CN110574686A - In-vitro cultivation method of Dendrobium Nanyue - Google Patents
In-vitro cultivation method of Dendrobium Nanyue Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
the invention discloses an in vitro cultivation method of dendrobium candidum, which is characterized by comprising the following steps: (1) sterilizing and disinfecting the south yue curved stem dendrobium explant, inoculating the south yue curved stem dendrobium explant into an explant induction culture medium for culture, selecting uninfected new buds, and sending the new buds to the next step; (2) transferring the uninfected buds obtained in the step (1) into a proliferation culture medium, establishing a sterile system, and obtaining proliferation tissue culture seedlings after the culture is finished; (3) transferring the proliferation tissue culture seedling obtained in the step (2) to a rooting culture medium for rooting culture to form a rooting tissue culture seedling; (4) and (3) hardening the rooted tissue culture seedlings under the condition of natural scattered light, and then transferring the hardened seedlings into a greenhouse substrate for cultivation to obtain the normal production seedlings of the Nanyue bent stem dendrobium. The method completes the process from wild to artificial propagation of the Dendrobium Nanyue, and provides support for regional resource rapid propagation and industrial development of Dendrobium Nanyue.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to an in vitro cultivation method of Dendrobium Nanyue.
background
Dendrobium kojiri (Dendrobium flexicaule Z.H.Tsi, S.C.Sunet L.G.xu) is a new extremely endangered species of dendrobe newly discovered in 1986 in China, is a perennial Orchidaceae herbaceous plant and is a national secondary protective plant. Wild resources are listed in the protection scope of International trade convention on endangered species of wild animals and plants (CITES). Systematic examination is carried out by Mingxing Jia of traditional Chinese medicine research institute in Chongqing, and the famous dendrobium nobile medicinal material is firstly demonstrated to be dendrobium nobile (Mingxing Jia, famous examination of dendrobium nobile [ J ]. Chinese traditional medicine journal, 2016, 41 (10): 1956-1964; Mingxing Jia, and the like, based on morphological Dendrobium nobile, famous examination of Dendrobium nobile [ J ]. Chinese traditional medicine journal), and the medicinal dendrobium nobile recorded in the history materials is definitely: 3 species such as dendrobium nobile, dendrobium officinale, dendrobium huoshanense and the like. The dendrobium aspergillum reported in China only has a Qin bar mountain area and a Henan Funiu mountain area, and in the beginning of 2015 4 months, when an applicant surveys domestic wild dendrobium resources, the variety is collected from local farmers in the south of Hunan province for the first time and is identified as dendrobium aspergillum after identification. Dendrobium kojiense is used as a medicine by stems, and is a strong tonic medicinal material of Chinese herbal medicines. The pharmacological actions mainly include promoting the production of body fluid, removing toxic substances, inhibiting cancer, reducing blood sugar, improving immunity, resisting cataract and resisting bacteria, and is called as the first of the Chinese immortal grass and anticancer pioneer. At present, the selling price of fresh products in the market reaches 4 ten thousand yuan per kilogram. In recent years, due to predatory excavation and the habitat limitation of dendrobium, seeds are difficult to develop under natural conditions, the reproduction rate is extremely low, and dendrobium kojimi is in an extremely endangered place. Dendrobium candidum is well known in folk, but few research institutions and researchers exist, and no successful cultivation example has been seen so far (history of resource discovery of the jujuanjuan, mingxincha, endangered Dendrobium candidum [ J ]. Chongqing Chinese herbal medicine research, 2019, phase 1 (75 th total)). In terms of seedling breeding, although the influence of the combination of royal plum (royal plum, etc., basic medium and hormone on the proliferation of dendrobium stem protocorm [ J ]. Guangxi agricultural science, 2006.37 vol.I: 10-12), Zhang Ming Yue (Zhang Ming Yue, etc., research on tissue culture and propagation of dendrobium kojim [ J ]. Nenjiang academy of academic faculty of Neighenry, 2018, 33 vol.10, No. 76-79), Yangli (Yangli river, etc., research on rapid propagation of dendrobium kojim [ J ]. Chinese medicinal journal, 2002, 27 vol.6), dawn (Yuli, etc., research on isolated culture and regeneration system of wild dendrobium kojim [ J ]. Wuhan Industrial academy of academic, 2010, 29 vol.I: 21-24) four studies on isolated culture of dendrobium stem, the propagation coefficient is low, and there are differences in the species.
at present, no report is found about the natural ecological environment, distribution area and artificial propagation of the dendrobium candidum in the south Yue. Therefore, in order to protect the precious regional species resource and realize the resource protection and industrial application of the dendrobium candidum, the tissue culture technology optimization research of salvageability on the dendrobium candidum in south Yue needs to be carried out.
Disclosure of Invention
The invention provides an in vitro cultivation method of Dendrobium Nanyue, which comprises the following steps:
(1) Sterilizing and disinfecting the south yue curved stem dendrobium explant, inoculating the south yue curved stem dendrobium explant into an explant induction culture medium for culture, selecting uninfected new buds, and sending the new buds to the next step;
(2) transferring the uninfected buds obtained in the step (1) into a proliferation culture medium, establishing a sterile system, and obtaining proliferation tissue culture seedlings after the culture is finished;
(3) Transferring the proliferation tissue culture seedling obtained in the step (2) to a rooting culture medium for rooting culture to form a rooting tissue culture seedling;
(4) and (3) hardening the rooted tissue culture seedlings under the condition of natural scattered light, and then transferring the hardened seedlings into a greenhouse substrate for cultivation to obtain the normal production seedlings of the Nanyue bent stem dendrobium.
According to the method of the invention, in step (1), the sterilization process comprises: soaking the south yue curved stem dendrobium explant with a small amount of vegetable detergent (such as 1-2 drops) for 2-3 minutes, then washing with clear water for 20-40 minutes, and placing the south yue curved stem dendrobium explant into an ultra-clean workbench for further sterilization. Wherein the sterilization method comprises the following steps: sterilizing the cleaned explant material with 65-75% alcohol for 30-50 s, and then 0.08-0.15% Hgcl2sterilizing for 5-15 min.
According to the method of the present invention, in step (1), the induction medium comprises an inorganic culture, an organic culture and a growth regulating substance;
wherein the inorganic culture comprises: (a) macroelements: potassium nitrate 2810-2850 mg/L, ammonium sulfate 453-470mg/L, monopotassium phosphate 380-420mg/L, magnesium sulfate 175-195mg/L, calcium chloride 156-176 mg/L; (b) trace elements: 20-25mg/L of manganese sulfate, 5-10mg/L of zinc sulfate, 4-8 mg/L of boric acid, 0.5-1mg/L of potassium iodide, 0.15-0.4mg/L of sodium molybdate and 0.01-0.04mg/L of copper sulfate; (c) iron salt: 23-30 mg/L, EDTA 32-40 mg/L ferrous sulfate;
Wherein the organic culture comprises: 50-100 mg/L inositol, 40-60 mg/L thiamine sulfate, 0.8-1.5 mg/L pyridoxine sulfate, 0.3-0.6 mg/L, Vc 4.0.0-6.0 mg/L nicotinic acid, 1-5.0 mg/L compound nitro, 100-200 mg/L choline chloride;
The growth regulating substance comprises: 2.0-3.0 mg/L, NAA 0.5.5-1.0 mg/L of 6-BA and 0.3-0.6 mg/L, KT 0.5.5-1.0 mg/L of 2, 4-D.
preferably, the complex nitro group comprises: at least two of 0.5-1.5mg/L of 5-nitroguaiacol sodium, 1-2.5mg/L of p-nitrophenol sodium and 1-2.5mg/L of o-nitrophenol sodium; illustratively, the complex nitro group includes: 1.0mg/L of 5-nitroguaiacol sodium, 2.0mg/L of p-nitrophenol sodium and 2.0mg/L of o-nitrophenol sodium.
According to the method of the invention, in the step (1), the induction medium further comprises agar and white sugar. Further, the pH value of the induction medium is 5.5-6.0, preferably 5.8. Further, the preparation process of the induction medium comprises the following steps: adding the components into water per liter according to the dosage of the components, then adding 4-8 g of agar and white sugar, mixing and boiling until the solid is completely dissolved, adjusting the pH value of the system to 5.5-6.0, then using an autoclave to keep at the temperature of 119 ℃ and 122 ℃ for 20-40 minutes, and standing for later use after sterilization is completed; wherein the adding amount of white sugar accounts for 2-4% of the mass of the induction culture medium.
according to the method of the invention, in the step (1), the explants are placed in a culture room for culture for 30-50 days at the temperature of 23-28 ℃ under the illumination of 2000-3000LX after being inoculated on a sterile operating platform.
According to the method of the present invention, in step (2), the proliferation medium comprises: inorganic cultures, organic cultures, growth regulating substances and growth supplements;
Wherein the inorganic culture and the organic culture have the meaning as described above;
the growth regulating substance comprises: 1.0-2.0 mg/L, NAA 0.5.5-7.0 mg/L of 6-BA;
The growth supplements include: 15-25 ml/L of aloe and cactus biological fermentation liquid and 100g/L of banana or carrot. Furthermore, each milliliter of the cactus biological fermentation liquid contains 3 hundred million bacteria.
wherein the aloe and cactus biological fermentation liquid is prepared from aloe, cactus, water, white sugar and EM bacteria according to the weight ratio of (1.5-3) to (2-5) to 1:1, preferably, the weight ratio is 2:2:4:1: 1.
According to the method, in the step (2), the proliferation medium further comprises agar and white sugar. Further, the pH value of the proliferation medium is 5.5 to 6.0, preferably 5.8. Further, the preparation process of the proliferation medium comprises the following steps: adding the components into water per liter according to the dosage of the components, then adding 4-8 g of agar and white sugar, mixing and boiling until the solid is completely dissolved, adjusting the pH value of the system to 5.5-6.0, then using an autoclave to keep at the temperature of 119 ℃ and 122 ℃ for 20-40 minutes, and standing for later use after sterilization is completed; wherein the adding amount of white sugar accounts for 2-4% of the mass of the induction culture medium.
according to the method of the invention, in the step (2), the proliferation culture process comprises: transferring the tissue culture seedling of Dendrobium Nanyue to a proliferation culture medium in a sterile operation table, culturing at 23-28 deg.C under illumination of 2000-3000LX for 50-60 days.
According to the method of the present invention, in step (3), the rooting medium comprises: inorganic cultures, organic cultures, growth regulating substances and growth supplements;
Wherein the inorganic culture comprises: (a) macroelements: potassium nitrate 1405-1425 mg/L, ammonium sulfate 226.5-235mg/L, potassium dihydrogen phosphate 190-210mg/L, magnesium sulfate 87.5-97.5mg/L, calcium chloride 78-88 mg/L; (b) trace elements: 20-25mg/L of manganese sulfate, 5-10mg/L of zinc sulfate, 4-8 mg/L of boric acid, 0.5-1mg/L of potassium iodide, 0.15-0.4mg/L of sodium molybdate and 0.01-0.04mg/L of copper sulfate; (c) iron salt: 23-30 mg/L, EDTA 32-40 mg/L ferrous sulfate;
the organic culture and growth supplements have the meaning as described above;
The growth regulating substance comprises: IBA 0.5-1.0 mg/L, NAA 0.2.2-5.0 mg/L.
according to the method, in the step (3), the rooting medium further comprises agar and white sugar. Further, the pH value of the rooting medium is 5.5-6.0, preferably 5.8. Further, the preparation process of the proliferation medium comprises the following steps: adding the components into water per liter according to the dosage of the components, then adding 4-8 g of agar and white sugar, mixing and boiling until the solid is completely dissolved, adjusting the pH value of the system to 5.5-6.0, then using an autoclave to keep at the temperature of 119 ℃ and 122 ℃ for 20-40 minutes, and standing for later use after sterilization is completed; wherein the adding amount of white sugar accounts for 2-4% of the mass of the induction culture medium.
According to the method, in the step (3), the rooting culture process comprises the following steps: transferring the propagation tissue culture seedling of the dendrobium candidum in the south Yue Qu stem to a rooting culture medium in a sterile operation table, and culturing for 20-30 days at the temperature of 23-28 ℃ under the illumination of 2000-3000LX to form the rooting tissue culture seedling.
according to the method, in the step (4), the seedling exercising time is 7-15 days.
the "Nanyue Quhuhou dendrobe" in the present application refers to the Quhuhou dendrobe produced in the Hunan Nanyue region.
The invention has the beneficial effects that:
The method provided by the invention is applied to rescue of endangered regional species, namely the Nanyue bent stem dendrobium, and the inventor forms a technical system aiming at the artificial propagation optimization of the Nanyue bent stem dendrobium by screening through a tissue culture method. Can make resources return to the original place, protect local resources, enrich precious medicinal material variety resources and improve local economic income.
The inventor compares and verifies the effect of the tissue culture technology on the basis of the original research of the dendrobium candidum, and finds out the tissue culture technology difference of the dendrobium candidum in different regions. The optimal tissue culture technical scheme of the south Yue Qu stem dendrobium is determined by screening and optimizing a tissue culture technical method, particularly determining formulas and concentrations of an inorganic culture, an organic culture, a growth regulating substance and an organic additive, and the support is provided for the regional resource rapid propagation and industrial development of the south Yue Qu stem dendrobium. In addition, the Chinese medicinal material resources are enriched, and basic conditions are created for further research, development and utilization.
The dendrobium kojim is in the abrogation of the nature due to harsh growth conditions, slow growth speed, low natural reproduction rate and over-picking of pesticide farmers. The inventor obtains the optimized tissue culture technology of the application through the implementation and screening of a tissue culture technical test scheme for three years, completes the process from wild to artificial propagation, and the propagation efficiency is optimal at present:
1. Through induction culture, the sterile bud induction rate is 80%, and the bud induction multiplication coefficient is 3.2 times;
2. Through proliferation culture, the bud proliferation multiple reaches 8.3, which is 2.4 times higher than the highest level of the current research, and the method can meet the technical conditions of industrial tissue culture seedling culture;
3. through rooting culture, the rooting rate reaches 98 percent, is 7 percent higher than the highest level of the current research, has good seedling strengthening quality, and is suitable for artificial field culture.
4. Over 10 million strains of tissue culture seedlings have been obtained after two years of research.
drawings
FIG. 1 shows the dendrobii officmalis caulis sprout obtained in step (1) of the method of example 1.
FIG. 2 shows the propagated seedling of Dendrobium Nanyue Quadrature obtained in step (2) of example 1.
FIG. 3 is the rooted seedling of Dendrobium Nanyue Quadrature obtained in step (3) of the method of example 1.
FIG. 4 is the seedling obtained by the step (4) of the method of example 1.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
example 1
the tissue culture method of the dendrobium nobile lindl comprises the following steps: the tissue culture seedling complete plant is formed by three processes of sterile line induction culture, proliferation culture and rooting culture.
1. and (3) sterile system induction: the tissue culture sterile line is established by sterilizing the living dendrobium kojimi material and preparing and sterilizing a culture medium.
1.1, sterilizing the dendrobium kojiense explant: soaking the active material of Dendrobium loddigesii Rolfe in 1-2 drops of vegetable detergent for 2-3 min, washing with clear water for 30 min, and sterilizing in a clean bench. The sterilization method comprises the following steps: the washed explant material is sterilized by 70% alcohol for 30 seconds, then sterilized by 0.1% Hgcl2 bacteria for 10 minutes, and inoculated into a special explant induction culture medium.
1.2, performing induced culture preparation on dendrobium kojiense: consists of inorganic culture, organic culture and growth regulating matter.
1.2.1, inorganic culture: macroelements: 2830 mg/L of potassium nitrate, 463 mg/L of ammonium sulfate, 400 mg/L of monopotassium phosphate, 180mg/L of magnesium sulfate and 165 mg/L of calcium chloride; trace elements: 22.3 mg/L of manganese sulfate, 8.6 mg/L of zinc sulfate, 6.2 mg/L of boric acid, 0.83 mg of potassium iodide, 0.25 mg/L of sodium molybdate and 0.025 mg/L of copper sulfate; iron salt: 27.8 mg/L, EDTA 37.3.3 mg/L ferrous sulfate;
1.2.2, organic culture preparation: 50-100 mg/L inositol, 50 mg/L thiamine sulfate, 1.0mg/L pyridoxine sulfate, 0.5mg/L, Vc 5.0.0 mg/L nicotinic acid, 1.0mg/L compound nitro solution, and 100 mg/L choline chloride;
The compound nitro solution consists of 1.0mg/L of 5-nitroguaiacol sodium, 2.0mg/L of p-nitrophenol sodium and 2.0mg/L of o-nitrophenol sodium.
1.2.3, growth regulating substances: 3.0 mg/L, NAA 1.0.0 mg/L of 6-BA and 0.5mg/L, KT 0.5.5 mg/L of 2, 4-D;
1.2.4, mixing the inorganic culture, the organic culture and the growth regulating substance, adding water according to a unit proportion (per L), adding 4-8 g of agar and 3% of white sugar, mixing, boiling and completely dissolving, adjusting the pH value to 5.8, using an autoclave, keeping at 121 ℃ for 20 minutes, and sterilizing for later use.
1.3 after inoculation in the sterile operating platform, the culture room is cultured for 30-50 days at the temperature of 23-28 ℃ under the illumination of 2000-3000 LX. The uninfected shoots (as shown in FIG. 1) were then transferred to a growth medium to establish a sterile line.
2. and (3) proliferation culture: the proliferation culture medium comprises inorganic culture, organic culture, growth regulating substance, and growth additive.
2.1, inorganic and organic cultures (induction medium as above);
2.2, the growth regulating substances comprise: 2.0mg/L, NAA 0.5.5 mg/L of 6-BA;
2.3, growth addition: 20ml/L of aloe and cactus biological fermentation liquid (each milliliter contains 3 hundred million bacteria) and 100g of carrot; the aloe and cactus biological fermentation liquid is prepared from aloe, cactus, water, white sugar and EM bacteria according to the weight ratio of 2:2:4:1: 1.
2.4, mixing 2.1, 2.2 and 2.3, adding 4-8 g of agar and 3% of white sugar, mixing, boiling, completely dissolving, adjusting pH to 5.8, using an autoclave, keeping at 121 ℃ for 20 minutes, sterilizing and cooling for later use.
2.5, transferring the dendrobium candidum tissue culture seedling to a propagation culture medium in an aseptic operation platform, culturing for 50-60 days at the temperature of 23-28 ℃ under the illumination of 2000-3000LX to obtain a propagation culture seedling (as shown in figure 2), and transferring to a rooting culture medium for rooting culture.
3. rooting culture: the rooting culture medium consists of inorganic culture, organic culture, growth regulating matter and growth additive.
3.1, inorganic culture: inorganic cultures (same as induction medium above, but with halved macroelements), organic cultures (same as induction medium above);
3.2, the growth regulating substances comprise: IBA 0.5mg/L, NAA 0.2 mg/L;
3.3, mixing 3.1 and 3.2, adding 4 to 8 grams of agar and 2 percent of white sugar, mixing, boiling, completely dissolving, adjusting the pH value to 5.8, using an autoclave, keeping at the temperature of 121 ℃ for 20 minutes, sterilizing and cooling for later use.
3.4, transferring the dendrobium candidum proliferation tissue culture seedling to a rooting culture medium in an aseptic operation platform, culturing for 20-30 days at the temperature of 23-28 ℃, and illuminating 2000-3000LX to form the rooting tissue culture seedling (as shown in figure 3).
4. Hardening the rooted tissue culture seedling for 7-15 days under the condition of natural scattered light, and transferring into greenhouse matrix for cultivation to form normal production seedling of Dendrobium kojiense (shown in figure 4).
in the embodiment, through induction culture, the sterile bud induction rate is 80%, and the bud induction multiplication coefficient is 3.2 times; through proliferation culture, the bud proliferation multiple reaches 8.3, which is 2.4 times higher than the highest level of the current research, and the method can meet the technical conditions of industrial tissue culture seedling culture; through rooting culture, the rooting rate reaches 98 percent, is 7 percent higher than the highest level of the current research, has good seedling strengthening quality, and is suitable for artificial field culture. Over 10 million strains of tissue culture seedlings have been obtained after two years of research.
the embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An in vitro cultivation method of Dendrobium Nanyue Quadrature is characterized by comprising the following steps:
(1) Sterilizing and disinfecting the south yue curved stem dendrobium explant, inoculating the south yue curved stem dendrobium explant into an explant induction culture medium for culture, selecting uninfected new buds, and sending the new buds to the next step;
(2) Transferring the uninfected buds obtained in the step (1) into a proliferation culture medium, establishing a sterile system, and obtaining proliferation tissue culture seedlings after the culture is finished;
(3) Transferring the proliferation tissue culture seedling obtained in the step (2) to a rooting culture medium for rooting culture to form a rooting tissue culture seedling;
(4) and (3) hardening the rooted tissue culture seedlings under the condition of natural scattered light, and then transferring the hardened seedlings into a greenhouse substrate for cultivation to obtain the normal production seedlings of the Nanyue bent stem dendrobium.
2. the method of claim 1, wherein in step (1), the sterilization process comprises: soaking the south yue curved stem dendrobium explant with a small amount of vegetable detergent for 2-3 minutes, then washing with clear water for 20-40 minutes, and placing the south yue curved stem dendrobium explant into an ultra-clean workbench for further sterilization;
wherein the sterilization method comprises the following steps: sterilizing the cleaned explant material with 65-75% alcohol for 30-50 s, and then 0.08-0.15% Hgcl2Sterilizing for 5-15 min.
3. the method according to claim 1, wherein in step (1), the induction medium comprises an inorganic culture, an organic culture and a growth regulating substance;
Wherein the inorganic culture comprises: (a) macroelements: potassium nitrate 2810-2850 mg/L, ammonium sulfate 453-470mg/L, monopotassium phosphate 380-420mg/L, magnesium sulfate 175-195mg/L, calcium chloride 156-176 mg/L; (b) trace elements: 20-25mg/L of manganese sulfate, 5-10mg/L of zinc sulfate, 4-8 mg/L of boric acid, 0.5-1mg/L of potassium iodide, 0.15-0.4mg/L of sodium molybdate and 0.01-0.04mg/L of copper sulfate; (c) iron salt: 23-30 mg/L, EDTA 32-40 mg/L ferrous sulfate;
Wherein the organic culture comprises: 50-100 mg/L inositol, 40-60 mg/L thiamine sulfate, 0.8-1.5 mg/L pyridoxine sulfate, 0.3-0.6 mg/L, Vc 4.0.0-6.0 mg/L nicotinic acid, 1-2.0 mg/L compound nitro, 100-200 mg/L choline chloride;
The growth regulating substance comprises: 2.0-3.0 mg/L, NAA 0.5.5-1.0 mg/L of 6-BA and 0.3-0.6 mg/L, KT 0.5.5-1.0 mg/L of 2, 4-D.
4. the method according to claim 3, wherein in the step (1), the induction medium further comprises agar and white sugar;
The pH value of the induction culture medium is 5.5-6.0;
the preparation process of the induction medium comprises the following steps: adding the components into water per liter according to the dosage of the components, then adding 4-8 g of agar and white sugar, mixing and boiling until the solid is completely dissolved, adjusting the pH value of the system to 5.5-6.0, then using an autoclave to keep at the temperature of 119 ℃ and 122 ℃ for 20-40 minutes, and standing for later use after sterilization.
5. The method as claimed in claim 1, wherein in step (1), the explant is cultured in a culture room for 30-50 days at 23-28 ℃ under illumination of 2000 LX after being inoculated in a sterile operating platform.
6. The method according to claim 1, wherein in step (2), the propagation medium comprises: inorganic cultures, organic cultures, growth regulating substances and growth supplements;
Wherein the inorganic and organic cultures have the meaning as defined in claim 3;
the growth regulating substance comprises: 1.0-2.0 mg/L, NAA 0.5.5-7.0 mg/L of 6-BA;
The growth supplements include: 15-25 ml/L of aloe and cactus biological fermentation liquid and 100g/L of banana or carrot.
7. The method according to claim 6, wherein in the step (2), the proliferation medium further comprises agar and white sugar;
the pH value of the proliferation culture medium is 5.5-6.0;
The preparation process of the proliferation culture medium comprises the following steps: adding the components into water per liter according to the dosage of the components, then adding 4-8 g of agar and white sugar, mixing and boiling until the solid is completely dissolved, adjusting the pH value of the system to 5.5-6.0, then using an autoclave to keep at the temperature of 119 ℃ and 122 ℃ for 20-40 minutes, and standing for later use after sterilization is completed;
Preferably, in step (2), the proliferation culturing process comprises: transferring the tissue culture seedling of Dendrobium Nanyue to a proliferation culture medium in a sterile operation table, culturing at 23-28 deg.C under illumination of 2000-3000LX for 50-60 days.
8. The method of claim 1, wherein in step (3), the rooting medium comprises: inorganic cultures, organic cultures, growth regulating substances and growth supplements;
Wherein the inorganic culture comprises: (a) macroelements: potassium nitrate 1405-1425 mg/L, ammonium sulfate 226.5-235mg/L, potassium dihydrogen phosphate 190-210mg/L, magnesium sulfate 87.5-97.5mg/L, calcium chloride 78-88 mg/L; (b) trace elements: 20-25mg/L of manganese sulfate, 5-10mg/L of zinc sulfate, 4-8 mg/L of boric acid, 0.5-1mg/L of potassium iodide, 0.15-0.4mg/L of sodium molybdate and 0.01-0.04mg/L of copper sulfate; (c) iron salt: 23-30 mg/L, EDTA 32-40 mg/L ferrous sulfate;
The organic culture has the meaning as claimed in claim 3;
The growth supplements have the meaning as claimed in claim 6;
The growth regulating substance comprises: IBA 0.5-1.0 mg/L, NAA 0.2.2-5.0 mg/L.
9. the method according to claim 8, wherein in step (3), the rooting medium further comprises agar and white sugar;
The pH value of the rooting culture medium is 5.5-6.0;
the preparation process of the proliferation culture medium comprises the following steps: adding the components into water per liter according to the dosage of the components, then adding 4-8 g of agar and white sugar, mixing and boiling until the solid is completely dissolved, adjusting the pH value of the system to 5.5-6.0, then using an autoclave to keep at the temperature of 119 ℃ and 122 ℃ for 20-40 minutes, and standing for later use after sterilization.
10. the method of claim 1, wherein in step (3), the process of rooting culture comprises: transferring the propagation tissue culture seedling of the dendrobium candidum in the south Yue Qu stem to a rooting culture medium in a sterile operation table, and culturing for 20-30 days at the temperature of 23-28 ℃ under illumination of 2000-3000LX to form a rooting tissue culture seedling;
Preferably, in the step (4), the seedling exercising time is 7-15 days.
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| CN112056197A (en) * | 2020-09-07 | 2020-12-11 | 贵州沿河乌江生物科技发展有限公司 | Rapid tree culture method for dendrobium officinale tissue culture seedlings |
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| CN112056197A (en) * | 2020-09-07 | 2020-12-11 | 贵州沿河乌江生物科技发展有限公司 | Rapid tree culture method for dendrobium officinale tissue culture seedlings |
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Application publication date: 20191217 |