Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method

A genetic transformation method, the technology of Agrobacterium rhizogenes, applied in the direction of botany equipment and methods, the use of vectors to introduce foreign genetic material, plant products, etc., can solve the problem of low efficiency of soybean genetic transformation, to improve the transformation rate and increase the infection rate. efficiency effect

Inactive Publication Date: 2012-11-07
JILIN UNIV
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  • Abstract
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Problems solved by technology

[0009] The purpose of the present invention is to overcome the difficulty of the low efficiency of the existing soybean genetic transformation, to pro

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  • Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method
  • Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method
  • Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method

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Embodiment Construction

[0042] The content of each step of the inventive method is described in further detail now:

[0043] 1. Selection, disinfection and germination of soybean seeds

[0044] Select well-developed and disease-free "Jidou No. 2" soybean seeds, sterilize by fumigation with chlorine gas at room temperature (chlorine gas is produced by 100ml5.24%NaCIO+3.5ml12mol / L HCI), and fumigate in a desiccator for 14 hours; here Prepare aseptic soil during this period, mix plant ash and vermiculite at a ratio of 1:1, add an appropriate amount of 1 / 4MS liquid medium (without sucrose) to moisten, stir evenly, and then sterilize under high temperature and high pressure (121°C, 20min). Arranged in a flowerpot with a diameter of 15cm. Take out the sterile soybeans in the ultra-clean workbench, connect them to the place 1-2cm below the surface of the sterile wet vermiculite, seal them with plastic wrap, and place them in a dark culture room at 25°C for bud-inducing cultivation. After 5 days, prepare A...

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Abstract

The invention relates to an agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method, and belongs to the technical fields of molecular biology and genetic engineering. The agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method comprises the following steps of: selecting genotypes of soybean seeds, disinfecting the soybean seeds, and germinating; preparing bacterial liquid, which contains anti-nematode bacillus thuringiensis (Bt) genes, of agrobacterium rhizogene K599; transforming the agrobacterium rhizogenes-mediated soybeans; performing histochemical staining analysis on transgenic roots; and performing polymerase chain reaction (PCR) detection on the transgenic roots. According to the method, an agrobacterium rhizogenes-mediated and vacuum infiltration-assisted transformation method is applied to the soybeans for the first time, and hairy roots of soybean roots are transformed successfully; the disinfected soybean seeds are planted in sterile vermiculite and are germinated, the injury of epidermic meristematic cells is caused at the upper end of hypocotyl, the agrobacterium rhizogene is infected under the vacuum condition, and genetic transformation is performed to obtain the soybean transgenic hairy roots containing the anti-nematode BT genes. By implementing the agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method, the opportunity of the invasion of the agrobacterium rhizogenes can be improved fully, the infection efficiency of the agrobacterium rhizogenes can be improved obviously, and the transformation rate of the soybeans is improved effectively.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to a method and application of soybean genetic transformation mediated by Agrobacterium rhizogenes mediated by vacuum infiltration. Background technique [0002] Soybean is an important food crop and economic crop, but affected by various diseases, insect pests and various abiotic adversities, the quality and yield of soybean cannot meet people's needs. Soybean cyst nematode (soybean cyst nematode, referred to as SCN), caused by soybean cyst nematode infestation, is one of the most serious diseases harmful to soybean production in the world. It is characterized by wide distribution, serious damage, wide host range, It is a soil-borne disease that is extremely difficult to control due to its multiple transmission routes and long survival time of dormant bodies (cysts). It is more common in Northeast my country and in Huang, Huai, and Hai ...

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 刘金亮冯頔谢丽霞贾保磊魏毅张世宏潘洪玉
Owner JILIN UNIV
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