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Process for abstracting root protoplast of rosaceous plants

A technology of protoplasts and Rosaceae, which is applied in the field of extracting root protoplasts of Rosaceae plants, can solve problems such as wasting time, increasing the number of ruptures, and rupture of protoplasts, and achieves the effect of great application prospects

Inactive Publication Date: 2008-10-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the enzyme concentration is too high, it will lead to protoplast poisoning and protoplast rupture; and if the enzymatic hydrolysis time is too long, it will not only lead to the rupture of protoplasts released earlier, but also waste time.

Method used

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  • Process for abstracting root protoplast of rosaceous plants
  • Process for abstracting root protoplast of rosaceous plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, the cultivation of material

[0019] In this example, salt-tolerant Malus zumi Mats were used as experimental materials.

[0020] The media used are as follows:

[0021] (1) MS medium:

[0022] Element

molecular weight

Use concentration (mg / L)

Macronutrients

Potassium nitrate KNO3

Ammonium nitrate NH4NO3

Potassium dihydrogen phosphate KH2PO4

Magnesium Sulfate MgSO4.7H2O

Calcium chloride CaCl2.2H2O

101.11

80.04

136.09

246.47

147.02

1900

1650

170

370

440

trace elements

Potassium iodide KI

Boric acid H3BO3

Manganese Sulfate MnSO4.4H2O

Zinc sulfate ZnSO4.7H2O

Sodium molybdate Na2MoO4.2H2O

Copper Sulfate CuSO4.5H2O

Cobalt chloride CoCl2.62

166.01

61.83

223.01

287.54

241.95

249.68

237.93

0.83

6.2

22.3

8.6

0.25

0.025...

Embodiment 2

[0037] Embodiment 2, extract root protoplast

[0038] 1. Preparation of enzymatic hydrolysis solution

[0039] Enzyme base solution: 2mmol / LCaCl 2 , 1mmol / L MgCl 2 , 0.05mg / mL bovine serum albumin (BSA), 0.044mg / mL ascorbic acid (VC), 0.5mg / mL polyvinylpyrrolidone (PVP), and the rest is water.

[0040] Enzyme solution I: Add Cellulysin (Shanghai Beiji Biotechnology Co., Ltd., XS-16010) to the enzyme base solution, so that the concentration of cellulase is 280U / ml enzymatic solution, and adjust the enzymatic solution The pH value is 5.8. The enzymatic solution was sterilized by filtration through a 0.22 μm mixed cellulose pore filter.

[0041] Enzymolysis solution II: add cellulase RS (Cellulase RS) (Yakult Company, L0011) to the enzyme base solution; and pectinase Y-23 (Pectolase Y-23) (Shanghai Maikun Chemical Co., Ltd., 25M0460), The concentration of cellulase RS is 240U / ml enzymolysis solution, the concentration of pectinase Y-23 is 2U / ml enzymolysis solution, and the ...

Embodiment 3

[0051] Embodiment 3, the extraction of protoplast

[0052] 1. Preparation of enzymatic hydrolysis solution

[0053] Enzyme base solution: 0.5mmol / LCaCl 2 , 0.5mmol / L MgCl 2 , 0.15mg / mL bovine serum albumin (BSA), 0.022mg / mL ascorbic acid (VC), 0.25mg / mL PVP, and the rest is water.

[0054] Enzyme solution I: Add Cellulysin (Shanghai Beiji Biotechnology Co., Ltd., XS-16010) to the enzyme base solution, so that the concentration of cellulase is 210U / ml enzymatic solution, and adjust the enzymatic solution The pH value is 5.8. The enzymatic solution was sterilized by filtration through a 0.22 μm mixed cellulose pore filter.

[0055] Enzymolysis solution II: add cellulase RS (Cellulase RS) (Yakult company, L0011) to the enzyme base solution; and pectinase Y-23 (Pectolase Y-23) (Shanghai Maikun Chemical Co., Ltd., 25M0460), The concentration of cellulase RS is 80 U / ml enzymolysis solution, the concentration of pectinase Y-23 is 1 U / ml enzymolysis solution, and the pH value of ...

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Abstract

The invention discloses a method for extracting a rosaceous plant root protoplast. The method for extracting the rosaceous plant root protoplast is as follows: enzymolysis of a rosaceous plant root is performed at first by utilization of enzymolysis liquid I and then performed by utilization of enzymolysis liquid II, and the rosaceous plant root protoplast is obtained; the enzymolysis liquid I is solution containing 210 to 420U / ml cellulose and the enzymolysis liquid II is solution containing 80 to 320U / ml cellulose and 1 to 4U / ml pectase. Both the output and the vitality of protoplasts obtained by the method are higher than those of the one-step enzymolysis method, and the extraction time is obviously shorter than that of the one-step enzymolysis method and generally requires 2 to 3 hours to complete the process, thereby damage of the method on the protoplasts is relatively small, consequently the method has wide application prospect.

Description

technical field [0001] The invention relates to a method for extracting root protoplasts of Rosaceae plants. Background technique [0002] Protoplast refers to the naked part of a plant cell from which the cell wall has been removed. The use of protoplasts for cell fusion can realize distant genetic recombination and create new genotypes; it can transfer stress resistance traits and improve crop quality; it can transfer traits controlled by cytoplasmic genes. Protoplasts can also be used as recipients for gene recombination and transfer, to cultivate new germplasm of high-quality and high-yield crops, and to increase the content of secondary metabolites in plant cells. Establishing a stable and efficient protoplast extraction system is the key to basic theoretical research on protoplasts and protoplast culture. [0003] In the extraction of plant protoplasts, the yield and vitality of the extracted protoplasts are closely related to the source of raw materials and physiolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
Inventor 韩振海于立杰许雪峰李天忠王忆
Owner CHINA AGRI UNIV
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