CN107593444A - A kind of tissue cultivation rapid breeding method of dutchmanspipe root - Google Patents
A kind of tissue cultivation rapid breeding method of dutchmanspipe root Download PDFInfo
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- CN107593444A CN107593444A CN201710971638.3A CN201710971638A CN107593444A CN 107593444 A CN107593444 A CN 107593444A CN 201710971638 A CN201710971638 A CN 201710971638A CN 107593444 A CN107593444 A CN 107593444A
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Abstract
The invention discloses a kind of tissue cultivation rapid breeding method of dutchmanspipe root, and dutchmanspipe root rapid propagation in vitro system is established by explant of dutchmanspipe root stem segment with axillary bud, is laid the foundation to solve dutchmanspipe root large-scale production.The present invention is realized the tissue-culturing quick-propagation of dutchmanspipe root by processes such as inducing clumping bud, Multiplying culture, offspring induction and test tube seedling rooting cultures, technical support is provided for the industrialized development of dutchmanspipe root using dutchmanspipe root stem segment with axillary bud as explant.With simple technological means, obtain the rapid propagation system of dutchmanspipe root, seedling planting percent is high, fast-growth, when cyclopentadienyl root strengthen, high yield of having a good harvest.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, is related to a kind of dutchmanspipe root
Tissue cultivation rapid breeding method.
Background technology
Dutchmanspipe root also known as：Herba aristolochiae, the water banksia rose, acute diseases such as cholera and sunstroke medicine.Belong to herbaceous perennial vine.Root cylinder, 0.5 ~ 1.5 li of diameter
Rice, surface taupe or taupe brown.Stem nascent is upright, after climb up by holding on to.Leaf alternate, blade triangular shape Long Circle, the pleurapophysis of base portion two
Go out such as ear.Spend single leave armpit, oblique infundibulate, purple green, falciform bending.9 ~ October result, fruit is subsphaeroidal, is about 6 centimetres, directly
, there are 6 ribs in about 4 centimetres of footpath, are ftractureed when ripe.Seed is flat, obtuse triangle, and long and wide about 4 centimetres, edge has white film quality wide
Wing.Root is excavated in season in spring and autumn two, is dried standby.Summer adopts cane leaves, dries.It is grown on each provinces and regions and Henan, mountain on the south the Yangtze river basin
East is distributed.It is born in field, hillside, roadside, mountain valley, limes marginis more.Dutchmanspipe root acrid flavour, hardship, it is cold in nature.With promoting qi circulation and relieving pain, removing toxic substances
Detumescence, hypotensive and other effects.Traditional dutchmanspipe root acquisition methods are excavation wild plant resources, cause soil erosion and serious soil
Earth desertification.In addition, dutchmanspipe root seed, essentially from wild, its variet complexity, seed is rare, extremely low, wild dutchmanspipe root of germinateing
Resource has not adapted to the needs of scale and standardized production, turns into the maximum bottleneck for hindering dutchmanspipe root industry development.It is domestic
External plant resources, chemical composition analysis, Pharmacognosy Studies, pharmacological action and clinical practice, callus tissue culture and break up again
Aspect has done substantial amounts of work, and these researchs have established certain basis for the clinical practice using dutchmanspipe root as base source preparation.But
It is to still have many theory and practice urgent problems in dutchmanspipe root production.In order to seek the development of dutchmanspipe root industry,
Dutchmanspipe root industry is led to move towards high-tech, high standard, the road of the science and technology of high level, dutchmanspipe root tissue cultures have become to be ground at present
The focus studied carefully and developed.Therefore, it is highly desirable to establish dutchmanspipe root vitro Regeneration System, is provided for the industrialized development of dutchmanspipe root
Technical support.
The content of the invention
It is explant it is an object of the invention to provide a kind of stem segment with axillary bud, passes through inducing clumping bud, Multiplying culture, root
The process such as seedling induction and test tube seedling rooting culture realizes the tissue-culturing quick-propagation of dutchmanspipe root, so as to realize the present invention's
Purpose.Technical method is simple, and operation is easy, and seedling is sturdy, fast growing, a kind of tissue culture rapid for the dutchmanspipe root increased income of getting bumper crops
Breeding method.
A kind of tissue cultivation rapid breeding method of dutchmanspipe root of the present invention, including following processing step：
（1）The processing of explant：Choose and the stem section with axillary bud cut on healthy and strong dutchmanspipe root plant as explant, cut off blade,
4h is first rinsed in the section with 3 axillary buds for being cut into 2 length with washing powder water, then gently scrubs explant surface by its surface with hair
Dust and part thalline remove, and are placed in after then rinsing 4h with running water in superclean bench, first with after 75% ethanol disinfection 32s
12min is sterilized with sterile washing 6 times, then with 0.1% mercuric chloride solution, surface is dried with aseptic filter paper again 8 times with aseptic water washing
It is standby after the globule;
（2）Multiple Buds are led：By step（1）Stem segment with axillary bud after processing is cut into 3.5cm segments and is inoculated on inducing culture,
First full light culture 6 days under the conditions of 24 DEG C, are subsequently placed in daily illumination 15 hours, intensity of illumination 3500lx, until induction shape
Into Multiple Buds;
（3）Multiplying culture：By step（2）The tender shoots that culture obtains cuts off and is transferred on proliferated culture medium when growing to about 3cm and carries out
Squamous subculture, first full light culture 5 days under the conditions of 24 DEG C after inoculation, is subsequently placed in daily illumination 18 hours, intensity of illumination is
2900lx, it is placed under conditions of cultivation temperature is 29 DEG C and cultivates, repeatedly cutting carries out squamous subculture repeatedly, to obtain more clumps
Sprout;
（4）Culture of rootage：Work as step（2）Or（3）Multiple Buds length to 2.0cm it is high when, Multiple Buds are cut into single seedling and are seeded to
Culture of rootage is carried out in root media, first full light culture 5 days under the conditions of 24 DEG C after inoculation, it is small to be subsequently placed in daily illumination 18
When, intensity of illumination 1900lx, culture is until send out roots under conditions of cultivation temperature is 24 DEG C;
（5）Test tube transplantation of seedlings：When the new root system that seedling is grown is up to 4cm or so, and lateral root is grown, during more than height of seedling 6cm,
Culture bottle cap is opened after being placed in natural lighting lower refining seedling 8 days, test tube seedling is taken out from blake bottle, wash root culture off
Base, root is not injured as far as possible, in the vermiculite for planting sterilized mistake.
Step（2）Described inducing culture is：MS+ 2mg/L NAA+ 0.4mg/L 6-BA+ 1.2mg/L KT+
The activated carbon of+0.25% agar of 2.5% sucrose+0.12%, pH value 5.3.
Step（3）Described proliferated culture medium is：The fine jade of MS+3.5mg/L 6-BA+1.5mg/L NAA+1.5% sucrose+0.3%
The activated carbon of fat+0.12%, pH value 5.3.
Step（5）Described root media is：MS+12mg/L KH2PO4The activity of+1.0%+0.3% agar of sucrose+0.12%
Charcoal, pH value 5.3.
It is an advantage of the invention that:Dutchmanspipe root rapid propagation in vitro system is established by explant of dutchmanspipe root stem segment with axillary bud, for solution
Certainly dutchmanspipe root large-scale production lays the foundation.The present invention is trained using stem segment with axillary bud as explant by inducing clumping bud, propagation
The process such as foster, offspring induction and test tube seedling rooting culture realizes the tissue-culturing quick-propagation of dutchmanspipe root, is dutchmanspipe root
Industrialized development provides technical support.Technology easily support is held, easy to operate, and obtained seedling is healthy and strong, and growth germination is fast, variety pure
Just, germination percentage is high, can meet market needs.
Embodiment
Following examples are the further explanations to the present invention, are not limitations of the present invention.
Embodiment 1：
（1）The processing of explant：Choose and the stem section with axillary bud cut on healthy and strong dutchmanspipe root plant as explant, cut off blade,
4h is first rinsed in the section with 3 axillary buds for being cut into 2.0 length with washing powder water, then gently scrubs explant surface with banister brush and incite somebody to action
The dust and part thalline on its surface remove, and are placed in superclean bench after then rinsing 2h with running water, are first disappeared with 70% ethanol
With sterile washing 4 times after malicious 6s, then with 0.1% mercuric chloride solution 6min is sterilized, dried again with aseptic filter paper for 5 times with aseptic water washing
It is standby after the globule on surface.
（2）Multiple Buds are led：By step（1）Dutchmanspipe root stem segment with axillary bud after processing is cut into 1.25cm segments and is inoculated into and lures
Lead on culture medium, first full light culture 5 days under the conditions of 26 DEG C, are subsequently placed in daily illumination 9 hours, intensity of illumination is 2600lx bars
21 days each axillary bud stem sections are cultivated under part 2 Multiple Buds.Inducing culture is：MS+1mg/L NAA+ 2mg/L 6-BA+
The activated carbon of+0.55% agar of 1.0mg/L KT+1.25% sucrose+0.2%, pH value 5.6.
（3）Multiplying culture：By step（2）The tender shoots that culture obtains cuts off and is transferred on proliferated culture medium when growing to about 3cm
Squamous subculture is carried out, first full light culture 4 days under the conditions of 26 DEG C after inoculation, is subsequently placed in daily illumination 12 hours, intensity of illumination
For 2500lx, it is placed in cultivation temperature and reaches 8 times to cultivate 18 days proliferation times under conditions of 26 DEG C.Repeatedly repeatedly cutting carry out after
It is commissioned to train foster, to obtain more Multiple Buds.Described proliferated culture medium is：MS+1.2mg/L 6-BA+0.6mg/L NAA+3.0%
The activated carbon of the agar of sucrose+0.6%+0.3%, pH value 5.6.
（4）Culture of rootage：Work as step（2）Or（3）Multiple Buds length to 2.0cm it is high when, Multiple Buds are cut into single seedling and connect
Kind culture of rootage is carried out into root media, first full light culture 2 days under the conditions of 26 DEG C after inoculation, be subsequently placed in daily illumination
12 hours, intensity of illumination 1900lx, cultivation temperature was cultivated 4 days i.e. visible formation, inductivity and reached under conditions of being 26 DEG C
98.5%, mean elements is up to 6, and root system is light brown, elongated, branch amount is few, and root long averagely can reach a 6cm left sides after cultivating 21 days
It is right.Described root media is：MS+13mg/L KH2PO4The activated carbon of+0.6% agar of+2.0% sucrose+0.12%, pH value are
5.6。
（5）Test tube transplantation of seedlings：When the new root system that seedling is grown is up to 4cm or so, and grow lateral root, height of seedling 4cm with
When upper, culture bottle cap opened after being placed in natural lighting lower refining seedling 4 days, test tube seedling is taken out from blake bottle, washes root off
Portion's culture medium, does not injure root as far as possible, in the vermiculite for planting sterilized mistake, survival rate more than 98%.
Embodiment 2：
（1）The processing of explant：Choose and the stem section with axillary bud cut on healthy and strong dutchmanspipe root plant as explant, cut off blade,
5h is first rinsed in the section with 4 axillary buds for being cut into 6.0 length with washing powder water, then gently scrubs explant surface with banister brush and incite somebody to action
The dust and part thalline on its surface remove, and are placed in superclean bench after then rinsing 4h with running water, are first disappeared with 75% ethanol
With sterile washing 6 times after malicious 12s, then with 0.1% mercuric chloride solution 8min is sterilized, dried again with aseptic filter paper for 6 times with aseptic water washing
It is standby after the globule on surface.
（2）Multiple Buds are led：By step（1）Stem segment with axillary bud after processing is cut into 2.8cm segments and is inoculated into Fiber differentiation
On base, first full light culture 5 days under the conditions of 26 DEG C, are subsequently placed in daily illumination 11 hours, under the conditions of intensity of illumination is 1900lx
22 days each axillary bud stem sections of culture have 3 Multiple Buds.Inducing culture is：MS+ 2.8mg/L NAA+ 1.5mg/L 6-BA+
The activated carbon of+0.6% agar of 0.9mg/L KT+3.6% sucrose+0.06%, pH value 5.6.
（3）Multiplying culture：By step（2）The tender shoots that culture obtains cuts off and is transferred on proliferated culture medium when growing to about 3cm
Squamous subculture is carried out, first full light culture 4 days under the conditions of 26 DEG C after inoculation, is subsequently placed in daily illumination 12 hours, intensity of illumination
For 2900lx, it is placed in cultivation temperature and reaches 8.2 times to cultivate 22 days proliferation times under conditions of 26 DEG C.Repeatedly cutting is carried out repeatedly
Squamous subculture, to obtain more Multiple Buds.Described proliferated culture medium is：MS+1.6mg/L 6-BA+0.8mg/L NAA+
The activated carbon of+0.6% agar of 3.6% sucrose+0.2%, pH value 5.6.
（4）Culture of rootage：Work as step（2）Or（3）Multiple Buds length to 4cm it is high when, Multiple Buds are cut into single seedling and are inoculated with
Culture of rootage is carried out into root media, first full light culture 3 days under the conditions of 26 DEG C after inoculation, is subsequently placed in daily illumination 11
Hour, intensity of illumination 1900lx, cultivation temperature cultivate 6 days i.e. visible and formed under conditions of being 26 DEG C, inductivity up to 100%,
Mean elements is up to 6, and root system is light brown, elongated, branch amount is few, and root long averagely can reach 6cm or so after cultivating 22 days.It is described
Root media be：MS+13mg/L KH2PO4The activated carbon of+0.6% agar of+2.6% sucrose+0.2%, pH value 5.6.
（5）Test tube transplantation of seedlings：When the new root system that seedling is grown is up to 5cm or so, and grow lateral root, height of seedling 6cm with
When upper, culture bottle cap opened after being placed in natural lighting lower refining seedling 8 days, test tube seedling is taken out from blake bottle, washes root off
Portion's culture medium, does not injure root as far as possible, in the vermiculite for planting sterilized mistake, survival rate more than 94%.
Claims (4)
1. a kind of tissue cultivation rapid breeding method of dutchmanspipe root, it is characterised in that comprise the following steps that：
（1）The processing of explant：Choose and the stem section with axillary bud cut on healthy and strong dutchmanspipe root plant as explant, cut off blade,
4h is first rinsed in the section with 3 axillary buds for being cut into 2 length with washing powder water, then gently scrubs explant surface by its surface with hair
Dust and part thalline remove, and are placed in after then rinsing 4h with running water in superclean bench, first with after 75% ethanol disinfection 32s
12min is sterilized with sterile washing 6 times, then with 0.1% mercuric chloride solution, surface is dried with aseptic filter paper again 8 times with aseptic water washing
It is standby after the globule;
（2）Multiple Buds are led：By step（1）Stem segment with axillary bud after processing is cut into 3.5cm segments and is inoculated on inducing culture,
First full light culture 6 days under the conditions of 24 DEG C, are subsequently placed in daily illumination 15 hours, intensity of illumination 3500lx, until induction shape
Into Multiple Buds;
（3）Multiplying culture：By step（2）The tender shoots that culture obtains cuts off and is transferred on proliferated culture medium when growing to about 3cm and carries out
Squamous subculture, first full light culture 5 days under the conditions of 24 DEG C after inoculation, is subsequently placed in daily illumination 18 hours, intensity of illumination is
2900lx, it is placed under conditions of cultivation temperature is 29 DEG C and cultivates, repeatedly cutting carries out squamous subculture repeatedly, to obtain more clumps
Sprout;
（4）Culture of rootage：Work as step（2）Or（3）Multiple Buds length to 2.0cm it is high when, Multiple Buds are cut into single seedling and are seeded to
Culture of rootage is carried out in root media, first full light culture 5 days under the conditions of 24 DEG C after inoculation, it is small to be subsequently placed in daily illumination 18
When, intensity of illumination 1900lx, culture is until send out roots under conditions of cultivation temperature is 24 DEG C;
（5）Test tube transplantation of seedlings：When the new root system that seedling is grown is up to 4cm or so, and lateral root is grown, during more than height of seedling 6cm,
Culture bottle cap is opened after being placed in natural lighting lower refining seedling 8 days, test tube seedling is taken out from blake bottle, wash root culture off
Base, root is not injured as far as possible, in the vermiculite for planting sterilized mistake.
A kind of 2. tissue cultivation rapid breeding method of dutchmanspipe root according to claim 1, it is characterised in that step（2）It is described
Inducing culture be：The agar of MS+ 2mg/L NAA+ 0.4mg/L 6-BA+ 1.2mg/L KT+2.5% sucrose+0.25%+
0.12% activated carbon, pH value 5.3.
A kind of 3. tissue cultivation rapid breeding method of dutchmanspipe root according to claim 1, it is characterised in that step（3）It is described
Proliferated culture medium be：The activated carbon of+0.3% agar of MS+3.5mg/L 6-BA+1.5mg/L NAA+1.5% sucrose+0.12%, pH value
For 5.3.
A kind of 4. tissue cultivation rapid breeding method of dutchmanspipe root according to claim 1, it is characterised in that step（5）It is described
Root media be：MS+12mg/L KH2PO4The activated carbon of+0.3% agar of+1.0% sucrose+0.12%, pH value 5.3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109496842A (en) * | 2018-11-20 | 2019-03-22 | 广州中医药大学(广州中医药研究院) | A kind of abductive approach of murraya paniculataJack callus |
| CN111149705A (en) * | 2020-03-13 | 2020-05-15 | 云南珍逸德农业科技有限公司 | Tissue culture and rapid propagation method for stem segments of rhubarb vines |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104686345A (en) * | 2015-02-24 | 2015-06-10 | 陈桂容 | Tissue culture rapid propagation method of liquorice |
-
2017
- 2017-10-18 CN CN201710971638.3A patent/CN107593444A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104686345A (en) * | 2015-02-24 | 2015-06-10 | 陈桂容 | Tissue culture rapid propagation method of liquorice |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109496842A (en) * | 2018-11-20 | 2019-03-22 | 广州中医药大学(广州中医药研究院) | A kind of abductive approach of murraya paniculataJack callus |
| CN109496842B (en) * | 2018-11-20 | 2021-09-10 | 广州中医药大学(广州中医药研究院) | Induction method of calli of murraya paniculata |
| CN111149705A (en) * | 2020-03-13 | 2020-05-15 | 云南珍逸德农业科技有限公司 | Tissue culture and rapid propagation method for stem segments of rhubarb vines |
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