High efficiency transformation of tall fescue with Agrobacterium tumefaciens

1. High efficiency transformation of tall fescue withAgrobacterium tumefaciens Shujie Dong, Rongda Qu * Plant Science 168 (2005) 1453–1458 生科四甲 李廷原 91390604

2. High efficiency transformation of tall fescue with Agrobacterium tumefaciens Plant Science 168 (2005) 1453–1458 Shujie Dong, Rongda Qu * Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA Received 22 December 2004; accepted 14 January 2005 Available online 2 February 2005

3. Introduction Improvement on important agronomic traits, such as disease resistance and abiotic stress tolerance, would be very helpful for agronomic. Genetic engineering has opened new avenues to the modification,and provides us an alternative approach to meet specific breeding goals . Establishment of high frequency transformation system would greatly facilitate the efforts for grass improvement via genetic engineering. First attempts to introduce foreign DNA into tall fescue were through the protoplast and Agrobacterium tumefaciens-mediated transformation of tall fescue .

4. Materials and methods • Plant materials and tissue culture condition • Agrobacterium strains and vectors • Transformation procedure • GUS histochemical assay and Southern analysis • Inheritance study of the transgenic plant

5. Materials and methods • Plant materials and tissue culture condition • Agrobacterium strains and vectors • Transformation procedure • GUS histochemical assay and Southern analysis • Inheritance study of the transgenic plant

6. http://www.cambia.org/daisy/cambia/585.html

7. Materials and methods • Plant materials and tissue culture condition • Agrobacterium strains and vectors • Transformation procedure • GUS histochemical assay and Southern analysis • Inheritance study of the transgenic plant

8. 切位 有興趣的基因 農桿菌載體 構築 插入載體中 送入大腸桿菌，並選拔對kan具抗性者 大腸桿菌 農桿菌 與農桿菌交配 農桿菌 藉由DNA同原片段之交換作用，將大腸桿菌T-DNA質體送入農桿菌Ti質體 大腸桿菌 具重組質體之農桿菌 感染植物

9. Ti質體 傷口釋放出訊息分子 Vir蛋白質 植物細胞 農桿菌 VirA及VirG蛋白質活化其他Vir基因 T-DNA由RB切下 Vir基因產物協助T-DNA轉移 T-DNA由LB切下 T-DNA進入植物細胞 T-DNA插入植物染色體中 Ti質體DNA經由DNA複製進行修補

10. Materials and methods • Plant materials and tissue culture condition • Agrobacterium strains and vectors • Transformation procedure • GUS histochemical assay and Southern analysis • Inheritance study of the transgenic plant

12. Materials and methods • Plant materials and tissue culture condition • Agrobacterium strains and vectors • Transformation procedure • GUS histochemical assay and Southern analysis • Inheritance study of the transgenic plant

13. Results (A) Transient GUS expression in the infected cells; (B) a resistant callus growing on hyg B selection; (C) stable GUS gene expression in an hyg B resistant callus; (D) plantlet regeneration under hyg B selection;

14. (E) root development of a hyg B resistant plantlet in the selection rooting medium; (F) various levels of GUS gene expression in different transgenic plants; (G) transgenic plants growing in soil; (H) response of progenies of transgenic plant ETR3-6 to hyg B

15. Results of three experiments of Agrobacterium-mediated tall fescuetransformation (cv.‘‘Matador’’)

16. Discussion • Establishment of an efficient Agrobacterium-mediated transformation system of tall fescue Agrobacterium-mediated transformation is often preferred over other plant transformation systems because of the simplicity, the low cost, andlower transgene copies integrated into plant genome • Analyses of factors that may affect tall fescue transformation efficiency pTOK47 do not play a substantial role in the observed high transformation frequency, 2,4-D concentration in the medium play an important role in the efficient transformation.