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A method for freeing apple pulp protoplasts and its special kit

A technology of protoplasts and kits, applied in the field of plant biology, can solve the problems of few genotypes for successful regeneration and limited sources of protoplasts

Inactive Publication Date: 2015-10-28
CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This stage is the breakthrough stage of apple protoplast culture and regeneration, but there are few genotypes that successfully regenerate, and the source of protoplasts is also limited.

Method used

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  • A method for freeing apple pulp protoplasts and its special kit
  • A method for freeing apple pulp protoplasts and its special kit
  • A method for freeing apple pulp protoplasts and its special kit

Examples

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Embodiment 1

[0052] Embodiment 1, the method that apple pulp protoplast is dissociated

[0053]Since the cultivar used for apple whole genome sequencing was Golden Delicious, the fruit of Golden Delicious was selected as the research object for the convenience of subsequent research. The procedure of free apple pulp protoplasts is roughly: enzyme solution dissociation - nylon mesh filtration - elution suspension - activity detection, so experiments are designed from each step to determine the best system for free apple pulp protoplasts.

[0054] The classic method of dissociating leaf and callus protoplasts is to immerse the material in the enzyme solution and lightly vacuum, so that the enzyme solution can penetrate into the intercellular space and fully dissociate. Therefore, in this experiment, the material treatment is divided into light pumping. Vacuum and no vacuum.

[0055] 1. Isolation of apple pulp protoplasts

[0056] 1. Enzymatic hydrolysis and optimization of its enzymatic so...

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Abstract

The invention discloses a method and dedicated kit for dissociating an apple pulp protoplast and provides a kit for dissociating a pulp protoplast. The kit comprises an enzyme solution, wherein the enzyme solution is composed of a solute A and a solvent, the solute A is composed of cellulase, macerozyme, mannitol, 2-N-morpholinoethanesulfonic acid, ascorbic acid, CaCl2 and BSA (Bovine Serum Albumin), and the proportion of the cellulose to the macerozyme to the mannitol to the 2-N-morpholinoethanesulfonic acid to the ascorbic acid to the CaCl2 to the BSA is (0.3-0.7)g:(0.3-0.7)g:(200-600)mmol:10mmol:5.7mmol:5mmol:0.1g. Proved by experiments, the method disclosed by the invention can be used for dissociating more higher-quality protoplasts.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a method for freeing apple pulp protoplasts and a special kit thereof. Background technique [0002] Apple is a tree species that has been studied earlier in the isolation and culture of protoplasts in deciduous fruit trees. In the past, protoplast free materials were limited to young leaves, petioles, anthers and callus of tissue culture seedlings, and fruits were also limited to culturing suspended cells to free protoplasts. In 1983, Huang et al. first isolated protoplasts from the mesophyll of complete plants. After the technical improvement of different researchers, the isolation of mesophyll protoplasts achieved satisfactory results, and the isolated protoplasts were cultured to obtain callus. Since then, there have been some reports of apple protoplast regeneration. So far, more than 12 apple genotypes have been regenerated from protoplasts, most of which originated fro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
Inventor 贾文锁韩瑜黄丛林吴忠义李自超李金杰李冰冰李兴亮
Owner CHINA AGRI UNIV
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