Method for in-situ transgenosis of corn mature embryo

A technology of transgenic and mature embryos, which is applied in the field of in situ transgenic corn mature embryos, can solve the problems of low transformation efficiency, low seed setting rate, cumbersome operation, etc., and achieve the effects of high transformation rate, short production cycle and simple operation

Inactive Publication Date: 2011-09-07
TIANJIN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, using mature corn embryos as materials, that is, transgenic corn mature embryos, there are few reports of transgenic development by obtaining embryo tips, bud points, etc.
The former is mostly limited by time, and the operation is cumbersome. It needs to rely on the mature corn regenera

Method used

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  • Method for in-situ transgenosis of corn mature embryo
  • Method for in-situ transgenosis of corn mature embryo
  • Method for in-situ transgenosis of corn mature embryo

Examples

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Embodiment 1

[0041] 1) Obtaining the infusion solution of Agrobacterium tumefaciens EHA105 for the in situ transgenic method of maize

[0042] ① The process of constructing vector pCambia2300-Bar and transforming Agrobacterium tumefaciens EHA105 competent cells is as follows:

[0043] Using the Bar gene as a template, the two ends of the upstream and downstream primers were respectively introduced into the Xho I restriction site for PCR amplification. Then, the PCR product and the pCambia2300 plasmid were digested with Xho I, respectively, and the digestion products of the two were ligated: 16°C for 16 hours. The ligation product was transformed into TOP10 Competent Cell (purchased from Tiangen Biochemical Technology Co., Ltd. Cat#CB104, Lot#J8713), and spread on the LB plate containing kanamycin. PCR was performed using the target gene as a template to obtain a 552bp product, and a 1576bp target band was identified by digestion with BamH I and Sal I. The final sequencing verification res...

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Abstract

The invention relates to a method for in-situ transgenosis of corn mature embryos. The method comprises the following steps: obtaining germs of mature embryos needed for in-situ transgenosis of the corn mature embryos; treating the germs to expose growing points and slightly damaging the growing points; carrying out in-situ transgenosis in the growing points of the corn by utilizing impregnating fluid; coculturing impregnated cells at the growth points and agrobactrium tumefaciens to enable Bar genes to be integrated into corn chromosomes by virtue of T-DNA (triplex-deoxyribonucleic acid) integration of the agrobactrium tumefaciens and rejuvenating the Bar genes; carrying out herbicide screening to initially obtain a herbicide resistant plant for in-situ transgenosis and transplanting the plant; obtaining genetically modified corn seeds by selfing pollination of a PCR (polymerase chain reaction) positive plant of the in-situ genetically modified corn. According to the method provided by the invention, transgenosis is carried out on the growth points of the corn mature embryos and the method of spraying or coating herbicide on corn lamina to screen transgenic plants and carrying out molecular detection to further verify the PCR positive plants is adopted. The method provided by the invention is simple and efficient and can secure a conversion rate of 4%, thus being of great significance for genetic engineering and inheritance breeding of corn.

Description

technical field [0001] The invention relates to a method for transgenicing plants in genetic engineering, in particular to a method for transgenicing mature corn embryos, in particular to a method for in situ transgenic corn mature embryos. Background technique [0002] Transferring exogenous genes or modified endogenous genes into plants to change a certain characteristic of plants is an important way to improve crop quality. Since the world's first transgenic plant, the transgenic tobacco, was first bred in 1983, scientists have tried different transgenic methods, introducing exogenous genes or endogenous genes modified in vitro into plants, improving plant traits, and explored A variety of plant transgenic methods. At present, there are two main types of gene transfer methods in maize: genetic transformation mediated by Agrobacterium tumefaciens and transformation methods of direct DNA introduction, the latter mainly including gene gun method, pollen tube passage method,...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68A01H1/02A01H5/00
Inventor 季静王罡武卫党关春峰
Owner TIANJIN UNIV
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