Method for transforming genes by using efficient living body of chemical male sterilant

A kind of andricide and chemical technology, which is applied in the field of high-efficiency transformation of genes using chemical andricide, can solve the problems of low transformation efficiency, high cost, unclear time of cells and transformation, etc., to achieve improved transformation efficiency and low cost , Conducive to the effect of Agrobacterium infection

Inactive Publication Date: 2011-10-12
SOUTHWEST UNIVERSITY
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Problems solved by technology

This method is also difficult to apply directly to other crops, because other crops do not have thin petal walls like Arabidopsis, so it is difficult to infect
In addition, the transformed cells and the timing of the transformation are still not well understood
7 Although the gene gun transformation method is not limited by species, it is used in many species, but its cost is high, expensive equipment is required, and the transformation efficiency is low, and many researchers are disappointed

Method used

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Examples

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Embodiment Construction

[0026] Take Brassica napus as an example:

[0027] 1) Take the homozygous Brassica napus Zhongshuang 11, and drill it in two plots A and B, each plot is 3×3m2 ;

[0028] 2) In the early stage of budding, the plants in area A were chemically killed with 0.6 μg / mL ESP (chemical andricide, 98% of all sterile plants), and the plants in area B were bagged;

[0029] 3) Cultivation of Agrobacterium: Pick a single colony of Agrobacterium preserved on the plate and inoculate it in YEP culture medium (additional rifampicin 50mg / L, streptomycin 25mg / L and kanamycin 100mg / L), shake at 28°C Bacteria for 16-18 hours (200r / min), take 1-5 mL of bacterial liquid and connect to 100 mL of YEP culture medium (containing 100 μmol / L of acetophenone, 25 mg / L of rifampicin, 12.5 mg / L of streptomycin and kanamycin 50 mg / L) to expand the culture for about 2 to 4 hours until the logarithmic growth phase of Agrobacterium (OD 600 = 0.3~0.5), centrifuge at 4800 r / min for 6 min to collect the bacteria, r...

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Abstract

The invention relates to chemical male sterilization, pollen-tube pathway and pollen-mediated transformation technologies, and belongs to the technical field of molecular breeding and gene engineering. The method comprises the following steps of: performing chemical male sterilization treatment on a crop T0 of a budding stage; cutting stigmas of buds; mixing fertile pollen by using the pollen-tube pathway technology, namely using target gene-containing agrobacterium-mediated infection solution (added with 5 percent of sucrose and 500muL/L of SilwetL-77) with OD600 of 0.8; and dipping an absorbent cotton ball in the bacterial solution, coating the bacterial solution to the stigma cut ovaries, performing shading treatment for three days to realize normal growth, and finally collecting selfing seeds. Herbicide resistance screening is performed on the generation T1, polymerase chain reaction (PCR) detection is performed on resistant plants, and positive plants are determined. The method has the characteristics of low cost, simplicity in operation, no need of expensive instruments and equipment, high transformation rate (1 to 5 percent) and the like, is suitable for any flowering plant, and can be applied to large-scale crop genetic transformation.

Description

technical field [0001] The invention belongs to the technical fields of molecular breeding and genetic engineering, and in particular relates to a method for efficiently transforming genes in vivo using chemical andricides. Background technique [0002] Plant genetic engineering began in the 1980s. It is a technology that is gradually developed on the basis of plant tissue culture technology, cell culture technology and DNA in vitro recombination technology, combined with other technologies. The current transgenic technology mainly relies on dedifferentiated cells for bacterial liquid infection, and then differentiates and regenerates to realize the transfer of exogenous genes, so as to obtain transgenic plants. This approach has its unique advantages, such as good repeatability, basically not limited by seasons, strong reference for the transformation technology of various crops, and high conversion rate, etc., but this approach also has its shortcomings, such as tissue cul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12Q1/68A01H5/00
Inventor 付东辉魏大勇钱伟李加纳
Owner SOUTHWEST UNIVERSITY
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