1. High efficiency transformation of tall fescue with Agrobacterium 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. pCAMBIAR-gene plants R-gene bacteria PolylinkerReporter Gene T-DNA size (bp) Reading Frame or lacZ Vector Vector family 1301hptII kanpUC18gusA 5607 -GIS 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. 農桿菌載體 構築 切位 有興趣的基因 插入載體中 送入大腸桿菌，並 選拔對 kan 具抗性者 與農桿菌交配 農桿菌 大腸桿菌 具重組質體之農桿菌 感染植物 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

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

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 fescue transformation (cv.‘‘Matador’’) ExperimentTotal no. of callus infected Hyg B resistant callus (%) Regenerated plants GUS+ plantsOverall transformation frequency (%) 112033.30115.83 29033.30771.10 39036.7016 17.78 Total30034.3.24 8.00

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.