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A method of increasing the cotton regeneration and conversion efficiency and applications

A genetic transformation method and cotton technology, applied in the field of improving cotton regeneration and transformation efficiency, can solve the problems of low transformation efficiency, long transformation cycle, genotype aging, etc., and achieve the effects of improving regeneration ability and shortening breeding cycle.

Active Publication Date: 2017-06-06
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the defects of genotype aging, low transformation efficiency and long transformation cycle in the existing genetic transformation process of cotton, and provide a method and application for improving cotton regeneration and transformation efficiency

Method used

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  • A method of increasing the cotton regeneration and conversion efficiency and applications
  • A method of increasing the cotton regeneration and conversion efficiency and applications
  • A method of increasing the cotton regeneration and conversion efficiency and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preliminary evaluation test of cotton regeneration ability under in vitro culture conditions

[0051] In order to verify the technical route of the present invention, four cotton varieties Y668 (Crop Research Institute of Henan Academy of Agricultural Sciences), Kezi Cotton 312 (Cotton Research Group of Huazhong Agricultural University) and YZ-1 (Cotton Research Institute of Chinese Academy of Agricultural Sciences) Comparing regeneration ability (control) with Simian No. 3 (Cotton Research Group of Huazhong Agricultural University) (these four materials are conventional cotton strain materials, the public can buy or obtain from relevant units), cotton somatic embryogenesis The technical process of process and regeneration capacity comparison is as follows: figure 1 shown.

[0052] The steps are as follows:

[0053] (1) Cultivation of aseptic seedlings: peel off the seed coats of cotton seeds after delinting with sulfuric acid and soak them in 0.1% mercuric ...

Embodiment 2

[0060] Example 2: Using Y668 as the transformation recipient, Agrobacterium-mediated GFP genetic transformation

[0061] Agrobacterium strain EHA105 and plasmid vector pBIN m-gfp5-ER (see Jin et al., 2012) are used for genetic transformation of cotton, and the T-DNA structure of the plasmid is shown in figure 2 . The procedure for transforming cotton hypocotyls with Agrobacterium is as follows: pick plump and healthy cotton seeds and peel off their seed coats, soak them in 0.1% HgCl2 for 10 minutes, and wash them three times with sterile water. Inoculate the obtained sterilized cotton hypocotyls on the germination medium of sterile seedlings, cultivate them in a 28°C incubator for 4-6 days under dark conditions, and cut the hypocotyls of sterile seedlings into 0.5-0.8cm segments , inoculated in 0.5OD Agrobacterium suspension suspension with MGL, after infecting 10mim, blot the bacterial solution on the hypocotyl surface with sterile filter paper, and inoculate the hypocotyl ...

Embodiment 3

[0062] Example 3 The breeding process of Jin 668 and the comparison of transformation and regeneration efficiency of different generations of Jin 668

[0063] Seeds harvested from selfing of Null plants were used as targets for domestication of regenerative capacity. Pick plump and healthy cotton seeds and peel off their seed coats, soak them in 0.1% HgCl2 for 10 minutes, and wash them with sterile water for 3 times. Inoculate on the germination medium of sterile seedlings, culture in a 28°C incubator for 4-6 days under dark conditions, take the hypocotyls of sterile seedlings and cut them into 0.5-0.8cm sections and inoculate them on callus induction medium (MSB +2,4-D0.1mg / L+KT 0.1mg / L+3% Glucose+0.25% Phytagel, pH5.9), subculture once every 1 month until embryogenic callus (or somatic cell embryo). Then the embryogenic callus or somatic embryos were transferred to differentiation medium until regenerated seedlings were obtained. The obtained seedlings were transferred to...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method of increasing the cotton regeneration and conversion efficiency and applications. A cotton transgenic receptor material Y668 having excellent agronomic traits is obtained through regeneration capacity comparing and screening, a female single plant Null in a segregation population T1 generation obtained by transferring a GFP gene through adopting the Y668 as a receptor is adopted as a tissue culture receptor material, an R2 population is obtained by somatic cell regeneration for successive two times, and finally a conversion receptor material Jin 668 the regeneration capacity of which is 3 times improved than regeneration capacity of a parent is obtained by typical single plant selection and selfing. The invention also discloses applications of the Jin 668 in cotton transgenosis. The cultivated Jin 668 is adopted as a transgenic receptor material and a plurality of insect-proof and oil component increasing genes are introduced into a cotton host cell to obtain a transgenic cotton plant expressing exogenous genes.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method and application for improving cotton regeneration and transformation efficiency. Background technique [0002] A successful genetic transformation system of cotton depends on efficient and stable transformation and plant regeneration process. With the widespread application of Agrobacterium-mediated genetic transformation and biolistic transformation, the transformation technology of cotton has become increasingly mature, and transgenic insect-resistant cotton has also achieved great success, but the regeneration frequency of transformed cells and the genotype of recipient cotton are limited. remains the most important limiting factor for cotton transgenics. Previous studies have shown that cotton tissue culture plant regeneration mainly through two pathways: one is the organogenesis pathway, and the other is the somatic embryogenesis pathw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H1/02A01H4/00A01H5/00
Inventor 金双侠涂礼莉张献龙
Owner HUAZHONG AGRI UNIV
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