Method for acquiring disease-resistance expression of agrobacterium-mediated potato transgenic hrap

An Agrobacterium-mediated, potato-based technology, applied in other methods of inserting foreign genetic materials, horticultural methods, genetic engineering, etc., can solve the problems of restricting production and processing capacity, easy loss of resistance, rapid mutation of pathogenic races, etc.

Inactive Publication Date: 2008-02-06
贵州省生物技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, potato is an autotetraploid crop, and it is difficult to directly use it due to reproductive barriers, long breeding cycle, and complicated process to improve varieties by conventional breeding methods.
At the same time, the vertical resistance gene of the wild species is easy to lose resistance due to the rapid variation of the pathogenic race; the horizontal resistance gene alone is not enough to effectively resist the invasion of late blight, and is easily affected by environmental co

Method used

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  • Method for acquiring disease-resistance expression of agrobacterium-mediated potato transgenic hrap
  • Method for acquiring disease-resistance expression of agrobacterium-mediated potato transgenic hrap

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] 1. Transformation and cultivation of explants

[0109] (1) Bacterial solution preparation: Inoculate the Agrobacterium LBA4404 bacterial solution carrying the plasmid pBI hrap into the liquid LB medium supplemented with 100mg / L Kan, 100mg / L Sm and 100mg / L Rif, and culture at a moderate speed at 28°C on a shaker to OD 600 The value is 0.4, the Agrobacterium bacterium liquid is obtained, and it is set aside;

[0110] (2) Pre-cultivation: take the leaves of "Burbank" tissue culture seedlings that have been cultivated for 4 weeks and grow robustly, and cut them into 0.4cm 2 Inoculate the small pieces face down in the y2 medium composed of MS+2.0mg / L 6BA+0.2mg / L NAA+0.2mg / L 2,4D+16g / L sucrose+6g / L agar, at 22 Cultivate in dark for 2 days;

[0111] (3) Diffusion: the leaves of the pre-cultured "Burbank" tissue-cultured seedlings were inoculated into the bacterial liquid of Agrobacterium LBA4404 carrying the plasmid pBI hrap, and the leaves of the tissue-cultured seedlings ...

Embodiment 2

[0118] Embodiment 2: Transformation and cultivation of explants: the Agrobacterium LBA4404 bacterium fluid that will preserve, carrying plasmid pBI hrap is inoculated in the liquid LB culture medium that adds 100mg / L Kan, 100mg / L Sm and 100mg / L Rif, in Cultivate at medium speed at 28°C on a shaker until OD 600 The value is 0.5, and the Agrobacterium bacterial liquid is obtained for use; the leaves of the "Atlantic" tissue culture seedlings that have been cultivated for 3 weeks and grown vigorously are cut off the edge and cut into 0.5cm 2 Inoculate the small pieces face down in the y2 medium composed of MS+2.0mg / L 6BA+0.2mg / L NAA+0.2mg / L 2,4D+16g / L sucrose+6g / L agar, at 22 Cultivate in dark at ℃ for 2 days; inoculate the leaves of the tissue cultured seedlings into the bacterial solution of Agrobacterium LBA4404 carrying the plasmid pBI hrap, and make the leaves of the tissue cultured seedlings fully contact with the bacterial solution of Agrobacterium bacterium for 10 minutes...

Embodiment 3

[0119] Embodiment 3: Transformation and cultivation of explants: the Agrobacterium LBA4404 bacterium liquid that will preserve, carrying plasmid pBI hrap is inoculated in the liquid LB culture medium that adds 100mg / L Kan, 100mg / L Sm and 100mg / L Rif, in Cultivate at medium speed at 28°C on a shaker until OD 600 The value is 0.5, and the Agrobacterium bacterial liquid is obtained, and it is used for later use; the leaves of "Russet Burbank" potato tissue culture seedlings that have been cultivated for 4 weeks and grown robustly are cut off the edges and cut into 0.4cm 2 Inoculate the small piece of the small piece in y2 medium face down, and culture it in dark at 23°C for 2 days; the leaves of the tissue cultured seedlings were inoculated in the bacterial solution of Agrobacterium LBA4404 carrying the plasmid pBI hrap, and the leaves of the tissue cultured seedlings were mixed with Agrobacterium Fully contact with the solution for 20 minutes; take out the leaves of the tissue c...

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Abstract

The present invention discloses a method for attaining potato plates which are disease resistant by utilizing agrobacterium-mediated hrap gene. After the combined inoculation of the method and the bacteria allergic protein harpinPss, the allergic reaction caused by harpinPss can be promoted in the intercellular space of all inoculated plates, and the polymeride of harpinPss is dissociated, so the disease resisting effect is better, and the disease resistance of the potato plates can be promoted. The hrap gene is introduced in the potato plates through the agrobacterium-mediated, thus resulting that the potato plates have the ability to resist the late blight and the bacterial wilt. The present invention has the technical proposal that the pre-cultured explants are inoculated in the agrobacterium germ liquid to be soaked, explants after being soaked select the tube seedling which can root after two times of root selections by co-culturing, callus of induce, bud inducting and rootage cultivating and selecting, and the potted transformed plants are transferred after the mass propagation, the transformed plants select positive plants by the methods of the PCR detection and the Southern hybridization detection, and then the potato plates which are disease resistant can be obtained by the in-vivo inoculation resistance identification. The present invention improves the callus of inducing and the bud inducing of the explants, and enhances the agrobacterium-mediated ability. That the roots of the explants are selected by two times can eliminate the false positive plates in the transformation process , avoid the continuing cultivation to the false positive plates, save the labour force and decrease the manufacturing cost.

Description

technical field [0001] The invention relates to a method for using agrobacterium to mediate the introduction of hrap gene in potato to obtain the expression of disease resistance. Background technique [0002] Potato is the fourth most important food crop in the world, and it is also a vegetable, feed, processing and industrial raw material crop. Now there are 148 countries that grow potatoes in the world, with a total output of 300 million tons. Research by the International Potato Center (CIP) shows that worldwide demand for potatoes will be expected to increase by 20% by 2020, surpassing the growth of rice, wheat, and corn; times. [0003] Our country has unique advantages in producing potatoes in terms of geographical and climatic conditions. The perennial planting area of ​​potatoes in my country is about 4.7 million ha 2 , with a total output of more than 70 million tons. The output and area account for one-fifth and one-fourth of the world's total respectively, r...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N15/33C12N5/04A01H4/00C12Q1/68C12N15/84
Inventor 黄先群林忠平蒋敏华李丽陈坚毛堂芬董颖苹冯腾永
Owner 贵州省生物技术研究所
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