CN111616140A - Vitrification ultralow-temperature preservation method and application of actinidia arguta dormant bud - Google Patents

Vitrification ultralow-temperature preservation method and application of actinidia arguta dormant bud Download PDF

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CN111616140A
CN111616140A CN202010529260.3A CN202010529260A CN111616140A CN 111616140 A CN111616140 A CN 111616140A CN 202010529260 A CN202010529260 A CN 202010529260A CN 111616140 A CN111616140 A CN 111616140A
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dormant
vitrification
actinidia arguta
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秦红艳
白晓雪
李昌禹
范书田
杨义明
赵滢
张宝香
许培磊
刘迎雪
韩先焱
舒楠
王月
路文鹏
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Institute Special Animal and Plant Sciences CAAS
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract

The invention discloses a vitrification ultra-low temperature preservation method and application of actinidia arguta dormant buds, which relate to the technical field of plant tissue culture, and comprise the steps of stripping the dormant buds from actinidia arguta plants, and putting the actinidia arguta dormant buds into a pre-culture medium for pre-culture for 1-5 days; transferring the dormant bud into a freezing tube under aseptic condition, loading at room temperature for 10-60min, treating with vitrification liquid at 0 deg.C for 90-150min, adding small amount of fresh vitrification liquid, rapidly adding into liquid nitrogen, and freezing; taking out, thawing in water bath immediately, washing with MS unloading liquid, unloading, drying, transferring to recovery culture medium, dark culturing for 1-3d, and culturing under light; the material used by the method is derived from actinidia arguta variety, the operation is simple and effective, special instruments and complicated treatment procedures are not needed, the almond after being stored at ultralow temperature recovers well, the survival rate can reach 86.30%, and the method is reliable.

Description

Vitrification ultralow-temperature preservation method and application of actinidia arguta dormant bud
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a vitrified ultralow-temperature preservation method and application of actinidia arguta dormant buds.
Background
Actinidia arguta (Actinidia Sieb. et Zucc.) is a precious cold-resistant fruit tree resource in China and is a large-scale fallen leaf vine plant of Actinidia (Actinidiaceae) Actinidia. The actinidia arguta has extremely strong resistance and rich nutrition, is rich in vitamin C, has important nutritive value and economic value, is one of species with important utilization value in actinidia, and is an important germplasm resource for improving actinidia varieties. In recent years, due to forest clearing and tending and artificial predatory picking, a large amount of excellent resources of wild actinidia arguta are lost. In order to store precious germplasm resources, some scientific research departments and production units successively establish actinidia arguta germplasm resource gardens for in-vivo planting and storage of garden fields, but the land and capital are limited, so that the stored resources are few; some units carry out tissue culture to store a large amount of resources, but due to the increase of the number of subcultures, the tissue culture germplasm material possibly has the risk of accumulating variation; in the ultra-low temperature preservation technology developed in recent years, equipment is simple, the quantity of preserved materials is large, the risk of accumulation and variation can be avoided, an ultra-low temperature preservation warehouse is built for a plurality of plants, and thousands of precious germplasm resources are preserved.
Ultra-low temperature preservation is a resource preservation technology developed on the basis of isolated culture, and refers to a biological technology for preserving plant organs, tissues, cells and the like under the condition of liquid nitrogen (196 ℃ below zero). Under the temperature condition, the growth and metabolic activities in the plant material cells are basically stopped and are in a relatively stable biological state (the 'state of organism pause'), so that the metabolic aging process can be greatly slowed down or even stopped, the genetic stability of the material is maintained, and the aim of long-term storage is fulfilled. Therefore, cryopreservation is regarded as the most ideal and effective method for long-term preservation of plant germplasm resources. The vitrification method established by Sakai et al is a simple and efficient ultralow temperature preservation method with simple equipment, simple and convenient material processing steps and good effect and repeatability. The dormant bud is a protective organ formed by the plant in order to adapt to the severe cold environment, and the survival rate of the plant after being stored at ultralow temperature is improved through natural low-temperature domestication. Until now, dormant buds of plants such as apples, persimmons, almonds and the like are successfully preserved by utilizing a vitrification method, but the survival rate is extremely low when the methods are directly applied to actinidia arguta, and the treatment mode and the treatment time in the vitrification method are seen, so that different species have great difference. The reports on the vitrification ultra-low temperature preservation technology of the actinidia arguta dormant bud are less. The actinidia arguta variety Kuilu' approved by the crop variety approval Committee of Jilin province in 1995 is a good variety bred by the specialty research institute of Chinese academy of agricultural sciences for more than 10 years, has extremely strong stress resistance, can safely live through the winter at the temperature of-38 ℃, and does not have serious diseases and insect pests in growing seasons.
Disclosure of Invention
The invention aims to provide a vitrification ultra-low temperature preservation method and application of actinidia arguta dormant bud, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a vitrified ultralow temperature preservation method for actinidia arguta dormant buds, which comprises the following steps:
(1) stripping dormant buds from actinidia arguta plants, and placing the actinidia arguta plants into a pre-culture solution for pre-culture for 1-6 days;
(2) loading dormant buds at room temperature for 10-60min under aseptic condition, treating with vitrification liquid at 0 deg.C for 30-300min, adding small amount of fresh vitrification liquid, quickly adding into liquid nitrogen, and freezing;
(3) taking out, thawing in water bath, washing with MS unloading liquid, drying, transferring to recovery culture medium, dark culturing for 1-5 days, and culturing under light.
Further, in the step (1), before the dormant bud is stripped from the actinidia arguta plant, sterilization needs to be carried out, and the method specifically comprises the following steps:
cleaning dormant buds with water, cutting into 1-3cm single bud stem segments, soaking and cleaning with detergent for 30min, stirring for 1 time every 10min, washing with water for 30min, transferring to a clean bench, peeling, soaking in 75% ethanol for 30s for surface sterilization, washing with sterile water for 4-5 times, and washing with 0.1% HgCl2And (5) vibrating and sterilizing for 30min, washing with sterile water for 4-5 times, and stripping to obtain dormant buds.
Further, in the step (1), the pre-culture solution is MS culture medium containing 0.3-0.7mol/L of sucrose and/or 0.5-3mol/L of glycerol.
Further, in the step (2), the loaded culture solution comprises: MS, 2mol/L glycerol and 0.4mol/L sucrose.
Further, in the step (2), the vitrification liquid is PVS1、PVS2、PVS3Or PVS4。
Further, in the step (2), the vitrification liquid is PVS2。
Further, in the step (3), the temperature of the water bath for thawing the water bath is 38 ℃.
Further, in the step (3), the MS unloading liquid contains 1.2mol/L of sucrose; the recovery medium is MS medium containing 0.01-0.02mg/LNAA and 1-2 mg/L6-BA.
The invention also provides application of the method in-vitro preservation of the actinidia arguta dormant bud.
The invention discloses the following technical effects:
the material of the invention is selected from the dormant bud of actinidia arguta, the dormant bud is acclimated in a winter natural environment, the low-temperature acclimation process can be reduced during the ultralow-temperature preservation, the step of culturing tissue culture seedlings is reduced, the operation is simpler and more convenient, the culture solution is loaded after the direct pre-culture, and the vitrification solution PVS is used for loading2And (5) treating and performing ultralow temperature freezing preservation.
The material used by the method is derived from actinidia arguta variety, the operation is simple and effective, special instruments and complicated treatment procedures are not needed, the almond after being stored at ultralow temperature recovers well, the survival rate can reach 86.30%, and the method is reliable.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing the effect of different pre-culture solutions on the survival rate of frozen dormant buds;
FIG. 2 is a graph showing the effect of pre-culture time on survival rate of frozen dormant buds;
FIG. 3 is a graph of the effect of loading time on survival of frozen dormant buds;
FIG. 4 is a PVS2Treatment time to survival rate of frozen dormant budThe influence of (a);
FIG. 5 is a diagram showing the restoration culture of frozen dormant buds and normal tissue culture seedlings, wherein a the dormant buds start to revive after thawing, b the dormant buds survive after freezing, c the dormant buds grow into plants after freezing, and d the dormant buds which are not preserved at ultra-low temperature grow into plants;
FIG. 6 is a ploidy evaluation of regenerated plants after cryopreservation of 'Quilgreen' dormant buds, wherein a plants developed from dormant buds which were not cryopreserved, and b regenerated plants from dormant buds which were cryopreserved.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
1 materials and methods
1.1 test materials
The test material is actinidia arguta 'kuilgreen' (the invention is not limited to the actinidia arguta) dormant bud, the tree age is 25 years, cutting seedlings are collected from actinidia arguta germplasm resource gardens of special local institute of academy of agriculture and sciences in 2018 and 12 months.
1.2 test methods
1.2.1 Sterilization of materials
Cleaning the dormant branches with tap water, cutting into single-bud stem segments with length of about 2cm, soaking and cleaning with detergent for 30min, stirring for 1 time every 10min, and washing with tap water for 30 min. Transferring to a superclean bench, peeling single bud stem, soaking in 75% alcohol for 30s for surface disinfection, washing with sterile water for 4-5 times, and washing with 0.1% HgCl2And (5) vibrating and sterilizing for 30min, washing with sterile water for 4-5 times, and stripping dormant buds for vitrification cryopreservation.
1.2.2 vitrification ultra-low temperature preservation method
Basic procedure for cryopreservation of dormant buds by vitrification: the dormant bud is peeled at 0.3mol ∙ L-1Sucrose +1mol ∙ L-1Shake culturing in glycerol preculture solution for 2d, transferring dormant bud into 2ml freezing tube under aseptic condition, repeating for 3 times, with 10 buds per tube. Loading at room temperature for 20min, and loading with PVS at 0 deg.C2Dehydrating for 120min, and replacing fresh PVS2Then quickly adding liquid nitrogen for freezing and storing for 24h, taking out and immediately placing into 38 ℃ water bath for thawing for 2min, and using a liquid containing 1.2mol of ∙ L-1Washing the sucrose MS unloading solution for 3 times, each time for 10min, and inoculating to MS +2mg ∙ L after sterile filter paper is blotted-16-BA+0.02mg∙L-1Culturing in dark for 3 days in NAA recovery medium under lightAnd (5) culturing, and counting the survival rate after 3 weeks (taking the standard that the dormant buds appear obvious green). Single factor experiments were performed by varying the single factor based on the basic procedure (as in table 1).
TABLE 1 vitrification cryopreservation of 'Kuilu' dormant bud Single factor test
Figure BDA0002534639390000071
1.2.3 ploidy identification of regenerated plants
And (3) identifying the ploidy level of the plant regenerated from the dormant bud after vitrification ultralow temperature storage by taking the plant developed from the dormant bud without ultralow temperature storage. Take 1cm2The young leaves were placed in A clean petri dish, 0.4ml of Partec HR-A lysate was added, and the tissue was minced with A razor blade. After 5min, the sample was filtered into a small tube with a 100 μm sieve, and then 1.6ml of Partec HR-B solution was added. After storage in the dark for 2min, the samples were analyzed in a Partec ploidy analyzer.
1.3 data statistics and processing
Survival rate (number of dormant buds appearing obvious green/number of non-polluted dormant buds preserved at ultralow temperature) multiplied by 100%;
data statistics were performed using Excel software, data analysis was performed using SAS9.2, and significance analysis was performed using Duncan's multiple comparison (P ═ 0.05).
2 results and analysis
2.1 Effect of Pre-culture solution on survival Rate of 'Kuilu' dormant bud after ultra-Low temperature preservation
The high-concentration sucrose can induce the dehydration of cells, thereby increasing the freezing resistance of the cells, and freezing protective agents DMSO, glycerol and the like can be added into the culture solution to improve the freezing survival rate. Sucrose and glycerol were selected as osmoprotective substances in the pre-culture solution, and the dormant buds were placed in MS pre-culture solution containing sucrose and glycerol at different concentrations for shake culture, with the results shown in FIG. 1. Test results show that the pre-culture solution of sucrose and glycerol with different concentrations has great influence on the survival rate of 'Kuilu' dormant buds, and the survival rate of the dormant buds is reduced along with the increase of the sucrose concentration in the pre-culture solutionThe trend is reduced. At 0.3mol of ∙ L-1Sucrose and 0.3mol ∙ L-1Sucrose +1mol ∙ L-1The survival rate of the dormant buds cultured in the MS preculture solution of glycerol is the highest and is 75.93 percent and 84.72 percent respectively, and the survival rate are not obviously different. Containing 0.7mol of ∙ L-1The survival rate of the dormant buds cultured in MS pre-culture solution containing sucrose and glycerol with different concentrations is lower. Analysis results show that the sucrose concentration is a main factor influencing the survival rate of dormant buds, and the glycerol plays a part of roles as an osmotic protective solution. Containing 2mol of ∙ L-1The survival rate of the dormant buds cultured in MS pre-culture solution containing glycerol and sucrose with different concentrations is low, probably because the concentration of the osmoprotective substance is high, osmotic stress is generated on the dormant buds, the cell structure is damaged, and a part of cells lose activity.
2.2 Effect of Pre-incubation time on survival Rate of 'Kuilu' dormant bud after ultra-Low temperature preservation
Research shows that the pre-culture for a certain time is beneficial to improving the survival rate of the preserved materials. The survival rate of the dormant buds after different times of preculture and ultra-low temperature preservation is found to be different (see figure 2). When the bud is pre-cultured for 2 days, the survival rate of the frozen dormant buds is the highest and reaches 74.24%, the survival rate of the dormant buds is gradually reduced along with the increase of the pre-culture time, and the survival rate of the dormant buds is reduced to 7.04% after the bud is pre-cultured for 6 days. The pretreatment time is too long, and the cells are damaged due to excessive dehydration, so that the survival rate is reduced. At 0.3mol of ∙ L-1Sucrose +1mol ∙ L-1The survival rate of dormant buds pre-cultured for 2d in the MS pre-culture solution of glycerol is the highest, and the ultralow-temperature preservation effect is the best.
2.3 Effect of survival Rate of 'Kuilu' dormant bud after ultralow temperature storage at Loading time
To further protect the dormant buds from dehydration, the buds were treated with loading solution for various periods of time and the results are shown in FIG. 3. The liquid loading is processed for 10-50min, the survival rate of the frozen dormant buds is higher, and after the liquid loading is processed for 60min, the survival rate of the dormant buds is remarkably reduced to 32.50%. The treatment with the loading liquid can reduce the freezing point and the over-saturation point of water, so that the damage of ice crystals to cells in the freezing process can be avoided, but the over-dehydration can also damage the cells, thereby reducing the survival rate of the frozen dormant buds. The survival rate reaches 75.93% when the fertilizer is loaded for 20min, and the effect is better.
2.4 Effect of vitrification protective solution on survival rate of 'Kuilu' dormant bud after ultralow temperature preservation
The vitrification process is an important step in the ultra-low temperature preservation process, so the selection of the type of the vitrification liquid and the processing time of the vitrification liquid are also important. The effect of different types of vitrification liquids on survival rate of 'kuilu' dormant buds after cryopreservation is shown in table 2. The test results show that PVS2The survival rate of the dormant bud after treatment is obviously higher than that of other three vitrification liquids, PVS1、PVS3、PVS4The survival rate of the dormant buds after treatment has no obvious difference.
TABLE 2 Effect of different vitrification liquids on survival rates of dormant buds after cryopreservation
Figure BDA0002534639390000091
Note: values are mean ± SD, with different letters indicating significant differences at the 0.05 level. The same applies below.
At 0 deg.C, using PVS2The effect of vitrification protective solution treatment for different time on the survival rate of dormant buds after ultra-low temperature preservation is shown in figure 4. Following PVS2The survival rate of dormant buds after ultralow temperature storage is increased and then decreased due to the increase of the processing time. PVS2The survival rate is remarkably improved every 30min after the dehydration treatment, the highest survival rate is 84.72% when the dehydration time is 120min, and after the dehydration time exceeds 120min, due to PVS2The survival rate of dormant buds gradually decreases due to toxic action.
2.5 Effect of recovery Medium on survival Rate of 'Kuilu' dormant bud after ultra-Low temperature preservation
The ` Quilgreen ` dormant bud was passed through 0.3mol ∙ L-1Sucrose +1mol ∙ L-1Culturing in glycerol MS preculture solution for 2 days, loading for 20min, and PVS2Dehydrating for 120min, and replacing fresh PVS2The cells were immersed in liquid nitrogen for cryopreservation, thawed and transferred to different recovery media with the results shown in Table 3. The survival rate in the culture medium without hormone is as low as 28.33%, and the survival rate is obvious by adding NAA and 6-BA with different concentrationsAnd (4) improving. When 0.01mg ∙ L is added-1In NAA, the concentration of 6-BA has no obvious influence on survival; when 6-BA in the recovery medium was quantified, 0.02mg ∙ L was added-1The survival rate of the NAA dormant bud is obviously higher than that of the added 0.01mg ∙ L-1NAA. In recovery Medium MS +0.02mg ∙ L-1NAA+2mg∙L-1On 6-BA, the survival rate of the 'Kuilu' dormant bud after ultralow temperature storage is the highest and reaches 86.30%. On this medium, the dormant bud started to erupt green and survive after being stored at ultralow temperature for 7 days (FIGS. 5a and 5b), and the viable dormant bud could grow into a normal plant after 10 weeks (FIG. 5 c). Experiments also find that the dormant bud directly develops into a plant when regenerating, and the dormant bud does not pass through stages such as callus, so that the probability of genetic variation is greatly reduced. FIG. 5d is a plant developing a ` Quinogreen ` dormant bud under normal light without cryopreservation, with no apparent difference in morphology.
TABLE 3 Effect of recovery Medium on survival Rate of dormant buds after cryopreservation
Figure BDA0002534639390000101
2.6 ploidy identification of dormant bud regenerated plant after ultralow temperature preservation
The chromosome ploidy of the regenerated plants obtained by cryopreservation of 23 plants was examined by a flow cytometer using a plant developed from a 'kuilu' dormant bud which was not subjected to cryopreservation as a control, and the results are shown in fig. 6. The fluorescence curves of the 23 plants are consistent with the control, which shows that the chromosome ploidy of the plant regenerated by the frozen dormant bud is not changed.
3 results
The selection of the material plays a decisive role in the survival rate of the plants after the ultralow temperature preservation. Compared with the stem tip of the tissue culture seedling, the dormant bud is subjected to severe cold acclimation in the natural environment in winter, the low-temperature acclimation process can be reduced during ultralow-temperature storage, and the step of culturing the tissue culture seedling is reduced, so that the operation is simpler and more convenient. According to the invention, the actinidia arguta kumquat dormant bud is selected as a test material to carry out vitrification cryopreservation research, so that a higher survival rate after freezing is obtained, and the survival rate can reach 86.30%.
The vitrification method is an ultralow temperature preservation method that plant materials are subjected to a series of protective dehydration processes of pre-culture, loading and vitrification liquid treatment, and finally fresh vitrification protective liquid is replaced and directly put into liquid nitrogen for cryopreservation. The pre-culture can enhance the freezing resistance of the plant material, and is a key step for the success of the ultra-low temperature preservation technology. The pre-culture adopts osmotic substance sucrose, and the survival rate of the frozen material can be improved by adding the cryoprotectant. The commonly used cryoprotectants comprise dimethyl sulfoxide, glycerol and the like, the glycerol greatly improves the regeneration rate of the materials after ultralow-temperature storage, and the addition of the dimethyl sulfoxide into the pre-culture solution also improves the survival rate. Because dimethyl sulfoxide has toxic action on dormant buds, sucrose and glycerol are selected to play a role in penetration protection. Experiments show that the higher survival rate of the dormant buds after freezing can be obtained by pre-culturing in MS pre-culture solution of 0.3mol of ∙ L-1 sucrose and 0.3mol of ∙ L-1 sucrose +1mol of ∙ L-1 glycerol. Due to different genotypes of the materials, the glycerol does not obviously improve the survival rate of the frozen dormant buds and only plays a role in slightly improving. The pre-culture time also has great influence on the ultra-low temperature preservation effect, and the survival rate after freezing is in the trend of increasing firstly and then decreasing along with the extension of the pre-culture time. The present study shows that 2d is the optimal pre-incubation time. The cells are protectively dehydrated by the penetration of the pre-culture solution into the cells, the pre-culture time is insufficient, the dehydration degree of the cells is insufficient, the cells are easily frozen, the pre-culture time is too long, the cells are damaged by excessive dehydration, and the survival rate is reduced.
Vitrification is to convert free water in plant cell from liquid state to vitrified state, and this can increase cell viscosity and avoid formation of ice crystal, so as to raise the freezing resistance of cell. The concentration of the vitrification solution is high, and the vitrification solution is easy to damage cells, so that loading treatment is needed to adapt the cells to the vitrification solution before vitrification. The research of the invention shows that the loading time is 20min, and the effect is better. When the glass transition metal is treated by the glass transition metal, the type of the glass transition metal and the treatment time of the glass transition metal are important factors influencing the ultra-low temperature preservation effect. PVS2Ultralow-temperature preservation effect of actinidia arguta dormant bud as vitrification protection liquidMost preferably. The invention explores PVS2The influence of different treatment times on the survival rate of the dormant buds after ultralow-temperature preservation shows that the effect is better when the dehydration time is 120 min. According to the invention, the influence of the recovery medium on the survival rate of the dormant bud after ultralow-temperature preservation is researched, and the result shows that the dormant bud is MS +0.02mg ∙ L-1NAA+2mg∙L-1The 6-BA can normally tell green to survive on a recovery culture medium, and can be directly regenerated into plants without callus and other stages. The shapes of the plant seedlings regenerated by the recovery culture medium and the tissue culture seedlings have no obvious difference, and the ploidy of the regenerated plants is not changed through the identification of a flow cytometer.
4 conclusion
The system suitable for ultralow-temperature preservation of actinidia arguta 'kuilgreen' dormant bud by a vitrification method is as follows: the sterilized dormant bud is 0.3mol ∙ L-1Sucrose +1mol ∙ L-1Culturing in glycerol preculture solution for 2 days, loading at room temperature for 20min, dehydrating at 0 deg.C for 120min with PVS2, changing fresh PVS2 protective solution, and quickly freezing in liquid nitrogen. Taking out, soaking in 38 deg.C water bath for thawing for 2min, and thawing with solution containing 1.2mol ∙ L-1Washing with sucrose MS unloading solution for 30min, inoculating to MS +2mg ∙ L-16-BA+0.02mg∙L-1And (4) performing dark culture on the NAA recovery culture medium for 3d, and culturing under normal illumination, wherein the survival rate reaches 86.30%.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. A vitrification ultra-low temperature preservation method for actinidia arguta dormant buds is characterized by comprising the following steps:
(1) stripping dormant buds from dormant actinidia arguta branches, and putting the dormant buds into a pre-culture solution for pre-culture for 1-5 days;
(2) loading dormant buds at room temperature for 10-60min under aseptic condition, treating with vitrification liquid at 0 deg.C for 90-150min, adding small amount of fresh vitrification liquid, quickly adding into liquid nitrogen, and freezing;
(3) taking out, thawing in water bath, washing with MS unloading liquid, unloading, drying, transferring to recovery culture medium, dark culturing for 1-3 days, and culturing under light.
2. The method for storing actinidia arguta dormant bud at a vitrification ultra-low temperature according to claim 1, wherein in the step (1), before the dormant bud is stripped from the actinidia arguta dormant branch, sterilization is required, and the method comprises the following specific steps:
cleaning dormant branches with water, cutting into 1-3cm single bud stem segments, soaking and cleaning with detergent for 30min, stirring for 1 time every 10min, washing with water for 30min, transferring to a clean bench, peeling, soaking in 75% alcohol for 30s for surface sterilization, washing with sterile water for 4-5 times, and washing with 0.1% HgCl2And (5) vibrating and sterilizing for 30min, washing with sterile water for 4-5 times, and stripping to obtain dormant buds.
3. The method for storing actinidia arguta dormant bud at a vitrification ultra-low temperature as in claim 1, wherein in the step (1), the pre-culture solution is MS liquid culture medium containing 0.3-0.7mol/L of sucrose and/or 1-2mol/L of glycerol.
4. The method for storing actinidia arguta resting bud at vitrification ultra-low temperature as claimed in claim 1, wherein in step (2), the loaded culture solution is MS +2mol/L glycerol +0.4mol/L sucrose.
5. The method for storing actinidia arguta dormancy bud at a vitrification ultra-low temperature as claimed in claim 1, wherein in the step (2), the vitrification liquid is PVS1、PVS2、PVS3Or PVS4。
6. The method for storing actinidia arguta dormancy bud at a vitrification ultra-low temperature as claimed in claim 5, wherein in the step (2), the vitrification liquid is PVS2。
7. The method for vitrification cryopreservation of actinidia arguta dormant bud, according to claim 1, wherein in the step (3), the temperature of the water bath for thawing in the water bath is 38 ℃.
8. The method for storing actinidia arguta dormancy bud at a vitrification ultra-low temperature as set forth in claim 1, wherein in the step (3), the MS discharging liquid contains 1.2mol/L sucrose; the recovery culture medium is MS solid culture medium containing 0.01-0.02mg/LNAA and 1-2 mg/L6-BA.
9. Use of the method of any one of claims 1-8 for the ex vivo preservation of quiescent shoots of actinidia arguta.
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