CN102499085B - Method for raising directly transplantable seedlings of dendrobium candidum - Google Patents
Method for raising directly transplantable seedlings of dendrobium candidum Download PDFInfo
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- CN102499085B CN102499085B CN 201110332813 CN201110332813A CN102499085B CN 102499085 B CN102499085 B CN 102499085B CN 201110332813 CN201110332813 CN 201110332813 CN 201110332813 A CN201110332813 A CN 201110332813A CN 102499085 B CN102499085 B CN 102499085B
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Abstract
The invention relates to a tissue culture method, in particular to a method for tissue culture of dendrobium candidum, which belongs to the technical field of biology and solves the problem that method for raising directly transplantable seedlings of dendrobium candidum needs to be provided. In order to solve the problem, an existing culture medium formula is modified, a special tissue culture cup with good light permeability, low infection rate and sufficient air is creatively provided, and a clean and appropriate natural light environment is provided. By means of the formula, the cup and the environment, capsule seeds of a plant of dendrobium candidum can be subjected to tissue culture for 7-8 months to breed 200,000 tissue culture seedlings of dendrobium candidum, the tissue culture seedlings can be directly transplanted outdoor with no need of traditional domestication (seedling exercising) for 3-5 months, and transplant survival rate reaches more than 98%.
Description
Technical field
The present invention relates to a kind of tissue culture method, especially at the tissue culture method of dendrobium candidum, belong to biological technical field.
Background technology
Having in the Dendrobium plant much is rare Chinese medicine, and the stem of noble dendrobium is listed in top grade in Compendium of Material Medica.There are nearly 40 kinds to do medicinal in 76 kinds of Dendrobium plants of China, famous have HERBA DENDROBII, dendrobium candidum, a Dendrobidium huoshanness etc., have reinforcing yin essence benefit essence, the beneficial power of kidney tonifying, the effect such as prolong life of committing suicide, its medicinal and health care is excavated day by day, its range of application enlarges just rapidly.Simultaneously, the Dendrobium plant has 1/4th can supply to view and admire approximately, and is gorgeous and celebrated to spend, and as the ornamental plants development rapidly, formed independent industry in recent years.In view of integrating medicinal and value that view and admire, make it excessive felling utilization, its body natural propagation rate is low in addition, has caused the pressure between the stem of noble dendrobium supply and demand, and China has classified it as one of shielded herbal species that is on the brink of to die out.
Since the vegetative propagation technique that nineteen sixty Frenchman Gmorel utilizes stem of noble dendrobium base point tissue to cultivate virus-free plant is founded, stem of noble dendrobium tissue culture technique is progressively deepened, especially in recent years stem of noble dendrobium tissue cultivating and seedling progressively tends to industrialization, the optimum condition that stem of noble dendrobium tissue is cultivated, comprise suitable illumination, temperature, humidity, pH and how to select explant for use how to dispose nutrition suitable culture liquid etc. and all obtain considerable progress.But by traditional stem of noble dendrobium tissue culture method, particularly the seed that cultivates of candidum tissue culturing method is because sturdy not enough, vitality a little less than, therefore all need in booth, carry out 3-5 month instructionization (hardening) before in being transplanted to natural environment, and the growth cycle after its transplanting is longer, causes seedling cost higher.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of group of training seedling and need not instructionization, and the candidum tissue culturing method that can directly transplant, reproductive speed, the quality of production, the transplanting survival rate of candidum tissue culturing seedling have been improved, and shortened the growth cycle of transplanting the back dendrobium candidum, promoted the quality of the adult stem of noble dendrobium.
For addressing the above problem, dendrobium candidum seedling-cultivating method of the present invention comprises the following steps:
1) with the capsule seed of dendrobium candidum after sterilization, broadcast in special culture media I+dedicated set training cup+special-purpose culturing room, sprout cultivation, cultivation temperature is 24-26 ℃, humidity is 65-75%, and illuminance is 1000-2000lx, and light application time is 10-12 hour/day, cultivated 45-60 days, the explant rudiment forms green protocorm;
2) protocorm is changed in special culture media II+dedicated set training cup+special-purpose culturing room, carry out protocorm propagation, differentiation cultivation, cultivation temperature is 22-28 ℃, and humidity is 65-75%, illuminance is 1500-2500lx, light application time is 10-12 hour/day, cultivates 60 days, cultivates into the 1-2CM seedling by protocorm, simultaneous has the new protocorm of part to produce, seedling is changed under same group training cup, medium and the environment again and continue to cultivate 1 month, seedling is cultivated into middle seedling, height of seedling is 3-4cm;
3) middle seedling is changed in special culture media III+dedicated set training cup+special-purpose culturing room, cultivation temperature is 20-30 ℃, and humidity is 65-75%, illuminance is 2000-4000lx, and light application time is 10-12 hour/day, cultivates 60 days, cultivating into height by seedling is 5-9CM, and stem slightly is the strong sprout of 0.4-0.5CM;
Described special culture media I comprises 1/2MS minimal medium+taro juice 15-25% (V/V)+agar 4-6g/L, and pH value is 5.2-5.8;
Described special culture media II comprises MS minimal medium+BA (6-Bian aminoadenine) 0.1-0.2mg/L+ agar 4-6g/L+ murphy juice 6-10% (V/V)+bananas juice 10-14% (V/V), and pH value is 5.4-5.8;
Described special culture media III comprises MS minimal medium+NAA (α-Nai Yisuan) 0.1-0.2mg/L+ bananas juice 8-10% (V/V)+murphy juice 5-7% (V/V)+taro juice 8-12% (V/V)+agar 4-6g/L, and pH value is 5.4-5.8;
The transparent plastics cup of described dedicated set training cup for having appropriate spatial volume, described plastics cup rim of a cup arranges filter membrane by transparent plastic foil plastic packaging on the described plastic foil;
Described special-purpose culturing room is sterilization culturing room, and described sterilizing chamber adopts natural lighting, and its top arranges daylighting watt and sunshade net, and is provided with the temperature humidity conditioning equipment.
Further prioritization scheme of the present invention is characterized in that described plastics cup is cylindrical, and the rim of a cup diameter is 9-10cm, and cup end diameter is 5-6cm, and cup is high to be 9-10cm, wall thickness 0.15-0.25mm.
The described group of candidum tissue culturing method that the training seedling can directly be transplanted is characterized in that described special culture media I comprises 1/2MS minimal medium+taro juice 20% (V/V)+agar 5g/L, and pH value is 5.5.
The described group of candidum tissue culturing method that the training seedling can directly be transplanted, it is characterized in that described special culture media II comprises MS minimal medium+BA (6-Bian aminoadenine) 0.15mg/L+ agar 5g/L+ murphy juice 8% (V/V)+bananas juice 12% (V/V), pH value is 5.5.
The described group of candidum tissue culturing method that the training seedling can directly be transplanted, it is characterized in that described special culture media III comprises MS minimal medium+NAA (α-Nai Yisuan) 0.15mg/L+ bananas juice 9% (V/V)+murphy juice 6% (V/V)+taro juice 10% (V/V)+agar 5g/L, pH value is 5.5.
The present invention has improved existing culture medium prescription, satisfied group training seedling at the needs and equilibrium of each vegetative stage to various nutritions, created that to have light transmission good, the dedicated set training cup of the low air abundance of infection rate, and provide cleaning suitable natural light environment, under three's acting in conjunction, can be cultivated by 7-8 month tissue by the capsule seed of a dendrobium candidum, cultivate the stem of noble dendrobium group training seedling of 200,000 strains, this group training seedling need not traditional 3-5 month instruction process, can directly be transplanted to outdoor, transplanting survival rate on 98% with.
The inventive method has not only improved reproductive speed, the quality of production, the transplanting survival rate of candidum tissue culturing seedling, and shortened the dendrobium candidum booth effectively and cultivated the time, promote the quality of the dendrobium candidum of growing up, this has very positive meaning to resources of medicinal plant regeneration in short supply in imminent danger and sustainable utilization.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the structural representation of dedicated set training cup of the present invention.
Among the figure: 1-transparent plastic cup, 2-plastic foil plastic packaging, 3-filter membrane.
Embodiment
Embodiment 1.
The 1st stage, the capsule seed of the dendrobium candidum that maturation is sturdy is after sterilization, in an aseptic environments, the transparent plastic cup that the medium of 1/2MS minimal medium+taro juice 15% (V/V)+agar 4g/L is housed in it is broadcast is sprouted cultivation for 1 li, plastics cup 1 rim of a cup arranges filter membrane 3 by transparent plastic foil 2 plastic packagings on the plastic foil 2.Send into sterilization at last and cultivate in the culturing room, the room, sterilizing chamber adopts natural lighting, and its top arranges daylighting watt and sunshade stratum reticulare, and inside is provided with the temperature humidity conditioning equipment.Cultivation temperature is 24-26 ℃, and humidity is 65-75%, and illuminance is 1000-2000lx, and light application time is 10-12 hour/day, cultivates 45-60 days, and the explant rudiment forms green protocorm.
The 2nd stage, with protocorm in an aseptic environments, 1 li of the transparent plastic cup that the medium of MS minimal medium+BA (6-Bian aminoadenine) 0.1mg/L+ agar 4g/L+ murphy juice 6% (V/V)+bananas juice 10% (V/V) is housed in it is changed over to carries out protocorm propagation, differentiation is cultivated, plastics cup 1 rim of a cup arranges filter membrane 3 by transparent plastic foil 2 plastic packagings on the plastic foil 2.Send at last in the aseptic culture chamber and cultivate, the desinfection chamber roof arranges daylighting watt and sunshade stratum reticulare, and cultivation temperature is 22-28 ℃, and humidity is 65-75%, illuminance is 1500-2500lx, light application time is 10-12 hour/day, cultivates 60 days, cultivates into the 1-2CM seedling by protocorm, simultaneous has the new protocorm of part to produce, seedling is changed under same group training cup, medium and the environment again and continue to cultivate 1 month, seedling is cultivated into middle seedling, height of seedling is 3-4cm.
The 3rd stage, with middle seedling in an aseptic environments, the transparent plastic cup that the medium that changes MS minimal medium+NAA (α-Nai Yisuan) 0.1mg/L+ bananas juice 8% (V/V)+murphy juice 6% (V/V)+taro juice 8% (V/V)+agar 4g/L over to is housed in it is changed over to carries out strong seedling culture for 1 li, plastics cup 1 rim of a cup arranges filter membrane 3 by transparent plastic foil 2 plastic packagings on the plastic foil 2.Send at last in the aseptic culture chamber and cultivate, the desinfection chamber room adopts natural lighting, its top arranges daylighting watt and sunshade stratum reticulare, cultivation temperature is 20-30 ℃, and humidity is 65-75%, and illuminance is 2000-4000lx, light application time is 10-12 hour/day, cultivated 60 days, cultivating into height by seedling is 5-9CM, and stem slightly is the strong sprout of 0.4-0.5CM.
Further optimization method, in the above-mentioned steps, the pH value of medium is 5.4, and plastics cup 1 is cylindricality, and the rim of a cup diameter is 9-10cm, and a cup end diameter is 5-6cm, cup is high to be 9-10cm, during wall thickness 0.15-0.25mm, the most suitable.
Embodiment 2:
In embodiment 1, when special culture media I prescription volume ratio is 1/2MS minimal medium+taro juice 25% (V/V)+agar 6g/L, pH value is 5.8 or is 1/2MS minimal medium+taro juice 20% (V/V)+agar 5g/L that pH value is 5.5;
Or special culture media II prescription volume ratio is MS minimal medium+BA (6-Bian aminoadenine) 0.2mg/L+ agar 6g/L+ murphy juice 10% (V/V)+bananas juice 14% (V/V), pH value is 5.8 or is MS minimal medium+BA (6-Bian aminoadenine) 0.15mg/L+ agar 5g/L+ murphy juice 8% (V/V)+bananas juice 12% (V/V) that pH value is 5.5;
Or special culture media III prescription volume ratio is MS minimal medium+NAA (α-Nai Yisuan) 0.2mg/L+ bananas juice 10% (V/V)+murphy juice 7% (V/V)+taro juice 12% (V/V)+agar 6g/L, pH value is 5.8 or is MS minimal medium+NAA (α-Nai Yisuan) 0.15mg/L+ bananas juice 9% (V/V)+murphy juice 6% (V/V)+taro juice 10% (V/V)+agar 5g/L, pH value is 5.5 o'clock, also can reach the effect of embodiment 1.
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all is included within protection scope of the present invention.
Claims (2)
1. the dendrobium candidum seedling-cultivating method that can directly transplant is characterized in that this seedling-cultivating method comprises the following steps:
1) with the capsule seed of dendrobium candidum after sterilization, broadcast in special culture media I+dedicated set training cup+special-purpose culturing room, sprout cultivation, cultivation temperature is 24-26 ℃, humidity is 65-75%, and illuminance is 1000-2000lx, and light application time is 10-12 hour/day, cultivated 45-60 days, the explant rudiment forms green protocorm;
2) protocorm is changed in special culture media II+dedicated set training cup+special-purpose culturing room, carry out protocorm propagation, differentiation cultivation, cultivation temperature is 22-28 ℃, and humidity is 65-75%, illuminance is 1500-2500lx, light application time is 10-12 hour/day, cultivates 60 days, cultivates into the 1-2CM seedling by protocorm, simultaneous has the new protocorm of part to produce, seedling is changed under same group training cup, medium and the environment again and continue to cultivate 1 month, seedling is cultivated into middle seedling, height of seedling is 3-4cm;
3) middle seedling is changed in special culture media III+dedicated set training cup+special-purpose culturing room, cultivation temperature is 20-30 ℃, and humidity is 65-75%, illuminance is 2000-4000lx, and light application time is 10-12 hour/day, cultivates 60 days, cultivating into height by seedling is 5-9CM, and stem slightly is the strong sprout of 0.4-0.5CM;
Described special culture media I comprises 1/2MS minimal medium+20% volume ratio taro juice+agar 5g/L, and the pH value is 5.5;
Described special culture media II comprises MS minimal medium+BA (6-benzyl aminoadenine) 0.15mg/L+ agar 5g/L+ 8% volume ratio murphy juice+12% volume ratio bananas juice, and the pH value is 5.5;
Described special culture media III comprises MS minimal medium+NAA (α-Nai Yisuan) 0.15mg/L+ 9% volume ratio bananas juice+6% volume ratio murphy juice+10% volume ratio taro juice+agar 5g/L, and the pH value is 5.5;
Described dedicated set training cup is for having the transparent plastics cup (1) of appropriate volume, and described plastics cup (1) rim of a cup arranges filter membrane (3) by transparent plastic foil (2) plastic packaging on the described plastic foil (2);
Described special-purpose culturing room is sterilization culturing room, and described sterilization culturing room adopts natural lighting, and its top arranges daylighting watt and sunshade net, and inside is provided with the temperature humidity conditioning equipment.
2. the dendrobium candidum seedling-cultivating method that can directly transplant according to claim 1 is characterized in that described plastics cup (1) is cylindricality, and the rim of a cup diameter is 9-10cm, and cup end diameter is 5-6cm, and cup is high to be 9-10cm, wall thickness 0.15-0.25mm.
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| CN 201110332813 CN102499085B (en) | 2011-10-28 | 2011-10-28 | Method for raising directly transplantable seedlings of dendrobium candidum |
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| CN 201110332813 CN102499085B (en) | 2011-10-28 | 2011-10-28 | Method for raising directly transplantable seedlings of dendrobium candidum |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103168683A (en) * | 2012-09-28 | 2013-06-26 | 刘宏源 | Construction of novel simulated natural Dendrobium candidum tissue culture room |
| CN103355167A (en) * | 2013-07-17 | 2013-10-23 | 红河州巨丰生物科技有限公司 | Dendrobium officinale tissue culture method with plastic milk tea cup as tissue culture container |
| CN104488705A (en) * | 2014-12-03 | 2015-04-08 | 柳州市泓吉农业科技有限公司 | Method for cultivating tissue culture seedlings of dendrobium nobile lindl |
Citations (4)
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| CN2483951Y (en) * | 2001-04-19 | 2002-04-03 | 许惠贞 | Tearable plant-tissue cultuvation container |
| CN1608424A (en) * | 2004-11-24 | 2005-04-27 | 上海增靓生物科技有限公司 | Large-area cultivation of officinal dendrobium |
| CN2741354Y (en) * | 2004-09-05 | 2005-11-23 | 张纪勋 | Sterile culture cup |
| CN102119655A (en) * | 2010-07-29 | 2011-07-13 | 云南红土生源药用生物科技开发有限公司 | Natural light rapid breeding method for dendrobium officinale |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2483951Y (en) * | 2001-04-19 | 2002-04-03 | 许惠贞 | Tearable plant-tissue cultuvation container |
| CN2741354Y (en) * | 2004-09-05 | 2005-11-23 | 张纪勋 | Sterile culture cup |
| CN1608424A (en) * | 2004-11-24 | 2005-04-27 | 上海增靓生物科技有限公司 | Large-area cultivation of officinal dendrobium |
| CN102119655A (en) * | 2010-07-29 | 2011-07-13 | 云南红土生源药用生物科技开发有限公司 | Natural light rapid breeding method for dendrobium officinale |
Non-Patent Citations (1)
| Title |
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| 张治国等.名贵中药——铁皮石斛.《名贵中药——铁皮石斛》.上海科学技术文献出版社,2006,第64-65页. * |
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