CN102948370A - Rapid propagation method of nothapodytes pittosporoides - Google Patents
Rapid propagation method of nothapodytes pittosporoides Download PDFInfo
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Abstract
The invention relates to a rapid propagation method of nothapodytes pittosporoides, and the method is characterized in that tissues such as tender shoot sections are cultivated on a murashige and skoog (MS) culture medium, a tender shoot stem section with one to two sprouts is cut as explants, the explants are grafted in MS+6-butyl acrylate (BA) 1.2mg/L+ indole butyric acid (IBA)0.2mg/L culture medium after being sterilized, the bud is germinated in 10 to 15 days after the grafting, seedlings with leaves are formed in 30 days, the seedlings are cut off and transferred onto the MS+6-BA1.2mg/L+IBA0.4mg/L culture medium to keep the high-speed increase, and the propagation coefficient is more than 5. The MS+6-BA0.5mg/L+ naphthyl acetic acid (NAA)1.5mg/L is selected as rooting culture medium, the rooting rate reaches more than 95 percent, and a complete plant can be formed in 30 days. Test-tube plantlets are transplanted in sandy soil, attention is paid on the moisture preservation, the transplanting survival rate can reach up to 93 percent after the test-tube plantlets are transplanted for 45 days, and a high-frequency stable renewable system is established. The method has the advantages of good stability, simplicity and convenience in operation, fast propagation speed, low production cost, industrialization and the like.
Description
Technical field
The present invention relates to bioengineering field, relate to plant tissue culture technique, specifically a kind of method for tissue culture of mappia foetida.
Background technology
Plant is the natural treasure-house of medicine, and people utilize the historical of long standing and well established of medicinal plant, and approximately there is 75% population in the whole world with the medicament sources (Xing Jianmin, 2001) of plant as the Prevention disease.The mankind have found many medicines with height physiologically active from plant, the medicine from plant origin accounts for more than 25% of medicine total amount (Zheng Guangzhi, 1987) at present.The history in existing thousands of years of the Chinese traditional herbs of China is still widely used in China and many countries and regions so far.But because traditional Chinese herbal medicine acquisition methods is that to gather and consume a large amount of wild plant resources be cost, when gathering and consumption when surpassing the regeneration capacity of natural resources, will inevitably causes the in imminent danger of species even become extinct.Along with the day by day destruction of natural ecological environment, also further cause the scarcity of resources of medicinal plant simultaneously.Biotechnology flourish for the production that fundamentally changes traditional medicine provides a brand-new method, the plant tissue and cell culture technology then is one of them important means.China is since professor Luo Shiwei in 1964 has reported that at first ginseng tissue is cultivated the achievement in research that succeeds, and many scientists successively have been engaged in the Study on tissue culture of multiple medicinal plant.So far, the Study on tissue culture of China's medicinal plant develops rapidly.At present, countries in the world are all attached great importance to the medicinal R and D of plant, with regard to the U.S., have 47% to make (Xie Qikun, 1986) take plant as raw material in its patent medicine.In order to solve the imbalance between supply and demand of medicinal plant, people adopt tame method to enlarge the medicine source.But in tame medicinal plant, there is the production cycle such as many rare medicinal herbss longer, such as ginseng, the coptis, if with the conventional method breeding or grow seedlings, needs the cost long time; Other has some medicinal plants such as the bulb of fritillary, Crocus sativus etc., and, consumption little because of reproduction coefficient planted amount greatly, causes breeding speed very slow, and production cost increases.Utilize the sexual propagation mode of plant larger to its active constituent content fluctuation ratio, so utilize plant regeneration and the breeding problem of plant tissue culture technique solution medicinal plant extremely urgent.In recent years, do a lot of work in this respect both at home and abroad, the medicinal plant that successful cultured in vitro obtains test tube plantlet has 200 kinds at least, as: Yunnan ribbed hedyotis herb, trilliaceae, sachalin rhodiola rhizome, curcuma zedoary, Saussurea medusa, star flower meadow sweet, rabdosia lophanthide, Radix Mussaendae, east area of the Liao River, coextensive with eastern and southern Liaoning Province pomegranate wood etc.; Has the rare plant of anticancer effective component comprising some, such as Chinese yew, mappia foetida, Chinese littleleaf box, euphorbia, catharanthus roseus, Aglaia odorata, paris polyphylla, dog tartar and Chinese torreya etc.
Mappia foetida has another name called public yellow pearl, starflowerlike loosestrife root or herb, belongs to the Icacinaceae plant, perennial brushwood, high 2-3(-10) m.Branch has rib, and by pubescence, rear change is without hair, and bud is by pubescence.Leaf alternate or branch top are closely to life; The long 1-3cm of petiole, the wide dark essence of upper mask, in the groove by strigose; Blade Long Circle or the lanceolar that falls, long 7-24cm, wide 2-4.5cm, the long gradually point of tip, the base portion wedge shape, full edge, above dirty-green, tool gloss; Middle arteries and veins is recessed, and lateral vein 6-7 pair, arc Qu Shangsheng is away from leaf margin place anastomose.Flower both sexes or polygamy, give birth on the cyme top, is about 7cm, and total stalk, branch, rhachis are usually flat, by thick volt hair, the long 1-2mm of bennet; Calyx is green, and is bell, is about 2mm, and the rare quilt in outside slightly lies prostrate hair, 5 carnassial tooths, carnassial tooth triangle; Petal is yellow, and linear, warp is about 7mm, wide approximately 2mm; Stamen 5 is about 5mm, the long 4-5mm of filigree, and base portion is slightly thick, and flower pesticide is avette; Ovary is subsphaeroidal, and by long bristle, style is green, is about 2mm, the column cap head; The floral disc meat, the irregular sliver of tool or dark knuckle-tooth, dredge by long bristle the inside, and deposit the place during fruit.Drupe is oval, and is slightly flat, and young fruit is green, turns yellow, is red when ripe, long 1-2cm, and footpath 0.6-0.8cm, the obvious tool squama of tip navel has the calyx place to deposit.The florescence 4-6 month, the fruit phase 6-8 month.Ecotope: be born in height above sea level (150) 450-(2500) in the woods of m.Resource distribution: be distributed in the ground such as Gansu, Hubei, Hunan, Guangdong, Hainan, Guangxi, Sichuan, Guizhou.
The methoxy derivatives that contains camptothecine and camptothecine in the mappia foetida root skin, its active ingredient are more than three times of Common Camptotheca Fruit, and the content of camptothecine has reached 0.8%.The mappia foetida seed is difficult to germinate, and mainly with the breeding of rhizome bud, wild mappia foetida content in the root skin is unstable in addition for wild mappia foetida, and its active constituent content is relevant with the growth year of wild mappia foetida, and is general more consistent at the wild mappia foetida content more than 10 years.So the extraction with wild mappia foetida can cause resource exhaustion, cause in addition the output of its camptothecine unstable.Publication number is the technical method that the Chinese patent of CN102071233A discloses the synthetic camptothecine of a kind of artificial induction's form nothapodytes endophyte, be the ratio of 50-200g/L weight in wet base according to inoculum concentration with the form nothapodytes immature cell, place the growth regulator methyl α-naphthyl acetate (NAA) that added 50-80 μ mol/L or the MS minimal medium of 5-10 μ mol/L benayl aminopurine (BA), in 25-30 ℃, cultivate; At the cell index early growth period, adding concentration is the abiotic derivant of 1-2000 μ mol/L and sucrose or the glucose of 20-50g/L; Grow mid-term at cell index, adding concentration is the abiotic derivant of 1-2000 μ mol/L again.Though this invention has utilized the plant form nothapodytes that is rich in camptothecine, by the artificial induction, the method that tissue is cultivated realizes the synthetic of camptothecine, can improve to a certain extent the output of camptothecine, because due to camptothecine accumulates in its root skin for a long time, so be difficult to suitability for industrialized production.
Therefore, in order to satisfy the medicinal demand that day by day increases, seed harvest yield is little; the seminal propagation emergence rate is extremely low, and the cultivating seeds process is loaded down with trivial details, and growth rate is slow; protect simultaneously the wild resource of mappia foetida, utilize group culturation rapid propagating technology, realize that artificial cultivation is best solution.
Up to now, there is not yet report about the success of mappia foetida tissue culture quick breeding at home and abroad.We find out the ripe method of a cover finally by lot of experiments, have successfully realized the tissue-culturing rapid propagation of mappia foetida, can go out a large amount of seedling by quickly breeding, provide possibility for realizing industrialization artificial cultivation mappia foetida.The successful foundation of the group culturation rapid propagating technology method of mappia foetida can provide seedling for extensive artificial cultivation medicinal plant mappia foetida, for the modern science and technology agricultural provides a kind of practicable exploration project.
Summary of the invention
The method for quickly breeding that the purpose of this invention is to provide mappia foetida, the method are fit to suitability for industrialized production very much, and the prescription effect is good, and bud ratio is high, and incubation time is short, and plant strain growth is vigorous, and is workable, the using value advantages of higher.Has less investment, the characteristics that output is high.
The method for quickly breeding of mappia foetida of the present invention, its concrete operation step is as follows:
1) the choosing and sterilizing of explant: select on the tender stem of mappia foetida sprout as culture materials, clean through liquid detergent and running water, put and wash after 10-30 minute under the flowing water, place 75% alcohol to process 10-30 second, and then after the sterilization of 0.1% mercuric chloride, use aseptic water washing, cut after the part of explant variable color for subsequent use;
2) sprouting of sprout: the sprout after the sterilization treatment is seeded on the Ms+6-BA1.2mg/L+IBA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 12 hours, intensity of illumination 1000LX, the sprouting of 10-15 days sprouts after the inoculation forms the seedling with leaf about 30 days;
3) inducing clumping bud and propagation are cultivated: seedling is downcut transfer on the Ms+6-BA1.2mg/L+IBA0.4mg/L medium, until formation Multiple Buds, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 15 hours, intensity of illumination 1000LX is a growth cycle about 25 days, increase progressively at a high speed keeping, growth coefficient is more than 5;
4) culture of rootage: select Ms+6-BA0.5mg/L+NAA 1.5mg/L as root media, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 16 hours, intensity of illumination 1000LX, rooting rate reach more than 95%, and only needs formed whole plant in about 30 days;
5) transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water the agar of root is cleaned up, transfer is noted the water conservation heat and moisture preserving to sandy soil or rich supporting in the soil, transfer 45 days, and the transfer survival rate reaches more than 92%.
In a specific embodiments, adopt 0.1% mercuric chloride to carry out disinfection in the step 1, disinfecting time is controlled preferably at 12-15 minute.
In another embodiment, field-transplanting humidity is controlled at 40-70% in the step 5; Temperature is controlled at 20-30 ℃.
Technique effect:
(1) utilize mappia foetida band bud rhizome section to be explant, be easier to so that explant is drawn materials, all can draw materials throughout the year, and quantity be many, adopt the rhizome sprout to do explant, guaranteed genetic stability, overcome simultaneously in the tissue cultivation, callus stops growing gradually along with the differentiation of bud on differential medium, lose the weakness of multiplication capacity, can prevent in the injured tissue Subculture, along with the increase of cultivating algebraically, the phenomenon of variation appears in differentiation seedling out.
(2) the inventive method utilizes explant to set up, and propagation this technology path of synchronization of taking root only needs to form whole plant in about 30 days, obtains a large amount of whole plants in 4-6 month, realizes that the large-scale industrialized of mappia foetida grow seedlings.
(3) matrix that adopts when transfer of the test-tube plantlet that obtains of the inventive method is utilized sandy soil, and method is simple, and greatly reduces the transfer cost.This cultural method sprouts soon, and the rate of increase is high, and method is simple, and production cost is low, can be mass, and using value is high.
(4) production of mappia foetida can be carried out under the manual control condition, is not subjected to the restriction of the factors such as season, weather conditions and soil, can get rid of the invasion and attack of damage by disease and insect and the impact of residue of pesticide, strictly controls the quality of mappia foetida.Growth rate is fast, and is with short production cycle, and equipment is simple, and floor space is few, can save human and material resources etc., is convenient to batch production production.
The mappia foetida seedling of (5) using group culturation rapid propagating technology provided by the invention to obtain carries out artificial cultivation, can obtain individual difference little, the mappia foetida finished product of quality homogeneous.
(6) can preserve the germ plasm resource of mappia foetida, be conducive to simultaneously protect the mappia foetida wild resource, reduce the destruction to natural resources.
Embodiment
Below, the present invention will be further detailed with embodiment, but it is not limited to any or the similar example of these embodiment.
Embodiment 1
Draw materials: the mappia foetida stem segment with axillary bud is explant
1, the choosing and sterilizing of explant: the tender tissues such as the fresh and tender leaf stem section of mappia foetida that pick up from Cili, Hunan, as culture materials, clean through liquid detergent and running water, put and wash after 15 minutes under the flowing water, place 75% alcohol to process 10-30 second, and then use aseptic water washing through the sterilization of 0.1% mercuric chloride after 12 minutes, cut after the part of explant variable color for subsequent use.
2, the sprouting of sprout: the sprout after the sterilization treatment is seeded on the Ms+6-BA1.2mg/L+IBA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 25-27 ℃, and illumination every day 12 hours, intensity of illumination 1000LX; Inoculate the sprouting of rear 10 days sprouts, form the seedling with leaf about 30 days.
3, inducing clumping bud and propagation are cultivated: seedling is downcut transfer on the Ms+6-BA1.2mg/L+IBA0.4mg/L medium, until form Multiple Buds, the pH value is 5.8, cultivation temperature 25-27 ℃, and illumination every day 15 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping, growth coefficient is more than 5.
4, culture of rootage: select Ms+6-BA0.5mg/L+NAA 1.5mg/L as root media, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 16 hours, intensity of illumination 1000LX, rooting rate reach more than 95%, and only needs formed whole plant in about 30 days.
5, the transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water the agar of root is cleaned up, transfer is in sandy soil, and humidity is controlled at 60%; Temperature is controlled at 25 ℃, and transfer is after 45 days, and the transfer survival rate reaches more than 96%.
Embodiment 2
Draw materials: the mappia foetida stem segment with axillary bud is explant
1, the choosing and sterilizing of explant: the tender tissues such as the fresh and tender leaf stem section of mappia foetida that pick up from Cili, Hunan, as culture materials, clean through liquid detergent and running water, put and wash after 10 minutes under the flowing water, place 75% alcohol to process 10-30 second, and then use aseptic water washing through the sterilization of 0.1% mercuric chloride after 15 minutes, cut after the part of explant variable color for subsequent use.
2, the sprouting of sprout: the sprout after the sterilization treatment is seeded on the Ms+6-BA1.2mg/L+IBA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 25-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX; Inoculate the sprouting of rear 15 days sprouts, form the seedling with leaf about 30 days.
3, inducing clumping bud and propagation are cultivated: seedling is downcut transfer on the Ms+6-BA1.2mg/L+IBA0.4mg/L medium, until form Multiple Buds, the pH value is 5.8, cultivation temperature 25-27 ℃, and illumination every day 15 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping, growth coefficient is more than 5.
4, culture of rootage: select Ms+6-BA0.5mg/L+ NAA1.5mg/L as root media, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 16 hours, intensity of illumination 1000LX, rooting rate reach more than 95%, and only needs formed whole plant in about 30 days.
5, the transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water the agar of root is cleaned up, transfer is in sandy soil, and humidity is controlled at 50%; Temperature is controlled at 28 ℃, and transfer is after 45 days, and the transfer survival rate reaches more than 94%.
Embodiment 3
Draw materials: the mappia foetida stem segment with axillary bud is explant
1, the choosing and sterilizing of explant: the tender tissues such as the fresh and tender leaf stem section of mappia foetida that pick up from Sangzhi, Hunan, as culture materials, clean through liquid detergent and running water, put and wash after 30 minutes under the flowing water, place 75% alcohol to process 10-30 second, and then use aseptic water washing through the sterilization of 0.1% mercuric chloride after 13 minutes, cut after the part of explant variable color for subsequent use.
2, the sprouting of sprout: the sprout after the sterilization treatment is seeded on the Ms+6-BA1.2mg/L+IBA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 25-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX; Inoculate the sprouting of rear 12 days sprouts, form the seedling with leaf about 30 days.
3, inducing clumping bud and propagation are cultivated: seedling is downcut transfer on the Ms+6-BA1.2mg/L+IB0.4mg/L medium, until form Multiple Buds, the pH value is 5.8, cultivation temperature 25-27 ℃, and illumination every day 15 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping, growth coefficient is more than 5.
4, culture of rootage: select Ms+6-BA0.5mg/L+NAA1.5mg/L as root media, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 16 hours, intensity of illumination 1000LX, rooting rate reach more than 95%, and only needs formed whole plant in about 30 days.
5, the transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water the agar of root is cleaned up, transfer is in sandy soil, and humidity is controlled at 70%; Temperature is controlled at 20 ℃, transfer 45 days, and the transfer survival rate reaches more than 93%.
Claims (3)
1. the method for quickly breeding of a mappia foetida, its concrete operation step is as follows:
1) the choosing and sterilizing of explant: select on the tender stem of mappia foetida sprout as culture materials, clean through liquid detergent and running water, put and wash after 10-30 minute under the flowing water, place 75% alcohol to process 10-30 second, and then after the sterilization of 0.1% mercuric chloride, use aseptic water washing, cut after the part of explant variable color for subsequent use;
2) sprouting of sprout: the sprout after the sterilization treatment is seeded on the Ms+6-BA1.2mg/L+IBA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 12 hours, intensity of illumination 1000LX, the sprouting of 10-15 days sprouts after the inoculation forms the seedling with leaf about 30 days;
3) inducing clumping bud and propagation are cultivated: seedling is downcut transfer on the Ms+6-BA1.2mg/L+IBA0.4mg/L medium, until formation Multiple Buds, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 15 hours, intensity of illumination 1000LX is a growth cycle about 25 days, increase progressively at a high speed keeping, growth coefficient is more than 5;
4) culture of rootage: select Ms+6-BA0.5mg/L+NAA 1.5mg/L as root media, the pH value is 5.8, cultivation temperature 25-27 ℃, illumination every day 16 hours, intensity of illumination 1000LX, rooting rate reach more than 95%, and only needs formed whole plant in about 30 days;
5) transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water the agar of root is cleaned up, transfer is noted the water conservation heat and moisture preserving to sandy soil or rich supporting in the soil, transfer 45 days, and the transfer survival rate reaches more than 93%.
2. the method for claim 1 adopts 0.1% mercuric chloride to carry out disinfection in the step 1, and disinfecting time is controlled preferably at 12-15 minute.
3. the method for claim 2, field-transplanting humidity is controlled at 40-70% in the step 5; Temperature is controlled at 20-30 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104686147A (en) * | 2015-02-16 | 2015-06-10 | 唐忠海 | Large-scale mappia pittosporoides seedling and cultivation method |
| CN107258546A (en) * | 2017-08-04 | 2017-10-20 | 黄小燕 | The tissue culture propagation of centering rattan |
| CN108029558A (en) * | 2017-12-28 | 2018-05-15 | 长沙湘资生物科技有限公司 | A kind of rapid propagation method of salvia chinensis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104686147A (en) * | 2015-02-16 | 2015-06-10 | 唐忠海 | Large-scale mappia pittosporoides seedling and cultivation method |
| CN107258546A (en) * | 2017-08-04 | 2017-10-20 | 黄小燕 | The tissue culture propagation of centering rattan |
| CN108029558A (en) * | 2017-12-28 | 2018-05-15 | 长沙湘资生物科技有限公司 | A kind of rapid propagation method of salvia chinensis |
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Application publication date: 20130306 |