CN107616092B - A kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt - Google Patents
A kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt Download PDFInfo
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Abstract
The present invention is a kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt, selection including explant and disinfection, the induction of callus, the induction of adventitious bud and strengthening seedling and rooting and etc..The present invention is to take out Roripa smalt seedling leaves as explant, the tissue culturing system for taking out Roripa smalt is explored for the first time, establish the acquisition of sterilizable material, the induction of callus, the induction of adventitious bud, strong sprout and the ideal condition of culture such as take root, it is finally successfully established the vegetative propagation system for taking out Roripa smalt, to taking out the certain theory of offers and the technical basis such as the making full use of of Roripa smalt herb resource, the cultivation of genetic improvement and clone nursery stock.Cultural method advantage of the invention is that the induction of callus and one step of differentiation of adventitious bud are completed, easy to operate, and the time of culture is greatly shortened while saving workload, a large amount of tissue-cultured seedling can be produced in the short time, not only breeding potential is high, but also seedling is high-quality, is suitble to the factorial production.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to it is a kind of take out Roripa smalt tissue cultures and Regeneration in Vitro side
Method.
Background technique
Taking out Roripa smalt (Clerodendrum subscaposum) is the perennial fallen leaves of Verenaceae Clerodendron or evergreen filling
Wood, complete stool are used as medicine, and flower can calm the nerves, stop blooding, and cure mainly palpitation and insomnia, hemorrhoid hemorrhage；Leaf energy wind-dispelling dissipates the stasis of blood, removing toxicity for detumescence, cures mainly
Migraine is beaten the stasis of blood swell, carbuncle sore tumefacting virus；Root, stem can clearing lung-heat heat, diuresis, cooling blood and hemostasis, be clinically used for cough with lung heat, heat gonorrhea,
The treatment of difficult urination, hemoptysis, hematuria, hemorrhoid hemorrhage, treating rheumatic ostealgia.
It is upright to take out Roripa smalt stem, not branch or few branch, four prismatic of sprig.Leaf interaction has long handle, leaf to life, Bao Gezhi
Piece heart or oval, the rapid point or tapering in top, base portion is heart-shaped, and edge has serration, the scattered small thick volt hair in blade face.Total shape circular cone flower
Sequence basidixed, to side deflection.Spend small, but filigree is long；Calyx, corolla, bennet are equal, are bright-coloured peony, the florescence is long, and drupe is close
Spherical shape, black-and-blue when ripe, outsourcing is with red place calyx；Flower, the fruiting period 5-11 month.
Property pyrophilous, wet, half climatic environment covered, the deep acid soil of happiness soil layer, resistance to concealment is barren-resistant, avoids
Arid avoids flood, and chilly is cold, and growth thermophilic is 23~30 DEG C.Mainly originate in China Jiangsu, Southern Zhejiang, South Jiangxi, Hunan,
Fujian, Taiwan, Guangdong, Guangxi, Sichuan, Guizhou and Yunnan；India northeast, Bangladesh, Sillim, Bhutan, South East Asia Mainland, Ma Laixi
Sub-, Japan is also distributed, and is often born in Plain, mountain valley, small stream side or sparse woods or cultivates in flower garden.
Pertinent literature is consulted, Roripa smalt is taken out by tissue cultures and realizes that fast numerous correlation technique has not yet to see report, it is therefore, anxious
It need to explore and a kind of be able to achieve rapid propagation method to meet the needs of modern production.
Summary of the invention
The object of the present invention is to provide a kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt, to take out Roripa smalt blade
The tissue culture rapid propagation system for taking out Roripa smalt is set up by experimental exploring for the first time for explant, to take out filling for Roripa smalt medicine resource
Utilization is divided to provide certain theory and technology foundation.
To achieve the above object, the technical solution adopted by the present invention is that: a kind of tissue cultures that taking out Roripa smalt and it is in vitro again
Generation method, including following steps in sequence:
(1) explant select and disinfection: select to take out Roripa smalt seedling leaves be explant, clean soil is put into beaker
In, water and a small amount of washing powder is added, jiggles 5min, then rinse 2h with clear water, washing powder is rinsed well；It is turning lastly to surpass
On net workbench, 3-5s is sterilized with the ethanol solution of 75% (v/v), sterile washing 2 times utilizes 0.1% liter containing tween
Mercury solution sterilizes 4-5min, and sterile washing 5-6 times spare；
(2) induction of callus: under sterile working, it is 1-1.5cm that the blade disinfected, which is cut into area,2Sheet,
It is laid flat and is seeded in callus inducing medium in the upward mode of face of blade, 23 ± 2 DEG C of temperature, relative air humidity
50%-60%, intensity of illumination 2000Lx, 12 hour/day of light application time are cultivated 20 days, make to grow 2-6mm thickness at paddle cutout
A circle dense form, light green, blocky callus；
(3) differentiation of adventitious bud: callus continuation continues culture about 10-12 days under above-mentioned condition of culture, makes callus
Tissue differentiates the budlet that bottle green, state are normal, quantity is extremely more；
(4) strong seedling culture of bud: the adventitious bud differentiated is cut in time together with callus, is accessed in strong seedling culture base,
23 ± 2 DEG C of temperature, relative air humidity 50%-60%, intensity of illumination 2000Lx, 12 hour/day of light application time, cultivated days
25-30 angel's height of seedling is 3-5cm；
(5) culture of rootage: seedling single plant of step (4) training after strong is cut, is accessed in root media, temperature 23 ± 2
DEG C, relative air humidity 50%-60%, intensity of illumination 2000Lx, 12 hour/day of light application time, culture 20-30 angel seedling is raw
Long 3-4 item, the adventitious root that length is 2-4cm.
Further, in step (2), when blade is put on culture medium, only incision contacts culture medium, rest part are not embedded to
Culture medium.
Further, the differentiation used medium formula of the callus induction and the adventitious bud are as follows: WPM, MS or
1/2MS culture medium+TDZ 1.5-2.5mg/L+NAA 0.1-0.3mg/L+ agar 4.5g/L+ sucrose 20g/L, the medium pH
It is 5.8；
Further, the strong seedling culture based formulas are as follows: 1/2MS or MS culture medium+6-BA 0.05-0.2mg/L+NAA
0.02-0.1mg/L+ agar 4.5g/L+ sucrose 30g/L, the medium pH are 5.8；
Further,
The prescription of rooting medium are as follows: 1/2MS culture medium+NAA 0.5-1.0mg/L+IBA 0.2-0.5mg/L+ agar
4.5g/L+ sucrose 30g/L, the medium pH are 5.8.
In the present invention, WPM culture medium be basic composition is: NH4NO3 400mg/L、Ca(NO3)2·4H2O 556mg/L、
K2SO4 990mg/L、CaCl2·2H2O 96mg/L、KH2PO4 170mg/L、Na2MoO4·2H2O 0.25mg/L、MgSO4·
7H2O 370mg/L、MnSO4·H2O 22.4mg/L、ZnSO4·7H2O 8.6mg/L、CuSO4·5H2O 0.25mg/L、
FeSO4·7H2O 27.8mg/L、Na2- EDTA 37.3mg/L, inositol 100mg/L, vitamin B1 1.0mg/L, niacin 0.5mg/
L, vitamin B6 0.5mg/L, glycine 2.0mg/L.
The method have the benefit that: the present invention to take out Roripa smalt blade as explant, by the disinfection of explant,
The processes such as the induction of callus, the differentiation of adventitious bud and strengthening seedling and rooting culture, which obtain, takes out Roripa smalt Regeneration in Vitro plant,
It effectively establishes and takes out Roripa smalt tissue culture rapid propagation system, and the induction of pumping Roripa smalt callus and one step of differentiation of adventitious bud are complete
At, it is easy to operate, the time of culture is greatly shortened while saving workload, and a large amount of tissue-cultured seedling can be produced in the short time,
Not only breeding potential is high, but also seedling is high-quality, is suitble to the factorial production.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
A kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt, step include:
(1) it the disinfection of explant: selects to take out Roripa smalt seedling leaves to be explant, soil is cleaned with tap water, is put into
In beaker, water and a small amount of washing powder is added, solution immersion is made, 5min is jiggled, then rinse 2h with tap water, by washing powder
It rinses well；It is turning lastly on superclean bench, sterilizes 3s with 75% ethanol solution, sterile washing 2 times contains using 0.1%
There is the mercuric chloride solution disinfection 4min of tween (2 drop), sterile washing 6 times is cleaned spare.
(2) under sterile working, the blade disinfected the induction of callus and the differentiation of adventitious bud: is cut into 1cm2Greatly
Small blade is laid flat in the upward mode of face of blade and is seeded to callus inducing medium (WPM culture medium+TDZ
2.0mg/L+NAA 0.2mg/L+ agar 4.5g/L+ sucrose 20g/L, pH=5.8) in, make incision contacts culture medium, avoids simultaneously
Blade is totally submerged in the medium, is placed in culturing room and is cultivated, and 23 DEG C of temperature, relative air humidity 50%, intensity of illumination
2000Lx, 12 hour/day of light application time are cultivated 22 days, it is seen that it is blocky that paddle cutout director goes out the thick circle dense form of about 0.5cm
Callus, color are light green.It is changed without culture medium and does not change condition of culture, continue culture 10 days, divide on callus
Dissolve the budlet that bottle green, state are normal, quantity is extremely more.
(3) strong seedling culture of bud: the adventitious bud differentiated in step (2) is cut in time together with a small amount of callus, is connect
Enter strong seedling culture base (1/2MS culture medium+6-BA 0.05mg/L+NAA 0.02mg/L+ agar 4.5g/L+ sucrose 30g/L, pH=
5.8) in, 23 ± 2 DEG C of temperature, relative air humidity 60%, intensity of illumination 2000Lx, 12 hour/day of light application time is cultivated 25 days
Keep budlet long to 3cm high；
(4) culture of rootage: seedling single plant of step (3) training after strong is cut, access root media (1/2MS culture medium+
NAA 0.5mg/L+IBA 0.2mg/L+ agar 4.5g/L+ sucrose 30g/L, pH=5.8) in, 23 DEG C of temperature, relative air humidity
50%, intensity of illumination 2000Lx, 12 hour/day of light application time, the adventitious root that culture 25 angel's growth of seedling, 3 length are 4cm
Seedling.
Embodiment 2
A kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt, step include:
(1) it the disinfection of explant: selects to take out Roripa smalt seedling leaves to be explant, soil is cleaned with tap water, is put into
In beaker, water and a small amount of washing powder is added, solution immersion is made, 5min is jiggled, then rinse 2h with tap water, by washing powder
It rinses well；It is turning lastly on superclean bench, sterilizes 5s with 75% ethanol solution, sterile washing 2 times contains using 0.1%
There is the mercuric chloride solution disinfection 5min of tween (2 drop), sterile washing 6 times spare；
(2) under sterile working, the blade disinfected the induction of callus and the differentiation of adventitious bud: is cut into 1.2cm2
Size is laid flat in the upward mode of face of blade and is seeded to callus inducing medium MS culture medium+TDZ 1.5mg/L+NAA
0.1mg/L+ agar 4.5g/L+ sucrose 20g/L, pH=5.8) in, make incision contacts culture medium and blade full wafer is avoided to be immersed in
In culture medium, it is placed in culturing room and cultivates, 25 DEG C of temperature, relative air humidity 55%, intensity of illumination 2000Lx, light application time 12
It cultivates 20 days in hour/day, it is seen that paddle cutout director goes out the thick circle dense form bulk callus of about 0.4cm, and color is light
Green；It is changed without culture medium and does not change condition of culture, continue culture 11 days, differentiated on absinthe-green callus many small
Bud, color are bottle green, and the state of bud is normal, and quantity is extremely more；
(3) strong seedling culture of bud: the adventitious bud differentiated in step (2) is cut in time together with a small amount of callus, is connect
Enter strong seedling culture base (1/2MS culture medium+6-BA0.2mg/L+NAA0.1mg/L+ agar 4.5g/L+ sucrose 30g/L, pH=5.8)
In, 23 ± 2 DEG C of temperature, relative air humidity 55%, intensity of illumination 2000Lx, it is small to cultivate 27 angels for 12 hour/day of light application time
Bud is long to 4cm high；
(4) culture of rootage: seedling single plant of step (3) training after strong is cut, access root media (1/2MS culture medium+
NAA 1.0mg/L+ agar 4.5g/L+ sucrose 30g/L, pH=5.8) in, 23 ± 2 DEG C of temperature, relative air humidity 55%, illumination
Intensity 2000Lx, 12 hour/day of light application time, the adventitious root seedling that culture 27 angel's growth of seedling, 4 length are 3cm.
Embodiment 3
A kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt, step include:
(1) it the disinfection of explant: selects to take out Roripa smalt seedling leaves to be explant, soil is cleaned with tap water, is put into
In beaker, water and a small amount of washing powder is added, solution immersion is made, 5min is jiggled, then rinse 2h with tap water, by washing powder
It rinses well；It is turning lastly on superclean bench, sterilizes 3s, sterile washing 2 times with 75% ethanol solution, 0.1% contain is spat
The mercuric chloride solution of temperature (2 drop) sterilizes 4min, and sterile washing 6 times is cleaned spare；
(2) under sterile working, the blade disinfected the induction of callus and the differentiation of adventitious bud: is cut into 1.5cm2
Size is laid flat in the upward mode of face of blade and is seeded to callus inducing medium (1/2MS culture medium+TDZ 2.5mg/L
+ NAA 0.3mg/L+ agar 4.5g/L+ sucrose 20g/L, pH=5.8) in, make incision contacts culture medium and ensures that blade full wafer soaks
Enter in culture medium, is placed in culturing room and cultivates, 21 DEG C of temperature, relative air humidity 60%, intensity of illumination 2000Lx, light application time
It cultivates 22 days 12 hours/day, it is seen that paddle cutout director goes out the thick circle dense form bulk callus of about 0.6cm, and color is
Light green；It is changed without culture medium and does not change condition of culture, continue culture 12 days, differentiate many on absinthe-green callus
Budlet, color are bottle green, and the state of bud is normal, and quantity is extremely more.
(3) strong seedling culture of bud: the adventitious bud differentiated in step (2) is cut in time together with a small amount of callus, is connect
Enter strong seedling culture base (MS culture medium+6-BA 0.1mg/L+NAA 0.05mg/L+ agar 4.5g/L+ sucrose 30g/L, pH=5.8)
In, 21 DEG C of temperature, relative air humidity 60%, intensity of illumination 2000Lx, 30 angel budlets are cultivated in 12 hour/day of light application time
It grows to 5cm high；
(4) culture of rootage: seedling single plant of step (3) training after strong is cut, and access root media 1/2MS culture medium+
NAA 0.5mg/L+IBA 0.5mg/L+ agar 4.5g/L+ sucrose 30g/L, pH=5.8) in, 23 ± 2 DEG C of temperature, air is opposite
Humidity 50%, intensity of illumination 2000Lx, 12 hour/day of light application time, culture 30 angel's growth of seedling, 4 length be 2cm not
Determine root seedling.
| Explant blade area (cm2) | Incubation time (day) | Seedling numbers (strain) | |
| Embodiment 1 | 1 | 82 | 4 |
| Embodiment 2 | 1.2 | 85 | 6 |
| Embodiment 3 | 1.5 | 94 | 5 |
It takes out Roripa smalt by the method for the invention, can realize the purpose quickly bred at three months or so, greatly shorten culture
Time, a large amount of tissue-cultured seedling can be produced in the short time, not only breeding potential is high, but also seedling is high-quality, is suitble to factory's metaplasia
It produces.
It should be noted last that: the above embodiments are only used to illustrate and not limit the technical solutions of the present invention, although ginseng
It is described the invention in detail according to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that: it still can be to this
Invention is modified or replaced equivalently, without departing from the spirit or scope of the invention, or any substitutions,
It is intended to be within the scope of the claims of the invention.
Claims (2)
1. a kind of tissue cultures and in-vitro regeneration method for taking out Roripa smalt, which is characterized in that including following steps in sequence:
(1) explant select and disinfection: select to take out Roripa smalt seedling leaves be explant, clean soil is put into beaker, adds
Enter water and a small amount of washing powder, jiggles 5min, then rinse 2h with clear water, washing powder is rinsed well；It is turning lastly to ultra-clean work
Make on platform, sterilizes 3-5s with the ethanol solution of 75% (v/v), sterile washing 2 times is molten using 0.1% mercuric chloride containing tween
Liquid disinfectant 4-5min, sterile washing 5-6 times spare；
(2) induction of callus: under sterile working, it is 1-1.5cm that the blade disinfected, which is cut into area,2Sheet, with blade
The mode to face up, which is laid flat, to be seeded in callus inducing medium, 23 ± 2 DEG C of temperature, relative air humidity 50%-
60%, intensity of illumination 2000Lx, 12 hour/day of light application time are cultivated 20-25 days, make to grow 2-6mm thickness at paddle cutout
One circle dense form, light green, blocky callus；
(3) differentiation of adventitious bud: callus continuation continues culture 10-12 days under above-mentioned condition of culture, makes on callus
Differentiate the budlet that bottle green, state are normal, quantity is extremely more；
(4) strong seedling culture of bud: the adventitious bud differentiated is cut in time together with callus, is accessed in strong seedling culture base, temperature
23 ± 2 DEG C, relative air humidity 50%-60%, intensity of illumination 2000Lx, 12 hour/day of light application time, cultivated days 25-30
Angel's height of seedling is 3-5cm；
(5) culture of rootage: seedling single plant of step (4) training after strong is cut, is accessed in root media, 23 ± 2 DEG C of temperature, sky
20-30 angel growth of seedling 3-4 is cultivated in gas relative humidity 50%-60%, intensity of illumination 2000Lx, 12 hour/day of light application time
Item, the adventitious root that length is 2-4cm；
The differentiation used medium formula of the callus induction and the adventitious bud are as follows: WPM, MS or 1/2MS culture medium+
TDZ 1.5-2.5mg/L+NAA 0.1-0.3mg/L+ agar 4.5g/L+ sucrose 20g/L, the medium pH are 5.8；
The strong seedling culture based formulas are as follows: 1/2MS or MS culture medium+6-BA 0.05-0.2mg/L+NAA 0.02-0.1mg/L+
Agar 4.5g/L+ sucrose 30g/L, the medium pH are 5.8；
The prescription of rooting medium are as follows: 1/2MS culture medium+NAA 0.5-1.0mg/L+IBA 0.2-0.5mg/L+ agar
4.5g/L+ sucrose 30g/L, the medium pH are 5.8.
2. taking out the tissue cultures and in-vitro regeneration method of Roripa smalt according to claim 1, which is characterized in that in step (2),
When blade is put on culture medium, only incision contacts culture medium, rest part are not embedded to culture medium.
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| CN105918132A (en) * | 2016-05-26 | 2016-09-07 | 南京林业大学 | Rapid breeding method of clerodendrum trichotomum thunb |
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| CN105918132A (en) * | 2016-05-26 | 2016-09-07 | 南京林业大学 | Rapid breeding method of clerodendrum trichotomum thunb |
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