Method for inducing vigna unguiculata transgenic hairy roots and excellent genotype used therein

A hairy root and genetically modified technology, which is applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., to achieve the effects of reducing costs, saving space, and reducing pollution rates

Inactive Publication Date: 2018-09-28
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The present invention aims at the many shortcomings of the existing cowpea hairy root genetic transformation system when the rock wool block is used as the carrier, and the existing system has not screened the transformation efficiency of different genotypes of long cowpea subspecies, etc., through the optimization of the induction culture system and genotype screening, provide a method for efficiently inducing the formation of long cowpea transgenic hairy roots based on seed germination bags, and provide technical support for long cowpea functional genome research

Method used

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  • Method for inducing vigna unguiculata transgenic hairy roots and excellent genotype used therein
  • Method for inducing vigna unguiculata transgenic hairy roots and excellent genotype used therein
  • Method for inducing vigna unguiculata transgenic hairy roots and excellent genotype used therein

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Experimental program
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Effect test

Embodiment 1

[0025] Example 1, the seed germination bag was used as the growth vector to successfully transform the drought-induced expression gene p35S:UP12_8740 in long cowpea with unknown function. The expression level of UP12_8740 gene in roots increased by 7.4 times after drought treatment (Xu et al., 2015);

[0026]1) Long cowpea grains are sterilized and cultivated to obtain the explants of the aerial part;

[0027] Select mature long cowpea seeds that are free from diseases and insect pests, disinfect them with 70% alcohol for 10 minutes, wash them twice with sterile water, and sow them in the matrix (the preparation of the matrix: mix vermiculite and peat in a ratio of 3 to 1, 121 ° C, 20 minutes under high pressure use after sterilization) and poured water, placed in a light incubator for cultivation, the temperature at night is 25°C, the temperature during the day is 28°C, and the photoperiod is 16h / 8h (light / dark). After about one week of growth, before the three compound leav...

Embodiment 2

[0044] Example 2, using the seed germination bag to successfully transform the unique unknown gene p35S:UP12_11094 of long cowpea. After drought treatment, the expression level of this gene in roots was moderately up-regulated (Xu et al., 2015);

[0045] 1) Long cowpea grains are sterilized and cultivated to obtain the explants of the aerial parts;

[0046] Utilize the method in embodiment one to obtain above-ground part explant

[0047] 2) Infect the aerial parts with R1000 Agrobacterium rhizogenes, and use the seed germination bag to obtain combined seedlings;

[0048] Take 10 μL of R1000 Agrobacterium rhizogenes glycerol (the binary expression vector pCAMBIA1301 carrying the UP12_11094 gene) and add it into 1 mL of YEB (50 mg / L Kan) liquid medium, culture it in a constant temperature incubator at 28 °C at a speed of 180 rpm for 12 h, and then Add 200 μL of Agrobacterium into 200 mL of YEB (containing 50 mg / L Kan), shake overnight at 28 ° C on a shaker at 180 rpm, and cult...

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Abstract

The invention discloses a method for inducing vigna unguiculata transgenic hairy roots and an excellent genotype used therein. Various vigna unguiculata genotype materials are selected for hairy rootinducing. The method includes following steps: 1), acquiring explant; 2), infecting; 3), utilizing a seed germination bag as a growth carrier to induce hairy root formation; recording average rootingnumber and conversion rate of each vigna unguiculata genotype material, performing difference significance analysis, and comprehensively comparing to screen out an excellent genotype vigna unguiculatagenotype material for inducing hairy root formation. The method is low in cost and space-saving, pollution rate is lowered, convenience and quickness in experiment operation are improved, and the excellent genotype having high conversion efficiency can be acquired by screening.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a method for inducing long cowpea transgenic hairy roots and an excellent genotype used. Background technique [0002] Cowpea (Vigna unguiculata L.Walp) is an important bean crop in the world. Cowpea is mainly divided into two subspecies: long cowpea (V. unguiculata ssp. Sesquipedalis) and common cowpea (V. unguiculatassp. Unguiculata). Long cowpea is one of the important summer vegetable crops in my country, and is deeply loved by people everywhere; common cowpea is mainly used as grain, and it is planted more in Africa. Cowpea is highly resistant to salt and drought, but the genes that control these traits are unknown. Transgenic technology is an important means to study plant gene function and its mechanism of action, and it is also one of the ways to improve traits. At present, the most widely used transgenic method is mainly Agrobacterium-mediated method, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06A01H6/54
CPCC12N15/8205C12N15/8261
Inventor 徐沛周雯吴新义汪宝根胡耀文李国景吴晓花鲁忠富
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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