A method for ultralow temperature storage of sugarcane protoplast

Abstract

A method for ultralow temperature storage of sugarcane protoplast is disclosed. Young leaves of sugarcane are enzymatically hydrolyzed with 2% cellulase, 0.5% of pectinase, 0.1% of segregase and 0.3%hemicellulase at room temperature for 4 h, 65%-75% of a KM8P medium, 20% of bovine serum albumin and 5%-15% of dimethyl sulfoxide are used as cryopreservation solution. The cryopreservation tube withcell suspension is put into 4 DEG C for 40 min, and then put into -20 DEG C for 30-60 min, and then place at -80 DEG C overnight and finally place at -80 DEG C refrigerator or -196 DEG C liquid nitrogen tank. After thawing and resuscitation, the protoplasts thaw by taking out the cryopreserved tubes and shaken in a constant temperature water bath at 37 DEG C, and then is cultured by KM8P medium after steps of centrifugally discarding the cryopreserved solution, and washing with 9% mannitol three times. The method of the invention can optimize the cryopreservation condition of the sugarcane protoplast, reduce the toxicity of the cryopreservation liquid, improve the cryopreservation effect, retain the biological characteristics of the protoplast to the maximum extent, guarantee the hereditary stability of the sugarcane, and improve the recovery vigor after preservation.

Description

technical field [0001] The invention belongs to the technical field of cell preservation of plant germplasm resources, in particular to a method for cryopreservation of sugarcane protoplasts. Background technique [0002] Plant somatic cell fusion breeding is based on the principle of cell pluripotency and cell membrane fluidity. The cell wall is removed by enzymatic hydrolysis, and the protoplasts of different plant species, genera, and even families are artificially induced to fuse, and then cultured in vitro. A technique for regenerating hybrid plants. It breaks the boundary of incompatibility of sexual hybridization, extensively combines various genotypes, thus forming new hybrid plants that cannot be obtained by sexual hybridization, and cell fusion technology avoids separation, purification, shearing, Genetic operations such as splicing, the transferred gene basically does not have genetic safety issues, because the transferred gene itself exists in sugarcane, the req...

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Problems solved by technology

The disadvantages of this method are: enzymatic hydrolysis takes a long time; unused protoplasts can only be cultivated or discarded, and the efficiency is low; seasonality of materials causes some seasonal experiments to be shelved due to lack of materials

[0005] Since sugarcane not only has a long growth period, but also is a subtropical crop, its growth is limited by seasons and regions. If there is no suitable method for freezing sugarcane protoplasts, it will lead to research on fusion breeding of sugarcane protoplasts not in the sugarcane growing season and subtropical regions. There are no research materials, and a large amount of experimental materials are wasted during the growing season because they cannot be used in time and stored for a long time, which greatly limits the smooth progress of sugarcane body fusion breeding research.

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