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Agrobacterium tumefaciens-mediated moss stable genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of plant transgenics, can solve problems such as cases of no successful transformation

Pending Publication Date: 2021-05-28
XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far there are no examples of successful conversion

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  • Agrobacterium tumefaciens-mediated moss stable genetic transformation method
  • Agrobacterium tumefaciens-mediated moss stable genetic transformation method
  • Agrobacterium tumefaciens-mediated moss stable genetic transformation method

Examples

Experimental program
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Effect test

Embodiment 1

[0047] The method for the genetic transformation of Physcomitrella patens, concrete operation is carried out according to the following steps:

[0048] Physcomitrella patens protonema culture:

[0049] a. Cultivate the protonema of Physcomitrella patens in the laboratory, adopt the grinding method, put the protonema of Physcomitrella patens into a sterilized 50 ml centrifuge tube, add 20 ml of sterile water, and grind for 1 minute with a grinder , grind into a homogenate, absorb 2ml of Physcomitrella patens protonema homogenate and add it to the conventional PPNH4 solid medium. The medium is evenly covered with sterile cellophane, sealed with a 3M gas-permeable membrane, and grown for 6 days as a transformation material. .

[0050] Agrobacterium cultivation:

[0051] b. Put the single colony of Agrobacterium containing the target gene into the Agrobacterium culture solution, and culture it with shaking at 28°C and 200rpm. When the OD value of the Agrobacterium solution is 0....

Embodiment 2

[0061] The method for the genetic transformation of Physcomitrella patens, concrete operation is carried out according to the following steps:

[0062] Culture of Physcomitrella patens gametophytes:

[0063] a. Cultivate Physcomitrella patens gametophytes in the laboratory, adopt grinding method, get Physcomitrella patens gametophytes 0.1g and put into a sterilized 50ml centrifuge tube, add 20ml of improved KNOP liquid medium, grind for 1 minute with a grinder, Grind into a homogenate, draw 2ml of gametophyte homogenate and add it to the conventional modified KNOP solid medium. The medium is evenly covered with sterile cellophane, and grow for 30 days as the transformation material;

[0064] Agrobacterium cultivation:

[0065] b. Put the single colony of Agrobacterium containing the target gene into the Agrobacterium culture solution, and culture it with shaking at 28°C and 200rpm. When the OD value of the Agrobacterium solution is 0.4-0.6, centrifuge at 5500rpm for 10 minute...

Embodiment 3

[0073] The method for the genetic transformation of the silver leaf moss, concrete operation is carried out according to the following steps:

[0074] Cultivation of the protonema of Moss argentifolius:

[0075]a. Cultivate the protonema of the silver leaf moss in the laboratory, adopt the grinding method, take 0.1g of the silver leaf protonema and put it into a sterilized 50ml centrifuge tube, add 20ml of the improved KNOP liquid medium, and grind it with a grinder Grind for 1 minute, grind into a homogenate, draw 2 ml of the homogenate, add it to the PPNH4 medium, seal it with a 3M air-permeable membrane, and cultivate it for 6 days as a transformation material;

[0076] Agrobacterium cultivation:

[0077] b. Put the single colony of Agrobacterium containing the target gene into the Agrobacterium culture solution, and culture it with shaking at 28°C and 200rpm. When the OD value of the Agrobacterium solution is 0.4-0.6, centrifuge at 5500rpm for 10 minutes and collect The ...

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Abstract

The invention relates to an agrobacterium tumefaciens-mediated moss stable genetic transformation method. According to the method, the moss is the protonemata or gametophyte of the Physcomitrella patens and the Bryum argenteum. The method comprises the steps of culture of moss protonema and gametophyte, genetic transformation and co-culture, screening culture and identification. By taking moss protonema and gametophyte as infection materials, the agrobacterium engineering bacterium liquid carrying a target gene is filled into a culture dish for genetic transformation; the moss is directly infected with agrobacterium, the transformation period is short, and a large number of transgenic materials can be obtained in a short time. Compared with a protoplast transformation method, the method is simple and easy to operate and control, short in period, high in transformation efficiency, good in expression intensity and low in cost, and lays a foundation for gene function research.

Description

technical field [0001] The invention relates to the technical field of plant transgenesis, in particular to a method for moss stable genetic transformation mediated by Agrobacterium. Background technique [0002] Bryophytes (bryophytes) are the earliest terrestrial plants in existence. They are a group of higher plants transitioning from aquatic to terrestrial. They are the lowest group of higher plants. They have short plants, no vascular tissue, and no flowers. The structure, taxonomically between algae (algae) and ferns (pteridophytes), has a unique evolutionary position. Moss has a short growth cycle and is easy to cultivate, which can be used as an important material for studying gene functions. [0003] Physcomitrella patens is a moss of the genus Physcomitrium in the family Funariaceae of the order Funariales. Genomics and molecular biology research conducted on Physcomitrella patens as an object found that in order to adapt to land life, Physcomitrella patens acqui...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/84A01H11/00
CPCC12N15/8205C07K14/415Y02A40/22
Inventor 张道远周平刘秀瑾张燕李小双杨红兰
Owner XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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