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Soybean conversion method

A technology for soybeans and soybean plants, applied in biochemical equipment and methods, botany equipment and methods, angiosperms/flowering plants, etc., can solve the problems of difficult emergence and low transformation efficiency, and achieve simple and convenient Agrobacterium infection process , high conversion efficiency and increased efficiency

Active Publication Date: 2014-01-22
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

This method avoids the influence of human factors that require careful manipulation when the cotyledon nodes are scratched, and makes the operation easier; but this transformation method is difficult to emerge and the transformation efficiency is low

Method used

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no. 1 example

[0044] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0045] 1. Construct a recombinant cloning vector containing the target gene

[0046] The PAT nucleotide sequence was connected to 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; PAT is glufosinate Acetyltransferase gene nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0047] Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 co...

no. 2 example

[0054] The second embodiment, the acquisition of transgenic soybean plants

[0055] Soybean seed germination: Inoculate 100 mature soybean seeds (Zhonghuang 13) on soybean germination medium (B5 salt 3.1g / L, B5 vitamin, sucrose 20g / L, agar 8g / L, pH5.6) for germination , the culture conditions are: temperature 25±1°C, photoperiod 16 / 8h.

[0056] Pretreatment: 1 day after germination, the cotyledon and the first true leaf were removed, and the exposed meristem was inoculated into a pretreatment medium containing cytokinin (MS salt 4.3g / L, B5 vitamin, sucrose 20g / L, Agar 8g / L, 2-morpholineethanesulfonic acid (MES) 4g / L, zeatin (ZT) 2mg / L, 6-benzyl adenine (6-BAP) 1mg / L, acetosyringone (AS) 40mg / L, pH5.3), in this step, cytokinin was added to make the cells in the meristem area active, and AS was also added to the medium, pretreated for 3 days, and the pretreated tissue block Use the back of a scalpel to injure 5 times, and perform ultrasonic treatment for 3 minutes after the i...

no. 3 example

[0063] The third embodiment, using TaqMan to verify the soybean plant transferred to the PAT nucleotide sequence

[0064]About 100 mg of leaves of soybean plants with PAT nucleotide sequence were taken as a sample, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of PAT gene was detected by fluorescent quantitative PCR method of Taqman probe. 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 invention relates to a soybean conversion method which comprises the following steps: cotyledon and the first euphylla of germinated soybean seeds are removed, and naked meristem is obtained; the naked meristem is inoculated to a pretreatment culture medium containing cytokinin for pretreatment; meristem blocks after the pretreatment are infected by agrobacteria. According to the soybean conversion method, the soybean meristem blocks after the pretreatment are used as explant materials for the first time; the explant materials are subjected to high-concentration cytokinin treatment before agrobacterium infection so that the meristem of soybeans can be in an active state and exogenous genes can be inserted easily; meanwhile, the meristem is multiplied and enlarged, so that the infection process is more convenient to operate; in the process of agrobacterium infection, ultrasonic treatment is added, so that the efficiency of insertion of the exogenous genes in a plant genome is greatly improved, and the conversion rate of the soybeans can reach 10%; meanwhile, the conversion period is short, the number of chimera is small, and conversion cost is low.

Description

technical field [0001] The invention relates to a method for plant transformation, in particular to a method for transforming soybean tissue. Background technique [0002] Soybean (Glycine max) originated in China and is the main source of plant protein. It is also an important oil crop and high-protein grain and feed crop in China. The planting area is second only to rice, corn and wheat, ranking fourth. In order to improve soybean herbicide tolerance, resistance to pests and diseases, and physiological adversity, improve soybean quality, and increase soybean yield, in addition to traditional breeding methods, genetic engineering improvement can be achieved through genetic engineering. [0003] The genetic transformation methods of soybean mainly include Agrobacterium-mediated method, gene gun bombardment method, electric shock method, PEG method, microinjection method, ultrasonic-assisted method, and pollen tube introduction method, among which Agrobacterium-mediated metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 贾志伟刘雁华
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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