Genetic transformation method of larch embryonic cells

A genetic transformation method, embryogenic cell technology, applied in sterilization methods, biochemical equipment and methods, methods of supporting/immobilizing microorganisms, etc., can solve the problems of lack of system, slow progress of genetic transformation research, lack of regeneration system, etc. question

Inactive Publication Date: 2017-06-27
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because most conifer species lack good regeneration systems, the research on genetic transformation is slow and lacks systematic

Method used

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  • Genetic transformation method of larch embryonic cells
  • Genetic transformation method of larch embryonic cells
  • Genetic transformation method of larch embryonic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1, Construction of recombinant plasmid pSuper::MIR156 and preparation of recombinant Agrobacterium GV3101 / MIR156

[0081] 1. Construction of recombinant plasmid pSuper::MIR156

[0082] 1. Design primer pairs

[0083] The coding sequence of the miR156 precursor is shown in Sequence 1 of the Sequence Listing, and a primer pair (composed of M156F and M156R) was designed at a position greater than 90 bp at both ends of the stem-loop structure sequence.

[0084] M156F: 5'-CCC AAGCTT TTGTACTCAGCCGACAGAA-3';

[0085] M156R: 5'-GG ACTAGT CCTCTAGCGGTAAATCTCAA-3'.

[0086] 2. Genomic DNA of Japanese larch was extracted as a template, and the composition of M156F and M156R was used for PCR amplification to obtain PCR amplification products.

[0087] 3. Digest the PCR amplification product obtained in step 2 with restriction endonucleases Hind III and Spe I, and recover the digested product.

[0088] 4. Digest the Super1300 vector with restriction endonucleases Hind...

Embodiment 2

[0092] Embodiment 2, genetic transformation of larch

[0093] For the flow chart of genetic transformation, see figure 1 .

[0094] 1. Preparation and pre-cultivation of receptor materials

[0095] 1. Inoculate Japanese larch embryogenic cells into S 0 On the culture medium, cultured statically at 23°C and in the dark for 15 days.

[0096] 2. After completing step 1, pick about 1g of embryogenic cells with vigorous growth on the surface, and add 92ml Y 0 In a 250ml Erlenmeyer flask with culture medium, shake culture at 100rpm under 23°C and dark conditions. Subculture once every 7 days (the method of each subculture is as follows: get about 1g of embryogenic cells, add 92ml Y 0 medium in a 250ml Erlenmeyer flask, 23°C, 100rpm shaking culture under dark conditions). On the 7th day of subculture, 1 mg of acetosyringone was added to each flask, and the timing was started from the addition of acetosyringone. After 12 hours, embryogenic cells were taken as the recipient mater...

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Abstract

The invention discloses a genetic transformation method of larch embryonic cells. The method sequentially comprises the following steps: (a1) taking larch embryonic cells, and suspending in a recombinant agrobacterium infection solution to perform infection; (a2) after completing the step (a1), collecting the larch embryonic cellsa, and carrying out recovery culture; and (a3) after completing the step (a2), taking the larch embryonic cells, and removing bacteria by a vacuum-filtration bacterium remover. The method disclosed by the invention can be used for carrying out rapid oriented genetic improvement on larch by gene engineering means, and provides a technical platform for researching the control on expression of exogenous genes in larch, and functional verification and embryonic development.

Description

technical field [0001] The invention relates to a method for genetic transformation of larch embryogenic cells. Background technique [0002] Larch (Larix Leptolepis) is a fast-growing coniferous tree species used for afforestation in northern my country and subtropical middle and high mountain areas. It is an afforestation tree species that both state-owned forest farms and farmers attach great importance to, and the area of ​​artificial afforestation is expanding. Therefore, carrying out larch breeding research is an important field of conifer improvement in my country. At present, the genetic improvement of larix is ​​mainly carried out in my country through traditional breeding methods such as artificial introduction, breeding of improved varieties, and interspecific hybridization. Although remarkable progress has been made in this field, from a technical point of view, these traditional breeding methods have problems such as large manpower and material resources, being...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12M1/12
CPCC12M29/04C12M29/20C12N15/8205
Inventor 张立峰韩素英齐力旺李水根徐海燕
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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