Agrobacterium tumefaciens-medicated genetic transformation method of wheat

An Agrobacterium-mediated, Agrobacterium-mediated technology, applied in botanical equipment and methods, using vectors to introduce foreign genetic material, angiosperms/flowering plants, etc., can solve the problems of plant regeneration, limit transgenic wheat, etc., and achieve the transformation cycle. Short, simple operation, wide adaptability effect

CN102533845AInactive Publication Date: 2012-07-04HARBIN NORMAL UNIVERSITY
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2012-07-04
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses an agrobacterium tumefaciens-medicated genetic transformation method of wheat. The agrobacterium tumefaciens-medicated genetic transformation method comprises the following steps of: firstly, inoculating recombinant Agrobacterium tumefaciens containing a target DNA (Deoxyribonucleic Acid) into wheat immature embryos; and then co-culturing the inoculated wheat immature embryos for enabling the T-DNA of the recombinant Agrobacterium tumefaciens to be integrated with a chromosome of the wheat; and secondly, directly inoculating the wheat immature embryos co-cultured in the first step in a long-seedling culture medium for culturing to obtain transgenic wheat seedlings with roots, stems and leaves. The method disclosed by the invention has the advantages that the applicability is wide, so that the method is suitable for transforming various wheat species; the method is simple in operation, and thus a transgenic plant can be obtained without inducing a tissue culture and regenerating the plant; and the transformation period is short, i.e. the process from the infection to the obtaining of the transgenic wheat seedlings with the roots, the stems and the leaves only requires 11-14 days. The method has very important significance for genetic improvement on the wheat species and the obtaining of new species of the transgenic wheat.
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Description

technical field

[0001] The invention relates to a wheat genetic transformation method mediated by Agrobacterium. Background technique

[0002] Wheat is one of the important cereal crops that human beings depend on for survival. Its planting area and total output exceed other crops in the world. Its genetic improvement has always been an important goal of people's efforts. The traditional wheat breeding technology has limitations such as long breeding cycle and limited yield increase potential. With the development of biotechnology, the use of genetic engineering technology to improve wheat varieties has attracted more and more attention. Compared with other cereal crops such as rice and corn, wheat shows lag and difficulty in the research of plant gene transformation system. Wheat genetic transformation research began in the late 1980s. Since wheat was not a natural host for Agrobacterium and the regeneration of wheat protoplasts was relatively difficult, it was not until 1...

Examples

Embodiment 1

[0039] Embodiment 1, utilizing Agrobacterium rhizogenes to carry out chitinase gene transformation to wheat

[0040] 1. Construction of recombinant Agrobacterium rhizogenes containing target DNA

[0041] The recombinant vector used to transform Agrobacterium rhizogenes is pMLH7133-Chi, the target gene on the vector is chitinase gene (Chi), and the marker selection gene on the vector is hygromycin phosphotransferase (HPT) gene.

[0042] The recombinant vector pMLH7133-Chi was transformed into Agrobacterium rhizogenes strain R1000 (Agrobacterium rhizogenes strain R1000) to obtain recombinant Agrobacterium rhizogenes R1000 / pMLH7133-Chi containing chitinase gene. The specific method is as follows: (1) Pick a single colony of Agrobacterium rhizogenes R1000 from the YEB solid plate containing 50 mg / L streptomycin, and inoculate it in a test tube containing 10 ml of YEB liquid medium (containing 50 mg / L streptomycin) , culture overnight at 28°C with shaking; (2) Take 400 μl of bacte...