Method for obtaining transgenic cotton through agrobacterium-mediated living immature embryo transformation

An Agrobacterium-mediated, living immature embryo technology, applied in biochemical equipment and methods, horticultural methods, genetic engineering, etc., can solve the problem of low multi-copy integration probability of foreign genes, different cotton regeneration ability, long tissue growth cycle, etc. problems, to achieve the effect of overcoming the long growth cycle, overcoming the limitations of cotton lines, and having a short growth cycle

Inactive Publication Date: 2015-06-24
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the Agrobacterium-infected cotyledon hypocotyl transformation method is not limited by time and season, low probability of multi-copy integration of exogenous genes, and high transformation efficiency. It is favored by many researchers and is currently a commonly used cotton transgenic method. , but this method also has certain limitations, such as: easy to be limited by cotton strains, and the regeneration ability of cotton of different strains is different; the tissue growth cycle is long, and it usually takes 8 to 18 months from hypocotyls to regenerated plants; and There are many deformed seedlings, which are prone to mutations such as cell variation and organ variation; defects such as low seed vigor of regenerated plants

Method used

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  • Method for obtaining transgenic cotton through agrobacterium-mediated living immature embryo transformation
  • Method for obtaining transgenic cotton through agrobacterium-mediated living immature embryo transformation
  • Method for obtaining transgenic cotton through agrobacterium-mediated living immature embryo transformation

Examples

Experimental program
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Effect test

Embodiment 1

[0035] The method for obtaining transgenic cotton by germline transformation of the present embodiment consists of the following steps:

[0036] 1. Sterile embryo stripping

[0037] Mix 10mL of 30% hydrogen peroxide and 90mL of sterile water in a sterilized Erlenmeyer flask to prepare a disinfectant solution. Put the cotton seeds into the disinfectant solution after delinting, soak at 25°C for 24 hours, take out the seeds and put them in a petri dish, cut them The seed coat and 1 / 2 cotyledons are removed, and the remaining cotton embryos are put into a petri dish containing basal medium, and the 1000mL basal medium is composed of the following raw materials:

[0038]

[0039] Mix evenly, adjust the pH to 5.8-6.3, sterilize in a high-pressure steam sterilizer at 0.07 MPa and 115°C for 15 minutes, divide into petri dishes, cool naturally, and prepare a basal medium.

[0040] 2. Preparation of transformation solution

[0041] Add 20 μL of 50 mg / mL kanamycin, 10 μL of 50 mg / m...

Embodiment 2

[0066] The method for obtaining transgenic cotton by germline transformation of the present embodiment consists of the following steps:

[0067] In step 1 of aseptic embryo stripping, mix 10mL of 30% hydrogen peroxide and 90mL of sterile water in a sterilized Erlenmeyer flask to prepare a disinfectant solution, put the cotton seeds into the disinfectant solution, soak at 25°C for 24 hours, The seeds were taken out and placed in a petri dish, the seed coat and 1 / 3 cotyledons were cut off, and the remaining cotton embryos were put into a petri dish containing a basal medium. The raw material ratio and preparation method of the basal medium were the same as in Example 1.

[0068] In step 2 of preparing the transformation solution, add 20 μL of 50 mg / mL kanamycin, 10 μL of 50 mg / mL rifampicin, 20 μL of 50 mg / mL streptomycin, and 1000 μL of Agrobacterium LBA4404 containing the GFP expression vector into 10 mL of LB liquid medium, The GFP expression vector is transformed into Agroba...

Embodiment 3

[0072] The method for obtaining transgenic cotton by germline transformation of the present embodiment consists of the following steps:

[0073] In step 1 of aseptic embryo stripping, mix 10mL of 30% hydrogen peroxide and 90mL of sterile water in a sterilized Erlenmeyer flask to prepare a disinfectant solution, put the cotton seeds into the disinfectant solution, soak at 25°C for 24 hours, The seeds were taken out and placed in a petri dish, the seed coat and 1 / 2 cotyledons were cut off, and the remaining cotton embryos were put into a petri dish containing a basal medium. The raw material ratio and preparation method of the basal medium were the same as in Example 1.

[0074] In step 2 of preparing the transformation solution, add 20 μL of 50 mg / mL kanamycin, 10 μL of 50 mg / mL rifampicin, 20 μL of 50 mg / mL streptomycin, and 1000 μL of Agrobacterium LBA4404 containing the GFP expression vector into 10 mL of LB liquid medium, The GFP expression vector is transformed into Agroba...

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Abstract

The invention discloses a method for obtaining transgenic cotton through agrobacterium-mediated living immature embryo transformation. The method comprises the steps of stripping of a sterile embryo, preparation of a transformation liquid, oscillation infection, preparation of the transgenic cotton and identification of a transgenic plant. In the step of stripping of the sterile embryo, 30 percent hydrogen peroxide and sterile water are mixed to form a disinfection liquid; cotton seeds are detinted and put into the disinfection liquid to be soaked for 24 hours at the temperature of 25 DEG C; the seeds are taken out and put in a culture dish; seed peels and 1 / 3-1 / 2 of cotyledons are cut off; the surplus cotton embryos are put into the culture dish including a basic culture medium to enable the cotton embryos to be damaged; in the step of preparation of the transformation liquid, an inducing liquid is prepared from acetosyringone and magnesium sulfate, the acetosyringone can be used for inducing an agrobacterium Vir gene to be activated so as to improve the integration efficiency of an exogenous gene; and in the step of oscillation infection, Silwet L-77 and MS liquid culture media are added into the transformation liquid to ensure that the agrobacterium covers the cotton embryos in a large range. The method has the advantages of easiness for screening and operation, short growth period of the cotton, high transformation efficiency and the like.

Description

technical field [0001] The invention belongs to the technical field of transgenic cotton, and in particular relates to the establishment of a method for obtaining transgenic cotton by transforming live immature embryos mediated by Agrobacterium. Background technique [0002] Cotton is an important fiber crop, and its fiber production accounts for 40% of the world's total fiber production, and the planting amount of upland cotton reaches 90% of the total cotton. Cottonseed is an oil-containing plant seed second only to soybeans and rapeseeds, so cotton plays an important role in economic crops. [0003] Improving the quality of cotton fiber and increasing the oil yield of cottonseed through transgenic technology are important means for cultivating new cotton varieties. In 1987, transgenic cotton plants were obtained for the first time by Agrobacterium-infected cotyledon hypocotyl transformation method, followed by particle bombardment embryo suspension cell method and pollen...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 俞嘉宁蔡云巧
Owner SHAANXI NORMAL UNIV
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