CN104285791B - A kind of method of Chimonanthusn itens Oliv. tissue culture and rapid proliferation - Google Patents
A kind of method of Chimonanthusn itens Oliv. tissue culture and rapid proliferation Download PDFInfo
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Abstract
A kind of method that the invention discloses Chimonanthusn itens Oliv. tissue culture and rapid proliferation, comprises the following steps successively: the acquisition of aseptic seedling: takes the Chimonanthusn itens Oliv. fruit of maturation, strips out the seed with intact seed coats；The induction of callus and the induction of adventitious bud: cotyledon is cut, is divided into 1cm with dissecting knife2Fritter, be inoculated in minimal medium；The propagation of adventitious bud and strong sprout: the bud differentiated is cut from callus, proceed to proliferated culture medium and carry out enrichment culture；Taking root without root: when adventitious bud grows 3-4cm height, cut the adventitious bud of stalwartness, be inoculated in root media；The seedling exercising of tissue culture regeneration plant and transplanting: the height of seedling 5cm of tissue cultured seedling regeneration plant to be taken root, radical are more than 3, when root length is more than 5cm, can carry out seedling exercising, after 3d, smashs culture medium to pieces and takes out plant, imbedded in compost by regeneration plant root.The method cultivate Chimonanthusn itens Oliv. take root soon, vitrification, melting brown rate low, survival rate height.
Description
Technical field
The invention belongs to technical field of plant culture, be specifically related to the method for quickly breeding of Chimonanthus Nitens.
Background technology
Chimonanthusn itens Oliv. (ChimonanthusnitensOliv.), have another name called smelly wax prunus mume (sieb.) sieb.et zucc., autumn wax prunus mume (sieb.) sieb.et zucc., bright leaf wax prunus mume (sieb.) sieb.et zucc., wild wax prunus mume (sieb.) sieb.et zucc. etc., for 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, name Chimonanthusn itens Oliv., different name Ailanthus altissima(Mill.)Swingle, hair Flos Camelliae Japonicae, Maackia amurensis Rupr et Maxim plum, and it is warm in nature, acrid in the mouth, micro-hardship, has expelling pathogenic wind from the body surface, 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.Leaf of Chimonanthus Nitens has strong fragrance, and its derived essential oil not only has analgesia, antitussive, phlegm-dispelling functions, moreover it is possible to appetite-suppressing, hence it is evident that the body weight slowing down mice increases.Utilize Chimonanthusn itens Oliv. tea flu victims tool significant curative effect clinically, especially to the most notable by the fever fear of cold that causes of flu, conjunctival congestion curative effect.Because of containing olefines chemical composition in Chimonanthusn itens Oliv., such as caryophyllene, germacrene etc., can be widely used for spice, food industry, medicinal intermediates etc.；Volatile alcohol compounds has Rose Essentielle abnormal smells from the patient infusive, harmonicity, and has the characteristics such as anti-corrupt, anti-filterable virus, and such as linalool, geraniol and nerolidol etc., cedrol then has Cupressus funebris Endl. fragrance.Containing a large amount of eucalyptus oils in leaf of Chimonanthus Nitens volatile oil, it is widely used in important essence and flavoring agent, food additive, cosmetics, medicine etc..Additionally, Chimonanthusn itens Oliv. Detoxication Decoction can treat herpes simplex keratitis, develop the product such as Chimonanthusn itens Oliv. pectoral syrup, Chimonanthusn itens Oliv. drop pill at present clinically.Additionally, because the florescence is 9～December, therefore Chimonanthusn itens Oliv. is also graceful evergreen ornamental shrub and the decorative flower in autumn with ornamental value.The breeding of Chimonanthusn itens Oliv. can pass through the Traditional breeding processes such as grafting, press strip, cuttage and seed, it is apparent that tissue culture for its Fast-propagation, introduce a fine variety the maintenance with merit there is considerable effect.Wax-cakes bait is 3 kind of plant only, and is China's special product.Only having wax prunus mume (sieb.) sieb.et zucc. (C.praecox) at present and have a small amount of report, what outer implant 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 base mainly adopts is MS, improvement MS and 1/2MS, that callus induction 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 what take root employing is IBA, NAA or both combinations.But in general, La Mei tissue culture also exists easy brownization, vitrification and the problems such as difficulty of taking root.
Summary of the invention
The technical problem to be solved is: the deficiency existed for prior art, it is provided that one is taken root soon, vitrification, melting brown rate are low, the Chimonanthusn itens Oliv. method for quickly breeding that survival rate is high
For realizing the purpose of the present invention, it is achieved by the following technical solutions: a kind of method of Chimonanthusn itens Oliv. tissue culture and rapid proliferation, comprises the following steps successively:
(1) acquisition of aseptic seedling: take the Chimonanthusn itens Oliv. fruit of maturation, strip out its oblong achene, with operating scissors, achene two ends are respectively cut off a little, strip out the seed with intact seed coats, be soaked in the HgCl of mass fraction 0.2% in superclean bench2Sterilization 17-40min, sterilized water soaking flushing 3 times after taking-up, each 5min in solution；It is inoculated in and with the addition of GA3In the Nitsch minimal medium of 0.1mg/L+6-BA2.0mg/L+NAA0.2mg/L.Sucrose addition in this culture medium is 30g/L, and agar addition is 7g/L, pH is 5.8-6.0, and culture medium is autoclave sterilization 20min at 121 DEG C once；After cultivating 7d in the dark, proceeding to illumination cultivation, cultivation temperature is 25 ± 1 DEG C；Cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark is proceeded to after every illumination cultivation 14h2S)；After seed germination, when two panels cotyledon launches and turns green, desirable cotyledon is as outer implant, or takes the stem section of aseptic seedling after bud grows as outer implant；
(2) induction of the induction of callus and adventitious bud:
Cotyledon is cut, is divided into 1cm with dissecting knife2The fritter of left and right size, it is inoculated in the Nitsch minimal medium that with the addition of TDZ0.1-2.0mg/L+NAA0-2.0mg/L+PVP0-5.0mg/L+LH0-1.0mg/L, it is incubated at illumination cultivation indoor, after every illumination cultivation 14h, proceeds to cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark2S)；
(3) propagation of adventitious bud and strong sprout:
The bud differentiated is cut from callus, proceeds to proliferated culture medium Nitsch+TDZ0-2.0mg/L+6-BA0-5.0mg/L+CH0-5.0mg/L+PVP1.0mg/L and carry out enrichment culture；
(4) taking root without root:
When adventitious bud grows 3-4cm height, cut the adventitious bud of stalwartness, be inoculated in root media 12.5-100%Nitsch+IAA0-1.0mg/L+NAA0-0.5mg/L+AC0-2.0mg/L+CH 2.0mg/L；
(5) seedling exercising of tissue culture regeneration plant and transplanting:
The height of seedling 5cm of tissue cultured seedling regeneration plant to be taken root, radical are more than 3, when root length is more than 5cm, can carry out seedling exercising, and seedling exercising carries 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 12h to cover half bottleneck, the space of half accepts the irradiation of daylight lamp, after 12h, take off tissue culture bottle lid, allow daylight lamp irradiate regeneration plant completely, after 3d, with Glass rod, culture medium is smashed to pieces, carefully regeneration plant is taken out, in clear water, carefully rinse remaining agar, the regeneration plant root of clean agar is imbedded in compost, transparent vessel on face shield on plant, plant field planting to be regenerated can take off container after extracting new blade out.
Preferably: the Nitsch in described step (2) substantially cultivates and with the addition of TDZ0.5mg/L+NAA0.2mg/L+PVP1.0mg/L+LH0.5mg/L.
Preferably: the proliferated culture medium in described step (3) is Nitsch+TDZ0.3mg/L+6-BA1.0mg/L+CH2.0mg/L+PVP1.0mg/L.
Preferably: the root media in described step (4) is 50%Nitsch+IAA0.5mg/L+NAA0.1mg/L+AC0.2mg/L+CH2.0mg/L.
Preferably: the induction being Multiple Buds in described step (2): when growing to 4cm until the bud of aseptic seedling, cut in the culture medium being inoculated in Nitsch+TDZ0-2.0mg/L+6-BA0-5.0mg/L+PVP0-5.0mg/L+LH0-1.0mg/L, it is incubated at illumination cultivation indoor, cultivation temperature is 25 ± 1 DEG C, cultivation 10h, intensity of illumination 30-40 μm of ol/ (m s) in dark is proceeded to after every illumination cultivation 14h.
Preferably: the culture medium in described step (2) is Nitsch+TDZ0.6mg/L+6-BA2.0mg/L+PVP1.0mg/L+LH0.5mg/L.
Compared with prior art, the invention has the beneficial effects as follows: cultivate Chimonanthusn itens Oliv. by the method for the present invention, it is possible to make Chimonanthusn itens Oliv. take root soon, vitrification, melting brown rate low, survival rate height.
Detailed description of the invention
Embodiment 1
A kind of method of Chimonanthusn itens Oliv. tissue culture and rapid proliferation, comprises the following steps successively:
(1) acquisition of aseptic seedling:
Take the Chimonanthusn itens Oliv. fruit of maturation, 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 with intact seed coats by fingernail carefully, superclean bench is soaked in 0.2%HgCl2Sterilization 17-40min(spends the short time and easily pollutes in solution, long is easily caused seed death), sterilized water soaking flushing 3 times after taking-up, each 5min, it is inoculated in Nitsch+GA30.1mg/L+6-BA2.0mg/L+NAA0.2mg/L("+" mean mixing) and culture medium in.Sucrose addition in this culture medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, culture medium is autoclave sterilization 20min at 121 DEG C in advance, after cultivating 7d in dark, proceeding to illumination cultivation, cultivation temperature is (25 ± 1) DEG C, cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark is proceeded to after every illumination cultivation 14h2S), seed germination after 18d, when two panels cotyledon launches and turns green, desirable cotyledon is as outer implant.
(2) induction of the induction of callus and adventitious bud
Cotyledon is cut, is divided into 1cm with dissecting knife2The fritter of left and right size, it is inoculated in the culture medium of Nitsch+TDZ0.5mg/L+NAA0.2mg/L+PVP1.0mg/L+LH0.5mg/L, it is incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark is proceeded to after every illumination cultivation 14h2S).Expanding occurs in 8d cotyledon, and 11d occurs that yellow green dense callus, 28d Calli Differentiation go out 3-5 adventitious bud, callus induction rate 100%, differentiation rate 65%, and melting brown rate is 5%.
(3) propagation of adventitious bud and strong sprout
Being cut from callus by the bud differentiated, proceed to proliferated culture medium Nitsch+TDZ0.3mg/L+6-BA1.0mg/L+CH2.0mg/L+PVP1.0mg/L and carry out enrichment culture, 25d proliferation times reaches 7.0.Vitrification phenomenon not occurring, and blastogenesis length is normal, leaf color is normal.
(4) taking root without root
When adventitious bud grows 3-4cm height, cut the adventitious bud of stalwartness, be inoculated in root media 50%Nitsch+IAA0.5mg/L+NAA0.1mg/L+AC0.2mg/L+CH2.0mg/L, taking root without root 8d the earliest, 25d rooting rate is 88%, radical 3.8, root length 4.7cm, root rugosity is moderate.
Wherein 50% in 50%Nitsch, refers to the concentration of Nitsch minimal medium in culture medium, and the concentration of other material is unaffected.50% i.e. 1/2 concentration, it is original 1/2 that 50%Nitsch refers to all substances consumption in Nitsch minimal medium formula,
(5) seedling exercising of tissue culture regeneration plant and transplanting
The height of seedling 5cm of tissue cultured seedling regeneration plant to be taken root, radical are more than 3, when root length is more than 5cm, can carry out seedling exercising, and seedling exercising carries 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 12h to cover half bottleneck, and the space of half accepts the irradiation of daylight lamp, after 12h, take off tissue culture bottle lid, allowing daylight lamp irradiate regeneration plant completely, after 3d, culture medium is smashed to pieces by useable glass rod, careful by regeneration plant taking-up, remaining agar, the root hair of attached regeneration plant as tight in a small amount of agar, the careful outwash of banister brush is carefully rinsed in clear water.Root can be imbedded in compost by the regeneration plant cleaning agar, for keeping moistening, can on plant transparent vessel such as glass beaker on face shield, plant field planting to be regenerated can take off container after extracting new blade out, and this method transplanting survival rate is up to more than 95%.
Literary composition relates to the implication of initialism:
AC activated carbon
6-BA6-benzyladenine
CH caseinhydrolysate
GA3Gibberellins
IAA heteroauxing
LH lactoalbumin hydrolysate
NAA α-naphthaleneacetic acid
PVP polyvinylpyrrolidone
It is grand that TDZ fills in benzene
Embodiment 2
The impact on the induction of callus and the induction of adventitious bud of the different culture media additive
The present embodiment and embodiment 1 are distinctive in that step (2): cut by cotyledon, be divided into 1cm with dissecting knife2The fritter of left and right size, it is inoculated in the culture medium of Nitsch+TDZ0.1-2.0mg/L+NAA0-2.0mg/L+PVP0-5.0mg/L+LH0-1.0m g/L, it is incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark is proceeded to after every illumination cultivation 14h2S).During 8-15d, cotyledon starts to expand, and 11-25d starts yellow green dense callus occur, and during 28-40d, Calli Differentiation goes out adventitious bud, callus induction rate is 70-100%, differentiation rate is 28-70%, has the outer implant of part, after producing callus, brownization, melting brown rate 5-45% occur.Nitsch+TDZ0.5mg/L+NAA0.2mg/L+PVP1.0mg/L+LH0.5mg/L is optimal medium, expanding occurs in 8d cotyledon, there is yellow green dense callus in 11d, 28d Calli Differentiation goes out 3-5 adventitious bud, callus induction rate 100%, differentiation rate 65%, melting brown rate is 5%, illustrates that PVP1.0mg/L+LH0.5mg/L is highly effective for alleviating brownization.
The impact that table 1 below is each group of culture medium additive on the induction of callus and the induction of adventitious bud.
Table 1
Embodiment 3
The different enrichment culture based additives propagation on adventitious bud and the impact in strong sprout
The present embodiment and embodiment 1 are distinctive in that step (3): cut from callus by the bud differentiated, proceeding to proliferated culture medium Nitsch+TDZ0-2.0mg/L+6-BA0-5.0mg/L+CH0-5.0mg/L+PVP1.0mg/L and carry out enrichment culture, 25d proliferation times is up to 3.5-7.2.Wherein with the best results of Nitsch+TDZ0.3mg/L+6-BA1.0mg/L+CH2.0mg/L+PVP1.0mg/L, proliferation times 7.0, it does not have vitrification phenomenon occurring, and blastogenesis length is normal, leaf color is normal.Though the interpolation of CH does not significantly improve the rate of increase, but the rate of increase is had slight facilitation by it, and the strong sprout of adventitious bud is also had facilitation.On the whole, hormone concentration used is too high to occur vitrified reason to be in that, as long as hormonal regulation is in proper level, it is possible to control vitrification degree.
Table 2 below is each group of enrichment culture based additive propagation on adventitious bud and the impact in strong sprout.
Table 2
Embodiment 4
Taking root without root
The present embodiment and embodiment 1 are distinctive in that step (4): when adventitious bud grows 3-4cm height, cut the adventitious bud of stalwartness, it is inoculated in root media 12.5-100%Nitsch+IAA0-1.0mg/L+NAA0-0.5mg/L+AC0-2.0mg/L+CH 2.0mg/L, take root without root 8-15d the earliest, 25d rooting rate is 67-88%, along with root media and auxin concentration increase, root is thicker to shorten, wherein with 50%Nitsch+IAA0.5mg/L+NAA0.1mg/L+AC0.2mg/L+CH2.0mg/L for best root media, rooting rate 88%, radical 3.8, root length 4.7cm, root rugosity is moderate.The concentration of minimal medium is had inducing action by heterotrophism to autotrophy for group training thing, but too low nutrition is also easily caused group, and training thing blade turns yellow, and root is elongated, thus losing vigor.Activated carbon (AC) has primarily served the effect of dark in taking root, but also can play absorption aldehydes matter and nutrient substance to acting on, and the former can alleviate browning, and the latter is easy for causing the malnutrition 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 fertilizer, and during transplanting, survival rate is low.In culture medium, the concentration (10-50g/L) of sugar is notable to rooting efficiency, Considering experimental effect and cost, this experiment employing 20g/L.
Table 3 below is the experimental data that root of hair time the earliest, rooting rate and growing state are affected by each group of culture medium.
Table 3
Aforementioned four embodiment is compared, and embodiment 1 all have employed the culture medium additive of optimum proportioning in each growth stage of Chimonanthusn itens Oliv., and the survival rate making Chimonanthusn itens Oliv. is the highest, is most suitable as carrying out the reference of large-scale production.
Embodiment 5
A kind of method of Chimonanthusn itens Oliv. tissue culture and rapid proliferation, comprises the following steps successively:
(1) acquisition of aseptic seedling:
Take the Chimonanthusn itens Oliv. fruit of maturation, 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 with intact seed coats by fingernail carefully, superclean bench is soaked in 0.2%HgCl2Sterilization 17-40min(spends the short time and easily pollutes in solution, long is easily caused seed death), sterilized water soaking flushing 3 times after taking-up, each 5min, it is inoculated in Nitsch+GA3In the culture medium of 0.1mg/L+6-BA2.0mg/L+NAA0.2mg/L.Sucrose addition in this culture medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, culture medium is autoclave sterilization 20min at 121 DEG C in advance, after cultivating 7d in dark, proceeding to illumination cultivation, cultivation temperature is (25 ± 1) DEG C, proceed to after every illumination cultivation 14h in dark and cultivate 10h(hour), intensity of illumination 30-40 μm of ol/ (m2S), 18d(days) seed germination afterwards, after bud grows, take the stem section of aseptic seedling as outer implant.
(2) induction of Multiple Buds
When the bud of aseptic seedling grows to 4cm, cut and be inoculated in the Nitsch minimal medium adding TDZ0-2.0mg/L+6-BA0-5.0mg/L+PVP0-5.0mg/L+LH0-1.0mg/L, it is incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark is proceeded to after every illumination cultivation 14h2S), finding when statistics adventitious bud number and growing state after cultivating 25d, the adventitious bud number of above-mentioned culture 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+TDZ0.6mg/L+6-BA2.0mg/L+PVP1.0mg/L+LH0.5mg/L is optimal medium, and the adventitious bud number of generation is 6.3, and melting brown rate is 5%, and the adventitious bud blade edge of generation, without yellow or vitrification phenomenon.
Table 4 below is the impact on the induction of Multiple Buds of each group of culture medium additive.
Table 4
| Culture medium | Adventitious bud induction frequency (%) | Melting brown rate (%) | Adventitious bud number | Growing state |
| TDZ 0.6 +6-BA 2.0+PVP 1.0 +LH 0.5 | 93 | 5 | 6.3 | Indefinite bud-leaf color is 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 color pale green, still can grow, and stem nobleness can |
| TDZ 0.6 | 60 | 22 | 5.1 | Indefinite bud-leaf color pale green, still can grow, and stem nobleness can |
| 6-BA 2.0+PVP 1.0 +LH 0.5 | 35 | 6 | 2.8 | Indefinite bud-leaf color pale green, growth is general |
| TDZ 2.0 +6-BA 5.0+PVP 1.0 +LH 0.5 | 88 | 10 | 5.9 | The indefinite water stain shape of bud-leaf color, deformity |
(3) propagation of adventitious bud and strong sprout
Being cut from callus by the bud differentiated, proceed to proliferated culture medium Nitsch+TDZ0.3mg/L+6-BA1.0mg/L+CH2.0mg/L+PVP1.0mg/L and carry out enrichment culture, 25d proliferation times reaches 7.0.Vitrification phenomenon not occurring, and blastogenesis length is normal, leaf color is normal.
(4) taking root without root
When adventitious bud grows 3-4cm height, cut the adventitious bud of stalwartness, be inoculated in root media 50%Nitsch+IAA0.5mg/L+NAA0.1mg/L+AC0.2mg/L+CH2.0mg/L, taking root without root 8d the earliest, 25d rooting rate is 88%, radical 3.8, root length 4.7cm, root rugosity is moderate.
(5) seedling exercising of tissue culture regeneration plant and transplanting
The height of seedling 5cm of tissue cultured seedling regeneration plant to be taken root, radical are more than 3, when root length is more than 5cm, can carry out seedling exercising, and seedling exercising carries 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 12h to cover half bottleneck, and the space of half accepts the irradiation of daylight lamp, after 12h, take off tissue culture bottle lid, allowing daylight lamp irradiate regeneration plant completely, after 3d, culture medium is smashed to pieces by useable glass rod, careful by regeneration plant taking-up, remaining agar, the root hair of attached regeneration plant as tight in a small amount of agar, the careful outwash of banister brush is carefully rinsed in clear water.Root can be imbedded in compost by the regeneration plant cleaning agar, for keeping moistening, can on plant transparent vessel such as glass beaker on face shield, plant field planting to be regenerated can take off container after extracting new blade out, and this method transplanting survival rate is up to more than 95%.
Claims (1)
1. the method for a Chimonanthusn itens Oliv. tissue culture and rapid proliferation, it is characterised in that comprise the following steps successively:
(1) acquisition of aseptic seedling:
Take the Chimonanthusn itens Oliv. fruit of maturation, 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 with intact seed coats by fingernail carefully, superclean bench is soaked in 0.2%HgCl2In solution, sterilization 17-40min, sterilized water soaking flushing 3 times after taking-up, each 5min, be inoculated in Nitsch+GA3In the culture medium of 0.1mg/L+6-BA2.0mg/L+NAA0.2mg/L；Sucrose addition in this culture medium is 30g/L, agar addition is 7g/L, pH is 5.8-6.0, culture medium is autoclave sterilization 20min at 121 DEG C in advance, illumination cultivation is proceeded to after dark cultivates 7d, cultivation temperature is (25 ± 1) DEG C, proceeds to cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark after every illumination cultivation 14h2S), seed germination after 18d, when two panels cotyledon launches and turns green, take cotyledon as outer implant；
(2) induction of the induction of callus and adventitious bud
Cotyledon is cut, is divided into 1cm with dissecting knife2The fritter of left and right size, it is inoculated in the culture medium of Nitsch+TDZ0.5mg/L+NAA0.2mg/L+PVP1.0mg/L+LH0.5mg/L, it is incubated at illumination cultivation indoor, cultivation temperature is (25 ± 1) DEG C, cultivation 10h, intensity of illumination 30-40 μm of ol/ (m in dark is proceeded to after every illumination cultivation 14h2S)；Expanding occurs in 8d cotyledon, and 11d occurs that yellow green dense callus, 28d Calli Differentiation go out 3-5 adventitious bud, callus induction rate 100%, differentiation rate 65%, and melting brown rate is 5%；
(3) propagation of adventitious bud and strong sprout
Being cut from callus by the bud differentiated, proceed to proliferated culture medium Nitsch+TDZ0.3mg/L+6-BA1.0mg/L+CH2.0mg/L+PVP1.0mg/L and carry out enrichment culture, 25d proliferation times reaches 7.0；Vitrification phenomenon not occurring, and blastogenesis length is normal, leaf color is normal；
(4) taking root without root
When adventitious bud grows 3-4cm height, cut the adventitious bud of stalwartness, be inoculated in root media 50%Nitsch+IAA0.5mg/L+NAA0.1mg/L+AC0.2mg/L+CH2.0mg/L, taking root without root 8d the earliest, 25d rooting rate is 88%, radical 3.8, root length 4.7cm, root rugosity is moderate；
Wherein 50% in 50%Nitsch, refers to the concentration of Nitsch minimal medium in culture medium, and the concentration of other material is unaffected；50% i.e. 1/2 concentration, it is original 1/2 that 50%Nitsch refers to all substances consumption in Nitsch minimal medium formula,
(5) seedling exercising of tissue culture regeneration plant and transplanting
The height of seedling 5cm of tissue cultured seedling regeneration plant to be taken root, radical are more than 3, when root length is more than 5cm, carry out seedling exercising, and seedling exercising carries 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, making bottle cap cover half bottleneck after 12h, the space of half accepts the irradiation of daylight lamp, after 12h, take off tissue culture bottle lid, allow daylight lamp irradiate regeneration plant completely, after 3d, with Glass rod, culture medium is smashed to pieces, careful by regeneration plant taking-up, remaining agar, the root hair of attached regeneration plant as tight in a small amount of agar, the careful outwash of banister brush is carefully rinsed in clear water；Root is imbedded in compost by the regeneration plant cleaning agar, and for keeping moistening, transparent vessel on face shield on plant, plant field planting to be regenerated takes off container after extracting new blade out.
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| CN110786156A (en) * | 2018-08-03 | 2020-02-14 | 江西佑美制药有限公司 | Chimonanthus nitens breeding method |
| CN110574685B (en) * | 2019-10-09 | 2021-08-27 | 北部湾大学 | Aseptic seedling induction method for Saraca indica |
| 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 |
| 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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