CN109673515A - Asparagus filicicum Ham ultralow temperature cultural method - Google Patents
Asparagus filicicum Ham ultralow temperature cultural method Download PDFInfo
- Publication number
- CN109673515A CN109673515A CN201910113311.1A CN201910113311A CN109673515A CN 109673515 A CN109673515 A CN 109673515A CN 201910113311 A CN201910113311 A CN 201910113311A CN 109673515 A CN109673515 A CN 109673515A
- Authority
- CN
- China
- Prior art keywords
- asparagus
- culture
- solution
- stem tip
- asparagus fern
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000005340 Asparagus officinalis Nutrition 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 17
- 241000234427 Asparagus Species 0.000 title claims abstract 12
- 239000007788 liquid Substances 0.000 claims abstract description 73
- 238000004017 vitrification Methods 0.000 claims abstract description 56
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 52
- 230000018044 dehydration Effects 0.000 claims abstract description 29
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 29
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 26
- 238000005138 cryopreservation Methods 0.000 claims abstract description 22
- 239000000243 solution Substances 0.000 claims description 90
- 241000432824 Asparagus densiflorus Species 0.000 claims description 77
- 238000011084 recovery Methods 0.000 claims description 57
- 238000004321 preservation Methods 0.000 claims description 29
- 244000248539 Asparagus cochinchinensis Species 0.000 claims description 28
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 27
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 235000009292 Asparagus cochinchinensis Nutrition 0.000 claims description 26
- 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000005720 sucrose Substances 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 18
- 238000007710 freezing Methods 0.000 claims description 15
- 230000008014 freezing Effects 0.000 claims description 15
- 238000012136 culture method Methods 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- 238000005192 partition Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000002250 absorbent Substances 0.000 claims description 8
- 230000002745 absorbent Effects 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- VRIVJOXICYMTAG-IYEMJOQQSA-L iron(ii) gluconate Chemical compound [Fe+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O VRIVJOXICYMTAG-IYEMJOQQSA-L 0.000 claims description 8
- 238000005507 spraying Methods 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims description 4
- 239000011491 glass wool Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 241000432767 Asparagus setaceus Species 0.000 claims description 3
- 238000012258 culturing Methods 0.000 abstract description 22
- 230000008929 regeneration Effects 0.000 abstract description 11
- 238000011069 regeneration method Methods 0.000 abstract description 11
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 208000005156 Dehydration Diseases 0.000 description 24
- 238000005286 illumination Methods 0.000 description 19
- 244000003416 Asparagus officinalis Species 0.000 description 16
- 235000011187 glycerol Nutrition 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 7
- 240000008790 Musa x paradisiaca Species 0.000 description 7
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 5
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 4
- 235000017491 Bambusa tulda Nutrition 0.000 description 4
- 241001330002 Bambuseae Species 0.000 description 4
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 4
- 239000011425 bamboo Substances 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241001448336 Asparagus filicinus Species 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 2
- 235000009854 Cucurbita moschata Nutrition 0.000 description 2
- 240000001980 Cucurbita pepo Species 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
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 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
- 241000894007 species Species 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 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
- 201000004681 Psoriasis Diseases 0.000 description 1
- 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
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000000044 effect on lymphoma Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
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)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of asparagus filicicum Ham ultralow temperature cultural methods, include: at room temperature will length of the culture in pre-culture solution be 1-2mm asparagus filicicum Ham 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 asparagus filicicum Ham 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.By means of the present invention, the anabiosis rate of asparagus filicicum Ham and regeneration rate are very high, provide technology for asparagus filicicum Ham Germ-plasma resources protection in imminent danger and guarantee, new varieties will be cultivated to Asparagus and using Plant Diversity and provide an effective way.
Description
Technical Field
The invention relates to the technical field of germplasm resource preservation, in particular to an ultra-low temperature culture method for asparagus fern.
Background
Asparagus fern (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 asparagus fern contains rich 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. Radix asparagi can clear heat, promote diuresis, promote blood circulation, and eliminate stagnation, and is mainly used for treating hepatitis, psoriasis, hyperlipidemia, and hyperplasia of mammary glands, and also has certain curative effect on lymphoma, bladder cancer, breast cancer, skin cancer, etc.
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 asparagus fern ultra-low temperature preservation technology is not 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 culture method for asparagus fern, which has high recovery rate and regeneration rate, provides technical guarantee for storing endangered asparagus fern germplasm resources, and provides an effective way for asparagus and a new variety culture by utilizing plant diversity.
To achieve these objects and other advantages in accordance with the present invention, there is provided an ultra-low temperature culture method of asparagus fern, comprising: placing the asparagus cochinchinensis stem tip with the length of 1-2mm cultured in a pre-culture solution in a shaking table oscillator for dark culture for 1-3d at room temperature, treating with a loading solution for 20-60min, taking out the asparagus cochinchinensis stem tip, transferring into a vitrification solution with the temperature of 0 ℃ for dehydration for 30-50min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Preferably, the method further comprises the following steps: taking out the cryopreservation tube from liquid nitrogen, absorbing the vitrification solution, washing the stem tip of the asparagus fern by using unloading liquid for 1-3 times, and transferring the stem tip to a recovery culture medium for recovery culture.
Preferably, after the vitrification solvent is absorbed, the asparagus fern stem tip is firstly placed in a water bath with the temperature of 35-45 ℃ for soaking for 1-5min, then the asparagus fern stem tip is washed by unloading liquid for 1-3 times, then the surface of the asparagus fern stem tip is washed by sterile water, and finally the asparagus fern stem tip is transferred into 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 a asparagus cochinchinensis tip, 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,
wherein, the vitrification solution of 0 ℃ of splendid attire in the arc wall, contain the asparagus stem tip of sheep tooth in the objective housing, the vitrification solution submergence of 0 ℃ of splendid attire in the arc wall holds the shell, and when vitrification solution of 0 ℃ is full of through four feed liquor holes, a plurality of through-holes behind the objective housing, motor control the pivot is clockwise rotation quarter round earlier, and anticlockwise rotation quarter round again, so that hold the shell and swing in the arc wall and end up to the dehydration, just hold the shell submergence all the time in vitrification solution.
Preferably, the asparagus fern stem tips are further treated with the following treatment before being treated with the loading solution: taking the stem tip of the asparagus fern out of a shaking table oscillator, soaking the stem tip of the asparagus fern in distilled water for 30s, placing the stem tip of the asparagus fern on absorbent paper, covering the absorbent paper with two layers of gauze, spraying 0.5mL of 0.5% sodium chloride aqueous solution to the gauze, standing for 1min, spraying 0.3mL of 5ppm abscisic acid, standing for 1min, washing the surface of the stem tip of the asparagus fern with distilled water, and treating with a loading solution.
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 ultra-low temperature preservation and culture method for asparagus fern, which provides technical guarantee for preservation of endangered species resources of asparagus fern and provides an effective way for asparagus and for breeding new species by utilizing plant diversity.
The recovery rate of the stem tips of the asparagus fern cultured by the ultralow-temperature preservation method is higher than 94%, the regeneration rate is higher than 90%, and the recovery rate and the regeneration rate are almost the same as those of stem tips directly cut from asparagus fern plants. Particularly, the invention adopts the dehydration device during the dehydration treatment of the asparagus fern stem tip, prevents the formation of crystal nucleus inside the asparagus fern stem tip during dehydration, promotes the asparagus fern stem tip to form a uniform vitrification state, is beneficial to 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 an asparagus fern plant for culture.
The stem tip of the asparagus fern is pretreated before being put into the pre-culture solution, active substances in the stem tip of the asparagus fern are activated through the pretreatment, all substances in the whole cell are in a uniform dynamic state, the stem tip of the asparagus fern has good activity capability before the pre-culture, and the stem tip of the asparagus fern is beneficial to full dehydration after the pre-culture, so that the recovery rate and the regeneration rate of the stem tip of the asparagus fern 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 an ultra-low temperature preservation and culture method of asparagus fern, which comprises the following steps: placing the cochinchnese asparagus 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 20min by using a loading solution, taking out the cochinchnese asparagus stem tip, transferring the cochinchnese asparagus stem tip into a vitrification solution with the temperature of 0 ℃, dehydrating for 25min, placing the cochinchnese asparagus stem tip 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 an ultra-low temperature preservation and culture method of asparagus fern, which comprises the following steps: placing the asparagus fern stem tips with the length of 2mm cultured in the 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 asparagus fern stem tips, transferring into a vitrification solution with the temperature of 0 ℃ for dehydration for 40min, then 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 an ultra-low temperature preservation and culture method of asparagus fern, which comprises the following steps: placing the asparagus fern stem tips with the length of 2mm cultured in the pre-culture solution in a shaking table oscillator for dark culture for 3d at room temperature, treating for 460min with a loading solution, taking out the asparagus fern stem tips, transferring into a vitrification solution with the temperature of 0 ℃ for dehydration for 60min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultra-low 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 cryopreservation tube of example 1 was taken out of liquid nitrogen, the vitrification solution was aspirated, and the stem tips of Asparagus ferox were washed 2 times with the unloading solution and 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 cryopreservation tube of example 2 was taken out of liquid nitrogen, the vitrification solution was aspirated, and the stem tips of Asparagus ferox were washed 2 times with the unloading solution and 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 cryopreservation tube of example 3 was taken out of liquid nitrogen, the vitrification solution was aspirated, and the stem tips of Asparagus ferox were washed 2 times with the unloading solution and 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 asparagus fern shoot tips were immersed in a water bath at 35 ℃ for 1min, washed with a liquid-releasing agent for 3 times, then 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, and after the vitrification solvent was absorbed, the stem tip of Asparagus cochinchinensis was immersed in a water bath at 40 ℃ for 3min, then washed with an unloading liquid for 3 times, then washed with sterile water, and finally transferred to a recovery medium for recovery 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 9>
The cryopreservation tube in example 3 was taken out of liquid nitrogen, and after the vitrification solvent was absorbed, the stem tip of Asparagus cochinchinensis was immersed in a water bath at 45 ℃ for 5min, and then washed with a liquid-releasing agent for 3 times, and then the surface of the stem tip of Asparagus cochinchinensis was washed with sterile water, and finally transferred to a recovery medium for recovery 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 an ultra-low temperature preservation and culture method of asparagus fern, which comprises the following steps: placing the asparagus cochinchinensis 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 30min by using a loading solution, taking out the asparagus cochinchinensis stem tip, transferring into a vitrification solution with the temperature of 0 ℃ for dehydration for 40min, then placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
Taking the cryopreservation tube out of liquid nitrogen, sucking the vitrified solution, washing the stem tip of the asparagus fern for 2 times by using unloading liquid, and transferring the stem tip to 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 an ultra-low temperature preservation and culture method of asparagus fern, which comprises the following steps: placing the asparagus cochinchinensis 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 30min by using a loading solution, taking out the asparagus cochinchinensis stem tip, transferring into a vitrification solution with the temperature of 0 ℃ for dehydration for 40min, then 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, removing vitrified solution, soaking the asparagus fern stem tip in water bath at 40 ℃ for 5min, washing the asparagus fern stem tip with unloading liquid for 2 times, washing the surface of the asparagus fern stem tip with sterile water, and transferring to 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 asparagus fern stem tips of example 10 was carried out 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 asparagus fern stem tips, 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;
wherein, the vitrification solution of 0 ℃ of splendid attire in the arc wall 12, the thing section of thick bamboo 252 is held the fern asparagus stem tip of sheep tooth, the vitrification solution submergence of 0 ℃ of splendid attire in the arc wall 12 hold the shell 24, when the vitrification solution of 0 ℃ is full of through four feed liquor holes 241, a plurality of through-holes 253 the thing section of thick bamboo 252 back is filled with, motor control the pivot 22 rotates quarter round clockwise earlier, rotates quarter round anticlockwise again, so that hold shell 24 and swing in the arc wall 12 until the dehydration is finished, just hold shell 24 submergence all the time in vitrification solution.
< example 13>
The dehydration operation of the asparagus fern stem tips of example 11 was carried out 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 asparagus fern stem tips, 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;
wherein, the vitrification solution of 0 ℃ of splendid attire in the arc wall 12, the thing section of thick bamboo 252 is held the fern asparagus stem tip of sheep tooth, the vitrification solution submergence of 0 ℃ of splendid attire in the arc wall 12 hold the shell 24, when the vitrification solution of 0 ℃ is full of through four feed liquor holes 241, a plurality of through-holes 253 the thing section of thick bamboo 252 back is filled with, motor control the pivot 22 rotates quarter round clockwise earlier, rotates quarter round anticlockwise again, so that hold shell 24 and swing in the arc wall 12 until the dehydration is finished, just hold shell 24 submergence all the time in vitrification solution.
< example 14>
In example 10, the asparagus fern stem tips of step one were further treated as follows before being treated with the loading solution: taking the stem tip of the asparagus fern out of a shaking table oscillator, soaking the stem tip of the asparagus fern in distilled water for 30s, placing the stem tip of the asparagus fern on absorbent paper, covering the absorbent paper with two layers of gauze, spraying 0.5mL of 0.5% sodium chloride aqueous solution to the gauze, standing for 1min, spraying 0.3mL of 5ppm abscisic acid, standing for 1min, washing the surface of the stem tip of the asparagus fern with distilled water, and treating with a loading solution.
< example 15>
Example 11 in step one, the asparagus fern stem tips were further treated as follows before being treated with the loading solution: taking the stem tip of the asparagus fern out of a shaking table oscillator, soaking the stem tip of the asparagus fern in distilled water for 30s, placing the stem tip of the asparagus fern on absorbent paper, covering the absorbent paper with two layers of gauze, spraying 0.5mL of 0.5% sodium chloride aqueous solution to the gauze, standing for 1min, spraying 0.3mL of 5ppm abscisic acid, standing for 1min, washing the surface of the stem tip of the asparagus fern with distilled water, and treating with a loading solution.
< Standard control group >
Directly cut the asparagus cochinchinensis stem tip with the length of 2mm from the asparagus cochinchinensis plant and directly put the asparagus cochinchinensis stem tip into a culture medium for culture, wherein the culture medium is as follows: 1/2MS, 1.0mg/L of 6-BA, 0.5mg/mL of NAA.
< comparative example 1>
The prior art does not have a method for culturing asparagus fern after cryopreservation, and the known ultralow-temperature culture method is selected for culturing the asparagus fern, and the specific method comprises the following steps: the stem tip of the asparagus fern with the length of 2mm is placed in a position S101: pre-culturing 3.0-6.0 cm banana seedlings in a culture medium containing 0.4mol/L sucrose for 2 days; stripping the stem tip with 1-2 leaf primordia at room temperature; loading the stem tip with a loading solution for 30-60 min, and treating with a vitrification solution (PVS2) at 0 ℃ for 50 min; transferring the stem tip to an aluminum foil strip with the width of 0.5cm and the length of 2cm, on which PVS2 liquid drops are dropped, dipping the stem tip in liquid nitrogen, quickly filling the stem tip into a freezing tube filled with liquid nitrogen, and immersing the stem tip into the liquid nitrogen for storage; after being preserved in liquid nitrogen for at least 1h, the mixture is thawed in 1.2mol/L sucrose culture solution; washing with 1.2mol/L sucrose culture solution for 2 times, each time for 10 min; and transferring the stem tip after the treatment into an MS culture medium containing 0.3mol/L sucrose, culturing in a dark room for 2 days, transferring into an MS culture medium containing 0.5 mg/L6-BA, recovering and culturing for 30-35 days, and inoculating the seed source recovering normal growth into a new growth recovery culture medium for proliferation and rapid propagation.
< 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 the components which are helpful for the radication and germination of asparagus fern in the prior art, such as: 1/2MS +0.6mg/L NAA +0.4 mg/L6-BA.
100 of the stem tips of Asparagus ferox of examples 1-13, 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 | 80％ | 76％ |
| Example 2 | 82％ | 75％ |
| Example 3 | 81％ | 76％ |
| Example 4 | 83％ | 76％ |
| Example 5 | 82％ | 75％ |
| Example 6 | 82％ | 75％ |
| Example 7 | 84％ | 76％ |
| Example 8 | 84％ | 76％ |
| Example 9 | 83％ | 75％ |
| Example 10 | 85％ | 78％ |
| Example 11 | 86％ | 77％ |
| Example 12 | 88％ | 77％ |
| Example 13 | 87％ | 79％ |
| Example 14 | 85％ | 76％ |
| Example 15 | 86％ | 77％ |
| Standard control group | 89％ | 79％ |
| Comparative example 1 | 51％ | 42％ |
As can be seen from Table 1, the recovery rate and the regeneration rate of the stem tips of Asparagus cochinchinensis cultured by the cryopreservation method of the present invention are both higher than 90% and higher than 85%, which are comparable to those of stem tips directly cut from Asparagus cochinchinensis plants. In particular, in the embodiments 12 and 13 of the present invention, the dehydration device is used to dehydrate the asparagus cochinchinensis tips, when dehydrating, the asparagus cochinchinensis tips contained in the object carrying cylinder 252 reciprocate in the arc-shaped containing groove to form a single pendulum type swing, and when swinging, the liquid inlet hole 241 and the through hole 253 are arranged in such a way that the asparagus cochinchinensis tips are not affected by a larger liquid flow, the vitrification solution around the asparagus cochinchinensis tips forms a tiny disturbance, which is helpful for the water separated from the asparagus cochinchinensis to flow with the vitrification solution, so that more pure vitrification solution surrounds the asparagus cochinchinensis tips to be fully dehydrated, and the tiny drift formed by swinging is matched with the dehydration process, thereby promoting the movement of cells inside the asparagus cochinchinensis, preventing the formation of crystal nuclei inside the asparagus cochinchinensis tips, promoting the asparagus cochinchinensis tips to form a uniform vitrification state, the recovery rate and the regeneration rate are high when the culture medium is taken out from the liquid nitrogen for recovery culture (see table 1).
In addition, in the embodiments 14 and 15 of the invention, the asparagus fern stem tips are subjected to pre-dehydration treatment before being treated by the loading liquid, so that the asparagus fern stem tips are in a dehydrated state, the dehydration in a dehydration device at the later stage is facilitated, the dehydration is thorough, and finally the recovery rate and the regeneration rate of the asparagus fern stem tips 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 asparagus fern is characterized by comprising the following steps: placing the asparagus cochinchinensis stem tip with the length of 1-2mm cultured in a pre-culture solution in a shaking table oscillator for dark culture for 1-3d at room temperature, treating with a loading solution for 20-60min, taking out the asparagus cochinchinensis stem tip, transferring into a vitrification solution with the temperature of 0 ℃ for dehydration for 30-50min, placing into a freezing tube, and storing the freezing tube in liquid nitrogen for ultralow-temperature preservation.
2. The method for ultralow-temperature preservation and culture of asparagus fern as claimed in claim 1, further comprising the following steps: taking out the cryopreservation tube from liquid nitrogen, absorbing the vitrification solution, washing the stem tip of the asparagus fern by using unloading liquid for 1-3 times, and transferring the stem tip to a recovery culture medium for recovery culture.
3. The method for ultralow-temperature preservation and culture of Asparagus ferox according to claim 2, wherein after the vitrification solvent is absorbed, the stem tip of Asparagus ferox is first soaked in a water bath at 35-45 ℃ for 1-5min, then the stem tip of Asparagus ferox is washed with the unloading liquid for 1-3 times, then the surface of the stem tip of Asparagus ferox is washed with sterile water, and finally the stem tip is transferred to the recovery culture medium for recovery culture.
4. The cryopreservation culture method of Astragus cochinchinensis, as claimed in claim 1, wherein the pre-culture solution is MS culture solution comprising 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 ultralow-temperature preservation and culture of asparagus fern 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 asparagus fern 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 cryopreservation culture method of asparagus fern 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 Asparagus fern according to claim 2 or 3, wherein during the recovery culture, the Asparagus fern is cultured for 3 days in the dark, and then is cultured for 7 days in the light, wherein the light time is 12h/d, the light intensity is 1800lux, the temperature is 25 ℃, and the humidity is 40%.
9. The cryopreservation culture method of Asparagus cochinchinensis as claimed in claim 1, wherein dehydration is performed in a dehydration apparatus comprising a first container for containing a vitrification solution at 0 ℃ and a second container for containing the tip of Asparagus cochinchinensis, 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,
wherein, the vitrification solution of 0 ℃ of splendid attire in the arc wall, contain the asparagus stem tip of sheep tooth in the objective housing, the vitrification solution submergence of 0 ℃ of splendid attire in the arc wall holds the shell, and when vitrification solution of 0 ℃ is full of through four feed liquor holes, a plurality of through-holes behind the objective housing, motor control the pivot is clockwise rotation quarter round earlier, and anticlockwise rotation quarter round again, so that hold the shell and swing in the arc wall and end up to the dehydration, just hold the shell submergence all the time in vitrification solution.
10. The method for ultralow-temperature preservation and culture of asparagus fern as claimed in claim 1, wherein the asparagus fern stem tips are further treated as follows before being treated with the loading solution: taking the stem tip of the asparagus fern out of a shaking table oscillator, soaking the stem tip of the asparagus fern in distilled water for 30s, placing the stem tip of the asparagus fern on absorbent paper, covering the absorbent paper with two layers of gauze, spraying 0.5mL of 0.5% sodium chloride aqueous solution to the gauze, standing for 1min, spraying 0.3mL of 5ppm abscisic acid, standing for 1min, washing the surface of the stem tip of the asparagus fern with distilled water, and treating with a loading solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910113311.1A CN109673515A (en) | 2019-02-13 | 2019-02-13 | Asparagus filicicum Ham ultralow temperature cultural method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910113311.1A CN109673515A (en) | 2019-02-13 | 2019-02-13 | Asparagus filicicum Ham ultralow temperature cultural method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109673515A true CN109673515A (en) | 2019-04-26 |
Family
ID=66194261
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910113311.1A Pending CN109673515A (en) | 2019-02-13 | 2019-02-13 | Asparagus filicicum Ham ultralow temperature cultural method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109673515A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116458427A (en) * | 2023-04-14 | 2023-07-21 | 广西壮族自治区农业科学院 | Method for producing root-knot potatoes in asparagus bottle |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102803507A (en) * | 2009-06-12 | 2012-11-28 | 精密公司 | Compositions and methods for dehydrated storage of on-board reagents in microfluidic devices |
| CN104012524A (en) * | 2014-06-18 | 2014-09-03 | 中国农业科学院作物科学研究所 | Ultralow temperature storage and regeneration culture method of in vitro stem tip of Jerusalem artichoke |
| CN104488854A (en) * | 2014-12-02 | 2015-04-08 | 山东省果树研究所 | Vitrification ultralow-temperature preservation method for prunus avium dwarf rootstock gisela |
| CN204520290U (en) * | 2015-03-29 | 2015-08-05 | 宋新军 | A kind of boiling peanut digester |
| CN105519522A (en) * | 2016-03-01 | 2016-04-27 | 中国科学院合肥物质科学研究院 | Method for preserving sapium sebiferum stem tips through vitrification at ultralow temperature |
| CN106998675A (en) * | 2014-07-30 | 2017-08-01 | 多伦多大学董事局 | System and method for the automatic glass of biomaterial |
| CN107997624A (en) * | 2017-12-29 | 2018-05-08 | 浙江帝恒实业有限公司 | A kind of deep fryer of automatic stirring |
-
2019
- 2019-02-13 CN CN201910113311.1A patent/CN109673515A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102803507A (en) * | 2009-06-12 | 2012-11-28 | 精密公司 | Compositions and methods for dehydrated storage of on-board reagents in microfluidic devices |
| CN104012524A (en) * | 2014-06-18 | 2014-09-03 | 中国农业科学院作物科学研究所 | Ultralow temperature storage and regeneration culture method of in vitro stem tip of Jerusalem artichoke |
| CN106998675A (en) * | 2014-07-30 | 2017-08-01 | 多伦多大学董事局 | System and method for the automatic glass of biomaterial |
| CN104488854A (en) * | 2014-12-02 | 2015-04-08 | 山东省果树研究所 | Vitrification ultralow-temperature preservation method for prunus avium dwarf rootstock gisela |
| CN204520290U (en) * | 2015-03-29 | 2015-08-05 | 宋新军 | A kind of boiling peanut digester |
| CN105519522A (en) * | 2016-03-01 | 2016-04-27 | 中国科学院合肥物质科学研究院 | Method for preserving sapium sebiferum stem tips through vitrification at ultralow temperature |
| CN107997624A (en) * | 2017-12-29 | 2018-05-08 | 浙江帝恒实业有限公司 | A kind of deep fryer of automatic stirring |
Non-Patent Citations (3)
| Title |
|---|
| 李翠等: ""药用植物种质资源的超低温保存"", 《中国现代中药》 * |
| 郭仰东等: "《植物生物技术实验教程》", 30 June 2015, 中国农业大学出版社 * |
| 陈世昌等: "《植物组织培养》", 30 June 2016, 重庆大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116458427A (en) * | 2023-04-14 | 2023-07-21 | 广西壮族自治区农业科学院 | Method for producing root-knot potatoes in asparagus bottle |
| CN116458427B (en) * | 2023-04-14 | 2024-01-26 | 广西壮族自治区农业科学院 | Method for producing root-knot potatoes in asparagus bottle |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109644874A (en) | Meadow sweet cryopreservation cultural method | |
| WO2016180259A1 (en) | Dry animal-derived collagen fiber tissue material and preparation method and bioprosthesis thereof | |
| CN103392694A (en) | Method for preparing tropical plant preserving humid preparation | |
| CN109673515A (en) | Asparagus filicicum Ham ultralow temperature cultural method | |
| CN109315356A (en) | One kind being suitable for aleyrodid class or parasitic wasp class insects hatch, feeding device and its application method | |
| CN113767895A (en) | Composite hair follicle tissue preservation solution, preparation method thereof and preservation method for maintaining hair follicle in-vitro activity | |
| CN100372576C (en) | Composite collagen nerve ductus for promoting neural regeneration, and method for forming filature from hollow wet process | |
| CN106310384A (en) | Xenogeneic acellular dermis and preparation method thereof | |
| CN103444309B (en) | Gibberellin and filter paper combine the method making paris polyphylla seed quick-speed germination | |
| CN107853291B (en) | Vitrification cryopreservation and thawing method for stem tips of curcuma zedoary | |
| CN109792954A (en) | Asparagus cryopreservation cultural method | |
| CN109379921A (en) | A method of accelerating rhizoma polygonati seed and sprouts | |
| CN108294051A (en) | A kind of low temperature season evergreen tree callus cladding material and its processing method | |
| CN111771450A (en) | Low-temperature preservation process for taxus mairei seeds | |
| CN105746496A (en) | Method for in-vitro conservation of almond dormant buds by means of vitrification ultralow temperature | |
| JP4688987B2 (en) | Preservation method of isolated mammalian animals | |
| CN105285071A (en) | Living body fresh-keeping method for desertliving cistanche | |
| CN111616140A (en) | Vitrification ultralow-temperature preservation method and application of actinidia arguta dormant bud | |
| CN106854636A (en) | A kind of preparation method of cells of pilose antler organism epimatrix | |
| CN206506895U (en) | A kind of dendrobium candidum save set | |
| CN100463963C (en) | Freeze preservating method for domestic pig skin tissue | |
| CN117158324B (en) | In-vitro preservation method of coconut germplasm resources | |
| CN109362878A (en) | Extend the method on corn fresh-keeping date | |
| CN107637215A (en) | Break the method for ardisia gigantifolia Stapf seed dormancy | |
| CN115074315B (en) | High-quality pig ear tissue sampling and long-term high-efficiency preservation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190426 |