CN107980630A - A kind of preparation method of STEVIA REBAUDIANA callus - Google Patents
A kind of preparation method of STEVIA REBAUDIANA callus Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention belongs to culture plant cell field, and in particular to a kind of preparation method of STEVIA REBAUDIANA callus.The step of preparation method of STEVIA REBAUDIANA callus provided by the invention includes the Stomacal guard cell of STEVIA REBAUDIANA being subject to Fiber differentiation, suspension culture and induction of callus.The preparation method of STEVIA REBAUDIANA callus provided by the invention can quickly obtain substantial amounts of epidermis fragment (containing Stomacal guard cell), the quick plant cell suspension cultures for establishing high-purity, shorten cell screening and incubation time, the callus quality of acquisition is high, the percentage of embryo callus is high, and weightening is fast.
Description
Technical field
The invention belongs to culture plant cell field, and in particular to a kind of preparation method of STEVIA REBAUDIANA callus.
Background technology
STEVIA REBAUDIANA (Stevia rebaudiana Bertoni) is composite family sweetleaf Dendranthema, originates in the Ba La of South America
Gui, introduces China in 1976 from Japan.Now, China has become the country of STEVIA REBAUDIANA cultivated area maximum in the world, Jiangsu,
There is large area plantation on the ground such as Fujian, Guangdong, Zhejiang, Shandong, Henan, Anhui, Xinjiang.STEVIA REBAUDIANA blade is rich in stevia
Glycosides, sugariness high (for 350-400 times of sucrose), heat low (for the 1/300 of sucrose), it is safe and non-toxic, be known as " being good for third place in the world
Health sugar source ".It is the quantity-unlimiting natural sweetener used that ministry of Health of China ratified stevioside respectively in 1985 and nineteen ninety
With pharmaceutical sweetener auxiliary material.In addition, stevioside has the function that to control blood glucose, reduces blood pressure, boosts metabolism, also
Have treating diabetes, obesity, adjust hydrochloric acid in gastric juice, recover the effect of neural fatigue.STEVIA REBAUDIANA can also lift domestic animal, horse racing and pet
Appetite, treatment animal chronic disease and cattle infertility, have potential using value in herding and Feed Manufacturing.
STEVIA REBAUDIANA is unsuitable for seedling breeding because seed is minimum, mass of 1000 kernel is low (about 0.02g).Tissue-culturing rapid propagation and suspension are thin
Born of the same parents' culture becomes the major measure that stevia is factory produced.STEVIA REBAUDIANA cultured in vitro is carried out early in twentieth century both at home and abroad
Research, callus has been induced from explants such as seed, stem apex, blade, petiole and stem sections.(the Ferreira such as Ferreia
M.C, Handro W.Production, maintenance and plant regeneration from cell
suspension cultures of Stevia rebaudiana(Bert.)Bertoni.1988Plant Cell
Reports, 7 (2):The culture that suspends 123-126) is established using the callus from STEVIA REBAUDIANA induction, improves STEVIA REBAUDIANA cell
Reproduction speed；Utilize the suspension cell culture of STEVIA REBAUDIANA, it is possible to realize large-scale industrialized production stevioside.Select
Suitable coercing cultivation base, additionally it is possible to improve the stevioside content more than 5% of suspension cell.However, STEVIA REBAUDIANA callus group
The cell type knitted mixes, and particularly the difference of splitting ability and the speed of growth is greatly, it is necessary to which very long cultivation cycle, could screen
Go out the suspension cell line that purity is high, disruptive force is strong, growth rate is fast.Domestic research and report to STEVIA REBAUDIANA cell suspension cultures
It is considerably less, therefore, research and develop in relation to quickly establishing high-purity, the new technology right and wrong of high-content stevioside suspended culture cell system
It is often necessary.
Stomata is the main thoroughfare that plant carries out gas and exchange of moisture with external environment, usually by 2 guard cell's groups
Into.Guard cell is more in isometrical shape, size is close, nucleoplasmic ratio is big, kytoplasm is dense, cell high homogenous, is to establish to suspend carefully
The preferable explant of born of the same parents' culture.However, guard cell is considered as the specific cell of terminal differentiation for a long time, division is lost
Ability.At present, separation and cultured in vitro in relation to STEVIA REBAUDIANA blade Stomacal guard cell, both at home and abroad without report.Current section
In grinding and putting into practice, it is short to be required to a kind of separative efficiency is high, obtains cell purity height, the establishment cycle of suspended culture cell system
The cultural method of STEVIA REBAUDIANA Stomacal guard cell.
The content of the invention
The present invention provides a kind of preparation method of STEVIA REBAUDIANA callus, this method is thin using the stomatal guard of STEVIA REBAUDIANA
The splitting ability of born of the same parents, callus is formed by Stomacal guard cell through tissue cultures.
A kind of preparation method of STEVIA REBAUDIANA callus, this method include being induced the Stomacal guard cell of STEVIA REBAUDIANA
The step of culture, suspension culture and induction of callus.
In a preferred embodiment, the Fiber differentiation include by the epidermis fragment of STEVIA REBAUDIANA in inducing culture into
Row Fiber differentiation, to obtain the operation of small cell cluster；Wherein, the inducing culture is：Based on B5 minimal mediums, add
It is 2%-4% to add sucrose to final weight percentage, adds 6-BA to final concentration of 0.5-5mg/L, and addition NAA is dense to end
Spend for 0.05-0.5mg/L, addition glutamine to final concentration of 100-200mg/L.
In a preferred embodiment, the culture that suspends includes existing the small cell cluster obtained by the Fiber differentiation
The operation of suspension culture is carried out in suspension medium；Wherein, the suspension medium is：Based on B5 minimal mediums, add
It is 2%-4% to add sucrose to final weight percentage, adds 6-BA to final concentration of 0.05-0.50mg/L, addition NAA is extremely
Final concentration of 0.5-5mg/L, addition glutamine to final concentration of 100-200mg/L.
In a preferred embodiment, the induction of callus includes to obtain by the culture that suspends small
Cell mass carries out the operation of induction of callus in callus inducing medium；Wherein, the callus induction
Culture medium is：Based on MS minimal mediums, addition sucrose to final weight percentage is 2%-4%, adds 6-BA
To final concentration of 0.1-3.0mg/L, NAA to final concentration of 0.5-5mg/L, addition agar to final weight percentage are added
For 0.4%-1.0%.
In a preferred embodiment, the Fiber differentiation carries out under non-illuminated conditions, and the temperature of culture is 20-30
℃；The culture density of the Fiber differentiation is 0.1-1mL PCV/mL culture mediums.
In a preferred embodiment, the Fiber differentiation further includes the operation for adding liquid feeding culture medium twice；Wherein,
Once add liquid feeding culture medium operation be：At the 7-10 days of the Fiber differentiation, according to liquid feeding culture medium and Fiber differentiation
The volume ratio of base is (0.5-5):10 ratio adds liquid feeding culture medium into inducing culture；Second of addition liquid feeding culture medium
Operation be：It is (1-5) according to the volume ratio of liquid feeding culture medium and inducing culture at the 15-21 days of the Fiber differentiation:
10 ratio adds liquid feeding culture medium into inducing culture；Wherein, the liquid feeding culture medium is：Using B5 minimal mediums as base
Plinth, addition sucrose to final weight percentage is 2%-4%, adds 6-BA to final concentration of 0.2-1.0mg/L, addition
NAA to final concentration of 0.2-0.8mg/L, addition glutamine to final concentration of 100-200mg/L.
In a preferred embodiment, the culture that suspends carries out under non-illuminated conditions, and the temperature of culture is 20-30
DEG C, cultivation cycle is 7-8 days, co-cultures 2-6 cycle；
In a preferred embodiment, the culture that suspends is shake culture.
In a preferred embodiment, the operation of the induction of callus is：First in 22-28 DEG C, no light
Under the conditions of cultivate 7-21 days, be then transferred under illumination condition continue culture 20-45 days.
In a preferred embodiment, the condition cultivated under the illumination condition is：Intensity of illumination is 500-1500LUX,
When light application time 14-18 is small/day.
Compared with prior art, the present invention has the advantages that：
1st, the present invention utilizes plant tissue separator, and substantial amounts of epidermis fragment is isolated from the blade of STEVIA REBAUDIANA tissue culture plant
(containing Stomacal guard cell), its separative efficiency improve 40-50 times than manual partition method, and the time shortens 20-30 times.
2nd, present invention firstly discovers that the Stomacal guard cell of STEVIA REBAUDIANA has splitting ability, by Fiber differentiation, suspend and train
Support and induction of callus operation, establish a whole set of induction in relation to guard cell, division, cell mass formed, more
Injured tissue is grown and the in-vitro culture method and operation sequence of development, and a new way has been opened up to obtain the callus of STEVIA REBAUDIANA
Footpath.
3rd, the small cell cluster that the present invention is further produced using STEVIA REBAUDIANA guard cell, quickly establishes the suspension of high-purity
Cell culture system, shortens 2-3 times of cell screening and incubation time, for beneficial metabolics such as the biofermentation of the species and synanthrin glycosides
The extraction of product provides a kind of new method.
4th, compared with other prior arts, separative efficiency of the invention it is good (than manual method improve 20-30 times), acquisition
Cell purity height (5-6 times higher than other methods), the establishment cycle of suspended culture cell system are short (than other methods shortening 2-3
Times), technology is unique, quick practical.
5th, the present invention is directly established using the small cell cluster obtained from Stomacal guard cell Fiber differentiation becomes the culture that suspends,
Prolonged callus induction, screening and purge process are avoided, substantially reduces the time of cell mass selection and culture, than
Other cell deriveds and screening technique shorten 3-6 months；In addition, colony's ratio of suspension guard cell uses callus induction
Suspension cell line it is more homozygous and stablize.
6th, act synergistically between each step and parameter of the invention, further increase being cured for guard cell's formation jointly
The quality and quantity of injured tissue.
Other features and advantages of the present invention are by following specific embodiment part detailed description.
Brief description of the drawings
The accompanying drawings which form a part of this application are used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its explanation are used to explain the present invention, do not form inappropriate limitation of the present invention.
Fig. 1 is the micro-structure diagram (times magnification for the epidermis fragment (containing Stomacal guard cell) that acquisition is separated in embodiment 1
Number：400 times).
Fig. 2 is that the micro-structure diagram of the cell obtained in embodiment 1 after Fiber differentiation (shows Stomacal guard cell
Splitting status, amplification factor：400 times).
Fig. 3 is the micro-structure diagram (amplification factor of the small cell cluster obtained in embodiment 1 after suspending and cultivating：300
Times).
Fig. 4 is the photo of the callus obtained in embodiment 1 after induction of callus.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with implementation of the attached drawing to the present invention
Mode is described in further detail.It should be appreciated that the specific embodiments described herein are only used for describing and explaining
The present invention, is not intended to limit the invention.
The present invention provides a kind of preparation method of STEVIA REBAUDIANA callus, this method is included the stomatal guard of STEVIA REBAUDIANA
The step of cell is by Fiber differentiation, suspension culture and induction of callus.
According to the present invention, the Stomacal guard cell of STEVIA REBAUDIANA can be obtained by using the method included the following steps：
First, the leaf fragments of STEVIA REBAUDIANA are obtained.
The acquisition modes of the leaf fragments of STEVIA REBAUDIANA can be：Aseptically (for example, in superclean bench), will
After STEVIA REBAUDIANA blade removes master pulse and leaf margin with scalpel, blade is cut into the leaf fragments of 5-10mm square, and be soaked in 5-
In the isolation medium of 10mL.The leaf fragments cut using this method, leaf thickness is open and flat, vascular tissue is few, is separation epidermis
The preferable explant of fragment (containing Stomacal guard cell).
Then, separation obtains epidermis fragment from the leaf fragments of STEVIA REBAUDIANA (containing Stomacal guard cell).
Wherein, above-mentioned isolation medium can be：Based on 1/2MS minimal mediums, 6-BA is to final concentration of for addition
0.1-0.3mg/L (such as can be in 0.1,0.12,0.15,0.17,0.19,0.2,0.22,0.25,0.28 and 0.3mg/L
Scope between any one or two, such as 0.15-0.25mg/L, are preferably 0.2mg/L), addition sucrose to final weight
Amount percentage composition be 2%-4% (such as can be 2%, 2.2%, 2.4%, 2.5%, 2.7%, 2.8%, 3%, 3.2%,
3.5%th, the scope between any one in 3.7%, 3.8% and 4% or two, such as 2.5%-3.5%, are preferably
3%), add PVP-40 (Polyvinylpyrrolidone polyvinylpyrrolidones) to final concentration of 0.5-1.5g/L (such as
Can be the model between any one in 0.5,0.6,0.7,0.8,0.9,1,1.1,1.2,1.3,1.4 and 1.5g/L or two
Enclose, such as 0.8-1.2g/L, be preferably 1g/L), addition VC to final concentration of 30-70mg/L (such as can be 30,35,40,
45th, the scope between any one in 50,55,60,65 and 70mg/L or two, such as 45-55mg/L, are preferably 50mg/
L), 121 DEG C of sterilizings cool down spare after twenty minutes.
Wherein, STEVIA REBAUDIANA blade preferably uses STEVIA REBAUDIANA tissue culture sterile plant leaf.
STEVIA REBAUDIANA tissue culture sterile plant can use the method included the following steps to obtain：
Stevia seed is cultivated to sprouting in culture medium for cultivating, obtains STEVIA REBAUDIANA aseptic seedling.Selection growth is vigorous, stem is thick
Leaf is thick, the big healthy and strong STEVIA REBAUDIANA aseptic seedling plant open and flat, leaf color is bud green of leaf, is transferred in new culture medium for cultivating and carries out 1-3
The squamous subculture in squamous subculture cycle.Wherein, each squamous subculture cycle can be 27-28 days, and each squamous subculture cycle is equal
The culture medium for cultivating more renewed.Chosen from the STEVIA REBAUDIANA of the above-mentioned squamous subculture by 1-3 squamous subculture cycle and be used as this
The STEVIA REBAUDIANA tissue culture sterile plant of invention.
Wherein, culture medium for cultivating can be：Based on MS minimal mediums, addition sucrose to final weight percent contains
Measure for 2%-4% (such as can be 2%, 2.2%, 2.4%, 2.5%, 2.7%, 2.8%, 3%, 3.2%, 3.5%,
3.7%th, the scope between any one in 3.8% and 4% or two, such as 2.5%-3.5%, are preferably 3%) addition
Agar to final weight percentage be 3-10% (such as can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% in
Any one or two between scope, such as 5%-8% is preferably 7%), to add 6-BA to final concentration of 0.06-
0.14mg/L (such as can be any one in 0.06,0.07,0.08,0.09,0.1,0.11,0.12,0.13 and 0.14mg/L
It is a or two between scope, such as 0.08-0.12mg/L, is preferably 0.1mg/L), add NAA to final concentration of 0.03-
0.07mg/L (such as can be the scope between any one in 0.03,0.04,0.05,0.06 and 0.07mg/L or two,
Such as 0.04-0.06mg/L, it is preferably 0.05mg/L), 121 DEG C of sterilizings cool down spare after twenty minutes.
The condition of above-mentioned squamous subculture can be：23-27 DEG C of temperature (such as can be 23 DEG C, 24 DEG C, 25 DEG C, 26 DEG C and
The scope between any one or two in 27 DEG C, such as 24 DEG C -26 DEG C, preferably 25 DEG C), intensity of illumination 2500-3500LUX
(such as can be any in 2500,2600,2700,2800,2900,3000,3100,3200,3300,3400 and 3500LUX
Scope between one or two, such as 2800-3200LUX, preferably 3000LUX), when light application time 14-18 is small/day (such as
When can be that 14,15,16,17 and 18 are small/day in any one or two between scope, it is excellent such as when 15-17 is small/day
Select 16 it is small when/day).
Wherein, from the leaf fragments of STEVIA REBAUDIANA separation obtain the operation of epidermis fragment (containing Stomacal guard cell) can be with
Carried out using high speed homogenizer；Such as WARING separators (BLENDER 8011EB, the model of the production of the original-pack U.S. can be used
HGB2WT) carry out, the speed of service of the separator is divided into two kinds of " low speed " (1800 revs/min) and " high speed " (22000 revs/min).
From STEVIA REBAUDIANA leaf fragments separation obtain epidermis fragment (containing Stomacal guard cell) operation can include such as
Lower step：
The isolation medium of 5-10mL is poured into separate cup, freezes precooling (such as 5 minutes), then will with the tweezers of sterilizing
Above-mentioned STEVIA REBAUDIANA leaf fragments are sandwiched in separate cup.
Separate cup is inserted into using the above-mentioned separator (for example, separator can be placed in precooling on dry ice) Jing Guo precooling
In, run under " low speed " (1800 revs/min) pattern 0.5-5 seconds (for example, 0.5 second, 1.0 seconds, 1.5 seconds, 2.0 seconds, 2.5 seconds,
The scope between any one or two in 3.0 seconds, 3.5 seconds, 4.0 seconds, 4.5 seconds and 5.0 seconds, such as 2-4 seconds, preferably 3
Second).
By above-mentioned separator be adjusted to run under " high speed " (22000 revs/min) pattern 5-30 seconds (for example, 5 seconds, 10 seconds,
The scope between any one or two in 15 seconds, 20 seconds, 25 seconds and 30 seconds, preferably 15-25 seconds, more preferably 20
Second), obtain separation product.
Separation product is subjected to filtration treatment, collects the filtration product for obtaining epidermis fragment (containing Stomacal guard cell).
The filtration treatment for example can be that to use 50-150 mesh (such as can be 50,60,70,80,90,100,110,120,130,140
Scope between 150 any one or two in mesh, such as 80-120 mesh etc.) sieve (for example, stainless steel mesh)
Carry out filtration treatment.
Filtration product is subjected to rinsing processing with above-mentioned isolation medium, obtains rinsing product.It is for instance possible to use 200-
The isolation medium of 300mL, blows and beats filtration product repeatedly, removes the impurity without Stomacal guard cell with rinsing, then slightly
Sediment is collected by centrifugation.Rinsing processing can carry out repeatedly as needed, such as 2-5 times.
Rinsing product is subjected to centrifugal treating, sediment is collected, obtains epidermis fragment (containing Stomacal guard cell).Should be from
The heart processing for example can be, will above-mentioned rinsing product move into 15mL sterile centrifugation tubes in, add 8-15mL (such as can be 8,
10th, the scope between any one in 12,14 and 15 or two, such as 8-12mL, are preferably 10mL) isolation medium,
2-8 DEG C (such as can be scope between any one or two in 2,4,6 and 8 DEG C, such as 2-6 DEG C, preferably 4 DEG C),
500-2000rpm (such as can be any one in 500,800,1000,1200,1500,1700 and 2000rpm or two
Between scope, such as 800-1200rpm, preferably 1000rpm) under centrifugal treating 10-30min (minute) (such as can be 10,
15th, the scope between any one in 20,25 and 30min or two, such as 15-25min, are preferably 20min).
After the completion of centrifugal treating, sterile glass pipette can be used to absorb supernatant fluid, collect and detect and be deposited in centrifugation
The epidermis fragment (containing Stomacal guard cell) of bottom of the tube.
It can calculate with mL PCV (Packed Cell Volume overstock cell volume) for unit and use aforesaid operations side
The separation yield for the epidermis fragment (containing Stomacal guard cell) that method obtains.For ten separating resultings, epidermis fragment (is protected containing stomata
Guard cell) separation yield be 0.8-1.2mL PCV/g leaves, average value is 1.0mL PCV/g leaves.Contaminated with 0.01%FDA fluorescence
The percentage of color method detection Stomacal guard cell living, the survival rate for measuring Stomacal guard cell is 45%-53%, and average value is
49%.Compared with routine " hand is torn " method, epidermis fragment (an epidermis fragment comes from the leaf fragments of a 5-10mm square,
Every epidermis fragment containing about 10-93 Stomacal guard cell) more than 100 times of separation output increased, the time shortens 20-30
Times.
According to the present invention, Fiber differentiation is carried out to Stomacal guard cell to be included (protecting the epidermis fragment of acquisition containing stomata
Guard cell) Fiber differentiation is carried out in inducing culture, to obtain the operation of inducing cell group.
Wherein, inducing culture can be：Based on B5 minimal mediums, addition sucrose to final weight percent contains
Measure for 2%-4% (such as can be 2%, 2.2%, 2.4%, 2.5%, 2.7%, 2.8%, 3%, 3.2%, 3.5%,
3.7%th, the scope between any one in 3.8% and 4% or two, such as 2.5%-3.5%, are preferably 3%) addition
6-BA to final concentration of 0.5-5mg/L (such as can be 0.5,0.8,1.0,1.2,1.5,1.8,2.0,2.2,2.5,2.8,
3.0th, the scope between any one in 3.2,3.5,3.8,4.0,4.2,4.5,4.8 and 5mg/L or two, such as 0.8-
2.0mg/L, preferably 1.0mg/L), addition NAA to final concentration of 0.05-0.5mg/L (such as can be 0.05,0.08,0.10,
0.12nd, 0.15,0.18,0.20,0.22,0.25,0.28,0.30,0.32,0.35,0.38,0.40,0.42,0.45,0.48 and
The scope between any one or two in 0.5mg/L, such as 0.08-0.20mg/L, preferably 0.10mg/L), add paddy ammonia
Acid amides to final concentration of 100-200mg/L (such as can be 100,110,120,130,140,150,160,170,180,190 and
The scope between any one or two in 200mg/L, such as 130-170mg/L, preferably 146mg/L), 121 DEG C of sterilizings 20
Minute postcooling is spare.
The culture density of the Fiber differentiation can be 0.1-1mL PCV/mL culture mediums (such as can be 0.1,0.2,
0.3rd, the scope between any one in 0.4,0.5,0.6,0.7,0.8,0.9 and 1.0mL PCV/mL culture mediums or two,
Such as 0.1-0.5mL PCV/mL culture mediums, preferred 0.2mL PCV/mL culture mediums).
The Fiber differentiation can fill inducing culture culture dish (culture dish can be purchased from the U.S.
The aseptic plastic culture dish of " CORNING " company, diameter 3-10cm, high 2-3cm) in, after being sealed with sealed membrane, carry out induction training
Support.
The Fiber differentiation can carry out under non-illuminated conditions, and the temperature of culture, which can be 20-30 DEG C, (such as can be
20th, the scope between any one in 21,22,23,24,25,26,27,28,29 and 30 DEG C or two, such as 22-28 DEG C,
Preferably 25 DEG C).
In general, Stomacal guard cell cultivated in inducing culture 7-8 days start first division, the 8-12 days into
Enter vigorous division stage, form small cell cluster (containing 2-8 cell) within the 13-14 days.Preferably, lured to Stomacal guard cell
During leading culture, the operation for adding liquid feeding culture medium twice can also be included.
Wherein, the operation of addition liquid feeding culture medium can be for the first time：In the vigorous division stage of Stomacal guard cell, that is, lure
Lead culture the 7-10 days, are (0.5-5) according to the volume ratio of liquid feeding culture medium and inducing culture:10 (such as can be
0.5:10、1.0:10、1.5:10、2.0:10、2.5:10、3.0:10、3.5:10、4.0:10、4.5:10 and 5.0:Appointing in 10
The scope anticipated between one or two, such as (0.5-2.0):10, preferably 1.0:10) ratio is added into inducing culture to be added
Liquid culture medium.
Adding the operation of liquid feeding culture medium for the second time can be：In the growth of small cell cluster and growth rate than very fast
When, i.e. the 15-21 days of Fiber differentiation are (1-5) according to the volume ratio of liquid feeding culture medium and inducing culture:10 (such as can
Think 1:10、1.5:10、2.0:10、2.5:10、3.0:10、3.5:10、4.0:10、4.5:10 and 5.0:Any one in 10
Or the scope between two, such as (2-3):10) ratio adds liquid feeding culture medium into inducing culture.
Wherein, liquid feeding culture medium can be：Based on B5 minimal mediums, addition sucrose to final weight percent contains
Measure for 2%-4% (such as can be 2%, 2.2%, 2.4%, 2.5%, 2.7%, 2.8%, 3%, 3.2%, 3.5%,
3.7%th, the scope between any one in 3.8% and 4% or two, such as 2.5%-3.5%, are preferably 3%) addition
6-BA to final concentration of 0.2-1.0mg/L (such as can be in 0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 and 1mg/L
Any one or two between scope, such as 0.4-0.6mg/L, preferably 0.5mg/L), add NAA to final concentration of 0.2-
0.8mg/L (such as can be the model between any one in 0.2,0.3,0.4,0.5,0.6,0.7 and 0.8mg/L or two
Enclose, such as 0.3-0.7mg/L, preferably 0.5mg/L), addition glutamine to final concentration of 100-200mg/L (such as can be
100th, the scope between any one in 110,120,130,140,150,160,170,180,190 and 200mg/L or two,
Such as 130-170mg/L, preferably 146mg/L), 121 DEG C of sterilizings cool down spare after twenty minutes.
Using above-mentioned Fiber differentiation operating method, 1.1%-3.5% can be obtained, the gas of average 2.3% (five repetitions)
Hole guard cell's division rate；Contained number of cells is 2-20 in the small cell cluster of acquisition, and average value is about 11；Efficiency of plating is
0.1-0.2%, average out to 0.15%.
By the operation of above-mentioned Fiber differentiation, Stomacal guard cell division forms inducing cell group, wherein most
Inducing cell group is small cell cluster (containing 10-20 cell), only a few for maxicell group (about containing 50-100 cell,
Have more than 100 cells).These small cell clusters are needed by the culture that suspends, to obtain more small cell clusters.It is if big thin
The negligible amounts of born of the same parents group (ratio for accounting for all cell mass quantity is less than 10%), it may not be necessary to remove maxicell group；If quantity
More (accounting for the ratio of all cell mass quantity more than 10%) can remove maxicell by using the stainless steel mesh of 200 mesh
Group.
According to the present invention, the small cell cluster that the culture that suspends includes to obtain by Fiber differentiation is in suspension medium
Carry out the operation of suspension culture.The small cell cluster obtained by Fiber differentiation is carried out suspension culture purpose be to expand it is small thin
The quantity of born of the same parents group, so that the quantity of the small cell cluster obtained afterwards by the culture that suspends reaches target, for follow-up
Induction of callus.
Wherein, suspension medium can be：Based on B5 minimal mediums, addition sucrose to final weight percent contains
Measure for 2%-4% (such as can be 2%, 2.2%, 2.4%, 2.5%, 2.7%, 2.8%, 3%, 3.2%, 3.5%,
3.7%th, the scope between any one in 3.8% and 4% or two, such as 2.5%-3.5%, are preferably 3%) addition
6-BA to final concentration of 0.05-0.50mg/L (such as can be 0.05,0.08,0.10,0.12,0.15,0.18,0.20,
0.22nd, any one in 0.25,0.28,0.30,0.32,0.35,0.38,0.40,0.42,0.45,0.48 and 0.50mg/L
Or the scope between two, such as 0.08-0.20mg/L, preferably 0.10mg/L), add NAA to final concentration of 0.5-5mg/L
(such as can be 0.5,0.8,1.0,1.2,1.5,1.8,2.0,2.2,2.5,2.8,3.0,3.2,3.5,3.8,4.0,4.2,
4.5th, the scope between any one in 4.8 and 5.0mg/L or two, such as 0.8-2.0mg/L, preferably 1.0mg/L), add
Add glutamine to final concentration of 100-200mg/L (such as can be 100,110,120,130,140,150,160,170,
180th, the scope between any one in 190 and 200mg/L or two, such as 130-170mg/L, preferably 146mg/L), 121
DEG C sterilizing cool down after twenty minutes it is spare.Using the suspension medium, small cell cluster growth division is fast, in a cultivation cycle (7-8
My god, see below) in growth coefficient can reach 2.1 to 2.5.
The suspension culture can be shake culture, such as shake culture can be carried out on tablet rotary shaker, will be flat
The adjustment of rotational speed of plate rotary shaker is 50-200rpm, is preferably 120rpm.
The suspension culture can carry out under non-illuminated conditions, and the temperature of culture, which can be 20-30 DEG C, (such as can be
20th, the scope between any one in 21,22,23,24,25,26,27,28,29 and 30 DEG C or two, such as 22-28 DEG C,
Preferably 25 DEG C).
The cultivation cycle of the culture that suspends can be 7-8 days, that is, per 7-8 days secondary cultures once.Suspension culture can
(such as can be model between any one or two in 2,3,4,5 and 6 cultivation cycles to carry out 2-6 cultivation cycle
Enclose, for example, 3-5 cycle, preferably 4 cycles).
The suspension culture can be：Produced according to suspension medium and the Fiber differentiation obtained after above-mentioned Fiber differentiation
Thing (inducing culture containing small cell cluster) is (5-20) according to volume ratio:1 (such as can be 5:1、7:1、9:1、10:1、
12:1、14:1、16:1、18:1 and 20:The scope between any one or two in 1, such as 9:1 (such as 5mL can be taken to pass through
The suspension medium for crossing after above-mentioned Fiber differentiation the Fiber differentiation product obtained and 45mL mixes)) ratio mix, then according to
Above-mentioned suspension culture mode of operation carries out suspension culture.
, can be by the small cell cluster originating from Stomacal guard cell by 21- using above-mentioned suspension culture operating method
Suspension cell system is obtained after the shake culture of 28 days (3-4 cultivation cycle), 80%-95% is small in the suspension cell system
Cell mass (i.e. the neat degree of small cell cluster is 80-95%, and colony's homozygosity of small cell cluster is 80-95% in other words), and
And small cell cluster, in cultivation cycle (7-8 days) internal breeding 1.8-2.5 times (fresh weight), colony's homozygosity of small cell cluster is at least
For 90%.
The present invention is more to obtain by the way that the small cell cluster originating from Stomacal guard cell to be carried out to above-mentioned suspension culture
Small cell cluster, avoid prolonged callus induction, screening and purge process, substantially reduce small cell cluster selection and
The time of culture, can shorten operating time 3-6 month compared to other cell deriveds and screening technique.In addition, the culture that suspends
Stomacal guard cell colony than using callus induction suspension cell line it is more homozygous and stablize.
According to the present invention, the small cell cluster that the induction of callus includes to obtain by the culture that suspends is in callus
Organize the operation of progress induction of callus in inducing culture.
Wherein, callus inducing medium can be：Based on MS minimal mediums, addition sucrose to final weight
Amount percentage composition is 2%-4%, addition 6-BA to final concentration of 0.1-3.0mg/L (such as can be 0.1,0.3,0.5,0.8,
1.0th, the scope between any one in 1.2,1.5,1.8,2.0,2.2,2.5,2.7 and 3.0mg/L or two, such as 0.2-
0.8mg/L, preferably 0.50mg/L), addition NAA to final concentration of 0.5-5mg/L (such as can be 0.5,0.8,1.0,1.2,
1.5th, any one in 1.8,2.0,2.2,2.5,2.8,3.0,3.2,3.5,3.8,4.0,4.2,4.5,4.8 and 5.0mg/L
Or the scope between two, such as 0.8-2.0mg/L, preferably 1.0mg/L), addition agar to final weight percentage is
0.4%-1.0% (such as can be any one in 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1.0%
Or the scope between two, such as 0.6%-0.8%, it is preferably that 0.7%), addition sucrose to final weight percentage is
2%-4% (such as can be 2%, 2.2%, 2.4%, 2.5%, 2.7%, 2.8%, 3%, 3.2%, 3.5%, 3.7%,
The scope between any one or two in 3.8% and 4%, such as 2.5%-3.5%, are preferably 3%) 121 DEG C of sterilizings 20
Minute postcooling is spare.
The operation of the induction of callus can be：First 22-28 DEG C (such as can be 22,23,24,25,
26th, the scope between any one in 27 and 28 DEG C or two, such as 23-27 DEG C, is preferably 25 DEG C) train under non-illuminated conditions
Support 7-21 days (such as can for any one in 7,8,9,10,11,12,13,14,15,16,17,18,19,20 and 21 days or
Scope between two, such as 10-18 days, preferably 14 days), be then transferred under illumination condition continue culture 20-45 days (such as
Can be 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,
43rd, the scope between any one in 44 and 45 days or two, such as 25-35 days or 30-45 days, preferably 30 days)；Illumination
Intensity can be 500-1500LUX (such as can be 500,600,700,800,900,1000,1100,1200,1300,1400
Scope between any one or two in 1500LUX, such as 800-1200LUX, preferably 1000LUX), light application time
When 14-18 is small/day (such as when can be that 14,15,16,17 and 18 are small/day in any one or two between scope, example
As 15-17 it is small when/day, preferably 16 it is small when/day).
Induction of callus can operate in the following way：The suspension culture that will be obtained by the culture that suspends
(culture of culture to the 7th day after generally passing on) is small by centrifugation (can be with 1000rpm centrifugations 10 minutes or so) extraction
Cell mass, after removing fluid nutrient medium raffinate using aseptic filter paper, above-mentioned induction of callus is transferred to by small cell cluster
In base, induction of callus is carried out according to the method described above.
After above-mentioned induction of callus (for example, cultivating 30 days), the callus group of 1.5-2.0cm can be formed
Knit, wherein the percentage of embryo callus can reach 3%-5%, and fresh weight increases 1.9-2.5 times.
Above-mentioned MS minimal mediums, 1/2MS minimal mediums, the component of B5 minimal mediums are that this area is routinely selected
Select, these three minimal mediums can be prepared voluntarily, can also be bought by market；Preferably, above-mentioned three kinds of minimal mediums
Component is as follows：
The component of above-mentioned MS minimal mediums (without sucrose and agar) is：
A great number of elements, including：
Potassium nitrate (KNO3)：1900mg/L, ammonium nitrate (NH4NO3)：1650mg/L, magnesium sulfate (MgSO4·7H2O)：
370mg/L, potassium dihydrogen phosphate (KH2PO4)：170mg/L, calcium chloride (CaCl2·2H2O)：440mg/L；
Trace element, including：
Manganese sulfate (MnSO4·H2O)：16.9mg/L, zinc sulfate (ZnSO4·7H2O)：8.6mg/L, boric acid (H3BO3)：
6.2mg/L, potassium iodide (KI)：0.83mg/L, sodium molybdate (Na2MoO4·2H2O)：0.25mg/L, copper sulphate (CuSO4·5H2O)：
0.025mg/L, cobalt chloride (CoCl2·6H2O)：0.025mg/L；
Molysite, including：
Two ethylenediamine hydrate tetraacethyl disodium (Na2-EDTA)：37.3mg/L, ferrous sulfate (FeSO4·4H2O)：
27.8mg/L；
Organic matter, including：
Glycine：2.0mg/L, puridoxine hydrochloride：0.5mg/L, thiamine hydrochloride：0.1mg/L, nicotinic acid：0.5mg/L, flesh
Acid：100mg/L；
The component of above-mentioned 1/2MS minimal mediums (without sucrose and agar) is (to be free of sugarcane in above-mentioned MS minimal mediums
Sugar and agar) on the basis of, the dosage of a great number of elements is reduced to 1/2.
The component of above-mentioned B5 minimal mediums (without sucrose and agar) is：
A great number of elements, including：
Potassium nitrate (KNO3)：2500mg/L, magnesium sulfate (MgSO4·7H2O)：250mg/L, calcium chloride (CaCl2·2H2O)：
150mg/L, ammonium sulfate (NH4)2SO4：134mg/L, sodium dihydrogen phosphate NaH2PO4·H2O：150mg/L；
Trace element, including：
Potassium iodide (KI)：0.75mg/L, manganese sulfate (MnSO4·4H2O)：10mg/L, boric acid (H3BO3)：3mg/L, sulfuric acid
Zinc (ZnSO4·7H2O)：2mg/L, sodium molybdate (Na2MoO4·2H2O)：0.25mg/L, cobalt chloride (CoCl2·6H2O)：
0.025mg/L；Copper sulphate (CuSO4·5H2O)：0.025mg/L；
Molysite, including：
Two ethylenediamine hydrate tetraacethyl disodium (Na2-EDTA)：37.3mg/L, ferrous sulfate (FeSO4·4H2O)：
27.8mg/L；
Organic matter, including：
Inositol：100mg/L, nicotinic acid：1.0mg/L, puridoxine hydrochloride：1.0mg/L, Tyiamine Hd：10mg/L.
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments be only used for the present invention without
For limiting the scope of the invention.Externally it is to be understood that after present disclosure has been read, those skilled in the art are to this hair
Bright to make various changes or modifications, these equivalent forms also fall within the scope of the appended claims of the present application.
Embodiment 1
The present embodiment is used for the preparation method for illustrating the STEVIA REBAUDIANA callus of the present invention.
(1) acquisition of the epidermis fragment (containing Stomacal guard cell) of STEVIA REBAUDIANA
The epidermis fragment (containing Stomacal guard cell) of STEVIA REBAUDIANA is obtained according to following mode of operation：
Stevia seed is cultivated to sprouting in culture medium for cultivating, obtains STEVIA REBAUDIANA aseptic seedling.Selection growth is vigorous, stem is thick
Leaf is thick, the big healthy and strong STEVIA REBAUDIANA aseptic seedling plant open and flat, leaf color is bud green of leaf, is transferred in new culture medium for cultivating and carries out first
The squamous subculture in squamous subculture cycle (28 days), is then transferred in new culture medium for cultivating and continues second squamous subculture week
The squamous subculture of phase (28 days), is then transferred in new culture medium for cultivating and continues the 3rd squamous subculture cycle (28 days) again
Squamous subculture, the sweetleaf used in the present embodiment is chosen in the STEVIA REBAUDIANA for the squamous subculture that cultivation cycle is sent it to from above three
Chrysanthemum tissue culture sterile plant.
Wherein, culture medium for cultivating is：Based on MS minimal mediums, addition sucrose to final weight percentage is
3%, addition agar to final weight percentage be 7%, add 6-BA to final concentration of 0.1mg/L, addition NAA to eventually it is dense
Spend and cool down after twenty minutes spare for 0.05mg/L, 121 DEG C of sterilizings.The condition of squamous subculture is：25 DEG C of temperature, intensity of illumination
3000LUX, when light application time 16 is small/day.
Under the aseptic condition of superclean bench, the blade of above-mentioned STEVIA REBAUDIANA tissue culture sterile plant is removed with scalpel
After removing master pulse and leaf margin, blade is cut into the leaf fragments of 5mm square, and is soaked in the isolation medium of 5-10mL.Wherein,
Isolation medium is：Based on 1/2MS minimal mediums, 6-BA to final concentration of 0.2mg/L is added, addition sucrose is to final
Weight percentage be 3%, add PVP-40 to final concentration of 1g/L, add VC to final concentration of 50mg/L, 121 DEG C sterilizing
Cool down after twenty minutes spare.
The leaf fragments cut according to aforesaid operations mode, leaf thickness is open and flat, vascular tissue is few, is separation epidermis fragment
The preferable explant of (containing Stomacal guard cell).
Then, the WARING separators (BLENDER 8011EB, model HGB2WT) produced using the original-pack U.S. are obtained from above-mentioned
Isolated epidermis fragment (containing Stomacal guard cell) in the leaf fragments of the STEVIA REBAUDIANA obtained, the speed of service point of the separator
For two kinds of " low speed " (1800 revs/min) and " high speed " (22000 revs/min).Concrete operations mode is as follows：
Pour into the above-mentioned isolation medium of 10mL into separate cup, first freeze precooling in 5 minutes at -20 DEG C, then with sterilizing
Tweezers sandwich above-mentioned STEVIA REBAUDIANA leaf fragments in separate cup.
Above-mentioned separator is placed on dry ice after precooling and is inserted into separate cup, is transported under " low speed " (1800 revs/min) pattern
Row 3 seconds.
Above-mentioned separator is adjusted to run 20 seconds under " high speed " (22000 revs/min) pattern, obtains separation product.
Separation product is subjected to filtration treatment with the stainless steel mesh (being placed on glass funnel) of 100 mesh, collects and obtains table
The filtration product of skin graft section (containing Stomacal guard cell).
The above-mentioned isolation medium of 200-300mL is added in filtration product, gently piping and druming carries out rinsing processing repeatedly, with
Rinsing removes the impurity without Stomacal guard cell, and then gentle centrifugation collects sediment, obtains rinsing product.
Above-mentioned rinsing product is moved into 15mL sterile centrifugation tubes, the isolation medium of 15mL is added, in 4 DEG C, 1000rpm
Lower centrifugal treating 20min, collects sediment, obtains epidermis fragment (containing Stomacal guard cell).The epidermis fragment of acquisition (contains
Stomacal guard cell) as shown in Figure 1 (400 times of amplification).
After the completion of centrifugal treating, supernatant fluid is absorbed using sterile glass pipette, collects and detects and be deposited in centrifuge tube bottom
The epidermis fragment (containing Stomacal guard cell) in portion.
In units of mL PCV, point of the epidermis fragment (containing Stomacal guard cell) obtained using aforesaid operations method is calculated
It is 1.2mL PCV/g leaves from yield；With the percentage of 0.01%FDA fluorescence colours detection Stomacal guard cell living, measure
The survival rate of Stomacal guard cell is 53%.Compared with routine " hand is torn " method, (an epidermis fragment comes from one to epidermis fragment
The leaf fragments of 5-10mm square, containing about 10-93 Stomacal guard cell of every epidermis fragment) separation output increased 100
More than times, the time shortens 20-30 times.
(2) Fiber differentiation
By the epidermis fragment (containing Stomacal guard cell) of the STEVIA REBAUDIANA obtained according to aforesaid operations method according to 0.2mL
The culture density of PCV/mL culture mediums be transferred to fill inducing culture " CORNING " culture dish (Corning Incorporated production
Aseptic plastic culture dish, diameter 3-10cm, high 2-3cm) after, culture dish is sealed with sealed membrane, is put into 25 DEG C, under non-illuminated conditions
Fiber differentiation (static gas wave refrigerator) is carried out, to obtain inducing cell group.
Wherein, inducing culture is：Based on B5 minimal mediums, addition sucrose to final weight percentage is
3%, 6-BA to final concentration of 1.0mg/L is added, adds NAA to final concentration of 0.10mg/L, addition glutamine to final concentration
For 146mg/L.
Stomacal guard cell is cultivated in inducing culture starts first division for 7-8 days, enters vigorous point within the 8-12 days
The phase is split, forms small cell cluster (containing 2-8 cell) within the 13-14 days.
At the 8th day of Fiber differentiation, cell entered division animated period gradually, at this time according to liquid feeding culture medium and Fiber differentiation
The volume ratio of base is 1:10 ratio adds liquid feeding culture medium (addition liquid feeding culture medium for the first time)；The 15th of Fiber differentiation the
My god, the growth of small cell cluster and growth rate than it is very fast when, be 3 according to the volume ratio of liquid feeding culture medium and inducing culture:
10 ratio adds liquid feeding culture medium (second of addition liquid feeding culture medium).Wherein, liquid feeding culture medium is：With B5 minimal mediums
Based on, addition sucrose to final weight percentage is 3%, adds 6-BA to final concentration of 0.5mg/L, addition NAA is extremely
Final concentration of 0.5mg/L, addition glutamine to final concentration of 146mg/L, 121 DEG C of sterilizings cool down spare after twenty minutes.
Using the operation of above-mentioned Fiber differentiation, the division rate of Stomacal guard cell can reach 3.5%；The cellule of acquisition
Contained number of cells is 11 in group；Efficiency of plating is 0.2%.The cell obtained after Fiber differentiation (amplification as shown in Figure 2
400 times).
(3) suspend culture
By the Fiber differentiation product (containing small cell cluster) and suspension medium that are obtained by above-mentioned Fiber differentiation with 9:1
After ratio mixing (taking 5mL Fiber differentiations product to be mixed with 45mL suspension mediums), under no light and 25 DEG C of temperature conditionss,
Concussion suspension culture is carried out with 120rpm on tablet rotary shaker.Wherein, suspension medium is：Using B5 minimal mediums as base
Plinth, addition sucrose to final weight percentage be 3%, add 6-BA to final concentration of 0.10mg/L, addition NAA to eventually it is dense
Spend for 1.0mg/L, addition glutamine to final concentration of 146mg/L.Every 7 days secondary cultures once, are passed on 4 times and (that is, shaken altogether
Suspend culture 28 days), five suspension cell lines are obtained, every 7 days propagation 2.5 times (fresh weights) of small cell cluster, growth is very fast,
For the cell quantity that multiple small cell clusters contain between 15-30, colony's homozygosity of small cell cluster is 95%, is presented than more typical
Suspension cell line character：Cellular morphology is similar, nucleoplasmic ratio is big, kytoplasm is dense, without vacuolization degree is low, cell is highly pure
Conjunction, growth rate are fast.The cell obtained after suspending and cultivating is as shown in Figure 3 (300 times of amplification).
(4) induction of callus
After the 4th secondary culture the 7th day (that is, concussion suspend culture the 28th day), will concussion suspension culture with
Small cell cluster is extracted after 1000rpm centrifugations 10min, after removing fluid nutrient medium raffinate with aseptic filter paper, is transferred to callus
Induction of callus is carried out in inducing culture.Wherein, callus inducing medium is：Using MS minimal mediums as base
Plinth, adds 6-BA to final concentration of 0.50mg/L, adds NAA to final concentration of 1.0mg/L, addition sucrose to final weight hundred
It is 3% to divide content, and addition agar to final weight percentage is 0.7%, and 121 DEG C of sterilizings cool down spare after twenty minutes.
Induction of callus operates as follows：
Cultivated 14 days first under 25 DEG C of non-illuminated conditions, be then transferred to intensity of illumination as 1000LUX, light application time 16
Continue culture 30 days under the illumination condition in hour/day.
By above-mentioned induction of callus, multiple callus between 1.5-2.0cm, wherein embryo can be obtained
The percentage of callus reaches 5%, and fresh weight increases by 2.5 times.The callus obtained after induction of callus is such as
Shown in Fig. 4.
Embodiment 2
The present embodiment is used for the preparation method for illustrating the STEVIA REBAUDIANA callus of the present invention.
In addition to following operating parameter, remaining is and embodiment 1 is identical.
(1) acquisition of the epidermis fragment (containing Stomacal guard cell) of STEVIA REBAUDIANA
Separator is run 1 second under " low speed " pattern, is run 30 seconds under " high speed " pattern.
(2) Fiber differentiation
Inducing culture is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 0.5mg/L is added, adds NAA to final concentration of 0.5mg/L, addition glutamine is to final concentration of
100mg/L。
The division rate of Stomacal guard cell can reach 3.0%；Contained number of cells is 10 in the small cell cluster of acquisition；
Efficiency of plating is 0.15%.
(3) suspend culture
Fiber differentiation product (containing small cell cluster) is with suspension medium with 5:After 1 ratio mixing, shaken with 50rpm
Swing suspension culture.
Suspension medium is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 0.05mg/L is added, adds NAA to final concentration of 2.0mg/L, addition glutamine is to final concentration of
100mg/L。
Colony's homozygosity of the small cell cluster obtained after secondary culture is 92%.
(4) induction of callus
Callus inducing medium is：Based on MS minimal mediums, 6-BA to final concentration of 0.10mg/L is added,
NAA to final concentration of 5.0mg/L is added, addition sucrose to final weight percentage is 3%, addition agar to final weight
It is 0.5% to measure percentage composition.
By above-mentioned induction of callus, multiple callus between 1.5-2.0cm, wherein embryo can be obtained
The percentage of callus reaches 3%, and fresh weight increases by 2.5 times.
Embodiment 3
The present embodiment is used for the preparation method for illustrating the STEVIA REBAUDIANA callus of the present invention.
In addition to following operating parameter, remaining is and embodiment 1 is identical.
(1) acquisition of the epidermis fragment (containing Stomacal guard cell) of STEVIA REBAUDIANA
Separator is run 1 second under " low speed " pattern, is run 30 seconds under " high speed " pattern.
(2) Fiber differentiation
Inducing culture is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 5.0mg/L is added, adds NAA to final concentration of 0.05mg/L, addition glutamine is to final concentration of
200mg/L。
Addition liquid feeding culture medium for the first time：The volume ratio of liquid feeding culture medium and former culture medium is 0.5:10；
Second of addition liquid feeding culture medium：The volume ratio of liquid feeding culture medium and former culture medium is 1:10.
The division rate of Stomacal guard cell can reach 2.4%；Contained number of cells is 6 in the small cell cluster of acquisition；
Efficiency of plating is 0.15%.
(3) suspend culture
Fiber differentiation product (containing small cell cluster) is with suspension medium with 15:After 1 ratio mixing, carried out with 200rpm
Concussion, which suspends, cultivates.
Suspension medium is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 0.5mg/L is added, adds NAA to final concentration of 0.5mg/L, addition glutamine is to final concentration of
200mg/L。
During secondary culture, every 7 days propagation 2.4 times (fresh weights) of small cell cluster, colony's homozygosity of small cell cluster is 92%.
(4) induction of callus
Callus inducing medium is：Based on MS minimal mediums, 6-BA to final concentration of 3.0mg/L is added,
NAA to final concentration of 0.5mg/L is added, addition sucrose to final weight percentage is 3%, addition agar to final weight
It is 1.0% to measure percentage composition.
By above-mentioned induction of callus, multiple callus between 1.5-2.0cm, wherein embryo can be obtained
The percentage of callus reaches 4.5%, and fresh weight increases by 2.2 times.
Embodiment 4
The present embodiment is used for the preparation method for illustrating the STEVIA REBAUDIANA callus of the present invention.
In addition to following operating parameter, remaining is and embodiment 1 is identical.
(1) acquisition of the epidermis fragment (containing Stomacal guard cell) of STEVIA REBAUDIANA
Separator is run 5 seconds under " low speed " pattern, is run 10 seconds under " high speed " pattern；Rinse product 4 DEG C,
Centrifugal treating 8min under 1500rpm.
(2) Fiber differentiation
Inducing culture is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 3.0mg/L is added, adds NAA to final concentration of 0.05mg/L, addition glutamine is to final concentration of
200mg/L。
Addition liquid feeding culture medium for the first time：The volume ratio of liquid feeding culture medium and former culture medium is 5:10；
Second of addition liquid feeding culture medium：The volume ratio of liquid feeding culture medium and former culture medium is 5:10.
The division rate of Stomacal guard cell can reach 2.3%；Contained number of cells is 8 in the small cell cluster of acquisition；
Efficiency of plating is 0.15%.
(3) suspend culture
Fiber differentiation product (containing small cell cluster) is with suspension medium with 15:After 1 ratio mixing, carried out with 200rpm
Concussion, which suspends, cultivates.
Suspension medium is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 0.5mg/L is added, adds NAA to final concentration of 0.5mg/L, addition glutamine is to final concentration of
200mg/L。
During secondary culture, every 7 days propagation 2.2 times (fresh weights) of small cell cluster, colony's homozygosity of small cell cluster is 92%.
(4) induction of callus
Callus inducing medium is：Based on MS minimal mediums, 6-BA to final concentration of 3.0mg/L is added,
NAA to final concentration of 0.5mg/L is added, addition sucrose to final weight percentage is 3%, addition agar to final weight
It is 1.0% to measure percentage composition.
By above-mentioned induction of callus, multiple callus between 1.5-2.0cm, wherein embryo can be obtained
The percentage of callus reaches 4.5%, and fresh weight increases by 2.3 times.
Embodiment 5
The present embodiment is used for the preparation method for illustrating the STEVIA REBAUDIANA callus of the present invention.
In addition to following operating parameter, remaining is and embodiment 1 is identical.
(1) acquisition of the epidermis fragment (containing Stomacal guard cell) of STEVIA REBAUDIANA
Separator is run 4 seconds under " low speed " pattern, is run 25 seconds under " high speed " pattern；Product is rinsed in 4 DEG C, 800rpm
Lower centrifugal treating 25min.
(2) Fiber differentiation
Inducing culture is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 0.8mg/L is added, adds NAA to final concentration of 0.45mg/L, addition glutamine is to final concentration of
180mg/L。
Addition liquid feeding culture medium for the first time：The volume ratio of liquid feeding culture medium and former culture medium is 1:10；
Second of addition liquid feeding culture medium：The volume ratio of liquid feeding culture medium and former culture medium is 1.5:10.
The division rate of Stomacal guard cell can reach 3.1%；Contained number of cells is 7 in the small cell cluster of acquisition；
Efficiency of plating is 0.15%.
(3) suspend culture
Fiber differentiation product (containing small cell cluster) is with suspension medium with 9:After 1 ratio mixing, shaken with 80rpm
Swing suspension culture.
Suspension medium is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 3%,
6-BA to final concentration of 0.45mg/L is added, adds NAA to final concentration of 4.5mg/L, addition glutamine is to final concentration of
200mg/L。
During secondary culture, every 7 days propagation 2.5 times (fresh weights) of small cell cluster, colony's homozygosity of small cell cluster is 94%.
(4) induction of callus
Callus inducing medium is：Based on MS minimal mediums, 6-BA to final concentration of 0.8mg/L is added,
NAA to final concentration of 3.0mg/L is added, addition sucrose to final weight percentage is 3%, addition agar to final weight
It is 1.0% to measure percentage composition.
By above-mentioned induction of callus, multiple callus between 1.5-2.0cm, wherein embryo can be obtained
The percentage of callus reaches 4.3%, and fresh weight increases by 2.2 times.
Embodiment 6
In addition to following operating parameter, remaining is and embodiment 1 is identical.
During Fiber differentiation, liquid feeding culture medium is changed into inducing culture.
It was found that by after Fiber differentiation, the division rate of Stomacal guard cell is only 1.1%；In the small cell cluster of acquisition
Contained number of cells is 22；Efficiency of plating is 0.1%, not ideal for follow-up suspension culture, therefore is not continued
Follow-up suspension culture and induction of callus.
Comparative example 1
In addition to following operating parameter, remaining is and embodiment 1 is identical.
Inducing culture in embodiment 1 is changed to following inducing culture：
Based on MS minimal mediums, addition sucrose to final weight percentage is 3%, and addition 6-BA is dense to end
Spend for 1.0mg/L, addition NAA to final concentration of 0.10mg/L, addition glutamine to final concentration of 146mg/L.
It turns out that using the inducing culture of this comparative example, substantially the epidermis fragment of STEVIA REBAUDIANA (cannot be contained gas
Hole guard cell) induction obtain inducing cell group.
Comparative example 2
In addition to following operating parameter, remaining is and embodiment 1 is identical.
Inducing culture in embodiment 1 is changed to following inducing culture：
Based on 1/2MS minimal mediums, addition sucrose to final weight percentage is 3%, and addition 6-BA is extremely
Final concentration of 0.5mg/L, adds NAA to final concentration of 0.10mg/L, addition glutamine to final concentration of 146mg/L.
It turns out that using the inducing culture of this comparative example, the epidermis fragment of STEVIA REBAUDIANA (can only be contained stomatal guard
Cell) induction obtain minimal amount of inducing cell group, Stomacal guard cell division rate only has 0.2%, can not meet subsequently to suspend
Culture and the demand of induction of callus.
, can be quick by above-described embodiment 1-5 it can be found that using the preparation method of the STEVIA REBAUDIANA callus of the present invention
The plant cell suspension cultures of high-purity are established, shorten cell screening and incubation time, and separative efficiency is good, the cell of acquisition
Purity is high；The division rate of Stomacal guard cell is high, and number of cells of the small cell cluster of acquisition more and in small cell cluster is reasonable, plants plate
Rate is high；Propagation is fast when small cell cluster is cultivated through suspending, and colony's homozygosity of small cell cluster is high；The callus quality of acquisition is high,
The percentage of embryo callus is high, and weightening is fast.
The inducing culture used in the present invention it can be seen from the comparison of embodiment 1 and comparative example 1 and 2 can be efficient
Promotion Stomacal guard cell division, obtain the rational small cell cluster of number of cells, and the efficiency of plating of small cell cluster is high, is
Extraordinary basis has been laid in follow-up suspension culture.
Include addition liquid feeding culture medium twice using in the present invention it can be seen from the comparison of embodiment 1 and embodiment 6
Fiber differentiation operation, and using the present invention liquid feeding culture medium, higher Stomacal guard cell division rate can be obtained, and
And the celliferous number of institute is more reasonable in the small cell cluster obtained, efficiency of plating is high, is follow-up suspension culture and callus
Fiber differentiation is laid a good foundation.
As known by the technical knowledge, the present invention can pass through the embodiment party of other essence without departing from its spirit or essential feature
Case is realized.Therefore, embodiment disclosed above, all things considered, is all merely illustrative, not the only.Institute
Have within the scope of the present invention or be included in the invention in the change being equal in the scope of the present invention.
Claims (10)
1. a kind of preparation method of STEVIA REBAUDIANA callus, this method includes the Stomacal guard cell of STEVIA REBAUDIANA being induced training
The step of foster, suspension culture and induction of callus.
2. according to the method described in claim 1, it is characterized in that：
The Fiber differentiation includes the epidermis fragment of STEVIA REBAUDIANA carrying out Fiber differentiation in inducing culture, to obtain cellule
The operation of group；Wherein,
The inducing culture is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 2%-
4%, 6-BA to final concentration of 0.5-5mg/L is added, adds NAA to final concentration of 0.05-0.5mg/L, addition glutamine is extremely
Final concentration of 100-200mg/L.
3. according to the method described in claim 1, it is characterized in that：
The culture that suspends includes the small cell cluster obtained by the Fiber differentiation carrying out suspension training in suspension medium
Foster operation；Wherein,
The suspension medium is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 2%-
4%, 6-BA to final concentration of 0.05-0.50mg/L is added, adds NAA to final concentration of 0.5-5mg/L, addition glutamine is extremely
Final concentration of 100-200mg/L.
4. according to the method described in claim 1, it is characterized in that：
The induction of callus includes training the small cell cluster obtained by the culture that suspends in callus induction
Support the operation that induction of callus is carried out in base；Wherein,
The callus inducing medium is：Based on MS minimal mediums, addition sucrose to final weight percent contains
Measure as 2%-4%, addition 6-BA to final concentration of 0.1-3.0mg/L, addition NAA to final concentration of 0.5-5mg/L, addition agar
It is 0.4%-1.0% to final weight percentage.
5. according to the method described in claim 2, it is characterized in that：
The Fiber differentiation carries out under non-illuminated conditions, and the temperature of culture is 20-30 DEG C；
The culture density of the Fiber differentiation is 0.1-1mL PCV/mL culture mediums.
6. according to the method described in claim 5, it is characterized in that：
The Fiber differentiation further includes the operation for adding liquid feeding culture medium twice；Wherein,
For the first time addition liquid feeding culture medium operation be：At the 7-10 days of the Fiber differentiation, according to liquid feeding culture medium and lure
The volume ratio for leading culture medium is (0.5-5):10 ratio adds liquid feeding culture medium into inducing culture；
The operation of second addition liquid feeding culture medium is：At the 15-21 days of the Fiber differentiation, according to liquid feeding culture medium and lure
The volume ratio for leading culture medium is (1-5):10 ratio adds liquid feeding culture medium into inducing culture；Wherein,
The liquid feeding culture medium is：Based on B5 minimal mediums, addition sucrose to final weight percentage is 2%-
4%, 6-BA to final concentration of 0.2-1.0mg/L is added, adds NAA to final concentration of 0.2-0.8mg/L, addition glutamine is extremely
Final concentration of 100-200mg/L.
7. according to the method described in claim 3, it is characterized in that：
The culture that suspends carries out under non-illuminated conditions, and the temperature of culture is 20-30 DEG C, and cultivation cycle is 7-8 days, is co-cultured
2-6 cycle.
8. according to the method described in claim 7, it is characterized in that：
The culture that suspends is shake culture.
9. according to the method described in claim 4, it is characterized in that：
The operation of the induction of callus is：Cultivated 7-21 days first under 22-28 DEG C, non-illuminated conditions, Ran Houzhuan
Move under illumination condition and continue culture 20-45 days.
10. according to the method described in claim 9, it is characterized in that：
The condition cultivated under the illumination condition is：Intensity of illumination is 500-1500LUX, when light application time 14-18 is small/day.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624547A (en) * | 2018-05-22 | 2018-10-09 | 天津农学院 | A method of separation monocotyledon guard cell |
| CN108812317A (en) * | 2018-06-27 | 2018-11-16 | 东台润洋甜叶菊高科有限公司 | A method of STEVIA REBAUDIANA RM content is improved using callus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103766222A (en) * | 2014-02-12 | 2014-05-07 | 四川农业大学 | Stevia rebaudiana tissue culture method and culture medium thereof |
| CN103858757A (en) * | 2012-12-11 | 2014-06-18 | 丰益(上海)生物技术研发中心有限公司 | Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof |
-
2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103858757A (en) * | 2012-12-11 | 2014-06-18 | 丰益(上海)生物技术研发中心有限公司 | Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof |
| CN103766222A (en) * | 2014-02-12 | 2014-05-07 | 四川农业大学 | Stevia rebaudiana tissue culture method and culture medium thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624547A (en) * | 2018-05-22 | 2018-10-09 | 天津农学院 | A method of separation monocotyledon guard cell |
| CN108624547B (en) * | 2018-05-22 | 2021-08-13 | 天津农学院 | Method for separating monocotyledon guard cells |
| CN108812317A (en) * | 2018-06-27 | 2018-11-16 | 东台润洋甜叶菊高科有限公司 | A method of STEVIA REBAUDIANA RM content is improved using callus |
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