experiment wasundertaken to study the response of grasspea varietiesto phosphorus and molybdenum in a rainfed alluvialsoil (entisol).

Material and MethodsThe field experiment was conducted during the winterseason of 1998/99, 1999/2000 and 2000/01 at theZonal Adaptive Research Station, Krishnagar, WestBengal, India (88.31°E, 23.24°N and 15 m above sealevel). The soil was a Gangetic alluvial (entisol),having 0.53% organic carbon, 26 kg/ha availableP2O5, 148 kg/ha available K2O, with pH 7.5. Thetreatments consisted of 2 grasspea varieties viz.Nirmal and Biol-212; 4 phosphorus levels (0, 20, 40and 60 kg/ha of P2O5); 2 levels of foliar spray ofmolybdenum (no foliar spray and 0.05% Mo) weretested in a factorial design with 3 replications inwinter rice fallow land. Treatments allotted to plotswere fixed for 3 seasons on the same site. Seeds were

treated with rhizobial inoculant (Rhizobiumleguminosarum) before sowing. The crop was sownduring the second fortnight of November each yearafter harvest of winter rice. A basal dose of 20 kg/haN and 40 kg/ha K2O, along with P2O5 as per treatmentwere applied at sowing. Dilute solutions of 0.05% Mowere applied as ammonium molybdate at the rate of800 L/ha of water as foliar spray during thepreflowering stage. Nodulation was studied with 2plants from each plot at post flowering stage. Datawere recorded on 10 randomly selected plants fromeach plot for yield attributes, and yields werecomputed after harvest of the crop from each plot. Atotal rainfall of 64.5, 56.0 and 49.5 mm was recordedin the winter season of 1998/99, 1999/2000 and2000/01, respectively.

Results In general, the yield level was higher in 1999/2000than in 1998/99 and 2000/01.

The number as well as dry weight of nodules/plantwere significantly higher in variety Nirmal. Thevariety Nirmal outyielded Biol-212 during all theyears and in pooled data (Table 1). The number ofnodules/plant, and dry weight significantly increasedwith the increasing level of P up to 60 kg/ha P2O5. Theyield attributes and seed yield increased with theincrease in each successive level of P up to 60 kg/haP2O5. There was a 31.5% mean increase in seed yieldwith 60 kg/ha P2O5 over 0 P application.

Foliar spray of 0.05% Mo increased number ofnodules/plant, dry weight of nodules and alsoimproved yield attributes and seed yield over no Mospray. Such a foliar spray of Mo resulted in 24.0%higher seed yield over the control in the pooled data.


37

Table 1. Nodulation, yield attributes and seed yield of two grasspea varieties under P fertilisation and foliarspray of Mo (pooled data for three years: 1998/99, 1999/2000 and 2000/01).

Seed yield (t/ha)Treatment

Nodules/plant

Dry weightnodules(mg/plant)

Pods/plant

Podlength(cm)

Seeds/pod

1000 seedweight (g) 1998/

991999/2000

2000/01

Pooled

Variety:Nirmal 9.42 57.04 26.08 3.62 3.06 65.81 0.91 1.38 0.93 1.07Biol-212 8.73 48.85 23.71 3.05 3.05 70.92 0.86 1.24 0.82 0.98LSD (P<0.05) 0.60 3.52 1.69 0.21 NS 2.00 0.04 0.05 0.09 0.05Phosphorus (P2O5 kg/ha)0 6.60 28.92 21.18 3.16 2.78 66.27 0.77 1.09 0.76 0.7620 6.85 45.66 23.77 3.26 3.05 68.20 0.83 1.22 0.82 0.8240 6.90 61.33 25.70 3.47 3.11 69.00 0.95 1.41 0.92 0.9260 7.00 75.89 28.93 3.48 3.25 69.93 0.99 1.54 1.00 1.00LSD (P<0.05) 0.28 4.99 2.31 0.25 0.27 2.80 0.05 0.07 0.13 0.06Molybdenum 0 8.12 44.54 23.57 3.59 2.97 68.23 0.78 1.17 0.80 0.910.05% Mo 10.01 61.06 26.21 3.08 3.13 68.50 0.99 1.46 0.95 1.14LSD (P<0.05) 0.60 3.53 1.64 0.21 NS NS 0.04 0.05 0.09 0.07

DiscussionHigher yield of grasspea in 1999/2000 is attributed toclimatic factors in this year. Grasspea variety Nirmalperformed better than Biol-212, the higher seed yieldfor Nirmal could be attributed to efficient nodulationfor N- fixation and its assimilation.

Application of P increased the yield attributes andshowed a marked increase in yield of grasspea. Theimprovement in yield attributes and yield by Papplication may be attributed to profuse nodulation,leading to increased N-fixation which in turn hadpositive effect on photosynthetic organs and rate (7).Improvement in number and dry weight ofnodules/plant due to foliar spray of Mo may beascribed to the significant increase in nitrogenaseactivity by Mo. Foliar spray of 0.05% Mo resulted inmarked increase in seed yield and overallimprovement in yield attributes. The increase in yieldby Mo could be attributed to greater nitrogenaseactivity which led to increases in yield attributes andconsequent higher yields. Higher seed yield ingrasspea with foliar spray of Mo might possibly bedue to greater assimilation of carbohydrate and proteinsynthesis as Mo acts in N-assimilation (2) andincreases intensity of photosynthesis (6).

References1. Hruska J, Luskoniny SZN. 1956. Legume. SZN,

Praha.2. Nicholas DJD, Nason A, McLeroy WD. 1954.

Molybdenum and nitrate reductase. Ind J BiolChem 207, 241-251.

3. Saraf CS. 1983. Advances in fertilizermanagement for rainfed pulses. Fertilizer News28, 91-98.

4. Sarno R, Stringi L. 1979. Proc. Cong.“Prospective della proteaginose in Italia”,Perugia, pp. 365-370.

5. Singh K, Singh S, Singh V. 1999. Molybdenumnutrition of cowpea in relation to potassium andmolybdenum fertilization. Ind J Plant Physiol 3,227-228.

6. Sovoleva AV. 1959. Combined effect of the traceelements Mo, Mn, Cu on photosynthesis and Ncontent in the leaves of Helianthus annuus on oilformation in seeds. Doulady Aked Nauk, USSRProc Acad of Sci 129, 950-952.

7. Srivastava TK, Ahlawat IPS. 1995. Response ofpea (Pisum sativum) to phosphorus, molybdenumand biofertilisers. Ind J Agron 40, 630-635.


38

Effect of chickling vetch (Lathyrus sativus L.) or alfalfa (Medicago sativa) hay ingestating ewe diets

C. Poland1*, T. Faller2 and L. Tisor1

1. Dickinson Research Extension Center, North Dakota State University, 1089 State Avenue, Dickinson, ND 58601, USA

2. Hettinger Research Extension Center, North Dakota State University


Originally published in the 2003 Sheep Day Report, Hettinger RE Centerhttp://www.ag.ndsu.nodak.edu/hettinge/livestock/2003sheepday/chicklingvetch.pdf

Accessed 12th December 2003.

Introduction Lathyrus sativus (grasspea or chickling vetch, guayain Ethiopia, khesari in India) (5) is a common foodlegume widely grown and eaten throughout manyparts of the world (5,6,7). The nutritional composition ofL. sativus and L. cicera (two closely related species) issimilar to that of other feed grain legumes e.g. fieldpea (Pisum sativum), faba bean (Vicia faba), lupin(Lupinus angustifolius) (4,8). However, Lathyrus spp.can contain a large number of antinutritionalsubstances that can reduce their potential as a raw,unprocessed feedstuffs (2,3). Most notable is aneurotoxin, 3-N-oxalyl-L-2,3-diaminopropionic acid(acronymns: -oxalyl-diamino-propionic acid or ODAPand -oxalyl-amino-alanine or BOAA), which cancause a paralysis of the lower limbs known as"lathyrism" (3,4).

In Canada, the annual creeping vine grass pea hasbeen used primarily as a green manure alternative tosummer fallow in small grain production systems toreduce wind and water erosion and increase soilnitrogen concentrations (7). AC-Greenfix is a variety ofL. sativus developed at the Semiarid PrairieAgricultural Research Centre (SPARC) in SwiftCurrent, Saskatchewan, Canada (1). It is marketed inthe US by Dakota Frontier Seeds.

The objective of this preliminary study was to test theforage quality and general safety of L. sativus haycompared to alfalfa (Medicago sativa) hay in gestatingewes.

Material and MethodsTwenty pregnant, whitefaced ewes (BW = 77.5 ± 6.1kg; condition score = 3.0 ± 0.33) were randomlyallotted into one of four groups (5 ewes/group).Groups were then assigned to one of two dietary

treatments. Treatments were ad libitum access toeither alfalfa (ALFA) or chickling vetch (var. AC-Greenfix provided by Dakota Frontier Seeds, Flasher,ND; ACGF) hay. Standard supplementation (e.g.grain, minerals, vitamins) practices for gestating ewesat HREC were also provided uniformly to each group.Ewes were fed hays for 56 d prior to lambing. Eweswere weighed and condition scored (1 = very thin and5 = obese) on days 0, 28 and 56. Ewes were shornduring the first 28 d on feed. Individual fleece weightaveraged 5.5 kg. Liveweights during the study werenot adjusted for fleece removal. Feed deliveries andrefusals were recorded daily and weekly, respectively.Feed disappearance was the differences between haydelivery and refusal. Feed efficiency was calculated asliveweight gain divided by feed disappearance.

One ewe on the alfalfa treatment lambed on day 18 ofthe treatment phase. This ewe and lambs weremaintained in their respective pen for the remainder ofthe phase to allow for calculation of feeddisappearance. Gain data from this ewe was excludedfrom the data set and feed parameters adjusted to a perhead basis for comparison purposes. Four ewes (twofrom each treatment) had not lambed by springturnout. Calculations of lambs born and weanedreflect only those ewes that lambed after the treatmentphase and before spring turnout to pasture. Data wereanalysed as a completely random design using pen asthe experimental unit for feed and efficiency data andanimal as the experimental unit for liveweight andcondition score data.

Results and DiscussionNutritional composition of alfalfa and chickling vetchhay are reported in Table 1. Dry matter and crudeprotein concentrations were similar between the twohay types. Chickling vetch tended to have more acid-and neutral-detergent fibre and lower calculated


39

energy concentrations compared to alfalfa. Despitethese differences, both hays were nutrient denseforage of high quality. One visible difference was thepresence of a small amount of corn stover in thechickling vetch hay. Stover contamination was theresult of corn being grown on the same field in thepreceding year.

Body weight or gain and condition score or changewere not affected (P > 0.1) by dietary treatment (Table2). Ewes lost an average of 6.3 kg in the first 28 d. Amajority of this loss was fleece weight. Ewes gainedapproximately 11.5 kg over the last 28 d. Ewes gainedapproximately 5.2 kg and 0.5 condition score unitsover the 56 d feeding period. Lambs born and weanedwere also not affected by dietary treatment (Table 2).Ewes produced 1.4 lambs/ewe prior to spring turnoutand weaned 1.3 lambs/ewe.

Table 1. Nutrient composition of chickling vetch(var. AC-Greenfix; ACGF) and alfalfa (ALFA) hayfed to gestating ewes.

Item ACGF ALFADry matter (DM) 87.4 88.6Crude Protein (CP), %DM 18.2 18.1Acid Detergent Fiber (ADF),%DM

36.3 35.0

Neutral Detergent Fiber (NDF),%DM

48.6 44.6

Energies:Total Digestible Nutrients(TDN), %DM

60.6 61.7

Net Energy for Maintenance(NEm), MJ/kg DM

5.5 5.7

Net Energy for Gain (NEg),MJ/kg DM

3.1 3.3

Table 2. Effect of hay source on body weight and condition when fed to gestating ewes.

Itemb Treatmentsa

ACGF ALFA SEc P-valued

Initial Weight 78.5 76.2 2.0 0.43 Condition 2.9 3.1 0.10 0.17Day 0 -28 Weight 72.4 69.8 1.7 0.31 Gain -6.17 -6.44 0.68 0.77 Daily gain -0.22 -0.23 0.02 0.77Day 28 - 56 Weight 82.8 82.2 2.0 0.85 Gain 10.5 12.4 1.0 0.19 Daily gain 0.37 0.44 0.04 0.19Day 0 - 56 Gain 4.3 6.0 1.0 0.25 Daily gain 0.08 0.11 0.02 0.25 Condition 3.3 3.7 0.16 0.12 - change 0.40 0.56 0.16 0.52Number of lambs per ewee

Born 1.4 1.4 - -Weaned 1.4 1.2 - -

a Treatments include ad libitum access to chickling vetch (var. AC-Greenfix; ACGF) and alfalfa (ALFA) hay for 56 d during gestation. b Body weight and gain are expressed in kg and daily gain in kg/d. Body condition scored on a 5-point scale (1-very thin and 5-obese). c Standard error. d Probability of statistical significance. e Two lambs in each treatment lambed after ewes and lambs went to pasture. Thus, lambs born and weaned for these ewes were unknown andrecorded as 0. Also, one lamb in the alfalfa treatment lambed early during the gestation feeding period and lamb data from this ewe was removedfrom analysis.

Hay delivery (P < 0.01), refusal (P < 0.01) anddisappearance (P = 0.02) in the first 28 d was affectedby dietary treatment (Table 3). ACGF had greater haydeliveries (77 g/d) and refusals (150 g/d) and lessdisappearance (68 g/d) compared to ALFA. Feedefficiency (P = 1.0) during the first 28 d was not

affected by dietary treatment. During the second 28 d,feed delivery (P = 0.08) and refusals (P < 0.01) wereincreased 154 and 236 g/d, respectively, by ACGF.Hay disappearance (P = 0.35) and feed efficiency (P =0.70) were not affected by dietary treatment in thesecond 28 d. Overall hay delivery (P = 0.04) and


40

refusal (P < 0.01) were increased 118 and 172 g/d,respectively, by ACGF. Overall feed disappearance (P= 0.19) and efficiency (P = 0.54) were not affected bydietary treatment.

Chickling vetch produces a hay that is comparable toalfalfa hay in nutrient composition. No adverse affectswere observed in gestating ewes fed chickling vetch

hay compared to alfalfa hay. Observed increases inhay delivery and refusal of chickling vetch hay wereprobably related to the presence of corn stover in thehay bale that ewes tended to sort out and not consume.These preliminary data suggest no overt problemsfrom feeding chickling vetch hay to sheep and thatchickling vetch (var. AC-Greenfix) is comparable toalfalfa hay in gestating ewe diets.

Table 3. Effect of hay source on hay deliveries, refusal and disappearance and feed efficiency when fed togestating ewes.

Treatmentsa

Itemb ACGF ALFA SEc P-valued

Days 0 - 28 Delivered 2.39 2.31 0.003 <.01 Refusal -0.22 -0.07 0.007 <.01 Disappearance 2.17 2.24 0.007 .02 Efficiency -10.1 -10.1 1.29 1.0Days 28 - 56 Delivered 2.60 2.45 0.034 .08 Refusal -0.27 -0.03 0.015 <.01 Disappearance 2.33 2.41 0.046 .35 Efficiency 16.0 18.1 3.39 .70Days 0 - 56 Delivered 2.50 2.38 0.019 .04 Refusal -0.25 -0.05 0.009 <.01 Disappearance 2.25 2.33 0.027 .19 Efficiency 3.41 4.47 1.03 .54

a Treatments include ad libitum access to chickling vetch (var. AC-Greenfix; ACGF) and alfalfa (ALFA) hay for 56 d during gestation. b Hay delivery, refusal and disappearance are expressed as kg/d. Efficiency is body weight gain (Table 1) expressed as a percentage of haydisappearance. c Standard error. d Probability of statistical significance.

References

1. DFS. 2003. AC Greenfix. Dakota Frontier Seeds,Flasher, ND. (www.acgreenfix.com/main.html).

2. Foster JG, Turner KE, Ernst CL and Ernst AL.1996. Performance of feeder cattle offered a dietcontaining early-bloom stage flatpea silage. JProd Agric 9, 415-418.

3. Grela ER, Studzinski T and Matras J. 2001.Antinutritional factors in seeds of Lathyrussativus cultivated in Poland. Lathyrus LathyrismNewsletter 2, 101-104.

4. Hanbury CD, White CL, Mullan BP and SiddiqueKHM. 2000. A review of the potential ofLathyrus sativus L. and L. cicera L. grain for useas animal feed. Lathyrus Lathyrism Newsletter. 1,34-35.

5. IPBO, 2003. IPBO Lathyrus Research. PlantBiotechnology Institute for Developing Countries.(www.ipbo.rug.ac.be/Ourresearch/Lathyrus.html)

6. Jaby El-Haramein F, Abd-El Moneim A andNakkoul H. 1998. Prediction of the neuro-toxinbeta-N-oxalyl-amino-L-alanine in Lathyrusspecies, using near infrared reflectancespectroscopy. J Near Infrared Spectrosc 6, 93-96.

7. Small E. 1999. New crops for Canadianagriculture. In: Janick J (ed.), Perspectives on newcrops and new uses. ASHS Press, Alexandria,VA. USA. pp.15-52.

8. White C, Hanbury C and K. Siddique K. 2001.The nutritional value of Lathyrus cicera andLupinus angustifolius grain for sheep. LathyrusLathyrism Newsletter 2, 49-50.


41

Effect of antinutritional factors in khesari seeds (Lathyrus sativus) on thebiological performance of chicks

Archana Sharma*, M. Kalia and S.R. Malhotra

Department of Food Science and Nutrition, College of Home ScienceHimachal Pradesh Agricultural University (CSKKV), Palampur; H.P. 176 062, India


Introduction To meet the protein requirement of the ever growingand predominantly vegetarian human population inIndia, efforts are in progress in the country to increasepulse production and to make those pulsesconsumable, which may otherwise adversely affecthuman health. Pulses constitute a primary source ofproteins, add flavour and variety to a well-plannedcourse of meals. These pulses are commonly calleddhals and vary in their qualitative composition.Although the people consume pulses in most meals,excessive consumption of some pulses has beenreported to result in adverse effects on human healthdue to the presence of certain anti-nutritional factors.Sometimes the pulses are found to be adulteratedeither intentionally or unintentionally. Among theseadulterants, khesari (Lathyrus sativus) is the mostimportant in pulses. In spite of restrictions on khesaricultivation, many poor and marginal people/farmersstill grow khesari because it is a hardy crop, requiresvery little irrigation and other inputs and is resistant todrought and floods. Khesari contains 29-32% protein(4) but various reports have confirmed that prolongedconsumption of this pulse afflicts the central nervoussystem, characterised by weakness and paralysis of legmuscles and death in extreme cases (neurolathyrism).The causative agent of lathyrism is thought to be thepresence of β-N-oxalylamino-L-alanine (BOAA orODAP), additionally there are other antinutritionalfactors in khesari such as tannins and trypsininhibitors.

With this consideration in mind, the presentinvestigation with a local variety of khesari seed wasundertaken with the following objectives:1. To analyse the antinutritional factors.2. To study the effect of treatments on anti-

nutritional factors.3. To assess the effect of khesari on the biological

performance of chicks.

Material and MethodsPreparation/ procurement of samples:

The seeds of khesari were procured from localfarmers, cleaned of dust, dirt or any other foreignmaterial, dried and packed in polyethylene bags.Because of the presence of antinutritional factors inkhesari some treatments were also given for furtheranalysis.

Treatments:Soaking: khesari seeds were soaked overnight in waterat room temperature making sure that all the seedswere completely covered with water.Autoclaving: khesari seeds were autoclaved for 1 hourat 0.7 kg.cm-2 pressure.

Chemical analysis: The following antinutritional factors were studiedusing standard methods:Total phenolic compounds/tannins (7).Trypsin inhibitor activities (6).

Animal experiment:

Table 1. Composition of diets used.

Diets, % vegetable proteinreplaced by khesari

Ingredients* 0 25% 50%Premix (%) 30 22.5 15.0Khesari (%) - 11.1 22.2Maize (%) 32.8 31.1 21.7Wheat (%) 30.0 28.2 34.0Fish meal (%) 5.0 5.0 5.0Di-calcium phosphate (%) 2 2 2Trace min./ vit. (%) 0.156 0.156 0.156Total crude protein 22.3 21.9 21.9*Premix (Ground nut cake, sunflower cake, soya flakesproviding 13% crude protein or CP in total), khesari(26.25% CP), maize (9.0% CP), wheat (13.1% CP) and fishmeal (48% CP).

To assess the toxic effect of khesari a feeding trial onday old male layer chicks (Anderson Thomson Strain),obtained from Embrocia Hatcheries in Pathankot, wasconducted in the Department of Animal Nutrition,CSKKV. Random samples of 7 day old chicks


42

(according to average body weight) were housed ingroups, in brooder compartments and care was takento feed the chicks on isonitrogenous diets. The diets (2)

were prepared using different levels of khesari (0, 25& 50%; Table 1) and fed to the chicks for 4 weeks.

Results and DiscussionTotal phenolic compounds/ tannin contents of thesamples analyzed were a maximum in raw khesari(0.96g/100g) followed by soaked and autoclaved seeds(Table 2). Statistically significant differences wereobserved between the treatments and could be due tothe water-soluble nature of these antinutritionalfactors. The tannin contents present in were observedhigher from those found in khesari in other studies (1). Trypsin inhibitor activities (TIA), commonly presentin Leguminosae, have an antinutritional effect. Anattempt was made to estimate these inhibitoryactivities in khesari (treated as well as untreated;Table 2). Maximum TIA was observed in untreatedkhesari samples (18.61mg/g), in the soaking andautoclaving treatments lower activities were observed(9.61 and 2.63mg/g, respectively), this could be due tothe water soluble nature of these protease inhibitors.Mean TIA in advanced Lathyrus sativus lines hasbeen found to be 18.16 ± 2.38 mg/g (1).

Changes in physical activity: In Table 3 some effectcan be seen on the chicks fed 25% and 50% khesari.The group fed on 25% level of khesari showed goodgrowth with no symptom of dysentery/ diarrhoea anda normal gait, however, the feathers were found to beslightly ruffled and feed intake slightly reduced. Thegroup fed 50% khesari showed stunted growth with nosymptoms of dysentery/ diarrhoea and a hopping gaitwas observed, the feathers were found to be ruffledand feed intake reduced. A few birds showed paralysisof legs (unable to bear weight on legs), hopping gait,lack of coordination (Fig. 1) in contrast to the controlbirds. The reasons for this kind of behaviour may bedue to the neurotoxin and other antinutritional factorspresent in the khesari as reported previously (3).

Table 2. Antinutritional factors (tannins andtrypsin inhibitor activities or TIA) in khesari seedtreated in various ways.

Treatments Tannins (g/100g) TIA (mg/g)Raw 0.96 18.61Soaked 0.72 9.61Autoclaved 0.52 2.62LSD (P<0.05) 0.04 0.88

Table 3. Observations on physical activities of chicks.

Treatment Growth Dysentery/Diarrhoea

Feathers Feed intake Gait

Control Excellent No Normal Normal NormalKhesari (25%) Good No Slightly ruffled Slightly affected NormalKhesari (50%) Stunted No Ruffled Affected Hopping

Gain in body weight: The chicks fed on control dietgained maximum weight (225.7 ± 5.2 g) where as thechicks fed on 25% and 50% khesari diets showedlower gain of 150.5 ± 0.2 and 138.9 ± 6.0g,respectively (Table 4). Neurotoxins, trypsin inhibitors,phenolic compounds etc. may be responsible for thegrowth retardation in the chicks fed on different levelsof khesari. It was also clear from the study that byincreasing the concentration of protein in theexperimental chick’s diet the gain in body weight wasreduced as compared to the chicks fed on the controldiet.

Feed efficiency ratio (FER): FER represents theweight gain of an animal per weight of feed consumedunder specific conditions (Table 4). Determination ofFER in experimental subjects fed on two levels (25%and 50%) of khesari, (Table 4) revealed that the FERvalues were the lowest for group fed on khesari dietsand this difference could be due to the poordigestibility of the increased khesari concentration inthe diet. Fig. 1. Affected legs of chickens fed 50% khesari.


43

Protein intake: Protein intake for control diet wasmaximum (114.0 g) followed by 93.2 g and 89.9 g incase of 25% and 50% khesari respectively. FromTable 4 it can be concluded that chicks consumed lessprotein level in case of 50% khesari because of thepoor digestibility owing to the presence of neurotoxinsand other antinutritional factors.

Protein efficiency ratio (PER): PER is the weight gainof an animal per gram of protein consumed undercertain specific conditions. PER in the chicks was 1.62and 1.54 in case of 25% and 50% khesari diets,respectively (Table 4), however control fed birds

showed the maximum value (1.98). It has beenpreviously shown that (5) broiler chickens fed on 37%raw khesari seed showed decreased PER because ofthe presence of antinutritional factors.

Digestibility coefficient: Mean digestibility coefficientfor chicks fed on control diet was found to be 79.7, thevalues for chicks fed on 25% and 50% khesari dietswere significantly (P<0.05) lower (71.9 and 68.6respectively).

Table 4. Biological performance of experimental chicks fed khesari at varying proportions of the diet.

DietParameters 0% khesari 25% khesari 50% khesari LSD (P<0.05)Average initial body weight (g) 27.2 ± 0.22 27.2 ± 0.00 27.2 ± 0.22 -Average gain in body weight (g) 225.7 ± 5.2 150.5 ± 0.2 138.9 ± 6.0 8.6Feed efficiency ratio (FER) 0.44 0.36 0.34 0.014Protein intake 114.0 93.2 89.9 -Protein efficiency ratio (PER) 1.98 1.62 1.54 -Mean digestibility coefficient 79.7 71.9 68.6 4.9

ConclusionsBecause of the presence of antinutritional factors suchas trypsin inhibitors, tannins and some neurotoxins; itwas attempted to analyse trypsin-inhibitor and tannincontents as well as study different parameters ofbiological significance. Trypsin inhibitor activities(TIA) and tannin contents were greatest in raw seedsof khesari and lowest after autoclaving, with soakingin water intermediate.

The biological studies involving different levels (25and 50%) of the khesari, isonitrogenous diet was fedto each group of chicks ad libitum in an in vivo study.Chicks fed on khesari revealed stunted growth, ruffledfeathers, abnormal gait and significant reductions inthe following: feed efficiency ratio (FER), gain inbody weight, protein efficiency ratio (PER),digestibility co-efficient, protein intake in comparisonto the chicks fed on the control diet.

The effects of antinutritional factors cannot beseparated from each other and BOAA was notmeasured. However, it can be concluded that thevarious antinutritional factors have probablycontributed to the poorer performance of chicks fedkhesari. The abnormal gait exhibited is consistent withknown effects of BOAA.

It has therefore been concluded from the study onchicks that the local variety of khesari could adverselyaffect human health if consumed extensively.

References1. Aletor VA, Abd El-Moneim A and Goodchild

AV. 1994. Evaluation of the seeds of selectedlines of three Lathyrus spp. for β-N-oxalyl-amino-l-alanine (BOAA) tannins, trypsin inhibitoractivities and certain in-vitro characteristics. J SciFood Agric 65, 143-151.

2. BIS Poultry Feeds Specifications. 1992. Bureauof Indian Standards, New Delhi.

3. Ganapathy KT, Dwivedi MP, Nagrajan V andDikshitulu VN. 1963. Experiments on chicks fedon Lathyrus sativus. Ind J Med Res 51, 865-869.

4. Kothari S. 1995. Renowned nutritious khesari dalturned poisonous and untouchable. Academy ofNutrition Improvement, Soyamilk Complex,Wardha Road, Sitabuldi, Nagpur-440012, India.

5. Latif MA, Morris TR, Jayne-William DJ. 1976.Use of khesari (Lathyrus sativus) in chicks diets.British Poult Sci 17, 539-546.

6. Roy DM and Rao PS. 1971. Evidence, isolation,prepitation and some properties of trypsininhibitor in Lathyrus sativus. J Agric Food Chem19, 257-259.

7. Singh V, Jambunathan, R. Swain and Hillis,(1981). Studies on desi and kabuli chickpeacultivars, level of protease 1, level ofpolyphenolic compounds and in-vitrodigestibility. J Food Sci 46, 1364-1467.


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Lathyrus cicera as quality feed for laying hens

Colin Hanbury1 and Bob Hughes2

1. CLIMA, University of Western Australia35 Stirling Hwy, Crawley WA 6009, Australia

2. Nutrition Research Laboratory, Pig and Poultry Production Institute,Roseworthy Campus, University of Adelaide

Roseworthy SA 5371, Australia

1. Email: [email protected]

Introduction Research at CLIMA has indicated that Lathyrus ciceraand L. sativus (grasspea) have potential as grainlegumes on 100 000 to 300 000 ha of neutral toalkaline soils in low-to-medium rainfall areas ofsouthern Australia. They do not have serious diseaseproblems and are envisaged as low maintenance/lowcost crops for the purposes of green manure, managingherbicide resistant weeds, forage, hay and grainprincipally for animal feed. Particularly L. sativus is ahuman food in large parts of the Indian sub-continentand Ethiopia.

Both of these Lathyrus species have been usedextensively in the past for animal feed. The neurotoxin3-(-N-oxalyl)-L-2,3-diamino propionic acid (ODAP)is found in L. cicera and L. sativus grain and ifconsumed in large amounts can produce a paralysis ofthe hind legs known as “lathyrism”. ODAP wasidentified in the 1960’s and since that time plantbreeding has produced lines with low toxin levels (2).Due to the presence of ODAP Lathyrus species havealmost disappeared from many regions where theywere once cultivated extensively, such as Europe. Thenewer lines with low toxin levels have not beenwidely evaluated for animal feed and the present studyis the only one to our knowledge that has evaluatedlow toxin lines in poultry.

Since one of the goals of establishing Lathyruscultivation in Australia was to develop animal feedmarkets it was decided to investigate the use of L.cicera cultivar ‘Chalus’ in trials with laying hens.Chalus was released by CLIMA in 1998 and wasshown in extensive studies to have low levels ofODAP (Table 1), about 70% lower than what is foundin fields in India for example. Chalus has shown goodadaptation across southern Australia and is the first ina series of cultivars that are to be released by CLIMA.

One part of the study was to establish Chalus as safefor both the laying hens and for any consumers ofeggs or bird tissue. Since little was known about the

fate of ODAP in hens, one aim was to investigatewhether after feeding with Chalus that ODAP couldbe found in eggs or body tissue of hens. The secondaim was to demonstrate that Chalus was a goodquality feed capable of replacing, for example, fieldpeas (Pisum sativum) in laying hen diets withoutpenalty in egg production.


Intense short term feeding studyThere were 6 dietary treatments of wheat-based dietswith Chalus included at 0, 5, 10, 15, 25 and 30%Chalus (Chalus composition see Table 1). Each dietwas fed to 16 replicates of 8 birds each for a period of8 weeks. Bird weights were recorded at the beginningand end of the experiment. Bird behaviour wasobserved daily for neurotoxic symptoms. Theexperimental diets were commenced when the birdswere 26 weeks old. Feed consumption was monitoredweekly.

In the seventh week excreta from each cage wascollected for moisture determination. In the final weekegg weight of all eggs laid in a 3 day period wasrecorded. Shell characteristics and yolk colour ofapproximately 600 eggs (100 eggs per treatment) weremeasured. The proportion of dirty eggs was visuallyassessed. On the final day four samples (pooled) ofeggs, breast meat, whole brain and liver per treatmentwere sampled for analysis of ODAP.

Long term feeding trial A long term feeding study was conducted on somebirds retained after the 8 week feeding study describedabove. Each of the six dietary treatments was given tothree birds for a further period of 24 weeks. Eggswere analysed for ODAP after a further 12 weeks;eggs and tissues were analysed for ODAP at the endof the study as done previously.


45

Table 1. Chemical composition of Lathyrus cicera cv Chalus.

Component Amount Component Amount% g/16g N

Moisture 10.6 Essential amino acidsFat 0.7 Cystine 1.23Protein 27.6 Methionine 0.82Ash 3.1 Threonine 3.68NDF (enzyme modified) 24.5 Valine 4.57ADF 10.7 Isoleucine 4.01Starch 42 Leucine 7.36Lignin 0.2 Phenylalanine 4.46NFE (nitrogen free extractives) 61.8 Lysine 7.13In vitro digestibility (IVD) 80 Histidine 2.45

Arginine 8.81MineralsP 0.33 AntinutritionalsK 0.91 %Na 0.07 ODAP 0.09Ca 0.25 Tannins (total) 1.08Mg 0.13 Tannin, catechin 0.51S 0.17 Oligosaccharides 4.12

mg/kg Phytic acid 0.91Fe 156 g/kgMn 11 Trypsin inhibitor activity 2.07Zn 20 Chymotrypsin inhibitor activity 3.46Cu 9

Results and DiscussionNo ODAP was detected in egg white at any time. Eggyolk (Table 2), breast meat and liver showed tracelevels of ODAP, but this was not consistent. After the32 weeks of feeding the brain tissue showed the mostconsistent traces of ODAP (Table 3) but levels were20 times less than that shown in previous studies ofrats when lathyrism symptoms were evident (1). Thetrace levels detected in hens and egg yolk were 300times less than the ODAP levels in the Chalus grainthey were fed. Any consumption of these low levels ofODAP in the hens and the eggs would be too low toaffect humans or animals. Studies of humanconsumption of ODAP have shown that regularconsumption of grain at levels 3000 times the levelsfound in this study are sufficient to cause lathyrismsymptoms, only if the grain is consumed exclusivelyand under circumstances of malnourishment. Hencethe possibility that consuming eggs or hen tissue asshown here is extremely unlikely to pose any problem.The hens showed no signs of neurotoxicity and deathsin the experiment were minimal and not related to feedtype.

Egg production and quality was as good as the fieldpea based diet and in some cases showed smallimprovements and was always at a level expected forthe age and breed of the hens. Feed intake wasmarginally greater for Chalus than field pea. Feed

conversion was also marginally better for Chalus thanfield pea (Fig. 1). Soiling of eggs was shown to beslightly better in Chalus. Egg shell thickness andproportion were unaffected by inclusion of Chalus inthe diet, as was the egg weight. However the yolkcolour was significantly improved by the inclusion ofChalus.

Table 2. Effect of Chalus content of diet onconcentration of ODAP (ppm) in egg yolk sampleover an extended feeding period of 32 weeks in thesecond long term feeding experiment.

Time on diets (weeks)Chalus (%) 0 8 20 32

0 0.0 0.0 0.0 0.05 0.0 0.1 0.1 0.010 0.0 0.5 0.0 0.015 0.0 0.8 0.7 0.025 0.0 0.0 0.2 0.130 0.0 0.0 0.2 0.0


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Table 3. Effect of Chalus content of diet onconcentration of ODAP in brain sample after anextended feeding period of 32 weeks in the secondlong term feeding experiment.

Chalus (%) ODAP (ppm)0 0.05 0.5

10 0.815 0.425 0.730 0.7

1.90

1.95

2.00

2.05

2.10

2.15

2.20

0 5 10 15 25 30

Chalus content (%)

Feed

to e

ggs

(kg:

kg)

Fig. 1. Effect of Chalus content of diet onconversion rate of feed to eggs at 34 weeks of age(means ± standard errors).

ConclusionsIn comparison to field pea Chalus was as good a feedingredient, with no shown detrimental effects. Onesignificant advantage of Chalus is however the likelygrain price. Due to the low maintenance nature of thecrop it is expected that grain price is likely to bearound $AUS140 per tonne. This compares veryfavourably with field peas at $AUS220 per tonne andeven lupins (Lupinus angustifolius) at $AUS190 pertonne. Chalus certainly seems to be a better qualityingredient for layers than lupins are currently.Composition-wise Chalus is almost identical to fieldpeas but has about 2% higher protein levels at about26%.

The very pleasing results with laying hens bode wellfor the continued adoption of Lathyrus species in low-to-medium rainfall farming systems of southernAustralia, especially as the initial problem ofestablishing cultivation has been the lack of existingmarkets. Other cultivars are planned with greateradaptation and better agronomic characteristics, theadoption of these cultivars will lead to a stable supplyof grain for the egg industry and thus ensuring accessto a low cost, high quality feed ingredient.

Wide dissemination of the results of the layer feedingtrial will go a long way to establishing Lathyrusspecies as a choice for farmers wishing to grow agrain legume in their crop rotation system. Previouspleasing feeding results with pigs (3,4) and sheep (5) inAustralia will encourage increased local production ofgrain which will become available for the eggindustry.

AcknowledgementsFunding was kindly provided by Rural IndustriesResearch and Development Corporation (RIRDC),Egg Program- now Australian Egg CorporationLimited (AECL) http://www.aecl.org

References1. Cheema PS, Malathi K, Padmanaban G and

Sarma PS. 1969. The neurotoxicity of β-N-oxalyl-αβ-diaminopropionic acid, the neurotoxin fromthe pulse Lathyrus sativus. Biochem J 112, 29-33.

2. Hanbury CD, White CL, Mullan BP and SiddiqueKHM. 2000. A review of the potential ofLathyrus sativus L. and L. cicera L. grain for useas animal feed. Animal Feed Sci Tech 87, 1-27

3. Mullan BP, Hanbury CD, Hooper JA, NichollsRR, Hagan CR and Siddique KHM. 1999.Lathyrus (Lathyrus cicera): a potential newingredient in pig grower diets. In Corbett JL (ed)Recent Advances in Animal Nutrition in Australia12, 12A. University of New England, Armidale.

4. Trezona M, Mullan BP, Pluske JR, Hanbury CD,Siddique KHM. 2000. Evaluation of Lathyrus(Lathyrus cicera) as an ingredient in diets forweaner pigs. Proc Nutrition Soc Aust Vol 24.24th Annual Scientific meeting Fremantle,Western Australia (3-6 December 2000). pp. 119.

5. White CL, Hanbury CD, Young P, Phillips N,Wiese SC, Milton JB, Davidson RH and SiddiqueKHM. 2002. The nutritional value of Lathyruscicera and Lupinus angustifolius grain for sheep.Animal Feed Sci Tech 99, 45-64.


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A new amperometric ß-ODAP biosensor

Negussie W. Beyene*, Helmut Moderegger and Kurt Kalcher

Institute of Chemistry, Analytical Chemistry, Karl-Franzens University of Graz, Universitaetsplatz 1, A-8010 Graz, Austria

* Current address: Department of Chemistry, University of Pretoria, Pretoria 0002, South Africa.


Introduction The Lathyrus/lathyrism challenge is broad-based byits nature and requires multi-disciplinary efforts ofspecialists in the field of epidemiology, neurology,biochemistry, chemistry, nutrition, and agronomy. Therole of chemists is in systematic determination ofminor natural products in L. sativus seeds so that thesole responsibility of ß-ODAP for neurolathyrism isascertained (1). Moreover, analytical chemists play apivotal role in the development of a simple andreliable analytical method for ß-ODAP quantitation inseeds, food preparations, and biological samples takenfrom victims since lack of such a method hindered, inone way or another, research undertakings in theaforementioned disciplines. Our work addresses thelatter role of targeting the development of anamperometric biosensor for ß-ODAP. This ß-ODAPbiosensor is based on the pioneering work of Mogesand Johannson that reported the activity of glutamateoxidase (GlOD) towards ß-ODAP (2). One of theoxidation products, hydrogen peroxide, reduces thetetravalent manganese (modifier in the screen printedcarbon electrode, SPCE) to lower oxidation states thatreoxidize again electrochemically producing a currentproportional to the concentration of ß-ODAP.

Material and MethodsMnO2 bulk-modified SPCEs were produced inaccordance to previous reports and the flow systemand the electrochemical analyzer used were basicallythe same (3). GlOD was immobilized by entrapment inneutralized Nafion® film as effected by drop coatingthe enzyme-polymer mixture onto the surface of theSPCE.

Results and DiscussionOperational parameters were assessed using the mainsubstrate glutamate. An applied potential of 440 mVvs. Ag/AgCl, flow rate of 0.2 mL min-1, and pH 7.75of the carrier (0.1 mol L-1 phosphate buffer) werefound to give the best signal as well as better samplethroughput. These parameters were used for ß-ODAPbiosensor except the flow rate. Flow rate of 0.1 mLmin-1 was chosen in this case because of the slowerreaction kinetics of the toxin towards the enzyme(equations 1 and 2) (2, 4).

Linear relation between concentration of ß-ODAP andcurrent response was observed in the range 50-500 mgL-1 (i [nA] = 0.25 c [mg L-1] + 42.12, r2 = 0.996). Thedetection limit (as 3σ values) from 6 injections of 100µL standard ß-ODAP solution (50 mg L-1) was foundto be 29 mg L-1 and a relative standard deviation of4.5% was recorded at this concentration of ß-ODAP.In comparison to previous reports (5, 6) the linear range

between signal and concentration of ß-ODAP in thiswork was far better though the detection limit was abit higher. The higher detection limit could beattributed to the diffusion barrier created by theNafion-enzyme layer. As the thickness of layersincreases the linear range extends but the detectionlimit becomes higher as reported elsewhere (7-10).

L-glutamate + O2 + H2O L-glutamate oxidase α−ketoglutarate + NH4+ + H2O2 (1)

ß-ODAP + O2 + H2O L-glutamate oxidase α−ketoacid + NH4+ + H2O2 (2)

fast

slow


48

To destroy glutamate inherent in grass pea seedsamples the enzyme glutamate decarboxylase (GlDC)was used. Incubation of glutamate solution with GlDCat 37 oC for 3 hours caused almost complete loss ofthe glutamate signal indicating the effectiveness andspecificity of the enzyme in destroying glutamate (seereaction below).

L-glutamate GlDC γ-aminobutyric acid + CO2

It was also observed that GlDC showed no activity atpH 7.75 (no effect on the glutamate concentration) butwas effective at a pH between 4 and 5 (which is alsothe pH of distilled water) as recommended by themanufacturer. Wodajo et al. showed that extraction ofß-ODAP from grass pea seed powder could be madein distilled water as effectively as in phosphate buffer(11). Thus, a solution of dihydrogen phosphate (pH 4.5)can be used for the extraction as well as for samplepre-treatment with GlDC; the solution can be adjustedto pH 7.75 using disodium phosphate solution beforeinjecting it to the FI biosensor system.

The decarboxylase has no effect on ß-ODAP aschecked by incubating 500 mg L-1 ß-ODAP solutionovernight at 37 oC. There was no difference (relativeerror 2%) between the signals of ß-ODAP injectionwith and without GlDC treatment. Moreover, therewas a significant difference (162%) in the responsebetween grass pea extracts untreated and treated withGlDC.

Spiking glutamate (50 mg L-1) to ß-ODAP solution(100 mg L-1) and treating the mixture with GlDC didnot make any difference in the ß-ODAP signal. Thus,

determination of ß-ODAP in grass pea seed was donein accordance to this finding. Recovery test by spiking50 mg L-1 standard ß-ODAP to one of the samplesgave 98.6 ± 3.2 %.

Moreover, the biosensor exhibited extraordinarystability retaining 50% of the original response evenafter 65 days on-line in the FI system as monitored byinjection of standard glutamate solution regularly. Italso showed sufficient activity for glutamate whenstored in the working buffer for more than 2 months.

To our knowledge, this is the first ß-ODAP biosensorproduced using SPCEs. Interferences from glutamatepresent in grass pea seed extracts have beeneliminated using the enzyme GlDC. GlDC has noeffect on ß-ODAP (which is also reported for the firsttime) but completely destroys glutamate in the sampleafter 3 hours incubation. Extraction of ß-ODAP andelimination of glutamate has been effected indihydrogen phosphate solution (0.1 mol L-1). The off-line sample pre-treatment is a bit time consuming.However, it can further be improved by addingsodium chloride that is known to activate GlDC (12, 13).It should be noted that the same amount of sodiumchloride should be added in the carrier solution toavoid a change in the ionic strength that mayotherwise affect the current response. Addition ofchloride solution to the carrier can also have theadditional advantage of maintaining the stability of thereference electrode, which is chloride concentrationdependent. Once, sodium chloride is introduced theenzymatic decarboxylation of glutamate can be fasterthan observed in this work and the sample pre-treatment can be done on-line by using dual channelflow system as shown in Fig. 1.

Fig. 1. Proposed dual flow system for improvement of ß-ODAP biosensor.


49

The first flow channel (I) is to propel the dihydrogenphosphate solution (pH 4.5) at a very low flow rate(Fig. 1) and the second channel (II) to propel disodiumphosphate solution (pH 9.2) at higher flow rate. Theinjection port can be placed somewhere in channel 1before the GlDC reactor (column). The two channelscombine in the mixing tee (M) adjusting the pH to7.75 and then pass to the GlOD electrode for the mainanalytical reaction. One further advantage of usingGlDC is that it is cheaper and can be produced easilyfrom green pepper (14). Further research couldinvestigate simple sensors with multi-enzymatic layerconfigurations where the upper most layer containsGlDC to destroy glutamate and the bottom layercontains GlOD with enough activity to oxidize ß-ODAP.

In conclusion, this work demonstrated thatimmobilization of GlOD in a Nafion® film on MnO2bulk-modified carbon electrodes (screen printed) canbe used for constructing biosensors for thedetermination of ß-ODAP. The biosensor exhibited awider linear range than biosensors of previous studiesas well as extraordinary stability. Furthermore, thiswork showed the effectiveness of GlDC in removingany interference from inherent glutamate that may bepresent in grass pea seeds.

AcknowledgementsN.W. Beyene acknowledges the Austrian AcademicExchange Service (ÖAD) for the scholarship grant.

References1. Lambein F. 2000. Lathyrus Lathyrism Newsletter

1, 4-5.2. Moges G, Johannson G. 1994. Anal Chem 66,

3834-3839.3. Turkušić E, Kalcher K, Schachl K, Komersova A,

Bartos M, Moderegger H, Svancara I, Vytras K.2001. Anal Lett 34, 2633-2647.

4. Moges G, Solomon T, Johansson G. 1994. AnalLett 27, 2207-2221.

5. Yigzaw Y, Larsson L, Gorton L, Ruzgas T,Solomon T. 2001. J Chromatogr A 929, 13-21.

6. Yigzaw Y, Gorton L, Solomon T. 2002. CurrSeparations 19, 119-125.

7. Maines A, Ashworth D, Vadgama P. 1996. AnalChim Acta 333, 223-231.

8. Harrison DJ, Turner RFB, Baltes HP. 1988. AnalChem 60, 2002-2007.

9. Mullen WH, Keedy FH, Churchouse SJ,Vadgama PM. 1986. Anal Chim Acta 183, 59-66.

10. Maines A., Prodromidis MI, Tzouwara-KarayanniSM, Karayannis MI, Ashworth D, Vadgama P.2000. Electroanalysis 12, 1118-1123.

11. Wodajo N. [Beyene NW], Moges G, Solomon T.1997. Bull Chem Soc Ethiop 11, 151-154.

12. Worthington V. Worthington Enzyme Manual:enzymes and related biochemicals, WorthingtonBiochemical Corporation, www.worthington-biochem.com, accessed on 02/05/2001.

13. O’Leary M, Brummund W. 1974. J Biol Chem249, 3737-3740.

14. Oliveira MIP, Pimentel MC, MontenegroMCBSM, Araujo AN, Pimentel MF, da Silva VL.2001. Anal Chim Acta 448, 207-213.

http://www.worthington-biochem.com/

http://www.worthington-biochem.com/


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Items of InterestIncludes any items of interest to Lathyrus and lathyrism researchers. Please send any suggested inclusions forfuture editions to the editor. Conferences, meetings or other items will be included.

Lathyrus discussion on-line

A Lathyrus Email discussion group has been initiated. To join send an Email to:[email protected] notify Colin Hanbury at [email protected] and you will be added to the list.Alternatively if you visit:

http://groups.yahoo.com/group/Lathyrusand follow the prompts under “Join This Group!”, you will be able to join and keep up to datewith what is happening world-wide in Lathyrus related areas.

Fifth European Conference on Grain Legumes with theSecond International Conference on Legume Genomics and Genetics

Legumes for the benefit of agriculture, nutrition and the environment: their genomics, theirproducts, and their improvement. 7–11 June 2004 in Dijon, France

Conference secretariat: AEP Executive Secretariat, 12 avenue George V 75 008 Paris, FranceTel: +33 1 40 69 49 09 Fax: +33 1 47 23 58 72 Email: [email protected] http://www.grainlegumes.comPoster in PDF format:http://www.grainlegumes.com/ulf/AEP/AEP_cata/paragraphe/affiche_dijon_377_1357.PDF

Lathyrus publications on-line from IPGRI.

1. Lathyrus Genetic Resources in Asia: Proceedings of a Regional Workshop, 27-29 December1995, Indira Gandhi Agricultural University, Raipur, India. 1996. Available at:http://www.ipgri.cgiar.org/publications/pubfile.asp?ID_PUB=656

2. Lathyrus Genetic Resources Network: Proceedings of a IPGRI-ICARDA-ICAR RegionalWorking Group Meeting, 8-10 December 1997, National Bureau of Plant Genetic Resources,New Delhi. 1998. Available at:http:// www.ipgri.cgiar.org /publications/pubfile.asp?ID_PUB=94

3. Grass Pea. Lathyrus sativus L. Promoting the conservation and use of underutilized andneglected crops. 18. 1997. Available at:http:// www.ipgri.cgiar.org /publications/pubfile.asp?ID_PUB=430

4. Descriptors for Lathyrus spp. 2000. Available at:http:// www.ipgri.cgiar.org /publications/pubfile.asp?ID_PUB=547

For any further information on these publications contact [email protected]

volume 3 (1), december 2003 · 2010. 7. 5. · radovici, societe medicale des hospitaux de...

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