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Agrobacterium tumefaciens-mediated genetic transformation method for Jatropha curcas

A genetic transformation method and Agrobacterium-mediated technology, which can be used in the fields of Agrobacterium-mediated genetic transformation of Jatropha curcas and Agrobacterium-mediated genetic transformation, which can solve the problems of low transformation rate and long time.

Active Publication Date: 2014-10-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of agricultural method applied to Jatropha curcas cotyledon explant transformation acceptor system for existing Agrobacterium-mediated Jatropha curcas cotyledon explant genetic transformation method. Bacillus-mediated genetic transformation method

Method used

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  • Agrobacterium tumefaciens-mediated genetic transformation method for Jatropha curcas
  • Agrobacterium tumefaciens-mediated genetic transformation method for Jatropha curcas
  • Agrobacterium tumefaciens-mediated genetic transformation method for Jatropha curcas

Examples

Experimental program
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Effect test

Embodiment 1

[0025] S1. Preparation of Agrobacterium liquid for infecting and treating Jatropha curcas cotyledon explants

[0026] Use a sterilized inoculation loop to dip a little bacterial solution from the small tube containing the Agrobacterium strain (containing the recombinant plasmid of the plant binary expression vector), and after streaking on the solid YEP medium, place the petri dish at 28 Cultivate in a biochemical constant temperature incubator for 3 days. After a single colony grows on the medium, pick a single colony with an inoculation needle and inoculate it into a 50 mL centrifuge tube (sterilized) containing 20 mL of liquid YEP medium. After culturing on a shaker (28°C, 200 rpm) for 24 hours, centrifuge at 4000 rpm for 10 min, retain the precipitate and pour off the supernatant, then add 20 mL of liquid co-culture medium to the large centrifuge tube, suspend the precipitate, and place on a constant temperature shaker ( 28°C, 200 rpm) for 1 hour, adding liquid co-cu...

Embodiment 2

[0067] Example 2 Effects of pre-cultivation and co-cultivation time on the regeneration efficiency of induced cotyledon explant resistant shoots

[0068] In this study, the aseptic seedlings of Jatropha curcas seeds with a seedling age of 5 days were cut to obtain cotyledon explants, which were treated with a short-term high-concentration TDZ solution (same as in Example 1); After removing excess liquid from the explants, the explants were inoculated onto the pre-medium for 0, 1, 2 and 4 days of pre-cultivation; using OD 600 =0.1~0.2 Agrobacterium bacteria solution soaked and infected the explants for 30 seconds, blotted the excess bacteria solution on the explants on sterile absorbent paper, and then inoculated it on the surface of the medium and placed a sterile On the filter paper co-culture medium, after 0, 1, 2, 3 and 6 days of co-cultivation, the explants were inoculated on the selection and recovery medium and cultured for 30 days. The experimental results are s...

Embodiment 3

[0073] Example 3 Optimization of the PCR Detection System for the Obtained Resistant Bud Strips

[0074] In this study, the aseptic seedlings of Jatropha curcas seeds with a seedling age of 5 days were cut to obtain cotyledon explants, which were treated with short-term TDZ solution (same as in Example 1); After the excess liquid on the explants, inoculate the explants on the pre-medium for 1 day pre-culture; use OD 600 =0.1~0.2 of the Agrobacterium bacteria solution to soak and infect the explants for 30 seconds, then absorb the excess bacteria solution on the explants on sterile absorbent paper, without the step of cleaning the explants with antibiotic solution, Then inoculated on the co-culture medium with sterile filter paper placed on the surface of the medium, after 2 days of dark culture, the explants were inoculated on the selection and recovery medium and cultured for 30 days, and then the explants with resistant buds were inoculated The plant was inoculated to t...

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Abstract

The invention belongs to the field of plant genetic engineering technology and specifically discloses an Agrobacterium tumefaciens-mediated genetic transformation method applied to cotyledon explant transformation receptor system of Jatropha curcas. According to the invention, high-concentration TDZ (thidiazuron) is used for short-term soaking of cotyledon explant, then low-concentration Agrobacterium tumefaciens bacterial liquid is used for short-term invasion of the cotyledon explant, so the genetic transformation rate of Agrobacterium tumefaciens can be substantially improved; the invaded explant is subjected to regeneration culture, resistant buds directly undergo PCR identification, and then micro-grafting can be carried out so as to obtain a transgenic plant. The method provided by the invention greatly improves the genetic transformation rate and substantially shortens a period for obtainment of a completely transformed plant.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a method for genetic transformation of Jatropha curcas mediated by Agrobacterium curcas, and more specifically to an Agrobacterium-mediated genetic transformation method applied to a Jatropha curcas cotyledon explant transformation receptor system conversion method. Background technique [0002] Plant genetic transformation is an important and advanced breeding technology. Due to the high oil content (40-60%) in its seeds, Jatropha curcas can be developed into "biodiesel", thus becoming a very promising energy plant, but its distribution area is narrow and the seed setting rate is low , the quality of seed oil also needs to be further improved. In order to expand the planting area of ​​Jatropha curcas, new varieties of Jatropha curcas with high yield and high quality have been bred, and genetic transformation technology can be used to achieve these goals. At ...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 刘振兰刘颖杨跃生郑少燕庄楚雄李静陈晓阳
Owner SOUTH CHINA AGRI UNIV
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