Preparation method of cotton protoplast

A protoplast and cotton technology, applied in the field of plant cell biology, can solve the problems of low preparation efficiency, long time consumption, and affecting the vitality of protoplasts, and achieve the effect of short enzymatic hydrolysis time, shortening time, and ensuring the vitality of protoplasts

Active Publication Date: 2020-06-05
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the defects of the above-mentioned existing cotton protoplast preparation using young leaves as materials, the preparation process takes a long time, the preparation efficiency is low, and further affects the vitality of the protoplast, the present invention proposes a cotton protoplast preparation method

Method used

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  • Preparation method of cotton protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A method for preparing cotton protoplasts, comprising the steps of:

[0031] (1) Take cotton flowers, peel off the petals and place them in a petri dish, cut the petals into filaments with a blade, the cotton flowers are fresh cotton flowers on the day of flowering, and the width of the filaments is 0.8mm;

[0032] (2) Put the filament in a 6-well cell culture plate, put a petal in each hole, add 5ml of enzymatic hydrolysis solution to each well, then place the cell culture plate on a shaker for enzymatic hydrolysis, after adding the enzymatic hydrolysis solution, Place the cell plate on a shaker at 28°C, shake at 15 rpm, and perform enzymatic hydrolysis for 1 hour. The enzymatic hydrolysis solution includes 0.9gg cellulase, 0.3gg isolated enzyme, 6.558gg mannitol, 0.1491g KCl, 0.3905 g of MES, 0.111g CaCl 2 , 0.5g of PVP, and dilute to 100ml with sterile water;

[0033] (3) After the enzymolysis ends, the enzymolysis reaction solution is filtered, and after the enzym...

Embodiment 2

[0037] A method for preparing cotton protoplasts, comprising the steps of:

[0038] (1) Take cotton flowers, peel off the petals and place them in a petri dish, cut the petals into filaments with a blade, the cotton flowers are fresh cotton flowers on the day of flowering, and the width of the filaments is 1mm;

[0039] (2) Place the filaments in a 6-well cell culture plate, put a petal in each well, add 6.5ml of enzymatic hydrolysis solution to each well, then place the cell culture plate on a shaker for enzymatic hydrolysis, after adding the enzymatic hydrolysis solution , place the cell plate on a shaker at 28°C, shake at 15 rpm, and perform enzymolysis for 1.25 hours. The enzymolysis solution includes 1.05g cellulase, 0.4g isolated enzyme, 8.016g mannitol, 0.1491g KCl, 0.3905g of MES, 0.111g of CaCl 2 , 0.5g of PVP, and dilute to 100ml with sterile water;

[0040] (3) After the enzymolysis ends, the enzymolysis reaction solution is filtered, and after the enzymolysis end...

Embodiment 3

[0044] A method for preparing cotton protoplasts, comprising the steps of:

[0045] (1) Take cotton flowers, peel off the petals and place them in a petri dish, cut the petals into filaments with a blade, the cotton flowers are fresh cotton flowers on the day of flowering, and the width of the filaments is 1.2mm;

[0046] (2) Place the filaments in a 6-well cell culture plate, put a petal in each hole, add 8ml of enzymatic hydrolysis solution to each well, then place the cell culture plate on a shaker for enzymatic hydrolysis, after adding the enzymatic hydrolysis solution, Place the cell plate on a shaker at 28°C, shake at 15 rpm, and perform enzymatic hydrolysis for 1.5 hours. The enzymatic hydrolysis solution includes 1.2g cellulase, 0.5g isolated enzyme, 9.473g mannitol, 0.1491g KCl, 0.3905 g of MES, 0.111g CaCl 2 , 0.5g of PVP, and dilute to 100ml with sterile water;

[0047] (3) After the enzymolysis ends, the enzymolysis reaction solution is filtered, and after the en...

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Abstract

The invention provides a preparation method of cotton protoplast, and belongs to the technical field of plant cell biology. The preparation method comprises the following steps of (1) taking cotton flowers, and cutting petals into filaments with a blade; (2) placing the filaments in a 6-hole cell culture plate, placing one petal in each hole, adding 5-8ml of enzymatic hydrolysate to each hole, andthen performing enzymolysis; (3) after the enzymolysis is finished, filtering enzymolysis reaction liquid; (4) taking filtrate, adding the taken filtrate into a centrifugal tube, and performing centrifuging for the first time; and (5) absorbing supernatant in the centrifugal tube with a gun head, adding a first W5 solution to protoplast precipitate, performing centrifuging for the second time, absorbing supernatant with the gun head once again, and then adding a second W5 solution once again for re-suspending the protoplast precipitate, so as to obtain protoplast re-suspension. The preparation of the cotton protoplast can be completed within 1.5-2 hours, the time required for preparing the cotton protoplast is greatly shortened, guaranteeing the vitality of the protoplast is facilitated,the quantity can also meet subsequent transient transformation requirements, and the preparation efficiency of the cotton protoplast is greatly improved.

Description

technical field [0001] The invention relates to the technical field of plant cell biology, in particular to a method for preparing cotton protoplasts. Background technique [0002] Protoplast is an important tool for plant cell biology research. Protoplasts are used as receptors for transient transformation, and can be widely used for subcellular localization of target gene proteins, promoter and protein activity detection, and BIFC (bimolecular fluorescence complementation) Gene function research such as protein-protein interaction. The preparation of protoplasts has been studied more in Arabidopsis, tobacco and other plants, but the research on the preparation and application of protoplasts in cotton is less. The subcellular localization of cotton genes, BIFC and other gene function analysis are often achieved by transiently transforming onion epidermis or tobacco using Agrobacterium-mediated or particle gun methods. However, due to the genetic differences of different s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 张雪刘传亮孙瑞斌王少辉马丹栗祎琳刘志红
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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