CN103918551A - Imitation wild cultivation method of mycorrhizated dendrobium plant tea tree - Google Patents
Imitation wild cultivation method of mycorrhizated dendrobium plant tea tree Download PDFInfo
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- Y02P60/216—
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Abstract
The invention belongs to the technical field of plantation, and concretely relates to an imitation wild cultivation method of mycorrhizated dendrobium plant tea tree. The cultivation method comprises the steps of fermentation culturing of mycorrhizal fungi, culturing of dendrobium tissue culture plants, symbiotic cultivation of dendrobium plants and mycorrhizal fungi, hardening seedling of the mycorrhizated dendrobium plants, and epiphytic cultivation of the dendrobium tissue culture plants and tea tree. The method improves the medical values and he economic benefit of dendrobium.
Description
Technical field
The invention belongs to planting technology field, be specifically related to a kind of Mycorrhizal stem of noble dendrobium seedling tea tree pseudo-wild cultivating method.
Background technology
The stem of noble dendrobium is China's tradition rare traditional Chinese medicine, and it is all on the books that edition pharmacopeia is gone through by China.The former plant of the Chinese medicine stem of noble dendrobium is the orchid family (Orchidaceae) Dendrobium (Dendrobium Sw.) herbaceos perennial.For many years, the commodity stem of noble dendrobium mainly relies on Wild Medicinal Dendrobium Plants, and along with deepening continuously and the increase year by year of demand both at home and abroad 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 that gradually the wild plant of the stem of noble dendrobium is carried out to house and plant cultivation, high-yield culture technique etc. and put into practice.
In current artificial cultivation, breeding is main relies on division propagation and cottage propagation, but the method reproduction coefficient is low, both uneconomical, has limited again the yield potentiality of the stem of noble dendrobium, and inconvenient Planting management is promoted, thereby expansion cultivated area is suppressed.Many places have adopted tissue culture propagation method to obtain fast a large amount of seedling; Because the stem of noble dendrobium carries out when the tissue culture propagation in artificial environment, its medical value is far away from wild dendrobium candidum, therefore the wild method plantation dendrobium candidum of necessary employing.
Summary of the invention
Technical problem to be solved by this invention is: the deficiency of the medical value having for the pure artificial planting stem of noble dendrobium, a kind of imitating wild planting process is provided, and to improve the medical value of the stem of noble dendrobium, increase economic efficiency.
The present invention is achieved through the following technical solutions.
A Mycorrhizal stem of noble dendrobium seedling tea tree pseudo-wild cultivating method, its feature comprises the following steps:
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, root segment is immersed to 1-10min in 1% calcium hypochlorite solution and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 20-30 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, after 2-30 ℃ of constant temperature culture 5-50 days, at colony edge, punch into bacterium sheet, directly use or as mycorrhizal fungi seed bacterial classification, further access liquid or solid fermentation medium and amplify cultivation;
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, the hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, the agar 1-30gg/L that ground, pH value 4-9;
Described fermentation medium is: potato 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 bacterial classification is accessed to triangular flask or the vibration of other containers or the dark cultivation of fermentation tank ventilation for fermentation medium, and cultivation finishes rear direct use or smashes cultured products with refiner standby; Fermentation condition is: inoculum concentration 6%-18%, temperature 20-30 ℃, incubation time 5-90h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 6%-18%, temperature 20-30 ℃ of bottom fermentation cultivated 2-50 days, obtains mycorrhizal fungi solid bacterium piece when mycelia 50%～100% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: gather wild Dendrodium and belong to the young stem with axillalry bud, with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 1-10 minute, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L that 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+ ground; PH is 4-9, and at 20-30 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 10-90 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting; Maybe the protocorm inducing is inoculated into alternate culture on shoot proliferation liquid nutrient medium and shoot proliferation solid culture medium, it 10-60 days is a subculture cycle that propagation is cultivated, cultivation temperature 20-30 ℃, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ active carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9;
Described shoot proliferation solid 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+ active carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9;
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ banana homogenate 20-200g/L+ sucrose 5-50g/L+ active carbon 0.1-10g/L+ agar 3-30g/L that 1/2MS+NAA0.1-10mg/L+ ground; Cultivation temperature 20-30 ℃, pH4-9, cultivates 3-90 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated to 1-90 days, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial to 1-15 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 3-10 minute, the bog moss that the soaking sterilization of take was processed and/or hypodontia alpine rush or palm-bark rain cape moss are cultivation matrix, (1/3-1/10) knop5-30g/L+ sucrose 1-30g/L+ ammonium tartrate 0.01-30g/L+ mycorrhizal fungi zymotic fluid 5-100mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 10-38 ℃, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, hardening time 1-12 month,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. the selection of tea tree: select to be positioned at nature obscurity at 60%-80%, illumination is scattered light, the tea tree trunk of intensity of illumination under 1000-5000Lx environment;
B. stem of noble dendrobium companion liver moss is pasted tree planting: at the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 1-20cm place around, thickness is 0.1-10cm, cultivate after 5-20 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at sunny slope tea tree limb liver moss position, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium is bundled on tea tree, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once wetting plant root and near liver moss;
Described liver moss mixture (w/w) is by (1/3-1/10) knop5-30, glutinous rice 5-20, fermentation animal wastes 10-70, liver moss 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30;
C. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, and while gathering, root stays the long root head of 2-3cm, cut always stay tender.
A in described step 1 is:
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 1-10min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 20-30 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, after 2-30 ℃ of constant temperature culture 5-50 days, at colony edge, punch into bacterium sheet, directly use or as mycorrhizal fungi seed bacterial classification, further access liquid or solid fermentation medium and amplify cultivation;
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, the hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, the agar 1-30gg/L that ground, pH value 4-9;
Described fermentation medium is: potato 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 shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 5-15 minute on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 1-10 minute, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L that 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+ ground; PH is 4-9, and at 20-30 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 10-90 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting; Maybe the protocorm inducing is inoculated into alternate culture on liquid and solid multiplication medium, it 10-60 days is a subculture cycle that propagation is cultivated, cultivation temperature 20-30 ℃, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ active carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9;
Described shoot proliferation solid 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+ active carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9;
Described step 5 is:
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the trunk place nature obscurity of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx;
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is 10-April 10 annual March, or takes second place September; At the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 1-20cm place around, thickness is 0.1-10cm, after cultivating 5-20d, the stem of noble dendrobium seedling in little plastic basin is shifted out and tied at the more position of sunny slope tea tree limb liver moss together with liver moss matrix, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium is bundled on tea tree, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture (w/w) is by (1/3-1/10) knop5-30, glutinous rice 5-20, fermentation animal wastes 10-70, liver moss 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30;
C. bionical dendrobium tea ground Cultivate administration: answer every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 1-12 time; The water of spraying every day 1-2 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, suitable harvest period be after the Beginning of Winter to before Clear and Bright, while gathering, root stays the long root head of 2-3cm, cuts always to stay tenderly, can continue to gather in the crops more than 8 years.
Described liver moss comprises bog moss, hypodontia alpine rush or palm-bark rain cape moss, the knob moss that comes into leaves, intend one or more of broad-leaved gravelstone moss.
Described binding material is one or more of nonwoven, shade net, fishing line, straw rope, rattan.
Embodiment
Embodiment 1
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 3min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 27 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, in 5 ℃ of constant temperature culture, after 40 days, at colony edge, punch into bacterium sheet, directly use;
Described isolation medium is: glucose 20g/L, peptone 8g/L, streptomycin 0.5g/L, the hypodontia alpine rush or palm-bark rain cape moss 25g/L, casein hydrolysate 3g/L, the agar 19g/L that ground, pH value 4;
Described fermentation medium is: potato 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 bacterial classification access fermentation medium is cultivated with triangular flask, with refiner, smashed cultured products standby; Fermentation condition is: inoculum concentration 12%, 25 ℃ of temperature, incubation time 30h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 15%, 25 ℃ of bottom fermentations of temperature are cultivated 40 days, obtain mycorrhizal fungi solid bacterium piece when mycelia 90% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 5-15 minute on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 7 minutes, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 50g/L+ potato juice 105g/L+ sucrose 9g/L that 1/2MS+6-BA4mg/L+NAA0.5mg/L+ ground; PH is 6, and at 22 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 70 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA3mg/L+NAA5mg/L+ potato liquor 80g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 110g/L+ active carbon 6g/L+ sucrose 37g/L, pH6;
Described shoot proliferation solid culture 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+ active carbon 3g/L+ sucrose 29g/L+ agar 20g/L, pH7;
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 150g/L+ banana homogenate 120g/L+ sucrose 30g/L+ active carbon 5g/L+ agar 20g/L that 1/2MS+NAA3mg/L+ ground; 25 ℃ of cultivation temperature, pH5, cultivates 10 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated 30 days, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial to 1-15 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 8 minutes, the hypodontia alpine rush or palm-bark rain cape moss that the soaking sterilization of take was processed is cultivation matrix, the 1/7knop20g/L+ sucrose 28g/L+ ammonium tartrate 22g/L+ mycorrhizal fungi zymotic fluid 27mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 10-38 ℃, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, hardening time 1-12 month,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the trunk place nature obscurity of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx;
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is 10-April 10 annual March, at the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 10cm place around, thickness is 3cm, cultivate after 10 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at the more position of sunny slope tea tree limb liver moss, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium being bundled on tea tree with nonwoven, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture is by 1/9knop10g, glutinous rice 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 every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 5 times; The water of spraying every day 1-2 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, suitable harvest period be after the Beginning of Winter to before Clear and Bright, while gathering, root stays the long root head of 2-3cm, cuts always to stay tenderly, can continue to gather in the crops more than 8 years.
Embodiment 2
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 7min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 26 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, in 8 ℃ of constant temperature culture, after 40 days, at colony edge, punch into bacterium sheet, as mycorrhizal fungi seed bacterial classification, further access liquid fermentation medium and amplify cultivation;
Described isolation medium is: glucose 15g/L, peptone 13g/L, streptomycin 0.3g/L, the hypodontia alpine rush or palm-bark rain cape moss 10g/L, casein hydrolysate 3g/L, the agar 23g/L that ground, pH value 4.5;
Described fermentation medium is: potato 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 bacterial classification access fermentation medium is cultivated with fermentation tank ventilation is dark, cultivated and finish rear direct use; Fermentation condition is: inoculum concentration 10%, 25 ℃ of temperature, incubation time 70h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 10%, 28 ℃ of bottom fermentations of temperature are cultivated 30 days, obtain mycorrhizal fungi solid bacterium piece when mycelia 80% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 5-15 minute on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 3 minutes, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 110g/L+ potato juice 160g/L+ sucrose 14g/L that 1/2MS+6-BA2mg/L+NAA5mg/L+ ground; PH is 6, and at 27 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 50 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting; It 20 days is a subculture cycle that propagation is cultivated, 23 ℃ of cultivation temperature, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA3mg/L+NAA7mg/L+ potato liquor 160g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 180g/L+ active carbon 5g/L+ sucrose 32g/L, pH5;
Described shoot proliferation solid 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+ active carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 130g/L+ banana homogenate 170g/L+ sucrose 19g/L+ active carbon 3g/L+ agar 17g/L that 1/2MS+NAA7mg/L+ ground; 25 ℃ of cultivation temperature, pH8, cultivates 40 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated 50 days, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial 8 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 3-10 minute, the bog moss that the soaking sterilization of take was processed and hypodontia alpine rush or palm-bark rain cape moss are cultivation matrix, the 1/8knop20g/L+ sucrose 21g/L+ ammonium tartrate 3g/L+ mycorrhizal fungi zymotic fluid 70mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 10-38 ℃, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, hardening time 1-12 month,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the trunk place nature obscurity of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx;
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is September; At the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 18cm place around, thickness is 6cm, cultivate after 16 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at the more position of sunny slope tea tree limb liver moss, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium being bundled on tea tree with shade net, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture is by 1/4knop18g, glutinous rice 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 every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 7 times; The water of spraying every day 1-2 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, suitable harvest period be after the Beginning of Winter to before Clear and Bright, while gathering, root stays the long root head of 2-3cm, cuts always to stay tenderly, can continue to gather in the crops more than 8 years.
Embodiment 3
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 1-10min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 20-30 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, in 23 ℃ of constant temperature culture, after 30 days, at colony edge, punch into bacterium sheet, as mycorrhizal fungi seed bacterial classification, further access liquid;
Described isolation medium is: glucose 8g/L, peptone 4g/L, streptomycin 0.02g/L, the hypodontia alpine rush or palm-bark rain cape moss 18g/L, casein hydrolysate 3g/L, the agar 7g/L that ground, pH value 4;
Described fermentation medium is: potato 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
By mycorrhizal fungi seed bacterial classification access fermentation medium triangular flask, with refiner, smash cultured products standby; Fermentation condition is: inoculum concentration 8%, 28 ℃ of temperature, incubation time 59h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 8%, 23 ℃ of bottom fermentations of temperature are cultivated 20 days, obtain mycorrhizal fungi solid bacterium piece when mycelia 60% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 12 minutes on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 8 minutes, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 50g/L+ potato juice 100g/L+ sucrose 8g/L that 1/2MS+6-BA8mg/L+NAA4mg/L+ ground; PH is 8, and at 20-30 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 20 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA3mg/L+NAA4mg/L+ potato liquor 80g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 60g/L+ active carbon 9g/L+ sucrose 30g/L, pH7;
Described shoot proliferation solid culture 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+ active carbon 3g/L+ sucrose 20g/L+ agar 15g/L, pH6;
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 10g/L+ banana homogenate 120g/L+ sucrose 10g/L+ active carbon 2g/L+ agar 5g/L that 1/2MS+NAAlmg/L+ ground; 25 ℃ of cultivation temperature, pH5, cultivates 30 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated 10 days, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial 10 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 5 minutes, the hypodontia alpine rush or palm-bark rain cape moss that the soaking sterilization of take was processed is cultivation matrix, the 1/8knop10g/L+ sucrose 5g/L+ ammonium tartrate 1g/L+ mycorrhizal fungi zymotic fluid 50mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 10-38 ℃, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, 8 months hardening time,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the natural obscurity in trunk place of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx:
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is September; At tea root, be coated with one deck liver moss mixture in diameter 10cm place around, thickness is 1cm, cultivate after 10 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at the more position of sunny slope tea tree limb liver moss, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium being bundled on tea tree with fishing line, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture is by 1/5knop15g, glutinous rice 8g, fermentation animal wastes 60g, come into leaves knob moss 13g, sucrose 3g, ammonium tartrate 1d, mycorrhizal fungi powder 13g;
C. bionical dendrobium tea ground Cultivate administration: answer every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 10 times; The water of spraying every day 1-2 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, suitable harvest period be after the Beginning of Winter to before Clear and Bright, while gathering, root stays the long root head of 2-3cm, cut always stay tender.
Embodiment 4
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 10min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 20 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, in 2 ℃ of constant temperature culture, after 50 days, at colony edge, punch into bacterium sheet, as mycorrhizal fungi seed bacterial classification, further access solid fermentation medium and amplify cultivation;
Described isolation medium is: glucose 30g/L, peptone 1g/L, streptomycin 1g/L, the hypodontia alpine rush or palm-bark rain cape moss 10g/L, casein hydrolysate 0.01g/L, the agar 1g/L that ground, pH value 4;
Described fermentation medium is: potato 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 bacterial classification access fermentation medium is cultivated with fermentation tank ventilation is dark, cultivated and finish rear direct use; Fermentation condition is: inoculum concentration 6%%, 20 ℃ of temperature, incubation time 90h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 18%, temperature 20-30 ℃ of bottom fermentation cultivated 50 days, obtains mycorrhizal fungi solid bacterium piece when mycelia 50%% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 5-15 minute on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 1 minute, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 10g/L+ potato juice 10g/L+ sucrose 2g/L that 1/2MS+6-BA10mg/L+NAA0.01mg/L+ ground; PH is 4, at 20-30 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 10 days, axillalry bud place can be clump bud shape by pumping shoot, the protocorm inducing is inoculated into alternate culture on liquid and solid multiplication medium, and it 10 days is a subculture cycle that propagation is cultivated, cultivation temperature 20-30 ℃, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA10mg/L+NAA0.1mg/L+ potato liquor 20g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 10g/L+ active carbon 1g/L+ sucrose 25g/L, pH4.9;
Described shoot proliferation solid culture 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+ active carbon 10g/L+ sucrose 2g/L+ agar 1g/L, pH4;
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 200g/L+ banana homogenate 20g/L+ sucrose 50g/L+ active carbon 0.1g/L+ agar 30g/L that 1/2MS+NAA0.1mg/L+ ground; Cultivation temperature 20-30 ℃, pH4, cultivates 3 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated 1 day, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial to 1-15 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 3 minutes, the bog moss that the soaking sterilization of take was processed is cultivation matrix, the 1/10knop30g/L+ sucrose 30g/L+ ammonium tartrate 0.01g/L+ mycorrhizal fungi zymotic fluid 5mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 10-38 ℃, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, 1 month hardening time,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the trunk place nature obscurity of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx;
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is 10-April 10 annual March; At the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 1cm place around, thickness is 10cm, cultivate after 5 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at the more position of sunny slope tea tree limb liver moss, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium being bundled on tea tree with straw rope, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture is by 1/10knop30g, glutinous rice 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 every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 1 time; The water of spraying every day 1 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: plant and gather before clear and bright for latter 2 years, while gathering, root stays the long root head of 2-3cm, cut always stay tender.
Embodiment 5
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 10min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 30 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, in 30 ℃ of constant temperature culture, after 5 days, at colony edge, punch into bacterium sheet, directly use;
Described isolation medium is: glucose 30g/L, peptone 1g/L, streptomycin 0.001g/L, the hypodontia alpine rush or palm-bark rain cape moss 1g/L, casein hydrolysate 10g/L, the agar 30g/L that ground, pH value 9;
Described fermentation medium is: potato 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
By mycorrhizal fungi seed bacterial classification access fermentation medium triangular flask shaken cultivation, cultivate and finish rear direct use; Fermentation condition is: inoculum concentration 6%, 20 ℃ of temperature, incubation time 90h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 6%%, 20 ℃ of bottom fermentations of temperature are cultivated 50 days, obtain mycorrhizal fungi solid bacterium piece when mycelia 100% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 5-15 minute on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 10 minutes, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 200g/L+ potato juice 1g/L+ sucrose 2g/L that 1/2MS+6-BA10mg/L+NAA0.01mg/L+ ground; PH is 9, at 30 ℃, intensity of illumination is 1500-2500Lx, illumination 12h/d, cultivate 90 days, axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, and the protocorm inducing is inoculated into alternate culture on liquid and solid multiplication medium, it 60 days is a subculture cycle that propagation is cultivated, cultivation temperature 20-30 ℃, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA10mg/L+NAA0.1mg/L+ potato liquor 200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1/L+ active carbon 0.1g/L+ sucrose 2g/L, pH4;
Described shoot proliferation solid culture 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+ active carbon 10g/L+ sucrose 2g/L+ agar 30g/L, pH9;
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 200g/L+ banana homogenate 20g/L+ sucrose 5g/L+ active carbon 0.1g/L+ agar 30g/L that 1/2MS+NAA10mg/L+ ground; 30 ℃ of cultivation temperature, pH4, cultivates 90 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated 90 days, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial 15 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 3-10 minute, the bog moss that the soaking sterilization of take was processed is cultivation matrix, the 1/3knop5g/L+ sucrose 1g/L+ ammonium tartrate 0.01g/L+ mycorrhizal fungi zymotic fluid 5mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 38 ℃, air humidity is 85%, shade density 70%, illuminance 1000-5000Lx, water and take spray process, 12 months hardening time,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the trunk place nature obscurity of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx;
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is 10-April 10 annual March; At the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 20cm place around, thickness is 0.1cm, cultivate after 20 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at the more position of sunny slope tea tree limb liver moss, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium being bundled on tea tree with rattan, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture is by 1/3knop5g, glutinous rice 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 every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 12 times; The water of spraying every day 1 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: gather while planting rear 1 year the Beginning of Winter, while gathering, root stays the long root head of 2-3cm, cut always stay tender.
Test example: the inhibitory action to toes carrageenan induced Acute inflammation
The preparation method of water-soluble extractive: adopt the stem of noble dendrobium and the commercially available artificial cultivation stem of noble dendrobium of embodiment 1～5 gained to squeeze the juice.
Experimental technique: rat random packet, every group 10, first 1 hour gastric infusion (2g crude drug/kg) of Yu Zhiyan, sterile working, after giving Rat Right, toes aponeurosis (aponeuroses) hemostasis 10% carrageenan 0.1m1 causes inflammation, with drainage measure cause scorching before and cause scorching after the right back toes volume of different time so that the sufficient volume difference before and after scorching is swelling value, according to swelling value, calculates swelling rate and inhibiting rate.Experimental result sees the following form 1:
Swelling rate %=E
n-E
0/ E
n* 100
E
n=cause scorching after the swelling value of different time
E
0=cause the swelling value before inflammation
Inhibiting rate %=C-T/C * 100
The swelling rate of C=control group
The swelling rate of T=treatment group
The inhibitory action of the different stem of noble dendrobium water-soluble extractives of table 1 to rat toes carrageenan induced Acute inflammation
*：p<0.05；**：p<0.01
Above experimental result shows: adopt the relative commercially available product of water-soluble extractive that the gained stem of noble dendrobium of the present invention makes to have acute inflammation effect better.
Claims (6)
1. a Mycorrhizal stem of noble dendrobium seedling tea tree pseudo-wild cultivating method, its feature comprises the following steps:
Step 1: the fermented and cultured of mycorrhizal fungi
A. choose the fresh feeding root of wild Dendrodium, root segment is immersed to 1-10min in 1% calcium hypochlorite solution and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 20-30 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, after 2-30 ℃ of constant temperature culture 5-50 days, at colony edge, punch into bacterium sheet, directly use or as mycorrhizal fungi seed bacterial classification, further access liquid or solid fermentation medium and amplify cultivation;
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, the hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, the agar 1-30gg/L that ground, pH value 4-9;
Described fermentation medium is: potato 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 bacterial classification is accessed to triangular flask or the vibration of other containers or the dark cultivation of fermentation tank ventilation for fermentation medium, and cultivation finishes rear direct use or smashes cultured products with refiner standby; Fermentation condition is: inoculum concentration 6%-18%, temperature 20-30 ℃, incubation time 5-90h;
C. solid fermentation is cultivated
Mycorrhizal fungi seed bacterial classification is seeded in solid fermentation medium, inoculum concentration 6%-18%, temperature 20-30 ℃ of bottom fermentation cultivated 2-50 days, obtains mycorrhizal fungi solid bacterium piece when mycelia 50%～100% is covered with medium;
Step 2: the cultivation of dendrobe tissue culture seedling
A. protocorm induction and shoot proliferation are cultivated: gather wild Dendrodium and belong to the young stem with axillalry bud, with after 75% alcohol-pickled 50 ± 10 seconds, the mercuric chloride solution that is 1g/L by concentration again soaks 1-10 minute, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L that 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+ ground; PH is 4-9, and at 20-30 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 10-90 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting; Maybe the protocorm inducing is inoculated into alternate culture on shoot proliferation liquid nutrient medium and shoot proliferation solid culture medium, it 10-60 days is a subculture cycle that propagation is cultivated, cultivation temperature 20-30 ℃, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ active carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9;
Described shoot proliferation solid 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+ active carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9;
B. Rooting and hardening-off culture: be transferred on strengthening seedling and rooting medium and cultivate when 2-3 centimetre of unrooted height of seedling; Strengthening seedling and rooting medium is: the hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ banana homogenate 20-200g/L+ sucrose 5-50g/L+ active carbon 0.1-10g/L+ agar 3-30g/L that 1/2MS+NAA0.1-10mg/L+ ground; Cultivation temperature 20-30 ℃, pH4-9, cultivates 3-90 days, and now illumination is strengthened to 2500-4000Lx, illumination 10-14 hour/day;
Step 3: the symbiotic cultivation of stem of noble dendrobium seedling and mycorrhizal fungi: under aseptic condition, symbiosis in the normal 3-4 leaf of root system group training seedling access mycorrhizal fungi solid bacterium piece is cultivated to 1-90 days, cultivate under field conditions (factors);
Step 4: the stem of noble dendrobium method for refining seeding of Mycorrhizal
A. under constant temperature, test-tube plantlet is refined in vial to 1-15 days;
B. stem of noble dendrobium seedling is taken out from vial, clean the medium of root, in 0.1% liquor potassic permanganate, soak airing after 3-10 minute, the bog moss that the soaking sterilization of take was processed and/or hypodontia alpine rush or palm-bark rain cape moss are cultivation matrix, (1/3-1/10) knop5-30g/L+ sucrose 1-30g/L+ ammonium tartrate 0.01-30g/L+ mycorrhizal fungi zymotic fluid 5-100mL/L of take is symbiotic cultivation nutrient solution, take little plastic basin as cultivating container classification hardening, getting 3-5 strain is one clump, root is planted in cultivation matrix, hardening adopts warm canopy, temperature is controlled at 10-38 ℃, air humidity is 40%-85%, shade density 50-70%, illuminance 1000-5000Lx, water and take spray process, hardening time 1-12 month,
Step 5: dendrobe tissue culture seedling and the tea tree cultivation method of growing nonparasitically upon another plant
A. the selection of tea tree: select to be positioned at nature obscurity at 60%-80%, illumination is scattered light, the tea tree trunk of intensity of illumination under 1000-5000Lx environment;
B. stem of noble dendrobium companion liver moss is pasted tree planting: at the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 1-20cm place around, thickness is 0.1-10cm, cultivate after 5-20 days and the stem of noble dendrobium seedling in little plastic basin is shifted out together with liver moss matrix and tie at sunny slope tea tree limb liver moss position, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium is bundled on tea tree, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once wetting plant root and near liver moss;
Described liver moss mixture (w/w) is by (1/3-1/10) knop5-30, glutinous rice 5-20, fermentation animal wastes 10-70, liver moss 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30;
C. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, and while gathering, root stays the long root head of 2-3cm, cut always stay tender.
2. cultivation method according to claim 1, the A described in its feature in step 1 is:
A. choose the fresh feeding root of wild Dendrodium, with flowing water, rinse out root surface humus and foreign material; Root segment is immersed to 1-10min in 1% calcium hypochlorite solution on superclean bench and carry out surface sterilizing, then use aseptic water washing 3-5 time; Root segment is cut into the long segment of 0.5-3mm, be placed on isolation medium, at 20-30 ℃, secretly cultivate, until mycelia from growing up in root segment after bacterium colony, picking mycelia carries out transferring in solid fermentation medium after culture purified on isolation medium, after 2-30 ℃ of constant temperature culture 5-50 days, at colony edge, punch into bacterium sheet, directly use or as mycorrhizal fungi seed bacterial classification, further access liquid or solid fermentation medium and amplify cultivation;
Described isolation medium is: glucose 1-30g/L, peptone 1-10g/L, streptomycin 0.001-1g/L, the hypodontia alpine rush or palm-bark rain cape moss 1-30g/L, casein hydrolysate 0.01-10g/L, the agar 1-30gg/L that ground, pH value 4-9;
Described fermentation medium is: potato 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.
3. cultivation method according to claim 1, the A described in its feature in step 2 is:
A. protocorm induction and shoot proliferation are cultivated: in the 3-4 the first tenday period of a month month, gather wild Dendrobium with the young stem of axillalry bud, with flowing water, rinse after 5-15 minute on superclean bench with after 75% alcohol-pickled 50 ± 10 seconds, with the mercuric chloride solution that concentration is 1 grams per liter, soak 1-10 minute again, aseptic water washing 3-5 time, cuts on the agar medium that 0.1-3cm stem section is inoculated into the hypodontia alpine rush or palm-bark rain cape moss 1-200g/L+ potato juice 1-200g/L+ sucrose 2-20g/L that 1/2MS+6-BA0.01-10mg/L+NAA0.01-10mg/L+ ground; PH is 4-9, and at 20-30 ℃, intensity of illumination is 1500-2500Lx, and illumination 12h/d cultivates 10-90 days, and axillalry bud place can be clump bud shape by pumping shoot, and shoot can be cut and repeat to cultivate, until grow up to complete test-tube plantlet for transplanting; Maybe the protocorm inducing is inoculated into alternate culture on liquid and solid multiplication medium, it 10-60 days is a subculture cycle that propagation is cultivated, cultivation temperature 20-30 ℃, luminous intensity 1500-4000Lx, illumination 10-14 hour/day;
Described shoot proliferation liquid nutrient medium is: 1/2MS+6-BA0.1-10mg/L+NAA0.1-10mg/L+ potato liquor 2-200g/L+ hypodontia alpine rush or palm-bark rain cape moss abrasive material 1-200g/L+ active carbon 0.1-10g/L+ sucrose 2-50g/L, pH4-9;
Described shoot proliferation solid 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+ active carbon 0.1-10g/L+ sucrose 2-50g/L+ agar 1-30g/L, pH4-9.
4. cultivation method according to claim 1, step 5 is described in its feature:
A. near the selection of tea tree: need to meet the water source of national grade ii standard tea ground, mean temperature of air is at 10 ℃ to 20 ℃ throughout the year, and air relative temperature is between 45% to 85%, and average annual rainfall is 800 millimeters to 1800 millimeters; The tea place domatic gradient that faces south is greater than 5-20 degree, the 10 years above tea places of growing; Before field planting, first will carry out suitably pruning and beating leaf, make the trunk place nature obscurity of cultivating the stem of noble dendrobium at 60%-80%, illumination is scattered light, and intensity of illumination is at 1000-5000Lx;
B. stem of noble dendrobium companion liver moss is pasted tree planting: the plantation time is 10-April 10 annual March, or takes second place September; At the nearly root trunk of tea tree and tea root, be coated with one deck liver moss mixture in diameter 1-20cm place around, thickness is 0.1-10cm, after cultivating 5-20d, the stem of noble dendrobium seedling in little plastic basin is shifted out and tied at the more position of sunny slope tea tree limb liver moss together with liver moss matrix, make seedling root system be adjacent to trunk liver moss, trunk liver moss mixture coating is around close in matrix bottom; One clump of 3 to 5 strain, every clump at a distance of more than 10 centimetres; After the stem of noble dendrobium is bundled on tea tree, within continuous 7 days, with symbiotrophy liquid, plant is sprayed once before 8 in the morning, wetting plant root and near liver moss, guarantee that liver moss can not ponding;
Described liver moss mixture (w/w) is by (1/3-1/10) knop5-30, glutinous rice 5-20, fermentation animal wastes 10-70, liver moss 1-30, sucrose 1-10, ammonium tartrate 0.01-10, mycorrhizal fungi powder 1-30;
C. bionical dendrobium tea ground Cultivate administration: answer every year weeding 2-3 time, remove weeds and dry branches and fallen leaves; Annual spray symbiotrophy liquid 1-12 time; The water of spraying every day 1-2 time, humidity is controlled at 60-80%, and winter-spring season cuts off the overstocked branch of attached main plant in good time, to guarantee that obscurity is at 70-80%, can spray one time plant resource biological insecticides when damage by disease and insect is occurred frequently per ten days;
D. gathering of the stem of noble dendrobium: after plantation, 1-2 can gather, suitable harvest period be after the Beginning of Winter to before Clear and Bright, while gathering, root stays the long root head of 2-3cm, cuts always to stay tenderly, can continue to gather in the crops more than 8 years.
5. according to the cultivation method described in claim 1,4, it is characterized in that: described liver moss comprises bog moss, hypodontia alpine rush or palm-bark rain cape moss, the knob moss that comes into leaves, intend one or more of broad-leaved gravelstone moss.
6. according to the cultivation method described in claim 1,4, it is characterized in that: described binding material is one or more of nonwoven, shade net, fishing line, straw rope, rattan.
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