CN105519416A - Artificial breeding method for symbiotic germchit of Dendrobium devonianum - Google Patents
Artificial breeding method for symbiotic germchit of Dendrobium devonianum Download PDFInfo
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- 241001264165 Dendrobium devonianum Species 0.000 title claims abstract description 51
- 238000009395 breeding Methods 0.000 title claims abstract description 15
- 239000000725 suspension Substances 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000011159 matrix material Substances 0.000 claims abstract description 11
- 241000233866 Fungi Species 0.000 claims abstract description 7
- 239000008223 sterile water Substances 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 33
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 claims description 24
- 230000002538 fungal effect Effects 0.000 claims description 13
- 230000035784 germination Effects 0.000 claims description 13
- 239000004677 Nylon Substances 0.000 claims description 10
- 239000004744 fabric Substances 0.000 claims description 10
- 229920001778 nylon Polymers 0.000 claims description 10
- 239000007921 spray Substances 0.000 claims description 10
- 241000737241 Cocos Species 0.000 claims description 8
- 235000013162 Cocos nucifera Nutrition 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000001186 cumulative effect Effects 0.000 claims description 2
- 230000003203 everyday effect Effects 0.000 claims description 2
- 238000005507 spraying Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 20
- 230000004083 survival effect Effects 0.000 abstract description 4
- 241000574106 Tulasnella sp. Species 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 238000009400 out breeding Methods 0.000 abstract 1
- 230000008569 process Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241001523681 Dendrobium Species 0.000 description 1
- 241000026010 Dendrobium candidum Species 0.000 description 1
- 240000004638 Dendrobium nobile Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- 230000003816 axenic effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000442 meristematic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000027272 reproductive process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses an artificial breeding method for symbiotic germchit of Dendrobium devonianum. The method comprises the steps of subjecting Tulasnella sp., of which the collection number is CGMCC No. 9551, to amplified fermenting culture, filtering mycelia, adding a proper volume of sterile water into the mycelia, carrying out crushing by a refiner so as to prepare a fungus suspension, adding Dendrobium devonianum seeds into the fungus suspension so as to prepare a seed-fungus suspension, preparing a seedling raising matrix from coco coir, tree leaves and tree barks, sprinkling 50-500ml of the seed-fungus suspension to the matrix per square meter, and carrying out breeding for 45 days while maintaining the moisture content of the seedling raising matrix to 50-90%, thereby obtaining a large number of symbiotic young seedlings. According to the method, the seedling forming time of the seeds can be remarkably shortened, the seedling raising cost can be reduced, a relatively large number of Dendrobium devonianum young seedlings can be obtained in one time, and the growth speed and natural-environment-transplanted survival rate of the symbiotic young seedlings are improved and are higher than those of asymbiotic-germinated young seedlings. The method achieves a new way for obtaining the seed seedlings from the Dendrobium devonianum seeds and has important practical popularization and application values.
Description
Technical field
The invention belongs to dendrobe cultivation technical field, specifically relate to a kind of fungal biodegradation dendrobium devonianum seeds that utilizes to obtain the artificial breeding method of a large amount of dendrobium devonianum symbiosis seedling fast.
Background technology
Dendrobium devonianum (Dendrobiumdevonianum), as traditional Chinese medicine material, has high medical value, and its cultivation scale, inferior to dendrobium candidum, even becomes pillar industry in some area (as Longling County, Yunnan Province).The Main Cultivation pattern of the current stem of noble dendrobium has the intensive cropping of enterprise and " company+base+peasant household " pattern of employing thereof, also have part small enterprise or peasant household to adopt simple and easy frame intensive cropping on a small scale, and the Ecology that enterprise or individual carry out on a small quantity is planted.The source of seedling of various cultivation mode is mainly plantlet in vitro, and small part is Wild plant, rare symbiosis seedling.Main cause is that growing dendrobium seedlings is relatively stable by tissue culture technology flow process, is easy to batch production large-scale production.Although dendrobe tissue culture seedling growing process develops relative maturity, but still also exist that cost is high, the cycle is long, the operated by personnel that the larger needs of technical difficulty are special, many defects such as transplanting survival rate is on the low side under field conditions (factors), and later period growth and development is slow.Source of seedling becomes the bottleneck of dendrobe cultivation industry development, has had a strong impact on the health of industry, sustainable development.
Basic biological characteristics based on dendrobium devonianum: seed is very tiny, ripe seed does not have endosperm or cotyledon, and only have the embryo of ateliosis, seed germination needs to rely on specific symbiotic effects to obtain nutriment completely under field conditions (factors).If a large amount of symbiosis seedling can be obtained by dendrobium devonianum seeds symbiotic germination technology, then can overcome the defect of existing tissue culture, not only can improve seedling quantity, simplify seedling production process, shorten the seed seedling time, obvious reduction production cost, the more important thing is that can significantly improve seedling revert to the survival rate after in natural environment and growth of seedling speed, strengthens the environmental suitability of seedling.But still successfully do not report in prior art.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, the method of a kind of quick acquisition a large amount of dendrobium devonianum symbiosis seedling is provided, to shorten the dendrobium devonianum seedling fostering time, reduces seedling fostering cost, improve seedling quantity, improve the environmental suitability of dendrobium devonianum seedling.
Object of the present invention is achieved by the following technical programs.
Except as otherwise noted, percentage of the present invention is mass percent.
An artificial breeding method for dendrobium devonianum symbiosis seedling, comprises the following steps:
(1) gather field naturally solid and ripe but really the still uncracked dendrobium devonianum seeds of pod save backup;
(2) making of fungal suspension liquid:
The making of 2.1 medium: bottled by PDA liquid nutrient medium with the amount of conical flask 10% volume, load autoclave after sealing, in 121 DEG C, under 115KPa condition, sterilizing 20min, for subsequent use;
2.2 by the activation culture glued membrane bacterium (Tulasnellasp.) of 3 ~ 10 days, and CGMCCNo.9551 is inoculated in PDA liquid nutrient medium for subsequent use, and in 23 ~ 28 DEG C, 120 ~ 200rpm condition bottom fermentation is cultivated 5 ~ 10 days;
2.3 obtain mycelium with filtered through gauze zymotic fluids, with aseptic water washing mycelium 3 ~ 5 times and draining to remove residual glucose, the sterile water of mycelium and 5 ~ 50 times of quality is put into refiner and smashes and make fungal suspension liquid;
(3) making of seed-fungal suspension liquid: dendrobium devonianum seeds and fungal suspension liquid are mixed and makes seed-fungal suspension liquid, controlling great-hearted dendrobium devonianum seeds density in seed-fungal suspension liquid is 20 ~ 200/ml;
(4) seedling frame and seedling dish prepare:
4.1 install the shade net that shade rate is 60 ~ 90% above seedbed;
Coconut palm chaff, bark and leaf soak one week with clear water by 4.2 respectively, and the ratio then accounting for matrix cumulative volume 50% ~ 95% in coconut palm chaff mixes, and in 121 DEG C, sterilizing 20min under 115KPa condition, as seedling medium;
Seedling medium is loaded seedling dish by 4.3 uniformly, thickness 1.5 ~ 2.0cm, then seedling dish is spilt slightly larger than the nylon net cloth of seedling dish to prevent seed at the stromal surface one piece of area that tiles, the aperture of nylon net cloth is 20 ~ 40 μm, the seedling medium of uniform fold a layer thickness 0.4 ~ 0.6cm again on nylon net cloth, spray water saturated to matrix, for subsequent use;
(5) dendrobium devonianum seeds and fungi nursery symbiotic germination are sowed:
The seed made-fungal suspension liquid is evenly sprayed at Miao Panshang for subsequent use according to the ratio of every square meter of substrate sprinkling 50 ~ 500ml by 5.1;
After 5.224 hours, under non-solar direct projection condition, spray atomized water every day to keep the water content of seedling medium 50% ~ 90%;
During 5.3 seed symbiotic germination to 45 day, a large amount of symbiosis seedling can be formed.
Further, the condition of culture described in step (2.2) is preferably 28 DEG C, 150rpm, 7 days.
The quality of the making fungal suspension liquid sterile water used described in step (2.3) is mycelial 10 times.
Dendrobium devonianum seeds density described in step (3) is 185/ml.
Shade net shade rate described in step (4.1) is 75%.
In seedling medium described in step (4.2), the volume ratio of coconut palm chaff, bark and leaf is 8:1:1.
The aperture of the nylon net cloth described in step (4.3) is preferably 30 μm.
In step (5.1), every square meter of substrate sprays the seed-fungal suspension liquid of 300ml;
In step (5.2), the water content keeping seedling medium is 70% ~ 80%.
In step (5.2), the time of spraying atomized water is before every morning 9 or after 6 pm.
Compared with prior art, the present invention has the following advantages:
1, compare seed asepsis sprouting technology, the present invention significantly can reduce seedling cost, only needs the numerous fungi of preparation expansion PDA liquid nutrient medium used and seedling medium can complete sapling multiplication process.Not needing in reproductive process through repeatedly transferring, without the need to hardening, significantly shorten the seed seedling time yet.
2, under same culture environment and condition, the dendrobium devonianum seeds that check experiment and axenic germination are sowed can not be sprouted and form seedling, the germination rate of Orchid Seeds under the natural conditions of bibliographical information field is had to be no more than 0.03%, and germination rate improves greatly after applying the present invention, a large amount of symbiosis seedlings can be obtained in the shorter time.
3, simultaneously, the symbiotic relation that Early seedling stage has been set up with fungi, therefore the growth rate of symbiosis seedling and the survival rate be transplanted in natural environment are all higher than non-symbiosis germination seedling.
4, present invention achieves the new way that dendrobium devonianum seeds obtains seedling, there are important real promotion and application and be worth, for industry development provides brand-new thinking.
The explanation of preservation biomaterial
Bacterial strain of the present invention, on 07 18th, 2014, has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCG); This centre address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica.This strain classification called after glued membrane bacterium (Tulasnellasp.), preserving number is CGMCCNo.9551.
Embodiment
The present invention is described in further detail by the following examples, but embodiment is not the restriction to technical solution of the present invention.
Embodiment 1:
1, the acquisition of tooth lobe kind matter and preservation
Gather naturally solid and the ripe but still uncracked dendrobium devonianum seeds in field to save backup.Dendrobium devonianum seeds used picks up from the Wild population of Longling County, Baoshan, Yunnan city.
2, the making of the suspension of fungi
The making of 2.1 medium: PDA liquid nutrient medium Normal practice, in right amount.Load 70 bottles of 500ml triangular flasks with every bottle of 50ml average mark, wrap up bottleneck after sealing with newspaper, load autoclave in 121 DEG C, under 115KPa condition, sterilizing 20min, for subsequent use.
2.2 by the activation culture bacterial strain of 7 days-glued membrane bacterium (Tulasnellasp.) CGMCCNo.9551, and be inoculated in the triangular flask containing 50ml liquid nutrient medium of above-mentioned sterilizing, in 28 DEG C, 150rpm condition of culture bottom fermentation cultivates 7 days.
2.3 obtain mycelium with the cultured zymotic fluid of filtered through gauze also uses aseptic water washing mycelium 4 times, and each draining is to remove residual glucose.Put into refiner for the draining mycelium of 1:10 and sterile water in mass ratio to smash and make fungal suspension liquid.
The making of 3, seed-fungal suspension liquid
3.1 routinely TTC method detect dendrobium devonianum seeds vigor for subsequent use, to confirm that seed vitality has that it's too late the ratio of vigor seed, confirm seed consumption according to there being the ratio of vigor seed.In the present embodiment, vigourous seed ratio is 79.6%.
3.2 by dendrobium devonianum fruit pod about 30% seed add the mixing of fungal suspension liquid and make seed-suspension.Get 6 the free-hand film-makings of 200 μ l suspension and detect seed number under the microscope, namely seed amount contained by calculating every milliliter obtains suspension seed density.This obtains seed-fungal suspension liquid altogether and amounts to 360ml, and seed density is 147/ml.
3.3 prepare CK blank simultaneously: the seed that the aqueous agar solution of 100ml0.1% adds in a dendrobium devonianum fruit pod about 20% makes blank suspension, and seed density measures 6 times altogether, seed density 375/ml.
4, the preparation of simple and easy seedling frame and seedling dish
Process of sheltering from heat or light is done with the shade net that shade density is 75% in space high for about 2m above seedbed by 4.1, floor space about 15 square meter.
The bark collected in coconut palm chaff and the mountain forest near nursery, fallen leaves soak one week by 4.2 in advance, then mix in the ratio of coconut palm chaff, bark and leaf volume 8:1:1 and make seedling medium, proceed at 121 DEG C in container, under 115KPa condition, sterilizing 20min, for subsequent use.
4.3 ready matrix are taken out, uniform flat overlay on thickness on seedling dish and be about 1.5-2.0cm (seedling dish size is: length × wide × height=53 × 26 × 6cm), be now that the nylon net cloth of 30 μm spills seedling dish to prevent seed in stromal surface one piece of aperture slightly larger than seedling disc area of tiling, then order is in the nylon net cloth flat same matrix covered a layer thickness and be about 0.5cm uniformly, spray running water saturated to water content of substrate, for subsequent use.
5,06 ~ July in 2015 carry out dendrobium devonianum seeds and mycosymbiosis and sprout test at Chinese Academy of Sciences's Xishuangbanna tropical botanical garden seedling nursery, mean temperature of air 23 DEG C ~ 31 DEG C.
5.1 get seed-fungal suspension liquid watering can that 40ml made is evenly sprayed at the Miao Panshang that matrix is housed.Meanwhile, spray agar-seed suspension liquid and test in contrast, contrast seedling dish places distance symbiosis seedling culture more than dish 50m in case fungal contamination.
Within 5.2 second days, rise, before every morning 9 or spray appropriate atomized water after 6 pm to keep the humidity of artificial substratum.
Start to note to observe the developmental state of protocorm, quantity carry out experimental record when 5.330 days.Get part Experiment sample when finding protocorm to take back laboratory and carry out microexamination to it: the mycorrhizal morphology in protocorm, structure, hypha body color etc.
6, data analysis
Find that there is a large amount of protocorms and seedling during seed-mycosymbiosis sprouting experiment 45 days, even have protocorm protomeristem to be differentiated to form two panels leaf further, now the protocorm quantity of each seedling dish is in table 1.The blank group not connecing bacterium has not observed protocorm or seedling formation.The protocorm formation rate 4.95 ± 3.48% (n=9) of inoculating strain is significantly higher than blank process.Interpretation of result shows, dendrobium devonianum seeds only connects bacterium process and could sprout, and forms protocorm and seedling.Relative to blank test, mycosymbiosis Germination Technique of the present invention is sprouted promotion dendrobium devonianum seeds and is had remarkable effect.
Table 1 is experiment process and control experiment protocorm quantity respectively
Table1Theprotcormnumberofeachseedlingplateandcontroltestplate
Embodiment 2
Repeat embodiment 1, have following difference:
Within 2015 01 ~ 02 month, a simple and easy seedbed is built in the vacant lot in Xishuangbanna tropical botanical garden scientific research center of the Chinese Academy of Sciences, is that process of sheltering from heat or light is done in space high for about 1m above seedbed by the shade net of 75% by shade density, this seedbed floor space about 9 square meter.Mean temperature of air 13 DEG C ~ 24 DEG C.
Get seed-fungal suspension liquid watering can that 100ml made and be evenly sprayed at the Miao Panshang that matrix is housed, the seed density of suspension is 185/ml, sows 4 seedling dishes altogether.Meanwhile, spray agar-seed suspension liquid and test in contrast, seed density is 320/ml, and contrast seedling dish places distance symbiosis seedling culture more than dish 30m in case fungal contamination, sows 2 seedling dishes altogether.All the other condition of culture are consistent.
Seed-mycosymbiosis is sprouted when testing about 50 days and is found that there is a large amount of protocorms and seedling, and even have part protocorm meristematic tissue to be differentiated to form two panels leaf further, the protocorm quantity of each seedling dish is in table 2.The protocorm formation rate 5.34 ± 1.80% (n=4) of inoculating strain is significantly higher than control treatment.Interpretation of result shows, dendrobium devonianum seeds only connects bacterium process and could sprout, and forms protocorm and seedling.Relative to check experiment, mycosymbiosis Germination Technique of the present invention is sprouted promotion dendrobium devonianum seeds and is had remarkable effect.
Table 2 is experiment process and control experiment protocorm quantity respectively
Table2Theprotcormnumberofeachseedlingplateandcontroltestplateinsummer
The present invention sprouts dendrobium devonianum seeds and mycosymbiosis and has carried out research under open environment condition, successfully obtain a large amount of symbiosis seedlings, (be respectively: 4.95 ± 3.48% (n=9) and 5.34 ± 1.80% (n=4) and each seedling dish sprouting protocorm quantity heterogeneity although average germination rate is lower, but, in view of dendrobium devonianum fruit pod seed content pole is nearly tens thousand of to hundreds thousand of (this laboratory was once measured numerical value and is about 18.3 ten thousand), and the method sowing operating process is simple, with low cost and without the need to plurality of advantages such as hardenings, therefore the germination rate that this sowing obtains still has important using value, for the acquisition of dendrobium devonianum cultivation industry seedling provides feasible method.
Claims (10)
1. an artificial breeding method for dendrobium devonianum symbiosis seedling, comprises the following steps:
(1) gather field naturally solid and ripe but really the still uncracked dendrobium devonianum seeds of pod save backup;
(2) making of fungal suspension liquid:
The making of 2.1 medium: bottled by PDA liquid nutrient medium with the amount of conical flask 10% volume, load autoclave after sealing, in 121 DEG C, under 115KPa condition, sterilizing 20min, for subsequent use;
2.2 by the activation culture glued membrane bacterium (Tulasnellasp.) of 3 ~ 10 days, and CGMCCNo.9551 is inoculated in PDA liquid nutrient medium for subsequent use, and in 23 ~ 28 DEG C, 120 ~ 200rpm condition bottom fermentation is cultivated 5 ~ 10 days;
2.3 obtain mycelium with filtered through gauze zymotic fluids, with aseptic water washing mycelium 3 ~ 5 times and draining to remove residual glucose, the sterile water of mycelium and 5 ~ 50 times of quality is put into refiner and smashes and make fungal suspension liquid;
(3) making of seed-fungal suspension liquid: dendrobium devonianum seeds and fungal suspension liquid are mixed and makes seed-fungal suspension liquid, controlling great-hearted dendrobium devonianum seeds density in seed-fungal suspension liquid is 20 ~ 200/ml;
(4) seedling frame and seedling dish prepare:
4.1 install the shade net that shade rate is 60 ~ 90% above seedbed;
Coconut palm chaff, bark and leaf soak one week with clear water by 4.2 respectively, and the ratio then accounting for matrix cumulative volume 50% ~ 95% in coconut palm chaff mixes, and in 121 DEG C, sterilizing 20min under 115KPa condition, as seedling medium;
Seedling medium is loaded seedling dish by 4.3 uniformly, thickness 1.5 ~ 2.0cm, then seedling dish is spilt slightly larger than the nylon net cloth of seedling dish to prevent seed at the stromal surface one piece of area that tiles, the aperture of nylon net cloth is 20 ~ 40 μm, the seedling medium of uniform fold a layer thickness 0.4 ~ 0.6cm again on nylon net cloth, spray water saturated to matrix, for subsequent use;
(5) dendrobium devonianum seeds and fungi nursery symbiotic germination are sowed:
The seed made-fungal suspension liquid is evenly sprayed at Miao Panshang for subsequent use according to the ratio of every square meter of substrate sprinkling 50 ~ 500ml by 5.1;
After 5.224 hours, under non-solar direct projection condition, spray atomized water every day to keep the water content of seedling medium 50% ~ 90%;
During 5.3 seed symbiotic germination to 45 day, symbiosis seedling can be formed.
2. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: the condition of culture described in step (2.2) is preferably 28 DEG C, 150rpm, 7 days.
3. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: the quality of the making fungal suspension liquid sterile water used described in step (2.3) is mycelial 10 times.
4. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: the dendrobium devonianum seeds density described in step (3) is 185/ml.
5. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: the shade net shade rate described in step (4.1) is 75%.
6. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: in the seedling medium described in step (4.2), the volume ratio of coconut palm chaff, bark and leaf is 8:1:1.
7. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: the aperture of the nylon net cloth described in step (4.3) is preferably 30 μm.
8. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: in step (5.1), and every square meter of substrate sprays the seed-fungal suspension liquid of 300ml.
9. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: in step (5.2), and the water content keeping seedling medium is 70% ~ 80%.
10. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, is characterized in that: in step (5.2), and the time of spraying atomized water is before every morning 9 or after 6 pm.
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