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 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






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






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











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.

Documents

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 with Agrobacterium tumefaciens Shujie Dong, Rongda Qu * Plant Science 168 (2005) 1453–1458 生科四甲李廷原 91390604