1 Wei-Ying Chien National Tainan Teachers College Department of Natural Sciences Education 紅豆組織培養暨基因轉殖研究

2/37 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…

3/37 Can we improve the quality of legume? Conventional breeding Tissue culture and transformation 基改 ─ 黃豆 育種 ─ 彩色海芋

4/37 My object of study Azuki bean

5/37 My object of study When we joined WTO… What is our local advantage? Azuki bean

6/37 My object of study 主要栽種地：高屏、雲嘉南 秋裡作物 Azuki bean

7/37 production and marketing of azuki bean in Taiwan

8/37 Methods of plant tissue culture

9/37 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…

10/37 Methods of plant tissue culture Meristem culture Pionerr: Morel, 1960, leaf primordia Material : meristem Disease-free plant (particularly viral diseases)

11/37 Methods of plant tissue culture Micropropatation Material : buds, stem, … repid propagation of a superior plant while maintation the genotype. Germplasm storage

12/37 Methods of plant tissue culture Somatic embryoenesis somatic embryo ? embryo? Callus? Plant tissue callus somatic embryo Regenerated plant

13/37 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

14/37 Methods of plant tissue culture In vitro selection Somaclonal variation Ex.Colchicine Anther culture To produce haploid plant

15/37 The factors effected tissue culture Explant source Age of culture Hormonal factor Genotypic factor Environment factor salt, carbohydrate...

16/37 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: , 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.

17/37 Regeneration from protoplasts K. L. Ge et al., 1989 Protoplast cultures from sterilized leaves of azuki bean plasmid : pBI121 Marker gene : GUS, npt- Ⅱ

18/37 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

19/37 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

20/37 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

21/37 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

22/37 Transformation by Agrobacterium Yamada et al., 2000 Agrobacterium strains : LBA4404, AGL1, EHA105 vector: pIG121 and pSG65T Tissue culture : epicotyl

23/37 Transformation by Agrobacterium

24/37 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

25/37 Transformation by Agrobacterium Results plant regeneration from azuki bean epicotyls BDADHDESKO

26/37 Transformation by Agrobacterium Results Effect of acetosyringone and BA conc. GUS expression was tested 2 weeks after 2-d co-cultivation.

27/37 Transformation by Agrobacterium Results Screening of efficient A. tumefaciens strains

28/37 Transformation by Agrobacterium Results Establishment of transgenic plants

29/37 Transformation by Agrobacterium Results Analysis of transformed plants sGFP protein expressedGUS expressed

30/37 Transformation by Agrobacterium Southern blot analysis of genomic DNA c

31/37 The first GM azuki bean AR-9 Year of approval :1999 Developer :Japan, Nat’l Agr. Res. Ctr. Characteristics: insect resistant

32/37 My work -NOW and FUTURE Strains:

33/37 My work -NOW and FUTURE Tissue culture : epicotyl

34/37 My work -NOW and FUTURE

35/37 My work -NOW and FUTURE Plasmid ? Gene transfer : Agrobacterium strain --LBA4404, EHA105 … Analysis of transformed plants…

36/37 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: , 1989 M. K. Smith and R. A. Drew. Current applications of tissue culture in plant propagation and improvement. Aust. J. Physiol. 17: , 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.

37/37 Thank you for your attention.