Rape transgenic method

A technology of transgenic and rapeseed, applied in the field of bioengineering, can solve the problems of dependence on tissue culture, low transformation efficiency, and low cost

Inactive Publication Date: 2006-08-23
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0025] The purpose of the present invention is to provide a method for genetic transformation of rapeseed, which solves the problems of low transformation efficiency, small flux, and dependence on tissue culture and genotype in the currently commonly used rapeseed genetic transformation method. The method not only has high transformation efficiency, low cost, There is no genotype dependence, and the tissue culture work during the transformation operation is avoided, and the problems of vernalization and oversummering in the propagation of transgenic plants are avoided

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] Example 1: Observation on the process of pollen tube elongation after pollination of Brassica napus variety "Zhongshuang Nine"

[0164] Brassica napus variety "Zhongshuang No. 9" (cultivated by the Institute of Oil Crops, Chinese Academy of Agricultural Sciences, also known as "Almighty 628"). This variety is a double-low (low erucic acid and low glucosinolate) high-quality rapeseed variety, resistant to viral diseases and sclerotinia, and is one of the main cultivated varieties in the Yangtze River Basin. Sow "Zhongshuang No. 9" in autumn (September 26-September 30), with a box width of 2 meters and a row spacing of 0.3 meters. According to normal cultivation management and fertilizer and water measures, it will bloom in the spring of the next year. After flowering, the ovary of "Zhongshuangjiu" after self-pollination was sampled every 1 hour to determine the microscopic observation of pollen tube elongation, so as to determine the best time for plasmid introduction. ...

Embodiment 2

[0186] Example 2: Extraction preparation and enzyme digestion identification of exogenous DNA plasmids

[0187] The exogenous DNA to be transformed is the plasmid pGreen0229 plant expression vector carrying the herbicide glufosinate resistance (bar gene) (the Escherichia coli strain DH5α carrying the plasmid pGreen0229 was donated by Liu Yuhui, Institute of Biotechnology, Chinese Academy of Agricultural Sciences). For the nucleotide sequence and restriction endonuclease map of plasmid pGreen0229, see the attached figure 1 and http: / / www.pgreen.ac.uk / jit / pG0229.htm .

[0188] (1), the extraction steps of exogenous DNA plasmid are as follows:

[0189] 1. Take 50 microliters of Escherichia coli DH5α strains with plasmid pGreen0229, insert them into a Erlenmeyer flask containing 500 milliliters of LB medium, and cultivate them on a constant temperature shaker at 37°C at a speed of 200 rpm. After 16 hours of incubation, the medium became cloudy.

[0190] 2. Extraction of plas...

Embodiment 3

[0268] Example 3: Transformation and seed setting rate analysis of Brassica napus variety "Zhongshuang Nine"

[0269] Sow "Zhongshuang No. 9" in autumn (September 26-September 30), with a box width of 2 meters and a row spacing of 0.3 meters. According to normal cultivation management and fertilizer and water measures, it will bloom in the spring of the next year.

[0270] In the blooming stage, choose sunny weather, choose unopened flower buds, leave 10 to 15 inflorescences, and manually emasculate before transformation. Knock off the small flower buds that are not emasculated at the top of the inflorescence, remove the siliques and flowers that have opened, and put a sulfuric acid paper bag of appropriate size. At the same time, another inflorescence is selected, the flowers that have been opened are knocked out, bagged, and pollen is prepared to provide newly opened flowers and stamens for pollination the next day. Hang a tag on the inflorescence shaft below the infloresc...

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Abstract

The rape transgenic method of the present invention adopts the in-situ reproduction organ as transgenic receptor. On the basis of cytological observation of pollinated rape style and ovary, the transforming time is determined. The transformation includes cutting off the style head, dropping exogenous DNA solution, transforming the reproductive cell with the gene carrying exogenous gene and glufosinate resistance marker gene to obtain transgenic T1 generation zygocyte in the transformation period; and screening the T1 generation seedling with low concentration glufosinate solution to obtain transgenic positive rape plant. The present invention has transgenic efficiency of 2-10 %, high transformation flux, low cost, less work, no genotype dependence and other advantages.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for transgenic rapeseed, which is applicable to all rapeseed varieties, strains or germplasm resource materials. Background technique [0002] Plant gene transformation technology transfers the target gene (exogenous gene) isolated from animals, microorganisms or plants to the genome of the recipient plant through various transformation systems, so that the exogenous gene can be transferred to the genome of the recipient plant. Stable inheritance in somatic plants, and endow plants with new agronomic traits, such as insect resistance, disease resistance, stress resistance, high yield, high quality, etc. [0003] Rapeseed (seed rape) is an important oil source plant and feed protein source, and is the main oil crop in my country. Variety improvement and genetics research by means of bioengineering and transgene are important contents of rapeseed gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/04A01H1/04A01G7/00
Inventor 刘胜毅郭学兰董彩华刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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