CN103918551B - A kind of Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method - Google Patents
A kind of Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method Download PDFInfo
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Classifications
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- Y02P60/216—
Abstract
The invention belongs to planting technology field, it is specifically related to a kind of Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method, described cultural method includes: the fermentation culture of mycorrhizal fungi, the cultivation of dendrobe tissue culture Seedling, Herba Dendrobii Seedling and the symbiotic cultivation of mycorrhizal fungi, the Herba Dendrobii method for refining seeding of Mycorrhizal, dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant five steps of cultural method, adopt the medical value of the inventive method Herba Dendrobii and economic benefit all to be improved.
Description
Technical field
The invention belongs to planting technology field, be specifically related to a kind of Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method.
Background technology
Herba Dendrobii is China's tradition rare Chinese medicine, and it is all on the books that edition pharmacopeia is gone through by China.The former plant of Chinese medicine Herba Dendrobii is the orchid family (Orchidaceae) Dendrobium (DendrobiumSw.) herbaceos perennial.For many years, commodity Herba Dendrobii relies primarily on Wild Medicinal Dendrobium Plants, and along with the increase year by year with domestic and international demand that deepens continuously that medicinal dendrobium develops, China's Wild Medicinal Dendrobium Plants has suffered heavy damage, and some area even faces exhaustion.In recent years, people start the wild plant to Herba Dendrobii gradually and carry out house and plant cultivation, high-yield culture technique etc. and put into practice.
In current artificial culture, breeding relies primarily on division propagation and cottage propagation, but the method breeding coefficient is low, both uneconomical, again limit the yield potentiality of Herba Dendrobii, and inconvenience Planting management is promoted, thus expands cultivated area and be suppressed.Many places have employed tissue culture propagation method quickly to obtain a large amount of seedling；Owing to Herba Dendrobii carries out in artificial environment when tissue culture propagation, its medical value is far away from wild Herba Dendrobii, the method plantation Herba Dendrobii that therefore necessary employing is wild.
Summary of the invention
The technical problem to be solved is: for the deficiency of the medical value that pure artificial growth Herba Dendrobii has, it is provided that a kind of imitating wild planting process, to improve the medical value of Herba Dendrobii, increases economic efficiency.
The present invention is achieved through the following technical solutions.
A kind of Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method, its feature comprises the following steps:
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, root segment is immersed 1-10min in 1% calcium hypochlorite solution and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 20-30 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 2-30 DEG C after constant temperature culture 5-50 days, directly use or access liquid or solid fermentation medium further as mycorrhizal fungi seed strain and be amplified cultivating；
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, ground hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, agar 1-30gg/L, pH value 4-9；
Described fermentation medium is: Rhizoma Solani tuber osi 20-300g/L, glucose 2-300g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 20-300g/L, peptone 1-30g/L, ammonium sulfate 0.1-20g/L, potassium dihydrogen phosphate 0.1-20g/L, magnesium sulfate 0.1-20g/L, manganese chloride 0.1-20g/L, agar 1-30g/L, pH value 4-9；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain is accessed fermentation medium triangular flask or the vibration of other containers or fermentation tank ventilation light culture, cultivates and directly use after terminating or to smash cultured products with refiner standby；Fermentation condition is: inoculum concentration 6%-18%, temperature 20-30 DEG C, incubation time 5-90h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 6%-18%, temperature 20-30 DEG C of bottom fermentation is cultivated 2-50 days, obtains mycorrhizal fungi solid truffle when mycelia 50%～100% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: gather wild Dendrodium and belong to the young stem with axillalry bud, after alcohol-pickled 50 ± 10 seconds of 75%, soak 1-10 minute with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L ground for 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+；PH is 4-9, and at 20-30 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 10-90 days, and axillalry bud place can pumping shoot be clump bud shape, and shoot can be cut and repeat to cultivate, until growing up to complete test tube Seedling for transplanting；The protocorm induced maybe is inoculated into alternate culture on subculture multiplication fluid medium and subculture multiplication solid medium, and enrichment culture 10-60 days is a subculture cycle, cultivation temperature 20-30 DEG C, light intensity 1500-4000Lx, illumination 10-14 hour/day；
Described subculture multiplication fluid medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ Rhizoma Solani tuber osi liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9；
Described subculture multiplication solid medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato juice 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9；
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ Fructus Musae homogenate 20-200g/L+ sucrose 5-50g/L+ activated carbon 0.1-10g/L+ agar 3-30g/L ground for 1/2MS+NAA0.1-10mg/L+；Cultivation temperature 20-30 DEG C, pH4-9, to cultivate 3-90 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 1-90 days in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 1-15 days in vial；
nullB. Herba Dendrobii seedling is taken out from vial，Clean the culture medium of root，Airing after soaking 3-10 minute in the potassium permanganate solution of 0.1%，The bog moss and/or the hypodontia alpine rush or palm-bark rain cape moss that processed with soaking sterilization are cultivation matrix，With (1/3-1/10) knop5-30g/L+ sucrose 1-30g/L+ ammonium tartrate 0.01-30g/L+ mycorrhizal fungi fermentation liquid 5-100mL/L for symbiotic cultivation nutritional solution，With little plastic tub for cultivating container classification seedling exercising，Taking 3-5 strain is one clump，Root is planted in cultivation matrix，Seedling exercising adopts temperature canopy，Temperature controls at 10-38 DEG C，Air humidity is 40%-85%，Shade density 50-70%，Illuminance 1000-5000Lx，Water and take spray process，Seedling exercising time 1-12 month；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: selecting to be positioned at nature obscurity at 60%-80%, illumination is scattering light, intensity of illumination Camellia sinensis trunk under 1000-5000Lx environment；
B. Herba Dendrobii companion lichen patch tree planting: diameter 1-20cm place is coated with one layer of lichen mixture around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, thickness is 0.1-10cm, after cultivating 5-20 days, the Herba Dendrobii Seedling in little plastic tub is removed together with lichen substrate and tie at sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss position, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii is bundled in Camellia sinensis, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once, moistening plant root and neighbouring lichen；
Described lichen mixture (w/w) is by (1/3-1/10) knop5-30, Oryza glutinosa 5-20, fermentation animal wastes 10-70, lichen 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30；
C. the gathering of Herba Dendrobii: after plantation, 1-2 can gather, and when gathering, root stays the long root head of 2-3cm, cut always stay tender.
A in described step 1 is:
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 1-10min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 20-30 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 2-30 DEG C after constant temperature culture 5-50 days, directly use or access liquid or solid fermentation medium further as mycorrhizal fungi seed strain and be amplified cultivating；
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, ground hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, agar 1-30gg/L, pH value 4-9；
Described fermentation medium is: Rhizoma Solani tuber osi 20-300g/L, glucose 2-300g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 20-300g/L, peptone 1-30g/L, ammonium sulfate 0.1-20g/L, potassium dihydrogen phosphate 0.1-20g/L, magnesium sulfate 0.1-20g/L, manganese chloride 0.1-20g/L, agar 1-30g/L, pH value 4-9；
A in described step 2 is:
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Dendrobium young stem with axillalry bud, with after running water 5-15 minute on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 1-10 minute with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L ground for 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+；PH is 4-9, and at 20-30 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 10-90 days, and axillalry bud place can pumping shoot be clump bud shape, and shoot can be cut and repeat to cultivate, until growing up to complete test tube Seedling for transplanting；The protocorm induced maybe is inoculated into alternate culture in liquid and solid multiplication culture medium, and enrichment culture 10-60 days is a subculture cycle, cultivation temperature 20-30 DEG C, light intensity 1500-4000Lx, illumination 10-14 hour/day；
Described subculture multiplication fluid medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ Rhizoma Solani tuber osi liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9；
Described subculture multiplication solid medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato juice 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9；
Described step 5 is:
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx；
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is 10-April 10 annual March, or JIUYUE is taken second place；Around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, diameter 1-20cm place is coated with one layer of lichen mixture, thickness is 0.1-10cm, cultivating to be removed together with lichen substrate by the Herba Dendrobii Seedling in little plastic tub after 5-20d ties at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii is bundled in Camellia sinensis, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture (w/w) is by (1/3-1/10) knop5-30, Oryza glutinosa 5-20, fermentation animal wastes 10-70, lichen 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 1-12 time；Spraying every day 1-2 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, can spray a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: after plantation, 1-2 can gather, suitable harvest period is that when gathering, root stays the long root head of 2-3cm to before Clear and Bright after the Beginning of Winter, cut always stay tender, it is possible to continue results more than 8 years.
Described lichen include bog moss, hypodontia alpine rush or palm-bark rain cape moss, the knob moss that comes into leaves, intend broad-leaved gravelstone moss one or more.
Described binding material be non-woven fabrics, shade net, fishing line, straw rope, rattan one or more.
Embodiment
Embodiment 1
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 3min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 27 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 5 DEG C of constant temperature culture after 40 days, directly use；
Described isolation medium is: glucose 20g/L, peptone 8g/L, streptomycin 0.5g/L, ground hypodontia alpine rush or palm-bark rain cape moss 25g/L, casein hydrolysate 3g/L, agar 19g/L, pH value 4；
Described fermentation medium is: Rhizoma Solani tuber osi 120g/L, glucose 180g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 100g/L, peptone 8g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 8g/L, magnesium sulfate 12g/L, manganese chloride 02g/L, agar 10g/L, pH value 9；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain accesses fermentation medium triangular flask cultivate, smash cultured products with refiner standby；Fermentation condition is: inoculum concentration 12%, temperature 25 DEG C, incubation time 30h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 15%, 25 DEG C of bottom fermentations of temperature are cultivated 40 days, obtain mycorrhizal fungi solid truffle when mycelia 90% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Dendrobium young stem with axillalry bud, with after running water 5-15 minute on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 7 minutes with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 50g/L+ potato juice 105g/L+ sucrose 9g/L ground for 1/2MS+6-BA4mg/L+NAA0.5mg/L+；PH is 6, and at 22 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 70 days, and axillalry bud place can pumping shoot be clump bud shape, and shoot can be cut and repeat to cultivate, until growing up to complete test tube Seedling for transplanting；
Described subculture multiplication fluid medium is: 1/2MS+6-BA3mg/L+NAA5mg/L+ Rhizoma Solani tuber osi liquor 80g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 110g/L+ activated carbon 6g/L+ sucrose 37g/L, pH6；
Described subculture multiplication solid medium is: 1/2MS+6-BA3mg/L+NAA7mg/L+ potato juice 110g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 90g/L+ activated carbon 3g/L+ sucrose 29g/L+ agar 20g/L, pH7；
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 150g/L+ Fructus Musae homogenate 120g/L+ sucrose 30g/L+ activated carbon 5g/L+ agar 20g/L ground for 1/2MS+NAA3mg/L+；Cultivation temperature 25 DEG C, pH5, to cultivate 10 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 30 days in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 1-15 days in vial；
B. Herba Dendrobii seedling is taken out from vial, clean the culture medium of root, airing after soaking 8 minutes in the potassium permanganate solution of 0.1%, the hypodontia alpine rush or palm-bark rain cape moss processed with soaking sterilization is for cultivation matrix, with 1/7knop20g/L+ sucrose 28g/L+ ammonium tartrate 22g/L+ mycorrhizal fungi fermentation liquid 27mL/L for symbiotic cultivation nutritional solution, with little plastic tub for cultivating container classification seedling exercising, taking 3-5 strain is one clump, root is planted in cultivation matrix, seedling exercising adopts temperature canopy, temperature controls at 10-38 DEG C, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, seedling exercising time 1-12 month；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx；
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is 10-April 10 annual March, around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, diameter 10cm place is coated with one layer of lichen mixture, thickness is 3cm, after cultivating 10 days, the Herba Dendrobii Seedling in little plastic tub is removed together with lichen substrate and tie at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii being bundled in Camellia sinensis with non-woven fabrics, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture is by 1/9knop10g, Oryza glutinosa 14g, fermentation animal wastes 40g, bog moss 25g, sucrose 3g, ammonium tartrate 2g, mycorrhizal fungi powder 25g；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 5 times；Spraying every day 1-2 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, can spray a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: after plantation, 1-2 can gather, suitable harvest period is that when gathering, root stays the long root head of 2-3cm to before Clear and Bright after the Beginning of Winter, cut always stay tender, it is possible to continue results more than 8 years.
Embodiment 2
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 7min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 26 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 8 DEG C of constant temperature culture after 40 days, access liquid fermentation medium further as mycorrhizal fungi seed strain and be amplified cultivating；
Described isolation medium is: glucose 15g/L, peptone 13g/L, streptomycin 0.3g/L, ground hypodontia alpine rush or palm-bark rain cape moss 10g/L, casein hydrolysate 3g/L, agar 23g/L, pH value 4.5；
Described fermentation medium is: Rhizoma Solani tuber osi 250g/L, glucose 100g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 220g/L, peptone 21g/L, ammonium sulfate 11g/L, potassium dihydrogen phosphate 14g/L, magnesium sulfate 10g/L, manganese chloride 10g/L, agar 18g/L, pH value 7；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain is accessed fermentation medium fermentation tank ventilate light culture, cultivate and directly use after terminating；Fermentation condition is: inoculum concentration 10%, temperature 25 DEG C, incubation time 70h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 10%, 28 DEG C of bottom fermentations of temperature are cultivated 30 days, obtain mycorrhizal fungi solid truffle when mycelia 80% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Dendrobium young stem with axillalry bud, with after running water 5-15 minute on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 3 minutes with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 110g/L+ potato juice 160g/L+ sucrose 14g/L ground for 1/2MS+6-BA2mg/L+NAA5mg/L+；PH is 6, and at 27 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 50 days, and axillalry bud place can pumping shoot be clump bud shape, and shoot can be cut and repeat to cultivate, until growing up to complete test tube Seedling for transplanting；Enrichment culture 20 days is a subculture cycle, cultivation temperature 23 DEG C, light intensity 1500-4000Lx, illumination 10-14 hour/day；
Described subculture multiplication fluid medium is: 1/2MS+6-BA3mg/L+NAA7mg/L+ Rhizoma Solani tuber osi liquor 160g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 180g/L+ activated carbon 5g/L+ sucrose 32g/L, pH5；
Described subculture multiplication solid medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato juice 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 130g/L+ Fructus Musae homogenate 170g/L+ sucrose 19g/L+ activated carbon 3g/L+ agar 17g/L ground for 1/2MS+NAA7mg/L+；Cultivation temperature 25 DEG C, pH8, to cultivate 40 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 50 days in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 8 days in vial；
B. Herba Dendrobii seedling is taken out from vial, clean the culture medium of root, airing after soaking 3-10 minute in the potassium permanganate solution of 0.1%, the bog moss and the hypodontia alpine rush or palm-bark rain cape moss that processed with soaking sterilization are cultivation matrix, with 1/8knop20g/L+ sucrose 21g/L+ ammonium tartrate 3g/L+ mycorrhizal fungi fermentation liquid 70mL/L for symbiotic cultivation nutritional solution, with little plastic tub for cultivating container classification seedling exercising, taking 3-5 strain is one clump, root is planted in cultivation matrix, seedling exercising adopts temperature canopy, temperature controls at 10-38 DEG C, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, seedling exercising time 1-12 month；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx；
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is JIUYUE；Around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, diameter 18cm place is coated with one layer of lichen mixture, thickness is 6cm, after cultivating 16 days, the Herba Dendrobii Seedling in little plastic tub is removed together with lichen substrate and tie at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii being bundled in Camellia sinensis with shade net, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture is by 1/4knop18g, Oryza glutinosa 13g, fermentation animal wastes 50g, hypodontia alpine rush or palm-bark rain cape moss 8g, sucrose 5g, ammonium tartrate 3g, mycorrhizal fungi powder 7g；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 7 times；Spraying every day 1-2 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, can spray a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: after plantation, 1-2 can gather, suitable harvest period is that when gathering, root stays the long root head of 2-3cm to before Clear and Bright after the Beginning of Winter, cut always stay tender, it is possible to continue results more than 8 years.
Embodiment 3
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 1-10min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 20-30 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 23 DEG C of constant temperature culture after 30 days, access liquid further as mycorrhizal fungi seed strain；
Described isolation medium is: glucose 8g/L, peptone 4g/L, streptomycin 0.02g/L, ground hypodontia alpine rush or palm-bark rain cape moss 18g/L, casein hydrolysate 3g/L, agar 7g/L, pH value 4；
Described fermentation medium is: Rhizoma Solani tuber osi 100g/L, glucose 120g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 100g/L, peptone 1-30g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 2g/L, magnesium sulfate 7g/L, manganese chloride 5g/L, agar 10g/L, pH value 4；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain is accessed fermentation medium triangular flask, smashes cultured products with refiner standby；Fermentation condition is: inoculum concentration 8%, temperature 28 DEG C, incubation time 59h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 8%, 23 DEG C of bottom fermentations of temperature are cultivated 20 days, obtain mycorrhizal fungi solid truffle when mycelia 60% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Dendrobium young stem with axillalry bud, with after running water 12 minutes on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 8 minutes with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 50g/L+ potato juice 100g/L+ sucrose 8g/L ground for 1/2MS+6-BA8mg/L+NAA4mg/L+；PH is 8, and at 20-30 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 20 days, and axillalry bud place can pumping shoot be clump bud shape, and shoot can be cut and repeat to cultivate, until growing up to complete test tube Seedling for transplanting；
Described subculture multiplication fluid medium is: 1/2MS+6-BA3mg/L+NAA4mg/L+ Rhizoma Solani tuber osi liquor 80g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 60g/L+ activated carbon 9g/L+ sucrose 30g/L, pH7；
Described subculture multiplication solid medium is: 1/2MS+6-BA3mg/L+NAA2mg/L+ potato juice 10g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 50g/L+ activated carbon 3g/L+ sucrose 20g/L+ agar 15g/L, pH6；
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 10g/L+ Fructus Musae homogenate 120g/L+ sucrose 10g/L+ activated carbon 2g/L+ agar 5g/L ground for 1/2MS+NAAlmg/L+；Cultivation temperature 25 DEG C, pH5, to cultivate 30 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 10 days in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 10 days in vial；
B. Herba Dendrobii seedling is taken out from vial, clean the culture medium of root, airing after soaking 5 minutes in the potassium permanganate solution of 0.1%, the hypodontia alpine rush or palm-bark rain cape moss processed with soaking sterilization is for cultivation matrix, with 1/8knop10g/L+ sucrose 5g/L+ ammonium tartrate 1g/L+ mycorrhizal fungi fermentation liquid 50mL/L for symbiotic cultivation nutritional solution, with little plastic tub for cultivating container classification seedling exercising, taking 3-5 strain is one clump, root is planted in cultivation matrix, seedling exercising adopts temperature canopy, temperature controls at 10-38 DEG C, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, 8 months seedling exercising time；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx:
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is JIUYUE；Around Radix Camelliae sinensis, diameter 10cm place is coated with one layer of lichen mixture, thickness is 1cm, after cultivating 10 days, the Herba Dendrobii Seedling in little plastic tub is removed together with lichen substrate and tie at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii being bundled in Camellia sinensis with fishing line, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture is by 1/5knop15g, Oryza glutinosa 8g, fermentation animal wastes 60g, the knob moss 13g that comes into leaves, sucrose 3g, ammonium tartrate 1d, mycorrhizal fungi powder 13g；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 10 times；Spraying every day 1-2 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, can spray a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: after plantation, 1-2 can gather, suitable harvest period is that when gathering, root stays the long root head of 2-3cm to before Clear and Bright after the Beginning of Winter, cut always stay tender.
Embodiment 4
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 10min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 20 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 2 DEG C of constant temperature culture after 50 days, access solid fermentation culture medium further as mycorrhizal fungi seed strain and be amplified cultivating；
Described isolation medium is: glucose 30g/L, peptone 1g/L, streptomycin 1g/L, ground hypodontia alpine rush or palm-bark rain cape moss 10g/L, casein hydrolysate 0.01g/L, agar 1g/L, pH value 4；
Described fermentation medium is: Rhizoma Solani tuber osi 20g/L, glucose 20g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 20g/L, peptone 10g/L, ammonium sulfate 0.2g/L, potassium dihydrogen phosphate 0.2g/L, magnesium sulfate 0.1g/L, manganese chloride 20g/L, agar 30g/L, pH value 4；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain is accessed fermentation medium fermentation tank ventilate light culture, cultivate and directly use after terminating；Fermentation condition is: inoculum concentration 6%%, temperature 20 DEG C, incubation time 90h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 18%, temperature 20-30 DEG C of bottom fermentation is cultivated 50 days, obtains mycorrhizal fungi solid truffle when mycelia 50%% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Dendrobium young stem with axillalry bud, with after running water 5-15 minute on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 1 minute with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 10g/L+ potato juice 10g/L+ sucrose 2g/L ground for 1/2MS+6-BA10mg/L+NAA0.01mg/L+；PH is 4, at 20-30 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 10 days, axillalry bud place can pumping shoot be clump bud shape, the protocorm induced is inoculated into alternate culture in liquid and solid multiplication culture medium, and enrichment culture 10 days is a subculture cycle, cultivation temperature 20-30 DEG C, light intensity 1500-4000Lx, illumination 10-14 hour/day；
Described subculture multiplication fluid medium is: 1/2MS+6-BA10mg/L+NAA0.1mg/L+ Rhizoma Solani tuber osi liquor 20g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 10g/L+ activated carbon 1g/L+ sucrose 25g/L, pH4.9；
Described subculture multiplication solid medium is: 1/2MS+6-BA0.1mg/L+NAA0.1mg/L+ potato juice 200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1g/L+ activated carbon 10g/L+ sucrose 2g/L+ agar 1g/L, pH4；
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 200g/L+ Fructus Musae homogenate 20g/L+ sucrose 50g/L+ activated carbon 0.1g/L+ agar 30g/L ground for 1/2MS+NAA0.1mg/L+；Cultivation temperature 20-30 DEG C, pH4, to cultivate 3 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 1 day in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 1-15 days in vial；
B. Herba Dendrobii seedling is taken out from vial, clean the culture medium of root, airing after soaking 3 minutes in the potassium permanganate solution of 0.1%, the bog moss processed with soaking sterilization is for cultivation matrix, with 1/10knop30g/L+ sucrose 30g/L+ ammonium tartrate 0.01g/L+ mycorrhizal fungi fermentation liquid 5mL/L for symbiotic cultivation nutritional solution, with little plastic tub for cultivating container classification seedling exercising, taking 3-5 strain is one clump, root is planted in cultivation matrix, seedling exercising adopts temperature canopy, temperature controls at 10-38 DEG C, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, 1 month seedling exercising time；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx；
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is 10-April 10 annual March；Around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, diameter 1cm place is coated with one layer of lichen mixture, thickness is 10cm, after cultivating 5 days, the Herba Dendrobii Seedling in little plastic tub is removed together with lichen substrate and tie at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii being bundled in Camellia sinensis with straw rope, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture is by 1/10knop30g, Oryza glutinosa 5g, fermentation animal wastes 70g, plan broad-leaved gravelstone moss 30g, sucrose 10g, ammonium tartrate 10g, mycorrhizal fungi powder 1g；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 1 time；Spraying every day 1 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, can spray a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: gathering before planting latter 2 year Clear and Bright, when gathering, root stays the long root head of 2-3cm, cut always stay tender.
Embodiment 5
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 10min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 30 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 30 DEG C of constant temperature culture after 5 days, directly use；
Described isolation medium is: glucose 30g/L, peptone 1g/L, streptomycin 0.001g/L, ground hypodontia alpine rush or palm-bark rain cape moss 1g/L, casein hydrolysate 10g/L, agar 30g/L, pH value 9；
Described fermentation medium is: Rhizoma Solani tuber osi 300g/L, glucose 2g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 20g/L, peptone 1g/L, ammonium sulfate 0.1g/L, potassium dihydrogen phosphate 20g/L, magnesium sulfate 0.1g/L, manganese chloride 0.1g/L, agar 30g/L, pH value 4-9；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain is accessed fermentation medium triangular flask shaken cultivation, cultivates and directly use after terminating；Fermentation condition is: inoculum concentration 6%, temperature 20 DEG C, incubation time 90h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 6%%, 20 DEG C of bottom fermentations of temperature are cultivated 50 days, obtain mycorrhizal fungi solid truffle when mycelia 100% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Dendrobium young stem with axillalry bud, with after running water 5-15 minute on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 10 minutes with the mercuric chloride solution that concentration is 1g/L again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 200g/L+ potato juice 1g/L+ sucrose 2g/L ground for 1/2MS+6-BA10mg/L+NAA0.01mg/L+；PH is 9, at 30 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivate 90 days, axillalry bud place can pumping shoot be clump bud shape, and shoot can be cut and repeat to cultivate, and the protocorm induced is inoculated into alternate culture in liquid and solid multiplication culture medium, enrichment culture 60 days is a subculture cycle, cultivation temperature 20-30 DEG C, light intensity 1500-4000Lx, illumination 10-14 hour/day；
Described subculture multiplication fluid medium is: 1/2MS+6-BA10mg/L+NAA0.1mg/L+ Rhizoma Solani tuber osi liquor 200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1/L+ activated carbon 0.1g/L+ sucrose 2g/L, pH4；
Described subculture multiplication solid medium is: 1/2MS+6-BA0.1mg/L+NAA10mg/L+ potato juice 200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 200g/L+ activated carbon 10g/L+ sucrose 2g/L+ agar 30g/L, pH9；
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 200g/L+ Fructus Musae homogenate 20g/L+ sucrose 5g/L+ activated carbon 0.1g/L+ agar 30g/L ground for 1/2MS+NAA10mg/L+；Cultivation temperature 30 DEG C, pH4, to cultivate 90 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 90 days in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 15 days in vial；
B. Herba Dendrobii seedling is taken out from vial, clean the culture medium of root, airing after soaking 3-10 minute in the potassium permanganate solution of 0.1%, the bog moss processed with soaking sterilization is for cultivation matrix, with 1/3knop5g/L+ sucrose 1g/L+ ammonium tartrate 0.01g/L+ mycorrhizal fungi fermentation liquid 5mL/L for symbiotic cultivation nutritional solution, with little plastic tub for cultivating container classification seedling exercising, taking 3-5 strain is one clump, root is planted in cultivation matrix, seedling exercising adopts temperature canopy, temperature controls at 38 DEG C, air humidity is 85%, shade density 70%, illuminance 1000-5000Lx, water and take spray process, 12 months seedling exercising time；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx；
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is 10-April 10 annual March；Around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, diameter 20cm place is coated with one layer of lichen mixture, thickness is 0.1cm, after cultivating 20 days, the Herba Dendrobii Seedling in little plastic tub is removed together with lichen substrate and tie at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii being bundled in Camellia sinensis with rattan, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture is by 1/3knop5g, Oryza glutinosa 20g, fermentation animal wastes 10g, plan broad-leaved gravelstone moss 1g, hypodontia alpine rush or palm-bark rain cape moss 4g, sucrose 1g, ammonium tartrate 0.01g, mycorrhizal fungi powder 30g；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 12 times；Spraying every day 1 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, can spray a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: gathering when planting rear 1 year the Beginning of Winter, when gathering, root stays the long root head of 2-3cm, cut always stay tender.
Test example: the inhibitory action to toes carrageenin induced Acute inflammation
The preparation method of water solubility extract: adopt the Herba Dendrobii of embodiment 1～5 gained and commercially available artificial culture Herba Dendrobii to squeeze the juice.
Experimental technique: rat random packet, often group 10, first 1 hour gastric infusion (2g crude drug/kg) of Yu Zhiyan, sterile working, inflammation is caused to toes aponeurosis (aponeuroses) hemostasis 10% carrageenin 0.1m1 after Rat Right, measure before causing inflammation and cause after inflammation toes volume behind the right side of different time with drainage, so that the sufficient volume difference before and after scorching is swelling value, calculate swelling rate and suppression ratio according to swelling value.Experimental result is shown in table 1 below:
Swelling rate %=En-E0/ En×100
En=cause the swelling value of different time after inflammation
E0=cause the swelling value before inflammation
Suppression ratio %=C-T/C × 100
The swelling rate of C=matched group
The swelling rate of T=treatment group
The different Herba Dendrobii water solubility extract inhibitory action to rat toes carrageenin induced Acute inflammation of table 1
*: p < 0.05；*: p < 0.01
Above experimental result shows: adopt the relative commercially available product of water solubility extract that gained Herba Dendrobii of the present invention prepares to have acute inflammation effect better.
Claims (2)
1. a Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method, its feature comprises the following steps:
Step 1: the fermentation culture of mycorrhizal fungi
A. choose the fresh Nutrient root of wild Dendrodium, fall root surface humus and foreign material with running water；Root segment is immersed 1-10min in 1% calcium hypochlorite solution by superclean bench and carries out surface sterilizing, then use aseptic water washing 3-5 time；Root segment is cut into the segment of 0.5-3mm length, it is placed on isolation medium, light culture at 20-30 DEG C, after mycelia grows up to bacterium colony in root segment, picking mycelia is transferred after carrying out culture purified on isolation medium in solid fermentation culture medium, punch into bacterium sheet at colony edge in 2-30 DEG C after constant temperature culture 5-50 days, directly use or access liquid or solid fermentation medium further as mycorrhizal fungi seed strain and be amplified cultivating；
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, ground hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, agar 1-30g/L, pH value 4-9；
Described fermentation medium is: Rhizoma Solani tuber osi 20-300g/L, glucose 2-300g/L, hypodontia alpine rush or palm-bark rain cape moss abrasive material 20-300g/L, peptone 1-30g/L, ammonium sulfate 0.1-20g/L, potassium dihydrogen phosphate 0.1-20g/L, magnesium sulfate 0.1-20g/L, manganese chloride 0.1-20g/L, agar 1-30g/L, pH value 4-9；
B. liquid fermentation and culture
Mycorrhizal fungi seed strain is accessed liquid fermentation medium triangular flask or the vibration of other containers or fermentation tank ventilation light culture, cultivates and directly use after terminating or to smash cultured products with refiner standby；Fermentation condition is: inoculum concentration 6%-18%, temperature 20-30 DEG C, incubation time 5-90h；
C. solid fermentation is cultivated
Being seeded in solid fermentation culture medium by mycorrhizal fungi seed strain, inoculum concentration 6%-18%, temperature 20-30 DEG C of bottom fermentation is cultivated 2-50 days, obtains mycorrhizal fungi solid truffle when mycelia 50%-100% covers with culture medium；
Step 2: the cultivation of dendrobe tissue culture Seedling
A. protocorm induction and subculture multiplication are cultivated: in the 3-4 the first tenday period of a month month, gather the wild Herba Dendrobii Seedling young stem with axillalry bud, with after running water 5-15 minute on superclean bench behind with 75% alcohol-pickled 50 ± 10 seconds, soak 1-10 minute with the mercuric chloride solution that concentration is 1 g/l again, aseptic water washing 3-5 time, cuts 0.1-3cm stem section and is inoculated on the agar culture medium of hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L ground for 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+；PH is 4-9, and at 20-30 DEG C, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivates 10-90 days, and axillalry bud place can pumping shoot be clump bud shape, and shoot cutting repeats to cultivate, until growing up to complete test tube Seedling for transplanting；The protocorm induced maybe is inoculated into alternate culture in liquid and solid multiplication culture medium, and enrichment culture 10-60 days is a subculture cycle, cultivation temperature 20-30 DEG C, light intensity 1500-4000Lx, illumination 10-14 hour/day；
Described liquid proliferated culture medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ Rhizoma Solani tuber osi liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss grinds 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9；
Described solid multiplication culture medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato juice 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ activated carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9；
B. Rooting and hardening-off culture: be transferred to when unrooted height of seedling 2-3 centimetre in strengthening seedling and rooting culture medium and cultivate；Strengthening seedling and rooting culture medium is: hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ Fructus Musae homogenate 20-200g/L+ sucrose 5-50g/L+ activated carbon 0.1-10g/L+ agar 3-30g/L ground for 1/2MS+NAA0.1-10mg/L+；Cultivation temperature 20-30 DEG C, pH4-9, to cultivate 3-90 days, now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day；
Step 3: the symbiotic cultivation of Herba Dendrobii Seedling and mycorrhizal fungi: aseptically normal for root system 3-4 leaf tissue cultured seedling is accessed symbiosis culture 1-90 days in mycorrhizal fungi solid truffle, cultivates under field conditions (factors)；
Step 4: the Herba Dendrobii method for refining seeding of Mycorrhizal
A. under constant temperature, test tube Seedling is refined 1-15 days in vial；
B. Herba Dendrobii seedling is taken out from vial, clean the culture medium of root, dry after the potassium permanganate solution of 0.1% soaks 3-10 minute, the bog moss and/or the hypodontia alpine rush or palm-bark rain cape moss that processed with soaking sterilization are cultivation matrix, with 1/3-1/10knop5-30g/L+ sucrose 1-30g/L+ ammonium tartrate 0.01-30g/L+ mycorrhizal fungi fermentation liquid 5-100mL/L for symbiotic cultivation nutritional solution, with little plastic tub for cultivating container classification seedling exercising, taking 3-5 strain is one clump, root is planted in cultivation matrix, seedling exercising adopts temperature canopy, temperature controls at 10-38 DEG C, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, seedling exercising time 1-12 month；
Step 5: dendrobe tissue culture Seedling and Camellia sinensis are grown nonparasitically upon another plant cultural method
A. the selection of Camellia sinensis: need to meet the water source of national grade ii standard near tea ground, long-term temperature on average is at 10 DEG C to 20 DEG C, and relative air humidity is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters；Tea place faces south the domatic gradient more than 5-20 degree, grows the tea place of more than 10 years；First carrying out before field planting suitably prunes beats leaf, makes the trunk place nature obscurity of cultivation Herba Dendrobii at 60%-80%, and illumination is scattering light, and intensity of illumination is at 1000-5000Lx；
B. Herba Dendrobii companion lichen patch tree planting: the plantation time is 10-April 10 annual March, or JIUYUE is taken second place；Around the nearly root trunk of Camellia sinensis and Radix Camelliae sinensis, diameter 1-20cm place is coated with one layer of lichen mixture, thickness is 0.1-10cm, cultivating to be removed together with cultivation matrix by the Herba Dendrobii Seedling in little plastic tub after 5-20d ties at the more position of sunny slope tea branch Enteromorpha clathrata (Roth) Grev. Emend bliding moss, making seedling root system be adjacent to trunk lichen, lichen mixture coating is close to around trunk in substrate bottom；One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres；After Herba Dendrobii is bundled in Camellia sinensis, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, moistening plant root and neighbouring lichen, it is ensured that lichen can not hydrops；
Described lichen mixture is made up of 1/3-1/10knop5-30, Oryza glutinosa 5-20, fermentation animal wastes 10-70, lichen 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30 by w/w；
C. bionical dendrobium tea ground Cultivate administration: answer weeding 2-3 time every year, remove weeds and dry branches and fallen leaves；Annual spray symbiotrophy liquid 1-12 time；Spraying every day 1-2 water, humid control is at 60-80%, and winter-spring season cuts off the branch that attached main plant is overstocked in good time, to ensure that obscurity is at 70-80%, sprays a plant source biopesticide when pest and disease damage is occurred frequently per ten days；
D. the gathering of Herba Dendrobii: after plantation, 1-2 can gather, suitable harvest period is that when gathering, root stays the long root head of 2-3cm to before Clear and Bright after the Beginning of Winter, cuts and always stays tender, continues results more than 8 years.
2. cultural method according to claim 1, it is characterised in that: described binding material be non-woven fabrics, shade net, fishing line, straw rope, rattan one or more.
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