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Method for isolating efficiently protoplast from switchgrass leaves

A technology of protoplasts and leaves, which is applied in the field of suspension cell lines, can solve the problems of hard leaves and other problems, and achieve the effects of easy operation, high yield and simple operation

Inactive Publication Date: 2012-09-26
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, switchgrass leaves contain a lot of cellulose and the leaves are hard, which has become the biggest obstacle to the isolation of protoplasts

Method used

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  • Method for isolating efficiently protoplast from switchgrass leaves
  • Method for isolating efficiently protoplast from switchgrass leaves
  • Method for isolating efficiently protoplast from switchgrass leaves

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1) Preparation of preplasma wall separation solution, buffer solution and enzymatic hydrolysis solution:

[0028] 0.4 mol mannitol and 8 mmol CaCl are contained in each 1L of preplasma separation liquid 2 ;

[0029] 6 mmol CaCl per 1 L of plasmolysis buffer 2 , 0.4mol mannitol;

[0030] Each 100ml enzymatic solution contains 3g cellulase, 0.2g pectinase, 0.6mmol CaCl 2 , 0.04 mol mannitol, pH=5~6.

[0031] 2) Treatment of switchgrass leaves:

[0032] Collect switchgrass leaves in different periods (one month, half a year), wipe the surface of the leaves with alcohol with a volume fraction of 70%, rinse them with distilled water, and dry them in the air. Gently scrape off the epidermis of the switchgrass leaves with a clean blade, cut them into 0.5 mm filaments, and put them in a 50ml beaker.

[0033] 3) Enzymolysis:

[0034] Firstly, let stand in the pre-plasma separation solution in the dark for 1 hour, suck out the liquid with a pipette gun, and wash the leaves...

Embodiment 2

[0041] 6) Preparation of preplasma wall separation solution, buffer solution and enzymatic hydrolysis solution:

[0042] 0.5 mol mannitol and 10 mmol CaCl are contained in each 1L of preplasma separation liquid 2 ;

[0043] 7 mmol CaCl per 1 L of plasmolysis buffer 2 , 0.5mol mannitol;

[0044] Each 100ml enzymatic solution contains 4g cellulase, 0.2g pectinase, 0.6mmol CaCl 2 , 0.04 mol mannitol, pH=5~6.

[0045] 7) Treatment of switchgrass leaves:

[0046] Collect switchgrass leaves in different periods (one month, half a year), wipe the surface of the leaves with alcohol with a volume fraction of 70% to 80%, rinse them with distilled water, and dry them in the air. Gently scrape off the epidermis of the switchgrass leaves with a clean blade, cut them into 0.5 mm filaments, and put them in a 50ml beaker.

[0047] 8) Enzymolysis:

[0048] Firstly, let it stand in the dark for 1.5 hours in the pre-plasma separation solution, suck out the liquid with a pipette gun, and w...

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PUM

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Abstract

The invention relates to a method for isolating efficiently protoplast from switchgrass leaves, belonging to the technical filed of suspension cell lines. In the method, switchgrass leaves in different periods are selected, and the steps mainly comprise cleaning leave surfaces through wiping with 75% alcohol, cutting the leaves into filaments about 5mm width, putting the filaments into a beaker, keeping the filaments in a pre-plasmolysis liquid standing for 1 hour under dark conditions, adding an enzyme solution, carrying out a low-speed enzymatic hydrolysis for 2 hours, keeping standing for 12 hours, split-charging after-separating liquid into 1.5 ml centrifuge tubes to carry out a centrifugation in 800 rpm for 10 min after which supernatant is abandoned, and using a buffer solution for washing for three times. Obtained lower deposition is dissociated somatic cell protoplast. The method of the invention has advantages of simple and easy operation, and low requirements for equipment and culturing conditions.

Description

technical field [0001] The invention belongs to the technical field of suspension cell lines, and in particular relates to a method for separating protoplasts from switchgrass leaves. Background technique [0002] Protoplasts are viable "naked cells" that have had their cell walls removed and surrounded by a plasma membrane. Since protoplasts have no cell walls, they can be processed under artificial control in many ways, such as cell fusion, cell hybridization, gene control, genetic It is an ideal recipient for gene transformation. Plant protoplast culture is the premise of cell fusion, which can be used to overcome some obstacles in distant hybridization, to combine various favorable genetic traits of plants more extensively, and can be used for organelle transplantation and cell mutant screening. An important part and basic work of technical research has become a new way of improving plant varieties. [0003] In the past 40 years since the successful enzymatic hydrolysi...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 杜道林张欣黄萍薛永来杨冉田远飞
Owner JIANGSU UNIV
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