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Wheat anther callus gene gun genetic transformation method

A genetic transformation method and callus technology, applied in the field of agricultural biology, can solve the problems of poor regeneration characteristics of somatic callus and low genetic transformation efficiency, and achieve the effect of high regeneration frequency and improved efficiency

Inactive Publication Date: 2010-06-23
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In view of the defects or deficiencies in the above-mentioned prior art, the object of the present invention is to provide a method for gene gun transformation of wheat anther callus, which utilizes the high regeneration frequency of wheat anther callus and doubles the regenerated strains to be homozygous two The characteristics of ploidy, through gene gun bombardment, wheat anther callus regeneration and chromosome doubling of regenerated strains, quickly and efficiently carry out wheat genetic transformation, overcoming the poor regeneration characteristics of somatic cell callus in traditional wheat somatic genetic transformation , the defect of low genetic transformation efficiency

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Embodiment 1

[0026] The method for gene gun genetic transformation of wheat anther callus of the present invention comprises the following steps:

[0027] 1) Wheat anther callus induction: select wheat young ears whose wheat pollen cells have developed to the middle to late mononucleate stage, and place them at a low temperature at 2°C for 2 days;

[0028] The young ears of wheat placed at low temperature were routinely sterilized, the anther tissue was stripped and inoculated on the anther callus induction medium, and the wheat anther callus was induced by shading culture at 25°C;

[0029] The wheat anther callus induction medium is based on the general medium W14, and the following substances are added: 2,4-dichlorophenoxyacetic acid (2,4-D): 2.0mg / L, casein: 500mg / L, Biotin: 0.5mg / L, sucrose: 100000mg / L, agar powder: 5000mg / L;

[0030] Adjust the pH value of anther callus induction medium to 5.6, place in an autoclave (pressure: 1.0kg / cm 2 ) to sterilize for 20 minutes.

[0031] The ...

Embodiment 2

[0043] Embodiment 2: The steps of this embodiment are the same as those of Example 1, the difference is that the temperature of the low-temperature storage in step 1) of this embodiment is 4°C, and the young ears of wheat after the low-temperature storage are cultured in shading at 27°C to induce wheat anthers Callus; in step 5), the culture temperature for differentiation and selection is 25°C.

Embodiment 3

[0044] Embodiment 3: The steps of this embodiment are the same as those of Example 1, the difference is that the temperature of the low-temperature storage in step 1) of this embodiment is 2°C, and the young ears of wheat after the low-temperature storage are cultured in shading at 26°C to induce wheat anthers Callus; in step 5), the culture temperature for differentiation and selection is 27°C.

[0045] Two, the experimental research and the result of the method of the present invention

[0046] In 2008, the inventor used the F 1 Substitute material, via F 1 Substitute anther tissues were cultured in vitro to induce callus, and 180 pieces of anther callus with dense texture were selected and transformed by gene gun bombardment with the method of the present invention, and 69 regenerated seedlings were obtained after recovery culture and regeneration, which were detected by RT-PCR , obtained 15 transgene positive plants, and the transformation efficiency reached 8.3%.

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Abstract

The invention discloses a wheat anther callus gene gun genetic transformation method, which comprises the steps of anther callus induction, hyperosmotic cultivation, gene gun bombardment transformation, renewal cultivation, differentiation and screening cultivation, strong seedling and rooting cultivation, chromosome doubling and molecular detection and screening. Compared with the conventional somatic cell callus gene gun transformation, the anther callus has high regeneration frequency after the bombardment of the gene gun, and transgenosis homozygous stable plants can be obtained directly after the chromosome of the regenerated plant is doubled, so the technical shortcomings that the callus has low regeneration frequency and transformant gene heterozygosis and homozygote is selected via selfing two generations in the conventional somatic cell callus gene gun transformation are overcome, the wheat genetic transformation and molecular breeding efficiency is increased, and the transformation efficiency is above 8 percent.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a gene gun genetic transformation method of wheat anther callus. Background technique [0002] Wheat is one of the important food crops. Since Vasil et al. introduced the GUS / Bar gene into wheat to obtain the first transgenic wheat in 1992, the research on transgenic wheat has made great progress. The common methods of wheat transgenic are microprojectile bombardment, pollen Tube channel method (polly tube pathway) and Agrobacterium mediated transformation (Agrobacterium mediated transformation). Since wheat is not the natural host of Agrobacterium and there is no damage response phenomenon in cereal plants, biolistic transformation has always been the main method for genetic transformation of wheat. Although there are many methods for genetic transformation of wheat, the transgenic efficiency of various methods is very low. The important reason is that the ...

Claims

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Application Information

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IPC IPC(8): C12N15/87A01H4/00A01H1/08
Inventor 陈耀锋史勇陈鑫李春莲任慧莉崔志刚权军利白延红
Owner NORTHWEST A & F UNIV
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