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Extraction method of pear leaf protoplast

A protoplast and extraction method technology, applied in the field of pear leaf protoplast extraction, can solve the problems that it is difficult to suppress browning, protoplasts are not easy to succeed, and browning is easy to occur

Pending Publication Date: 2021-09-14
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since pear tissue is a material that is prone to browning, in the process of preparing protoplasts, cutting pear leaves into filaments is very easy to cause browning, and it is difficult to inhibit browning even if antioxidants are added.
This is also the reason why it is not easy to succeed in preparing protoplasts from pears.

Method used

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  • Extraction method of pear leaf protoplast
  • Extraction method of pear leaf protoplast
  • Extraction method of pear leaf protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] (1) Take Xinli No. 7 tissue culture seedlings grown for 35 days, fully expanded and healthy leaves 2 to 3 nodes from the shoot tip (such as figure 1 shown). To prevent deactivation from loss of moisture, soak it in water (such as figure 2 shown).

[0039] (2) Enzyme solution configuration:

[0040] Cellulase 1.0% (wt / vol), isolated enzyme 0.4% (wt / vol), pectinase 0.1% w / v, mannitol 0.5mol / L, 2-(N-morpholino)ethanesulfonic acid (MES) 20mMpH5.8, potassium chloride 20mM mixed, put in a 55 ℃ water bath for 10min, after cooling to room temperature, add 10mM calcium chloride and 0.1% bovine serum albumin, filter into the Erlenmeyer flask with a 0.45μm filter head, and obtain the enzymatic hydrolysis liquid for use.

[0041] (3) Take the leaves of the tissue cultured seedlings and cut them into 0.5-1mm wide filaments with a scalpel blade. The mass volume ratio to the enzymolysis solution is 1:10.

[0042] (4) Enzymatic hydrolysis at 25°C for 12 hours in the dark. Obser...

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Abstract

The invention discloses an extraction method of pear leaf protoplast, and belongs to the technical field of protoplast. The method adopts pear tissue culture seedling leaves, solves the problems that field seedlings are easy to brown and complete protoplast cannot be prepared, and breaks the bottleneck of pear protoplast preparation; the enzymatic hydrolysate is adopted, so that the extraction efficiency is improved and the structural integrity of the protoplast is ensured; and the pear leaf protoplast prepared by the method can improve the concentration of the protoplast and maintain the integrity and activity of the protoplast, and the concentration of the prepared pear leaf protoplast can reach 106 parts / ml.

Description

technical field [0001] The invention relates to the technical field of protoplasts, in particular to a method for extracting pear leaf protoplasts. Background technique [0002] Pear belongs to Rosaceae (Rosaceae) Pyrus (Pyrus L.) plant, is one of important fruit tree species in my country, has very high economic value and health care function. At present, new varieties of pears are mainly bred through traditional methods such as natural selection and selection of offspring through sexual hybridization. These methods are time-consuming and have low breeding efficiency. With the increasing maturity of biotechnology, biotechnology breeding combined with genomics and molecular biology gradually breaks the limitations of some conventional breeding and becomes a new breeding method. However, there is still no efficient and stable genetic system for pear, which directly hinders the development of biotechnology breeding and functional genomics. [0003] Protoplast is a general te...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04A01H6/7481
Inventor 许建锋高成昱马辉张海霞于新乐李英丽张江红张玉星
Owner HEBEI AGRICULTURAL UNIV.
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