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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in ^ ew riter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper aligmnent can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor M3 48106-1346 USA 313/761-4700 800/521-0600 GENETIC ENGINEERING APPROACHES TO IMPROVE AGRONOMIC TRAITS IN CASSAVA (MANIHOT ESCULENTA CRANTZ) DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Diana Isabel Arias-Garzon, B.S. ***** The Ohio State University 1997 Dissertation Committee; Dr. Richard T. Sayre, Adviser Apjjroved by: Dr. Zhenbiao Yang Dr. Michael Evans Advis Plant Biology Department I3MI Number: 9801635 UMI Microform 9801635 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT Cassava (Manihot esculenta Crantz) is a tropical tuber crop that is grown for its starchy, thickened roots. The cassava roots are used mainly as a food source or for animal feed. Due to the presence of cyanogenic glycosides, however, cassava is potentially toxic. This and other aspects of cassava are potentially amenable to improvement through genetic manipulation. Cassava is a highly heterozygous plant with low natural fertility which makes genetic manipulation via traditional breeding methods very long and difficult. Genetic engineering is an alternative approach to circumvent this problem and modify some aspects of cassava such as: starch quality and quantity, resistance to pests and diseases, and reduction of cyanogenic potential. Linamarin, a cyanogenic glycoside, is stored in the root, stems and leaves of cassava and upon tissue damage (such as food preparation) is broken down by linamarase to produce acetone cyanohydrin. Acetone cyanohydrin can break down to produce acetone and hydrogen cyanide either spontaneously or by the action of hydroxynitrile lyase (HNL). O f these three cyanogens (linamarin, acetone cyanohydrin, and hydrogen cyanide) acetone cyanohydrin is the main contributor to consumer cyanide exposure. 11 Here, we report for the first time the stable transformation of cassava via Agrobacterium-vaed^dXod. system with a gene of agronomic interest. We have cloned a HNL cDNA into an Agrobacterium binary vector under the control of a double CaMV 35S promoter. The modified binary vector was transformed into two different strains o f Agrobacterium, LB A 4404 and EHA105 which were used for stable transformation of cassava. In vitro apical leaves and germinated somatic embryos of a cassava cultivar, Mcol 2215, were used to regenerate transgenic cassava plants resistant to paromomycin after co-cultivation with Agrobacterium. The overall efficiency of transformation was approximately 2.8%, however, when only apical leaves were co­ cultivated with the modified LBA4404, the transformation efficiency increases to 5.5%. All plant DNA evaluated so far by PCR amplification of specific introduced genes indicates integration of the selectable marker, nptfL, and the gene of interest, HNL. Hydroxynitrile lyase fi'om leaves and stems of untransformed plants had an activity of 1.7 mmol HCN/mg protein/h versus 2.4, 3.8,4.0 mmol HCN/mg protein/h firom three different transformed plants. Western blots of untransformed and transformed leaf-stem total protein support the higher activity of hydroxynitrile lyase in at least one of the transformed plants. However, HNL has not yet been detected in root tissues of transformed plants. Ill Dedicated to Mauricio and my parents, Luis Elder and Margarita. IV ACKNOWLEDGMENTS I wish to thank my adviser. Dr. Richard T. Sayre, for his guidance, support, and encouragement. His insight and patience made this dissertation possible. I would also like to thank the members of my committee. Dr. Mike Evans and Dr. Zhenbiao Yang for their critical review of my dissertation. I would also like to acknowledge the remarkable support that I received from the people in my lab, Ron Hutchison, Evangeline Ricks, Wanda White, Stuart Ruffle, Jennifer McMahon, Svetlana Makova, Xiao-Hua Cai, Uzoma Diemere, Dimuth Siritunga, Chris Brown, and last but not least. Sue Lawrence. I would like to thank Dr. Pablo Jourdan for his collaboration at the initial steps of the establishment of the tissue cultures protocols, as well as Rodrigo Sarria for his advice about cassava transformation. I am grateful to Anton and Claire Bartolo, Angel Arroyo, Maria Claudia Sanchez and my Colombian friends. Thanks to all them for their invaluable friendship and making my life outside of the university more enjoyable. I would like to thanks those who prepared me for my work here at OSU. I thank the faculty of “Universidad del Valle”, specially members of the Biology Department. Special thanks goes to Dr. William Roca and the members of the Biotechnology Research Unit at CIAT for introducing me into the world of plant research. I thank my family for their support and encouragement through all these years of studying. Lastly, I extend my gratitude and acknowledgments to my husband, Mauricio, for his constant support, scientific discussions, companionship and assistance not only doing this dissertation but also during all these years of graduate school. VI VITA January 4,1963 .......................................Bora - Cali, Valle, Colombia. 1987 ........................................................ B.S. Plant Biology, Universidad del Valle, Cali, Valle, Colombia. 1986 - 1990.............................................Research Assistant, International Center for Tropical Agriculture, Cali, Colombia. 1991- present .......................................... Graduate Teaching and Research Associate, The Ohio State University. PUBLICATIONS Research Publication 1. Arias, D.I., 1987. Effect of sucrose and mannitol levels on the in vitro growth of six cassava varieties. Thesis. 2. Chavez, R., Roca, W.M. Arias D.I., Withers, L., Williams, T., 1988. Cooperative EBPGR and CIAT pilot project tests feasibility of in vitro conservation. Diversity 16:8-10. 3. Roca, W.M., Chavez, R., Marin, M.L., Arias D.I., Mafia, G., Reyes, R., 1989. In vitro methods of germplasm conservation. Genome 31: 813-817. Vll 4. Roca, W.M., Arias, D.I., Chavez, R., 1991. Metodos de conservacion in vitro de germoplasma. In: Cultive de tejidos en la agricultura: Fundamentos y Aplicaciones. Roca W.M. y Mroginski L.A. (eds.). Centro Intemacional de Agricultura Tropical. Cali, Colombia, p. 697-713. 5. Angel, P., Arias, D.I., Tohme J., Iglesias C., Roca W.M., 1993. Toward the construction of a molecular map of cassava (Manihot esculenta Crantz): comparison of restriction enzymes and probe sources in detecting RFLP's. J. of Biotech., 31: 103-113. 6. Arias-Garzon, D.I., Sayre, R.T., 1993. Tissue specific inhibition of transient gene expression in cassava (Manihot esculenta Crantz). Plant Science, 93:121-130. 7. Arias-Garzon, D.I., Sayre, R.T. 1992. Correlation between the frequency of transient gene expression and DNase activity in cassava tissues. Roca, W.M. and Thro, A.M. (eds.). Proceedings of the First International Scientific Meeting of the Cassava Biotechnology network, Cartagena, Colombia, August 1992. Cali, Colombia: Centro Intemacional de Agricultura Tropical pp 239-243. 8. Arias-Garzon, D.I., Sarria, R., Gelvin S., Sayre, R.T., 1994. New Agrobacterium tumefaciens plasmids for cassava transformation. Proceedings of the Second International Scientific Meeting, Bogor, Indonesia, August 1994. pp 245-256. 9. Arias-Garzon, D.I., Sayre, R.T., 1995. Optimization of transient transformation in cassava (Manihot esculenta, Crantz). Plant Physiology 108:152. FIELDS OF STUDY Major Field: Plant Biology V lll TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication ...........................................................................................................................iv Acknowledgments ............................................................................................................... v Vita...................................................................................................................................
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