EFFORTS TO ACCELERATE DOMESTICATION OF WINGED BEAN (PSOPHOCARPUSTETRAGONOLOBUS (L.) DC.) BY MEANS OF INDUCED MUTATIONS AND TISSUE CULTURE Promotor: Dr. Ir. E. Jacobsen Hoogleraar in de plantenveredeling, in het bijzonder de genetische variatie en reproductie Co-promotor: Dr. Ir. A.M. van Harten Universitair hoofddocent plantenveredeling MfJoSzoJ , ZIS^ George.Y.P.KIu EFFORTS TO ACCELERATE DOMESTICATION OF WINGED BEAN (PSOPHOCARPUSTETRAGONOLOBUS (L.) DC.) BY MEANS OF INDUCED MUTATIONS AND TISSUE CULTURE Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van de Landbouwuniversiteit Wageningen, Dr. C.M.Karssen, in het openbaar te verdedigen op dinsdag 22 oktober 1996 des namiddags te vier uur in de Aula. gIBLiOTHEEX LANDBOUWUNIVERSITHT WAGENfNC.FN Cover: A proposed "ideal" winged bean plant for grain and tuber production on a background of the winged bean plant as depicted by Rumphius (1747). pNO?-?°', ^^ Propositions (Stellingen) 1. Induced mutations can be used to accelerate the domestication of winged bean (Psophocarpustetragonolobus (L.) DC). (This Thesis). 2. In the genetic improvement of winged bean (Psophocarpus tetragonolobus (L.) DC, chimerism is much more of an asset than aproblem . (This Thesis). 3. Legumes, including winged bean, are the cheapest and often the most easily available source of proteinfo r the under- privilegedperson . Efforts into legume improvement constitute a lasting investment for the people in the poverty-striken parts of the world. (This Thesis). 4. Development of effective selectionscheme stoward s specifictrait s ina particula r crop plant is acontributio n to the reduction of the "it isb ychance " stigma labelled against plant breeding by induced mutations. (This Thesis). 5. Seed coat colour mutants in winged bean are an indirect way of obtaining plants with an altered nodulation. (This Thesis). 6. The number of people involved in farming in a country is not a good yardstick for determining the significance of the contribution of farming to the economy of that country. 7. Genetic improvement of crops and their multiplication for Africa must be carried out in Africa by Africans. 8. Sandwiches, particularly those of McDonald's, are universally enjoyed. However, there is aspecia l type of sandwich that requires hardwork , commitment and good strategy to give satisfaction to the consumer. This refers to the "sandwich" that can be found on the plate of the many PhD students in Wageningen Agricultural University. 9. "Reinforcement of ....patent rights should stimulate investment in research and development and accelerate the creation of promising biotechnology inventions". However, as much as patenting rewards the biotechnology companies, it may be a serious draw-back to the potential end-user who may be financially handicapped. 10. Modern soccer has positively evolved tremendously indifferen t ways. However, it gives reminiscence of the dark periods of human trade in the history of Africa. George Y.P. Klu. 1996 ACKNOWLEDGEMENTS The Executive Board of the Wageningen Agricultural University, The Netherlands, gave me admission to this sandwich PhD programme with a scholarship. Funds were made available for return trips, for living expenses and for putting this thesis together. Without allthese , this PhDthesi s would not have seen the light of day. To the Members of this Board and to all who madethi s possible Ia mdeepl y indebted. Iwoul dparticularl y liket othan k Mr. C.M.M.va n Heijst for his administrative support. The assistance received from the Ghana Atomic Energy Commision, where the bulk of the research work leading to this thesis was carried out, isdeepl y acknowledged. Ia m particularly thankful toth e Chairmenan d Memberso f Managements ofth e GhanaAtomi c EnergyCommissio n and the Biotechnology and Nuclear Agriculture Research Institute, my employers, for the permission granted me to undertake this sandwich programme. I would like to thank Prof. Dr. Ir. J.E. Parlevliet. for the kind reception he gave me when I first entered the Vakgroep Plantenveredeling in 1991. Prof. Dr. Ir. E.Jacobsen ,an d Dr. Ir.A.M .va n Harten were notjus t my Promotor and Co-Promotor respectively, but they really cared. Your combined patience, tolerance, concern and critical discussions of my manuscripts induced met o success. Iwoul d also like to express my sincere thanks to you for the ticket providedfo r my wife to comet o Wageningen. "Prof.Evert" , I would liket o thank youver y muchfo r your lasting interest inth e subject of mythesis ,you r stimulating suggestions and the time you invested in me. Ton, your visit to Ghana for discussions with me in 1990 gave me the needed filip for the studies at WAU. The times you set aside for casual discussions, both in the office and in your house, were very refreshing and gave me alo t of encouragement. Youals otranslate dth e summaryo f thisthesi s into Dutch and provided stimulating ideastha t culminated inth e design onth e cover of this document. Ia mver y grateful to you for all these. Ingeneral , I am overwhelmed byth e support I receivedfro mth e Vakgroep Plantenveredeling. Iwoul d liket o express mydeepes t appreciationt o the magement andal lstaf f of the department for their assistance. Firsto f all, Iwoul d liket othan k MsIr .Josk a Buitendyk who took meroun dt o dosom e shopping on the very first day I arrived in Wagenigen. Without this I would have been locked up in my room for the rest of that weekend with no one to talk to. Joska, I am also grateful to you for accepting to act as my "Paranimf" . Ms. Annie Marchai and Ms. Letty Dijker-Lefers, you both linked up so many things and tied up many loose ends for me. I very much appreciate how readily you responded to the many requests that I made. Ms. Annie Schouten, you willingly kept me up to date with literature by quickly responding to my numerous requests for some literature to be posted to me in Ghana. Annie, Letty and Annie thanks very much. Thanks are due also to Mr. Han Dorenstouter for his friendliness and all the administrative assistance he gave me. I would also like to thank Dr. Ir. C.J.J.M Raemakers. Krit, your willingness to set time aside for discussions on this thesis and to act as my "Paranimf" is very much appreciated. Thanks are also due to thank Dr. Ir. J.J. Bos, Curator of the Botanic Gardens, Wageningen, for the warm-hearted assistance he gave me in tracing the records of Rumphius at the Taxonomy Department, WAU. I have been deeply touched by the life of Georgius Everhardus Rumphius (1628-1702), who worked as a merchant and a botanist in Amboina. Rumphius became blind during his profession but visual disability was not a handicap to him. He worked hard as a blind man to produce a document that was published (1741-1747) as "Het Amboinsch kruidboek". His illustration of "De vierkante Peul-Plant" - the winged bean - formed the background of the cover design of this thesis. The assistance received from my colleagues at the Ghana Atomic Energy Commission particularly Messrs Edison Amanor, M. Quanor-Addy and B. Quaye is also acknowledged. Mon ami, Jean Dah-Dovonon from the Republic of Benin, the times we spent together at the "Studentenflat" Marijkeweg,Wageninge n were very stimulating. You received me very warmly and you did not get bothered by the very bad attempts I made to speak some "broken" French with you. Your company duringth e last phase of "consuming my sandwich" cannot be forgotten. The role of my wife, Liz and our children in this work has been immense. This project took me from home for long periods. Thank you very much for your support and patience when this work took the greater part of my life. With you I share this success. The investigations described in this thesis were carried out at the Biotechnology and Nuclear Agriculture Research Institute, Kwabenya. Ghana. CIP-DATA KONINKLIJKE BIBLIOTHEEK DEN HAAG George Y.P.KIu Efforts to accelerate domestication of winged bean (Psophocarpus tetragonolobus (L.) DC) by means of induced mutations and tissue culture. Thesis Landbouwuniversiteit Wageningen, with réf. ISBN 90-5485-604-1 Bibliographic Abstract: This thesis describes mutation breeding and tissue culture techniques developed for accelerated domestication of winged bean (Psophocarpus tetragonolobus (L.) D C). Thetissu e culture techniques, which areth e first steps towards genetic transformation of the crop, include: (1) direct adventitious shoot formation from the axes of cotyledon expiants; (2) direct simultaneous regeneration of adventitious shoots and somatic embryos; and (3) direct somatic embryogenesis on the wounds of cotyledon explants. An optimised mutation breedingtechniqu e for economic significance, basedo nth e earlyselectio no f chlorophyll mutations generatedfro m gamma-radiate d seeds,ha sbee ndeveloped . The useo f this scheme has resulted in the recovery of seed coat colour mutants which have succesfully served as an indirect methodfo r selecting changes in tannin content and nodulation. Adesire d mutant with reduced tannin content and improved nodulation was selected. CONTENTS CHAPTER 1. General Introduction. 1 CHAPTER 2. Status and potential for genetic improvement of winged bean (Psophocarpus tetragonolobus (L.) DC). 8 CHAPTER 3. Tissue culture techniques for improvement of winged bean (Psophocarpus tetragonolobus (L.) DC): Direct organogenesis and somatic embryogenesis in mature cotyledon expiants. 25 CHAPTER 4. Optimisation of mutant recovery from plants obtained from gamma radiated seeds of winged bean (Psophocarpus tetragonolobus (L.) DC). 37 CHAPTER 5. Induced mutations for improvement of winged bean (Psophocarpus tetragonolobus (L.) DC.) towards an improved ideotype for grain production with low tannin content. 47 CHAPTER 6. Testing of induced mutants of winged bean (Psophocarpus tetragonolobus (L.) DC.) for nodulation and phenotypic petrformance. 61 CHAPTER 7. General discussion. 72 Summary.