Methods for Improving Transformation Efficiency of Soybean

A technology for soybeans and transgenic plants, applied in biochemical equipment and methods, botany equipment and methods, plant products, etc., can solve the problems of high false positive plant rate and low transformation efficiency

Active Publication Date: 2020-11-03
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for improving soybean transformation efficiency, which can effectively overcome the technical defects of high false-positive plant rate and low transformation efficiency in the prior art of configuring ALS inhibitors in the culture medium, and provide a new method for large-scale genetic transformation. and breeding plants with herbicide tolerance traits provides new options

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  • Methods for Improving Transformation Efficiency of Soybean
  • Methods for Improving Transformation Efficiency of Soybean
  • Methods for Improving Transformation Efficiency of Soybean

Examples

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Effect test

no. 1 example

[0140] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0141] 1. Construct a recombinant cloning vector containing the target gene

[0142] The SULE nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, and its construction process Such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; SULE is the sulfonylurea Herbicide-like hydrolase gene nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0143]Then, the recombinant cloning vector DBN01-T was transformed into Escheri...

no. 2 example

[0150] The second embodiment, the acquisition of transgenic soybean plants

[0151] Disinfection of soybean seeds: Take completely dried mature soybean seeds (Zhonghuang 13) and place them in a petri dish, accounting for about 1 / 3 of the petri dish. Place it in a desiccator in a fume hood, and place a 250ml beaker in the desiccator with 120ml of sodium hypochlorite inside. Add 6ml of concentrated hydrochloric acid dropwise along the wall of the beaker, close the desiccator and seal the lid, and close the glass of the fume hood at the same time, so that the soybean seeds are exposed to the chlorine gas in the fume hood to sterilize for 3 hours; Take out and shake for 2-3 minutes. Repeat the above process once, and put the Petri dish containing the soybean seeds in a fume hood to sterilize overnight.

[0152] Soybean seed germination: inoculate 15 soybean seeds after disinfection on the soybean germination medium (B5 salt 3.1g / L, B5 vitamin, sucrose 20g / L, agar 8g / L, pH5.6) to...

no. 3 example

[0176] The third embodiment, using TaqMan to verify the soybean plant transferred to the SULE nucleotide sequence

[0177] Take about 100 mg of the leaves of the above 12 kinds of treated soybean plants transferred to the SULE nucleotide sequence as samples, extract the genomic DNA with Qiagen's DNeasy Plant Maxi Kit, and detect the copy of the SULE gene by the Taqman probe fluorescence quantitative PCR method number. At the same time, the wild-type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

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Abstract

The present invention relates to a method for improving soybean transformation efficiency. The method for selecting transformed plant cells comprises: using a recombinant vector containing a target gene and a gene encoding a sulfonylurea herbicide hydrolase to transform plant cells; The above-mentioned transformed plant cells are screened and cultivated for ALS inhibitors, and the gene encoding sulfonylurea herbicide hydrolase is used as a selection marker; plant cells that are not killed and / or not inhibited are selected. The present invention proposes for the first time that the selection agent is added to the proliferation medium and the differentiation medium by means of external application (especially dripping) during the plant transformation process, and the effective screening concentration range of the selection agent is optimized, and the proportion of positive plants obtained by its offspring and the transformation efficiency are significantly increased; meanwhile, the transgenic plants obtained through the transformation using the sulfonylurea herbicide hydrolase gene as the selection marker in the present invention have high commercial value, good resistance and stable genetics.

Description

technical field [0001] The invention relates to a method for plant transformation, in particular to a method for improving soybean transformation efficiency by externally applying a screening agent. Background technique [0002] With the rapid increase of transgenic plant species and planting area, the biosafety of selectable marker genes in transgenic plants has become one of the problems that people generally pay attention to. Appropriate marker genes can provide a powerful judgment basis for obtaining true transformants, and play a crucial role in the process of plant genetic transformation. At present, marker genes widely used in plant genetic transformation include antibiotic resistance genes (such as NPTⅡ gene, HPT gene, etc.) and herbicide resistance genes (such as PAT gene, EPSPS gene, bar gene, etc.). Success is no longer useful, and even there is a potential threat to the ecological environment and food safety, so the development of biosafety marker genes is very ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/54
CPCC12N15/8205C12N15/821C12N15/8274C12N15/8275C12N15/8277C12N15/8278
Inventor 刘雁华王媛媛贾志伟宋庆芳
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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