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Method and dedicated kit for dissociating apple pulp protoplast

A protoplast and kit technology, applied in the field of plant biology, can solve the problems of limited source of protoplasts and few genotypes with successful regeneration.

Inactive Publication Date: 2013-10-30
CHINA AGRI UNIV +1
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  • Abstract
  • Description
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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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  • Method and dedicated kit for dissociating apple pulp protoplast
  • Method and dedicated kit for dissociating apple pulp protoplast
  • Method and dedicated kit for dissociating apple pulp protoplast

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0052] Embodiment 1, the free method of apple pulp protoplast

[0053]Since the variety used for apple genome sequencing is Golden Delicious, for the convenience of follow-up research, the fruit of Golden Delicious was selected as the research object. The process of dissociated apple pulp protoplasts is roughly: dissociation with enzyme solution-nylon mesh filtration-elution suspension-activity detection, so design experiments from each step to determine the best system for dissociated apple pulp protoplasts.

[0054] The classic method of freeing leaf and callus protoplasts is to immerse the material in the enzyme solution and lightly pump the 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 With and without vacuum.

[0055] 1. Isolation of apple pulp protoplasts

[0056] 1. Enzymatic hydrolysis and optimization of enzyme solution component...

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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 an earlier tree species in the study of protoplast isolation and culture in deciduous fruit trees. In the past, the free protoplast materials were limited to the young leaves, petioles, anthers and callus of tissue culture seedlings, and the fruit was also limited to culture suspension cells to free protoplasts. In 1983, Huang et al. isolated protoplasts from the mesophyll of intact plants for the first time. After technical improvements by different researchers, the separation of mesophyll protoplasts achieved satisfactory results, and the isolated protoplasts could be cultured to obtain callus. Since then, there have been some reports of apple protoplast regenerated plants. So far, there have been more than 12 apple genotype protoplast regenerated plants, most of wh...

Claims

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Application Information

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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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