CN102613082A - Modified medium for improving propagation of stems of Dendrobium officinale and propagation method - Google Patents
Modified medium for improving propagation of stems of Dendrobium officinale and propagation method Download PDFInfo
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- CN102613082A CN102613082A CN2012100972381A CN201210097238A CN102613082A CN 102613082 A CN102613082 A CN 102613082A CN 2012100972381 A CN2012100972381 A CN 2012100972381A CN 201210097238 A CN201210097238 A CN 201210097238A CN 102613082 A CN102613082 A CN 102613082A
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Abstract
The invention discloses a modified medium for improving propagation of stems of Dendrobium officinale and a propagation method. The medium comprises MS (Murashige and Skoog) basal medium, cane sugar 20g/l, agar 6g/L, 6-BA (benayl aminopurine) 2mg/l, NAA (alpha-naphthaleneacetic acid) 0.2-0.5mg/l, banana mash 15-20g/l, potato mash 15-20g/l, and common tap water replacing distilled water. The modified MS medium is used to propagate the stems with axillary buds of Dendrobium officinale, alkaloid and polysaccharide content of Dendrobium officinale is evidently increased, and Dendrobium officinale grow normally. By the method, consumption of cane sugar is reduced, the tap water is used to replace the distilled water, and accordingly tissue culture technology for Dendrobium officinale is more convenient, more efficient and resource-saving.
Description
Technical field
The invention belongs to agricultural technology field, be specifically related to a kind of dendrobium candidum stem section propagation that promotes and expand numerous improved culture medium and utilize this medium to breed expanding propagation method.
Background technology
Dendrobium candidum (Dendrobium officinale) is a herbaceos perennial, belongs to the epiphytic orchid class, the popular name Tiepi Fengdou; Because of the outer black in color of its old stem; Have another name called and be ribbed hedyotis herb, the stem of noble dendrobium is the rare Chinese medicine of using always, and applicating history is long; In Shennong's Herbal, classified as top grade, confirmed at present it have nourishing Yin and clearing heat, the beneficial stomach that promotes the production of body fluid, relieving cough and moistening lung, wet one's whistle make eye bright, improve immune function of human body, anti-curing oncoma and angiocardiopathy and improve sleep, prevent and treat cataract, effect such as promote longevity.But limit to distributing because growing environment is special, and excavate for a long time, natural resources is exhausted day by day, has been classified as the natural crude drugs of focused protection by country.
Dendrobium candidum mainly is distributed in the subtropical zone, and China is distributed in southwest and southern coastal provinces, and the dendrobium candidum seed is minimum; No endosperm needs under the natural conditions could sprout with some mycosymbiosis, is difficult to cultivate with seedling; And the reproduction rate of modes such as traditional plant division, cuttage is extremely low, adds and excessively excavates artificially, and is endangered; In order to keep the merit of kind, breeding in a large number fast produces protocorm through the stem-tip tissue culture technique again; Carry out enrichment culture in the protocorm stage; Make protocorm breed into plantlet again, obtain to produce through successive transfer culture constantly again and use seedling, could directly plant through refining seedling but such process must spend the time in 1 year at least.In order to improve productivity effect; Save production cost and the production time, we adopt directly the propagation through dendrobium candidum stem section (selection has the stem section of axillalry bud) to expand and numerously obtain production and use seedling, are that the approach of breeding of explant is simple relatively with the stem section; Only need 30～50 days from the stem section → bud → whole plant of growing thickly; Leaf is dark green, and seedling growth is healthy and strong, need not pass through can directly be used to take root strong sprout.So not only save time, and the cultivation survival rate behind the refining seedling is higher, is more suitable for large-scale batch production production.
Because dendrobium candidum has huge medical value, has caused long-term immoderate harvesting, causes resource to be on the verge of exhaustion.Country in 1987 has classified dendrobium candidum as and has laid special stress on protecting wild rare medicinal material kind, and country's first " Chinese rare and endangered protective plant register " is also listed it wherein in.In order to protect the dendrobium candidum wild resource better, become wild and plant into family, should greatly develop the large-scale artificial cultivation, ensure the dendrobium candidum Sustainable utilization of resources conscientiously.From the seventies in 20th century; Appropriate authority has just begun the R&D work of dendrobium candidum both at home and abroad; Especially in the tissue culture technique field of dendrobium candidum, but culture medium preparation needs a large amount of distilled water and sucrose in the tissue culture procedures, and producing of the preparation of distilled water and sucrose all needs special instrument and power consumption very big; Increase cost burden, caused very big waste.
Summary of the invention
To the problems referred to above that exist on the prior art, the object of the present invention is to provide a kind of improved culture medium and propagation expanding propagation method thereof of practicing thrift more, save time, promoting easily that the expansion of dendrobium candidum stem section propagation is numerous.Medium of the present invention reduces waste of material and time loss, environmental protection more, and group training is more efficiently produced, and producing for large-scale group training provides advantage.
The objective of the invention is to realize in the following manner:
A kind of dendrobium candidum stem section propagation that promotes expands numerous medium, and this medium comprises following component: MS minimal medium (claiming the MS medium again, commercially available routine); Sucrose 20g/l; Agar 6g/l, 6-BA (benayl aminopurine) 2mg/l, NAA (NAA) 0.2～0.5mg/l; Banana puree 15～20g/l, mashed potatoes 15～20g/l; PH5.6～5.8, this medium is by the running water constant volume.
Described mashed potatoes is fresh potato with banana puree and smashs into the mud acquisition to pieces with fresh banana, and more natural, nutrient component is abundanter, promotes the propagation of dendrobium candidum stem section to expand numerous.
Utilizing above-mentioned medium to carry out the numerous method of dendrobium candidum propagation expansion may further comprise the steps: the dendrobium candidum stem section of getting axillalry bud; In above-mentioned medium; Under 23～27 ℃ of cultivation temperature, illuminance 1500～2000lx, light application time 12h/d condition, cultivated 30～50 days, preferably cultivated 30～35 days.The stem section selects to have the stem section of axillalry bud, like this can be so that inductivity reaches more than 95%.
Culture medium preparation is extremely important in the dendrobium candidum tissue culture procedures, and medium not only requires under aseptic condition, the most important thing is to give the growth of dendrobium candidum that required element and suitable growing environment is provided.The more important thing is that medium satisfying under the condition of plant growing, can reduce production costs, environmental protection more is adapted to the production demand.Tradition MS culture medium preparation needs macroelement, trace element, organic matter, molysite, sucrose, agar and distilled water.And distilled water produce the equipment that needs specialty, not only power consumption, and will waste more professional equipment, electric resources and time and just can obtain distilled water.Medium of the present invention needing to have replaced the distilled water of professional equipment preparation with common running water; Not only dendrobium candidum stem section propagation is expanded numerous influence that has no; And reduce production costs greatly, practice thrift the production time, reached efficient, safe, controlled requirement in the traditional Chinese medicine production process.And; The present invention adopt promote dendrobium candidum stem section propagation expand numerous improved culture medium with cane sugar content 30g/l from being reduced to 20g/l; Expand in numerous group training process in dendrobium candidum stem section propagation, the dendrobium candidum growth does not change, and dendrobium polysaccharide and alkaloids content are still stable.
With prior art beneficial effect more of the present invention: the present invention directly adopts the modified MS medium of running water preparation to replace the general medium of distilled water preparation; Reduced cane sugar content; The propagation of stem section in improved culture medium of selecting to be fit to expands numerous be convenient to more produce needs, mature and feasible on tissue culture technology, considerable benefit economically; Benefit the nation and the people, adopt this technology to make the tissue culture technique of dendrobium candidum convenient, economize on resources simultaneously efficiently.
Description of drawings
Fig. 1 expands numerous comparison diagram for the dendrobium candidum stem section propagation of the MS medium that the modified MS medium and the distilled water of the preparation of employing running water are prepared.
Wherein, right bottle expands numerous for the present invention adopts the dendrobium candidum stem section of the modified MS medium of running water preparation to breed, and left side bottle expands numerous for the dendrobium candidum stem section propagation of the MS medium of employing distilled water preparation.
Embodiment
Below in conjunction with embodiment the present invention is further specified.
The dendrobium candidum stem section that axillalry bud is arranged that embodiment 1 gets the long length that 3～4 stem segments are arranged under aseptic condition be 1.5～2cm is as explant (deriving from the aseptic seedling of dendrobium candidum); After the improved culture medium for preparing (pH5.8) carried out high-temperature sterilization; With this dendrobium candidum stem section in the improved culture medium of running water preparation; In temperature is (25 ± 2) ℃, and illuminance 1500～2000lx cultivated 30 days under the condition of light application time 12h/d; Dendrobium officinale polysaccharide content in the improved culture medium of mensuration and the preparation of record ordinary tap water is cultivated and is carried out after 30 days carrying out acclimatization and transplants behind the successive transfer culture next time.
The improved culture medium component (running water is settled to 1L) of running water preparation:
| Constituent | Quantity (mg/l) | Constituent | Quantity (mg/l) |
| Ammonium nitrate | 1650 | Sodium molybdate | 0.25 |
| Potassium nitrate | 1900 | Copper sulphate | 0.025 |
| Calcium chloride | 440 | Cobalt chloride | 0.025 |
| Magnesium sulfate | 370 | Iron sulfate | 27.8 |
| Potassium dihydrogen phosphate | 170 | Agar | 6000 |
| KI | 0.83 | Sucrose | 20000 |
| Boric acid | 5.2 | 6-BA (benayl aminopurine) | 2 |
| Magnesium sulfate | 22.3 | NAA (NAA) | 0.5 |
| Zinc sulphate | 3.6 | Banana puree | 20000 |
| Mashed potatoes | 20000 |
The dendrobium candidum stem section that axillalry bud is arranged that embodiment 2 gets the long length that 3～4 stem segments are arranged under aseptic condition be 1.5～2cm is as explant (deriving from the aseptic seedling of dendrobium candidum); After the improved culture medium for preparing (pH5.6) carried out high-temperature sterilization; With this dendrobium candidum stem section in the improved culture medium of running water preparation; In temperature is (25 ± 2) ℃, and illuminance 1500～2000lx cultivated 35 days under the condition of light application time 12h/d; Measure and write down Dendrobium officinale polysaccharide content in the improved culture medium of ordinary tap water preparation respectively, cultivate and carry out after 30 days carrying out acclimatization and transplants behind the successive transfer culture next time.
The improved culture medium component (running water is settled to 1L) of running water preparation:
The dendrobium candidum stem section of axillalry bud that will have embodiment 3 adopts the medium of distilled water preparation to carry out tissue culture according to the method for embodiment 1, incubation time 40～50 days.
The MS nutrient media components (distilled water is settled to 1L) of distilled water preparation:
Extracting dendrobium officinale polysaccharide method: get the dendrobium candidum stem section drying sample after the tissue culture, accurately claim fixed in right amount,, filter through benzinum (60～90 ℃) backflow degreasing 1h; Fling to solvent, extract 1h, take advantage of heat filtering, filter residue is added water refluxing extraction 1h with 80% alcohol reflux; Take advantage of heat filtering, filtrating concentrates, and adds 0.1% activated carbon decolorizing, filters; Add 95% ethanol and make solution contain alcohol 80%, hold over night is filtered; With absolute ethyl alcohol, acetone washs 8 times successively, and the gained polysaccharide is in 60 ℃ of dry for standby.The accurate 0.5ml that draws presses operation under the calibration curve item, obtains concentration of glucose in each polysaccharide liquid, is calculated as follows polyoses content:
Polyoses content %=Cs.D.f/W * 1000
Cs is need testing solution concentration of glucose (ug/ml) in the formula, and D is the dilution factor of need testing solution, and f is a conversion factor, and W is a test sample weight.
Dendrobium polysaccharide composition, growth and development state comparative result are seen table 1
The comparison sheet of table 1 embodiment 1-3 culture polyoses content and upgrowth situation
Experimental result shows: there is a big difference for the polyoses content of the dendrobium candidum in the improved culture medium of ordinary tap water preparation and the common distilled water medium; This gap is mainly derived under the normal condition of dendrobium candidum upgrowth situation; The increase of the reduction of cane sugar content and stem section growth coefficient can better promote photosynthesis and improve the photosynthetic carbon assimilation speed of plant, its dendrobium polysaccharide content is increased.
Embodiment 4 measures and writes down total alkaloid content in the embodiment 1-3 culture.
Dendrobium candidum total alkaloid contents assay method: material is cleaned with distilled water, placed 105 ℃ in baking oven, the 30min that completes, 60 ℃ are spent the night, dry to constant weight.Subsequent use with mortar grinding (crossing 60 mesh sieves).Take by weighing sample 0.400g through drying and crushing in 250ml ground conical flask, wetting with an amount of concentrated ammonia liquor, close plug 30min adds chloroform 10.0ml, weighs, and puts reflux 2h in the water-bath, and the cooling back replenishes chloroform to original weight.Filter, get subsequent filtrate 2.0ml, add chloroform, shake up, be liquid to be measured.Draw above-mentioned liquid to be measured in separatory funnel, be diluted to 10.0ml, add the buffer solution 5.0ml of pH4.6 with chloroform, add again 1.0ml0.04% to smell cresols green, shaken leaves standstill.Filter, get subsequent filtrate 6.0ml, add 0.01molL
-1, NaoH solution shakes up.Other gets the same operation repetitive of 10.0ml chloroform give, as blank.Survey absorbance in the 630nm wavelength.Experiment replication 3 times.
Alkaloid in the sample: alkaloid (%)=C * D/W * 10
-6* 100%
The sample alkaloid concentration (ug/ml) of C for calculating in the formula according to calibration curve; D is the extension rate of sample solution; W is the weight (g) of sample.
Chromatographic peak relative integral area and total alkaloid content comparative result are seen table 2
The comparison sheet of table 2 embodiment 1-3 culture total alkaloid content
Experimental result shows: the culture total alkaloid content after employing ordinary tap water improved culture medium and the distilled water medium tissue culture has very big difference.This growth conditions with dendrobium candidum is relevant; The cane sugar content of low concentration is more suitable for the photosynthesis of dendrobium candidum and has improved the photosynthetic carbon assimilation speed of plant; Its dendrobium polysaccharide content is increased; And improved culture medium more help the accumulation of plant total alkaloid be because the TA amount of dendrobium candidum stem section itself just greater than other position, and ordinary tap water can provide more mineral element with respect to distilled water, makes the TA amount content increase of dendrobium candidum.
The comparison that embodiment 5 embodiment 1-3 culture stem sections propagation expands numerous coefficient.
Through comparing, the effect of in proliferated culture medium, adding an amount of mashed potatoes and banana puree is fine, makes growth coefficient high, and the aseptic seedling growth is normal, situation such as stem tubbiness, vitreous, yellow and water stain shape do not occur, and is long and sturdy between stipes.
Dendrobium candidum stem section propagation in the general medium that the improved culture medium and the distilled water of running water preparation are prepared expands the comparative result of numerous coefficient and sees table 3
Table 3 embodiment 1-3 culture stem section propagation expands the comparison sheet of numerous coefficient
The result shows: the stem section is grown faster in the ordinary tap water improved culture medium; Growth coefficient is higher; The growth and the formation that utilize root are more arranged, and dendrobium candidum can absorb more nutrient through the root that grows on the stem section like this, thereby accumulates more polysaccharide; Can shorten the production time through improving the propagation multiplication technique simultaneously, enhance productivity.
Claims (4)
1. one kind promotes dendrobium candidum stem section propagation to expand numerous improved culture medium, it is characterized in that consisting of of this medium: MS minimal medium, sucrose 20g/l; Agar 6g/l, benayl aminopurine 2mg/l, NAA 0.2～0.5mg/l; Banana puree 15～20g/l, mashed potatoes 15～20g/l; PH5.6～5.8, this medium is by the running water constant volume.
2. promotion dendrobium candidum stem section propagation according to claim 1 expands numerous improved culture medium, it is characterized in that described banana puree and mashed potatoes are respectively fresh potato and smash into mud to pieces with fresh banana.
3. one kind is utilized the described medium of claim 1 to carry out the numerous method of dendrobium candidum propagation expansion; It is characterized in that getting the dendrobium candidum stem section of axillalry bud; In the described medium of claim 1, under 23～27 ℃ of cultivation temperature, illuminance 1500～2000lx, light application time 12h/d condition, cultivated 30～50 days.
4. method according to claim 3 is characterized in that described incubation time is 30～35 days.
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Cited By (14)
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| CN102972299A (en) * | 2012-12-10 | 2013-03-20 | 黄怀毅 | Rapid propagation method for dendrobium officinale stem tissue culture |
| CN103039368A (en) * | 2013-01-27 | 2013-04-17 | 云南天泉生物科技股份有限公司 | Sugar-free tissue culture method of dendrobium officinale |
| CN103053425A (en) * | 2013-01-22 | 2013-04-24 | 杨宝明 | Rapid propagation method for tissue cultivation of dendrobium candidum stem |
| CN103229718A (en) * | 2013-04-24 | 2013-08-07 | 浙江韵芝堂生物科技有限公司 | Tissue culture rapid propagation method of Dendrobium officinale |
| CN103229719A (en) * | 2013-04-24 | 2013-08-07 | 浙江韵芝堂生物科技有限公司 | Liquid suspension culture method of Dendrobium officinale |
| CN103408370A (en) * | 2013-08-14 | 2013-11-27 | 宁波枫康生物科技有限公司 | Dendrobium officinale imitate-wild cultivation seedling medium formula |
| 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 |
| CN103733990A (en) * | 2013-12-16 | 2014-04-23 | 莫文油 | Horsewhip dendrobium nobile tissue culturing method |
| CN104206282A (en) * | 2014-10-10 | 2014-12-17 | 江苏神草生物科技有限公司 | Culture medium for dendrobium tissue cultivation |
| CN104509440A (en) * | 2014-12-22 | 2015-04-15 | 广西南宁桂知科技有限公司 | Culture medium for culturing dendrobium officinale tissue cultured seedling and method for preparing culture medium |
| CN104969863A (en) * | 2015-07-08 | 2015-10-14 | 厦门加晟生物科技有限公司 | Dendrobium officinale tissue culture propagation method |
| CN105230489A (en) * | 2015-11-02 | 2016-01-13 | 大新县生产力促进中心 | Tissue culture fast propagation method for dendrobium candidum |
| CN108684524A (en) * | 2018-05-16 | 2018-10-23 | 芜湖市三山区绿色食品产业协会 | The method for tissue culture of dendrobium candidum |
| CN109293668A (en) * | 2018-11-01 | 2019-02-01 | 仲恺农业工程学院 | A kind of extracting method of alkaloid |
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| CN102972299A (en) * | 2012-12-10 | 2013-03-20 | 黄怀毅 | Rapid propagation method for dendrobium officinale stem tissue culture |
| CN103053425B (en) * | 2013-01-22 | 2014-11-12 | 杨宝明 | Rapid propagation method for tissue cultivation of dendrobium candidum stem |
| CN103053425A (en) * | 2013-01-22 | 2013-04-24 | 杨宝明 | Rapid propagation method for tissue cultivation of dendrobium candidum stem |
| CN103039368A (en) * | 2013-01-27 | 2013-04-17 | 云南天泉生物科技股份有限公司 | Sugar-free tissue culture method of dendrobium officinale |
| CN103039368B (en) * | 2013-01-27 | 2014-02-05 | 云南天泉生物科技股份有限公司 | Sugar-free tissue culture method of dendrobium officinale |
| CN103229718A (en) * | 2013-04-24 | 2013-08-07 | 浙江韵芝堂生物科技有限公司 | Tissue culture rapid propagation method of Dendrobium officinale |
| CN103229719A (en) * | 2013-04-24 | 2013-08-07 | 浙江韵芝堂生物科技有限公司 | Liquid suspension culture method of Dendrobium officinale |
| CN103229718B (en) * | 2013-04-24 | 2016-04-13 | 浙江韵芝堂生物科技有限公司 | A kind of tissue culture and rapid propagation method of dendrobium candidum |
| CN103229719B (en) * | 2013-04-24 | 2016-04-13 | 浙江韵芝堂生物科技有限公司 | A kind of method that dendrobium candidum liquid suspension is cultivated |
| CN103408370A (en) * | 2013-08-14 | 2013-11-27 | 宁波枫康生物科技有限公司 | Dendrobium officinale imitate-wild cultivation seedling medium formula |
| 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 |
| CN103733990A (en) * | 2013-12-16 | 2014-04-23 | 莫文油 | Horsewhip dendrobium nobile tissue culturing method |
| CN104206282A (en) * | 2014-10-10 | 2014-12-17 | 江苏神草生物科技有限公司 | Culture medium for dendrobium tissue cultivation |
| CN104509440A (en) * | 2014-12-22 | 2015-04-15 | 广西南宁桂知科技有限公司 | Culture medium for culturing dendrobium officinale tissue cultured seedling and method for preparing culture medium |
| CN104509440B (en) * | 2014-12-22 | 2016-10-05 | 广西南宁桂知科技有限公司 | A kind of candidum tissue culturing seedling culture medium and preparation method thereof |
| CN104969863A (en) * | 2015-07-08 | 2015-10-14 | 厦门加晟生物科技有限公司 | Dendrobium officinale tissue culture propagation method |
| CN105230489A (en) * | 2015-11-02 | 2016-01-13 | 大新县生产力促进中心 | Tissue culture fast propagation method for dendrobium candidum |
| CN108684524A (en) * | 2018-05-16 | 2018-10-23 | 芜湖市三山区绿色食品产业协会 | The method for tissue culture of dendrobium candidum |
| CN109293668A (en) * | 2018-11-01 | 2019-02-01 | 仲恺农业工程学院 | A kind of extracting method of alkaloid |
| CN109293668B (en) * | 2018-11-01 | 2020-03-31 | 仲恺农业工程学院 | Method for extracting alkaloid |
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