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Ultralow-temperature preservation method for Eucommia ulmoides suspension cells

A technology for ultra-low temperature preservation and suspension cells, which is applied in the field of ultra-low temperature preservation of eucommia suspension cells to achieve the effect of reducing free water content, simple equipment and improving cell survival.

Inactive Publication Date: 2019-11-22
徐宁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, any kind of material can only find out the suitable cryopreservation scheme through experiments

Method used

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  • Ultralow-temperature preservation method for Eucommia ulmoides suspension cells
  • Ultralow-temperature preservation method for Eucommia ulmoides suspension cells
  • Ultralow-temperature preservation method for Eucommia ulmoides suspension cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] A method for cryopreservation of Eucommia suspension cells, the method comprising the following steps:

[0030] (1) To establish a cell suspension culture line: select Eucommia calli with vigorous growth, loose structure, and easy dispersion to inoculate the suspension medium (composition: B 5 Liquid medium+NAA0.5mg / L+6-BA1.0mg / L+Phenylpeptamine 0.2mg / L+Dithiothreitol 5.0mg / L+Hydrolyzed casein 80mg / L+Sucrose 30g / L, pH 5.6) medium, the inoculum amount was 35g / L fresh weight, and placed on a constant temperature shaker for shaking culture; after 10 days of culture, filter with a 40-mesh sieve to remove large callus pieces and larger cell clusters; culture every 20~25d Subculture 1 time, subculture 3~4 times to obtain a cell suspension culture line containing only single cells or small cell clusters; the conditions for shaking culture are: shaker speed 110r / min, culture temperature 23°C, light intensity 800lx, light time 11h / d.

[0031] (2) Pre-cultivation: Eucommia ulmo...

Embodiment 2

[0037] Example 2 The impact of pre-culture time on cell viability after cryopreservation:

[0038] This experiment compared the effect of different preculture time on the cryopreservation effect of Eucommia suspension cells. According to the method of Example 1, in step (2), the Eucommia suspension cells were pre-cultured for 0d, 1d, 3d, 5d, 7d, and 9d, and the other steps were the same as in Example 1, and the cell survival rate was calculated. The test results are shown below Table 1.

[0039]

[0040] As shown in Table 1, the cell survival rate of suspension cells without pre-culture was the lowest, only 32.43%, indicating that the cell viability was low and the antifreeze ability was weak; during the 1-5 days of pre-culture, the cell survival rate decreased with The prolongation of the pre-cultivation time increases, and the antifreeze ability also gradually improves; when the pre-culture time is 5 days, the cell survival rate reaches the highest, which is 88.62%; but ...

Embodiment 3

[0041] Example 3 Effect of pre-medium on cell viability after cryopreservation

[0042] This experiment compared the effect of different pre-medium on cryopreservation effect of Eucommia suspension cells. According to the method of Example 1, the following pre-culture media were used in step (2), and the steps were the same as in Example 1, and the cell survival rate was calculated. The test results are shown in Table 2 below.

[0043] Pre-medium 1: Pre-medium of the present invention in Example 1; Pre-medium 2: B 5 Liquid medium + 5% DMSO, reference "Cryogenic preservation of Eucommia callus"; pre-medium 3: MS liquid medium + 5% DMSO, reference "Vitrification method of suspension culture cells of Rhodiola longifera Research on cryopreservation"; pre-medium 4: MS liquid medium + 700mg / L hydrolyzed milk protein + 8% sucrose, reference "Research on cryopreservation of American ginseng suspension cells".

[0044]

[0045] The purpose of pre-culture is to improve the antifree...

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Abstract

The invention relates to an ultralow-temperature preservation method of Eucommia ulmoides suspension cells. The method comprises the following steps: (1) a step of establishing a cell suspension culture system; (2) a pre-culture step, namely a step of inoculating the suspension cells into a pre-culture medium, and culturing the cells at 4-6 DEG C for 5 days; (3) a pretreatment step, namely a stepof treating the suspension cells with a pre-cooled vitrification solution and a cryoprotectant in sequence; (4) a cryopreservation step, namely a step of adding a pre-cooled cryoprotectant into the suspension cells, and directly putting the suspension cells into liquid nitrogen for preservation; (5) a thawing and washing step, namely a step of thawing the cells in a water bath at 40 DEG C, and repeatedly washing the cells for 3-5 times; (6) a recovery culture step. Aiming at characteristics of the Eucommia ulmoides suspension cells, the proper pre-culture medium and the proper cryoprotectant are explored, the whole technical process is optimized, the survival rate of the Eucommia ulmoides suspension cells preserved through the method can reach 88% or above, needed equipment is simple, treatment steps are convenient, repeatability is good, cell preservation is safe and stable for a long time, and the cost is low.

Description

technical field [0001] The invention relates to a method for ultra-low temperature preservation of eucommia suspension cells, belonging to the technical field of medicinal plant cell preservation. Background technique [0002] Eucommia ( Eucommia ulmoides Oliv.), also known as Sizhong, Sixian, etc., is a perennial deciduous tree of the Eucommia family Eucommia, with a tree height of up to 20 meters and a breast diameter of about 50 centimeters. Eucommia ulmoides likes a warm and cool climate. It is a light-loving tree species. It can grow well under strong light and full light conditions. It has strong adaptability and has no strict selection of soil. However, the soil is deep, loose, fertile, moist and well-drained. best. The growth rate of Eucommia ulmoides is relatively slow in its infancy, and the fast-growing period appears in 7 to 20 years. After 20 years, the growth rate decreases year by year. After 50 years, the growth of the tree height basically stops, and the ...

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 徐宁
Owner 徐宁
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