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A method for preparing transgenic cotton by using cotton cotyledon axillary buds

A transgenic cotton technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of high mutation rate of tissue culture, difficult regeneration of main plant varieties, etc., and achieve high transformation efficiency, The effect of short conversion cycles

Inactive Publication Date: 2015-10-28
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The disadvantage of Agrobacterium tumefaciens-mediated tissue culture cotton genetic transformation is that it relies on cotton tissue culture technology, the tissue culture mutation rate is high, and limited by genotype, most of the varieties (or materials) that can be regenerated now are eliminated in production However, it is not easy to regenerate the main plant species. It is necessary to obtain transgenic regenerated plants by transferring model materials, and then transfer the target gene to an excellent variety by hybridization or backcrossing.

Method used

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  • A method for preparing transgenic cotton by using cotton cotyledon axillary buds
  • A method for preparing transgenic cotton by using cotton cotyledon axillary buds
  • A method for preparing transgenic cotton by using cotton cotyledon axillary buds

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Experimental program
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Effect test

Embodiment 1

[0059] Embodiment 1, the preparation of recombinant agrobacterium

[0060] 1. Preparation of LBA4404 Competent State

[0061] (1) Pick a little Agrobacterium LBA4404 from the inoculation loop, inoculate in 5ml YEP liquid medium (containing 50mg / L rifampicin), and culture overnight at 28°C and 200r / mim.

[0062] (2) Take 2ml of the culture and continue to culture it in YEP liquid medium (containing 50mg / L rifampicin) until the OD600 is about 0.5.

[0063] (3) Place the culture in step (2) in an ice bath for 30 minutes, centrifuge at 5000 r / min at 4°C for 5 minutes, and discard the supernatant.

[0064] (4) Suspend the bacterial liquid with 10ml of cold 0.1mol / L NaCl solution.

[0065](5) Centrifuge the suspended bacterial liquid obtained in step (4) at 4°C and 5000r / min for 5min, and discard the supernatant.

[0066] (6) Suspend the bacterial liquid with 1ml of cold 10mM CaCl2 solution, aliquot into 50μl / tube, freeze in liquid nitrogen and store at -80°C.

[0067] 2. Transf...

Embodiment 2

[0074] Embodiment 2, the preparation of infection liquid

[0075] 1. Bacterial liquid expansion

[0076] Pick a single colony of recombinant Agrobacterium obtained in Example 1 and place it in 50 mL of LB medium containing hygromycin (30 mg / L) and rifampin (25 mg / L), culture at 28° C. with shaking at 300 r / min until OD 600 The value is 2.0-2.5, centrifuge at 8000r / min for 5min at 4°C, and collect the bacteria.

[0077] 2. Preparation of infection solution

[0078] Infection reagent: the reagent is composed of solvent and solute; the solvent is 1 / 2MS medium; the solute is SILWET L-77, sucrose, acetosyringone and KT; the volume percentage of SILWET L-77 in the infection reagent is 0.02%-0.04%, the concentration of sucrose in the infection reagent is 0.4% (% means the percentage of mass volume, g / 100ml), the concentration of acetosyringone in the infection reagent is 50mg / L, and the concentration of KT in the infection reagent The concentration in the reagent is 0.1mg / L.

[0...

Embodiment 3

[0080] Embodiment 3, the preparation of transgenic cotton

[0081] 1. Preparation of cotton seedlings: Select the plump seeds of Baimian No. 1, sow them on seedling trays, germinate at a room temperature of 25°C, and seedlings will emerge in about 3 days.

[0082] 2. Under the condition of 25°C, Baimian No. 1 is about 7-9 days from the time when the two cotyledons unfold to the first true leaf grows. After the two cotyledons unfold and the first true leaf emerges, it is the best transformation period. Cotton The axillary buds of the cotyledons are in a dormant state at this stage and generally do not grow. Only by destroying the apical buds of the stem can the inhibition of the axillary buds be released.

[0083] Gently pull a cotyledon with one hand to make the gap between the two cotyledons slightly larger. Use a scalpel to gently move the young stem tip between the two cotyledons first to destroy the apex bud and ensure the integrity of the cotyledon. Primordia split and g...

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Abstract

The invention discloses a method for preparing genetically modified cotton from an axillary bud of a cotton cotyledon. An infection reagent disclosed by the invention comprises a solvent and a solute, wherein the solvent is a 1 / 2MS culture medium, and the solute includes SILWET L-77, cane sugar, acetosyringone and KT. The method has the following advantages: 1, infection is conducted directly in the special stage of the cotyledon period of a cotton seedling to avoid the problem that the culture and regeneration of a cotton tissue is restricted by genotype, so that all cotton types can be transformed; 2, the transformation period is short, a genetically modified cotton plant can be obtained within only one cotton growth period of about four months; 3, the transformation efficiency is as high as 30%.

Description

technical field [0001] The invention relates to a method for preparing transgenic cotton by using cotton cotyledon axillary buds. Background technique [0002] At present, in the transformation technology of cotton transgenic research, the transgenic method of tissue culture plant regeneration mediated by Agrobacterium tumefaciens, gene gun method, and pollen tube passage method have been established. [0003] Agrobacterium-mediated transgenic method is currently the most researched method with the clearest mechanism and the most mature technology. It is a natural genetic transformation system in nature. After Agrobacterium-mediated exogenous genes enter plant cells, they are mostly integrated on the chromosome in the form of a single copy. The transgenic offspring have good genetic stability and less silencing. Most of the offspring conform to Mendelian inheritance laws, which is convenient for subsequent research. At the same time, the T-DNA of the Ti plasmid can accommod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 胡根海王清连李成奇
Owner HENAN INST OF SCI & TECH
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