CN104094845A - In-vitro culture method for dendranthema indicum var aromaticum - Google Patents
In-vitro culture method for dendranthema indicum var aromaticum Download PDFInfo
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Abstract
The invention discloses an in-vitro culture method for dendranthema indicum var aromaticum. The in-vitro culture method comprises the following steps: using sterilized tender stem segments with buds as an explant, and then performing induction of cluster buds, strong seedling culture, rooting, hardening and transplanting to obtain dendranthema indicum var aromaticum regeneration plants, wherein the tender stem segments with the buds is cleaned before sterilization, that is a detergent solution with the concentration of 0.2 to 0.8% is used to soak the tender stem segments with the buds for 20-40min, then washing off is carried out, and the tender stem segments with the buds is flushed with water for 1-2h; an arundinella anomala extract and an activated carbon are added into a medium during the strong seedling culture and rooting culture, and the regeneration plants are subjected to acoustical wave stimulation during the rooting culture. According to the method provided by the invention, a large number of cluster buds of the dendranthema indicum var aromaticum can be induced in high frequency, so that the survival rate of the regeneration plants is high, and can reach 100%, and a good foundation is laid for improving the propagation coefficient of the dendranthema indicum var aromaticum as well as the germplasm conservation and genetic transformation of the dendranthema indicum var aromaticum.
Description
Technical field
The present invention relates to the propagation technique of Dendranthema indicum, relate more specifically to a kind of cultured in vitro method of Dendranthema indicum.
Background technology
Dendranthema indicum (Dendranthema indicum (L.) Des Moul.var.aromaticum Q.H.Liu et S.F.Zhang Var.Nov.) is a kind of New resource plants of Wuhan Institute of Zoology Mr. Liu Qihong nineteen eighty-two first finding and name at Shennongjia, perennial herb, blade is compared with little and thick, bottle green, vein obviously swells above, the lower mask minimum body of gland that caves in, capitulum is less, general diameter is no more than 1.5 centimetres, is that with the main distinction of mother chrysanthemum flower, leaf and root all have special strong fragrance.Dendranthema indicum is concentrated face south open hillside, the roadside that are distributed in Shennongjia height above sea level 2600-2700 rice, a new variant for the distinctive composite family of Shennongjia (Asteraceae) Chrysanthemum (Dendranthema), likeness in form mother chrysanthemum (Dendranthema indicum (L.) Des Moul.).The Mountain Area of Western Hubei Province flower drying in the shade with Dendranthema indicum among the people, leaf are used for the treatment of flu, abscess of throat, red eye, swell pain, carbuncle pin sore furuncle etc.The volatile oil component that chemical composition analysis shows Dendranthema indicum is mainly taking the monoterpene in terpene and sequiterpene and containing oxygen derivative thereof as main.Terpenoid in Dendranthema indicum volatile oil has firpene, terpineol, thujene, lauro lene, borneol, 1, the compounds such as 8-cineole, Bronyl acetate, pharmacological testing analysis shows that these compositions have antibacterial, antiviral, antispastic, eliminate the phlegm, the effect such as relieving cough and asthma; Volatile oil not only, pharmaceutically having important effect, also can be used in the spices such as beverage, cigarette and cosmetics and daily chemical industry, and its utilization rate is high, and economic worth is by fairly obvious.
The narrow distribution range of Dendranthema indicum, and resource is very limited.For this reason, all positive artificial propagation of carrying out Dendranthema indicum and cultivations of a lot of researchers.Technical staff has carried out introduction and cultivation to Dendranthema indicum respectively, can obtain fast a large amount of regeneration individualities by plant tissue culture technique, the direct development ways of organ obtains regeneration plant can effectively be shortened cultivation cycle and reduce variation link, to expanding the reproduction coefficient of these species a kind of effective ways of can yet be regarded as.Wherein there is spire taking Dendranthema indicum as explant and micro cuttage mode, set up Dendranthema indicum and in vitro breed system.Taking Flower Bud of Dendranthema indicum var. aromaticum as explant, successfully induce Flower Bud of Dendranthema indicum var. aromaticum callus and obtain regeneration plant, but limited by material source.Also have and sprout axillalry bud evoking adventive bud in vitro by Dendranthema indicum and set up Dendranthema indicum vitro Regeneration System, but its group training seedling is thin and delicate and problem excessive growth is never effectively solved, the optimization system in its strong sprout also has problems.Based on the problems referred to above, in the urgent need to passing through the screening of explant, plant growth regulator, minimal medium and condition of culture, set up the Dendranthema indicum regenerating system that breeding cycle, short, inheritance stability high-frequency occurred, thereby provide good experimental system for the research of growing under the biotechnology breeding of Dendranthema indicum, germ plasm resource preservation, Fast-propagation, manual control condition.
Summary of the invention
The object of this invention is to provide a kind of cultured in vitro method of Dendranthema indicum, can induce to high-frequency a large amount of Dendranthema indicum Multiple Buds, after strengthening seedling and rooting, regeneration plant survival rate is high, can reach 100%.
The cultured in vitro method of Dendranthema indicum of the present invention, taking the band bud point tender stem segments through sterilizing as explant, through induction, the strong seedling culture of Multiple Buds, take root, obtain Dendranthema indicum regeneration plant after hardening and transplanting, wherein:
Described band bud point tender stem segments cleans before sterilizing, and described cleaning step is: the liquid detergent solution that working concentration is 0.2-0.8% soaks 20-40min, scrub afterwards, then water rinses 1-2h; Because the tender stem segments volatile oil content of Dendranthema indicum is higher, the easily too much dust of bonding of bud point position especially on tender stem segments, and be not easy to clean.So, in cleaning, add micro-liquid detergent can effectively remove the dust boning on tender stem segments, guarantee to be with bud point tender stem segments cleaner, for inducing clumping bud becomes to provide basis.
Arundinella hirta (Thunb.) Tanaka extract and active carbon when described strong seedling culture and culture of rootage, all in its medium, are added;
The strong seedling culture base using in described strong seedling culture step is on the basis of MS minimal medium, to have added active carbon, Arundinella hirta (Thunb.) Tanaka extract, 6-benzyladenine (6-BA) and α-naphthaleneacetic acid (NAA);
Preferably, in described strong seedling culture base, the concentration of 6-benzyladenine is 0-2.0mg/L, and the concentration of α-naphthaleneacetic acid is 0-2.0mg/L, and the concentration of active carbon is 0.2-0.8%, and the concentration of Arundinella hirta (Thunb.) Tanaka supernatant is 5-10%;
The preparation method of described Arundinella hirta (Thunb.) Tanaka supernatant adds 5ml distilled water for getting 1g Arundinella hirta (Thunb.) Tanaka dried powder, leaves standstill at 4 DEG C after 20h, and the extract refrigerated centrifuge of described 0.2g/ml is extracted, and obtains Arundinella hirta (Thunb.) Tanaka supernatant.
Preferably, described strong seedling culture comprises the following steps: described strong seedling culture base is packed in conical flask, described inducing clumping bud is cultivated to the Multiple Buds obtaining to be inoculated on described strong seedling culture base, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, cultivates 20-60 days under the condition of intensity of illumination 2000-2500lux.
Preferably, described culture of rootage comprises the following steps: the Multiple Buds that described strong seedling culture step is obtained is isolated individual plant and is transferred on root media, in 25 ± 2 DEG C of temperature, light application time 12-16h/ days, root induction under intensity of illumination 2000-2500lux condition, after 5 days, start, carry out sound stimulation every day one time, time 30-60min, continues 10-15 days, sound wave frequency used is 1000Hz, and the sound intensity is 100dB.Within 20-30 days, cultivate and grow to obtain regrowth; Described root media is the 1/4-3/4MS medium that has added the 1/2MS medium of Arundinella hirta (Thunb.) Tanaka extract and active carbon or added active carbon.
Preferably, described inducing clumping bud is cultivated and is comprised the following steps: the induction step of described Multiple Buds is specially: by cutting into the short stem section of single bud point and being inoculated on inducing clumping bud medium with bud point tender stem segments through sterilizing, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, cultivates 10-15 days under intensity of illumination 2000-2500lux condition;
Described inducing clumping bud medium is on the basis of MS minimal medium, to add the 6-benzyladenine of 0-2.0mg/L, the α-naphthaleneacetic acid of 0-2.0mg/L.
Preferably, described hardening step specifically comprises: when the regeneration plant obtaining through culture of rootage grows 6-8cm, open gradually bottle cap, regeneration plant is contacted, at 20-25 DEG C of lower refining seedling 5-10 days with natural air.
Preferably, after described hardening step, transplant, be about to plant in the mixed-matrix of sandy soil and shredded coconut stuffing through the Transplantation of Regenerated Plantlets of hardening, obtain Dendranthema indicum and survive regeneration plant, the mixed proportion of described sandy soil and shredded coconut stuffing is 1: 0.25-1.
In an embodiment, the cultured in vitro method of Dendranthema indicum of the present invention specifically comprises the following steps therein:
(1) explant sterilization: choosing the young tender stem with bud of new germinating is explant; The method of explant being carried out to sterilizing is: Dendranthema indicum tender stem segments is removed to blade, in the detergent liquid for example dripping containing 4-8 for every 100ml in concentration, soak and for example after 20-40min, scrub, water rinses 1-2h again, then be placed on superclean bench and soak 10-30s in 75% alcohol, drip subsequently the 0.1%HgCl2 solution sterilization 15min of soil temperature-40 containing 2-4 with every 100ml, by sterile water wash 4 times, each 1-3min.Described detergent is commercially available common without phosphorus detergent, the carving board detergent that such as Nice Group Co., Ltd. produces etc.
(2) induction of Multiple Buds: taking the Dendranthema indicum stem section with bud point after sterilization treatment as explant, be placed on induced bundle on inducing clumping bud medium and sprout; 25 ± 2 DEG C of temperature, light application time 12-16h/ days, cultivates 10-15 days under intensity of illumination 2000-2500lux condition.Described inducing clumping bud medium is on the basis of MS minimal medium, to have added 6-benzyladenine (6-BA) and α-naphthaleneacetic acid (NAA); Wherein the concentration of 6-benzyladenine is 0-2.0mg/L, is preferably 2.0mg/L, and the concentration of α-naphthaleneacetic acid is 0-2.0mg/L, is preferably 0.05mg/L.
(3) strong seedling culture: described inducing clumping bud is cultivated to the Multiple Buds obtaining and be inoculated on strong seedling culture base, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, cultivates 20-60 days under the condition of intensity of illumination 2000-2500lux, preferably 30 days.And after 5-15 days, start, carry out sound stimulation every day one time, time 30-60min, sound wave frequency used is 1000Hz, the sound intensity is 100dB.Wherein said strong seedling culture base is: on the basis of MS minimal medium, added active carbon, Arundinella hirta (Thunb.) Tanaka extract, 6-benzyladenine (6-BA) and α-naphthaleneacetic acid (NAA).In the process of field investigation, find in the eugonic place of Dendranthema indicum, often association has Arundinella hirta (Thunb.) Tanaka, find that after deliberation Arundinella hirta (Thunb.) Tanaka plays the positive effect that helps to change to growing of Dendranthema indicum, so, in strong seedling culture base, add Arundinella hirta (Thunb.) Tanaka extract can promote growing of Multiple Buds, be conducive to obtain healthy and strong Multiple Buds, for the survival rate that improves regeneration plant lays the first stone.
Preferably, in described strong seedling culture base, the concentration of 6-benzyladenine is 0-2.0mg/L, is preferably 2.0mg/L, and the concentration of α-naphthaleneacetic acid is 0-2.0mg/L, is preferably 0.05mg/L; The concentration of active carbon is 0.2-0.8%, is preferably 0.5%, and the concentration of Arundinella hirta (Thunb.) Tanaka supernatant is 5-10%, is preferably 10%.
(4) take root and plant regeneration: the healthy and strong Multiple Buds after strong seedling culture is isolated to individual plant and be placed in root induction on root media, obtain Dendranthema indicum complete regenerated plant; Described root media is to have added the 1/4-3/4MS medium of active carbon or at the 1/2MS medium that has added active carbon and Arundinella hirta (Thunb.) Tanaka extract.Wherein, described root media is for example for adding 0.2% active carbon and 5% Arundinella hirta (Thunb.) Tanaka extract in 1/2MS medium.
Condition of culture is: 25 ± 2 DEG C of temperature, light application time 12-16h/ days, intensity of illumination 2000-2500lux.After 5 days, indefinite bud base portion starts to take root, and starts after 5 days, carries out sound stimulation every day one time, and time 30-60min continues 10-15 days, and sound wave frequency used is 1000Hz, and the sound intensity is 100dB.Cultivated and develop into whole plant, rooting rate 100% through 20-30 days.For improving the survival rate after Transplantation of Regenerated Plantlets, can carry out hardening to the regeneration plant obtaining in step (4) after culture of rootage, method is: in the time that regrowth grows 6-8cm, open gradually bottle cap, regeneration plant is contacted with natural air, after 20-25 DEG C of lower refining seedling 5-10 days, can transplant.Sound stimulation can promote the growth of Dendranthema indicum root, promotes its Rapid Rooting, is conducive to obtain the regeneration plant of well developed root system, thereby improves survival rate.
(5) hardening and transplanting: the regeneration plant obtaining after culture of rootage in step (4) is carried out to transplanting method is: in the time of height of seedling 6-8cm, the intensive stalwartness of root system, open gradually cultivation bottle cap, regeneration plant is contacted with natural air, can transplant after 5-10 days at 20-25 DEG C of temperature lower refining seedling.Transplantation of Regenerated Plantlets through hardening is planted in the mixed-matrix of the sandy soil that mix and shredded coconut stuffing, and in mixed-matrix, the ratio of sandy soil and shredded coconut stuffing is: 1: 0.25-1.Obtain Dendranthema indicum and survive regeneration plant, survival rate reaches 98%.
The invention provides a kind of extracorporeal culturing method of rare resources plant Dendranthema indicum.The method is to be with the tender stem section of bud as explant, obtains regeneration plant by inducing clumping bud approach.The advantage of carrying out Regeneration in Vitro taking stem section as explant is to draw materials conveniently, available material abundance.The method of the invention can induce to high-frequency a large amount of Dendranthema indicum Multiple Buds, and after strengthening seedling and rooting, regeneration plant survival rate is high, can reach 100%, and has advantages of that by adventitious shoot regeneration Variations of Regenerated Plants is little and genetic stability is higher.The present invention is the reproduction coefficient of raising Dendranthema indicum, haves laid a good foundation for germplasm preservation and the genetic transformation thereof of these species, has a extensive future.
Brief description of the drawings
Fig. 1 is the Dendranthema indicum Multiple Buds that the cultured in vitro method medium-high frequency of Dendranthema indicum of the present invention occurs;
Fig. 2 is the Multiple Buds through strong seedling culture in the cultured in vitro method of Dendranthema indicum of the present invention;
Fig. 3 is the individual plant seedling that in the cultured in vitro method of Dendranthema indicum of the present invention, Dendranthema indicum is taken root;
Fig. 4 is Dendranthema indicum regeneration plant root system in the cultured in vitro method of Dendranthema indicum of the present invention;
Fig. 5 is the regeneration plant of Dendranthema indicum transplant survival in the cultured in vitro method of Dendranthema indicum of the present invention.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to specification word.
The cultured in vitro of embodiment 1, Dendranthema indicum
MS minimal medium: can reference literature (Murashige T, Skoog F.A revised medium for rapid grouth and bioassays with tobacco tissue cultures.Physiol.Plant, 1962,15:473-497) preparation.
The cultured in vitro method of Dendranthema indicum of the present invention, comprises the following steps:
(1) sterilizing of stem explants
First Dendranthema indicum tender stem segments is removed to blade, in the concentration detergent liquid (the carving board detergent that Nice Group Co., Ltd. produces) that to be every 100ml drip containing 4-8, after immersion 20-40min, scrub, water rinses 1-2h again, then be placed on superclean bench and soak 10-30s in 75% alcohol, drip subsequently the 0.1%HgCl2 solution sterilization 15min of soil temperature-40 containing 2-4 with every 100ml, by sterile water wash 4-6 time, each 1-3min.
(2) induction of Multiple Buds
Inducing clumping bud medium: added 6-BA 2.0mg/L and NAA0.05mg/L on the basis of MS minimal medium.
Tender stem segments taking step (1) with bud point is as explant, be placed on described inducing clumping bud medium, for example in each blake bottle, inoculate 6 sections, 10 blake bottles of each combination inoculation, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, under intensity of illumination 2000-2500lux, induced bundle is sprouted.
As shown in Figure 1, latter the 3rd day of explant inoculation, axillalry bud grows.Inoculate after 7 days, axillalry bud base portion has graininess projection, inoculates after 10 days, and clump base portion is sprouted to germinate and grown.
(3) strong seedling culture
Strong seedling culture base: added 6-BA 2.0mg/L and NAA 2.0mg/L on the basis of MS minimal medium, added the activated carbon powder of 0.5% concentration and 10% Arundinella hirta (Thunb.) Tanaka extract simultaneously.
The Multiple Buds that induction obtains for 15 days afterwards in step (2) is transferred on the above-mentioned medium that contains variable concentrations active carbon and carried out strong seedling culture, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, under intensity of illumination 2000-2500lux, cultivate, cultivating robust growth (Fig. 2) after 30 days.
(4) take root and plant regeneration
Root media: the Arundinella hirta (Thunb.) Tanaka extract that has added 0.2% active carbon and 5% on the basis of 1/2MS medium (macro-and microelements comprising be MS minimal medium full dose 1/2).
The Multiple Buds that step (3) is obtained is isolated individual plant and is transferred on root media 25 ± 2 DEG C of temperature, light application time 12-16h/ days, root induction under intensity of illumination 2000-2500lux, after 5 days, indefinite bud base portion starts to take root, and starts after 5 days, carries out sound stimulation every day one time, time 40min, continue 10 days, sound wave frequency used is 1000Hz, and the sound intensity is 100dB.As shown in Figure 3, Figure 4, cultivated and develop into whole plant, rooting rate 100% through 20-30 days.
(5) hardening and transplanting
The regeneration plant obtaining after culture of rootage in step (4) is carried out to transplanting method is: in the time of height of seedling 6-8cm, the intensive stalwartness of root system, open gradually cultivation bottle cap, regeneration plant is contacted with natural air, can transplant after 5-10 days at 20-25 DEG C of temperature lower refining seedling.By through the Transplantation of Regenerated Plantlets of hardening in planting in geometric ratio (the 1/4-3/4 ratio all can) sandy soil of mixing and the mixed-matrix of shredded coconut stuffing, obtain Dendranthema indicum and survive regeneration plant, survival rate reaches 98% (Fig. 5).
The cultured in vitro of embodiment 2, Dendranthema indicum
MS minimal medium: can reference literature (Murashige T, Skoog F.A revised medium for rapid grouth and bioassays with tobacco tissue cultures.Physiol.Plant, 1962,15:473-497) preparation.
The cultured in vitro method of Dendranthema indicum of the present invention, comprises the following steps:
(1) sterilizing of stem explants
First Dendranthema indicum tender stem segments is removed to blade, in the concentration detergent liquid (the carving board detergent that Nice Group Co., Ltd. produces) that to be every 100ml drip containing 4-8, after immersion 20-40min, scrub, water rinses 1-2h again, then be placed on superclean bench and soak 10-30s in 75% alcohol, drip subsequently the 0.1%HgCl2 solution sterilization 15min of soil temperature-40 containing 2-4 with every 100ml, by sterile water wash 4-6 time, each 1-3min.
(2) induction of Multiple Buds
Inducing clumping bud medium: added 6-BA 2.0mg/L and NAA0.08mg/L on the basis of MS minimal medium.
Tender stem segments taking step (1) with bud point is as explant, be placed on described inducing clumping bud medium, for example in each blake bottle, inoculate 6 sections, 10 blake bottles of each combination inoculation, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, under intensity of illumination 2000-2500lux, induced bundle is sprouted.
As shown in Figure 1, latter the 3rd day of explant inoculation, axillalry bud grows.Inoculate after 7 days, axillalry bud base portion has graininess projection, inoculates after 10 days, and clump base portion is sprouted to germinate and grown.
Inducing clumping bud medium is except the medium explant callusization being 1.0-2.0mg/L containing NAA concentration is serious, and in the time of 6-BA 2.0mg/L and NAA 0.05mg/L, the inductivity of Multiple Buds is the highest, is that 83.3%, 1 axillalry bud base portion can germinate 6-8 indefinite bud.On the MS medium that contains high concentration NAA (1.0-2.0mg/L), callusization is serious, can not induce Multiple Buds.Show that, in the Induction Process of Dendranthema indicum Multiple Buds, 6-BA is essential, NAA has certain inhibitory action, therefore inducing clumping bud medium is decided to be: on the basis of MS minimal medium, added 6-BA 0-2.0mg/L and NAA 0-1.0mg/L.If the Multiple Buds that induction produces continues to cultivate on former medium, the further intensive germinating of Multiple Buds, but easily there is vitrification phenomenon, cause indefinite bud anamorphosis undesired.
(3) strong seedling culture
Strong seedling culture base: added 6-BA 2.0mg/L and NAA 2.0mg/L on the basis of MS minimal medium, added the activated carbon powder of 0.5% concentration and 8% Arundinella hirta (Thunb.) Tanaka extract simultaneously.
The Multiple Buds that induction obtains for 15 days afterwards in step (2) is transferred on the above-mentioned medium that contains variable concentrations active carbon and carried out strong seedling culture, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, under intensity of illumination 2000-2500lux, cultivate, cultivating robust growth (Fig. 2) after 30 days.
Research is found, is adding on the strong seedling culture base of 0.5% active carbon, and thin and delicate slight vitrified Multiple Buds is being cultivated robust growth after 30 days, can be for taking root and plant regeneration.Indefinite bud poor growth on the active carbon medium of excessive concentrations may be due to due to too much activated carbon powder absorption plant growth regulator and nutrient component.
(4) take root and plant regeneration
Root media: the Arundinella hirta (Thunb.) Tanaka extract that has added 0.2% active carbon and 5% on the basis of 1/2MS medium (macro-and microelements comprising be MS minimal medium full dose 1/2).
The Multiple Buds that step (3) is obtained is isolated individual plant and is transferred on root media 25 ± 2 DEG C of temperature, light application time 12-16h/ days, root induction under intensity of illumination 2000-2500lux, after 5 days, indefinite bud base portion starts to take root, and starts after 5 days, carries out sound stimulation every day one time, time 30min, continue 15 days, sound wave frequency used is 1000Hz, and the sound intensity is 100dB.As shown in Figure 3, Figure 4, cultivated and develop into whole plant, rooting rate 100% through 20-30 days.
(5) hardening and transplanting
The regeneration plant obtaining after culture of rootage in step (4) is carried out to transplanting method is: in the time of height of seedling 6-8cm, the intensive stalwartness of root system, open gradually cultivation bottle cap, regeneration plant is contacted with natural air, can transplant after 5-10 days at 20-25 DEG C of temperature lower refining seedling.By through the Transplantation of Regenerated Plantlets of hardening in planting in geometric ratio (the 1/4-3/4 ratio all can) sandy soil of mixing and the mixed-matrix of shredded coconut stuffing, obtain Dendranthema indicum and survive regeneration plant, survival rate reaches 98%.
Although embodiment of the present invention are open as above, but it is not restricted to listed utilization in specification and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend of describing.
Claims (7)
1. a cultured in vitro method for Dendranthema indicum, taking the band bud point tender stem segments through sterilizing as explant, through induction, the strong seedling culture of Multiple Buds, take root, obtain Dendranthema indicum regeneration plant after hardening and transplanting, wherein:
Described band bud point tender stem segments cleans before sterilizing, and described cleaning step is: the liquid detergent solution that working concentration is 0.2-0.8% soaks 20-40min, scrub afterwards, then water rinses 1-2h;
Arundinella hirta (Thunb.) Tanaka extract and active carbon when described strong seedling culture and culture of rootage, all in its medium, are added;
The strong seedling culture base using in described strong seedling culture step is on the basis of MS minimal medium, to have added active carbon, Arundinella hirta (Thunb.) Tanaka extract, 6-benzyladenine and α-naphthalene second
acid.
2. Dendranthema indicum cultured in vitro method as claimed in claim 1, it is characterized in that, in described strong seedling culture base, the concentration of 6-benzyladenine is 0-2.0mg/L, and the concentration of α-naphthaleneacetic acid is 0-2.0mg/L, the concentration of active carbon is 0.2-0.8%, and the concentration of Arundinella hirta (Thunb.) Tanaka supernatant is 5-10%;
The preparation method of described Arundinella hirta (Thunb.) Tanaka supernatant is the ratio lixiviate that adds 5ml distilled water according to 1g Arundinella hirta (Thunb.) Tanaka dried powder, leaves standstill at 4 DEG C after 20h, and the extract refrigerated centrifuge of described 0.2g/ml is extracted, and obtains Arundinella hirta (Thunb.) Tanaka supernatant.
3. Dendranthema indicum cultured in vitro method as claimed in claim 2, it is characterized in that, described strong seedling culture comprises the following steps: described inducing clumping bud is cultivated to the Multiple Buds obtaining and be inoculated on described strong seedling culture base, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, cultivates 20-60 days under the condition of intensity of illumination 2000-2500lux.
4. Dendranthema indicum cultured in vitro method as claimed in claim 1, it is characterized in that, described culture of rootage comprises the following steps: the Multiple Buds that described strong seedling culture step is obtained is isolated individual plant and is transferred on root media, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, root induction under intensity of illumination 2000-2500lux condition started after 5 days, carried out sound stimulation every day one time, time 30-60min, continue 10-15 days, sound wave frequency used is 1000Hz, and the sound intensity is 100dB; Within 20-30 days, cultivate and grow to obtain regeneration plant; Described root media is the 1/4-3/4MS medium that has added the 1/2MS medium of Arundinella hirta (Thunb.) Tanaka extract and active carbon or added active carbon.
5. Dendranthema indicum cultured in vitro method as claimed in claim 1, it is characterized in that, described inducing clumping bud is cultivated and is comprised the following steps: by cutting into the short stem section of single bud point and being inoculated on inducing clumping bud medium with bud point tender stem segments through sterilizing, 25 ± 2 DEG C of temperature, light application time 12-16h/ days, cultivates 10-15 days under intensity of illumination 2000-2500lux condition;
Described inducing clumping bud medium is on the basis of MS minimal medium, to add the 6-benzyladenine of 0-2.0mg/L, the α-naphthaleneacetic acid of 0-2.0mg/L.
6. Dendranthema indicum cultured in vitro method as claimed in claim 1, it is characterized in that, described hardening step specifically comprises: when the regeneration plant obtaining through culture of rootage grows 6-8cm, open gradually bottle cap, regeneration plant is contacted, at 20-25 DEG C of lower refining seedling 5-10 days with natural air.
7. Dendranthema indicum cultured in vitro method as claimed in claim 6, it is characterized in that, after described hardening step, transplant, be about to plant in the mixed-matrix of sandy soil and shredded coconut stuffing through the Transplantation of Regenerated Plantlets of hardening, obtain Dendranthema indicum and survive regeneration plant, the mixed proportion of described sandy soil and shredded coconut stuffing is 1: 0.25-1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104304022A (en) * | 2014-10-24 | 2015-01-28 | 柳州市天姿园艺有限公司 | Special culture medium for fertilized chrysanthemum |
| CN104351049A (en) * | 2014-10-24 | 2015-02-18 | 柳州市天姿园艺有限公司 | Special culture medium for cineraria |
| CN104351050A (en) * | 2014-10-24 | 2015-02-18 | 柳州市天姿园艺有限公司 | Special culture medium for chrysanthemum chanetii |
| CN105475144A (en) * | 2016-02-26 | 2016-04-13 | 邓珂 | Huai-pearl chrysanthemum tissue culture medium |
| CN112167058A (en) * | 2020-09-29 | 2021-01-05 | 四川云辰园林科技有限公司 | Rapid propagation method for improved plant tissue culture |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104304022A (en) * | 2014-10-24 | 2015-01-28 | 柳州市天姿园艺有限公司 | Special culture medium for fertilized chrysanthemum |
| CN104351049A (en) * | 2014-10-24 | 2015-02-18 | 柳州市天姿园艺有限公司 | Special culture medium for cineraria |
| CN104351050A (en) * | 2014-10-24 | 2015-02-18 | 柳州市天姿园艺有限公司 | Special culture medium for chrysanthemum chanetii |
| CN104304022B (en) * | 2014-10-24 | 2016-06-15 | 柳州市天姿园艺有限公司 | Chrysanthemum special culture media after fertilizer |
| CN104351049B (en) * | 2014-10-24 | 2016-08-17 | 柳州市天姿园艺有限公司 | Cineraria special culture media |
| CN104351050B (en) * | 2014-10-24 | 2016-08-24 | 柳州市天姿园艺有限公司 | Little red chrysanthemum special culture media |
| CN105475144A (en) * | 2016-02-26 | 2016-04-13 | 邓珂 | Huai-pearl chrysanthemum tissue culture medium |
| CN112167058A (en) * | 2020-09-29 | 2021-01-05 | 四川云辰园林科技有限公司 | Rapid propagation method for improved plant tissue culture |
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