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Agrobacterium tumefaciens gene transformation method of hybrid poplar

A technology of gene transformation and Agrobacterium, applied in the field of genetic engineering, can solve the problems of relatively little research on Baiyangpai and its hybrid clones, and the inapplicability of Baiyangpai, achieving the effect of short cycle time and high transformation efficiency

Inactive Publication Date: 2012-08-15
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the genetic transformation of Populus has been gradually deepened, these studies are mostly applied to species such as Populus euphratica, European black poplar, Populus trichocarpa, and Populus microphylla, and there are relatively few studies on poplar and its hybrid clones; and The currently used Agrobacterium-mediated method is not suitable for poplar

Method used

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  • Agrobacterium tumefaciens gene transformation method of hybrid poplar
  • Agrobacterium tumefaciens gene transformation method of hybrid poplar
  • Agrobacterium tumefaciens gene transformation method of hybrid poplar

Examples

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Effect test

Embodiment 1

[0044] An agrobacterium gene transformation method for hybrid poplar, comprising the steps of:

[0045] (1) Pre-cultivation:

[0046] The axillary buds of 717 poplar (P.tremula×P.alba) were subcultured on the basic medium, and cultured for 4 weeks to obtain tissue cultured seedlings; cut off the 0.8-1.5 cm stem section without axillary buds of the tissue cultured seedlings, and cut along the The wounds were drawn twice in the direction of the longitudinal axis of the stem section, placed in M1' medium, and pre-cultured at 24°C for 2 days in the dark;

[0047] (2) Activation and cultivation of bacteria:

[0048] Pick up Agrobacterium C58 / pMP90 / pH7GWIWG2(I) stored at -80°C with a pipette tip onto the YEB solid medium containing rifampicin, gentamicin and spectinomycin, and incubate in the dark at 27°C for 20 hours ;Pick the cultured Agrobacterium to streak on another YEB solid medium containing the same antibiotic as before, and culture it upside down at 27°C for 48h in the da...

Embodiment 2

[0073] An agrobacterium gene transformation method for hybrid poplar, comprising the steps of:

[0074] (1) Pre-cultivation:

[0075] Place the terminal buds of 717 poplar (P.tremula×P.alba) on the basic medium for subculture, and cultivate them for 5 weeks to obtain tissue cultured seedlings; 2 The petioles of the leaves were placed in the M1' medium and pre-cultured for 3 days at 26°C in the dark;

[0076] (2) Activation and cultivation of bacteria:

[0077] Use a pipette tip to pick up Agrobacterium C58 / pMP90 / pH7GWIWG2(I) stored at -80°C on YEB solid medium containing rifampicin, gentamicin and spectinomycin, and culture in the dark at 28°C for 24 hours ;Pick the cultured Agrobacterium and streak it on another YEB solid medium containing the same antibiotic as before, and culture it upside down at 28°C for 40h in the dark;

[0078] Pick a single colony into a sterile test tube containing 3mL of YEB liquid medium containing the same antibiotic as before, and incubate in t...

Embodiment 3

[0102] An agrobacterium gene transformation method for hybrid poplar, comprising the steps of:

[0103] (1) Pre-cultivation:

[0104] The axillary buds of 717 poplar (P.tremula×P.alba) were subcultured on the basic medium, and cultured for 6 weeks to obtain tissue cultured seedlings; the 0.8-1.5 cm stem section without axillary buds of the tissue cultured seedlings was cut, and used The razor blade was drawn 3 times along the longitudinal axis of the stem section to cause wounds, placed in M1' medium, and pre-cultured for 3 days under dark conditions at 25°C;

[0105] (2) Activation and cultivation of bacteria:

[0106] Use a pipette tip to pick Agrobacterium C58 / pMP90 / pH7GWIWG2(I) stored at -80°C on YEB solid medium containing rifampicin, gentamicin and spectinomycin, and incubate in the dark at 29°C for 28 hours ; Pick the cultured Agrobacterium and streak it on another YEB solid medium containing the same antibiotic as before, and culture it in the dark at 29°C for 36 hou...

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Abstract

The invention discloses an agrobacterium tumefaciens gene transformation method of hybrid poplar. The method comprises the following steps of: (1) pre-culture; (2) strain activation and culture; (3) infection; (4) co-culture; (5) delayed selection; (5) adventitious bud induction; (7) extension culture; and (8) root induction and detection. In the method provided by the invention, NAA and 2ip act together to induce the agrobacterium tumefaciens-mediated stem section or leaf stalk to generate callus; under the effect of cytokinin TDZ, the callus redifferentiation is promoted to form adventitious buds; the bud in a vitrified state extends under the induction of BAP (Benzo A Pyrene), and the form gradually becomes normal; and the resistant regenerated poplar roots in a 1 / 2MS (Murashige and Skoog) culture medium containing antibiotic hygromycin. The resistant genetically-modified poplar obtained by the method has the advantages of short period and high transformation efficiency, thereby laying firm foundation and theoretical support for the practical breeding of the genetically-modified poplar.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. Background technique [0002] There are more than 100 species of Populus L. in the world, widely distributed in Eurasia and North America. There are about 62 species in my country, which is the country with the most poplar germplasm resources in the world. Populus plants not only play an important role in soil and water conservation and maintaining ecological balance due to their fast growth, strong adaptability and wide range of uses, but also have a wide range of industrial uses and are important raw materials for pulp and paper, plywood, packaging materials, etc. [0003] For a long time, the improvement of poplar has been mainly carried out through cross breeding, but because its growth and development are affected by many factors such as light, water and temperature, compared with other crops and horticultural plants, it has many unique biological characteristics, such as longer...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 王洁华王旭
Owner TIANJIN UNIV
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