CN102150615A - Method for culturing, planting and mycorrhizal production of dendrobium officinale kimura et migo - Google Patents
Method for culturing, planting and mycorrhizal production of dendrobium officinale kimura et migo Download PDFInfo
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Abstract
The invention relates to a method for culturing, planting and mycorrhizal production of dendrobium officinale kimura et migo, comprising the following steps of: (1) culturing mycorrhizal fungi by utilizing a solid or liquid culture medium, wherein the mycorrhizal fungi are mycorrhizal fungi LP2 deuteromycotina Fusariumsp, mycorrhizal fungi LP3, or mixed strain deuteromycotina Verticilliumsp of the mycorrhizal fungi LP2 deuteromycotina Fusariumsp and the mycorrhizal fungi LP3; (2) culturing tissue culture seedlings of the dendrobium officinale kimura et migo; (3) preparing a nutrient solution; (4) carrying out water culture and training the tissue culture seedlings; and (5) carrying out planting and mycorrhizalculturing of the dendrobium officinale kimura et migo. The method for culturing, planting and mycorrhizal production of the dendrobium officinale kimura et migo, provided by the invention, can promote growth of dendrobium officinale kimura et migo seedlings obviously; and compared with a contrast plant, the dendrobium officinale kimura et migo cultured by inoculating pathogen has increased rooting numbers, high survival rate, longer plant length, better growth vigor, obviously expanded internodes, obviously big stems, improved fresh weight and dry weight and obviously enhanced stress resistance of a pathogen-inoculated plant.
Description
Technical field
The present invention relates to a kind of officinal dendrobium stem plantation plantation mycorrhizal method.Be specifically related to the application method of a kind of mycorrhizal fungi in officinal dendrobium stem plantation, the cultivation, dendrobium candidum that it comprises the mycorrhizal fungi that the present invention is used by protocorm grow to seedling and successive transfer culture thereof, mycorrhizal fungi in officinal dendrobium stem plantation application method and to dendrobium candidum growth promotion effect and to the influence of metabolic activity, resistance, active constituent content.
Background technology
Dendrobium candidum (
Dendrobium officinale Kimura et Migo) the rare high added value medicinal material in imminent danger of genus, its health care result of treatment is definite, and is all on the books in " Taoist Scriptures ", Shennong's Herbal, Compendium of Material Medica, " modern Chinese herbal medicine voluminous dictionary ", medical ancient books and records such as the version Pharmacopoeia of the People's Republic of China in 2010.Tang opens Taoist school's classical works in the first year " Taoist Scriptures " and includes Chinese nine immortal grass (dendrobium candidum, Herba Saussureae Involueratae, three double gensengs, 120 years tubers of multiflower knotweed, the Poria cocos of cycle of sixty years, desert cistanche, remote mountains glossy ganoderma, seabed pearl, Cordyceps sinensis), because of dendrobium candidum enriching yin qi-restoratives effect is remarkable, be listed in position first of " Chinese nine immortal grass ".The record of Li Shizhen (1518-1593 A.D.) Compendium of Material Medica: " the reinforcing yin essence benefit is smart.Obey for a long time, thick stomach, never sufficient in the benefit, flat stomach Qi, longue meat by skin heat symptoms caused by an exopathgen miliaria gas, a little less than the painful cold numbness of pin knee, is decided intelligence except that frightened, makes light of one's life by commiting suicide and prolongs life.Benefit gas heat extraction, strong sun, cold for a long time in the bone by the skin wandering arthritis, kidney tonifying benefit power.Control the heating spontaneous perspiration, plug in the ulcer apocenosis." single-row in one one of the version Pharmacopoeia of the People's Republic of China in 2010, distinguish with common stem of noble dendrobium kind such as other dendrobium loddigesii Rolfe, stem of Eyeshaped Dendrobium, HERBA DENDROBII, HERBA DENDROBII and the drumstick stem of noble dendrobium, its function with cure mainly: " reinforcing stomach reg fluid, nourishing Yin and clearing heat.Be used for the moon and hinder body fluid deficiency, the dry polydipsia, food is few retches, abnormal heat after being ill, order is secretly not clear.”
The resource medicinal material of in imminent danger because of belonging to, rare, high added value, dendrobium candidum is put into (CITES) appendix II of " special-protection-by-the-State natural crude drugs species register " (1987), " Chinese Plants Red Data Book " (1992), " CITS ".2010, dendrobium candidum production technology and industrialization were put into the technical field of first developing of state key new product planning and State Torch Program once more.The stem of noble dendrobium is as a kind of conventional Chinese medicine material, and market demand is bigger, how to solve the contradiction between demand and the resource? only approach is an artificial cultivation.In all age, people are exploring the artificial cultivation of the stem of noble dendrobium always, but because the reproduction coefficient of Dendrobium plant is low, and annual production is limited, enter that to abound with time phase long, can not form scale, and output is extremely limited.The present low situation of ubiquity tissue cultivating seedling transplanting survival rate.How to improve the survival rate of officinal dendrobium stem plantation, become a big obstacle of restriction stem of noble dendrobium production development.Under nature, orchid will form mycorhiza with mycosymbiosis, just can make the growth of orchid stalwartness.According to these characteristics, we took to the research of separation, cultivation and the officinal dendrobium stem plantation plantation mycorrhizal method of dendrobium candidum mycorrhizal fungi from 1992.
Summary of the invention
The object of the invention is to provide a kind of method of officinal dendrobium stem plantation plantation Mycorrhizal.This method can solve dendrobium candidum breeding and the cultivation survival rate is low, and cultivating and growing is slow, and the cycle is long, and active constituent content is low, problems such as drug effect difference.
The method that the present invention adopts is to cultivate earlier mycorrhizal fungi respectively, mycorrhizal fungi and dendrobium candidum stem is induced organize the refinery seedling symbiotic cultivation of training out again.
The step of the method for the invention is as follows:
(1) cultivation of mycorrhizal fungi
With the hybrid bacterial strain of mycorrhizal fungi LP2, mycorrhizal fungi LP3 or two kinds from the slant tube of low-temperature preservation behind actication of culture, be connected in the PDA plate, in 23 ~ 28 ℃ of (preferred 24 ~ 26 ℃) constant temperature culture 10 ~ 15 d (preferred 11 ~ 13 d), punch into bacterium sheet (preferred Φ=8 mm) at colony edge, and insert solid culture medium or liquid nutrient medium with fritter; Mycorrhizal fungi LP2 be the Deuteromycotina Fusarium (
Fusarium sp.), mycorrhizal fungi LP3 be the Deuteromycotina Verticillium (
Verticillium sp.); These two kinds of culture presevation are in China medicinal microorganism fungus kind preservation administrative center, and deposit number is respectively CPCC810233 and CPCC421139;
The solid culture medium of mycorrhizal fungi contains nutrients such as lignin, cellulose, carbon, nitrogen, form by being selected from raw material cotton seed hulls, wood chip, broken corncob, broad-leaved tree sawdust, the wheat bran one or more, add water, the water yield is 150 ~ 200 wt% (preferred 160 ~ 180 wt%) of raw material; Insert respectively behind the autoclaving through cultivating gained mycorrhizal fungi LP1, per 100 g medium connect 2 ~ 3 bacterium sheets; Fungi is left standstill dark cultivation with vessel such as test tube, conical flask or blake bottles, takes out standby when treating the mycelia major part or all covering with medium;
The liquid nutrient medium of mycorrhizal fungi contains glucose, inorganic salts composition and natural additive; The preparation of the liquid nutrient medium water of mycorrhizal fungi, glucose 18 ~ 22 g/L (preferred 20 g/L) wherein, KH
2PO
418 ~ 22 g/L (preferred 20 g/L), MgSO
41.2 ~ 1.8 g/L (preferred 1.5 g/L), Cobastab
18 ~ 12 mg/L (preferred 10 mg/L), natural goods: wheat bran 25 ~ 35 g/L (preferred 30 g/L) (liquor) or potato 180 ~ 220 g/L (preferred 200 g/L) (using its leachate), the medium pH value is 5.6 ~ 6.5 (preferred pH=6.0); Insert behind the autoclaving through cultivating gained mycorrhizal fungi LP1, per 100 mL medium insert 1 ~ 2 bacterium sheet; Fungi is secretly cultivated with triangular flask or the ventilation of other containers, and containing quantity is 50%, places 23 ~ 25 ℃ of vibrations, and rotating speed 100 ~ 120 rpm gather in the crops behind cultivation 20 ~ 30 d; Obtain liquid mycorrhizal fungi after cultivate finishing, can directly use or to smash cultured products with refiner standby;
(2) candidum tissue culturing seedling is cultivated
Get the sprouting bud on well-grown dendrobium candidum plant, get its stem section, sterilization is inoculated in inducing culture (1/2-1) MS+BA 2.0 mg/L+NAA 0.5 mg/L+3 wt% sucrose+0.75 wt% agar; Through inducing the yellowish green protocorm of formation to transfer in proliferation and differentiation medium (1/2-1) MS+BA 1.0 mg/L+NAA 0.5 mg/L+10 wt% bananas juices+3 wt% sucrose+0.75 wt% agar, 2 months subcultures once obtain the seedling that root has leaf; To there be root to have the seedling of leaf to be inoculated in medium (1/2-1) MS+10 wt% bananas juice+NAA 2.0 mg/L+3 wt% sucrose+0.75 wt% agar again, make its growth;
(3) preparation nutrient solution
Nutrient solution is made of following component: potassium nitrate 1.0 ~ 1.2 wt ‰ (preferred 1.1 wt ‰), ammonium sulfate 0.18 ~ 0.22 wt ‰ (preferred 0.2 wt ‰), ammonium nitrate 0.08 ~ 0.12 wt ‰ (preferred 0.1 wt ‰), magnesium sulfate 0.1 ~ 0.14 wt ‰ (preferred 0.12 wt ‰), potassium dihydrogen phosphate 0.08 ~ 0.12 wt ‰ (preferred 0.10 wt ‰), calcium chloride 0.04 ~ 0.08 wt ‰ (preferred 0.06 wt ‰), manganese sulphate 0.01 ~ 0.03 wt ‰ (preferred 0.02 wt ‰), ferrous sulfate 0.02 ~ 0.04 wt ‰ (preferred 0.03 wt ‰), zinc sulphate 0.004 ~ 0.006 wt ‰ (preferred 0.005 wt ‰), all the other are water;
(4) the water planting hardening of tissue cultivating seedling
The dendrobium candidum that obtains is cultivated seedling to be taken out, flushing with clean water with 15 ~ 20 ℃ (preferred 17 ℃), drop removes excessive moisture, plant in the hole of the cystosepiment of accomplishing fluently aperture of frame on the nutrition liquid bath, nutrient solution is injected the nutrition liquid bath, make 40 ~ 60% of nutrient solution submergence root system, start the water circulating pump of nutrition liquid bath, flow velocity 4 ~ 6 m/s; Ventilate, wind speed 0.3 ~ 0.5 m/s carries out the seedling refinery; Behind 18 ~ 22 d, the seedlings of Dendrobium officinale well developed root system, regenerated root increases, and stem is long to be increased slightly, and it is green that blade turns, and thickening promptly can be extracted and carry out field-transplanting;
(5) dendrobium candidum plantation Mycorrhizal cultivation
1. candidum tissue culturing seedling is transplanted
The tissue cultivating seedling root of the process water planting refinery that can transplant cleans with clear water, notes the stem that fractures of trying not, damaged blade during cleaning; Cultivation matrix adopts glutenite powder and soil (You Xuan Lang mountain glutenite powder Yu Lang mountain soil) be the mixture of 1:1 ~ 2 by weight; Before the transplanting, place the wheat bran sugar leaf (Fagaceae) that 5 ~ 10 g pulverize at the base portion of every young plant, place the liquid mycorrhizal fungi of 1.5 ~ 2.5 mL abovementioned steps preparation or the solid mycorrhizal fungi of 1.5 ~ 2.5 g abovementioned steps preparation on it again, the root of dendrobium candidum is contacted with bacterium; During cultivation root is embedded in the matrix, stromal surface is exposed on stem top, will water normal root water after planting, and matrix keeps 75% moisture; Seedling is planted in seedling dish or the flowerpot, and the individual plant seedling gets final product when planting, and does not need many strains to be planted in together, and the cultivation distance is 3 ~ 5 cm;
2. the control of temperature and humidity
Have the seedling dish of tissue cultivating seedling or flowerpot to move into the film booth transplanting, the growth temperature of dendrobium candidum is controlled at 25 ~ 30 ℃, adopts 75% sunshade net covering summer, plastic film is raised, in order to cooling; Winter, the sunshade net with 30% covered, and perhaps removes the sunshade net plastic film is put down; The dendrobium candidum transplanting initial stage requires high humidity, and air humidity remains on 75 ~ 80%, and matrix keeps moistening and ponding not;
3. the results of dendrobium candidum
Dendrobium candidum is planted back 10 ~ 18 months (preferred 16 months) and can be gathered in the crops, and can connect root during results and take out, and cleans cultivation matrix, removes the moisture that sticks in the surface because of cleaning.
Officinal dendrobium stem plantation of the present invention plantation mycorrhizal method can obviously promote the dendrobium candidum growth of seedling, and the dendrobium candidum comparison that connects bacteria cultivation increases according to the plant quantity of taking root, length, the growing way of plant are better, internode obviously expands, and stem is obviously sturdy, and fresh weight and dry weight are improved.The resistance that connects the bacterium plant also obviously strengthens.Chemical composition contents such as the alkaloid of seedlings of Dendrobium officinale also are increased to wild stem of noble dendrobium contents level, have guaranteed the quality of medicinal material of dendrobium candidum after the mycorhiza symbiosis, for mycorrhizal fungi is applied to the dendrobium candidum scale, standardized planting provides scientific basis.Problems such as it is slow to have solved the dendrobium candidum plant strain growth, and survival rate is low.In addition, the present invention does not execute any chemical fertilizer and agricultural chemicals, environmental protection, and cultivation method is simple, small investment.
Embodiment:
Below in conjunction with embodiment the present invention is further specified.
Embodiment 1
The operating procedure of LP2 group is as follows:
1, the cultivation of mycorrhizal fungi
Present embodiment adopts mycorrhizal fungi LP2 bacterial strain behind the slant tube actication of culture of low-temperature preservation, is connected in the PDA plate, in 25 ℃ of constant temperature culture 12 d, punches into bacterium sheet (Φ=8 mm) at colony edge, and inserts liquid nutrient medium with fritter.Mycorrhizal fungi LP2 be the Deuteromycotina Fusarium (
Fusarium sp.), this bacterial classification is preserved in Chinese medicinal microorganism fungus kind preservation administrative center at present, and deposit number is CPCC810233.
Mycorrhizal fungi is adopted liquid nutrient medium, culture medium preparation: water preparation, glucose 20 g/L, KH
2PO
420 g/L, MgSO
41.5 g/L, Cobastab
110 mg/L, natural goods: wheat bran 30 g/L (liquor), medium pH=6.0.Insert above-mentioned fungi behind the autoclaving, per 100 mL medium insert 2 bacterium sheets (Φ=8 mm); Fungi is cultivated with triangular flask ventilation is dark, and containing quantity is 50%, places 25 ℃ of vibrations, and rotating speed 100 rpm gather in the crops after cultivating 25 d.Obtain liquid mycorrhizal fungi after cultivate finishing, can directly use or smash cultured products, obtain the liquid mycorrhizal fungi with refiner.
2, candidum tissue culturing seedling is cultivated
Get the sprouting bud on well-grown dendrobium candidum plant, get its stem section, sterilization is inoculated in inducing culture (1/2-1) MS+BA 2.0 mg/L+NAA 0.5 mg/L+3 wt% sucrose+0.75 wt% agar; Transfer in proliferation and differentiation medium (1/2-1) MS+BA 1.0 mg/L+NAA 0.5 mg/L+10 wt% bananas juices+3 wt% sucrose+0.75 wt% agar through inducing the yellowish green protocorm of formation), 2 months subcultures once obtain the seedling that root has leaf; To there be root to have the seedling of leaf to be inoculated in medium (1/2-1) MS+10 wt% bananas juice+NAA 2.0 mg/L+3 wt% sucrose+0.75 wt% agar again, make its growth.
3, preparation nutrient solution
Nutrient solution is made of following component: potassium nitrate 1.1 wt ‰, ammonium sulfate 0.2 wt ‰, ammonium nitrate 0.1 wt ‰, magnesium sulfate 0.12 wt ‰, potassium dihydrogen phosphate 0.10 wt ‰, calcium chloride 0.06 wt ‰, manganese sulphate 0.02 wt ‰, ferrous sulfate 0.03 wt ‰, zinc sulphate 0.005 wt ‰, all the other are water.
4, the water planting refinery of tissue cultivating seedling
Prepare the nutrition liquid bath, cystosepiment (the thick 5 mm) frame that will accomplish fluently aperture (Φ=5 mm) is on the nutrition liquid bath; Dendrobium candidum is cultivated seedling take out flushing with clean water with 17 ± 1 ℃, drop removes excessive moisture, plants in the hole of foam kickboard, and nutrient solution is injected the nutrition liquid bath, makes nutrient solution submergence root system 50%, starts the water circulating pump of nutrition liquid bath, flow velocity 5 meter per seconds; Ventilate, wind speed 0.4 m/s carries out the seedling refinery.Behind 20 d, the seedlings of Dendrobium officinale well developed root system, regenerated root increases, and stem is long to be increased slightly, and it is green that blade turns, and thickening promptly can be extracted and transplant.
5, dendrobium candidum plantation Mycorrhizal cultivation
(1) candidum tissue culturing seedling is transplanted
The tissue cultivating seedling root of the process water planting refinery that can transplant cleans with clear water, notes the stem that fractures of trying not, damaged blade during cleaning; Cultivation matrix Cai Yong Lang mountain glutenite powder is Yu Lang mountain soil is 1:2 by weight.Before the transplanting, place the wheat bran sugar leaf (Fagaceae) that 8 g pulverize, place the liquid mycorrhizal fungi of 2.5 mL abovementioned steps preparation on it again, the root of dendrobium candidum is contacted with bacterium at the base portion of every young plant; During cultivation root is embedded in the matrix, stromal surface is exposed on stem top, will water normal root water after planting, and matrix keeps 75% moisture; Seedling is planted in seedling dish or the flowerpot, and the individual plant seedling gets final product when planting, and does not need many strains to be planted in together, and the cultivation distance is 4 cm.
(2) control of temperature and humidity
Have the seedling dish of tissue cultivating seedling or flowerpot to move into the film booth transplanting, the growth temperature of dendrobium candidum is controlled at 27 ± 1 ℃, adopts 75% sunshade net covering summer, plastic film is raised, in order to cooling; Winter, the sunshade net with 30% covered, and perhaps removes the sunshade net plastic film is put down; The dendrobium candidum transplanting initial stage requires high humidity, and air humidity remains on 80%, and matrix keeps moistening and ponding not.
(3) results of dendrobium candidum
Dendrobium candidum is planted and can be gathered in the crops in back 16 months, can connect root during results and take out, and cleans cultivation matrix, removes the moisture that sticks in the surface because of cleaning.
The control group plant does not connect the bacterium self-sow, and other institute's bacterium that connects in steps groups are consistent.
Present embodiment experimental result such as table 1:
" * " expression is compared difference with control group and is reached significance level, and " * * " expression is compared difference with control group and reached utmost point significance level.Alkaloid adopts determination of acid-basetitration; Respiratory rate adopts photosynthetic analyzer to measure.
Embodiment 2
Present embodiment basic operation condition is with embodiment 1, and difference is:
1, the mycorrhizal fungi used of present embodiment is mycorrhizal fungi LP2 and mycorrhizal fungi LP3.Mycorrhizal fungi LP2 be the Deuteromycotina Fusarium (
Fusarium sp.), mycorrhizal fungi LP3 be the Deuteromycotina Verticillium (
Verticillium sp.).These two kinds of bacterial classifications are preserved in Chinese medicinal microorganism fungus kind preservation administrative center at present, and deposit number is respectively CPCC810233 and CPCC421139.
2, fungal bacterial strain is cultivated and adopted solid culture, and the solid culture medium of mycorrhizal fungi consists of: cotton seed hulls adds water with the corncob of smashing after 1:1 mixes, amount of water is 170 wt% of raw material weight.Insert above-mentioned fungi behind the autoclaving respectively, per 100 g medium connect 2 bacterium sheets (Φ=8 mm).Fungi is left standstill dark cultivation with conical flask, takes out standby when treating the mycelia major part or all covering with medium.
The control group plant does not connect the bacterium self-sow, and other institute's bacterium that connects in steps groups are consistent.
Experimental result such as table 2:
" * " expression is compared difference with control group and is reached significance level, and " * * " expression is compared difference with control group and reached utmost point significance level.Alkaloid adopts determination of acid-basetitration; Respiratory rate adopts photosynthetic analyzer to measure.
After dendrobium candidum connect bacterium, the initial stage was similar to the contrast mode of appearance.Connect vaccine and grow behind 30 d, begin long newborn feeding root, the seedling color and luster is bud green, and old root begins jaundice, and newly root is light green, grows fine; Connect the vaccine newborn feeding root of the amount of growing up behind 65 d, height of seedling increases obviously, and part stem, leaf redden, and the seedling growth reaches optimum state, and the no new root of contrast grows, and plant is withered thin and weak, and aetiolation is serious, and growth potential is obviously weak.Dendrobium candidum amount of growth measurement result is after meeting bacterium 65 d: inoculation mycorrhizal fungi LP2 bacterial strain and the clean growth rate of the inoculation mycorrhizal fungi LP2+ mycorrhizal fungi LP3 average fresh weight of bacterial strain dendrobium candidum all are higher than control group.The plant respiratory rate and a series of physiological metabolism activity that connect bacterium are accelerated, and connect the bacterium plant than not connecing the bacterium plant and have stronger metabolic capability, thereby shortened the time of vegetation growth of plant.The resistance that connects the bacterium plant also obviously strengthens, and chemical composition contents such as alkaloid also significantly improve.
Claims (8)
1. an officinal dendrobium stem plantation is planted the Mycorrhizal production method, it is characterized in that step is as follows:
(1) cultivation of mycorrhizal fungi
With the hybrid bacterial strain of mycorrhizal fungi LP2, mycorrhizal fungi LP3 or two kinds from the slant tube of low-temperature preservation behind actication of culture, be connected in the PDA plate, in 23 ~ 28 ℃ of constant temperature culture 10 ~ 15 d, punch into the bacterium sheet at colony edge, and insert solid culture medium or liquid nutrient medium with fritter; Mycorrhizal fungi LP2 be the Deuteromycotina Fusarium (
Fusarium sp.), mycorrhizal fungi LP3 be the Deuteromycotina Verticillium (
Verticillium sp.); These two kinds of culture presevation are in China medicinal microorganism fungus kind preservation administrative center, and deposit number is respectively CPCC810233 and CPCC421139;
The solid culture medium of mycorrhizal fungi contains nutrients such as lignin, cellulose, carbon, nitrogen, forms by being selected from raw material cotton seed hulls, wood chip, broken corncob, broad-leaved tree sawdust, the wheat bran one or more, adds water, and the water yield is 150 ~ 200 wt% of raw material; Insert respectively behind the autoclaving through cultivating gained mycorrhizal fungi LP1, per 100 g medium connect 2 ~ 3 bacterium sheets; Fungi is left standstill dark cultivation with vessel such as test tube, conical flask or blake bottles, takes out standby when treating the mycelia major part or all covering with medium;
The liquid nutrient medium of mycorrhizal fungi contains glucose, inorganic salts composition and natural additive; The preparation of the liquid nutrient medium water of mycorrhizal fungi, glucose 18 ~ 22 g/L wherein, KH
2PO
418 ~ 22 g/L, MgSO
41.2 ~ 1.8 g/L, Cobastab
18 ~ 12 mg/L, natural goods: wheat bran 25 ~ 35 g/L (liquor) or potato 180 ~ 220 g/L (using its leachate), the medium pH value is 5.6 ~ 6.5; Insert behind the autoclaving through cultivating gained mycorrhizal fungi LP1, per 100 mL medium insert 1 ~ 2 bacterium sheet; Fungi is secretly cultivated with triangular flask or the ventilation of other containers, and containing quantity is 50%, places 23 ~ 25 ℃ of vibrations, and rotating speed 100 ~ 120 rpm gather in the crops behind cultivation 20 ~ 30 d; Obtain liquid mycorrhizal fungi after cultivate finishing, can directly use or to smash cultured products with refiner standby;
(2) candidum tissue culturing seedling is cultivated
Get the sprouting bud on well-grown dendrobium candidum plant, get its stem section, sterilization is inoculated in inducing culture (1/2-1) MS+BA 2.0 mg/L+NAA 0.5 mg/L+3 wt% sucrose+0.75 wt% agar; Through inducing the yellowish green protocorm of formation to transfer in proliferation and differentiation medium (1/2-1) MS+BA 1.0 mg/L+NAA 0.5 mg/L+10 wt% bananas juices+3 wt% sucrose+0.75 wt% agar, 2 months subcultures once obtain the seedling that root has leaf; To there be root to have the seedling of leaf to be inoculated in medium (1/2-1) MS+10 wt% bananas juice+NAA 2.0 mg/L+3 wt% sucrose+0.75 wt% agar again, make its growth;
(3) preparation nutrient solution
Nutrient solution is made of following component: potassium nitrate 1.0 ~ 1.2 wt ‰, ammonium sulfate 0.18 ~ 0.22 wt ‰, ammonium nitrate 0.08 ~ 0.12 wt ‰, magnesium sulfate 0.1 ~ 0.14 wt ‰, potassium dihydrogen phosphate 0.08 ~ 0.12 wt ‰, calcium chloride 0.04 ~ 0.08 wt ‰, manganese sulphate 0.01 ~ 0.03 wt ‰, ferrous sulfate 0.02 ~ 0.04 wt ‰, zinc sulphate 0.004 ~ 0.006 wt ‰, all the other are water;
(4) the water planting hardening of tissue cultivating seedling
The dendrobium candidum that obtains is cultivated seedling to be taken out, flushing with clean water with 15 ~ 20 ℃, drop removes excessive moisture, plant in the hole of the cystosepiment of accomplishing fluently aperture of frame on the nutrition liquid bath, nutrient solution is injected the nutrition liquid bath, make 40 ~ 60% of nutrient solution submergence root system, start the water circulating pump of nutrition liquid bath, flow velocity 4 ~ 6 m/s; Ventilate, wind speed 0.3 ~ 0.5 m/s carries out the seedling refinery; Behind 18 ~ 22 d, the seedlings of Dendrobium officinale well developed root system, regenerated root increases, and stem is long to be increased slightly, and it is green that blade turns, and thickening promptly can be extracted and carry out field-transplanting;
(5) dendrobium candidum plantation Mycorrhizal cultivation
1. candidum tissue culturing seedling is transplanted
The tissue cultivating seedling root of the process water planting refinery that can transplant cleans with clear water, notes the stem that fractures of trying not, damaged blade during cleaning; It is the mixture of 1:1 ~ 2 by weight that cultivation matrix adopts glutenite powder and soil; Before the transplanting, place the wheat bran sugar leaf (Fagaceae) that 5 ~ 10 g pulverize at the base portion of every young plant, place the liquid mycorrhizal fungi of 1.5 ~ 2.5 mL abovementioned steps preparation or the solid mycorrhizal fungi of 1.5 ~ 2.5 g abovementioned steps preparation on it again, the root of dendrobium candidum is contacted with bacterium; During cultivation root is embedded in the matrix, stromal surface is exposed on stem top, will water normal root water after planting, and matrix keeps 75% moisture; Seedling is planted in seedling dish or the flowerpot, and the individual plant seedling gets final product when planting, and does not need many strains to be planted in together, and the cultivation distance is 3 ~ 5 cm;
2. the control of temperature and humidity
Have the seedling dish of tissue cultivating seedling or flowerpot to move into the film booth transplanting, the growth temperature of dendrobium candidum is controlled at 25 ~ 30 ℃, adopts 75% sunshade net covering summer, plastic film is raised, in order to cooling; Winter, the sunshade net with 30% covered, and perhaps removes the sunshade net plastic film is put down; The dendrobium candidum transplanting initial stage requires high humidity, and air humidity remains on 75 ~ 80%, and matrix keeps moistening and ponding not;
3. the results of dendrobium candidum
Dendrobium candidum is planted the back and can be gathered in the crops in 10 ~ 18 months, can connect root during results and take out, and cleans cultivation matrix, removes the moisture that sticks in the surface because of cleaning.
2. officinal dendrobium stem plantation according to claim 1 plantation Mycorrhizal production method, it is characterized in that described with the mycorrhizal fungi pipe behind actication of culture, be connected in the PDA plate, in 24 ~ 26 ℃ of constant temperature culture 11 ~ 13 d.
3. officinal dendrobium stem plantation plantation Mycorrhizal production method according to claim 1, the amount of water that it is characterized in that the solid culture medium of described mycorrhizal fungi is 160 ~ 180 wt% of raw material.
4. officinal dendrobium stem plantation according to claim 1 plantation Mycorrhizal production method is characterized in that the liquid nutrient medium water preparation of described mycorrhizal fungi, glucose 20 g/L wherein, KH
2PO
420 g/L, MgSO
41.5 g/L, Cobastab
110 mg/L, natural goods: wheat bran 30 g/L (liquor) or potato 200 g/L (using its leachate), the medium pH value is 6.0.
5. officinal dendrobium stem plantation plantation Mycorrhizal production method according to claim 1, it is characterized in that described nutrient solution is made of following component: potassium nitrate 1.1 wt ‰, ammonium sulfate 0.2 wt ‰, ammonium nitrate 0.1 wt ‰, magnesium sulfate 0.12 wt ‰, potassium dihydrogen phosphate 0.10 wt ‰, calcium chloride 0.06 wt ‰, manganese sulphate 0.02 wt ‰, ferrous sulfate 0.03 wt ‰, zinc sulphate 0.005 wt ‰, all the other are water.
6. officinal dendrobium stem plantation plantation Mycorrhizal production method according to claim 1 when it is characterized in that the water planting hardening of described tissue cultivating seedling, is cultivated the seedling taking-up with the dendrobium candidum that abovementioned steps obtains, and with 17 ℃ flushing with clean water, drop removes excessive moisture.
7. officinal dendrobium stem plantation plantation Mycorrhizal production method according to claim 1, when it is characterized in that described candidum tissue culturing seedling is transplanted, cultivation matrix Cai Yong Lang mountain glutenite powder is Yu the mixture of Lang mountain soil.
8. officinal dendrobium stem plantation according to claim 1 plantation Mycorrhizal production method is characterized in that described dendrobium candidum is planted can gather in the crops in back 16 months.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105826429A CN102150615A (en) | 2010-12-10 | 2010-12-10 | Method for culturing, planting and mycorrhizal production of dendrobium officinale kimura et migo |
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| CN102428874A (en) * | 2011-12-27 | 2012-05-02 | 杭州师范大学 | Method for inducing protocorms by utilizing dendrobe tissues |
| CN102668957A (en) * | 2012-06-01 | 2012-09-19 | 凌云县长生仙草生物科技开发有限公司 | Method for preparing dendrobium officinale culture substrate |
| CN102715058A (en) * | 2012-06-29 | 2012-10-10 | 浙江农林大学 | Method for improving garden plant transplanting survival rate |
| CN103881928A (en) * | 2014-03-13 | 2014-06-25 | 浙江农林大学 | Application of mortierella mycorrhiza fungi to culture of tissue culture seedling of dendrobium officinale |
| CN103918551A (en) * | 2014-01-16 | 2014-07-16 | 贵州清控置业有限公司 | Imitation wild cultivation method of mycorrhizated dendrobium plant tea tree |
| CN103918552A (en) * | 2014-01-16 | 2014-07-16 | 贵州清控置业有限公司 | Method for rapid tissue culture of dendrobium seedlings |
| CN104195057A (en) * | 2014-09-10 | 2014-12-10 | 江苏农林职业技术学院 | Umbelopsis nana and applications thereof in preventing leaf spot disease of dendrobium officinale |
| CN107018883A (en) * | 2017-01-06 | 2017-08-08 | 广西壮族自治区农业科学院微生物研究所 | A kind of method for producing banana mycorhiza tissue culture of sprout |
| CN107125005A (en) * | 2017-06-12 | 2017-09-05 | 贵州省林业科学研究院 | The method of Paphiopedilum micranthum mycorrhizal seedling raising |
| CN109197586A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum Protocorm base and its application |
| CN109197585A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of method of dendrobium candidum sprouting and rooting |
| CN109197584A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum root media and its application |
| CN114766341A (en) * | 2022-06-07 | 2022-07-22 | 浙江省亚热带作物研究所(浙南林业科学研究院) | Application of mycorrhizal fungi in cultivation of dendrobium officinale tissue culture seedlings |
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| CN102428874A (en) * | 2011-12-27 | 2012-05-02 | 杭州师范大学 | Method for inducing protocorms by utilizing dendrobe tissues |
| CN102668957A (en) * | 2012-06-01 | 2012-09-19 | 凌云县长生仙草生物科技开发有限公司 | Method for preparing dendrobium officinale culture substrate |
| CN102715058B (en) * | 2012-06-29 | 2016-05-11 | 浙江农林大学 | A kind of method that improves ornamental plant transplanting survival rate |
| CN102715058A (en) * | 2012-06-29 | 2012-10-10 | 浙江农林大学 | Method for improving garden plant transplanting survival rate |
| CN103918552B (en) * | 2014-01-16 | 2016-05-11 | 贵州涵龙生物科技有限公司 | A kind of quick tissue is cultivated the method for stem of noble dendrobium seedling |
| CN103918552A (en) * | 2014-01-16 | 2014-07-16 | 贵州清控置业有限公司 | Method for rapid tissue culture of dendrobium seedlings |
| CN103918551A (en) * | 2014-01-16 | 2014-07-16 | 贵州清控置业有限公司 | Imitation wild cultivation method of mycorrhizated dendrobium plant tea tree |
| CN103918551B (en) * | 2014-01-16 | 2016-07-06 | 贵州涵龙生物科技有限公司 | A kind of Mycorrhizal Herba Dendrobii Seedling Camellia sinensis pseudo-wild cultivating method |
| CN103881928A (en) * | 2014-03-13 | 2014-06-25 | 浙江农林大学 | Application of mortierella mycorrhiza fungi to culture of tissue culture seedling of dendrobium officinale |
| CN104195057A (en) * | 2014-09-10 | 2014-12-10 | 江苏农林职业技术学院 | Umbelopsis nana and applications thereof in preventing leaf spot disease of dendrobium officinale |
| CN104195057B (en) * | 2014-09-10 | 2016-09-21 | 江苏农林职业技术学院 | A kind of short and small umbrella is mould and application in Herba Dendrobii disease-resistanting leaf spot |
| CN107018883A (en) * | 2017-01-06 | 2017-08-08 | 广西壮族自治区农业科学院微生物研究所 | A kind of method for producing banana mycorhiza tissue culture of sprout |
| CN107125005A (en) * | 2017-06-12 | 2017-09-05 | 贵州省林业科学研究院 | The method of Paphiopedilum micranthum mycorrhizal seedling raising |
| CN107125005B (en) * | 2017-06-12 | 2020-03-13 | 贵州省林业科学研究院 | Mycorrhizal seedling raising method for paphiopedilum harderi |
| CN109197586A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum Protocorm base and its application |
| CN109197585A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of method of dendrobium candidum sprouting and rooting |
| CN109197584A (en) * | 2018-08-03 | 2019-01-15 | 南京龙源生态农业有限公司 | A kind of dendrobium candidum root media and its application |
| CN114766341A (en) * | 2022-06-07 | 2022-07-22 | 浙江省亚热带作物研究所(浙南林业科学研究院) | Application of mycorrhizal fungi in cultivation of dendrobium officinale tissue culture seedlings |
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