Application of novel rooting method in soybean transgenic technology

A technology of transgenic and rooting medium, applied in application, horticultural methods, recombinant DNA technology, etc., can solve the problems of low rooting rate of transformed plants, slow growth of clump buds, and low survival rate of transplanting

Inactive Publication Date: 2011-09-07
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Soybean is recognized as a difficult plant to transform. The cotyledonary node transformation system and embryo tip transformation system mediated by Agrobacterium have been widely used in soybean genetic transformation research, but the resistant buds obtained by these two systems have the problem of rooting difficulties. After the two stages of rooting and seedling hardening, the survival rate of transplanting is very low, about 20% and 10% respectively, which seriously restricts the research and development of soybean genetic transformation
[0003] In the Agrobacterium-mediated soybean genetic transformation system, it is necessary to use antibiotics to inhibit the growth of Agrobacterium, but antibiotics will inevitably have a serious impact on the growth of soybean explants while inhibiting Agrobacterium, resulting in slow growth of clustered shoots Difficulty in rooting regenerated seedlings
In the cotyledonary node transformation system and embryo tip transformation system, in order to improve the rooting rate of regenerated seedlings, the method of gradually reducing the concentration of antibiotics or removing antibiotics is generally used, but it often causes the problems of low rooting rate of transformed plants and contamination of transformed plants by Agrobacterium re-growth, which is difficult. Fundamentally solve the effect of antibiotics and other bacteriostatic agents on the rooting of transplanted plants

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Using Adventitious Buds Transformed from Cotyledonary Nodes to Obtain Regenerated Plants by Rooting on Double Layer Medium

[0017] 1- Applied Materials

[0018] 1) Plant material: The soybean variety is Jidou 16, which was purchased from the Grain and Oil Crops Institute of Hebei Academy of Agricultural Sciences;

[0019] 2) Bacterial strains and plasmids: the Agrobacterium strain is EHA105 (purchased from the market), the vector is pCAMBIA3301 (purchased from the market), and the screening marker gene is the bar gene;

[0020] 2- Acquisition of Adventitious Buds

[0021] 1) Seed disinfection: Pick soybean seeds with a smooth surface and no cracks for chlorine fumigation for 16-20 hours;

[0022] 2) Inoculate sterile soybean seeds into germination medium: B5+30g / L sucrose+7.0g / L agar+0.4g / L MES, pH 5.8, 23°C, light culture for 5 days;

[0023] 3) Obtaining of cotyledon node explants: when the cotyledons turn green, separate the cotyledons with a scalpel, remove the ...

Embodiment 2

[0039] Using the Adventitious Buds Transformed from Embryo Tips to Obtain Regenerated Plants by Rooting on Double Layer Medium

[0040] 1- Applied Materials

[0041] 1) Plant material: The soybean variety is Wuxing No. 2, purchased from the Grain and Oil Crops Institute of Hebei Academy of Agricultural Sciences;

[0042] 2) Bacterial strains and plasmids: the Agrobacterium strain is GV3101 (purchased from the market), the vector is pCAMBIA1300 (purchased from the market), and the screening marker gene is the hpt gene;

[0043] 2- Acquisition of Adventitious Buds

[0044] 1) Seed disinfection: Pick soybean seeds with a smooth surface and no cracks for chlorine fumigation for 16-20 hours;

[0045] 2) Obtaining of explants: Soak sterilized soybean seeds overnight in sterile water, remove cotyledons, remove true leaves, collect embryo tips, inoculate into pre-medium, MS+30g / L sucrose+0.4 g / L MES+3.5mg / L 6-BAP+7.0% agar, light at 23℃, pre-cultivate for 30h;

[0046] 3) Agrobact...

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Abstract

The invention relates to an agrobacterium tumfaciens-mediated transgenic soybean regenerated seedling rooting method, and belongs to the technical fields of soybean tissue culture and transgenic research. Research of soybean cotyledon node and embryo genetic transformation is performed by using an agrobacterium tumfaciens mediating method; a novel transgenic plant rooting method is adopted for the first time; and even if a double-layer rooting culture medium is adopted, the upper layer contains antibiotics and the lower layer does not contain antibiotics, the growth of agrobacterium tumfaciens can be inhibited, adverse influence of the antibiotics on transgenic regenerated plants can be greatly reduced, and the transplanting survival rates of the agrobacterium tumfaciens infected plants reach 90.70 percent and 88.10 percent respectively. The application of the method has significance for improving the agrobacterium tumfaciens-mediated soybean genetic transformation efficiency, and has good reference effect on genetic transformation of other plants.

Description

technical field [0001] The invention relates to a method for rooting transgenic soybean regenerated seedlings mediated by Agrobacterium, and belongs to the technical field of soybean tissue culture and transgene research. Background technique [0002] The genetic improvement of crops using exogenous genes has become one of the important means of plant breeding. At present, Agrobacterium-mediated and particle gun bombardment methods are mostly used for the introduction of exogenous genes. Especially, the Agrobacterium-mediated method has become a popular method for monocotyledonous and dicotyledonous plant transgenic breeding due to its simple method, high efficiency, and low copy number. The preferred method of [Horsh R B]. Soybean is recognized as a difficult plant to transform. The cotyledonary node transformation system and embryo tip transformation system mediated by Agrobacterium have been widely used in soybean genetic transformation research, but the resistant buds o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 郑文永张洁王冬梅
Owner HEBEI AGRICULTURAL UNIV.
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