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Method for obtaining switchgrass transgene plant

A technology of transgenic plants, applied in the biological field, can solve the problems of inconsistency between regenerable cell parts and transformation competent cells, general application limitations of embryogenic callus, strong dependence on tissue culture technology, etc., to shorten the transformation cycle, transform Effect of improved genotype range, simplified procedure

Inactive Publication Date: 2008-03-19
NORTHWEST A & F UNIV
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Problems solved by technology

[0004] Similar to the genetic transformation of other plants, the Agrobacterium-mediated transformation of switchgrass also has a strong dependence on tissue culture technology, a long transformation cycle, and genotype restrictions (currently only individual genotypes of switchgrass are successfully transformed), and regenerated cells The disadvantages such as inconsistency between the site and the competent cells for transformation have greatly limited its general application in switchgrass, which is currently difficult to obtain a large amount of embryogenic callus through tissue culture techniques

Method used

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Embodiment

[0062] Three switchgrass cultivars GA9930, N. Switchgrass and Blackwell were transformed with Agrobacterium containing leaf senescence delaying gene PsAG12-ipt. The specific conversion process is:

[0063] (1) Sterilization of seeds

[0064] a. Take the mature seeds of GA9930, N.Switchgrass and Blackwell 3 varieties of switchgrass, and use 70% alcohol for surface disinfection for 1 minute; take 5000 seeds for each variety, and transform 5 times in total in the experiment, with 1000 seeds transformed each time;

[0065] b. Use 3% calcium hypochlorite (add 0.1% Tween 80), 500 rpm / min, and sterilize for 2 hours:

[0066] c. Rinse 3 times with sterile water, and culture in dark for 12 hours at 4°C;

[0067] d. Secondary sterilization with 3% calcium hypochlorite for 1.5 hours;

[0068] e. Rinse 3 times with sterile water.

[0069] (2) Germination of seeds

[0070] Put the seeds on 5 layers of sterile filter paper fully wetted with sterile water, and culture in dark at 24°C un...

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Abstract

The present invention discloses a method that a switchgrass transgenic plant is obtained by adopting agrobacterium-mediated transformation. When the genetic transformation of the switchgrass is operated, a seed which burgeons after being sterilized is used as a transformation acceptor, and the agrobacterium transformation and the co-culture are adopted; by screening on culture mediums of SMBP1, SMBP2 and SMBP3 in sequence, a resistant bud is obtained; the resistant bud is cultivated into a seedling in a rooting culture medium SMO; the seedling is transplanted to a greenhouse or the field to perform the detection of the molecular biological method, and a transgenic plant containing the exogenous aim is obtained by screening according to the molecular biological method and the target trait. The transgenic method of the switchgrass established by the present invention overcomes the disadvantage that the dependance of the agrobacterium-mediated transformation to the tissue culture condition is strong, widens the genotype range of the genetic transformation, and simplifies the transformation process, to provides a new approach for the genetic improvement of the switchgrass by adopting the transgenic technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the gene transformation of bioenergy plant switchgrass, and further relates to a method for obtaining switchgrass transgenic plants by transformation through the transformation method mediated by Agrobacterium. Background technique [0002] Using transgenic technology to genetically improve energy plants is a new breakthrough in the application of modern genetic engineering technology in the field of agriculture. Switchgrass is an important new bioenergy plant and has become one of the main targets of genetic transformation. Among them, the use of transgenic technology to enhance the stress resistance of switchgrass, improve photosynthetic efficiency, increase biological yield, reduce lignin content and increase ethanol yield has become a research hotspot and shows good application prospects. [0003] The Agrobacterium-mediated gene transformation system has become the preferred method fo...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00
Inventor 奚亚军刘曙东
Owner NORTHWEST A & F UNIV
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