CN109644874A - Meadow sweet cryopreservation cultural method - Google Patents
Meadow sweet cryopreservation cultural method Download PDFInfo
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- CN109644874A CN109644874A CN201910113034.4A CN201910113034A CN109644874A CN 109644874 A CN109644874 A CN 109644874A CN 201910113034 A CN201910113034 A CN 201910113034A CN 109644874 A CN109644874 A CN 109644874A
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- 235000016622 Filipendula ulmaria Nutrition 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 21
- 244000061544 Filipendula vulgaris Species 0.000 title 1
- 239000007788 liquid Substances 0.000 claims abstract description 78
- 238000004017 vitrification Methods 0.000 claims abstract description 55
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 50
- 238000004321 preservation Methods 0.000 claims abstract description 35
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- 244000308505 Filipendula ulmaria Species 0.000 claims description 86
- 238000011084 recovery Methods 0.000 claims description 56
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 29
- 229930006000 Sucrose Natural products 0.000 claims description 26
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 26
- 229960004793 sucrose Drugs 0.000 claims description 26
- 238000005286 illumination Methods 0.000 claims description 22
- 241000681535 Spiraea salicifolia Species 0.000 claims description 19
- 239000005720 sucrose Substances 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 14
- 238000007710 freezing Methods 0.000 claims description 14
- 230000008014 freezing Effects 0.000 claims description 14
- 239000011521 glass Substances 0.000 claims description 13
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- 238000005192 partition Methods 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 7
- 239000004471 Glycine Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000011491 glass wool Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 abstract description 23
- 241001092387 Spiraea Species 0.000 abstract description 14
- 230000008929 regeneration Effects 0.000 abstract description 10
- 238000011069 regeneration method Methods 0.000 abstract description 10
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 241000957095 Spiraea alba Species 0.000 abstract 6
- 235000004789 Rosa xanthina Nutrition 0.000 abstract 1
- 241000220222 Rosaceae Species 0.000 abstract 1
- 208000005156 Dehydration Diseases 0.000 description 19
- 244000187976 Spiraea japonica Species 0.000 description 12
- 235000011187 glycerol Nutrition 0.000 description 11
- 241000234295 Musa Species 0.000 description 6
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 6
- 235000005340 Asparagus officinalis Nutrition 0.000 description 5
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- 241000234427 Asparagus Species 0.000 description 3
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- 238000000338 in vitro Methods 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 244000003416 Asparagus officinalis Species 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 2
- 240000004244 Cucurbita moschata Species 0.000 description 2
- 235000009854 Cucurbita moschata Nutrition 0.000 description 2
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 208000004880 Polyuria Diseases 0.000 description 2
- 101100484967 Solanum tuberosum PVS1 gene Proteins 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
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- 230000035619 diuresis Effects 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 235000015136 pumpkin Nutrition 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 1
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- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
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- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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Classifications
-
- 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Dentistry (AREA)
- Toxicology (AREA)
- Agronomy & Crop Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a kind of meadow sweet cryopreservation cultural methods, at room temperature will length of the culture in pre-culture solution be 1-2mm meadow sweet stem apex be placed in shaking table oscillator and be protected from light dark culturing 1-3d after, 20-60min is handled with loading liquid, it takes out meadow sweet stem apex and is transferred to temperature to be put into cryopreservation tube after dehydration 30-50min in 0 DEG C of vitrification solution, then cryopreservation tube is stored in liquid nitrogen and carries out cryopreservation.The anabiosis rate and regeneration rate of the meadow sweet of culture of the present invention are very high, technology is provided for meadow sweet Germ-plasma resources protection in imminent danger to guarantee, to rosaceae, meadow sweet subfamily, the preservation of Spiraea resource and will cultivate new varieties using Plant Diversity one effective way is provided.
Description
Technical Field
The invention relates to the technical field of germplasm resource preservation, in particular to an ultra-low temperature preservation and culture method for spiraea ulmaria.
Background
Spiraea salicifolia (Asparagus officinalis) is a perennial herb of Asparagus of Liliaceae, also called Asparagus, Asparagus and the like, has slightly sweet taste and mild property, and has the effects of clearing heat, promoting diuresis, promoting blood circulation, resolving masses and the like. According to the report of the literature, the spiraea japonica contains abundant mineral substances, amino acids, asparagus saponin, polysaccharide, flavone and other bioactive components, and has the pharmacological activities of resisting oxidation, tumors, fungi and blood fat. Spiraea ulmaria can clear heat and promote diuresis, promote blood circulation and dissipate stagnation, is mainly used for treating hepatitis, psoriasis, hyperlipidemia and hyperplasia of mammary glands, and has certain curative effect on lymphoma, bladder cancer, breast cancer, skin cancer and the like.
Cryopreservation (Cryopreservation) 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 activity of the substance in the preserved material cell are almost completely stopped and are in a relatively stable biological state, so that 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. Vitrification cryopreservation is to place cells or tissues in a Vitrification solution composed of a certain proportion of permeable and impermeable protective agents, solidify the materials and the Vitrification solution into an amorphous Vitrification state at a sufficiently fast cooling rate, and store the materials and the Vitrification solution in the glass state at a low temperature. The technology for ultralow-temperature preservation of spiraea ulmaria has not been reported at home and abroad.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide an ultra-low temperature preservation and culture method for the spiraea, the recovery rate and the regeneration rate of the spiraea are high, technical guarantee is provided for preservation of endangered spiraea germplasm resources, and an effective way is provided for preservation of rosaceous, spiraea subfamily and spiraea plant resources and cultivation of new varieties by utilizing plant diversity.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for cryopreservation culture of spiraea ulmaria, comprising: placing spiraea ulmaria stem tip or axillary bud with length of 1-2mm cultured in pre-culture solution in shaking table oscillator under dark condition in dark place for 1-3d at room temperature, treating with loading solution for 20-60min, taking out spiraea ulmaria stem tip or axillary bud, transferring into vitrification solution with temperature of 0 deg.C, dehydrating for 30-50min, placing into freezing tube, and storing in liquid nitrogen for ultra-low temperature preservation.
Preferably, the method further comprises the following steps: taking out the cryopreservation tube from liquid nitrogen, absorbing and removing the vitrification solution, washing the spiraea ulmaria stem tip with unloading liquid for 1-3 times, and transferring to a recovery culture medium for recovery culture.
Preferably, after the vitrification solvent is absorbed, the spiraea ulmaria stem tip or the axillary bud is firstly placed in a water bath with the temperature of 35-45 ℃ for soaking for 1-5min, then the spiraea ulmaria stem tip or the axillary bud is washed by unloading liquid for 1-3 times, then the surface of the spiraea ulmaria stem tip or the axillary bud is washed by sterile water, and finally the spiraea ulmaria stem tip or the axillary bud is transferred to a recovery culture medium for recovery culture.
Preferably, the pre-culture solution is MS culture solution which comprises DMSO with the mass fraction of 5%, 6-BA with the mass fraction of 1.0mg/mL and NAA with the mass fraction of 0.5 mg/mL.
Preferably, the loading solution comprises the following components: MS, 2mol/L of glycerol and 0.4mol/L of cane sugar.
Preferably, the vitrification solution includes: MS, 30% of glycerol by mass, 15% of DMSO by mass, 15% of ethylene glycol by mass and 0.4mol/L of sucrose.
Preferably, the unloading liquid comprises: MS, 1.0mol/L sucrose; the recovery medium comprises: 1/2MS, 1.0mg/L of 6-BA, 0.5mg/mL of NAA.
Preferably, during the recovery culture, the culture is firstly carried out for 3 days under the dark condition, and then the culture is carried out for 7 days under the illumination, the illumination time is 12h/d, the illumination intensity is 1800lux, the temperature is 25 ℃, and the humidity is 40%.
Preferably, the dehydration is carried out in a dehydration apparatus comprising a first container for containing a vitrification solution of 0 ℃ and a second container for containing spiraea ulmaria stem tip or axillary bud, wherein,
the first container is a cuboid shell with an open top, the first container is horizontally placed, an arc-shaped partition plate with an upward concave surface is clamped inside the first container, two linear edges of the arc-shaped partition plate are respectively sealed with a wide edge at the top of the first container to divide the first container into an arc-shaped groove at the upper part and a closed cavity at the lower part, and superfine glass wool is filled in the closed cavity; the second container comprises a support, a rotating shaft, a connecting rod and a containing shell, the support is positioned outside the first container, the rotating shaft is rotatably and horizontally arranged on the support, the rotating shaft is driven by a motor, one end of the connecting rod is fixedly connected to the rotating shaft, the other end of the connecting rod is fixedly connected with the top of the containing shell, the containing shell is of a spherical structure with a sample inlet at the bottom, four liquid inlet holes are arranged on the containing shell at intervals, two of the liquid inlet holes are close to the top of the containing shell and are symmetrical relative to the rotating shaft, the remaining two liquid inlet holes are close to the sample inlet and are symmetrical relative to the rotating shaft, sponge is attached to the inner wall of the containing shell, the sponge does not shield the four liquid inlet holes, the containing shell is not contacted with the arc-shaped partition plate, a cover body is detachably arranged on the sample inlet, a fixed column is fixedly arranged on the cover body, the fixed column and the object carrying cylinder are both positioned in the accommodating shell, the top of the object carrying cylinder props against sponge in the accommodating shell, a plurality of through holes are arranged on the side wall of the object carrying cylinder at intervals, and the axis of any through hole is vertical to the axis of any liquid inlet hole; when the accommodating shell is positioned at the right center of the arc-shaped groove, the connecting rod, the object carrying cylinder and the fixing column are coaxially arranged in the vertical direction,
the improved multifunctional container comprises an arc-shaped groove, a containing shell, a rotating shaft, a containing shell and a containing shell, wherein the arc-shaped groove is internally filled with 0 ℃ vitrification solution, the containing shell is filled with spiraea ulmaria stem tips or axillary buds, the 0 ℃ vitrification solution filled in the arc-shaped groove is immersed in the containing shell, after the 0 ℃ vitrification solution is filled in the containing shell through four liquid inlet holes and a plurality of through holes, the rotating shaft is controlled by a motor to rotate clockwise for a quarter circle first, and then rotate counterclockwise for a quarter circle, so that the containing shell swings in the arc-shaped groove until the dehydration is finished, and the containing shell is immersed in the vitrification solution all the time.
Preferably, the spiraea ulmaria stem tip or axillary bud is pretreated before being placed in the pre-culture solution, and the pretreatment comprises the following steps: the spiraea ulmaria stem tip or axillary bud is placed into 1.2mol/L glycine to be soaked for 30s, then the spiraea ulmaria stem tip or axillary bud is buried in a transparent glass filled with agar, an incandescent lamp is adopted for irradiation, the transparent device is rotated by taking the incandescent lamp as the center and taking the distance of 10cm from the incandescent lamp as the radius during irradiation, the transparent device is rotated clockwise for 30s and then rotated anticlockwise for 30s, the plane where the transparent device is rotated is opposite to the incandescent lamp, wherein the transparent glass is a cylinder with the diameter and the height of 1cm, the spiraea ulmaria stem tip or axillary bud is located at the central point of the transparent glass after being buried in the agar, and the color temperature of the incandescent.
The invention at least comprises the following beneficial effects:
the method has the advantages of simple and convenient operation, low cost, wide suitable preservation variety, stable heritability of the preserved material and the like, and is a preferred method for long-term preservation of excellent germplasm resources in the last decade.
The invention provides an ultralow-temperature preservation and culture method for spiraea, provides technical guarantee for preservation of endangered spiraea germplasm resources, and provides an effective way for preservation of rosaceous, spiraea subfamily and spiraea plant resources and cultivation of new varieties by utilizing plant diversity.
The recovery rate and the regeneration rate of the spiraea ulmaria stem tip cultured by the ultralow temperature preservation method are respectively higher than 94% and higher than 90%, and are almost the same as those of the spiraea ulmaria plant directly cut from the spiraea ulmaria plant. Particularly, the invention adopts the dehydration device during the dehydration treatment of the spiraea ulmaria stem tip, prevents the inner part of the spiraea ulmaria stem tip from forming crystal nucleus during the dehydration, promotes the spiraea ulmaria stem tip to form a uniform vitrification state, is beneficial to the subsequent preservation in liquid nitrogen, has high recovery rate and regeneration rate when being taken out from the liquid nitrogen for recovery culture, and has better germination and survival conditions than the stem tip directly cut from a spiraea ulmaria plant for culture.
The spiraea japonica stem tip is pretreated before being put into the pre-culture solution, active substances in the spiraea japonica stem tip are activated through the pretreatment, substances in the whole cell are in a uniform dynamic state, the spiraea japonica stem tip has good activity capability before the pre-culture, and the spiraea japonica stem tip is beneficial to full dehydration after the pre-culture, so that the recovery rate and the regeneration rate of the spiraea japonica stem tip are improved finally.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
Fig. 1 is a schematic structural diagram of the dewatering device in one embodiment of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the drawings and examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof. It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials, if not otherwise specified, are commercially available; in the description of the present invention, the terms "transverse," "longitudinal," "axial," "radial," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like refer to orientations or positional relationships that are illustrated in the accompanying drawings, which are used for convenience in describing the present invention and to simplify the description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be considered limiting of the present invention.
< example 1>
The invention provides a method for ultralow-temperature preservation and culture of spiraea ulmaria, which comprises the following steps: placing the spiraea ulmaria stem tip with the length of 1mm cultured in a pre-culture solution in a shaking table oscillator for dark culture for 1d at room temperature, treating for 30min by using a loading solution, taking out the spiraea ulmaria stem tip, transferring into a vitrification solution with the temperature of 0 ℃, dehydrating for 20min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Wherein the pre-culture solution is as follows: 1/2MS +0.8mol/L sucrose +7g/L agar;
the loading liquid comprises: 1/2MS +2mol/L propanetriol +0.5mol/L cane sugar
The vitrification solution is: PVS1
< example 2>
The invention provides a method for ultralow-temperature preservation and culture of spiraea ulmaria, which comprises the following steps: placing the spiraea ulmaria stem tip with the length of 2mm cultured in a pre-culture solution in a shaking table oscillator for dark culture for 2d at room temperature, treating for 40min by using a loading solution, taking out the spiraea ulmaria stem tip, transferring into a vitrification solution with the temperature of 0 ℃, dehydrating for 40min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Wherein the pre-culture solution is as follows: MS +1.2mol/L glucose +0.2mg/mL NAA;
the loading liquid comprises: 1/2MS +2mol/L propanetriol +0.7mol/L cane sugar
The vitrification solution is: PVS3
< example 3>
The invention provides a method for ultralow-temperature preservation and culture of spiraea ulmaria, which comprises the following steps: placing the spiraea ulmaria stem tip with the length of 2mm cultured in a pre-culture solution in a shaking table oscillator for dark culture in the dark for 3d at room temperature, treating for 50min by using a loading solution, taking out the spiraea ulmaria stem tip, transferring into a vitrification solution with the temperature of 0 ℃, dehydrating for 60min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Wherein the pre-culture solution is as follows: MS +1.2mol/L glucose + 5% DMSO +1.0mg/mL6-BA
The loading liquid comprises: 1/2MS +2mol/L propanetriol +0.7mol/L cane sugar
The vitrification solution is: PVS1
< example 4>
The cryopreserved tube of example 1 was removed from liquid nitrogen, the vitrification solution was aspirated, the spiraea ulmaria shoot tips were washed 2 times with the unloading solution, and then transferred to a recovery medium for resuscitation culture.
Wherein the recovery medium is: 1/2MS +0.1mg/L NAA +0.5 mg/L6-BA +10g/L banana +5g/L agar;
the liquid unloading liquid comprises the following steps: 1/2MS +1.2mol/L sucrose
During recovery culture, culturing for 2 days in the dark, and then culturing for 5 days in the light at the illumination intensity of 1500lux and the temperature of 25 ℃ and the humidity of 40 percent, wherein the illumination time is 8 h/d.
< example 5>
The cryopreserved tube of example 2 was removed from liquid nitrogen, the vitrification solution was aspirated, the spiraea ulmaria shoot tips were washed 2 times with the unloading solution, and then transferred to a recovery medium for resuscitation culture.
Wherein the recovery medium is: 1/2MS +0.2mg/L NAA +5g/L pumpkin powder +10g/L banana +5g/L agar;
the liquid unloading liquid comprises the following steps: 1/2MS +1.2mol/L sucrose
During recovery culture, culturing for 3 days in the dark, and then culturing for 7 days in the light at the illumination intensity of 1200lux and the temperature of 25 ℃ and the humidity of 40 percent, wherein the illumination time is 8 h/d.
< example 6>
The cryopreserved tube of example 3 was removed from liquid nitrogen, the vitrification solution was aspirated, the spiraea ulmaria shoot tips were washed 2 times with the unloading solution, and then transferred to a recovery medium for resuscitation culture.
Wherein the recovery medium is: 1/2MS +0.5 mg/L6-BA +8g/L banana +3g/L agar;
the liquid unloading liquid comprises the following steps: 1/2MS +1.2mol/L sucrose
During recovery culture, culturing for 3 days in the dark, and then culturing for 7 days in the light at the illumination intensity of 1800lux and the humidity of 40% at the illumination time of 8 h/d.
< example 7>
The cryopreservation tube in example 1 was taken out of liquid nitrogen, after the vitrification solvent was absorbed, the spiraea japonica stem tip was immersed in a water bath at 35 ℃ for 1min, then washed with a liquid-releasing agent for 3 times, and then the surface of the spiraea japonica stem tip was washed with sterile water, and finally transferred to a recovery medium for recovery culture.
Wherein the recovery medium is: 1/2MS +0.1mg/L NAA +0.5 mg/L6-BA +10g/L banana +5g/L agar;
the liquid unloading liquid comprises the following steps: 1/2MS +1.2mol/L sucrose
During recovery culture, culturing for 2 days in the dark, and then culturing for 5 days in the light at the illumination intensity of 1500lux and the temperature of 25 ℃ and the humidity of 40 percent, wherein the illumination time is 8 h/d.
< example 8>
The cryopreservation tube in example 2 was taken out of liquid nitrogen, after the vitrification solvent was absorbed, the spiraea japonica stem tip was immersed in a water bath at 40 ℃ for 3min, then washed with the unloading liquid for 3 times, then washed with sterile water again, and finally transferred to a recovery medium for resuscitation.
Wherein the recovery medium is: 1/2MS +0.2mg/L NAA +5g/L pumpkin powder +10g/L banana +5g/L agar;
the liquid unloading liquid comprises the following steps: 1/2MS +1.2mol/L sucrose
During recovery culture, culturing for 3 days in the dark, and then culturing for 7 days in the light at the illumination intensity of 1200lux and the temperature of 25 ℃ and the humidity of 40 percent, wherein the illumination time is 8 h/d.
< example 9>
The cryopreservation tube in example 3 was taken out of liquid nitrogen, after the vitrification solvent was absorbed, the spiraea japonica stem tip was immersed in a water bath at 45 ℃ for 5min, then washed with a liquid-releasing agent for 3 times, and then the surface of the spiraea japonica stem tip was washed with sterile water, and finally transferred to a recovery medium for resuscitation culture.
Wherein the recovery medium is: 1/2MS +0.5 mg/L6-BA +8g/L banana +3g/L agar;
the liquid unloading liquid comprises the following steps: 1/2MS +1.2mol/L sucrose
During recovery culture, culturing for 3 days in the dark, and then culturing for 7 days in the light at the illumination intensity of 1800lux and the humidity of 40% at the illumination time of 8 h/d.
< example 10>
The invention provides a method for ultralow-temperature preservation and culture of spiraea ulmaria, which comprises the following steps: placing the spiraea ulmaria stem tip with the length of 2mm cultured in a pre-culture solution in a shaking table oscillator for dark culture for 1d at room temperature, treating for 40min by using a loading solution, taking out the spiraea ulmaria stem tip, transferring into a vitrification solution with the temperature of 0 ℃, dehydrating for 40min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Taking out the cryopreservation tube from liquid nitrogen, absorbing and removing the vitrification solution, washing the spiraea ulmaria stem tip with unloading liquid for 2 times, and transferring the spiraea ulmaria stem tip into a recovery culture medium for recovery culture.
Wherein,
the preculture solution is MS culture solution which comprises DMSO with the mass fraction of 5%, 6-BA with the mass fraction of 1.0mg/mL and NAA with the mass fraction of 0.5 mg/mL.
The loading liquid comprises the following components: MS, 2mol/L of glycerol and 0.4mol/L of cane sugar.
The vitrification solution includes: MS, 30% of glycerol by mass, 15% of DMSO by mass, 15% of ethylene glycol by mass and 0.4mol/L of sucrose.
The liquid unloading carrier comprises: MS, 1.0mol/L sucrose; the recovery medium comprises: 1/2MS, 1.0mg/L of 6-BA, 0.5mg/mL of NAA.
During recovery culture, culturing for 3 days in the dark, and then culturing for 7 days in the light at the illumination intensity of 1800lux and the humidity of 40% at the illumination time of 12 h/d.
< example 11>
The invention provides a method for ultralow-temperature preservation and culture of spiraea ulmaria, which comprises the following steps: placing the spiraea ulmaria stem tip with the length of 2mm cultured in a pre-culture solution in a shaking table oscillator for dark culture for 1d at room temperature, treating for 40min by using a loading solution, taking out the spiraea ulmaria stem tip, transferring into a vitrification solution with the temperature of 0 ℃, dehydrating for 40min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Taking out the frozen tube from liquid nitrogen, removing vitrified solution, soaking spiraea ulmaria stem tip in water bath at 40 ℃ for 5min, washing the spiraea ulmaria stem tip with unloading liquid for 2 times, cleaning the surface of the spiraea ulmaria stem tip with sterile water, and transferring the spiraea ulmaria stem tip into a recovery culture medium for recovery culture.
Wherein,
the preculture solution is MS culture solution which comprises DMSO with the mass fraction of 5%, 6-BA with the mass fraction of 1.0mg/mL and NAA with the mass fraction of 0.5 mg/mL.
The loading liquid comprises the following components: MS, 2mol/L of glycerol and 0.4mol/L of cane sugar.
The vitrification solution includes: MS, 30% of glycerol by mass, 15% of DMSO by mass, 15% of ethylene glycol by mass and 0.4mol/L of sucrose.
The liquid unloading carrier comprises: MS, 1.0mol/L sucrose; the recovery medium comprises: 1/2MS, 1.0mg/L of 6-BA, 0.5mg/mL of NAA.
During recovery culture, culturing for 3 days in the dark, and then culturing for 7 days in the light at the illumination intensity of 1800lux and the humidity of 40% at the illumination time of 12 h/d.
< example 12>
The dehydration operation of the spiraea ulmaria stem tip of example 10 was performed in a dehydration apparatus, as shown in fig. 1, comprising a first container for containing a vitrification solution of 0c and a second container for containing the spiraea ulmaria stem tip, wherein,
the first container is a cuboid shell with an open top, the first container is horizontally placed, an arc-shaped partition plate 11 with an upward concave surface is clamped inside the first container, two linear edges of the arc-shaped partition plate 11 are respectively sealed with a wide edge at the top of the first container to divide the first container into an arc-shaped groove 12 positioned above and a closed cavity 13 positioned below, and the closed cavity 13 is filled with superfine glass wool; the second container includes support, pivot 22, connecting rod 23, holds shell 24, the support is located the first container outside, the rotatable level of pivot 22 sets up on the support, pivot 22 passes through motor drive, the one end rigid coupling of connecting rod 23 is in pivot 22, the other end with the top rigid coupling that holds shell 24, hold shell 24 and have the spheroid structure of introduction port for the bottom, it is equipped with four feed liquor holes 241 to hold shell 24 last interval, wherein two feed liquor holes 241 are close to hold shell 24's top and about pivot 22 is symmetrical, and remaining two feed liquor holes 241 are close to the introduction port and about pivot 22 is symmetrical, hold attached sponge 242 on shell 24's the inner wall, sponge 242 does not shelter from four feed liquor holes 241, hold shell 24 not with arc baffle 11 contacts, introduction port detachably is equipped with lid 25, a fixed column 251 is fixedly arranged on the cover body 25, an object carrying cylinder 252 with an open top is fixedly arranged at the top end of the fixed column 251, the fixed column 251 and the object carrying cylinder 252 are both positioned in the accommodating shell 24, the top of the object carrying cylinder 252 abuts against the sponge 242 in the accommodating shell 24, a plurality of through holes 253 are arranged on the side wall of the object carrying cylinder 252 at intervals, and the axis of any one through hole 253 is perpendicular to the axis of any one liquid inlet hole 241; when the accommodating case 24 is located at the midpoint of the arc-shaped slot 12, the connecting rod 23, the object carrying cylinder 252 and the fixing column 251 are coaxially arranged in the vertical direction;
the arc-shaped groove 12 is filled with 0 ℃ vitrification solution, the object carrying cylinder 252 is filled with spiraea ulmaria stem tips, the object carrying cylinder 252 is filled with the 0 ℃ vitrification solution filled in the arc-shaped groove 12, and after the object carrying cylinder 252 is filled with the 0 ℃ vitrification solution through the four liquid inlet holes 241 and the through holes 253, the motor controls the rotating shaft 22 to rotate clockwise for a quarter of a circle and then rotate anticlockwise for a quarter of a circle, so that the object carrying cylinder 24 swings in the arc-shaped groove 12 until the dehydration is finished, and the object carrying cylinder 24 is always immersed in the vitrification solution.
< example 13>
The dehydration operation of the spiraea ulmaria stem tip of example 11 was performed in a dehydration apparatus, as shown in fig. 1, comprising a first container for containing a vitrification solution of 0c and a second container for containing the spiraea ulmaria stem tip, wherein,
the first container is a cuboid shell with an open top, the first container is horizontally placed, an arc-shaped partition plate 11 with an upward concave surface is clamped inside the first container, two linear edges of the arc-shaped partition plate 11 are respectively sealed with a wide edge at the top of the first container to divide the first container into an arc-shaped groove 12 positioned above and a closed cavity 13 positioned below, and the closed cavity 13 is filled with superfine glass wool; the second container includes support, pivot 22, connecting rod 23, holds shell 24, the support is located the first container outside, the rotatable level of pivot 22 sets up on the support, pivot 22 passes through motor drive, the one end rigid coupling of connecting rod 23 is in pivot 22, the other end with the top rigid coupling that holds shell 24, hold shell 24 and have the spheroid structure of introduction port for the bottom, it is equipped with four feed liquor holes 241 to hold shell 24 last interval, wherein two feed liquor holes 241 are close to hold shell 24's top and about pivot 22 is symmetrical, and remaining two feed liquor holes 241 are close to the introduction port and about pivot 22 is symmetrical, hold attached sponge 242 on shell 24's the inner wall, sponge 242 does not shelter from four feed liquor holes 241, hold shell 24 not with arc baffle 11 contacts, introduction port detachably is equipped with lid 25, a fixed column 251 is fixedly arranged on the cover body 25, an object carrying cylinder 252 with an open top is fixedly arranged at the top end of the fixed column 251, the fixed column 251 and the object carrying cylinder 252 are both positioned in the accommodating shell 24, the top of the object carrying cylinder 252 abuts against the sponge 242 in the accommodating shell 24, a plurality of through holes 253 are arranged on the side wall of the object carrying cylinder 252 at intervals, and the axis of any one through hole 253 is perpendicular to the axis of any one liquid inlet hole 241; when the accommodating case 24 is located at the midpoint of the arc-shaped slot 12, the connecting rod 23, the object carrying cylinder 252 and the fixing column 251 are coaxially arranged in the vertical direction;
the arc-shaped groove 12 is filled with 0 ℃ vitrification solution, the object carrying cylinder 252 is filled with spiraea ulmaria stem tips, the object carrying cylinder 252 is filled with the 0 ℃ vitrification solution filled in the arc-shaped groove 12, and after the object carrying cylinder 252 is filled with the 0 ℃ vitrification solution through the four liquid inlet holes 241 and the through holes 253, the motor controls the rotating shaft 22 to rotate clockwise for a quarter of a circle and then rotate anticlockwise for a quarter of a circle, so that the object carrying cylinder 24 swings in the arc-shaped groove 12 until the dehydration is finished, and the object carrying cylinder 24 is always immersed in the vitrification solution.
< example 14>
Example 10. spiraea ulmaria stem tips were pretreated before being placed in a pre-culture solution, the pretreatment comprising: the spiraea ulmaria stem tip or axillary bud is placed into 1.2mol/L glycine to be soaked for 30s, then the spiraea ulmaria stem tip or axillary bud is buried in a transparent glass filled with agar, an incandescent lamp is adopted for irradiation, the transparent device is rotated by taking the incandescent lamp as the center and taking the distance of 10cm from the incandescent lamp as the radius during irradiation, the transparent device is rotated clockwise for 30s and then rotated anticlockwise for 30s, the plane where the transparent device is rotated is opposite to the incandescent lamp, wherein the transparent glass is a cylinder with the diameter and the height of 1cm, the spiraea ulmaria stem tip or axillary bud is located at the central point of the transparent glass after being buried in the agar, and the color temperature of the incandescent.
< example 15>
Example 11. spiraea ulmaria stem tips were pretreated before being placed in a pre-culture solution, the pretreatment comprising: the spiraea ulmaria stem tip or axillary bud is placed into 1.2mol/L glycine to be soaked for 30s, then the spiraea ulmaria stem tip or axillary bud is buried in a transparent glass filled with agar, an incandescent lamp is adopted for irradiation, the transparent device is rotated by taking the incandescent lamp as the center and taking the distance of 10cm from the incandescent lamp as the radius during irradiation, the transparent device is rotated clockwise for 30s and then rotated anticlockwise for 30s, the plane where the transparent device is rotated is opposite to the incandescent lamp, wherein the transparent glass is a cylinder with the diameter and the height of 1cm, the spiraea ulmaria stem tip or axillary bud is located at the central point of the transparent glass after being buried in the agar, and the color temperature of the incandescent.
< Standard control group >
Directly cutting a spiraea ulmaria stem tip with the length of 2mm from a spiraea ulmaria plant, and directly culturing in a culture medium, wherein the culture medium comprises: 1/2MS, 1.0mg/L of 6-BA, 0.5mg/mL of NAA.
< comparative example 1>
In the prior art, no method for culturing the spiraea subjected to ultralow-temperature preservation exists, and the known ultralow-temperature culture method is selected for culturing the spiraea, and the specific method comprises the following steps: putting the in-vitro stem tip containing 2 leaf primordia and having the stem tip length of 2-3 mm into a pre-culture solution for treatment, and pre-culturing the MS solution with the optimal sucrose concentration of 0.3mol/L in the pre-culture solution for 3d at the temperature of 4 ℃; placing the pre-cultured in-vitro stem tip in LS loading liquid, and loading for 10-50 min at room temperature of 25 ℃; the loading liquid comprises the following components: 2M glycerol +0.4mol/L sucrose + MS solution; placing the loaded stem tips in a PVS2 protective solution, and dehydrating for 20-100 min at the temperature of 0 ℃; the PVS2 protective solution comprises the following components: 30% glycerol, 15% glycol, 15% dimethyl sulfoxide, 0.4mol/L sucrose and MS; replacing the stem tip treated by the PVS2 protective solution with fresh PVS2 protective solution, then quickly putting into liquid nitrogen, and preserving for 24 h; taking out the freezing pipe after the liquid nitrogen preservation for defrosting, wherein the defrosting mode is as follows: thawing the stem tip in 40 deg.C water bath for 70 s; the recovery culture specifically comprises the step of inoculating the stem tip washed by the unloading liquid for 20min on a recovery culture medium for recovery culture; the unloading liquid comprises the following components: 1.2mol/L sucrose + MS solution; the recovery culture medium comprises: MS + KT0.5mg/L + NAA0.1mg/L + GA31.0mg/L; the recovery culture conditions are as follows: culturing in 25 deg.C incubator for 14 days, and culturing under normal light.
< test comparison >
The frozen tubes of examples 1, 2 and 3 were thawed in a water bath at 40 ℃ for 10min, washed with sterile water for 3 times, and then placed in a recovery medium for recovery culture, wherein the recovery medium comprises components which are helpful for the rooting and germination of the spiraea ulmaria in the prior art, such as: 1/2MS +0.6mg/L NAA +0.4 mg/L6-BA.
100 of the spiraea japonica shoot tips of examples 1 to 13, the standard control group and comparative example 1 were subjected to recovery culture, and the recovery rate after 10 days and the regeneration rate after 30 days were recorded as shown in Table 1 below.
TABLE 1
| Rate of resuscitation | Regeneration rate | |
| Example 1 | 83％ | 75％ |
| Example 2 | 84％ | 76％ |
| Example 3 | 83％ | 77％ |
| Example 4 | 84％ | 76％ |
| Example 5 | 85％ | 75％ |
| Example 6 | 85％ | 77％ |
| Example 7 | 86％ | 79％ |
| Example 8 | 86％ | 78％ |
| Example 9 | 87％ | 79％ |
| Example 10 | 86％ | 79％ |
| Example 11 | 87％ | 80％ |
| Example 12 | 88％ | 80％ |
| Example 13 | 89％ | 91％ |
| Example 14 | 88％ | 78％ |
| Example 15 | 87％ | 79％ |
| Standard control group | 89％ | 75％ |
| Comparative example 1 | 41％ | 35％ |
As can be seen from Table 1, the recovery rate and regeneration rate of the spiraea ulmaria stem tip cultured by the cryopreservation method of the invention are both higher than 80% and higher than 78%, which are comparable to those of the spiraea ulmaria plant directly cut from the stem tip. In particular, in embodiments 12 and 13 of the present invention, the dehydration device is used during the dehydration process of the spiraea ulmaria stem tip, the spiraea ulmaria stem tip accommodated in the object carrying barrel 252 during the dehydration process reciprocates in the arc-shaped accommodating groove to form a single pendulum type swing, and during the swing, the liquid inlet hole 241 and the through hole 253 are arranged in such a way that the spiraea ulmaria stem tip is not affected by a larger liquid flow, the vitrification solution around the spiraea ulmaria stem tip forms a tiny disturbance, which is helpful for the water separated from the spiraea ulmaria to flow with the vitrification solution, so that more pure vitrification solution surrounds the spiraea ulmaria stem tip for sufficient dehydration, and the tiny drift formed by the swing is matched with the dehydration process, thereby promoting the movement of cells inside the spiraea ulmaria stem tip, preventing the formation of crystal nucleus inside the spiraea stem tip, promoting the spiraea stem tip to form a uniform vitrification state, and being beneficial for subsequent storage in liquid nitrogen, when taken out of liquid nitrogen for resuscitation culture, the resuscitation and regeneration rates were high (see table 1).
In addition, in examples 14 and 15 of the present invention, the spiraea ulmaria stem tip is pretreated before being placed in the pre-culture solution, and in the process of irradiating the incandescent lamp, substances in cells of the spiraea ulmaria stem tip soaked by glycine are in a uniform dynamic state, so that the spiraea ulmaria stem tip has good activity capability before the pre-culture, and is beneficial to full dehydration after the pre-culture, and finally, the recovery rate and the regeneration rate of the spiraea ulmaria stem tip are improved.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. The method for ultralow temperature preservation and culture of spiraea salicifolia is characterized in that spiraea salicifolia stem tips or axillary buds which are cultured in a pre-culture solution and have the length of 1-2mm are placed in a shaking table oscillator to be dark-shielded and cultured for 1-3 days at room temperature, then the spiraea salicifolia stem tips or the axillary buds are treated with a loading solution for 20-60min, the spiraea salicifolia stem tips or the axillary buds are taken out and transferred into a vitrification solution with the temperature of 0 ℃ to be dehydrated for 30-50min and then placed in a freezing tube, and the freezing tube is stored in liquid nitrogen to be ultralow temperature preserved.
2. The method for cryopreservation and culture of spiraea ulmaria as claimed in claim 1, further comprising: taking out the frozen tube from liquid nitrogen, removing the vitrified solution by suction, washing the spiraea ulmaria stem tip or axillary bud with unloading liquid for 1-3 times, and transferring to a recovery culture medium for recovery culture.
3. The method for ultralow-temperature preservation and culture of spiraea salicifolia as claimed in claim 2, wherein after the vitrification solvent is absorbed, the spiraea salicifolia stem tip or axillary bud is firstly placed in a water bath with the temperature of 35-45 ℃ for soaking for 1-5min, then the spiraea salicifolia stem tip or axillary bud is washed by using a liquid unloading agent for 1-3 times, then the surface of the spiraea salicifolia stem tip or axillary bud is washed by using sterile water, and finally the spiraea salicifolia stem tip or axillary bud is transferred to a recovery culture medium for recovery culture.
4. The method for ultralow-temperature preservation and culture of spiraea salicifolia as claimed in claim 1, wherein the preculture solution is an MS culture solution which comprises DMSO with a mass fraction of 5%, 6-BA with a mass fraction of 1.0mg/mL and NAA with a mass fraction of 0.5 mg/mL.
5. The method for cryopreservation and culture of spiraea ulmaria as claimed in claim 1, wherein the loading solution comprises the following components: MS, 2mol/L of glycerol and 0.4mol/L of cane sugar.
6. The method for cryopreservation and culture of spiraea ulmaria as claimed in claim 1, wherein the vitrification solution comprises: MS, 30% of glycerol by mass, 15% of DMSO by mass, 15% of ethylene glycol by mass and 0.4mol/L of sucrose.
7. The method for cryopreservation culture of spiraea salicifolia as claimed in claim 2 or 3, wherein the unloading liquid comprises: MS, 1.0mol/L sucrose; the recovery medium comprises: 1/2MS, 1.0mg/L of 6-BA, 0.5mg/mL of NAA.
8. The method for ultralow-temperature preservation and culture of spiraea salicifolia as claimed in claim 2 or 3, wherein during the recovery culture, the spiraea salicifolia is cultured for 3 days in the dark, and then is transferred to the illumination culture for 7 days, the illumination time is 12h/d, the illumination intensity is 1800lux, the temperature is 25 ℃, and the humidity is 40%.
9. The method for the cryopreservation culture of spiraea salicifolia as claimed in claim 1, wherein the dehydration is performed in a dehydration device comprising a first container for containing a vitrification solution at 0 ℃ and a second container for containing spiraea salicifolia stem tip or axillary bud, wherein,
the first container is a cuboid shell with an open top, the first container is horizontally placed, an arc-shaped partition plate with an upward concave surface is clamped inside the first container, two linear edges of the arc-shaped partition plate are respectively sealed with a wide edge at the top of the first container to divide the first container into an arc-shaped groove at the upper part and a closed cavity at the lower part, and superfine glass wool is filled in the closed cavity; the second container comprises a support, a rotating shaft, a connecting rod and a containing shell, the support is positioned outside the first container, the rotating shaft is rotatably and horizontally arranged on the support, the rotating shaft is driven by a motor, one end of the connecting rod is fixedly connected to the rotating shaft, the other end of the connecting rod is fixedly connected with the top of the containing shell, the containing shell is of a spherical structure with a sample inlet at the bottom, four liquid inlet holes are arranged on the containing shell at intervals, two of the liquid inlet holes are close to the top of the containing shell and are symmetrical relative to the rotating shaft, the remaining two liquid inlet holes are close to the sample inlet and are symmetrical relative to the rotating shaft, sponge is attached to the inner wall of the containing shell, the sponge does not shield the four liquid inlet holes, the containing shell is not contacted with the arc-shaped partition plate, a cover body is detachably arranged on the sample inlet, a fixed column is fixedly arranged on the cover body, the fixed column and the object carrying cylinder are both positioned in the accommodating shell, the top of the object carrying cylinder props against sponge in the accommodating shell, a plurality of through holes are arranged on the side wall of the object carrying cylinder at intervals, and the axis of any through hole is vertical to the axis of any liquid inlet hole; when the accommodating shell is positioned at the right center of the arc-shaped groove, the connecting rod, the object carrying cylinder and the fixing column are coaxially arranged in the vertical direction,
the improved multifunctional container comprises an arc-shaped groove, a containing shell, a rotating shaft, a containing shell and a containing shell, wherein the arc-shaped groove is internally filled with 0 ℃ vitrification solution, the containing shell is filled with spiraea ulmaria stem tips or axillary buds, the 0 ℃ vitrification solution filled in the arc-shaped groove is immersed in the containing shell, after the 0 ℃ vitrification solution is filled in the containing shell through four liquid inlet holes and a plurality of through holes, the rotating shaft is controlled by a motor to rotate clockwise for a quarter circle first, and then rotate counterclockwise for a quarter circle, so that the containing shell swings in the arc-shaped groove until the dehydration is finished, and the containing shell is immersed in the vitrification solution all the time.
10. The method for cryopreservation culture of spiraea salicifolia as claimed in claim 1, wherein the spiraea salicifolia stem tip or axillary bud is pretreated before being put into the pre-culture solution, and the pretreatment comprises: the spiraea ulmaria stem tip or axillary bud is placed into 1.2mol/L glycine to be soaked for 30s, then the spiraea ulmaria stem tip or axillary bud is buried in a transparent glass filled with agar, an incandescent lamp is adopted for irradiation, the transparent device is rotated by taking the incandescent lamp as the center and taking the distance of 10cm from the incandescent lamp as the radius during irradiation, the transparent device is rotated clockwise for 30s and then rotated anticlockwise for 30s, the plane where the transparent device is rotated is opposite to the incandescent lamp, wherein the transparent glass is a cylinder with the diameter and the height of 1cm, the spiraea ulmaria stem tip or axillary bud is located at the central point of the transparent glass after being buried in the agar, and the color temperature of the incandescent.
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