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Preparation method of rape mesophyll protoplast

A technology for protoplasts and rapeseed leaves, applied in plant cells and other directions, can solve the problems of few research reports, the lag in separation and purification of protoplast homeopathic expression systems, and the inability to achieve convenience, efficiency and speed, and achieve the effect of improving preparation efficiency.

Inactive Publication Date: 2020-09-18
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the genetic transformation system of rapeseed is becoming more and more perfect, the homeopathic expression system of protoplasts, especially its isolation and purification, is obviously lagging behind, and it cannot achieve convenient, efficient and fast results, and there are relatively few related research reports

Method used

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  • Preparation method of rape mesophyll protoplast
  • Preparation method of rape mesophyll protoplast
  • Preparation method of rape mesophyll protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0040] 1. Experiment preparation

[0041] 1. Material preparation

[0042] 3-4 weeks old hydroponic rapeseed (take normal and fully expanded leaves).

[0043] 2. Mother liquor preparation:

[0044] 1M mannitol (MW:182.17), 18.22g dissolved in ddH 2 O, dilute to 100mL, heat to dissolve;

[0045] 1M MgCl 2 ·6H 2 O (MW:203.3), 20.33g dissolved in ddH 2 O, dilute to 100mL;

[0046] 0.1M MES (MW:195.2), 1.95g dissolved in ddH 2 O (heating to dissolve), set the volume to 100mL, adjust the pH to 5.7 with KOH;

[0047] 1M MES (MW:195.2), 19.52g dissolved in ddH 2 O (heating to dissolve), set the volume to 100mL, adjust the pH to 5.7 with KOH;

[0048] 1M CaCl 2 (MW:110.98), 11.1g dissolved in ddH 2 O, dilute to 100mL;

[0049] 0.2M KCl (MW:74.55), 1.49g dissolved in ddH 2 O, dilute to 100mL;

[0050] 1.54M NaCl (MW:58.44), 8.99g dissolved in ddH 2O, dilute to 100mL.

[0051] 3. Solution A (with two kinds: unadjusted pH value and pH=6.0)

[0052]

[0053] ddH 2 0 ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of rape mesophyll protoplast. According to the technology, young rape leaves are used as testmaterials, the influence of different Mannitol concentrations on mesophyll protoplast separation is analyzed, and the optimum concentration is determined; in a purification process, a 20% Ficoll solution is utilized, the complete protoplast stays between a Solution A and the 20% Ficoll solution, the damaged protoplast sinks into the bottom of a tube, and then the Solution A is used for two times of resuspension, so that the integrity of the obtained rape mesophyll protoplast is effectively guaranteed, and meanwhile, the quantity can meet the requirement of subsequent instantaneous conversion;and a convenient, rapid and efficient rape protoplast preparation system is established.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for preparing rapeseed mesophyll protoplasts. Background technique [0002] The remaining plant cells after removal of the cell wall in plant cell engineering are called protoplasts. In the field of biological research, protoplasts are often used as transient expression systems for plants, which have the characteristics of simple transformation and fast detection speed. [0003] Rapeseed is one of the oil crops with the highest oil production efficiency in China, and its research in the field of molecular biology is also deepening, and a large number of important functional genes have been excavated. However, although the genetic transformation system of rapeseed is becoming more and more perfect, the homeopathic expression system of protoplasts, especially its isolation and purification lags behind obviously, and cannot achieve convenient, efficient and fast results, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 丁广大张浩李玉石磊汪社亮徐芳森
Owner HUAZHONG AGRI UNIV
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