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Protoplast preparation and genetic transformation method of Chinese cabbage

A technology of protoplast and genetic transformation, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, plant cells, etc. In order to achieve the effect of uniform size, good living condition, and simple and easy method, the problems of body culture and genetic transformation are difficult and other problems.

Active Publication Date: 2019-11-19
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although plant protoplasts have such great research value and application value, due to the difficulty and poor repeatability of protoplast culture and genetic transformation, the potential application value of protoplasts has not been effectively tapped and fully brought into play.

Method used

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  • Protoplast preparation and genetic transformation method of Chinese cabbage
  • Protoplast preparation and genetic transformation method of Chinese cabbage
  • Protoplast preparation and genetic transformation method of Chinese cabbage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Preparation of Chinese cabbage protoplasts and observation of subcellular localization of GFP protein

[0048] 1. Preparation of protoplasts

[0049] 1, get the mesophyll tissue of the yellowing inner leaf of the flat-headed Chinese cabbage Degao No. 16 Chinese cabbage that has been knotted and the mesophyll tissue of the true leaves of the flat-headed Chinese cabbage Degao No. 16 Chinese cabbage seedlings that have grown for two weeks (as a contrast ), were cut into thin strips <1mm, put into 10mL enzymatic hydrolysis solution, and enzymatically hydrolyzed at 25°C for 3 hours in the dark to obtain the enzymatic hydrolysis solution;

[0050] Wherein, the enzymolysis solution is composed of cellulase R-10, isolated enzyme R-10, MES, mannitol, KCl and water (solvent), and the content of each component in the solute is 10g / L cellulase R- 10. 2.5g / L isolated enzyme R-10, 0.02mol / L MES (pH5.7), 0.4mol / L mannitol and 0.02mol / L KCl.

[0051] 2. Filter the enzymoly...

Embodiment 2

[0060] Example 2. Preparation of Chinese cabbage protoplasts and observation of subcellular localization of nuclear localized GFP fusion protein

[0061] 1. Preparation of protoplasts

[0062] 1, get the mesophyll tissue of the yellowing inner leaf of the flat-headed Chinese cabbage Degao No. 16 Chinese cabbage that has been knotted and the mesophyll tissue of the true leaves of the flat-headed Chinese cabbage Degao No. 16 Chinese cabbage seedlings that have grown for two weeks (as a contrast ), were cut into thin strips <1mm, put into 10mL enzymatic hydrolysis solution, and enzymatically hydrolyzed at 25°C for 5 hours in the dark to obtain the enzymatic hydrolysis solution;

[0063] Wherein, the enzymolysis solution is composed of cellulase R-10, isolated enzyme R-10, MES, mannitol, KCl and water (solvent), and the content of each component in the solute is 10g / L cellulase R- 10. 2.5g / L isolated enzyme R-10, 0.02mol / L MES (pH5.7), 0.4mol / L mannitol and 0.02mol / L KCl.

[006...

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PUM

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Abstract

The invention discloses a protoplast preparation and genetic transformation method of Chinese cabbage. The invention firstly discloses a method for preparing protoplasts of Chinese cabbage. The methodcomprises the step of acquiring the Chinese cabbage protoplasts from yellowing inner leaves of Chinese cabbage. The invention further discloses a genetic transformation method of the Chinese cabbageprotoplasts. The Chinese cabbage protoplast preparation method takes mesophyll tissues of the yellowing inner leaves of flat-headed Chinese cabbage as materials. Compared with other materials, the method can rapidly obtain a large amount of protoplasts and effectively carry out genetic transformation, and is suitable for exogenous expression and functional verification of Chinese cabbage genes.

Description

technical field [0001] The invention relates to the field of biological breeding, in particular to the field of plant cell biology technology, and more specifically to a method for preparing protoplasts and genetic transformation of Chinese cabbage. Background technique [0002] Chinese cabbage is a brassicaceae, Brassica is a biennial herb, up to 60 cm high, the whole plant is glabrous, the basal leaves are numerous, large, obovate oblong to wide obovate, the top is blunt, the edge shrinks, Wavy, petiole white, flat, bright yellow flowers, sepals oblong or ovate-lanceolate, erect, light green to yellow. Chinese cabbage can be divided into loose-leaf varieties, semi-headed varieties, flower-centered varieties and headed varieties. There are three types of knot varieties: oval, flat, and straight. [0003] Since the 1970s, the research work of plant protoplasts has been widely valued, and its important value has been fully demonstrated in both basic research and applied res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8201
Inventor 苏同兵辛晓云李佩荣张凤兰于栓仓王单
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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