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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
High efficiency transformation of tall fescue withAgrobacterium tumefaciens
Plant Science 168 (2005) 1453–1458
Shujie Dong, Rongda Qu *Department of Crop Science, North Carolina State Uni
versity, Raleigh, NC 27695-7620, USAReceived 22 December 2004; accepted 14 January 20
05Available online 2 February 2005
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 greatlyfacilitate 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 .
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
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
pCAMBIA R-gene plants
R-gene bacteria
Polylinker Reporter
Gene
T-DNA size (bp)
Reading Frame or lacZ Vector
Vector
family
1301 hptII kan pUC18 gusA 5607 - GIS
http://www.cambia.org/daisy/cambia/585.html
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
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
農桿菌
大腸桿菌
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
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
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
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
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;
(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
Results of three experiments of Agrobacterium-mediated tall fescuetransformation (cv.‘‘Matador’’)
Experiment Total no. of callus infected
Hyg B resistant callus (%)
Regenerated plants
GUS+ plants Overall
transformation
frequency (%)
1 120 33.30 1 1 5.83
2 90 33.30 7 7 1.10
3 90 36.70 16 16 17.78
Total 300 34.3. 24 24 8.00
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.