紅豆組織培養暨基因轉殖研究

1. 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

2. 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…

3. 基改─黃豆 育種─彩色海芋 Can we improve the quality of legume? • Conventional breeding • Tissue culture and transformation

4. My object of study Azuki bean

5. My object of study • When we joined WTO… • What is our local advantage? Azuki bean

6. My object of study • 主要栽種地：高屏、雲嘉南 • 秋裡作物 Azuki bean

7. production and marketing of azuki bean in Taiwan

8. Methods of plant tissue culture

9. 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…

10. Methods of plant tissue culture • Meristem culture • Pionerr: Morel, 1960, leaf primordia • Material : meristem • Disease-free plant (particularly viral diseases)

11. Methods of plant tissue culture • Micropropatation • Material : buds, stem, … • repid propagation of a superior plant while maintation the genotype. • Germplasm storage

12. Methods of plant tissue culture • Somatic embryoenesis • somatic embryo ? embryo? Callus? callus Regenerated plant Plant tissue somatic embryo

13. 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

14. Methods of plant tissue culture • In vitro selection • Somaclonal variation • Ex.Colchicine • Anther culture • To produce haploid plant

15. The factors effected tissue culture • Explant source • Age of culture • Hormonal factor • Genotypic factor • Environment factor • salt, carbohydrate...

16. 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.

17. Regeneration from protoplasts • K. L. Ge et al., 1989 • Protoplast cultures from sterilized leaves of azuki bean • plasmid : pBI121 • Marker gene : GUS, npt-Ⅱ

18. Regeneration from protoplasts • Materials and methods: Sterilized seedOMS, 14days 16 first leavescut into pieces 13% CPW13M enzyme solution, 14h Filter and spinprotoplast culture medium Gene transfer (protoplasts) Transient expression

19. 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

20. Regeneration from protoplasts • Results: • protoplast-derived calli • 1 month after subculture on solid medium • Ⅲ-S-2 • regenerated plantlet • 196 days after transferring to medium 108

21. 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

22. Transformation by Agrobacterium • Yamada et al., 2000 • Agrobacterium strains : LBA4404, AGL1, EHA105 • vector: pIG121 and pSG65T • Tissue culture : epicotyl

23. Transformation by Agrobacterium

24. 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

25. BD AD HD ES KO Transformation by Agrobacterium • Results • plant regeneration from azuki bean epicotyls

26. Transformation by Agrobacterium • Results • Effect of acetosyringone and BA conc. GUS expression was tested 2 weeks after 2-d co-cultivation.

27. Transformation by Agrobacterium • Results • Screening of efficient A. tumefaciens strains

28. Transformation by Agrobacterium • Results • Establishment of transgenic plants

29. Transformation by Agrobacterium • Results • Analysis of transformed plants GUS expressed sGFP protein expressed

30. Transformation by Agrobacterium c Southern blot analysis of genomic DNA

31. The first GM azuki bean • AR-9 • Year of approval :1999 • Developer :Japan, Nat’l Agr. Res. Ctr. • Characteristics: insect resistant

32. My work -NOW and FUTURE • Strains:

33. My work -NOW and FUTURE • Tissue culture : epicotyl

34. My work -NOW and FUTURE

35. My work -NOW and FUTURE • Plasmid ? • Gene transfer : Agrobacterium strain --LBA4404, EHA105 … • Analysis of transformed plants…

36. 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.

37. Thank you for your attention.