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Encapsulation-vitrification ultra-low temperature preservation method for dendrobium protocorm

A technology of ultra-low temperature preservation and vitrification, which is applied in the preservation of plants, botanical equipment and methods, and applications, etc., can solve the problems of loss of germplasm resources, large amount of Dendrobium fruit seeds, and decreased growth of progeny, etc. Preservation, reduced mutation and growth vigor, reduced poisoning effect

Inactive Publication Date: 2012-10-03
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the reproductive ability of Dendrobium orchid itself is extremely weak, and the fruit of Dendrobium has large seeds and no endosperm. Under wild conditions, it must be combined with specific orchid bacteria to germinate.
Coupled with the increasing demand for Dendrobium in the flower market and medicinal market, similar wild resources have been over-harvested, making Dendrobium listed as an endangered species. The use of Dendrobium tissue culture technology has become the main means of Dendrobium mass reproduction, but the long-term Subculture leads to the loss of germplasm resources, the decline of offspring growth, and the decline of morphogenetic ability and genetic stability

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Obtaining Protocorms of Dendrobium: Protocorms of Protocorms of Protocorms of Protocorms of Dendrobium nobile tissues were obtained through protocorm induction technology of Protocorms of Protocorms of Protocorms of Protocorms of Dendrobium nobile (see patent application with application number 201110445130.2), and young protocorms with a diameter of 3~4cm were obtained.

[0027] (2) Pre-cultivation: the young protocorms obtained by the above method were placed in the pre-culture medium, and cultured in the dark at 4°C for 7 days to obtain pre-cultured dendrobium protocorms; the pre-culture medium was added with a final concentration of 4g / L agar powder and 1 / 2MS culture solution with a final concentration of 0.4mol / L sorbitol, pH5.8;

[0028] (3) Two-step embedding: Soak the pre-cultured dendrobium protocorms in step (2) in sodium alginate embedding solution at room temperature (25°C) for 20 minutes, then remove the dendrobium protocorms and sodium alginate-coated ...

Embodiment 2

[0034] A method for cryopreserving dendrobium protocorms by embedding vitrification:

[0035] (1) Acquisition of protocorms: Protocorms of Dendrobium nobile tissue were induced to obtain sterile protocorms of Dendrobium nobile for 30 days (the acquisition method was the same as in Example 1), and young protocorms with a diameter of 3-4 cm were obtained.

[0036] (2) Pre-cultivation: The pre-culture medium is 1 / 2MS culture solution with a final concentration of 4g / L agar powder and a final concentration of 0.4mol / L sucrose, pH 5.8, and other operations are the same as step (2) of Example 1 to obtain the pre-culture Dendrobium protocorms after cultivation;

[0037] (3) Two-step embedding: the sodium alginate embedding solution is MS basic culture with the addition of 20 g / L sodium alginate, 2 mol / L glycerol and 4% (40 g / L) sucrose at the final mass concentration solution; the calcium ion embedding solution is a 1 / 2MS culture solution with a final concentration of 0.3mol / L calci...

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Abstract

The invention discloses an encapsulation-vitrification ultra-low temperature preservation method for dendrobium protocorm. The encapsulation-vitrification ultra-low temperature preservation method comprises the following steps of: taking and placing young and tender the dendrobium protocorm in a pre-culture medium, respectively carrying out encapsulation by using a sodium alginate encapsulation liquid and a calcium ion encapsulation liquid after culturing for 7 days in the darkness at the temperature of 4 DEG C, placing the embedding particles of the dendrobium protocorm in a PVS2 mixed solution with 60% of mass concentration, processing for 30-60min at the temperature of 0 DEG C, removing the PVS2 mixed solution, adding a PVS2 solution, balancing for 2-3h at the temperature of 0 DEG C, rapidly placing the loaded encapsulated particles in cryovials and preserving the cryovials in liquid nitrogen; and when the particles are used, taking out the cryovials preserved in the liquid nitrogen, placing the unfrozen encapsulated particles in the MS basic media to restore the culturing of the particles, and realizing the ultra-low temperature preservation of dendrobium protocorm seeds. The encapsulation-vitrification ultra-low temperature preservation method has the advantages of being capable of effectively reducing the poison of dimethyl sulfoxide caused by a vitrification method to tissues, better preserving the dendrobium protocorm genetic resources and solving the problems of mutation, reduction of growth vigor and the like generated in the repeated subculture process.

Description

(1) Technical field [0001] The invention relates to a method for ultra-low temperature preservation of plant tissues, in particular to a method for ultra-low temperature preservation of dendrobium protocorms by embedding vitrification. (2) Background technology [0002] Dendrobium is the second largest family in the Orchidaceae, which is a perennial herb. The origin is mainly distributed in subtropical and tropical regions, and there are more than 1,000 species, of which there are about 76 species in my country. Is there any Dendrobium orchid posture? The flower color is gorgeous, the flowering period is long, and most of them are aromatic. It is a popular ornamental flower. At the same time, Dendrobium has extremely high medicinal value. Among the 76 species of dendrobium in my country, 40 species can be used for medicinal purposes. Among them, the more famous ones are Dendrobium nobile, Dendrobium officinale and Dendrobium Huoshanense. It is recorded in ancient books...

Claims

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Application Information

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IPC IPC(8): A01N3/00
Inventor 薛大伟朱涵毅王慧中
Owner HANGZHOU NORMAL UNIVERSITY
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