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Agrobacterium-mediated cotton transgenic method independent of tissue culture

A technique of Agrobacterium-mediated tissue culture, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., to achieve the effect of avoiding somatic clone variation and simple transformation system

Inactive Publication Date: 2010-04-07
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

So far, there is no report on genetic transformation of cotton style by drip-coating cotton style with Agrobacterium-sucrose solution after pollination

Method used

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  • Agrobacterium-mediated cotton transgenic method independent of tissue culture
  • Agrobacterium-mediated cotton transgenic method independent of tissue culture
  • Agrobacterium-mediated cotton transgenic method independent of tissue culture

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Embodiment Construction

[0027] 1) Using the popular variety Simian No. 3 in the Yangtze River Basin as the experimental receptor, during the flowering period in July and August, select the flower buds that will flower the next day for selfing for transformation.

[0028] 2) The Agrobacterium strain is EHA105, and the plasmid is pKF111 (commonly known and public, Ni M, Tepperman J, Quail P. PIF3, aPhytochrome-lnteracting Factor Necessary for Normal Photoinduced Signal Transduction, Is a Novel Basic Helix-Loop-Helix Protein. Cell, 1998 , 95: 657-668). The plasmid T-DNA region contains the bar gene for Basta herbicide resistance. The engineered Agrobacterium containing the bar gene is stored in an additional 50mg l -1 Kanamycin and 20mg l -1 Rifampicin on solid LB medium. Pick 2-3 clones of engineered Agrobacterium in 50mg l -1Incubate overnight at 28°C in 50ml of liquid LB with kanamycin. Centrifuge at room temperature at 4000rpm for 5min, discard the supernatant, resuspend the Agrobacterium preci...

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Abstract

The invention relates to an agrobacterium-mediated cotton transgenic method independent of tissue culture, belonging to the biotechnology field. The method comprises the following steps: dispensing agrobacterium-sucrose solution with 0.05% of surfactant Silwet L-77 and 40mg of 1-1acetosyringone on stylus in the afternoon of cotton fertilization or in the morning on the next day; preserving moisture for two days; harvesting seeds and performing resistance authentication; extracting DNA and performing PCR and Southern blot detection; and obtaining transgenic plants by multiple treatments. The transformation ratio ranges from 0.05% to 0.65%, thus proving the repeatability and reliability of transformation systems of agrobacterium-dipped cotton stylus. Compared with the traditional transgenic method dependent on tissue culture, the transformation method of the agrobacterium-dipped cotton stylus is simpler, more convenient, more economical and efficient, and simultaneously can avoid problems of limitation by cotton genetype and easy occurrence of somaclonal variation and the like in the process of tissue culture.

Description

(1) Technical field: [0001] The invention relates to an Agrobacterium-mediated cotton transgenic method independent of tissue culture, which is specially used for genetic transformation of crops (cotton) and selection of new transgenic varieties, and belongs to the field of biotechnology. (two) background technology: [0002] Cotton is one of the main textile raw materials. After years of systematic selection, the genetic base of cotton varieties is rather narrow. It is one of the effective ways to increase the genetic diversity of cotton by introducing useful exogenous genes into recipient materials and stably expressing them through transgenic methods. The report of genetic transformation of cotton began in 1987, and transgenic cotton also entered commercial production in the 1990s. To date, genetically modified cotton is one of the most successful crops to be commercialized. But the genetic transformation of cotton is still a difficult problem. Agrobacterium-mediated ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/84
Inventor 张天真陈天子郭旺珍
Owner NANJING AGRICULTURAL UNIVERSITY
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