Asiatic cotton protoplast separation and exogenous gene transformation method

A protoplast and exogenous gene technology, applied in the fields of cell biology and molecular biology, can solve the problems of large damage, severe operating conditions, and low yield, so as to improve the separation efficiency, save unnecessary waste, and improve the integrity high effect

Active Publication Date: 2019-05-28
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The preparation of protoplasts mainly includes mechanical methods and enzymatic hydrolysis methods, among which the prep...

Method used

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  • Asiatic cotton protoplast separation and exogenous gene transformation method
  • Asiatic cotton protoplast separation and exogenous gene transformation method
  • Asiatic cotton protoplast separation and exogenous gene transformation method

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Experimental program
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Embodiment 1

[0033] (1) Two groups of 30 full Asian cotton 972 seeds were selected and planted in nutrient soil. One group was cultured under normal light, and the other group was cultured in the dark. The temperature was set at 16h28°C / 8h25°C. About 8-10 days old, when the cotyledons are fully unfolded but the true leaves have not yet grown, samples are taken, and watered 2-3 times during this period.

[0034] (2) Preparations before protoplast preparation: scissors, tweezers, 100ml Erlenmeyer flask, 50ml round-bottom centrifuge tube, 2ml round-bottom centrifuge tube, cut off the top 1ml blue tip and 200ul yellow tip, measuring cylinder and other experimental consumables Sterilize, and configure sterile ultrapure water and filter-sterilized mother liquor.

[0035] Table 1 Mother liquor formula

[0036] Reagent

concentration

product

Remark

NaCl

2M

import sigma

KCl

2M

import sigma

CaCl 2

1M

import sigma

MgC...

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Abstract

The invention discloses an Asiatic cotton protoplast separation and exogenous gene transformation method. The method comprises the steps of Asiatic cotton seed germination, enzymolysis separation of protoplast, centrifugal collection of the protoplast, culture, exogenous gene transformation, microscopic observation and identification and the like. The concentration of the protoplast which is separated from leaves growing in a dark environment meets the experimental requirements, and the integrity is high so that the intact successfully-transformed protoplast can be obtained; therefore, by means of the method for separating the protoplast from the leaves cultured in the dark environment, the purposes of Asiatic cotton protoplast separation and exogenous gene transformation are achieved better. By means of Asiatic cotton protoplast separation and successful exogenous gene transformation, basic help can be provided for carrying out large-scale cell culture of Asiatic cotton, transferringexogenous genes on purpose and carrying out further innovation and quality improvement work.

Description

technical field [0001] The invention belongs to the fields of cell biology and molecular biology, and specifically relates to a method for isolating Asian cotton protoplasts and transforming exogenous genes. Background technique [0002] Plant protoplasts refer to naked cells covered by plasma membranes with the cell walls removed. They are pluripotent and can regenerate complete plants. At the same time, protoplasts are also an ideal single-cell system. Protoplast The protoplast transformation technology can be used for transient gene expression research, mutant materials can be obtained, and exogenous target genes can also be introduced to improve plant germplasm resources. Therefore, protoplasts can provide favorable test materials for many aspects of modern biotechnology research, and have been widely used in basic theoretical research and genetic improvement of plants. The preparation of protoplasts mainly includes mechanical method and enzymatic hydrolysis method, and...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
Inventor 杜雄明王丽媛王晓阳傅国勇何守朴潘兆娥陈保军贾银华耿晓丽王立如庞保印李洪戈
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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