Agrobacterium-mediated jatropha curcas gene transformation method

An Agrobacterium-mediated and gene transformation technology is applied in the field of obtaining A. oleifera transgenic plants through the Agrobacterium-mediated gene transformation technology, which can solve the problems of chimera generation and gene silencing, and achieve the effect of simplifying the procedure and shortening the transformation period.

Inactive Publication Date: 2011-01-19
XISHUANGBANNA TROPICAL BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some problems in gene gun bombardment. For example, multiple copies of exogenous genes are often randomly integrated into the recipient genom...

Method used

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  • Agrobacterium-mediated jatropha curcas gene transformation method
  • Agrobacterium-mediated jatropha curcas gene transformation method
  • Agrobacterium-mediated jatropha curcas gene transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]Take mature seeds of tung tree, peel off the outer testa, soak in sterile water for 10 hours, then sterilize with 75% alcohol for 30 seconds, rinse with sterile water for 2 to 3 times, and then treat with 1.0% sodium hypochlorite solution for 10 minutes , rinsed with sterile water 3-4 times, placed on sterile filter paper to remove excess water; peeled off the endosperm with sterile tweezers and a scalpel, took out the embryo, and cut off 1 / 3-1 / 4 of the cotyledons at the end of the hypocotyl , and the remaining cotyledons are used as transformation recipients ( figure 1 A).

[0036] Pick the LB4404 Agrobacterium single clone containing the pCAMBIA 2301 plasmid and activate it in the YEP liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin, shake it at 28°C at 200 rpm for 12 hours, transfer and merge Concentration of secondary activated Agrobacterium to OD 600 =0.4, collect the cells by centrifugation at 4000rpm at 4°C for 5 minutes, discard the s...

Embodiment 2

[0043] Repeat Example 1, with the following differences: the time for callus induction culture is 3 weeks, and then subcultured to the selection medium containing kanamycin sulfate for culture.

Embodiment 3

[0045] Repeat Example 1, with the following differences: the callus induction culture time is 2 weeks, and then subcultured to culture on the selection medium containing kanamycin sulfate.

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Abstract

The invention discloses an agrobacterium-mediated jatropha curcas gene transformation method. A material after 1/3 to 1/4 of base is cut from the cotyledon of a mature seed of jatropha curcas is used as an agrobacterium infection receptor. The method comprises the following steps of: after co-culturing, inoculating the transformed receptor and the agrobacterium to a culture medium without a screening agent to perform callus induced culture for 4 weeks; and succeeding the receptor and the agrobacterium to a selective culture medium and screening resistant buds by using kanamycin sulfate as a screening agent, sub-culturing the resistant buds obtained by screening to obtain resistant plants, and obtaining transgenic jatropha curcas plants by molecular detection. Compared with the conventional jatropha curcas transformation method, the method can obtain high regenerative bud induction rate, simplifies the transformation program, shortens the transformation period, greatly improves the transformation efficiency and has good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the transgenic technology of tung tree, in particular to a method for obtaining the transgenic plant of tung tree through the gene transformation technology mediated by Agrobacterium. Background technique [0002] Jatropha curcas L., also known as Jatropha curcas L., is a perennial shrub or small tree of the Euphorbiaceae (Euphorbiaceae) genus Jatropha. It is widely distributed in tropical and subtropical regions. A multi-purpose tree species has good application prospects in biodiesel, pharmaceutical development, biopesticides and fertilizers. The oil content of its seeds is generally 30-40%, and it is currently recognized as one of the energy plants with the most development potential in the world. However, the yield of tung tree planted around the world is generally too low to meet the large demand for raw materials for the development of the biodiesel industry. Due to the long genera...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
CPCC12N15/8205
Inventor 徐增富潘竟丽付乾堂
Owner XISHUANGBANNA TROPICAL BOTANICAL GARDEN CHINESE ACAD OF SCI
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