CN104285791A - Method applied to tissue culture and rapid propagation of chimonanthus nitens - Google Patents
Method applied to tissue culture and rapid propagation of chimonanthus nitens Download PDFInfo
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Abstract
The invention discloses a method applied to tissue culture and rapid propagation of chimonanthus nitens. The method sequentially comprises the following steps: obtaining aseptic seedlings; taking mature chimonanthus nitens fruits and peeling seeds with complete seed coat; inducing callus and inducing adventitious buds; cutting off cotyledon, cutting the cotyledon into small slices with area of 1 square centimeter by a scalpel, inoculating the small slices in a minimal medium; proliferating and seedling the adventitious buds; cutting off the differentiated buds from the callus, and transferring the differentiated buds into a proliferation culture medium to carry out proliferation culture; rooting rootless seedlings; cutting off strong adventitious buds when the adventitious buds grow to be 3-4cm high, and inoculating the strong adventitious buds in a rooting culture medium; hardening and transplanting seedlings of tissue culture regeneration plants; hardening the seedlings when the seedlings of to-be-rooted tissue culture regeneration plants are 5cm high, the root number is greater than 3 and the root length is greater than 5cm, pounding the culture medium and taking out the plants after three days and burying the roots of the regeneration plants in the culture soil. The chimonanthus nitens cultured by the method is capable of quickly rooting and is low in vitrifying and browning rate and high in survival rate.
Description
Technical field
The invention belongs to technical field of plant culture, be specifically related to the method for quickly breeding of chimonanthea.
Background technology
Mountain wax plum (
chimonanthus nitensoliv.), have another name called smelly wax plum, the autumn wax plum, bright leaf wax plum, wild wax plum etc., be Calycanthaceae wax-cakes bait evergreen shrubs, originate in the provinces and regions such as Anhui, Zhejiang, Jiangsu, Jiangxi, Fujian, Hubei, Hunan, Guangxi, Yunnan, Guizhou and Shaanxi.Leaf is used as medicine, famous mountain wax plum, different name shineing wintersweet flower, hair camellia, Maackia amurensis Rupr et Maxim plum, and it is warm in nature, pungent, the micro-hardship of taste, and tool expels pathogenic wind from the body surface, the effect of removing dampness by means of aromatics, the disease such as cure mainly influenza, heatstroke, chronic bronchitis, wet tired uncomfortable in chest and mosquito ant bite.Folium Chimonanthi Nitentis has strong fragrance, and its derived essential oil not only has analgesia, antibechic, phlegm-dispelling functions, can also appetite-suppressing, obviously slows down the body weight gain of mouse.Utilize mountain wax plum tea flu victims tool significant curative effect clinically, especially remarkable to the fever chilly, the conjunctival congestion curative effect that cause by catching a cold.Because of containing olefines chemical composition in the wax plum of mountain, as caryophyllene, germacrene etc., spices, food industry, medicinal intermediates etc. can be widely used in; Volatile alcohol compounds has Rose Essentielle smell that is infusive, harmonicity, and has the characteristics such as anti-corruption, anti-filterable virus, and as linalool, geraniol and nerolidol etc., cedrol then has cypress fragrance.Containing a large amount of eucalyptus oil in Folium Chimonanthi Nitentis volatile oil, be widely used in important essence and flavoring agent, food additives, cosmetics, medicine etc.In addition, mountain wax plum Detoxication Decoction can treat herpes simplex keratitis, has developed the product such as wax plum pectoral syrup of coming out of retirement and taking up an official post, mountain wax plum dripping pill at present clinically.In addition, because the florescence is 9 ~ December, therefore mountain wax plum is also graceful evergreen ornamental shrub and the decorative flower in autumn with ornamental value.The breeding of mountain wax plum can pass through the Traditional breeding processes such as grafting, press strip, cuttage and seed, but obviously tissue cultures for its Fast-propagation, introduce a fine variety, with the maintenance of merit, there is very important effect.Wax-cakes bait is 3 Plants only, and are China's special product.Only have at present wax plum (
c. praecox) there is a small amount of report, what explant mainly adopted is stem section or stem apex, and only a few adopts Cotyledon and embryo, and what walk is adventitious organogenesis, or first generation callus breaks up again, or goes out Shoot propagation; That basic cultivation sends out that base mainly adopts is MS, improvement MS and 1/2MS, that evoked callus and differentiation adopt is 6-BA+KT+2,4-D or 6-BA+KT+NAA combines, what the Initial culture of stem section or stem apex mainly adopted is 6-BA+NAA or 6-BA+IBA combination, what propagation adopted is the combination of 6-BA+NAA, and taking root, what adopt is IBA, NAA or both combinations.But in general, wax plum tissue cultures also exists easy brownization, vitrifying and the problems such as difficulty of taking root.
Summary of the invention
Technical problem to be solved by this invention is: the deficiency existed for prior art, provides that one is taken root soon, vitrifying, melting brown rate be low, the mountain wax plum method for quickly breeding that survival rate is high
For realizing the object of the present invention, be achieved by the following technical solutions: the method for a kind of mountain wax plum tissue culture and rapid proliferation, comprises the following steps successively:
(1) acquisition of aseptic seedling: get ripe mountain wax plum fruit, strip out its oblong achene, respectively cut off a little at achene two ends with operating scissors, strips out the seed of band intact seed coats, is soaked in the HgCl of mass fraction 0.2% in superclean bench
2sterilization 17-40 min in solution, sterile water soaking flushing 3 times after taking out, each 5 min; Be inoculated in and with the addition of GA
3in the Nitsch minimal medium of 0.1 mg/L+6-BA 2.0 mg/L+NAA 0.2mg/L.Sucrose addition in this medium is 30 g/L, and agar addition is 7 g/L, pH is 5.8-6.0, and medium is autoclave sterilization 20 min at 121 DEG C once; After cultivating 7 d in the dark, proceed to illumination cultivation, cultivation temperature is 25 ± 1 DEG C; Proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s); After seed germination, when two panels cotyledon launches and turns green, desirable cotyledon is as explant, or the stem section of getting aseptic seedling after bud grows is as explant;
(2) induction of callus and the induction of indefinite bud:
Cotyledon is cut, is divided into 1 cm with scalpel
2the fritter of left and right size, be inoculated in the Nitsch minimal medium that with the addition of TDZ 0.1-2.0 mg/L+NAA 0-2.0 mg/L+PVP 0-5.0 mg/L+LH 0-1.0 mg/L, be incubated at illumination cultivation indoor, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s);
(3) propagation of indefinite bud and strong sprout:
The bud differentiated is cut from callus, proceeds to proliferated culture medium Nitsch+TDZ 0-2.0 mg/L+6-BA 0-5.0 mg/L+CH 0-5.0 mg/L+ PVP 1.0 mg/L and carry out Multiplying culture;
(4) taking root without offspring:
When indefinite bud growth 3-4 cm height, cut healthy and strong indefinite bud, be inoculated in root media 12.5-100%Nitsch+IAA 0-1.0 mg/L+NAA0-0.5 mg/L+AC 0-2.0 mg/L+ CH 2.0 mg/L;
(5) hardening of tissue culture regeneration plant and transplanting:
Height of seedling 5 cm of plantlet in vitro regeneration plant to be taken root, radical more than 3, root long more than 5 cm time, can carry out hardening, hardening is carried out in illumination cultivation indoor, first by the bottle cap unscrewing of tissue culture bottle, inner air and outer air is allowed to exchange, a small amount of clear water is added in bottle, bottle cap can be made after 12 h to cover half bottleneck, the space of half accepts the irradiation of fluorescent lamp, after 12 h, take off tissue culture bottle lid, fluorescent lamp is allowed to irradiate regeneration plant completely, after 3 d, with glass bar, medium is smashed to pieces, carefully regeneration plant is taken out, the remaining agar of careful rinsing in clear water, the regeneration plant root of clean agar is imbedded in compost, transparent vessel on face shield on plant, after new blade is extracted in plant field planting to be regenerated out, container can be taken off.
Preferably: the Nitsch in described step (2) substantially cultivates and with the addition of TDZ 0.5 mg/L+NAA 0.2 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L.
Preferably: the proliferated culture medium in described step (3) is Nitsch+TDZ 0.3 mg/L+6-BA 1.0 mg/L+CH 2.0 mg/L+ PVP 1.0 mg/L.
Preferably: the root media in described step (4) is 50% Nitsch+IAA 0.5 mg/L+NAA 0.1 mg/L+AC 0.2 mg/L+ CH 2.0 mg/L.
Preferably: the induction being Multiple Buds in described step (2): when growing to 4 cm until the bud of aseptic seedling, cut in the medium being inoculated in Nitsch+TDZ 0-2.0 mg/L+6-BA 0-5.0 mg/L+ PVP 0-5.0 mg/L+LH 0-1.0 mg/L, be incubated at illumination cultivation indoor, cultivation temperature is 25 ± 1 DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (ms).
Preferably: be Nitsch+ TDZ 0.6 mg/L+6-BA 2.0 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L at the medium of described step (2).
Compared with prior art, the invention has the beneficial effects as follows: cultivate mountain wax plum by method of the present invention, can make that mountain wax plum takes root soon, vitrifying, melting brown rate be low, survival rate is high.
Embodiment
embodiment 1
A method for mountain wax plum tissue culture and rapid proliferation, comprises the following steps successively:
(1) acquisition of aseptic seedling:
Get ripe mountain wax plum fruit, strip out its oblong achene, respectively cut off a little with operating scissors achene two ends, note not breaking seed coat or injury seed, strip out the seed of band intact seed coats with finger nail carefully, in superclean bench, be soaked in 0.2% HgCl
2the 17-40 min(that sterilizes in solution spends the short time and easily pollutes, and the long seed that easily causes is dead), sterile water soaking flushing 3 times after taking out, each 5 min, are inoculated in Nitsch+GA
30.1 mg/L+6-BA 2.0 mg/L+NAA 0.2mg/L("+" means mixing) medium in.Sucrose addition in this medium is 30 g/L, agar addition is 7 g/L, pH is 5.8-6.0, medium is autoclave sterilization 20 min at 121 DEG C in advance, after cultivating 7 d in dark, proceed to illumination cultivation, cultivation temperature is (25 ± 1) DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s), seed germination after 18 d, when two panels cotyledon launches and turns green, desirable cotyledon is as explant.
(2) induction of callus and the induction of indefinite bud
Cotyledon is cut, is divided into 1 cm with scalpel
2the fritter of left and right size, be inoculated in the medium of Nitsch+TDZ 0.5 mg/L+NAA 0.2 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L, be incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s).Expanding appears in 8 d cotyledons, and yellow green dense callus appears in 11 d, and 28 d Calli Differentiations go out 3-5 indefinite bud, callus induction rate 100%, differentiation rate 65%, and melting brown rate is 5%.
(3) propagation of indefinite bud and strong sprout
Cut from callus by the bud differentiated, proceed to proliferated culture medium Nitsch+TDZ 0.3 mg/L+6-BA 1.0 mg/L+CH 2.0 mg/L+ PVP 1.0 mg/L and carry out Multiplying culture, 25 d proliferation times reach 7.0.Vitrification phenomenon does not occur, and blastogenesis is long normal, leaf look normal.
(4) taking root without offspring
When indefinite bud growth 3-4 cm height, cut healthy and strong indefinite bud, be inoculated in root media 50% Nitsch+IAA 0.5 mg/L+NAA 0.1 mg/L+AC 0.2 mg/L+ CH 2.0 mg/L, without offspring the earliest 8d take root, 25 d rooting rates are 88%, radical 3.8, long 4.7 cm of root, root rugosity is moderate.
Wherein 50% in 50%Nitsch, refers to the concentration of Nitsch minimal medium in medium, and the concentration of other material is unaffected.50% i.e. 1/2 concentration, 50% Nitsch just refers to that all substances consumption in Nitsch minimal medium formula is original 1/2,
(5) hardening of tissue culture regeneration plant and transplanting
Height of seedling 5 cm of plantlet in vitro regeneration plant to be taken root, radical more than 3, root long more than 5 cm time, can carry out hardening, hardening is carried out in illumination cultivation indoor.First by the bottle cap unscrewing of tissue culture bottle, allow inner air and outer air exchange, in bottle, add a small amount of clear water, bottle cap can be made after 12 h to cover half bottleneck, and the space of half accepts the irradiation of fluorescent lamp, after 12 h, take off tissue culture bottle lid, allow fluorescent lamp irradiate regeneration plant completely, after 3 d, medium is smashed to pieces by useable glass rod, carefully regeneration plant is taken out, the remaining agar of careful rinsing in clear water, the root hair of attached regeneration plant as tight in a small amount of agar, the careful outwash of banister brush.Cleaning the regeneration plant of agar can imbed in compost by root, for keeping moistening, can on plant on face shield transparent vessel as glass beaker, after new blade is extracted in plant field planting to be regenerated out, can container be taken off, this method transplanting survival rate can reach more than 95%.
The implication of initialism is related in literary composition:
AC active carbon
6-BA 6-benzyladenine
CH caseinhydrolysate
GA
3gibberellin
IAA heteroauxin
LH lactoalbumin hydrolysate
NAA α-naphthaleneacetic acid
PVP polyvinylpyrrolidone
It is grand that TDZ fills in benzene
embodiment 2
Different culture media additive is on the impact of the induction of callus and the induction of indefinite bud
The difference of the present embodiment and embodiment 1 is step (2): cut by cotyledon, be divided into 1 cm with scalpel
2the fritter of left and right size, be inoculated in the medium of Nitsch+TDZ 0.1-2.0 mg/L+NAA 0-2.0 mg/L+PVP 0-5.0 mg/L+LH 0-1.0 mg/L, be incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s).During 8-15 d, cotyledon starts to expand, and 11-25 d starts to occur yellow green dense callus, and during 28-40 d, Calli Differentiation goes out indefinite bud, callus induction rate is 70-100%, differentiation rate is 28-70%, has part explant brownization to occur, melting brown rate 5-45% after generation callus.Nitsch+TDZ 0.5 mg/L+NAA 0.2 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L is optimal medium, expanding appears in 8 d cotyledons, there is yellow green dense callus in 11 d, 28 d Calli Differentiations go out 3-5 indefinite bud, callus induction rate 100%, differentiation rate 65%, melting brown rate is 5%, illustrates that PVP 1.0 mg/L+LH 0.5 mg/L is very effective for alleviating brownization.
Following table 1 is each group of culture medium additive on the impact of the induction of callus and the induction of indefinite bud.
Table 1
embodiment 3
the different propagation of Multiplying culture based additive on indefinite bud and the impact in strong sprout
The difference of the present embodiment and embodiment 1 is step (3): cut from callus by the bud differentiated, proceed to proliferated culture medium Nitsch+TDZ 0-2.0 mg/L+6-BA 0-5.0 mg/L+CH 0-5.0 mg/L+ PVP 1.0 mg/L and carry out Multiplying culture, 25 d proliferation times can reach 3.5-7.2.Wherein with the best results of Nitsch+TDZ 0.3 mg/L+6-BA 1.0 mg/L+CH 2.0 mg/L+ PVP 1.0 mg/L, not there is vitrification phenomenon in proliferation times 7.0, and blastogenesis is long normal, and leaf look normal.Though the interpolation of CH does not significantly improve the rate of increase, it has slight facilitation to the rate of increase, and also has facilitation to the strong sprout of indefinite bud.On the whole, there is vitrified reason and be that hormone concentration used is too high, as long as hormone control is in proper level, just can control vitrifying degree.
Following table 2 is the propagation of each group of Multiplying culture based additive on indefinite bud and the impact in strong sprout.
Table 2
embodiment 4
Taking root without offspring
The difference of the present embodiment and embodiment 1 is step (4): when indefinite bud grows 3-4 cm height, cut healthy and strong indefinite bud, be inoculated in root media 12.5-100%Nitsch+IAA 0-1.0 mg/L+NAA0-0.5 mg/L+AC 0-2.0 mg/L+ CH 2.0 mg/L, without offspring the earliest 8-15 d take root, 25 d rooting rates are 67-88%, along with root media and auxin concentration increase, root is thicker to shorten, wherein with 50% Nitsch+IAA 0.5 mg/L+NAA 0.1 mg/L+AC 0.2 mg/L+ CH 2.0 mg/L for best root media, rooting rate 88%, radical 3.8, long 4.7 cm of root, root rugosity is moderate.The concentration of minimal medium has inducing action by heterotrophism to autotrophy for group training thing, but too low nutrition also easily causes group training thing, and blade turns yellow, and root is elongated, thus loses vigor.Active carbon (AC) mainly serves dark effect in taking root, but also can play and absorb aldehydes matter and nutriment to effect, and the former can alleviate browning, and the latter just easily causes the nutritional deficiency of seedling of taking root and the undergrowth that arrives.Two kinds of growths have superposition, and too high auxin concentration can be too low to quality of rooting, and main manifestations is that root is many, thick, short, and this type of root is difficult to water suction and inhales fertile, and during transplanting, survival rate is low.In medium, the concentration (10-50 g/L) of sugar is not remarkable to rooting efficiency, Considering experimental effect and cost, this experiment employing 20 g/L.
Following table 3 is each group of medium on the experimental data of root of hair time the earliest, rooting rate and growing state impact.
Table 3
Above-mentioned four embodiments are compared, and embodiment 1 all have employed the culture medium additive of optimum proportioning at the mountain each vegetative stage of wax plum, making the survival rate of mountain wax plum the highest, being most suitable for the reference as carrying out large-scale production.
embodiment 5
A method for mountain wax plum tissue culture and rapid proliferation, comprises the following steps successively:
(1) acquisition of aseptic seedling:
Get ripe mountain wax plum fruit, strip out its oblong achene, respectively cut off a little with operating scissors achene two ends, note not breaking seed coat or injury seed, strip out the seed of band intact seed coats with finger nail carefully, in superclean bench, be soaked in 0.2% HgCl
2the 17-40 min(that sterilizes in solution spends the short time and easily pollutes, and the long seed that easily causes is dead), sterile water soaking flushing 3 times after taking out, each 5 min, are inoculated in Nitsch+GA
3in the medium of 0.1 mg/L+6-BA 2.0 mg/L+NAA 0.2mg/L.Sucrose addition in this medium is 30 g/L, agar addition is 7 g/L, pH is 5.8-6.0, medium is autoclave sterilization 20 min at 121 DEG C in advance, after cultivating 7 d in dark, proceed to illumination cultivation, cultivation temperature is (25 ± 1) DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h(hour), intensity of illumination 30-40 μm of ol/ (m
2s), 18 d(days) seed germination afterwards, after bud grows, get the stem section of aseptic seedling as explant.
(2) induction of Multiple Buds
When the bud of aseptic seedling grows to 4 cm, cut in the Nitsch minimal medium being inoculated in and adding TDZ 0-2.0 mg/L+6-BA 0-5.0 mg/L+ PVP 0-5.0 mg/L+LH 0-1.0 mg/L, be incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s), find when adding up indefinite bud number and growing state after cultivating 25 d, the indefinite bud number of above-mentioned medium is between 2.6-6.3, and adventitious bud induction frequency is 62-93%, and melting brown rate, at 3%-35%, shows the trend raised along with the increase of hormone concentration.Nitsch+ TDZ 0.6 mg/L+6-BA 2.0 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L is optimal medium, and the indefinite bud number of generation is 6.3, and melting brown rate is 5%, and the indefinite bud blade edge of generation, without yellow or vitrification phenomenon.
Following table 4 is that each group of culture medium additive is on the impact of the induction of Multiple Buds.
Table 4
| Medium | Adventitious bud induction frequency (%) | Melting brown rate (%) | Indefinite bud number | Growing state |
| TDZ 0.6 +6-BA 2.0+PVP 1.0 +LH 0.5 | 93 | 5 | 6.3 | Indefinite bud-leaf look dark green, well-grown, and stem height is higher |
| TDZ 0.6 +PVP 1.0 +LH 0.5 | 75 | 5 | 5.2 | Indefinite bud-leaf look pale green, still can grow, stem nobleness can |
| TDZ 0.6 | 60 | 22 | 5.1 | Indefinite bud-leaf look pale green, still can grow, stem nobleness can |
| 6-BA 2.0+PVP 1.0 +LH 0.5 | 35 | 6 | 2.8 | Indefinite bud-leaf look pale green, growth is general |
| TDZ 2.0 +6-BA 5.0+PVP 1.0 +LH 0.5 | 88 | 10 | 5.9 | Indefinite bud-leaf look water stain shape, deformity |
(3) propagation of indefinite bud and strong sprout
Cut from callus by the bud differentiated, proceed to proliferated culture medium Nitsch+TDZ 0.3 mg/L+6-BA 1.0 mg/L+CH 2.0 mg/L+ PVP 1.0 mg/L and carry out Multiplying culture, 25 d proliferation times reach 7.0.Vitrification phenomenon does not occur, and blastogenesis is long normal, leaf look normal.
(4) taking root without offspring
When indefinite bud growth 3-4 cm height, cut healthy and strong indefinite bud, be inoculated in root media 50% Nitsch+IAA 0.5 mg/L+NAA 0.1 mg/L+AC 0.2 mg/L+ CH 2.0 mg/L, without offspring the earliest 8d take root, 25 d rooting rates are 88%, radical 3.8, long 4.7 cm of root, root rugosity is moderate.
(5) hardening of tissue culture regeneration plant and transplanting
Height of seedling 5 cm of plantlet in vitro regeneration plant to be taken root, radical more than 3, root long more than 5 cm time, can carry out hardening, hardening is carried out in illumination cultivation indoor.First by the bottle cap unscrewing of tissue culture bottle, allow inner air and outer air exchange, in bottle, add a small amount of clear water, bottle cap can be made after 12 h to cover half bottleneck, and the space of half accepts the irradiation of fluorescent lamp, after 12 h, take off tissue culture bottle lid, allow fluorescent lamp irradiate regeneration plant completely, after 3 d, medium is smashed to pieces by useable glass rod, carefully regeneration plant is taken out, the remaining agar of careful rinsing in clear water, the root hair of attached regeneration plant as tight in a small amount of agar, the careful outwash of banister brush.Cleaning the regeneration plant of agar can imbed in compost by root, for keeping moistening, can on plant on face shield transparent vessel as glass beaker, after new blade is extracted in plant field planting to be regenerated out, can container be taken off, this method transplanting survival rate can reach more than 95%.
Claims (6)
1. a method for mountain wax plum tissue culture and rapid proliferation, is characterized in that comprising the following steps successively:
(1) acquisition of aseptic seedling: get ripe mountain wax plum fruit, strip out its oblong achene, respectively cut off a little at achene two ends with operating scissors, strips out the seed of band intact seed coats, is soaked in the HgCl of mass fraction 0.2% in superclean bench
2sterilization 17-40 min in solution, sterile water soaking flushing 3 times after taking out, each 5 min; Be inoculated in and with the addition of GA
3in the Nitsch minimal medium of 0.1 mg/L+6-BA 2.0 mg/L+NAA 0.2mg/L; Sucrose addition in this medium is 30 g/L, and agar addition is 7 g/L, pH is 5.8-6.0, and medium is autoclave sterilization 20 min at 121 DEG C once; After cultivating 7 d in the dark, proceed to illumination cultivation, cultivation temperature is 25 ± 1 DEG C; Proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s); After seed germination, when two panels cotyledon launches and turns green, desirable cotyledon is as explant, or the stem section of getting aseptic seedling after bud grows is as explant;
(2) induction of callus and the induction of indefinite bud:
Cotyledon is cut, is divided into 1 cm with scalpel
2the fritter of left and right size, be inoculated in the Nitsch minimal medium that with the addition of TDZ 0.1-2.0 mg/L+NAA 0-2.0 mg/L+PVP 0-5.0 mg/L+LH 0-1.0 mg/L, be incubated at illumination cultivation indoor, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (m
2s);
(3) propagation of indefinite bud and strong sprout:
The bud differentiated is cut from callus, proceeds to proliferated culture medium Nitsch+TDZ 0-2.0 mg/L+6-BA 0-5.0 mg/L+CH 0-5.0 mg/L+ PVP 1.0 mg/L and carry out Multiplying culture;
(4) taking root without offspring:
When indefinite bud growth 3-4 cm height, cut healthy and strong indefinite bud, be inoculated in root media 12.5-100%Nitsch+IAA 0-1.0 mg/L+NAA0-0.5 mg/L+AC 0-2.0 mg/L+ CH 2.0 mg/L;
(5) hardening of tissue culture regeneration plant and transplanting:
Height of seedling 5 cm of plantlet in vitro regeneration plant to be taken root, radical more than 3, root long more than 5 cm time, can carry out hardening, hardening is carried out in illumination cultivation indoor, first by the bottle cap unscrewing of tissue culture bottle, inner air and outer air is allowed to exchange, a small amount of clear water is added in bottle, bottle cap can be made after 12 h to cover half bottleneck, the space of half accepts the irradiation of fluorescent lamp, after 12 h, take off tissue culture bottle lid, fluorescent lamp is allowed to irradiate regeneration plant completely, after 3 d, with glass bar, medium is smashed to pieces, carefully regeneration plant is taken out, the remaining agar of careful rinsing in clear water, the regeneration plant root of clean agar is imbedded in compost, transparent vessel on face shield on plant, after new blade is extracted in plant field planting to be regenerated out, container can be taken off.
2. the method for a kind of mountain according to claim 1 wax plum tissue culture and rapid proliferation, is characterized in that: the Nitsch in described step (2) substantially cultivates and with the addition of TDZ 0.5 mg/L+NAA 0.2 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L.
3. the method for a kind of mountain according to claim 1 wax plum tissue culture and rapid proliferation, is characterized in that: the proliferated culture medium in described step (3) is Nitsch+TDZ 0.3 mg/L+6-BA 1.0 mg/L+CH 2.0 mg/L+ PVP 1.0 mg/L.
4. the method for a kind of mountain according to claim 1 wax plum tissue culture and rapid proliferation, is characterized in that: the root media in described step (4) is 50% Nitsch+IAA 0.5 mg/L+NAA 0.1 mg/L+AC 0.2 mg/L+ CH 2.0 mg/L.
5. the method for a kind of mountain according to claim 1 wax plum tissue culture and rapid proliferation, it is characterized in that: the induction being Multiple Buds in described step (2): when growing to 4 cm until the bud of aseptic seedling, cut in the medium being inoculated in Nitsch+TDZ 0-2.0 mg/L+6-BA 0-5.0 mg/L+ PVP 0-5.0 mg/L+LH 0-1.0 mg/L, be incubated at illumination cultivation indoor, cultivation temperature is 25 ± 1 DEG C, proceed in dark after every illumination cultivation 14 h and cultivate 10 h, intensity of illumination 30-40 μm of ol/ (ms).
6. the method for a kind of mountain according to claim 5 wax plum tissue culture and rapid proliferation, is characterized in that: be Nitsch+ TDZ 0.6 mg/L+6-BA 2.0 mg/L+PVP 1.0 mg/L+LH 0.5 mg/L at the medium of described step (2).
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| CN107006370A (en) * | 2017-05-03 | 2017-08-04 | 中国科学院合肥物质科学研究院 | A kind of wax plum plant high-efficiency in-vitro regeneration method |
| CN110574685A (en) * | 2019-10-09 | 2019-12-17 | 北部湾大学 | Aseptic seedling induction method for Saraca indica |
| CN110786156A (en) * | 2018-08-03 | 2020-02-14 | 江西佑美制药有限公司 | Chimonanthus nitens breeding method |
| CN110999788A (en) * | 2019-12-11 | 2020-04-14 | 中国科学院合肥物质科学研究院 | Method for rapidly propagating wintersweet plants |
| CN114467757A (en) * | 2022-03-07 | 2022-05-13 | 中国科学院合肥物质科学研究院 | Method for reducing formation of basal callus in chimonanthus nitens stem tissue culture regeneration |
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| CN100407904C (en) * | 2006-08-29 | 2008-08-06 | 四川大学 | Calyx canthus small hole pricking air layering method |
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| CN102599060B (en) * | 2012-03-29 | 2014-03-12 | 常熟市润丰农业有限公司 | Calyx canthus tissue culture rapid propagation method |
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| CN107006370A (en) * | 2017-05-03 | 2017-08-04 | 中国科学院合肥物质科学研究院 | A kind of wax plum plant high-efficiency in-vitro regeneration method |
| CN107006370B (en) * | 2017-05-03 | 2018-10-09 | 中国科学院合肥物质科学研究院 | A kind of wax plum plant high-efficiency in-vitro regeneration method |
| CN110786156A (en) * | 2018-08-03 | 2020-02-14 | 江西佑美制药有限公司 | Chimonanthus nitens breeding method |
| CN110574685A (en) * | 2019-10-09 | 2019-12-17 | 北部湾大学 | Aseptic seedling induction method for Saraca indica |
| CN110574685B (en) * | 2019-10-09 | 2021-08-27 | 北部湾大学 | Aseptic seedling induction method for Saraca indica |
| CN110999788A (en) * | 2019-12-11 | 2020-04-14 | 中国科学院合肥物质科学研究院 | Method for rapidly propagating wintersweet plants |
| CN110999788B (en) * | 2019-12-11 | 2021-08-24 | 中国科学院合肥物质科学研究院 | Method for rapidly propagating wintersweet plants |
| CN114467757A (en) * | 2022-03-07 | 2022-05-13 | 中国科学院合肥物质科学研究院 | Method for reducing formation of basal callus in chimonanthus nitens stem tissue culture regeneration |
| CN114467757B (en) * | 2022-03-07 | 2022-12-27 | 中国科学院合肥物质科学研究院 | Method for reducing formation of basal callus in chimonanthus nitens stem tissue culture regeneration |
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