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Method for preparing novel downy-mildew-resistant common head cabbage germplasm through protoplast asymmetric fusion

A technology of protoplasts and cabbage, applied in the field of agricultural biotechnology engineering, can solve the problems of long cycle, difficult resistance gene technology, and sexual hybridization, and achieve the effect of overcoming hybrid incompatibility

Inactive Publication Date: 2011-09-14
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many disease resistances are controlled by multiple genes. Only one gene is isolated and introduced, and the disease resistance obtained by transgenic materials is often unsatisfactory.
Moreover, the analysis, positioning, cloning and transformation of resistance genes have problems such as high technical difficulty, long cycle, high investment, and market access and acceptance.
Another approach is to introduce resistance genes into cultivars through sexual crossing, but reproductive isolation between species makes routine sexual crossing very difficult or impossible

Method used

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  • Method for preparing novel downy-mildew-resistant common head cabbage germplasm through protoplast asymmetric fusion
  • Method for preparing novel downy-mildew-resistant common head cabbage germplasm through protoplast asymmetric fusion
  • Method for preparing novel downy-mildew-resistant common head cabbage germplasm through protoplast asymmetric fusion

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Firstly, it was determined that the susceptible downy mildew of cabbage was caused by cruciferous downy mildew, and the cruciferous downy mildew was collected from Jiangsu and Zhejiang provinces for identification. Simultaneously inoculate the different downy mildews collected on the disease-resistant Brussels sprouts material, and carry out resistance identification, and its specific method is as follows:

[0051] According to the disease symptoms of natural diseased cabbage materials in Yixing, Taizhou, Nanjing, Hangzhou and Haining, Zhejiang, and the fresh diseased leaves were treated and cultivated, a large amount of moldy mold layer was found on the back of the leaves, and the surface was observed under a microscope. Pathogen identification: cyst peduncles are solitary or in clusters, colorless, without septa, the base of the main branch is slightly enlarged, repeated dichotomous branches, 4 to 8 dichotomous acute-angled branches at the top, and small stalks at the ...

Embodiment 2

[0054] The present invention relates to asymmetric fusion of protoplasts. Firstly, the hypocotyl protoplasts of the recipient downy mildew-resistant Brussels sprouts and the mesophyll protoplasts of the downy mildew-resistant Brussels sprouts were obtained, and the isolation and purification of the hypocotyls and mesophyll protoplasts of Brassica plants established in this laboratory Methods to obtain pure protoplasts. Then utilize UV ​​rays to process donor mesophyll protoplasts, the specific steps are as follows:

[0055] 1. Isolation and purification of head cabbage hypocotyl protoplasts: using head cabbage with excellent economic and agronomic traits bred by Jiangsu Academy of Agricultural Sciences as the recipient, the seeds of this variety were surface-sterilized with 70% ethanol for 30 s, and then With 0.1%HgCI 2 Sterilize the solution for 15 minutes, rinse with sterile water for 3 to 5 times, blot dry the filter paper, inoculate on the hormone-free MS basic medium fo...

Embodiment 3

[0059] The fusion of asymmetric protoplasts involved in the present invention adopts polyethylene glycol (PEG) fusion method, and the steps are as follows: adjust the protoplast concentrations of the donor Brussels sprouts and the acceptor Brussels sprouts to 2×10 respectively with the pre-fusion solution. 6 ·mL -1 , mixed uniformly at a ratio of 1:1 to obtain a protoplast suspension. Evenly distribute 7 drops of mixed protoplast solution in a sterile petri dish with a diameter of 6 cm, 40 μL per drop, and let it stand still for 10 min to make the protoplasts settle on the bottom of the petri dish, then add 60 μL containing 40 % polyethylene glycol (PEG1450) fusion solution, stand still for 5 min to promote the fusion of protoplasts, slightly tilt the culture dish, absorb the mixture in the culture dish along the edge of the protoplast suspension, then carefully add 2 mL PEG cleaning solution Ⅰ After 5 minutes of resting, suck it off, then carefully add 2 mL of PEG cleaning s...

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Abstract

The invention discloses a method for creating a novel downy-mildew-resistant common head cabbage germplasm through a protoplast asymmetric fusion technology, solves the problems that a common head cabbage variety which has high resistance against downy mildew of cruciferae and is even immune is substantially not available in production at present and overcomes the defects of incompatible distant hybridization, large difficulty and high investment of a genetic engineering technology and the like. The method for preparing the novel downy-mildew-resistant common head cabbage germplasm through the protoplast asymmetric fusion is characterized in that: a brussels sprout carrying downy-mildew-resistant gene is used as a donor; protoplast of the donor is treated through UV (Ultraviolet) ray; the common head cabbage which is sensitive to the downy mildew and has other superior economical and agronomic characters is used as a receptor; and the novel downy-mildew-resistant common head cabbage germplasm is created through a protoplast asymmetric fusion method. In the method, the downy-mildew-resistant gene of the brussels sprout is transferred into a common head cabbage, so that a system for quickly breeding the novel disease-resistant germplasm is established by using a protoplast fusion technology, and a modern cabbage germplasm resource innovation and disease-resistant breeding high-efficiency technical platform is established.

Description

[0001] technical field [0002] The invention belongs to agricultural biotechnical engineering, and specifically relates to a breeding approach for asymmetric fusion of protoplasts to create new downy mildew-resistant cabbage germplasm. Background technique [0003] Brassica vegetables include head cabbage, cauliflower, broccoli, kohlrabi, kale, Brussels sprouts, kale, etc. It is one of the main vegetable groups loved and widely cultivated by our people. Cabbage downy mildew is a worldwide disease caused by obligate parasitic downy mildew. This fungal disease often causes the leaves of seedlings and adult plants to turn yellow, resulting in reduced yield and quality, which brings great harm to cabbage production. Loss. Moreover, there are basically no head cabbage varieties that are highly resistant or even immune to downy mildew in production at present, so it is imminent to breed head cabbage varieties that are resistant to downy mildew. [0004] Under long-term natural ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/05A01H5/00
Inventor 王神云刘凡王红宗梅李建斌
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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