Method for obtaining transgenic cotton through transformation of live immature embryos mediated by Agrobacterium

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, and long tissue growth cycle. problems, to achieve the effect of overcoming the long growth cycle, overcoming the limitations of cotton lines, and short growth cycles

Inactive Publication Date: 2018-07-10
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 transformation of live immature embryos mediated by Agrobacterium
  • Method for obtaining transgenic cotton through transformation of live immature embryos mediated by Agrobacterium
  • Method for obtaining transgenic cotton through transformation of live immature embryos mediated by Agrobacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0033] The method for obtaining transgenic cotton by germline transformation of the present embodiment consists of the following steps:

[0034] 1. Sterile embryo stripping

[0035] 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:

[0036]

[0037] 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.

[0038] 2. Preparation of transformation solution

[0039] Add 20 μL of 50 mg / mL kanamycin, 10 μL of 50 mg / m...

Embodiment 2

[0064] The method for obtaining transgenic cotton by germline transformation of the present embodiment consists of the following steps:

[0065] 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.

[0066] 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 Agrob...

Embodiment 3

[0070] The method for obtaining transgenic cotton by germline transformation of the present embodiment consists of the following steps:

[0071] 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.

[0072]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 by transforming live immature embryos mediated by Agrobacterium, which consists of the steps of peeling off aseptic embryos, preparing transformation liquid, shaking and infecting, preparing transgenic cotton, and identifying transgenic plants. In the aseptic embryo stripping, 30% hydrogen peroxide and sterile water are prepared into a disinfectant solution, the cotton seeds are plucked and put into the disinfectant solution, soaked at 25°C for 24 hours, the seeds are taken out and placed in a petri dish, and the seed coat and 1 / 3 to 1 / 2 cotyledons, and the remaining cotton embryos are placed in a petri dish containing basal medium to injure the cotton embryos; in the step of preparing the transformation solution, the induction solution is made of acetosyringone and magnesium sulfate, and acetosyringone Ketones can induce the activation of the Vir gene of Agrobacterium and improve the efficiency of exogenous gene integration; in the shaking infection step, Silwet L‑77 and MS liquid medium were added to the transformation solution to make Agrobacterium cover cotton embryos in a wider range. The invention has the advantages of easy screening, short cotton growth cycle, easy operation, 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H4/00A01H5/00A01H6/60
Inventor 俞嘉宁蔡云巧
Owner SHAANXI NORMAL UNIV
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