CN101878736A - Method for producing dendrobium candicum - Google Patents
Method for producing dendrobium candicum Download PDFInfo
- Publication number
- CN101878736A CN101878736A CN2010102027937A CN201010202793A CN101878736A CN 101878736 A CN101878736 A CN 101878736A CN 2010102027937 A CN2010102027937 A CN 2010102027937A CN 201010202793 A CN201010202793 A CN 201010202793A CN 101878736 A CN101878736 A CN 101878736A
- Authority
- CN
- China
- Prior art keywords
- protocorm
- culture
- dendrobium candidum
- agar
- sucrose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for culturing dendrobium candicum protocorm-like bodies by using solid tissues. The method comprises three links, namely selective induction of explant, multiplication culture and subculture, wherein the induction and multiplication culture medium comprises MS, 20g/L of sucrose, 4.5g/L of agar, 0.5mg/L of NAA and 2mg/L of 6-BA. The pH is adjusted to 5.8. The culture temperature is controlled to be 25+/-1 DEG C, the light intensity is 50mu mol/m<2>/s, and illumination is performed for 12 hours every day. The subculture medium comprises MS, 1.0mg/ml of NAA, 0.5mg/ml of 6-BA, 20g/L of sucrose and 4.5g/L of agar; and the culture conditions comprise that: the illumination time is 12 hours every day, the light intensity is 100mu mol/m<2>/s, the culture temperature is 25+/-1 DEG C and the pH is 5.8. The method has the advantages of easy operation, high effective ingredient content, low production cost and environmental protection, and can reach industrialized level. The dendrobium candicum protocorm-like bodies produced by the method have the advantages of denaturing resistance, high yield, low cost and short period.
Description
Technical field
The invention belongs to medical technical field, relate to a kind of method of producing dendrobium candidum, be specifically related to use plant tissue culture technique to produce the method for dendrobium candidum.
Background technology
Dendrobium candidum (Dendrobium candidum Wall.ex Lind1.) is the perennial herbaceous plant that grows nonparasitically upon another plant of the orchid family (Orchidaceae) Dendrobium (Dendrobium), has another name called ribbed hedyotis herb, is China's rare traditional Chinese medicine in imminent danger.In the Shennong's Herbal of the Eastern Han Dynasty, put down in writing the stem of noble dendrobium " in main the wound, remove numbness, the therapeutic method to keep the adverse qi flowing downward, tonifying five zang organs consumptive disease win thin, reinforcing yin essence, 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 title of " a thousand pieces of gold grass " is arranged.Modern pharmacology studies show that, the plurality of active ingredients that dendrobium candidum contains has antitumor, hypoglycemic, hypotensive, reducing blood lipid, and effects such as anti-oxidant and antimutagenic.
Because dendrobium candidum belongs to the medicinal material of growing nonparasitically upon another plant, plant 20～30cm height, it is very slow to grow, and seed germination rate is lower than 5% under field conditions (factors).Therefore, under common cultivation condition, dendrobium candidum needs 3～4 years by seed to blooming, and need 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.
Discover that by the growing multiplication of dendrobium candidum plan protocorm, produce a large amount of plan protocorms, it is feasible obtaining its medicinal ingredient.It is just at the somatic embryo of Growth and Differentiation that dendrobium candidum is intended protocorm (protocorm-like bodies, i.e. PLBs), has metabolism identical with former plant and morphological development potential.By intending the proliferation and differentiation of protocorm, produce a large amount of tissue cultivating seedling, make dendrobium candidum realize the breeding of low-cost high-efficiency, be one of main path that solves at present the dendrobium candidum shortage of resources.
Wild dendrobium candidum has been subjected to the focused protection of the world and the Chinese government as endangered plant species, forbid to pluck.Study dendrobium candidum for this reason and intend the cultivation of protocorm, realize that industrialization seems very important.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of producing dendrobium candidum, to overcome the problem of dendrobium candidum shortage of resources.
The present invention is achieved through the following technical solutions:
Provided by the present invention is that the method that dendrobium candidum is intended protocorm is cultivated by a kind of solid tissue, and it comprises the steps:
(1) selection of aseptic seedling and processing
Take out the seedling of the about 3～10cm of plant height from aseptic bottle, aseptic condition is cut into the explant that the long band of 1cm saves with dissecting the stem of scissors with plant down, and leaf or root are cut into the long explant of 1cm.
(2) intend inducing and breeding of protocorm
The explant that cuts kept flat to be inoculated into intend in the protocorm inducing culture, each combination connects 10 bottles, 5 explants of every blake bottle inoculation.Induce and the medium that rises in value is MS+ sucrose 20g/L+ agar 4.5g/L+NAA 0.5mg/L+6-BA 2mg/L.Adjusting pH is 5.8.The control cultivation temperature is (25 ± 1) ℃, light intensity 50 μ molm
-2S
-1, illumination every day 12h.
(3) intend protocorm successive transfer culture method
With the plan protocorm that induces in former inducing culture subculture once after, change successive transfer culture on the subculture medium over to, condition of culture MS+NAA 1.0mg/ml+6-BA 0.5mg/ml+ sucrose 20g/L+ agar 4.5g/L, light application time 12h/d, light intensity 100 μ molm
-2S
-1, cultivation temperature: 25 ± 1 ℃, pH=5.8.
Cultivate the technical scheme that the method for protocorms of dendrobium candidum is more optimized as solid tissue of the present invention, the formula rate and the condition of culture of medium is optimized selection.
Described plan protocorm is induced and the cultivation of rising in value: cultivation cycle: 20～40 days, and cultivation temperature: 20～30 ℃, light application time: 8～24 hours/day, intensity of illumination: 50～80 μ mol/m
-2S
-1Described 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 successive transfer culture: cultivation cycle: 20～40 days, cultivation temperature: 20～25 ℃, light application time: 8～24 hours/day, intensity of illumination: 50～80 μ mol/m
-2S
-1Described large-scale culture base is: MS medium+sucrose 30～40g/L+ agar 4～6g/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.
Successive transfer culture is an important technical links of the present invention, comprises the steps:
(1) get colors bud greenly, growth is intended protocorm switching synchronously, rapidly on subculture medium;
(2) intend the approximate S type of protocorm growth curve, dendrobium candidum is intended protocorm and was growth-delaying at 0～10 day, be logarithmic growth in 10～40 days, be in vigorous splitting status from the 14th day beginning cell of cultivating, it is very rapid to intend the protocorm increment in this stage, after enter and grow into stationary phase, intending protocorm fresh weight propagation multiple is more than 5 times.Intending the protocorm dry weight from about the 30th day beginning dendrobium candidum of cultivating increases slowly, and reduces gradually.
(3) intend in initial 20 days of the content of polysaccharide in the protocorm in rising trendly, reach maximum 85.6mg/g at the 20th day polyoses content.At the 20th day to the 35th day content held stationary, polyoses content began to descend after 35 days subsequently.The growth of protocorm and the accumulation of polysaccharide are intended in comprehensive examination, and best subculture and collecting time are 30 days.
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, intend the inductivity average out to 99% of protocorm, polyoses content improves more than 5 times than wild dendrobium candidum.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: the method that dendrobium candidum is intended protocorm is cultivated by a kind of solid tissue, and it comprises the steps:
(1) selection of aseptic seedling and processing
Take out the seedling of the about 3cm of plant height from aseptic bottle, aseptic condition is cut into the explant that the long band of 1cm saves with dissecting the stem of scissors with plant down, and leaf or root are cut into the long explant of 1cm.
(2) intend inducing and breeding of protocorm
The explant that cuts kept flat to be inoculated into intend in the protocorm inducing culture, each combination connects 10 bottles, 5 explants of every blake bottle inoculation.Inducing culture is MS+ sucrose 20g/L+ agar 4.5g/L+NAA0.5mg/L+6-BA 2mg/L.Adjust pH5.8.Control cultivation temperature (25 ± 1) ℃, light intensity 50 μ molm
-2S
-1, illumination every day 12h.
(3) intend protocorm successive transfer culture method
With the plan protocorm that induces in former inducing culture subculture once after, change successive transfer culture on the subculture medium over to, condition of culture MS+NAA 1.5mg/ml+6-BA 1.5mg/ml+ sucrose 22g/L+ agar 4.5g/L, light application time 12h/d, light intensity 100 μ molm
-2S
-1, cultivation temperature: 25 ± 1 ℃, pH=5.8.
Embodiment 2: a kind of method of tissue culture protocorms of dendrobium candidum, it comprises the steps:
(1) selection of aseptic seedling and processing
Take out the seedling of the about 5cm of plant height from aseptic bottle, aseptic condition is cut into the explant that the long band of 1.5cm saves with dissecting the stem of scissors with plant down, and leaf or root are cut into the long explant of 1.5cm.
(2) intend inducing and breeding of protocorm
The explant that cuts kept flat to be inoculated into intend in the protocorm inducing culture, each combination connects 10 bottles, 8 explants of every blake bottle inoculation.Inducing culture is MS+ sucrose 25g/L+ agar 5.0g/L+NAA1.5mg/L+6-BA 2.5mg/L.Adjust pH5.6.Control cultivation temperature (25 ± 1) ℃, light intensity 60 μ molm
-2S
-1, illumination every day 12h.
(3) intend protocorm successive transfer culture method
With the plan protocorm that induces in former inducing culture subculture once after, change successive transfer culture on the subculture medium over to, inoculum concentration 80 ± 10g/L, condition of culture MS+NAA 1.0mg/ml+6-BA 0.5mg/ml+ sucrose 20g/L+ agar 5.5g/L, light application time 12h/d, light intensity 100 μ molm
-2S
-1, cultivation temperature: 25 ± 1 ℃, pH=5.8.
Embodiment 3: wild dendrobium candidum, embodiment 1 and embodiment 2 are cultivated the dendrobium candidum plan protocorm dendrobium polysaccharide content that obtains detect, concrete detection method comprises the steps:
(1) extract: taking by weighing wild dendrobium candidum or dendrobium candidum, to intend protocorm an amount of, 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 adds 5% phenol solution 1mL, shakes up, and adds concentrated sulfuric acid 5mL rapidly, shakes up, and puts in 100 ℃ of water-baths and heats 15min, and 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 (μ g/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 answers＞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 5 times than the dendrobium polysaccharide content of wild dendrobium candidum.
Embodiment 4: adopt MS medium, White medium and inducing culture of the present invention respectively the dendrobium candidum fritter to be organized in and organize inducing culture in the inducing culture, experimental result is as follows:
Different medium induction ratios
Experimental result proves, adopts inducing culture of the present invention to carrying out inducing culture by the little block organization of dendrobium candidum, organizes fresh weight and inductivity to significantly improve.
Claims (3)
1. method of producing dendrobium candidum is characterized in that: described method is that the method that dendrobium candidum is intended protocorm is cultivated by solid tissue, comprises that the selection of explant is induced, rises in value and cultivate and successive transfer culture, specifically comprises the steps:
(1) selection of aseptic seedling and processing
Take out the seedling of the about 3～10cm of plant height from aseptic bottle, aseptic condition is cut into the explant that the long band of 1cm saves with dissecting the stem of scissors with plant down, and leaf or root are cut into the long explant of 1cm;
(2) intend inducing and breeding of protocorm
With the explant that cuts keep flat be inoculated into intend that protocorm is induced and the medium that rises in value in, each combination connects 10 bottles, 5～10 explants of every blake bottle inoculation, induce and the medium that rises in value is MS+ sucrose 20g/L+ agar 4.5g/L+NAA 0.5mg/L+6-BA 2mg/L, adjust pH=5.8, control cultivation temperature (25 ± 1) ℃, light intensity 50 μ molm
-2S
-1, illumination every day 12h;
(3) intend protocorm successive transfer culture method
With the plan protocorm that induces in former inducing culture subculture once after, change successive transfer culture on the subculture medium over to, condition of culture MS+NAA 1.0mg/ml+6-BA 0.5mg/ml+ sucrose 20g/L+ agar 4.5g/L, light application time 12h/d, light intensity 100 μ molm
-2S
-1, cultivation temperature: 25 ± 1 ℃, pH=5.8.
2. according to the method for the described production dendrobium candidum of claim 1, it is characterized in that described plan protocorm is induced and rised in value and cultivates: cultivation cycle: 20～40 days, cultivation temperature: 20～30 ℃, light application time: 8～24 hours/day, intensity of illumination: 50～80 μ mol/m
-2S
-1Described 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, the pH value is 5.5～6.5.
3. according to the method for the described production dendrobium candidum of claim 1, it is characterized in that, in the described plan protocorm successive transfer culture: cultivation cycle: 20～40 days, cultivation temperature: 20～25 ℃, light application time: 8～24 hours/day, intensity of illumination: 50～80 μ mol/m
-2S
-1Described large-scale culture base is (as above not mentioning the large-scale culture base in the claim): MS medium+sucrose 30～40g/L+ agar 4～6g/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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102027937A CN101878736A (en) | 2010-06-18 | 2010-06-18 | Method for producing dendrobium candicum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102027937A CN101878736A (en) | 2010-06-18 | 2010-06-18 | Method for producing dendrobium candicum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101878736A true CN101878736A (en) | 2010-11-10 |
Family
ID=43050991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102027937A Pending CN101878736A (en) | 2010-06-18 | 2010-06-18 | Method for producing dendrobium candicum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101878736A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102265788A (en) * | 2011-07-18 | 2011-12-07 | 常熟理工学院 | Method for increasing proliferation multiple of dendrobium candidum protocorms |
| CN102428870A (en) * | 2011-09-22 | 2012-05-02 | 澄思源生物科技（上海）有限公司 | Preparation method for artificial seeds of dendrobium candidum |
| CN103609446A (en) * | 2013-11-27 | 2014-03-05 | 苏州田园农业技术开发有限公司 | Dendrobium officinale propagation method capable of shortening vegetative propagation cycle of Dendrobium officinale |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715733A (en) * | 2010-01-19 | 2010-06-02 | 烟台汇鹏生物科技有限公司 | Tissue culturing method of protocorms of dendrobium candidum |
-
2010
- 2010-06-18 CN CN2010102027937A patent/CN101878736A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715733A (en) * | 2010-01-19 | 2010-06-02 | 烟台汇鹏生物科技有限公司 | Tissue culturing method of protocorms of dendrobium candidum |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102428870A (en) * | 2011-09-22 | 2012-05-02 | 澄思源生物科技（上海）有限公司 | Preparation method for artificial seeds of dendrobium candidum |
| CN103609446A (en) * | 2013-11-27 | 2014-03-05 | 苏州田园农业技术开发有限公司 | Dendrobium officinale propagation method capable of shortening vegetative propagation cycle of Dendrobium officinale |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103828715B (en) | Bletilla striata tissue culture breeding method | |
| CN102119655B (en) | Natural light rapid breeding method for dendrobium officinale | |
| CN102369881B (en) | Rapid propagation technique of Dendrobium candidum axillary buds | |
| CN103190343B (en) | Key technology of organic additive for roxburgh anoectochilus terminal bud industrialization intermediate propagation | |
| CN102144555B (en) | Rapid propagation method for radix puerariae | |
| CN103931492A (en) | Tissue-culture rapid seedling growing method for apple rootstock M9 | |
| CN104396742B (en) | The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss | |
| CN101715733A (en) | Tissue culturing method of protocorms of dendrobium candidum | |
| CN105850728B (en) | A kind of Jing Banxia seedling stems rapid propagation method | |
| CN101773071A (en) | Callus culture method of snow lotus herb | |
| CN108770691A (en) | A method of induction camphor tree leaf blueberry tissue culture seedling leaf directly generates adventitious root | |
| CN103404437B (en) | Novel method for tissue culture rapid propagation of acer paimatum | |
| CN103451146A (en) | Rapid propagation method of astragalus membranaceus calluses and astragalus membranaceus suspension cells | |
| CN101878736A (en) | Method for producing dendrobium candicum | |
| CN101946698A (en) | Fast propagation technology for wild buckwheat rhizome | |
| CN104221862B (en) | The method of medicinal Dioscorea camposita group training forming seedling through one step culture mass production | |
| CN102138527B (en) | Method for culturing tissue culture seedlings of glabrous greenbrier rhizome | |
| CN103609452B (en) | Tissue-culture rapid propagation method of cymbidium | |
| CN104737906A (en) | Building method of rosemarinus officinalis tissue culture system | |
| CN104304002B (en) | Inducing method of lonicera confusa leaf adventitious bud | |
| CN102823499B (en) | Factorized breeding method of blueberry tissue-cultured seedlings | |
| CN109644875A (en) | A kind of method of celery microspore callus differentiation and regeneration plant | |
| CN103695365A (en) | Buckwheat cell culture method for improving buckwheat cell synchronization | |
| CN104094840A (en) | Establishment method for Fraxinus rhynchophylla Hance. suspension culture system | |
| CN106718940A (en) | A kind of tissue culture and rapid propagation method suitable for tung oil tree |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20101110 |