Ultra-low temperature storage method for shoot tips of euonymus bungeanus

A technology of cryopreservation and Euonymus peach leaf, which is applied in the field of long-term preservation of shoot tips, to achieve the effects of strong cell regeneration ability, high genetic stability, and convenient material extraction

Inactive Publication Date: 2017-08-01
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The cryopreservation of shoot tip of Euonymus peach leaf has not been reported yet

Method used

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  • Ultra-low temperature storage method for shoot tips of euonymus bungeanus
  • Ultra-low temperature storage method for shoot tips of euonymus bungeanus

Examples

Experimental program
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Effect test

Embodiment 1

[0027] A method for ultra-low temperature preservation of the stem tip of Euonymus peach, the specific operation is as follows:

[0028] 1) Under aseptic conditions, the in vitro stem apex with the apex length of 2~3mm extracted from the healthy growing sterile Euonymus lanceolata tissue culture seedling;

[0029] 2) Put the stripped stem tips into the pre-culture solution, the optimum concentration of sucrose in the pre-culture solution is 0.4mol / L MS salt solution, and cultivate in the dark in an artificial climate box at 25℃ for 2 days;

[0030] 3) Transfer the pre-cultured stem apex to the loading solution and load it at room temperature for 10-30 minutes; the composition of the loading solution is: MS salt solution + 2M glycerol + 0.4M sucrose;

[0031] 4) Put the loaded stem tip into 100% PVS2 vitrification solution and treat it at 0°C for 30 minutes; the composition of the PVS2 vitrification solution is: MS salt solution + 30% glycerin + 15% ethylene glycol + 15 % Dimethyl sulf...

Embodiment 2

[0035] Example 2 Obtaining sterile seedlings

[0036] During the vegetative growth period (May to August) of Euonymus vulgaris, the explants with stem tips are brought back to the laboratory to be washed with detergent and rinsed under running water for 1 to 2 hours. After rinsing, place the explants on a clean bench for sterilization. First, use 75% alcohol to sterilize the surface for 10 seconds, and rinse with sterile water for 3 to 4 times; then use different sterilizing agents for different time for deep sterilization (as shown in Table 1), and finally rinse with sterile water 6 to 8 times. Then the stripped shoot tips were inoculated on MS+6-BA2.0mg / L+NAA0.5mg / L+30g / L sucrose+7g / L agar, pH 5.8 medium. After 15 days, the pollution rate, death rate and survival rate in each bottle were counted. At the same time, in order to study the most suitable medium for callus, the shoot tips were inoculated on the medium of different concentrations, MS as the basic medium, 30g / L sucr...

Embodiment 3

[0047] The effect of the length of the stem tip on the ultra-low temperature storage of the stem tip of Euonymus vulgaris was tested: the apex of the healthy sterile euonymus lanceolata tissue cultured seedlings was peeled off under aseptic conditions, and the symmetrical shape of the apex of the euonymus vulgaris can be observed through a stereo microscope. Therefore, during the peeling process, the length of the stem tip can be determined according to the logarithm of the remaining young leaves. A total of 1~2mm (excluding leaf primordium), 2~3mm (including a pair of leaf primordia), 3~5mm (including two pairs of leaf primordia) ) Three gradients. The research results show that (such as figure 1 Shown) The length of the stem tip of Euonymus peach has a significant effect on the survival rate of the stem tip after cryopreservation. When the length of the shoot tip is 2 to 3 mm, the survival rate of the shoot tip after cryopreservation can reach 83.33%, which is much greater th...

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Abstract

The invention relates to an ultra-low temperature storage method for shoot tips of euonymus bungeanus. The method sequentially comprises the steps of stripping of the shoot tips of euonymus bungeanus, shoot tips pre-treating, loading, PVS2 vitrification treatment, liquid nitrogen storage, unfreezing, unloading and renewal cultivation. The method has the advantages of being simple in operation, occupying a small space, being safe and stable, and being low in storage cost. The shoot tips stored at ultra-low temperature can keep integral, stable inheritance. In addition, a low temperature environment can prevent introduction of diseases and insects. The ultra-low temperature storage method for shoot tips of euonymus bungeanus has practice and promotion significance in crossbreeding of euonymus bungeanus and molecular research.

Description

Technical field [0001] The invention relates to a long-term preservation method of stem tips, in particular to a method for ultra-low temperature preservation of peach leaf Euonymus stem tips. Background technique [0002] Peach-leaf Euonymus, also known as Baidu and Silk Mianmu, is a small tree of the genus Euonymus of the family Euonymus, and is one of the common ornamental garden tree species in my country. Its leaves are beautiful, the plants are luxuriant, and the red aril is covered The fruit of the tree can live on the branches for a long time. In winter, the white snow and the red fruit are very beautiful and add a lot of color to the northern gardens in winter. Euonymus japonicus has good adaptability in terms of cold tolerance, drought resistance, wind resistance, etc.; Euonymus japonicus fruit total saponins have obvious inhibitory effects on human liver cancer cell line-SMMC, human cervical cancer cell line-HeLa and breast cancer cell line-MCF7 The seeds of Euonymus p...

Claims

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Application Information

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IPC IPC(8): A01N3/00
CPCA01N3/00
Inventor 宋红李享苏晴
Owner NORTHEAST FORESTRY UNIVERSITY
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