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Method for separating protoplasts of different tissues and organs of peanuts and application of method

A protoplast, tissue and organ technology, applied in the field of biotechnology and plant molecular biology, can solve the problem of low research efficiency and achieve the effect of good cell activity and high acquisition rate

Active Publication Date: 2020-09-29
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no uniform configuration standard for the enzymatic solution for removing cell walls. It needs to be prepared according to the differences in plant species and the osmotic pressure of different plant cells, resulting in low research efficiency.

Method used

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  • Method for separating protoplasts of different tissues and organs of peanuts and application of method
  • Method for separating protoplasts of different tissues and organs of peanuts and application of method
  • Method for separating protoplasts of different tissues and organs of peanuts and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 A kind of method for separating peanut tissue organ protoplast

[0039] The method specifically includes the following steps:

[0040] S1. Cut the peanut leaves into fine tissues smaller than 2mm, and then freeze and thaw repeatedly 3 times, wherein, freeze at 0°C for 2 hours, and thaw at room temperature for 1 hour;

[0041] S2. Add enzymatic hydrolysis solution to the frozen-thawed tissues and organs according to the solid-liquid ratio (g / mL) of 1:7, then place in a constant temperature shaker, and enzymolyze at 28°C at a speed of 60g / min for 2.5h , the enzymatic hydrolyzate comprises the following components:

[0042] The mass concentration of cellulase is 3%, the mass concentration of isolated enzyme is 1%, the mass concentration of collapse enzyme is 0.8%, the mass concentration of ferulic acid esterase is 0.4%, the mass concentration of saponin is 0.08%, The mass concentration of sorbitol is 2%, the mass concentration of mannitol is 4.5%, the volume...

Embodiment 2

[0045] Embodiment 2 A kind of method for separating peanut tissue organ protoplast

[0046] The method specifically includes the following steps:

[0047] S1. Cut the peanut stem into small tissues smaller than 2 mm, and then freeze and thaw twice, wherein, freeze at 0°C for 2 hours, and thaw at room temperature for 1 hour;

[0048] S2. Add enzymatic hydrolysis solution to the frozen-thawed tissues and organs according to the solid-liquid ratio (g / mL) of 1:5, and then place them in a constant temperature shaker, and perform enzymatic hydrolysis at 28°C at a speed of 50g / min for 2 hours. The enzymolysis solution includes the following components:

[0049] The mass concentration of cellulase is 1%, the mass concentration of isolated enzyme is 0.5%, the mass concentration of collapse enzyme is 0.5%, the mass concentration of ferulic acid esterase is 0.3%, the mass concentration of saponin is 0.05%, The mass concentration of sorbitol is 1%, the mass concentration of mannitol is ...

Embodiment 3

[0052] Embodiment 3 A kind of method for separating peanut tissue organ protoplast

[0053] The method specifically includes the following steps:

[0054] S1. Cut the young peanut seeds and fruit needles into fine tissues less than 2mm, and then freeze and thaw repeatedly 4 times, wherein, freeze at 4°C for 2 hours, and thaw at room temperature for 1 hour;

[0055] S2. Add enzymatic hydrolysis solution to the frozen-thawed tissues and organs according to the solid-liquid ratio (g / mL) of 1:10, and then place them in a constant temperature oscillator, and perform enzymatic hydrolysis at a speed of 70g / min at 28°C for 3 hours. The enzymolysis solution includes the following components:

[0056] The mass concentration of cellulase is 5%, the mass concentration of isolated enzyme is 2%, the mass concentration of collapse enzyme is 1%, the mass concentration of ferulic acid esterase is 0.5%, the mass concentration of saponin is 0.1%, The mass concentration of sorbitol is 3%, the m...

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Abstract

The invention belongs to the field of biotechnology and plant molecular biology, particularly relates to a method for separating protoplasts of different tissues and organs of peanuts and an application of the method. In order to facilitate separation of the protoplasts of the different tissues and organs of the peanuts, the invention provides the method for separating the protoplasts of the different tissues and organs of the peanuts according to specific structures of tissue cells of the peanuts. The method is a universal method suitable for separating different peanut tissue cells; protoplast cells of different peanut tissues and organs can be rapidly and efficiently prepared; the separated peanut protoplasts are high in yield and good in cell activity; and the method can be applied topeanut molecular biology related researches such as fusion of peanut somatic cells, development of a peanut protein transient expression system, sequencing of peanut single cells and knockout target detection based on CRISPR / Cas9 and CRISPR / Cpf1.

Description

technical field [0001] The invention belongs to the fields of biotechnology and plant molecular biology, and particularly relates to a method for isolating protoplasts of different tissues and organs of peanuts and an application thereof. Background technique [0002] Plant protoplasts are cells surrounded by plasma membranes after the cell wall is removed. A large number of genetically homogeneous cells can be obtained in a short period of time, and they can directly ingest foreign plasmids and DNA. Therefore, they become good recipients of plant genetic transformation. material, which is of great significance to the study of gene function; protoplasts contain all the genetic information of an individual, and can produce important secondary metabolites, and their separation, extraction and detection can provide new ideas for the production of metabolites; protoplasts are also widely used in Somatic cell distant hybridization can overcome hybridization barriers and is an eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N5/14
CPCC12N5/04C12N5/14
Inventor 刘浩陈小平洪彦彬梁炫强
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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