CN106106190A - A kind of tissue cultivation rapid breeding method of Radix Zanthoxyli - Google Patents
A kind of tissue cultivation rapid breeding method of Radix Zanthoxyli Download PDFInfo
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- CN106106190A CN106106190A CN201610735090.8A CN201610735090A CN106106190A CN 106106190 A CN106106190 A CN 106106190A CN 201610735090 A CN201610735090 A CN 201610735090A CN 106106190 A CN106106190 A CN 106106190A
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- radix zanthoxyli
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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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- 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 the tissue cultivation rapid breeding method of a kind of Radix Zanthoxyli, feature is to be obtained the method for culturing seedlings of the stable seedling of strain by tissue culture technique.The present invention is outer implant with Radix Zanthoxyli belt segment rattan, is achieved the tissue-culturing quick-propagation of Radix Zanthoxyli seedling by stages such as evoking adventive bud, adventitious buds proliferation cultivation, rooting of vitro seedling and acclimatization and transplantses.Solve the seedling supply of Radix Zanthoxyli implant mass, also provide raw material and ensure for making the pharmaceutical production of raw material with Radix Zanthoxyli medical material.
Description
Technical field
The present invention relates to the fast breeding technique field of a kind of Radix Zanthoxyli, feature is the tissue culture quick breeding side of a kind of Radix Zanthoxyli
Method.
Background technology
Radix Zanthoxyli is rutaceae, the dry root of Radix Zanthoxyli Zanthoxylum nitidm cRoxbjdc.Main product is wide
The ground such as east, Guangxi, Fujian, Yunnan, Guizhou, Zhejiang, Taiwan, adopt the whole year, take root, remove branch and leaf, earth, clean, section,
Drying, its pharmacological property is, root is that class is cylindrical, and long 5 ~ 20cm, diameter 0.5 ~ 7cm, surface is brown color or faint yellow, hole skin
Similar round, yellow.Commodity are cut into irregular piece of sheet or section more.Not of uniform size, thick 1 ~ 4mm, the crust place that comes off is light brown.Cut
Dough cover portion becomes brown, and woody part is light yellow, it is seen that proper alignment ring grain and intensive aperture, matter is hard.Feeble QI is fragrant, bitter in the mouth and pungent, have
Numb feeling in the tongue., abnormal smells from the patient dense person thick with root bark is preferred.Main Ingredients and Appearance: root and root bark are containing nitidine, the root bark also Folium Citri tangerinae glycosides Han cloth.Merit
With curing mainly: promoting the circulation of QI to relieve pain, blood circulation promoting and blood stasis dispelling.For rheumatic osteopathia, sore throat, stomachache, toothache, traumatic injury, scald, venom.
Summary of the invention
The purpose of the present invention is provided with a kind of by induction importing normal bud, adventitious buds proliferation, rooting of vitro seedling, acclimatization and transplants
Deng, make Radix Zanthoxyli obtain Guan Miao by the rapid propagation in vitro mode of Multiple Buds, then through transplanting the group of a kind of Radix Zanthoxyli succeeded
Knit cultivation quick-breeding method.
The technical solution of the present invention is such, the tissue cultivation rapid breeding method of a kind of Radix Zanthoxyli, it is characterised in that
Comprise the following steps that:
(1) evoking adventive bud: choosing Radix Zanthoxyli belt segment rattan is outer implant, after 16 seconds kinds of 75% alcohol disinfecting, rushes with sterilized water
Wash 6 times and be placed in the mercuric chloride solution of 0.4% sterilization 32 minutes, then use aseptic water washing 7 times, through aseptic filter paper suck dry moisture
After be inoculated on inducing culture, full light culture under the conditions of 29 DEG C, until induced synthesis adventitious bud；
(2) adventitious buds proliferation is cultivated: is inoculated on proliferated culture medium by adventitious bud and carries out enrichment culture, and inoculation is placed on every daylight
According to 16 hours, intensity of illumination was 1600lx, and cultivation temperature is cultivated under conditions of being 29 DEG C, and switching in 19 days is once；
(3) rooting of vitro seedling: carry out root induction, after inoculation by growing to the Multiple Buds of 5cm to cut to be inoculated on root media
First full light culture 15 days, then illumination every day 16 hours under the conditions of 29 DEG C, intensity of illumination is 3100lx, and cultivation temperature is 29 DEG C
Under conditions of cultivate to taking root；
(4) acclimatization and transplants: after the culture bottle bottle cap of the long rooting tube plantlet to 5cm is opened natural lighting lower refining seedling 8 days, will examination
Guan Miaocong culture bottle takes out, washes root culture medium off, plant in the substrate being mixed into by peat soil and river sand (1:1) and field planting
In big Tanaka.
The outer implant of Radix Zanthoxyli band rattan described in step (1) needed to carry out pretreatment before carrying out Induce aerosor, processed
Method be: be placed on the potassium permanganate of 0.06% by rinsing silt well from the Radix Zanthoxyli band rattan tap water of field acquisition
Solution soaks 9 hours, then rinses with tap water that to be placed on 6 DEG C of refrigerator overnight with glue plastic bag sealing after 6 times standby.
Inducing culture described in step (1) is: with GS as minimal medium, and additional 3.6% sucrose 0.6% agar 0.2% is lived
Property charcoal 0.6% acidic hydrolysis casein and 2.2mg/L KT2.2mg/L IBA and 1.2mg/L 2,4-D, pH value is 5.3.
Proliferated culture medium described in step (2) is: with MS as minimal medium, additional 3.2mg/L 6-BA1.6mg/L
NAA 7.0mg/L riboflavin 0.6mg/L D-VB5 calcium 7.0mg/L ascorbic acid 6.0mg/L Cys and 3.6% sucrose
0.6% agar 0.2% activated carbon, pH value is 5.3.
Root media described in step (3) is: with 1/2MS as minimal medium, additional 0.9mg/L IBA1.2mg/L
GGR2.6mg/L Cys and 2.6% sucrose 0.8% agar 0.2% activated carbon, pH value is 5.3.
Compared with prior art the prominent effect of the present invention is: technical matters route is simple, feasible, processing ease, is bred as
Test tube shoot survival percent reach more than 95%, provide for market the Radix Zanthoxyli seedling of high-quality to beat in a large number and determine basis.
Detailed description of the invention
Following example are to further illustrate the present invention, are not limitations of the present invention.
Embodiment 1
1, outer implant pretreatment: be placed on the height of 0.02% by rinsing silt well from the Radix Zanthoxyli band rattan tap water gathered
Potassium manganate solution soaks 6 hours, then rinses with tap water that to be placed on 2 DEG C of refrigerator overnight with glue plastic bag sealing after 6 times standby
With.
2, incubation:
(1) evoking adventive bud: Radix Zanthoxyli belt segment rattan after 10 seconds kinds of 75% alcohol disinfecting, with after aseptic water washing 4 times through 0.1%
Mercuric chloride solution in sterilize 20 minutes, then with aseptic water washing 5 times, after aseptic filter paper suck dry moisture, be inoculated into inducing culture
On base, full light culture under the conditions of 26 DEG C, form adventitious bud after cultivating 46 days.The inducing culture used is: GS+1.6mg/
LKT+0.2mg/LIBA and 0.5mg/L 2,4-D+3.3% sucrose+0.30% agar+0.06% activated carbon+0.2% acidic hydrolysis cheese
Albumen, pH value is 5.6;
(2) adventitious buds proliferation is cultivated: is inoculated on proliferated culture medium by adventitious bud and carries out enrichment culture, and inoculation is placed on every daylight
According to 16 hours, intensity of illumination was 1100lx, and cultivation temperature is cultivated under conditions of being 26 DEG C, and once, growth coefficient is in switching in 26 days
4.The proliferated culture medium used is: MS+1.6mg/L 6-BA+0.6mg/L NAA+1.2mg/L riboflavin+0.6mg/L D-
Calcium pantothenate+1.2mg/L ascorbic acid+1.2mg/L Cys+3.5% sucrose+0.30% agar+0.2% activated carbon, pH value
It is 5.6；
(3) rooting of vitro seedling: carry out root induction, after inoculation by growing to the Multiple Buds of 5cm to cut to be inoculated on root media
First full light culture 8 days, then illumination every day 16 hours under the conditions of 26 DEG C, intensity of illumination is 3100lx, and cultivation temperature is 26 DEG C
Under conditions of cultivate and take root for 21 days, rooting rate reaches 96%.The root media used is: 1/2MS+0.2mg/L IBA+
0.5mg/L GGR+1.2mg/L Cys+2.2%% sucrose+0.5% agar+0.2% activated carbon, pH value is 5.6；
(4) acclimatization and transplants: after the culture bottle bottle cap of the long rooting tube plantlet to 5cm is opened natural lighting lower refining seedling 6 days, will examination
Guan Miaocong culture bottle takes out, washes root culture medium off, plant into the substrate being mixed into by peat soil and river sand (1:1) and be colonizated in
Big Tanaka, survival rate 95%.
Embodiment 2
1, outer implant pretreatment: be placed on 0.02% by rinsing silt well from the Radix Zanthoxyli band rattan tap water of field acquisition
Potassium permanganate solution in soak 9 hours, then rinse after 4 times with tap water and be placed on mistake in 6 DEG C of refrigerators with glue plastic bag sealing
Night is standby.
2, incubation:
(1) evoking adventive bud: Radix Zanthoxyli belt segment rattan after 6 seconds kinds of 75% alcohol disinfecting, with after aseptic water washing 6 times through 0.3%
Mercuric chloride solution in sterilize 12 minutes, then with aseptic water washing 6 times, after aseptic filter paper suck dry moisture, be inoculated into inducing culture
On base, full light culture under the conditions of 26 DEG C, form adventitious bud after cultivating 41 days.The inducing culture used is: GS+0.9mg/
L KT+0.3mg/L IBA and 0.3mg/L 2,4-D+3.2% sucrose+0.5% agar+0.2% activated carbon+0.6% acidic hydrolysis cheese
Albumen, pH value is 5.5;
(2) adventitious buds proliferation is cultivated: is inoculated on proliferated culture medium by adventitious bud and carries out enrichment culture, and inoculation is placed on every daylight
According to 13 hours, intensity of illumination was 1250lx, and cultivation temperature is cultivated under conditions of being 26 DEG C, and once, growth coefficient is in switching in 29 days
7.The proliferated culture medium used is: MS+0.9mg/L 6-BA+0.4mg/L NAA+4.0mg/L riboflavin+0.4mg/L D-
Calcium pantothenate+3.6mg/L ascorbic acid+4.6mg/L Cys+3.6% sucrose+0.6% agar+0.06% activated carbon, pH value
It is 5.5；
(3) rooting of vitro seedling: carry out root induction, after inoculation by growing to the Multiple Buds of 5cm to cut to be inoculated on root media
First full light culture 11 days under the conditions of 26 DEG C, are subsequently placed in illumination every day 13 hours, and intensity of illumination is 3000lx, and cultivation temperature is
Cultivating under conditions of 26 DEG C and take root for 29 days, rooting rate reaches 95%.The root media used is: 1/2MS+0.6mg/L IBA+
1.2mg/L GGR+2.6mg/L Cys+2.6%% sucrose+0.5% agar+0.06% activated carbon, pH value is 5.5；
(4) acclimatization and transplants: after the culture bottle bottle cap of the long rooting tube plantlet to 5cm is opened natural lighting lower refining seedling 8 days, will examination
Guan Miaocong culture bottle takes out, washes root culture medium off, plant into the substrate being mixed into by peat soil and river sand (1:1) and be colonizated in
Big Tanaka, survival rate 96%.
Embodiment 3
1, outer implant pretreatment: be placed on 0.05% by rinsing silt well from the Radix Zanthoxyli band rattan tap water of field acquisition
Potassium permanganate solution in soak 4 hours, then rinse after 5 times with tap water and be placed on mistake in 5 DEG C of refrigerators with glue plastic bag sealing
Night is standby.
2, incubation:
(1) evoking adventive bud: Radix Zanthoxyli belt segment rattan, after 5 seconds kinds of 75% alcohol disinfecting, is placed on for 4 times with aseptic water washing
The mercuric chloride solution of 0.1% is sterilized 13 minutes, then uses aseptic water washing 5 times, after aseptic filter paper suck dry moisture, be inoculated into induction
In culture medium, full light culture under the conditions of 25 DEG C, form adventitious bud after cultivating 48 days.The inducing culture used is: GS+
2.0mg/L KT+0.8mg/L IBA and 0.4mg/L 2,4-D+3.5% sucrose+0.4% agar+0.05% activated carbon+0.3% are acid
Caseinhydrolysate, pH value is 5.4;
(2) adventitious buds proliferation is cultivated: is inoculated on proliferated culture medium by adventitious bud and carries out enrichment culture, and inoculation is placed on every daylight
According to 13 hours, intensity of illumination was 1200lx, and cultivation temperature is cultivated under conditions of being 27 DEG C, and once, growth coefficient is 3 in switching in 26 days
～6.The proliferated culture medium used is: MS+1.5mg/L 6-BA+0.6mg/L NAA+5.0mg/L riboflavin+0.5mg/L
D-VB5 calcium+6.0mg/L ascorbic acid+4.0mg/L Cys+3.5% sucrose+0.5% agar+0.1% activated carbon, pH value
It is 5.4；
(3) rooting of vitro seedling: carry out root induction, after inoculation by growing to the Multiple Buds of 6cm to cut to be inoculated on root media
First full light culture 13 days under the conditions of 25 DEG C, are subsequently placed in illumination every day 13 hours, and intensity of illumination is 3000lx, and cultivation temperature is
Cultivating under conditions of 25 DEG C and take root for 20 days, rooting rate reaches 95%.The root media used is: 1/2MS+0.2mg/L IBA+
0.8mg/L GGR+1.5mg/L Cys+2.5%% sucrose+0.5% agar+0.1% activated carbon, pH value is 5.4；
(4) acclimatization and transplants: the culture bottle bottle cap of the long rooting tube plantlet to 5cm is opened and is placed in natural lighting lower refining seedling 6 days
After, test tube Seedling is taken out from culture bottle, washes root culture medium off, plant into the substrate being mixed into by peat soil and river sand (1:1)
In and be colonizated in big Tanaka, survival rate 95%.
Claims (5)
1. the tissue cultivation rapid breeding method of a Radix Zanthoxyli, it is characterised in that comprise the following steps that:
Step (1) evoking adventive bud: choosing Radix Zanthoxyli belt segment rattan is outer implant, after 16 seconds kinds of 75% alcohol disinfecting, with aseptic
Water rinses in the mercuric chloride solution being placed on 0.4% for 6 times and sterilizes 32 minutes, then uses aseptic water washing 7 times, blots through aseptic filter paper
It is inoculated into after moisture on inducing culture, full light culture under the conditions of 29 DEG C, until induced synthesis adventitious bud；
Step (2) adventitious buds proliferation is cultivated: is inoculated on proliferated culture medium by adventitious bud and carries out enrichment culture, and inoculation is placed on every
It illumination 16 hours, intensity of illumination is 1600lx, and cultivation temperature is cultivated under conditions of being 29 DEG C, and switching in 19 days is once；
Step (3) rooting of vitro seedling: carry out root induction by growing to the Multiple Buds of 5cm to cut to be inoculated on root media, connect
First full light culture 15 days, then illumination every day 16 hours under the conditions of 29 DEG C after Zhong, intensity of illumination is 3100lx, and cultivation temperature is
Cultivate to taking root under conditions of 29 DEG C；
Step (4) acclimatization and transplants: after the culture bottle bottle cap of the long rooting tube plantlet to 5cm is opened natural lighting lower refining seedling 8 days,
Test tube Seedling is taken out from culture bottle, washes root culture medium off, plant into the substrate being mixed into by peat soil and river sand (1:1) and determine
Plant in big Tanaka.
The tissue cultivation rapid breeding method of a kind of Radix Zanthoxyli the most according to claim 1, it is characterised in that described in step (1)
The outer implant of Radix Zanthoxyli band rattan needed to carry out pretreatment before carrying out Induce aerosor, and the method for process is: will be from field acquisition
Radix Zanthoxyli band rattan tap water rinse well silt be placed in the potassium permanganate solution of 0.06% immersion 9 hours, then with oneself
It is standby that water is placed on 6 DEG C of refrigerator overnight with glue plastic bag sealing after rinsing 6 times.
The tissue cultivation rapid breeding method of a kind of Radix Zanthoxyli the most according to claim 1, it is characterised in that described in step (1)
Inducing culture is: with GS as minimal medium, additional 3.6% sucrose 0.6% agar 0.2% activated carbon 0.6% acidic hydrolysis cheese egg
White and 2.2mg/L KT2.2mg/L IBA and 1.2mg/L 2,4-D, pH value is 5.3.
The tissue cultivation rapid breeding method of a kind of Radix Zanthoxyli the most according to claim 1, it is characterised in that described in step (2)
Proliferated culture medium is: with MS as minimal medium, additional 3.2mg/L 6-BA1.6mg/L NAA 7.0mg/L riboflavin 0.6mg/
L D-VB5 calcium 7.0mg/L ascorbic acid 6.0mg/L Cys and 3.6% sucrose 0.6% agar 0.2% activated carbon, pH value
It is 5.3.
The tissue cultivation rapid breeding method of a kind of Radix Zanthoxyli the most according to claim 1, it is characterised in that described in step (3)
Root media is: with 1/2MS as minimal medium, additional 0.9mg/L IBA1.2mg/L GGR2.6mg/L Cys
With 2.6% sucrose 0.8% agar 0.2% activated carbon, pH value is 5.3.
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| CN201610735090.8A CN106106190A (en) | 2016-08-28 | 2016-08-28 | A kind of tissue cultivation rapid breeding method of Radix Zanthoxyli |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106508683A (en) * | 2016-11-24 | 2017-03-22 | 广州中医药大学 | A kind of tissue culture culture medium of Radix Zanthoxyli regrowth and cultural method |
| CN106718879A (en) * | 2016-11-24 | 2017-05-31 | 华南农业大学 | A kind of inducing culture of Radix zanthoxyli callus and abductive approach and application |
| CN107027634A (en) * | 2017-06-22 | 2017-08-11 | 玉林市林业科学研究所 | A kind of tissue cultivation rapid breeding method of high-quality shatian pomelo |
| CN109380119A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of green pepper purpose tissue cultivation rapid breeding method |
-
2016
- 2016-08-28 CN CN201610735090.8A patent/CN106106190A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 时群: ""两面针组培技术试验"", 《林业科技开发》 * |
| 韦大器等: ""两面针的组织培养和快速繁殖"", 《植物生理学通讯》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106508683A (en) * | 2016-11-24 | 2017-03-22 | 广州中医药大学 | A kind of tissue culture culture medium of Radix Zanthoxyli regrowth and cultural method |
| CN106718879A (en) * | 2016-11-24 | 2017-05-31 | 华南农业大学 | A kind of inducing culture of Radix zanthoxyli callus and abductive approach and application |
| CN106508683B (en) * | 2016-11-24 | 2018-03-20 | 广州中医药大学 | The tissue culture culture medium and cultural method of a kind of Radix zanthoxyli regrowth |
| CN107027634A (en) * | 2017-06-22 | 2017-08-11 | 玉林市林业科学研究所 | A kind of tissue cultivation rapid breeding method of high-quality shatian pomelo |
| CN109380119A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of green pepper purpose tissue cultivation rapid breeding method |
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