Establishment method of high efficient transient transformation system of exogenous genes of cunninghamia lanceolata

A technology of exogenous gene and transient transformation, applied in the field of plant cytology, can solve the problems of immature separation and purification system, and achieve the effects of cost saving, broad application prospects and high-efficiency expression

Active Publication Date: 2019-10-18
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In some woody plants, protoplasts can be successfully isolated and purified, but the separation and purification system of most woody plant protoplasts is not yet mature, even poplar with better transformation efficiency in existing related rep...

Method used

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  • Establishment method of high efficient transient transformation system of exogenous genes of cunninghamia lanceolata
  • Establishment method of high efficient transient transformation system of exogenous genes of cunninghamia lanceolata

Examples

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Effect test

Embodiment 1

[0021] Choose healthy annual Chinese fir seedlings, cut the main stem into 9cm stem segments, and peel off the bark. Put the peeled stem segments prepared above into a 50mL tube containing 40mL of enzymatic hydrolysis solution, put 4 stem segments of 9cm in one tube, and incubate for 4 hours in the dark and avoid light at a temperature of 25°C to obtain the enzymatic hydrolysis solution. stem segments. Transfer the enzymatically treated stem section from the enzymolysis solution to the MMG solution, shake gently horizontally for 2 minutes to release the protoplasts, and the MMG solution becomes turbid to obtain a protoplast suspension; fold the protoplast suspension with a 70 μm filter for 2 Layer was filtered, the filtrate was collected, the filtrate was centrifuged at 300 g for 5 min, the supernatant was sucked off, and the precipitate remained, and the precipitate was resuspended with 1 mL MMG to obtain a protoplast suspension. Take 10 μL of isolated protoplasts and add th...

Embodiment 2

[0028] Choose healthy annual Chinese fir seedlings, cut the main stem into 10cm stem segments, and peel off the bark. Put the peeled stem sections prepared above into a 50mL tube containing 40mL of enzymatic hydrolysis solution, put 4 stem sections of 10cm in one tube, and incubate for 3 hours in the dark and away from light at a temperature of 25°C to obtain the enzymatic hydrolysis solution. stem segments. Transfer the enzymatically treated stem section from the enzymolysis solution to the MMG solution, shake gently horizontally for 2 minutes to release the protoplasts, and the MMG solution becomes turbid to obtain a protoplast suspension; fold the protoplast suspension with a 70 μm filter for 2 Layer was filtered, the filtrate was collected, the filtrate was centrifuged at 300 g for 5 min, the supernatant was sucked off, and the precipitate remained, and the precipitate was resuspended with 1 mL MMG to obtain a protoplast suspension. Take 10 μL of isolated protoplasts and ...

Embodiment 3

[0035] Choose healthy annual Chinese fir seedlings, cut the main stem into 8cm stem segments, and peel off the bark. Put the peeled stem segments prepared above into a 50 mL tube containing 40 mL of enzymatic hydrolysis solution, put 4 stem segments of 8 cm in one tube, and incubate for 2 hours in the dark and away from light at a temperature of 26°C to obtain enzymatic hydrolysis Liquid-treated stem segments. Transfer the enzymatically treated stem section from the enzymolysis solution to the MMG solution, shake gently horizontally for 1 min to release the protoplasts, and the MMG solution becomes turbid to obtain a protoplast suspension; fold the protoplast suspension with a 70 μm filter for 2 Layer was filtered, the filtrate was collected, the filtrate was centrifuged at 300g for 5min, the supernatant was sucked off, and the precipitate remained, and the precipitate was resuspended with 1 mL MMG to obtain a protoplast suspension. Take 10 μL of isolated protoplasts and add ...

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Abstract

The invention discloses an establishment method of a high efficient transient transformation system of exogenous genes of cunninghamia lanceolata, and belongs to the technical field of plant cytology.The method includes the following steps that cunninghamia lanceolata stem segments are subjected to digestion and enzymolysis through an enzymolysis approach to release protoplasts, the protoplasts are purified, PEG mediates the transformation of exogenous gene plasmid DNA into the protoplasts, high efficient transient transformation of the exogenous genes in the gymnosperm, namely cunninghamia lanceolata, is achieved, and the transformation efficiency of the cunninghamia lanceolata is improved greatly, and the method provides a technical support for molecular biology research and genetic transformation of main candidate genes of cunninghamia lanceolata.

Description

technical field [0001] The invention belongs to the technical field of plant cytology, and mainly relates to a method for establishing a high-efficiency transient transformation system of foreign gene of Chinese fir. Background technique [0002] Chinese fir( Cunninghamia lanceolata ), also known as sand wood, belongs to Cypress order, fir tree, which has the characteristics of fast growth, good material properties, high yield, and wide application. It is one of the important commodity tree species in my country, and its fir root, fir bark, fir, fir leaves, seeds, etc. all have certain medicinal value. There is no research report on the genetic transformation of Chinese fir. As a morphological structural unit of cells, protoplast is a naked cell without cell wall coating. Since there is no cell wall, it can be used as an ideal material for genetic transformation. The principle of protoplast transformation is to introduce foreign genes by changing the permeability of the cel...

Claims

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Application Information

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IPC IPC(8): C12N15/82
CPCC12N15/8206
Inventor 顾连峰魏文桃胡凯强陈凯高鹏飞丁家治王慧慧刘旭庆张千悦
Owner FUJIAN AGRI & FORESTRY UNIV
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