CN107494282A - A kind of Yellowmouth Dutchmanspipe Root method for tissue culture - Google Patents
A kind of Yellowmouth Dutchmanspipe Root method for tissue culture Download PDFInfo
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- CN107494282A CN107494282A CN201710967526.0A CN201710967526A CN107494282A CN 107494282 A CN107494282 A CN 107494282A CN 201710967526 A CN201710967526 A CN 201710967526A CN 107494282 A CN107494282 A CN 107494282A
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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
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
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Abstract
The invention discloses a kind of Yellowmouth Dutchmanspipe Root method for tissue culture, Yellowmouth Dutchmanspipe Root is aristolochiaceae plant, and the four seasons can harvest, but preferably autumn quarrier.Silt and fibrous root are removed after taking root, is shone to half-dried, diameter is half-and-half rived in more than 2.5cm, is dried.Medicinal Materials Characters：For root in cylinder, crust light brown or light brown yellow, it is ecru to scrape off crust.Matter is solid, not easily broken, and section is uneven, thorn-like, cross section canescence to yellow-white, mealiness, and skin zone is thicker.Yellowmouth Dutchmanspipe Root bitter, micro-puckery.Main Ingredients and Appearance：Containing aristolochic acid, sitosterol, magnoflorine, allantoin etc..Function cures mainly：Wind-dispelling Li Shui, dehumidifying analgesia.For head edema of limbs, waist and knee arthralgia function.The present invention is induced, the process such as bud Multiplying culture, culture of rootage, hardening and transplanting realizes the Regeneration in Vitro of Yellowmouth Dutchmanspipe Root using Yellowmouth Dutchmanspipe Root stem with bud as explant by bud, and technical support is provided for its a large amount of seed selection, quick breeding, new varieties popularization.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in Plant Biotechnology, specifically, is related to a kind of Yellowmouth Dutchmanspipe Root
Method for tissue culture.
Background technology
Yellowmouth Dutchmanspipe Root is aristolochiaceae plant, main product Hanzhong Area of Shaanxi.In addition, the ground such as Hubei, Hunan, Sichuan, Yunnan.
The four seasons can harvest, but preferably section season quarrier.Silt and fibrous root are removed after taking root, scrapes off or does not scrape off cork, is shone extremely
Half-dried, diameter is half-and-half rived in more than 2.5cm, is dried.Medicinal Materials Characters：Root is in cylinder, and how many bendings, there is depth in knee
Fall into traverse furrow, long 6 ~ 15cm, 1.5 ~ 3cm of diameter.Crust light brown or light brown yellow, it is ecru to scrape off crust.Matter is solid, no
Easily broken, section is uneven, and thorn-like, cross section canescence to yellow-white, mealiness, skin zone is thicker, woody part beam lark, radial
Arrangement.Bitter, micro-puckery.So that block is big, skin depth, rich mealiness person are preferred.Main Ingredients and Appearance：Containing aristolochic acid ,-sitosterol, magnolia
Alkali, allantoin etc..Function cures mainly：Wind-dispelling Li Shui, dehumidifying analgesia.For head edema of limbs, waist and knee arthralgia etc..At present,
Yellowmouth Dutchmanspipe Root is mainly grown using cuttage, but survival rate is low, far can not meet needs of the large-scale production to Yellowmouth Dutchmanspipe Root seedling.
Also, difference is deposited in effective component between seminal propagation offspring, is unfavorable for the popularization of improved seeds.Utilize Plant Tissue Breeding
Technology can effectively solve the problems, such as rareness species rescue in imminent danger and wild plant resource scarcity.Therefore the training of Yellowmouth Dutchmanspipe Root tissue is established
The technology of supporting, laid the foundation for its large-scale industrialized production, be current urgent problem, and solve the one of the market demand
Big method.
The content of the invention
It is an object of the invention to provide one kind using Yellowmouth Dutchmanspipe Root stem with bud as explant, induced by bud, bud propagation
The processes such as culture, culture of rootage, hardening and transplanting realize the Regeneration in Vitro of Yellowmouth Dutchmanspipe Root.Percentage of seedgermination is high, and seedling is sturdy,
Fast growing, a kind of big Yellowmouth Dutchmanspipe Root method for tissue culture of income.
A kind of Yellowmouth Dutchmanspipe Root method for tissue culture of the present invention, it is characterised in that comprise the following steps：
（1）Bud inducement cultivation：Using Yellowmouth Dutchmanspipe Root stem with bud as explant, blade is cut off, be cut into 4 centimeter lengths carries 3
10min is first soaked in the section of axillary bud with washing powder solution, then scrubs explant surface by the dust on its surface and part with hairbrush
Thalline removes, and is placed in after then rinsing 4h with running water in superclean bench, first washes 4 with sterile with after 76% ethanol disinfection 32s
It is secondary, then 28min is sterilized with 0.15% mercuric chloride solution, with being inoculated with after 6 globules for drying surface with aseptic filter paper again of aseptic water washing
Bud induction is carried out to bud inducement cultivation base, first full light culture 6 days under the conditions of 25 DEG C after inoculation, it is small to be subsequently placed in daily illumination 16
When, intensity of illumination is cultivated 35 days under the conditions of being 3100lx, counts its bud induction rate and growing state；
（2）Multiplying culture：By step（1）Cultivate that obtained growth is normal, has obvious stem, the sprout of impeller structure is cut and is transferred to increasing
Grow and squamous subculture is carried out on culture medium, first full light culture 6 days under the conditions of 25 DEG C after inoculation, it is small to be subsequently placed in daily illumination 16
When, intensity of illumination 2600lx, it is placed under conditions of cultivation temperature is 25 DEG C after cultivating 27 days and observes growing state and bud propagation
Situation, sprout is repeatedly cut repeatedly and carries out squamous subculture, obtains Multiple Buds；
（3）Culture of rootage：By step（1）Or（2）Process obtain height be about 7cm, well-grown, without browning, vitrifying and
The adventitious bud cutting of albinism, which is inoculated on root media, carries out culture of rootage, the first complete dark training under the conditions of 25 DEG C after inoculation
Support 6 days, be subsequently placed in daily illumination 16 hours, intensity of illumination 3600lx, cultivation temperature is cultivated 38 days under conditions of being 25 DEG C
Situation of taking root is counted afterwards；
（4）Acclimatization and transplantses：By step（3）Obtain high about 8cm well-grown and stalwartness rooting tube plantlet go bottle cap to be placed in
After natural lighting lower refining seedling 8 days, test tube seedling is taken out from blake bottle, washes root culture medium off, plants into by Nutrition Soil：Perlite
=1:3 matrix being mixed into, it is placed in illumination box and cultivates, watered daily with 1/2MS macro-element nutrients liquid to seedling, is protected
Humidity is held, is transplanted again after seedling survives.
Step（1）Described bud inducement cultivation base is：MS+6mg/L6-BA+3mg/L NAA+31g/L sucrose+6.0g/L fine jades
Fat, pH 5.8.
Step（2）Described proliferated culture medium is：MS+1.2mg/LNAA+8mg/L 6-BA+32g/L sucrose+6.0g/L fine jades
Fat, pH 5.8.
Step（3）Described root media is：1/4MS+1.0mg/L NAA+1.0mg/L IBA+31g/L sucrose+
6.0g/L agar, pH 5.8.
Compared with prior art it is an advantage of the invention that：The present invention in the plant tissue culture technique short time by quickly obtaining
The method for obtaining a large amount of Yellowmouth Dutchmanspipe Root improved seeds nursery stocks.Using Yellowmouth Dutchmanspipe Root stem with bud as explant, induced by bud, bud propagation
The processes such as culture, culture of rootage, hardening and transplanting realize the Regeneration in Vitro of Yellowmouth Dutchmanspipe Root, be its a large amount of seed selection, quick breeding,
New varieties, which are promoted, provides technical support.Technical method is simple, and operation is easy, and percentage of seedgermination is high, and seedling early growth is good, yield
It is high.
Embodiment
Following examples are the further explanations to the present invention, are not limitations of the present invention.
Embodiment 1
（1）Bud inducement cultivation：Using Yellowmouth Dutchmanspipe Root stem with bud as explant, blade is cut off, be cut into 6 centimeter lengths carries 3
5min is first soaked in the section of axillary bud with washing powder solution, then gently scrubbed with banister brush explant surface by the dust on its surface and
Part thalline removes, and is placed in after then rinsing 1h with running water in superclean bench, first with using sterilized water after 75% ethanol disinfection 5s
Wash 3 times, then 10min is sterilized with 0.1% mercuric chloride solution, be followed by with 5 globules for drying surface with aseptic filter paper again of aseptic water washing
Kind bud induction is carried out to bud inducement cultivation base, first full light culture 4 days under the conditions of 26 DEG C after inoculation, be subsequently placed in daily illumination 11
Hour, intensity of illumination is cultivated 28 days under the conditions of being 1200lx, counts its bud induction rate and growing state, bud induction rate 89%.Institute
The bud inducement cultivation base stated is：MS+4mg/L6-BA+0.5mg/L NAA+25g/L sucrose+3.4g/L agar, pH 5.6.
（2）Multiplying culture：By step（1）Cultivate that obtained growth is normal, have obvious stem, the sprout of impeller structure is cut and turned
Enter and squamous subculture is carried out on proliferated culture medium, first full light culture 2 days under the conditions of 26 DEG C after inoculation, be subsequently placed in daily illumination 10
Hour, intensity of illumination 1000lx, it is placed under conditions of cultivation temperature is 28 DEG C after cultivating 29 days and observes growing state and bud increasing
Grow situation.Sprout is repeatedly cut repeatedly and carries out squamous subculture, to obtain more Multiple Buds, newborn sprout robust growth, propagation
Coefficient is 4.8.Described proliferated culture medium is MS+0.25mg/LNAA+1.0mg/L 6-BA+27g/L sucrose+5.5g/L agar,
PH is 5.8.
（3）Culture of rootage：By step（1）Or（2）Process obtain height be about 3cm, well-grown, without browning, glass
Change and the cutting of the adventitious bud of albinism is inoculated on root media and carries out culture of rootage, it is first complete under the conditions of 26 DEG C after inoculation
Light culture 2 days, it is subsequently placed in daily illumination 12 hours, intensity of illumination 2000lx, cultivation temperature is cultivated under conditions of being 26 DEG C
Situation of taking root, rooting rate 93% are counted after 28 days.Described root media is：1/4MS+0.5mg/L NAA+0.10mg/L
IBA+11g/L sucrose+3.0g/L agar, pH 5.6.
（4）Acclimatization and transplantses：By step（3）Obtain high about 11cm well-grown and stalwartness rooting tube plantlet remove bottle cap
After being placed in natural lighting lower refining seedling 6 days, test tube seedling is taken out from blake bottle, washes root culture medium off, plants into by Nutrition Soil：It is precious
Zhu Yan=1:3 matrix being mixed into, it is placed in illumination box and cultivates, poured daily with 1/2MS macro-element nutrients liquid to seedling
Water, keep humidity.Transplant crop field, transplanting survival rate 90% again after seedling survives.
Embodiment 2
（1）Bud inducement cultivation：Using Yellowmouth Dutchmanspipe Root stem with bud as explant, blade is cut off, be cut into 3 length carries 6 axillary buds
Section first soak 16min with washing powder solution, then with banister brush gently scrub explant surface by the dust on its surface and portion
Point thalline removes, and is placed in after then rinsing 3h with running water in superclean bench, first with using sterilized water after 75% ethanol disinfection 15s
Wash 6 times, then 15min is sterilized with 0.1% mercuric chloride solution, be followed by with 5 globules for drying surface with aseptic filter paper again of aseptic water washing
Kind bud induction is carried out to bud inducement cultivation base, first full light culture 3 days under the conditions of 28 DEG C after inoculation, be subsequently placed in daily illumination 10
Hour, intensity of illumination is cultivated 30 days under the conditions of being 1500lx, counts its bud induction rate and growing state, bud induction rate 85%.Institute
The bud inducement cultivation base stated is：MS+5mg/L6-BA+0.1mg/L NAA+28g/L sucrose+3.2g/L agar, pH 5.6.
（2）Multiplying culture：By step（1）Cultivate that obtained growth is normal, have obvious stem, the sprout of impeller structure is cut and turned
Enter and squamous subculture is carried out on proliferated culture medium, first full light culture 4 days under the conditions of 28 DEG C after inoculation, be subsequently placed in daily illumination 15
Hour, intensity of illumination 1800lx, it is placed under conditions of cultivation temperature is 28 DEG C after cultivating 31 days and observes growing state and bud increasing
Grow situation.Sprout is repeatedly cut repeatedly and carries out squamous subculture, to obtain more Multiple Buds, newborn sprout robust growth, propagation
Coefficient is 7.0.Described proliferated culture medium is MS+0.3mg/LNAA+8mg/L 6-BA+28g/L sucrose+5.7g/L agar, pH
For 5.6.
（3）Culture of rootage：By step（1）Or（2）Process obtain height be about 3cm, well-grown, without browning, glass
Change and the cutting of the adventitious bud of albinism is inoculated on root media and carries out culture of rootage, it is first complete under the conditions of 29 DEG C after inoculation
Light culture 2 days, it is subsequently placed in daily illumination 10 hours, intensity of illumination 2500lx, cultivation temperature is cultivated under conditions of being 28 DEG C
Situation of taking root, rooting rate 96% are counted after 30 days.Described root media is：1/4MS+1.0mg/L NAA+0.9mg/L
IBA+19g/L sucrose+2.9g/L agar, pH 5.6.
（4）Acclimatization and transplantses：By step（3）Obtain high about 12cm well-grown and stalwartness rooting tube plantlet remove bottle cap
After being placed in natural lighting lower refining seedling 8 days, test tube seedling is taken out from blake bottle, washes root culture medium off, plants into by Nutrition Soil：It is precious
Zhu Yan=1:3 matrix being mixed into, it is placed in illumination box and cultivates, poured daily with 1/2MS macro-element nutrients liquid to seedling
Water, keep humidity.Transplanted again after seedling survives, transplanting survival rate 95%.
Claims (4)
1. a kind of Yellowmouth Dutchmanspipe Root method for tissue culture, it is characterised in that comprise the following steps：
（1）Bud inducement cultivation：Using Yellowmouth Dutchmanspipe Root stem with bud as explant, blade is cut off, be cut into 4 centimeter lengths carries 3
10min is first soaked in the section of axillary bud with washing powder solution, then scrubs explant surface by the dust on its surface and part with hairbrush
Thalline removes, and is placed in after then rinsing 4h with running water in superclean bench, first washes 4 with sterile with after 76% ethanol disinfection 32s
It is secondary, then 28min is sterilized with 0.15% mercuric chloride solution, with being inoculated with after 6 globules for drying surface with aseptic filter paper again of aseptic water washing
Bud induction is carried out to bud inducement cultivation base, first full light culture 6 days under the conditions of 25 DEG C after inoculation, it is small to be subsequently placed in daily illumination 16
When, intensity of illumination is cultivated 35 days under the conditions of being 3100lx, counts its bud induction rate and growing state；
（2）Multiplying culture：By step（1）Cultivate that obtained growth is normal, has obvious stem, the sprout of impeller structure is cut and is transferred to increasing
Grow and squamous subculture is carried out on culture medium, first full light culture 6 days under the conditions of 25 DEG C after inoculation, it is small to be subsequently placed in daily illumination 16
When, intensity of illumination 2600lx, it is placed under conditions of cultivation temperature is 25 DEG C after cultivating 27 days and observes growing state and bud propagation
Situation, sprout is repeatedly cut repeatedly and carries out squamous subculture, obtains Multiple Buds；
（3）Culture of rootage：By step（1）Or（2）Process obtain height be about 7cm, well-grown, without browning, vitrifying and
The adventitious bud cutting of albinism, which is inoculated on root media, carries out culture of rootage, the first complete dark training under the conditions of 25 DEG C after inoculation
Support 6 days, be subsequently placed in daily illumination 16 hours, intensity of illumination 3600lx, cultivation temperature is cultivated 38 days under conditions of being 25 DEG C
Situation of taking root is counted afterwards；
（4）Acclimatization and transplantses：By step（3）Obtain high about 8cm well-grown and stalwartness rooting tube plantlet go bottle cap to be placed in
After natural lighting lower refining seedling 8 days, test tube seedling is taken out from blake bottle, washes root culture medium off, plants into by Nutrition Soil：Perlite
=1:3 matrix being mixed into, it is placed in illumination box and cultivates, watered daily with 1/2MS macro-element nutrients liquid to seedling, is protected
Humidity is held, is transplanted again after seedling survives.
A kind of 2. Yellowmouth Dutchmanspipe Root method for tissue culture according to claim 1, it is characterised in that step（1）Described bud
Inducing culture is：MS+6mg/L6-BA+3mg/L NAA+31g/L sucrose+6.0g/L agar, pH 5.8.
A kind of 3. Yellowmouth Dutchmanspipe Root method for tissue culture according to claim 1, it is characterised in that step（2）Described increasing
Growing culture medium is：MS+1.2mg/LNAA+8mg/L 6-BA+32g/L sucrose+6.0g/L agar, pH 5.8.
A kind of 4. Yellowmouth Dutchmanspipe Root method for tissue culture according to claim 1, it is characterised in that step（3）Described life
Root culture medium is：1/4MS+1.0mg/L NAA+1.0mg/L IBA+31g/L sucrose+6.0g/L agar, pH 5.8.
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| CN201710967526.0A CN107494282A (en) | 2017-10-17 | 2017-10-17 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
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| CN201710967526.0A CN107494282A (en) | 2017-10-17 | 2017-10-17 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109247237A (en) * | 2018-11-22 | 2019-01-22 | 林登淞 | A kind of construction method of river Ciliatenerve Knotweed Root regenerating system |
| CN109258472A (en) * | 2018-11-22 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of powder tetrandra root |
| CN109496852A (en) * | 2018-11-27 | 2019-03-22 | 钟天路 | A kind of tissue culture technique of mountain tortoise |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104082138A (en) * | 2014-06-28 | 2014-10-08 | 玉林师范学院 | Tissue-culture rapid propagation method of Aristolochia fordiana Hemsl |
| CN104686329A (en) * | 2015-02-21 | 2015-06-10 | 杨业云 | Tissue culture method for Eucommia ulmoides Oliv. |
| CN105265320A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Tissue culture propagation method for herba aristolochia mollissima |
| CN106172016A (en) * | 2016-08-31 | 2016-12-07 | 李军 | A kind of Cortex Acanthopancis tissue culture and rapid propagation method |
-
2017
- 2017-10-17 CN CN201710967526.0A patent/CN107494282A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104082138A (en) * | 2014-06-28 | 2014-10-08 | 玉林师范学院 | Tissue-culture rapid propagation method of Aristolochia fordiana Hemsl |
| CN104686329A (en) * | 2015-02-21 | 2015-06-10 | 杨业云 | Tissue culture method for Eucommia ulmoides Oliv. |
| CN105265320A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Tissue culture propagation method for herba aristolochia mollissima |
| CN106172016A (en) * | 2016-08-31 | 2016-12-07 | 李军 | A kind of Cortex Acanthopancis tissue culture and rapid propagation method |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109247237A (en) * | 2018-11-22 | 2019-01-22 | 林登淞 | A kind of construction method of river Ciliatenerve Knotweed Root regenerating system |
| CN109258472A (en) * | 2018-11-22 | 2019-01-25 | 林登淞 | A kind of tissue culture technique of powder tetrandra root |
| CN109496852A (en) * | 2018-11-27 | 2019-03-22 | 钟天路 | A kind of tissue culture technique of mountain tortoise |
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Application publication date: 20171222 |