CN101473792B - Tissue culture of Ypsilandra thibetica and planting method - Google Patents
Tissue culture of Ypsilandra thibetica and planting method Download PDFInfo
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- CN101473792B CN101473792B CN2009100940571A CN200910094057A CN101473792B CN 101473792 B CN101473792 B CN 101473792B CN 2009100940571 A CN2009100940571 A CN 2009100940571A CN 200910094057 A CN200910094057 A CN 200910094057A CN 101473792 B CN101473792 B CN 101473792B
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Abstract
The invention provides a method for in vitro tissue culture and nuisance-free planting by taking maternal tissue leaves of Ypsilandra thibetica as explant materials. Large quantities of tissue culture seedlings can be obtained by the method, which meets the planting requirement. In the method, plantation in a natural forest is adopted to overcome pollution caused by pesticides and heavy metals, the survival rate reaches 95.5%, new medicinal materials can be supplied within 3 years, weight of an individual plant is twice more than that of wild medicinal materials and non-proliferated medicinal materials at the same year, the content of effective ingredients which can induce uterus contraction and arrest bleeding is higher than that of the wild medicinal materials at the same year, thus meeting the requirement of pharmaceutical enterprises for raw medicinal materials. The method helps culture the tissue culture seedlings in batches and plant the tissue culture seedlings at a large area, and obtain the nuisance-free medicinal materials 2 years earlier, therefore, the method has popularization value.
Description
Technical field:
The invention belongs to Plant Tissue Breeding and transplant the planting technology field, particularly, relate to the tissue culture technique and the non-polluted planting method of the new medicinal material of liliaceous plant Ypsilandra.
Background technology:
The Ypsilandra plant has heat-clearing, detoxifcation, dissipating bind, diuresis at title Shi Fengdan among the people, red with oneself, cures mainly consumptive disease pestilence, oedema, and this platymiscium has four kinds, is respectively Ypsilandra (Y.thibetica); Yunnan Ypsilandra (Y.yunnanensis var.micranthaHand-Mazz); High mountain Ypsilandra (Y.alpina wang et Tang); Little Ypsilandra (Y.cavaleriei Levl et Vant) all belongs to the unlisted Chinese medicine in market at home, is listed in Yunnan Province's traditional Chinese medicine standard by Yunnan Province's food and medicine Surveillance Authority, 2005 version Yi nationality, distributed over Yunnan, Sichuan and Guizhou medicine (III) in 2009 the issue.It now is natural plants.It is raw material that Chinese patent CN1081036C discloses with the Ypsilandra herb, extracts acquisition furan steroid class steroid saponin and has active anticancer, and this patent of invention is also recorded, and (Kawabc.et.al.CS 1996,124:325368y) in U.S. chemical abstract.Chinese patent is 03117694.1 to disclose the effect that steroid saponin in the Ypsilandra platymiscium has the treatment gynecology hemorrhagic disease.
After the pharmacy corporation Ypsilandra was the new drug of raw material production for treating gynecologic blood diseases, supply Ypsilandra traditional Chinese medicine was estimated every year and is not less than the 4000-5000 ton on the market.Only rely on wild Ypsilandra resource, can't solve and satisfy the needs that consume.Be badly in need of plantation, realize the regeneration and the sustainable utilization of Ypsilandra resource.
Because the Ypsilandra pollen tube enters blastular by the hole of bead and discharges two sperms and belong to the double fertilization process, zygote resting stage of after fertilization is longer, embryonic development is slower, embryo not differentiation as yet during seed maturity, embryo still needs further slowly to grow, so fail successfully with seminal propagation, immature Ypsilandra is not found at Ypsilandra week Wall in the field yet.Chinese patent application " a kind of artificial breeding method of Ypsilandra " number of patent application is 200610048655.1 for this reason, and disclosing with the Ypsilandra leaf is that material carries out artificial propagation techniques research achieving success, and emergence rate is up to 67%.Yet this technology is because emergence rate is the highest only reaches 67%, and emergence rate is still lower, and this method artificial planting needs the soil, shades time-consuming and has high input, and can not satisfy establishing in large scale and to the wilderness demand of medicinal material.
Summary of the invention:
At the above-mentioned deficiency of present technology, the invention provides a kind of Ypsilandra tissue cultivating and non-polluted planting technology, adopt method of the present invention, can obtain large quantities of tissue cultivating seedling and satisfy the plantation needs; And adopt the natural forest area plantation to overcome agricultural chemicals and heavy metal pollution, and survival rate reached in 95.5%, three year can provide new medicinal material, and individual plant weight is higher than 2 times of wild and no numerous plantation medicinal materials in the same year, and the oxytocin bleeding active constituent content is higher than natural crude drugs in the same year; And satisfy the demand of pharmacy corporation to crude drug, but not only can cultivate in batch but also the establishing in large scale tissue cultivating seedling, and obtained nuisanceless medicinal material in 2 years ahead of time, have practical value.
In order to realize purpose of the present invention, the invention provides following technical scheme:
The group training of Ypsilandra and kind method for planting comprise and get the Ypsilandra explant that sterilization is cleaned, callus induction and differentiation, adventitious bud proliferation, root induction, hardening, non-polluted planting step.
Described tissue cultivating and seedling is to grow seedlings with leaf texture's cultured in vitro, cut blade, be seeded in after the sterilization on the MS medium and cultivated 30 days, form callus and be divided into clump bud, the single incision switching of the indefinite bud of cultivating enrichment culture on medium, the seedling that forms paraphyte is transferred in the medium of 1/2MS+IBA 0.2mg/L, takes root in 15-21 days, transplants behind the white silk seedling.
The preferred blade of Ypsilandra explant, through washing, 70% ethanol and mercuric chloride sterilization, sterile water is cut into small pieces after cleaning, and carries out callus induction and differentiation.
Callus induction and differentiation are under aseptic condition, cut 1 square centimeter of leaflet piece from Ypsilandra sterilization rear blade, being seeded in the MS medium adds and contains 6-BA 0.25-0.5mg/L, on the NAA 0.2-0.3mg/L medium, at 20-25 ℃, illumination 1000Lx, illumination 10-12 hour, humidity produces the clump bud greater than directly inducing under 70% condition, and 30-40 days begin to sprout; There was clump bud to occur again through 8-14 days; The clump bud came into leaves in 7-15 days.
Adventitious bud proliferation is with callus induction and the single incision of clump bud that differentiates, and adds at the MS medium and contains 6-BA 0.5mg/L, carries out enrichment culture on the NAA 0.3-0.4mg/L medium; Just can form seedling in 40-60 days; The new clump bud that enrichment culture produces, it is green that leaf turns, and during 2-3cm, further enrichment culture or successive transfer culture are carried out in switching.
Root induction is the seedling of taking out the high 4-5cm that enrichment culture grows, and single incision is transferred in the 1/2MS+IBA 0.2mg/L medium, and switching back 15-21 days begins to take root; Treat that 40-60 days roots reach more than 3, leaf reaches the 6-10 sheet, practices seedling up to the examination villous themeda seedling taking-up of 6-8cm.
Hardening is examination villous themeda seedling to be taken out from group training chamber be placed on the warm canopy, placed 5-7 days, taking out seedling cleans and is implanted in that detritus soil and laterite 1: 1 to be housed and to add a little detritus leaf be in the nutritious bag of matrix behind the medium, water, in 1000-2000Lx illumination 8-12 hour, humidity, is planted up to 6-8cm time shift plantation when leaf reaches the 6-10 sheet greater than practicing seedling 1 month under 70% condition.
Non-polluted planting is to plant under natural endowment, selects height above sea level 800 ~ 2,500m, sylvan life, limes marginis, waterfall both sides or the waste soil of the condition of sheltering from heat or light is arranged; Satisfy the annual at sunshine 6 hours/day, annual maximum temperature are not higher than 18 ℃, monthly mean of daily maximum temperature 11 ~ 23 ℃, the minimum condition that reaches 0 ℃; Satisfy underground 5cm soil temperature annual maximum temperature and be not higher than 16 ℃, monthly mean of daily maximum temperature 9 ~ 20 ℃, the minimum condition that reaches below 9 ℃; Set up isolated area, the anti-oxen and horses enter and step on bad.
Particularly, technical solution of the present invention can be described as:
1, material:
Gathering wild or tame kind of Ypsilandra complete stool culture transferring in firework, is the material of explant with wild or tame kind of Ypsilandra parent leaf.
2, method:
1). the explant sterilization:: cut the wild Ypsilandra blade of planting in box, maternal tissue's blade of water flushing Ypsilandra, ethanol through 70% and mercuric chloride sterilization, sterile water is cut into 1cm after cleaning
2Blockage is put 4-6 piece explant for every bottle, covers tight bottle cap, seals bottleneck with freshness protection package.
2) group training:
1. medium preparation: with MS is minimal medium, and other adds the plant hormone of variable concentrations.Grow body outward by blade and induce the generation callus, and evoked callus produces indefinite bud, make adventitious bud proliferation, indefinite bud forms seedling, carries out culture of rootage, adopts to add to contain 6-BA 0.25-0.5mg/L (6-benzyladenine), NAA 0.2-0.4mg/L (Naphthalene acetc acid, methyl) and add and contain IBA 0.2mg/L medium, process for preparation is according to conventional method, and blake bottle is vial or transparent plastic bottle.20 minutes high pressure moist heat sterilizations of 121 ℃ of sterilizations are adopted in sterilization.
2. condition of tissue culture:
At 20-25 ℃, illumination 1000Lx, illumination 10-12 hour, humidity was greater than under 70% condition:
Growing body outward induces the generation callus to form generation indefinite bud: 30-40 days.Back 8-14 days appearance clumps bud begins to sprout; Come into leaves after 7-15 days clump buds.
Adventitious bud proliferation: take out the single incision of clump bud of having come into leaves and carry out just can forming seedling in enrichment culture 40-60 days.The seedling of getting its high 4-5cm carries out culture of rootage.The new clump bud that enrichment culture produces, it is green that leaf turns, and 2-3cm can one advances to transfer and carries out enrichment culture or successive transfer culture.
Culture of rootage: take out the seedling that enrichment culture grows high 4-5cm, single incision is transferred to and contains in the IBA 0.2mg/L nutrient chemical, and switching back 15-21 days begins to take root.Root reached more than 3 in 40-60 days, leaf 6-10 sheet, and the examination villous themeda seedling of high 6-8cm can take out experienced seedling.
3. hardening: will try the villous themeda seedling and place warm canopy, and under control illumination and the humidity, place 5-7 days, and take out examination villous themeda seedling and clean medium with clear water and be implanted in and be mixed with in the matrix nutrition bag, and water clear water, and survive the back transplanting January of preserving moisture, can gather in 2 years big Tanaka.
3, plantation:
Non-polluted planting under the natural endowment.Plantation under the natural endowment: select height above sea level 800 ~ 2,500m, sylvan life, limes marginis, a day as requested is satisfied: annual 6 hours/day in waterfall both sides or the waste soil of the condition of sheltering from heat or light is arranged; The annual maximum temperature is not higher than 18 ℃, and monthly mean of daily maximum temperature is at 11 ~ 23 ℃, minimumly reaches 0 ℃.Underground 5cm soil temperature: the annual maximum temperature is not higher than 16 ℃, and monthly mean of daily maximum temperature is at 9 ~ 20 ℃, minimum reaching below 9 ℃.
Natural endowment is plantation down, sets up isolated area, and the anti-oxen and horses enter and step on bad.Natural endowment of the present invention is plantation down, 4 years results, fresh weight 5-10 gram/strain/year, active constituent content 1.3% (greater than wild 1.07%); Bionical attitude plantation can be gathered in the crops fresh weight 15-30 gram/strain/year, active constituent content 1.6% in 3 years.
Beneficial effect of the present invention is:
Adopt method of the present invention, can obtain large quantities of tissue cultivating seedling and satisfy the plantation needs; And adopt the natural forest area plantation to overcome agricultural chemicals and heavy metal pollution, survival rate reached in 95.5%, three year can provide new medicinal material, and individual plant weight is higher than 2 times of wild and no numerous plantation medicinal materials in the same year, and the oxytocin bleeding active constituent content is higher than natural crude drugs in the same year, has the practical value of popularization.Can satisfy the demand of pharmacy corporation, but not only can cultivate in batch but also the establishing in large scale tissue cultivating seedling, and obtain nuisanceless medicinal material in 2 years ahead of time, have practical value crude drug.
Description of drawings:
Fig. 1 represents that Ypsilandra maternal plant leaf of the present invention makes explant;
Fig. 2 represents evoked callus figure of the present invention;
Fig. 3 represents callus differentiation figure of the present invention;
Fig. 4 represents that the present invention takes root;
Fig. 5 represents hardening of the present invention;
Fig. 6 represents that box of the present invention plantation plants;
Fig. 7 represents natural nuisance-free plantation of the present invention.
Embodiment:
Below in conjunction with accompanying drawing, by embodiments of the invention, foregoing of the present invention is described in further detail again, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technical schemes that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1:
1, the taking and handling of explant material:
Select the anosis maternal plant New Development young leaflet tablet leaf of Ypsilandra 3-8 month riotous growth for growing the body material outward.Gather wild or tame kind of Ypsilandra complete stool culture transferring in firework, wash leaf, treat that young leaves grows with clear water.Cutting blades from maternal plant washed 15 minutes with running water, remove the dust and the fine hair of leaf surface, blade is put into 20 seconds of 70% ethanol upset, remove ethanol with the flushing with clean water blade face after removing the waxy substance of leaf surface, put into 0.1% mercuric chloride liquid and soak sterilization in 15 minutes, sterile water is cut into 1cm after cleaning
2Blockage.Leaf back lies against in the medium, puts 4-6 piece explant for every bottle.Cover tight bottle cap, seal bottleneck with freshness protection package.On the sterile working platform, finish inoculation.
2, callus induction and differentiation, adventitious bud proliferation and root induction:
2.1 callus induction and differentiation: under aseptic condition, cut 1 square centimeter of leaflet piece from the sterilization rear blade, being seeded in the MS medium adds and contains 6-BA 0.25-0.5mg/L (6-benzyladenine), on NAA 0.2-0.3mg/L (Naphthalene acetc acid, the methyl) medium, at 20-25 ℃, illumination 1000Lx, illumination 10-12 hour, humidity was greater than under 70% condition: directly induce to produce the clump bud, 30-40 days begin to sprout; There was clump bud to occur again through 8-14 days; The clump bud came into leaves in 7-15 days.
2.2 adventitious bud proliferation: take out the single incision of clump bud of having come into leaves, add at the MS medium and contain 6-BA0.5mg/L, on the NAA 0.3-0.4mg/L medium, carry out enrichment culture.Just can form seedling in 40-60 days.The seedling of getting its high 4-5cm carries out culture of rootage.The new clump bud that enrichment culture produces, it is green that leaf turns, and 2-3cm can one advances to transfer and carries out enrichment culture or successive transfer culture.
2.3 root induction: take out the seedling that enrichment culture grows high 4-5cm, single incision is transferred in the 1/2MS+IBA 0.2mg/L cultivation, and switching back 15-21 days begins to take root.Root reached more than 3 in 40-60 days, leaf 6-10 sheet, and the examination villous themeda seedling of high 6-8cm can take out experienced seedling.
3, hardening:
To try the villous themeda seedling is placed on the warm canopy from group training chamber taking-up, placed 5-7 days, and took out seedling and be implanted in the nutritious bag, detritus soil and laterite are housed in the nutritious bag are mixed at 1: 1 with the clean medium of clear water, adding a little detritus leaf is matrix, around clear water, the control illumination 1000-2000Lx that preserves moisture, illumination 8-12 hour, humidity is greater than under 70% condition, practice seedling and survive January and to reach 92.5%, leaf 6-10 sheet, high 6-8cm transplants big Tanaka.Can gather in 2 years.Transplant according to the condition of embodiment 2 after practicing seedling, fresh weight 12 gram/strains in the time of 6 months, after 1 year, it is medicinal that the content of mensuration oxytocin bleeding composition reaches 1.6%, two year and can gather in acrial part.
4, plantation:
Natural endowment is plantation down:
Select height above sea level 800 ~ 2,500m, sylvan life, limes marginis, waterfall both sides or one-tenth that the condition of sheltering from heat or light arranged are all over the soil.Satisfy a day as requested: 0 ~ 8 hour/day, annual 6 hours/day, seeding row spacing is decided according to environment, can be rare, can be close.
Weather conditions: temperature: the annual maximum temperature is not higher than 18 ℃, and monthly mean of daily maximum temperature is at 11 ~ 23 ℃, minimumly reaches 0 ℃.Underground 5cm soil temperature: the maximum temperature annual is not higher than 16 ℃, and monthly mean of daily maximum temperature is at 9 ~ 20 ℃, minimum reaching below 9 ℃.Humidity: be up to 90%, minimumly be not less than 70%, monthly average humidity is between 70 ~ 90%, and natural tree is sheltered from heat or light.Natural endowment is plantation down, sets up isolated area, and the anti-oxen and horses enter and step on bad.
To sum up, cultivation method emergence rate height provided by the invention, survival rate is high by 90%, has realized that wild Ypsilandra man plants.For Ypsilandra becomes new traditional Chinese medicine, a large amount of practical methods that provide of using.
Claims (8)
1. tissue culture of Ypsilandra thibetica and plant method for planting, comprise and get the Ypsilandra explant, sterilization is cleaned, add 6-BA 0.25-0.5mg/L at the MS medium, carry out callus induction and differentiation among the NAA 0.2-0.3mg/L, add 6-BA 0.5mg/L at the MS medium, carry out adventitious bud proliferation among the NAA 0.3-0.4mg/L, in 1/2MS+IBA 0.2mg/L medium, carry out root induction, hardening, non-polluted planting is promptly planted step under natural endowment.
2. method according to claim 1, it is characterized in that: described group of training is to grow seedlings with leaf texture's cultured in vitro, cut blade, be seeded in the MS medium after the sterilization and add 6-BA0.25-0.5mg/L, cultivated 30 days among the NAA 0.2-0.3mg/L, form callus and be divided into clump bud, the single incision switching of the indefinite bud of cultivating adds 6-BA0.5mg/L at the MS medium, carry out enrichment culture among the NAA 0.3-0.4mg/L, form the seedling of paraphyte, be transferred in the medium of 1/2MS+IBA 0.2mg/L, took root, and transplanted after the hardening in 15-21 days.
3. the method for claim 1 is characterized in that the Ypsilandra explant selects blade for use, through washing, and 70% ethanol and mercuric chloride sterilization, sterile water is cut into small pieces after cleaning, and carries out callus induction and differentiation.
4. the method for claim 1, it is characterized in that callus induction and differentiation are under aseptic condition, cut 1 square centimeter of leaflet piece from Ypsilandra sterilization rear blade, be seeded in the MS medium and add and contain 6-BA 0.25-0.5mg/L, on the NAA 0.2-0.3mg/L medium, at 20-25 ℃, illumination 1000Lx, illumination 10-12 hour, humidity produced the clump bud greater than directly inducing under 70% condition, and 30-40 days begin to sprout; There was clump bud to occur again through 8-14 days; The clump bud came into leaves in 7-15 days.
5. the method for claim 1 is characterized in that adventitious bud proliferation is with callus induction and the single incision of clump bud that differentiates, and adds at the MS medium and contains 6-BA 0.5mg/L, carries out enrichment culture on the NAA 0.3-0.4mg/L medium; Formed seedling in 40-60 days; The new clump bud that enrichment culture produces, it is green that leaf turns, and during 2-3cm, further enrichment culture or successive transfer culture are carried out in switching.
6. the method for claim 1 is characterized in that root induction is the seedling of taking out the high 4-5cm that enrichment culture grows, and single incision is transferred in the 1/2MS+IBA 0.2mg/L medium, and switching back 15-21 days begins to take root; Treat that 40-60 days roots reach more than 3, leaf reaches the 6-10 sheet, up to the test-tube plantlet taking-up hardening of 6-8cm.
7. as claim 1 or 6 described methods, it is characterized in that hardening is test-tube plantlet to be taken out from group training chamber be placed on the warm canopy, placed 5-7 days, taking out seedling cleans and is implanted in that detritus soil and laterite 1: 1 to be housed and to add a little detritus leaf be in the nutritious bag of matrix behind the medium, water, in 1000-2000Lx illumination 8-12 hour, humidity was greater than 70% condition lower refining seedling 1 month, when leaf reaches the 6-10 sheet, plant up to 6-8cm time shift plantation.
8. the method for claim 1 is characterized in that non-polluted planting is to plant under natural endowment, selects height above sea level 800～2,500m; Sylvan life, limes marginis, waterfall both sides or the waste soil of the condition of sheltering from heat or light is arranged; Satisfied the annual at sunshine 6 hours/day, 18 ℃ of annual maximum temperatures, the condition that 11～23 ℃ of monthly mean of daily maximum temperature and lowest temperature are 0 ℃; Satisfy 16 ℃ of underground 5cm soil temperature annual maximum temperatures, 9～20 ℃ of monthly mean of daily maximum temperature and minimum 9 ℃ condition; Satisfy humidity and be up to 90%, minimum 70%, the condition of monthly average humidity between 70～90%; Set up isolated area, the anti-oxen and horses enter and step on bad.
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| CN105684852A (en) * | 2016-02-26 | 2016-06-22 | 邓珂 | Soilless culture method for narcissuses |
| CN105660238A (en) * | 2016-02-26 | 2016-06-15 | 邓珂 | Seedling hardening method for hoya carnosa |
| CN107155552A (en) * | 2017-04-25 | 2017-09-15 | 马山县盛世农业发展有限责任公司 | Use the implantation methods of the elegant jessamine of tissue-cultured seedling |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1537570A (en) * | 2003-04-15 | 2004-10-20 | 中国科学院昆明植物研究所 | Application of Ypsilandra herbs for preparing medicines for treating hemorrhagic diseases |
| CN1918973A (en) * | 2006-09-05 | 2007-02-28 | 红河千山生物工程有限公司 | Artificial breeding method for common forkstamenflower |
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2009
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1537570A (en) * | 2003-04-15 | 2004-10-20 | 中国科学院昆明植物研究所 | Application of Ypsilandra herbs for preparing medicines for treating hemorrhagic diseases |
| CN1918973A (en) * | 2006-09-05 | 2007-02-28 | 红河千山生物工程有限公司 | Artificial breeding method for common forkstamenflower |
Non-Patent Citations (2)
| Title |
|---|
| 赵桦.丫蕊花(Ypsilandra thibetica Franch.)胚胎发育的研究Ⅰ.大孢子,雌配子体的发生以及淀粉粒的动态.《四川大学学报》.1980,(第4期),183-189. * |
| 赵桦.丫蕊花(YpsilandrathibeticaFranch.)胚胎发育的研究Ⅰ.大孢子 雌配子体的发生以及淀粉粒的动态.《四川大学学报》.1980 |
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