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A kind of Arabidopsis thaliana mesophyll cell protoplast and its preparation method and application

A protoplast, Arabidopsis leaf technology, applied in the field of cell biology, can solve the problems of easy death and aggregation, difficult to operate in large quantities, and low transformation efficiency, and achieve the effects of broadening sources, high transformation rate, and improving transformation efficiency.

Active Publication Date: 2022-02-11
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, the protoplast preparation and transformation methods of Wu et al. also have defects, such as protoplasts are easy to die and aggregate under light culture conditions, low transformation efficiency and difficult to operate in large quantities.

Method used

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  • A kind of Arabidopsis thaliana mesophyll cell protoplast and its preparation method and application
  • A kind of Arabidopsis thaliana mesophyll cell protoplast and its preparation method and application
  • A kind of Arabidopsis thaliana mesophyll cell protoplast and its preparation method and application

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experiment example

[0038] 1. Materials and methods

[0039] 1.1 Planting of Arabidopsis plants

[0040] Seeds of wild type Arabidopsis thaliana (Col-0) (Arabidopsis thaliana) were sterilized with 10% sodium hypochlorite solution (containing 0.1% SDS) for 12 minutes, centrifuged briefly to discard the sodium hypochlorite solution, washed 5 times with sterile water, and then transferred to MS medium (containing 100mg / ml Timentin). After 10 days, the germinated Arabidopsis seedlings were transplanted into a cultivation pot containing nutrient soil (nutrient soil: vermiculite: perlite = 3:1:1), covered with plastic wrap, and placed in an incubator for cultivation. The cultivation conditions were as follows: 16h light / 8h dark, the light intensity is 10000 LUX, the temperature under light and dark conditions are 22±1℃ and 20±1℃ respectively, and the relative humidity is 80%. Two days later, the plastic wrap was removed, and the cultivation was continued for 3 weeks.

[0041] 1.2 The formula and pre...

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Abstract

The invention discloses a method for preparing Arabidopsis thaliana mesophyll cell protoplasts. The specific steps are as follows: cut the Arabidopsis leaves of 3-5 weeks and fix the upper epidermis and the lower surface of the leaves with inelastic paper tape or cloth tape Epidermis; Gently tear off the inelastic paper tape or cloth tape on the lower epidermis of the leaves, put the cut leaves into a petri dish containing enzymatic hydrolysis solution; place the petri dish with leaves on a shaker for cultivation 30-90min or until most or all of the protoplasts are detached from the leaves; transfer the enzymolysis solution containing the protoplasts to a round-bottomed centrifuge tube, and collect the protoplasts by centrifugation; wash the protoplasts with pre-cooled W5 solution, Resuspend protoplasts with W5 solution and place on ice for a minimum of 30 minutes. The invention cultures the protoplasts under dark conditions, effectively solves the phenomenon of cell death and aggregation caused by light culture, and can greatly improve the transformation efficiency.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to an Arabidopsis thaliana mesophyll cell protoplast and its preparation method and application. Background technique [0002] Plant protoplasts are clusters of cells that have had their cell walls removed but remain viable. Due to the lack of protection and support of the cell wall, protoplasts can absorb macromolecules such as microorganisms, organelles and nucleic acids, which makes it an important multipurpose cell system in basic theoretical research, in plant viruses, plant physiology and crop breeding research. There are applications. The protoplasts isolated from different tissues and plants retain the original physiological activity and regulatory functions, and the metabolic mode and physiological environment are closer to the state of the cells on the whole plant, which provides a more efficient cell than bacteria, yeast, insects and mammals. Good cell ...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82C12N5/10
CPCC12N5/04C12N15/82C12N2510/00
Inventor 史勇金维环陈彦惠任真真刘姣姣王林会
Owner HENAN AGRICULTURAL UNIVERSITY
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