Transcript ADZUKI BEAN
紅豆組織培養暨基因轉殖研究 Wei-Ying Chien National Tainan Teachers College Department of Natural Sciences Education 2004-6-25 1 Importance of legumes Nutritional source for humans and animals Protein, oil, minerals, carbohydrate… To improve the soil When legumes meet the challenges of the 21st century… 2/37 Can we improve the quality of legume? Conventional breeding Tissue culture and transformation 基改─黃豆 育種─彩色海芋 3/37 My object of study Azuki bean 4/37 My object of study Azuki bean When we joined WTO… What is our local advantage? 5/37 My object of study Azuki bean 主要栽種地：高屏、雲嘉南 秋裡作物 6/37 production and marketing of azuki bean in Taiwan 公噸 12,000 10,000 8,000 年產量 6,000 進口量 出口量 4,000 2,000 0 民81 民82 民83 民84 民85 民86 民87 民88 民89 民90 年 7/37 Methods of plant tissue culture 8/37 Methods of plant tissue culture Embryo culture the first tissue culture techniques to be applied to plant breeding.(1976) Material : late-stage or early-stage embryos Ex.cotton, tomato, barley, rice… 9/37 Methods of plant tissue culture Meristem culture Pionerr: Morel, 1960, leaf primordia Material : meristem Disease-free plant (particularly viral diseases) 10/37 Methods of plant tissue culture Micropropatation Material : buds, stem, … repid propagation of a superior plant while maintation the genotype. Germplasm storage 11/37 Methods of plant tissue culture Somatic embryoenesis somatic embryo ? embryo? Callus? callus Regenerated plant Plant tissue somatic embryo 12/37 Methods of plant tissue culture Protoplast culture protoplasts : cells that the cell wall has been removed To incorporate potentially useful genes from one plant species to another To transfer specific genes into protoplasts 13/37 Methods of plant tissue culture In vitro selection Somaclonal variation Ex.Colchicine Anther culture To produce haploid plant 14/37 The factors effected tissue culture Explant source Age of culture Hormonal factor Genotypic factor Environment factor salt, carbohydrate... 15/37 Gene transformation of azuki bean From protoplasts K. L. Ge, Y. Z. Wang, X. M. Yuan, P. M. Huang, J. S. Yang, J. Q. Huang, C. C. Tan, N. Lee and D. Testa. Plantlet regeneration from protoplasts isolated from mesophyll cells of adsuki bean (Phaseolus angularis, Wight). Plant science, 63:209-216, 1989 From epicotyls T. Yamada, M. Teraishi, K. Hattori and M. Ishimoto. Transformation of azuki bean by Agrobacterium tumefaciens. Plant cell, tissue and organ culture. 64:47-54, 2001. 16/37 Regeneration from protoplasts K. L. Ge et al., 1989 Protoplast cultures from sterilized leaves of azuki bean plasmid : pBI121 Marker gene : GUS, npt-Ⅱ 17/37 Regeneration from protoplasts Materials and methods: Sterilized seedOMS, 14days 16 first leavescut into pieces 13% CPW13M enzyme solution, 14h Filter and spinprotoplast culture medium Gene transfer (protoplasts) Transient expression 18/37 Regeneration from protoplasts Results: • First divesion • 4-5 days after culture in I-3 medium • second divesion • 7-8 days after culture in I-3 medium • cell cluster • 20 days after culture in I-3 medium 19/37 Regeneration from protoplasts Results: • protoplast-derived calli • regenerated plantlet • 1 month after subculture on solid medium • 196 days after transferring to medium 108 20/37 Ⅲ-S-2 Regeneration from protoplasts Results: GUS : 5.4% of the protoplasts showed transient expression 3 days after gene transfer Transformants could be selected on selective medium containing 50mg/l kanamycin 21/37 Transformation by Agrobacterium Yamada et al., 2000 Agrobacterium strains : LBA4404, AGL1, EHA105 vector: pIG121 and pSG65T Tissue culture : epicotyl 22/37 Transformation by Agrobacterium 23/37 Transformation by Agrobacterium Meterials and methods Elongated epicotyls cut into pieces about 10mm Co-cultivation medium:10mg/l BA , acetosyringone, Bacterial cells were placed on the wounded site. 2d Explants were plated on MS medium w/ 1mg/l BA, 100mg/l kanamycin, and 500mg/l lilacillin . Analysis of transformed plants 24/37 Transformation by Agrobacterium Results plant regeneration from azuki bean epicotyls BD AD HD ES KO 25/37 Transformation by Agrobacterium Results Effect of acetosyringone and BA conc. GUS expression was tested 2 weeks after 2-d co-cultivation. 26/37 Transformation by Agrobacterium Results Screening of efficient A. tumefaciens strains 27/37 Transformation by Agrobacterium Results Establishment of transgenic plants 28/37 Transformation by Agrobacterium Results Analysis of transformed plants GUS expressed sGFP protein expressed 29/37 Transformation by Agrobacterium c Southern blot analysis of genomic DNA 30/37 The first GM azuki bean AR-9 Year of approval :1999 Developer :Japan, Nat’l Agr. Res. Ctr. Characteristics: insect resistant 31/37 My work -NOW and FUTURE Strains: 32/37 My work -NOW and FUTURE Tissue culture : epicotyl 33/37 My work -NOW and FUTURE 34/37 My work -NOW and FUTURE Plasmid ? Gene transfer : Agrobacterium strain --LBA4404, EHA105 … Analysis of transformed plants… 35/37 References K. L. Ge, Y. Z. Wang, X. M. Yuan, P. M. Huang, J. S. Yang, J. Q. Huang, C. C. Tan, N. Lee and D. Testa. Plantlet regeneration from protoplasts isolated from mesophyll cells of adsuki bean (Phaseolus angularis, Wight). Plant science, 63:209-216, 1989 M. K. Smith and R. A. Drew. Current applications of tissue culture in plant propagation and improvement. Aust. J. Physiol. 17:267-289, 1990 R. Walden and J. Schell. Tissue culture and the use of transgenic plants to study plant development. In Vitro Cell. Dev. Biol. 27:1-10, 1991 T. Yamada, M. Teraishi, K. Hattori and M. Ishimoto. Transformation of azuki bean by Agrobacterium tumefaciens. Plant cell, tissue and organ culture. 64:47-54, 2001. 36/37 Thank you for your attention. 37/37