CN103688860B - 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 the propagation method of a kind of medicinal plants seedling, relate to specifically
And the tissue culture method of a kind of Herba Dendrobii Fast-propagation.
Background technology
Herba Dendrobii, also known as Herba hedyotis costatae, is a kind of rare medicinal plants and famous and precious decorative indoor plant.Belong to country one
The medical material valuable in imminent danger of level protection, successive dynasties medical science classics all give it for " top grade in medicine ".The Compendium of Material Medica of Ming Dynasty's Li Shizhen (1518-1593 A.D.)
Described in: the Herba Dendrobii eliminating impediment therapeutic method to keep the adverse QI flowing downwards, benefit internal organs asthenia are won thin, reinforcing YIN-essence benefit essence.Clothes for a long time, thick the intestines and stomach, the most sufficient in benefit.Carry out gastric qi, long
Muscle, Fructus Alpiniae Oxyphyllae, except frightened, made light of one's life by commiting suicide and is prolonged life.In Taoism Medicine classical " Taoist Scriptures ", Herba Dendrobii ranks first of China's " nine big Herba mesonae chinensis ".Ferrum
The medicinal effects of skin Herba Dendrobii is fresh or dry stem, and modern medicine study shows: Herba Dendrobii can significantly improve immunity of organism merit
Can, defying age, resisting fatigue, anoxia enduring, there is the effects such as auxiliary suppression tumor.
Seeds of Dendrobium Candidum is thin such as dust, and seed contained by a capsule reaches 1,000,000 more than.Owing to lacking endosperm, plant
Son could need to be sprouted with mycosymbiosis, germination rate the lowest (less than 5%) under natural conditions.Herba Dendrobii natural propagation power pole
Low, Sterile culture is the most difficult, and for many years the collection capacity of Herba Dendrobii is much larger than its increment, and dendrobium officinale is natural
Breeding has been on the verge of disappearance.Meeting the market demand for development artificial culture, some appreciable varieties in medicinal dendrobium have become current
The emphasis of tissue rapid propagation research.
At present the Study on tissue culture about Herba Dendrobii has had a relevant report, but the cost mistake needed for tissue culture
High and test tube seedling cycle length problem is the bottleneck of the restriction big production of Herba Dendrobii industrialization.Therefore needing one badly can be the most numerous
Grow the tissue culturing system of Herba Dendrobii, establish theory and practice base for promoting artificial culture technology and large-scale industrialized nursery
Plinth.
Summary of the invention
Tissue culture for current Herba Dendrobii is relatively costly, obtains longer the asking of process of seedling from aseptic sowing seeds to
Topic, it is an object of the invention to design a kind of can Fast-propagation Herba Dendrobii and can the tissue culturing system of whole year production, for
Industrial seedling rearing provides technical support.
Of the present invention a kind of by the special culture media series of protocorms of dendrobium candidum Fast-propagation seedling, including sprouting
Sending out culture medium A, proliferation-inducing culture medium B, division culture medium C, strong seedling culture base D or E and root media F, each culture medium is each
Component content in 1L culture medium is respectively as follows:
(1) culture medium A: NH4NO3:1600-2000mg、KNO3:1800-2300mg、MgSO4·7H2O:330-380mg、
KH2PO4:150-180mg、CaCl2:330-360mg、KI:0.65-0.85mg、H3BO3:6.0-6.6mg、MnSO4·4H2O:
22.0-25.0mg、ZnSO4·7H2O:8.5-9.0mg、Na2MoO4·2H2O:0.18-0.25mg、CuSO4·5H2O:0.015-
0.025mg、CoCl2·6H2O:0.015-0.025mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg、
Inositol 80-150mg, nicotinic acid 0.2-0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine
1.7-2.5mg, agar 6.5-7.5g, white sugar 20-30g.
(2) culture medium B:(NH4)2SO4:460-480mg、KNO3:2800-2850mg、MgSO4·7H2O:175-185mg、
KH2PO4:400-450mg、CaCl2:125-140mg、KI:0.65-0.85mg、H3BO3:1.6-1.9mg、MnSO4·4H2O:
4.0-4.5mg、ZnSO4·7H2O:1.3-1.5mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg, cigarette
Acid 0.2-0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, naphthalene acetic acid
0.1-0.5mg, 6-benzyl aminopurine 0.2-1.0mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g.
(3) culture medium C: (NH4)2SO4:460-480mg、KNO3:2800-2850mg、MgSO4·7H2O:175-185mg、
KH2PO4:400-450mg、CaCl2:125-140mg、KI:0.65-0.85mg、H3BO3:1.6-1.9mg、MnSO4·4H2O:
4.0-4.5mg、ZnSO4·7H2O:1.3-1.5mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg, cigarette
Acid 0.2-0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, naphthalene acetic acid
0.1-0.5mg, 6-benzyl aminopurine 0.1-0.5mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g.
(4) culture medium D:1-5g spends treasured two, 0.1-0.8g caseinhydrolysate, naphthalene acetic acid 0.1-0.5mg, 6-benzyl amino fast
Purine 0.1-0.5mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g.
(5) culture medium E:(NH4)2SO4:128-135mg、KNO3:1800-2500mg、MgSO4·7H2O:250-300mg、
NaH2PO4·H2O:130-160mg、CaCl2:150-170mg、KI:0.65-0.85mg、H3BO3:2.3-3.0mg、MnSO4·
4H2O:9.5-11.0mg、ZnSO4·7H2O:1.6-2.2mg、Na2MoO4·2H2O:0.18-0.25mg、CuSO4·5H2O:
0.015-0.025mg、CoCl2·6H2O:0.015-0.025mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-
28.5mg, inositol 80-150mg, nicotinic acid 0.9-1.6mg, pyridoxine hydrochloride 1.0-1.8mg, thiamine hydrochloride 9.6-10.5mg, naphthalene
Acetic acid 0.3-0.8mg, 6-benzyl aminopurine 0.4-1.0mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g.
(6) culture medium F:NH4NO3:800-830mg、KNO3:950-1000mg、MgSO4·7H2O:175-190mg、
KH2PO4:60-85mg、CaCl2:160-250mg、KI:0.65-0.85mg、H3BO3:6.0-6.6mg、MnSO4·4H2O:22.0-
25.0mg、ZnSO4·7H2O:8.5-9.0mg、Na2MoO4·2H2O:0.18-0.25mg、CuSO4·5H2O:0.015-
0.025mg、CoCl2·6H2O:0.015-0.025mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg、
Inositol 80-150mg, nicotinic acid 0.2-0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine
1.7-2.5mg, naphthalene acetic acid 0.1-0.8mg, heteroauxing 0.2-1.0mg, activated carbon 0.5-1.5mg, Fructus Musae 100-150g, agar
6.5-7.5g, white sugar 20-30g.
The special culture media series formula of above-mentioned protocorms of dendrobium candidum Fast-propagation seedling, this formula process for preparation is such as
Under (1L culture medium):
(1) measure about 500ml water put in water bath and heat；
(2) 6.5-7.5g agar powder is weighed；
(3) when water temperature reaches 80 DEG C, add agar powder, stir, be heated to boiling；
(4) medicine required for every kind of culture medium is added；
(5) weigh 80-150g Rhizoma Solani tuber osi or Fructus Musae during preparation culture medium B, C, D, E, F, be cut into fritter and use juice extractor
Squeeze the juice；
(6) murphy juice squeezed or bananas juice are filtered to water bath；
(7) activated carbon 0.5-1.5mg is added during preparation culture medium F；
(8) white sugar 20-30g is added；
(9) add water after medicine and white sugar all dissolve and be settled to 1L and stir；
(10) pH value is adjusted to be 5.8-6.2 with pH reagent paper；
(11) subpackage, pours the culture medium prepared into tissue culture bottle, and pouring volume is depending on tissue culture bottle amount of capacity, generally
About the 1/5 of container；
(12) high pressure steam sterilization, 121 DEG C, 1.5Mpa, 20min；
(13) culture medium of bacterium of having gone out is put into transfer room and is stood, and allows and solidifies after its cooling.
The invention also discloses and use above-mentioned culture medium series to be trained by the group of protocorms of dendrobium candidum Fast-propagation seedling
Method, its step is as follows:
(1) axenic germination of Seeds of Dendrobium Candidum
Put in superclean bench, at the aseptic bar of superclean bench after ripe seed tap water is rinsed well
Under part, seed is put into successively in the alcoholic solution of 75% immersion 1min, the liquor natrii hypochloritis of 4% soaks 20min, then uses nothing
Bacterium water rinses 3 times, each 1min.The seed dissecting knife handled well is at one end cut an osculum, clamps seed with tweezers
Powder embryo is uniformly sowed in culture medium A by the other end.It is 25 DEG C that tissue culture bottle is placed in temperature, carries out in the environment of unglazed photograph
Cultivate and every day is to its culture environment ozone sterilization 1h.About about 30 days, seed germination, tissue culture bottle is now placed in temperature was
25 DEG C, illumination 2000lx, to cultivate in the environment of illumination every day 12h, every day is to its culture environment ozone sterilization 1h.45 days left
The right side, the Seed Development protocorm of sprouting.
(2) propagation of Herba Dendrobii protocorm
The protocorm formed by seed germination with spoon on superclean bench is transferred in culture medium B uniformly.By group
It is 25 DEG C that training bottle is placed in temperature, illumination 2000lx, carries out cultivating also every day smelly to its culture environment in the environment of illumination every day 12h
Oxygen sterilizing 1h.Protocorm in tissue culture bottle be can be observed and grow faint yellow callus, within about 45 days, callus constantly divides
Propagation forms protocorms (as shown in Figure 2).
(3) differentiation of protocorms of dendrobium candidum
Protocorms is chosen with spoon by superclean bench and is transferred to uniformly in culture medium C.Tissue culture bottle is put
It is 25 DEG C in temperature, illumination 2000lx, carry out cultivation every day in the environment of illumination every day 12h to its culture environment ozone sterilization
1h.About 60-90 days it is observed that the protocorms in tissue culture bottle is through being differentiated to form the seedling of high about 1-2cm (such as Fig. 3 institute
Show).
(4) strong sprout of Herba Dendrobii seedling
Superclean bench goes out, with the folder that tweezers are careful, the seedling that protocorms is differentiated to form be placed on and sterilized in advance
Big culture dish on, and seedling is inserted in culture medium D.It is 25 DEG C that tissue culture bottle is placed in temperature, illumination 2000lx, illumination every day
Cultivation every day is carried out to its culture environment ozone sterilization 1h in the environment of 12h.About 60-90 days it is observed that in tissue culture bottle
Seedling form the seedlings (as shown in Figure 4) of the basically identical high about 3-4cm of regularity through strong sprout.
(5) the taking root of Herba Dendrobii seedlings
Superclean bench goes out Herba Dendrobii seedlings with the folder that tweezers are careful and is placed on the big culture dish sterilized in advance
On, choosing seedlings one one and inserting in culture medium F.It is 25 DEG C that tissue culture bottle is placed in temperature, illumination 2000lx, every day
Cultivation every day is carried out to its culture environment ozone sterilization 1h in the environment of illumination 12h.About 60-90 days it is observed that group is trained
Seedlings in Ping grow fine, and regularity is suitable, and blade is thick greatly, in oblong, and dark green leaf color, every Seedling at least 3-4
The root system that bar prosperity is healthy and strong, plant height reaches 6-8cm(as shown in Figure 5).
(6) seedling exercising of candidum tissue culturing seedling and transplanting
Seedling in tissue culture bottle being taken out bottle Seedling at natural light lower refining seedling and clean root culture medium after one week, transplanting is to mud
Charcoal soil is that in the mixed-matrix of 1:1, survival rate is more than 90% with lichen volume ratio.
In order to ensure to obtain more seedling in process of production simultaneously in order to realize whole year production, step (2) can
To repeat, protocorms constantly can produce new protocorms by division growth in Subculture.
In step (4), in order to obtain more seedling in process of production, can select protocorms in step (3)
The seedling being differentiated to form inserts in culture medium E.This culture medium can remarkably promote the generation of seedling Multiple Buds, thus obtains more
Seedlings.
Above-mentioned is a kind of by the tissue culture method of protocorms of dendrobium candidum Fast-propagation seedling, and design object is clear and definite, improves
The rate of increase of Herba Dendrobii clone's Seedling and transplanting survival rate.The method of the present invention not only shortens the cultivation week of Herba Dendrobii
Phase, and simple to operate, whole year production can not be limited by time place, development Herba Dendrobii industry is significant.
Accompanying drawing explanation
Fig. 1 is group training flow chart.
Fig. 2 is protocorms of dendrobium candidum photo.
Fig. 3 is Herba Dendrobii Seedling (before strong sprout) photo.
Fig. 4 is Herba Dendrobii Seedling (after strong sprout) photo.
Fig. 5 is Herba Dendrobii seedling photo.
Detailed description of the invention
The present invention is further illustrated below by way of experimental example.
Experiment one:
1, preparation culture medium A, B, C, D, E, F.
2, choose dendrobium officinale mature seed, after being rinsed well by seed tap water, put into superclean bench
In, under the aseptic condition of superclean bench, seed is put into successively in the alcoholic solution of 75% immersion 1min, the hypochlorous acid of 4%
Sodium solution soaks 20min, then with rinsed with sterile water 3 times, each 1min.The seed dissecting knife handled well is cut one little
Mouthful, with tweezers, the kind grain of powder is sowed in containing NH4NO3:1600mg、KNO3:1800mg、MgSO4·7H2O:330mg、
KH2PO4:150mg、CaCl2:330mg、KI:0.65mg、H3BO3:6.0mg、MnSO4·4H2O:22.0mg、ZnSO4·7H2O:
8.5mg、Na2MoO4·2H2O:0.18mg、CuSO4·5H2O:0.015mg、CoCl2·6H2O:0.015mg、Na2-EDTA:
37.0mg、FeSO4·7H2O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, pyridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg,
Glycine 1.7mg, agar 6.5g, white sugar 30g culture medium A in.Being placed in temperature is 25 DEG C, trains in the environment of unglazed photograph
Support.After a period of time, seed germination, it is 25 DEG C that culture medium is now placed in temperature, illumination 2000lx, the ring of illumination every day 12h
Cultivate under border.About 70 days, the Seed Development protocorm of sprouting.
3, the protocorm that seed germination is formed aseptically is proceeded to containing (NH4)2SO4:460mg、KNO3:2800mg、
MgSO4·7H2O:175mg、KH2PO4:400mg、CaCl2:125mg、KI:0.65mg、H3BO3:1.6mg、MnSO4·4H2O:
4.0mg、ZnSO4·7H2O:1.3mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, nicotinic acid 0.2mg, pyridoxine hydrochloride
0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, naphthalene acetic acid 0.2mg, 6-benzyl aminopurine 0.6mg, Rhizoma Solani tuber osi 80g, agar
6.5g, white sugar 30g proliferated culture medium B in.Being placed in temperature is 25 DEG C, and illumination 2000lx enters in the environment of illumination every day 12h
Row is cultivated.The protocorm faint yellow callus of generation observed in tissue culture bottle for about 20 days, the continuous division growth of callus,
Within about 70 days, protocorm is bred at double as protocorms.
4, the protocorms by protocorm Callus formation is aseptically proceeded to containing (NH4)2SO4:460mg、
KNO3:2800mg、MgSO4·7H2O:175mg、KH2PO4:400mg、CaCl2:125mg、KI:0.65mg、H3BO3:1.6mg、
MnSO4·4H2O:4.0mg、ZnSO4·7H2O:1.3mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, nicotinic acid
0.2mg, pyridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, naphthalene acetic acid 0.1mg, 6-benzyl aminopurine
0.1mg, Rhizoma Solani tuber osi 80g, agar 6.5g, white sugar 30g division culture medium C in.Being placed in temperature is 25 DEG C, illumination 2000lx, often
Cultivate in the environment of day illumination 12h.The protocorms observed in tissue culture bottle for about 90 days is through being differentiated to form high about 1-
The seedling of 2cm.
5, aseptically proceed to the seedling of differentiation spend treasured two, 0.2g caseinhydrolysate, naphthalene acetic acid containing 2g
0.1mg, 6-benzyl aminopurine 0.1mg, Rhizoma Solani tuber osi 80g, agar 6.5g, white sugar 20g strong seedling culture base D in.Being placed in temperature is
25 DEG C, illumination 2000lx, cultivates in the environment of illumination every day 12h.The seedling observed in tissue culture bottle for about 90 days passes through
Form the seedlings of regularity basically identical high about 3-4cm strong sprout.
6, the seedlings formed through strong sprout are aseptically transferred to containing NH4NO3:800mg、KNO3:950mg、
MgSO4·7H2O:175mg、KH2PO4:60mg、CaCl2:160mg、KI:0.65mg、H3BO3:6.0mg、MnSO4·4H2O:
22.0mg、ZnSO4·7H2O:8.5mg、Na2MoO4·2H2O:0.18mg、CuSO4·5H2O:0.015mg、CoCl2·6H2O:
0.015mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, pyridoxine hydrochloride 0.2mg,
Thiamine hydrochloride 0.08mg, glycine 1.7mg, naphthalene acetic acid 0.2mg, heteroauxing 0.2mg, activated carbon 0.5mg, Fructus Musae 100g,
Agar 6.5g, white sugar 30g root media F in.Being placed in temperature is 25 DEG C, illumination 2000lx, the environment of illumination every day 12h
Under cultivate.The seedlings observed in tissue culture bottle for about 90 days grow fine, and regularity is suitable, and blade is thick greatly, in long ellipse
Circle, dark green leaf color, the root system that every Seedling at least 3-4 bar prosperity is healthy and strong, plant height reaches 6-8cm.
7, the seedling in tissue culture bottle being taken out bottle Seedling at natural light lower refining seedling and clean root culture medium after one week, transplanting is extremely
In peat soil and mixed-matrix that lichen volume ratio is 1:1, survival rate is more than 90%.
Experiment two:
1, preparation culture medium A, B, C, D, E, F.
2, choose the Herba Dendrobii mature seed of artificial culture, after being rinsed well by seed tap water, put into ultra-clean work
In station, under the aseptic condition of superclean bench, seed is put into successively in the alcoholic solution of 75% immersion 1min, 4% time
Sodium chlorate solution soaks 20min, then with rinsed with sterile water 3 times, each 1min.The seed dissecting knife handled well is cut one
Osculum, sows the kind grain of powder in containing NH with tweezers4NO3:1600mg、KNO3:1800mg、MgSO4·7H2O:330mg、
KH2PO4:150mg、CaCl2:330mg、KI:0.65mg、H3BO3:6.0mg、MnSO4·4H2O:22.0mg、ZnSO4·7H2O:
8.5mg、Na2MoO4·2H2O:0.18mg、CuSO4·5H2O:0.015mg、CoCl2·6H2O:0.015mg、Na2-EDTA:
37.0mg、FeSO4·7H2O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, pyridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.08mg,
Glycine 1.7mg, agar 6.5g, white sugar 30g culture medium A in.Being placed in temperature is 25 DEG C, trains in the environment of unglazed photograph
Support.After a period of time, seed germination, it is 25 DEG C that culture medium is now placed in temperature, illumination 2000lx, the ring of illumination every day 12h
Cultivate under border.About 70 days, the Seed Development protocorm of sprouting.
3, the protocorm that seed germination is formed aseptically is proceeded to containing (NH4)2SO4:460mg、KNO3:2800mg、
MgSO4·7H2O:175mg、KH2PO4:400mg、CaCl2:125mg、KI:0.65mg、H3BO3:1.6mg、MnSO4·4H2O:
4.0mg、ZnSO4·7H2O:1.3mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, nicotinic acid 0.2mg, pyridoxine hydrochloride
0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, naphthalene acetic acid 0.2mg, 6-benzyl aminopurine 0.6mg, Rhizoma Solani tuber osi 80g, agar
6.5g, white sugar 30g proliferated culture medium B in.Being placed in temperature is 25 DEG C, and illumination 2000lx enters in the environment of illumination every day 12h
Row is cultivated.The protocorm faint yellow callus of generation observed in tissue culture bottle for about 20 days, the continuous division growth of callus,
Within about 70 days, protocorm is bred at double as protocorms.
4, the protocorms by protocorm Callus formation is aseptically proceeded to containing (NH4)2SO4:460mg、
KNO3:2800mg、MgSO4·7H2O:175mg、KH2PO4:400mg、CaCl2:125mg、KI:0.65mg、H3BO3:1.6mg、
MnSO4·4H2O:4.0mg、ZnSO4·7H2O:1.3mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, nicotinic acid
0.2mg, pyridoxine hydrochloride 0.2mg, thiamine hydrochloride 0.8mg, glycine 1.7mg, naphthalene acetic acid 0.1mg, 6-benzyl aminopurine
0.1mg, Rhizoma Solani tuber osi 80g, agar 6.5g, white sugar 30g division culture medium C in.Being placed in temperature is 25 DEG C, illumination 2000lx, often
Cultivate in the environment of day illumination 12h.The protocorms observed in tissue culture bottle for about 90 days is through being differentiated to form high about 1-
The seedling of 2cm.
5, the seedling of differentiation is aseptically proceeded to containing (NH4)2SO4:120mg、KNO3:1800mg、MgSO4·
7H2O:230mg、NaH2PO4·H2O:150mg、CaCl2:150mg、KI:0.65mg、H3BO3:2.3mg、MnSO4·4H2O:
9.5mg、ZnSO4·7H2O:1.6mg、Na2MoO4·2H2O:0.18mg、CuSO4·5H2O:0.015mg、CoCl2·6H2O:
0.015mg、Na2-EDTA:37.00mg、FeSO4·7H2O:27.5mg, inositol 80mg, nicotinic acid 0.9mg, pyridoxine hydrochloride
1.0mg, thiamine hydrochloride 9.6mg, naphthalene acetic acid 0.5mg, 6-benzyl aminopurine 0.1mg, Rhizoma Solani tuber osi 80g, agar 6.5g, white sugar
In the strong seedling culture base E of 30g.Being placed in temperature is 25 DEG C, and illumination 2000lx cultivates in the environment of illumination every day 12h.90 days
Left and right observes that the seedling in tissue culture bottle forms the seedlings of the basically identical high about 3-4cm of regularity, and every strain seedlings through strong sprout
At least produce 2-3 differentiation Seedling out.
6, the seedlings formed through strong sprout are aseptically transferred to containing NH4NO3:800mg、KNO3:950mg、
MgSO4·7H2O:175mg、KH2PO4:60mg、CaCl2:160mg、KI:0.65mg、H3BO3:6.0mg、MnSO4·4H2O:
22.0mg、ZnSO4·7H2O:8.5mg、Na2MoO4·2H2O:0.18mg、CuSO4·5H2O:0.015mg、CoCl2·6H2O:
0.015mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, pyridoxine hydrochloride 0.2mg,
Thiamine hydrochloride 0.08mg, glycine 1.7mg, naphthalene acetic acid 0.2mg, heteroauxing 0.2mg, activated carbon 0.5mg, Fructus Musae 100g,
Agar 6.5g, white sugar 30g root media culture medium E in.Being placed in temperature is 25 DEG C, illumination 2000lx, illumination every day 12h
In the environment of cultivate.The seedlings observed in tissue culture bottle for about 900 days grow fine, and regularity is suitable, and blade is thick greatly,
In oblong, dark green leaf color, the root system that every Seedling at least 3-4 bar prosperity is healthy and strong, plant height reaches 6-8cm.
7, the seedling in tissue culture bottle being taken out bottle Seedling at natural light lower refining seedling and clean root culture medium after one week, transplanting is extremely
In peat soil and mixed-matrix that lichen volume ratio is 1:1, survival rate is more than 90%.
Experiment three:
1, preparation culture medium A, B, C, D, E, F.
2, choose the Herba Dendrobii mature seed of artificial culture, after being rinsed well by seed tap water, put into ultra-clean work
In station, under the aseptic condition of superclean bench, seed is put into successively in the alcoholic solution of 75% immersion 1min, 4% time
Sodium chlorate solution soaks 20min, then with rinsed with sterile water 3 times, each 1min.The seed dissecting knife handled well is cut one
Osculum, sows the kind grain of powder in containing NH with tweezers4NO3:1600mg、KNO3:1800mg、MgSO4·7H2O:370mg、
KH2PO4:180mg、CaCl2:330mg、KI:0.85mg、H3BO3:6.0mg、MnSO4·4H2O:22.0mg、ZnSO4·7H2O:
8.5mg、Na2MoO4·2H2O:0.25mg、CuSO4·5H2O:0.025mg、CoCl2·6H2O:0.025mg、Na2-EDTA:
37.3mg、FeSO4·7H2O:27.8mg, inositol 100mg, nicotinic acid 0.5mg, pyridoxine hydrochloride 0.5mg, thiamine hydrochloride
0.08mg, glycine 1.8mg, agar 6.5g, white sugar 30g culture medium A in.Being placed in temperature is 25 DEG C, the environment of unglazed photograph
Under cultivate.After a period of time, seed germination, it is 25 DEG C that culture medium is now placed in temperature, illumination 2000lx, illumination every day
Cultivate in the environment of 12h.About 50 days, the Seed Development protocorm of sprouting.
3, the protocorm that seed germination is formed aseptically is proceeded to containing (NH4)2SO4:460mg、KNO3:2800mg、
MgSO4·7H2O:180mg、KH2PO4:400mg、CaCl2:125mg、KI:0.80mg、H3BO3:1.6mg、MnSO4·4H2O:
4.3mg、ZnSO4·7H2O:1.6mg、Na2-EDTA:37.3mg、FeSO4·7H2O:27.8mg, nicotinic acid 0.5mg, pyridoxine hydrochloride
0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, naphthalene acetic acid 0.2mg, 6-benzyl aminopurine 0.6mg, Rhizoma Solani tuber osi 100g, agar
6.5g, white sugar 30g proliferated culture medium B in.Being placed in temperature is 25 DEG C, and illumination 2000lx enters in the environment of illumination every day 12h
Row is cultivated.The protocorm faint yellow callus of generation observed in tissue culture bottle for about 15 days, the continuous division growth of callus,
Within about 60 days, protocorm is bred at double as protocorms.
4, the protocorms by protocorm Callus formation is aseptically proceeded to containing (NH4)2SO4:460mg、
KNO3:2800mg、MgSO4·7H2O:180mg、KH2PO4:400mg、CaCl2:125mg、KI:0.80mg、H3BO3:1.6mg、
MnSO4·4H2O:4.3mg、ZnSO4·7H2O:1.6mg、Na2-EDTA:37.3mg、FeSO4·7H2O:27.8mg, nicotinic acid
0.5mg, pyridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, naphthalene acetic acid 0.2mg, 6-benzyl aminopurine
0.2mg, Rhizoma Solani tuber osi 100g, agar 6.5g, white sugar 30g division culture medium C in.Being placed in temperature is 25 DEG C, illumination 2000lx, often
Cultivate in the environment of day illumination 12h.The protocorms observed in tissue culture bottle for about 60 days is through being differentiated to form high about 1-
The seedling of 2cm.
5, aseptically proceed to the seedling of differentiation spend treasured two, 0.4g caseinhydrolysate, naphthalene acetic acid containing 3g
0.2mg, 6-benzyl aminopurine 0.2mg, Rhizoma Solani tuber osi 100g, agar 6.5g, white sugar 20g strong seedling culture base D in.Being placed in temperature is
25 DEG C, illumination 2000lx, cultivates in the environment of illumination every day 12h.The seedling observed in tissue culture bottle for about 70 days passes through
Form the seedlings of regularity basically identical high about 3-4cm strong sprout.
6, the seedlings formed through strong sprout are aseptically transferred to containing NH4NO3:815mg、KNO3:950mg、
MgSO4·7H2O:180mg、KH2PO4:85mg、CaCl2:165mg、KI:0.85mg、H3BO3:6.0mg、MnSO4·4H2O:
22.0mg、ZnSO4·7H2O:8.5mg、Na2MoO4·2H2O:0.25mg、CuSO4·5H2O:0.025mg、CoCl2·6H2O:
0.025-mg、Na2-EDTA:37.3mg、FeSO4·7H2O:27.8mg, inositol 100mg, nicotinic acid 0.5mg, pyridoxine hydrochloride
0.5mg, thiamine hydrochloride 0.08mg, glycine 1.8mg, naphthalene acetic acid 0.4mg, heteroauxing 0.4mg, activated carbon 0.5mg, Fructus Musae
100g, agar 6.5g, white sugar 30g root media F in.Being placed in temperature is 25 DEG C, illumination 2000lx, illumination every day 12h
In the environment of cultivate.The seedlings observed in tissue culture bottle for about 60 days grow fine, and regularity is suitable, and blade is thick greatly,
In oblong, dark green leaf color, the root system that every Seedling at least 3-4 bar prosperity is healthy and strong, plant height reaches 6-8cm.
7, the seedling in tissue culture bottle being taken out bottle Seedling at natural light lower refining seedling and clean root culture medium after one week, transplanting is extremely
In peat soil and mixed-matrix that lichen volume ratio is 1:1, survival rate is more than 90%.
Experiment four:
1, preparation culture medium A, B, C, D, E, F.
2, choose dendrobium officinale mature seed, after being rinsed well by seed tap water, put into superclean bench
In, under the aseptic condition of superclean bench, seed is put into successively in the alcoholic solution of 75% immersion 1min, the hypochlorous acid of 4%
Sodium solution soaks 20min, then with rinsed with sterile water 3 times, each 1min.The seed dissecting knife handled well is cut one little
Mouthful, with tweezers, the kind grain of powder is sowed in containing NH4NO3:1600mg、KNO3:1800mg、MgSO4·7H2O:370mg、
KH2PO4:180mg、CaCl2:330mg、KI:0.85mg、H3BO3:6.0mg、MnSO4·4H2O:22.0mg、ZnSO4·7H2O:
8.5mg、Na2MoO4·2H2O:0.25mg、CuSO4·5H2O:0.025mg、CoCl2·6H2O:0.025mg、Na2-EDTA:
37.3mg、FeSO4·7H2O:27.8mg, inositol 100mg, nicotinic acid 0.5mg, pyridoxine hydrochloride 0.5mg, thiamine hydrochloride
0.08mg, glycine 1.8mg, agar 6.5g, white sugar 30g culture medium A in.Being placed in temperature is 25 DEG C, the environment of unglazed photograph
Under cultivate.After a period of time, seed germination, it is 25 DEG C that culture medium is now placed in temperature, illumination 2000lx, illumination every day
Cultivate in the environment of 12h.About 50 days, the Seed Development protocorm of sprouting.
3, the protocorm that seed germination is formed aseptically is proceeded to containing (NH4)2SO4:460mg、KNO3:2800mg、
MgSO4·7H2O:180mg、KH2PO4:400mg、CaCl2:125mg、KI:0.80mg、H3BO3:1.6mg、MnSO4·4H2O:
4.3mg、ZnSO4·7H2O:1.6mg、Na2-EDTA:37.3mg、FeSO4·7H2O:27.8mg, nicotinic acid 0.5mg, pyridoxine hydrochloride
0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, naphthalene acetic acid 0.2mg, 6-benzyl aminopurine 0.6mg, Rhizoma Solani tuber osi 100g, agar
6.5g, white sugar 30g proliferated culture medium B in.Being placed in temperature is 25 DEG C, and illumination 2000lx enters in the environment of illumination every day 12h
Row is cultivated.The protocorm faint yellow callus of generation observed in tissue culture bottle for about 15 days, the continuous division growth of callus,
Within about 60 days, protocorm is bred at double as protocorms.
4, the protocorms by protocorm Callus formation is aseptically proceeded to containing (NH4)2SO4:460mg、
KNO3:2800mg、MgSO4·7H2O:180mg、KH2PO4:400mg、CaCl2:125mg、KI:0.80mg、H3BO3:1.6mg、
MnSO4·4H2O:4.3mg、ZnSO4·7H2O:1.6mg、Na2-EDTA:37.3mg、FeSO4·7H2O:27.8mg, nicotinic acid
0.5mg, pyridoxine hydrochloride 0.5mg, thiamine hydrochloride 0.8mg, glycine 1.8mg, naphthalene acetic acid 0.2mg, 6-benzyl aminopurine
0.2mg, Rhizoma Solani tuber osi 100g, agar 6.5g, white sugar 30g division culture medium C in.Being placed in temperature is 25 DEG C, illumination 2000lx, often
Cultivate in the environment of day illumination 12h.The protocorms observed in tissue culture bottle for about 60 days is through being differentiated to form high about 1-
The seedling of 2cm.
5, the seedling of differentiation is aseptically proceeded to containing (NH4)2SO4:130mg、KNO3:2500mg、MgSO4·
7H2O:250mg、NaH2PO4·H2O:150mg、CaCl2:150mg、KI:0.65mg、H3BO3:2.8mg、MnSO4·4H2O:
9.5mg、ZnSO4·7H2O:1.8mg、Na2MoO4·2H2O:0.25mg、CuSO4·5H2O:0.025mg、CoCl2·6H2O:
0.025mg、Na2-EDTA:37.3mg、FeSO4·7H2O:27.8mg, inositol 100mg, nicotinic acid 1.0mg, pyridoxine hydrochloride
1.0mg, thiamine hydrochloride 9.6mg, naphthalene acetic acid 0.5mg, 6-benzyl aminopurine 0.5mg, Rhizoma Solani tuber osi 100g, agar 6.5g, white sugar
In the strong seedling culture base E of 30g.Being placed in temperature is 25 DEG C, and illumination 2000lx cultivates in the environment of illumination every day 12h.70 days
Left and right observes that the seedling in tissue culture bottle forms the seedlings of the basically identical high about 3-4cm of regularity, and every strain seedlings through strong sprout
At least produce 2-3 differentiation Seedling out.
6, the seedlings formed through strong sprout are aseptically transferred to containing NH4NO3:800mg、KNO3:950mg、
MgSO4·7H2O:175mg、KH2PO4:60mg、CaCl2:160mg、KI:0.65mg、H3BO3:6.0mg、MnSO4·4H2O:
22.0mg、ZnSO4·7H2O:8.5mg、Na2MoO4·2H2O:0.18mg、CuSO4·5H2O:0.015mg、CoCl2·6H2O:
0.015mg、Na2-EDTA:37.0mg、FeSO4·7H2O:27.5mg, inositol 80mg, nicotinic acid 0.2mg, pyridoxine hydrochloride 0.2mg,
Thiamine hydrochloride 0.08mg, glycine 1.7mg, naphthalene acetic acid 0.2mg, heteroauxing 0.2mg, activated carbon 0.5mg, Fructus Musae 100g,
Agar 6.5g, white sugar 30g root media culture medium F in.Being placed in temperature is 25 DEG C, illumination 2000lx, illumination every day 12h
In the environment of cultivate.The seedlings observed in tissue culture bottle for about 70 days grow fine, and regularity is suitable, and blade is thick greatly,
In oblong, dark green leaf color, the root system that every Seedling at least 3-4 bar prosperity is healthy and strong, plant height reaches 6-8cm.
7, the seedling in tissue culture bottle being taken out bottle Seedling at natural light lower refining seedling and clean root culture medium after one week, transplanting is extremely
In peat soil and mixed-matrix that lichen volume ratio is 1:1, survival rate is more than 90%.
Claims (4)
1. one kind by the series special culture medium of protocorms of dendrobium candidum Fast-propagation seedling, it is characterised in that this serial culture
Base includes germination medium A, proliferation-inducing culture medium B, division culture medium C, strong seedling culture base D or E and root media F, institute
Stating in each culture medium that component is in terms of 1L culture medium, content is respectively as follows:
A. culture medium A: NH4NO3:1600-2000mg、KNO3:1800-2300mg、MgSO4·7H2O:330-380mg、KH2PO4:
150-180mg、CaCl2:330-360mg、KI:0.65-0.85mg、H3BO3:6.0-6.6mg、MnSO4·4H2O:22.0-
25.0mg、ZnSO4·7H2O:8.5-9.0mg、Na2MoO4·2H2O:0.18-0.25mg、CuSO4·5H2O:0.015-
0.025mg、CoCl2·6H2O:0.015-0.025mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg、
Inositol 80-150mg, nicotinic acid 0.2-0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine
1.7-2.5mg, agar 6.5-7.5g, white sugar 20-30g；
B. culture medium B:(NH4)2SO4:460-480mg、KNO3:2800-2850mg、MgSO4·7H2O:175-185mg、KH2PO4:
400-450mg、CaCl2:125-140mg、KI:0.65-0.85mg、H3BO3:1.6-1.9mg、MnSO4·4H2O:4.0-4.5mg、
ZnSO4·7H2O:1.3-1.5mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg, nicotinic acid 0.2-
0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, naphthalene acetic acid 0.1-
0.5mg, 6-benzyl aminopurine 0.2-1.0mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g；
C. culture medium C: (NH4)2SO4:460-480mg、KNO3:2800-2850mg、MgSO4·7H2O:175-185mg、KH2PO4:
400-450mg、CaCl2:125-140mg、KI:0.65-0.85mg、H3BO3:1.6-1.9mg、MnSO4·4H2O:4.0-4.5mg、
ZnSO4·7H2O:1.3-1.5mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg, nicotinic acid 0.2-
0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.8-1.0mg, glycine 1.7-2.5mg, naphthalene acetic acid 0.1-
0.5mg, 6-benzyl aminopurine 0.1-0.5mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g；
D. culture medium D:1-5g spends treasured two, 0.1-0.8g caseinhydrolysate, naphthalene acetic acid 0.1-0.5mg, 6-benzyl aminopurine
0.1-0.5mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g；
E. culture medium E:(NH4)2SO4:128-135mg、KNO3:1800-2500mg、MgSO4·7H2O:250-300mg、
NaH2PO4·H2O:130-160mg、CaCl2:150-170mg、KI:0.65-0.85mg、H3BO3:2.3-3.0mg、MnSO4·
4H2O:9.5-11.0mg、ZnSO4·7H2O:1.6-2.2mg、Na2MoO4·2H2O:0.18-0.25mg、CuSO4·5H2O:
0.015-0.025mg、CoCl2·6H2O:0.015-0.025mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-
28.5mg, inositol 80-150mg, nicotinic acid 0.9-1.6mg, pyridoxine hydrochloride 1.0-1.8mg, thiamine hydrochloride 9.6-10.5mg, naphthalene
Acetic acid 0.3-0.8mg, 6-benzyl aminopurine 0.4-1.0mg, Rhizoma Solani tuber osi 80-150g, agar 6.5-7.5g, white sugar 20-30g；
F. culture medium F:NH4NO3:800-830mg、KNO3:950-1000mg、MgSO4·7H2O:175-190mg、KH2PO4:60-
85mg、CaCl2:160-250mg、KI:0.65-0.85mg、H3BO3:6.0-6.6mg、MnSO4·4H2O:22.0-25.0mg、
ZnSO4·7H2O:8.5-9.0mg、Na2MoO4·2H2O:0.18-0.25mg、CuSO4·5H2O:0.015-0.025mg、
CoCl2·6H2O:0.015-0.025mg、Na2-EDTA:37.0-38.0mg、FeSO4·7H2O:27.5-28.5mg, inositol 80-
150mg, nicotinic acid 0.2-0.6mg, pyridoxine hydrochloride 0.2-0.5mg, thiamine hydrochloride 0.08-0.15mg, glycine 1.7-
2.5mg, naphthalene acetic acid 0.1-0.8mg, indolebutyric acid 0.2-1.0mg, activated carbon 0.5-1.5mg, Fructus Musae 100-150g, agar 6.5-
7.5g, white sugar 20-30g.
2. the compound method of serial culture base described in a claim 1, it is characterised in that process for preparation, in terms of 1L culture medium, walks
Rapid as follows:
A. measure about 500ml water put in water bath and heat；
B. 6.5-7.5g agar powder is weighed；
C. when water temperature reaches 80 DEG C, add agar powder, stir, be heated to boiling；
D. all groups in addition to agar powder, activated carbon, white sugar and Rhizoma Solani tuber osi or Fructus Musae of each culture medium in claim 1 are added
Point；
E. weigh 80-150g Rhizoma Solani tuber osi, 100-150g Fructus Musae, be cut into fritter and squeeze the juice with juice extractor；
F. the murphy juice squeezed, bananas juice are filtered to water bath；
G. activated carbon 0.5-1.5mg is added；
H. white sugar 20-30g is added；
I. add water after medicine and white sugar all dissolve and be settled to 1L and stir；
J. pH value is adjusted to be 5.8-6.2 with pH reagent paper；
K. subpackage, pour the culture medium prepared into tissue culture bottle, pouring volume depending on tissue culture bottle amount of capacity, generally container
About 1/5；
L. high pressure steam sterilization, 121 DEG C, 1.5Mpa, 20min；
M. the culture medium of bacterium of having gone out is put into transfer room and is stood, and allows and solidifies after its cooling；
Step e to be carried out, f when preparing culture medium B, C, D, E, F；
Step g to be carried out when preparing culture medium F.
3. one kind uses serial culture base described in claim 1 by the group training side of protocorms of dendrobium candidum Fast-propagation seedling
Method, its feature comprises the following steps:
(1) axenic germination of Seeds of Dendrobium Candidum
A., Seeds of Dendrobium Candidum is used on superclean bench alcohol-pickled 1min successively, and liquor natrii hypochloritis sterilizes 20min, nothing
Bacterium water rinses 3 times, each 1min；
B. the seed after thorough disinfection rinsing is taken out, cut from one end of seed, after incision, by uniform for powder embryo
Sow in culture medium A；
C. being placed in temperature is 25 DEG C, cultivates in the environment of unglazed photograph；
D. every day is to its culture environment ozone sterilization 1h；
E.30, after the seed germination of left and right, sky, it is 25 DEG C that tissue culture bottle is placed in temperature, illumination 2000lx, the ring of illumination every day 12h
Cultivating under border, every day is to its culture environment ozone sterilization 1h；
F.45 about sky, the Seed Development protocorm of sprouting；
(2) propagation of Herba Dendrobii protocorm
A. the protocorm formed by seed germination with spoon on superclean bench is transferred in culture medium B uniformly；
B. being placed in temperature is 25 DEG C, and illumination 2000lx cultivates in 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 45 days, the continuous division growth of callus became protocorms；
(3) differentiation of protocorms of dendrobium candidum
A. with spoon protocorms chosen on superclean bench and be transferred to uniformly in culture medium C；
B. being placed in temperature is 25 DEG C, and illumination 2000lx cultivates in the environment of illumination every day 12h；
C. every day is to its culture environment ozone sterilization 1h；
D.60-90 sky, protocorms is divided into seedling；
(4) strong sprout of Herba Dendrobii seedling
A. on superclean bench, go out, with the folder that tweezers are careful, protocorms is differentiated to form seedling to be placed on and sterilized in advance
On big culture dish, and seedling is inserted in culture medium D or E；
B. being placed in temperature is 25 DEG C, and illumination 2000lx cultivates in the environment of illumination every day 12h；
C. every day is to its culture environment ozone sterilization 1h；
D.60-90 sky, seedling grows into the Herba Dendrobii seedlings grown fine；
(5) the taking root of Herba Dendrobii seedlings
A. on superclean bench, go out Herba Dendrobii seedlings with the folder that tweezers are careful to be placed on the big culture dish sterilized in advance；
B. choosing seedlings one one and inserting in culture medium F；
C. being placed in temperature is 25 DEG C, and illumination 2000lx cultivates in the environment of illumination every day 12h；
D. every day is to its culture environment ozone sterilization 1h；
E.60-90 sky, seedlings grow into complete candidum tissue culturing seedling；
(6) seedling exercising of candidum tissue culturing seedling and transplanting
A. the seedling in tissue culture bottle is placed on nature light lower refining seedling one week；
B., after bottle Seedling being taken out and clean root culture medium, transplant to peat soil and mixed-matrix that lichen volume ratio is 1:1,
Survival rate is more than 90%.
It is the most according to claim 3 by the tissue culture method of protocorms of dendrobium candidum Fast-propagation seedling, it is characterised in that
Seeds of Dendrobium Candidum collection described in described step (1) is the Herba Dendrobii of dendrobium officinale or artificial culture.
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