CN104082138B - A kind of tissue cultivation rapid breeding method of Radix Aristolochiae Tagalae - Google Patents
A kind of tissue cultivation rapid breeding method of Radix Aristolochiae Tagalae Download PDFInfo
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Abstract
A kind of tissue cultivation rapid breeding method of Radix Aristolochiae Tagalae, relate to Radix Aristolochiae Tagalae (Aristolochia fordianaHemsl) method for culturing seedlings of the stable seedling of strain is obtained by tissue culture technique.The present invention is outer implant with Radix Aristolochiae Tagalae belt segment rattan, is achieved the tissue-culturing quick-propagation of Radix Aristolochiae Tagalae 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 Aristolochiae Tagalae implant mass, also provide raw material for the pharmaceutical production with Radix Aristolochiae Tagalae medical material as raw material and ensure.
Description
Technical field
The present invention relates to the method for plant tissue culture in agricultural biotechnologies, specifically, relate to a kind of Radix Aristolochiae Tagalae
Tissue cultivation rapid breeding method.
Background technology
Wither in Radix Aristolochiae Tagalae (Aristolochia fordianaHemsl) five tiger Tongcheng again, centering grass, natural grass, blood, Caulis seu Radix Schisandrae Henryi
Secretly disappear, Rhizoma Begoniae Willsonii, for Aristolochiaceae Aristolochia (Aristolochia) liana medicinal plants, be Guangxi famous medical herbs among the people,
There is good analgesic activity, the treatment for diseases such as wound bitterly, snakebite, high heat among the people.Radix Aristolochiae Tagalae is rich in Aristolochic Acid
(aristolochia acid), its rhizome Aristolochic Acid content is up to 0.6%, and Aristolochic Acid pharmacology at home and abroad and clinic are ground
Study carefully and all prove have the most antibacterial, antiinflammatory, analgesia and the effect of enhancing human body immunity function, and commercially produce, as
" Tardolyt " and domestic power of the biting acid sheet of external clinical practice, is Aristolochic Acid total acid.
Radix Aristolochiae Tagalae is distributed mainly on the ground such as Guangxi, Yunnan, owing to Eucalyptus plantation, refining mountain afforestation are widelyd popularize in various places in recent years
Frequently and the reason such as herbicide use, wild resource drastically reduces, it is impossible to meet the needs of medicine.Development artificial growth is to solve
Certainly one of approach of Radix Aristolochiae Tagalae imbalance between supply and demand, current Radix Aristolochiae Tagalae sapling multiplication rely primarily on seminal propagation and point root, layer of vine and
Staying the root and stem of certain plants to breed, modes of reproduction speed is slow, efficiency is low for these, it is impossible to meet the Radix Aristolochiae Tagalae implantation in large scale demand to high quality seedling,
Therefore, it is highly desirable to set up the Radix Aristolochiae Tagalae tissue culture quick breeding system of complete set.At present, Radix Aristolochiae Tagalae tissue culture technique is still
There are no report.
Summary of the invention
It is an object of the invention to provide the tissue cultivation rapid breeding method of a kind of Radix Aristolochiae Tagalae, by evoking adventive bud, grow thickly
In the stages such as Shoot propagation, rooting of vitro seedling, acclimatization and transplants, Radix Aristolochiae Tagalae is made to obtain a large amount of test tube by the rapid propagation in vitro mode of Multiple Buds
Seedling, it is achieved thereby that the purpose of the present invention.
A kind of tissue cultivation rapid breeding method of the Radix Aristolochiae Tagalae of the present invention, the processing step including following:
(1) evoking adventive bud: choosing Radix Aristolochiae Tagalae belt segment rattan is outer implant, through 75%～80% alcohol disinfecting 5～15 seconds kinds
After, it is placed in the mercuric chloride solution of 0.1%～0.3% sterilization 10～30 minutes with aseptic water washing 2～5 times, then uses sterilized water
Rinse 3～6 times, be inoculated on inducing culture after aseptic filter paper suck dry moisture, full light culture under the conditions of 25～28 DEG C, directly
To 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
It illumination 12～15 hours, intensity of illumination is 1000～1500lx, and cultivation temperature is cultivated under conditions of being 25～28 DEG C, 20～30
It switching is once.
(3) rooting of vitro seedling: cut to be inoculated on root media by the long Multiple Buds to 2～4cm and carry out root induction,
First full light culture 7～14 days under the conditions of 25～28 DEG C after inoculation, be subsequently placed in illumination every day 12～15 hours, and intensity of illumination is
2000～3000lx, cultivation temperature is cultivated to taking root under conditions of being 25～28 DEG C.
(4) acclimatization and transplants: the culture bottle bottle cap of the rooting tube plantlet of length to 6～8cm is opened and is placed under natural lighting
Seedling exercising 5～after 7 days, takes out test tube Seedling from culture bottle, washes root culture medium off, plants and mixes into by peat soil and river sand (1:1)
Synthesis substrate in and be colonizated in big Tanaka.
Outside the Radix Aristolochiae Tagalae band rattan that above-mentioned steps (1) is described, implant needed to carry out pre-place before carrying out Induce aerosor
Reason, the method for process is: by from the Radix Aristolochiae Tagalae band rattan tap water of field acquisition rinse well silt be placed on 0.01%～
The potassium permanganate solution of 0.05% soaks 4～8 hours, then with tap water rinse glue plastic bag sealing after 3～5 times be placed on 3～
5 DEG C of refrigerator overnight are standby.
Inducing culture described in above-mentioned steps (1) is: with GS as minimal medium, additional 2.0%～3.5% sucrose,
0.35%～0.5% agar, 0.05%～0.1% activated carbon, 0.05%～0.5% acidic hydrolysis casein and 0.5～2.0mg/L
KT, 0.1～2.0mg/L IBA and 0.1～1.0mg/L 2,4-D, pH value is 5.4～5.8;
Proliferated culture medium described in above-mentioned steps (2) is: with MS as minimal medium, additional 0.2～3.0mg/L 6-BA,
0.1～1.5mg/L NAA, 1.0～6.0mg/L riboflavin, 0.1～0.5mg/L D-VB5 calcium, 1.0～6.0mg/L Vitamin C
Acid, 1.0～5.0mg/L Cys and 2.5%～3.5% sucrose, 0.35%～0.5% agar, 0.05%～0.1% activated carbon,
PH value is 5.4～5.8；
Root media described in above-mentioned steps (3) is: with 1/2MS as minimal medium, additional 0.1～0.8mg/L
IBA, 0.1～1.0mg/L GGR(note: double gill-GGR, purchased from Beijing Ai Bidi research and development centre), 0.5～2.5mg/L
Cys and 2.0%～2.5% sucrose, 0.4%～0.6% agar, 0.05%～0.1% activated carbon, pH value is 5.4～5.8；
The present invention has the special feature that, the present invention utilizes plant tissue culture technique to carry out Radix Aristolochiae Tagalae seedling large-scale production,
Establish the tissue cultivation rapid breeding method of Radix Aristolochiae Tagalae, and the present invention has simple, easy, economic feature.Educated by the present invention
The Radix Aristolochiae Tagalae test tube transplantation of seedlings survival rate become, to more than 93%, can be that Radix Aristolochiae Tagalae implantation in large scale provides seedling guarantee.
Detailed description of the invention
Following example are to further illustrate the present invention, are not limitations of the present invention.
Embodiment one
1, outer implant pretreatment
By from the Radix Aristolochiae Tagalae band rattan tap water of field acquisition rinse well silt be placed on 0.03% potassium permanganate molten
Liquid soaks 5 hours, then rinsing glue plastic bag sealing after 5 times with tap water, to be placed on 3 DEG C of refrigerator overnight standby.
2, incubation
(1) evoking adventive bud: Radix Aristolochiae Tagalae belt segment rattan is after 8 seconds kinds of 75% alcohol disinfecting, rearmounted with aseptic water washing 3 times
Sterilize 15 minutes in the mercuric chloride solution of 0.1%, then use aseptic water washing 4 times, be inoculated into after aseptic filter paper suck dry moisture and lure
Lead in culture medium, full light culture under the conditions of 28 DEG C, form adventitious bud after cultivating 45 days.The inducing culture used is: GS+
1.5mg/L KT+0.3mg/L IBA and the acid of 0.4mg/L 2,4-D+3.5% sucrose+0.35% agar+0.08% activated carbon+0.3%
Property caseinhydrolysate, pH value is 5.8;
(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 15 hours, intensity of illumination is 1000lx, and cultivation temperature is cultivated under conditions of being 28 DEG C, and once, propagation is in switching in 25 days
Number is 5～7.The proliferated culture medium used is: MS+1.5mg/L 6-BA+0.5mg/L NAA+1.0mg/L riboflavin+
0.5mg/L D-VB5 calcium+1.0mg/L ascorbic acid+1.0mg/L Cys+3.0% sucrose+0.35% agar+0.1% is lived
Property charcoal, pH value is 5.8；
(3) rooting of vitro seedling: cut to be inoculated on root media by the long Multiple Buds to 2～4cm and carry out root induction,
After inoculation, first full light culture 7 days under the conditions of 28 DEG C, are subsequently placed in illumination every day 15 hours, and intensity of illumination is 3000lx, cultivate
Temperature is cultivated under conditions of being 28 DEG C and is taken root for 20 days, and rooting rate reaches 95%.The root media used is: 1/2MS+0.3mg/L
IBA+0.6mg/L GGR+1.0mg/L Cys+2.0%% sucrose+0.4% agar+0.1% activated carbon, pH value is 5.8；
(4) acclimatization and transplants: the culture bottle bottle cap of the rooting tube plantlet of length to 6～8cm is opened and is placed under natural lighting
After seedling exercising 5 days, test tube Seedling is taken out from culture bottle, wash root culture medium off, plant and be mixed into into by peat soil and river sand (1:1)
Substrate in and be colonizated in big Tanaka, survival rate 92%.
Embodiment two
1, outer implant pretreatment
By from the Radix Aristolochiae Tagalae band rattan tap water of field acquisition rinse well silt be placed on 0.01% potassium permanganate molten
Liquid soaks 8 hours, then rinsing glue plastic bag sealing after 3 times with tap water, to be placed on 5 DEG C of refrigerator overnight standby.
2, incubation
(1) evoking adventive bud: Radix Aristolochiae Tagalae belt segment rattan is after 5 seconds kinds of 78% alcohol disinfecting, rearmounted with aseptic water washing 5 times
Sterilize 10 minutes in the mercuric chloride solution of 0.2%, then use aseptic water washing 5 times, be inoculated into after aseptic filter paper suck dry moisture and lure
Lead in culture medium, full light culture under the conditions of 25 DEG C, form adventitious bud after cultivating 42 days.The inducing culture used is: GS+
0.8mg/L KT+0.2mg/L IBA and 0.2mg/L 2,4-D+3.0% sucrose+0.4% agar+0.1% activated carbon+0.5% are acid
Caseinhydrolysate, 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
It illumination 12 hours, intensity of illumination is 1300lx, and cultivation temperature is cultivated under conditions of being 25 DEG C, and once, propagation is in switching in 28 days
Number is 4～6.The proliferated culture medium used is: MS+0.8mg/L 6-BA+0.3mg/L NAA+3.0mg/L riboflavin+
0.3mg/L D-VB5 calcium+3.5mg/L ascorbic acid+4.5mg/L Cys+3.5% sucrose+0.5% agar+0.08% is lived
Property charcoal, pH value is 5.6；
(3) rooting of vitro seedling: cut to be inoculated on root media by the long Multiple Buds to 2～4cm and carry out root induction,
After inoculation, first full light culture 10 days under the conditions of 25 DEG C, are subsequently placed in illumination every day 12 hours, and intensity of illumination is 2500lx, cultivate
Temperature is cultivated under conditions of being 25 DEG C and is taken root for 28 days, and rooting rate reaches 92%.The root media used is: 1/2MS+0.5mg/L
IBA+1.0mg/L GGR+2.5mg/L Cys+2.5%% sucrose+0.4% agar+0.05% activated carbon, pH value is 5.6；
(4) acclimatization and transplants: the culture bottle bottle cap of the rooting tube plantlet of length to 6～8cm is opened and is placed under natural lighting
After seedling exercising 7 days, test tube Seedling is taken out from culture bottle, wash root culture medium off, plant and be mixed into into by peat soil and river sand (1:1)
Substrate in and be colonizated in big Tanaka, survival rate 95%.
Embodiment three
1, outer implant pretreatment
By from the Radix Aristolochiae Tagalae band rattan tap water of field acquisition rinse well silt be placed on 0.05% potassium permanganate molten
Liquid soaks 4 hours, then rinsing glue plastic bag sealing after 5 times with tap water, to be placed on 5 DEG C of refrigerator overnight standby.
2, incubation
(1) evoking adventive bud: Radix Aristolochiae Tagalae belt segment rattan is after 5 seconds kinds of 80% alcohol disinfecting, rearmounted with aseptic water washing 4 times
Sterilize 13 minutes in the mercuric chloride solution of 0.1%, then use aseptic water washing 5 times, be inoculated into after aseptic filter paper suck dry moisture and lure
Lead in culture medium, full light culture under the conditions of 25 DEG C, form adventitious bud after cultivating 47 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
It illumination 13 hours, intensity of illumination is 1200lx, and cultivation temperature is cultivated under conditions of being 27 DEG C, and once, propagation is in switching in 26 days
Number is 3～6.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% is lived
Property charcoal, pH value is 5.4；
(3) rooting of vitro seedling: cut to be inoculated on root media by the long Multiple Buds to 2～4cm and carry out root induction,
After inoculation, 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, cultivate
Temperature is cultivated under conditions of being 25 DEG C and is taken root for 20 days, and 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 rooting tube plantlet of length to 6～8cm is opened and is placed under natural lighting
After seedling exercising 6 days, test tube Seedling is taken out from culture bottle, wash root culture medium off, plant and be mixed into into by peat soil and river sand (1:1)
Substrate in and be colonizated in big Tanaka, survival rate 94%.
Claims (2)
1. the tissue cultivation rapid breeding method of a Radix Aristolochiae Tagalae, it is characterised in that comprise the following steps that:
(1) evoking adventive bud: choosing Radix Aristolochiae Tagalae belt segment rattan is outer implant, after 75%～80% alcohol disinfecting 5～15 seconds kinds, uses
Aseptic water washing 2～sterilize 10～30 minutes, then with aseptic water washing 3 in being placed on the mercuric chloride solution of 0.1%～0.3% for 5 times
～6 times, it being inoculated on inducing culture after aseptic filter paper suck dry moisture, full light culture under the conditions of 25～28 DEG C, until luring
Leading formation adventitious bud, described inducing culture is: with GS as minimal medium, additional 2.0%～3.5% sucrose, 0.35%～
0.5% agar, 0.05%～0.1% activated carbon, 0.05%～0.5% acidic hydrolysis casein and 0.5～2.0mg/L KT, 0.1～
2.0mg/L IBA and 0.1～1.0mg/L 2,4-D, pH value is 5.4～5.8；
(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 12～15 hours, intensity of illumination was 1000～1500lx, and cultivation temperature is cultivated under conditions of being 25～28 DEG C, within 20～30 days, turned
Connecing once, described proliferated culture medium is: with MS as minimal medium, additional 0.2～3.0mg/L 6-BA, 0.1～1.5mg/L
NAA, 1.0～6.0mg/L riboflavin, 0.1～0.5mg/L D-VB5 calcium, 1.0～6.0mg/L ascorbic acid, 1.0～
5.0mg/L Cys and 2.5%～3.5% sucrose, 0.35%～0.5% agar, 0.05%～0.1% activated carbon, pH value is
5.4～5.8；
(3) rooting of vitro seedling: cut to be inoculated on root media by the long Multiple Buds to 2～4cm and carry out root induction, inoculation
Rear elder generation full light culture 7～14 days under the conditions of 25～28 DEG C, be subsequently placed in illumination every day 12～15 hours, and intensity of illumination is 2000
～3000lx, cultivation temperature cultivates to taking root under conditions of being 25～28 DEG C, and described root media is: with 1/2MS as base
Basal culture medium, additional 0.1～0.8mg/L IBA, 0.1～1.0mg/L GGR, 0.5～2.5mg/L Cys and 2.0%
～2.5% sucrose, 0.4%～0.6% agar, 0.05%～0.1% activated carbon, pH value is 5.4～5.8；
(4) acclimatization and transplants: the culture bottle bottle cap of the rooting tube plantlet of length to 6～8cm is opened and is placed in natural lighting lower refining seedling 5
～after 7 days, test tube Seedling is taken out from culture bottle, wash root culture medium off, plant and mix into by the peat soil that ratio is 1:1 and river sand
Synthesis substrate in and be colonizated in big Tanaka.
The tissue cultivation rapid breeding method of a kind of Radix Aristolochiae Tagalae the most according to claim 1, it is characterised in that described in step (1)
The outer implant of Radix Aristolochiae Tagalae belt segment rattan needed to carry out pretreatment before carrying out Induce aerosor, and the method for process is: will adopt from field
The Radix Aristolochiae Tagalae belt segment rattan tap water of collection is rinsed silt well and is placed in the potassium permanganate solution of 0.01%～0.05% immersion 4
～8 hours, then rinsing glue plastic bag sealing after 3～5 times with tap water, to be placed on 3～5 DEG C of refrigerator overnight standby.
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| CN104770296A (en) * | 2015-04-03 | 2015-07-15 | 广西壮族自治区药用植物园 | Method for rapidly propagating through adoption of aristolochia fordiana leaves |
| CN104798684B (en) * | 2015-04-25 | 2017-04-05 | 玉林师范学院 | A kind of tissue cultivation rapid breeding method of astral oil rattan |
| 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 |
| CN107494282A (en) * | 2017-10-17 | 2017-12-22 | 李正美 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
| CN109380084A (en) * | 2018-11-23 | 2019-02-26 | 张世燊 | A kind of tissue cultivation rapid breeding method of the seed of feather cockscomb |
| CN109496844A (en) * | 2018-11-23 | 2019-03-22 | 韦宇 | A kind of tissue cultivation rapid breeding method of Changshan |
| CN109380118A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of tissue cultivation rapid breeding method of radish seed |
| CN109329063A (en) * | 2018-11-27 | 2019-02-15 | 广西玉林市华睿茶业有限公司 | A kind of tissue cultivation rapid breeding method of moellendorf spikemoss herb |
| CN116458429A (en) * | 2023-04-28 | 2023-07-21 | 广西壮族自治区南宁良凤江国家森林公园 | Application and method for tissue culture propagation of sargentgloryvine stem seeds and embryo |
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| CN103168693A (en) * | 2013-04-12 | 2013-06-26 | 广西橡胶研究所 | Proliferation culture medium for aristolochia longgonensis tissue culture and application of proliferation culture medium |
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| CN103168693A (en) * | 2013-04-12 | 2013-06-26 | 广西橡胶研究所 | Proliferation culture medium for aristolochia longgonensis tissue culture and application of proliferation culture medium |
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