CN103875533A - Method and culture medium for proliferation storage of somatic embryos of dendrobiumcandidum - Google Patents
Method and culture medium for proliferation storage of somatic embryos of dendrobiumcandidum Download PDFInfo
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- CN103875533A CN103875533A CN201410113720.9A CN201410113720A CN103875533A CN 103875533 A CN103875533 A CN 103875533A CN 201410113720 A CN201410113720 A CN 201410113720A CN 103875533 A CN103875533 A CN 103875533A
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Abstract
The invention discloses a method and a culture medium for proliferation storage of somatic embryos of dendrobiumcandidum. The method comprises the following steps: (1) disinfecting explants; (2) performing induction culture on protocorm; (3) inducing the somatic embryos; (4) performing proliferation storage culture on the somatic embryos; (5) performing recovery growth and proliferation culture on the somatic embryos; (6) germinating the somatic embryos; and (7) performing rooting culture on strong test-tube plantlets. According to the method for the proliferation storage culture of the somatic embryos of the dendrobiumcandidum, the culture can be continuously stored for 12 months and can be proliferated by 40 times without subculture during storage, the operation is easy, the cost is low, the recovery culture reaction is quick, and a large quantity of the somatic embryos can be proliferated or the somatic embryos can be stored under the control of the proliferation speed within a relatively short time according to market and production needs.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of propagation store method and medium of dendrobium candidum somatic embryo.
Background technology
Dendrobium candidum (Dendrobiumcandidum), is commonly called as Tiepi Fengdou, is the orchid family Dendrobium herbaceos perennial, for famous and precious traditional Chinese medicine, the medical value that tool is important, has nourishing Yin and clearing heat, to promote the production of body fluid beneficial stomach, relieving cough and moistening lung effect, has much the market development and is worth.In recent years, because people excessively excavate it, cause its wild resource sharply to reduce, increasingly in imminent danger, be now listed in National Wild Conservative Plants.Because dendrobium candidum requires highly to ecotope, under natural conditions, the low growth rate of dendrobium candidum reproduction rate is slow, adopts conventional method to breed dendrobium candidum efficiency very low.Therefore the Study on tissue culture of dendrobium candidum has obtained paying attention to and having obtained larger progress.
Fu Chuanming etc. have carried out the research of dendrobium candidum axenic sowing industrialization raising technology, adopt the quick seedling of approach of seed → protocorm → whole plant → transplanting to carry out batch production production, each stage medium is screened, and some other factor of influence compares research.Result shows: the Seeds of Dendrobium Candidum of growing after artificial pollination 60～180 days all can be sprouted in vitro, the sprouting effect of 150～180 days seeds of wherein pollinating is best, germination rate is 87.2%～94.4%, and suitable germination medium is MS+6-BA1.0mg/L+NAA0.1mg/L+ potato juice 200g/L+AC1.0g/L; The optimal medium of protocorm propagation is MS+6-BA1.5mg/L+NAA0.1mg/L+ bananas juice 100g/L+AC1.0g/L; Protocorm breaks up cultivation on MS+6-BA1.0mg/L+NAA0.1mg/L+ potato juice 200g/L+AC1.0g/L medium, when differentiation, can also carry out certain propagation; The bud seedling having broken up was transferred to after strong seedling culture base MS+6-BA0.5mg/L+NAA0.2mg/L+ bananas juice 100g/L+AC1.0g/L 1 generation of upper cultivation, be transferred on root media 1/2MS+NAA0.8mg/L+ mineral salt A0.2-0.5mg/L+ bananas juice 100g/L+AC1.0g/L, cultivate after 50～70 days, rooting rate 100%, mineral salt A can control callus or protocormal formation effectively, obviously improves the quality and quantity of the seedling of taking root.
The Primary Study that Li Dan etc. have carried out black joint dendrobium candidum protocorm induction, again differentiation and axillary bud sprouting and taken root, thinks, the disinfection way of black joint dendrobium candidum stem with bud the best is to process 20min with 0.1%HgCl2 again after 70% alcohol is processed 30s.The optimal medium of black joint dendrobium candidum stem with bud protocorm induction is MS+KT1mg/L+NAA0.1mg/L.The optimal medium that black joint dendrobium candidum stem with bud protocorm breaks up is again MS+KT2mg/L+NAA0.5mg/L.The optimal medium of black joint dendrobium candidum stem with bud axillary bud sprouting is MS+KT2mg/L+NAA0.5mg/L.These experimental results are that technical foundation has been established in the batch production production of black joint dendrobium officinale test-tube plantlet.
Pan Chaomei etc. get the subculture cultivation dendrobium candidum class protocorm of 30 days and make inoculation material, taking N6 as minimal medium, add plant hormone, and make elicitor with fungal extract, and class protocorm and callus are induced to cultivation.Microexamination result shows that the formation of dendrobium candidum somatic embryo can be produced by the epidermal cell of callus or inner cell.Thinking that induction that dendrobium candidum passes through class protocorm and callus is cultivated can obtain a large amount of clumps of buds.
Zhan Zhonggen etc. are taking the in vitro tip of a root of dendrobium candidum as explant, study under different condition of culture the in vitro tip of a root through callus organizator blast and directly produced the cytology process of somatic embryo regeneration plant, having thought that dendrobium candidum class protocorm is the real somatic embryo of unicellular origin.
Although cultivating, the tissue of dendrobium candidum obtains larger success, the fast breeding that utilizes tissue culture technique to carry out seedlings of Dendrobium officinale has not had very large technical problem, but market demand is often inconsistent with the condition of production, how making to produce applicable market, is the key that tissue culture technology successfully applies to produce and obtain higher economic benefit.For this reason, scientist begins one's study and organizes the Vitro Preservation of culture materials.Chen Yong etc. have reported the cryopreservation by vitrification research of dendrobium candidum germ plasm resource, protoplast, protocorm to dendrobium candidum have carried out cryopreservation by vitrification research, survival rate reach respectively 48% and 88%. experimental result show: material, pretreatment, the method for thawing, freeze the factors such as front pre-culture medium and all survival rate had to considerable influence.
Shi Yongzhong etc. have reported the in vitro research of preserving of iron sheet stone solution germplasm room temperature, think: dendrobium officinale test-tube plantlet is at 1/2MS, on the medium of additional NAA0.5mg/L, 20g/L sucrose, 0.5mg/L active carbon and 7g/L agar, under common culturing room condition (25 2 DEG C of scholars), preserve 12 months continuously, survival rate reaches 100%.In medium, add low concentration NAA, sucrose and active carbon, can obviously change the state of preserving material.Although the report results such as Shi Yongzhong are thought, recover growth comparatively fast through the dendrobium officinale test-tube plantlet of its preservation.But due to during preserving test-tube plantlet, its base portion starts to take root, and is just unfavorable for its propagation.Can recover quickly growth although preserve material, if it is numerous to preserve the expansion of material, need wipe out root system, the startup that re-starts propagation is cultivated.Therefore the method is only suitable for the preservation of dendrobium candidum, and is unfavorable for recovering propagation after dendrobium candidum is preserved.
So far have no, the research report of dendrobium candidum somatic embryo propagation preservation aspect.
Summary of the invention
The object of the present invention is to provide a kind of propagation store method and medium of dendrobium candidum somatic embryo, make explant with eight points of ripe seeds of dendrobium candidum, induction protocorm is set up aseptic strain, aseptic strain subculture is cultivated inductor blast a large amount of propagation, by the allotment of medium component and plant growth regulator, reach the object that somatic embryo propagation is preserved.
The propagation store method of dendrobium candidum somatic embryo of the present invention, comprises the following steps:
1) sterilization of explant: get dendrobium candidum capsule, on superclean bench, with 75% alcohol disinfecting after 45～60 seconds, then use 15～20%(volume ratio) aqueous sodium hypochlorite solution soaks 15-25 minute, aseptic water washing 3-5 time;
2) induction of protocorm is cultivated: the moisture on the dendrobium candidum capsule surface after sterilization is blotted in sterile working, cut capsule with scalpel, its seed of picking, be inoculated in and on protocorm inducing culture, carry out low light level photograph, after 20～30 days, be transferred under illumination and cultivate, continue to cultivate after 25～35 days, explant is sprouted and is formed cone shape protocorm, 25 ± 2 DEG C of temperature;
Protocorm inducing culture formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.5～1.5g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L;
3) induction of somatic embryo and propagation: protocorm subculture is incubated in the induction and proliferated culture medium of somatic embryo, after 30 days, protocorm produces gradually somatic embryo and constantly breeds;
Somatic embryo inducement and proliferation culture medium formula are: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L;
4) propagation of somatic embryo is preserved and is cultivated: somatic embryo switching is incubated to somatic embryo propagation Storaged media, after 2 months, reach peak of proliferation, if carry out Fast-propagation, should transfer and breed again cultivation in fresh somatic embryo propagation Storaged media, carry out Fast-propagation if do not needed, and to carry out germplasm preservation, do not transfer reaching after peak of proliferation, continue to cultivate 3～4 months on former propagation Storaged media, somatic embryo still can slowly be bred, and there is a small amount of somatic embryo to start to sprout, cultivate after 10～12 months, medium greatly reduces, culture flocks together, in semistarvation state, but still keep good vitality,
Somatic embryo propagation Storaged media formula is; MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L;
5) recovery of somatic embryo growth is cultivated with propagation: propagation is preserved to the somatic embryo of cultivating, dispersion culture recovers growth and proliferated culture medium in fresh somatic embryo, and after 1 week, somatic embryo starts to recover growth, after 2 weeks, breed gradually, obtain dendrobium candidum somatic embryo;
Somatic embryo recovers growth: MS minimal medium, 6-benzyl aminoadenine 0.8～1.2mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.5～1.0g/L, banana puree 80～120g/L, sucrose 2～4%, agar powder 6～7g/L;
6) sprouting of somatic embryo: propagation is cultivated to the dendrobium candidum somatic embryo switching obtaining and be incubated at somatic embryo germination medium, after 2 weeks, somatic embryo is sprouted into seedling gradually;
The culture medium prescription that somatic embryo is sprouted is: MS minimal medium, 6-benzyl aminoadenine 0.3～0.6mg/L, 6-glycosyl aminopurine 0.3～0.6mg/L, methyl α-naphthyl acetate 0.1～0.2mg/L, caseinhydrolysate 0.6～0.8g/L, active carbon 0.3～0.4g/L, sucrose 2～4%, agar powder 6～7g/L;
7) strengthening seedling and rooting is cultivated: dendrobium candidum seedling is incubated to strengthening seedling and rooting medium, within 30 days, grows afterwards for complete candidum tissue culturing seedling;
The culture medium prescription of strengthening seedling and rooting is: 1/2MS minimal medium, methyl α-naphthyl acetate 0.3～0.5mg/L, active carbon 0.3～0.5g/L, sucrose 2～4%, agar powder 6～7g/L;
The pH of all medium is 5.4～5.8.
Preferably, the maturity of the dendrobium candidum capsule described in step 1) be eight points ripe.
Preferably, step 2) described low light level photograph, intensity of illumination is 300～500 luxs.
Preferably, step 2) described illumination cultivation, intensity of illumination is 2000～2500 luxs.
Preferably, the cultivation temperature described in step 4) is 20～25 DEG C.
A kind of medium for dendrobium candidum somatic embryo inducement and propagation of the present invention, its formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L.
A kind of medium of preserving for dendrobium candidum somatic embryo propagation of the present invention, its formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L.
The MS using in medium of the present invention or 1/2MS minimal medium are grown the inorganic nutritive elements such as N, P, K are provided for dendrobium candidum culture, to meet the primary demand of its growth;
Wherein, described MS minimal medium formula is as shown in table 1 below, and mg/L represents the milligram number containing each composition in every liter of MS medium.
Table 1
The caseinhydrolysate using in medium of the present invention, English name caseinhydrolysace(is abbreviated as CH, below repeat no more) be taking crude milk albumen as raw material, through hydrochloric acid hydrolysis, decolouring, desalination, the dry product forming of spraying, contain 18 kinds of free amino acids, amino acid is the basis of synthetic protein, it is one of the most basic nutrient component of plant, plant growth is there is to the effect of long-acting benefit nitrogen, can Ao close trace element, promote plant to absorb.
The active carbon using in medium of the present invention, English name activatedcarbon(is abbreviated as AC, below repeats no more) there is the effect of the noxious material of metabolism in the growth of absorption culture.
The 6-benzyl aminoadenine using in medium of the present invention, English name 6-Benzylaminopurine(is abbreviated as 6-BA, below repeat no more), molecular formula is C12H11N5, belong to broad spectrum activity plant growth regulator, can promote plant cell growth, suppress the degraded of plant chlorophyll, improve amino acid whose content, Delaying Leaf-Senescence etc.
The methyl α-naphthyl acetate using in medium of the present invention, English name 1-Naphthaleneaceticacid(is abbreviated as NAA, below repeat no more), molecular formula is C10H7CH2CO2H, be broad spectrum type plant growth regulator, can promote cell division and expansion, induction forms adventive root and increases setting, prevent shedding, change female, male flower ratio etc.
The 6-glycosyl aminopurine using in medium of the present invention, English name 6-Furfurylamino-purine(is abbreviated as 6-KT, below repeats no more), molecular formula is C
10h
9n
5o, except having the fissional effect of promotion, also has the excised leaf of delaying and cuts flower withering, the effect of induced bud Differentiation and development and increase stomatal aperture.
In the composition of various medium of the present invention, the effect maximum of plant growth regulator, minimal medium only ensures the existence of culture and minimum physiological activity, only have and coordinate suitable plant growth regulator inducing cell division to start, the growth of callus growth and root, sprout blast etc. conforms with desirable growth.In dendrobium candidum tissue cultivation, important plant growth regulator is 6-BA, NAA and 6-KT, suitable plant growth regulator proportioning impels dendrobium candidum culture to develop to different directions, the induction of its somatic embryo and propagation need have the participation of NAA, low concentration proportioning is impelled culture seedling differentiation, suitably improves concentration proportioning and impels protocorms propagation.
The present invention is successfully inducing on the basis of dendrobium candidum somatic embryo, and the method that dendrobium candidum somatic embryo propagation is preserved has been grasped in research, and the molecular breeding, Fast-propagation and in vitro preservation that can be dendrobium candidum provide basic material.
Beneficial effect of the present invention:
1) method that dendrobium candidum somatic embryo of the present invention propagation is preserved, is used eight points of ripe capsules, plants shell not firmly, do not ftracture, and is easy to disinfection, avoids polluting, and is also conducive to operate; Utilize method of the present invention, cultivation program is simple, and reproduction coefficient is large, and seedling quality is high, and culture can be preserved 12 months continuously, between storage life, can breed 40 times in the situation that not needing subculture, can overcome in prior art the defects such as reproduction coefficient is low.
2) somatic embryo inducement of utilization of the present invention and proliferated culture medium are taking MS as minimal medium, to meet the primary demand of dendrobium candidum somatic embryo growth, and be equipped with 6-BA, 6-KT and the NAA of suitable concentration, ensure that somatic embryo division starts, breeds and grow; Add CH and AC supplements the nutrients and adsorbs the Toxic of discharging in its metabolism for somatic embryo growth provides simultaneously.Consequent dendrobium candidum somatic embryo cultivates that to have quantity many, and speed is fast, and the feature of structural integrity, for dendrobium candidum carries out genetic manipulation and breed improvement provides reliable foundation and effective way on cellular level.
Because somatic embryo is the artificial seed that plant produces under condition of tissue culture, so all have wide practical use in research fields such as plant developmental biology, plant highly efficient regeneration, genetic transformation, secondary metabolites production, cell engineerings.
3) the somatic embryo propagation Storaged media that the present invention utilizes, in the time dendrobium candidum somatic embryo being bred to preservation cultivation, can make culture in earlier stage carry out normal proliferate, after 2 months because medium reduces gradually, culture growth slows down, cultivate as proceeded normal propagation, should be transferred in time in fresh somatic embryo propagation Storaged media.But the condition of production and the market demand are inconsistent sometimes, need to postpone propagation, now culture can not transferred, continue to cultivate under former medium and environment, play the effect that a propagation is preserved, due to the minimizing of nutrition and the reduction of hormone, culture is in semistarvation state, culture proliferation and poor growth even stop growing, thereby have played the effect of a preservation.Preserve and compare with low temperature with ultralow temperature, the present invention preserves more convenient and cheap at normal temperatures, and breeds in preservation process, is more conducive to preserve the recovery growth of material and further breed.
4) the present invention has avoided the method that traditional low temperature is preserved, and has advantages of that normal temperature is cultivated power saving, renewal cultivation reacts fast, also can breed to meet market demand in a large number in the short period.
Brief description of the drawings
Fig. 1 is eight points of ripe dendrobium candidum capsules.
Fig. 2 is the protocorms that the induction of dendrobium candidum immature seed forms.
Fig. 3 is that propagation is cultivated the dendrobium candidum somatic embryo obtaining.
Fig. 4 is that dendrobium candidum somatic embryo forms leaf primordium.
Fig. 5 is that propagation is preserved the dendrobium candidum somatic embryo of cultivating 6 wheat harvesting periods.
Fig. 6 is the bud of dendrobium candidum embryo differentiate.
Fig. 7 is the seedling of dendrobium candidum embryo differentiate.
Embodiment
Below in conjunction with specific embodiments and the drawings, technical scheme of the present invention is described in further detail, but described embodiment does not limit the scope of the invention.
Embodiment 1
1) sterilization of explant: get eight points of ripe dendrobium candidum capsules (Fig. 1), on superclean bench, with 75% alcohol disinfecting after 45 seconds, then use 15～20%(volume ratio) aqueous sodium hypochlorite solution soaks 16 minutes, aseptic water washing 3-5 time;
2) induction of protocorm is cultivated: the moisture on the dendrobium candidum capsule surface after sterilization is blotted in sterile working, cut capsule with scalpel, its seed of picking, be inoculated on protocorm inducing culture and carry out the low light level according to cultivating, intensity of illumination 500 left and right, lux, were transferred under illumination and cultivate after 25 days, intensity of illumination is 2000 luxs, continue to cultivate after 20～25 days, explant is sprouted and is formed cone shape protocorm (Fig. 2), and protocorm inductivity reaches 95%;
Protocorm inducing culture formula is: MS minimal medium, 6-BA1.0mg/L, NAA0.5mg/L, CH1.0g/L, AC0.5g/L, sucrose 3%+ agar powder 6～7g/L;
3) induction of somatic embryo and propagation: protocorm subculture is incubated in the induction and proliferated culture medium of somatic embryo, after 30 days, protocorm produces somatic embryo and breeds gradually (Fig. 3); The rate of increase of 50 days reaches 18.7 times;
Induction and the proliferation culture medium formula of somatic embryo are: MS minimal medium, 6-BA1.0mg/L, 6-KT0.5mg/L, NAA0.5mg/L, CH0.5g/L, AC0.5g/L, sucrose 3%, agar powder 6～7g/L;
4) propagation of somatic embryo is preserved and is cultivated: somatic embryo switching is incubated to somatic embryo propagation Storaged media, after 2 months, reach peak of proliferation, appreciation rate reaches 20 times, continue to cultivate culture and start slow propagation, after 3～4 months, have a small amount of somatic embryo to start to sprout (Fig. 4), cultivate after 10-12 month, medium greatly reduces, the culture of a large amount of propagation flocks together, and in semistarvation state, but substantially keeps good vitality (Fig. 5);
The culture medium prescription that somatic embryo propagation is preserved is: MS minimal medium, 6-BA1.0mg/L, 6-KT0.5mg/L, NAA0.5mg/L, CH0.5g/L, AC0.5g/L, sucrose 3%, agar powder 6～7g/L;
5) recovery of somatic embryo growth is cultivated with propagation: propagation is preserved to the somatic embryo of cultivating, dispersion culture recovers growth and proliferated culture medium in fresh somatic cell, and after 1 week, somatic embryo starts to recover growth, after 2 weeks, breed gradually, obtain dendrobium candidum somatic embryo;
Somatic cell recovers growth: MS minimal medium, 6-BA1.0mg/L, 6-KT0.5mg/L, NAA0.5mg/L, CH0.5g/L, banana puree 100g/L, sucrose 3%, agar powder 6～7g/L;
6) sprouting of somatic embryo: propagation is cultivated to the dendrobium candidum somatic embryo switching obtaining and be incubated at somatic embryo germination medium, after 2～3 weeks, somatic embryo is sprouted into seedling (Fig. 6, Fig. 7) gradually;
The culture medium prescription that somatic embryo is sprouted is: MS minimal medium, 6-BA0.5mg/L, 6-KT0.5mg/L, NAA0.2mg/L, CH0.6g/L, AC0.3g/L, sucrose 3%, agar powder 6～7g/L;
7) strengthening seedling and rooting is cultivated: dendrobium candidum seedling is incubated to strengthening seedling and rooting medium, within 30 days, grows afterwards for complete candidum tissue culturing seedling;
Strengthening seedling and rooting culture medium formula is: 1/2MS minimal medium, NAA0.5mg/L, AC0.5g/L, sucrose 2%, agar powder 6～7g/L;
PH=5.4～5.6 of above-mentioned medium.
Finally should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.
Claims (7)
1. a propagation store method for dendrobium candidum somatic embryo, comprises the following steps:
1) sterilization of explant:
Get dendrobium candidum capsule, on superclean bench, with 75% alcohol disinfecting after 45～60 seconds, then the aqueous sodium hypochlorite solution that is 15～20% by volume ratio soaks 15～25 minutes, aseptic water washing 3～5 times;
2) induction of protocorm is cultivated:
The moisture on the dendrobium candidum capsule surface after sterilization is blotted in sterile working, cut capsule with scalpel, its seed of picking, be inoculated on protocorm inducing culture, first carry out the low light level according to cultivating, after 20～30 days, be transferred to illumination cultivation, continue to cultivate after 25～35 days, explant is sprouted into cone shape protocorm, and cultivation temperature is 25 ± 2 DEG C; Protocorm inducing culture formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.5～1.5g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L;
3) induction of somatic embryo and propagation:
Protocorm subculture is incubated in the induction and proliferated culture medium of somatic embryo, after 30 days, protocorm produces somatic embryo and breeds gradually;
Somatic embryo inducement and proliferation culture medium formula are: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L;
4) propagation of somatic embryo is preserved and is cultivated:
Somatic embryo switching is incubated to somatic embryo propagation Storaged media, after 2 months, reaches peak of proliferation; If carry out Fast-propagation, should transfer and breed again cultivation in fresh somatic embryo propagation Storaged media; Carry out Fast-propagation if do not needed, and to carry out germplasm preservation, and do not transfer reaching after peak of proliferation, continue to cultivate 3～4 months on former propagation Storaged media, somatic embryo still can slowly be bred, and have a small amount of somatic embryo to start to sprout, and to cultivate after 10～12 months, medium greatly reduces, culture flocks together, in semistarvation state, but substantially keep good vitality, cultivation temperature is 25 ± 2 DEG C;
Somatic embryo propagation Storaged media formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L;
5) recovery of somatic embryo growth is cultivated with propagation:
Propagation is preserved to the somatic embryo of cultivation, dispersion culture recovers growth and proliferated culture medium in fresh somatic embryo, and after 1 week, somatic embryo starts to recover growth, after 2 weeks, breeds gradually, obtains dendrobium candidum somatic embryo;
Somatic embryo recovers growth: MS minimal medium, 6-benzyl aminoadenine 0.8～1.2mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.5～1.0g/L, banana puree 80～120g/L, sucrose 2～4%, agar powder 6～7g/L;
6) sprouting of somatic embryo:
Propagation is cultivated to the dendrobium candidum somatic embryo switching obtaining and be incubated at somatic embryo germination medium, after 2～3 weeks, somatic embryo is sprouted into seedling gradually;
The culture medium prescription that somatic embryo is sprouted is: MS minimal medium, 6-benzyl aminoadenine 0.3～0.6mg/L, 6-glycosyl aminopurine 0.3～0.6mg/L, methyl α-naphthyl acetate 0.1～0.2mg/L, caseinhydrolysate 0.6～0.8g/L, active carbon 0.3～0.4g/L, sucrose 2～4%, agar powder 6～7g/L;
7) strengthening seedling and rooting is cultivated:
Dendrobium candidum seedling is incubated to strengthening seedling and rooting medium, within 30 days, grows afterwards for complete candidum tissue culturing seedling;
The culture medium prescription of strengthening seedling and rooting is: 1/2MS minimal medium, methyl α-naphthyl acetate 0.3～1.0mg/L, active carbon 0.3～0.5g/L, sucrose 2～4%, agar powder 6～7g/L;
The pH of all medium is 5.4～5.8.
2. the propagation store method of dendrobium candidum somatic embryo according to claim 1, is characterized in that, the maturity of the dendrobium candidum capsule described in step 1) be eight points ripe.
3. the propagation store method of dendrobium candidum somatic embryo according to claim 1, is characterized in that step 2) intensity of illumination of described low light level photograph is 300～500 luxs.
4. the propagation store method of dendrobium candidum somatic embryo according to claim 1, is characterized in that step 2) intensity of illumination of described illumination cultivation is 2000～2500 luxs.
5. the propagation store method of dendrobium candidum somatic embryo according to claim 1, is characterized in that, the cultivation temperature described in step 4) is 20～25 DEG C.
6. the medium for dendrobium candidum somatic embryo inducement and propagation, its formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L.
7. a medium of preserving for dendrobium candidum somatic embryo propagation, its formula is: MS minimal medium, 6-benzyl aminoadenine 0.5～1.5mg/L, 6-glycosyl aminopurine 0.2～0.8mg/L, methyl α-naphthyl acetate 0.2～0.6mg/L, caseinhydrolysate 0.4～0.8g/L, active carbon 0.3～0.6g/L, sucrose 2～4%, agar powder 6～7g/L.
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