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Vitrification cryopreservation method for vitis amurensis callus tissues

A vitrification ultra-low temperature and ultra-low temperature preservation technology, which is applied in horticultural methods, botanical equipment and methods, horticulture and other directions, can solve the problems of difficult rooting of cutting propagation, low propagation coefficient of layering propagation, and limitation of grafting propagation, etc., so as to avoid the loss of germplasm. Or destroy, avoid genetic variation or pollution, reduce the effect of labor intensity

Inactive Publication Date: 2015-11-11
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The propagation coefficient of layering propagation is low; grafting propagation is limited by the source of scion; cutting propagation is not easy to take root; and in seed propagation, Vitis vinifera is a polygenic hybrid, sowing seedlings will produce about 90% low-yielding single plants

Method used

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  • Vitrification cryopreservation method for vitis amurensis callus tissues
  • Vitrification cryopreservation method for vitis amurensis callus tissues
  • Vitrification cryopreservation method for vitis amurensis callus tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Vitis vine callus vitrification cryopreservation technology

[0043] Experimental materials: from the National Germplasm Resource Garden of Vitis vinifera, the germplasm number is '75122'.

[0044] The specific steps of the experimental method are as follows:

[0045] 1. Material acquisition

[0046] Inoculate vine grape stems with 2 mg L of BA (6-benzylaminoadenine) in MS medium -1 , NAA (naphthalene acetic acid) 0.1mg L -1 , sucrose 30g·L -1 , agar 7g·L -1 medium, the temperature is 25±2°C, the light intensity is 1200Lux~1600Lux, and the callus is induced under the condition of 16h of light every day;

[0047] 2. Callus Dehydration

[0048] Cut the callus into small pieces of 0.2*0.2cm and put them in the ultra-clean workbench for 30 minutes to blow dry the callus properly to dehydrate the callus, which can prevent the formation of ice crystals in the tissue during ultra-low temperature storage.

[0049] 3. Loading with loading solution and cryopre...

Embodiment 2

[0057] The effect of embodiment 2 drying time on the viability of mountain grape callus

[0058] Only the drying time of the callus in the ultra-clean workbench was changed, and the rest of the steps were the same as in Example 1, to study the effect of the drying time on the survival rate of the callus of Vitis vinifera in cryopreservation. The result is as figure 1 shown.

[0059] From figure 1 It can be seen that after the fresh mountain grape callus is frozen, the survival rate is 5%; as the drying time prolongs, the callus survival rate increases. When the drying time is 40min and the water content of the callus is 40.7%. , the highest survival rate was 90%. However, if the drying time is too long, the callus will lose water seriously, and the survival rate of the callus will decrease.

Embodiment 3

[0060] The impact of embodiment 3 loading liquid on the viability of grape callus

[0061] Only change the composition of the loading solution, and the rest of the steps are the same as in Example 1, to study the impact of the loading solution on the cryopreservation survival rate of the grape callus, wherein S1 is ultrapure water with 10% dimethyl sulfoxide (DMSO)+10% B Diol + 8% glucose, S2 is ultrapure water plus 12.5% ​​DMSO + 0.25% hydrolyzed casein, S3 is ultrapure water plus 10% DMSO + 10% sorbitol, S4 is PVS2. The result is as figure 2 As shown, when the S1 loading solution was used to load the vine callus and it was quickly frozen, the vine callus survival rate was the highest, reaching 90%, followed by the S4 loading solution for slow freezing, and the vine callus The survival rate was 75%. When using S2 and S3 loading solution and carrying out step freezing, the survival rate of grape callus was the lowest, both were 20%.

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Abstract

The invention provides a vitrification cryopreservation method for vitis amurensis callus tissues. The vitrification cryopreservation method comprises the steps of callus tissue induction, air drying, cryopreservation and defrosting. The vitis amurensis callus tissues preserved according to the vitrification cryopreservation method are high in reproduction rate; the dangers that germplasms are lost or destroyed because preservation in field nurseries are easily impacted by natural disasters, and that inheritable characters are mutated or polluted because of serial subcultures during tissue culture and preservation of test-tube plantlets can be avoided; through adoption of the vitrification cryopreservation method, long-term succeeding preservation is not needed, and the manual labor intensity is reduced.

Description

technical field [0001] The invention relates to the field of ultra-low temperature preservation, in particular to a vitrification ultra-low temperature preservation method of grape callus. Background technique [0002] Cryopreservation is a modern in vitro preservation technology for germplasm resources developed in the 1970s. Usually stored in liquid nitrogen, the substance metabolism and growth activities in the preserved material cells are almost completely stopped, and they are in a relatively stable biological state, achieving the purpose of long-term preservation of germplasm. Ultra-low temperature preservation is currently the only method that does not require continuous subculture. Medium and long-term preservation method. Vitrification cryopreservation is to place cells or tissues in a vitrification solution composed of a certain proportion of permeable and non-permeable protective agents, so that the material and its vitrification solution solidify into an amorpho...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 孙丹艾军秦红艳赵滢李昌禹杨义明范书田
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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