CN109769802B - Cryopreservation method and thawing method of vitis amurensis stem section and thawed vitis amurensis stem section - Google Patents
Cryopreservation method and thawing method of vitis amurensis stem section and thawed vitis amurensis stem section Download PDFInfo
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Abstract
The invention relates to the technical field of agriculture, and provides an ultralow-temperature preservation method for vitis amurensis stems, which comprises the following steps: placing the vitis amurensis stem section with the dormant bud and the water content of 28-32% in a vacuum container; and cooling the vacuum container with the vitis amurensis stem segments to-200 to-160 ℃ at a cooling rate of 0.2 to 0.6 ℃/min, and then putting the vacuum container into a liquid nitrogen environment for storage. The method has long storage time and high survival rate. A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps: the vitis amurensis stem segments preserved by the preservation method are taken out from a liquid nitrogen environment, naturally unfrozen at room temperature for 1.5-2.5 hours, and then soaked in water for 22-26 hours. The method has high survival rate of the thawed Vitis amurensis stem. A vitis amurensis stem section unfrozen after being preserved at ultralow temperature is obtained by adopting the unfreezing method. The vitis amurensis stem has high survival rate.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to a method for preserving and unfreezing vitis amurensis stem sections at ultralow temperature and the unfrozen vitis amurensis stem sections.
Background
The Vitis amurensis Rupr is a perennial deciduous vine berry of Vitis of vitidae, which originates from northeast and far east russia in China and is distributed in the korean peninsula. The wild grape variety basically does not have white rot, anthracnose and spike stalk brown blight, downy mildew is light or does not occur, pesticides do not need to be sprayed to prevent and control diseases, the 'green wild grape wine' is brewed by the wild grape berry, and the fact that the brewing of the high-grade dry wild grape wine is realized from the brewing of the low-grade sweet wild grape wine to the brewing of the high-grade dry wild grape wine is realized because the sugar content (soluble solid content) of the wild grape berry can reach 18% -23%. With the continuous development of the wine brewing industry of the amur grape, the demand of human beings on raw materials is larger and larger, the activities of over-development and natural resource utilization are intensified, the living environment of wild amur grape is damaged, the population quantity of amur grape is sharply reduced, and the amur grape is positioned at the edge of extinction or being on extinction. Therefore, the preservation of germplasm resources can ensure that the germplasm resources of the amur grape are not lost any more, and is vital to germplasm innovation of the amur grape and production of high-quality seedlings.
In the prior art, field nursery storage or test-tube plantlet storage is generally adopted, the field nursery storage is easily influenced by natural disasters to cause germplasm loss or destruction, and the test-tube plantlet storage is easily subjected to genetic character variation or pollution caused by multiple subcultures. Therefore, the preservation or long-term preservation of vitis amurensis stem segments for breeding is particularly important.
At present, ultra-low temperature preservation is a modern germplasm resource in vitro preservation technology developed in the last 70 th century. The germ plasm is preserved in liquid nitrogen, the metabolism and growth of the material in the cell are almost completely stopped, and the material is in a relatively stable biological state, so the genetic stability of the germ plasm can be maintained, the purpose of preserving the germ plasm for a long time is achieved, and the ultra-low temperature preservation is the only medium-long term preservation mode which does not need continuous subculture at present. However, the existing vitis amurensis stem segments are basically not preserved at ultralow temperature, or even if the vitis amurensis stem segments are preserved at ultralow temperature, the survival rate is low and is difficult to exceed 50 percent.
In view of this, the present application is specifically made.
Disclosure of Invention
The invention provides an ultralow-temperature preservation method of vitis amurensis stems, which aims to prolong the preservation time of the vitis amurensis stems and has higher germination rate after being thawed under the condition of long-time preservation.
The invention provides a method for unfreezing vitis amurensis stem sections preserved at an ultralow temperature, which is used for unfreezing vitis amurensis stem sections preserved by the method for ultralow temperature preservation of vitis amurensis stem sections, and the unfrozen vitis amurensis stem sections have high germination rate.
The invention also provides a vitis amurensis stem section which is unfrozen after being preserved at ultralow temperature, and the vitis amurensis stem section has high germination rate.
The invention is realized by the following steps:
a cryopreservation method of vitis amurensis stem segments comprises the following steps:
placing the vitis amurensis stem sections with dormant buds with the water content of 28% -32% in a vacuum container;
and (3) cooling the vacuum container with the vitis amurensis stem segments to-200 to-160 ℃ at a cooling rate of 0.2 to 0.6 ℃/min, and then putting the vacuum container into a liquid nitrogen environment for storage.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem segments preserved by the preservation method are taken out from a liquid nitrogen environment, naturally unfrozen at room temperature for 1.5-2.5 hours, and then soaked in water for 22-26 hours.
A vitis amurensis stem section unfrozen after being preserved at ultralow temperature is obtained by adopting the unfreezing method.
The invention has the beneficial effects that: according to the method for preserving the vitis amurensis stem at the ultralow temperature, due to the adoption of liquid nitrogen ultralow temperature preservation, the metabolism of vitis amurensis stem cells is almost completely stopped in the preservation process, the method can realize the long-term preservation of the vitis amurensis stem, the water content of the vitis amurensis stem is reduced to be within the range of 28% -32% before preservation, the vitis amurensis stem with the water content is subjected to vacuum packaging, and is immediately put into a liquid nitrogen environment after being cooled to-200 to-160 ℃ at the cooling rate of 0.2-0.6 ℃/min, so that the germination rate of the vitis amurensis stem obtained by unfreezing after ultralow temperature cryopreservation can reach 65% or more.
The thawing method of the vitis amurensis stem section subjected to the ultralow-temperature preservation, which is designed by the invention, is used for thawing the vitis amurensis stem section frozen and preserved by the ultralow-temperature preservation method provided by the invention, and the germination rate of the thawed vitis amurensis stem section is high.
The vitis amurensis stem section which is obtained through the design and is subjected to ultralow temperature preservation is obtained by adopting the unfreezing method provided by the invention, and the vitis amurensis stem section has high germination rate.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following provides a detailed description of the cryopreservation method of vitis amurensis stem, the thawing method of the vitis amurensis stem after cryopreservation, and the vitis amurensis stem thawed after cryopreservation.
A cryopreservation method of vitis amurensis stem segments comprises the following steps:
s1, placing the vitis amurensis stems with the dormant buds with the water content of 28% -32% in a vacuum container.
Specifically, the vitis amurensis branches with the diameter of 0.6 cm-0.8 cm are cut into vitis amurensis stem sections with the length of 8 cm-12 cm in the month of January in winter, and the number of dormant buds on each stem section is one. And then, placing the vitis amurensis stems in a refrigerator, and adjusting the temperature to 2-5 ℃ so that the vitis amurensis stems are dried to 28% -32%. Then the vitis amurensis stems are placed in an aluminum foil bag, and the aluminum foil bag is vacuumized and sealed.
The preferred vitis amurensis stem section with the size is more suitable for ultralow temperature storage, the germination rate after storage is relatively high, and the preferred reason is that each stem section is provided with a dormant bud so as to ensure that the subsequent germination rate can be higher. And preferably, the vitis amurensis stem section is dried at the temperature of 2-5 ℃, so that the cell activity can be reduced on the premise of not damaging the cell structure of the stem section, and the cells lose water to a proper range.
S2, cooling the vacuum container with the vitis amurensis stems to-200 to-160 ℃ at a cooling rate of 0.2 to 0.6 ℃/min, and then putting the vacuum container into a liquid nitrogen environment for storage.
Specifically, the aluminum foil bag vacuum bag filled with the vitis amurensis stem segments is placed in a programmed cooling instrument, cooled to-200 to-160 ℃ at a cooling rate of 0.2 to 0.6 ℃/min and then rapidly put into a liquid nitrogen tank for storage so as to avoid temperature change in the transfer process.
According to the method for ultralow-temperature preservation of the vitis amurensis stem segments, the metabolism of vitis amurensis stem segment cells is almost completely stopped in the preservation process due to the adoption of liquid nitrogen preservation, the method can realize the long-term preservation of the vitis amurensis stem segments, the water content of the vitis amurensis stem segments is in the range of 28% -32%, the water content is not too low, so that the cells are easy to die due to low water content, and the water content is not too high, so that the cells die due to the change of a large amount of water state due to too high water content in the subsequent ultralow-temperature environment. The vitis amurensis stem section is arranged in the vacuum container, can be isolated from air and is not directly contacted with liquid nitrogen after being arranged in the liquid nitrogen, so that the cell death of the vitis amurensis stem section can be effectively avoided when the vitis amurensis stem section is arranged in the liquid nitrogen. The vitis amurensis stem section is cooled to a proper temperature range at the cooling rate provided by the invention, so that the vitis amurensis stem section can gradually adapt to a low-temperature environment, and the survival rate is improved. The cooling rate can not be too high or too low, the germination rate of the vitis amurensis stem segments can be greatly reduced when the cooling rate is too high or too low, the vitis amurensis stem segments are thrown into a liquid nitrogen environment when the reduced temperature does not reach the range required by the invention, and the germination rate can be greatly reduced when the vitis amurensis stem segments are not adapted to low temperature when the temperature is too high.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem segments preserved by the preservation method are taken out from a liquid nitrogen environment, naturally unfrozen at room temperature for 1.5-2.5 hours, and then soaked in water for 22-26 hours.
After the vitis amurensis stem section preserved at ultralow temperature is thawed by adopting the thawing method provided by the invention, the germination rate of the vitis amurensis stem section is high.
The method comprises the following steps of soaking the seeds in water for 22-26 hours in order to further improve the germination rate: changing water every 50-60 min for the first 5-7 h, and changing water every 5-7 h for the rest time in 22-26 h. Preferably, the water used for soaking is purified water.
The vitis amurensis stem section which is subjected to ultralow temperature preservation is obtained by adopting the unfreezing method provided by the invention, and the vitis amurensis stem section has high germination rate.
The present invention provides a method for cryopreservation of vitis amurensis stem, a method for thawing a vitis amurensis stem after cryopreservation, and a vitis amurensis stem thawed after cryopreservation, which are described in detail below with reference to specific embodiments.
Example 1
The present embodiment provides a method for cryopreservation of vitis amurensis stem segments, a method for thawing the vitis amurensis stem segments subjected to cryopreservation, and a vitis amurensis stem segment thawed after cryopreservation.
A cryopreservation method of vitis amurensis stem segments comprises the following steps:
in January in winter, 0.6 cm-0.8 cm-diameter vitis amurensis branches are cut into vitis amurensis stem sections with the length of 8cm, and the number of dormant buds on each stem section is one. The vitis amurensis stem sections were then placed in a refrigerator and the temperature was adjusted to 4 ℃ so that the vitis amurensis stem sections were dried to a humidity of 30%. Then the vitis amurensis stems are placed in an aluminum foil bag, and the aluminum foil bag is vacuumized and sealed.
Placing the aluminum foil bag vacuum bag filled with the vitis amurensis stem segment in a programmed cooling instrument, cooling to-180 ℃ at a cooling rate of 0.5 ℃/min, and then quickly putting into a liquid nitrogen tank for storage so as to avoid temperature change in the transfer process.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem section preserved by the cryopreservation method of the vitis amurensis stem section provided by the embodiment is taken out from a liquid nitrogen tank, is naturally thawed at room temperature for 2 hours, is then soaked in water for 24 hours, is changed with water every 1 hour in the first 6 hours, is changed with water every 6 hours in the last 18 hours, is taken out after soaking, is thawed, and is finished, and the thawed vitis amurensis stem section is the vitis amurensis stem section which is thawed after cryopreservation and provided by the embodiment.
Example 2
The present embodiment provides a method for cryopreservation of vitis amurensis stem segments, a method for thawing the vitis amurensis stem segments subjected to cryopreservation, and a vitis amurensis stem segment thawed after cryopreservation.
A cryopreservation method of vitis amurensis stem segments comprises the following steps:
in January in winter, 0.6 cm-0.8 cm-diameter vitis amurensis branches are cut into vitis amurensis stem sections with the length of 12cm, and the number of dormant buds on each stem section is one. The vitis amurensis stem sections were then placed in a refrigerator and the temperature was adjusted to 2 ℃ so that the vitis amurensis stem sections were dried to a humidity of 28%. Then the vitis amurensis stems are placed in an aluminum foil bag, and the aluminum foil bag is vacuumized and sealed.
And (3) placing the aluminum foil bag vacuum bag filled with the vitis amurensis stem segments in a programmed cooling instrument, cooling to-160 ℃ at a cooling rate of 0.2 ℃/min, and then quickly putting into a liquid nitrogen tank for storage so as to prevent the temperature from changing in the transferring process.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem section preserved by the cryopreservation method of the vitis amurensis stem section provided by the embodiment is taken out from a liquid nitrogen tank, is naturally thawed at room temperature for 1.5 hours, is then soaked in water for 22 hours, water is changed every 1 hour for the first 7 hours, water is changed every 5 hours for the next 15 hours, the vitis amurensis stem section is taken out after soaking, and is thawed, wherein the thawed vitis amurensis stem section is the vitis amurensis stem section which is thawed after cryopreservation and provided by the embodiment.
Example 3
The present embodiment provides a method for cryopreservation of vitis amurensis stem segments, a method for thawing the vitis amurensis stem segments subjected to cryopreservation, and a vitis amurensis stem segment thawed after cryopreservation.
A cryopreservation method of vitis amurensis stem segments comprises the following steps:
in January in winter, 0.6 cm-0.8 cm-diameter vitis amurensis branches are cut into vitis amurensis stem sections with the length of 8cm, and the number of dormant buds on each stem section is one. The vitis amurensis stem sections were then placed in a refrigerator and the temperature was adjusted to 5 ℃ so that the vitis amurensis stem sections were dried to a humidity of 32%. Then the vitis amurensis stems are placed in an aluminum foil bag, and the aluminum foil bag is vacuumized and sealed.
And (3) placing the aluminum foil bag vacuum bag filled with the vitis amurensis stem segments in a programmed cooling instrument, cooling to-200 ℃ at a cooling rate of 0.6 ℃/min, and then quickly putting into a liquid nitrogen tank for storage so as to prevent the temperature from changing in the transferring process.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem section preserved by the cryopreservation method of the vitis amurensis stem section provided by the embodiment is taken out from a liquid nitrogen tank, is placed at room temperature for natural thawing for 2.5 hours, is then placed in water for soaking for 26 hours, is changed with water every 50min for the first 5 hours, is changed with water every 7 hours for the next 21 hours, is taken out after soaking, is thawed, and is finished, wherein the thawed vitis amurensis stem section is the vitis amurensis stem section which is thawed after cryopreservation and provided by the embodiment.
Example 4
The present embodiment provides a method for cryopreservation of vitis amurensis stem segments, a method for thawing the vitis amurensis stem segments subjected to cryopreservation, and a vitis amurensis stem segment thawed after cryopreservation.
A cryopreservation method of vitis amurensis stem segments comprises the following steps:
in January in winter, 0.6 cm-0.8 cm-diameter vitis amurensis branches are cut into vitis amurensis stem sections with the length of 9cm, and the number of dormant buds on each stem section is one. The vitis amurensis stem sections were then placed in a refrigerator and the temperature was adjusted to 3 ℃ so that the vitis amurensis stem sections were dried to a humidity of 31%. Then the vitis amurensis stems are placed in an aluminum foil bag, and the aluminum foil bag is vacuumized and sealed.
And (3) placing the aluminum foil bag vacuum bag filled with the vitis amurensis stem segments in a programmed cooling instrument, cooling to-190 ℃ at a cooling rate of 0.3 ℃/min, and then quickly putting into a liquid nitrogen tank for storage so as to prevent the temperature from changing in the transferring process.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem section preserved by the cryopreservation method of the vitis amurensis stem section provided by the embodiment is taken out from a liquid nitrogen tank, is placed at room temperature for natural thawing for 2.3 hours, is then placed in water for soaking for 25 hours, the water changing time period of the first 7 hours is 60min, 55min, 50min, 55min, 50min and 50min, the water changing time period of the second 18 hours is 6, 5 and 7, the vitis amurensis stem section is taken out after soaking is finished, thawing is finished, and the vitis amurensis stem section after thawing is the vitis amurensis stem section which is thawed after cryopreservation and provided by the embodiment.
Example 5
The present embodiment provides a method for cryopreservation of vitis amurensis stem segments, a method for thawing the vitis amurensis stem segments subjected to cryopreservation, and a vitis amurensis stem segment thawed after cryopreservation.
A cryopreservation method of vitis amurensis stem segments comprises the following steps:
in January in winter, 0.6 cm-0.8 cm-diameter vitis amurensis branches are cut into vitis amurensis stem sections with the length of 11cm, and the number of dormant buds on each stem section is one. The vitis amurensis stem sections were then placed in a refrigerator and the temperature was adjusted to 4 ℃ so that the vitis amurensis stem sections were dried to a humidity of 29%. Then the vitis amurensis stems are placed in an aluminum foil bag, and the aluminum foil bag is vacuumized and sealed.
And (3) placing the aluminum foil bag vacuum bag filled with the vitis amurensis stem segments in a programmed cooling instrument, cooling to-170 ℃ at a cooling rate of 0.4 ℃/min, and then quickly putting into a liquid nitrogen tank for storage so as to prevent the temperature from changing in the transferring process.
A thawing method of a cryopreserved vitis amurensis stem section comprises the following steps:
the vitis amurensis stem section preserved by the cryopreservation method of the vitis amurensis stem section provided by the embodiment is taken out from a liquid nitrogen tank, is placed at room temperature for natural thawing for 1.8 hours, is then placed in water for soaking for 23 hours, the water changing time period for the first 7 hours is 50min, 60min, 55min, 50min, 55min, 50min, the water changing time period for the second 16 hours is 5, 6, 5, the vitis amurensis stem section is taken out after soaking is finished, thawing is finished, and the vitis amurensis stem section after thawing is the vitis amurensis stem section which is thawed after cryopreservation provided by the embodiment.
Comparative example 1
This comparative example is essentially the same as example 1 except that the vitis amurensis stem sections were dried to a water content of 20% prior to freezing.
Comparative example 2
This comparative example is essentially the same as example 1 except that the vitis amurensis stem sections were dried to a water content of 40% prior to freezing.
Comparative example 3
This comparative example is essentially the same as example 1 except that the vitis amurensis stem sections were dried to a water content of 50% before freezing.
Comparative example 4
This comparative example is essentially the same as example 1, except that the vitis amurensis stem segments were placed in 50ml screw-top tubes and then placed in a liquid nitrogen tank for cryopreservation without direct contact with liquid nitrogen, but in a non-vacuum environment.
Comparative example 5
This comparative example is essentially the same as example 1, except that the vitis amurensis stem segments are stored directly in a liquid nitrogen tank, in contact with liquid nitrogen.
Comparative example 6
The comparative example is substantially the same as example 1, except that the vitis amurensis stem segments are placed in a programmed cooling instrument to be cooled to-180 ℃, and the cooling rate is 1 ℃/min.
Comparative example 7
The comparative example is substantially the same as example 1, except that the vitis amurensis stem segments are placed in a programmed cooling instrument to be cooled to-80 ℃, and the cooling rate is 0.5 ℃/min.
Experimental example 1
40 stem segments are taken from each group according to the freezing method provided by the examples 1-5 and the comparative examples 1-8 and the thawing method provided by the examples and the comparative examples after 7 days of frozen storage, after thawing, germination experiments are carried out, and the germination rate is recorded after 30 days. The experiment was repeated 3 times and the average germination rate was recorded for each group to tables 1 and 2. (the examples are abbreviated to S and the comparative examples are abbreviated to D)
TABLE 1 Germination Rate of Amur grape Stem segments of each example
| Group of | S1 | S2 | S3 | S4 | S5 |
| The germination rate% | 70.83 | 65.83 | 65 | 66.67 | 68.33 |
As can be seen from table 1, the germination rates of the vitis amurensis stems thawed by the thawing method provided by the present invention after the cryopreservation method provided by the embodiments of the present invention are all 65% or more. This is a higher germination rate for low or ultra-low temperature cold fields.
TABLE 2 germination rates of the stem segments of example 1 and comparative vitis amurensis
| Group of | S1 | D1 | D2 | D3 | D4 | D5 | D6 | D7 |
| The germination rate% | 70.83 | 0 | 12.5 | 0 | 22.5 | 10 | 17.5 | 22.5 |
By comparing S1, D1, D2 and D3 in Table 2, it can be shown that the water content of the vitis amurensis stem segments before freezing and storing can not be too low, and when the water content is lower than the range required by the invention, the vitis amurensis stem segments obtained by thawing after freezing do not germinate; before freezing and storing, the water content of the vitis amurensis stem section cannot be too high, and when the water content is higher than the range required by the invention, the vitis amurensis stem section obtained by thawing after freezing has low germination rate or does not germinate. Comparing S1, D4 and D5, it can be shown that the germination rate is higher after the vitis amurensis stem segments are placed in liquid nitrogen for preservation after being sealed and vacuum-sealed and then placed in liquid nitrogen for frozen preservation and unfreezing. Comparing S1 with D6 and D7, it can be shown that the cooling rate is not too high in the cooling process before ultra-low temperature freezing, when the cooling rate is higher than the range of the cooling rate required by the invention, the germination rate of the thawed amur grape stem is lower, and when the temperature is reduced to a value higher than the range required by the invention before ultra-low temperature freezing preservation, the germination rate of the thawed amur grape stem is also lower.
Experimental example 2
According to the freezing method provided in the embodiment 1, the stem segments are respectively frozen and stored for 2 months, one year and 5 years, 100 stem segments are taken from each group, unfreezing is carried out according to the unfreezing method provided in the embodiment 1 after freezing and storage, a germination experiment is carried out after unfreezing, and the germination rate is recorded to be shown in table 3 after 30 days.
TABLE 3 germination rates of Ampelopsis grossedentata stem segments of example 1 and comparative examples
| Freezing time | 2 month | 1 year | 5 years old |
| The germination rate% | 70 | 67 | 63 |
When the germination rates of the groups in the table 3 are compared with the germination rates of the groups S1 in the table 1, the germination rates of the groups after being frozen for 7 days are basically the same as the germination rates of the groups after being frozen for 2 months, and the survival rates after being frozen for 1 year or even 5 years are reduced but not greatly reduced. Therefore, the ultralow temperature freezing method provided by the invention is suitable for long-term preservation of the vitis amurensis stems.
In summary, according to the ultralow temperature preservation method of the vitis amurensis stem section provided by the invention, due to the adoption of liquid nitrogen ultralow temperature preservation, the metabolism of vitis amurensis stem section cells is almost completely stopped in the preservation process, the method can realize the long-term preservation of the vitis amurensis stem section, the water content of the vitis amurensis stem section is reduced to be within the range of 28% -32% before preservation, the vitis amurensis stem section with the water content is packaged in the air, and is immediately put into a liquid nitrogen environment after being cooled to-200 to-160 ℃ at the cooling rate of 0.2 to 0.6 ℃/min, so that the germination rate of the vitis amurensis stem section obtained by unfreezing after ultralow temperature cryopreservation can reach 65% or more.
The thawing method of the vitis amurensis stem section preserved at the ultralow temperature is used for thawing the vitis amurensis stem section preserved by the cryopreservation method provided by the invention, and the thawed vitis amurensis stem section has high germination rate.
The vitis amurensis stem section preserved at ultralow temperature is obtained by adopting the unfreezing method provided by the invention, and the vitis amurensis stem section has high germination rate.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. An ultra-low temperature preservation method of vitis amurensis stem segments is characterized by comprising the following steps:
drying the vitis amurensis stem segments with the dormant buds in an environment with the temperature of 2-5 ℃ until the water content is 28% -32%;
placing the vitis amurensis stem section with the dormant bud and the water content of 28-32% in a vacuum container;
and cooling the vacuum container in which the vitis amurensis stem segments are placed to-200 to-160 ℃ at a cooling rate of 0.2 to 0.6 ℃/min, and then putting the vacuum container into a liquid nitrogen environment for storage.
2. The method for cryopreservation of vitis amurensis stem segments as claimed in claim 1, wherein the number of dormant buds attached to the vitis amurensis stem segments is 1.
3. The method for cryopreservation of vitis amurensis stem segments as claimed in claim 1, wherein the vacuum container is an evacuated aluminum foil bag.
4. The method for ultralow-temperature preservation of vitis amurensis stem segments according to claim 1, wherein the vacuum container in which the vitis amurensis stem segments are placed is put into a liquid nitrogen environment after being cooled to-200 to-160 ℃ in a programmed cooling instrument at a cooling rate of 0.2 to 0.6 ℃/min.
5. The method for the ultra-low temperature preservation of vitis amurensis stem segments according to claim 1, wherein the vitis amurensis stem segments have a diameter of 0.6cm to 0.8cm and a length of 8cm to 12 cm.
6. A thawing method of a vitis amurensis stem section preserved at ultralow temperature is characterized by comprising the following steps:
taking the vitis amurensis stem segments preserved by the preservation method according to any one of claims 1 to 5 out of the liquid nitrogen environment, naturally thawing the vitis amurensis stem segments at room temperature for 1.5 to 2.5 hours, and then soaking the vitis amurensis stem segments in water for 22 to 26 hours.
7. The thawing method of the vitis amurensis stem segments preserved at ultralow temperature according to claim 6, wherein the step of soaking the vitis amurensis stem segments in water for 22-26 hours comprises the following steps: changing water every 50-60 min for the first 5-7 h, and changing water every 5-7 h for the rest time in 22-26 h.
8. The method of thawing the cryopreserved vitis amurensis stem sections according to claim 6, wherein the water is purified water.
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| CN105028195A (en) * | 2015-06-25 | 2015-11-11 | 中国农业科学院特产研究所 | Vitrification cryopreservation method for vitis amurensis callus tissues |
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| WO2004026222A3 (en) * | 2002-09-20 | 2004-06-03 | Af Consulting | Compositions which are used to care for skin suffering from a hormonal imbalance and which contain one or more resveratrol oligomers, particularly $g(e)-viniferin, and/or certain derivatives thereof |
| CN104304238A (en) * | 2014-09-12 | 2015-01-28 | 贵州大学 | Embedding drying ultralow temperature preservation method of vitis heyneana stem tip |
| CN105028195A (en) * | 2015-06-25 | 2015-11-11 | 中国农业科学院特产研究所 | Vitrification cryopreservation method for vitis amurensis callus tissues |
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