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Genetic transformation process of Chinese white poplar

A genetic transformation method and technology of Populus tomentosa, applied in the directions of botanical equipment and methods, introduction of foreign genetic material using vectors, and plant genetic improvement, etc.

Inactive Publication Date: 2011-05-11
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the different plasmids used and various factors such as the screening of antibiotics, this secondary transformation method cannot be directly applied to Populus tomentosa; Secondary genetic transformation method

Method used

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  • Genetic transformation process of Chinese white poplar
  • Genetic transformation process of Chinese white poplar
  • Genetic transformation process of Chinese white poplar

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A kind of genetic transformation method of Populus tomentosa, its specific implementation steps are as follows:

[0040] a. Take the healthy leaves of Populus tomentosa, sterilize them in mercuric dichloride with a concentration of 0.1g / l for 10 minutes, then rinse them with sterile water for 4 times, and then cut the aseptic leaves into 1 square centimeter hairs. poplar leaf disc;

[0041] b. Inoculate the Agrobacterium strain LBA4404 containing the p5-GhGA20ox1 plasmid onto the YEB solid medium plate with kanamycin 50mg / L and streptomycin 125mg / L; after cultivating for 48 hours at 28°C; pick the agrobacterium Bacillus single colony, inoculated in YEB liquid medium containing 50 mg / L kanamycin and 125 mg / L streptomycin, cultured with shaking at 28°C overnight, and obtained the Agrobacterium bacterium liquid with the OD600 value of the bacterial liquid concentration reaching 0.6;

[0042] C, immerse the Populus tomentosa leaf disk gained in step a into the bacterium li...

Embodiment 2～4

[0048] A kind of genetic transformation method of Populus tomentosa, its implementation steps are the same as in Example 1, the difference is that the plant binary expression vector used for one transformation in Example 2 is pCAMBIA, and the plant binary expression vector used for secondary transformation is pCAMBIA1301 The plant binary expression vector used for one transformation in embodiment 3 is pZIP, and the plant binary expression vector used for secondary transformation is pCAMBIA1301; The plant binary expression vector used for transformation is pCAMBIA1301; see Table 1 for specific parameters.

[0049] Table 1

[0050]

[0051]

Embodiment 5

[0053] A kind of genetic transformation method of Populus tomentosa, its specific implementation steps are as follows:

[0054] a. Take the healthy leaves of Populus tomentosa, sterilize them in mercuric dichloride of 0.1g / L for 10 minutes, then wash them with sterile water for 4 times, and then cut the aseptic leaves into 1 square centimeter hairs. poplar leaf disc;

[0055] b. Inoculate the Agrobacterium strain LBA4404 containing the p5-GhGA20ox1 plasmid onto the YEB solid medium plate with kanamycin 50mg / L and streptomycin 125mg / L; after cultivating for 48 hours at 28°C; pick the agrobacterium A single colony of bacillus was inoculated in YEB liquid medium containing 50 mg / L kanamycin and 125 mg / L streptomycin, cultured overnight at 28°C with shaking; the Agrobacterium cultured overnight was transferred at a ratio of 1:50 Add 500μg / L final concentration of acetosyringone liquid YEB medium, secondary activation culture for 3 to 5 hours, to obtain the Agrobacterium bacterium...

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Abstract

The invention provides a method for carrying out genetic transformation of agrobacterium-mediated Chinese white poplar. The method is characterized in that: an agrobacterium-mediated method is adopted to carry out genetic transformation of Chinese white poplar twice so as to introduce two exogenous genes, wherein the primary transformation adopts a plant binary expression vector containing neomycin phosphotransferase gene NptII and beta-glucuronic acid carbohydrase gene, and takes kalamycin as antibiotic; and the secondary transformation adopts a plant binary expression vector containing hygromycin phosphotransferase gene, and takes hygromycin as antibiotic. The method can effectively introduce the two exogenous genes into Chinese white poplar so as to improve plant characters better.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic transformation method of Populus tomentosa. Background technique [0002] Poplar is one of the most important afforestation tree species widely cultivated in the world. Poplar has the characteristics of fast growth and high quality, short rotation period, strong adaptability, easy reproduction, and wide use. Poplar is an important tree species for afforestation and afforestation in many countries in the world and our country, and it is also the main tree species of ecological protection forests. It is also an important industrial raw material for pulping and papermaking, plywood, and packaging materials (Zhang Qiwen et al. 1999). Compared with other forest trees, poplar grows fast. It is easy for asexual reproduction and genetic transformation. Various saturated linkage maps have been constructed, and a large number of molecular markers relate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H1/00A01H5/00
Inventor 邓伟李义李正国吕立堂杨迎武
Owner CHONGQING UNIV
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