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A method for genetic transformation of soybeans with vacuum-assisted Agrobacterium infection

A genetic transformation method, vacuum-assisted technology, applied in the field of differentiation culture, can solve the problems of low transformation efficiency, long transformation cycle, heavy workload, etc., and achieve the effect of short cycle, fast gene transformation and less chimeras

Inactive Publication Date: 2020-01-03
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of genetic transformation, the usual practice is to use soybean cotyledon node explants for genetic transformation operations. This method has many chimeras, low transformation efficiency, and heavy workload.
Although there are also reports on the use of immature soybean embryos for genetic transformation (Qian Xueyan, Guo Dongquan, Yang Xiangdong, et al. Anhui Agricultural Sciences, 2012, 40(2): 658-661), but this method has a long transformation cycle and a large workload , low transformation efficiency, few transformants

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Select the tender soybean pods of Tianlong No. 1 25-30 days after flowering that are free from diseases and insect pests, rinse with tap water for 15 minutes, disinfect with 75% ethanol for 1 minute, and then rinse with sterile water for 3 times. Peel off the pod in a sterile table, take out the immature seeds with tweezers, peel off 2 immature embryos, and cut 7 cuts on the back of the immature embryos, with a depth of about 0.5mm and a length of about 2mm.

[0028] (2) Three days before the infection transformation test, take Agrobacterium (containing the exogenous gene bar to be transformed) from the -80 refrigerator and make a plate. Cultivate at 28°C for two days, pick a single colony on the plate to 2ml YEP liquid medium (containing corresponding antibiotics), shake the bacteria overnight at 28°C. According to the ratio of 1:50, take the bacterial solution and add it to the new YEP liquid medium (containing corresponding antibiotics), shake the bacteria to OD ...

Embodiment 2

[0037] (1) Pick the young soybean pods of Williams 82 free from diseases and insect pests 25-30 days after flowering, rinse with tap water for 15 minutes, disinfect with 75% ethanol for 1 minute, and then rinse with sterile water for 3 times. Peel off the pod in a sterile table, take out the immature seeds with tweezers, peel off 2 pieces of immature embryos, cut 8 knives on the back of the immature embryos, the depth is about 0.5mm, and the length is about 2mm.

[0038] (2) Three days before the infection transformation test, take Agrobacterium (containing the exogenous gene bar to be transformed) from the -80 refrigerator and make a plate. Cultivate at 28°C for two days, pick a single colony on the plate to 2ml YEP liquid medium (containing corresponding antibiotics), shake the bacteria overnight at 28°C. According to the ratio of 1:50, take the bacterial solution and add it to the new YEP liquid medium (containing corresponding antibiotics), shake the bacteria to OD 600 =0...

Embodiment 3

[0047] (1) Pick young soybean pods free from diseases and insect pests 25-30 days after Jack flowering, rinse with tap water for 15 minutes, disinfect with 75% ethanol for 1 minute, and then rinse with sterile water for 3 times. Peel off the pod in a sterile table, take out the immature seeds with tweezers, peel off 2 immature embryos, and cut 7 cuts on the back of the immature embryos, with a depth of about 0.5mm and a length of about 2mm.

[0048] (2) Three days before the infection transformation test, take Agrobacterium (containing the exogenous gene bar to be transformed) from the -80 refrigerator and make a plate. Cultivate at 28°C for two days, pick a single colony on the plate to 2ml YEP liquid medium (containing corresponding antibiotics), shake the bacteria overnight at 28°C. According to the ratio of 1:50, take the bacterial solution and add it to the new YEP liquid medium (containing corresponding antibiotics), shake the bacteria to OD 600 =0.6-0.8. After centrifu...

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Abstract

The invention discloses a vacuum-assisted agrobacterium infected soybean genetic transformation method. The method comprises the following steps: 1) obtaining an immature embryo of a soybean; 2) transforming the immature embryo from step 1) with vacuum-assisted agrobacterium infection and transferring the immature embryo to a cocultivation medium for cultivation; 3) washing the immature embryo produced from step 2) with a sterilization medium, and transferring the immature embryo after washing to an induction medium containing screening agents for induction and formation of a resistant callus; 4) using a differential medium containing screening agents to differentiate the resistant callus produced from step 3) to form induced buds, and obtaining regenerated seedlings by subculture and rooting; 5) exercising and transplanting the regenerated seedlings obtained from step 4) to harvest the seeds of the transgenic plants. Compared with the prior art, the method shortens the genetic transformation period of the immature embryo of the soybean, obtains a large number of transgenic plants in the current generation, and the genetic stability of the offspring is achieved, thus having broad application prospect in the research of soybean breeding or its gene function.

Description

technical field [0001] The invention relates to plant tissue culture and genetic transformation methods, in particular to a method of using immature soybean embryos as explants to conduct vacuum-assisted Agrobacterium infection to induce resistant soybean calli, and then through differentiation culture to produce Method for transgenic soybean plants. Background technique [0002] Soybean is an annual leguminous herb, which is an important source of edible oil and vegetable protein in the world. China has a long history of soybean planting. Improving soybean germplasm resources through molecular breeding and transgenic means has become the trend of soybean breeding. At present, transgenic technology has been widely used in the fields of soybean biotechnology breeding and gene function identification. The main methods of gene transformation include Agrobacterium-mediated transformation, gene gun transformation method and pollen tube passage method, among which Agrobacterium-m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/54
CPCA01H4/00C12N15/8205
Inventor 王彪苏彤邢茂玉卢欣欣姚陆铭李瑶武天龙
Owner SHANGHAI JIAOTONG UNIV
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