CN103688860A - Culture medium for rapid propagation and seedling of dendrobium officinale protocorm like-bodies and tissue culture method - Google Patents
Culture medium for rapid propagation and seedling of dendrobium officinale protocorm like-bodies and tissue culture method Download PDFInfo
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Abstract
The invention relates to a culture medium for rapid propagation and seedling of dendrobium officinale protocorm like-bodies and a tissue culture method. The culture medium series includes a germination culture medium A, a propagation inducing culture medium B, a differentiation culture medium C, a strong seedling culture medium D or E and a rooting culture medium F. The tissue culture method disclosed by the invention comprises the steps of cultivating seeds in the germination culture medium, and after protocorms are germinated from the seeds, inducing in the protocorm propagation inducing culture medium to generate a callus tissue which further develops into protocorm like-bodies; differentiating the protocorm like-bodies to obtain young seedlings; transferring the young seedlings to the strong seedling culture medium until obtaining strong seedlings; and cultivating the strong seedlings in the rooting culture medium to obtain at least 3-4 developed strong root systems which develop into dendrobium officinale seedlings which are 6-8cm high. The culture medium disclosed by the invention can shorten cultivation duration of dendrobium officinale, and is not only simple to operate but also free from time and space limitation; and the protocorm like-bodies continuously divide and propagate to guarantee annual production, so as to facilitate industrial production and application on a large scale.
Description
Technical field
The invention belongs to plant biotechnology field, relate to a kind of propagation method of medicinal plant seedling, relate in particular to a kind of tissue culture method of dendrobium candidum Fast-propagation.
Background technology
Dendrobium candidum claims again ribbed hedyotis herb, is a kind of rare medicinal plant and famous and precious decorative indoor plant.The medicinal material valuable in imminent danger that belongs to first-grade state protection, successive dynasties medical science classics are all given it for " product in medicine ".In the < < Compendium of Materia Medica > > of Ming Dynasty's Li Shizhen (1518-1593 A.D.), record: the stem of noble dendrobium removes gas under numbness, the win of benefit internal organ consumptive disease is thin reinforcing yin essence benefit essence.Take for a long time, thick stomach, never sufficient in benefit.Carry out stomach Qi, longue meat, intelligence development, except frightened, is made light of one's life by commiting suicide and is prolonged life.In the classical < < of Taoism medical science Taoist Scriptures > >, dendrobium candidum ranks first of China's " nine immortal grass ".The medicinal part of dendrobium candidum is fresh or dry stem, and modern medicine study shows: dendrobium candidum can significantly improve body's immunity, anti-ageing, antifatigue, and resistance to anoxic, has the auxiliary effects such as tumour that suppress.
Seeds of Dendrobium Candidum is thin as dust, and in a capsule, contained seed reaches 1,000,000 more than.Owing to lacking endosperm, seed need could be sprouted with mycosymbiosis, germination rate very low (less than 5%) under natural conditions.Dendrobium candidum natural propagation power is extremely low, and Sterile culture is more difficult again, and for many years to the collection capacity of dendrobium candidum much larger than its amount of growth, wild dendrobium candidum natural propagation be on the verge of disappearance.For development artificial cultivation, meet the need of market, some appreciable varieties in medicinal dendrobium have become the emphasis of current tissue rapid propagation research.
Study on tissue culture about dendrobium candidum has had relevant report at present, but tissue is cultivated required high cost and long problem of test tube seedling cycle is the bottleneck of the large production of restriction dendrobium candidum industrialization.Therefore need a kind of tissue culturing system that can Fast-propagation dendrobium candidum badly, for promoting artificial culture technology and large-scale industrialized growing seedlings established theory and practice basis.
Summary of the invention
For the tissue of current dendrobium candidum, cultivate cost higher, from aseptic sowing seeds to, obtain the longer problem of process of seedling, the object of the invention is to the tissue culturing system that designs a kind of energy Fast-propagation dendrobium candidum and can produce in the anniversary, for factorial seedling growth provides technical support.
A kind of series of the special culture media by protocorms of dendrobium candidum Fast-propagation seedling of the present invention, comprise germination medium A, proliferation-inducing medium B, differential medium C, strong seedling culture base D or E and root media F, the content of each component of each medium in 1L medium is respectively:
(1) culture medium A: NH
4nO
3: 1600-2000mg, KNO
3: 1800-2300mg, MgSO
47H
2o:330-380mg, KH
2pO
4: 150-180mg, CaCl
2: 330-360mg, KI:0.65-0.85mg, H
3bO
3: 6.0-6.6mg, MnSO
44H
2o:22.0-25.0mg, ZnSO
47H
2o:8.5-9.0mg, Na
2moO
42H
2o:0.18-0.25mg, CuSO
45H
2o:0.015-0.025mg, CoCl
26H
2o:0.015-0.025mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, inositol 80-150mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine 1.7-2.5mg, agar 6.5-7.5g, white granulated sugar 20-30g.
(2) medium B:(NH
4)
2sO
4: 460-480mg, KNO
3: 2800-2850mg, MgSO
47H
2o:175-185mg, KH
2pO
4: 400-450mg, CaCl
2: 125-140mg, KI:0.65-0.85mg, H
3bO
3: 1.6-1.9mg, MnSO
44H
2o:4.0-4.5mg, ZnSO
47H
2o:1.3-1.5mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, methyl α-naphthyl acetate 0.1-0.5mg, 6-benzyl aminopurine 0.2-1.0mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g.
(3) culture medium C: (NH
4)
2sO
4: 460-480mg, KNO
3: 2800-2850mg, MgSO
47H
2o:175-185mg, KH
2pO
4: 400-450mg, CaCl
2: 125-140mg, KI:0.65-0.85mg, H
3bO
3: 1.6-1.9mg, MnSO
44H
2o:4.0-4.5mg, ZnSO
47H
2o:1.3-1.5mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, methyl α-naphthyl acetate 0.1-0.5mg, 6-benzyl aminopurine 0.1-0.5mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g.
(4) medium D:1-5g spends No. two, treasured, 0.1-0.8g caseinhydrolysate, methyl α-naphthyl acetate 0.1-0.5mg, 6-benzyl aminopurine 0.1-0.5mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g.
(5) medium E:(NH
4)
2sO
4: 128-135mg, KNO
3: 1800-2500mg, MgSO
47H
2o:250-300mg, NaH
2pO
4h
2o:130-160mg, CaCl
2: 150-170mg, KI:0.65-0.85mg, H
3bO
3: 2.3-3.0mg, MnSO
44H
2o:9.5-11.0mg, ZnSO
47H
2o:1.6-2.2mg, Na
2moO
42H
2o:0.18-0.25mg, CuSO
45H
2o:0.015-0.025mg, CoCl
26H
2o:0.015-0.025mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, inositol 80-150mg, nicotinic acid 0.9-1.6mg, puridoxine hydrochloride 1.0-1.8mg, thiamine hydrochloride 9.6-10.5mg, methyl α-naphthyl acetate 0.3-0.8mg, 6-benzyl aminopurine 0.4-1.0mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g.
(6) medium F:NH
4nO
3: 800-830mg, KNO
3: 950-1000mg, MgSO
47H
2o:175-190mg, KH
2pO
4: 60-85mg, CaCl
2: 160-250mg, KI:0.65-0.85mg, H
3bO
3: 6.0-6.6mg, MnSO
44H
2o:22.0-25.0mg, ZnSO
47H
2o:8.5-9.0mg, Na
2moO
42H
2o:0.18-0.25mg, CuSO
45H
2o:0.015-0.025mg, CoCl
26H
2o:0.015-0.025mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, inositol 80-150mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine 1.7-2.5mg, methyl α-naphthyl acetate 0.1-0.8mg, heteroauxin 0.2-1.0mg, active carbon 0.5-1.5mg, banana 100-150g, agar 6.5-7.5g, white granulated sugar 20-30g.
The special culture media series formula of above-mentioned protocorms of dendrobium candidum Fast-propagation seedling, this formulated process following (1L medium):
(1) measure about 500ml water and put into water bath heating;
(2) take 6.5-7.5g agar powder;
(3) when water temperature reaches 80 ℃, add agar powder, stir, be heated to boiling;
(4) add every kind of needed medicine of medium;
(5) while preparing medium B, C, D, E, F, take 80-150g potato or banana, be cut into fritter and squeeze the juice with juice extractor;
(6) murphy juice having squeezed or bananas juice are filtered in water bath;
(7) while preparing medium F, add active carbon 0.5-1.5mg;
(8) add white granulated sugar 20-30g;
(9) after medicine and the whole dissolvings of white granulated sugar, adding water is settled to 1L and stirs;
(10) with pH test paper adjust pH, be 5.8-6.2;
(11) packing, pours the medium preparing into tissue culture bottle, and pouring volume, depending on tissue culture bottle amount of capacity, is generally 1/5 left and right of container;
(12) high pressure steam sterilization, 121 ℃, 1.5Mpa, 20min;
(13) gone out that to put into transfer room standing for the medium of bacterium, allowed its cooling after coagulation.
The invention also discloses and use above-mentioned medium series by the tissue culture method of protocorms of dendrobium candidum Fast-propagation seedling, its step is as follows:
(1) axenic germination of Seeds of Dendrobium Candidum
After being rinsed well with running water, ripe seed puts into superclean bench, under the aseptic condition of superclean bench, seed is put into successively in 75% alcoholic solution and soaks 1min, in 4% liquor natrii hypochloritis, soak 20min, use again rinsed with sterile water 3 times, each 1min.The seed of handling well is at one end cut to an osculum with scalpel, with tweezers, clamp the other end of seed Powdered embryo is evenly sowed in culture medium A.It is 25 ℃ that tissue culture bottle is placed in to temperature, under the environment of unglazed photograph, cultivate and every day to its culture environment ozone sterilization 1h.About about 30 days, seed germination, now tissue culture bottle being placed in to temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h, and every day is to its culture environment ozone sterilization 1h.About 45 days, the Seed Development protocorm of sprouting.
(2) propagation of dendrobium candidum protocorm
On superclean bench, with the protocorm that spoon forms seed germination, be transferred to uniformly on medium B.It is 25 ℃ that tissue culture bottle is placed in to temperature, illumination 2000lx, under the environment of illumination every day 12h, cultivate and every day to its culture environment ozone sterilization 1h.The protocorm can be observed in tissue culture bottle grows faint yellow callus, and within 45 days, callus continuous division growth in left and right forms protocorms (as shown in Figure 2).
(3) differentiation of protocorms of dendrobium candidum
On superclean bench, with spoon, protocorms chosen and be transferred in culture medium C uniformly.It is 25 ℃ that tissue culture bottle is placed in to temperature, illumination 2000lx, under the environment of illumination every day 12h, cultivate and every day to its culture environment ozone sterilization 1h.The protocorms that can observe about 60-90 days in tissue culture bottle passes through the seedling (as shown in Figure 3) that is differentiated to form high about 1-2cm.
(4) strong sprout of dendrobium candidum seedling
On superclean bench, with the careful folder of tweezers, go out the seedling that protocorms is differentiated to form and be placed on prior large culture dish of sterilizing, and seedling is inserted in medium D.It is 25 ℃ that tissue culture bottle is placed in to temperature, illumination 2000lx, under the environment of illumination every day 12h, cultivate and every day to its culture environment ozone sterilization 1h.The seedling that can observe about 60-90 days in tissue culture bottle passes through the seedlings (as shown in Figure 4) that form the about 3-4cm of the basically identical height of regularity strong sprout.
(5) dendrobium candidum seedlings takes root
On superclean bench, with the careful folder of tweezers, go out dendrobium candidum seedlings and be placed on prior large culture dish of sterilizing, choosing and inserting in medium F one one of seedlings.It is 25 ℃ that tissue culture bottle is placed in to temperature, illumination 2000lx, under the environment of illumination every day 12h, cultivate and every day to its culture environment ozone sterilization 1h.The seedlings that can observe in tissue culture bottle about 60-90 days grow fine, and regularity is suitable, and blade is large and thick, is oblong, dark green leaf color, and every seedling has the flourishing healthy and strong root system of 3-4 bar at least, plant height reaches 6-8cm(as shown in Figure 5).
(6) hardening of candidum tissue culturing seedling and transplanting
Seedling in tissue culture bottle taken out after natural daylight lower refining seedling one week to bottle seedling and clean root medium, transplanting to peat soil and the liver moss volume ratio mixed-matrix that is 1:1, survival rate is more than 90%.
In order guarantee to obtain in process of production more seedling simultaneously for the anniversary of can realizing produces, step (2) can repeat, protocorms in Subculture constantly division growth produce new protocorms.
In step (4), in order to obtain in process of production more seedling, can select the seedling that protocorms in step (3) is differentiated to form to insert in medium E.This medium can significantly promote the generation of seedling Multiple Buds, thereby obtains more seedlings.
Above-mentioned a kind of tissue culture method by protocorms of dendrobium candidum Fast-propagation seedling, design object is clear and definite, has improved the rate of increase and the transplanting survival rate of dendrobium candidum clone seedling.Method of the present invention has not only shortened the cultivation cycle of dendrobium candidum, and simple to operate, not produced by the restriction anniversary in time place, and development dendrobium candidum industry is significant.
Accompanying drawing explanation
Fig. 1 is group training flow chart.
Fig. 2 is protocorms of dendrobium candidum photo.
Fig. 3 is dendrobium candidum seedling (before strong sprout) photo.
Fig. 4 is dendrobium candidum seedling (after strong sprout) photo.
Fig. 5 is dendrobium candidum seedling photo.
Embodiment
Below example further illustrates the present invention by experiment.
Experiment one:
1, preparation culture medium A, B, C, D, E, F.
2, choose wild dendrobium candidum mature seed, after being rinsed well with running water, seed puts into superclean bench, under the aseptic condition of superclean bench, seed is put into successively in 75% alcoholic solution and soaks 1min, in 4% liquor natrii hypochloritis, soak 20min, use again rinsed with sterile water 3 times, each 1min.The seed of handling well is cut to an osculum with scalpel, with tweezers, pulverous kind of grain sowed in containing NH
4nO
3: 1600mg, KNO
3: 1800mg, MgSO
47H
2o:330mg, KH
2pO
4: 150mg, CaCl
2: 330mg, KI:0.65mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.18mg, CuSO
45H
2o:0.015mg, CoCl
26H
2o:0.015mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the culture medium A of O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg, glycine 1.7mg, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, under the environment of unglazed photograph, cultivates.After a period of time, seed germination, now medium being placed in to temperature is 25 ℃, illumination 2000lx cultivates under the environment of illumination every day 12h.About 70 days, the Seed Development protocorm of sprouting.
3, protocorm seed germination being formed proceeds to containing (NH under aseptic condition
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:175mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.65mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.0mg, ZnSO
47H
2o:1.3mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the proliferated culture medium B of O:27.5mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.6mg, potato 80g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorm of observing about 20 days in tissue culture bottle produces faint yellow callus, the continuous division growth of callus, and within 70 days, left and right protocorm is bred at double as protocorms.
4, the protocorms being formed by protocorm callus is proceeded under aseptic condition containing (NH
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:175mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.65mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.0mg, ZnSO
47H
2o:1.3mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the differential medium C of O:27.5mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, methyl α-naphthyl acetate 0.1mg, 6-benzyl aminopurine 0.1mg, potato 80g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorms of observing about 90 days in tissue culture bottle passes through the seedling that is differentiated to form high about 1-2cm.
5, the seedling of differentiation is proceeded under aseptic condition containing 2g and spend in the strong seedling culture base D of No. two, treasured, 0.2g caseinhydrolysate, methyl α-naphthyl acetate 0.1mg, 6-benzyl aminopurine 0.1mg, potato 80g, agar 6.5g, white granulated sugar 20g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedling of observing about 90 days in tissue culture bottle passes through the seedlings that form the about 3-4cm of the basically identical height of regularity strong sprout.
6, the seedlings that form through strong sprout are transferred to containing NH under aseptic condition
4nO
3: 800mg, KNO
3: 950mg, MgSO
47H
2o:175mg, KH
2pO
4: 60mg, CaCl
2: 160mg, KI:0.65mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.18mg, CuSO
45H
2o:0.015mg, CoCl
26H
2o:0.015mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the root media F of O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg, glycine 1.7mg, methyl α-naphthyl acetate 0.2mg, heteroauxin 0.2mg, active carbon 0.5mg, banana 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedlings of observing in tissue culture bottle about 90 days grow fine, and regularity is suitable, and blade is large and thick, are oblong, dark green leaf color, and every seedling has the flourishing healthy and strong root system of 3-4 bar at least, and plant height reaches 6-8cm.
7, the seedling in tissue culture bottle taken out after natural daylight lower refining seedling one week to bottle seedling and clean root medium, transplanting to peat soil and the liver moss volume ratio mixed-matrix that is 1:1, survival rate is more than 90%.
Experiment two:
1, preparation culture medium A, B, C, D, E, F.
2, choose the dendrobium candidum mature seed of artificial culture, after being rinsed well with running water, seed puts into superclean bench, under the aseptic condition of superclean bench, seed is put into successively in 75% alcoholic solution and soaks 1min, in 4% liquor natrii hypochloritis, soak 20min, use again rinsed with sterile water 3 times, each 1min.The seed of handling well is cut to an osculum with scalpel, with tweezers, pulverous kind of grain sowed in containing NH
4nO
3: 1600mg, KNO
3: 1800mg, MgSO
47H
2o:330mg, KH
2pO
4: 150mg, CaCl
2: 330mg, KI:0.65mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.18mg, CuSO
45H
2o:0.015mg, CoCl
26H
2o:0.015mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the culture medium A of O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg, glycine 1.7mg, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, under the environment of unglazed photograph, cultivates.After a period of time, seed germination, now medium being placed in to temperature is 25 ℃, illumination 2000lx cultivates under the environment of illumination every day 12h.About 70 days, the Seed Development protocorm of sprouting.
3, protocorm seed germination being formed proceeds to containing (NH under aseptic condition
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:175mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.65mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.0mg, ZnSO
47H
2o:1.3mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the proliferated culture medium B of O:27.5mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.6mg, potato 80g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorm of observing about 20 days in tissue culture bottle produces faint yellow callus, the continuous division growth of callus, and within 70 days, left and right protocorm is bred at double as protocorms.
4, the protocorms being formed by protocorm callus is proceeded under aseptic condition containing (NH
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:175mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.65mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.0mg, ZnSO
47H
2o:1.3mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the differential medium C of O:27.5mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, methyl α-naphthyl acetate 0.1mg, 6-benzyl aminopurine 0.1mg, potato 80g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorms of observing about 90 days in tissue culture bottle passes through the seedling that is differentiated to form high about 1-2cm.
5, the seedling of differentiation is proceeded under aseptic condition containing (NH
4)
2sO
4: 120mg, KNO
3: 1800mg, MgSO
47H
2o:230mg, NaH
2pO
4h
2o:150mg, CaCl
2: 150mg, KI:0.65mg, H
3bO
3: 2.3mg, MnSO
44H
2o:9.5mg, ZnSO
47H
2o:1.6mg, Na
2moO
42H
2o:0.18mg, CuSO
45H
2o:0.015mg, CoCl
26H
2o:0.015mg, Na
2-EDTA:37.00mg, FeSO
47H
2in the strong seedling culture base E of O:27.5mg, inositol 80mg, nicotinic acid 0.9mg, puridoxine hydrochloride 1.0mg, thiamine hydrochloride 9.6mg, methyl α-naphthyl acetate 0.5mg, 6-benzyl aminopurine 0.1mg, potato 80g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedling of observing about 90 days in tissue culture bottle passes through the seedlings that form the about 3-4cm of the basically identical height of regularity strong sprout, and every strain seedlings at least produce 2-3 differentiation seedling out.
6, the seedlings that form through strong sprout are transferred to containing NH under aseptic condition
4nO
3: 800mg, KNO
3: 950mg, MgSO
47H
2o:175mg, KH
2pO
4: 60mg, CaCl
2: 160mg, KI:0.65mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.18mg, CuSO
45H
2o:0.015mg, CoCl
26H
2o:0.015mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the root media medium E of O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg, glycine 1.7mg, methyl α-naphthyl acetate 0.2mg, heteroauxin 0.2mg, active carbon 0.5mg, banana 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedlings of observing in tissue culture bottle about 900 days grow fine, and regularity is suitable, and blade is large and thick, are oblong, dark green leaf color, and every seedling has the flourishing healthy and strong root system of 3-4 bar at least, and plant height reaches 6-8cm.
7, the seedling in tissue culture bottle taken out after natural daylight lower refining seedling one week to bottle seedling and clean root medium, transplanting to peat soil and the liver moss volume ratio mixed-matrix that is 1:1, survival rate is more than 90%.
Experiment three:
1, preparation culture medium A, B, C, D, E, F.
2, choose the dendrobium candidum mature seed of artificial cultivation, after being rinsed well with running water, seed puts into superclean bench, under the aseptic condition of superclean bench, seed is put into successively in 75% alcoholic solution and soaks 1min, in 4% liquor natrii hypochloritis, soak 20min, use again rinsed with sterile water 3 times, each 1min.The seed of handling well is cut to an osculum with scalpel, with tweezers, pulverous kind of grain sowed in containing NH
4nO
3: 1600mg, KNO
3: 1800mg, MgSO
47H
2o:370mg, KH
2pO
4: 180mg, CaCl
2: 330mg, KI:0.85mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.25mg, CuSO
45H
2o:0.025mg, CoCl
26H
2o:0.025mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the culture medium A of O:27.8mg, inositol 100mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.08mg, glycine 1.8mg, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, under the environment of unglazed photograph, cultivates.After a period of time, seed germination, now medium being placed in to temperature is 25 ℃, illumination 2000lx cultivates under the environment of illumination every day 12h.About 50 days, the Seed Development protocorm of sprouting.
3, protocorm seed germination being formed proceeds to containing (NH under aseptic condition
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:180mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.80mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.3mg, ZnSO
47H
2o:1.6mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the proliferated culture medium B of O:27.8mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.6mg, potato 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorm of observing about 15 days in tissue culture bottle produces faint yellow callus, the continuous division growth of callus, and within 60 days, left and right protocorm is bred at double as protocorms.
4, the protocorms being formed by protocorm callus is proceeded under aseptic condition containing (NH
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:180mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.80mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.3mg, ZnSO
47H
2o:1.6mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the differential medium C of O:27.8mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.2mg, potato 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorms of observing about 60 days in tissue culture bottle passes through the seedling that is differentiated to form high about 1-2cm.
5, the seedling of differentiation is proceeded under aseptic condition containing 3g and spend in the strong seedling culture base D of No. two, treasured, 0.4g caseinhydrolysate, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.2mg, potato 100g, agar 6.5g, white granulated sugar 20g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedling of observing about 70 days in tissue culture bottle passes through the seedlings that form the about 3-4cm of the basically identical height of regularity strong sprout.
6, the seedlings that form through strong sprout are transferred to containing NH under aseptic condition
4nO
3: 815mg, KNO
3: 950mg, MgSO
47H
2o:180mg, KH
2pO
4: 85mg, CaCl
2: 165mg, KI:0.85mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.25mg, CuSO
45H
2o:0.025mg, CoCl
26H
2o:0.025-mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the root media F of O:27.8mg, inositol 100mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.08mg, glycine 1.8mg, methyl α-naphthyl acetate 0.4mg, heteroauxin 0.4mg, active carbon 0.5mg, banana 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedlings of observing in tissue culture bottle about 60 days grow fine, and regularity is suitable, and blade is large and thick, are oblong, dark green leaf color, and every seedling has the flourishing healthy and strong root system of 3-4 bar at least, and plant height reaches 6-8cm.
7, the seedling in tissue culture bottle taken out after natural daylight lower refining seedling one week to bottle seedling and clean root medium, transplanting to peat soil and the liver moss volume ratio mixed-matrix that is 1:1, survival rate is more than 90%.
Experiment four:
1, preparation culture medium A, B, C, D, E, F.
2, choose wild dendrobium candidum mature seed, after being rinsed well with running water, seed puts into superclean bench, under the aseptic condition of superclean bench, seed is put into successively in 75% alcoholic solution and soaks 1min, in 4% liquor natrii hypochloritis, soak 20min, use again rinsed with sterile water 3 times, each 1min.The seed of handling well is cut to an osculum with scalpel, with tweezers, pulverous kind of grain sowed in containing NH
4nO
3: 1600mg, KNO
3: 1800mg, MgSO
47H
2o:370mg, KH
2pO
4: 180mg, CaCl
2: 330mg, KI:0.85mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.25mg, CuSO
45H
2o:0.025mg, CoCl
26H
2o:0.025mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the culture medium A of O:27.8mg, inositol 100mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.08mg, glycine 1.8mg, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, under the environment of unglazed photograph, cultivates.After a period of time, seed germination, now medium being placed in to temperature is 25 ℃, illumination 2000lx cultivates under the environment of illumination every day 12h.About 50 days, the Seed Development protocorm of sprouting.
3, protocorm seed germination being formed proceeds to containing (NH under aseptic condition
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:180mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.80mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.3mg, ZnSO
47H
2o:1.6mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the proliferated culture medium B of O:27.8mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.6mg, potato 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorm of observing about 15 days in tissue culture bottle produces faint yellow callus, the continuous division growth of callus, and within 60 days, left and right protocorm is bred at double as protocorms.
4, the protocorms being formed by protocorm callus is proceeded under aseptic condition containing (NH
4)
2sO
4: 460mg, KNO
3: 2800mg, MgSO
47H
2o:180mg, KH
2pO
4: 400mg, CaCl
2: 125mg, KI:0.80mg, H
3bO
3: 1.6mg, MnSO
44H
2o:4.3mg, ZnSO
47H
2o:1.6mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the differential medium C of O:27.8mg, nicotinic acid 0.5mg, puridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, methyl α-naphthyl acetate 0.2mg, 6-benzyl aminopurine 0.2mg, potato 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The protocorms of observing about 60 days in tissue culture bottle passes through the seedling that is differentiated to form high about 1-2cm.
5, the seedling of differentiation is proceeded under aseptic condition containing (NH
4)
2sO
4: 130mg, KNO
3: 2500mg, MgSO
47H
2o:250mg, NaH
2pO
4h
2o:150mg, CaCl
2: 150mg, KI:0.65mg, H
3bO
3: 2.8mg, MnSO
44H
2o:9.5mg, ZnSO
47H
2o:1.8mg, Na
2moO
42H
2o:0.25mg, CuSO
45H
2o:0.025mg, CoCl
26H
2o:0.025mg, Na
2-EDTA:37.3mg, FeSO
47H
2in the strong seedling culture base E of O:27.8mg, inositol 100mg, nicotinic acid 1.0mg, puridoxine hydrochloride 1.0mg, thiamine hydrochloride 9.6mg, methyl α-naphthyl acetate 0.5mg, 6-benzyl aminopurine 0.5mg, potato 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedling of observing about 70 days in tissue culture bottle passes through the seedlings that form the about 3-4cm of the basically identical height of regularity strong sprout, and every strain seedlings at least produce 2-3 differentiation seedling out.
6, the seedlings that form through strong sprout are transferred to containing NH under aseptic condition
4nO
3: 800mg, KNO
3: 950mg, MgSO
47H
2o:175mg, KH
2pO
4: 60mg, CaCl
2: 160mg, KI:0.65mg, H
3bO
3: 6.0mg, MnSO
44H
2o:22.0mg, ZnSO
47H
2o:8.5mg, Na
2moO
42H
2o:0.18mg, CuSO
45H
2o:0.015mg, CoCl
26H
2o:0.015mg, Na
2-EDTA:37.0mg, FeSO
47H
2in the root media medium F of O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, puridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg, glycine 1.7mg, methyl α-naphthyl acetate 0.2mg, heteroauxin 0.2mg, active carbon 0.5mg, banana 100g, agar 6.5g, white granulated sugar 30g.Being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h.The seedlings of observing in tissue culture bottle about 70 days grow fine, and regularity is suitable, and blade is large and thick, are oblong, dark green leaf color, and every seedling has the flourishing healthy and strong root system of 3-4 bar at least, and plant height reaches 6-8cm.
7, the seedling in tissue culture bottle taken out after natural daylight lower refining seedling one week to bottle seedling and clean root medium, transplanting to peat soil and the liver moss volume ratio mixed-matrix that is 1:1, survival rate is more than 90%.
Claims (4)
1. the special culture media by protocorms of dendrobium candidum Fast-propagation seedling is serial, it is characterized in that this medium series comprises germination medium A, proliferation-inducing medium B, differential medium C, strong seedling culture base D or E and root media F, in described each medium, component is in 1L medium, and content is respectively:
A. culture medium A: NH
4nO
3: 1600-2000mg, KNO
3: 1800-2300mg, MgSO
47H
2o:330-380mg, KH
2pO
4: 150-180mg, CaCl
2: 330-360mg, KI:0.65-0.85mg, H
3bO
3: 6.0-6.6mg, MnSO
44H
2o:22.0-25.0mg, ZnSO
47H
2o:8.5-9.0mg, Na
2moO
42H
2o:0.18-0.25mg, CuSO
45H
2o:0.015-0.025mg, CoCl
26H
2o:0.015-0.025mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, inositol 80-150mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine 1.7-2.5mg, agar 6.5-7.5g, white granulated sugar 20-30g;
B. medium B:(NH
4)
2sO
4: 460-480mg, KNO
3: 2800-2850mg, MgSO
47H
2o:175-185mg, KH
2pO
4: 400-450mg, CaCl
2: 125-140mg, KI:0.65-0.85mg, H
3bO
3: 1.6-1.9mg, MnSO
44H
2o:4.0-4.5mg, ZnSO
47H
2o:1.3-1.5mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, methyl α-naphthyl acetate 0.1-0.5mg, 6-benzyl aminopurine 0.2-1.0mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g;
C. culture medium C: (NH
4)
2sO
4: 460-480mg, KNO
3: 2800-2850mg, MgSO
47H
2o:175-185mg, KH
2pO
4: 400-450mg, CaCl
2: 125-140mg, KI:0.65-0.85mg, H
3bO
3: 1.6-1.9mg, MnSO
44H
2o:4.0-4.5mg, ZnSO
47H
2o:1.3-1.5mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, methyl α-naphthyl acetate 0.1-0.5mg, 6-benzyl aminopurine 0.1-0.5mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g;
D. medium D:1-5g spends No. two, treasured, 0.1-0.8g caseinhydrolysate, methyl α-naphthyl acetate 0.1-0.5mg, 6-benzyl aminopurine 0.1-0.5mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g;
E. medium E:(NH
4)
2sO
4: 128-135mg, KNO
3: 1800-2500mg, MgSO
47H
2o:250-300mg, NaH
2pO
4h
2o:130-160mg, CaCl
2: 150-170mg, KI:0.65-0.85mg, H
3bO
3: 2.3-3.0mg, MnSO
44H
2o:9.5-11.0mg, ZnSO
47H
2o:1.6-2.2mg, Na
2moO
42H
2o:0.18-0.25mg, CuSO
45H
2o:0.015-0.025mg, CoCl
26H
2o:0.015-0.025mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, inositol 80-150mg, nicotinic acid 0.9-1.6mg, puridoxine hydrochloride 1.0-1.8mg, thiamine hydrochloride 9.6-10.5mg, methyl α-naphthyl acetate 0.3-0.8mg, 6-benzyl aminopurine 0.4-1.0mg, potato 80-150g, agar 6.5-7.5g, white granulated sugar 20-30g;
F. medium F:NH
4nO
3: 800-830mg, KNO
3: 950-1000mg, MgSO
47H
2o:175-190mg, KH
2pO
4: 60-85mg, CaCl
2: 160-250mg, KI:0.65-0.85mg, H
3bO
3: 6.0-6.6mg, MnSO
44H
2o:22.0-25.0mg, ZnSO
47H
2o:8.5-9.0mg, Na
2moO
42H
2o:0.18-0.25mg, CuSO
45H
2o:0.015-0.025mg, CoCl
26H
2o:0.015-0.025mg, Na
2-EDTA:37.0-38.0mg, FeSO
47H
2o:27.5-28.5mg, inositol 80-150mg, nicotinic acid 0.2-0.6mg, puridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine 1.7-2.5mg,
Methyl α-naphthyl acetate 0.1-0.8mg, heteroauxin 0.2-1.0mg, active carbon 0.5-1.5mg, banana 100-150g, agar 6.5-7.5g, white granulated sugar 20-30g.
2. a compound method for medium series described in claim 1, is characterized in that process for preparation is in 1L medium, and step is as follows:
A. measure about 500ml water and put into water bath heating;
B. take 6.5-7.5g agar powder;
C. when water temperature reaches 80 ℃, add agar powder, stir, be heated to boiling;
D. add in claim 1 medicine described in each medium;
E. take 80-150g potato or banana, be cut into fritter and squeeze the juice with juice extractor;
F. the murphy juice having squeezed or bananas juice are filtered in water bath;
G. add active carbon 0.5-1.5mg;
H. add white granulated sugar 20-30g;
I. after medicine and the whole dissolvings of white granulated sugar, adding water is settled to 1L and stirs;
J. with pH test paper adjust pH, be 5.8-6.2;
K. packing, pours the medium preparing into tissue culture bottle, and pouring volume, depending on tissue culture bottle amount of capacity, is generally 1/5 left and right of container;
L. high pressure steam sterilization, 121 ℃, 1.5Mpa, 20min;
M. it is standing that the medium of bacterium of having gone out is put into transfer room, allows its cooling after coagulation;
Composition in when the medicine that steps d adds when preparation culture medium A, B, C, D, E, F corresponds to respectively culture medium A claimed in claim 1, B, C, D, E, F;
When preparation medium B, C, D, E, F, to carry out step e, f;
When preparation medium F, to carry out step g.
3. described in right to use requirement 1, medium series is by a tissue culture method for protocorms of dendrobium candidum Fast-propagation seedling, and its feature comprises the following steps:
(1) axenic germination of Seeds of Dendrobium Candidum
A. on superclean bench, Seeds of Dendrobium Candidum is used to alcohol-pickled 1min successively, the liquor natrii hypochloritis 20min that sterilizes, rinsed with sterile water 3 times, each 1min;
B. the seed after thorough disinfection rinsing is taken out, from one end of seed, cut, after incision, Powdered embryo is sowed in culture medium A uniformly;
C. being placed in temperature is 25 ℃, under the environment of unglazed photograph, cultivates;
D. every day is to its culture environment ozone sterilization 1h;
E.30 after day left and right seed germination, it is 25 ℃ that tissue culture bottle is placed in to temperature, and illumination 2000lx cultivates under the environment of illumination every day 12h, and every day is to its culture environment ozone sterilization 1h;
F.45 day left and right, the Seed Development protocorm of sprouting;
(2) propagation of dendrobium candidum protocorm
A. on superclean bench, with the protocorm that spoon forms seed germination, be transferred to uniformly on medium B;
B. being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h;
C. every day is to its culture environment ozone sterilization 1h;
D. protocorm grows faint yellow callus, and about 45 days, the continuous division growth of callus becomes protocorms;
(3) differentiation of protocorms of dendrobium candidum
A. on superclean bench, with spoon, protocorms chosen and be transferred to uniformly in culture medium C;
B. being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h;
C. every day is to its culture environment ozone sterilization 1h;
D.60-90 about sky, protocorms is divided into seedling;
(4) strong sprout of dendrobium candidum seedling
A. on superclean bench, with the careful folder of tweezers, go out the seedling that protocorms is differentiated to form and be placed on prior large culture dish of sterilizing, and seedling is inserted in medium D or E;
B. being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h;
C. every day is to its culture environment ozone sterilization 1h;
D.60-90 about sky, seedling is grown into the dendrobium candidum seedlings that grow fine;
(5) dendrobium candidum seedlings takes root
A. on superclean bench, with the careful folder of tweezers, going out dendrobium candidum seedlings is placed on prior large culture dish of sterilizing;
B. choosing and inserting in medium F one one of seedlings;
C. being placed in temperature is 25 ℃, and illumination 2000lx cultivates under the environment of illumination every day 12h;
D. every day is to its culture environment ozone sterilization 1h;
E.60-90 about sky, seedlings are grown into complete candidum tissue culturing seedling;
(6) hardening of candidum tissue culturing seedling and transplanting
A. the seedling in tissue culture bottle is placed on to natural daylight lower refining seedling one week;
B. bottle seedling is taken out and cleaned after root medium, transplant to peat soil and the liver moss volume ratio mixed-matrix that is 1:1, survival rate is more than 90%.
4. the tissue culture method by protocorms of dendrobium candidum Fast-propagation seedling according to claim 3, what it is characterized in that Seeds of Dendrobium Candidum collection that described step (1) is described is the dendrobium candidum of wild dendrobium candidum or artificial culture.
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| CN104542309A (en) * | 2015-02-06 | 2015-04-29 | 江西万茂科技有限公司 | Special culture medium for rooting and seedling strengthening of dendrobium officinale |
| CN105325287A (en) * | 2015-11-24 | 2016-02-17 | 长江大学 | Disinfection and inoculation method of microphyte seeds and pollen |
| CN106472306A (en) * | 2016-09-30 | 2017-03-08 | 南京仙草堂生物科技有限公司 | One kind begins to flourish Herba Dendrobii high quality seedling asexual clonal method for quickly breeding |
| CN110178734A (en) * | 2019-07-16 | 2019-08-30 | 中国农业科学院特产研究所 | A kind of the post-directed training base and post-directed training seedling establishment method of mountain orchid germination seed |
| CN110959528A (en) * | 2019-07-15 | 2020-04-07 | 浙江省农业科学院 | Culture medium for improving differentiation rate of protocorm-like bodies of dendrobium officinale and efficient seedling culture method |
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| CN104542309A (en) * | 2015-02-06 | 2015-04-29 | 江西万茂科技有限公司 | Special culture medium for rooting and seedling strengthening of dendrobium officinale |
| CN105325287A (en) * | 2015-11-24 | 2016-02-17 | 长江大学 | Disinfection and inoculation method of microphyte seeds and pollen |
| CN106472306A (en) * | 2016-09-30 | 2017-03-08 | 南京仙草堂生物科技有限公司 | One kind begins to flourish Herba Dendrobii high quality seedling asexual clonal method for quickly breeding |
| CN106472306B (en) * | 2016-09-30 | 2018-09-25 | 南京仙草堂生物科技有限公司 | One kind begins to flourish stem of noble dendrobium high quality seedling asexual clonal rapid propagation method |
| CN110959528A (en) * | 2019-07-15 | 2020-04-07 | 浙江省农业科学院 | Culture medium for improving differentiation rate of protocorm-like bodies of dendrobium officinale and efficient seedling culture method |
| CN110178734A (en) * | 2019-07-16 | 2019-08-30 | 中国农业科学院特产研究所 | A kind of the post-directed training base and post-directed training seedling establishment method of mountain orchid germination seed |
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