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Gene gun living quick transformation method of cotton

A technology of gene transformation and gene gun, applied in the field of rapid transformation of cotton by gene gun, can solve the problems of difficult transformation, imperfect transformation technology, complicated transformation procedures, etc. Effect

Inactive Publication Date: 2013-05-01
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

Although the Agrobacterium-mediated method is a relatively complete transformation technology in theory and practice, and has been widely used in the transformation of dicotyledonous plants, this method is limited by the range of host genotypes, and it is difficult for many elite varieties (lines) to This method is transformed, and the transformation procedure is cumbersome and complicated
Although the pollen tube channel transformation method has no genotype and species control, its transformation technology is not perfect, and there is a lack of systematic research on the transformation mechanism, and its application is often limited to the flowering stage

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0022] 1. Preparation of recombinant vectors containing exogenous genes

[0023] The foreign gene is GhTrx gene.

[0024] Construction of recombinant vectors containing foreign genes: Digest vector pBI121 with XmaI and BamHI to collect large vector fragments; connect large vector fragments and gene fragments with T4 ligase to obtain recombinant vectors.

[0025] 2. Gene gun bullet preparation:

[0026] Gold powder treatment

[0027] (1) Take 30mg of gold powder and put it in a centrifuge tube, add 1ml of 70% ethanol, shake it fully for 3-5min, let it stand for 15min,

[0028] (2) Centrifuge at 12000rpm for 30s, remove the supernatant,

[0029] (3) Add 1ml of sterile water to resuspend and centrifuge at 12000rpm for 5s, remove the supernatant,

[0030] (4) Repeat the above steps 5-7 times, suspend the gold powder in 500 μl sterile distilled water, and store at 4°C or room temperature.

[0031] DNA processing

[0032] (1) Take 100 μl of gold powder suspension and add 10 μl...

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PUM

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Abstract

The invention relates to a gene gun living quick transformation method of cotton. The method comprises the following steps of (1) using a gene gun bombarding method to bombard a carrier containing a foreign gene into pollen of paternal cotton; (2) pollinating paternal pollen bombarded by the gene gun into living maternal cotton; (3) using the seeds produced by the pollinated maternal cotton as the seeds of transgenic cotton, so as to realize the gene transformation of the cotton. The method has the advantages that the tissue culture step is not required, and the gene transformation is directly realized on the living cotton; the microtrauma is formed for buds and pollen during living transformation by the third-generation portable gene gun and the optimization and transformation parameters, and the period for obtaining transgenic plants is greatly shortened; and the practicality is realized, the operation is easy, the dependency on the tissue culture is avoided, and the stability is high.

Description

technical field [0001] The invention relates to a method for rapidly transforming living organisms of cotton with a gene gun. Background technique [0002] Among the cotton transgenic technologies developed so far, Agrobacterium-mediated genetic transformation is the most widely used. Since Umbeck et al. successfully obtained the transgenic plants of Cotton cotton through the Agrobacterium-mediated method in 1987, the Agrobacterium-mediated method has gradually become the most common transformation method in the world, and has made great progress. The Cotton Research Institute of the Chinese Academy of Agricultural Sciences has successfully established an efficient and stable large-scale transformation system for more than 20 cotton varieties, realized assembly line operation, and established an efficient and industrialized cotton transgenic technology system. Although the Agrobacterium-mediated method is a relatively complete transformation technology in theory and practic...

Claims

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Application Information

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IPC IPC(8): A01H1/02A01H1/00
Inventor 叶武威王德龙阴祖军王俊娟王帅樊宝香王桂峰孔静静宋贵方张德超陆许可
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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