CN102599065A - Quick propagation method for humulus scandens - Google Patents
Quick propagation method for humulus scandens Download PDFInfo
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Abstract
The invention discloses a quick propagation method for humulus scandens. The method comprises the following steps of: culturing tissues, such as stem section of the humulus scandens, on an LS culture medium; accurately cutting and taking the stem section with 1-2 axillary buds as an explant; disinfecting and then inoculating in an LS+6-BA 1.2mg/L+NAA 0.2mg/L culture medium for culturing; wherein the explant buds after being inoculated for about 6-8 days and grows into a plantlet with leaves after being inoculated for about 10 days; cutting off the plantlet and transferring to an LS+6-BA 0.5mg/L+IAA 0.4mg/L culture medium, so as to keep high-speed increasing and a propagation coefficient above 5; selecting LS+6-BA 0.5mg/L+IAA 0.4mg/L+NAA 0.5mg/L as a rooting culture medium, wherein the rooting rate is above 90% and the complete plant can be obtained only after about 25 days; and transplanting a test-tube plantlet into sandy soil, carefully keeping water and humidity, and establishing a set of high-frequency stable regeneration system after the transplanting survival rate is above 90% after transplanting for 1 month. The method provided by the invention has the advantages of excellent stability, convenience in operation, high propagation speed and low production cost, achieves industrial level, 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 cairo morningglory root or leaf.
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.
Cairo morningglory root or leaf has another name called native cortex acanthopanacis, Herba Fici Simplicissimae, belongs to Moraceae.Shrub or dungarunga, high 2-8m. complete stool has milk; Tender skill hollow, branch, leaf, petiole and receptacle of inflorescence (banyan fruit) are all by the long bristle of the wide exhibition of golden yellow.Single leaf alternate; Petiole is sturdy, long 2-7cm; Ask leaf egg shape by aciculiform, long 1-3cm, membranous, redness is by pubescence; Blade is many types of, the ovum shape is oval, oval shape by aciculiform or the shape of falling the ovum by aciculiform, long 8-25cm, wide 4-18cm; Tip is point or short point gradually, and base portion is narrow, perfectly round shape or heart, and there is sawtooth at the edge; All fronts or 3-5 drastic crack, leaf surface is coarse, dredges and gives birth to short bristle, below except that the long bristle of golden yellow; Sometimes close living pubescence, base are given birth to arteries and veins 3-7 bar, and lateral vein 5-7 is right.The axil of the branch of falling leaves is given birth to or be born in to the paired armpit of hypanthodium, spherical in shape or oval sphere, and stockless or nearly stockless, diameter 8-20mm, the top bract forms boss during the children, and base is given birth to bract ovum shape and is draped over one's shoulders profile, long 1-3cm, redness is by long bristle; Male flower, gall flower are born in the same receptacle of inflorescence, and male flower and given birth to nearly oral area, stalk is arranged, tepal 4, stamen 2-3; Gall flower tepal and male flower are with number, and ovary is spherical, and style is short, adnation; Female flower is born in another plant receptacle of inflorescence, sphere, and tepal 4 has stalk or does not have stalk.Achene, surface have the tubercle body, the style adnation, and column cap is bar-shaped.Flower, the fruit phase 3-11 month.Mainly be that its root is used as medicine, contain organic acid, amino acid, triterpene, coumarin, alkaloid etc. in the root, be mainly used in and dispel rheumatism; Eliminating stasis to resolve swelling.The main air impotence numbness that wets; Pain in waist and lower extremities; Dysentery; Oedema; Band down; Scrofula; Traumatic injury; Through closing; Breast is few.
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; The part of being used as medicine mainly is a rhizome, wild cairo morningglory root or leaf is destroyed seriously, simultaneously also in order to protect the wild resource of cairo morningglory root or leaf; 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 cairo morningglory root or leaf 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 cairo morningglory root or leaf, can go out a large amount of seedling by quickly breeding, for realizing industrialization artificial cultivation cairo morningglory root or leaf possibility are provided.The successful foundation of the group culturation rapid propagating technology method of cairo morningglory root or leaf can be cultivated the medicinal plant cairo morningglory root or leaf 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 cairo morningglory root or leaf, 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 cairo morningglory root or leaf according to the invention, its concrete operations step is following:
1) explant selection and sterilization: the stem section of selecting cairo morningglory root or leaf 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 is sterilized, use aseptic water washing, cut after the part of explant variable color subsequent use;
2) sprouting of sprout: the stem section after the sterilization treatment is seeded on the LS+6-BA1.2mg/L+NAA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 12 hours; Intensity of illumination 1000LX, the sprouting of the 6-8 days stem segment with axillary buds 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 LS+6-BA0.5mg/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 LS+6-BA0.5mg/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 noted the water conservation heat and moisture preserving to sandy soil or rich supporting in the soil, and transfer is after 1 month, and the transfer survival rate reaches more than 90%;
In a specific embodiments, adopt 0.1% mercuric chloride to carry out disinfection in the step 1, disinfecting time is controlled preferably at 7-15 minute.
Technique effect:
(1) utilizes cairo morningglory root or leaf 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 stem section 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) 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, and thing obtains a large amount of whole plants in 4-6 month, realizes that the large-scale industrialized of cairo morningglory root or leaf 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 cairo morningglory root or leaf 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 cairo morningglory root or leaf.
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 cairo morningglory root or leaf 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 cairo morningglory root or leaf finished product of quality homogeneous.
(6) can preserve the germ plasm resource of cairo morningglory root or leaf, help protecting the cairo morningglory root or leaf 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: the cairo morningglory root or leaf stem segment with axillary bud is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender leaf stem section of cairo morningglory root or leaf that pick up from the Zhangjiajie, 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 LS+6-BA1.2mg/L+NAA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃, and illumination every day 12 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 LS+6-BA0.5mg/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 LS+6-BA0.5mg/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 noted the water conservation heat and moisture preserving in sandy soil, and transfer is after 1 month, and the transfer survival rate reaches more than 90%.
6, final-period management: after transplanting the land for growing field crops about April; 6-9 month covers with two-layer sunshade net, and adopts the dropper mode to carry out moisturizing, and entering is incubated water conservation with mulch film after November; To about in March, second, remove mulch film, adopt and carry out the cairo morningglory root or leaf cultivation management with upper type.If black spot takes place, spray with 1000 times of liquid of 70% thiophanate methyl WP.Root knot nematode disease harm is arranged sometimes, irritate with 3% carbofuran Granules and execute basin soil.If spending the back to execute 1 time phosphate fertilizer to mid-August during fertilizer deficiency.
Embodiment 2:
Draw materials: cairo morningglory root or leaf band bud rhizome section is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender leaf stem section of cairo morningglory root or leaf that pick up from the Zhangjiajie, 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 LS+6-BA1.2mg/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 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 LS+6-BA0.5mg/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 LS+6-BA0.5mg/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 supported in the soil to richness, and the attention water conservation is preserved moisture, and transfer is after 1 month, and the transfer survival rate reaches more than 90%.
6, final-period management: after transplanting the land for growing field crops about April; 6-9 month covers with two-layer sunshade net, and adopts the dropper mode to carry out moisturizing, and entering is incubated water conservation with mulch film after November; To about in March, second, remove mulch film, adopt and carry out the cairo morningglory root or leaf cultivation management with upper type.If black spot takes place, spray with 1000 times of liquid of 70% thiophanate methyl WP.Root knot nematode disease harm is arranged sometimes, irritate with 3% carbofuran Granules and execute basin soil.If spending the back to execute 1 time phosphate fertilizer to mid-August during fertilizer deficiency.
Embodiment 3:
Draw materials: cairo morningglory root or leaf band bud rhizome section is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender leaf stem section of cairo morningglory root or leaf that pick up from the Zhangjiajie, 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 LS+6-BA1.2mg/L+NAA0.2mg/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 LS+6-BA0.5mg/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 LS+6-BA0.5mg/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%.
6, final-period management: after transplanting the land for growing field crops about April; 6-9 month covers with two-layer sunshade net, and adopts the dropper mode to carry out moisturizing, and entering is incubated water conservation with mulch film after November; To about in March, second, remove mulch film, adopt and carry out the cairo morningglory root or leaf cultivation management with upper type.If black spot takes place, spray with 1000 times of liquid of 70% thiophanate methyl WP.Root knot nematode disease harm is arranged sometimes, irritate with 3% carbofuran Granules and execute basin soil.If spending the back to execute 1 time phosphate fertilizer to mid-August during fertilizer deficiency.
Claims (3)
1. the method for quickly breeding of a cairo morningglory root or leaf, its concrete steps are following:
1) explant selection and sterilization: select for use on the pseudobulb of cairo morningglory root or leaf 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 is sterilized, 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 LS+6-BA1.2mg/L+NAA0.2mg/L medium, and the pH value is 5.8, cultivation temperature 23-27 ℃; Illumination every day 12 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 LS+6-BA0.5mg/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 LS+6-BA0.5mg/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 noted the water conservation heat and moisture preserving to sandy soil or rich supporting in the soil, and transfer is after 1 month, and the transfer survival rate reaches more than 90%;
6) final-period management: after transplanting the land for growing field crops about April; 6-9 month covers with two-layer sunshade net, and adopts the dropper mode to carry out moisturizing, and entering is incubated water conservation with mulch film after November; To about in March, second, remove mulch film, adopt and carry out the cairo morningglory root or leaf cultivation management with upper type; If black spot takes place, spray with 1000 times of liquid of 70% thiophanate methyl WP; If root knot nematode disease harm takes place, irritate with 3% carbofuran Granules and execute basin soil; If spending the back to execute 1 time phosphate fertilizer to mid-August during fertilizer deficiency.
2. the process of claim 1 wherein and adopt in the step 1 0.1% mercuric chloride to carry out disinfection, disinfecting time is controlled preferably at 7-15 minute.
3. the method for claim 2, wherein land for growing field crops humidity is controlled at 40-70% in the step 6; Temperature is controlled at 10-30 ℃.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283596A (en) * | 2013-05-20 | 2013-09-11 | 董爱文 | Rapid propagation and standardized cultivation method of ampelopsis grossedentata |
| CN104106468A (en) * | 2014-06-26 | 2014-10-22 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method of radix fici simplicissimae |
| 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 |
| CN109169288A (en) * | 2018-10-29 | 2019-01-11 | 海南大学 | A method of by water spinach stalk the first internode regeneration induction plant |
| CN115226629A (en) * | 2022-07-29 | 2022-10-25 | 武汉轻工大学 | Tissue culture technique breeding method for ipomoea cairica |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283596A (en) * | 2013-05-20 | 2013-09-11 | 董爱文 | Rapid propagation and standardized cultivation method of ampelopsis grossedentata |
| CN103283596B (en) * | 2013-05-20 | 2015-06-24 | 吉首大学 | Rapid propagation and standardized cultivation method of ampelopsis grossedentata |
| CN104106468A (en) * | 2014-06-26 | 2014-10-22 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method of radix fici simplicissimae |
| CN104106468B (en) * | 2014-06-26 | 2015-09-16 | 广西壮族自治区药用植物园 | The quick breeding method for tissue culture of a kind of radix fici simplicissimae |
| 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 |
| CN109169288A (en) * | 2018-10-29 | 2019-01-11 | 海南大学 | A method of by water spinach stalk the first internode regeneration induction plant |
| CN115226629A (en) * | 2022-07-29 | 2022-10-25 | 武汉轻工大学 | Tissue culture technique breeding method for ipomoea cairica |
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Application publication date: 20120725 |