CN109792955A - A kind of preparation method of fast-growing Luo Han Guo embryogenic callus - Google Patents
A kind of preparation method of fast-growing Luo Han Guo embryogenic callus Download PDFInfo
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- CN109792955A CN109792955A CN201910161105.8A CN201910161105A CN109792955A CN 109792955 A CN109792955 A CN 109792955A CN 201910161105 A CN201910161105 A CN 201910161105A CN 109792955 A CN109792955 A CN 109792955A
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- callus
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- siraitia grosvenorii
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 146
- 238000002360 preparation method Methods 0.000 title claims abstract description 43
- 230000000408 embryogenic effect Effects 0.000 title 1
- 241001409321 Siraitia grosvenorii Species 0.000 claims abstract description 43
- 235000011171 Thladiantha grosvenorii Nutrition 0.000 claims abstract description 43
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 43
- 239000002609 medium Substances 0.000 claims abstract description 25
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 50
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims description 30
- 229960001231 choline Drugs 0.000 claims description 29
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 25
- 229930003268 Vitamin C Natural products 0.000 claims description 25
- 235000019154 vitamin C Nutrition 0.000 claims description 25
- 239000011718 vitamin C Substances 0.000 claims description 25
- 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 21
- 229930006000 Sucrose Natural products 0.000 claims description 21
- 239000005720 sucrose Substances 0.000 claims description 21
- 229920001817 Agar Polymers 0.000 claims description 17
- 239000008272 agar Substances 0.000 claims description 17
- 239000007787 solid Substances 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 7
- 229960000367 inositol Drugs 0.000 claims description 7
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000004069 differentiation Effects 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 2
- 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 2
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 2
- 229910052603 melanterite Inorganic materials 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- 239000011686 zinc sulphate Substances 0.000 claims description 2
- LFDGRWDETVOGDT-UHFFFAOYSA-N 1h-pyrrole;hydrochloride Chemical compound Cl.C=1C=CNC=1 LFDGRWDETVOGDT-UHFFFAOYSA-N 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims 1
- 206010044565 Tremor Diseases 0.000 claims 1
- 229910052564 epsomite Inorganic materials 0.000 claims 1
- 229960003512 nicotinic acid Drugs 0.000 claims 1
- 235000001968 nicotinic acid Nutrition 0.000 claims 1
- 239000011664 nicotinic acid Substances 0.000 claims 1
- 239000000725 suspension Substances 0.000 abstract description 12
- 230000012010 growth Effects 0.000 abstract description 11
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 229940064880 inositol 100 mg Drugs 0.000 description 12
- 229930182470 glycoside Natural products 0.000 description 10
- 150000002338 glycosides Chemical class 0.000 description 10
- 230000035876 healing Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
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- 238000011105 stabilization Methods 0.000 description 7
- 235000013399 edible fruits Nutrition 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 235000009815 Momordica Nutrition 0.000 description 4
- 241000218984 Momordica Species 0.000 description 4
- 235000009508 confectionery Nutrition 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000006641 stabilisation Effects 0.000 description 4
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 244000269722 Thea sinensis Species 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000000232 gallbladder Anatomy 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
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- 235000019504 cigarettes Nutrition 0.000 description 1
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- 210000002808 connective tissue Anatomy 0.000 description 1
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of preparation methods of the Siraitia grosvenorii embryo callus of fast-growth, comprising: stablizes the preparation of callus: inducing the seed, blade or stem section of Siraitia grosvenorii, obtains and stablize callus；The preparation of newborn callus: will stablize callus tissue culture 70-80 days, takes the cell mass of its browning to be seeded in induced medium and carries out squamous subculture, obtain newborn callus；Can suspend the preparation of embryo callus: selection fast growing, yellow-white newborn callus subculture carry out squamous subculture into new culture medium, acquisition can suspend newborn callus.The present invention induces on the basis of stablizing callus and squamous subculture, shortens the growth lag phase and suspension cultivation cycle of callus, improves the increment of Siraitia grosvenorii seedling.
Description
Technical field
The present invention relates to technical field of tissue culture, are cured more particularly to a kind of Siraitia grosvenorii embryo of fast-growth
The preparation method of injured tissue.
Background technique
Siraitia grosvenorii is the perennial liana of Curcurbitaceae.Main sweet ingredient is momordica glycoside V in Lo Han Guo fruit, it
Sugariness be 300 times of sucrose, sugariness curve is similar to sucrose, because the features such as high sugariness, low in calories, good mouthfeel and widely
Masses like.It is the optimum sweetener of the special populations such as obesity, hypertension, diabetic and health care product, is had wide
General application value.
Momordica glycoside V is mainly extracted from natural Lo Han Guo fruit and is obtained.But the intrinsic arhat in natural Siraitia grosvenorii
Fruit sweet tea glycosides ingredient is extremely low, and the prior art is not high for the utilization rate of Siraitia grosvenorii and the recovery rate of mogroside, causes
Great wastage of material.Simultaneously as the influence of the natural causes such as land resource, weather, the yield of natural Siraitia grosvenorii are limited
System, has been unable to meet ever-increasing market demand.Currently, the method that the cultivation of Siraitia grosvenorii takes tissue cultures mostly, it will be steady
Determine callus to be cultivated, but stationary level Callus material slow growth, the inoculating cell work of preparation suspension culture
Amount is big, and suspending, cell yield when cultivating is few, the period that suspends is long, low efficiency, so that the yield of Siraitia grosvenorii is not able to satisfy city still
Field demand.
Therefore, how to provide the short Siraitia grosvenorii embryo callus of a kind of fast growing, cultivation period is art technology
The problem of personnel's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides a kind of preparation methods of the Siraitia grosvenorii embryo callus of fast-growth, steady
Determine to carry out induction and squamous subculture on the basis of callus, shortens the growth lag phase of callus, shorten suspension training
The period is supported, the increment of Siraitia grosvenorii seedling is improved.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth, includes the following steps:
1) stablize the preparation of callus: the seed, blade or stem section of Siraitia grosvenorii are induced, obtain and stablize callus group
It knits；
2) preparation of newborn callus: it will stablize obtained in the step 1) callus tissue culture 70-80 days, take it
The cell mass of browning, which is seeded in induced medium, carries out squamous subculture, obtains newborn callus；
3) can suspend the preparation of embryo callus: select the newborn callus of fast growing in the step 2), yellow-white
Tissue subculture carries out squamous subculture into new culture medium, and acquisition can suspend newborn callus.
The present invention that above technical scheme reached have the technical effect that, which discloses a kind of acquisition Siraitia grosvenorii and can suspend, cultivates fast fast-growing
The method of long embryo callus.The resulting primary callus growth of new life callus comparison is quick, a large amount of to suspend
Cell inoculation material is easily prepared, and cultivation cycle was reduced to 15 days by 21 days, and growth lag phase is short, and increment, which also has, substantially to be mentioned
It rises.It can also be with well-grown in a small amount of inoculum concentration.When same inoculum concentration 50g/L (fresh weight), after cultivating a period, primary is cured
The dry cell weight for hurting cell is 6.73g/L, and newborn healing cell dry weight is up to 15.77g/L.The arhat after inducer acts on simultaneously
Fruit sweet tea glycosides V yield is suitable with primary callus.
Optionally, the detailed process induced in the step 1) includes: the preparation of (1) primary callus: by sieve
After Chinese fruit seed, blade or stem section disinfection, access in the primary induced medium that pH is 5-7, it is for 20-30 DEG C and unglazed in temperature
Fiber differentiation 15-21 days according under conditions of obtain primary callus；(2) stablize the preparation of callus: by the step
(1) the primary callus obtained in carries out squamous subculture, the stable callus of acquired character.
Optionally, induced medium includes: containing choline 0.5-2.0g/L, vitamin C 0.5- in the step 2)
3.0mg/L, sucrose 25-35g/L, agar 4-5g/L, inositol 80-120mg/L, 6-BA 0.5-2mg/L and NAA 0.5-1.5mg/
The B5 solid medium of L.
What above technical scheme reached has the technical effect that choline is positively charged tetravalence base, to plant growth, produces
The important physiological action such as amount, product quality, photosynthetic performance, membrane permeability, plant resistance to environment stress has Beneficial Effect.Gallbladder is provided with to plant
Alkali can be improved its resistance to frost ability, and reduce Phase transition temperature.Meanwhile choline is not only that film rouge ingredient provides choline group,
And there is varied organisms effect to plant as plant hormone, it needs in certain Valid concentration (0.5-2.0g/
L) its best adjustment of competence exertion acts on；Vitamin C is a kind of antioxidant, can help plant resistant arid, ozone and ultraviolet
Line bring pressure, meanwhile, vitamin C can play one to the adjustment effect of plant as a kind of substance that expression activitiy is high
Fixed help, in plant in adverse circumstance and during aging, work that vitamin C can help plant to play protection and delay
With.
Optionally, in the step 3) squamous subculture condition are as follows: temperature is 25-30 DEG C, light intensity 4000lux, illumination
Time is 12h/d.
Optionally, the primary induced medium is by the basis B5 saline solution culture medium, sucrose 25-35g/L, agar 4-
5g/L, inositol 80-120mg/L, 6-BA 0.5-2mg/L and NAA 0.5-1.5mg/L composition
Optionally, the basis the B5 saline solution culture medium is by KNO3 2500mg/L、MgSO4·7H2O250mg/L、
CaCl2·2H2O 150mg/L、(NH4)2SO4 134mg/L、NaH2PO4.H2O 150mg/L、KI 0.75mg/L、H3BO3
3.0mg/L、MnSO4·4H2O 10mg/L、ZnSO4·7H2O 2.0mg/L、Na2MoO4·2H2O 0.25mg/L、CoCl2·
6H2O 0.025mg/L、CuSO4·5H2O 0.025mg/L、Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L, cigarette
Sour 1.0mg/L, puridoxine hydrochloride 1.0mg/L and Tyiamine Hd element 10mg/L composition
It can be seen via above technical scheme that compared with prior art, the present disclosure provides one kind can fast-growth
Siraitia grosvenorii embryo callus preparation method, what is reached has the technical effect that
1) on the basis of primary callus, continue to induce it, the resulting embryo callus pair that suspends
Quicker than primary callus growth, a large amount of suspension cell inoculation material is easily prepared, and cultivation cycle was reduced to 15 days by 21 days,
And growth lag phase is short, increment is also substantially improved.
It 2) can also be with well-grown in a small amount of inoculum concentration.When same inoculum concentration 50g/L (fresh weight), a period is cultivated
Afterwards, the dry cell weight of former healing cell is 6.73g/L, and newborn healing cell dry cell weight is up to 15.77g/L.
3) choline and vitamin C be joined in induced medium, improve the anti-adversity ability of plant.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is the stabilization callus schematic diagram that the embodiment of the present invention 3 is obtained through Seed inducement；
Fig. 2 attached drawing is the schematic diagram that the embodiment of the present invention 3 stablizes callus browning cell mass；
Fig. 3 attached drawing is the schematic diagram of the newborn callus of the embodiment of the present invention 3；
Fig. 4 attached drawing is that the embodiment of the present invention 3 can suspend the schematic diagram of embryo callus；
Fig. 5 attached drawing is that the embodiment of the present invention 4 can suspend the schematic diagram of embryo callus.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The present embodiment specifically discloses a kind of fast-growth for judging choline and ascorbic best concentration ratio
The preparation method of Siraitia grosvenorii embryo callus, includes the following steps:
1) preparation of primary callus: after the Siraitia grosvenorii seed disinfection, access pH is 6, water-soluble by the basis MS salt
The induction of liquid culture medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L and NAA 0.5mg/L composition
In culture medium, Fiber differentiation 15 days is under conditions of temperature is 27 DEG C and no light to get to primary callus；
2) stablize the preparation of callus: the primary callus obtained in the step 1) being subjected to squamous subculture, is obtained
Obtain the stable callus of character；
3) preparation of newborn callus: it will stablize obtained in the step 2) callus tissue culture 75 days, take its brown
The cell mass of change is seeded to contains a. choline 0g/L, vitamin C 0mg/L respectively；B. choline 0.5g/L, vitamin C 0mg/L；c.
Choline 1g/L, vitamin C 0mg/L；D. choline 2g/L, vitamin C 0mg/L；E. choline 0g/L, vitamin C 0.5mg/L；F. gallbladder
Alkali 0.5g/L, vitamin C 0.5mg/L；G. choline 1g/L, vitamin C 0.5mg/L；H. choline 2g/L, vitamin C 0.5mg/L；i
Choline 0g/L, vitamin C 1mg/L；J. choline 0.5g/L, vitamin C 1mg/L；K. choline 1g/L, vitamin C 1mg/L；L. gallbladder
Alkali 2g/L, vitamin C 1mg/L；M. choline 0g/L, Catergen mg/L；N. choline 0.5g/L, Catergen mg/L；O. choline
1g/L, Catergen mg/L；P. choline 2g/L, vitamin C .2mg/L；Q. choline 0g/L, vitamin C 3mg/L；R. choline
0.5g/L, vitamin C 3mg/L；S. choline 1g/L, vitamin C 3mg/L；T. choline 2g/L, vitamin C .3mg/L；Other components
Content is the B5 solid culture of sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA 1.0mg/LNAA 0.5mg/L
Squamous subculture is carried out in base, obtains newborn callus.
4) can suspend the preparation of embryo callus: select the newborn callus of fast growing in the step 3), yellow-white
It organizes subculture into new culture medium, is carried out under conditions of temperature is 27 DEG C, light intensity 4000lux, light application time are 12h/d
Squamous subculture, acquisition can suspend newborn callus.
The inductivity of obtained newborn callus and the embryonal connective tissue that can suspend is shown in Table one:
Newborn healing cell inductivity=new life healing cell group number/browning healing cell rolls into a ball number × 100%
Can suspend the embryo callus subculture cell inductivity=embryo callus subculture cell mass number that can suspend/new life healing cell group number ×
100%
Table one
Embodiment 2
The present embodiment carries out suspension training by the stabilization callus that seed, blade and stem section induce respectively for comparing
It supports the embryo callus that suspends obtained with technical solution of the present invention and carries out the technical effect difference cultivated and obtained that suspends, specifically
Steps are as follows:
1) Siraitia grosvenorii seed, blade and stem section are sterilized respectively, and to seed decladding, takes embryo；Respectively by embryo, disinfection
Blade and disinfection stem section access pH value be 6 induced medium in, temperature be 20-30 DEG C and no light under conditions of lure
Culture is led 15 days to get primary callus is arrived, wherein induced medium is by the basis MS saline solution culture medium, sucrose 30g/
L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L, NAA 1mg/L composition.
2) the primary callus of three kinds obtained in step 1) is taken, sucrose 30g/L, agar 4.6g/L, inositol are being contained
100mg/L, 6-BA 1.0/L, NAA 0.5mg/L, pH are squamous subculture on 6.0 B5 solid medium, choose continuous subculture 4
It is secondary and growth it is vigorous, quality is loose, in stable condition three kinds of uniform stable callus.
3) three kinds of stable callus obtained in step 2) is taken to cultivate 75 days respectively and be seeded to containing vitamin
C0.5mg/L, choline 1g/L, sucrose 30/L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L, NAA 0.5mg/L, pH
For squamous subculture on 6 B5 solid medium.
4) the newborn callus of fast growing in the 6-10 days in three culture mediums, yellow-white is selected in step 3) respectively
Subculture is into new culture medium (with the culture medium in step 1), and continuous passage 4 times, acquisition quality is loose, grows vigorous and stablizes
The faint yellow embryo callus subculture that suspends knit.
5) callus will be stablized obtained in step 2) and step 4) and embryo callus can be suspended respectively with inoculation
It is 5.5- that amount 50g/L, which is transferred to 2 groups to contain sucrose 30g/L, inositol 100mg/L, 6-BA 1.0/L, NAA 0.5mg/L, pH range,
In 6.0 fluid nutrient medium, and revolving speed is 150r/min, cultivation temperature is 27 DEG C, light intensity 4000lux, light application time are
It is cultivated in the shaking table of 12h/d, obtains 4 kinds of Siraitia grosvenorii suspension cell systems primary.
6) it is trained ripe Siraitia grosvenorii suspension cell with 4 kinds of Siraitia grosvenorii suspension cell systems primary obtained by step 5), cultivated
6th day addition methyl jasmonate, 120 μm of ol/L in the process.
7) in culture the 0-24 days, Siraitia grosvenorii suspension cell is every other day extracted, Siraitia grosvenorii suspension cell is transferred to and is contained
It is filtered in the bottle,suction for having 8 μm of filter membranes, during which three times with aseptic water washing cell, is filtered until filter paper no longer drips.
Weight at this time is the fresh weight (FCW) of callus；By filtered cell as drying to constant weight in 60 DEG C of baking ovens, weight at this time
Amount is the dry weight (DCW) of callus, and dry weight content is specifically shown in Table two:
Table two
By table two it can be seen that the stabilization callus obtained by stem section, blade, Seed inducement is when suspending culture, the 21st
Its cell concentration reaches maximum；The embryo callus that can suspend in the 15th day dry cell weight reaches maximum, and incubation time shortens.And
And the growth index for the embryo callus that can suspend is 0.8778g/L.d, than stem section-stabilization callus 0.1849g/
L.d, blade-stabilization callus 0.2270g/L.d, seed-stabilization callus 0.1914g/Ld are improved respectively
4.7,3.8,4.6 times.
8) 1g Siraitia grosvenorii cellular dry weight is weighed, after methanol ultrasonic extraction 1h is added with the solid-liquid ratio of 10:1, in 60 DEG C of conditions
Lower mechanical shaking extraction 2h, combined extract carries out being concentrated into 1ml under nitrogen evaporator after extracting 3 times repeatedly.Concentrate is used
0.22 μm of membrane filtration carries out qualitative and quantitative analysis with high performance liquid chromatograph.
Chromatographic condition: chromatographic column is the reversed column of C18,4.6mm × 250mm；Mobile phase is water-second eyeball=78-22, flow velocity
1.0ml/min, Detection wavelength 203nm.
Influence of the Siraitia grosvenorii callus of separate sources to the culture momordica glycoside V synthesis that suspends is shown in Table 3.
Table 3
The callus cell in variant source is in suspension incubation, the content of momordica grosvenori glycoside V not plus when inducer
It is extremely low.After adding inducer, the content of momordica glycoside V is obviously improved.The arhat of newborn callus as can be seen from Table 3
Fruit sweet tea glycosides V content can be handled through inducer to be obviously improved.Combination cell growing state, by stem section, blade, Seed inducement
Obtained callus suspension cultivation cycle can be taken as 21 days, and newborn healing cell suspension cultivation cycle is 15 days, fermentation index
Respectively 0.0864,0.1146,0.1342,0.3445mg/Ld.The fermentation index of newborn healing cell is than stem section, blade, kind
The callus cell of son induction promotes 4 times, 3 times, 2.5 times respectively.
Embodiment 3
Present embodiment discloses a kind of preparation methods of the Siraitia grosvenorii embryo callus of fast-growth, comprising:
1) preparation of primary callus: after the Siraitia grosvenorii seed disinfection, access pH is 5, water-soluble by the basis B5 salt
The induction of liquid culture medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L and NAA 0.5mg/L composition
In culture medium, primary is obtained to get to primary callus within Fiber differentiation 15 days under conditions of temperature is 20 DEG C and no light
Callus；
2) stablize the preparation of callus: the primary callus obtained in the step 1) being subjected to squamous subculture, is obtained
The stable callus of character is obtained, concrete outcome is referring to attached drawing 1；
3) preparation of newborn callus: it will stablize obtained in the step 2) callus tissue culture 75 days, take its brown
The cell mass (referring to attached drawing 2) of change is seeded to contains choline 0.5g/L, vitamin C 1.0mg/L, sucrose 30g/L, agar respectively
4.6g/L, inositol 100mg/L, 6-BA 1.0mg/L, NAA 0.5mg/L B5 solid medium in carry out squamous subculture, obtain
Newborn callus；Referring to attached drawing 3.
4) can suspend the preparation of embryo callus: select the newborn callus of fast growing in the step 3), yellow-white
It organizes subculture into new culture medium, is carried out under conditions of temperature is 25 DEG C, light intensity 4000lux, light application time are 12h/d
Squamous subculture, acquisition can suspend embryo callus, referring to fig. 4.
Embodiment 4
Present embodiment discloses a kind of preparation methods of the Siraitia grosvenorii embryo callus of fast-growth, comprising:
1) preparation of primary callus: after the Siraitia grosvenorii seed disinfection, access pH is 5.5, by the basis B5 salt water
What hydroponics base, sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L and NAA 0.5mg/L were formed lures
It leads in culture medium, is obtained just to get to primary callus within Fiber differentiation 15 days under conditions of temperature is 30 DEG C and no light
Grade callus；
2) stablize the preparation of callus: the primary callus obtained in the step 1) being subjected to squamous subculture, is obtained
Obtain the stable callus of character；
3) preparation of newborn callus: it will stablize obtained in the step 2) callus tissue culture 75 days, take its brown
The cell mass of change is seeded to contains choline 0.5g/L, vitamin C 1.0mg/L, sucrose 30g/L, agar 4.6g/L, inositol respectively
100mg/L, 6-BA 1.0mg/LNAA, 0.5mg/L B5 solid medium in carry out squamous subculture, obtain newborn callus；
4) can suspend the preparation of embryo callus: select the newborn callus of fast growing in the step 3), yellow-white
It organizes subculture into new culture medium, is carried out under conditions of temperature is 30 DEG C, light intensity 4000lux, light application time are 12h/d
Squamous subculture, acquisition can suspend embryo callus, referring to attached drawing 5.
Embodiment 5
The present embodiment is for comparing the stabilization callus tissue culture 15-20 induced respectively by seed, blade and stem section
The fresh callus of its non-browning is compared with the inducing effect of the browning callus of culture 70-80 days.The technology of the present invention side
What case obtained suspend, and embryo callus carries out suspends the technical effect difference that culture obtains, the specific steps are as follows:
1) Siraitia grosvenorii seed, blade and stem section are sterilized respectively, and to seed decladding, takes embryo；Respectively by embryo, disinfection
Blade and disinfection stem section access pH value be 6 induced medium in, temperature be 20-30 DEG C and no light under conditions of lure
Culture is led 15 days to get primary callus is arrived, wherein induced medium is by the basis B5 saline solution culture medium, sucrose 30g/
L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L, NAA 1mg/L composition.
2) the primary callus of three kinds obtained in step 1) is taken, sucrose 30g/L, agar 4.6g/L, inositol are being contained
100mg/L, 6-BA 1.0/L, NAA 0.5mg/L, pH are squamous subculture on 6.0 B5 solid medium, choose continuous subculture 4
It is secondary and growth it is vigorous, quality is loose, in stable condition three kinds of uniform stable callus.
3) three kinds of stable callus obtained in step 2) is taken to cultivate 75 days respectively and be seeded to containing vitamin
C0.5mg/L, choline 1g/L, sucrose 30/L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L, NAA 0.5mg/L, pH
For squamous subculture on 6 B5 solid medium.
4) the newborn callus of fast growing in the 6-10 days in three culture mediums, yellow-white is selected in step 3) respectively
Subculture is into new culture medium (with the culture medium in step 1), and continuous passage 4 times, acquisition quality is loose, grows vigorous and stablizes
The faint yellow embryo callus that suspends.
5) three kinds of stable callus obtained in step 2) is taken to cultivate 15 days respectively and be seeded to containing vitamin
C0.5mg/L, choline 1g/L, sucrose 30/L, agar 4.6g/L, inositol 100mg/L, 6-BA1.0mg/L, NAA 0.5mg/L, pH
For squamous subculture on 6 B5 solid medium.
6) select in step 5) the 6-10 days in three culture mediums fresh callus subcultures to new culture medium respectively
In (with the culture medium in step 1), continuous passage 4 times, acquisition quality is loose, grows the vigorous and stable faint yellow embryo that suspends
Property callus is knitted.
7) callus will be stablized obtained in step 2), step 4) and step 6) and the embryo callus difference that can suspend
2, which are transferred to, with inoculum concentration 50g/L contains sucrose 30g/L, inositol 100mg/L, 6-BA1.0/L, NAA 0.5mg/L, pH range
For in the B5 fluid nutrient medium of 5.5-6.0, and revolving speed is 150r/min, cultivation temperature is 27 DEG C, light intensity 4000lux, light
According to cultivating in the shaking table that the time is 12h/d, 4 kinds of Siraitia grosvenorii suspension cell systems primary are obtained.Culture surveys its dry weight, glycosides V in 24 days
Content be shown in Table 4.
Table 4
It follows that new induced medium only has significant inducing action to the callus of culture to browning, and it is right
The growth of fresh callus and the synthesis of glycosides V are not obviously improved effect.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so being described relatively simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (6)
1. a kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth, which is characterized in that include the following steps: 1) steady
Determine the preparation of callus: the seed, blade or stem section of Siraitia grosvenorii are induced, obtains and stablize callus；
2) preparation of newborn callus: it will stablize obtained in the step 1) callus tissue culture 70-80 days, take its browning
Cell mass be seeded in induced medium and carry out squamous subculture, obtain newborn callus；
3) can suspend the preparation of embryo callus: select the newborn callus of fast growing in the step 2), yellow-white
Subculture carries out squamous subculture into new culture medium, and acquisition can suspend newborn callus.
2. a kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth according to claim 1, feature exist
In the detailed process induced in the step 1) includes:
(1) preparation of primary callus: after the disinfection of the Siraitia grosvenorii seed, blade or stem section, the primary that pH is 5-7 is accessed
In induced medium, Fiber differentiation 15-21 days under conditions of temperature is 20-30 DEG C and no light, primary callus is obtained；
(2) stablize the preparation of callus: the primary callus obtained in the step (1) being subjected to squamous subculture, is obtained
The stable callus of character.
3. a kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth according to claim 1, feature exist
In induced medium includes: containing choline 0.5-2.0g/L, vitamin C 0.5-3.0mg/L, sucrose 25- in the step 2)
The B5 solid culture of 35g/L, agar 4-5g/L, inositol 80-120mg/L, 6-BA 0.5-2mg/L and NAA 0.5-1.5mg/L
Base.
4. a kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth according to claim 1, feature exist
In the condition of squamous subculture in the step 3) are as follows: temperature is 25-30 DEG C, light intensity 4000lux, light application time 12h/d.
5. a kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth according to claim 2, feature exist
In the primary induced medium is by the basis B5 saline solution culture medium, sucrose 25-35g/L, agar 4-5g/L, inositol 80-
120mg/L, 6-BA 0.5-2mg/L and NAA 0.5-1.5mg/L composition.
6. a kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth according to claim 5, feature exist
In the basis the B5 saline solution culture medium is by KNO32500mg/L、MgSO4·7H2O 250mg/L、CaCl2·2H2O
150mg/L、(NH4)2SO4134mg/L、NaH2PO4.H2O 150mg/L、KI 0.75mg/L、H3BO33.0mg/L、MnSO4·
4H2O 10mg/L、ZnSO4·7H2O 2.0mg/L、Na2MoO4·2H2O 0.25mg/L、CoCl2·6H2O 0.025mg/L、
CuSO4·5H2O 0.025mg/L、Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L, niacin 1.0mg/L, hydrochloric acid pyrrole
The alcohol 1.0mg/L and Tyiamine Hd element 10mg/L that trembles is formed.
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| Title |
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| 胡颂平等: "《植物细胞组织培养技术》", 31 August 2014, 中国农业大学出版社 * |
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