CN1586168A - Tissue cultivation quick breeding method for Dysosma versipellis - Google Patents
Tissue cultivation quick breeding method for Dysosma versipellis Download PDFInfo
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Abstract
The present invention discloses the tissue cultivation process for quickly breeding Dysosma versipellis. The process includes selection and processing of explant, inducing culture, proliferating culture, rooting culture, transplanting and other steps. The present invention uses MS culture medium with added auxiliary agents including active carbon, 6-benzyl adenine, gibberellin and corn extract for two key stages including the inducing growth of fascicular seedling and rooting of robust seedling. The present invention solves the technological problems in fast breeding and stable rooting of Dysosma versipellis, and has the advantages of high breeding coefficient, stable technology, short seedling growth period and low cost.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the tissue culture quick propagation culturing method of a kind of Dysosma versipellis.
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 and consumption when surpassing the regeneration capacity of natural resources because 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.Biotechnology flourish for the production that fundamentally changes traditional medicinal material provides a brand-new method, 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 made (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 as 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.So utilize the 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, Liao Dong Aralia wood etc.; Has the rare plant of anticancer effective component comprising some, as Chinese yew, Chinese mugwort, Chinese littleleaf box, euphorbia, catharanthus roseus, Aglaia odorata, dog tartar and Chinese torreya etc.
Dysosma versipellis Dysosma versipellis (Hance) M.Cheng is the herbaceos perennial of Berberidaceae Berberidaceae Dysosma Dysosma, is distributed in ground such as China Hunan, Hubei, Zhejiang, Jiangxi, Yunnan, Guizhou, Sichuan, Henan and Shaanxi.Grow under the cheuch shaw of height above sea level 400-1400 rice hillside in moistening place or the dark and damp trench thick grass.The florescence 4-6 month, the fruit phase 7-9 month.Dysosma versipellis is a famous medicinal plant in China's traditional Chinese medicine, mainly gets its root-like stock hyoscine, has and reduces phlegm and clearing heat and detoxicating effect, and the stasis of blood that is widely used in dispelling detoxifcation is controlled traumatic injury, joint pain, worm snake bite, diseases such as abscess of throat and sore furuncle.In the modern medicine field, Dysosma versipellis is the primary raw material of famous cancer therapy drug VP-16, VM-26.
Studies show that, root-like stock of Dysosma versipellis and root are rich in active ingredients such as podophyllotoxin, dehydropodophyllotoxin and deoxidation podophyllotoxin (the long army 1997 of Liu, Chen Min henry 1979), wherein main active ingredient is podophyllotoxin (Podophyllotoxin), content can reach 1.4% in plant corpus, has multiple physiologically actives such as antitumor, antiviral.But the toxic and side effect of natural component podophyllotoxin is too big, be not suitable for directly clinical medicinal (King1946), but by cancer therapy drugs such as chemical modification (Creaven.1975) generation podophyllotoxin derivative VP-16, VM-26, these two kinds of clinical drug tests have the broad spectrum anticancer activity, multiple cancers such as carcinoma of testis, leucocyte cancer, lymph meat cancer, glioma, Huo Jiejinshi disease there is special efficacy, has been widely used in clinical now.But because Dysosma versipellis is strict to the habitat, the destruction of ecotope for many years in addition, the environment that is fit to its growth is fewer and feweri; Because it has important medical value, recruit to dig excessively disorderly and adopt, excessively utilize again, the mechanism of breeding of adding itself is bad, causes distribution of wild Dysosma versipellis and quantity to fall sharply, and is on the brink of extinction.Dysosma versipellis has been listed in three grades of protective plants in imminent danger of country now.Existing Dysosma versipellis wild resource is difficult to satisfy the needs of growing field of medicaments at all.Therefore,, protect the wild resource of Dysosma versipellis simultaneously, utilize group culturation rapid propagating technology, realize that artificial cultivation is best solution in order to satisfy the medicinal demand that increases day by day.Yet because wild Dysosma versipellis requires harshness to the habitat, reproduction mechanisms is bad, is not only to cause its reason in imminent danger, and causes conventional tissue culture technique extremely to be difficult to it is carried out numerous soon.Up to now, do not see report at home and abroad as yet about the success of Dysosma versipellis tissue culture quick breeding.We last 3 years and find out the ripe method of a cover finally by a large amount of tests, have successfully realized the tissue-culturing rapid propagation of Dysosma versipellis, can go out a large amount of seedling by quickly breeding, provide possibility for realizing industrialization artificial cultivation Dysosma versipellis.The successful foundation of the group culturation rapid propagating technology method of Dysosma versipellis can provide seedling for extensive artificial cultivation medicinal plant Dysosma versipellis, for the modern science and technology agricultural provides a kind of practicable exploration project.
List of references
Xing Jianmin .2000 such as Zhao Xiude, saussurea medusa bio-reactor suspension culture. Botany Gazette, 42 (1): 98-101.
Zheng Guang plants .1987, the new development of Secondary Metabolism of Plant cell engineering research: the 6th international plant tissue and cell culture meeting are commented. Plant Physiology Communications, (2): 75-79.
Thank to Qi Kun .1986, the medicinal plant tissue culture. Shanghai, Shanghai science tech publishing house. Liu Changjun, Hou Song gives birth to .1997, the progress of anti-cancer active matter Podophyllum emodi var chinense lignans. research and development of natural products, 9 (3): 81～89.
The prosperous .1979 of Chen Yu, the research of China's Podophyllum emodi var chinense class plant resources. Acta Pharmaceutica Sinica, 14 (2): 101-106King L S, et al.1946, The similarity of the effect of podophyllin and colchicine and
their?use?in?the?treatment?of?condylomata?acuminata?acuminata.Since，104：244.Creaven?P?J，etal.1975，PTG?a?new?antineoplastic?epipodophyllotoxin，ibid，18：227.
Damayanthi?Y.，Lown?JW，1998，Curr.Med.Chem.，5(3)：205
The meaning of mentioned abbreviation letter is seen following breviary vocabulary in the literary composition:
MS Murashige and Skoog medium MS medium
2,4-D 2,4-dichlorophenoxy acetic acid 2,4 dichlorophenoxyacetic acid
NAA α-naphthal acetic acid α-Nai Yisuan
IAA indole-3-acetic acid draws indolylbutyric acid
BA 6-benzylaminopurine 6-benzyl aminopurine
KT kinetin kinetin
GA gibberellic acid gibberellin
AC activated charcoal active carbon
PVP polyvinglypyrrolidon polyvinylpyrrolidone
ZT zeatin zeatin
IBA indolebutyic aci draws the diindyl butyric acid
Summary of the invention
The tissue culture quick propagation culturing method that the purpose of this invention is to provide a kind of Dysosma versipellis.
The step of method is as follows:
1) explant selection and processing: the bud of selecting Dysosma versipellis 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, use aseptic water washing in mercuric chloride (Hgcl) sterilization after 1-10 minute then through 0.1-0.15%, cut the part of explant variable color, be inoculated in the medium;
2) inducing culture: use MS+BA (0.2-0.5mg/l)+ZT (0-1.0mg/l) as inducing culture, selected bud is inoculated in the above-mentioned medium;
3) enrichment culture: will grow on the inducing culture explant and transfer and on MS+BA (0.2-1.0mg/l)+ZT (0-0.5mg/l)+GA (0-0.2mg/l)+PVP (0-0.5mg/l), carry out continuous culture.
4) culture of rootage: select 1/2 MS+BA (0.2-0.5mg/l)+ZT (0.1-0.5mg/L) as root media;
5) transplant: the plant that will take root is taken out, and is transplanted to perlite: peat soil (1: 1-1.5) on the matrix of Hun Heing.
Advantage of the present invention: the production of (1) Dysosma versipellis can be carried out under artificial controlled condition, is not subjected to 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 Dysosma versipellis.(2) 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.(3) carry out detoxification treatment in group training process, improve the Dysosma versipellis quality, effectively solve wild Dysosma versipellis quality instability, the problem that outward appearance and active ingredient are irregular is road, grown place medicinal material and processing sell in the postpartum condition that provides safeguard.(4) this technical method has solved Dysosma versipellis breeding and stable important technical links of taking root thereof fast, has reached consistent, and requirement that reproduction coefficient is high can be applicable to the purpose of suitability for industrialized production.(5) the Dysosma versipellis seedling of using group culturation rapid propagating technology provided by the invention to obtain carries out artificial cultivation, and it is little to obtain individual difference, and the Dysosma versipellis finished product of quality homogeneous can provide the raw material of stay in grade for producing podophyllotoxin.(6) can preserve the germ plasm resource of Dysosma versipellis, help protecting the Dysosma versipellis wild resource simultaneously, reduce destruction natural resources.
Embodiment
The group culturation rapid propagating technology of Dysosma versipellis comprises explant selection and processing, inducing culture, enrichment culture, culture of rootage, transplanting and other steps.Particularly in the operation, preferably the complete bud of Dysosma versipellis is a culture materials, clean through liquid detergent and running water, put and wash after 30 minutes under the flowing water, place 75% alcohol to handle 30 seconds, use aseptic water washing in mercuric chloride (Hgcl) sterilization after 8 minutes then, cut the part of explant variable color, be inoculated in and carry out inducing culture in the MS+BA1.0mg/L+ZT0.5mg/L medium through 0.1%.25 ± 2 ℃ of temperature, illumination every day 16 hours was cultivated 15-20 days under the condition of intensity of illumination 1000LX.Then a plurality of buds that obtain in every bottle are cut respectively, place the cultivation of rising in value of MS+BA1.0mg/l+ZT0.5mg/l+GA0.2mg/l+PVP0.5mg/l medium.Condition of culture is with above-mentioned inducing culture.Can obtain the green healthy and strong seedling of a strain after 20-30 days in each blake bottle, the seedling that needs are taken root is transferred on the 1/2 MS+BA 0.5mg/l+ZT0.5mg/L medium and carries out culture of rootage.Condition of culture is with above-mentioned inducing culture.Promptly take root about 25-30 days and finish.The plant that has taken root is taken out, placed 5-7 days down, open bottle cap, clean medium, perlite is arrived in transplantation of seedlings: peat soil (1: on mixed-matrix 1-1.5) in natural environment.Kept 25 ± 2 ℃ of environmental temperatures in preceding 7 days, humidity can carry out the transition to the environmental condition of nature subsequently gradually about 70%.Practice seedling and be transplanted to big Tanaka after one month, can reach 80% survival rate.
Induce, breed in the above-mentioned technology and the pH value of the step of taking root is 5.8,25 ± 2 ℃ of cultivation temperature, illumination every day 16 hours, intensity of illumination 1000LX.
After using the bud grafting kind of the present invention with Dysosma versipellis, can induce in 20-25 days to form the bud of growing thickly, general inductivity can reach 75-90%.Successive transfer culture on this basis, the bud of growing thickly can be bred in a large number, approximately per 25 days subcultures once, on average breeding multiple is 3.3 times.Transfer in the root media provided by the invention 25-35 days root of hair in a large number of the bud of growing thickly behind the subculture, go out the root rate about 80%, cultivating can the bottle outlet hardening after 30-45 days.
Adopt the present invention of above-mentioned measure, grasped inducing and growing of the bud of growing thickly in the Dysosma versipellis tissue culture, the technology of two critical stages of strengthening seedling and rooting, successfully solved the difficulties such as difficulty, appreciation rate is low, survival rate is low of taking root in the training of Dysosma versipellis group, made this important medicinal plant in imminent danger of Dysosma versipellis realize that fast seedling growing, factory culture become possibility.Use the present invention can protect this rare wild plant resource simultaneously effectively, reduce the private benign cycle that coyoting, formation sustainable use and reasonable development are coordinated mutually of digging.
Superiority of the present invention is embodied in: Plant Tissue Breeding and quick propagating technology that Dysosma versipellis is provided, found out the guardian technique of quick breeding and stable important step such as take root thereof, but explant 1 year just clonal propagation goes out tens thousand of strain offsprings, for the commercialization production of this medicinal plant provides a cover practicable production technology route, can be applied to the large-scale industrial production of Dysosma versipellis.
The present invention adopts following non-limiting embodiment to be further described.
Embodiment 1
Get the bud of the Dysosma versipellis of picking up from the Mount Emei, clean with running water, the flowing water flushing is carried out surface sterilization after half an hour again, sterilization method is: behind the 70% alcohol-pickled 30s, with twice of aseptic water washing, use 0.1% mercuric chloride 100ml (add 2-3 and drip tween T-20) sterilization 8min again, clean with aseptic water washing at last.Insert on the culture medium after sterilization MS+BA 0.5mg/l+ZT1.0mg/l.Temperature was cultivated 15 days for 25 ± 2 ℃, as seen had about 5 sproutings to occur on each explant.Have the explant of bud to take out under aseptic condition with long, each bud is separated with scalpel, place the culture vessel that above-mentioned identical medium is housed to cultivate respectively, after 20 days, the bud in each culture vessel is renewable to go out several sproutings.With each bud separately, transfer and on MS+BA1.0mg/l+ZT0.5mg/l+GA0.2mg/l+PVP0.5mg/l, carry out continuous culture again,, wherein add antioxidant PVP and prevent brownization to reach quick value-added purpose.Cultivate after 20 days, the Dysosma versipellis plant that needs are taken root is transferred on the 1/2 MS+BA 0.5mg/l+ZT0.5mg/L medium and carries out culture of rootage.Can take root about 30 days.The plant that has taken root is taken out, placed 5-7 days down, open bottle cap, clean medium, perlite is arrived in transplantation of seedlings: on the matrix that peat soil (1: 1) mixes in natural environment.Keep ambient humidity about 70%.Be transplanted to big Tanaka after one month, can reach 75% survival rate.
Embodiment 2
Get the bud of the Dysosma versipellis of picking up from the Mount Emei, clean with running water, again after flowing water flushing half an hour behind the 70% alcohol-pickled 30s, with twice of aseptic water washing, use 0.1% mercuric chloride 100ml (add 2-3 and drip tween T-20) sterilization 15min again, clean with aseptic water washing at last.Blot with aseptic paper, be cut into about 1 centimetre, be seeded among the medium MS+BA 0.5mg/l+NAA 0.2mg/l+GA 0.2mg/l.25 ± 2 ℃ of cultivation temperature, intensity of illumination 1500LX, light application time 10-12h/d.Cultivated 20 days, and had 5-7 sprouting to occur on the visible explant.Have the explant of several buds to take out under aseptic condition with long, with scalpel each bud is cut, being placed in transfers carries out culture of rootage on 1/2 MS+BA 0.2mg/l+IBA0.5mg/L+PVP0.05% medium.Cultivate and to take root in about 30 days.The plant that has taken root is taken out, placed 5-7 days down, open bottle cap, clean medium, perlite is arrived in transplantation of seedlings: on the matrix that peat soil (1: 1) mixes in natural temperature and illumination condition.Keep ambient humidity about 70%, avoid the direct light irradiation initial two weeks, practice seedling and just can be transplanted to big Tanaka after one month, can reach survival rate more than 75%.
Embodiment 3
Get the bud of the Dysosma versipellis of picking up from the Mount Emei, clean with running water, the flowing water flushing is carried out surface sterilization after half an hour again, sterilization method is: behind the 70% alcohol-pickled 30s, with twice of aseptic water washing, use 0.1% mercuric chloride 100ml (add 2-3 and drip tween T-20) sterilization 10min again, clean with aseptic water washing at last.Insert on the culture medium after sterilization MS+BA 0.5mg/l+ZT1.0mg/l.Dark condition was cultivated 1～2 day down, changed under the 40W daylight double to cultivate again, and light intensity is 2000lx, and temperature remains on 25 ± 2 ℃, cultivates 15 days, as seen had about 5 sproutings to occur on each explant.Have the explant of bud to take out under aseptic condition with long, each bud is separated with scalpel, place the culture vessel that above-mentioned continuous culture base is housed to cultivate respectively, in 10-20 days, the bud in each culture vessel is renewable to go out several sproutings.To grow on the inducing culture explant transfers carry out continuous culture on MS+BA0.5mg/l+ZT0.5mg/l+GA0.1mg/l+PVP0.5mg/l.The Dysosma versipellis plant that needs are taken root is transferred on the 1/2 MS+BA 0.5mg/l+ZT0.2mg/L medium and carries out culture of rootage.Can take root about 25 days.The plant that has taken root is taken out, placed 5-7 days down, open bottle cap, clean medium, perlite is arrived in transplantation of seedlings: on the matrix that peat soil (1: 1) mixes in natural environment.Keep ambient humidity about 70%.Be transplanted to big Tanaka after one month, can reach 75% survival rate.
Embodiment 4
Practice seedling and transplanting: the Dysosma versipellis aseptic seedling that embodiment 3 is obtained, in the uncork after 7 days of natural lighting lower refining seedling.Get strong sprout from the medium pincers, be transplanted to peat soil: perlite (1: 1) is the 12cm plastic basin of matrix, every basin 1 strain, and per 15 basins are a unit.Place green house, keep temperature in the highest daytime＜35 ℃, minimum night temperature＞18 ℃, relative moisture 50～80%, shading 75%.Transplant the back and cover with plastic foil, avoided the direct light photograph in initial 7 days, 7 day after carry out a foliage spray, and remove the plastic foil of covering 1 water of blade face spray every day.Later every two weeks waters the 1/2MS nutrient solution one time.Seedling percent reaches more than 80% after 1 month, can be transplanted to field planting.
Claims (5)
1. the tissue culture quick propagation culturing method of a Dysosma versipellis is characterized in that:
1) explant selection and processing: the bud of selecting Dysosma versipellis 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, use aseptic water washing in mercuric chloride (Hgcl) sterilization after 1-10 minute then through 0.1-0.15%, cut the part of explant variable color, be inoculated in the medium;
2) inducing culture: use MS+BA (0.2-0.5mg/l)+ZT (0-1.0mg/l) as inducing culture, selected bud is inoculated in the above-mentioned medium;
3) enrichment culture: will grow on the inducing culture explant and transfer and on MS+BA (0.2-1.0mg/l)+ZT (0-0.5mg/l)+GA (0-0.2mg/l)+PVP (0-0.5mg/l), carry out continuous culture;
4) culture of rootage: select 1/2 MS+BA (0.2-0.5mg/l)+ZT (0.1-0.5mg/L) as root media;
5) transplant: the plant that will take root is taken out, and is transplanted to perlite: peat soil (1: 1-1.5) on the matrix of Hun Heing.
2. the tissue culture quick propagation culturing method of a kind of Dysosma versipellis according to claim 1, it is characterized in that said use MS+BA (0.1-0.5mg/l)+ZT (0.5-1.0mg/l) is as inducing culture, selected bud is inoculated in the above-mentioned medium: the pH value 5.8 ± 0.1 of inducing culture, 25 ± 1 ℃ of cultivation temperature, every day is during light application time 12-16, intensity of illumination 1900-2300LX.
3. the tissue culture quick propagation culturing method of a kind of Dysosma versipellis according to claim 1, it is characterized in that the said explant that will grow on the inducing culture transfers carry out continuous culture on MS+BA (0.3-1.0mg/l)+ZT (0.1-0.5mg/l)+GA (0.1-0.2mg/l)+PVP (0.1-0.5mg/l) medium: be to cultivate 15-20 days, as seen there be 1-5 sprouting to occur on the explant, increase with incubation time, the number of bud increases.Have the explant of several buds to take out under aseptic condition with long, each bud is separated with scalpel, place above-mentioned continuous culture base to cultivate, in 10-20 days, the bud in each culture vessel is renewable to go out one to several sproutings, to reach the purpose of propagation.
4. the tissue culture quick propagation culturing method of a kind of Dysosma versipellis according to claim 1 is characterized in that said selection 1/2 MS+BA (0.2-0.5mg/l)+ZT (0.1-0.5mg/L) is as root media: be the plant in the continuous culture to be changed in the root media cultivate.At first under dark condition, cultivated 1-2 days, forward to subsequently to cultivate under the light and can induce the formation root in 10-15 days, cultivate again and can form flourishing root system in 15-25 days.
5. the tissue culture quick propagation culturing method of a kind of Dysosma versipellis according to claim 1, it is characterized in that the said plant taking-up that will take root, be transplanted to perlite: peat soil (1: 1-1.5) on the matrix of Hun Heing: be that the culture vessel that will the seedling that take root be housed places the environment of natural temperature, illumination to take exercise 2-5 days down, open bottle cap then, placed 2-10 days, the plant seedling that will take root is again taken out, clean the agar of root with clear water, be transplanted to perlite: peat soil (1: 1-1.5) cultivated 20-35 days on the mixed-matrix, can cultivate subsequently in the land for growing field crops.
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| CN105104199A (en) * | 2015-08-27 | 2015-12-02 | 广西壮族自治区药用植物园 | Production method of dysosma versipellis artificial seeds |
| CN105104199B (en) * | 2015-08-27 | 2017-04-19 | 广西壮族自治区药用植物园 | Production method of dysosma versipellis artificial seeds |
| CN108770626A (en) * | 2018-06-29 | 2018-11-09 | 黔南州司奇中草药种植有限公司 | A kind of artificial method for planting of Dysosma versipellis |
| CN109321611A (en) * | 2018-10-22 | 2019-02-12 | 覃家日 | The production method of taxol |
| CN109618805A (en) * | 2019-02-27 | 2019-04-16 | 湖南春光九汇现代中药有限公司 | A kind of implantation methods of evodia rutaecarpa |
| CN111657145A (en) * | 2020-06-28 | 2020-09-15 | 张家界欣农农业科技有限公司 | Method for rapidly cultivating dysosma versipellis seedlings |
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