CN104719168A - 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 belongs to the technical field of medicinal plant cultivation, and particularly relates to a method for cultivating bletilla striata seedlings by using an intermittent immersion bioreactor; 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, be specifically related to a kind of method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling.
Background technology
The bletilla striata (Bletilla striata(Thunb.) Reichb.f.) be the herbaceos perennial that the orchid family bletilla striata belongs to, be also " bletilla ", company and grass etc.Its dry tuber has the effect such as astringing to arrest bleeding, detumescence and promoting granulation, is usually used in treatment hemoptysis, haematemesis, chapped skin etc.There are some researches show that the bletilla striata has antibacterial and antineoplastic action.The bletilla striata, not only for tcm clinical practice, is also widely used in daily chemical products, as cosmetics, advanced ceramic manufacture, weaving, rubber production etc.
Along with the continuous expansion of bletilla striata application, its demand constantly increases.Cause wild bletilla striata quantity sharply to reduce, and the seed of the bletilla striata is small, provides without endosperm the required nutrient that germinates, in its natural state few Germination And Seedling.Traditional cultivation method mainly relies on division propagation, and the division propagation cycle is long, reproductive efficiency is low, consumption kind of amount is large, is difficult to the needs meeting cultivation.Since Kundson carries out the axenic germination of Orchid Seeds first, axenic germination has become the important means of orchid preserving seed and large-scaled propugation.Utilize the kind pod of excellent bletilla striata kind as expanding numerous material by tissue-culturing rapid propagation, the brood body that the important character such as hereditary basis and exterior quality shape is homogeneous can be obtained, and the problem such as can solve that the breeding cycle that traditional division propagation method brings is long, reproduction rate is low and consumption kind of amount is large, significantly reduces the cost of plantation.But the bletilla striata plantlet in vitro that traditional method for tissue culture obtains is thin and delicate short, needs strict acclimation conditions and bookkeeping when plantlet in vitro is applied to Production of Large Fields.When carrying out acclimatization and transplants, to comparatively large to its injury when plantlet in vitro adhering to the cleaning of agar, therefore hardening survival rate is lower, and bletilla striata pseudobulb just can be made to grow sprouting after growth a period of time, is unfavorable for the growth of bletilla striata plantlet in vitro.
In recent years, in order to meet the demand in market, cultivate the bletilla striata of high-quality, people have employed multiple method and cultivate the bletilla striata, wherein much also apply for patent, such as: application for a patent for invention (201310579744.9, 201410364717.4, 201210350910.3) all disclose the culturing and reproducing of the bletilla striata, main employing is the method that solid-based cultivates nursery, although successfully solve the problem of survival rate, but it is low to utilize conventional solid medium method for tissue culture to carry out the automatization level of seeling industry, and cost remains high, a wherein very large part is labour and energy consumption cost.
Interval submergence bioreactor culture mainly utilizes liquid nutrient medium to carry out interval submergence to the histoorgan of plant and cultivates, and supplies nutrition, provide enough oxygen during interval during submergence.Due to the Vitrification Occurred of plant tissue when this training method solves liquid culture preferably, and nutriment can obtain good transmission along with to the submergence of plant tissue, therefore the various difficult problems that can well solve in Plant Tissue Breeding are cultivated in interval submergence; And intermittent immersion cultivation mode can improve the gentle plant generation efficiency of Automated water greatly, and reducing the consumption of man power and material, is the effective method of one substituting tradition cultivation.
The research utilizing interval submergence cultivation reactor successfully to carry out other asexually propagated plant tissue cultures has been reported.Interval submergence bio-reactor is utilized to carry out roxburgh anoectochilus terminal bud tissue cultures expanding propagation method as Chinese patent (201310043181.1) discloses, it utilizes interval submergence bio-reactor to expand numerous introduction to the tissue cultures of roxburgh anoectochilus terminal bud, the method has cycle short, plantlet in vitro quality high, and the tissue-culturing quick-propagation for roxburgh anoectochilus terminal bud provides a kind of low cost, high efficiency method.But the cultivation that intermittent immersion cultivation mode is used for plant just as method for plant tissue culture to different Plant Tissue Breeding, need different conditions and processing mode when a kind of training method is applied to different plants.The cultural method related to during cultivation such as the various aspects such as the processing mode of the choosing of: material, medium formation, Immersion frequency, inoculum density, incubation time, external environment and different materials can not be obtained by simply limited experiment reasoning; And this patent reckon without plant and be directly transferred to liquid culture from solid culture and need laundering period over a long time, thus increase the growth cycle of roxburgh anoectochilus terminal bud plantlet in vitro; At present about utilizing intermittent immersed cultural method to carry out bletilla striata tissue cultures, take into account the laundering period phenomenon over a long time occurred when plant is transferred directly to liquid culture by solid culture in tissue culture procedures, and give processing method, have not been reported and open application simultaneously about this processing method.Injury during cleaning, seedling brought after the method simultaneously adopting liquid batch submergence training method to cultivate bletilla striata seedling solves and cultivated by solid-based; And by promoting that pseudobulb expands the survival rate obtaining and substantially increase kind of transplantation of seedlings with the bletilla striata seedling of larger pseudobulb.
In view of the production utilizing intermittent immersed cultural method to carry out bletilla striata seedling has no report and open application, therefore, be necessary to work out a kind of method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of growth cycle short, transplant that survival rate is high, cost is low, the automation size simple to operate and method of the cultivation bletilla striata seedling of the long laundering period phenomenon occurred when being transferred directly to liquid culture by solid culture can be solved.
For solving the problems of the technologies described above, the technical solution used in the present invention is: the method for bletilla striata seedling cultivated by this utilization interval submergence bio-reactor, comprise seed sterilisation step successively, seed germination step, Multiplying culture step, pseudobulb generates incubation step and pseudobulb and expands incubation step, utilizes the method for interval submergence bio-reactor fast culture bletilla striata seedling aseptically to carry out, and described Multiplying culture step comprises transition Multiplying culture step and propagation and culture of rootage step.
Pass through technique scheme, Multiplying culture process be divided into transition Multiplying culture step and breed and culture of rootage step two process, transition Multiplying culture step adopts shaking flask mode to carry out, thus transition Multiplying culture is eliminated or is shortened and is directly transferred to by solid culture the long laundering period phenomenon that interval submergence bioreactor culture occurs, thus obtain a large amount of for the production of bletilla striata seedling fast, the cultivation of then being expanded by pseudobulb obtains the bletilla striata seedling with larger pseudobulb, substantially increase the survival rate of kind of transplantation of seedlings, and the method adopting liquid batch submergence training method to cultivate bletilla striata seedling solves the injury brought seedling when cultivating rear cleaning by solid-based.
Further, also need an early-stage preparations step before described seed sterilisation step, described early-stage preparations step comprises: 1. select kind of a pod: select full breeding; 2. reactor and transparent utensil sterilizing: by reactor and transparent utensil wrapping, for subsequent use after HTHP moist heat sterilization; 3. medium and culture fluid is prepared;
Described seed sterilisation step is aseptically, carries out surface disinfection to bletilla seed pod, obtains aseptic seed;
Described seed germination step is evenly scattered by aseptic seed to carry out seed germination cultivation in solid culture medium;
Described transition Multiplying culture step is aseptically, is transferred in transparent utensil by the bletilla striata seedling after sprouting, and uses transition proliferated culture medium to carry out transition Multiplying culture by shaking flask mode at a constant temperature;
Described propagation and culture of rootage step are aseptically, be transferred in aseptic interval submergence bio-reactor, use propagation and culture of rootage liquid to carry out breeding and culture of rootage at a constant temperature by the bletilla striata transition propagation seedling in transparent utensil;
It is after propagation and culture of rootage terminate that described pseudobulb generates incubation step, cultivate in reactor in same interval submergence, changing culture fluid by propagation and culture of rootage fluid exchange is that miniature pseudobulb generates culture fluid, carries out cultivation at a constant temperature and obtains with miniature pseudobulb bletilla striata seedling;
It is after pseudobulb is formed that described pseudobulb expands incubation step, in same interval submergence culture bioreactors, change culture fluid and replace with by pseudobulb generation culture fluid the culture fluid promoting that pseudobulb expands, carrying out pseudobulb at a constant temperature and expand cultivation, obtaining with expanding pseudobulb bletilla striata seedling.
Further improve as the present invention and be, described seed sterilisation step comprises the steps:
1. surface decontamination: adopt running water current to rinse bletilla striata seeds, washing time is 0 ~ 60min, washes the dirty of kind of pod surface;
2. surface sterilization: aseptically, being put in soak time in the alcohol of 70 ~ 75% is 10 ~ 60s aseptic water washing 2 ~ 3 times;
3. surface disinfection: to put into concentration be 0.1 ~ 0.3% mercury chloride soak time is 1 ~ 20min, aseptic water washing 5 ~ 7 times; Obtain aseptic bletilla seed pod.
Further improve as the present invention and be, in described seed germination step, the formula of described solid culture medium is: adopt 1/2 MS liquid to be basic culture solution, add hormone NAA 0 ~ 2.0mg/L, mashed potatoes 10 ~ 50g/L, sucrose concentration 10 ~ 50g/L, agar concentration 1.0 ~ 7.0g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is: dark culturing 5 ~ 30d, cultivate under then proceeding to illumination condition, incubation time is 20 ~ 90d, intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, and temperature is 24 ± 1 DEG C.
Further improve as the present invention and be, in described transition Multiplying culture step, the material that described transparent utensil adopts is glass or plastics, and described transparent utensil is conical flask or gas bottle or wide-mouth bottle, and selected capacity is 250ml; Incubation time is 7 ~ 20d; The transition proliferated culture medium adopted is liquid or semisolid, and the formula of transition proliferated culture medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0 ~ 2.0mg/L, potato liquid 10 ~ 60g/L, sucrose 10 ~ 50g/L, agar 0 ~ 5g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is: be placed on shaking table by described transparent utensil, arranging rotating speed is 0 ~ 120rpm, intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, temperature 24 ± 1 DEG C.
Further improve as the present invention and be, in described propagation and culture of rootage step, the reaction condition that reactor is cultivated in described interval submergence is: interval Immersion frequency 1 ~ 10min/4h, inoculum density 10 ~ 50g/L, incubation time 30 ~ 90d; The formula of described propagation and culture of rootage liquid is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0 ~ 2.0mg/L, potato liquid 20 ~ 90g/L, sucrose 10 ~ 50g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, temperature 24 ± 1 DEG C.
Further improve as the present invention and be, described pseudobulb generates in incubation step, the formula that described pseudobulb generates culture fluid is: adopt 1/2 MS liquid to be basic culture solution, add hormone NAA 0 ~ 2.0mg/L, potato liquid 20 ~ 90g/L, bananas juice 10 ~ 90g/L, sucrose 10 ~ 50g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, temperature 24 ± 1 DEG C; The interval Immersion frequency that reactor is cultivated in described interval submergence is set to 1 ~ 10min/4h, incubation time 10 ~ 30d.
Further improve as the present invention and be, described pseudobulb expands in incubation step, described promotion pseudobulb expands the formula of culture fluid and is: adopt MS liquid to be basic culture solution, add hormone NAA 0 ~ 2.0mg/L, potato liquid 20 ~ 90g/L, bananas juice 10 ~ 80g/L, straw juice 20 ~ 120g/L, sucrose 10 ~ 50g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 8 ~ 18h/d, temperature 24 ± 1 DEG C; The interval Immersion frequency that reactor is cultivated in described interval submergence is set to 1 ~ 10min/6h, incubation time 20 ~ 90 days.
By a large amount of experiments confirm, by bletilla striata seeds sprout after transfer in transparent utensil carry out Shaking culture and by Shaking culture to reactor switch over operation process all more for convenience, greatly reduce the pollution rate in operating process; And liquid shaking bottle transition cultivation eliminates by solid culture directly to the long laundering period phenomenon that interval submergence bioreactor culture occurs, thus shorten the cultivation period of bletilla striata seedling; The submergence of employing interval simultaneously bioreactor culture mainly or interval submergence continuous by nutrient solution maintains a kind of training method of plant normal growth; Supply nutrition during submergence, during interval, provide enough oxygen; The Vitrification Occurred of plant tissue when this training method solves liquid culture preferably, and nutriment can obtain good transmission along with to the submergence of plant tissue, thus greatly improve the gentle plant generation efficiency of Automated water; In addition, promote that the incubation step that expands of pseudobulb carries out open cultivation in bio-reactor, this process can be equivalent to carry out hardening process to plantlet in vitro, thus the plantlet in vitro obtained is more healthy and stronger.Expand the step of cultivating owing to have passed through pseudobulb, the plantlet in vitro obtained can grow new root and tender shoots in the short time after transplanting.
With conventional solid medium for contrast, submergence bio-reactor in gap is utilized to cultivate the advantage of bletilla striata seedling as following table:
| Training method | The rate of increase | Required incubation time (d) | Survival rate (%) |
| This method | 1:40 | 50 | 95 |
| Interval submergence bio-reactor | 1:20 | 80 | 60 |
| Conventional solid medium | 1:6 | 120 | 50 |
This method is mainly with the difference of cultural method in the past: 1, increase transition and cultivate to shorten and cultivate the laundering period; 2, increase pseudobulb and expand step, the healthy and strong survival rate of seedling is high, and without the need to shortening cultivation cycle through hardening, reduces labour.
As preferred version of the present invention, the described step of described seed sterilization 1. in surface decontamination the deionized water rinsing time be 30 ~ 40min; Described step is 2. in surface sterilization, and described soak time is 30s; Described step is 3. in surface disinfection, and described soak time is 5 ~ 20min;
In described seed germination step, the formula of described solid culture medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0.5 ~ 1.0mg/L, mashed potatoes 20 ~ 30g/L, sucrose concentration 30 ~ 40g/L, agar concentration 5.0 ~ 6.5g/L, adjusted to ph 5.8 ~ 6.0; The condition of culture of setting is: dark culturing 7 ~ 15d, and cultivate under then proceeding to illumination condition, incubation time is 30 ~ 50d, light application time 10 ~ 12h/d;
In described transition Multiplying culture step, described incubation time is 10 ~ 15d; The formula of described medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, sucrose 30 ~ 40g/L, agar 0 ~ 3g/L, adjusted to ph 5.8 ~ 6.0; The rotating speed of shaking table is 60 ~ 90rpm, light application time 10 ~ 12h/d, and incubation time is 10 ~ 15 d;
In described propagation and culture of rootage step, described interval Immersion frequency 1 ~ 3min/4h, inoculum density 20 ~ 30g/L; Incubation time 40 ~ 60d; The formula of described propagation and culture of rootage liquid is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, sucrose 30g ~ 40/L, adjusted to ph 5.8 ~ 6.0, light application time 10 ~ 12h/d;
Described pseudobulb generates in incubation step, the formula that described pseudobulb generates culture fluid is: adopt 1/2 MS liquid to be basic culture solution, add hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, bananas juice 30 ~ 60g/L, sucrose 30g ~ 40/L, adjusted to ph 5.8 ~ 6.0, light application time 10 ~ 12h/d; Described interval Immersion frequency is set to 1 ~ 3min/4h;
Described pseudobulb expands in incubation step, described promotion pseudobulb expands the formula of culture fluid and is: adopt MS liquid to be basic culture solution, add hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, bananas juice 30 ~ 50g/L, straw juice 30 ~ 60g/L, sucrose 30 ~ 50g/L, adjusted to ph 5.8 ~ 6.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 12 ~ 14h/d; The interval Immersion frequency that reactor is cultivated in described interval submergence is set to 1 ~ 3min/6h, and incubation time is 30 ~ 60 d.
Adopt the present invention to produce the technical scheme of bletilla striata seedling, to be significant technique effect be its beneficial effect produced:
(1) rate of increase is high, and biological yield is large, and cultivation cycle is short.It is utilize liquid culture to carry out the cultivation of interval submergence to plant tissue organ that bioreactor culture is cultivated in interval submergence.Under this mode, the rate of increase of the bletilla striata is cultivated far above conventional solid base.The rate of increase of the bletilla striata namely utilizing the inventive method to obtain can reach 1:40, cultivates about 1:6 far above conventional solid base, and higher than directly being proceeded to the effect of interval submergence bioreactor culture 1:20 by solid culture.The higher rate of increase also provides the foundation for the plantlet in vitro obtaining some in the short time, thus reduces subculture number, reduces plantlet in vitro variation frequency.Utilize the mode of the inventive method can obtain the effect of propagation 40 times through the cultivation of about 50 days, directly proceeded in interval submergence bio-reactor by solid culture in the past and cultivate, and needed to obtain the effect of propagation 20 times through about 80 days cultivate one's ability, and conventional solid training method can only obtain the cultivation effect of 6 times through the cultivation of 100 days.
(2) plantlet in vitro quality is good, and seedling is healthy and strong, and cultivation survival rate is high.Supply nutrition during submergence, during interval, provide enough oxygen; The Vitrification Occurred of plant tissue when this training method solves liquid culture preferably, and nutriment can obtain good transmission along with to the submergence of plant tissue, thus greatly improve the gentle plant generation efficiency of Automated water; The incubation step that expands of promotion pseudobulb carries out open cultivation in bio-reactor, and this process can be equivalent to carry out hardening process to plantlet in vitro, and is equivalent to carry out hardening to plantlet in vitro, thus the plantlet in vitro obtained is more healthy and stronger.Owing to have passed through the step that pseudobulb expands, new root and tender shoots can be grown in the short time after the bletilla striata transplantation of seedlings obtained, and survival rate is more than 95%; And conventional solid medium culture and utilize interval submergence bioreactor culture mode to cultivate but expand without pseudobulb the plantlet in vitro that incubation obtains and need all to carry out hardening, and transplantation of seedlings process need at substantial manpower and high-level operation, transplanting survival rate is only 50% ~ 60% simultaneously.
(3) automatization level is high, saves a large amount of labour and consumes.Utilize submergence bio-reactor in gap to cultivate bletilla striata seedling, not only can reduce the consumption of blake bottle, and then reduce the manpower consumption of the aspects such as filling washing; Simultaneously bletilla striata seeds is transferred in conical flask and is carried out Shaking culture and all convenient and simple to the operating process of the switching of reactor by Shaking culture after sprouting, and reduces the pollution rate in operating process; And shaking flask transition Multiplying culture eliminates by the long laundering period phenomenon occurred in solid culture to interval submergence bioreactor culture, thus shorten the cultivation period of bletilla striata seedling.Its two, in a culture apparatus, form the seedling of about 2000, relative to the seedling of solid culture bottle every bottle of output about 10, obviously there is greatly efficiency difference.Its three, the inventive method can realize the control of automation in incubation, does not need plantlet in vitro of repeatedly transferring, and thus expends less manpower and shortens incubation time, thus saving human cost; The high rate of increase, higher seedling quality and high survival rate also reduce the production cost of seedling.
In sum, the present invention utilize cultivated by shaking flask transition, interval submergence bioreactor culture and pseudobulb expand the method for cultivating to bletilla striata seedling cultivate to have relative to traditional training method expand that numerous efficiency is high, incubation time is short, after seed germination switching convenient, eliminate or shorten by the long laundering period phenomenon of solid culture to the appearance of interval submergence bioreactor culture; Seedling is healthy and strong, cultivation survival rate is high and cost is low, automaticity is high, saves the advantages such as labour's consumption, is therefore more suitable for bletilla striata seedling large-scale cultivation and produces.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further details:
Fig. 1 is the growing state of bletilla striata seedling in each cultivation stage cycle;
Wherein: (a) is the growth conditions schematic diagram of bletilla striata seedling at the end of seed germination cultivation;
B () is the growth conditions schematic diagram of bletilla striata seedling at the end of transition Multiplying culture;
C () is the growth conditions schematic diagram of bletilla striata seedling at the end of propagation and culture of rootage in interval submergence bio-reactor;
(d) cultivate for pseudobulb in interval submergence reactor expands at the end of the large 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: by reactor and transparent utensil wrapping, for subsequent use after HTHP moist heat sterilization; Wherein reactor is gap submergence bio-reactor, 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, is put in 70 ~ 75%(v/v by cleaned bletilla seed pod) soak 30(s in alcohol), aseptic water washing 2 ~ 3 times; Surface disinfection: putting into concentration is again 0.1%(g/v) mercury chloride soaks 8min, aseptic water washing 5 ~ 7 times; Aseptic filter paper draws the moisture on kind of pod surface, adopts sterile working from bletilla seed pod one end-grain cutting one osculum;
(3) seed germination step: according to formula 1/2MS+NAA0.5mg/L+30g/L mashed potatoes+30g/L sucrose+agar 6.5g/L, adjusted to ph 5.8 prepares culture fluid; Bletilla striata seeds is shaken off uniformly and cultivates in bletilla striata seeds germination medium, under proceeding to illumination condition after first dark culturing 10d, cultivate 30d, intensity of illumination 1500 ~ 2000lx, light application time 10h/d, temperature 24 ± 1 DEG C;
(4) transition Multiplying culture step: according to formula 1/2MS+NAA0.5mg/L+50g/L potato liquid+30g/L sucrose, adjusted to ph 5.8 prepares culture fluid; Transfer in conical flask by the bletilla striata seedling (protocorm) after sprouting, be placed on by conical flask on shaking table, shaking speed is 60rmp, intensity of illumination 1500 ~ 2000lx, light application time 12h/d, temperature 24 ± 1 DEG C; 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, adjusted to ph 5.8 prepares propagation and culture of rootage liquid; Be transferred to by the bletilla striata seedling of transition Multiplying culture in conical flask in interval submergence culture bioreactors sterilized in advance, interval Immersion frequency 3min/4h, condition of culture is intensity of illumination 1500 ~ 2000lx, light application time 12h/d, temperature 24 ± 1 DEG C; Incubation time 50d;
(6) pseudobulb generates incubation step: according to formula 1/2MS+NAA0.5mg/L+50g/L potato liquid+bananas juice 40g/L+30g/L sucrose, adjusted to ph 5.8pH5.8 ~ 6.0 are prepared pseudobulb and generated culture fluid; In same interval submergence bio-reactor, culture fluid is replaced by pseudobulb and generates culture fluid, interval Immersion frequency 3min/4h intensity of illumination 1500 ~ 2000lx, light application time 10h/d, temperature 24 ± 1 DEG C, incubation time is 20 days;
(7) pseudobulb expands incubation step: according to formula MS+NAA0.5mg/L+50g/L potato liquid+bananas juice 50g/L+ straw juice 50g/L, the culture fluid that pH5.8 preparation short reason pseudobulb expands, in same interval submergence bio-reactor, culture fluid is replaced by the culture fluid promoting that pseudobulb expands, interval Immersion frequency 3min/6h; Intensity of illumination 1500 ~ 2000lx, light application time 12h/d, temperature 24 ± 1 DEG C; Incubation time 30d;
In a cultivation cycle, the laundering period of bletilla striata seedling in interval submergence bio-reactor shortens to 0 day, thus makes reactor proliferate efficiency reach more than 1:35; Incubation time 60 days, in a cultivation cycle, bletilla striata seedling pseudobulb expands more than 3.5 times, and the kind shoot survival percent obtained is more than 95%.
The growing state of bletilla striata seedling in each cultivation stage cycle is shown in Fig. 1.
Embodiment 2:
Interval submergence bio-reactor is utilized to cultivate bletilla striata seedling, concrete steps as described in Example 1, difference is Semi-solid cell culture by what adopt during transition Multiplying culture with it, selected culture medium prescription is 1/2MS+NAA0.5mg/L+50g/L potato liquid+30g/L sucrose+agar 3g/L, adjustment pH5.9, propagation and culture of rootage formula of liquid are 1/2MS+NAA1.0mg/L+50g/L potato liquid+30g/L sucrose, adjustment pH5.9, condition of culture is intensity of illumination 1500 ~ 2000lx, light application time 12h/d, temperature 24 ± 1 DEG C.In a cultivation cycle, the laundering period of bletilla striata seedling in interval submergence bio-reactor shortens to 0 day, thus makes reactor proliferate efficiency reach more than 1:30.
Embodiment 3:
Bio-reactor is utilized to cultivate bletilla striata seedling, concrete steps as described in Example 1, difference is Semi-solid cell culture by what adopt during transition Multiplying culture with it, selected culture medium prescription is 1/2MS+NAA0.5mg/L+50g/L potato liquid+30g/L sucrose+agar 2g/L, adjustment pH6.0, it is MS+NAA1.0mg/L+50g/L potato liquid+bananas juice 40g/L+ straw juice 60g/L that pseudobulb expands culture fluid, adjustment pH6.0, the laundering period of bletilla striata seedling in interval submergence bio-reactor shortens to 0.3 day, in a cultivation cycle, bletilla striata seedling pseudobulb expands about 2 ~ 3 times, the kind shoot survival percent obtained is more than 90%.
Embodiment 4:
Bio-reactor is utilized to cultivate bletilla striata seedling, concrete steps as described in Example 1, difference is that to expand culture fluid be MS+NAA1.0mg/L+50g/L potato liquid+bananas juice 50g/L+ straw juice 60g/L to pseudobulb with it, adjustment pH5.8, condition of culture is intensity of illumination 1500 ~ 2000lx, light application time 14h/d, temperature 24 ± 1 DEG C.The interval Immersion frequency 1min/6h incubation time of submergence of having a rest cultivation reactor 60 days, in a cultivation cycle, bletilla striata seedling pseudobulb expands more than 3 times, and the kind shoot survival percent obtained is more than 95%.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had, such as, change conical flask into other container; All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (9)
1. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling, comprise seed sterilisation step successively, seed germination step, Multiplying culture step, pseudobulb generates incubation step and pseudobulb and expands incubation step, it is characterized in that, utilize the method for interval submergence bio-reactor fast culture bletilla striata seedling aseptically to carry out, described Multiplying culture step comprises transition Multiplying culture step and breeds and culture of rootage step.
2. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 1, is characterized in that, also need an early-stage preparations step before described seed sterilisation step,
Described early-stage preparations step comprises: 1. select kind of a pod: select full breeding; 2. reactor and transparent utensil sterilizing: by reactor and transparent utensil wrapping, for subsequent use after HTHP moist heat sterilization; 3. medium and culture fluid is prepared;
Described seed sterilisation step is aseptically, carries out surface disinfection to bletilla seed pod, obtains aseptic seed;
Described seed germination is evenly scattered by aseptic seed to carry out seed germination cultivation in solid culture medium;
Described transition Multiplying culture is aseptically, is transferred in transparent utensil by the bletilla striata seedling after sprouting, and uses transition proliferated culture medium to carry out transition Multiplying culture by shaking flask mode at a constant temperature;
Described propagation and culture of rootage are aseptically, be transferred in aseptic interval submergence bio-reactor, use propagation and culture of rootage liquid to carry out breeding and culture of rootage at a constant temperature by the bletilla striata transition propagation seedling in transparent utensil;
It is after propagation and culture of rootage terminate that described pseudobulb generates cultivation, cultivate in reactor in same interval submergence, changing culture fluid by propagation and culture of rootage fluid exchange is that miniature pseudobulb generates culture fluid, carries out cultivation at a constant temperature and obtains with miniature pseudobulb bletilla striata seedling;
It is after pseudobulb is formed that described pseudobulb expands cultivation, in same interval submergence culture bioreactors, change culture fluid and replace with by miniature pseudobulb generation culture fluid the culture fluid promoting that pseudobulb expands, carrying out pseudobulb at a constant temperature and expand cultivation, obtaining with expanding pseudobulb bletilla striata seedling.
3. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 2, it is characterized in that, described seed sterilisation step comprises the steps:
1. surface decontamination: adopt running water current to rinse bletilla striata seeds, washing time is 0 ~ 60min, washes the dirty of kind of pod surface;
2. surface sterilization: aseptically, being put in soak time in the alcohol of 70 ~ 75% is 10 ~ 60s aseptic water washing 2 ~ 3 times;
3. surface disinfection: to put into concentration be 0.1 ~ 0.3% mercury chloride soak time is 1 ~ 20min, aseptic water washing 5 ~ 7 times; Obtain aseptic bletilla seed pod.
4. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 2, is characterized in that,
In described seed germination step, the formula of described solid culture medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0 ~ 2.0mg/L, mashed potatoes 10 ~ 50g/L, sucrose concentration 10 ~ 50g/L, agar concentration 1.0 ~ 7.0g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is: dark culturing 5 ~ 30d, cultivate under then proceeding to illumination condition, incubation time is 10 ~ 90d, intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, and temperature is 24 ± 1 DEG C.
5. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 2, is characterized in that,
In described transition Multiplying culture step, the material that described transparent utensil adopts is glass or plastics, and described transparent utensil is conical flask or gas bottle or wide-mouth bottle, and selected capacity is 100 ~ 500ml; Incubation time is 7 ~ 20d; The transition proliferated culture medium adopted is liquid or semisolid, and the formula of transition proliferated culture medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0 ~ 2.0mg/L, potato liquid 10 ~ 60g/L, sucrose 10 ~ 50g/L, agar 0 ~ 5g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is: be placed on shaking table by described transparent utensil, arranging rotating speed is 0 ~ 120rpm, intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, temperature 24 ± 1 DEG C.
6. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 3, is characterized in that,
In described propagation and culture of rootage step, the reaction condition that reactor is cultivated in described interval submergence is: interval Immersion frequency 1 ~ 10min/4h, inoculum density 10 ~ 50g/L, incubation time 30 ~ 90d; The formula of described propagation and culture of rootage liquid is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0 ~ 2.0mg/L, potato liquid 20 ~ 90g/L, sucrose 10 ~ 50g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, temperature 24 ± 1 DEG C.
7. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 4, is characterized in that,
Described pseudobulb generates in incubation step, and the formula that described pseudobulb generates culture fluid is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0 ~ 2.0mg/L, potato liquid 20 ~ 90g/L, bananas juice 10 ~ 90g/L, sucrose 10 ~ 50g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 1 ~ 18h/d, temperature 24 ± 1 DEG C; The interval Immersion frequency that reactor is cultivated in described interval submergence is set to 1 ~ 10min/4h, incubation time 10 ~ 30d.
8. the method utilizing interval submergence bio-reactor to cultivate bletilla striata seedling according to claim 4, is characterized in that,
Described pseudobulb expands in incubation step, described promotion pseudobulb expands the formula of culture fluid and is: adopt MS liquid to be basic culture solution, add hormone NAA 0 ~ 2.0mg/L, potato liquid 20 ~ 90g/L, bananas juice 10 ~ 80g/L, straw juice 20 ~ 120g/L, sucrose 10 ~ 50g/L, adjusted to ph 5.0 ~ 7.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 8 ~ 18h/d, temperature 24 ± 1 DEG C; The interval Immersion frequency that reactor is cultivated in described interval submergence is set to 1 ~ 10min/6h, incubation time 20 ~ 90 days.
9. the method for bletilla striata seedling cultivated by the utilization interval submergence bio-reactor according to any one of claim 2-8, it is characterized in that,
In described seed sterilisation step, described step 1. in surface decontamination the deionized water rinsing time be 30 ~ 40min; Described step is 2. in surface sterilization, and described soak time is 30s; Described step is 3. in surface disinfection, and described soak time is 5 ~ 20min;
In described seed germination step, the formula of described solid culture medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0.5 ~ 1.0mg/L, mashed potatoes 20 ~ 30g/L, sucrose concentration 30 ~ 40g/L, agar concentration 5.0 ~ 6.5g/L, adjusted to ph 5.8 ~ 6.0; The condition of culture of setting is: dark culturing 7 ~ 15d, and cultivate under then proceeding to illumination condition, incubation time is 30 ~ 50d, light application time 10 ~ 12h/d;
In described transition Multiplying culture step, described incubation time is 10 ~ 15d; The formula of described medium is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0.5 ~ 1.0mg/L, potato liquid 0 ~ 60g/L, sucrose 30 ~ 40g/L, agar 0 ~ 3g/L, adjusted to ph pH5.8 ~ 6.0; The rotating speed of shaking table is 60 ~ 90rpm, light application time 10 ~ 12h/, and incubation time is 10 ~ 15 d;
In described propagation and culture of rootage step, described interval Immersion frequency 1 ~ 3min/4h, inoculum density 20 ~ 30g/L; Incubation time 40 ~ 60d; The formula of described propagation and culture of rootage liquid is: adopt 1/2 MS liquid to be basic culture solution, adds hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, sucrose 30g ~ 40/L, adjusted to ph 5.8 ~ 6.0, light application time 10 ~ 12h/d;
Described pseudobulb generates in incubation step, the formula that described miniature pseudobulb generates culture fluid is: adopt 1/2 MS liquid to be basic culture solution, add hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, bananas juice 30 ~ 60g/L, sucrose 30g ~ 40/L, adjusted to ph 5.8 ~ 6.0, light application time 10 ~ 12h/d; Described interval Immersion frequency is set to 1 ~ 3min/4h;
Described pseudobulb expands in incubation step, described promotion pseudobulb expands the formula of culture fluid and is: adopt MS liquid to be basic culture solution, add hormone NAA 0.5 ~ 1.0mg/L, potato liquid 30 ~ 50g/L, bananas juice 30 ~ 50g/L, straw juice 30 ~ 60g/L, sucrose 30 ~ 50g/L, adjusted to ph 5.8 ~ 6.0; The condition of culture of setting is intensity of illumination 1500 ~ 2000lx, light application time 12 ~ 14h/d; The interval Immersion frequency that reactor is cultivated in described interval submergence is set to 1 ~ 3min/6h, and incubation time is 30 ~ 60 d.
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