Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants

A technology for cryopreservation and recovery culture, which is applied in the field of germplasm resource preservation and thawing cultivation, plant micro-shoot tip small droplet encapsulation and vitrification cryopreservation and thawing culture, which can solve the secondary mechanical damage of shoot tip and technical personnel requirements Higher problems, to achieve high plant regeneration rate, reduce mechanical damage, good economic and social effects

Inactive Publication Date: 2014-07-30
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, in the process of cryopreservation, it is necessary to perform several operations on the delicate shoot tip, which requires high technical personnel and easily causes secondary mechanical damage to the shoot tip

Method used

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  • Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The cryopreservation of embodiment 1 carnation

[0027] Material:

[0028] Carnation varieties: safflower varieties, purchased from the flower market. The flower stems are cut to propagate the test-tube plantlets, and the flower stem clones are obtained.

[0029] Pre-culture medium: containing 0.3mol·L -1 Sucrose MS Broth

[0030] Pretreatment solution: containing 2mol L -1 glycerol and 0.4mol L -1 Sucrose MS Broth

[0031] Vitrification protective agent: containing 3.26mol L -1 Glycerin, 2.42mol·L -1 Ethylene glycol, 1.9mol·L -1 DMSO and 0.4mol L -1 sucrose

[0032] method:

[0033] Select robust tissue-cultured seedlings of the above-mentioned carnation species, peel off the stem tips (1-2mm) of sterile seedlings under a dissecting microscope, dip them in 2%-5% sodium alginate solution without calcium ions, and use tweezers to Place the stem tip neatly on a 3mm×20mm sterile filter paper strip. Immerse the filter paper strip containing the stem tip into 0....

Embodiment 2

[0037] The cryopreservation of embodiment 2 chrysanthemums

[0038] Material:

[0039] Chrysanthemum species: cut chrysanthemum 'Shenma' (single chrysanthemum), purchased from the flower market. Cut out the receptacle to propagate the test-tube plantlets, and obtain the receptacle cloning line.

[0040] Pre-culture medium: containing 0.3mol·L -1 MS solid medium with sucrose

[0041] Pretreatment solution: containing 2mol L -1 glycerol and 0.4mol L -1 Sucrose MS Broth

[0042] Vitrification protective agent: containing 3.26mol L -1 Glycerin, 2.42mol·L -1 Ethylene glycol, 1.9mol·L -1 DMSO and 0.4mol L -1 sucrose

[0043] method:

[0044] Select robust tissue-cultured seedlings of the above-mentioned chrysanthemum species, cut off the stem tips (1.5-2mm) under a dissecting microscope, immerse them in 2%-5% sodium alginate solution without calcium ions, and place the stem tips neatly with tweezers. On 3 mm x 20 mm sterile filter paper strips. Immerse the filter paper ...

Embodiment 3

[0048] The cryopreservation of embodiment 3 lily

[0049] Material:

[0050] Edible Lily (Lilium davidii Var. Unicolor (Hoog) Cotton)

[0051] method:

[0052] Select the robust tissue culture seedlings of the above varieties, immerse 1-2mm small bulbs (including the growth point of the shoot tip) in 2%-5% calcium ion-free sodium alginate solution, and then use tweezers to place the shoot tip neatly. On 3 mm x 20 mm sterile filter paper strips. Immerse the filter paper strip containing the shoot tip in 0.1% calcium chloride solution, fix it for 20 minutes, put the filter paper strip with the shoot tip on the medium of MS+0.5mol·L-1 sucrose in 4 Pre-cultivate at low temperature for 5 days, transfer to 25°C pretreatment solution for 20 minutes, then treat with PVS2 in ice bath for 80 minutes, replace with fresh PVS2 and quickly put into liquid nitrogen.

[0053] Recultivation and survival rate inspection:

[0054] Take out the cryovial that has been frozen in liquid nitroge...

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Abstract

The invention provides a droplet encapsulation-vitrification ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants. Filter paper strips are used as operation carriers in the droplet encapsulation-vitrification ultralow-temperature storing, thawing and recultivating method. The droplet encapsulation-vitrification ultralow-temperature storing, thawing and recultivating method includes steps of stripping aseptic seedling stem tips under an anatomical lens, dipping the stripped aseptic seedling stem tips in 2-5% calcium-free ionic sodium alginate solution, arranging the stem tips on the aseptic filter paper strips, dipping the aseptic filter paper strips in 0.1% calcium chloride solution, fixing the aseptic filter paper strips in the calcium chloride solution for 10-20 minutes, placing the filter paper strips with the stem tips into 0.3-0.5mol L<-1> sugar MS (mass spectrometry) liquid, culturing the stem tips for 1-3 days, placing the filter paper strips in pretreatment solution to treat the stem tips for 20-40 minutes, transferring the filter paper strips into vitrification protective agents to treat the stem tips for 10-80 minutes, transferring the filter paper strips with the stem tips to aseptic cryovials, and storing the stem tips in liquid nitrogen; and taking the filter paper strips with the stem tips out of the cryovials when thawing is required, placing the filter paper strips in MS recultivating solution containing 1.2mol L<-1> sugar, thawing and washing the filter paper strips for 20 minutes, and placing the filter paper strips with the stem tips in recultivating media to recultivate the stem tips until the stem tips grow into plants. The droplet encapsulation-vitrification ultralow-temperature storing, thawing and recultivating method has the advantages that the filter paper strips are used as the operation carriers, so that the probability of direct contact of inoculation appliances and the tender stem tips of the plants is reduced, and mechanical damage is reduced.

Description

technical field [0001] The invention relates to a method for preservation and thawing cultivation of germplasm resources, in particular to a method for ultra-low temperature preservation and thawing cultivation of plant micro shoot tip droplets embedded in vitrification and belongs to the technical field of plant cell engineering. Background technique [0002] In a broad sense, there are two types of conservation of germplasm resources: on-site conservation and off-site conservation. For those plants that cannot be preserved with seeds for a long time, such as vegetative propagation plants and recalcitrant seed plants: Field preservation is easily affected by natural disasters, resulting in germplasm variation or destruction, and genetic traits caused by multiple subcultures in tissue culture preservation Risk of mutation or contamination. Under cryopreservation conditions, the physiological and biochemical activities of plants are almost stopped, and the physiological and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N3/00A01H4/00
Inventor 林田杨华龙萍朱天生刘灶长李天菲罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT
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