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Vitrification ultralow-temperature preservation and thawing method of agate red cherry embryonic calluses

An embryogenic callus, vitrification ultra-low temperature technology, applied in the preservation of plants, horticultural methods, botanical equipment and methods, etc. Low cost, low cost, and the effect of improving operational efficiency

Inactive Publication Date: 2020-11-24
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is: provide a kind of preservation and thawing method of agate red cherry embryogenic callus, to solve the low germination rate of existing agate red cherry planting natural sowing, embryogenic callus long-term continuous subculture Proliferation culture will cause the problem of loss of embryogenicity or reduced differentiation ability of cells, while restarting embryogenic callus induction is not only time-consuming and laborious, but also may cause somatic variation

Method used

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  • Vitrification ultralow-temperature preservation and thawing method of agate red cherry embryonic calluses
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  • Vitrification ultralow-temperature preservation and thawing method of agate red cherry embryonic calluses

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Experimental program
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Effect test

Embodiment 1

[0037] A method for vitrification cryopreservation of embryogenic callus of agate red cherry, comprising the following steps:

[0038] S1 pre-cultivation: inoculate the embryogenic callus of agate red cherry after passage 15 days into the medium of MS+50ml / LDS0+1.0mg / LTDZ+0.5mg / LIBA for pre-cultivation, and culture at 4°C for 4 days;

[0039] S2 loading solution treatment: the embryogenic callus of agate red cherry after the precultivation is treated with a volume fraction of 60% PVS2 (MS+30% glycerol+15% ethylene glycol+15%DMSO+0.4mol / L sucrose) at room temperature ) loaded for 20min;

[0040] S3 vitrification dehydration: soak and dehydrate the agate red cherry embryogenic callus with PVS2 at 0°C for 40 minutes;

[0041] S4 Storage in liquid nitrogen: soak the embryogenic callus of agate red cherry after dehydration in fresh PVS2, and then quickly store it in liquid nitrogen.

[0042]A method for thawing the embryogenic callus of agate cherry after vitrification cryopreser...

Embodiment 2

[0044] The operation procedure of embodiment 2 is the same as that of embodiment 1, except that the pre-cultivation time of the embryogenic callus of agate red cherry is respectively set to 0d, 1d, 2d, 3d, 5d and 6d. Determination of the impact of pre-cultivation time on the cryopreservation survival rate of embryogenic callus of agate red cherry, the results are as follows:

[0045] Table 1 Effect of pre-culture time on the relative survival rate of embryogenic callus of agate cherry

[0046]

[0047] Note: 20 calli were treated for each experiment, repeated 4 times

[0048] According to the test result of embodiment 1 and embodiment 2, it can be known that the pre-cultivation time has a greater impact on the survival rate of the cryopreservation of the embryogenic callus of agate cherry, and when the embryogenic callus of agate cherry was pre-cultivated for 0d, the cells contained If the amount of water is too high, a large number of ice crystals will be formed during cr...

Embodiment 3

[0050] The operation steps of embodiment 3 are the same as those of embodiment 1, except that the loading liquid treatment in step S2 and the vitrification dehydration in step 3 use different loading liquid and vitrification protection liquid components, and the vitrification protection liquids are respectively: PVS1 (22 % glycerol+13% ethylene glycol+13% polyethylene glycol+15% dimethyl sulfoxide); PVS2 (30% glycerol+15% ethylene glycol+15% dimethyl sulfoxide+0.4mol / L sucrose ); PVS3 (50% glycerol + 50% sucrose). The loading solution is used corresponding to the vitrification protection solution, and the volume fraction is 60% of the vitrification protection solution. The influence of loading solution and vitrification protection solution on the cryopreservation survival rate of embryogenic callus of agate cherry was determined, and the results are shown in Table 2:

[0051] Table 2 Effects of different vitrification solutions on the survival rate of embryogenic callus of ag...

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Abstract

The invention discloses a preservation and thawing method of agate red cherry embryonic calluses. The preservation and thawing method comprises the following steps of performing pre-culture, performing loading liquid treatment, performing vitrification dehydration, performing liquid nitrogen preservation, performing long-term cryopreservation on the agate red cherry embryonic calluses, performingthawing in a water bath, performing washing, and performing recovery culture to cultivate agate red cherry plants, so as to obtain the agate red cherry embryonic calluses. An ultralow-temperature preservation system for agate red cherry germplasm resources is established, the growth survival rate (61.5%) is far higher than the natural sowing germination rate (5%-20%), and the agate red cherry germplasm resources recovered, cultured and grown after unfreezing are good in hereditary stability. The method solves the problem of embryogenic loss of cells or embryo abortion caused by long-time continuous subculture multiplication culture of the red agate cherry embryogenic calluses, and provides a guarantee for the development of the red agate cherry industry.

Description

technical field [0001] The invention relates to an agate red cherry, in particular to a method for vitrified cryopreservation of embryogenic callus of agate red cherry, and belongs to the technical field of cryopreservation methods. Background technique [0002] Agate red cherry (P.pseudocerasus Lindl'Manaohong') is a new variety of local sour cherry variation found in Nayong County, Guizhou Province, belonging to Rosaceae and Prunus genus. In December 2011, it was approved by Guizhou Crop Variety Approval Committee (Chen Zuyao, 2013). This variety has high yield and high quality, sweet flesh, and good storage resistance. It is currently cultivated in a large area in Guizhou Province and promoted to surrounding provinces and regions. In the cultivation of cherries, due to long-term artificial directional selection, the seeds of many cultivars, especially early-maturing varieties, have weak vitality and insufficient development. After natural sowing, the germination rate is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N3/00A01H4/00
CPCA01H4/001A01H4/005A01N3/00
Inventor 文晓鹏李娅玲宋常美
Owner GUIZHOU UNIV
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