CN104542289A - Method for performing tissue culture of dendrobium candidum seedlings - Google Patents
Method for performing tissue culture of dendrobium candidum seedlings Download PDFInfo
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Abstract
The invention discloses a method for performing tissue culture of dendrobium candidum seedlings. The method comprises the following steps: sterilizing: sterilizing an inoculation chamber and inoculators; germinating: selecting dendrobium candidum capsules, disinfecting the capsules, longitudinally cutting up the capsules, taking out seeds by a pair of tweezers, uniformly spreading the seeds in a culture bottle containing germination nutrient solution to perform seed germination culture for 35-40 days, and forming green protocorms; transplanting: moving the protocorms in a culture bottle containing tissue culture nutrient solution to perform protocorm proliferation and differentiation culture for 20-25 days, and obtaining young seedlings; moving the young seedlings to a culture bottle containing the tissue culture nutrient solution to perform seedling proliferation and differentiation culture for 30-35 days, and obtaining plantlets; moving the plantlets to a culture bottle containing the tissue culture nutrient solution to perform strong seedling culture for 60-65 days, and obtaining middle-age seedlings; moving the middle-age seedlings to a culture bottle containing the tissue culture nutrient solution to perform strong seedling culture for 65-70 days, and obtaining the dendrobium candidum seedlings.
Description
Technical field
The present invention relates to plant biotechnology field, particularly relate to a kind of method of seedlings of Dendrobium officinale tissue cultures.
Background technology
Dendrobium candidum is famous stem of noble dendrobium kind, has unique medical value, ranks first of Chinese nine immortal grass.The Shennong's Herbal of Qin Han dynasty is recorded dendrobium candidum and " in main wound, except thin thin, the reinforcing yin essence of numbness, lower gas, tonifying five zang organs consumptive disease, is taken thick stomach for a long time ".Modern chemistry and pharmacological research show, the principle active component of dendrobium candidum is polysaccharide, and polyoses content is up to 20-30%.Because the composition such as dendrobium polysaccharide, alkaloid, amino acid, trace element contained by it has the immunocompetence strengthening body, anti-oxidant, anti-ageing, reduce blood sugar, nourishing Yin and promoting production of body fluid, strong kidney benefit essence, inhibition tumor cell, the unique effects such as alleviating physical fatigue.But because dendrobium officinale primary growth is on the overhanging cliff on high and steep mountains, because its seed is minimum, each capsule contains about 2000 seeds, thin as dust and without endosperm, germination rate is extremely low under field conditions (factors), and natural resources is very rare.Add long-term uncontrolled digging, wild resource is day by day exhausted, and natural production is difficult to meet existing market demand.
So far, the tissue-culturing quick-propagation of dendrobium candidum all selects stem apex, sprouting, sleeping bud to be explant mostly, cultivates by intending protocorm approach.Produce protocorm tissue culture method with aseptic seed and rarely have report.Although these methods optimize the medium component of traditional mode of production, easy and simple to handle, production cost is low, and medicinal ingredient, close to dendrobium officinale, exists reproduction rate lower, and differentiation is slow, is difficult to carry out the problem such as batch production and factorial praluction.
Summary of the invention
Based on the technical problem that background technology exists, the present invention proposes a kind of method of seedlings of Dendrobium officinale tissue cultures, the nutrient solution of different formulations is have employed in cultivation different phase, the nutrient component of its equilibrium can not only promote the quick growth of dendrobe cultivation seedling, and improve the quality of dendrobe cultivation seedling, and be not subject to seasonal restrictions, substantially reduce time and the cost of seedling tissue cultures, polyoses content in dendrobium candidum can also be improved.
The method of a kind of seedlings of Dendrobium officinale tissue cultures that the present invention proposes, comprises the steps:
S1, sterilizing: transfer room and vaccination equipment are carried out sterilizing;
S2, sprouting: the capsule choosing qualified dendrobium candidum, to capsule disinfection, capsule after disinfecting longitudinally is cut, evenly falls apart in being equipped with in the blake bottle sprouting nutrient solution with tweezers taking-up seed and carry out seed germination cultivation, cultivate and within 35-40 days, form green protocorm afterwards;
S3, transplanting: protocorm is moved in the blake bottle that group cultivation nutrient fluid is housed and carry out Protocorm Multiplication differentiation cultivation, cultivate and obtain seedling after 20-25 days; Seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carry out seedling proliferation differentiation cultivation, cultivate and obtain the seedling that height of seedling is more than 1cm after 30-35 days; Seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, cultivate after 60-65 days and obtain the middle seedling that height of seedling is 3-4cm; Middle seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, cultivate and obtain the seedlings of Dendrobium officinale that height of seedling is more than 5cm after 65-70 days.
The capsule of dendrobium candidum qualified in S2 is the fresh dendrobium candidum capsule do not split.
Preferably, in S1, described sterilizing is ultraviolet sterilization and/or ozone sterilization.
Preferably, in S2, described in disinfect for adopt 95% medicinal alcohol infiltrate capsule surface.
Preferably, in S2, the component sprouting nutrient solution comprises: manganese sulphate 0.007-0.012g/L, zinc sulphate 6.2-6.4 × 10
-3g/L, copper sulphate 5.3-5.4 × 10
-5g/L, ferrous sulfate 0.015-0.022g/L, magnesium sulfate 0.15-0.24g/L, ammonium nitrate 0.77-0.83g/L, potassium nitrate 0.9-1g/L, calcium chloride 0.2-0.24g/L, cobalt chloride 6.2-6.6 × 10
-5g/L, sodium molybdate 4.2-4.3 × 10
-4g/L, calcium dihydrogen phosphate 0.085-0.095g/L, potassium iodide 6.3-6.5 × 10
-4g/L, boric acid 7.3-7.5 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.03-0.05g/L, inositol 0.12-0.15g/L, nicotinic acid 3.1-3.4 × 10
-4g/L, puridoxine hydrochloride 6.4-6.5 × 10
-4g/L, thiamine hydrochloride 1.1-1.4 × 10
-4g/L, glycine 1.2-1.4 × 10
-3g/L, agar 4-4.2g/L, white sugar 38-43g/L.
Preferably, in S2, the component sprouting nutrient solution comprises: manganese sulphate 0.01g/L, zinc sulphate 6.30 × 10
-3g/L, copper sulphate 5.36 × 10
-5g/L, ferrous sulfate 0.02g/L, magnesium sulfate 0.19g/L, ammonium nitrate 0.81g/L, potassium nitrate 0.95g/L, calcium chloride 0.22g/L, cobalt chloride 6.43 × 10
-5g/L, sodium molybdate 4.29 × 10
-4g/L, calcium dihydrogen phosphate 0.09g/L, potassium iodide 6.39 × 10
-4g/L, boric acid 7.46 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.04g/L, inositol 0.13g/L, nicotinic acid 3.21 × 10
-4g/L, puridoxine hydrochloride 6.43 × 10
-4g/L, thiamine hydrochloride 1.29 × 10
-4g/L, glycine 1.29 × 10
-3g/L, agar 4.09g/L, white sugar 40g/L.
Preferably, in S2, the condition of culture that seed germination is cultivated is as follows: cultivation temperature is 23-24 DEG C, and cultivation intensity of illumination is 1500-1800Lux, and cultivation light application time is 12-13h/d, and average sterilization frequency is 1-1.5 times/day.
Preferably, in S3, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.007-0.012g/L, zinc sulphate 6.2-6.4 × 10
-3g/L, copper sulphate 5.3-5.4 × 10
-5g/L, ferrous sulfate 0.015-0.022g/L, magnesium sulfate 0.15-0.24g/L, ammonium nitrate 0.77-0.83g/L, potassium nitrate 0.9-1g/L, calcium chloride 0.2-0.24g/L, cobalt chloride 6.2-6.6 × 10
-5g/L, sodium molybdate 4.2-4.3 × 10
-4g/L, calcium dihydrogen phosphate 0.085-0.095g/L, potassium iodide 6.3-6.5 × 10
-4g/L, boric acid 7.3-7.5 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.03-0.05g/L, inositol 0.12-0.15g/L, nicotinic acid 3.1-3.4 × 10
-4g/L, puridoxine hydrochloride 6.4-6.5 × 10
-4g/L, thiamine hydrochloride 1.1-1.4 × 10
-4g/L, glycine 1.2-1.4 × 10
-3g/L, agar 4-4.2g/L, banana puree 67-72g/L, white sugar 38-43g/L.
Preferably, in S3, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.01g/L, zinc sulphate 6.30 × 10
-3g/L, copper sulphate 5.36 × 10
-5g/L, ferrous sulfate 0.02g/L, magnesium sulfate 0.19g/L, ammonium nitrate 0.81g/L, potassium nitrate 0.95g/L, calcium chloride 0.22g/L, cobalt chloride 6.43 × 10
-5g/L, sodium molybdate 4.29 × 10
-4g/L, calcium dihydrogen phosphate 0.09g/L, potassium iodide 6.39 × 10
-4g/L, boric acid 7.46 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.04g/L, inositol 0.13g/L, nicotinic acid 3.21 × 10
-4g/L, puridoxine hydrochloride 6.43 × 10
-4g/L, thiamine hydrochloride 1.29 × 10
-4g/L, glycine 1.29 × 10
-3g/L, agar 4.09g/L, banana puree 70g/L, white sugar 40g/L.
Preferably, in S3, the Transplanting den-sity in blake bottle is 18-20 strain/bottle.
Preferably, in S3, the condition of culture of Proliferation, Differentiation cultivation and strong seedling culture is as follows: cultivation temperature is 23-24 DEG C, and cultivation intensity of illumination is 1500-1800Lux, and cultivation light application time is 12-13h/d, and average sterilization frequency is 1-1.5 times/day.
The solvent of above-mentioned sprouting nutrient solution and group cultivation nutrient fluid is distilled water.
The present invention adopts method for tissue culture seedling, and have employed the nutrient solution of different formulations in cultivation different phase, the nutrient component of its equilibrium can not only promote the quick growth of dendrobe cultivation seedling, and improves the quality of dendrobe cultivation seedling, at protocorm induction period, sprout the inductivity that nutrient solution can effectively raise seed, in the protocorm differentiation stage, have employed group cultivation nutrient fluid, value-added coefficient is made to bring up to 10, solve the protocorm differentiation rate run into when all the time using capsule to breed low, the rate of increase is low, the problem that test-tube plantlet growing way is weak, and the present invention is not subject to seasonal restrictions, from Seeds of Dendrobium Candidum to being trained seedling, only need 7.5 months, substantially reduce time and the cost of seedling tissue cultures, compared with traditional seeding propagation, the reproductive speed of seedling is largely increased, seedling early growth is healthy and strong, leaf look dark green, damage by disease and insect is few, be easy to management, and gained seedling is in hardening, in the process of transplanting, survival rate can reach more than 95%, output can improve 25%, compared with the effective component content of common dendrobium candidum, the polysaccharide content of dendrobium candidum of gained of the present invention is higher than common dendrobium candidum, micronutrient levels a little more than or equal common dendrobium candidum content.
Embodiment
Below, by specific embodiment, technical scheme of the present invention is described in detail.
Embodiment 1
The method of a kind of seedlings of Dendrobium officinale tissue cultures that the present invention proposes, comprises the steps:
S1, sterilizing: transfer room and vaccination equipment are carried out ultraviolet sterilization;
S2, sprouting: the capsule choosing qualified dendrobium candidum, to capsule disinfection, 95% medicinal alcohol is infiltrated capsule surface, capsule after disinfecting longitudinally is cut, evenly fall apart in being equipped with in the blake bottle sprouting nutrient solution with tweezers taking-up seed and carry out seed germination cultivation, cultivation temperature is 23 DEG C, cultivation intensity of illumination is 1800Lux, cultivation light application time is 12h/d, average sterilization frequency is 1.5 times/day, cultivate and within 35 days, form green protocorm afterwards, the component wherein sprouting nutrient solution comprises: manganese sulphate 0.012g/L, zinc sulphate 6.2 × 10
-3g/L, copper sulphate 5.4 × 10
-5g/L, ferrous sulfate 0.015g/L, magnesium sulfate 0.24g/L, ammonium nitrate 0.77g/L, potassium nitrate 1g/L, calcium chloride 0.2g/L, cobalt chloride 6.6 × 10
-5g/L, sodium molybdate 4.2 × 10
-4g/L, calcium dihydrogen phosphate 0.095g/L, potassium iodide 6.3 × 10
-4g/L, boric acid 7.5 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.03g/L, inositol 0.15g/L, nicotinic acid 3.1 × 10
-4g/L, puridoxine hydrochloride 6.5 × 10
-4g/L, thiamine hydrochloride 1.1 × 10
-4g/L, glycine 1.4 × 10
-3g/L, agar 4g/L, white sugar 43g/L;
S3, transplanting: moved into by protocorm in the blake bottle that group cultivation nutrient fluid is housed and carry out Protocorm Multiplication differentiation cultivation, the Transplanting den-sity in blake bottle is 18 strains/bottle, cultivates and obtains seedling after 25 days; Moved into by seedling in the blake bottle that group cultivation nutrient fluid is housed and carry out seedling proliferation differentiation cultivation, the Transplanting den-sity in blake bottle is 18 strains/bottle, cultivates and obtains the seedling that height of seedling is more than 1cm after 35 days; Moved in the blake bottle that group cultivation nutrient fluid is housed by seedling and carry out strong seedling culture, the Transplanting den-sity in blake bottle is 18 strains/bottle, cultivates after 65 days and obtains the middle seedling that height of seedling is 3-4cm; Middle seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, Transplanting den-sity in blake bottle is 18 strains/bottle, cultivate and obtain the seedlings of Dendrobium officinale that height of seedling is more than 5cm after 70 days, wherein the condition of culture of Proliferation, Differentiation cultivation and strong seedling culture is as follows: cultivation temperature is 23 DEG C, cultivation intensity of illumination is 1800Lux, and cultivation light application time is 12h/d, and average sterilization frequency is 1.5 times/day, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.012g/L, zinc sulphate 6.2 × 10
-3g/L, copper sulphate 5.4 × 10
-5g/L, ferrous sulfate 0.015g/L, magnesium sulfate 0.24g/L, ammonium nitrate 0.77g/L, potassium nitrate 1g/L, calcium chloride 0.2g/L, cobalt chloride 6.6 × 10
-5g/L, sodium molybdate 4.2 × 10
-4g/L, calcium dihydrogen phosphate 0.095g/L, potassium iodide 6.3 × 10
-4g/L, boric acid 7.5 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.03g/L, inositol 0.15g/L, nicotinic acid 3.1 × 10
-4g/L, puridoxine hydrochloride 6.5 × 10
-4g/L, thiamine hydrochloride 1.1 × 10
-4g/L, glycine 1.4 × 10
-3g/L, agar 4g/L, banana puree 67g/L, white sugar 43g/L.
Embodiment 2
The method of a kind of seedlings of Dendrobium officinale tissue cultures that the present invention proposes, comprises the steps:
S1, sterilizing: transfer room and vaccination equipment are carried out ozone sterilization;
S2, sprouting: the capsule choosing qualified dendrobium candidum, to capsule disinfection, 95% medicinal alcohol is infiltrated capsule surface, capsule after disinfecting longitudinally is cut, evenly fall apart in being equipped with in the blake bottle sprouting nutrient solution with tweezers taking-up seed and carry out seed germination cultivation, cultivation temperature is 24 DEG C, cultivation intensity of illumination is 1500Lux, cultivation light application time is 13h/d, average sterilization frequency is 1 times/day, cultivate and within 40 days, form green protocorm afterwards, the component wherein sprouting nutrient solution comprises: manganese sulphate 0.007g/L, zinc sulphate 6.4 × 10
-3g/L, copper sulphate 5.3 × 10
-5g/L, ferrous sulfate 0.022g/L, magnesium sulfate 0.15g/L, ammonium nitrate 0.83g/L, potassium nitrate 0.9g/L, calcium chloride 0.24g/L, cobalt chloride 6.2 × 10
-5g/L, sodium molybdate 4.3 × 10
-4g/L, calcium dihydrogen phosphate 0.085g/L, potassium iodide 6.5 × 10
-4g/L, boric acid 7.3 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.05g/L, inositol 0.12g/L, nicotinic acid 3.4 × 10
-4g/L, puridoxine hydrochloride 6.4 × 10
-4g/L, thiamine hydrochloride 1.4 × 10
-4g/L, glycine 1.2 × 10
-3g/L, agar 4.2g/L, white sugar 38g/L;
S3, transplanting: moved into by protocorm in the blake bottle that group cultivation nutrient fluid is housed and carry out Protocorm Multiplication differentiation cultivation, the Transplanting den-sity in blake bottle is 20 strains/bottle, cultivates and obtains seedling after 20 days; Moved into by seedling in the blake bottle that group cultivation nutrient fluid is housed and carry out seedling proliferation differentiation cultivation, the Transplanting den-sity in blake bottle is 20 strains/bottle, cultivates and obtains the seedling that height of seedling is more than 1cm after 30 days; Moved in the blake bottle that group cultivation nutrient fluid is housed by seedling and carry out strong seedling culture, the Transplanting den-sity in blake bottle is 20 strains/bottle, cultivates after 60 days and obtains the middle seedling that height of seedling is 3-4cm; Middle seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, Transplanting den-sity in blake bottle is 20 strains/bottle, cultivate and obtain the seedlings of Dendrobium officinale that height of seedling is more than 5cm after 65 days, wherein the condition of culture of Proliferation, Differentiation cultivation and strong seedling culture is as follows: cultivation temperature is 24 DEG C, cultivation intensity of illumination is 1500Lux, and cultivation light application time is 13h/d, and average sterilization frequency is 1 times/day, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.007g/L, zinc sulphate 6.4 × 10
-3g/L, copper sulphate 5.3 × 10
-5g/L, ferrous sulfate 0.022g/L, magnesium sulfate 0.15g/L, ammonium nitrate 0.83g/L, potassium nitrate 0.9g/L, calcium chloride 0.24g/L, cobalt chloride 6.2 × 10
-5g/L, sodium molybdate 4.3 × 10
-4g/L, calcium dihydrogen phosphate 0.085g/L, potassium iodide 6.5 × 10
-4g/L, boric acid 7.3 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.05g/L, inositol 0.12g/L, nicotinic acid 3.4 × 10
-4g/L, puridoxine hydrochloride 6.4 × 10
-4g/L, thiamine hydrochloride 1.4 × 10
-4g/L, glycine 1.2 × 10
-3g/L, agar 4.2g/L, banana puree 72g/L, white sugar 38g/L.
Embodiment 3
The method of a kind of seedlings of Dendrobium officinale tissue cultures that the present invention proposes, comprises the steps:
S1, sterilizing: transfer room is carried out ultraviolet sterilization, carry out ozone sterilization to vaccination equipment;
S2, sprouting: the capsule choosing qualified dendrobium candidum, to capsule disinfection, 95% medicinal alcohol is infiltrated capsule surface, capsule after disinfecting longitudinally is cut, evenly fall apart in being equipped with in the blake bottle sprouting nutrient solution with tweezers taking-up seed and carry out seed germination cultivation, cultivation temperature is 23 DEG C, cultivation intensity of illumination is 1600Lux, cultivation light application time is 12h/d, average sterilization frequency is 1.4 times/day, cultivate and within 38 days, form green protocorm afterwards, the component wherein sprouting nutrient solution comprises: manganese sulphate 0.01g/L, zinc sulphate 6.30 × 10
-3g/L, copper sulphate 5.36 × 10
-5g/L, ferrous sulfate 0.02g/L, magnesium sulfate 0.19g/L, ammonium nitrate 0.81g/L, potassium nitrate 0.95g/L, calcium chloride 0.22g/L, cobalt chloride 6.43 × 10
-5g/L, sodium molybdate 4.29 × 10
-4g/L, calcium dihydrogen phosphate 0.09g/L, potassium iodide 6.39 × 10
-4g/L, boric acid 7.46 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.04g/L, inositol 0.13g/L, nicotinic acid 3.21 × 10
-4g/L, puridoxine hydrochloride 6.43 × 10
-4g/L, thiamine hydrochloride 1.29 × 10
-4g/L, glycine 1.29 × 10
-3g/L, agar 4.09g/L, white sugar 40g/L;
S3, transplanting: moved into by protocorm in the blake bottle that group cultivation nutrient fluid is housed and carry out Protocorm Multiplication differentiation cultivation, the Transplanting den-sity in blake bottle is 19 strains/bottle, cultivates and obtains seedling after 23 days; Moved into by seedling in the blake bottle that group cultivation nutrient fluid is housed and carry out seedling proliferation differentiation cultivation, the Transplanting den-sity in blake bottle is 19 strains/bottle, cultivates and obtains the seedling that height of seedling is more than 1cm after 32 days; Moved in the blake bottle that group cultivation nutrient fluid is housed by seedling and carry out strong seedling culture, the Transplanting den-sity in blake bottle is 19 strains/bottle, cultivates after 64 days and obtains the middle seedling that height of seedling is 3-4cm; Middle seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, Transplanting den-sity in blake bottle is 19 strains/bottle, cultivate and obtain the seedlings of Dendrobium officinale that height of seedling is more than 5cm after 68 days, wherein the condition of culture of Proliferation, Differentiation cultivation and strong seedling culture is as follows: cultivation temperature is 24 DEG C, cultivation intensity of illumination is 1700Lux, and cultivation light application time is 13h/d, and average sterilization frequency is 1.2 times/day, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.01g/L, zinc sulphate 6.30 × 10
-3g/L, copper sulphate 5.36 × 10
-5g/L, ferrous sulfate 0.02g/L, magnesium sulfate 0.19g/L, ammonium nitrate 0.81g/L, potassium nitrate 0.95g/L, calcium chloride 0.22g/L, cobalt chloride 6.43 × 10
-5g/L, sodium molybdate 4.29 × 10
-4g/L, calcium dihydrogen phosphate 0.09g/L, potassium iodide 6.39 × 10
-4g/L, boric acid 7.46 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.04g/L, inositol 0.13g/L, nicotinic acid 3.21 × 10
-4g/L, puridoxine hydrochloride 6.43 × 10
-4g/L, thiamine hydrochloride 1.29 × 10
-4g/L, glycine 1.29 × 10
-3g/L, agar 4.09g/L, banana puree 70g/L, white sugar 40g/L.
The above; be only the present invention's preferably embodiment; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to according to technical scheme of the present invention and inventive concept thereof and replace or change, all should be encompassed within protection scope of the present invention.
Claims (10)
1. a method for seedlings of Dendrobium officinale tissue cultures, is characterized in that, comprises the steps:
S1, sterilizing: transfer room and vaccination equipment are carried out sterilizing;
S2, sprouting: the capsule choosing qualified dendrobium candidum, to capsule disinfection, capsule after disinfecting longitudinally is cut, evenly falls apart in being equipped with in the blake bottle sprouting nutrient solution with tweezers taking-up seed and carry out seed germination cultivation, cultivate and within 35-40 days, form green protocorm afterwards;
S3, transplanting: protocorm is moved in the blake bottle that group cultivation nutrient fluid is housed and carry out Protocorm Multiplication differentiation cultivation, cultivate and obtain seedling after 20-25 days; Seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carry out seedling proliferation differentiation cultivation, cultivate and obtain the seedling that height of seedling is more than 1cm after 30-35 days; Seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, cultivate after 60-65 days and obtain the middle seedling that height of seedling is 3-4cm; Middle seedling is moved in the blake bottle that group cultivation nutrient fluid is housed and carries out strong seedling culture, cultivate and obtain the seedlings of Dendrobium officinale that height of seedling is more than 5cm after 65-70 days.
2. the method for seedlings of Dendrobium officinale tissue cultures according to claim 1, it is characterized in that, in S1, described sterilizing is ultraviolet sterilization and/or ozone sterilization.
3. the method for seedlings of Dendrobium officinale tissue cultures according to claim 1 or 2, is characterized in that, in S2, described in disinfect as adopting 95% medicinal alcohol to infiltrate capsule surface.
4. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-3, is characterized in that, in S2, the component sprouting nutrient solution comprises: manganese sulphate 0.007-0.012g/L, zinc sulphate 6.2-6.4 × 10
-3g/L, copper sulphate 5.3-5.4 × 10
-5g/L, ferrous sulfate 0.015-0.022g/L, magnesium sulfate 0.15-0.24g/L, ammonium nitrate 0.77-0.83g/L, potassium nitrate 0.9-1g/L, calcium chloride 0.2-0.24g/L, cobalt chloride 6.2-6.6 × 10
-5g/L, sodium molybdate 4.2-4.3 × 10
-4g/L, calcium dihydrogen phosphate 0.085-0.095g/L, potassium iodide 6.3-6.5 × 10
-4g/L, boric acid 7.3-7.5 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.03-0.05g/L, inositol 0.12-0.15g/L, nicotinic acid 3.1-3.4 × 10
-4g/L, puridoxine hydrochloride 6.4-6.5 × 10
-4g/L, thiamine hydrochloride 1.1-1.4 × 10
-4g/L, glycine 1.2-1.4 × 10
-3g/L, agar 4-4.2g/L, white sugar 38-43g/L.
5. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-4, is characterized in that, in S2, the component sprouting nutrient solution comprises: manganese sulphate 0.01g/L, zinc sulphate 6.30 × 10
-3g/L, copper sulphate 5.36 × 10
-5g/L, ferrous sulfate 0.02g/L, magnesium sulfate 0.19g/L, ammonium nitrate 0.81g/L, potassium nitrate 0.95g/L, calcium chloride 0.22g/L, cobalt chloride 6.43 × 10
-5g/L, sodium molybdate 4.29 × 10
-4g/L, calcium dihydrogen phosphate 0.09g/L, potassium iodide 6.39 × 10
-4g/L, boric acid 7.46 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.04g/L, inositol 0.13g/L, nicotinic acid 3.21 × 10
-4g/L, puridoxine hydrochloride 6.43 × 10
-4g/L, thiamine hydrochloride 1.29 × 10
-4g/L, glycine 1.29 × 10
-3g/L, agar 4.09g/L, white sugar 40g/L.
6. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-5, it is characterized in that, in S2, the condition of culture that seed germination is cultivated is as follows: cultivation temperature is 23-24 DEG C, cultivation intensity of illumination is 1500-1800Lux, cultivation light application time is 12-13h/d, and average sterilization frequency is 1-1.5 times/day.
7. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-6, is characterized in that, in S3, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.007-0.012g/L, zinc sulphate 6.2-6.4 × 10
-3g/L, copper sulphate 5.3-5.4 × 10
-5g/L, ferrous sulfate 0.015-0.022g/L, magnesium sulfate 0.15-0.24g/L, ammonium nitrate 0.77-0.83g/L, potassium nitrate 0.9-1g/L, calcium chloride 0.2-0.24g/L, cobalt chloride 6.2-6.6 × 10
-5g/L, sodium molybdate 4.2-4.3 × 10
-4g/L, calcium dihydrogen phosphate 0.085-0.095g/L, potassium iodide 6.3-6.5 × 10
-4g/L, boric acid 7.3-7.5 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.03-0.05g/L, inositol 0.12-0.15g/L, nicotinic acid 3.1-3.4 × 10
-4g/L, puridoxine hydrochloride 6.4-6.5 × 10
-4g/L, thiamine hydrochloride 1.1-1.4 × 10
-4g/L, glycine 1.2-1.4 × 10
-3g/L, agar 4-4.2g/L, banana puree 67-72g/L, white sugar 38-43g/L.
8. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-7, is characterized in that, in S3, the component of group cultivation nutrient fluid comprises: manganese sulphate 0.01g/L, zinc sulphate 6.30 × 10
-3g/L, copper sulphate 5.36 × 10
-5g/L, ferrous sulfate 0.02g/L, magnesium sulfate 0.19g/L, ammonium nitrate 0.81g/L, potassium nitrate 0.95g/L, calcium chloride 0.22g/L, cobalt chloride 6.43 × 10
-5g/L, sodium molybdate 4.29 × 10
-4g/L, calcium dihydrogen phosphate 0.09g/L, potassium iodide 6.39 × 10
-4g/L, boric acid 7.46 × 10
-3g/L, disodium ethylene diamine tetraacetate 0.04g/L, inositol 0.13g/L, nicotinic acid 3.21 × 10
-4g/L, puridoxine hydrochloride 6.43 × 10
-4g/L, thiamine hydrochloride 1.29 × 10
-4g/L, glycine 1.29 × 10
-3g/L, agar 4.09g/L, banana puree 70g/L, white sugar 40g/L.
9. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-8, it is characterized in that, in S3, the Transplanting den-sity in blake bottle is 18-20 strain/bottle.
10. the method for seedlings of Dendrobium officinale tissue cultures according to any one of claim 1-9, it is characterized in that, in S3, the condition of culture of Proliferation, Differentiation cultivation and strong seedling culture is as follows: cultivation temperature is 23-24 DEG C, cultivation intensity of illumination is 1500-1800Lux, cultivation light application time is 12-13h/d, and average sterilization frequency is 1-1.5 times/day.
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| CN106747817A (en) * | 2016-12-29 | 2017-05-31 | 广西壮族自治区亚热带作物研究所 | Sisal hemp floating seedlings nutrient solution and sisal hemp float seedling method |
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| CN109548625A (en) * | 2018-12-28 | 2019-04-02 | 苏州天成新农生物科技有限公司 | Fluid nutrient medium and preparation method thereof suitable for dendrobium candidum tender stem |
| CN109548626A (en) * | 2018-12-28 | 2019-04-02 | 苏州天成新农生物科技有限公司 | A kind of humic acid medium and preparation method thereof improving the dendrobium candidum tender stem speed of growth |
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