CN105706922A - Ultralow-temperature dendrobium nobile preserving and virus removing method - Google Patents
Ultralow-temperature dendrobium nobile preserving and virus removing method Download PDFInfo
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- 241000700605 Viruses Species 0.000 title abstract description 35
- 240000004638 Dendrobium nobile Species 0.000 title abstract 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 102
- 229930006000 Sucrose Natural products 0.000 claims description 99
- 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 99
- 239000005720 sucrose Substances 0.000 claims description 99
- 239000007788 liquid Substances 0.000 claims description 83
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 78
- 239000012530 fluid Substances 0.000 claims description 72
- 238000005286 illumination Methods 0.000 claims description 68
- 239000001963 growth medium Substances 0.000 claims description 54
- 229920001817 Agar Polymers 0.000 claims description 51
- 239000008272 agar Substances 0.000 claims description 51
- 229910052757 nitrogen Inorganic materials 0.000 claims description 39
- 238000004017 vitrification Methods 0.000 claims description 38
- 230000018044 dehydration Effects 0.000 claims description 37
- 238000006297 dehydration reaction Methods 0.000 claims description 37
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 35
- 239000002609 medium Substances 0.000 claims description 35
- 239000000661 sodium alginate Substances 0.000 claims description 35
- 235000010413 sodium alginate Nutrition 0.000 claims description 35
- 229940005550 sodium alginate Drugs 0.000 claims description 35
- 241000723826 Odontoglossum ringspot virus Species 0.000 claims description 30
- 238000001784 detoxification Methods 0.000 claims description 30
- 210000002257 embryonic structure Anatomy 0.000 claims description 26
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 23
- 229910052782 aluminium Inorganic materials 0.000 claims description 23
- 239000011888 foil Substances 0.000 claims description 23
- 238000010257 thawing Methods 0.000 claims description 18
- 238000012546 transfer Methods 0.000 claims description 18
- 238000007670 refining Methods 0.000 claims description 17
- 238000007710 freezing Methods 0.000 claims description 15
- 230000008014 freezing Effects 0.000 claims description 15
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 14
- 229910001424 calcium ion Inorganic materials 0.000 claims description 14
- 238000004321 preservation Methods 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 238000005138 cryopreservation Methods 0.000 claims description 7
- 238000003860 storage Methods 0.000 claims description 4
- 239000005030 aluminium foil Substances 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract 1
- 241000710019 Cymbidium mosaic virus Species 0.000 description 26
- 230000008030 elimination Effects 0.000 description 23
- 238000003379 elimination reaction Methods 0.000 description 23
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Classifications
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- 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
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
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Abstract
The invention relates to an ultralow-temperature dendrobium nobile preserving and virus removing method, and belongs to the technical field of plant virus prevention and control. The novel ultralow-temperature dendrobium nobile preserving and virus removing method is presented for protecting dendrobium nobile to be endangered more effectively, improving the seedling quality and producing virus-free seedlings, and the virus removing rate of the seedlings can be effectively increased. Compared with a traditional virus removing method, the ultralow-temperature virus removing method has the advantages that the virus removing rate is high and is not influenced by the stem tip size, the materials are convenient to obtain, operation is easy, no special instrument or equipment or expense drug reagent is needed, the cycle is short, the method can be used for germplasm resource preserving and virus removing simultaneously, and application and popularization are easy.
Description
Technical field
The invention belongs to plant virus controlling technical field, a kind of method being specifically related to Herba Dendrobii Excised Embryos detoxification.
Background technology
Herba Dendrobii (DendrobiumnobileLindl), has another name called lofty or bottomless palpus, bracketplant, Lin Lan etc., is the big genus in the orchid family, there is 1500-1600 initial species, being distributed mainly on tropical and subtropical region and the Oceania in Asia, China has 76 kinds, is distributed mainly on southwest and South China.Stem is upright, and meat shape is plump, and slightly flat cylinder is long 10-60 centimetre, slightly reaches 1.3 centimetres.Herba Dendrobii is famous Chinese crude drug, and property slightly sweet flavor is micro-salty, cold, returns stomach, kidney, lung meridian.Reinforcing stomach reg fluid, nourishing YIN and clearing away heat.Losing for cloudy impairment of body fluid, xerostomia excessive thirst, lack of appetite is retched, deficiency-heat after being ill, poor vision.Herba Dendrobii flower appearance is graceful, and exquisite lovely, pattern is bright-coloured, fragrant odour, is called one of " four view and admire greatly ocean flower ".
Due to Radix Stephaniae Tetrandrae platymiscium poor growth, seed is extremely small, and embryo has physiology after ripening, and when natural propagation, seed germination rate is less than 5%, illegally excavates for a long time in addition, causes multiple Radix Stephaniae Tetrandrae initial species endangered.Started from 1987; Radix Stephaniae Tetrandrae is successively listed in " People's Republic of China's rare or endangered species record ", " Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) " middle rank protection plant, and calendar year 2001 Radix Stephaniae Tetrandrae belongs to and is all put into " national key protected wild plants register (the 2nd batch of family) ".
Utilizing the stipes of Herba Dendrobii, bennet, spire point both at home and abroad is the fast numerous tissue cultured seedling of outer implant, has done substantial amounts of research work, but on market, Herba Dendrobii commodity medical material is still short in transplantation technique.Its reason is in that tissue cultured seedling transplanting survival rate is low, is subject to outside climatic impact, and easily band is viral, plant forms is caused to distort, producing the multiple symptoms such as shrinkage, floral leaf, assorted speckle, streak, plant is short and small, quality deteriorates, variety deterioration, also result in plant death time serious, yield declines.
So far, the virosis of orchid has been found that kind more than 30.The common virus infecting Herba Dendrobii has cymbidium mosaic virus (CyMV) and odontoglossum ring spot virus (ORSV).There is black necrosis roundlet speckle in the Radix Stephaniae Tetrandrae plant leaf positive and negative infecting CyMV virus, the streak of raw brown between vein, afterwards in striated floral leaf, flower leaf paresthesia is obvious on young leaves, ripe Helwingia himalaica leaf disease color is light so that plant leaf dysplasia or bloom abnormal, and sufferer is propagated rapidly, there is no effective treatment method at present, once find that suspicious plant can only isolate immediately or burn.Infecting the Herba Dendrobii of ORSV virus, the slough of leaf is concentric ring or has green district.Circle can be unified into variously-shaped, and leaf can be made to come off.
Seedling is the starting point of large-scale production, is the key point of protection resource and efficent use of resources, and the quality of seedling is guaranteed and could improve growing dendrobium and product quality.Therefore, seedling detoxification has important value and significance.Conventional poison-removing method has heat treatment detoxicity method, stem apex detoxicity method, heat treatment stem apex detoxicity method etc..Traditional poison-removing method virus elimination rate is low, and virus elimination rate is by the impact of stem apex size, difficulty of drawing materials.Therefore how overcoming the deficiencies in the prior art is the problem that current plant virus controlling technical field needs solution badly.
Summary of the invention
In order to more effective protection is by Herba Dendrobii in imminent danger, improves seedling quality, produce virus-free seedling, the invention provides a kind of novel Herba Dendrobii Excised Embryos poison-removing method, seedling virus elimination rate can be effectively improved.The method cycle is short, easy and simple to handle, it is not necessary to the medicine and reagent of the equipment of complexity, instrument and costliness, it is easy to popularization and application.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), selects the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the dendrobe tissue culture Seedling stem section 1.0-1.5cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedling 3-5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), strips the stem apex of 1-3mm length from the Seedling that step (1) is refined, then by stem apex preculture 1-3d under 4 DEG C of dark conditions；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 20-40min, stem apex is transferred to dehydration 30-60min in vitrification solution PVS2, stem apex is put into preservation 30min-1h in liquid nitrogen again, at room temperature proceed to stem apex rapidly afterwards to unload and carrier fluid thaws and unloads 20-30min, or stem apex defrosting 3-4min in 37 DEG C of water-baths proceeded to again unload unloading 20-30min in carrier fluid, stem apex after unloading is inoculated into and survives in culture medium, first light culture 1-3d, after proceed under normal illumination continue cultivate, it is thus achieved that Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.3-0.6mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.3-0.7mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.1-0.2ml/LNAA+0.1-2.0ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
It is further preferred that step (2) strips the stem apex of 1-3mm length under stereomicroscope.
Drop vitrification method or Vitrification is adopted when liquid nitrogen preserves it is further preferred that put into by stem apex.
Further, preferably described drop vitrification method is that aluminium-foil paper is cut into (0.8-1) cm*(2-2.2) aluminum foil strip of cm strip, stem apex after PVS2 processed is put in the one side that aluminum foil strip is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, then aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, tighten lid, finally put in liquid nitrogen and preserve.
It is further preferred that described Vitrification is that just stem apex after PVS2 processed is put in cryovial, adds PVS2 to cryovial and make stem apex be immersed in PVS2, directly cryovial is put in liquid nitrogen after covering tightly lid and preserve.
It is further preferred that the stem apex that preculture 1-3d under 4 DEG C of dark conditions obtains first is embedded, then reload, dehydration and Liquid nitrogen storage；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add to calcium ion embedding liquid, be placed under room temperature so that it is be fully frozen into embedding pearl；
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+2-3.5%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride；
The stem apex load time after embedding is 1.5-2h, and dewatering time is 4-5h, and the Liquid nitrogen storage time is 0.9-1.1h.
It is further preferred that the diameter of described embedding pearl is 4-5mm.
It is further preferred that the method for described Herba Dendrobii Excised Embryos detoxification, comprise the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.2cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 4 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 2mm length, then by stem apex preculture 2d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 30min, stem apex is transferred to dehydration 50min in vitrification solution PVS2, again stem apex is put in the one side that the aluminum foil strip of 0.9cm*2.1cm is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, afterwards aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, put into after tightening lid in liquid nitrogen and preserve 45min, at room temperature proceed to stem apex afterwards to unload and carrier fluid thaws and unloads 25min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.5mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.6mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.17ml/LNAA+1.5ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
It is further preferred that the method for described Herba Dendrobii Excised Embryos detoxification, comprise the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.3cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 3.5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 2mm length, then by stem apex preculture 1.8d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 25min, stem apex is transferred to dehydration 52min in vitrification solution PVS2, again stem apex is put in cryovial, adding PVS2 to cryovial makes stem apex be immersed in PVS2, directly cryovial is put into after liquid nitrogen preserves 43min after covering tightly lid, by cryovial defrosting 3.5min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload and carrier fluid unloads 26min defrosting, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2.5d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.47mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.55mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.15ml/LNAA+1.3ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
It is further preferred that the method for described Herba Dendrobii Excised Embryos detoxification, comprise the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.4cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 4.5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), the stem apex of 1.8mm length is stripped from the Seedling that step (1) is refined, then by stem apex preculture 2d under 4 DEG C of dark conditions, afterwards stem apex is first embedded, after adopting high sugar, with carrier fluid, stem apex is loaded 1.7h after embedding, stem apex is transferred to dehydration 4.5h in vitrification solution PVS2, stem apex is put into preservation 1h in liquid nitrogen again, afterwards by stem apex defrosting 3.6min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload and carrier fluid thaws and unloads 24min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.5mol/L sucrose+7g/L agar, pH5.8；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add and embed in liquid to calcium ion, be placed under room temperature 18min, form embedding pearl；The diameter of described embedding pearl is 4.5mm；
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+3%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride.
Described loading and dehydration all carry out on ice, and shaking table vibrates；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.4mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.15ml/LNAA+1ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
The successive transfer culture of indication in the present invention, it is simply that the seedling that the tissue cultured seedling subculture of formation breeds out in new culture medium, cultivates 1 month height of seedling 2-3cm, and culture medium not requirement of transferring, as long as make the well-grown culture medium of Seedling just passable.
It is to increase plant cell osmotic pressure that height sugar of the present invention loads, and protection plant is in the minimizing infiltration injury of next step During Vitrification in vitro and chemistry murder by poisoning；Also for reducing cell Free water, in liquid nitrogen, the freezing glassy state that formed is not likely to produce ice crystal injury cell, and cell easily survives.
Vitrification solution PVS2 processed is to be glassy state cell Free water by liquid state.The effect of uninstall process is to remove the vitrification solution of residual in cell.
Stem apex 30s in liquid nitrogen just can reach the effect of low temperature, and liquid nitrogen can reach the low temperature of-196 DEG C, and intracellular vegetative activity and metabolism almost stop at this low temperature, can indefinite preserve in theory, and detoxification only needs the of short duration time just can be effective.
Before the present invention unloads in unloading carrier fluid and thaws, addition water-bath is thawed, and after being because stem apex threading cryovial, volume is relatively big, and the direct normal temperature unfreezing time is long, it is easy to secondary injury, and the shortening thawing time that thaws in a water bath avoids secondary injury, and survival rate increases.
Loading and dehydration after present invention embedding all carry out on ice, and shaking table vibration, and this is to allow the full and uniform absorption liquid of material.
Preculture, loading, vitrification processes, unloading is all regulate the osmotic pressure of cell by the process time, reduces cell Free water, reduces cell and come to harm in liquid nitrogen process, it is easy to recovers to survive.In cell, Free water is more few, and cell more easily survives.Vegetable material survival rate is had Different Effects by the time difference that each process of diverse ways processes, but the material virus elimination rate impact survived is not notable.
As drop vitrification method, Vitrification, encapsulation-vitrification method do not carry out preculture, survival rate is low, and below 30%, preculture is critical step, carries out preculture and could improve survival rate.
Drop vitrification method preculture 5-8 days, loading processing 20-40min, PVS2 processes 30-60min, unloads 20-30mim, and survival rate is only 20.5%-30.4%, if to preculture 1-3 days, can obtain survival rate 50.25%-60.34%.
Encapsulation-vitrification method preculture 1-3 days, loading processing 60-120min, PVS2 processes 80-120min, unloads 30-40min, and survival rate is 25.42%-31.22%；
Encapsulation-vitrification method preculture 1-3 days loading processing 20-40min, PVS2 process 30-60min, unload 20-30min, and survival rate is 62.58-69.75%.
Compared with prior art, it has the beneficial effect that the present invention
1. virus elimination rate of the present invention is high, and virus elimination rate is up to 93.7%, and traditional stem apex detoxification virus elimination rate is only 53.8%.
2. the inventive method virus elimination rate is not by the impact of stem apex size.General stem apex detoxicity method, the size of stem apex becomes positive correlation with regeneration rate, and becomes negative correlation with virus elimination rate, and therefore, the virus elimination rate of Shoot Tip Culture is limited mainly by the impact of stem apex size.
And the inventive method is not by the impact of stem apex size.The long 1mm of stem apex, virus elimination rate is 93.7%, the long 5mm of stem apex, and virus elimination rate is 91.2%.
3. adopting this method method detoxification, virus elimination rate is not by the impact of ultralow temperature method, and adopting drop vitrification method, encapsulation-vitrification method, Vitrification is 80.5%-93.7%. to the virus elimination rate of CyMV and ORSV virus
4. the inventive method need not special instrument, equipment, and the cycle is short, it is only necessary to reagent used by conventional medium and equipment.
5. the inventive method can be simultaneously used for preservation and the detoxification of germ plasm resource, can be saved in liquid nitrogen for a long time.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete technology or condition person in embodiment, technology or condition described by the document in this area or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be and can pass through to buy the conventional products obtained.
Embodiment 1
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 3 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 1mm length, then by stem apex preculture 1d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 20min, stem apex is transferred to dehydration 30min in vitrification solution PVS2, again stem apex is put in the one side that the aluminum foil strip of 0.8cm*2cm is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, afterwards aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, put into after tightening lid in liquid nitrogen and preserve 30min, at room temperature proceed to stem apex afterwards to unload and carrier fluid thaws and unloads 20min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 1d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 90.4%.
Described pre-incubated culture medium is: 1/2MS+0.3mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.3mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.1ml/LNAA+0.1ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 2
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.5cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 3mm length, then by stem apex preculture 3d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 40min, stem apex is transferred to dehydration 60min in vitrification solution PVS2, again stem apex is put in the one side that the aluminum foil strip of 1cm*2.2cm is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, afterwards aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, put into after tightening lid in liquid nitrogen and preserve 60min, at room temperature proceed to stem apex afterwards to unload and carrier fluid thaws and unloads 30min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 3d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 93.5%.
Described pre-incubated culture medium is: 1/2MS+0.6mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.7mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.2ml/LNAA+2.0ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 3
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.2cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 4 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 2mm length, then by stem apex preculture 2d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 30min, stem apex is transferred to dehydration 50min in vitrification solution PVS2, again stem apex is put in the one side that the aluminum foil strip of 0.9cm*2.1cm is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, afterwards aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, put into after tightening lid in liquid nitrogen and preserve 45min, at room temperature proceed to stem apex afterwards to unload and carrier fluid thaws and unloads 25min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 93.7%.
Described pre-incubated culture medium is: 1/2MS+0.5mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.6mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.17ml/LNAA+1.5ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 4
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 3 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 1mm length, then by stem apex preculture 1d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 20min, stem apex is transferred to dehydration 30min in vitrification solution PVS2, again stem apex is put in cryovial, adding PVS2 to cryovial makes stem apex be immersed in PVS2, directly cryovial is put into after liquid nitrogen preserves 30min after covering tightly lid, by cryovial defrosting 3min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload and carrier fluid unloads 20min defrosting, stem apex after unloading is inoculated into and survives in culture medium, first light culture 1d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 90.0%.
Described pre-incubated culture medium is: 1/2MS+0.3mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.3mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.1ml/LNAA+0.1ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 5
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.5cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 3mm length, then by stem apex preculture 3d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 40min, stem apex is transferred to dehydration 60min in vitrification solution PVS2, again stem apex is put in cryovial, adding PVS2 to cryovial makes stem apex be immersed in PVS2, directly cryovial is put into after liquid nitrogen preserves 60min after covering tightly lid, by cryovial defrosting 4min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload and carrier fluid unloads 30min defrosting, stem apex after unloading is inoculated into and survives in culture medium, first light culture 3d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 89.9%.
Described pre-incubated culture medium is: 1/2MS+0.6mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.7mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.2ml/LNAA+2.0ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 6
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.3cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 3.5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 2mm length, then by stem apex preculture 1.8d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 25min, stem apex is transferred to dehydration 52min in vitrification solution PVS2, again stem apex is put in cryovial, adding PVS2 to cryovial makes stem apex be immersed in PVS2, directly cryovial is put into after liquid nitrogen preserves 43min after covering tightly lid, by cryovial defrosting 3.5min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload and carrier fluid unloads 26min defrosting, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2.5d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 90.2%.
Described pre-incubated culture medium is: 1/2MS+0.47mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.55mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.15ml/LNAA+1.3ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 7
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.0cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 3 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), the stem apex of 1mm length is stripped from the Seedling that step (1) is refined, then by stem apex preculture 1d under 4 DEG C of dark conditions, afterwards stem apex is first embedded, after adopting high sugar, with carrier fluid, stem apex is loaded 1.5h after embedding, stem apex is transferred to dehydration 4h in vitrification solution PVS2, stem apex is put into preservation 0.9h in liquid nitrogen again, afterwards by stem apex defrosting 3min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload in carrier fluid and thaw, and unload 20min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 1d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 89.5%.
Described pre-incubated culture medium is: 1/2MS+0.3mol/L sucrose+7g/L agar, pH5.8；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add and embed in liquid to calcium ion, be placed under room temperature 15min, form embedding pearl；The diameter of described embedding pearl is 4mm.
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+2%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride.
Described loading and dehydration all carry out on ice, and shaking table vibrates；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.3mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.1ml/LNAA+0.1ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 8
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.5cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), the stem apex of 3mm length is stripped from the Seedling that step (1) is refined, then by stem apex preculture 3d under 4 DEG C of dark conditions, afterwards stem apex is first embedded, after adopting high sugar, with carrier fluid, stem apex is loaded 2h after embedding, stem apex is transferred to dehydration 5h in vitrification solution PVS2, stem apex is put into preservation 1.1h in liquid nitrogen again, afterwards by stem apex defrosting 4min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload in carrier fluid and thaw, and unload 30min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 3d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 89.3%.
Described pre-incubated culture medium is: 1/2MS+0.6mol/L sucrose+7g/L agar, pH5.8；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add and embed in liquid to calcium ion, be placed under room temperature 20min, form embedding pearl；The diameter of described embedding pearl is 5mm.
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+3.5%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride.
Described loading and dehydration all carry out on ice, and shaking table vibrates；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.7mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.2ml/LNAA+2.0ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
Embodiment 9
A kind of method of Herba Dendrobii Excised Embryos detoxification, comprises the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.4cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 4.5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), the stem apex of 1.8mm length is stripped from the Seedling that step (1) is refined, then by stem apex preculture 2d under 4 DEG C of dark conditions, afterwards stem apex is first embedded, after adopting high sugar, with carrier fluid, stem apex is loaded 1.7h after embedding, stem apex is transferred to dehydration 4.5h in vitrification solution PVS2, stem apex is put into preservation 1h in liquid nitrogen again, afterwards by stem apex defrosting 3.6min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload in carrier fluid and thaw, and unload 24min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；After 60d, RT-PCR detects CyMV and ORSV, and virus elimination rate is 89.4%.
Described pre-incubated culture medium is: 1/2MS+0.5mol/L sucrose+7g/L agar, pH5.8；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add and embed in liquid to calcium ion, be placed under room temperature 18min, form embedding pearl；The diameter of described embedding pearl is 4.5mm.
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+3%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride.
Described loading and dehydration all carry out on ice, and shaking table vibrates；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.4mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.15ml/LNAA+1ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
The ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described.Skilled person will appreciate that of the industry; the present invention is not restricted to the described embodiments; described in above-described embodiment and description is that principles of the invention is described; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements both fall within the claimed scope of the invention.Claimed scope is defined by appending claims and equivalent thereof.
Claims (10)
1. the method for a Herba Dendrobii Excised Embryos detoxification, it is characterised in that comprise the steps:
Step (1), selects the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the dendrobe tissue culture Seedling stem section 1.0-1.5cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedling 3-5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), strips the stem apex of 1-3mm length from the Seedling that step (1) is refined, then by stem apex preculture 1-3d under 4 DEG C of dark conditions；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 20-40min, stem apex is transferred to dehydration 30-60min in vitrification solution PVS2, stem apex is put into preservation 30min-1h in liquid nitrogen again, at room temperature proceed to stem apex rapidly afterwards to unload and carrier fluid thaws and unloads 20-30min, or stem apex defrosting 3-4min in 37 DEG C of water-baths proceeded to again unload unloading 20-30min in carrier fluid, stem apex after unloading is inoculated into and survives in culture medium, first light culture 1-3d, after proceed under normal illumination continue cultivate, it is thus achieved that Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.3-0.6mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.3-0.7mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.1-0.2ml/LNAA+0.1-2.0ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
2. the method for Herba Dendrobii Excised Embryos detoxification according to claim 1, it is characterised in that strip the stem apex of 1-3mm length in step (2) under stereomicroscope.
3. the method for Herba Dendrobii Excised Embryos detoxification according to claim 1, it is characterised in that stem apex is put into and adopts drop vitrification method or Vitrification when preserving in liquid nitrogen.
4. the method for Herba Dendrobii Excised Embryos detoxification according to claim 3, it is characterized in that, described drop vitrification method is that aluminium-foil paper is cut into (0.8-1) cm*(2-2.2) aluminum foil strip of cm strip, stem apex after PVS2 processed is put in the one side that aluminum foil strip is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, then aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, tighten lid, finally put in liquid nitrogen and preserve.
5. the method for Herba Dendrobii Excised Embryos detoxification according to claim 3, it is characterized in that, described Vitrification is that just stem apex after PVS2 processed is put in cryovial, adding PVS2 to cryovial makes stem apex be immersed in PVS2, is directly put into by cryovial in liquid nitrogen and preserve after covering tightly lid.
6. the method for Herba Dendrobii Excised Embryos detoxification according to claim 1, it is characterised in that first embedded by the stem apex that preculture 1-3d under 4 DEG C of dark conditions obtains, then reloads, dehydration and Liquid nitrogen storage；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add to calcium ion embedding liquid, be placed under room temperature so that it is be fully frozen into embedding pearl；
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+2-3.5%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride；
The stem apex load time after embedding is 1.5-2h, and dewatering time is 4-5h, and the Liquid nitrogen storage time is 0.9-1.1h.
7. the method for Herba Dendrobii Excised Embryos detoxification according to claim 6, it is characterised in that the diameter of described embedding pearl is 4-5mm.
8. the method for Herba Dendrobii Excised Embryos detoxification according to claim 1, it is characterised in that comprise the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.2cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 4 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 2mm length, then by stem apex preculture 2d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 30min, stem apex is transferred to dehydration 50min in vitrification solution PVS2, again stem apex is put in the one side that the aluminum foil strip of 0.9cm*2.1cm is smooth, PVS2 is dripped on stem apex, make stem apex fully wrapped around in PVS2 small drops, afterwards aluminum foil strip is directly immersed in liquid nitrogen and carries out freezing, after freezing, aluminum foil strip is loaded in cryopreservation tube, put into after tightening lid in liquid nitrogen and preserve 45min, at room temperature proceed to stem apex afterwards to unload and carrier fluid thaws and unloads 25min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.5mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.6mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.17ml/LNAA+1.5ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
9. the method for Herba Dendrobii Excised Embryos detoxification according to claim 1, it is characterised in that comprise the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.3cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 3.5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), under stereomicroscope, strips the stem apex of 2mm length, then by stem apex preculture 1.8d under 4 DEG C of dark conditions from the Seedling that step (1) is refined；Then on superclean bench, after adopting high sugar, with carrier fluid, stem apex is loaded 25min, stem apex is transferred to dehydration 52min in vitrification solution PVS2, again stem apex is put in cryovial, adding PVS2 to cryovial makes stem apex be immersed in PVS2, directly cryovial is put into after liquid nitrogen preserves 43min after covering tightly lid, by cryovial defrosting 3.5min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload and carrier fluid unloads 26min defrosting, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2.5d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.47mol/L sucrose+7g/L agar, pH5.8；
Described loading and dehydration all carry out on ice；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.55mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.15ml/LNAA+1.3ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
10. the method for Herba Dendrobii Excised Embryos detoxification according to claim 1, it is characterised in that comprise the steps:
Step (1), takes the dendrobe tissue culture Seedling with CyMV and ORSV virus of seedling age in January, takes the stem section 1.4cm with simple bud, be inoculated on minimal medium, 4 DEG C of dark condition lower refining seedlings 4.5 weeks；Described January, seedling age was the dendrobe tissue culture Seedling in successive transfer culture January, and height of seedling degree is 2-3cm；
Described minimal medium is: 1/2MS+30g/L sucrose+7g/L agar, pH5.8；
Step (2), the stem apex of 1.8mm length is stripped from the Seedling that step (1) is refined, then by stem apex preculture 2d under 4 DEG C of dark conditions, afterwards stem apex is first embedded, after adopting high sugar, with carrier fluid, stem apex is loaded 1.7h after embedding, stem apex is transferred to dehydration 4.5h in vitrification solution PVS2, stem apex is put into preservation 1h in liquid nitrogen again, afterwards by stem apex defrosting 3.6min in 37 DEG C of water-baths, at room temperature proceed to stem apex afterwards to unload in carrier fluid and thaw, and unload 24min, stem apex after unloading is inoculated into and survives in culture medium, first light culture 2d, after proceed under normal illumination continue cultivate, obtain Herba Dendrobii detoxic seedling；
Described pre-incubated culture medium is: 1/2MS+0.5mol/L sucrose+7g/L agar, pH5.8；
Concretely comprising the following steps of described embedding: drip sodium alginate embedding liquid on stem apex, make stem apex fully wrapped around in sodium alginate embedding liquid, then sodium alginate containing stem apex embeds liquid drop add and embed in liquid to calcium ion, be placed under room temperature 18min, form embedding pearl；The diameter of described embedding pearl is 4.5mm；
Described sodium alginate embedding liquid is: 1/2MS+0.4mol/L sucrose+3%w/v sodium alginate；
Described calcium ion embedding liquid is: 1/2MS+0.4mol/L sucrose+0.1mol/l calcium chloride；
Described loading and dehydration all carry out on ice, and shaking table vibration；
Described high sugar with carrier fluid is: 1/2MS+2mol/L glycerol+0.4mol/L sucrose, pH5.8；
Described vetrifying solution PVS2:1/2MS+0.4mol/L sucrose+30%w/v glycerol+15%w/vDMSOpH5.8；
The described carrier fluid that unloads is: 1/2MS+1.2mol/L sucrose, pH5.8；
The described culture medium that survives is: 1/2MS+0.15ml/LNAA+1ml/L6-BA+50g/L Rhizoma Solani tuber osi+25g/L Fructus Musae+7g/L agar, pH5.8；
It is cultivate when temperature 25 ± 1 DEG C, intensity of illumination 1000-2000lx, illumination 12h/d that described normal illumination is cultivated.
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| CN107736244A (en) * | 2017-10-22 | 2018-02-27 | 威海市农业科学院 | Potato Shoot-tips peel off detoxifying fast breeding method |
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| CN110999783A (en) * | 2019-11-26 | 2020-04-14 | 河南省农业科学院园艺研究所 | Preservation and recovery culture method for melon haploid culture non-pollinated ovary |
| CN118000102A (en) * | 2024-04-10 | 2024-05-10 | 云南龙藏生物科技有限公司 | Ultralow-temperature detoxification method of passion fruits and rapid propagation method of detoxified seedlings |
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