CN102144553B - Method for rapidly propagating Paris polyphylla Smith - Google Patents
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Abstract
The invention provides a rapid propagation technology of Paris polyphylla Smith. The method comprises the following steps of: culturing tissues of the Paris polyphylla Smith such as rhizome sections and the like on a murashige and skoog (MS) culture medium, exactly taking a rhizome section with 1 to 2 buds as an explant, disinfecting, inoculating the explant into an MS plus 0.5mg/L 6-benzylamino adenine (BA) plus 0.4mg/L naphthylacetic acid (NAA) culture medium for culturing, ensuring the germination of the buds within about 6 to 8 days after the inoculation and the formation of seedlings with leaves within about 10 days, and cutting the seedlings and inoculating the seedlings into an MS plus 1.5mg/L 6-BA plus 0.4mg/L indoleacetic acid (IAA) culture medium to ensure that the high-speed increase is maintained and the multiplication factor is more than 5. MS plus 1.5mg/L 6-BA plus0.4mg/L IAA plus 0.5mg/L NAA is selected as a rooting culture medium, the rooting rate is over 90 percent, and an intact plant can be formed within about 25 days. A test tube seedling is transplanted into sand, the water retention and moisture retention are noticed, the transplant survival rate is over 90 percent after the seedling is transplanted for 1 month, and a set of high-frequency stable regeneration system is established. The method has the advantages of high stability, simple operation, high propagation speed, low production cost, industrialization level and the like.
Description
Technical field
The present invention relates to bioengineering field, relate to plant tissue culture technique, specifically the method for tissue culture of a kind of paris polyphylla.
Background technology
Plant is the natural treasure-house of medicine, and people utilize the historical of long standing and well established of medicinal plant, the whole world have approximately 75% population with plant as treatment prophylactic medicament sources (Xing Jianmin, 2001).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 traditional Chinese herbal medicine of China is still widely used in China and many countries and regions so far.But,, will inevitably cause the in imminent danger of species even become extinct when gathering when surpassing the regeneration capacity of natural resources with consumption because the traditional Chinese herbal medicine acquisition methods is to be cost to gather and to consume a large amount of wild plant resources.Along with the destruction day by day of natural ecological environment, also further cause the scarcity of resources of medicinal plant simultaneously.The flourish of biotechnology is that the production that fundamentally changes traditional medicinal material provides a brand-new method, and plant tissue and cell culture technology then are 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 of succeeing, 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 be that raw material is processed (Xie Qikun, 1986) with the plant 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,,, needs the cost long time if with the conventional method breeding or grow seedlings like genseng, the coptis; 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 bigger, so utilize the plant regeneration and the breeding problem of plant tissue culture technique solution medicinal plant extremely urgent to its active constituent content fluctuation ratio.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, like Chinese yew, Chinese mugwort, Chinese littleleaf box, euphorbia, catharanthus roseus, Aglaia odorata, dog tartar and Chinese torreya etc.Paris polyphylla has another name called schefflera arboricola, belongs to Liliaceae.Perennial herb, high 30～100cm.Stem is upright.5～8 wheels of leaf are born in the stem top, the oval shape lanceolar of blade, the shape of falling ovum lanceolar or the lanceolar that falls, long 7～17cm, wide 2.5～5cm.Bennet is extracted out from stem top, and is longer than leaf usually, and a flower is given birth on the top, wide 1～1.5mm, and long is the 1/3 isometric to closely of sepal; Stamen 8～10, the long 1.2～2cm of flower pesticide.Capsule is spherical.5～July of florescence, really 8～October of phase.Fruit is a capsule.Be born in the sylvan life of height above sea level 1800-3200 rice or the dark and damp place of thick grass of shady place of hillside sylvan life or limes marginis.China mainly is distributed in the southeast, Tibet, Yunnan, Hunan, Sichuan and Guizhou.Research shows that the dry rhizome of paris polyphylla has very high medicinal ingredient, mainly contains diosgenin, Pariphyllin A, B, Dioscin isoreactivity composition.Have certain antibacterial action: external all have inhibitory action to typhoid bacillus, paratyphoid bacillus A, will he and shigella flexneri.Kill the sperm effect, relieving asthma, antitussive action and pharmacological actions such as calmness, analgesic activity.Its dry rhizome is mainly widely applied in Yunnan Baiyao at present.Therefore, in order to satisfy the medicinal demand that increases day by day, 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 the wild resource of paris polyphylla simultaneously, utilize group culturation rapid propagating technology, realize that artificial cultivation is best solution.
Up to now, at home and abroad do not see as yet about the successful report of paris polyphylla tissue culture quick breeding.We find out the ripe method of a cover finally through a large amount of tests, have successfully realized the tissue-culturing rapid propagation of paris polyphylla, can go out a large amount of seedling by quickly breeding, for realizing industrialization artificial cultivation paris polyphylla possibility are provided.The successful foundation of the group culturation rapid propagating technology method of paris polyphylla can be cultivated the medicinal plant paris polyphylla for mass artificial seedling is provided, 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 paris polyphylla, this 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 paris polyphylla according to the invention, its concrete operations step is following:
1) explant selection and sterilization: select for use on the rhizome of paris polyphylla 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 handle 10-30 second, and then after 0.1% mercuric chloride was sterilized 10 minutes, use aseptic water washing, cut after the part of explant variable color subsequent use.
2) sprouting of sprout: the sprout after the sterilization treatment is seeded on the MS+6-BA0.5mg/L+NAA0.4mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 16 hours; Intensity of illumination 1000LX, the sprouting of the 6-8 days left and right sides sprouts in inoculation back forms the seedling of being with leaf about 10 days.
3) inducing clumping bud and enrichment culture: seedling downcut transfer on the MS+6-BA1.5mg/L+IAA0.4mg/L medium, until the formation bud of growing thickly, the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping that growth coefficient is more than 5.
4) culture of rootage: select MS+6-BA1.5mg/L+IAA0.4mg/L+NAA 0.5mg/L as root media, the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 16 hours; Intensity of illumination 1000LX, rooting rate reach more than 90%, and only needs formed whole plant in about 25 days.
5) transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water and clean up the agar of root, transfer is in sandy soil, and the attention water conservation is preserved moisture, and transfer is after 1 month, and the transfer survival rate reaches more than 90%.
Technique effect:
(1) utilizes paris polyphylla band bud rhizome section to be explant, make explant draw materials and be easier to, all can draw materials throughout the year; And quantity is many, adopts the rhizome sprout to do explant, has guaranteed genetic stability; Overcome simultaneously in the tissue culture, callus stops growing along with the differentiation of bud on differential medium gradually, loses the weakness of multiplication capacity; Can prevent in the injured tissue successive transfer culture process that along with the increase of cultivating algebraically, the phenomenon of variation appears in the seedling that differentiation is come out.
(2) this courage 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, and thing obtains a large amount of whole plants in 4-6 month, realizes that the large-scale industrialized of paris polyphylla grow seedlings.
(3) matrix that when transfer, adopted 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 produce in batches, and using value is high.
(4) production of paris polyphylla can be carried out under artificial controlled condition, does not receive the restriction of factors such as season, weather conditions and soil, can get rid of the invasion and attack of damage by disease and insect and the influence of residue of pesticide, the quality of strict control paris polyphylla.
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 paris polyphylla seedling of (5) using group culturation rapid propagating technology provided by the invention to obtain carries out artificial cultivation, and it is little to obtain individual difference, the paris polyphylla finished product of quality homogeneous.
(6) can preserve the germ plasm resource of paris polyphylla, help protecting the paris polyphylla wild resource simultaneously, reduce destruction natural resources.
Embodiment
Below, the present invention will further explain with embodiment, but it is not limited to any or the similar instance of these embodiment.
Embodiment 1:
Draw materials: paris polyphylla band bud rhizome section is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender bud rhizome of paris polyphylla section that pick up from the Cili, Hunan; As culture materials; Clean through liquid detergent and running water, put under the flowing water and wash after 10-30 minute, place 75% alcohol to handle 10-30 second; And then use aseptic water washing after 10 minutes through the sterilization of 0.1% mercuric chloride, cut after the part of explant variable color subsequent use.
2, the sprouting of sprout: the sprout after the sterilization treatment is seeded on the MS+6-BA0.5mg/L+NAA0.4mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX; The sprouting of the 6-8 days left and right sides sprouts in inoculation back forms the seedling of being with leaf about 10 days.
3, inducing clumping bud and enrichment culture: seedling downcut transfer on the MS+6-BA1.5mg/L+IAA0.4mg/L medium, until the formation bud of growing thickly, the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping that growth coefficient is more than 5.
4, culture of rootage: select MS+6-BA1.5mg/L+IAA0.4mg/L+NAA 0.5mg/L as root media, the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 16 hours; Intensity of illumination 1000LX, rooting rate reach more than 90%, and only needs formed whole plant in about 25 days.
5, the transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water and clean up the agar of root, transfer is in sandy soil, and the attention water conservation is preserved moisture, and transfer is after 1 month, and the transfer survival rate reaches more than 90%.
Embodiment 2:
Draw materials: paris polyphylla band bud rhizome section is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender bud rhizome of paris polyphylla section that pick up from the Cili, Hunan; As culture materials; Clean through liquid detergent and running water, put under the flowing water and wash after 10-30 minute, place 75% alcohol to handle 10-30 second; And then use aseptic water washing after 10 minutes through the sterilization of 0.1% mercuric chloride, cut after the part of explant variable color subsequent use.
2, the sprouting of sprout: the sprout after the sterilization treatment is seeded on the MS+6-BA0.5mg/L+NAA0.3mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX; The sprouting of the 7-8 days left and right sides sprouts in inoculation back forms the seedling of being with leaf about 12 days.
3, inducing clumping bud and enrichment culture: seedling downcut transfer on the MS+6-BA1.5mg/L+IAA0.4mg/L medium, until the formation bud of growing thickly, the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping that growth coefficient is more than 5.
4, culture of rootage: select MS+6-BA1.5mg/L+IAA0.4mg/L+NAA 0.5mg/L as root media, the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 16 hours; Intensity of illumination 1000LX, rooting rate reach more than 90%, and only needs formed whole plant in about 25 days.
5, the transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water and clean up the agar of root, transfer is in sandy soil, and the attention water conservation is preserved moisture, and transfer is after 1 month, and the transfer survival rate reaches more than 90%.
Embodiment 1:
Draw materials: paris polyphylla band bud rhizome section is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender bud rhizome of paris polyphylla section that pick up from the Cili, Hunan; As culture materials; Clean through liquid detergent and running water, put under the flowing water and wash after 10-30 minute, place 75% alcohol to handle 10-30 second; And then use aseptic water washing after 10 minutes through the sterilization of 0.1% mercuric chloride, cut after the part of explant variable color subsequent use.
2, the sprouting of sprout: the sprout after the sterilization treatment is seeded on the MS+6-BA0.5mg/L+NAA0.4mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX; The sprouting of the 6-8 days left and right sides sprouts in inoculation back forms the seedling of being with leaf about 10 days.
3, inducing clumping bud and enrichment culture: seedling downcut transfer on the MS+6-BA1.5mg/L+IAA0.4mg/L medium, until the formation bud of growing thickly, the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 16 hours, intensity of illumination 1000LX.Be a growth cycle about 30 days, increase progressively at a high speed keeping that growth coefficient is more than 5.
4, culture of rootage: select MS+6-BA1.5mg/L+NAA 0.5mg/L as root media, the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 16 hours; Intensity of illumination 1000LX, rooting rate reach more than 95%, and only needs formed whole plant in about 20 days.
5, the transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water and clean up the agar of root, transfer is in sandy soil, and the attention water conservation is preserved moisture, and transfer is after 1 month, and the transfer survival rate reaches more than 85%.
Claims (1)
1. the method for quickly breeding of a paris polyphylla is characterized in that its concrete operations step is following:
1) explant selection and sterilization: the band bud rhizome section of selecting paris polyphylla for use is 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 handle 10-30 second, and then after 0.1% mercuric chloride was sterilized 10 minutes, use aseptic water washing, cut after the part of explant variable color subsequent use;
2) sprouting of sprout: the band bud rhizome section after the sterilization treatment is seeded on the MS+6-BA0.5mg/L+NAA0.4mg/L medium; The pH value is 5.8; Cultivation temperature 23-27 ℃, illumination every day 16 hours, intensity of illumination 1000LX; The sprouting of the 6-8 days sprouts in inoculation back forms the seedling of being with leaf about 10 days;
3) inducing clumping bud and enrichment culture: seedling downcut transfer on the MS+6-BA1.5mg/L+IAA0.4mg/L medium, until the formation bud of growing thickly, the pH value is 5.8; Cultivation temperature 23-27 ℃; Illumination every day 16 hours, intensity of illumination 1000LX is 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-BA1.5mg/L+IAA0.4mg/L+NAA 0.5mg/L as root media, the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 16 hours; Intensity of illumination 1000LX, rooting rate reach more than 90%, and only needs formed whole plant in about 25 days;
5) transplanting of test-tube plantlet: choose the good test-tube plantlet of root growth, put into clear water and clean up the agar of root, transfer is in sandy soil, and the attention water conservation is preserved moisture, and transfer is after 1 month, and the transfer survival rate reaches more than 90%.
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| CN102550239B (en) * | 2011-11-18 | 2013-02-13 | 广州白云山和记黄埔中药有限公司 | Artificial domestication and cultivation method for Isodon Iophanthoides var. graciliflorus (Benth.) H.Hara, wild variety of Isodon serra |
| CN102870564B (en) * | 2012-09-26 | 2014-03-12 | 四川农业大学 | Fast efficient propagation method of Paris polyphylla |
| CN103270873B (en) * | 2013-05-20 | 2015-02-25 | 湖南农业大学 | Efficient interplanting method of paris polyphylla and maoyan berry |
| CN103548682B (en) * | 2013-10-31 | 2015-10-28 | 成都大学 | The method for quickly breeding of a kind of magnificent Paris polyphylla plant |
| CN103931502B (en) * | 2014-04-29 | 2016-03-02 | 中南民族大学 | Two step seedling methods of paris polyphylla seminal propagation |
| CN104160962B (en) * | 2014-09-03 | 2016-08-24 | 无锡中农科生物育种技术研究院有限公司 | The breeding method of autotetraploid Rhizoma Paridis plant |
| CN104904474B (en) * | 2015-06-07 | 2017-06-30 | 湖北济世药业有限责任公司 | A kind of asexual reproduction method of Paris polyphylla |
| CN105104213B (en) * | 2015-10-15 | 2017-05-03 | 湖南农业大学 | Rapid propagation and seedling culture method using rhizomes of paris polyphylla |
| CN105359971A (en) * | 2015-10-16 | 2016-03-02 | 金寨县绿野中药材专业合作社 | Paris polyphylla rhizome breeding method |
| CN106718901A (en) * | 2016-12-14 | 2017-05-31 | 淄博精诚专利信息咨询有限公司 | A kind of growth substrate and the method with its vegetative propagation medicinal plant Paris polyphylla |
| 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 |
| CN113951140B (en) * | 2021-11-11 | 2022-07-08 | 广西壮族自治区中国科学院广西植物研究所 | Method for promoting rapid propagation of seedlings of paris polyphylla young plants |
| CN116267610B (en) * | 2023-03-14 | 2024-03-15 | 广西医科大学 | Aseptic treatment and in-vitro preservation method for Paris polyphylla explant |
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| CN101461327A (en) * | 2008-10-24 | 2009-06-24 | 昆明优利丰园科技开发有限公司 | Rapid propagation technique of Paris polyphylla var. yunnanensis, seedling exercising and planting methods |
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