CN104719168B - Method for cultivating bletilla striata seedlings by using intermittent immersion bioreactor - Google Patents
Method for cultivating bletilla striata seedlings by using intermittent immersion bioreactor Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicinal materials, particularly relates to a method for cultivating bletilla striata seedlings by using an intermittent immersion bioreactor, and belongs to the technical field of medicinal plant cultivation; the method for culturing bletilla striata seedlings by using the intermittent immersion bioreactor sequentially comprises the steps of seed sterilization, seed germination, proliferation culture, pseudobulb generation culture and pseudobulb expansion culture, the method for rapidly culturing the bletilla striata seedlings by using the intermittent immersion bioreactor is carried out under the aseptic condition, and the proliferation culture comprises transitional proliferation culture and proliferation and rooting culture. According to the technical scheme, the proliferation culture process is divided into two processes of transitional proliferation culture and proliferation and rooting culture, and the transitional proliferation culture eliminates or shortens a long adaptation period phenomenon from solid culture to intermittent submerged bioreactor culture in a shaking mode, so that a large amount of bletilla striata seedlings are rapidly obtained, and then the pseudobulb seedlings are cultured in a swelling mode to obtain the method for producing the pseudobulb seedlings.
Description
Technical field
The invention belongs to medicinal plant Cultivating techniques field, and in particular to one kind is cultivated using interval submergence bioreactor
The method of bletilla striata seedling.
Background technology
The bletilla striata（Bletilla striata（Thunb.）Reichb.f.）The herbaceos perennial belonged to for the orchid family bletilla striata,
Also cry " bletilla ", even and grass etc..Its dry tuber has astringing to arrest bleeding, detumescence and promoting granulation and other effects, is usually used in treating hemoptysis, tells
Blood, chapped skin etc..There are some researches show the bletilla striata has antibacterial and antineoplastic action.The bletilla striata is applied not only to tcm clinical practice,
It is widely used in daily chemical products, such as manufacture of cosmetics, advanced ceramic, weaving, rubber production.
With the continuous expansion that the bletilla striata is applied, its demand is continuously increased.Wild bletilla striata quantity is caused drastically to reduce, and
And the seed of the bletilla striata is small, no endosperm provides nutrient needed for germination, in its natural state few Germination And Seedling.Traditional cultivation
Method relies primarily on division propagation, and the division propagation cycle is long, reproductive efficiency is low, consumption kind amount is big, it is difficult to meet the needs of cultivation.
Since Kundson carries out the axenic germination of Orchid Seeds first, axenic germination turn into orchid preserving seed and
The important means of large-scaled propugation.Pod is planted as numerous material is expanded by the use of excellent bletilla striata kind by tissue-culturing rapid propagation, can be obtained
The homogeneous brood body of the important character such as hereditary basis and exterior quality shape, and can solve traditional division propagation method institute
Breeding cycle length, low reproduction rate and the consumption kind brought measure the problems such as big, significantly reduce the cost of plantation.It is but traditional
The bletilla tissue culture seedlings that method for tissue culture obtains are thin and delicate short, tissue-cultured seedling be applied to need during Production of Large Fields strict acclimation conditions and
Management operation.It is larger to being injured when adhering to the cleaning of agar on tissue-cultured seedling to it when carrying out acclimatization and transplantses, therefore hardening survives
Rate is relatively low, and just bletilla striata pseudobulb can be made to grow sprouting after a period of time is grown, and is unfavorable for the growth of bletilla tissue culture seedlings.
In recent years, in order to meet the needs of market, the high-quality bletilla striata is cultivated, people employ a variety of methods and cultivate the bletilla striatas,
Wherein much also apply for patent, such as：Application for a patent for invention（201310579744.9 201410364717.4,
201210350910.3）The culturing and reproducing of the bletilla striata is disclosed, it is main using the method for being solid-based culture nursery, although
Successfully solve the problems, such as survival rate, but the automation of seeling industry is carried out using conventional solid culture medium method for tissue culture
Horizontal low and cost remains high, wherein a very big part is labour and energy consumption cost.
Interval submergence bioreactor culture is mainly to carry out interval leaching to the histoorgan of plant using fluid nutrient medium
Do not cultivate, supply nutrition during submergence, provide enough oxygen during interval.Because the training method preferably solves Liquid Culture
When plant tissue Vitrification Occurred, and nutriment can obtain good transmission with the submergence to plant tissue, because
This interval submergence culture can be very good the various problems for solving in Plant Tissue Breeding；And intermittent immersion cultivation mode can be with
The gentle plant generation efficiency of Automated water is greatly improved, and reduces the consumption of man power and material, is substitute tradition culture one
The effective method of kind.
The research that other asexually propagated plant tissue cultures are successfully carried out using interval submergence cultivation reactor is had been reported.
Such as Chinese patent（201310043181.1）Disclose numerous using interval submergence bioreactor progress roxburgh anoectochilus terminal bud tissue cultures expansion
Method, it is described using tissue cultures expansion of the interval submergence bioreactor to roxburgh anoectochilus terminal bud is numerous, and this method has the cycle
Short, the features such as tissue-cultured seedling quality is high, a kind of inexpensive, efficient side is provided for the tissue-culturing quick-propagation of roxburgh anoectochilus terminal bud
Method.But intermittent immersion cultivation mode be used for plant culture just as method for plant tissue culture to different Plant Tissue Breeding
Equally, a kind of training method is applied to need different condition and processing mode during different plants.The training being related to during culture
Foster method is such as：Selection, culture medium composition, Immersion frequency, inoculum density, incubation time, external environment and the different materials of material
The various aspects such as the processing mode of material can not be obtained by simple limited experiment reasoning；And the patent is not considered
Plant is directly transferred to Liquid Culture from solid culture needs the laundering period of long-term, so as to increase the life of roxburgh anoectochilus terminal bud tissue-cultured seedling
Long period；At present on utilizing intermittent immersed cultural method to carry out bletilla striata tissue cultures, it is contemplated that plant is in tissue cultures
During the laundering period phenomenon of long-term that occurs when being transferred directly to Liquid Culture by solid culture, and give processing side
Method, while having not been reported and openly applying on the processing method.Cultivated simultaneously using liquid batch submergence training method
To injury caused by seedling when the method for bletilla striata seedling is solved as being cleaned after solid-based culture；And by promoting pseudobulb
Expand and obtain the survival rate that the bletilla striata seedling with larger pseudobulb substantially increases kind of transplantation of seedlings.
In view of have no report using the production of intermittent immersed cultural method progress bletilla striata seedling and openly apply, therefore,
It is necessary to work out a kind of method for cultivating bletilla striata seedling using interval submergence bioreactor.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of growth cycle is short, transplanting survival rate is high, cost is low, operation is simple
Single automation size and the culture that can solve the long laundering period phenomenon occurred when being transferred directly to Liquid Culture by solid culture
The method of bletilla striata seedling.
In order to solve the above technical problems, the technical solution adopted by the present invention is：This utilizes interval submergence bioreactor training
The method of bletilla striata seedling is educated, successively including seed sterilisation step, seed sprouts step, Multiplying culture step, pseudobulb generation training
Support step and pseudobulb expands incubation step, the method that bioreactor fast culture bletilla striata seedling is submerged using interval is in nothing
Carried out under the conditions of bacterium, the Multiplying culture step includes transition Multiplying culture step and propagation and culture of rootage step.
Pass through above-mentioned technical proposal, Multiplying culture process is divided into transition Multiplying culture step and propagation and walked with culture of rootage
Rapid two processes, transition Multiplying culture step are carried out using shaking flask mode, so as to transition Multiplying culture eliminate or shorten by
Solid culture is directly transferred to the long laundering period phenomenon that interval submergence bioreactor culture occurs, a large amount of so as to quickly obtain
For producing bletilla striata seedling, the culture then expanded by pseudobulb obtains the bletilla striata seedling with larger pseudobulb, greatly
Add the survival rate of kind of transplantation of seedlings, and using liquid batch submergence training method cultivate bletilla striata seedling method solve by
To injury caused by seedling when being cleaned after solid-based culture.
Further, an early-stage preparations step, the early-stage preparations step bag are also needed to before the seed sterilisation step
Include：1. select kind of a pod：Select full breeding；2. reactor and transparent utensil sterilizing：Reactor and transparent utensil are wrapped up, it is high
It is standby after warm high pressure moist heat sterilization；3. prepare culture medium and nutrient solution；
The seed sterilisation step is aseptically, to carry out surface disinfection to bletilla seed pod, obtain aseptic seed；
It is uniformly to scatter aseptic seed to carry out seed sprouting culture in solid medium that the seed, which sprouts step,；
The transition Multiplying culture step is aseptically, the bletilla striata seedling after sprouting to be transferred into transparent utensil
In, transition Multiplying culture is carried out by shaking flask mode using transition proliferated culture medium at a constant temperature；
The propagation and culture of rootage step are aseptically, by the bletilla striata transition propagation seedling switching in transparent utensil
Into sterile interval submergence bioreactor, at a constant temperature using propagation and culture of rootage liquid is bred and culture of rootage；
After pseudobulb generation incubation step is propagation and culture of rootage terminates, cultivation reactor is submerged in same interval
In, replacing nutrient solution will breed and culture of rootage fluid exchange is that miniature pseudobulb generates nutrient solution, be cultivated at a constant temperature
Acquisition carries miniature pseudobulb bletilla striata seedling；
The pseudobulb expands incubation step, in same interval submerges culture bioreactors,
It is that pseudobulb generation nutrient solution is replaced with to the nutrient solution for promoting pseudobulb to expand to change nutrient solution, carries out pseudobulb at a constant temperature
Culture is expanded, obtains to carry and expands pseudobulb bletilla striata seedling.
It is that the seed sterilisation step comprises the following steps as further improve of the invention：
1. surface decontamination：Bletilla striata seeds are rinsed using running water current, washing time is 0 ~ 60min, washes kind of a pod table
Face it is dirty；
2. surface sterilization：Aseptically, it is 10 ~ 60s sterilized waters to be put in soak time in 70 ~ 75% alcohol
Rinse 2 ~ 3 times；
3. surface disinfection：It is that soak time is 1 ~ 20min in 0.1 ~ 0.3% mercury chloride to be put into concentration, aseptic water washing 5 ~ 7
It is secondary；Obtain sterile bletilla seed pod.
It is that the seed is sprouted in step, and the formula of the solid medium is as further improve of the invention：
1/2 MS liquid is used as basic culture solution, addition hormone 0 ~ 2.0mg/L of NAA, 10 ~ 50g/L of mashed potatoes, sucrose concentration 10 ~
50g/L, 1.0 ~ 7.0g/L of agar concentration, adjust pH value 5.0 ~ 7.0；The condition of culture set as：5 ~ 30d of dark culturing, then
It is transferred under illumination condition and cultivates, incubation time is 20 ~ 90d, 1500 ~ 2000lx of intensity of illumination, 1 ~ 18h/d of light application time, temperature
For 24 ± 1 DEG C.
It is as further improve of the invention, in the transition Multiplying culture step, what the transparent utensil used
Material is glass or plastics, and the transparent utensil is conical flask or gas bottle or wide-mouth bottle, and selected capacity is 250ml；
Incubation time is 7 ~ 20d；Used transition proliferated culture medium is liquid or semisolid, and the formula of transition proliferated culture medium is：
1/2 MS liquid is used as basic culture solution, adds hormone 0 ~ 2.0mg/L of NAA, potato 10 ~ 60g/L of liquid, 10 ~ 50g/L of sucrose,
0 ~ 5g/L of agar, adjust pH value 5.0 ~ 7.0；The condition of culture set as：The transparent utensil is placed on shaking table, rotating speed is set
For 0 ~ 120rpm, 1500 ~ 2000lx of intensity of illumination, 1 ~ 18h/d of light application time, 24 ± 1 DEG C of temperature.
It is as further improve of the invention, in the propagation and culture of rootage step, the interval submergence culture
The reaction condition of reactor is：Interval 1 ~ 10min/4h of Immersion frequency, 10 ~ 50g/L of inoculum density, 30 ~ 90d of incubation time；Institute
State propagation and the formula of culture of rootage liquid is：1/2 MS liquid is used as basic culture solution, adds hormone 0 ~ 2.0mg/L of NAA,
Potato 20 ~ 90g/L of liquid, 10 ~ 50g/L of sucrose, adjust pH value 5.0 ~ 7.0；The condition of culture set as intensity of illumination 1500 ~
2000lx, 1 ~ 18h/d of light application time, 24 ± 1 DEG C of temperature.
It is as further improve of the invention, in the pseudobulb generation incubation step, the pseudobulb generation training
The formula of nutrient solution is：1/2 MS liquid is used as basic culture solution, adds hormone 0 ~ 2.0mg/L of NAA, potato 20 ~ 90g/L of liquid,
10 ~ 90g/L of bananas juice, 10 ~ 50g/L of sucrose, adjust pH value 5.0 ~ 7.0；The condition of culture set as intensity of illumination 1500 ~
2000lx, 1 ~ 18h/d of light application time, 24 ± 1 DEG C of temperature；The interval Immersion frequency of the interval submergence cultivation reactor is arranged to
1 ~ 10min/4h, 10 ~ 30d of incubation time.
It is that the pseudobulb is expanded in incubation step as further improve of the invention, the promotion pseudobulb is swollen
The formula of big nutrient solution is：MS liquid is used as basic culture solution, adds hormone 0 ~ 2.0mg/L of NAA, potato 20 ~ 90g/L of liquid,
10 ~ 80g/L of bananas juice, 20 ~ 120g/L of straw juice, 10 ~ 50g/L of sucrose, adjust pH value 5.0 ~ 7.0；The condition of culture set as
1500 ~ 2000lx of intensity of illumination, 8 ~ 18h/d of light application time, 24 ± 1 DEG C of temperature；The interval of the interval submergence cultivation reactor
Immersion frequency is arranged to 1 ~ 10min/6h, incubation time 20 ~ 90 days.
By it is substantial amounts of it is experimentally confirmed that be transferred to after bletilla striata seeds are sprouted in transparent utensil carry out Shaking culture and by
The switch over operation process of Shaking culture to reactor is all more convenient, greatly reduces the pollution rate in operating process；And liquid
Body shaking flask transition culture eliminates the long laundering period phenomenon directly occurred by solid culture to interval submergence bioreactor culture,
So as to shorten the cultivation period of bletilla striata seedling；Mainly connected simultaneously using interval submergence bioreactor culture by nutrient solution
Continuous or interval is submerged to maintain a kind of training method of plant normal growth；Supply nutrition during submergence, provide during interval enough
Oxygen；The Vitrification Occurred of plant tissue when the training method preferably solves Liquid Culture, and nutriment is with right
The submergence of plant tissue can obtain good transmission, so as to be greatly enhanced the gentle plant generation efficiency of Automated water；In addition,
The incubation step that expands for promoting pseudobulb is that open culture is carried out in bioreactor, and this process can be equivalent to tissue-cultured seedling
Hardening treatment is carried out, tissue-cultured seedling is more healthy and stronger obtained from.Due to have passed through the step of pseudobulb expands culture, obtain
New root and tender shoots can be grown after tissue culture transplantation of seedlings in short time.
Using conventional solid culture medium as control, the advantage that bletilla striata seedling is cultivated using gap submergence bioreactor is as follows
Table：
| Training method | Proliferation rate | Required incubation time（d） | Survival rate（%） |
| This method | 1:40 | 50 | 95 |
| Interval submergence bioreactor | 1:20 | 80 | 60 |
| Conventional solid culture medium | 1:6 | 120 | 50 |
This method and the difference of conventional cultural method essentially consist in：1st, increase transition culture shortens the culture laundering period；2、
Increase pseudobulb expands step, and seedling stalwartness survival rate is high, and needs not move through hardening and shorten cultivation cycle, reduces labour.
As the preferred scheme of the present invention, the step of seed sterilization is 1. in surface decontamination during deionized water rinsing
Between be 30 ~ 40min；For the step 2. in surface sterilization, the soak time is 30s；The step is described 3. in surface disinfection
Soak time is 5 ~ 20min；
The seed is sprouted in step, and the formula of the solid medium is：1/2 MS liquid is used as basic culture solution,
Hormone 0.5 ~ 1.0mg/L of NAA, 20 ~ 30g/L of mashed potatoes, 30 ~ 40g/L of sucrose concentration, 5.0 ~ 6.5g/L of agar concentration are added, is adjusted
Whole pH value 5.8 ~ 6.0；The condition of culture set as：7 ~ 15d of dark culturing, then it is transferred under illumination condition and cultivates, incubation time
For 30 ~ 50d, 10 ~ 12h/d of light application time；
In the transition Multiplying culture step, the incubation time is 10 ~ 15d；The formula of the culture medium is：Using 1/
2 MS liquid are basic culture solution, add hormone 0.5 ~ 1.0mg/L of NAA, potato 30 ~ 50g/L of liquid, 30 ~ 40g/L of sucrose, agar
0 ~ 3g/L, adjust pH value 5.8 ~ 6.0；The rotating speed of shaking table is 60 ~ 90rpm, and 10 ~ 12h/d of light application time, incubation time is 10 ~ 15
d；
In the propagation and culture of rootage step, the interval Immersion frequency 1 ~ 3min/4h, 20 ~ 30g/L of inoculum density；
40 ~ 60d of incubation time；The propagation and the formula of culture of rootage liquid are：1/2 MS liquid is used as basic culture solution, addition swashs
Plain 0.5 ~ 1.0mg/L of NAA, potato 30 ~ 50g/L of liquid, sucrose 30g ~ 40/L, adjust pH value 5.8 ~ 6.0,10 ~ 12h/ of light application time
d；
In the pseudobulb generation incubation step, the formula of the pseudobulb generation nutrient solution is：Using 1/2 MS liquid
For basic culture solution, hormone 0.5 ~ 1.0mg/L of NAA are added, potato 30 ~ 50g/L of liquid, 30 ~ 60g/L of bananas juice, sucrose 30g ~
40/L, adjust pH value 5.8 ~ 6.0,10 ~ 12h/d of light application time；The interval Immersion frequency is arranged to 1 ~ 3min/4h；
The pseudobulb is expanded in incubation step, and the formula for promoting pseudobulb to expand nutrient solution is：Using MS liquid
For basic culture solution, hormone 0.5 ~ 1.0mg/L of NAA are added, potato 30 ~ 50g/L of liquid, 30 ~ 50g/L of bananas juice, straw juice 30 ~
60g/L, 30 ~ 50g/L of sucrose, adjust pH value 5.8 ~ 6.0；The condition of culture set is 1500 ~ 2000lx of intensity of illumination, during illumination
Between 12 ~ 14h/d；The interval Immersion frequency of interval submergence cultivation reactor is arranged to 1 ~ 3min/6h, and incubation time is 30 ~
60 d。
Using the technical scheme of present invention production bletilla striata seedling, its caused beneficial effect is significantly to have the technical effect that：
（1）Proliferation rate is high, and biological yield is big, and cultivation cycle is short.Interval submergence cultivation reactor culture is trained using liquid
Support the culture that interval submergence is carried out to plant tissue organ.The proliferation rate of the bletilla striata is trained far above conventional solid base under this mode
Support.The proliferation rate of the bletilla striata obtained using the inventive method can reach 1:40, far above conventional solid base culture 1:6 or so,
And submerge bioreactor culture 1 higher than interval is directly transferred to by solid culture:20 effect.Higher proliferation rate is also in short-term
In obtain a number of tissue-cultured seedling and provide the foundation, so as to reduce subculture number, reduce tissue-cultured seedling variation frequency.Utilize this
The mode of inventive method can obtain the effect of 40 times of propagation by the culture of 50 days or so, be directly transferred to by solid culture in the past
Cultivated in interval submergence bioreactor, need to obtain the effect for breeding 20 times by 80 days or so cultivate one's ability, and it is traditional
Solid culture mode can only obtain 6 times of cultivation effect by the culture of 100 days.
（2）Tissue-cultured seedling quality is good, and seedling is healthy and strong, and cultivation survival rate is high.Supply nutrition during submergence, provide during interval enough
Oxygen；The Vitrification Occurred of plant tissue when the training method preferably solves Liquid Culture, and nutriment is with right
The submergence of plant tissue can obtain good transmission, so as to be greatly enhanced the gentle plant generation efficiency of Automated water；Promote
The incubation step that expands of pseudobulb is that open culture is carried out in bioreactor, and this process can be carried out equivalent to tissue-cultured seedling
Hardening treatment, and equivalent to tissue-cultured seedling hardening has been carried out, tissue-cultured seedling is more healthy and stronger obtained from.Due to have passed through false squama
The step of stem expands, can grow new root and tender shoots in the short time after obtained bletilla striata transplantation of seedlings, and survival rate 95% with
On；And conventional solid medium culture and the culture of bioreactor culture mode but expanded using interval submergence without pseudobulb
The tissue-cultured seedling that incubation obtains needs all to carry out hardening, and transplantation of seedlings process needs to expend the behaviour of a large amount of manpowers and high standard
Make, while transplanting survival rate is only 50% ~ 60%.
（3）Automatization level is high, saves a large amount of labour's consumption.Bletilla seed is cultivated using gap submergence bioreactor
Seedling, the dosage of blake bottle can be not only reduced, and then reduce the manpower consumption of filling washing etc.；Bletilla striata seeds are through sprouting simultaneously
It is transferred to after hair and Shaking culture is carried out in conical flask and is facilitated by the operating process of the switching of Shaking culture to reactor
Simply, the pollution rate in operating process is reduced；And shaking flask transition Multiplying culture is eliminated by solid culture to interval submergence life
The long laundering period phenomenon occurred in thing bioreactor culture, so as to shorten the cultivation period of bletilla striata seedling.Second, in a culture
The seedling of 2000 or so is formed in device, relative to every bottle of output of solid culture bottle seedling of 10 or so, hence it is evident that have pole
The earth efficiency difference.Third, the control of automation can be achieved in the inventive method in incubation, it is not necessary to tissue culture of transferring repeatedly
Seedling, thus expend less manpower and shorten incubation time, so as to save human cost；High proliferation rate, higher kind
Seedling quality and high survival rate also reduce the production cost of seedling.
In summary, the present invention is swollen using shaking flask transition culture, interval submergence bioreactor culture and pseudobulb is passed through
The method cultivated greatly bletilla striata seedling is cultivated have relative to traditional training method expand numerous efficiency high, incubation time it is short,
The long laundering period occurred by solid culture to interval submergence bioreactor culture is shortened in convenient, elimination that seed is transferred after sprouting
Phenomenon；Seedling is healthy and strong, cultivation survival rate is high and cost is low, automaticity is high, saves the advantages that labour consumes, therefore more suitable
Close the production of bletilla striata seedling large-scale cultivation.
Brief description of the drawings
The embodiment of the present invention is described in further details below in conjunction with the accompanying drawings：
Fig. 1 is growing state of the bletilla striata seedling in each cultivation stage cycle；
Wherein：（a）The growth conditions schematic diagram of bletilla striata seedling at the end of cultivating is sprouted for seed；
（b）The growth conditions schematic diagram of bletilla striata seedling at the end of for transition Multiplying culture；
（c）The growth conditions signal of bletilla striata seedling at the end of intermittently to submerge in bioreactor propagation and culture of rootage
Figure；
（d）For intermittently submerge pseudobulb in reactor expand culture at the end of the big logotype of bletilla striata seedling pseudobulb；
Fig. 2 is the schematic diagram of bletilla seed pod.
Embodiment
Embodiment 1：As shown in figure 1,
（1）Early-stage preparations step：Reactor and transparent utensil sterilizing：Reactor and transparent utensil are wrapped up, HTHP
It is standby after moist heat sterilization；Wherein reactor is that gap submerges bioreactor, and transparent utensil is conical flask；
（2）Seed sterilisation step：Aseptic process is carried out to bletilla striata good species pod；
Surface decontamination：Running water current washing time is 30min；Surface sterilization：Then in superclean bench, will clean
The bletilla seed pod crossed is put in 70 ~ 75%（v/v）30 are soaked in alcohol（s）, aseptic water washing 2 ~ 3 times；Surface disinfection：Place into dense
Spend for 0.1%（g/v）8min, aseptic water washing 5 ~ 7 times are soaked in mercury chloride；Aseptic filter paper draws kind of the moisture on pod surface, uses
Sterile working cuts an osculum from bletilla seed pod one end；
（3）Seed sprouts step：According to formula 1/2MS+NAA0.5mg/L+30g/L mashed potatoess+30g/L sucrose+agar
6.5g/L, adjustment pH value 5.8 prepare nutrient solution；Bletilla striata seeds are uniformly shaken off and cultivated in bletilla striata seeds germination medium,
It is transferred to after first dark culturing 10d under illumination condition and cultivates 30d, 1500 ~ 2000lx of intensity of illumination, light application time 10h/d, temperature 24
±1℃；
（4）Transition Multiplying culture step：According to formula 1/2MS+NAA0.5mg/L+50g/L potato liquid+30g/L sucrose, adjust
Whole pH value 5.8 prepares nutrient solution；By the bletilla striata seedling after sprouting（Protocorm）Transfer in conical flask, conical flask is placed on
On shaking table, shaking speed 60rmp, 1500 ~ 2000lx of intensity of illumination, light application time 12h/d, 24 ± 1 DEG C of temperature；Incubation time
10d；
（5）Propagation and culture of rootage step：According to formula 1/2MS+NAA0.5mg/L+50g/L potato liquid+30g/L sucrose,
Adjust pH value 5.8 and prepare propagation and culture of rootage liquid；The bletilla striata seedling of transition Multiplying culture in conical flask is transferred in advance
In sterilized interval submergence culture bioreactors, interval Immersion frequency 3min/4h, condition of culture be intensity of illumination 1500 ~
2000lx, light application time 12h/d, 24 ± 1 DEG C of temperature；Incubation time 50d；
（6）Pseudobulb generates incubation step：According to formula 1/2MS+NAA0.5mg/L+50g/L potatoes liquid+bananas juice 40g/
Pseudobulb generation nutrient solution is prepared in L+30g/L sucrose, adjustment pH value 5.8pH5.8 ~ 6.0；Bioreactor is submerged in same interval
In, nutrient solution is replaced by pseudobulb generation nutrient solution, interval Immersion frequency 3min/4h 1500 ~ 2000lx of intensity of illumination, illumination
Time 10h/d, 24 ± 1 DEG C of temperature, incubation time are 20 days；
（7）Pseudobulb expands incubation step：According to formula MS+NAA0.5mg/L+50g/L potatoes liquid+bananas juice 50g/L+
Straw juice 50g/L, pH5.8, which is prepared, promotees the nutrient solution that reason pseudobulb expands, and in same interval submerges bioreactor, will cultivate
The nutrient solution that fluid exchange expands for promotion pseudobulb, interval Immersion frequency 3min/6h；1500 ~ 2000lx of intensity of illumination, during illumination
Between 12h/d, 24 ± 1 DEG C of temperature；Incubation time 30d；
In a cultivation cycle, laundering period of the bletilla striata seedling in interval submerges bioreactor shorten to 0 day, so as to
Reactor is set to breed efficiency up to 1:More than 35；Incubation time 60 days, bletilla striata seedling pseudobulb expands 3.5 in a cultivation cycle
More than times, obtained kind shoot survival percent is more than 95%.
Growing state of the bletilla striata seedling in each cultivation stage cycle is shown in Fig. 1.
Embodiment 2：
Bletilla striata seedling is cultivated using interval submergence bioreactor, specific steps are as described in Example 1, different therewith
It is by, using Semi-solid cell culture, selected culture medium prescription is 1/2MS+NAA0.5mg/L+ during transition Multiplying culture
50g/L potato liquid+30g/L sucrose+agar 3g/L, pH5.9 is adjusted, propagation and culture of rootage formula of liquid are 1/2MS+
NAA1.0mg/L+50g/L potato liquid+30g/L sucrose, pH5.9 is adjusted, condition of culture is 1500 ~ 2000lx of intensity of illumination, illumination
Time 12h/d, 24 ± 1 DEG C of temperature.In a cultivation cycle, laundering period of the bletilla striata seedling in interval submerges bioreactor
It shorten to 0 day, so that reactor breeds efficiency up to 1:More than 30.
Embodiment 3：
Using bioreactor cultivate bletilla striata seedling, specific steps as described in Example 1, therewith except that by transition
Using Semi-solid cell culture during Multiplying culture, selected culture medium prescription is 1/2MS+NAA0.5mg/L+50g/L potato liquid
+ 30g/L sucrose+agar 2g/L, pH6.0 is adjusted, it is MS+NAA1.0mg/L+50g/L potatoes liquid+perfume that pseudobulb, which expands nutrient solution,
Any of several broadleaf plants juice 40g/L+ straw juice 60g/L, pH6.0 is adjusted, laundering period of the bletilla striata seedling in interval submerges bioreactor shorten to
0.3 day, bletilla striata seedling pseudobulb expanded 2 ~ 3 times or so in a cultivation cycle, and obtained kind shoot survival percent is more than 90%.
Embodiment 4：
Using bioreactor cultivate bletilla striata seedling, specific steps as described in Example 1, therewith except that pseudobulb
It is MS+NAA1.0mg/L+50g/L potatoes liquid+bananas juice 50g/L+ straw juice 60g/L to expand nutrient solution, adjusts pH5.8, culture
Condition is 1500 ~ 2000lx of intensity of illumination, light application time 14h/d, 24 ± 1 DEG C of temperature.The interval leaching for submergence cultivation reactor of having a rest
Do not have frequency 1min/6h incubation times 60 days, bletilla striata seedling pseudobulb expands more than 3 times in a cultivation cycle, obtained kind
Shoot survival percent is more than 95%.
Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations, such as change conical flask into other container；The common skill of this area
All deformations that art personnel directly can export or associate from present disclosure, it is considered as the protection model of the present invention
Enclose.
Claims (2)
- A kind of 1. method for cultivating bletilla striata seedling using interval submergence bioreactor, successively including seed sterilisation step, seed Step, Multiplying culture step are sprouted, pseudobulb generation incubation step and pseudobulb expand incubation step, it is characterised in that utilize The method of interval submergence bioreactor fast culture bletilla striata seedling is aseptically carried out, the Multiplying culture step Including transition Multiplying culture step and propagation and culture of rootage step；An early-stage preparations are also needed to before the seed sterilisation step Step,The early-stage preparations step includes：1. select kind of a pod：Select full breeding；2. reactor and transparent utensil sterilizing：Will Reactor and transparent utensil are wrapped up, standby after HTHP moist heat sterilization；3. prepare culture medium and nutrient solution；The seed sterilisation step is aseptically, to carry out surface disinfection to bletilla seed pod, obtain aseptic seed；It is uniformly to scatter aseptic seed to carry out seed sprouting culture in solid medium that the seed, which is sprouted,；The transition Multiplying culture is aseptically, the bletilla striata seedling after sprouting to be transferred in transparent utensil, in constant temperature It is lower that transition Multiplying culture is carried out by shaking flask mode using transition proliferated culture medium；The propagation and culture of rootage are aseptically, the bletilla striata transition propagation seedling in transparent utensil to be transferred to sterile Interval submergence bioreactor in, at a constant temperature using propagation and culture of rootage liquid is bred and culture of rootage；After pseudobulb generation culture is propagation and culture of rootage terminates, in same interval submerges bioreactor, change Nutrient solution, will breed and culture of rootage fluid exchange is that miniature pseudobulb generates nutrient solution, carry out culture at a constant temperature and obtain band There is miniature pseudobulb bletilla striata seedling；It is after pseudobulb is formed that the pseudobulb, which expands culture, in same interval submerges bioreactor, changes nutrient solution, i.e., Miniature pseudobulb generation nutrient solution is replaced with to the nutrient solution for promoting pseudobulb to expand, pseudobulb is carried out at a constant temperature and expands training Support, obtain to carry and expand pseudobulb bletilla striata seedling；The seed sterilisation step comprises the following steps：1. surface decontamination：Bletilla seed pod is rinsed using running water current, washing time is 30~60min, washes kind of a pod surface It is dirty；2. surface sterilization：Aseptically, it is put in 70~75% alcohol, soak time is 10~60s, sterilized water Rinse 2~3 times；3. surface disinfection：It is that soak time is 1~20min in 0.1~0.3% mercury chloride to be put into concentration, aseptic water washing 5~7 It is secondary；Obtain sterile bletilla seed pod；The seed is sprouted in step, and the formula of the solid medium is：1/2MS liquid is used as basic culture solution, is added Hormone 0.5~2.0mg/L of NAA, 10~50g/L of mashed potatoes, 10~50g/L of sucrose concentration, 1.0~7.0g/L of agar concentration, adjust Whole pH value 5.0~7.0；The condition of culture set as：5~30d of dark culturing, then it is transferred under illumination condition and cultivates, during culture Between be 10~90d, 1500~2000lx of intensity of illumination, 1~18h/d of light application time, temperature is 24 ± 1 DEG C；In the transition Multiplying culture step, for glass or plastics, the transparent utensil is the material that the transparent utensil uses Conical flask or gas bottle, selected capacity are 100~500ml；Incubation time is 7~20d；Used transition propagation training It is liquid or semisolid to support base, and the formula of transition proliferated culture medium is：1/2MS liquid is used as basic culture solution, adds hormone 0.5~2.0mg/L of NAA, potato 10~60g/L of liquid, 10~50g/L of sucrose, 0~5g/L of agar, adjust pH value 5.0~7.0； The condition of culture set as：The transparent utensil is placed on shaking table, setting rotating speed is 60~120rpm, intensity of illumination 1500~ 2000lx, 1~18h/d of light application time, 24 ± 1 DEG C of temperature；In the propagation and culture of rootage step, the reaction condition of the interval submergence bioreactor is：Interval Immersion frequency 1 ~10min/4h, 10~50g/L of inoculum density, 30~90d of incubation time；The propagation and the formula of culture of rootage liquid are：Adopt It is basic culture solution with 1/2MS liquid, adds hormone 0.5~2.0mg/L of NAA, potato 20~90g/L of liquid, 10~50g/ of sucrose L, adjust pH value 5.0~7.0；The condition of culture set is 1500~2000lx of intensity of illumination, 1~18h/d of light application time, temperature 24±1℃；In the pseudobulb generation incubation step, the formula of the miniature pseudobulb generation nutrient solution is：Use 1/2MS liquid for Basic culture solution, adds hormone 0.5~2.0mg/L of NAA, potato 20~90g/L of liquid, 10~90g/L of bananas juice, and sucrose 10~ 50g/L, adjust pH value 5.0~7.0；The condition of culture set is 1500~2000lx of intensity of illumination, 1~18h/d of light application time, 24 ± 1 DEG C of temperature；The interval Immersion frequency of the interval submergence bioreactor is arranged to 1~10min/4h, incubation time 10 ~30d；The pseudobulb is expanded in incubation step, and the formula of the nutrient solution for promoting pseudobulb to expand is：Use MS liquid for Basic culture solution, add hormone 0.5~2.0mg/L of NAA, potato 20~90g/L of liquid, 10~80g/L of bananas juice, straw juice 20 ~120g/L, 10~50g/L of sucrose, adjust pH value 5.0~7.0；The condition of culture set as 1500~2000lx of intensity of illumination, 8~18h/d of light application time, 24 ± 1 DEG C of temperature；The interval Immersion frequency of interval submergence bioreactor is arranged to 1~ 10min/6h, incubation time 20~90 days.
- 2. the method according to claim 1 for cultivating bletilla striata seedling using interval submergence bioreactor, it is characterised in thatIn the seed sterilisation step, 1. washing time is 30~40min to the step in surface decontamination；The step 2. surface In sterilization, the soak time is 30s；For the step 3. in surface disinfection, the soak time is 5~20min；The seed is sprouted in step, and the formula of the solid medium is：1/2MS liquid is used as basic culture solution, is added Hormone 0.5~1.0mg/L of NAA, 20~30g/L of mashed potatoes, 30~40g/L of sucrose concentration, 5.0~6.5g/L of agar concentration, adjust Whole pH value 5.8~6.0；The condition of culture set as：7~15d of dark culturing, then it is transferred under illumination condition and cultivates, during culture Between be 30~50d, 10~12h/d of light application time；In the transition Multiplying culture step, the incubation time is 10~15d；The formula of the culture medium is：Using 1/2MS Liquid is basic culture solution, adds hormone 0.5~1.0mg/L of NAA, potato liquid 50g/L, 30~40g/L of sucrose, agar 0~ 3g/L, adjustment pH value pH5.8~6.0；The rotating speed of shaking table is 60~90rpm, light application time 10~12h/ d, incubation time 10 ~15d；In the propagation and culture of rootage step, the interval Immersion frequency 1~3min/4h, 20~30g/L of inoculum density；Training Support 40~60d of the time；The propagation and the formula of culture of rootage liquid are：1/2MS liquid is used as basic culture solution, adds hormone 0.5~1.0mg/L of NAA, potato 30~50g/L of liquid, sucrose 30g~40/L, adjustment pH value 5.8~6.0, light application time 10~ 12h/d；In the pseudobulb generation incubation step, the formula of the miniature pseudobulb generation nutrient solution is：Use 1/2MS liquid for Basic culture solution, addition hormone 0.5~1.0mg/L of NAA, potato 30~50g/L of liquid, 30~60g/L of bananas juice, sucrose 30g~ 40/L, adjust pH value 5.8~6.0,10~12h/d of light application time；The interval Immersion frequency is arranged to 1~3min/4h；The pseudobulb is expanded in incubation step, and the formula of the nutrient solution for promoting pseudobulb to expand is：Use MS liquid for Basic culture solution, add hormone 0.5~1.0mg/L of NAA, potato 30~50g/L of liquid, 30~50g/L of bananas juice, straw juice 30 ~60g/L, 30~50g/L of sucrose, adjust pH value 5.8~6.0；The condition of culture set as 1500~2000lx of intensity of illumination, 12~14h/d of light application time；The interval Immersion frequency of interval submergence bioreactor is arranged to 1~3min/6h, during culture Between be 30~60d.
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