CN109496860A - A method of suspend culture bletilla cell - Google Patents
A method of suspend culture bletilla cell Download PDFInfo
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- CN109496860A CN109496860A CN201811481695.4A CN201811481695A CN109496860A CN 109496860 A CN109496860 A CN 109496860A CN 201811481695 A CN201811481695 A CN 201811481695A CN 109496860 A CN109496860 A CN 109496860A
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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
- A01H4/008—Methods for regeneration to complete plants
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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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
This programme discloses a kind of method of suspension culture bletilla cell of Chinese medicine drug research technical field, using the callus obtained after bletilla Seed inducement and subculture as explant in this method, it is inoculated into suspension medium and carries out suspension culture, suspension medium is 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.45mg/L+2,4- dichlorphenoxyacetic acid ± 20g/L sucrose；Suspension medium pH is 5.9 ± 0.1；The volume ratio of explant inoculum concentration and culture medium is 1/80~1/20g/mL；The suspension culture of oscillation in 40~50 days is carried out after inoculation under the dark condition that revolving speed is 110 ± 10r/min, temperature is 25 ± 2 DEG C.The method that this programme is cultivated using cell tissue suspension; callus after bletilla Seed inducement and subculture is subjected to suspension culture as explant; this method not only can be cellular level pilot scale culture bletilla, further provide experimental basis and theoretical basis for bletilla industrialized production and the synthesis of chemical component high-performance bio.
Description
Technical field
The present invention relates to Chinese medicine drug research technical fields, and in particular to a method of suspend culture bletilla cell.
Background technique
Bletilla (Bletilla striata (Thunb.) Rchb.f.) orchid family bletilla category herbaceos perennial, is China
Traditional rare Chinese herbal medicine, and it is widely used in the industries such as industry and flowers greening, it is good as natural viscose glue and beauty
Product have good development and application prospect, and purposes is still constantly being expanded.Its pseudobulb has hemostasis convergence, detumescence life
The effect of flesh, the diseases such as interior external hemorrhage, tumour, ulcer are cured mainly, external application can control burn and scald, rhagadia manus et pedis, promote wound healing.It is modern
Pharmacological research is, it was also found that bletilla has dextran, marrow hemopoiesis, anti-aging, increases the health-care effects such as immunity of organisms.
With the serious destruction of the market demand sharply increased with the wild habitat of bletilla, traditional bletilla production model is remote
Far from the continuous rapidly growth expanded with the market demand for meeting its purposes.And plant cell suspension culture technology passes through completely
Artificially controllably meet cell Growth and Differentiation optimum condition, efficiently promotes cell Proliferation, produced currently with culture plant cell
The most promising biosynthesis mode of secondary metabolites has great importance in the medicinal ingredient synthesis of medicinal plant.State
It is inside and outside that the cell suspension cultures technology including a variety of medicinal plants such as Chinese yew, ginseng, catharanthus roseus has had been established, but about white
And the research of cell suspension cultures is but rarely reported, and its easy browning death in tissue culture procedures also increases suspension culture
Produce the difficulty of bletilla.
Summary of the invention
The invention is intended to provide a kind of method of culture bletilla cell that suspends, mainly trained with solving existing bletilla with solid
It supports, multiplication rate is lower, is easy browning, and cultivation cycle is long, and the technical problem that can not achieve large-scale production etc., (with proliferation
Rate is evaluated, i.e. proliferation rate=(aim parameter-primary quantity)/primary quantity x100%).
The method of one of this programme suspension culture bletilla cell, comprising the following steps:
Step 1: mature bletilla capsule is taken to carry out Aseptic sterilisation, and obtain bletilla seed；
Step 2: induction bletilla seed is sprouted and obtains callus: bletilla seed being put in sterile induced medium
It is cultivated under dark condition, cultivation temperature is 25 ± 2 DEG C, and incubation time is 60~90 days；Induced medium are as follows: MS+6- benzyl ammonia
Base purine 1.5 ± 0.5mg/L+2,4- dichlorphenoxyacetic acid 2.5 ± 0.5mg/L+30 ± 20g/L sucrose+7 ± 1g/L agar, lures
The pH for leading culture medium is 5.9 ± 0.1；
Step 3: inoculation: the callus that step 2 culture obtains is inoculated into subculture medium under dark condition
Culture, subculture medium are as follows: 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.5mg/L+2,4- dichlorphenoxyacetic acid
± 20g/L sucrose+7 ± 1g/L agar, the pH of subculture medium are 5.9 ± 0.1；The temperature of inoculated and cultured is 25 ± 2 DEG C, the time
It is to obtain explant after 25~45 days；
Step 4: the culture that suspends: the explant that step 3 is obtained, which is inoculated into suspension medium, carries out suspension culture, hangs
Floating culture medium is 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.45mg/L+2,4- dichlorphenoxyacetic acid ± 20g/L
Sucrose；The pH of suspension medium is 5.9 ± 0.1；The inoculum concentration of explant and the volume ratio of culture medium are 1/80~1/20g/
mL；Oscillation suspension culture is carried out after inoculation under the dark condition that revolving speed is 110 ± 10r/min, temperature is 25 ± 2 DEG C, is suspended
Incubation time is 40~50 days.
The advantageous effects of this programme are: method of this programme using cell tissue suspension culture, the progress of bletilla seed
Fiber differentiation obtains the callus of bletilla, then callus is carried out inoculation squamous subculture, obtains explant, finally using white
And explant carry out suspension culture, current bletilla tissue cultures can be overcome mainly with solid culture, multiplication rate is lower, hold
Easy browning, cultivation cycle is long, and the technical problem that can not achieve large-scale production etc..
Using the above method, experiment is established in the high-performance bio synthesis for the industrialized production and chemical component of bletilla biomass
Foundation and theoretical basis；Meanwhile this method provides ideal condition, method letter for the genetic manipulation of people on a cellular level
Just, cultivation cycle is short, can go out expected mutant in cell-based screening in the reproducible, short time, can be used directly to carry out
The separation of protoplast, culture with hybridize, the production of gene transfer and secondary metabolite the advantages that.
Further, in step 1, the aseptic sterilisation methods of bletilla capsule are as follows: first by capsule application alcohol disinfecting, then without
Bacterium water rinse, is subsequently carried out disinfection with mercuric chloride solution, is finally used sterile water rinse for several times again, is blotted capsule with sterile blotting paper
Fruit surface moisture.Further, suspend culture 45 days in step 4.
Detailed description of the invention
Fig. 1 is the flow chart that suspend culture bletilla cell growing method and cell growth curve building are established in embodiment；
Fig. 2 is suspended culture cell growth curve chart；
Fig. 3 is that the non-linear growth model of Logistic is fitted spy to the growth kinetics that suspended culture cell dry and wet changes again
Sign figure.
Specific embodiment
Below by the further details of explanation of specific embodiment:
A method of suspend culture bletilla cell, comprising the following steps:
Step 1: mature bletilla capsule is taken to carry out Aseptic sterilisation, and obtain bletilla seed；Sterilization method is first by capsule
Using alcohol disinfecting, then sterile water rinse is subsequently carried out disinfection with mercuric chloride solution, finally uses sterile water rinse 4 or 5 again
It is secondary, capsule surface moisture is blotted with sterile blotting paper；
Step 2: induction bletilla seed is sprouted and obtains callus: bletilla seed being put in sterile induced medium
It is cultivated under dark condition, cultivation temperature is 25 ± 2 DEG C, and preferably 25 DEG C, incubation time is 60~90 days, is preferably cultivated 70 days；
Induced medium are as follows: 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.5mg/L+2,4- dichlorphenoxyacetic acid ±
20g/L sucrose+7 ± 1g/L agar, the pH of induced medium are 5.9 ± 0.1, and preferably pH is 5.8；
Step 3: inoculation: the callus that step 2 culture obtains is inoculated into subculture medium under dark condition
Culture, subculture medium are as follows: 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.5mg/L+2,4- dichlorphenoxyacetic acid
± 20g/L sucrose+7 ± 1g/L agar, the pH of subculture medium are 5.9 ± 0.1, and preferably pH is 5.8；The temperature of inoculated and cultured is
25 ± 2 DEG C, preferably 25 DEG C, time are to obtain explant after 25~45 days, are preferably cultivated 45 days；
Step 4: the culture that suspends: quality is loose in selection step 3, the explant of strong and vigorous growth, bright yellow is inoculated into
Suspension culture is carried out in suspension medium, suspension medium is MS+6- benayl aminopurine 1.5 ± 0.45mg/L+2,4- dichloro-benzenes
2.5 ± 0.5mg/L+30 of fluoroacetic acid ± 20g/L sucrose；The pH of suspension medium is 5.9 ± 0.1, and preferably pH is 5.8；Explant
Inoculum concentration and culture medium volume ratio be 1/80~1/20g/mL；After inoculation revolving speed be 110 ± 10r/min, preferably
Oscillation suspension culture is carried out under 120r/min, the dark condition that temperature is 25 ± 2 DEG C, suspension incubation time is 40~50 days；It is excellent
Selecting temperature is 25 DEG C, and suspension incubation time is 45 days.
Test data sheet:
From starting to suspend and cultivating the same day, dry, weight in wet base measurement is carried out within every 3 days once, 45 days are a cycle, three groups of measurement
Data compare.The container that shaken cultivation is equipped with suspension cell liquid on shaking table is removed when measurement, whole culture solutions are gone to
In bottle,suction, filters when no culture solution drips, filter residue is weighed as weight in wet base；And it is placed in 50 DEG C of baking oven and dries
To constant weight, weighing is dry weight.Each data collection point takes 3 samples to measure, and each experimental data is measured 3 times and is averaged
Value.Record is done respectively, weight in wet base changes, and using incubation time as abscissa, dry, weight in wet base is the growth curve that ordinate draws cell.
According to suspension cell, dry, weight in wet base measurement data, non-using tri- kinds of Logistic, Boltzmann and DoseResp
Linear growth model is fitted and analyzes to growth curve, to assess the bletilla cell growth status for the culture that suspends.
Measurement data is shown in Table 1.
Table 1: the growth curve Model fitting result of suspended culture cell weight in wet base and dry weight
The available following knot from the overall process of the disinfection of bletilla capsule, the suspension culture of bletilla cell and growth curve
Fruit:
Fig. 2 is suspended culture cell growth curve chart, it can be seen that (dry cell weight and cell are fresh for 2 kinds of different growth indexes
Growth curve again) is almost the same, is in " S " type.The non-linear growth model of Logistic becomes suspended culture cell dry and wet again
After the growth kinetics fitting of change, Fig. 3 shows that the entire cultivation cycle of suspended culture cell can be divided into 6 periods, and the lag period refers to
Number phase, linear phase, deceleration period, resting stage and decline phase.Wherein, 0-6 days are the lag period, and suspended culture cell is dry during this period
Weight in wet base variation is slower, this is the process that a cell gradually adapts to environment；For exponential phase, cell is raw during this period within 6-12 days
Long speed is gradually accelerated, and cell proliferation rate reaches maximum；12-24 days be the linear phase, during this period callus proliferation rate by
Gradually stable, dry and wet weight variable quantity is linearly related to the time；24-36 days are deceleration period, and cell rate of rise is gradually during this period
Slow down；At 36 days, the dry and wet of cell weighed to maximum value；36-39 days be resting stage, cultivate cell dry and wet weight no longer occur it is bright
Aobvious variation.The dry weight and weight in wet base of cell start to be in be gradually reduced trend after 39 days.
Table 1 is the growth curve Model fitting result of suspended culture cell weight in wet base and dry weight, it is seen that the culture that suspends is thin
The weight in wet base of born of the same parents and Logistic, Boltzmann and DoseResp fit equation coefficient of determination of cultivated days be respectively 0.9817,
0.9231,0.9780；The coefficient of determination of suspended culture cell dry weight and 3 kinds of fit equations of cultivated days is respectively 0.9761,
0.8878,0.9453.By in fitting result it is found that more particularly suitable with the growth of Logistic model suspension cell biomass.Through
Further significance test is crossed, the F value (721.56294 and 613.94951) of Logistic equation is also above Boltzmann
Equation and DoseResp equation illustrate that Logistic equation curve and test data are more close.It cultivates after inoculation thin when 7d
The acceleration that born of the same parents increase reaches maximum, and in 13-14d, the growth rate of cell reaches maximum, and in 36d, the increment of cell reaches
It is maximum.
Culture medium preferred embodiment (using proliferation rate as evaluation index, i.e., (target fresh weight-starting fresh weight)/starting fresh weight ×
100%), pH value 5.8,25 DEG C of temperature, dark condition culture.It can promote the growth of cell by adding elicitor, it is related
Culture medium prescription is as follows:
D1:MS+1.0mg/L6-BA+3.0mg/L2,4-D+30g/L sucrose+7g/L agar；
D2:MS+1.0mg/L6-BA+3.0mg/L2,4-D+30g/L sucrose+7g/L agar+5g/L yeast extract；
D3:MS+1.0mg/L6-BA+3.0mg/L2,4-D+30g/L sucrose+7g/L agar+10mg/L salicylic acid；
D4:MS+1.0mg/L6-BA+3.0mg/L2,4-D+30g/L sucrose+7g/L agar (molysite be D1 10 times)；
Experimental result: the proliferation rate of D1 is 99.50%；The proliferation rate of D2 is 94.43%；The proliferation rate of D3 is 73.95%；
The proliferation rate of D4 is 70.21%.So D1 is that the present invention program is preferably formulated.
Preferred (using proliferation rate as evaluation index, the proliferation rate algorithm is same as above) pH value of condition of culture is 5.8, revolving speed 120r/
Min, 25 DEG C of temperature, dark condition culture.
1) with sucrose concentration (10g/L, 30g/L, 15g/L, 70g/L) be independent variable, MS+1.0mg/L6-BA+3.0mg/
L2,4-D is minimal medium.
Experimental result: the proliferation rate of 10g/L is 96.71%；The proliferation rate of 30g/L is 163.82%；The proliferation rate of 50g/L
It is 98.66%；The proliferation rate of 70g/L is 68.59%.
2) with culture volume (20mL, 35mL, 50mL, 65mL) be independent variable, MS+1.0mg/L6-BA+3.0mg/L2,
4-D+30g/L sucrose is minimal medium.
Experimental result: the proliferation rate of 20mL is 90.02%；The proliferation rate of 35mL is 132.00%；The proliferation rate of 50mL is
95.78%；The proliferation rate of 65mL is 118.17%.
3) with additive amount (0.5g/40mL, 1.0g/40mL, 1.5g/40mL, 2.0g/40mL) for independent variable, MS+1.0mg/
L6-BA+3.0mg/L2,4-D+30g/L sucrose is minimal medium.
Experimental result: the proliferation rate of 0.5g/40mL is 167.56%；1.0g/40mL proliferation rate be 139.48%；
1.5g/40mL proliferation rate be 178.96%；2.0g/40mL proliferation rate be 112.22%.
Claims (3)
1. a kind of method for the culture bletilla cell that suspends, which comprises the following steps:
Step 1: mature bletilla capsule is taken to carry out Aseptic sterilisation, and obtain bletilla seed；
Step 2: induction bletilla seed is sprouted and obtains callus: bletilla seed being put in sterile induced medium black
It is cultivated under dark condition, cultivation temperature is 25 ± 2 DEG C, and incubation time is 60~90 days；Induced medium are as follows: MS+6- benzyl amino is fast
Purine 1.5 ± 0.5mg/L+2,4- dichlorphenoxyacetic acid 2.5 ± 0.5mg/L+30 ± 20g/L sucrose+7 ± 1g/L agar, induction training
The pH for supporting base is 5.9 ± 0.1；
Step 3: inoculation: the callus that step 2 culture obtains is inoculated into subculture medium and is cultivated under dark condition,
Subculture medium are as follows: 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.5mg/L+2,4- dichlorphenoxyacetic acid ±
20g/L sucrose+7 ± 1g/L agar, the pH of subculture medium are 5.9 ± 0.1；The temperature of inoculated and cultured is 25 ± 2 DEG C, and the time is
Explant is obtained after 25~45 days；
Step 4: the culture that suspends: the explant that step 3 is obtained, which is inoculated into suspension medium, carries out suspension culture, and suspend training
Supporting base is 2.5 ± 0.5mg/L+30 of MS+6- benayl aminopurine 1.5 ± 0.45mg/L+2,4- dichlorphenoxyacetic acid ± 20g/L sugarcane
Sugar；The pH of suspension medium is 5.9 ± 0.1；The inoculum concentration of explant and the volume ratio of culture medium are 1/80~1/20g/mL；
Oscillation suspension culture is carried out after inoculation under the dark condition that revolving speed is 110 ± 10r/min, temperature is 25 ± 2 DEG C, suspend culture
Time is 40~50 days.
2. the method for the culture bletilla cell according to claim 1 that suspends, it is characterised in that: in step 1, bletilla capsule
Aseptic sterilisation methods are as follows: first by capsule application alcohol disinfecting, then sterile water rinse is subsequently disappeared with mercuric chloride solution
Poison finally uses sterile water rinse for several times again, blots capsule surface moisture with sterile blotting paper.
3. the method for the culture bletilla cell according to claim 2 that suspends, it is characterised in that: suspend culture 45 in step 4
It.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109874678A (en) * | 2019-04-18 | 2019-06-14 | 遵义医科大学 | A kind of method for inducing and cultivating of bletilla callus |
| CN110283775A (en) * | 2019-07-12 | 2019-09-27 | 遵义医科大学 | Secondary metabolite synthetic method in a kind of promotion bletilla suspended culture cell |
| CN111480572A (en) * | 2019-12-18 | 2020-08-04 | 长江大学 | Method for screening rice tissue culture material |
-
2018
- 2018-12-05 CN CN201811481695.4A patent/CN109496860B/en active Active
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109874678A (en) * | 2019-04-18 | 2019-06-14 | 遵义医科大学 | A kind of method for inducing and cultivating of bletilla callus |
| CN110283775A (en) * | 2019-07-12 | 2019-09-27 | 遵义医科大学 | Secondary metabolite synthetic method in a kind of promotion bletilla suspended culture cell |
| CN111480572A (en) * | 2019-12-18 | 2020-08-04 | 长江大学 | Method for screening rice tissue culture material |
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