CN108184672A - A kind of Stauntonia latifolia callus from stem segment method for inducing and cultivating - Google Patents
A kind of Stauntonia latifolia callus from stem segment method for inducing and cultivating Download PDFInfo
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- CN108184672A CN108184672A CN201810188629.1A CN201810188629A CN108184672A CN 108184672 A CN108184672 A CN 108184672A CN 201810188629 A CN201810188629 A CN 201810188629A CN 108184672 A CN108184672 A CN 108184672A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
A kind of Stauntonia latifolia callus from stem segment method for inducing and cultivating, including sport technique segments such as the anti-browning of explant, disinfection, axillary bud deriving culture, induction of callus and shoot proliferation cultures, method is just to sprout the band axillary bud tender stem segments of 7~15 days as explant, it impregnated with antioxidant vitamin C, disinfect mode with the improvement of the explant of potassium permanganate solution soaking disinfection again, significantly reduce the melting brown rate of explant and pollution rate, make explant just challenge inoculation survival rate up to 90%.By adding 1.5g.L in 1/2MS culture mediums‑1Activated carbon and 2.0 g.L‑1Polyvinylpyrrolidone with reference to the hormone prescription that different phase filters out, can make callus induction differentiation rate up to more than 90%, can shoot proliferation culture callus and Multiple Buds simultaneously, more than 3.0 growth coefficient.This method can provide Technical Reference to be utilized using callus tissue culture development new varieties mutagenesis cultivation and breeding fast seedling growing.
Description
Technical field
The present invention relates to the cultivations of crop, furthermore, are related to the induction of callus method of Stauntonia latifolia stem section.
Background technology
Stauntonia latifolia is being commonly called as Lardizabalaceae Three Akebia Decne Species threeleaf akebia (Holboetllia latifolia), perennial
Fallen leaves woody climber, is distributed widely in the provinces such as China Guizhou, Jiangxi, Zhejiang, Hunan, Sichuan, Hubei.Fruit is berry, purple brown
Color, oval tubular.Pulp is milky white, succulence, sweet, giving off a strong fragrance, sliding tender, unique flavor, rich in carbohydrate, unrighted acid, a variety of
The essential amino acid that 12 kinds of human bodies such as vitamin, minerals and trace element and valine, methionine cannot synthesize.Its rattan
There are the drug effects such as removing toxic substances, diuresis, dehumidifying, promoting menstruation, analgesia, apocenosis as traditional Chinese medicine, to treatment epersalgia cough, bruise bone
The illnesss such as folding, oedema, scald, scrofula, ureteral calculi have positive effect；Its florescence is longer, gives off a strong fragrance, the peculiar U.S. of fruit shape
It sees, great ornamental value.With Kiwi berry and claiming wild fruit king, it has also become the emerging water of river Hunan and Guizhou Mountain Debelopment plantation
Fruit.Fruit and seed is excessive, low edible rate is seriously to restrict the mortal wound that the characteristic fruit ranks among large fruit.
Induction of callus is to carry out the mutagenesis of Stauntonia latifolia new varieties to cultivate, and screens no seed or few seed new varieties, improves
The technical foundation of the new technology breedings such as edible rate research.Current Stauntonia latifolia sapling multiplication mainly with seminal propagation, root division and
Based on mound layering, tissue culture propagation technical research report is less.Li Shiling etc. (2008) is with MS minimal mediums to Stauntonia latifolia embryo
Breast carries out cultured in vitro research, it is found that Stauntonia latifolia endosperm culture browning phenomenon is serious, occur brown stain, brown stain in all culture mediums
Rate is up to 100%, minimum 10.2%, it is believed that control brown stain is that the important hand of endosperm vigor is ensured in Stauntonia latifolia endosperm culture
Section.Carry out the research of induction of callus using stem section, have not been reported at present.
About the planting technology of Stauntonia latifolia, there are some to apply for part in Chinese patent database, such as：2015106714315
Number《A kind of Stauntonia latifolia cottage method》, No. 2015105412341《A kind of method that Stauntonia latifolia and wild country melon are interplanted on salt-soda soil》、
No. 2016111636363《The Se accumulation cultural method of needle batt plant transfusion device and Stauntonia latifolia》, No. 2017105134383《One
The cultural method of kind Stauntonia latifolia》, No. 2017107609052《A kind of cuttage implantation methods of Stauntonia latifolia》Deng.So far, it there is no
The patent application of Stauntonia latifolia tissue cultures.
Invention content
The present invention is intended to provide a kind of Stauntonia latifolia callus from stem segment Fiber differentiation side that can significantly reduce Brown
Method establishes technical foundation to carry out mutation breeding and rapid seedling cultivation technical research.
The Stauntonia latifolia callus from stem segment method for inducing and cultivating that inventor provides, comprises the steps of：
(1) explant sterilizes：The Stauntonia latifolia young sprout of 7~15d has just been sprouted in acquisition, removes blade, stem segment with axillary bud is taken to do outer
Implant；After rinsing well, 5000mg.L is used-1Vitamin C distilled water solution impregnates 50min, then uses 3000mg.L-1Permanganic acid
Potassium distilled water solution impregnates 30min, with distilled water flushing 3~5 times, then uses 1000mg.L-1Mercuric chloride solution sterilizes 10min,
Distilled water flushing 5 times；
(2) minimal medium in Plant cell and tissue culture each stage is：1/2MS+12g.L-1Agar+30g.L-1Sugarcane
Sugar+1.5g.L-1AC (activated carbon)+2.0g.L-1PVP (polyvinylpyrrolidone), pH value 6.5；Each stage condition of culture is：Temperature
24 DEG C~26 DEG C of degree, illumination are 1500~2000Lux, and light application time is 12~14h/d.
(3) the first Fiber differentiation of axillary bud：By the explant after disinfection by growth polarity setting be seeded in minimal medium+
0.8mg.L-1KT (6 chaff adenine phosphate)+0.005mg.L-1The axillary bud of NAA (methyl α-naphthyl acetate) is just on inducing culture.
(4) induction of callus：It is inoculated with after the axillary bud that first Fiber differentiation generates is cut into the segment of length 0.5cm
In minimal medium+0.5mg.L-16-BA (6 Bian adenine phosphate)+0.1mg.L-12,4-D (2,4 dichlorophenoxyacetic acid)+
0.1mg.L-1~0.05mg.L-1On the callus tissue culture base of NAA, the green Lax callus of diameter 1.0cm can be generated.
(5) shoot proliferation culture：Gently callus is divided into after the fritter of diameter 0.5cm with tweezers and is seeded in basic training
Support base+2.5mg.L-1TDZ (Thidiazuron)+0.1mg.L-1In the subculture multiplication medium of IBA (indolebutyric acid), while differentiation is cured
Injured tissue and Multiple Buds, every piece of diameter 1.5cm or so, can break up 4~5 Multiple Buds.
1.5g.L is added in the minimal medium in (4) step in (2) of the above method, (3)-1AC (activated carbon) is with suction
The harmful substances such as attached phenols add 2g.L-1PVP (polyvinylpyrrolidone), will be brown to decompose and aldehydes matter is inhibited to generate
Rate is down to less than 10%.
Inventor points out：Stem section explant 5000mg.L-1Antioxidant vitamin C distilled water solutions impregnate 50min；With
3000mg.L-1Potassium permanganate distilled water solution impregnates 30min, without 75% ethanol disinfection, is remarkably improved explant vitality of subject, dirty
Dye rate can be reduced to less than 10%.
The innovative point of the present invention：
The method of Stauntonia latifolia stem section Fiber differentiation callus provided by the invention, it is easy to solve Stauntonia latifolia tissue culture propagation
Brown stain, the technical bottleneck of pollution.This method is impregnated by using antioxidant vitamin C distilled water solutions, is distilled with potassium permanganate
Aqueous solution soaking significantly improves explant vitality of subject, reduces pollution rate.By being added in the minimal medium in each stage
1.5g.L-1Activated carbon adsorbs harmful substance, adds 2g.L-1Polyvinylpyrrolidone distilled water solution inhibits and decomposes outer
The mode of the harmful substances such as the phenols of generation is secreted in implant incubation, so as to reach improve explant differentiation rate, reduce it is brown
The effect of variability.The hormone prescription in this method each stage is obtained by test statistics, point of energy significantly evoked callus
Change and squamous subculture can provide reliable technical foundation to carry out mutation breeding and rapid seedling cultivation technical research.
Specific embodiment
Embodiment 1：The experiment in March, 2015
(1) explant is disinfected：The Stauntonia latifolia young sprout 50 of 7d has just been sprouted in acquisition, removes blade, and tap water rinses dry
After net, distilled water flushing 2 times.Use 5000mg.L-1Vitamin C distilled water solution impregnates 50min, distilled water flushing 3 times；Ultra-clean
On workbench, stem section is cut into 1cm long stem segment with axillary bud, uses 3000mg.L-1Potassium permanganate distilled water solution impregnates 30min,
With distilled water flushing 3 times；1000mg.L is used again-1Mercuric chloride solution routine disinfection 10min, distilled water flushing 5 times.
(2) the first Fiber differentiation of axillary bud：By the explant after disinfection as growth polarity setting be seeded in axillary bud at the beginning of Fiber differentiation
On base, culture medium prescription is：1/2MS+12g.L-1Agar+30g.L-1Sucrose+1.5g.L-1AC+2g.L-1PVP+0.8mg.L-1KT
+0.005mg.L-1NAA, pH value 6.5；It is inoculated with 200 stem sections altogether.15d after inoculation, 20% axillary bud sprouting；25d after inoculation, 183
Explant sprouts 1 axillary bud, average long 1.0cm；17 Explant browning pollutions, melting brown rate 8.5%.
(3) induction of callus：First Fiber differentiation 40d, the averagely long 1.5cm of axillary bud or so, first Fiber differentiation is produced
Raw axillary bud is inoculated into after cutting into the segment of length 0.5cm on callus inducing medium.Culture medium prescription is：1/2MS+
12g.L-1Agar+30g.L-1Sucrose+1.5g.L-1AC+2g.L-1PVP+0.5mg.L-16-BA+0.1mg.L-12,4-D+
0.1mg.L-1NAA.After cultivating 35d, the differentiation of 95% stem section generates green Lax callus, and diameter is averaged 1.0cm.
(4) shoot proliferation culture：Switching is in 1/2MS+ after callus gently is divided into the fritter of diameter 0.5cm with tweezers
12g.L-1Agar+30g.L-1Sucrose+1.5g.L-1AC+2g.L-1PVP+2.5mg.L-1TDZ+0.1mg.L-1The shoot proliferation of IBA
On culture medium.After shoot proliferation culture 30d, every piece of diameter reaches 1.5cm or so, 3 times of proliferation times or more.
(5) each stage condition of culture is：24 DEG C~26 DEG C of temperature, illumination are 1500~2000Lux, light application time for 12~
14h/d。
The experiment in embodiment in March, 2 2016
(1) explant is disinfected：The Stauntonia latifolia young sprout 50 of 15d has just been sprouted in acquisition, removes blade, and tap water rinses dry
After net, distilled water flushing 2 times.Use 5000mg.L-1Vitamin C distilled water solution impregnates 50min, distilled water flushing 3 times；Ultra-clean
On workbench, stem section is cut into 1cm long stem segment with axillary bud, uses 3000mg.L-1Potassium permanganate distilled water solution impregnates 30min,
With distilled water flushing 5 times；1000mg.L is used again-1Mercuric chloride solution routine disinfection 10min, distilled water flushing 5 times.
(2) the first Fiber differentiation of axillary bud：By the explant after disinfection as growth polarity setting be seeded in axillary bud at the beginning of Fiber differentiation
On base.Culture medium prescription is：1/2MS+12g.L-1Agar+30g.L-1Sucrose+1.5g.L-1AC+2g.L-1PVP+0.8mg.L-1KT
+0.005mg.L-1NAA, pH value 6.5.It is inoculated with 200 stem sections altogether.15d after inoculation, 10% axillary bud sprouting；30d after inoculation, 179
Explant sprouts 1 axillary bud, average long 1.0cm；21 Explant browning pollutions, melting brown rate 10.5%.
(3) induction of callus：First Fiber differentiation 45d, the averagely long 1.5cm of axillary bud or so, first Fiber differentiation is produced
Raw axillary bud is inoculated into after cutting into the segment of length 0.5cm on callus inducing medium.Culture medium prescription is：1/2MS+
12g.L-1Agar+30g.L-1Sucrose+1.5g.L-1AC+2g.L-1PVP+0.5mg.L-16-BA+0.1mg.L-12,4-D+
0.1mg.L-1NAA.After cultivating 35d, the differentiation of 90% stem section generates green Lax callus, and diameter is averaged 0.9cm.
(4) shoot proliferation culture：Switching is 1/ after callus gently is divided into the fritter of diameter 0.5cm sizes with tweezers
2MS+12g.L-1Agar+30g.L-1Sucrose+1.5g.L-1AC+2g.L-1PVP+2.5mg.L-1TDZ+0.05mg.L-1The subculture of IBA
On proliferated culture medium.After shoot proliferation culture 45d, 4~5 buds at every piece of differentiation.
(5) each stage condition of culture is：24 DEG C~26 DEG C of temperature, illumination are 1500~2000Lux, light application time for 12~
14h/d。
Claims (2)
1. a kind of Stauntonia latifolia callus from stem segment method for inducing and cultivating, it is characterised in that include the following steps：
(1) explant sterilizes：The Stauntonia latifolia young sprout of 7~15d has just been sprouted in acquisition, removes blade, stem segment with axillary bud is taken to do explant；
After rinsing well, 5000mg.L is used-1Vitamin C distilled water solution impregnates 50min, then uses 3000mg.L-1Potassium permanganate distills
Aqueous solution soaking 30min with distilled water flushing 3~5 times, then uses 1000mg.L-1Mercuric chloride solution sterilizes 10min, distilled water
It rinses 5 times；
(2) minimal medium in Plant cell and tissue culture each stage is：1/2MS+12g.L-1Agar+30g.L-1Sucrose+
1.5g.L-1Activated carbon+2.0g.L-1Polyvinylpyrrolidone, pH value 6.5；Each stage condition of culture is：24 DEG C~26 DEG C of temperature,
Illumination is 1500~2000Lux, and light application time is 12~14h/d；
(3) the first Fiber differentiation of axillary bud：Explant after disinfection is seeded in minimal medium+0.8mg.L by growth polarity setting- 16-Furfurylaminopurine+0.005mg.L-1The axillary bud of methyl α-naphthyl acetate is just on inducing culture；
(4) induction of callus：Base is inoculated in after the axillary bud that first Fiber differentiation generates is cut into the segment of length 0.5cm
Basal culture medium+0.5mg.L-16- Bian adenine phosphates+0.1mg.L-12,4 dichlorophenoxyacetic acid+0.1mg.L-1~0.05mg.L-1Naphthalene
On the callus tissue culture base of acetic acid, the green Lax callus of diameter 1.0cm can be generated；
(5) shoot proliferation culture：Minimal medium is seeded in after callus gently is divided into the fritter of diameter 0.5cm with tweezers
+2.5mg.L-1Thidiazuron+0.1mg.L-1In the subculture multiplication medium of indolebutyric acid, while break up callus and Multiple Buds,
Every piece of diameter 1.5cm or so, can break up 4~5 Multiple Buds.
2. method for inducing and cultivating as described in claim 1, it is characterised in that (2) of method, (3), the basic training in (4) step
Foster base adds 1.5g.L-1Activated carbon adds 2g.L with harmful substances such as absorbing phenolics-1Polyvinylpyrrolidone, to decompose
It generated with inhibition aldehydes matter, reduce melting brown rate.
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