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Method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation

A transgenic soybean, Agrobacterium-mediated technology, applied in botanical equipment and methods, introduction of foreign genetic material using vectors, angiosperms/flowering plants, etc. Long cycle and other problems, to achieve the effect of simple operation, short conversion cycle and high conversion efficiency

Inactive Publication Date: 2013-07-31
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
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Problems solved by technology

Although the technology of Agrobacterium-mediated transformation of soybean has been continuously improved in recent years, most methods need to go through the process of dedifferentiation and redifferentiation of explants, and there are problems such as low transformation efficiency, poor genetic stability, and long transformation cycle.
These problems have seriously hindered the verification of functional genes and the progress of transgenic breeding

Method used

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  • Method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation
  • Method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation
  • Method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, Agrobacterium-mediated cultivation of transgenic soybean

[0028] Option I

[0029] 1. Preparation of recombinant Agrobacterium suspension

[0030] The plant binary expression vector PB2GW7 (Binary gateway overexpression vector PB2GW7) containing the herbicide resistance gene Bar (document: Dubin MJ, Bowler C, Benvenuto G.A modified Gateway cloning strategy for overexpressing tagged proteins in plants. Plant methods, 2008, 4 (3): 1-11. The public can obtain it from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences) into Agrobacterium tumefaciens EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.) to obtain a recombinant gene containing the target gene Agrobacterium tumefaciens EHA105 / PB2GW7.

[0031] A single colony of recombinant Agrobacterium tumefaciens EHA105 / PB2GW7 was treated with YEP culture solution containing spectinomycin 50mg / L and rifampicin 25mg / L (solvent is water, solute and its concentration a...

Embodiment 2

[0078] Embodiment 2, the impact of acetosyringone concentration on transformation efficiency in the Agrobacterium suspension

[0079] 1. Preparation of recombinant Agrobacterium suspension

[0080] The expression vector pCambia3301 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., containing the GUS reporter gene) was transformed into Agrobacterium tumefaciens EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.), and the GUS reporter gene was obtained. Gene recombinant Agrobacterium tumefaciens EHA105 / pCambia3301.

[0081] A single colony of recombinant Agrobacterium tumefaciens EHA105 / pCambia3301 was treated with YEP culture solution containing spectinomycin 50mg / L and rifampicin 25mg / L (solvent is water, solute and its concentration are: sodium chloride 5g / L, yeast extract 5g / L and tryptone 10g / L) at 28°C until OD 600 0.8—1.2; 4000rpm, centrifuge for 10min, resuspend to OD with the following liquid medium 600 Obtain a suspension of recombin...

Embodiment 3

[0096] Embodiment 3, the impact of different soybean varieties on transformation efficiency

[0097] Eight soybean varieties including Dongnong 50, Zhonghuang 35, Jindou 21, Zhongdou 33, Zhongdou 40, Tianlong 1, Kenfeng 16 and Heihe 48 were selected for transformation efficiency analysis. The specific method is as follows:

[0098] 1. Preparation of recombinant Agrobacterium suspension

[0099] The expression vector pCambia3301 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., containing the herbicide resistance gene Bar) was transformed into Agrobacterium tumefaciens EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.) to obtain Recombinant Agrobacterium tumefaciens EHA105 / pCambia3301 containing a GUS reporter gene.

[0100] A single colony of recombinant Agrobacterium tumefaciens EHA105 / pCambia3301 was treated with YEP culture solution containing spectinomycin 50mg / L and rifampicin 25mg / L (solvent is water, solute and its concentration are: ...

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Abstract

The invention discloses a method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation. The method is characterized by transferring a target DNA (deoxyribonucleic acid) into soybean cells through Agrobacterium tumefaciens mediated transformation, and comprises the following steps: 1) injecting a suspension of recombinant Agrobacterium tumefaciens containing the target DNA at cotyledonary node meristematic tissues of soybean seeds where seedlings are germinated, and then co-culturing the seedlings; and 2) directly performing long seedling culture on the seedlings co-cultured in the step 1) to obtain transgenic soybean plants having roots, stems and leaves. The invention has the advantages of simple operation process, short transformation period, high transformation efficiency (the maximum can be up to 15.3%), high economy and wide application range.

Description

technical field [0001] The invention relates to a method for cultivating transgenic soybeans mediated by Agrobacterium. Background technique [0002] Compared with traditional breeding technology, transgenic breeding technology has a short cycle, high efficiency, and strong purpose, and is an effective way to improve crop agronomic traits. At present, soybean transgenic methods mainly include Agrobacterium-mediated method, gene gun method, and pollen tube channeling method, among which Agrobacterium-mediated method is commonly used, which can insert T-DNA into plants in a single or low-copy form more completely. in the genome. Although the technology of Agrobacterium-mediated transformation of soybean has been continuously improved in recent years, most methods need to go through the process of dedifferentiation and redifferentiation of explants, and there are problems such as low transformation efficiency, poor genetic stability, and long transformation cycle. . These pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 陈李淼沙爱华张晓娟单志慧陈水莲张婵娟邱徳珍周蓉周新安
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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