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Agrobacterium-mediated soybean transgenic method

An Agrobacterium-mediated, transgenic technology, applied in the field of agricultural biology, can solve the problems of low transformation rate, low success rate, long cycle, etc., and achieve the effects of short transformation cycle, low cost and high seed setting rate

Inactive Publication Date: 2013-05-15
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

The currently widely used Agrobacterium-mediated soybean transgenic system includes cotyledonary node method, hypocotyl method, embryo tip method and other transformation methods, which have a long cycle. Generally, it takes 6-8 months to harvest seeds. The success rate of rooting and transplanting processes Low, and the conversion rate is less than 10%

Method used

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  • Agrobacterium-mediated soybean transgenic method
  • Agrobacterium-mediated soybean transgenic method
  • Agrobacterium-mediated soybean transgenic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Receptor material preparation: Pick 150 plump seeds of Dongnong 50, put them in a 12cm petri dish, spread double gauze on the upper and lower sides, add distilled water (about 100ml) just to cover the seeds, and germinate them in the dark at 25°C in an incubator. After 3 days, the elongated seedlings were transferred to the hydroponic box with MS1 culture solution ( figure 1 ), cultivated under light at 25°C, after 2 days (before the cotyledons unfold), carefully remove the terminal buds and lateral buds with a blade, lightly cut 3-5 wounds, gently close the cotyledons, and place the lower 1 / 2 of the cotyledons to the hypocotyl Wrap it with parafilm as a receptor.

[0048] Agrobacterium preparation: the plant expression vector pCAMBIA3301-GmFB (generated by replacing the GUS (β-glucuronidase, β-glucuronidase) in the vector pCAMBIA3301 with the GmFB gene) was transformed into Agrobacterium EHA105, EHA101 and LBA4404 by the freeze-thaw method to obtain engineering Bacter...

Embodiment 2

[0068] Analysis of transgenic efficiency of 16 different soybean genotypes (these soybean products can be purchased from the market).

[0069] Soybean transgenic receptors: Hefeng 35, Heihe 38, Dongnong 8004, Heinong 51, Hefeng 45, Dongnong 44, Suinong 28, Heinong 48, Dongnong 50, Heihe 48, Heinong 44, Kenfeng 16 , Hefeng 55, Dongnong 48, Dongnong 53, and Dongnong 47 were jointly provided by the Soybean Research Institute of Northeast Agricultural University, Heilongjiang Academy of Agricultural Sciences, and Heilongjiang Academy of Agricultural Sciences. Pick 50 plump seeds of each variety, put them in a 12cm petri dish, spread double layers of gauze on the upper and lower sides, add distilled water (about 100ml) just to cover the seeds, and germinate them in the dark at 25°C in an incubator, and take 3-day elongated seedlings Transfer to MS1 hydroponic box, cultivate under light at 25°C, and after 2 days (before the cotyledons unfold), carefully remove the terminal buds and ...

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Abstract

The invention discloses an Agrobacterium-mediated soybean transgenic method, and belongs to the technical field of agricultural organisms. The method comprises the following steps of: under liquid culture conditions, performing Agrobacterium infection on germinated soybean seeds by utilizing Agrobacterium, co-culturing, performing bud induction, screening, removing negative branches by utilizing a screening substance to obtain TO generation positive branches, and continuing culturing to harvest seeds. By the method, the transformation period is shortened, and only 3 to 4 months are required to obtain transgenic current generation seeds; moreover, the transformation efficiency is improved, and the T0 generation transformation efficiency is up to 15-34 percent. Meanwhile, the method is applicable to different soybean variety sources or genotypes, and the transformation efficiencies are all high.

Description

technical field [0001] The invention relates to a soybean transgenic method mediated by Agrobacterium, belonging to the field of agricultural biotechnology. Background technique [0002] Plant genetic engineering is an important way of soybean genetic improvement. In recent years, the number of transgenic plants has increased day by day, and the transformation methods have also been developed rapidly. Soybean transgenic methods mainly include Agrobacterium-mediated method, particle gun method, electric shock method, PEG (polyethylene glycol, polyethylene glycol) method, ultrasonic-assisted Agrobacterium transformation method and pollen tube passage method, etc. Soybean is recognized as a plant that is difficult to transform. The main difficulties of transgenics are low transformation rate, poor reproducibility, and genotype limitations. At present, transgenic plants are obtained mainly by a large number of transformed recipients. The currently widely used Agrobacterium-med...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 李文滨李永光赵琳
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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