CN102379281A - Ultralow-temperature preservation method and restoration culture method for carnation - Google Patents
Ultralow-temperature preservation method and restoration culture method for carnation Download PDFInfo
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- CN102379281A CN102379281A CN2011102351225A CN201110235122A CN102379281A CN 102379281 A CN102379281 A CN 102379281A CN 2011102351225 A CN2011102351225 A CN 2011102351225A CN 201110235122 A CN201110235122 A CN 201110235122A CN 102379281 A CN102379281 A CN 102379281A
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Abstract
The invention relates to an ultralow-temperature preservation method for carnations by using a droplet vitrification method. Pre-culture, pretreatment and vitrification protection treatment are conducted to the stem tips of the carnations and finally the carnations are put into liquid nitrogen for ultralow-temperature preservation. The invention additionally provides a restoration culture method for the carnations preserved at ultralow temperature. The ultralow-temperature preservation method for the carnations provided by the invention is simple, convenient, feasible, stable and reliable, the restoration growth condition of the preserved carnations is good and the regeneration rate of plants highly reaches more than 90 percent.
Description
Technical field
The present invention relates to the store method of plant ultralow temperature, be specifically related to cryopreservation method of a kind of carnation and cultural method again.
Background technology
Carnation (Diarahus caryophfllus L.) is the evergreen undershrub perennial root of Caryophyllaceae Carnation flowers, is one of world-renowned four big cut-flowers, and the resource of its cultivated species and wild species is effectively preserved most important to the production domesticization of carnation.Usually preserve the carnation resource through cuttage of kind garden and the regular subculture of tissue culture, but carnation is if infect virus, long-term vegetative propagation is prone to cause constantly accumulation in the virion, is difficult to effective long preservation germplasm, needs to seek the righttest store method.
The preservation of germ plasm resource broadly has terrain to preserve and the strange land is preserved two kinds.Carnation generally preserves through the field planting mode in numerous kind of garden or preservation garden as a kind of perennial root flowers that nourish and generate.This preserving type need be kept through cuttage year after year; Not only occupy cultivated land; And time and effort consuming again in the cultivation management; The danger that also has the influence of natural calamities such as receiving arid, flood, low temperature, heat evil, damage by disease and insect and cause germplasm to destroy; Do not fit into long preservation (Withers L.A.and Engels J.M.M.1990.The test tube genebank-a safe alternative to field conservation, IBPGR Newsletter for Asia and the Pacific 3,1-2.).Under this background, ultralow temperature is preserved the store method the most likely that is considered to the genetic resources long preservation that this type plant is present safely, effective, cost-effectiveness is worthwhile.Under the condition of ultralow temperature; Active being close to of the Physiology and biochemistry of plant stops; Physiology in the storage and hereditary variation can be controlled in the bottom line that (Engelmann is vitro conservation methods.In:Callow JA F.1997.In, Ford-Lloyd BV, Newbury HJ (eds) Biotechnology and plant genetic resources.CAB International; Oxford, pp 119-161.).
Prove that at present the stripped material of plant still can be bred rapidly in a large number under suitable condition after ultralow temperature is preserved, the plant that regeneration makes new advances, and keep original hereditary capacity.It has avoided the field to preserve being subject to the natural calamity influence and causing blastation or destruction, and tissue culture causes the danger of genetic character variation or pollution because of subculture repeatedly in preserving.Research about the preservation of plant ultralow temperature; After entering into the nineties in last century; The variation and the summary aspect of preserving technology constantly make progress; The plant species number that can preserve also increases constantly, but most to concentrate on potato class plants such as woody plant such as apple, cherry, citrus, banana, cassava and potato, Ipomoea batatas, strawberry be that the ultralow temperature of main plant genetic resources is preserved (Sakai A and Nishiyama is of winter vegetative buds of hardy fruit trees in liquid nitrogen.Hortic Sci 13:225-227. Y.1978.Cryopreservation).
Ultralow temperature is preserved several different methods; Recently often utilization with the vitrifying method of PVS2 as freezing preservation liquid, after promptly vegetable material is handled in the protection liquid of high concentration, drop in the liquid nitrogen rapidly; Thereby moisture formation vitrifying state is avoided forming ice crystal and is caused mechanical damage; Effectively the store method of protective plant is applied to the ultralow temperature preservation (Akira Sakai and Florent Engelmann, 2007) of 100 various plants different tissues.The droplet method that development is come out on conventional vitrifying method in recent years after vitrifying is handled, is dripped vegetable material into droplet on aluminium foil, directly drop into liquid nitrogen then and carry out freezing rapidly.Because frozen and thawing rate is fast, tissue is difficult for forming ice crystal, being prone to become to live regeneration, be a promising method.
The ultralow temperature research about carnation abroad starts from the seventies in 20th century, and its survival rate is merely 2% (Seibert, 1976).The carnation stem apex that Sakai handles 4-8 ℃ of domestication carries out the ultralow temperature preservation through freezing method (Uemura and Sakai, 1980) slowly, and survival rate is brought up to 70-80%.The back can make the survival rate of carnation reach 90% through two step freezings and programmed cooling method.(Dereuddre?et?al.，1987；Fukai，1989)。Along with the invention of vitrifying reagent, Langis adopts the vitrifying method successfully to preserve the carnation stem apex in 1990.After this, the droplet vitrifying method on conventional method for vitrification basis is preserved the way of carnation stem apex and is not seen report at present.Carry out frozenly Halmagyi handles the stem apex of 3 kinds of carnation with the sodium alginate to embed vitrifying after, survival rate can reach 60%～73%.
Summary of the invention
The objective of the invention is to preserve on the basis of research, the cryopreservation method of a kind of carnation is provided at existing ultralow temperature.
Above-mentioned store method may further comprise the steps:
(1) stem apex that cuts the carnation tissue cultivating seedling is cultivated in advance;
(2) stem apex after preparatory the cultivation is with containing 1～3molL
-1Glycerine and 0.2～0.6molL
-1The MS liquid nutrient medium preliminary treatment 20～40min of sucrose;
(3) pretreated stem apex changes in the vitrifying protectant and handles 60～80min;
(4) stem apex after step (3) is handled drops in the liquid nitrogen to be preserved.
In the said method, said step (2) for the stem apex after cultivating in advance with containing 2molL
-1Glycerine and 0.4molL
-1The MS liquid nutrient medium preliminary treatment 30min of sucrose; Said step (3) is handled 80min for pretreated stem apex changes in the vitrifying protectant.
Said step (1) is protected leaf encirclement stem apex for the carnation tissue cultivating seedling is successively peelled off outer blade to remaining 1～2, cuts 1～3mm stem apex and cultivates in advance, selects healthy and strong aseptic seedling when cutting.
Said preparatory cultivation can reduce stem-tip tissue cell free water content, and increases protective substance content such as soluble sugar, thereby improves the freeze proof power of stem apex, reduces or avoids freezing injury, reaches the purpose that improves survival rate.Culture medium can be the medium that contains Osmolyte regulators such as sucrose, mannitol, sorbierite in advance.The described preparatory cultural method of step (1) is that said stem apex is being contained 0.3～0.5molL
-1In the MS solid culture medium of sucrose, 0～10 ℃ of dark cultivation 1～5 day.
Because vitrifying protectant concentration is higher, can make the stem apex fast dewatering when changing over to stem apex in the vitrifying protectant, easily stem apex is damaged, thereby said stem apex need carry out preliminary treatment before the vitrifying protection.
The described vitrifying protectant of step (3) is 1～5molL
-1Glycerine, 1～5molL
-1Ethylene glycol, 1～5molL
-1Dimethyl sulfoxide (DMSO) and 0.1～0.5molL
-1The mixed solution that sucrose is formed.
Said step (4) is: said stem apex is transferred in the vitrifying protectant cryovial that precooling to 0～5 ℃ are housed, and every pipe is equipped with 5～15 stem apexs, said cryovial is dropped in the liquid nitrogen preserve again.
Said step (4) can also be the droplet method: earlier said stem apex and 3～8 μ l vitrifying protectants are formed droplet on aseptic aluminum foil strip; Then said aluminum foil strip input is full of in the ampoul tube of liquid nitrogen, more said ampoul tube is dropped in the liquid nitrogen at last and preserve.
The present invention also provides the cultural method again of the carnation after a kind of ultralow temperature is preserved, adopts above-mentioned store method to carry out ultralow temperature and preserves, and the carnation stem apex of earlier ultralow temperature being preserved thaws and washs, and can obtain the carnation plant through cultivating again.
The said process of thawing and washing was thawed 1～3 minute for said stem apex is put into 35～50 ℃ water-bath, then at room temperature with containing 1～2molL
-1The MS culture fluid washing of sucrose 1～3 time, each 5～10min; Or said stem apex directly put into contain 1～2molL
-1The MS culture fluid of sucrose thaws and washs 5～10min.
Said cultured method is to contain 0.3～0.6mgL
-1BA, 0.05～0.15mgL
-1NAA and 0.3～0.6mgL
-1The dark cultivation or the low light level were cultivated 2～5 days in the MS semisolid culturemedium of GA3, when stem apex returns green and the sign of sprouting is arranged, changed ordinary light cultivation in the MS solid culture medium again over to.
Do not particularly point out if having, solution related among the present invention is the aqueous solution; The precooling that relates among the present invention or the temperature of ice bath are meant about 4 ℃.
Technical scheme provided by the invention has realized that the ultralow temperature of carnation preserves, should method is simple, and reliable and stable, preserving the back carnation, to recover upgrowth situation good, and shoot regeneration frequency can be up to more than 90%.
Description of drawings
Fig. 1 is used to strip the carnation tissue cultivating seedling of stem apex and the stem apex that strips;
Fig. 2 is the carnation stem apex after the droplet method is handled;
To be different vitrifying processing time preserve the influence of survival rate to carnation ultralow temperature to Fig. 3, and wherein, abscissa is the time (min) of vitrifying protection agent treated, and ordinate is survival rate (%);
Fig. 4 recovers to cultivate the carnation stem apex after 7 days under the different frozen modes.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
The ultralow temperature of embodiment 1 carnation is preserved
Material:
The carnation kind: safflower variety, buy from the flowers market, Shanghai.Cut scape breeding test-tube plantlet, obtain scape clone.
Preparatory culture medium: contain 0.4molL
-1The MS solid culture medium of sucrose;
Preprocessing solution: contain 2molL
-1Glycerine and 0.4molL
-1The MS liquid nutrient medium of sucrose;
Vitrifying protectant: contain 3.26molL
-1Glycerine, 2.42molL
-1Ethylene glycol, 1.9molL
-1DMSO and 0.4molL
-1The solution of sucrose.
Method:
Choose the healthy and strong tissue cultivating seedling of above-mentioned carnation kind; (2～3mm), 4 ℃ of dark down cultivations 5 days are soaked 30min with stem apex under the room temperature in pretreatment fluid in preparatory culture medium to cut stem apex; Change over to then in the vitrifying protectant and handle 60～80min down at 4 ℃; A then: stem apex is transferred in the protectant cryovial of fresh vitrifying that the ice bath precooling is housed, and every pipe is adorned 10 stem apexs, drops in the liquid nitrogen and preserves; B: the drop (about 5 μ l) that contains 1 stem apex drops on the aseptic aluminum foil strip of 5mm * 20mm (Fig. 2), directly drops in the ampoul tube of filled with liquid nitrogen, drops into to preserve in the liquid nitrogen to get final product again.
Cultivate again and the survival rate investigation
A: get cryovial, put into 40 ℃ of water-baths immediately and thawed 2 minutes, stem apex is at room temperature with containing 1.2molL
-1The MS culture fluid washing of sucrose 3 times, each 5～10min; B: the aluminum foil strip input that directly will contain drop contains 1～2molL
-1Thaw in the MS culture fluid cleaning solution of sucrose and wash 5～10min.Stem apex after the washing blots with filter paper, moves to contain 0.5mgL
-1BA, 0.1mgL
-1NAA and 0.5mgL
-1In the MS semisolid culturemedium of GA3, secretly cultivated 3 days, wait stem apex to return green and change in the MS solid culture medium conventional illumination cultivation again over to when the sign of sprouting is arranged.
The result shows that it is good that stem apex is preserved the new plant recovery of back carnation upgrowth situation, and shoot regeneration frequency is 95%.
Sucrose concentration and pretreatment time are to the influence of preservation effect in the preparatory culture medium of the ultralow temperature preservation----of embodiment 2 carnations
The physiological status of experiment material has appreciable impact to freezing the back survival rate.Stem apex is cultivated through the short-term high concentration sucrose in advance, makes to organize to slough part moisture more lenitively, impels emiocytosis protection material simultaneously, helps freezing the back survival.Common method of operating is after cutting the preparatory cultivation of big stem-tip tissue, to cut suitable big or small stem apex again and carry out the ultralow temperature preservation.The carnation stem-tip tissue is prone to brownization after cutting, after the sucrose of high concentration is cultivated in advance, will cause the stem-tip tissue deliquescing again; Water stainization; Put to no little inconvenience for the operation that strips of back, stem apex is also very easily impaired, and in recovery is thereafter cultivated, the differentiation difficulty occurs.So technical scheme of the present invention changes into: under anatomical lens, stem apex is carefully successively peelled off outer blade earlier, surround stem apex, and be cut into suitable size (1-3mm, preferred 2-3mm), after cultivating in advance, directly carry out vitrifying and handle until there being the 1-2 sheet to protect leaf.
Material:
The carnation kind: Caryophyllaceae Carnation safflower carnation kind, buy from the flowers market, Shanghai City.Cut scape breeding test-tube plantlet, obtain holder clone.Cut holder breeding test-tube plantlet, obtain scape clone.
Preparatory culture medium: the MS solid culture medium of sucrose concentration enrichment, the dark cultivation;
Preprocessing solution: contain 2molL
-1Glycerine and 0.4molL
-1The MS liquid nutrient medium of sucrose;
Vitrifying protectant (PVS2): contain 3.26molL
-1Glycerine, 2.42molL
-1Ethylene glycol, 1.9molL
-1DMSO and 0.4molL
-1Sucrose.
Method:
Choose the healthy and strong tissue cultivating seedling of above-mentioned carnation kind, cut stem apex (2～3mm), be respectively 0.1molL at sucrose concentration
-1, 0.3molL
-1, 0.5molL
-1, 0.7molL
-1The MS medium in cultivate in advance 1,3,5,7d; Handle 30min with pretreatment fluid, handle 80min through the PVS2 ice bath again, transfer in the protectant cryovial of fresh vitrifying that the ice bath precooling is housed; Every pipe is adorned 10 stem apexs; Drop in the liquid nitrogen and preserve,, recover after the frozen week to cultivate with the method A of embodiment 1.
Cultivate again and the survival rate investigation
Get cryovial, put into 40 ℃ of water-baths immediately and thawed 2 minutes, stem apex is at room temperature with containing 1.2molL
-1The MS culture fluid washing of sucrose 3 times, each 5～10min, the stem apex after the washing blots with filter paper, moves to contain 0.5mgL
-1BA, 0.1mgL
-1NAA and 0.5mgL
-1In the MS semisolid culturemedium of GA3, secretly cultivated 3 days, wait stem apex to return green and change in the MS solid culture medium conventional illumination cultivation again over to when the sign of sprouting is arranged.
The result shows that the new plant recovery of stem apex preservation back carnation upgrowth situation is the different significant difference that occurs with sucrose concentration additional in the pre-culture medium.The result shows: with adding 0.5molL
-1The MS medium of sucrose was cultivated 5 days in advance, and survival rate is up to 88.3%; 0.3molL
-1Sucrose cultivate in advance and took second place 0.1～0.5molL in 5 days
-1Sucrose concentration is cultivated more than 3 days down in advance, and survival rate increases with sucrose concentration, but sucrose concentration is higher than 0.7molL
-1, stem apex is prone to brown stain and water stainization when cultivating in advance, and survival rate all obviously descends.When incubation time was greater than 7d in advance, survival rate descended.
Table 1 sucrose is cultivated concentration in advance and is cultivated the influence of fate to survival rate in advance
The ultralow temperature of embodiment 3 carnations is preserved the influence of----vitrifying processing time to preservation effect
Vitrifying reagent can make tissue slough moisture, and infiltrates tissue lenitively, in frozen, gets into the vitrifying state, protects cell structure effectively.But owing to contain toxic in the protectant composition and cause the composition (like dimethyl sulfoxide (DMSO)) that makes a variation, so need strict grasp.Guaranteeing have under the prerequisite of sufficiently high survival rate, shorten the processing time as far as possible.
Material:
The carnation kind: Caryophyllaceae Carnation safflower carnation kind, buy from the flowers market, Shanghai City.Cut scape breeding test-tube plantlet, obtain holder clone.Cut holder breeding test-tube plantlet, obtain scape clone.
Preparatory culture medium: contain 0.3molL
-1The MS solid culture medium of sucrose, the dark cultivation;
Preprocessing solution: the MS liquid nutrient medium that contains 2molL-1 glycerine and 0.4molL-1 sucrose;
Vitrifying protectant (PVS2): contain 3.26molL
-1Glycerine, 2.42molL
-1Ethylene glycol, 1.9molL
-1DMSO and 0.4molL
-1Sucrose.
Method:
Choose the healthy and strong tissue cultivating seedling of above-mentioned carnation kind, cut stem apex (2～3mm), be 0.3molL at sucrose concentration
-1MS medium dark situation under cultivate in advance 5d, handle 30min with pretreatment fluid, handle 15min, 30min, 60min, 80min, 120min respectively through PVS2 (0 ℃) again.Then stem apex is transferred in the protectant cryovial of fresh vitrifying that the ice bath precooling is housed, every pipe is adorned 10 stem apexs, drops into to preserve in the liquid nitrogen to get final product.
Cultivate again and the survival rate investigation
Get cryovial, put into 40 ℃ of water-baths immediately and thawed 2 minutes, stem apex is at room temperature with containing 1.2molL
-1The MS culture fluid washing of sucrose 3 times, each 5～10min, the stem apex after the washing blots with filter paper, moves to contain 0.5mgL
-1BA, 0.1mgL
-1NAA and 0.5mgL
-1In the MS semisolid culturemedium of GA3, secretly cultivated 3 days, wait stem apex to return green and change in the MS solid culture medium conventional illumination cultivation again over to when the sign of sprouting is arranged.
The recovery cultivation results shows: under this 5 kinds of vitrifying processing times, it is the highest that the 80min survival rate is handled in vitrifying, reaches 86.7%, prolongs survival rate thereafter in time and slightly descend.During 120min, survival rate still can reach 72.5%.(Fig. 3)
The ultralow temperature of embodiment 4 carnations is preserved the influence of----frozen mode to survival rate and plant regeneration
Freezing method can be divided into slow freezing and rapid freezing method because of freezing object and the freezing method difference of ultralow temperature.The vitrifying method that is widely used now is a snap frozen; Usually adopt and directly drop into liquid nitrogen to reach fast cooling; Be prone to most icing temperature range-140～-10 ℃ to escape, make the intracellular moisture ice crystal that also do not have enough time to form, just arrived-196 ℃ place of safety.When thawing, then thaw fast in the water-bath about 40 ℃, avoid that the recrystallization phenomenon pair cell damages in the course of defrosting.The droplet method that goes out according to the instant principle development of this quick-frozen, because volume is little, freezing and thawing rate is far above conventional vitrifying method; Ice crystal is difficult for forming, and recovers growth soon, and survival rate is high; Successful Application is preserved with the ultralow temperature of a lot of plant tissues at present; For example, more conventional vitrifying method, droplet method can make the survival rate of banana improve 40-50%.New way has been opened up in this ultralow temperature preservation for the plant that can not successfully preserve with conventional method.
Material:
The carnation kind: Caryophyllaceae Carnation safflower carnation kind, buy from the flowers market, Shanghai City.Cut scape breeding test-tube plantlet, obtain holder clone.Cut holder breeding test-tube plantlet, obtain scape clone.
Preparatory culture medium: contain 0.5molL
-1The MS solid culture medium of sucrose, the dark cultivation;
Preprocessing solution: the MS liquid nutrient medium that contains 2molL-1 glycerine and 0.4molL-1 sucrose;
Vitrifying protectant (PVS2): contain 3.26molL
-1Glycerine, 2.42molL
-1Ethylene glycol, 1.9molL
-1DMSO and 0.4molL
-1Sucrose.
Method:
Choose the healthy and strong tissue cultivating seedling of above-mentioned carnation kind, cut stem apex (2～3mm), be 0.5molL at sucrose concentration
-1MS medium dark situation under cultivate in advance 5d, handle 30min with pretreatment fluid, handle 80min through PVS2 (4 ℃) again.A then: stem apex is transferred in the protectant cryovial of fresh vitrifying that the ice bath precooling is housed, and every pipe is adorned 10 stem apexs, drops in the liquid nitrogen and preserves; B: the drop (about 5 μ l) that contains 1 stem apex drops on the aseptic aluminum foil strip of 5mm * 20mm (Fig. 2), directly drops in the ampere pipe of filled with liquid nitrogen, drops into to preserve in the liquid nitrogen to get final product.
Cultivate again and the survival rate investigation
A: get cryovial, put into 40 ℃ of water-baths immediately and thawed 2 minutes, stem apex is at room temperature with containing 1.2molL
-1The MS culture fluid washing of sucrose 3 times, each 5～10min; B: the aluminum foil strip input that directly will contain drop contains 1～2molL
-1Thaw in the MS culture fluid cleaning solution of sucrose and wash 5～10min.Stem apex after the washing blots with filter paper, moves to contain 0.5mgL
-1BA, 0.1mgL
-1NAA and 0.5mgL
-1In the MS semisolid culturemedium of GA3, secretly cultivated 3 days, wait stem apex to return green and change in the MS solid culture medium conventional illumination cultivation again over to when the sign of sprouting is arranged.
The recovery cultivation results shows: the droplet method is carried out frozen under optimization vitrifying program, and survival rate can reach 95%, is significantly higher than the processing of conventional vitrifying time.When the frozen recovery of droplet method was cultivated, the promptly visible stem apex of the fastest 1d was sprouted, and more conventional vitrifying method of recovery time will be lacked 3～5 days, and the plant that becomes to live all regenerates, the frozen regeneration plant of the more conventional vitrifying method of growing way strong (Fig. 4).
Claims (10)
1. the cryopreservation method of a carnation is characterized in that, may further comprise the steps:
(1) stem apex that cuts the carnation tissue cultivating seedling is cultivated in advance;
(2) stem apex after preparatory the cultivation is with containing 1～3molL
-1Glycerine and 0.2～0.6molL
-1The MS liquid nutrient medium preliminary treatment 20～40min of sucrose;
(3) pretreated stem apex changes in the vitrifying protectant and handles 60～80min;
(4) stem apex after step (3) is handled drops in the liquid nitrogen to be preserved.
2. store method according to claim 1 is characterized in that, said step (2) for the stem apex after cultivating in advance with containing 2molL
-1Glycerine and 0.4molL
-1The MS liquid nutrient medium preliminary treatment 30min of sucrose; Said step (3) is handled 80min for pretreated stem apex changes in the vitrifying protectant.
3. store method according to claim 1 and 2 is characterized in that, said step (1) is protected leaf encirclement stem apex for the carnation tissue cultivating seedling is successively peelled off outer blade to remaining 1～2, cuts 1～3mm stem apex and cultivates in advance.
4. store method according to claim 1 and 2 is characterized in that, the described preparatory cultural method of step (1) is that said stem apex is being contained 0.3～0.5molL
-1In the MS solid culture medium of sucrose, 0～10 ℃ of dark cultivation 1～5 day.
5. store method according to claim 1 and 2 is characterized in that, the described vitrifying protectant of step (3) is 1～5molL
-1Glycerine, 1～5molL
-1Ethylene glycol, 1～5molL
-1Dimethyl sulfoxide (DMSO) and 0.1～0.5molL
-1The mixed solution that sucrose is formed.
6. store method according to claim 5; It is characterized in that; Said step (4) is: said stem apex is transferred in the vitrifying protectant cryovial that precooling to 0～5 ℃ are housed, and every pipe is equipped with 5～15 stem apexs, said cryovial is dropped in the liquid nitrogen preserve again.
7. store method according to claim 5; It is characterized in that; Said step (4) is: earlier said stem apex and 3～8 μ l vitrifying protectants are formed droplet on aseptic aluminum foil strip; Then said aluminum foil strip input is full of in the ampoul tube of liquid nitrogen, more said ampoul tube is dropped in the liquid nitrogen at last and preserve.
8. the cultural method again of the carnation after each said cryopreservation method of claim 1-7 is preserved is characterized in that the carnation stem apex of earlier ultralow temperature being preserved thaws and washs, and can obtain the carnation plant through cultivating again.
9. cultural method more according to claim 8 is characterized in that, the said process of thawing and washing was thawed 1～3 minute for said stem apex is put into 35～50 ℃ water-bath, then at room temperature with containing 1～2molL
-1The MS culture fluid washing of sucrose 1～3 time, each 5～10min; Or said stem apex directly put into contain 1～2molL
-1The MS culture fluid of sucrose thaws and washs 5～10min.
10. according to Claim 8 or 9 described cultural methods again, it is characterized in that said cultured method is to contain 0.3～0.6mgL
-1BA, 0.05～0.15mgL
-1NAA and 0.3～0.6mgL
-1The dark cultivation or the low light level were cultivated 2～5 days in the MS semisolid culturemedium of GA3, when stem apex returns green and the sign of sprouting is arranged, changed ordinary light cultivation in the MS solid culture medium again over to.
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| CN108684657A (en) * | 2018-03-27 | 2018-10-23 | 中国医学科学院药用植物研究所海南分所 | A kind of dalbergia wood seed cryopreservation method |
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