CN107439377A - It is a kind of improve Momordica grosvenori can suspend culture frequency of embryonic callus induction method - Google Patents
It is a kind of improve Momordica grosvenori can suspend culture frequency of embryonic callus induction method Download PDFInfo
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- CN107439377A CN107439377A CN201710731754.8A CN201710731754A CN107439377A CN 107439377 A CN107439377 A CN 107439377A CN 201710731754 A CN201710731754 A CN 201710731754A CN 107439377 A CN107439377 A CN 107439377A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
Can suspend the method for cultivating Momordica grosvenori frequency of embryonic callus induction the invention discloses a kind of raising, belong to industrial biotechnology field.It comprises the following steps：After Momordica grosvenori seed disinfection, shell, take embryo, access in the inducing culture that pH value is 57, the Fiber differentiation 15 21 days under conditions of temperature is 20 30 DEG C and no light, obtain Momordica grosvenori embryo callus, wherein, the inducing culture by MS basal salt medias, the 35g/L of sucrose 25, the 5g/L of agar 4, the 120mg/L of inositol 80,6 BA 0.5 2mg/L, NAA 0.5 the 1.5mg/L and 6g/L of glycine betaine 4 form.The present invention realizes that the inductivity for the culture embryo callus that can suspend is 64% 93.6%, can shorten induction time, and provide callus in good condition for large-scale culture Momordica grosvenori cell.
Description
Technical field
Can suspend the method for cultivating Momordica grosvenori frequency of embryonic callus induction the present invention relates to a kind of raising, belong to industrial life
Thing technical field.
Background technology
Momordica grosvenori (Siraitia grosvenorii) derives from Curcurbitaceae Momordica (Momordica), is Guilin
A kind of distinctive precious medical and edible dual purpose plant.Mogroside (mogroside) is principle active component in Momordica grosvenori, is had clear
Hot moistening lung, relieving sore-throat open sound, the function of relaxing bowel.Recent research indicate that Momordica grosvenori is also anti-oxidant, anti-diabetic and anticancer
Effect.Momordica grosvenori taste is novel, the healthcare function of pure taste and uniqueness and it is very popular, be obesity, hypertension,
Diabetic best sweetener and health products, are with a wide range of applications.
The acquisition methods of existing momordica glycoside V, are mainly extracted from Momordica grosvenori, but from Lo Han Guo fruit
In extraction yield there was only 1%, and obtain and to be influenceed by region and season from fruit.It is next to that thin by Momordica grosvenori
Born of the same parents, which suspend, cultivates or obtains momordica glycoside V.Above two method will above all establish liquid suspension culture system, and this is just needed
Obtain a large amount of embryo callus in good condition, but on Momordica grosvenori cell embryo callus correlative study compared with
It is few.
Existing Momordica grosvenori embryonic callus induction is mainly by the seedling after seed sprouting, the stem section of plant, blade
And the part of root etc. carries out the induction of embryo callus as explant, but seed is sprouted this stage and then at least needed
The time of one month is taken, has elongated the time for establishing liquid suspension culture system significantly.Moreover, existing embryo callus
Inductivity is also than relatively low, the poor-performing of the cell suspendability culture derived.
In consideration of it, the application provides a kind of method that raising can suspend culture Momordica grosvenori frequency of embryonic callus induction, with
Make up above-mentioned deficiency.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided one kind improves the culture Momordica grosvenori embryo callus subculture that can suspend
The method for organizing inductivity.The present invention passes through the final induction for determining to be used as callus using embryo of the screening to explant
Body, Momordica grosvenori embryo callus is directly induced without sprouting, the inductivity for realizing the culture embryo callus that can suspend is
64%-93.6%, up to 93.6%, compared with the highest inductivity 76% of prior art, highest improves 17.6%, energy
Enough shorten induction time, and callus in good condition is provided for large-scale culture Momordica grosvenori cell.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：One kind improves the culture Momordica grosvenori embryo callus subculture that can suspend
The method for organizing inductivity, comprises the following steps：
After Momordica grosvenori seed disinfection, shell, take embryo, access pH value is in 5-7 inducing culture, is in temperature
Fiber differentiation 15-21 days, that is, obtain Momordica grosvenori embryo callus under conditions of 20-30 DEG C and no light, wherein, the induction
Culture medium is by MS bases saline solution culture medium, sucrose 25-35g/L, agar 4-5g/L, inositol 80-120mg/L, 6-BA 0.5-
2mg/L, NAA 0.5-1.5mg/L and glycine betaine 4-6g/L compositions.
The present invention principle be：Any explant body portion of Momordica grosvenori can inductive formation callus, but can realize
The efficiency of the embryo callus of suspension culture is significantly different.The present invention is using the embryo inside Momordica grosvenori seed as explant
Callus is directly made, by optimizing inducing culturing condition, the formation rate of obtained embryo callus cell is high, cell
The culture property that can suspend is strong so that the synthesis content of the mogroside finally given is high.
The inducing culture of the present invention, by MS bases saline solution culture medium, sucrose, agar, inositol, 6-BA, NAA and sweet tea
Dish alkali forms.Sucrose, agar, inositol, 6-BA, NAA are added in the saline solution culture medium of MS bases, you can obtain above-mentioned induction
Culture medium.Wherein, saline solution culture medium in MS bases provides the basic nutrition element of plant cell growth, is provided for cell growth
Necessary inorganic salts and micro-element nutrition；Sucrose provides the carbon source of plant cell growth；Agar is used as coagulating agent admittedly, in order to more
Injured tissue cell can carry out top layer growth and breeding；Inositol can promote cell metabolism, prevent the browning of healing cell；6-BA
As mitosin；NAA is used as growth hormone；Glycine betaine is used as the osmotic pressure regulator of callus cell, and contributes to it
The formation of embryo callus loose type, cultivated more suitable for suspending.The inducing culture of the present invention can promote Momordica grosvenori embryo
Property can suspend culture callus cell generation, the embryo callus cell of loose type can be rapidly and efficiently obtained, for hanging
Supernatant liquid state culture.
On the basis of above-mentioned technical proposal, the present invention can also do following improvement.
Further, the inducing culture is by MS bases saline solution culture medium, sucrose 30g/L, agar 4.6g/L, inositol
100mg/L, 6-BA 1mg/L, NAA 1mg/L and glycine betaine 5g/L compositions.
It is using above-mentioned further beneficial effect：Inducing culture uses above-mentioned composition, advantageously in induction arhat
Fruit explant forms embryo callus cell.
Further, the MS bases saline solution culture medium is by KNO31900mg/L、NH4NO31650mg/L、MgSO4·
7H2O 370mg/L、KH2PO4170mg/L、CaCl2·2H2O 440mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O
8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、
CoCL2·6H2O 0.025mg/L、Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L, glycine 2.0mg/L, hydrochloric acid
Pyridoxol 0.6mg/L, Tyiamine Hd element 0.1mg/L, remaining be water composition.
Further, the pH value of the inducing culture is 6.
Further, the temperature is 25 DEG C, and the time of the Fiber differentiation is 18 days.
Explanation of nouns：
6-BA, i.e. 6-benzyl aminopurine, it is the plant growth regulator to people, animal safety.
NAA, i.e. methyl α-naphthyl acetate, it is a kind of auxin analog, acts on methyl α-naphthyl acetate similar to auximone.
The beneficial effects of the invention are as follows：
(1) present invention is used as the inductor of callus by the final determination of screening to explant using embryo, without
Sprouting directly induction Momordica grosvenori embryo callus is crossed, the inductivity for realizing the culture embryo callus that can suspend is 64%-
93.6%, up to 93.6%, compared with the highest inductivity 76% of prior art, highest improves 17.6%, can contract
Short induction time, and provide callus in good condition for large-scale culture Momordica grosvenori cell.
(2) present invention develops the inducing culture for promoting Momordica grosvenori embryo to suspend culture callus cell generation,
The embryo callus cell of loose type can be rapidly and efficiently obtained, for suspension liquid culture.
(3) method of the invention is simple, wide market, is adapted to industrialized production.
Brief description of the drawings
Fig. 1 is the non embryogenic callus cell (Non-embryonic callus) of contrast test 1.
Fig. 2 is the vitrifying callus cell (Vitrification callus) of contrast test 2.
Fig. 3 is the embryo callus cell (embryonic call us) of contrast test 3.
Embodiment
The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Embodiment 1：
The raising of the present embodiment can suspend culture Momordica grosvenori frequency of embryonic callus induction method, comprise the following steps：
After 140 Momordica grosvenori seed disinfections, shell, take embryo, segment, obtain 410 embryo explants, access pH value
For in 5.0 inducing culture, Fiber differentiation 21 days, that is, obtain 282 arhats under conditions of temperature is 20 DEG C and no light
Fruit embryo callus cell mass, wherein, the inducing culture is by MS bases saline solution culture medium, sucrose 25g/L, agar
5g/L, inositol 80mg/L, 6-BA 2mg/L, NAA 1mg/L and glycine betaine 6g/L compositions.Wherein, MS bases saline solution
Culture medium is by KNO31900mg/L、NH4NO31650mg/L、MgSO4·7H2O 370mg/L、KH2PO4170mg/L、CaCl2·
2H2O 440mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、
Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCL2·6H2O 0.025mg/L、Na2-EDTA
37.3mg/L、FeSO4·7H2O 27.8mg/L, glycine 2.0mg/L, puridoxine hydrochloride 0.6mg/L, Tyiamine Hd element 0.1mg/
L, remaining is formed for water.
The inductivity of healing cell and the inductivity of embryo callus subculture cell are calculated respectively.Wherein, callus cell induces
Rate refers to that explant forms the production rate of healing cell on inducing culture；Embryo callus subculture cell induction rate is callus
The ratio of embryo callus subculture cell in Hemapoiesis total amount.
Calculation formula is as follows：
It is computed, the inductivity of the healing cell of the present embodiment is 100%, and the inductivity of embryo callus subculture cell is
68.7%.
Embodiment 2：
The raising of the present embodiment can suspend culture Momordica grosvenori frequency of embryonic callus induction method, comprise the following steps：
After 140 Momordica grosvenori seed disinfections, shell, take embryo, segment, obtain 410 embryo explants, access pH value
For in 6.0 inducing culture, Fiber differentiation 18 days, that is, obtain 385 arhats under conditions of temperature is 25 DEG C and no light
Fruit embryo callus cell mass, wherein, the inducing culture is by MS bases saline solution culture medium, sucrose 30g/L, agar
4.6g/L, inositol 100mg/L, 6-BA 1mg/L, NAA 1mg/L and glycine betaine 5g/L compositions.Wherein, MS bases salt is water-soluble
Liquid culture medium is by KNO31900mg/L、NH4NO31650mg/L、MgSO4·7H2O 370mg/L、KH2PO4170mg/L、CaCl2·
2H2O 440mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、
Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCL2·6H2O 0.025mg/L、Na2-EDTA
37.3mg/L、FeSO4·7H2O 27.8mg/L, glycine 2.0mg/L, puridoxine hydrochloride 0.6mg/L, Tyiamine Hd element 0.1mg/
L, remaining is formed for water.
It is computed, the inductivity of the healing cell of the present embodiment is 100%, and the average inductivity of embryo callus subculture cell is
94%.
Embodiment 3：
The raising of the present embodiment can suspend culture Momordica grosvenori frequency of embryonic callus induction method, comprise the following steps：
After 140 Momordica grosvenori seed disinfections, shell, take embryo, segment, obtain 410 embryo explants, access pH value
For in 7.0 inducing culture, Fiber differentiation 15 days, that is, obtain 304 arhats under conditions of temperature is 30 DEG C and no light
Fruit embryo callus cell mass, wherein, the inducing culture is by MS bases saline solution culture medium, sucrose 35g/L, agar
4g/L, inositol 160mg/L, 6-BA 0.5mg/L, NAA 3mg/L and glycine betaine 4g/L compositions.Wherein, MS bases salt is water-soluble
Liquid culture medium is by KNO31900mg/L、NH4NO31650mg/L、MgSO4·7H2O 370mg/L、KH2PO4 170mg/L、CaCl2·
2H2O 440mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、
Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCL2·6H2O 0.025mg/L、Na2-EDTA
37.3mg/L、FeSO4·7H2O 27.8mg/L, glycine 2.0mg/L, puridoxine hydrochloride 0.6mg/L, Tyiamine Hd element 0.1mg/
L, remaining is formed for water.
It is computed, the inductivity of the healing cell of the present embodiment is 100%, and the inductivity of embryo callus subculture cell is
74.2%.
Contrast test
It is used for the culture that can suspend to obtain preferable Momordica grosvenori embryo callus cell, the applicant have studied difference
Different hormones and accelerator beet alkali concn are to Callus formation in explant stem section, blade and embryo and culture medium
Influence.
The induction for the embryo callus that contrast test 1 is carried out with Momordica grosvenori stem section explant
As it can be seen from table 1 when containing 6-BA and NAA simultaneously in culture medium, the inductivity of stem section is higher, thus it is speculated that has
It is probably because the power of regeneration of stem section is stronger.The callus state induced under different 6-BA and NAA proportionings is essentially identical, greatly
Majority is all the broken thread non-embryonic callus (as shown in Figure 1) of platinum sponge, is only induced the vivid embryo of color on a small quantity
Dredge graininess callus.Although also performance structure is loose for the non embryogenic callus of this state, what microscopy was shown is all
There is no the vigor of the strip cell of obvious shape and callus weaker.The color of callus can add gradually after squamous subculture
Simultaneously there is browning in depth.Glycine betaine carries out embryonic callus induction DeGrain to stem section explant.From experimental result
Find out that only only a few stem section has been induced out embryo callus, callus is typically rendered as yellow-white, has certain
Metallic luster, callus surface are in granular form, and cell is uniform.Grown in the cells,primordial suspension incubation derived slow
Slowly, there is bulky grain aggregation and browning in solution.
Influence of the 1 different inductive conditions of table to stem section induced embryonic callus
The induction for the embryo callus cell that contrast test 2 is carried out with Momordica grosvenori blade explant
The inductive formation time using the blade progress callus cell of Momordica grosvenori is longer than the time of stem section, and blade exists
There is within 12-14 days a small amount of callus, and the callus of blade only occurs and grown in blade edge, can not make whole outer
Implant all is induced to form callus.Also it is the white flocculence of non-embryo mostly by the callus of blade induced synthesis,
Only it is induced on a small quantity as embryo callus.A kind of water stain shape is have also appeared in the callus that blade derives, it is transparent
The callus of curling, it is referred to as vitrified callus (as shown in Figure 2), grows shape after this callus squamous subculture
State is bad.
From table 2 it can be seen that when comprising only NAA in culture medium, blade can hardly be induced callus, but
When being NAA (1mg/L) containing higher level, there is a small amount of blade to produce callus.And when 6-BA is comprised only in culture medium,
Also there is same conclusion.When containing both hormones of 6-BA and NAA simultaneously in culture medium, inductivity can reach 100%.And
The high concentration of glycine betaine adds the generation for contributing to embryo callus, and estimation is to take part in the infiltration of regulation plant cell.But
When being using blade as explant, the inductivity for the culture embryo callus that can suspend is also very low, and it is not to lure to show blade
Lead the relatively good explant of embryo callus.
Influence of the 2 different inductive conditions of table to blade induced embryonic callus
Contrast test 3 carries out the induction of embryo callus cell with Momordica grosvenori embryo
The callus of embryo goes out more time corresponding concentration of hormone 6-BA and NAA typically in 12-15 days, culture medium and got over
Greatly, it is also more early to generate the time of callus for it.The forming process of seed-embryo calli is different from stem section and blade, is cured in formation
It is that first slowly expansion forms after callus and expands growth to surrounding whole embryo simultaneously before injured tissue.The callus of formation
The predominantly granular embryo callus of color cadmium yellow transparent surface (as shown in Figure 3), and embryo embryo callus lures
Conductance will be significantly larger than stem section and blade.Especially with the raising of beet alkali concn, embryo callus can be obviously improved
Transparent grain shape is presented in the production rate of cell, its callus cell formed more, more loosely, thin in liquid suspension culture
Intracellular growth state is preferable.
Influence of the 3 different inductive conditions of table to embryo induced embryonic callus
The induction result of embryo callus is different under different inductive conditions, when 6-BA concentration is that 1mg/L, NAA concentration are
When 1g/mL, beet alkali concn are 5g/L, frequency of embryonic callus induction has reached up to 93.6%.As can be seen here, 6-BA pairs
The inducing action of generation embryo callus will be significantly higher than NAA, and glycine betaine is only secondary to the inducing action of embryo callus subculture cell
In 6-BA, the induced efficiency of cells,primordial can be also obviously improved.
As can be seen here, embryo is the in vitro tissue type for being best suitable for induction Momordica grosvenori embryo callus, wherein, induction is cured
The optimal inducing culture of injured tissue is by MS bases saline solution culture medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg/
L, 6-BA 1mg/L, NAA 1mg/L and glycine betaine 5g/L compositions.The present invention is finally determined using kind by the screening to explant
Inductor of the embryo as callus, it can be suspended culturing embryo without directly induction Momordica grosvenori embryo callus, realization is sprouted
The inductivity of property callus reaches more than 93%, can shorten induction time, and provide for large-scale culture Momordica grosvenori cell
Callus in good condition.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (5)
- A kind of method of culture Momordica grosvenori frequency of embryonic callus induction 1. raising can suspend, it is characterised in that including following step Suddenly：After Momordica grosvenori seed disinfection, shell, take embryo, access pH value is in 5-7 inducing culture, is 20-30 in temperature DEG C and no light under conditions of Fiber differentiation 15-21 days, that is, obtain Momordica grosvenori embryo callus, wherein, the Fiber differentiation Base is by MS bases saline solution culture medium, sucrose 25-35g/L, agar 4-5g/L, inositol 80-120mg/L, 6-BA 0.5-2mg/ L, NAA 0.5-1.5mg/L and glycine betaine 4-6g/L compositions.
- The method of culture Momordica grosvenori frequency of embryonic callus induction 2. a kind of raising according to claim 1 can suspend, its It is characterised by, the inducing culture is by MS bases saline solution culture medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg/ L, 6-BA 1mg/L, NAA 1mg/L and glycine betaine 5g/L compositions.
- The method of culture Momordica grosvenori frequency of embryonic callus induction 3. a kind of raising according to claim 1 or 2 can suspend, Characterized in that, the MS bases saline solution culture medium is by KNO31900mg/L、NH4NO3 1650mg/L、MgSO4·7H2O 370mg/L、KH2PO4 170mg/L、CaCl2·2H2O 440mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、 CoCL2·6H2O 0.025mg/L、Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L, glycine 2.0mg/L, hydrochloric acid Pyridoxol 0.6mg/L, Tyiamine Hd element 0.1mg/L, remaining be water composition.
- The method of culture Momordica grosvenori frequency of embryonic callus induction 4. a kind of raising according to claim 1 or 2 can suspend, Characterized in that, the pH value of the inducing culture is 6.
- The method of culture Momordica grosvenori frequency of embryonic callus induction 5. a kind of raising according to claim 1 or 2 can suspend, Characterized in that, the temperature is 25 DEG C, the time of the Fiber differentiation is 18 days.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108251349A (en) * | 2018-01-15 | 2018-07-06 | 华东理工大学 | A kind of method that Siraitia grosvenorii suspension cell growth is promoted by salt stress and promotes sweet tea glycosides V content |
| CN109792955A (en) * | 2019-03-04 | 2019-05-24 | 华东理工大学 | A kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth |
| CN110651709A (en) * | 2019-08-22 | 2020-01-07 | 桂林莱茵生物科技股份有限公司 | Method for improving detoxification efficiency of siraitia grosvenorii seedlings |
| CN110684708A (en) * | 2018-07-06 | 2020-01-14 | 华东理工大学 | Method for amplifying and culturing fructus momordicae suspension cells |
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| CN102174463A (en) * | 2011-01-30 | 2011-09-07 | 桂林医学院 | Method for culturing siraitia grosvenorii callus cell suspension system |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251349A (en) * | 2018-01-15 | 2018-07-06 | 华东理工大学 | A kind of method that Siraitia grosvenorii suspension cell growth is promoted by salt stress and promotes sweet tea glycosides V content |
| WO2019136924A1 (en) * | 2018-01-15 | 2019-07-18 | 华东理工大学 | Method for promoting suspension cell growth of momordica grosvenori and improving content of mogroside v by means of salt stress |
| CN108251349B (en) * | 2018-01-15 | 2022-06-21 | 华东理工大学 | Method for promoting growth of momordica grosvenori suspension cells and increasing content of stevioside V through salt stress |
| CN110684708A (en) * | 2018-07-06 | 2020-01-14 | 华东理工大学 | Method for amplifying and culturing fructus momordicae suspension cells |
| CN110684708B (en) * | 2018-07-06 | 2023-05-16 | 华东理工大学 | Method for amplifying and culturing momordica grosvenori suspension cells |
| CN109792955A (en) * | 2019-03-04 | 2019-05-24 | 华东理工大学 | A kind of preparation method of the Siraitia grosvenorii embryo callus of fast-growth |
| CN109792955B (en) * | 2019-03-04 | 2022-08-23 | 华东理工大学 | Preparation method of rapidly-growing grosvenor momordica embryonic callus |
| CN110651709A (en) * | 2019-08-22 | 2020-01-07 | 桂林莱茵生物科技股份有限公司 | Method for improving detoxification efficiency of siraitia grosvenorii seedlings |
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