CN102599063A - Rapid propagation method of Bletilla striata - Google Patents
Rapid propagation method of Bletilla striata Download PDFInfo
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Abstract
The application relates to a rapid propagation method of Bletilla striata. The method comprises the following steps: firstly culturing a pseudobulb section and other tissues of Bletilla striata on an MS (Murashige and Skoog) culture medium, cutting a rootstock section with 1-2 buds, which is served as an explant; disinfecting, and inoculating in a culture medium of MS+6-BA (0.5mg/L)+NAA (0.4mg/L) and culturing, wherein the buds germinate about 6-8 days after inoculation and grow into seedlings with leaves about 10 days after inoculation; cutting down the seedlings and transferring to a culture medium of MS+6-BA (1.5mg/L)+IAA (0.4mg/L) so as to maintain high-speed multiplication, wherein the multiplication coefficient is higher than 5; using MS+6-BA (1.5mg/L)+IAA (0.4mg/L)+NAA (0.5mg/L) as a rooting culture medium, wherein the rooting rate is higher than 90% and the seedlings can grow into complete plants only about 25 days; and transplanting test-tube plantlets into sandy soil and maintaining proper water content and moisture, wherein transplanting survival rate is higher than 90% one month after transplanting and a set of high-frequency stable regeneration system is established. The method disclosed by the invention has the advantages of being good in stability, simple and convenient in operation, high in propagation speed, low in production cost and reaching the 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 bletilla striata.
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.
The bletilla striata has another name called and gives free of charge, belongs to the orchid family.Perennial herb is liked warm, shady and cool and more dark and damp environment, and is not cold-resistant, high 30～50cm.4～5 pieces on leaf, base portion cover mutually build up the stem shape, and central authorities extract scape out.A raceme tool number flower; Flower purple or pale red, about 5 centimetres of diameter is made up of the lip of 3 pieces of sepals, 2 pieces of petals and 1 piece of specialization; Lip 3 splits, and the typhloscle sheet is arranged above; Stamen and style symphysis form gynostemium, and there is a flower pesticide on the gynostemium top, and there is a column cap depression front upper place.Underground have a thick thick root-like stock, and like the chicken head shape, rich viscosity contains the bletilla colloid, i.e. bletilla mannan, but hyoscine have the effect of hemostasis tonifying lung, myogenic pain relieving, also can be made for thickener.The bletilla genus has 6 kinds, all originates in East Asia.Research shows that the pseudobulb of the bletilla striata has very high medicinal ingredient, mainly contains and is rich in starch, glucose, volatile oil, mucilaginous substance etc., and external application is embrocated, and the vestige under can eliminating as the acne on the face makes skin smooth seamless.Bulb contains bletilla colloid, starch, volatilization wet goods; Medicinal, have produce effects, effects such as tonifying lung hemostasis, detumescence, external application controls that traumatic bleeding, carbuncle are swollen, scald, furunculosis etc.; Spend beauty, cultivation supplies to view and admire.Astringing to arrest bleeding, detumescence and promoting granulation.Being mainly used in hemostasis, is a kind of famous and precious haemostatic medicament, is used for hemoptysis and spits blood traumatism and bleeding, sore swollen toxin, chapped skin; The pulmonary tuberculosis hemoptysis, ulcer haemorrhage.
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 the bletilla striata 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 bletilla striata 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 the bletilla striata, can go out a large amount of seedling by quickly breeding, for realizing the industrialization artificial cultivation bletilla striata possibility are provided.The successful foundation of the group culturation rapid propagating technology method of the bletilla striata can be cultivated the medicinal plant bletilla striata 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 the bletilla striata, 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 the bletilla striata according to the invention, its concrete operations step is following:
1) explant selection and sterilization: select for use on the pseudobulb of the bletilla striata 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 medium that contains NAA, 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 ℃, 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%;
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 bletilla striata 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.
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.
In a specific embodiments, the consumption of NAA is 0.1-0.3mg/L in the step 2.
In another embodiment, land for growing field crops humidity is controlled at 40-70% in the step 6; Temperature is controlled at 10-30 ℃.
Technique effect:
(1) utilizes bletilla striata 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) 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 the bletilla striata 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 the bletilla striata 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 the strict control bletilla striata.
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 bletilla striata 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 bletilla striata finished product of quality homogeneous.
(6) can preserve the germ plasm resource of the bletilla striata, help protecting bletilla striata 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: bletilla striata stem with bud is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender leaf stem section of the bletilla striata 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 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 bletilla striata 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: bletilla striata stem with bud is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender leaf stem section of the bletilla striata 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 LS+6-BA1.2mg/L+NAA0.3mg/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 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 bletilla striata 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: bletilla striata stem with bud is an explant
1, explant selection and sterilization: the tender tissues such as the fresh and tender leaf stem section of the bletilla striata 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 LS+6-BA1.2mg/L+NAA0.1mg/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 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 bletilla striata 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 (4)
1. the method for quickly breeding of a bletilla striata, its step comprises:
1) explant selection and sterilization: select for use on the pseudobulb of the bletilla striata 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 medium that contains NAA, 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 bletilla striata 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 the consumption of NAA is 0.1-0.3mg/L in the step 2.
4. the method for claim 3, 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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| CN102907323A (en) * | 2012-11-01 | 2013-02-06 | 建始县颐泰生物科技有限公司 | Method for aseptically producing miniature seed stems of common bletilla pseudobulb seeds |
| CN102972290A (en) * | 2012-11-15 | 2013-03-20 | 贵州师范大学 | Method for improving transplanting survival rate of rhizoma bletillae tissue culture seedling |
| CN103348913A (en) * | 2013-06-28 | 2013-10-16 | 江苏茅山地道中药材种植有限公司 | Rapid propagation method for bletilla striata |
| CN103875400A (en) * | 2014-03-14 | 2014-06-25 | 云南云蕊生物资源开发有限公司 | Method for accelerating growth and development of rhizoma bletillae |
| CN104885934A (en) * | 2015-05-11 | 2015-09-09 | 福建农林大学 | Method adopting pseudobulb to induce regeneration and propagation of malaxis microtatantha plants |
| CN106134666A (en) * | 2015-03-17 | 2016-11-23 | 重庆锦洪生态农业开发有限公司 | A kind of planting technology of the bletilla striata |
| CN106376328A (en) * | 2016-08-30 | 2017-02-08 | 云南金碧制药有限公司 | Wild bletila striata seed quick breeding method |
| CN106417011A (en) * | 2016-08-30 | 2017-02-22 | 云南金碧制药有限公司 | Wild bletilla striata tissue culture rapid propagation method |
| CN106613936A (en) * | 2015-11-02 | 2017-05-10 | 丹阳华都园艺有限公司 | Method for raising Bletilla striata seedlings by tissue culture |
| CN106973789A (en) * | 2017-03-29 | 2017-07-25 | 师宗县福禄农业科技开发有限公司 | A kind of root media and its method for culturing seedlings of bletilla striata tissue culture |
| CN107155880A (en) * | 2017-05-05 | 2017-09-15 | 江苏东郁园林科技有限公司 | A kind of medicinal bletilla striata tissue culture of sprout mating system |
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| CN108401910A (en) * | 2018-05-21 | 2018-08-17 | 陕西科技大学 | A kind of bletilla striata seedling method for tissue culture of quick raising bletilla striata seedling fostering quantity |
| CN108541593A (en) * | 2018-06-27 | 2018-09-18 | 芜湖东源新农村开发股份有限公司 | The method for tissue culture of white taro |
| CN109197591A (en) * | 2018-10-24 | 2019-01-15 | 河南云帮农业科技有限公司 | A kind of excellent bletilla striata tissue culture method |
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