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Non tissue culture gene transferring method by using half of peanut seed as acceptor

A technology of tissue culture and gene transformation, applied in biochemical equipment and methods, genetic engineering, plant gene improvement, etc., can solve the problems of long regeneration cycle, unstable transformation effect, low transformation and regeneration rate, and achieve simple and easy operation Good effect, stable transformation effect, high transformation efficiency and survival rate

Inactive Publication Date: 2011-09-21
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a non-tissue culture gene transformation method mediated by Agrobacterium using peanut half-slice mature seeds as the recipient, so as to solve the problem of regeneration in the existing peanut gene transformation method. Long period, low transformation and regeneration rate, unstable transformation effect and other problems, the method of the present invention has high throughput, good transformation effect, no genotype dependence, simple operation and low cost

Method used

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  • Non tissue culture gene transferring method by using half of peanut seed as acceptor
  • Non tissue culture gene transferring method by using half of peanut seed as acceptor
  • Non tissue culture gene transferring method by using half of peanut seed as acceptor

Examples

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Effect test

Embodiment 1

[0042] Embodiment 1: The genetic transformation method mediated by the Agrobacterium-mediated explant is used for the transformation of the herbicide-resistant (bar) gene with the peanut half slice mature seed

[0043] (1) Select mature and plump peanuts (Yueyou No. 7, Zhuzhuhong No. 1), shell the peanut seeds, rinse them with distilled water three times, and then soak them in distilled water for 30 minutes. Soak and sterilize with 75% (V / V) ethanol on the ultra-clean workbench for 30 seconds, rinse with sterile water 1-2 times, and place in 0.1% (W / V) HgCl 2 Soak in the solution for disinfection for 10 minutes, rinse with sterile water 4-5 times, blot the water on the surface with sterile filter paper, remove the seed coat, cut the seeds along the gap between the cotyledons, take half a seed containing the embryo, and then cut the seed along the cotyledon of the embryo. Node cut germ (eg figure 1 c. figure 2 shown), to obtain the explants to be transformed.

[0044] (2) tak...

Embodiment 2

[0056] Embodiment 2: The genetic transformation method mediated by the Agrobacterium mediated by peanut half slice mature seeds as explants is used for the transformation of soybean GmFAD3C gene

[0057] (1) Select mature and plump peanuts (Yueyou No. 7, Yueyou No. 13) seeds, shell the peanut seeds, wash them with distilled water three times, and then soak them in distilled water for 60 minutes. Soak and sterilize with 75% (V / V) ethanol on the ultra-clean workbench for 20 seconds, rinse with sterile water 1-2 times, and place in 0.1% (W / V) HgCl 2 Soak in the solution for disinfection for 8 minutes, rinse with sterile water 4-5 times, blot the water on the surface with sterile filter paper, remove the seed coat, cut the seeds along the gap between the cotyledon, take half a seed containing the embryo, and then cut the seed along the cotyledon of the embryo Node cut germ (eg figure 1 c. figure 2 shown), to obtain the explants to be transformed.

[0058] (2) Connect the NcoI ...

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Abstract

The invention discloses a non tissue culture gene transferring method by using half of a peanut seed as an acceptor, the method comprises: (1) shelling the peanut seed, flushing and soaking by distilled water; then disinfecting, shelling, cutting the seed, taking half of a seed containing embryo, cutting embryo along cotyledon nodes of the embryo to obtain an explant; (2) inoculating Agrobacterium to a YEP liquid nutrient solution and cultivating overnight; centrifuging to collect Agrobacterium thalline, hanging in a liquid MS medium containing acetosyringone to obtain an invasion, (3) immerging the explant into the invasion; taking out of the explant and laying in a co-culturing medium, culturing for 2 to 3 days in the dark; culturing for 7 to 10 days with light irradiation to obtain a co-culturing explant, (4) removing the co-cultured explant to disinfection sandy soil, culturing and acclimatizing, moving into a greenhouse for breeding. The method of the present invention has the advantages of short experimental period, high conversion efficiency and survival rate. The conversing effect is stable among the different genotype peanuts; the strict tissue culture technique required by a traditional co-culturing method can be avoided.

Description

technical field [0001] The invention belongs to the technical field of plant transgenics, and specifically relates to a gene transformation method, in particular to an Agrobacterium-mediated non-tissue culture gene transformation method using half-slice mature seeds of peanuts as receptors. Background technique [0002] Peanut (Arachis hypogaea L.) is an important oil crop and edible crop, and it is one of the main oil crops and economic crops in my country. Peanuts are rich in nutritional value. In my country, peanut production accounts for about 1 / 2 of the total output of oil crops. [0003] Peanut is a dicotyledonous plant and is the natural host of Agrobacterium, but it exhibits the same weakness as most leguminous crops—it is difficult to introduce foreign genes, which limits the transgenic breeding of peanuts and progresses slowly. [0004] Foreign countries began to study peanut tissue culture in the 1940s, and my country began to study it in the 1970s. At present, ...

Claims

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Application Information

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IPC IPC(8): C12N15/84
Inventor 温世杰刘海燕梁炫强
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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