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Novel method of soybean genetic transformation independent of tissue culture by taking soybean cotyledonary node as receptor

A technology of tissue culture and genetic transformation, which is applied in the field of improving the efficiency of genetic transformation of transgenic soybeans, and can solve the problems of long cycle, restriction of research and development of soybean genetic transformation, and low survival rate of transplanting.

Inactive Publication Date: 2011-11-16
JILIN NORMAL UNIV
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Problems solved by technology

However, the two stages of rooting and seedling hardening in this system are very laborious and time-consuming, and the survival rate of transplanting is very low, only about 10%, and the cycle from transformation to generation of T1 generation is long, about 6-8 months. time, which severely restricts the research and development of soybean genetic transformation

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  • Novel method of soybean genetic transformation independent of tissue culture by taking soybean cotyledonary node as receptor

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Effect test

Embodiment 1

[0018] Obtaining herbicide-resistant transgenic plants by transforming cotyledonary nodes independent of tissue culture

[0019] 1) Materials: soybean variety Tiefeng 31, collected in the experimental field of Jilin Normal University.

[0020] 2) Strains and plasmids: Agrobacterium strain EHA105 was preserved by the Institute of Ecology and Environment of Jilin Normal University, the transformation vector was CaMV 35S as a promoter, and the pCAMBIA 3301 plant binary expression vector (produced by Gift from researcher Zhao Guilan of Jilin Academy of Agricultural Sciences), introduced Agrobacterium tumefaciens strain EHA 105 as a genetically engineered bacterium to transform soybean cotyledon nodes. figure 1 is a diagram of the structure of a binary vector.

[0021] 3) Acquisition of seedlings: Sow soybean seeds into the soil matrix. After 5-7 days, after the cotyledons turn green and unfold, use a scalpel to clean the terminal buds and axillary buds, and then draw 5-6 knives a...

Embodiment 2

[0030] Transformation of Soybean with GUS Gene by Cotyledonary Node Transformation Method Independent of Tissue Culture

[0031] 1) Materials: soybean variety Tiefeng 31, collected in the experimental field of Jilin Normal University.

[0032] 2) Strains and plasmids: Agrobacterium strain LBA4404-PBI121 was preserved by the Institute of Ecology and Environment, Jilin Normal University, and the binary vector pBI121-GUS contains a 35s promoter, a GUS reporter gene, and karamycin as a selective resistance gene.

[0033] 3) Acquisition of seedlings: soybean seeds will be sown into the soil matrix on May 1. After 5-7 days, after the cotyledons turn green and unfold, use a scalpel to remove the terminal buds and axillary buds, and then draw 5 along the direction of the veins. - 6 knives, about 3-4mm long and 0.5mm deep.

[0034] 4) Pick a single colony from the Agrobacterium LBA4404-PBI121-GUS plate, inoculate it in YEB medium containing antibiotics, and culture it with shaking at ...

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Abstract

The invention provides a novel method of soybean genetic transformation independent of tissue culture by taking soybean cotyledonary node as a receptor, being applied to the technical fields of biology and modern agriculture. In the invention, on the basis of traditional cotyledonary node transformation, the genetic transformation of soybeans is improved by the transformation mode independent of tissue culture; on the basis of screening effective soybean cotyledonary node regeneration system, the transformation system independent of tissue culture is highlighted; through transformation verification of conventional soybean variety, transformation can be carried out directly under the condition of field cultivation, which can improve germination and growth of cluster buds at the transformation part; screening can be carried out effectively on positive branches through blade spraying or coating; the positive plants can directly obtain transgenic offspring 5 months after transformation with no need of the root generating process, thus improving soybean genetic transformation efficiency in a great degree, and dramatically shortening genetic transformation period of soybeans. The transgenic method can change the prior state of difficult soybean genetic transformation, and improve research and industrialized development of soybean genetic transformation.

Description

technical field [0001] The invention belongs to the technical field of plant transgenesis, and in particular relates to a method for improving the genetic transformation efficiency of transgenic soybeans. Background technique [0002] Soybean genetic transformation has become a model plant for many difficult-to-transform plants. At present, the cotyledon node transformation system mediated by Agrobacterium tumefaciens is the most widely recognized and applied soybean genetic transformation method. Several genetically modified soybean varieties that have been commercialized are mainly derived from this system. However, the two stages of rooting and seedling hardening in this system are very laborious and time-consuming, and the survival rate of transplanting is very low, only about 10%, and the cycle from transformation to generation of T1 generation is long, about 6-8 months. time, which seriously restricts the research and development of soybean genetic transformation. A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H1/00C12Q1/68
Inventor 程云清刘剑锋赵桂兰陈智文
Owner JILIN NORMAL UNIV
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