CN109819895A - A kind of method for tissue culture of kalanchoe daigremontiana - Google Patents
A kind of method for tissue culture of kalanchoe daigremontiana Download PDFInfo
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- CN109819895A CN109819895A CN201910227060.XA CN201910227060A CN109819895A CN 109819895 A CN109819895 A CN 109819895A CN 201910227060 A CN201910227060 A CN 201910227060A CN 109819895 A CN109819895 A CN 109819895A
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Abstract
The present invention relates to the technical fields of in vitro culture, more particularly to a kind of method for tissue culture of kalanchoe daigremontiana, it may be implemented high-volume and cultivates, convenient to control growing state, provide new approach for the quick breeding and the preservation of germ plasm resource of kalanchoe daigremontiana；The following steps are included: the acquisition of (1) material；(2) aseptic process；(3) callus induction；(4) callus breaks up；(5) adventitious bud proliferation；(6) culture of rootage；(7) hardening and transplanting；(8) transplanting culture.
Description
Technical field
The present invention relates to the technical fields of in vitro culture, more particularly to a kind of tissue cultures side of kalanchoe daigremontiana
Method.
Background technique
It is well known that kalanchoe daigremontiana is the perennial meat draft of Crassulaceae Bryophyllum, high 50-100cm, stem list
It is raw, uprightly, brown, base portion lignifying.For leaf to raw or verticillate, lower blade is larger, often embraces stem；Blade meat, Yangtze River Delta shape, tool
There is irregular brown purple speckle, leaf margin has bastard, and sawtooth director provides the seedling of 2-4 piece true leaf, touches landing, and root is deep
New pillar is produced in soil, it is therefore named to take root.Compound cyme, basidixed, multi-branched, floral clock shape is orange red, florescence 4-
June.The torrid areas from African Madagascar is originated in, the more ground in the present world can cultivate.
Contain cis-aconitic, ascorbic acid, p-Coumaric Acid, ferulic acid, syringic acid, coffee in the leaf of kalanchoe daigremontiana
Coffee acid, P-hydroxybenzoic acid and other organic acids also contain Quercetin, Kaempferol, that the Arabic glucoside of Quercetin -3- two, kaempferia galanga
Phenol -3- glycoside, 18a- oleanane, Ψ-taraxasterol, β-amyrin acetic acid esters, 24- ethyl -25- hydroxyl cholesteric
Alcohol, a, β-amyrin, decene base is luxuriant and rich with fragrance, and undecenyl is luxuriant and rich with fragrance, and take root sterol, and take root ketone, and take root ketenes, falls
Ground is taken root alcohol；Herb also contains cupreol, Quercetin -3- rhamnose-Arab's glucoside, and Bu Shadi glucoside member -3- acetic acid esters is fallen
Ground is taken root toxin A and B.
Therefore kalanchoe daigremontiana can be used as it is medicinal, with all herbal medicine, cure mainly function include cooling blood and hemostasis, it is clearing heat and detoxicating,
Main lobe blood, traumatic hemorrhage, traumatic injury, furunculosis carbuncle swells, acute mastitis, mammary cancer, erysipelas, ulcer, scald, stomachache, arthralgia, throat are swollen
Pain and cough with lung heat.
Since kalanchoe daigremontiana has good medical value, therefore realize to the large batch of culture of kalanchoe daigremontiana
The problem of as relatively meriting attention now, the cultural method of traditional kalanchoe daigremontiana are usually to choose kalanchoe daigremontiana
On the adventitious bud that newly grows directly carry out cuttage, contact it with soil, new individual can be become after taking root.
But when the cultural method culture new individual of existing kalanchoe daigremontiana, need to select the adventitious bud of health, it is right
The growing state of different adventitious buds is inconvenient to be controlled, and cuttage is not suitable for large batch of culture, therefore seeks one kind and be
The method that the quick breeding of kalanchoe daigremontiana and the preservation of germ plasm resource provide new approach is very necessary.
Tissue cultures be it is a kind of the tissue to suit the requirements, organ, cell or protoplast etc. are isolated from plant, pass through
Sterile working, cultivated under manual control condition obtained it is regenerated completion plant or production have economic value other
The technology of product.
Summary of the invention
High-volume may be implemented cultivate in order to solve the above technical problems, the present invention provides one kind, it is convenient to growing state into
Row control, for kalanchoe daigremontiana quickly breed and germ plasm resource preservation provide new approach kalanchoe daigremontiana group
Knit cultural method.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, comprising the following steps:
(1) acquisition of material: winning the fresh healthy leaves of kalanchoe daigremontiana, and surface 20min is rinsed under tap water；
(2) aseptic process: the blade after the completion of flushing is impregnated into 15s with 75% alcohol on aseptic operating platform, is impregnated
It uses aseptic water washing 1 time after the completion, is then soaked in 0.1% mercuric chloride solution and sterilizes 6min, again with sterile after the completion of disinfection
Water rinses 3-5 times, the moisture of blade surface is finally blotted with sterilized filter paper, then blade is cut into the size of about 1.0cm*1.0cm；
(3) callus induction: by the blade inoculation after the cutting after the completion of aseptic process to induction of callus
On base；
(4) callus breaks up: callus induction cuts robust growth callus after 4 weeks is transferred to callus
On differential medium, induction generates adventitious bud；
(5) adventitious bud proliferation: callus induction cuts adventitious bud callus after 4 weeks is transferred to bud proliferation
On culture medium, squamous subculture is carried out under certain cultivation temperature and illumination condition, after one month, sprout it is long to 5cm when, continue to select
The adventitious bud for taking robust growth, cut section be transferred on bud proliferated culture medium subculture expand it is numerous；
(6) culture of rootage: the sprout tender stem that bud Multiplying culture goes out is cut into the segment of 2-3cm, is seeded to root media
In, culture of rootage is carried out under certain cultivation temperature and illumination condition；
(7) hardening and transplanting: after most test tube seedlings bear 3-7 item root, by high 3cm and with the test tube of 3-7 item root
Seedling gradually move on to scattering light natural environment in carry out hardening 7 days after, seedling is taken out, the culture of seedling root is cleaned with warm water
Base, and will be in seedling replanting to the flowerpot of dress humus or hole tray；
(8) transplanting culture: after the completion of seedling replanting, it is 26 DEG C that the humidity in control culture place, which is 85% ± 2%, temperature,
Hardening is carried out at ± 1 DEG C again.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, the aseptic operating platform in the step (2) make
Sterilization 30min is carried out with needing to open ultraviolet germicidal lamp and blower before.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, 0.15% mercuric chloride in the step (2) are molten
It include the tween of 1-2 drop 20mg/L in liquid.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, the induced medium in the step (3) be by
What the NAA of MS culture medium, the 6-BA of 1.0mg/L and 1.0mg/L was prepared by mixing into, and wherein addition 2% sucrose and
0.65% agar powder.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, the subculture medium in the step (4) be by
What the NAA of MS culture medium, the 6-BA of 1.0mg/L and 0.1mg/L was prepared by mixing into, and wherein addition 2% sucrose and
0.65% agar powder.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, the root media in the step (5) be by
What the NAA of MS culture medium, the 6-BA of 0.2mg/L and 0.5mg/L was prepared by mixing into, and wherein addition 2% sucrose and
0.65% agar powder.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, the root media in the step (6) be by
What the NAA of MS culture medium and 0.5mg/L were prepared by mixing into, and wherein adding 1.5% sucrose and 0.65% agar powder.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, culture temperature of the step (3) into step (6)
Degree is 26 DEG C ± 2 DEG C.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, culture of the step (3) into step (6) are wet
Degree is 85% ± 1%.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, illumination item of the step (3) into step (6)
Part is to use daylight light irradiation 12h, intensity of illumination 1000-4000lx daily.
Compared with prior art the invention has the benefit that a kind of tissue cultures side of kalanchoe daigremontiana of the invention
Method is with the advantages of traditional direct cultivation:
1, its kalanchoe daigremontiana blade for being selected from health is adequately sterilized as propagation material, and to propagation material
And cleaning, so that its surface is no longer attached to miscellaneous bacteria；
2, it carries out adventitious bud inducing, indefinite to the mother cell in blade under certain temperature, humidity and illumination condition
Bud proliferation and culture of rootage, the growth conditions for a collection of sprout for cultivating the same period are essentially identical, and are individually cultivated sprout,
Choose unsound sprout in time, improve rooting rate, facilitates the growing state of control sprout；
3, the gradually normalization of the sprout vital sign after carrying out culture of rootage, by being moved after the hardening of certain time
It plants, carries out hardening after transplanting again, make the survival rate > 95% of sprout, reduce the generation of sick body, be suitable for large batch of culture
Kalanchoe daigremontiana plant, and new way can be provided for the quick breeding and the preservation of germ plasm resource of kalanchoe daigremontiana
Diameter.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
A kind of method for tissue culture of kalanchoe daigremontiana of the invention, comprising the following steps:
The acquisition of material: winning the fresh healthy leaves of kalanchoe daigremontiana, in the blade table that tap water undershoot washing takes
Face 20min；
Aseptic process: 30min opens the ultraviolet germicidal lamp of aseptic operating platform and blower before operation, and 10min is closed
Blower sufficiently sterilizes aseptic operating platform by ultraviolet germicidal lamp, after the completion of sterilization, the blade after the completion of flushing is existed
15s is impregnated with 75% alcohol on aseptic operating platform, is used aseptic water washing 1 time after the completion of impregnating, is then soaked in 0.1% liter
6min is sterilized in mercury solution, uses aseptic water washing 3-5 times again after the completion of disinfection, the water of blade surface is finally blotted with sterilized filter paper
Divide, then blade is cut into the size of about 1.0cm*1.0cm as shown；
Callus induction: the NAA of MS culture medium, the 6-BA of 1.0mg/L and 1.0mg/L is mixed into preparation first
At mixed liquor, and thereto, 2% sucrose of addition and 0.65% agar powder are prepared into callus inducing medium, will prepare
Callus inducing medium after the completion is dispensed to conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, and sterilizing is completed
Conical flask is taken out afterwards, solidifies induced medium natural cooling therein, by the blade after the cutting after the completion of aseptic process
It is seeded on callus inducing medium, callus induction is carried out under 26 DEG C of temperature, humidity 85%, it is every using fluorescent lamp
It provides the illumination of 12h, and callus appearance can be observed in intensity of illumination 1000-4000lx after 15 days；It is formed after 4 weeks bright
Aobvious more fluffy light green callus, inductivity > 90%；
Callus differentiation: the NAA of MS culture medium, the 6-BA of 1.0mg/L and 0.1mg/L is mixed into preparation first
At mixed liquor, and thereto, 2% sucrose of addition and 0.65% agar powder are prepared into callus differential medium, will prepare
Callus differential medium after the completion is dispensed to conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, and sterilizing is completed
Conical flask is taken out afterwards, callus differential medium natural cooling therein is solidified, cuts the callus of robust growth
It is transferred on callus differential medium, adventitious bud inducing is carried out under 26 DEG C of temperature, humidity 85%, it is daily using fluorescent lamp
The illumination of 12h is provided, intensity of illumination 1000-4000lx forms many green bud points in callus periphery after 10 days, and 4
Green bud point grows up to the adventitious bud of the green in color of meat after week；
Adventitious bud proliferation: the NAA of MS culture medium, the 6-BA of 0.2mg/L and 0.5mg/L is mixed be prepared into first
Mixed liquor, and 2% sucrose of addition and 0.65% agar powder are prepared into bud proliferated culture medium thereto, after the completion of preparation
Bud proliferated culture medium is dispensed to conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, takes out conical flask after the completion of sterilizing,
Solidify bud proliferated culture medium natural cooling therein, the adventitious bud that callus is differentiated to form is cut and accesses bud proliferation training
It supports in base, adventitious bud proliferation culture is carried out under 26 DEG C of temperature, humidity 85%, the illumination of 12h, light are provided daily using fluorescent lamp
It is 1000-4000lx according to intensity, adventitious bud proliferation is to 3-5 times after one month, and sprout is long to 5cm, can continue to choose at this time
The adventitious bud of robust growth, cut section be transferred on bud proliferated culture medium subculture expand it is numerous；
Culture of rootage: the NAA of MS culture medium and 0.5mg/L is mixed be prepared into mixed liquor first, and thereto
1.5% sucrose of addition and 0.65% agar powder are prepared into root media, and the root media after the completion of preparation is dispensed
To conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, conical flask is taken out after the completion of sterilizing, makes culture of rootage therein
The sprout tender stem that adventitious bud proliferation is turned out, is cut into the segment of 2-3cm, is seeded to root media by the solidification of base natural cooling
In, culture of rootage is carried out under 26 DEG C of temperature, humidity 85%, provides the illumination of 12h daily using fluorescent lamp, intensity of illumination is
1000-4000lx grows adventitious root after about 15 days, most of seedling grows 5-7 item root, rooting rate > 90% after 30 days；
Hardening and transplanting: filling humus in flowerpot or hole tray, and by high 3cm and seedling with 5-7 item root by
It walks after being carried out hardening 7 days in the natural environment for moving on to scattering light, seedling is taken out, cleans seedling root with 30 DEG C or so of warm water
Portion cleans up the culture medium of its root, and will be in seedling replanting to the flowerpot of dress humus or hole tray；
Transplanting culture: after the completion of seedling replanting, the humidity in control culture place be 85% ± 2%, temperature be 26 DEG C ±
Hardening is carried out under 1% again, seedling is allow to adapt to extraneous production environment, seedling percent > after culture 30 days completely
95%.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications, these improvements and modifications can also be made
Also it should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of method for tissue culture of kalanchoe daigremontiana, which comprises the following steps:
(1) acquisition of material: winning the fresh healthy leaves of kalanchoe daigremontiana, and surface 20min is rinsed under tap water；
(2) aseptic process: the blade after the completion of flushing is impregnated into 15s with 75% alcohol on aseptic operating platform, impregnates and completes
It uses aseptic water washing 1 time afterwards, is then soaked in 0.1% mercuric chloride solution and sterilizes 6min, rushed again with sterile water after the completion of disinfection
It washes 3-5 times, the moisture of blade surface is finally blotted with sterilized filter paper, then blade is cut into the size of about 1.0cm*1.0cm；
(3) callus induction: will be on the blade inoculation to callus inducing medium after the cutting after the completion of aseptic process；
(4) callus breaks up: callus induction cuts robust growth callus after 4 weeks is transferred to callus differentiation
On culture medium, induction generates adventitious bud；
(5) adventitious bud proliferation: callus induction cuts adventitious bud callus after 4 weeks is transferred to bud Multiplying culture
On base, squamous subculture is carried out under certain cultivation temperature and illumination condition, after one month, sprout it is long to 5cm when, continue to choose life
Long healthy and strong adventitious bud, cut section be transferred to subculture on bud proliferated culture medium expand it is numerous；
(6) culture of rootage: the sprout tender stem that adventitious bud proliferation is turned out is cut into the segment of 2-3cm, is seeded to root media
In, culture of rootage is carried out under certain cultivation temperature and illumination condition；
(7) hardening and transplanting: after most test tube seedlings bear 3-7 item root, by high 3cm and with 3-7 item root test tube seedling by
It walks after being carried out hardening 7 days in the natural environment for moving on to scattering light, seedling is taken out, the culture medium of seedling root is cleaned with warm water,
And it will be in seedling replanting to the flowerpot of dress humus or hole tray；
(8) transplanting culture: after the completion of seedling replanting, it is 26 DEG C ± 1 that the humidity in control culture place, which is 85% ± 2%, temperature,
Hardening is carried out at DEG C again.
2. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (2)
In aseptic operating platform need to open ultraviolet germicidal lamp and blower before the use and carry out sterilization 30min.
3. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (2)
In 0.1% mercuric chloride solution in include 1-2 drop 20mg/L tween.
4. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (3)
In callus inducing medium be prepared by mixing by the NAA of MS culture medium, the 6-BA of 1.0mg/L and 1.0mg/L, and
Wherein adding 2% sucrose and 0.65% agar powder.
5. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (4)
In callus differential medium be prepared by mixing by the NAA of MS culture medium, the 6-BA of 1.0mg/L and 0.1mg/L, and
Wherein adding 2% sucrose and 0.65% agar powder.
6. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (5)
In bud proliferated culture medium be to be prepared by mixing by the NAA of MS culture medium, the 6-BA of 0.2mg/L and 0.5mg/L, and wherein
Add 2% sucrose and 0.65% agar powder.
7. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (6)
In root media be prepared by mixing by the NAA of MS culture medium and 0.5mg/L, and wherein addition 1.5% sucrose
With 0.65% agar powder.
8. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (3)
Cultivation temperature into step (6) is 26 DEG C ± 2 DEG C.
9. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (3)
Culture humidity into step (6) is 85% ± 1%.
10. a kind of method for tissue culture of kalanchoe daigremontiana as described in claim 1, which is characterized in that the step (3)
Illumination condition into step (6) is to use daylight light irradiation 12h, intensity of illumination 1000-4000lx daily.
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Application publication date: 20190531 |