High-efficiency genetic transformation method for soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation

A genetic transformation method and vacuum infiltration technology, which is applied to the high-efficiency genetic transformation of soybean immature cotyledon regeneration system assisted by vacuum infiltration, and the field of improving soybean genetic traits. Efficiency, the effect of increasing the conversion rate

Inactive Publication Date: 2008-05-07
JILIN ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

[0006] The present invention discloses a high-efficiency genetic transformation method of vacuum infiltration assisted soybean immature cotyledon regeneration system, the purpose is to overcome the difficulty of low efficiency of existing soybean genetic transformation, using Soybean Immature Cotyledons as Explants Provide a Vacuum Infiltration-Assisted Transformation of Exogenous DNA in an Agrobacterium tumefaciens-Mediated Method

Method used

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  • High-efficiency genetic transformation method for soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation
  • High-efficiency genetic transformation method for soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Jilin 35 somatic embryogenesis and introduction of exogenous genes

[0025] Jilin 35 was planted in the field, and young soybean pods of 16-20 days after flowering were picked, rinsed once with sterile water, soaked in 75% ethanol for 2 minutes, and rinsed with sterile water for 3 times. Peel off the seed coat, select 3-5mm cotyledons to remove the hypocotyl, and inoculate the cotyledons with the adaxial side up in MS medium + 2,4-D 20mg / L + sucrose 3.0g / L + plant gel 3.0g / L (pH=6.0 ) in the dark at 25°C for 14-30 days.

[0026] Select bright and spherical green embryoid bodies, and move to MS medium + 2,4-D 5.0 ​​mg / L + asparagine 1.0 g / L + sucrose 3.0 g / L + plant gel 3.0 g / L (pH=5.8 ), cultivated under light at 25°C (16hr / 8hr), and subcultured once every two weeks.

[0027] The somatic embryos were transferred to MS medium + activated carbon 4.0g / L + maltose 60.0g / L + phytogel 3.0g / L (pH=6.4), and cultured under light at 25°C (16hr / 8hr). Passage every two weeks.

...

Embodiment 2

[0033] Somatic Embryogenesis and Introduction of Foreign Genes in Jilin 47

[0034] Jilin 47 was planted in the field, and young soybean pods that had bloomed for 16-20 days were picked, rinsed once with sterile water, soaked in 75% ethanol for 2 minutes, and rinsed three times with sterile water. Peel off the seed coat, select 3-5mm cotyledons to remove the hypocotyl, and inoculate the cotyledons with the adaxial side up in MS medium + 2,4-D 40mg / L + sucrose 3.0g / L + plant gel 3.0g / L (pH=6.0 ) in the dark at 25°C for 14-30 days.

[0035] Select bright and spherical green embryoid bodies, and move to MS medium + 2,4-D 5.0 ​​mg / L + asparagine 1.0 g / L + sucrose 3.0 g / L + plant gel 3.0 g / L (pH=5.8 ), cultivated under light at 25°C (16hr / 8hr), and subcultured once every two weeks.

[0036] The somatic embryos were transferred to MS medium + activated carbon 4.0g / L + maltose 60.0g / L + phytogel 3.0g / L (pH=6.4), and cultured under light at 25°C (16hr / 8hr). Passage every two weeks....

Embodiment 3

[0042] Somatic Embryogenesis and Introduction of Foreign Genes in Jilin 20

[0043] Jilin 20 was planted in the field, and young soybean pods that had bloomed for 16-20 days were picked, washed once with sterile water, soaked in 75% ethanol for 2 minutes, and rinsed three times with sterile water. Peel off the seed coat, select 3-5mm cotyledons to remove the hypocotyl, and inoculate the cotyledons with the adaxial side up in MS medium + 2,4-D 30mg / L + sucrose 3.0g / L + plant gel 3.0g / L (pH=6.0 ) in the dark at 25°C for 14-30 days.

[0044] Select bright and spherical green embryoid bodies, and move to MS medium + 2,4-D 5.0 ​​mg / L + asparagine 1.0 g / L + sucrose 3.0 g / L + plant gel 3.0 g / L (pH=5.8 ), cultivated under light at 25°C (16hr / 8hr), and subcultured once every two weeks.

[0045] The somatic embryos were transferred to MS medium + activated carbon 4.0g / L + maltose 60.0g / L + phytogel 3.0g / L (pH=6.4), and cultured under light at 25°C (16hr / 8hr). Passage every two weeks....

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Abstract

The invention discloses an efficient genetic transformation method of vacuum permeation assistance soybean immature cotyledon regeneration system, relating to the technical field of plant genetic engineering, comprising the following steps: (a) the induction and proliferation of soybean immature cotyledon somatic embryo; (b) the vacuum permeation assistance introduction of foreign DNA; (c) the regeneration of the somatic embryo with foreign DNA into a plant. Adopting the vacuum permeation assistance agrobacterium mediating method, under a certain vacuum condition, after treat for a certain time, a plurality of micromechanical wounds are made on the soybean somatic embryo, which fully raises the intruding opportunity for agrobacterium to obviously raise the infection efficiency of agrobacterium and the conversion of the soybean compared with the common agrobacterium mediating method. Soybean immature cotyledon is used as an explant to transform genetically, which avoids the problem that chimerism easily, appears with the cotyledonary node as the explant.

Description

Technical field: [0001] The invention relates to the field of plant genetic engineering, and discloses a high-efficiency genetic transformation method of vacuum infiltration assisted soybean immature cotyledon regeneration system, thereby improving the operation method of soybean genetic traits. Background technique: [0002] Soybean genetic transformation has always been one of the difficulties in the field of plant genetic engineering. Although there are many reports of successful soybean transgenic plants, there has been no breakthrough in soybean genetic transformation. The most important reason is that the conversion rate has not improved significantly. [0003] The methods currently applied to soybean genetic transformation are mainly divided into two categories: 1. Direct method: mainly including gene gun method, pollen tube channeling method, electric shock method, microinjection method, etc.; 2. Vector method: mainly including Agrobacterium tumefaciens mediated Law...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10A01H4/00
Inventor 赵桂兰郭东全杨向东钱雪艳
Owner JILIN ACAD OF AGRI SCI
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