CN1650696A - Inducting and culturing method of R. coccinea and R. quadrifida callus - Google Patents
Inducting and culturing method of R. coccinea and R. quadrifida callus Download PDFInfo
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- CN1650696A CN1650696A CN 200410018654 CN200410018654A CN1650696A CN 1650696 A CN1650696 A CN 1650696A CN 200410018654 CN200410018654 CN 200410018654 CN 200410018654 A CN200410018654 A CN 200410018654A CN 1650696 A CN1650696 A CN 1650696A
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- callus
- rhodiola
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- graceful
- rhodiola root
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- 238000000034 method Methods 0.000 title claims abstract description 14
- 206010020649 Hyperkeratosis Diseases 0.000 title claims description 132
- 241000130983 Rhodiola coccinea Species 0.000 title abstract description 3
- 241000269971 Rhodiola quadrifida Species 0.000 title abstract description 3
- 238000012258 culturing Methods 0.000 title abstract 2
- 230000001939 inductive effect Effects 0.000 claims abstract description 73
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 241001165494 Rhodiola Species 0.000 claims description 91
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 89
- 239000002609 medium Substances 0.000 claims description 68
- 244000042430 Rhodiola rosea Species 0.000 claims description 55
- 235000003713 Rhodiola rosea Nutrition 0.000 claims description 55
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- 239000007788 liquid Substances 0.000 claims description 42
- 239000007787 solid Substances 0.000 claims description 42
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 19
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims description 18
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims description 18
- 229960001669 kinetin Drugs 0.000 claims description 18
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- 239000012879 subculture medium Substances 0.000 claims description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 6
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- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 6
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 6
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
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- 239000011664 nicotinic acid Substances 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 6
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- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 claims description 2
- JLIDBLDQVAYHNE-LXGGSRJLSA-N 2-cis-abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\C1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-LXGGSRJLSA-N 0.000 claims description 2
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Abstract
A method for inducing and culturing the calli of rhodiola coccinea and rhodiola quadrifida and its culture medium are disclosed for improving their quality. Said culture medium is based on the basic culture medium MS and features addition use of the vegetative hormone composition and/or inductor.
Description
Technical field
The present invention relates to the cultural method and the used medium that gets of plant tissue, specifically, the present invention relates to a kind of method for tissue culture and medium of rhodiola root.
Background technology
Rhodida plant is described as " the refreshing grass in east ", " plateau genseng " because of its unique drug effect.Rhodiola root is at the existing very long applicating history of China, and " legendary god of farming's book on Chinese herbal medicine " and " four pharmacopeia " is all on the books, classifies top grade medicine (Liu Zhiwei, Wu Moucheng, Li Huiliang as.The development of extraction of water chestnut leaf rhodiola root active ingredient and health protection tea thereof.Food and fermentation industries, 2002,28 (5): 47-49), can be used for strengthening by means of tonics, eliminate fatigue, keep out the cold.Emperor Kangxi once removed army's fatigue with eliminating rhodiola root, and imperial family asks for rhodiola root during Qianrong as tribute.The Soviet Union in 1976 uses rhodiola root and is " adapting to former " sample medicine, is used for eliminations fatigues such as astronaut, pilot, diver, sportsman, enhances vigour.In recent years domestic with it as strong medicine, several formulations, health food and beverage have been developed, be used to delay senility, (bureau of drug policy ﹠ administration of Ministry of Health of the People's Republic of China, Nat'l Pharmaceutical ﹠ Biological Products Control Institute write, modern practical book on Chinese herbal medicine (first volume) for senile heart failure, calmness, diabetes etc.Beijing: the People's Health Publisher, 1997:393-397).
Rhodioloside (salidroside) is considered to most important composition with therapeutic activity in the Rhodida plant, if the drug main that utilizes rhodioloside to develop at present relies on from the root of natural Rhodida plant and rhizome and extracts (average content is 0.5%), (Zhourong's Chinese, natural resources of Chinese medicinal materials is learned to need to consume a large amount of natural plant resources.Beijing: Chinese Medicine science and technology publishing house, 1993:278-286).
The someone proposes with artificial cultivation as remedial pathway in recent years.Yet, because the rhodiola root habitat is narrow, suitable area seldom, rhodioloside yields poorly (being lower than 0.2%) under the artificial cultivation condition, therefore, artificial cultivation rhodiola root technology neither a desirable solution.
Cultivation by cell and tissue is directed produces rhodioloside and not influenced by the habitat and can improve rhodioloside output, is a kind of effective alternative route.
At present, the people is only arranged to sachalin rhodiola rhizome (Rhodiola sachalinenisis) (Xu J F, Zhao Y, Han Aiming, et al.Induction and culture of calli from Rhodiola sachalinenisis.A.Bor.Chin.J.Appl.Environ.Biol.1995,1 (1): 19-25.Xu J F, Xie J, Han A M, et al.Kineticand technical studies on large-scale culture of Rhodiola sachalinenisis compactcallus aggregates with air-lift reactors.Journal of Chemical Technology andBiotechnology, 1998,72 (3): the 227-234. plant callus particls suspending culture to produce rhodiola glycoside, Chinese patent, publication number CN1417343A, disclosed day on May 14th, 2003) and rose-red red-spotted stonecrop (Rhodiola rosea) (Miroslawa Furmanowa, Malgorzata Hartwich, August W.Alfermann, Wiktor Kozminski, Marian Olejnik.Rosacin as a product of glycosylation by Rhodiola rosea (roseroot) cell cultures.Plant Cell, Tissue and Organ Culture, 1999,56:105-110) carried out the Primary Study with tissue culture of inducing of callus, the similar research of other Rhodida plants does not appear in the newspapers as yet.
Summary of the invention
The object of the present invention is to provide graceful rhodiola root (R.coccinea) and four to split inducing and cultural method of red scape (R.quadrifida) callus.The invention provides for this reason:
A kind of graceful rhodiola root and four splits the abductive approach of rhodiola rosea callus, may further comprise the steps:
Get stem, leaf or bud, after flushing, the sterilization, sections, leaf that stem is cut into 0.5～1cm cut otch, bud and directly are inoculated on the solid inducing culture 23～24 ℃ of dark down cultivations.According to the method described above, induced the callus that graceful rhodiola root and four splits rhodiola root, and callus well-grown on inducing culture.
Plant hormone is a requisite material in plant cell, tissue, the organ cultured in vitro, and the plant hormone combination of variety classes, variable concentrations is very obvious to the influence of the inductivity of callus.The inventor has determined that by orthogonal experiment graceful rhodiola root and four splits the preferred scheme of rhodiola rosea callus abductive approach, is characterised in that used medium comprises the hormone of following concentration combination:
6-benzylaminopurine (6-BA) 0.5～3mg/L
And/or kinetin (KT) 0.1～1.0mg/L
In at least a, and
Methyl (NAA) 0.3～2mg/L
And/or 2,4 dichlorophenoxyacetic acid (2,4-D) 0.5～1.0mg/L
In at least a.
It is added in the basic MS medium (Murashige and Skoog, 1962), can induce graceful rhodiola root and four to split rhodiola rosea callus, the healing rate height, 30 days healing rate can reach 60%～100%.
The present invention also provides a kind of graceful rhodiola root and four to split the successive transfer culture method of rhodiola rosea callus, may further comprise the steps:
Graceful rhodiola root and four splits rhodiola rosea callus after cultivating 25～35 days on the solid inducing culture, selects grow fast, the fresh and tender callus of color, is seeded in the solid subculture medium 24 ℃ of cultivations.Go down to posterity after 3～5 times, the callus particle on the solid culture medium is inoculated in the liquid subculture medium, concussion is cultivated.According to the method described above, to split the callus growth of rhodiola root good for graceful rhodiola root and four.
The plant hormone of variety classes, variable concentrations also has the influence of highly significant to the cultivation of callus, through exploring, the inventor has determined that graceful rhodiola root and four splits the preferred scheme of rhodiola rosea callus culture method, it is characterized in that adding in the used medium hormone that comprises following concentration combination:
6-benzylaminopurine 3～8mg/L methyl (NAA) 0.5～1.0mg/L
(6-BA)
Abscisic acid (ABA) 0.01～0.1mg/L gibberellin (GA
3) 0.5～2.0mg/L
It is added in the basic MS medium, and graceful rhodiola root and four splits rhodiola rosea callus and increases comparatively fast, and dry weight can reach 18～25g/L during 20～25 days results; And the output of callus rhodioloside is higher, can reach 50～60mg/L.
Its preferred scheme is the elicitor that used medium comprises following concentration combination:
Salicylic acid 0.2～2mg/L
Methyl jasmonate 0.5～5mg/L
Yeast extract 0.1～1g/L
Elicitor plays the effect that plant is synthetic and accumulate secondary metabolite of coercing, this combination can improve the output that graceful rhodiola root and four splits rhodioloside in the rhodiola rosea callus, compare with the medium that does not add elicitor, salidroside content can improve 3～5 times.
The compositional optimization of medium is to improve secondary metabolite---one of important method of rhodioloside.The present invention also provides graceful rhodiola root and four to split the preferred scheme of rhodiola rosea callus culture method, it is characterized in that used medium is the MS medium of improvement, and its component and concentration are as follows:
Sucrose 30000～60000mg/L copper sulphate (CuSO
45H
2O) 0.025mg/L
Ammonium nitrate 1650mg/L cobalt chloride (CoCl
26H
2O) 0.025mg/L
Potassium nitrate 1900mg/L glycine 4～6mg/L
Potassium dihydrogen phosphate 170mg/L thiamine hydrochloride 0.8～2.0mg/L
Magnesium sulfate 370mg/L pyridoxine hydrochloride 1.0～2.0mg/L
Calcium chloride 440mg/L nicotinic acid 1.0～2.0mg/L
Manganese sulphate 22.3mg/L inositol 200～300mg/L
Zinc sulphate 8.6mg/L ferrous sulfate (FeSO
47H
2O) 55～85mg/L
Boric acid 6.2mg/L disodium ethylene diamine tetraacetate 74.6～111.9mg/L
Potassium iodide 0.83mg/L agar 0～2000mg/L
Sodium molybdate (Na
2MoO
42H
2O) 0.25mg/L
Solvent is a water.
The MS medium of this improvement is compared with the MS minimal medium, can make the output that graceful rhodiola root and four splits the rhodiola rosea callus rhodioloside improve 3～5 times.
Graceful rhodiola root and four splits the MS medium that the most preferred scheme of rhodiola rosea callus culture method is used improvement, form and content as follows:
Sucrose 30000mg/L copper sulphate (CuSO
45H
2O) 0.025mg/L
Ammonium nitrate 1650mg/L cobalt chloride (CoCl
26H
2O) 0.025mg/L
Potassium nitrate 1900mg/L glycine 4mg/L
Potassium dihydrogen phosphate 170mg/L thiamine hydrochloride 0.8mg/L
Magnesium sulfate 370mg/L pyridoxine hydrochloride 1.0mg/L
Calcium chloride 440mg/L nicotinic acid 1.0mg/L
Manganese sulphate 22.3mg/L inositol 200mg/L
Zinc sulphate 8.6mg/L ferrous sulfate (FeSO
47H
2O) 55.6mg/L
Boric acid 6.2mg/L disodium ethylene diamine tetraacetate 74.6mg/L
Potassium iodide 0.83mg/L agar 0～2000mg/L
Sodium molybdate (Na
2MoO
42H
2O) 0.25mg/L
All the other are water.
Above medium is by common medium compound method preparation.
Description of drawings
Accompanying drawing 1 is the growth kinetics (fresh weight) of graceful rhodiola rosea callus suspension culture
Accompanying drawing 2 is the growth kinetics (dry weight) of graceful rhodiola rosea callus suspension culture
Embodiment
Specify the present invention with embodiment below:
Embodiment 1
Inducing of graceful rhodiola root bud callus
The bud of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+0.6mg/L KT, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 32/50 after 30 days.
Embodiment 2
Inducing of graceful rhodiola root bud callus
The bud of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+0.2mg/L KT, 2.0mg/L NAA, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 30/50 after 30 days.
Embodiment 3
Inducing of graceful rhodiola root bud callus
The bud of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+0.8mg/L KT, 0.5mg/L NAA, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 33/50 after 30 days.
Embodiment 4
Inducing of graceful rhodiola root bud callus
The bud of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+1.0mg/L 6-BA, 0.5mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 49/50 after 30 days.
Inducing of graceful rhodiola root bud callus
The bud of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+3.0mg/L 6-BA, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 47/50 after 30 days.
Embodiment 6
Inducing of graceful rhodiola root leaf callus
The leaf of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 1.0mg/L NAA, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 42/50 after 30 days.
Embodiment 7
Inducing of graceful rhodiola root leaf callus
The leaf of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+3.0mg/L 6-BA, 0.3mg/L NAA, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 39/50 after 30 days.
Embodiment 8
Inducing of graceful rhodiola root leaf callus
The leaf of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 1.0mg/L NAA, 0.7mg/L 2,4-D, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 39/50 after 30 days.
Embodiment 9
Inducing of graceful rhodiola root leaf callus
The leaf of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+2.0mg/L 6-BA, 2.0mg/L NAA, 1.0mg/L 2,4-D, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 39/50 after 30 days.
Inducing of graceful rhodiola root leaf callus
The leaf of graceful rhodiola root washed 4 hours with running water, aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+3.0mg/L 6-BA, 1.0mg/L NAA, 0.5mg/L 2,4-D, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 39/50 after 30 days.
Embodiment 11
Inducing of graceful rhodiola root stem callus
From the base portion clip branch of graceful rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated into inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 0.5mg/L KT, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 41/50 after 30 days.
Embodiment 12
Inducing of graceful rhodiola root stem callus
From the base portion clip branch of graceful rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated into inducing culture: solid MS minimal medium+3.0mg/L 6-BA, 1.0mg/L KT, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 43/50 after 30 days.
Embodiment 13
Inducing of graceful rhodiola root stem callus
From the base portion clip branch of graceful rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated into inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 0.6mg/L KT, 2.0mg/L NAA, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 30/50 after 30 days.
Embodiment 14
Inducing of graceful rhodiola root stem callus
From the base portion clip branch of graceful rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated into inducing culture: solid MS minimal medium+2.0mg/L 6-BA, 1.0mg/L KT, 2.0mg/L NAA, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 31/50 after 30 days.
Four split inducing of rhodiola root stem callus
From the four base portion clip branches that split the rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated in inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 0.1mg/L KT, 0.3mg/L NAA, 0.7mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 40/50 after 30 days.
Embodiment 16
Four split inducing of rhodiola root stem callus
From the four base portion clip branches that split the rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated in inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 0.1mg/L KT, 2.0mg/L NAA, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 41/50 after 30 days.
Embodiment 17
Four split inducing of rhodiola root stem callus
From the four base portion clip branches that split the rhodiola root stem, running water flushing 5 hours, aseptic water washing 3 times, 75% alcohol disinfecting 30 seconds, aseptic water washing 3 times.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Remove blade, stem is cut into the sections of 0.5～1cm, be inoculated in inducing culture: solid MS minimal medium+0.5mg/L 6-BA, 1.0mg/L KT, 1.0mg/L NAA, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 7 days, projection is arranged around some stem section and grow callus gradually.The statistics healing rate is 38/50 after 30 days.
Embodiment 18
Four split inducing of rhodiola root leaf callus
Four leaves that split rhodiola root washed 4 hours aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times with running water.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+2.0mg/L 6-BA, 0.1mg/L KT, 0.3mg/L NAA, 1.0mg/L 2,4-D, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 36/50 after 30 days.
Embodiment 19
Four split inducing of rhodiola root leaf callus
Four leaves that split rhodiola root washed 4 hours aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times with running water.0.1%HgCl
2Sterilized aseptic water washing 3 times 10 minutes.Blade is sheared, be inoculated into inducing culture: solid MS minimal medium+2.0mg/L 6-BA, 1.0mg/L KT, 2.0mg/L NAA, 0.7mg/L 2,4-D, 23～24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around some blade and grow callus gradually, the statistics healing rate is 33/50 after 30 days.
Four split inducing of rhodiola root bud callus
Four buds that split rhodiola root washed 4 hours aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl with running water
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+3.0mg/L 6-BA, 0.1mg/L KT, 0.3mg/L NAA, 1.0mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 43/50 after 30 days.
Embodiment 21
Four split inducing of rhodiola root bud callus
Four buds that split rhodiola root washed 4 hours aseptic water washing 3 times, 70% alcohol disinfecting 30 seconds, aseptic water washing 3 times, 0.1%HgCl with running water
2Sterilized 10 minutes, and behind the aseptic water washing 3 times, be inoculated into inducing culture: solid MS minimal medium+3.0mg/L 6-BA, 1.0mg/L KT, 2.0mg/L NAA, 0.7mg/L 2,4-D, 24 ℃ of dark down cultivations.After inducing 5 days, projection is arranged around the bud and grow callus gradually.The statistics healing rate is 40/50 after 30 days.
Embodiment 22
The growth kinetics of graceful rhodiola rosea callus suspension culture
Graceful rhodiola rosea callus is after cultivating 25 days on the inducing culture, select grow fast, the fresh and tender graceful rhodiola root stem evoked callus of color, be inoculated in 20ml solid subculture medium according to 0.3g fresh weight/bottle: basic MS solid culture medium+3mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L ABA and 1.0mg/L GA
3, in the conical flask of 50ml, 24 ℃ of cultivations, intensity of illumination is 1000Lx, 8h/ days.Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+3mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L ABA and 1.0mg/L GA
3In, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good.Per four days results of callus are once surveyed fresh weight, dry weight.The results are shown in accompanying drawing 1 and accompanying drawing 2.The growth kinetics of rhodiola rosea callus suspension culture.As can be seen, callus just entered the logarithmic phase of quick growth since the 4th day, enter the stage of stable development in the time of 16 days from accompanying drawing 1 and accompanying drawing 2, and seasonings weighed the highest in 20 days.
Embodiment 23
The extraction of rhodioloside in the graceful rhodiola rosea callus
Get graceful rhodiola rosea callus pulverize in mortar of 0.2g oven dry, add 10ml methyl alcohol, ultrasonic (150W) handles 20min, and 12, the centrifugal 10min of 000rpm gets supernatant.Add the reprocessing of 10ml methyl alcohol in will precipitating again, merge supernatant and get the rhodioloside crude extract, with the membrane filtration of φ 0.22 μ m.
Embodiment 24
The HPLC of callus rhodioloside measures
Adopt Agilent 1100 series high performance liquid chromatographs, (250mm * 4.6mm, 5 μ l) carry out the constant speed wash-out at Hypersil C18 R-ODS post, and flowing phase is 20% methyl alcohol.Sample introduction 10 μ l, flow velocity 1ml/ minute, 30 ℃ of temperature, detect wavelength 276nm.
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+3mg/L 6-BA, 0.5mg/L NAA, 0.01mg/L ABA and 0.5mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 18.0g/L, salidroside content is 50mg/mL.
Embodiment 26
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+3mg/L 6-BA, 0.7mg/L NAA, 0.01mg/L ABA and 1.8mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 19.5g/L, salidroside content is 52mg/mL.
Embodiment 27
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+3mg/L 6-BA, 0.7mg/L NAA, 0.01mg/L ABA and 1.8mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 22.2g/L, salidroside content is 56mg/mL.
Embodiment 28
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+3mg/L 6-BA, 0.7mg/L NAA, 0.01mg/L ABA and 1.8mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 22.4g/L, salidroside content is 52mg/mL.
Embodiment 29
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+3mg/L 6-BA, 1.0mg/L NAA, 0.1mg/L ABA and 1.0mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 23.5g/L, salidroside content is 54mg/mL.
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+5mg/L 6-BA, 0.7mg/L NAA, 0.01mg/L ABA and 0.5mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 19.0g/L, salidroside content is 53mg/mL.
Embodiment 31
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 25.0g/L, salidroside content is 60mg/mL.
Embodiment 32
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+8mg/L 6-BA, 0.7mg/L NAA, 0.01mg/L ABA and 1.0mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 19.6g/L, salidroside content is 54mg/mL.
Embodiment 33
The liquid successive transfer culture of graceful rhodiola rosea callus
Rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium: basic MS liquid nutrient medium+8mg/L 6-BA, 0.7mg/L NAA, 0.08mg/L ABA and 2.0mg/L GA
3, 24 ℃ of room scattering light are cultivated down, and rotating speed is 90rpm.Callus growth is good, results in the time of 20 days, and dry weight is 20.2g/L, salidroside content is 53mg/mL.
Embodiment 34
Elicitor is to the influence of graceful rhodiola rosea callus rhodioloside output
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, added elicitor respectively at the 19th day: SA 0.2mg/L, MJ 0.5mg/L, YE 0.1g/L, 24 ℃ of following indoor natural lights are cultivated 20 days results down, and the survey dry weight is 22.5g/L, and HPLC mensuration content is 150mg/L behind the extraction rhodioloside.
Embodiment 35
Elicitor is to the influence of graceful rhodiola rosea callus rhodioloside output
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, added elicitor respectively at the 19th day: SA 0.2mg/L, MJ 0.5mg/L, YE 1.0g/L, 24 ℃ of following indoor natural lights are cultivated 20 days results down, and the survey dry weight is 23.6g/L, and HPLC mensuration content is 230mg/L behind the extraction rhodioloside.
Embodiment 36
Elicitor is to the influence of graceful rhodiola rosea callus rhodioloside output
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, added elicitor respectively at the 19th day: SA 0.2mg/L, MJ 3mg/L, YE 0.5g/L, 24 ℃ of following indoor natural lights are cultivated 20 days results down, and the survey dry weight is 24.7g/L, and HPLC mensuration content is 180mg/L behind the extraction rhodioloside.
Embodiment 37
Elicitor is to the influence of graceful rhodiola rosea callus rhodioloside output
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, added elicitor respectively at the 19th day: SA 1.8mg/L, MJ 0.5mg/L, YE 0.1g/L, 24 ℃ of following indoor natural lights are cultivated 20 days results down, and the survey dry weight is 24.0g/L, and HPLC mensuration content is 170mg/L behind the extraction rhodioloside.
Embodiment 38
Elicitor is to the influence of graceful rhodiola rosea callus rhodioloside output
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, added elicitor respectively at the 19th day: SA 2.0mg/L, MJ 0.5mg/L, YE 1.0g/L, 24 ℃ of following indoor natural lights are cultivated 20 days results down, and the survey dry weight is 25.0g/L, and HPLC mensuration content is 240mg/L behind the extraction rhodioloside.
Embodiment 39
Elicitor is to the influence of graceful rhodiola rosea callus rhodioloside output
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, added elicitor respectively at the 19th day: SA 2.0mg/L, MJ 5.0mg/L, YE 1.0g/L, 24 ℃ of following indoor natural lights are cultivated 20 days results down, and the survey dry weight is 23.8g/L, and HPLC mensuration content is 210mg/L behind the extraction rhodioloside.
Embodiment 40
Improve the medium of rhodioloside output in the graceful rhodiola root stem callus
Below be the MS liquid nutrient medium of improvement:
Sucrose 30000mg/L copper sulphate 0.025mg/L
(CuSO
4·5H
2O)
Ammonium nitrate 1650mg/L cobalt chloride 0.025mg/L
(CoCl
2·6H
2O)
Potassium nitrate 1900mg/L glycine 4mg/L
Potassium dihydrogen phosphate 170mg/L thiamine hydrochloride 0.8mg/L
Magnesium sulfate 370mg/L pyridoxine hydrochloride 1.0mg/L
Calcium chloride 440mg/L nicotinic acid 1.0mg/L
Manganese sulphate 22.3mg/L flesh-creatine 200mg/L
Zinc sulphate 8.6mg/L ferrous sulfate 55.6mg/L
(FeSO
4·7H
2O)
Boric acid 6.2mg/L disodium ethylene diamine tetraacetate 74.6mg/L
Potassium iodide 0.83mg/L
Sodium molybdate 0.25mg/L
(Na
2MoO
4·2H
2O)
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium in suspension culture, contain the 50ml medium in the conical flask of 250ml, each experiment has three bottles of repetitions, 24 ℃ of down dark cultivations.20 days results, surveying dry weight is 19.9, and HPLC measures content behind the extraction rhodioloside, and calculating output is 180mg/L.
Embodiment 41
Improve the medium of rhodioloside output in the graceful rhodiola root stem callus
Below be the MS liquid nutrient medium of improvement:
Sucrose 55000mg/L copper sulphate (CuSO
45H
2O) 0.025mg/L
Ammonium nitrate 1650mg/L cobalt chloride (CoCl
26H
2O) 0.025mg/L
Potassium nitrate 1900mg/L glycine 5mg/L
Potassium dihydrogen phosphate 170mg/L thiamine hydrochloride 1.5mg/L
Magnesium sulfate 370mg/L pyridoxine hydrochloride 2.0mg/L
Calcium chloride 440mg/L nicotinic acid 1.5mg/L
Manganese sulphate 22.3mg/L inositol 300mg/L
Zinc sulphate 8.6mg/L ferrous sulfate (FeSO
47H
2O) 80mg/L
Boric acid 6.2mg/L disodium ethylene diamine tetraacetate 100mg/L
Potassium iodide 0.83mg/L
Sodium molybdate (Na
2MoO
42H
2O) 0.25mg/L
Solvent is a water.
Graceful rhodiola root stem callus is seeded in respectively by 1.5g fresh weight/bottle contains 5mg/L 6-BA, 0.7mg/L NAA, 0.07mg/L ABA and 0.5mg/L GA
3The MS liquid nutrient medium of improvement in suspension culture, contain the 50ml medium in the conical flask of 250ml, each experiment has three bottles of repetitions, 24 ℃ of dark down cultivations.20 days results, surveying dry weight is 20.1, and HPLC measures content behind the extraction rhodioloside, and calculating output is 150mg/L.
Claims (7)
1. a graceful rhodiola root and four splits the abductive approach of rhodiola rosea callus, may further comprise the steps:
Get stem, leaf or bud, after flushing, the sterilization, sections, leaf that stem is cut into 0.5～1cm cut otch, bud and directly are inoculated on the solid inducing culture 23～24 ℃ of dark down cultivations.
2. the described graceful rhodiola root of claim 1 and four splits the abductive approach of rhodiola rosea callus, it is characterized in that used medium is to add in solid MS minimal medium:
6-benzylaminopurine (6-BA) 0.5～3mg/L
And/or kinetin (KT) 0.1～1.0mg/L
In at least a, and
Methyl (NAA) 0.3～2mg/L
And/or 2,4 dichlorophenoxyacetic acid (2,4-D) 0.5～1.0mg/L
In at least a.
3. a graceful rhodiola root and four splits the successive transfer culture method of rhodiola rosea callus, may further comprise the steps:
Callus is selected grow fast, the fresh and tender callus of color after cultivating 25～35 days on the solid inducing culture, be seeded in the solid subculture medium 24 ℃ of cultivations.Go down to posterity after 3～5 times, the rhodiola rosea callus particle on the solid culture medium is inoculated in the liquid subculture medium, concussion is cultivated.
4. the described graceful rhodiola root of claim 3 and four splits the cultural method of rhodiola rosea callus, it is characterized in that containing in the used medium:
6-benzylaminopurine (6-BA) 3～8mg/L methyl (NAA) 0.5～1.0mg/L
Abscisic acid (ABA) 0.01～0.1mg/L gibberellin (GA
3) 0.5～2.0mg/L
5. the described graceful rhodiola root of claim 4 and four splits the cultural method of rhodiola rosea callus, it is characterized in that also containing in the used medium;
Salicylic acid 0.2～2mg/L
Methyl jasmonate 0.5～5mg/L
Yeast extract 0.1～1g/L
6. claim 4 or 5 described graceful rhodiola roots and four split the cultural method of rhodiola rosea callus, it is characterized in that used medium is the MS medium of improvement, and its composition and content are as follows:
Sucrose 30000～60000mg/L copper sulphate (CuSO
45H
2O) 0.025mg/L
Ammonium nitrate 1650mg/L cobalt chloride (CoCl
26H
2O) 0.025mg/L
Potassium nitrate 1900mg/L glycine 4～6mg/L
Potassium dihydrogen phosphate 170mg/L thiamine hydrochloride 0.8～2.0mg/L
Magnesium sulfate 370mg/L pyridoxine hydrochloride 1.0～2.0mg/L
Calcium chloride 440mg/L nicotinic acid 1.0～2.0mg/L
Manganese sulphate 22.3mg/L inositol 200～300mg/L
Zinc sulphate 8.6mg/L ferrous sulfate (FeSO
47H
2O) 55～85mg/L
Boric acid 6.2mg/L disodium ethylene diamine tetraacetate 74.6～111.9mg/L
Potassium iodide 0.83mg/L agar 0～2000mg/L
Sodium molybdate 0.25mg/L
(Na
2MoO
4·2H
2O)
Solvent is a water.
7. the described graceful rhodiola root of claim 6 and four splits the cultural method of rhodiola rosea callus, it is characterized in that used medium is the MS medium of improvement, and its composition and content are as follows:
Sucrose 30000mg/L copper sulphate (CuSO
45H
2O) 0.025mg/L
Ammonium nitrate 1650mg/L cobalt chloride (CoCl
26H
2O) 0.025mg/L
Potassium nitrate 1900mg/L glycine 4mg/L
Potassium dihydrogen phosphate 170mg/L thiamine hydrochloride 0.8mg/L
Magnesium sulfate 370mg/L pyridoxine hydrochloride 1.0mg/L
Calcium chloride 440mg/L nicotinic acid 1.0mg/L
Manganese sulphate 22.3mg/L inositol 200mg/L
Zinc sulphate 8.6mg/L ferrous sulfate (FeSO
47H
2O) 55.6mg/L
Boric acid 6.2mg/L disodium ethylene diamine tetraacetate 74.6mg/L
Potassium iodide 0.83mg/L agar 0～2000mg/L
Sodium molybdate 0.25mg/L
(Na
2MoO
4·2H
2O)
Solvent is a water.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101444185B (en) * | 2006-12-15 | 2011-06-08 | 浙江大学 | Method for breeding heavy metal hyperaccumulative plant Sedum alfredii Hance germchit |
| CN103858756B (en) * | 2012-12-10 | 2016-01-20 | 吉林师范大学 | A kind of cultural method of rhodiola root aseptic seedling of well developed root system |
| CN105886452A (en) * | 2014-12-25 | 2016-08-24 | 廉美兰 | Method for improving active substances in protocorm of dendrobium officinale kimura et migo by virtue of abiotic elicitors |
| CN107034252A (en) * | 2016-02-03 | 2017-08-11 | 北京中医药大学 | The method that one kind induction suspension culture of Aquilaria sinensis healing cell produces 2- (2- phenethyls) chromone constituents |
| CN107347650A (en) * | 2017-08-28 | 2017-11-17 | 江苏丰收大地种业发展有限公司 | A kind of Radix Rhodiolae tissue culture propagation method |
| WO2018157335A1 (en) * | 2017-03-01 | 2018-09-07 | 桂林莱茵生物科技股份有限公司 | Method for increasing content of mogroside v in siraitia grosvenorii suspended cells |
| CN112997881A (en) * | 2021-02-02 | 2021-06-22 | 中信建设有限责任公司 | Sedum aizoon explant sterilization method, callus induction culture medium and induction method |
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2004
- 2004-02-04 CN CN 200410018654 patent/CN1650696A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101444185B (en) * | 2006-12-15 | 2011-06-08 | 浙江大学 | Method for breeding heavy metal hyperaccumulative plant Sedum alfredii Hance germchit |
| CN103858756B (en) * | 2012-12-10 | 2016-01-20 | 吉林师范大学 | A kind of cultural method of rhodiola root aseptic seedling of well developed root system |
| CN105886452A (en) * | 2014-12-25 | 2016-08-24 | 廉美兰 | Method for improving active substances in protocorm of dendrobium officinale kimura et migo by virtue of abiotic elicitors |
| CN107034252A (en) * | 2016-02-03 | 2017-08-11 | 北京中医药大学 | The method that one kind induction suspension culture of Aquilaria sinensis healing cell produces 2- (2- phenethyls) chromone constituents |
| CN107034252B (en) * | 2016-02-03 | 2020-09-11 | 北京中医药大学 | Method for inducing aquilaria sinensis callus cells to generate 2- (2-phenethyl) chromone components |
| WO2018157335A1 (en) * | 2017-03-01 | 2018-09-07 | 桂林莱茵生物科技股份有限公司 | Method for increasing content of mogroside v in siraitia grosvenorii suspended cells |
| CN107347650A (en) * | 2017-08-28 | 2017-11-17 | 江苏丰收大地种业发展有限公司 | A kind of Radix Rhodiolae tissue culture propagation method |
| CN112997881A (en) * | 2021-02-02 | 2021-06-22 | 中信建设有限责任公司 | Sedum aizoon explant sterilization method, callus induction culture medium and induction method |
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