Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method

A genetic transformation method and a technology for Brassica napus, applied in the field of plant tissue culture and transgenic, can solve the problems of low differentiation rate of resistant seedlings, poor transformation efficiency of target genes, etc., and achieve the effects of high differentiation rate of resistant seedlings and promoting development.

Active Publication Date: 2014-08-06
ZHEJIANG UNIV
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Problems solved by technology

[0005] The current genetic transformation technology of Brassica napus generally has low differentiation rate of resistant seedlings and poor transformation efficiency of the target ge

Method used

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  • Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method
  • Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method
  • Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method

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Embodiment 1

[0038] (1) Select an appropriate amount of "Zheda 619" rapeseed with full grains, wash it in 75% alcohol for 30 seconds, pour out the alcohol, add about 25mL of 5% sodium hypochlorite solution for disinfection for 15 minutes, and oscillate at the same time to make the disinfectant and rapeseed The seeds are in full contact, pour out the sodium hypochlorite solution, rinse with sterile water 4 to 5 times, and then evenly spread the sterilized seeds in the bottle containing the homogeneous medium.

[0039] (2) The solid medium is 1 / 2MS+18.0g / L sucrose+9.0g / L agar, the pH is 5.8. About 24 seeds are sown in each bottle of homologous medium, at 25-27°C, 16 hours of light, 8 hours of darkness, and a light intensity of 70 μmolm -2 the s -1 cultured under conditions.

[0040] (3) When the aseptic seedlings were cultivated for 8 days, the upper and lower ends of the hypocotyls of rapeseed seedlings were respectively cut on the ultra-clean workbench as explants, and inoculated onto pr...

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Abstract

The invention discloses an agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method. The method comprises the following steps: taking the hypocotyl of a cabbage type oilseed rape seedling, conducting preculture through adopting the lower end of the hypocotyl as an explant, and inducing for production of the callus; infecting the callus explant by adopting agrobacterium tumefaciens carrying target genes, and conducting the co-culture; after the co-culture is conducted on the explant, inducing the explant to generate resistance adventitious buds on a differential medium containing a selective agent; conducting the rooting culture to obtain a resistance seedling. According to the agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method, the lower end of the cabbage type oilseed rape hypocotyl serves as the explant, and the growth conditionis adopted, which is most suitable for cabbage type oilseed rape genetic transformation, and particularly includes the pH value and the high concentration glyphosate differentiation screening method and the low concentration glyphosate differentiation screening method in the co-culture medium, so that the transgenetic seedling with glyphosate resistance is obtained successfully; compared with the prior art, the method has higher resistance seedling differentiation rate, a high efficiency cabbage type oilseed rape regeneration system with glyphosate resistance is built, and a foundation is laid for studying about the transgenic technology of the agrobacterium tumefaciens mediated cabbage type oilseed rape.

Description

technical field [0001] The invention relates to an Agrobacterium-mediated genetic transformation method of Brassica napus with rape hypocotyls as explants, and belongs to the technical field of plant tissue culture and transgene. Background technique [0002] Rapeseed is an important oil crop widely planted in China, India, Canada, and Europe, and ranks third in global oil crop production. In 1974, Kartha et al first induced and cultivated plantlets from rapeseed stems. Since then, based on the principle of cell pluripotency, people have used different explant types of rapeseed to induce callus and obtain regenerated plants, such as stems, cotyledons, leaves, microspores, shoot tips, hypocotyls, roots and protoplasts of rapeseed. body etc. The establishment of rapeseed regeneration system is the basis of its genetic transformation, and the reduction of the regeneration frequency of rapeseed explants will result in the reduction of its genetic transformation efficiency. The...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 徐茜周伟军刘宏波李兰黄昌蓉许玲
Owner ZHEJIANG UNIV
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