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Pulse electrophoresis-assisted Agrobacterium-mediated plant transgenic method and device

An Agrobacterium-mediated, pulsed electrophoresis technology, applied in the field of bioengineering, can solve problems such as plants without integrated receptors, and achieve the effects of improving the efficiency of gene transformation and shortening the experimental period.

Active Publication Date: 2018-06-26
NANJING XIAOZHUANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports of pulse electrophoresis transgenics in plants such as rice and corn, but in these reports, the DNA was directly introduced into seeds or isolated tissues and cells, and was not integrated into the genome of the recipient plant.

Method used

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  • Pulse electrophoresis-assisted Agrobacterium-mediated plant transgenic method and device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, taking the Vinca minor plant as the recipient plant, converting the exogenous gene into the chitinase gene from tobacco

[0026] In this example, a pulse electrophoresis-assisted transgenic test was carried out on Vinca minor plants, and the transformed exogenous gene was chitinase gene from tobacco. The Vinca minor seedlings used in this example come from our laboratory.

[0027] This embodiment includes the following steps:

[0028] Step 1, excavating 20 robust vinca seedlings from the field as receptors;

[0029] Step 2, shaking and culturing the Agrobacterium solution containing the chitinase gene on a shaker, so that the OD value reaches 0.6;

[0030] The Agrobacterium solution obtained in step 3 and step 2 is centrifuged at 3500 rpm for 2 minutes, then resuspended with MS0 liquid medium with a pH of 5.2, and finally adding 200 μM acetosyringone to the suspension to obtain a transformation solution;

[0031] Step 4, inserting the rooted recipient plan...

Embodiment 2

[0037] Example 2, taking Mongolian leek as the recipient plant, converting the exogenous gene into the chitinase gene from tobacco

[0038] In this example, a pulse electrophoresis-assisted transgenic experiment was carried out on the plants of Chinese chives, and the exogenous gene to be transformed was the chitinase gene from tobacco. The Mongolian leek seedlings used in this example come from this laboratory.

[0039] This embodiment includes the following steps:

[0040] Step 1: Excavating 35 healthy Mongolian leek seedlings from the field as recipients;

[0041] Step 2, shaking and culturing the Agrobacterium solution containing the chitinase gene on a shaker to make the OD value reach 0.4;

[0042] The Agrobacterium solution obtained in step 3 and step 2 was centrifuged at 3400 rpm for 1.5 minutes, then resuspended with MS0 liquid medium with a pH of 5.1, and finally added 160 μM acetosyringone to the suspension to obtain a transformation solution;

[0043] Step 4, in...

Embodiment 3

[0049] Example 3, using Atractylodes macrocephala as the recipient plant, converting the exogenous gene into the chitinase gene from tobacco

[0050] In this example, a pulse electrophoresis-assisted transgenic test was carried out on Atractylodes macrocephala plants, and the transformed exogenous gene was chitinase gene from tobacco. The Atractylodes macrocephala seedlings used in this example come from our laboratory.

[0051] This embodiment includes the following steps:

[0052] Step 1, excavating 30 strong Atractylodes macrocephala seedlings from the field as recipients;

[0053] Step 2, shaking and culturing the Agrobacterium solution containing the chitinase gene on a shaker, so that the OD value reaches 0.8;

[0054] The Agrobacterium solution obtained in step 3 and step 2 was centrifuged at 3800 rpm for 3 minutes, then resuspended with MS0 liquid medium with a pH of 5.4, and finally 190 μM of acetosyringone was added to the suspension to obtain a transformation solu...

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Abstract

The invention discloses a pulse-electrophoresis-assisted agrobacterium-mediated plant transgenic method. According to the method, after agrobacterium comprising the target gene is put into an electric field, the negatively charged agrobacterium moves from the negative pole to the positive pole under the electric field intensity, thereby entering all tissue organs in the plant. The cell walls use fibers as the matrix and contain many pores, and thus, the agrobacterium can pass through the pores of the cell walls in the transfer process under the action of the electric field, and further enters plant cells via the cytomembranes. The method greatly enhances the gene transformation efficiency, shortens the experimental period especially for the plants which have not established the regeneration system, and thus, has certain application value.

Description

technical field [0001] The invention relates to the technical field of plant transgene in the field of bioengineering, in particular to a plant transgene method assisted by pulse electrophoresis mediated by Agrobacterium. Background technique [0002] As the core of biotechnology, transgenic technology has great development potential in plant improvement, medicine, animal husbandry and food industry. The application publication number is CN 104017825 A, and the name is the method of centrifugal sedimentation assisted plant transgenesis, which discloses that the plant receptor material is mixed with Agrobacterium and then subjected to centrifugal sedimentation treatment at a reasonable speed to obtain the gene conversion liquid, and the infected Plant recipient materials are screened under selection pressure, and positive transformants are finally obtained. Centrifugal sedimentation can not only increase the wound area of ​​the receptor material, but also allow Agrobacterium...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12M1/42A01H5/10
CPCC12M35/02C12N15/8205
Inventor 张边江陈全战唐宁
Owner NANJING XIAOZHUANG UNIV
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