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Transgenic method of agrobacterium-mediated rape field

An Agrobacterium-mediated, Agrobacterium-transformed technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of affecting transformation efficiency, low transformation efficiency, heavy flower damage, etc., to improve genetic transformation. efficiency, improve transformation efficiency, and maintain the effect of transformation vitality

Inactive Publication Date: 2010-06-23
HUNAN UNIV
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  • Summary
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  • Description
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Problems solved by technology

[0024] (3) The time for soaking the inflorescences with the transformed bacteria solution is 1 minute. If the time is too long, the damage to the flowers will be serious.
[0025] (4) Too many times of dipping the same inflorescence, and the interval time is short, which will seriously damage the flower
[0026] (5) Remove the paper bag on the inflorescence at the end of the flowering stage. It may be that the bagging time is too long, so that the fruit pods will not grow well due to lack of light, which will affect the quality of the seeds.
[0046] (1) The culture of Agrobacterium is liquid culture, which needs to be centrifuged and resuspended, which is troublesome to operate
[0047] (2) The transformation solution for configuring the Agrobacterium transformation solution is MS basic medium + 6BAP (0.01ng / L) + 5% sucrose + 0.05% Silwet-77; pH5.8, no addition to promote the entry of foreign genes into the plant genome substances, thus affecting the conversion efficiency to a certain extent
[0048] (3) The time for soaking the inflorescences in the bacterial solution is not clear, and the length of soaking time will also affect the transformation efficiency
[0049] (4) The interval between dipping the same inflorescence is too short, which is harmful to the flower
[0050] (5) Remove the paper bag 24 hours after the last dipping treatment. The bagging time is too short, which is not conducive to maintaining the transformation activity of Agrobacterium
[0051] In conclusion, due to the disadvantages mentioned above in this method, the conversion efficiency is low, only 2.03%

Method used

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  • Transgenic method of agrobacterium-mediated rape field
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  • Transgenic method of agrobacterium-mediated rape field

Examples

Experimental program
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Effect test

Embodiment 1

[0101] (1) Rapeseed planting

[0102] Rapeseed variety 1244W6 is mid-winter Brassica napus (B. napus, genome AACC, 2n=38). Its main features are: strong branching, luxuriant branches and leaves, well-developed fine roots; cold resistance, moisture resistance, fertilizer resistance, strong resistance to downy mildew, sclerotinia and viral diseases; large petals, yellow flowers, long siliques, There are many pods and the seeds are full; the oil content of the seeds is relatively high, generally 35% to 50%. The growth period is about 227 days, and the yield is about 340-350 catties / mu.

[0103] Rapeseed seeds are sown in seedbeds on September 15th, and the soil in the seedbeds should be fertile and moist. After about a month, the seedlings grow 3-4 true leaves, and at this time, the seedlings are transplanted to the field. The box width of Datian is 2 meters, the row spacing is 0.4 meters, the plant spacing is 0.3 meters, the distance between boxes is 0.3 meters, and the trenc...

Embodiment 2

[0126] (1) Rapeseed planting

[0127] Rapeseed variety N529 is mid-winter Brassica napus (B. napus, genome AACC, 2n=38).

[0128] Rapeseed seeds are sown in seedbeds on September 15th, and the soil in the seedbeds should be fertile and moist. After about a month, the seedlings grow 3-4 true leaves, and at this time, the seedlings are transplanted to the field. The box width of Datian is 2 meters, the row spacing is 0.4 meters, the plant spacing is 0.3 meters, the distance between boxes is 0.3 meters, and the trench depth is 0.2 meters. Plant 5,000 rapeseed plants, covering an area of ​​about 200 square meters. Rapeseed fertilizer and water management is carried out according to conventional methods, and the next spring (early March) blooms. At this point, choose the best time to transform Agrobacterium canola.

[0129] (2) Agrobacterium culture

[0130] See the T-DNA map of the pEGAD plasmid figure 2 . The T-DNA sequence contains the target gene, the herbicide resistan...

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Abstract

The invention discloses a transgenic method of an agrobacterium-mediated rape field. The method comprises the following steps: eluting an agrobacterium on a YEB solid plate with 1 / 2 MS fluid nutrient media which comprises 5% of sucrose, 0.05-0.1% of Silwet-77, 0.2-0.4% of acetosyringone and 0.0001-0.0002% of 6-BAP by concentration; removing blooming flowers from a main inflorescence and each branched inflorescence and only leaving buds when the buds of a principal branch and part of lateral branches of the rape and a plurality of flowers bloom before the conversion; bundling the inflorescences immediately after soaking in eluted conversion bacterium liquid for 30 seconds; continuously dip-dyeing one inflorescence for 3 times with an interval of 2-3 days; and removing a parchment bag on the inflorescence and the flowers and buds at the top end of the inflorescence. The method has the advantages of simple operation, low cost, high transgenic efficiency and no genotype dependence, thereby having excellent potential application in the aspects of culturing rapes of new species by utilizing the gene engineering means and establishing rape mutant libraries.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and relates to a field transgenic method of rapeseed mediated by Agrobacterium. Background technique [0002] Over the years, many breeders have tried to use traditional breeding methods to obtain rapeseed varieties, but the progress has been very slow. With the development of genetic engineering technology, people are more and more inclined to improve the yield, quality and stress resistance of rapeseed through genetic engineering. The transgenic technologies currently applied to rape mainly include: Agrobacterium-mediated transformation methods, gene gun methods, chemical methods (such as using polyethylene glycol) and physical methods (such as using electroporation). Among them, the Agrobacterium-mediated transformation method is widely used because of its simplicity and effectiveness. Using this method, a transformation system based on tissue culture has been established in rapeseed,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84
Inventor 刘选明赵小英林建中唐冬英廖红东林辰涛
Owner HUNAN UNIV
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