CN103270946A - Dendrobium officinale seedling production and hardening synchronization method - Google Patents
Dendrobium officinale seedling production and hardening synchronization method Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicinal materials, and relates to a of Dendrobium officinale seedling production and hardening synchronization method, more specifically, relates to a method for propagating Dendrobium officinale tissue culture seedlings and hardening seedlings in the same reactor by using an intermittent immersion type liquid culture system. According to the invention, aseptic Dendrobium officinale seeds, protocorms and tissue culture seedlings are used as materials, and a large amount of Dendrobium officinale tissue culture seedlings are obtained by inoculation culture in the intermittent immersion type liquid culture system, and the seedling hardening is completed in the same reactor. The invention provides a high efficiency and low cost method for producing Dendrobium officinale tissue culture seedlings.
Description
Technical field
The present invention relates to a kind of seedlings of Dendrobium officinale production and hardening method for synchronous, belong to the traditional Chinese medicine field of growing seedlings, be specifically related to a kind of immersion liquid culture systems of utilizing intermittently and expand numerous candidum tissue culturing seedling and the method for hardening synchronously.
Technical background
Dendrobium candidum (
Dedrobium candidumWall1 ex Lindl), claiming ribbed hedyotis herb again, belong to the orchid family epiphytic orchid class, is traditional rare traditional Chinese medicine, among the people being described as " first of the Chinese nine immortal grass ".In Shennong's Herbal, be listed in top gradely, have nourishing Yin and clearing heat, the beneficial stomach that promotes the production of body fluid, relieving cough and moistening lung, wet one's whistle make eye bright, improve human immunity, anti-curing oncoma and angiocardiopathy and improve sleep, prevent and treat cataract, effect such as promote longevity.
The seed of dendrobium candidum is very tiny, and embryonic development is incomplete, no endosperm tissue, and germination rate is extremely low under nature.Predatory excavating for a long time causes wild dendrobium candidum resource closely exhausted in addition, is in Critical Condition, can not satisfy the domestic and international market demand far away, causes market supply in short supply.Since the nineties, China begins this medicinal plant of large tracts of land artificial planting, and has formed the related industry chain.
The appliable plant tissue culture and rapid propagation method can obtain the brood body of important character homogeneous such as hereditary basis, exterior quality, and the pathogen that can remove the harm dendrobium candidum, be the method for the present numerous stem of noble dendrobium of using always of expansion, solved the phenomenon of stem of noble dendrobium high quality seedling shortage to a certain extent.But, traditional method has many shortcomings that are difficult to avoid for the acquisition of candidum tissue culturing seedling: 1) because sealed cultivation, can't carry out exchange gas in the incubation, cause candidum tissue culturing seedling vitrification phenomenon to occur, seedling quality is poor, transplanting survival rate is low; 2) need a large amount of switch over operations in the incubation, and need to handle discarded medium, increased labour cost; 3) the traditional plant tissue cultivate need be on solid culture medium by protocorm form, the bud of growing thickly expands numerous, plant division cultivation and process such as culture of rootage, needs bottle outlet after group training seedling becomes seedling, washes seedling, processes such as sterilization and substrate culture, the hardening survival rate is low, but complex operation, need specific facility and condition, cause production cost higher.
Use immersion liquid culture systems intermittently and carry out the defective that the production of dendrobium candidum can remedy produced in conventional processes, provide effective method for producing high-quality seedlings of Dendrobium officinale cheaply.
Parameter is set and is utilized intermittently submergence to cultivate the existing report of research that reactor successfully carries out other plant cultivation.Be the research of ZL 200910114406.1 " utilizing intermittent immersed submergence at intermittence culture systems to carry out tissue culturing and quick propagation of sugarcane " as the patent No., it is introduced the sugarcane cultivation of three kinds utilizing intermittently submergence to cultivate reactor, also provides the technology enlightenment for the cultivation that utilizes this kind training method to carry out other kind plant simultaneously.But, the different intermittently method of operating differences of submergence culture apparatuses of one side, employed culture apparatus is different with this patent in above-mentioned two patent documentations; Owing to the difference on various plant physiologies, the biochemistry, identical cultural method is cultivated different to different plants, need different condition of culture and processing mode on the other hand, can not obtain by simple limited experiment reasoning.The step of mentioning in the document patent and the concrete training method that wherein relates to and condition of culture can not be used for the cultivation of seedlings of Dendrobium officinale fully, and propagation cultivation, the culture of rootage of its operation and the seedling of the taking root domestication step of transplanting also with the present invention in the cultivation of the dendrobium candidum that relates to have diverse cultivation thinking.Therefore, be necessary to explore and set up this added value of dendrobium candidum than bioreactor culture, hardening and the cultivation method of higher plant tissue culture seedling.
Yet the method for utilizing intermittent immersed cultural method to carry out the dendrobium candidum tissue culture quick breeding is not appeared in the newspapers and open application.
The hardening of candidum tissue culturing seedling is a critical step in the dendrobium candidum artificial planting industry, and the success or not of hardening directly influences medicinal herb grower's harvest.So the research of relevant dendrobium candidum hardening is a lot, be that " a kind of hardening off method of candidum tissue culturing seedling ", the application number of ZL 200810195801.2 is 201110175709.1 " candidum tissue culturing seedling hardening domesticating method " as the patent No..Above-mentioned two kinds of dendrobium candidum hardening off method all are that the group cultivating seedling and propagating separates with hardening and carries out, and especially cultivate the seedling of acquisition rather than the dendrobium candidum seedling of plant bioreactor liquid culture based on conventional solid culture medium; The method that tradition obtains seedling and transplants from solid culture medium, the big survival rate of technical difficulty are difficult for grasping, and have increased labour's input, and group training seedling is had certain injury, the relevant report that does not also have candidum tissue culturing seedling production and hardening to carry out synchronously at present.
Seedlings of Dendrobium officinale production provided by the invention and hardening method for synchronous, not only on candidum tissue culturing seedling production efficiency far above traditional solid culture, and in reactor, continue to cultivate under the situation that does not shift out group training seedling and directly carry out hardening, saved the labour, shortened the hardening time.For production and the hardening of dendrobium candidum provides an efficient ways.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of seedlings of Dendrobium officinale production and hardening method for synchronous, this method has rate of increase height, group training seedling stalwartness, transplanting survival rate height and automaticity height, the labour consumes advantages such as few, especially mobile group is trained seedling in the hardening process, but replacing culture fluid, group training seedling preserves from, in the continued growth of hardening process, significantly improve production efficiency and training quality.
Technical scheme of the present invention is:
A kind of seedlings of Dendrobium officinale production and hardening method for synchronous comprise explant selection, seedling incubation step, hardening step, and utilization immersion system at intermittence carries out the seedlings of Dendrobium officinale cultivation and carries out synchronously with the hardening step.。
Particularly, described method may further comprise the steps:
(1) selection of explant: cultivate selected explant following any:
Seed;
Protocorm;
Group training seedling;
(2) seedling is cultivated: utilize intermittently immersion system, adopt the liquid culture mode, carry out the cultivation of seedlings of Dendrobium officinale;
(3) hardening: after candidum tissue culturing seedling was cultivated and finished, the culture fluid that will carry out the seedling cultivation at same intermittence in the immersion system was replaced with the hardening nutrient solution, does not change under the submergence frequency condition, finishes the hardening process after cultivation a period of time.
Further improvement of the present invention is:
(1) explant is selected: select three kinds of explants,
Dendrobium candidum seed: the aseptic stem of noble dendrobium seed that the fruit folder obtains after the surface sterilization sterilization treatment;
Protocorm: bred for four generations with interior nothing deformity and vitrified protocorm;
Group training seedling: bred for four generations and induce the candidum tissue culturing seedling that grows 1 ~ 3 leaf that obtains with interior protocorm.Wherein preferred group training group training seedling is as the explant of submergence cultivation at intermittence.
(2) seedling is cultivated: the device that seedling is cultivated be the immersion system at intermittence, and described intermittence, the culture parameters of bath unit was: the submergence frequency: greater than 3min/12h less than 20min/1h, preferred 5 ~ 10 min/6 ~ 8h; Inoculum density: 1.. seed: 0.05 ~ 2 fruit pod/L, preferred 0.1 ~ 0.5/L; 2.. protocorm: 2 ~ 8g/L, preferred 2.5 ~ 4g/L; 3.. group training seedling: 20 ~ 150 strains/L, preferred 50 ~ 80 strains/L; Incubation time: 60 ~ 120 d, preferred 75 ~ 90 d; Cultivation with the component of culture fluid prescription and content for but be not limited to the sucrose of 1/2Ms, 0.1 ~ 0.5mg/L methyl NAA, 30g/L, bananas juice or the murphy juice of 10 ~ 20g/L, pH:5.0 ~ 7.0, preferred 6.0 ~ 6.4; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 3 ℃ of temperature.
Described submergence frequency is when less than 3min/12h or greater than 20min/1h, and the dendrobium candidum brood body is withered or vitrifying is serious, can't be normally, healthy growth; The increase dendrobium candidum cultivation rate of increase and seedling quality along with the submergence frequency when the submergence frequency falls between increase, it is optimum that the submergence frequency reaches when 5 ~ 10 min/6 ~ 8h, when surpassing 5 ~ 10 min/6 ~ 8h, along with the increase dendrobium candidum of submergence frequency is cultivated the rate of increase and seedling quality weakens.Described inoculum density: the inoculum density of seed is at 0.1 ~ 2 fruit pod/L scope culture systems space availability ratio height, group training seedling normal growth, and it is low that inoculum density is lower than 0.1 fruit pod/L space availability ratio, is higher than 2 the crowded group training of fruit pod/L brood body seedling teratogenesis; When explant is that protocorm or group training seedling all have similar phenomenon.
(3) hardening: after finishing the cultivation of propagation and strong plantlets and rootage, tissue culture medium is replaced with the hardening culture fluid in the same reactor, and the submergence frequency of reactor is constant.
Described hardening is divided two stages: (1) is the laundering period gradually; (2) the domestication stage.
The hardening culture fluid that the described laundering period gradually uses is for reducing the culture fluid of the described liquid culture of sugared content gradually, and culture environment is: intensity of illumination 1800 ~ 2000 lux, light application time 10 ~ 14 h/d; Cultivation temperature is 24 ± 3 ℃, at interval with 20 ± 3 ℃; Incubation time is: 5 ~ 35d, preferred 15 ~ 25d.
The hardening culture fluid of described domestication stage use is mainly but is not limited to following several, (1) sheep excrement leachate, described sheep excrement leachate preparation: the sheep excrement that dries is ground, and behind 1/5 ~ 1/15 adding running water according to the shared mass fraction of sheep excrement, heating was boiled 0.5 ~ 4 hour, preferred 2 ~ 3 hours, the removal solid impurity is filtered with 10 ~ 60 purpose sub-sieves in cooling back, and preferred 32 ~ 50 orders are after adding water and supplying original liquor capacity, boil or 121 ℃ of 15 minutes moist heat sterilizations, use the cooling back.(2) macroelement culture fluid, the prescription of described macroelement culture fluid is:
NH4NO3……………………………1.65g/L
KNO3………………………………1.90g/L
MgSO4·7H2O?……………………0.37g/L
KH2PO4……………………………0.17g/L
The domestication environment is natural environment, 15 ~ 30 ℃ of temperature, and the domestication time is 10 ~ 30d, preferred 15 ~ 20d.
The present invention utilizes submergence at intermittence culture systems to carry out the production of candidum tissue culturing seedling, has obtained following significant effect:
(1) hardening survival rate height: utilize the characteristics of liquid culture, in time change culture fluid as required, and need not the taking-up group train seedling, directly in same reactor, carry out hardening; Compare with the agar that solid culture need the cleaning group be adhered on the training seedling, the group training seedling that obtains under this method liquid culture condition does not need to wash seedling, hardening is also not need mobile group training seedling, thereby for to group training seedling without any injury, can significantly improve the hardening survival rate, actual effect reaches 100% basically.
(2) rate of increase height.Intermittently the immersion liquid culture systems is to utilize pressure that filtrated air produces with the contacting of liquid nutrient medium and plant tissue culture seedling intermittence, to reach the method for fast culture.Its maximum characteristics are exactly rate of increase height, and the rate of increase that candidum tissue culturing seedling is cultivated can reach 50 ~ 80 times, exceeds the proliferate efficiency of about 10 times of traditional conventional solid tissue culture bottle cultural methods far away.
(3) automaticity height, the amount of labour are little.Because utilizing intermittently, the immersion liquid culture systems is that air pump provides power, time-controlled switch is controlled the cultivation chopper frequency, whole incubation comprises propagation, take root and hardening only needs once to inoculate, saved the operation of the frequent switching of culture and the can cleaning of tissue culture bottle in enormous quantities etc., reduce the amount of labour greatly, reduced production cost.
(4) become the seedling time short, reduce production cost.Utilize submergence at intermittence culture systems to carry out the material that the dendrobium candidum tissue is cultivated, once no longer switching after the inoculation, reduced subculture number, the expense of transit working and the consumption of reagent, medicine etc. have been reduced, save production cost, especially shortened group training seedling adaptation time, utilizing intermittently the submergence reactor to carry out dendrobium candidum, to cultivate the one-period time be 75 ~ 90 days, less than 90 ~ 120 days of conventional solid culture.And intermittently therefore the propagation multiple of submergence cultivation under the situation of the seedling that obtains same quantity, utilizes method of the present invention can shorten incubation time greatly again far above solid culture.
(5) group training seedling quality is good, and adaptability is strong.Because high-quality group of training of the production seedling that is reduced to of subculture number provides assurance, has reduced aberration rate to a certain extent; Intermittently the submergence culture systems is carried out the cultivation of dendrobium candidum tissue owing to utilize filtered air to be power, has strengthened exchange gas in the process of cultivation, and the simulating nature environment is grown, quality and the adaptive capacity to environment of enhancing group training seedling; The hardening process is with organic chemical fertilizer and nutrient solutions of substituting such as sheep excrement, and condition of culture can adjust as required, and directly hardening requires simply to facility in reactor, and easier seedling with high resistance to cold and diseases is more suitable for field production.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further details.
Fig. 1 is for being explant different times growth conditions in submergence at intermittence culture systems with the dendrobium candidum seed;
Fig. 2 is the comparison of different training methods;
Embodiment
The present invention proposes a kind of seedlings of Dendrobium officinale production and hardening method for synchronous, comprises explant selection, seedling incubation step, hardening step, and utilization immersion system at intermittence carries out the seedlings of Dendrobium officinale cultivation and carries out synchronously with the hardening step.
Concrete steps are as follows, the selection of explant: cultivate selected explant following any:
Seed;
Protocorm;
Group training seedling;
Seedling is cultivated: utilize intermittently immersion system, adopt the liquid culture mode, carry out the cultivation of seedlings of Dendrobium officinale;
Hardening: after candidum tissue culturing seedling was cultivated and finished, the culture fluid that will carry out the seedling cultivation at same intermittence in the immersion system was replaced with the hardening nutrient solution, does not change under the submergence frequency condition, finishes the hardening process after cultivation a period of time.
(1) explant is selected: the dendrobium candidum fruit that sterilization treatment is good cuts an osculum, seed is scattering into sterilize intermittently in the submergence culture apparatus.
(2) seedling is cultivated: intermittently the parameter of immersion system is: inoculum density: 0.5 fruit folder/L; Submergence frequency: 10 min/6h; Incubation time: 75 d; Cultivation is 1/2Ms, 0.2mg/L methyl NAA, 30g/L sucrose, 20g/L bananas juice or murphy juice with the culture fluid prescription, and pH 6.2; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
(3) hardening: the culture fluid of in same reactor aforesaid liquid being cultivated is replaced with and reduces the sucrose concentration culture fluid gradually, do not change the submergence frequency of reactor, at intensity of illumination 1800 ~ 2000 lux, light application time 10h/d, under the condition that temperature is 24 ± 3 ℃, cultivate 15d, finish the laundering period gradually of group training seedling.Group training seedling is replaced with sheep excrement leachate with culture fluid after the laundering period finishes gradually in same reactor, do not change the submergence frequency of reactor, cultivates 15d under natural environment, finishes the domestication stage of hardening.
After hardening is finished the dendrobium candidum seedling taken out directly to be transplanted to and carry out daily cultivation management in the dendrobe cultivation dedicated substrate.Survival rate is 100%.
Embodiment 2
(1) explant is selected: the dendrobium candidum fruit that sterilization treatment is good cuts an osculum, seed is scattering into sterilize intermittently in the submergence culture apparatus.
(2) liquid culture: intermittently the parameter of immersion system is: inoculum density: 0.5 fruit folder/L; Submergence frequency: 5 min/6h; Incubation time: 90 d; Cultivating with culture medium prescription is 1/2Ms, 0.2mg/L NAA, 30 g/L sucrose, 20 g/L bananas juice or murphy juices, and pH 6.2; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
(3) hardening: the culture fluid of in same reactor aforesaid liquid being cultivated is replaced with and reduces the sucrose concentration culture fluid gradually, do not change the submergence frequency of reactor, at intensity of illumination 1800 ~ 2000 lux, light application time 14 h/d, 24 ± 3 ℃ of temperature are cultivated the laundering period gradually that 25d finishes group training seedling.Group training seedling is replaced with sheep excrement leachate with culture fluid after the laundering period finishes gradually in same reactor, do not change the submergence frequency of reactor, cultivates 20d under natural environment, finishes the domestication stage of hardening.
After hardening is finished the dendrobium candidum seedling taken out directly to be transplanted to and carry out daily cultivation management in the dendrobe cultivation dedicated substrate.Survival rate is 100%.
Embodiment 3
(1) explant is selected: nothing deformity and vitrified protocorm of aseptic seed after planting being bred a generation are seeded in submergence at the intermittence culture apparatus.
(2) liquid culture: intermittently the parameter of immersion system is: inoculum density: 2.5g/L; Submergence frequency: 5 min/6h; Incubation time: 80d; Cultivating with culture medium prescription is 1/2Ms, 0.1mg/L NAA, 30 g/L sucrose, 20 g/L bananas juice or murphy juices, and pH 6.2; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
(3) hardening: the culture fluid of in same reactor aforesaid liquid being cultivated is replaced with and reduces the sucrose concentration culture fluid gradually, do not change the submergence frequency of reactor, at intensity of illumination 1800 ~ 2000 lux, light application time 11 h/d, 24 ± 3 ℃ of temperature, cultivate 17d, finish the laundering period gradually of group training seedling.Group training seedling is replaced with sheep excrement leachate with culture fluid after the laundering period finishes gradually in same reactor, do not change the submergence frequency of reactor, cultivates 16d under natural environment, finishes the domestication stage of hardening.
After hardening is finished the dendrobium candidum seedling taken out directly to be transplanted to and carry out daily cultivation management in the dendrobe cultivation dedicated substrate.Survival rate is 100%.
Embodiment 4
(1) explant is selected: will breed for four generations and induce the dendrobium candidum seedling that grows 1 ~ 3 leaf that obtains to be seeded in submergence at the intermittence culture apparatus with interior protocorm.
(2) liquid culture: intermittently the parameter of immersion system is: inoculum density: 60 strains/L; Submergence frequency: 5 min/6h; Incubation time: 75 d; Cultivating with culture medium prescription is 1/2Ms, 0.1mg/L NAA, 30 g/L sucrose, 10 g/L bananas juice or murphy juices, and pH 6.2; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
(3) hardening: the culture fluid of in same reactor aforesaid liquid being cultivated is replaced with and reduces the sucrose concentration culture fluid gradually, do not change the submergence frequency of reactor, at intensity of illumination 1800 ~ 2000 lux, light application time 12 h/d, 24 ± 3 ℃ of temperature, cultivate 20d, finish the laundering period gradually of group training seedling.Group training seedling is replaced with the macroelement culture fluid with culture fluid after the laundering period finishes gradually in same reactor, do not change the submergence frequency of reactor, cultivates 16d under natural environment, finishes the domestication stage of hardening.
After hardening is finished the dendrobium candidum seedling taken out directly to be transplanted to and carry out daily cultivation management in the dendrobe cultivation dedicated substrate.Survival rate is 100%.
Embodiment 5
(1) explant is selected: will breed for four generations and induce the dendrobium candidum seedling that grows 1 ~ 3 leaf that obtains to be seeded in submergence at the intermittence culture apparatus with interior protocorm.
(2) liquid culture: intermittently the parameter of immersion system is: inoculum density: 60 strains/L; Submergence frequency: 10min/6h; Incubation time: 90 d; Cultivating with culture medium prescription is 1/2Ms, 0.4mg/L NAA, 30 g/L sucrose, 20 g/L murphy juices, and pH 6.0; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
(3) hardening: the culture fluid of in same reactor aforesaid liquid being cultivated is replaced with and reduces the sucrose concentration culture fluid gradually, do not change the submergence frequency of reactor, at intensity of illumination 1800 ~ 2000 lux, light application time 13 h/d, 24 ± 3 ℃ of temperature, cultivate 22d, finish the laundering period gradually of group training seedling.Group training seedling is replaced with the macroelement culture fluid with culture fluid after the laundering period finishes gradually in same reactor, do not change the submergence frequency of reactor, cultivates 17d under natural environment, finishes the domestication stage of hardening.
After hardening is finished the dendrobium candidum seedling taken out directly to be transplanted to and carry out daily cultivation management in the dendrobe cultivation dedicated substrate.Survival rate is 100%.
Embodiment 6
(1) explant is selected: will breed for four generations and induce the dendrobium candidum seedling that grows 1 ~ 3 leaf that obtains to be seeded in submergence at the intermittence culture apparatus with interior protocorm.
(2) liquid culture: intermittently the parameter of immersion system is: inoculum density: 50 strains/L; Submergence frequency: 5 min/6h; Incubation time: 120 d; Cultivating with culture medium prescription is 1/2Ms, 0.2mg/L NAA, 30g/L sucrose, 10g/L murphy juice, and pH 6.2; Condition of culture is: intensity of illumination 1500-1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
(3) hardening: the culture fluid of in same reactor aforesaid liquid being cultivated is replaced with and reduces the sucrose concentration culture fluid gradually, do not change the submergence frequency of reactor, at intensity of illumination 1800 ~ 2000 lux, light application time 14 h/d, 24 ± 3 ℃ of temperature, cultivate 24d, finish the laundering period gradually of group training seedling.Group training seedling is replaced with the macroelement culture fluid with culture fluid after the laundering period finishes gradually in same reactor, do not change the submergence frequency of reactor, cultivates 19d under natural environment, finishes the domestication stage of hardening.
After hardening is finished the dendrobium candidum seedling taken out directly to be transplanted to and carry out daily cultivation management in the dendrobe cultivation dedicated substrate.Survival rate is 100%.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (9)
1. a seedlings of Dendrobium officinale production and hardening method for synchronous comprise explant selection, seedling incubation step, hardening step, it is characterized in that: utilize intermittently immersion system to carry out seedlings of Dendrobium officinale and cultivate with the hardening step and carry out synchronously.
2. a kind of seedlings of Dendrobium officinale production according to claim 1 and hardening method for synchronous, it is characterized in that: concrete steps are as follows, the selection of (1) explant: cultivate selected explant following any:
Seed;
Protocorm;
Group training seedling;
Seedling is cultivated: utilize intermittently immersion system, adopt the liquid culture mode, carry out the cultivation of seedlings of Dendrobium officinale;
Hardening: after candidum tissue culturing seedling was cultivated and finished, the culture fluid that will carry out the seedling cultivation at same intermittence in the immersion system was replaced with the hardening nutrient solution, does not change under the submergence frequency condition, finishes the hardening process after cultivation a period of time.
3. a kind of seedlings of Dendrobium officinale production according to claim 2 and hardening method for synchronous is characterized in that: the selection of explant in the described step (1): the aseptic stem of noble dendrobium seed that described seed obtains after the surface sterilization sterilization treatment for the fruit folder; Described protocorm is for breeding for four generations with interior nothing deformity and vitrified protocorm; Described group of training seedling then induced the dendrobium candidum seedling that grows 1 ~ 3 leaf that obtains for breeding for four generations with interior protocorm.
4. according to claim 2 seedlings of Dendrobium officinale production and hardening method for synchronous, it is characterized in that: the device that carries out described seedling cultivation is the immersion system at intermittence, described intermittence, the culture parameters of bath unit was: the submergence frequency: greater than 3min/12h less than 20min/1h, preferred 5 ~ 10 min/6 ~ 8h; Inoculum density: 1.. seed: 0.1 ~ 2 fruit pod/L, preferred 0.5 ~ 1/L; 2.. the preferred 2.5 ~ 4g/L of protocorm: 2 ~ 5g/L; 3.. group training seedling: 40 ~ 100 strains/L, preferred 50 ~ 80 strains/L; Incubation time: 60 ~ 120 d, preferred 75 ~ 90 d; Cultivation is the sucrose of 1/2Ms, 0.1 ~ 0.5mg/L methyl NAA, 30g/L, bananas juice or the murphy juice of 10 ~ 20g/L with component and the content of culture fluid prescription, pH:5.0 ~ 7.0, preferred 6.0 ~ 6.4; Condition of culture is: intensity of illumination 1500 ~ 1800 lux, light application time 12 h/d, 25 ± 1 ℃ of temperature.
5. a kind of seedlings of Dendrobium officinale production according to claim 2 and hardening method for synchronous, it is characterized in that: described step (3) hardening is divided two stages: (1) is the laundering period gradually; (2) the domestication stage.
6. a kind of seedlings of Dendrobium officinale production according to claim 6 and hardening method for synchronous, the described hardening stage (1) is the laundering period gradually, the hardening culture fluid that uses is for reducing the culture fluid of the described liquid culture of sugared content gradually, culture environment is: intensity of illumination 1800 ~ 2000 lux, light application time 10 ~ 14 h/d, 24 ± 3 ℃ of temperature, incubation time is: 5 ~ 35d, preferred 15 ~ 25d; Reactor submergence frequency is constant.
7. a kind of seedlings of Dendrobium officinale production according to claim 7 and hardening method for synchronous, in (2) the domestication stage in described domestication stage, the hardening culture fluid of use is mainly but is not limited to sheep excrement leachate or macroelement culture fluid.
8. according to claim 6-8 each described a kind of seedlings of Dendrobium officinale production and hardening method for synchronous, it is characterized in that: the parameter in domestication stage is set at: the domestication environment is natural environment, 15 ~ 30 ℃ of domestication times of temperature are 10 ~ 30d, preferred 15 ~ 20d, and reactor submergence frequency is constant.
9. a kind of seedlings of Dendrobium officinale production according to claim 8 and hardening method for synchronous, it is characterized in that: described sheep excrement leachate preparation: the sheep excrement that dries is ground, behind 1/5 ~ 1/15 adding running water according to the shared mass fraction of sheep excrement, heating was boiled 0.5 ~ 4 hour, preferred 2 ~ 3 hours, the cooling back was filtered with 10 ~ 60 purpose sub-sieves and is removed solid impurity, preferred 32 ~ 50 orders, boil or 121 ℃ of 15 minutes moist heat sterilizations, use the cooling back; The prescription of described macroelement culture fluid is:
NH
4NO
3……………………………1.65g/L
KNO
3………………………………1.90g/L
MgSO
4·7H
2O?……………………0.37g/L
KH
2PO
4……………………………0.17g/L。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104705188A (en) * | 2015-04-03 | 2015-06-17 | 南京工业大学 | Method for culturing dendrobium candidum protocorms |
| CN104823824A (en) * | 2015-05-04 | 2015-08-12 | 上海交通大学 | Temporary immersion type culture method and culture device for dendrobe |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104705188A (en) * | 2015-04-03 | 2015-06-17 | 南京工业大学 | Method for culturing dendrobium candidum protocorms |
| CN104823824A (en) * | 2015-05-04 | 2015-08-12 | 上海交通大学 | Temporary immersion type culture method and culture device for dendrobe |
| CN104823824B (en) * | 2015-05-04 | 2017-07-28 | 上海交通大学 | The intermittent immersed cultural method and culture apparatus of the stem of noble dendrobium |
| CN104855293A (en) * | 2015-06-15 | 2015-08-26 | 泉州正和堂生物科技有限公司 | Clone production process for dendrobium officinale kmuraet migo officinal protocorm by adopting large-scale bioreactor |
| CN105532473A (en) * | 2016-01-28 | 2016-05-04 | 遵义医学院 | Tissue culture method of dendrobium stem seedlings |
| CN106234188A (en) * | 2016-08-29 | 2016-12-21 | 江西九草铁皮石斛科技协同创新有限公司 | A kind of method of candidum tissue culturing seedling tidal type seedling exercising |
| CN110972944A (en) * | 2019-12-18 | 2020-04-10 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
| CN110972944B (en) * | 2019-12-18 | 2022-12-06 | 南京师范大学 | Method for intermittently immersing dendrobium nobile seedlings |
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