CN104969863A - Dendrobium officinale tissue culture propagation method - Google Patents
Dendrobium officinale tissue culture propagation method Download PDFInfo
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Abstract
The invention discloses a dendrobium officinale tissue culture propagation method including the steps: a step one, preparation of a culture medium; a step 2, explant treatment and inoculation adopting a naturally growing plant as an explant and comprising three steps of cleaning, disinfection and inoculation; a step three, culture comprising transferring an inoculated culture flask in a culture room, carrying out seedling induction and culture, wherein the culture conditions comprise that the temperature is 25+/-2 DEG C, a visible spectrum with the wavelength of 520-680 is used as a light source, illumination is carried out in a circulation and alternation mode (that is to say, after illumination is carried out for 6 hours, illumination is stopped for 6 hours, then illumination is carried out for 6 hours again, and illumination is carried out circularly and alternately), the illumination intensity is 1200-3000 lumens, the culture time is about 2-3 months, frequent checking is needed during the culture, and pollution is removed in time when found; and a step four, transplanting. With adopting of the 3-month tissue culture time, tissue-cultured seedlings with developed root systems and healthy and strong branches and leaves can be formed, and have the each seedling average weight of more than 5 grams heavier than that of traditional tissue-cultured seedlings.
Description
Technical field
The present invention relates to the technical field of growing dendrobium, particularly relate to a kind of tissue culture propagation method of dendrobium candidum.
Background technology
At present, along with the fast development of biotechnology, some rare mushrooms and rare plant move towards industrialization artificial domestication gradually and scale is planted, such as rare plant roxburgh anoectochilus terminal bud, the half light plant stem of noble dendrobium etc., dendrobium candidum is perennial Valuable Herbal Medicine, under wild environment reproduction rate and survival rate low, the dendrobium candidum that existing market is sold is mostly the ripe rear airing of artificial propagation cultivation, and dendrobium candidum requires it is also very harsh to the artificial growing environment cultivated, existing dendrobium candidum culture technique is placed on the culturing rack of culturing room by the culture vessel of dendrobium candidum, grow into after seedling until it, be transplanted to cultivation in booth again.
Chinese patent CN103053425B discloses a kind of rapid propagation method for tissue cultivation of dendrobium candidum stem, comprises the following steps: explant preparation, axillalry bud and protocorm induction, Multiplying culture and Rooting and hardening-off culture.Chinese patent CN103843662A discloses a kind of method promoting dendrobium plantlet in vitro strong sprout and take root, and comprises the steps: 1) dendrobium plantlet in vitro is proceeded to strong sprout and illumination cultivation in defined medium of taking root, strong sprout root induction; 2) plantlet in vitro of having taken root can be transplanted after hardening; 3) in the cultivation matrix dendrobium plantlet in vitro Transplantation of Regenerated Plantlets of having taken root mixed with vermiculite in bark.
In view of this, the present inventor studies and devises a kind of tissue culture propagation method of dendrobium candidum, and this case produces thus.
Summary of the invention
The object of the present invention is to provide a kind of tissue culture propagation method of dendrobium candidum, to obtain the dendrobium candidum that survival rate is high, growth is fast, output is high, nutrient component is high.
For achieving the above object, the present invention solves the technical scheme of its technical problem and is:
A tissue culture propagation method for dendrobium candidum, comprises the steps:
Step one, medium prepare:
Take MS as minimal medium, often liter is added 6-benzyl aminopurine 0.1-0.5mg, methyl α-naphthyl acetate 0.2-0.5mg, banana 100g, potato 25g and agar 6.5g, and the pH value of mixed medium is adjusted to 5.6; Medium after preparation is heated to agar dissolve completely, is sub-packed in clean blake bottle while hot equably, adds a cover after slightly cool; Pressure cooker sterilizing, sterilising conditions is: 121 DEG C, sterilizing 15 minutes under 105kPa, takes out blake bottle in time, cool for subsequent use after sterilizing;
Step 2, explant process and inoculation:
Adopt the plant of self-sow as explant, comprise cleaning, sterilization, inoculates three steps, wherein:
Cleaning: select self-sow, healthy and strong plant without damage by disease and insect, cleans plant, rear running water 2-3 hour with the water adding a little soap powder;
Sterilization: aseptically, removes blade, and after plants stems section being infiltrated in the alcohol of 75% 8-10 second, the mercuric chloride solution putting into 10% is sterilized 10-15 minute, with sterile water wash 3-6 time, drains away the water, puts into aseptic culture dish for subsequent use;
Inoculation: stem for subsequent use after sterilization is aseptically cut into long 8-12cm, some stem sections containing a stipes, is connected on described medium, every bottle graft 1 stem section;
Step 3, cultivation:
This step moves into connecting the blake bottle of planting induction and the cultivation that culturing room carries out seedling, condition of culture is: temperature 25 ± 2 DEG C, adopts wavelength at the visible spectrum of 520-680 as light source, and adopts the mode of cycle alternation to irradiate, namely irradiate after 6 hours, stop illumination 6 hours, then irradiate 6 hours, cycle alternation like this irradiates, intensity of illumination 1200-3000 lumen, about incubation time 2-3 month, want diligent inspection between culture period, what discovery was polluted will remove in time;
Step 4, transplanting:
Height of seedling 6-8cm, more than 3, tool leaf, stem foot diameter is selected to be about the test-tube plantlet of 2mm, root of hair 2-3 bar, hardening is after 4 days, cleans the medium be attached on plant, and transplants after drying washings and cultivate to greenhouse gardening matrix, plant by the standard of spacing in the rows 3-7cm, line-spacing 3-7cm, fill compaction.
As the preferred embodiment of embodiment, described planting matrix comprises inorganic matrix layer and organic substrate layer, and adopt peat soil as inorganic matrix layer, be exposed to the sun under burning sun 2-6 days; After being exposed to the sun, described peat soil is contained in standard planting tray, and thickness is 3-5cm; Above described peat soil, lay organic hypothallus again, thickness is 2-3cm; The preparation method of described machine hypothallus is: the raw material taking following parts by weight: cow dung 200-300, fallen leaves 20-30, stalk 20-35, sawdust 15-20 and bark 25-30; Mixed by above-mentioned raw materials, add appropriate water, inoculation yeast bacterium, heap fermentation 20-25 days, sterilization is obtained.
As the preferred embodiment of embodiment, the condition of greenhouse cultivation is:
1) control of temperature: described dendrobium candidum can normal growth in 15-30 DEG C, when temperature is more than more than 28 DEG C, is lowered the temperature by sprayer unit; Put down plastic film winter, be incubated to more than 6 DEG C;
2) control of humidity: described Growth of Dendrobium candidum air humidity is 55-80%;
3) control of luminosity: described dendrobium candidum luminosity is 1200-1800 lumen.
After the present invention adopts technique scheme, the present invention has following beneficial effect:
1. the present inventor is in once testing by accident, be surprised to find that and adopt wavelength at the visible spectrum of 520-680 as light source, and adopt the mode of cycle alternation to irradiate, greatly can improve growth rate and the output of dendrobium candidum, this may be relevant with exciting its somatotropin, the time of tradition candidum tissue culturing generally will more than 4 months, and the present invention 3 months, and the dendrobium candidum fresh weight of of the present invention group of training is all obviously greater than traditional plantlet in vitro, every seedling on average overweights traditional plantlet in vitro more than 5 grams;
2. add banana in the medium highly beneficial for promotion growth of seedling, the blade amount of having a net increase of, root of hair number and the fresh weight amount of having a net increase of are high;
3., through group training test, the rate of increase can reach 6.8 times.
Embodiment
embodiment 1
A tissue culture propagation method for dendrobium candidum, comprises the steps:
Step one, medium prepare:
Take MS as minimal medium, often liter is added 6-benzyl aminopurine 0.1mg, methyl α-naphthyl acetate 0.2mg, banana 100g, potato 25g and agar 6.5g, and the pH value of mixed medium is adjusted to 5.6; Medium after preparation is heated to agar dissolve completely, is sub-packed in clean blake bottle while hot equably, adds a cover after slightly cool; Pressure cooker sterilizing, sterilising conditions is: 121 DEG C, sterilizing 15 minutes under 105kPa, takes out blake bottle in time, cool for subsequent use after sterilizing;
Step 2, explant process and inoculation:
Adopt the plant of self-sow as explant, comprise cleaning, sterilization, inoculates three steps, wherein:
Cleaning: select self-sow, healthy and strong plant without damage by disease and insect, cleans plant, rear running water 2 hours with the water adding a little soap powder;
Sterilization: aseptically, removes blade, and after plants stems section is infiltrated 8 seconds in the alcohol of 75%, the mercuric chloride solution putting into 10% is sterilized 10 minutes, with sterile water wash 3-6 time, drains away the water, puts into aseptic culture dish for subsequent use;
Inoculation: stem for subsequent use after sterilization is aseptically cut into long 8cm, some stem sections containing a stipes, is connected on described medium, every bottle graft 1 stem section;
Step 3, cultivation:
This step moves into connecting the blake bottle of planting induction and the cultivation that culturing room carries out seedling, condition of culture is: temperature 25 ± 2 DEG C, adopt wavelength 520 visible spectrum as light source, and adopt the mode of cycle alternation to irradiate, namely irradiate after 6 hours, stop illumination 6 hours, then irradiate 6 hours, cycle alternation like this irradiates, intensity of illumination 1200 lumen, incubation time about 3 months, wants diligent inspection, finds will removing in time of pollution between culture period;
Step 4, transplanting:
Height of seedling 6-8cm, more than 3, tool leaf, stem foot diameter is selected to be about the test-tube plantlet of 2mm, root of hair 2-3 bar, hardening is after 4 days, cleans the medium be attached on plant, and transplants after drying washings and cultivate to greenhouse gardening matrix, plant by the standard of spacing in the rows 3-7cm, line-spacing 3-7cm, fill compaction.
As the preferred embodiment of embodiment, described planting matrix comprises inorganic matrix layer and organic substrate layer, and adopt peat soil as inorganic matrix layer, be exposed to the sun under burning sun 2-6 days; After being exposed to the sun, described peat soil is contained in standard planting tray, and thickness is 3-5cm; Above described peat soil, lay organic hypothallus again, thickness is 2-3cm; The preparation method of described machine hypothallus is: the raw material taking following parts by weight: cow dung 200-300, fallen leaves 20-30, stalk 20-35, sawdust 15-20 and bark 25-30; Mixed by above-mentioned raw materials, add appropriate water, inoculation yeast bacterium, heap fermentation 20-25 days, sterilization is obtained.
embodiment 2
A tissue culture propagation method for dendrobium candidum, comprises the steps:
Step one, medium prepare:
Take MS as minimal medium, often liter is added 6-benzyl aminopurine 0.5mg, methyl α-naphthyl acetate 0.5mg, banana 100g, potato 25g and agar 6.5g, and the pH value of mixed medium is adjusted to 5.6; Medium after preparation is heated to agar dissolve completely, is sub-packed in clean blake bottle while hot equably, adds a cover after slightly cool; Pressure cooker sterilizing, sterilising conditions is: 121 DEG C, sterilizing 15 minutes under 105kPa, takes out blake bottle in time, cool for subsequent use after sterilizing;
Step 2, explant process and inoculation:
Adopt the plant of self-sow as explant, comprise cleaning, sterilization, inoculates three steps, wherein:
Cleaning: select self-sow, healthy and strong plant without damage by disease and insect, cleans plant, rear running water 3 hours with the water adding a little soap powder;
Sterilization: aseptically, removes blade, and after plants stems section is infiltrated 10 seconds in the alcohol of 75%, the mercuric chloride solution putting into 10% is sterilized 15 minutes, with sterile water wash 3-6 time, drains away the water, puts into aseptic culture dish for subsequent use;
Inoculation: stem for subsequent use after sterilization is aseptically cut into long 12cm, some stem sections containing a stipes, is connected on described medium, every bottle graft 1 stem section;
Step 3, cultivation:
This step moves into connecting the blake bottle of planting induction and the cultivation that culturing room carries out seedling, condition of culture is: temperature 25 ± 2 DEG C, adopt wavelength 680 visible spectrum as light source, and adopt the mode of cycle alternation to irradiate, namely irradiate after 6 hours, stop illumination 6 hours, then irradiate 6 hours, cycle alternation like this irradiates, intensity of illumination 3000 lumen, incubation time about 2 months, wants diligent inspection, finds will removing in time of pollution between culture period;
Step 4, transplanting:
Height of seedling 6-8cm, more than 3, tool leaf, stem foot diameter is selected to be about the test-tube plantlet of 2mm, root of hair 2-3 bar, hardening is after 4 days, cleans the medium be attached on plant, and transplants after drying washings and cultivate to greenhouse gardening matrix, plant by the standard of spacing in the rows 3-7cm, line-spacing 3-7cm, fill compaction.
As the preferred embodiment of embodiment, described planting matrix comprises inorganic matrix layer and organic substrate layer, and adopt peat soil as inorganic matrix layer, be exposed to the sun under burning sun 2-6 days; After being exposed to the sun, described peat soil is contained in standard planting tray, and thickness is 3-5cm; Above described peat soil, lay organic hypothallus again, thickness is 2-3cm; The preparation method of described machine hypothallus is: the raw material taking following parts by weight: cow dung 200-300, fallen leaves 20-30, stalk 20-35, sawdust 15-20 and bark 25-30; Mixed by above-mentioned raw materials, add appropriate water, inoculation yeast bacterium, heap fermentation 20-25 days, sterilization is obtained.
embodiment 3
A tissue culture propagation method for dendrobium candidum, comprises the steps:
Step one, medium prepare:
Take MS as minimal medium, often liter is added 6-benzyl aminopurine 0.3mg, methyl α-naphthyl acetate 0.3mg, banana 100g, potato 25g and agar 6.5g, and the pH value of mixed medium is adjusted to 5.6; Medium after preparation is heated to agar dissolve completely, is sub-packed in clean blake bottle while hot equably, adds a cover after slightly cool; Pressure cooker sterilizing, sterilising conditions is: 121 DEG C, sterilizing 15 minutes under 105kPa, takes out blake bottle in time, cool for subsequent use after sterilizing;
Step 2, explant process and inoculation:
Adopt the plant of self-sow as explant, comprise cleaning, sterilization, inoculates three steps, wherein:
Cleaning: select self-sow, healthy and strong plant without damage by disease and insect, cleans plant, rear running water 2-3 hour with the water adding a little soap powder;
Sterilization: aseptically, removes blade, and after plants stems section is infiltrated 10 seconds in the alcohol of 75%, the mercuric chloride solution putting into 10% is sterilized 15 minutes, with sterile water wash 3-6 time, drains away the water, puts into aseptic culture dish for subsequent use;
Inoculation: stem for subsequent use after sterilization is aseptically cut into long 8cm, some stem sections containing a stipes, is connected on described medium, every bottle graft 1 stem section;
Step 3, cultivation:
This step moves into connecting the blake bottle of planting induction and the cultivation that culturing room carries out seedling, condition of culture is: temperature 25 ± 2 DEG C, adopt wavelength 580 visible spectrum as light source, and adopt the mode of cycle alternation to irradiate, namely irradiate after 6 hours, stop illumination 6 hours, then irradiate 6 hours, cycle alternation like this irradiates, intensity of illumination 2000 lumen, incubation time about 2.5 months, wants diligent inspection, finds will removing in time of pollution between culture period;
Step 4, transplanting:
Height of seedling 6-8cm, more than 3, tool leaf, stem foot diameter is selected to be about the test-tube plantlet of 2mm, root of hair 2-3 bar, hardening is after 4 days, cleans the medium be attached on plant, and transplants after drying washings and cultivate to greenhouse gardening matrix, plant by the standard of spacing in the rows 3-7cm, line-spacing 3-7cm, fill compaction.
As the preferred embodiment of embodiment, described planting matrix comprises inorganic matrix layer and organic substrate layer, and adopt peat soil as inorganic matrix layer, be exposed to the sun under burning sun 2-6 days; After being exposed to the sun, described peat soil is contained in standard planting tray, and thickness is 3-5cm; Above described peat soil, lay organic hypothallus again, thickness is 2-3cm; The preparation method of described machine hypothallus is: the raw material taking following parts by weight: cow dung 200-300, fallen leaves 20-30, stalk 20-35, sawdust 15-20 and bark 25-30; Mixed by above-mentioned raw materials, add appropriate water, inoculation yeast bacterium, heap fermentation 20-25 days, sterilization is obtained.
As the preferred embodiment of embodiment 1-3, the condition of greenhouse cultivation is:
1) control of temperature: described dendrobium candidum can normal growth in 15-30 DEG C, when temperature is more than more than 28 DEG C, is lowered the temperature by sprayer unit; Put down plastic film winter, be incubated to more than 6 DEG C;
2) control of humidity: described Growth of Dendrobium candidum air humidity is 55-80%;
3) control of luminosity: described dendrobium candidum luminosity is 1200-1800 lumen.
All distortion that those of ordinary skill in the art can directly derive from the disclosure of invention or associate, all should think protection scope of the present invention.
Claims (3)
1. a tissue culture propagation method for dendrobium candidum, is characterized in that: comprise the steps:
Step one, medium prepare:
Take MS as minimal medium, often liter is added 6-benzyl aminopurine 0.1-0.5mg, methyl α-naphthyl acetate 0.2-0.5mg, banana 100g, potato 25g and agar 6.5g, and the pH value of mixed medium is adjusted to 5.6; Medium after preparation is heated to agar dissolve completely, is sub-packed in clean blake bottle while hot equably, adds a cover after slightly cool; Pressure cooker sterilizing, sterilising conditions is: 121 DEG C, sterilizing 15 minutes under 105kPa, takes out blake bottle in time, cool for subsequent use after sterilizing;
Step 2, explant process and inoculation:
Adopt the plant of self-sow as explant, comprise cleaning, sterilization, inoculates three steps, wherein:
Cleaning: select self-sow, healthy and strong plant without damage by disease and insect, cleans plant, rear running water 2-3 hour with the water adding a little soap powder;
Sterilization: aseptically, removes blade, and after plants stems section being infiltrated in the alcohol of 75% 8-10 second, the mercuric chloride solution putting into 10% is sterilized 10-15 minute, with sterile water wash 3-6 time, drains away the water, puts into aseptic culture dish for subsequent use;
Inoculation: stem for subsequent use after sterilization is aseptically cut into long 8-12cm, some stem sections containing a stipes, is connected on described medium, every bottle graft 1 stem section;
Step 3, cultivation:
This step moves into connecting the blake bottle of planting induction and the cultivation that culturing room carries out seedling, condition of culture is: temperature 25 ± 2 DEG C, adopts wavelength at the visible spectrum of 520-680 as light source, and adopts the mode of cycle alternation to irradiate, namely irradiate after 6 hours, stop illumination 6 hours, then irradiate 6 hours, cycle alternation like this irradiates, intensity of illumination 1200-3000 lumen, about incubation time 2-3 month, want diligent inspection between culture period, what discovery was polluted will remove in time;
Step 4, transplanting:
Height of seedling 6-8cm, more than 3, tool leaf, stem foot diameter is selected to be about the test-tube plantlet of 2mm, root of hair 2-3 bar, hardening is after 4 days, cleans the medium be attached on plant, and transplants after drying washings and cultivate to greenhouse gardening matrix, plant by the standard of spacing in the rows 3-7cm, line-spacing 3-7cm, fill compaction.
2. the tissue culture propagation method of a kind of dendrobium candidum as claimed in claim 1, is characterized in that: described planting matrix comprises inorganic matrix layer and organic substrate layer, and adopt peat soil as inorganic matrix layer, be exposed to the sun under burning sun 2-6 days; After being exposed to the sun, described peat soil is contained in standard planting tray, and thickness is 3-5cm; Above described peat soil, lay organic hypothallus again, thickness is 2-3cm; The preparation method of described machine hypothallus is: the raw material taking following parts by weight: cow dung 200-300, fallen leaves 20-30, stalk 20-35, sawdust 15-20 and bark 25-30; Mixed by above-mentioned raw materials, add appropriate water, inoculation yeast bacterium, heap fermentation 20-25 days, sterilization is obtained.
3. the tissue culture propagation method of a kind of dendrobium candidum as claimed in claim 1, is characterized in that: the condition of greenhouse cultivation is:
1) control of temperature: described dendrobium candidum can normal growth in 15-30 DEG C, when temperature is more than more than 28 DEG C, is lowered the temperature by sprayer unit; Put down plastic film winter, be incubated to more than 6 DEG C;
2) control of humidity: described Growth of Dendrobium candidum air humidity is 55-80%;
3) control of luminosity: described dendrobium candidum luminosity is 1200-1800 lumen.
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| CN107637519A (en) * | 2017-09-27 | 2018-01-30 | 霍山宝信园石斛开发有限公司 | A kind of method for growing dendrobium seedlings |
| CN107646656A (en) * | 2017-11-13 | 2018-02-02 | 广西博白县顿谷镇旧塘家庭农场 | A kind of implantation methods of dendrobium candidum |
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Application publication date: 20151014 |