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A kind of cotton protoplast preparation method

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

Active Publication Date: 2022-07-29
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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  • A kind of cotton protoplast preparation method

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Experimental program
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Effect test

Embodiment 1

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

[0031] (1) get cotton flower, peel off petal and place in petri dish, with blade, petal is cut into filament, cotton flower is the fresh cotton flower on the day of blooming, and the width of filament is 0.8mm;

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

[0033] (3) filter the enzymolysis reaction solution after the end of the enzymolysis, and filter the enzymolysis reaction solution with six layers of 400 mesh nylon mesh after the e...

Embodiment 2

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

[0038] (1) get cotton flower, peel off petal and place in petri dish, with blade, petal is cut into filament, cotton flower is the fresh cotton flower on the day of blooming, and the width of filament is 1mm;

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

[0040] (3) filter the enzymolysis reaction solution after the end of the enzymolysis, and filter the enzymolys...

Embodiment 3

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

[0045] (1) get cotton flower, peel off petal and place in petri dish, with blade, petal is cut into filament, cotton flower is the fresh cotton flower on the day of blooming, and the width of filament is 1.2mm;

[0046] (2) Place the filaments in a 6-well cell culture plate, put one petal in each well, add 8 ml of enzymolysis solution to each well, then place the cell culture plate on a shaker for enzymolysis, and after adding the enzymolysis 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 1.2 g of cellulase, 0.5 g of isolated enzyme, 9.473 g of mannitol, 0.1491 g of KCl, and 0.3905 g of cellulase. g MES, 0.111 g CaCl 2 , 0.5g of PVP, and dilute to 100ml with sterile water;

[0047] (3) filter the enzymolysis reaction solution after the end of the enzymolysis, and filter the enzymolysis react...

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Abstract

The invention provides a method for preparing cotton protoplasts, which belongs to the technical field of plant cell biology and includes the following steps: (1) taking cotton flowers, and cutting the petals into filaments with a blade; (2) placing the filaments in 6 holes In the cell culture plate, the amount of one petal is placed in each hole, and 5-8ml of enzymolysis solution is added to each hole, followed by enzymolysis; (3) after the end of the enzymolysis, the enzymolysis reaction solution is filtered; (4) the filtrate is added to a centrifuge tube , the first centrifugation; (5) use a pipette tip to suck off the supernatant in the centrifuge tube, add the first W5 solution to the protoplast pellet, centrifuge for the second time, suck off the supernatant again with a pipette tip, and then add the second The protoplast pellet was resuspended in W5 solution, and the obtained protoplast was resuspended. The preparation of cotton protoplasts can be completed within 1.5-2 hours, which greatly shortens the time required for the preparation of cotton protoplasts, and is conducive to ensuring the vitality of protoplasts, and the quantity can also meet the needs of subsequent instantaneous transformation, greatly improving cotton protoplasts. Preparation efficiency.

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] Protoplasts are an important tool for plant cell biology research. Protoplasts are used as receptors for transient transformation, which can be widely used for subcellular localization of target gene proteins, detection of promoter and protein activity, and BIFC (bimolecular fluorescence complementation). Research on gene function such as protein-protein interaction. Protoplast preparation has been studied more in plants such as Arabidopsis thaliana and tobacco, but less research has been done on the preparation and application of protoplasts in cotton. Subcellular localization of cotton genes, BIFC and other gene function analysis are often achieved by transient transformation of onion epidermis or tobacco by Agrobacterium-mediated method or biolistic method. However, due to the genetic differe...

Claims

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Application Information

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Patent Type & Authority Patents(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