A Method for Quickly Obtaining Protoplasts from Herba halophyta Leaf

A protoplast and halophyte technology, applied in the direction of plant cells, can solve the problems of weak halophyte research, achieve good results, eliminate waste, and achieve large yields

Active Publication Date: 2018-08-03
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on halograss is relatively weak in my country, and the preparation of halograss biomass has not been reported. Therefore, it is of great significance to prepare halograss biomass with an efficient and economical method

Method used

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  • A Method for Quickly Obtaining Protoplasts from Herba halophyta Leaf
  • A Method for Quickly Obtaining Protoplasts from Herba halophyta Leaf
  • A Method for Quickly Obtaining Protoplasts from Herba halophyta Leaf

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: a kind of method of fast obtaining grass blade protoplast, its main feature is that the steps are:

[0030] (1) For the planting of seedlings, select full halophytic grass seeds and plant them in flower pots in April, and the volume ratio of vermiculite to sand is 1:3;

[0031] (2) The selection of blades, when the seedlings obtained in step (1) grow to 3 months, cut off the blades near the 2 / 3 of the plant base with a blade, rinse with distilled water three times, and stand by;

[0032] (3) Obtaining protoplasts, dry the leaves obtained in step (2) with filter paper, cut them longitudinally along the main veins of the leaves with a blade, then put them into the enzymatic hydrolysis solution, and perform enzymatic hydrolysis for 2 hours at 22°C in the dark;

[0033] (4) For the purification of protoplasts, add an equal volume of washing solution to the enzymolysis solution after step (3), place it in the dark at 22°C, on a horizontal shaker at 30rpm for 15...

Embodiment 2

[0042]Embodiment 2: A method for rapidly obtaining the protoplasts of halophytes leaves. Take the leaves of potted seedlings for 3 months, wash them three times with distilled water, dry the surface with filter paper, cut them longitudinally along the main veins, and then place them at 25°C in the dark. After 3 hours of enzymatic hydrolysis under the same conditions, add an equal volume of washing solution to the petri dish, then place it on a horizontal shaker at 25°C and 30 rpm for 15 minutes in the dark, and then use a 5ml pipette to suck the enzymolysis solution into 10ml Put the test tube into a centrifuge at 25°C and centrifuge at 2000r for 10 minutes, use a pipette to absorb 5ml of the supernatant, observe it under an inverted microscope, and finally gently blow and beat the above-mentioned protoplasts to make them evenly distributed, then use a pipette to absorb Put one drop on a hemocytometer, cover with a cover glass, count under an inverted microscope, count three ti...

Embodiment 3

[0046] Example 3: A method for rapidly obtaining the protoplasts of halophytes leaves. Take the leaves of potted seedlings for 2 months, wash them three times with distilled water, dry the surface with filter paper, cut them longitudinally along the main veins, and then place them at 25°C in the dark. After 2.30 hours of enzymatic hydrolysis under the same conditions, add an equal volume of washing solution to the petri dish, then place it in a horizontal shaker at 25°C and 30 rpm for 12 minutes in the dark, and then use a 5ml pipette gun to inhale 10ml of the enzymolysis solution Put it into a centrifuge at 25°C and centrifuge at 2500r for 10min, use a pipette to absorb 4ml of the supernatant, observe it under an inverted microscope, and finally gently blow the above protoplasts to make them evenly distributed, then use a pipette Take a drop on a hemocytometer, cover it with a cover glass, place it under an inverted microscope, and count three times in a row, and take the aver...

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Abstract

The present invention relates to a kind of preparation and purification method of saline steppe protoplast, which comprises the following steps: 1. Planting of seedling; 2. Selection of blade; 3. Obtaining of protoplast; Then put it into the enzymolysis solution; 4. Purification of protoplasts, after enzymolysis, add an equal volume of washing solution, and finally use a pipette to draw 4-5ml of the supernatant, which is the purer protoplasts; 5. The mensuration of protoplast yield, calculate the protoplast yield that every gram of blade obtains according to adding how many blades in the enzyme solution, calculation result expresses with the number of protoplasts per gram of blade (piece / g). The present invention is simple and easy, and the obtained protoplasts have high purity, and the number can reach 8.4×104 / g, and can also avoid tissue culture pollution, and can be used for hybridization of somatic cells, various organelles (such as nuclei, chloroplasts, mitochondria, vacuoles, etc.) ) separation, the construction of bioreactors and the fusion breeding of protoplasts lay the foundation.

Description

technical field [0001] The invention relates to a method for preparing and purifying saline grassland protoplasts, in particular to a method for directly obtaining relatively pure protoplasts by enzymolyzing leaves. Background technique [0002] Plant cell protoplast refers to the product of removing the cell wall. It is a real single cell, which can be applied to the fusion between distant parents, easy to introduce foreign genes, and also to separate various organelles (such as nucleus, chloroplast, mitochondria, vacuole, etc.) It is a good material and can provide technical support for the research of material transport and information exchange between cells. Halogeton glomeratus is an annual herbaceous dilute halophyte of the Chenopodiaceae genus Halogeratus, which is distributed in western Gansu, Qinghai, Xinjiang and Tibet. In recent years, it has been paid attention to because of its excellent drought resistance and salt tolerance. It contains a large number of genes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
Inventor 汪军成王化俊姚立蓉孟亚雄李葆春马小乐杨柯赖勇司二静任盼荣李吉睿
Owner GANSU AGRI UNIV
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