CN104094845B - A kind of in-vitro culture method of Dendranthema indicum - Google Patents
A kind of in-vitro culture method of Dendranthema indicum Download PDFInfo
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Abstract
The invention discloses the in-vitro culture method of a kind of Dendranthema indicum, it is outer implant with sterilized band bud point tender stem segments, through Multiple Buds induction, strong seedling culture, take root, obtain Dendranthema indicum regeneration plant after seedling exercising and transplanting, wherein: described band bud point tender stem segments is carried out before sterilization, concentration is liquid detergent solution soaking 20 40min of 0.2 0.8%, scrubs afterwards and rinses 1 2h with water again；Arundinella hirta's extracting solution and activated carbon is added the most in the medium when described strong seedling culture and root culture；And when root culture, regeneration plant is carried out sound stimulation.The method of the invention can induce to altofrequency substantial amounts of Dendranthema indicum Multiple Buds, regeneration plant survival rate is high, up to 100%, the breeding coefficient for raising Dendranthema indicum, preserving seed and the genetic transformation thereof for these species are had laid a good foundation, and have a extensive future.
Description
Technical field
The present invention relates to the reproduction technique of Dendranthema indicum, relate more specifically to the in vitro training of a kind of Dendranthema indicum
Breeding method.
Background technology
Dendranthema indicum (Dendranthema indicum (L.) Des Moul.var.aromaticum Q.H.Liu
Et S.F.Zhang Var.Nov.) be Wuhan Institute of Zoology Mr.'s Liu Qihong nineteen eighty-two at Shennongjia first
A kind of New resource plants found and name, perennial herb, blade is less and thick, bottle green, above leaf
The aobvious protuberance of veinclearing, the lower mask minimum body of gland of depression, head inflorescence is less, and general diameter is less than 1.5 lis
Rice, leaf colored with differring primarily in that of Herba Dendranthematis indici and root are respectively provided with special strong fragrance.Dendranthema indicum collection
In be distributed in the hillside the openst on the sunny side of Shennongjia height above sea level 2600-2700 rice, roadside, be that Shennongjia is peculiar
The new variant of Compositae (Asteraceae) Chrysanthemum (Dendranthema), be similar to Herba Dendranthematis indici
(Dendranthema indicum(L.)Des Moul.).Flower that Mountain Area of Western Hubei Province is among the people dries in the shade with Dendranthema indicum,
Leaf is used for treating flu, laryngopharynx swelling and pain, conjunctival congestion and swelling pain, carbuncle foot sore furuncle etc..Chemical composition analysis shows god
The volatile oil component of agriculture Folium Crossostephii Chinensis is mainly based on the monoterpene in terpenoid and sesquiterpene and containing oxygen derivative thereof.God
Terpenoid in agriculture Folium Crossostephii Chinensis volatile oil have pinene, terpinol, thujene, lauro lene, Borneolum Syntheticum, 1,8-
The compound such as cineole, borneol acetate, it is antibacterial, anti-that pharmacological testing analysis shows that these compositions have
Virus, spasmolytic, eliminate the phlegm, the effect such as relieving cough and asthma；Volatile oil not only pharmaceutically has important effect,
Can be additionally used in the spice such as beverage, medicated cigarette and cosmetics and daily chemical industry, its utilization rate is high,
Economic worth is by fairly obvious.
The narrow distribution range of Dendranthema indicum, and resource is extremely limited.To this end, a lot of research worker are the most positive
The artificial propagation carrying out Dendranthema indicum and cultivation.Dendranthema indicum has been carried out introducing a fine variety cultivation by technical staff respectively
Training test, can quickly obtain substantial amounts of regeneration individuality by plant tissue culture technique, and organ directly occurs
Approach obtains regeneration plant can effectively be shortened cultivation cycle and reduce variation link, to expanding these species
Breeding coefficient be can yet be regarded as a kind of effective ways.Wherein having with the spire of Dendranthema indicum is outer implant and micro-cuttage
Mode, sets up breeding system in Dendranthema indicum test tube.It is outer implant with Flower Bud of Dendranthema indicum var. aromaticum, successfully induces
Flower Bud of Dendranthema indicum var. aromaticum callus also obtains regeneration plant, but is limited by material source.Also have and pass through the legendary god of farming
Folium Crossostephii Chinensis germination in vitro axillary bud deriving adventitious bud sets up Dendranthema indicum vitro Regeneration System, but its tissue cultured seedling is thin and delicate
The most effectively being solved with the problem of excessive growth, the optimization system in its strong sprout there is also problem.Based on
The problems referred to above, in the urgent need to by outer implant, plant growth regulator, minimal medium and condition of culture
Screening, set up the Dendranthema indicum regenerating system that breeding cycle is short, inheritance stability altofrequency occurs, thus
Under the conditions of the biotechnology breeding of Dendranthema indicum, Germ-plasma resources protection, Fast-propagation, manual control
Growth promoter research provides good experimental system.
Summary of the invention
It is an object of the invention to provide the in-vitro culture method of a kind of Dendranthema indicum, can induce to altofrequency
Substantial amounts of Dendranthema indicum Multiple Buds, after strengthening seedling and rooting, regeneration plant survival rate is high, up to 100%.
The in-vitro culture method of Dendranthema indicum of the present invention, with sterilized band bud point tender stem segments for outward
Implant, through Multiple Buds induction, strong seedling culture, take root, obtain Dendranthema indicum again after seedling exercising and transplanting
Raw plant, wherein:
Described band bud point tender stem segments is carried out before sterilization, and described cleaning step is: concentration is
Liquid detergent solution soaking 20-40min of 0.2-0.8%, scrubs afterwards, then rinses 1-2h with water；Because it is refreshing
The tender stem segments volatile oil content of agriculture Folium Crossostephii Chinensis is higher, and on tender stem segments, especially bud point position easily bonds
Too much dust, and be not easy to clean.So, the liquid detergent adding trace cleaning when can have
Effect removes the dust of bonding on tender stem segments, it is ensured that band bud point tender stem segments is cleaner, lures for Multiple Buds
Offer basis is provided into.
In its culture medium, Arundinella hirta's extracting solution and activity is all added when described strong seedling culture and root culture
Charcoal；
The strong seedling culture base used in described strong seedling culture step is, on the basis of MS minimal medium
With the addition of activated carbon, Arundinella hirta's extracting solution, 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, α-naphthaleneacetic acid
Concentration be 0-2.0mg/L, the concentration of activated carbon is 0.2-0.8%, and the concentration of Arundinella hirta's supernatant is
5-10%；
The preparation method of described Arundinella hirta's supernatant adds 5ml distilled water for taking 1g Arundinella hirta's dried powder,
After standing 20h at 4 DEG C, the extract frozen centrifugation of described 0.2g/ml is extracted, obtains Arundinella hirta's supernatant.
Preferably, described strong seedling culture comprises the following steps: loaded by described strong seedling culture base in conical flask,
Described inducing clumping bud is cultivated the Multiple Buds obtained be inoculated on described strong seedling culture base, in temperature
25 ± 2 DEG C, light application time 12-16h/ days, cultivate under conditions of intensity of illumination 2000-2500lux
20-60 days.
Preferably, described root culture comprises the following steps: by growing thickly that described strong seedling culture step obtains
Bud is isolated individual plant and is transferred on root media, temperature 25 ± 2 DEG C, and light application time 12-16h/ days,
Root induction under the conditions of intensity of illumination 2000-2500lux, starts after 5 days, and every day carries out an infrasonic wave
Stimulating, time 30-60min, continue 10-15 days, the frequency of sound wave used is 1000Hz, and the sound intensity is 100dB.
Regrowth is grown to obtain in cultivation in 20-30 days；Described root media is for the addition of Arundinella hirta's extracting solution and activated carbon
1/2MS culture medium or with the addition of the 1/4-3/4MS culture medium of activated carbon.
Preferably, described inducing clumping bud is cultivated and is comprised the following steps: the induction step tool of described Multiple Buds
Body is: the short stem section that sterilized band bud point tender stem segments cuts into single bud point is inoculated into Multiple Buds
On inducing culture, in temperature 25 ± 2 DEG C, light application time 12-16h/ days, intensity of illumination 2000-2500lux
Under the conditions of cultivate 10-15 days；
Described inducing clumping bud culture medium is, adds 0-2.0mg/L's on the basis of MS minimal medium
6-benzyladenine, the α-naphthaleneacetic acid of 0-2.0mg/L.
Preferably, described seedling exercising step specifically includes: the regeneration plant obtained through root culture grows 6-8cm
Time, it is gradually opened bottle cap, makes regeneration plant contact with natural air, 20-25 DEG C of lower refining seedling 5-10 days.
Preferably, transplant after described seedling exercising step, will be through the Transplantation of Regenerated Plantlets of seedling exercising
Plant in the mixed-matrix of sandy soil and desiccated coconut, obtain Dendranthema indicum and survive regeneration plant, described sandy soil and
The mixed proportion of desiccated coconut is 1: 0.25-1.
Wherein in an embodiment, the in-vitro culture method of Dendranthema indicum of the present invention specifically include with
Lower step:
(1) outer implant sterilizing: choosing the young tender stem with bud of new germinating is outer implant；Externally implant is gone out
The method of bacterium is: Dendranthema indicum tender stem segments is removed blade, at for example, every 100ml of concentration containing 4-8
Scrub after the detergent liquid dripped soaks such as 20-40min, then rinse 1-2h with water, be then placed on
In 75% ethanol, soak 10-30s on superclean bench, drip soil temperature-40 with every 100ml containing 2-4 subsequently
0.1%HgCl2 solution sterilization 15min, with sterile water wash 4 times, each 1-3min.Described wash
Cleaning is commercially available common without phosphorus detergent, and the carving board that such as Nice Group Co., Ltd. produces is washed
Clean etc..
(2) induction of Multiple Buds: be outer implant with the Dendranthema indicum stem section of band bud point after sterilization treatment, will
It is placed in induced bundle in inducing clumping bud culture medium and sprouts；In temperature 25 ± 2 DEG C, light application time 12-16h/
My god, cultivate 10-15 days under the conditions of intensity of illumination 2000-2500lux.Described inducing clumping bud culture medium is
6-benzyladenine (6-BA) and α-naphthaleneacetic acid (NAA) is with the addition of on the basis of MS minimal medium；
Wherein the concentration of 6-benzyladenine is 0-2.0mg/L, preferably 2.0mg/L, and the concentration of α-naphthaleneacetic acid is
0-2.0mg/L, preferably 0.05mg/L.
(3) strong seedling culture: described inducing clumping bud is cultivated the Multiple Buds obtained and is inoculated into strong seedling culture base
On, temperature 25 ± 2 DEG C, light application time 12-16h/ days, under conditions of intensity of illumination 2000-2500lux
Carry out cultivate 20-60 days, preferably 30 days.And started after 5-15 days, carry out an infrasonic wave thorn every day
Swashing, time 30-60min, the frequency of sound wave used is 1000Hz, and the sound intensity is 100dB.Wherein said strong
Seedling culture medium is: with the addition of activated carbon, Arundinella hirta's extracting solution, 6-on the basis of MS minimal medium
Benzyladenine (6-BA) and α-naphthaleneacetic acid (NAA).During field investigation, find the legendary god of farming
The eugonic place of Folium Crossostephii Chinensis, is often associated with Arundinella hirta (Thunb.) Tanaka, it has been investigated that Arundinella hirta (Thunb.) Tanaka is to Dendranthema indicum
Growth promoter plays and positive helps to change effect, so, adding Arundinella hirta's extracting solution in strong seedling culture base can
To promote the growth promoter of Multiple Buds, be conducive to obtaining healthy and strong Multiple Buds, for improving the one-tenth of regeneration plant
Motility rate 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, the concentration of α-naphthaleneacetic acid is 0-2.0mg/L, preferably 0.05mg/L；The concentration of activated carbon is
0.2-0.8%, preferably 0.5%, the concentration of Arundinella hirta's supernatant is 5-10%, preferably 10%.
(4) take root and plant regeneration: the healthy and strong Multiple Buds after strong seedling culture is isolated individual plant and is placed in and takes root
Root induction in culture medium, obtains Dendranthema indicum complete regenerated plant；Described root media is for the addition of
The 1/4-3/4MS culture medium of activated carbon or cultivate at the 1/2MS that with the addition of activated carbon and Arundinella hirta's extracting solution
Base.Wherein, described root media for example, 1/2MS culture medium is added the activated carbon and 5% of 0.2%
Arundinella hirta's extracting solution.
Condition of culture is: temperature 25 ± 2 DEG C, light application time 12-16h/ days, intensity of illumination 2000-2500lux.
After 5 days, adventitious bud base portion starts to take root, and starts after 5 days, carries out a sound stimulation every day,
Time 30-60min, continues 10-15 days, and the frequency of sound wave used is 1000Hz, and the sound intensity is 100dB.Warp
Cross cultivation in 20-30 days and develop into whole plant, rooting rate 100%.For the one-tenth after raising Transplantation of Regenerated Plantlets
Motility rate, can carry out seedling exercising to the regeneration plant obtained after root culture in step (4), and method is: treat
When regrowth grows 6-8cm, it is gradually opened bottle cap, makes regeneration plant contact with natural air, at 20-25 DEG C
Lower refining seedling can be transplanted after 5-10 days.Sound stimulation can promote the growth of Dendranthema indicum root, promotes that it is fast
Fast-growing root, is conducive to obtaining the regeneration plant of well developed root system, thus improves survival rate.
(5) seedling exercising and transplanting: the regeneration plant obtained after root culture in step (4) is carried out transplanting side
Method is: until height of seedling 6-8cm, root system intensive healthy and strong time, be gradually opened cultivation bottle cap, make regeneration plant with
Natural air contacts, and at a temperature of 20-25 DEG C, seedling exercising can be transplanted after 5-10 days.By through seedling exercising again
Raw plantlet of transplant is planted in the mixed-matrix of the sandy soil mixed and desiccated coconut, sandy soil and desiccated coconut in mixed-matrix
Ratio be: 1: 0.25-1.Obtain Dendranthema indicum and survive regeneration plant, survival rate 98%.
The invention provides a kind of extracorporeal culturing method of rare resources plant Dendranthema indicum.The method be with
Band bud tender stem section, as outer implant, obtains regeneration plant by inducing clumping bud approach.With stem section as outer planting
Body carries out the advantage of Regeneration in Vitro and is draw materials convenience, available material abundance.The method of the invention
Can induce to altofrequency substantial amounts of Dendranthema indicum Multiple Buds, regeneration plant survival rate after strengthening seedling and rooting
Height, up to 100%, and it is little with hereditary stability relatively to have Variations of Regenerated Plants by adventitious shoot regeneration
High advantage.The present invention is to improve the breeding coefficient of Dendranthema indicum, for the preserving seed of these species and something lost thereof
Biography conversion is had laid a good foundation, and has a extensive future.
Accompanying drawing explanation
Fig. 1 is that the Dendranthema indicum of the in-vitro culture method medium-high frequency generation of Dendranthema indicum of the present invention is grown thickly
Bud；
Fig. 2 be Dendranthema indicum of the present invention in-vitro culture method in through the Multiple Buds of strong seedling culture；
Fig. 3 be Dendranthema indicum of the present invention in-vitro culture method in the individual plant Seedling taken root of Dendranthema indicum；
Fig. 4 be Dendranthema indicum of the present invention in-vitro culture method in Dendranthema indicum regeneration plant root system；
Fig. 5 be Dendranthema indicum of the present invention in-vitro culture method in the regeneration of Dendranthema indicum transplant survival
Plant.
Detailed description of the invention
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art's reference
Description word can be implemented according to this.
Embodiment 1, the isolated culture of Dendranthema indicum
MS minimal medium: can refer to document (Murashige T, Skoog F.A revised medium for
Rapid grouth and bioassays with tobacco tissue cultures.Physiol.Plant, 1962,15:
473-497) preparation.
The in-vitro culture method of Dendranthema indicum of the present invention, comprises the following steps:
(1) sterilizing of the outer implant of stem section
First Dendranthema indicum tender stem segments is removed blade, be the detergent that every 100ml drips containing 4-8 in concentration
Scrub after liquid (the carving board detergent that Nice Group Co., Ltd. produces) soaks 20-40min, then use
Water rinses 1-2h, is then placed on superclean bench and soaks 10-30s in 75% ethanol, uses subsequently
The 0.1%HgCl2 solution sterilization 15min that every 100ml drips soil temperature-40 containing 2-4, uses sterile water wash 4-6
Secondary, each 1-3min.
(2) induction of Multiple Buds
Inducing clumping bud culture medium: with the addition of on the basis of MS minimal medium 6-BA 2.0mg/L and
NAA0.05mg/L。
It is outer implant with the tender stem segments of step (1) band bud point, is placed on described inducing clumping bud culture medium
On, the most each culture bottle inoculates 6 sections, each combination 10 culture bottles of inoculation, temperature 25 ± 2 DEG C,
Light application time 12-16h/ days, under intensity of illumination 2000-2500lux, induced bundle is sprouted.
As it is shown in figure 1, outer implant inoculation after the 3rd day, axillalry bud grows.After inoculating 7 days, axillalry bud base portion
Having graininess projection, after inoculating 10 days, the germinating of sprouting of clump base portion grows.
(3) strong seedling culture
Strong seedling culture base: with the addition of 6-BA 2.0mg/L and NAA on the basis of MS minimal medium
2.0mg/L, adds the activated carbon powder of 0.5% concentration and Arundinella hirta's extracting solution of 10% simultaneously.
The Multiple Buds obtained after inducing 15 days in step (2) is transferred into above-mentioned containing variable concentrations activated carbon
Culture medium on carry out strong seedling culture, in temperature 25 ± 2 DEG C, light application time 12-16h/ days, intensity of illumination
Cultivate under 2000-2500lux, robust growth (Fig. 2) after cultivating 30 days.
(4) take root and plant regeneration
Root media: (macro-and microelements comprised is MS minimal medium to 1/2MS culture medium
The 1/2 of full dose) on the basis of with the addition of Arundinella hirta's extracting solution of 0.2% activated carbon and 5%.
The Multiple Buds that step (3) obtains is isolated individual plant and is transferred on root media temperature 25 ± 2 DEG C,
Light application time 12-16h/ days, root induction under intensity of illumination 2000-2500lux, indefinite bastem after 5 days
Portion starts to take root, and starts after 5 days, carries out a sound stimulation, time 40min every day, holds
Continuous 10 days, the frequency of sound wave used was 1000Hz, and the sound intensity is 100dB.As shown in Figure 3, Figure 4, warp
Cross cultivation in 20-30 days and develop into whole plant, rooting rate 100%.
(5) seedling exercising and transplanting
The regeneration plant obtained after root culture in step (4) is carried out method for transplanting is: treat height of seedling
During the intensive stalwartness of 6-8cm, root system, it is gradually opened cultivation bottle cap, makes regeneration plant contact with natural air,
At a temperature of 20-25 DEG C, seedling exercising can be transplanted after 5-10 days.By the Transplantation of Regenerated Plantlets through seedling exercising to pressing
Sandy soil that geometric ratio (1/4-3/4 ratio) mixes and the mixed-matrix of desiccated coconut are planted, obtains the legendary god of farming fragrant
Chrysanthemum survives regeneration plant, survival rate 98% (Fig. 5).
Embodiment 2, the isolated culture of Dendranthema indicum
MS minimal medium: can refer to document (Murashige T, Skoog F.A revised medium for
Rapid grouth and bioassays with tobacco tissue cultures.Physiol.Plant, 1962,15:
473-497) preparation.
The in-vitro culture method of Dendranthema indicum of the present invention, comprises the following steps:
(1) sterilizing of the outer implant of stem section
First Dendranthema indicum tender stem segments is removed blade, be the detergent that every 100ml drips containing 4-8 in concentration
Scrub after liquid (the carving board detergent that Nice Group Co., Ltd. produces) soaks 20-40min, then use
Water rinses 1-2h, is then placed on superclean bench and soaks 10-30s in 75% ethanol, uses subsequently
The 0.1%HgCl2 solution sterilization 15min that every 100ml drips soil temperature-40 containing 2-4, uses sterile water wash 4-6
Secondary, each 1-3min.
(2) induction of Multiple Buds
Inducing clumping bud culture medium: with the addition of on the basis of MS minimal medium 6-BA 2.0mg/L and
NAA0.08mg/L。
It is outer implant with the tender stem segments of step (1) band bud point, is placed on described inducing clumping bud culture medium
On, the most each culture bottle inoculates 6 sections, each combination 10 culture bottles of inoculation, temperature 25 ± 2 DEG C,
Light application time 12-16h/ days, under intensity of illumination 2000-2500lux, induced bundle is sprouted.
As it is shown in figure 1, outer implant inoculation after the 3rd day, axillalry bud grows.After inoculating 7 days, axillalry bud base portion
Having graininess projection, after inoculating 10 days, the germinating of sprouting of clump base portion grows.
Inducing clumping bud culture medium is except implant wound healing in addition to containing the culture medium that NAA concentration is 1.0-2.0mg/L
Changing beyond serious, when 6-BA 2.0mg/L and NAA 0.05mg/L, the inductivity of Multiple Buds is the highest, for
83.3%, 1 axillalry bud base portion can germinate 6-8 adventitious bud.Containing high concentration NAA (1.0-2.0mg/L)
MS culture medium on wound healingization serious, it is impossible to induce Multiple Buds.Show at Dendranthema indicum Multiple Buds
In Induction Process, 6-BA is required, and NAA has certain inhibitory action, therefore by inducing clumping bud
Culture medium is set to: with the addition of 6-BA 0-2.0mg/L and NAA on the basis of MS minimal medium
0-1.0mg/L.If the Multiple Buds that induction produces continues to cultivate in former culture medium, Multiple Buds is the most intensive
Germinating, but it is susceptible to vitrification phenomenon, cause adventitious bud anamorphosis abnormal.
(3) strong seedling culture
Strong seedling culture base: with the addition of 6-BA 2.0mg/L and NAA on the basis of MS minimal medium
2.0mg/L, adds the activated carbon powder of 0.5% concentration and Arundinella hirta's extracting solution of 8% simultaneously.
The Multiple Buds obtained after inducing 15 days in step (2) is transferred into above-mentioned containing variable concentrations activated carbon
Culture medium on carry out strong seedling culture, in temperature 25 ± 2 DEG C, light application time 12-16h/ days, intensity of illumination
Cultivate under 2000-2500lux, robust growth (Fig. 2) after cultivating 30 days.
Research finds, on the strong seedling culture base adding 0.5% activated carbon, and thin and delicate slight vitrified clump
Sprout and cultivating robust growth after 30 days, may be used for taking root and plant regeneration.The activated carbon of excessive concentrations
Adventitious bud poor growth in culture medium, it may be possible to due to too much activated carbon powder absorption plant growth regulator
Caused by nutritional labeling.
(4) take root and plant regeneration
Root media: (macro-and microelements comprised is MS minimal medium to 1/2MS culture medium
The 1/2 of full dose) on the basis of with the addition of Arundinella hirta's extracting solution of 0.2% activated carbon and 5%.
The Multiple Buds that step (3) obtains is isolated individual plant and is transferred on root media temperature 25 ± 2 DEG C,
Light application time 12-16h/ days, root induction under intensity of illumination 2000-2500lux, indefinite bastem after 5 days
Portion starts to take root, and starts after 5 days, carries out a sound stimulation, time 30min every day, holds
Continuous 15 days, the frequency of sound wave used was 1000Hz, and the sound intensity is 100dB.As shown in Figure 3, Figure 4, warp
Cross cultivation in 20-30 days and develop into whole plant, rooting rate 100%.
(5) seedling exercising and transplanting
The regeneration plant obtained after root culture in step (4) is carried out method for transplanting is: treat height of seedling
During the intensive stalwartness of 6-8cm, root system, it is gradually opened cultivation bottle cap, makes regeneration plant contact with natural air,
At a temperature of 20-25 DEG C, seedling exercising can be transplanted after 5-10 days.By the Transplantation of Regenerated Plantlets through seedling exercising to pressing
Sandy soil that geometric ratio (1/4-3/4 ratio) mixes and the mixed-matrix of desiccated coconut are planted, obtains the legendary god of farming fragrant
Chrysanthemum survives regeneration plant, survival rate 98%.
Although embodiment of the present invention are disclosed as above, but it is not restricted to description and embodiment party
Listed utilization in formula, it can be applied to various applicable the field of the invention completely, for being familiar with ability
For the personnel in territory, be easily achieved other amendment, therefore without departing substantially from claim and etc. homotype
Enclosing under limited general concept, the present invention is not limited to specific details and shown here as the figure with description
Example.
Claims (5)
1. an in-vitro culture method for Dendranthema indicum, is outer implant with sterilized band bud point tender stem segments,
Dendranthema indicum is obtained again after inducing clumping bud cultivation, strong seedling culture, root culture, seedling exercising and transplanting
Raw plant, wherein:
Described band bud point tender stem segments is carried out before sterilization, and the step of described cleaning is: concentration
For liquid detergent solution soaking 20-40min of 0.2-0.8%, scrub afterwards, then rinse 1-2h with water；
In its culture medium, Arundinella hirta's supernatant and activity is all added when described strong seedling culture and root culture
Charcoal；
The strong seedling culture base used in described strong seedling culture step is, on the basis of MS minimal medium
With the addition of activated carbon, Arundinella hirta's supernatant, 6-benzyladenine and α-naphthaleneacetic acid；
In described strong seedling culture base, the concentration of 6-benzyladenine is 2.0mg/L, and the concentration of α-naphthaleneacetic acid is
0.05-2.0mg/L, the concentration of activated carbon is 0.2-0.8%, and the concentration of Arundinella hirta's supernatant is 5-10%；
The culture medium that described inducing clumping bud is cultivated is, adds on the basis of MS minimal medium
The 6-benzyladenine of 2.0mg/L, the α-naphthaleneacetic acid of 0.05-2.0mg/L；
Described root culture comprises the following steps: isolated by the Multiple Buds that described strong seedling culture step obtains
Individual plant is transferred on root media, in temperature 25 ± 2 DEG C, light application time 12-16h/ days, intensity of illumination
Root induction under the conditions of 2000-2500lux, from the beginning of after 5 days, carries out a sound stimulation every day, time
Between 30-60min, continue 10-15 days, the frequency of sound wave used is 1000Hz, and the sound intensity is 100dB；20-30
Regeneration plant is grown to obtain in it cultivation, described root media be with the addition of 5% Arundinella hirta's supernatant and activity
The 1/2MS culture medium of charcoal；The preparation method of described Arundinella hirta's supernatant is according to 1g Arundinella hirta's xeraphium
End adds the ratio extraction of 5ml distilled water, after standing 20h, obtains the extract of 0.2g/ml at 4 DEG C；Will
The extract frozen centrifugation of 0.2g/ml extracts, and obtains Arundinella hirta's supernatant.
2. the in-vitro culture method of Dendranthema indicum as claimed in claim 1, it is characterised in that described strong
Seedling is cultivated and is comprised the following steps: described inducing clumping bud is cultivated the Multiple Buds obtained and is inoculated into described strong sprout
In culture medium, temperature 25 ± 2 DEG C, light application time 12-16h/ days, intensity of illumination 2000-2500lux
Under the conditions of carry out cultivate 20-60 days.
3. the in-vitro culture method of Dendranthema indicum as claimed in claim 1, it is characterised in that described clump
Inducing culture of sprouting comprises the following steps: sterilized band bud point tender stem segments cuts into the single bud point of band
Short stem section be inoculated in inducing clumping bud culture medium, temperature 25 ± 2 DEG C, light application time 12-16h/ days,
Cultivate 10-15 days under the conditions of intensity of illumination 2000-2500lux.
4. the in-vitro culture method of Dendranthema indicum as claimed in claim 1, it is characterised in that described refining
Seedling step specifically includes: when the regeneration plant that root culture obtains grows 6-8cm, be gradually opened bottle cap,
Regeneration plant is made to contact with natural air, 20-25 DEG C of lower refining seedling 5-10 days.
5. the in-vitro culture method of Dendranthema indicum as claimed in claim 4, it is characterised in that described
Transplant after seedling exercising step, will be through the Transplantation of Regenerated Plantlets of seedling exercising to sandy soil and the mixing of desiccated coconut
Planting in substrate, obtain Dendranthema indicum and survive regeneration plant, the mixed proportion of described sandy soil and desiccated coconut is
1:0.25-1。
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