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Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation

A technology of Agrobacterium rhizogenes and Agrobacterium, which is applied in the field of plant genetic engineering to achieve the effect of improving transformation frequency and growth state and increasing the number

Inactive Publication Date: 2010-05-12
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

[0005] The smear method transformation of Agrobacterium rhizogenes can be used for the research of symbiotic nitrogen fixation, mycorrhizal symbiosis and root physiology and biochemistry in soybean roots through the infection of Agrobacterium rhizogenes under sterile tissue culture conditions. Up to now there is no report

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  • Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation
  • Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation

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Embodiment 1

[0033] The method of the present invention mainly includes steps such as seed germination, Agrobacterium rhizogenes MSU440 bacterial plaque preparation, explant preparation and transgenic production of transgenic hairy roots. The methods reported by Dernier et al. using kanamycin as a selection agent for transformation of alfalfa truncation and Uhde-Stone et al. using kanamycin as a selection agent for transformation of white lupine. So far, there has been no report on the use of this transformation method for soybeans. The present invention has been summarized through a large number of long-term experiments, and has optimized the conditions of seed germination time, concentration of screening agents, and recipient soybean genotypes, and has successfully used this method to transform Soybeans produce transgenic hairy roots.

[0034] The details are as follows:

[0035] (1) Reagent

[0036] The used reagent and solution and the abbreviation of culture medium among the present...

Embodiment 2

[0098] GUS staining identification of embodiment 2 transgenic hairy roots

[0099] After the transformation, the hairy roots screened on the hairy root induction medium for 3 weeks were mixed with GUS staining solution, placed overnight at 37°C, washed 2 to 3 times with 70% alcohol, observed and photographed under a solid microscope, Record the number of GUS-positive hairy roots stained blue, and the results are shown in Table 1. The substrate of GUS staining solution is X-Gluc, which contains 0.5g / L X-Gluc, 100mmol / L phosphate buffer (pH7.0), 20% methanol, 0.5% (V / V) Triton X- 100.

[0100] Simultaneously, with other experimental conditions being the same, other three kinds of methods that screening method is different compare, I: the soybean seedling that earlier stage is cultivated on the hairy root induction medium that contains 2.5mg / L screening agent, the later stage still subcultures to new Cultivate in the hairy root induction medium containing 2.5mg / L screening agent;...

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Abstract

The invention discloses application of a method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation. The method for smearing and transforming agrobacterium rhizogen-mediated hypocotyl to is applied to soybeans for the first time, and hairy root transformation of soybean roots is successfully carried out. In this invention, the sprouting time of seeds and the concentration of screening agents are optimized, therefore not only increasing the quantity of transgenic hairy roots induced by each soybean explant, but also obviously enhancing the transformation frequency and growth conditions of hairy roots. A screening marker weed killer usually used for soybean transformation is successfully used for obtaining soybean compound plants in aseptic conditions, wherein each soybean compound plant has a transgenic root and a non-transgenic over-ground part and can be used as the ideal material for studying genes relative to the form of root systems, the symbiotic nitrogen fixation of roots and the formation of fungus roots.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of an Agrobacterium rhizogenes-mediated hypocotyl smear transformation method in soybean transformation. Background technique [0002] Agrobacterium is a Gram-negative soil bacterium. It has been found that there are two kinds of Agrobacterium related to plant gene transformation: one is Agrobacterium rhizogenes (Agrobacterium rhizogenes), which contains Ri (root inducing plasmid) plasmid, which can induce the infected plant cells to produce hairy roots ( hairy root); the second is Agrobacterium tumefaciens, which contains a Ti (tumor inducing plasmid) plasmid, which can induce the infected plant cells to form crown gall tumors. Compared with Agrobacterium tumefaciens, the hairy roots induced by the Ri plasmid of Agrobacterium rhizogenes grow faster, and the growth conditions can be artificially controlled and genetically stable, so i...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12R1/01
Inventor 王秀荣廖红严小龙
Owner SOUTH CHINA AGRI UNIV
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