CN101715733A - Tissue culturing method of protocorms of dendrobium candidum - Google Patents
Tissue culturing method of protocorms of dendrobium candidum Download PDFInfo
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- CN101715733A CN101715733A CN201010000764A CN201010000764A CN101715733A CN 101715733 A CN101715733 A CN 101715733A CN 201010000764 A CN201010000764 A CN 201010000764A CN 201010000764 A CN201010000764 A CN 201010000764A CN 101715733 A CN101715733 A CN 101715733A
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Abstract
The invention discloses a tissue culturing method of protocorms of dendrobium candidum through tissues, comprising three steps of selection and aseptic processing of a plant body, callus induced culture and mass culture, wherein the induced culture medium contains a MS culture medium, 20 to 30 g/L sucrose, 4 to 6 g/L agar, 2.0 to 3.0 mg/L alpha-naphthylacetic acid and 2.0 to 3.0 mg/L 6-benzyladenine, and the mass culture medium contains a MS culture medium, 20 tp 50 g/L sucrose, 0.1 to 5.0 mg/L alpha-naphthylacetic acid and 0.1 to 5.0 mg/L 6-benzyladenine. The method has the advantages of easy operation, high content of active ingredients, low production cost and environmental protection and can reach the industrial level.
Description
Technical field
The invention belongs to field of plant tissue culture technique, specifically is a kind of method of tissue culture protocorms of dendrobium candidum.
Background technology
Dendrobium candidum (Dendrobium candidum Wall.ex Lindl.) is the perennial herbaceous plant that grows nonparasitically upon another plant of the orchid family (Orchidaceae) Dendrobium (Dendrobium), because of old stem eustipes part is brown, have another name called ribbed hedyotis herb, be to view and admire and medicinal famous and precious rare herbaceous plant a kind of the collection, have unique medical value, be the superfine product of the stem of noble dendrobium.The stem of noble dendrobium is one of orchid of record the earliest during China's ancient Chinese prose is offered, its property slightly sweet flavor, little salty, and property belongs to clear and rich, benefit is arranged in clear, and bowl spares has clearly.In the Shennong's Herbal of the Eastern Han Dynasty, put down in writing the stem of noble dendrobium " in main the wound, remove thin thin, the reinforcing yin essence of numbness, the therapeutic method to keep the adverse qi flowing downward, tonifying five zang organs consumptive disease, obey thick stomach for a long time ".Become book Taoist school medical science classics " Taoist Scriptures " before more than 1,000 years dendrobium candidum to be classified as first of " Chinese nine immortal grass ", the treasure of dreaming of for the successive dynasties emperor, have the title of " a thousand pieces of gold grass ", have won fame both at home and abroad, be called " medicine circle giant panda " among the people being called " the celestial grass of help " by international medicinal plant circle.Modern pharmacology studies show that, dendrobium polysaccharide has the effect that strengthens T cell and macrophage immunity activity, be the gratifying Chinese medicine immunopotentiator of a kind of prospect, and have effects such as antitumor, hypoglycemic, anti-oxidant, antimutagenic, be subjected to the great attention of Chinese scholars.
Dendrobium candidum is often grown nonparasitically upon another plant on trees or rock, and plant is shorter and smaller, grows slowly, and blade area is little, photosynthetic area is little, intensity is low, and especially seed is minimum, embryo tool after ripening, so seed is difficult to sprout (germination rate is lower than 5%) under the natural conditions.Under common cultivation condition, dendrobium candidum needs 3～4 years by seed to blooming, and often needs could sprout with some mycosymbiosis, has been difficult to the seedling cultivation.And the survival rate of traditional vegetative propagation modes such as plant division cuttage is low, growth rate is slow, reproduction rate is low, and people excavate the predation formula of wild dendrobium candidum resource in recent years in addition, and its habitat is caused wild dendrobium candidum endangered by heavy damage.
Chinese scholars has been carried out wild dendrobium candidum has comprehensively been studied, and comprises chemical composition, pharmacologically active, the research of aspects such as artificial planting.And the cultivation of dendrobium candidum mainly concentrates on the aspects such as examination of callus induction, basic condition of culture screening and physical and chemical factor.The breeding of dendrobium officinale test-tube plantlet generally will be passed through protocorms (protocorm-like bodies, the i.e. PLBs) stage, and protocorms just comes down to have metabolism identical with former plant and morphological development potential at the somatic embryo of Growth and Differentiation.Given this, people propose to replace former plant to become the possibility of source new drugs with protocorms.Yet, existing result of study shows, the research that induces protocorms from the dendrobium candidum different parts exists also that inductivity is low, differentiation is fast and problems such as brown stain still do not solve, also do not find at present an efficiently and effectively culture technique in order to solve that protocorms of dendrobium candidum is induced and propagation and metabolite control in the doubtful point and the difficult point that exist, thereby influenced the further investigation and the exploitation of dendrobium candidum.
Natural dendrobium candidum has been subjected to the focused protection of the world and the Chinese government as endangered plant species, forbid to pluck.Study the cultivation of protocorms of dendrobium candidum for this reason, realize that industrialization seems very important.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of reproduction speed fast, the method for the tissue culture protocorms of dendrobium candidum that dendrobium polysaccharide content is high.
The object of the present invention is achieved like this: a kind of method of tissue culture protocorms of dendrobium candidum, it comprises the steps:
The selection of a, plant corpus and aseptic process:
Select the wild fresh plant of dendrobium candidum of growth in 6～August, remove root and leaf and clean and aseptic process, choose the young stem of seedling, young stem is cut into 0.3～0.5cm
2Size dendrobium candidum tissue;
B, organize inducing culture:
To be organized in through the dendrobium candidum after the aseptic process and organize inducing culture, cultivation cycle in the inducing culture: 10～24 days, cultivation temperature: 10～30 ℃, light application time: 1～24 hour/day, intensity of illumination: 0～100 μ mol/m
2S; Described inducing culture is: MS medium+sucrose 0～50g/L+ agar 0～10g/L+ α-Nai Yisuan 0.1～5.0mg/L+6-benayl aminopurine 0.1～5.0mg/L, and the pH value is 5.0～7.0;
C, large-scale culture:
The protocorms of dendrobium candidum that induces is carried out large-scale culture, cultivation cycle in the large-scale culture base: 10～24 days, cultivation temperature: 10～30 ℃, light application time: 0～24 hour/day, intensity of illumination: 0～100 μ mol/m
2S; Described large-scale culture base is: MS medium+sucrose 20～50g/L+ α-Nai Yisuan 0.1～5.0mg/L+6-benayl aminopurine 0.1～5.0mg/L, the pH value is 5.0～7.0.
The technical scheme of more optimizing as the method for tissue culture protocorms of dendrobium candidum of the present invention is optimized selection to the formula rate and the condition of culture of medium.
Described inducing clumping bud is cultivated: cultivation cycle: 15～20 days, and cultivation temperature: 20～25 ℃, light application time: 8～20 hours/day, intensity of illumination: 50～80 μ mol/m
2S; Described inducing culture is: MS medium+sucrose 20～30g/L+ agar 4～6g/L+ α-Nai Yisuan 2.0～3.0mg/L+6-benayl aminopurine 2.0～3.0mg/L, and the pH value is 5.5～6.5; Described large-scale culture: cultivation cycle: 15～20 days, cultivation temperature: 20～25 ℃, light application time: 8～20 hours/day, intensity of illumination: 50～80 μ mol/m
2S; Described large-scale culture base is: MS medium+sucrose 30～40g/L+ α-Nai Yisuan 2.0～3.0mg/L+6-benayl aminopurine 2.0～3.0mg/L, the pH value is 5.5～6.5.
Plant corpus aseptic process be important technical links of the present invention, the method for aseptic process comprises the steps:
A) with the young stem of the healthy seedling of the dendrobium candidum chosen, rinse well, use distilled water flushing again, put in the superclean bench with running water;
B) dendrobium candidum children stem was carried out surface sterilizing with 70～80% alcohol-pickled 20～40 seconds, clean with sterile water wash again;
C) will be through the dendrobium candidum children stem of alcohol sterilization with 0.05～0.15% mercuric chloride solution soaking disinfection 3～6 minutes, usefulness aseptic water washing 4～5 times blots sterile water with filter paper again;
D) under the aseptic condition the young stem of the dendrobium candidum of mercury chloride sterilization is cut into 0.3～0.5cm
2Size dendrobium candidum tissue is inoculated in the inducing culture 5 of every bottle graft kinds.
The invention has the advantages that: this method is operated easily, and production cost is low, and is free from environmental pollution, can realize producing in batches.Select the rational inducing culture of prescription for use, the inductivity average out to 99.6% of callus, by two sections best cultivation protocorms of dendrobium candidum output increase substantially and wild dendrobium candidum relatively dendrobium polysaccharide content improve more than 1 times.Produce protocorms of dendrobium candidum by the inventive method, consistency, output is big, and cost is low, and the cycle is short, has the market competitiveness.
Embodiment
The present invention will be further described below in conjunction with specific embodiment.
Embodiment 1: a kind of method of tissue culture protocorms of dendrobium candidum, it comprises the steps:
The selection of a, plant corpus and aseptic process:
A) the young stem of the wild healthy seedling of dendrobium candidum of selection growth in July is rinsed well with running water, uses distilled water flushing again, puts in the superclean bench; B) dendrobium candidum children stem was carried out surface sterilizing with 75% alcohol-pickled 30 seconds, clean with sterile water wash again; C) will be through the dendrobium candidum children stem of alcohol sterilization with 0.1% mercuric chloride solution soaking disinfection 5 minutes, usefulness aseptic water washing 5 times blots sterile water with filter paper again; D) under the aseptic condition the young stem of the dendrobium candidum of mercury chloride sterilization is cut into 0.3～0.5cm
2Size dendrobium candidum tissue is inoculated in the inducing culture 5 of every bottle graft kinds.
B, organize inducing culture:
To be organized in through the dendrobium candidum after the aseptic process and organize inducing culture, cultivation cycle in the inducing culture: 16 days, cultivation temperature: 20 ℃, light application time: 10 hours/day, intensity of illumination: 80 μ mol/m
2S; Described inducing culture is: MS medium+sucrose 28g/L+ agar 4.5g/L+ α-Nai Yisuan 2mg/L+6-benayl aminopurine 3mg/L, and the pH value is 5.5;
C, large-scale culture:
The protocorms of dendrobium candidum that induces is carried out large-scale culture, cultivation cycle in the large-scale culture base: 18 days, cultivation temperature: 22 ℃, light application time: 10 hours/day, intensity of illumination: 80 μ mol/m
2S; Described large-scale culture base is: MS medium+sucrose 40g/L+ α-Nai Yisuan 2mg/L+6-benayl aminopurine 3mg/L, the pH value is 5.5.
Embodiment 2: a kind of method of tissue culture protocorms of dendrobium candidum, it comprises the steps:
The selection of a, plant corpus and aseptic process:
A) the young stem of the wild healthy seedling of dendrobium candidum of selection growth in July is rinsed well with running water, uses distilled water flushing again, puts in the superclean bench; B) dendrobium candidum children stem was carried out surface sterilizing with 78% alcohol-pickled 30 seconds, clean with sterile water wash again; C) will be through the dendrobium candidum children stem of alcohol sterilization with 0.12% mercuric chloride solution soaking disinfection 5 minutes, usefulness aseptic water washing 4 times blots sterile water with filter paper again; D) under the aseptic condition the young stem of the dendrobium candidum of mercury chloride sterilization is cut into 0.3～0.5cm
2Size dendrobium candidum tissue is inoculated in the inducing culture 5 of every bottle graft kinds.
B, organize inducing culture:
To be organized in through the dendrobium candidum after the aseptic process and organize inducing culture, cultivation cycle in the inducing culture: 18 days, cultivation temperature: 25 ℃, light application time: 18 hours/day, intensity of illumination: 60 μ mol/m
2S; Described inducing culture is: MS medium+sucrose 25g/L+ agar 5g/L+ α-Nai Yisuan 3mg/L+6-benayl aminopurine 2mg/L, and the pH value is 6.0;
C, large-scale culture:
The protocorms of dendrobium candidum that induces is carried out large-scale culture, cultivation cycle in the large-scale culture base: 20 days, cultivation temperature: 25 ℃, light application time: 8 hours/day, intensity of illumination: 50 μ mol/m
2S; Described large-scale culture base is: MS medium+sucrose 30g/L+ α-Nai Yisuan 3mg/L+6-benayl aminopurine 2mg/L, the pH value is 6.0.
Embodiment 3: wild dendrobium candidum, embodiment 1 and embodiment 2 are cultivated the protocorms of dendrobium candidum dendrobium polysaccharide content that obtains detect, concrete detection method comprises the steps:
(1) extract: it is an amount of to take by weighing wild dendrobium candidum or protocorms of dendrobium candidum, adds the distilled water of 20 times of volumes, and lixiviate is 2 hours under 80 ℃ of water-baths, filter extract.Residue repeats to extract 2 times by above-mentioned steps, merges three times and extracts gained filtrate, and is stand-by.
The preparation of (2) 5% phenol solution: take by weighing phenol 100g, with aluminium flake 0.1g and sodium bicarbonate 0.05g, 182 ℃ of cuts are collected in air-distillation.Precision takes by weighing the about 10.0g of this cut and places the 200mL measuring bottle, is dissolved in water and is diluted to scale, shakes up the back transposition in brown reagent bottle, promptly gets 5% (W/V) phenol solution, is placed in the refrigerator standby.
(3) mensuration of sample: get said extracted liquid, by the proper proportion dilution, the accurate absorption in the 2ml tool plug scale test tube, add 5% phenol solution 1mL, shake up, add concentrated sulfuric acid 5mL rapidly, shake up, put heating 15mi n in 100 ℃ of water-baths, cold water is cooled to room temperature rapidly.Be operating as blank with 2mL distilled water with method, use ultraviolet specrophotometer, measuring wavelength is the absorbance A at 490nm place, and substitution calibration curve A=0.0153C-0.0122 obtains the concentration of glucose C (ug/ml) of sample polysaccharide,
Press polyoses content (%)=CDf/W sample * 100% and calculate polyoses content.
The W sample is an example weight in the formula; D is the dilution of sample multiple; F is a conversion factor.Gained sample polyoses content palpus＞30%.
As follows by this method to the testing result of the protocorms of dendrobium candidum sample dendrobium polysaccharide content of wild dendrobium candidum and above-mentioned two embodiment production:
The comparison of wild dendrobium candidum and two embodiment dendrobium polysaccharide content
Result of the test proves, adopts protocorms of dendrobium candidum that method of the present invention produces to exceed more than 2 times than the dendrobium polysaccharide content of wild dendrobium candidum.
Embodiment 4: adopt MS medium, B5 medium and inducing culture of the present invention to organize inducing culture in the inducing culture to being organized in by the dendrobium candidum fritter after the aseptic process respectively, result of the test is as follows:
Different medium induction ratios
Result of the test proves, adopts inducing culture of the present invention to carrying out inducing culture by the little block organization of the dendrobium candidum after the aseptic process, organizes fresh weight and inductivity to significantly improve.
Embodiment 5: adopt simple inducing culture, simple large-scale culture base and inducing culture of the present invention+large-scale culture base two-step method cultural method respectively, carry out tissue culture and produce the protocorms of dendrobium candidum test, except medium with cultivation cycle is different, other experimental condition is identical with embodiment 1, and result of the test is as follows:
Medium in different ways is to the influence of dendrobium candidum output
Result of the test proves, adopts the method for tissue culture protocorms of dendrobium candidum of the present invention to cultivate, use merely large-scale culture base cultivation output to increase more than 2 times than the simple inducing culture that uses.
Claims (3)
1. the method for a tissue culture protocorms of dendrobium candidum, it comprises the steps:
The selection of a, plant corpus and aseptic process:
Select the wild fresh plant of dendrobium candidum of growth in 6～August, remove root and leaf and clean and aseptic process, choose the young stem of seedling, young stem is cut into 0.3～0.5cm
2Size dendrobium candidum tissue;
B, organize inducing culture:
To be organized in through the dendrobium candidum after the aseptic process and organize inducing culture, cultivation cycle in the inducing culture: 10～24 days, cultivation temperature: 10～30 ℃, light application time: 1～24 hour/day, intensity of illumination: 0～100 μ mol/m
2S; Described inducing culture is: MS medium+sucrose 0～50g/L+ agar 0～10g/L+ α-Nai Yisuan 0.1～5.0mg/L+6-benayl aminopurine 0.1～5.0mg/L, and the pH value is 5.0～7.0;
C, large-scale culture:
The protocorms of dendrobium candidum that induces is carried out large-scale culture, cultivation cycle in medium: 10～24 days, cultivation temperature: 10～30 ℃, light application time: 0～24 hour/day, intensity of illumination: 0～100 μ mol/m
2S; Described large-scale culture base is: MS medium+sucrose 20～50g/L+ α-Nai Yisuan 0.1～5.0mg/L+6-benayl aminopurine 0.1～5.0mg/L, the pH value is 5.0～7.0.
2. according to the method for the described tissue culture protocorms of dendrobium candidum of claim 1, it is characterized in that: described inducing clumping bud is cultivated: cultivation cycle: 15～20 days, cultivation temperature: 20～25 ℃, light application time: 8～20 hours/day, intensity of illumination: 50～80 μ mol/m
2S; Described inducing culture is: MS medium+sucrose 20～30g/L+ agar 4～6g/L+ α-Nai Yisuan 2.0～3.0mg/L+6-benayl aminopurine 2.0～3.0mg/L, and the pH value is 5.5～6.5; Described large-scale culture: cultivation cycle: 15～20 days, cultivation temperature: 20～25 ℃, light application time: 8～20 hours/day, intensity of illumination: 50～80 μ mol/m
2S; Described large-scale culture base is: MS medium+sucrose 30～40g/L+ α-Nai Yisuan 2.0～3.0mg/L+6-benayl aminopurine 2.0～3.0mg/L, the pH value is 5.5～6.5.
3. according to the method for claim 1 or 2 described tissue culture protocorms of dendrobium candidum, it is characterized in that plant corpus the aseptic process method comprise the steps:
A) with the young stem of the healthy seedling of the dendrobium candidum chosen, rinse well, use distilled water flushing again, put in the superclean bench with running water;
B) dendrobium candidum children stem was carried out surface sterilizing with 70～80% alcohol-pickled 20～40 seconds, clean with sterile water wash again;
C) will be through the dendrobium candidum children stem of alcohol sterilization with 0.05～0.15% mercuric chloride solution soaking disinfection 3～6 minutes, usefulness aseptic water washing 4～5 times blots sterile water with filter paper again;
D) under the aseptic condition the young stem of the dendrobium candidum of mercury chloride sterilization is cut into 0.3～0.5cm
2Size dendrobium candidum tissue is inoculated in the inducing culture 5 of every bottle graft kinds.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101878736A (en) * | 2010-06-18 | 2010-11-10 | 沈阳药科大学 | Method for producing dendrobium candicum |
| 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 |
| CN102265788A (en) * | 2011-07-18 | 2011-12-07 | 常熟理工学院 | Method for increasing proliferation multiple of dendrobium candidum protocorms |
| CN102428874A (en) * | 2011-12-27 | 2012-05-02 | 杭州师范大学 | Method for inducing protocorms by utilizing dendrobe tissues |
| CN103155871A (en) * | 2013-03-07 | 2013-06-19 | 华中科技大学 | Dendrobium officinale sprout rapid propagation method with high efficiency |
| CN105613291A (en) * | 2015-12-31 | 2016-06-01 | 厦门涌泉科技有限公司 | Fast breeding method for dendrobium candidum protocorm tissue culture |
| CN116965335A (en) * | 2023-09-05 | 2023-10-31 | 河北农业大学 | Tissue culture and rapid propagation method for dendrobium candidum |
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2010
- 2010-01-19 CN CN201010000764A patent/CN101715733A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101878736A (en) * | 2010-06-18 | 2010-11-10 | 沈阳药科大学 | Method for producing dendrobium candicum |
| 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 |
| CN102119655B (en) * | 2010-07-29 | 2012-12-19 | 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 |
| CN102265788A (en) * | 2011-07-18 | 2011-12-07 | 常熟理工学院 | Method for increasing proliferation multiple of dendrobium candidum protocorms |
| CN102265788B (en) * | 2011-07-18 | 2012-07-25 | 常熟理工学院 | Method for increasing proliferation multiple of dendrobium candidum protocorms |
| CN102428874A (en) * | 2011-12-27 | 2012-05-02 | 杭州师范大学 | Method for inducing protocorms by utilizing dendrobe tissues |
| CN103155871A (en) * | 2013-03-07 | 2013-06-19 | 华中科技大学 | Dendrobium officinale sprout rapid propagation method with high efficiency |
| CN103155871B (en) * | 2013-03-07 | 2014-06-04 | 华中科技大学 | Dendrobium officinale sprout rapid propagation method with high efficiency |
| CN105613291A (en) * | 2015-12-31 | 2016-06-01 | 厦门涌泉科技有限公司 | Fast breeding method for dendrobium candidum protocorm tissue culture |
| CN116965335A (en) * | 2023-09-05 | 2023-10-31 | 河北农业大学 | Tissue culture and rapid propagation method for dendrobium candidum |
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