CN107535354A - A kind of scientific and effective river monkshood propagation method - Google Patents
A kind of scientific and effective river monkshood propagation method Download PDFInfo
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- CN107535354A CN107535354A CN201610458336.1A CN201610458336A CN107535354A CN 107535354 A CN107535354 A CN 107535354A CN 201610458336 A CN201610458336 A CN 201610458336A CN 107535354 A CN107535354 A CN 107535354A
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Abstract
The invention discloses a kind of scientific and effective river monkshood propagation method, including：The key step such as the selection of explant and sterilization, inoculation evoked callus, induction Multiple Buds, root induction, acclimatization and transplantses, by being that explant carries out tissue cultures, induction plant regeneration quickly breeds river monkshood seedling with river monkshood stem apex, blade, root, stem section, petiole and seed etc., its inductivity, differentiation rate and rooting rate are higher, and survival rate is high after moving into cultivation soil；The disease resistance that can effectively solve current race in the production of river monkshood is poor, yield poorly and the problems such as shakiness, variet complexity; the purification and rejuvenation of kind are realized by stem apex detoxification and using tissue cultures means etc.; river monkshood improved seeds are provided, batch production, the large-scale production for a large amount of high quality seedlings needed for river monkshood GAP bases etc. lay a good foundation.
Description
Technical field：
The invention belongs to the planting of medicinal materials technical field, more particularly to a kind of scientific and effective river monkshood propagation method.
Background technology：
Monkshood (Radix Aconiti Lateralis Preparata) is ranunculaceae plant rhizome of Chinese monkshood Aconitum
The processed goods of carmichaeli Debx. root, there is recuperating depleted yang, mends fire supporing yang, eliminating cold to stop pain and other effects；For yang depletion
Collapse, cold weak pulse of limb, impotence, Gong Leng, trusted subordinate's crymodynia, cold of insufficiency type vomiting and diarrhea, cold sensation of the genitalia oedema, deficiency of yang diseases caused by external factors, numbness and ache of cold dampness, cloudy subcutaneous ulcer sore
Deng.
Monkshood is one of Sichuan tradition genuine traditional Chinese medicine material, has important medicinal and economic value.Sichuan Province's Jiangyou City is made
For the Genuine producing area of monkshood, there is the plantation history of more than 1400 years, quality assay in 2006 obtains geography symbol product protection (2006
Year No. 41 is announced), it is national monkshood production commodity base, the Product Marketing whole nation and outlet.But given birth in recent years in quality assay
Occur some problems, the increasingly atrophy of monkshood cultivated area in production, medicinal herb grower's economic benefit declines year by year, and monkshood industrial prospect may
Sorrow.Main cause has at following 2 points：First, monkshood frequently changes kind and lacks unified management, cultivar exists different degrees of
Confusion and mix phenomenon, had a strong impact on the product quality and its medical value of monkshood.Second, monkshood production uses nothing for a long time
Sexual reproduction, disease accumulation, the wherein disease such as downy mildew, southern blight, root rot frequently occur, and cause monkshood yield reduction, produce
Cost increase, has a strong impact on and governs the development of monkshood production.
The artificial cultivation of the rhizome of Chinese monkshood is measured greatly mainly by sub- root propagation, consumption kind at present, and because the reasons such as virus infection cause
Kind sexual involution, yield and quality reduce, need to be reserved seed for planting in high mountain change kind every year, it is difficult to adapt to the need of scale and standardized production
Will, realize that seedling detoxification production is one of main path for solving the above problems by tissue cultures.
Group difference be present in the tissue cultures of tissue cultures particularly medicinal plant, or even the different tissues of same plant,
Different Organs all there may be larger difference in tissue cultures.Hu Yanyu (Plant Physiology Communications, 1985,16 (2) 37-40)
Regeneration plant, but used medium complicated component, growth cycle length are obtained using stem apex and stem segment with axillary bud culture.Woods is waited quietly
(Guizhou science, 1998,16 (2)：120) cultivated using Guizhou rhizome of Chinese monkshood blade and young stem, blade only induces callus
Stage and undifferentiated budding.(Chinese herbal medicine, 2003,34 (6) such as Guan Wenling：561-563) using the blade of Yunnan rhizome of Chinese monkshood test tube seedling as
Explant, regrowth is obtained by the approach of direct evoking adventive bud.(CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2005,30 (15) such as Wang Yuehua：
1197-1199) regrowth is obtained by explant of petiole.
The content of the invention
The main object of the present invention is for problem present in the production of above-mentioned monkshood, there is provided a kind of river monkshood tissue cultures
And rapid propagation method, mainly the disease resistance of current race is poor in the production of solution river monkshood, yields poorly and shakiness, variet complexity etc.
Problem, laid the foundation for river monkshood GAP productions.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of scientific and effective river monkshood propagation method, including following key steps：
The first step：The selection and sterilization of explant
March choose the stem apex of annual healthy and strong river monkshood (rhizome of Chinese monkshood) plant, blade, root, stem section, the stem section with axillary bud,
Seed that petiole or upper one year collect etc. (being preferred using stem apex, blade, stem section, the stem section with axillary bud) is explant (as connecing
Kind material), 4 DEG C of low temperature is handled 3 days；Rinsed well with running water, with 70~75% 15~20s of ethanol postincubation, 0.1% mercuric chloride liquid
10~12min is soaked, mercuric chloride liquid is removed, is changed clothes 4~5 times with sterilized water；Be cut into after stem apex is peeled off 0.2mm length, root, stem section,
Stem section and petiole with axillary bud are cut into 1.5cm or so (can 1.3~1.7cm), and blade is cut into 1cm × 1cm squares, and (seed is not required to
Cut), it is stand-by；
Second step：Inoculation, evoked callus
Using MS as minimal medium, NAA (α-naphthylacetic acid) 0.1~0.2mg/L+6-BA (6- are added in minimal medium
Benzyladenine) culture mediums of the 1.0~4.0mg/L as evoked callus, by the explant that the above-mentioned first step is handled well by
Conventional method inoculation carries out callus induction, dark culturing 28~32 days or so, obtains callus；
3rd step：Induce Multiple Buds
2.0~2.5mg/L of NAA0.1~0.2mg/L+6-BA are added in MS minimal mediums to lure as differential medium
Multiple Buds are led, the callus that above-mentioned second step obtains is transferred on the differential medium, carry out inducing clumping bud, illumination training
Support 28~32 days or so, induce Multiple Buds；
4th step：Root induction
IBA (indolebutyric acid) 1.0mg/L+AC (activity) 3.0g/L+PVP (polyethylene pyrroles are added in MS minimal mediums
Pyrrolidone) above-mentioned 3rd step-length to 3~5cm Multiple Buds as root media, is transferred to the root media by 3.0g/L
The formation of upper induction root, incubation time are 20~25 days or so, the test tube seedling taken root；
5th step：Acclimatization and transplantses
Cultivate to 4~5cm of root long, 6~7cm of plant, select on root media after the test tube seedling of above-mentioned 4th step
Healthy and strong seedling opens bottle cap and carries out hardening, and room temperature hardening cleans the culture medium on seedling, transplanting to use with running water after 2~3 days
The treated detritus soil of 0.1% carbendazim and (detritus soil is preferred with perlite using volume ratio as 2: 1) in perlite mixed-matrix,
It is placed in 22 ± 1 DEG C, periodicity of illumination 12h/d of temperature, in 1500~2000lx of intensity of illumination growth room, film covering, retaining ring
Border moistens, and removes film after one week and is transferred to field production.
Above-mentioned MS minimal mediums, preferably following MS culture mediums by improvement：Wherein mainly in original MS culture mediums
On the basis of increase KH2PO4The dosage of (potassium dihydrogen phosphate), dosage is 170mg/L in former MS culture mediums；Now increase is 200mg/L.
In addition, sucrose 3%, agar powder 0.4%；PH is the parameters such as 5.8 with former MS culture mediums.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention passes through long-term largely experimental study and analysis and summary, it is established that by with river monkshood stem apex, blade,
Root, stem section, petiole and seed etc. are that explant carries out tissue cultures, establishes the rapid propagation system of river monkshood, its inductivity, are divided
Rate and rooting rate are higher, and survival rate is high after moving into field production；Can effectively solve the disease-resistant of current race in the production of river monkshood
Property it is poor, yield poorly the problems such as unstable, variet complexity, realize carrying for kind by stem apex detoxification and using tissue cultures means etc.
Pure rejuvenation, to provide batch production, the large-scale production of a large amount of high quality seedlings needed for river monkshood improved seeds and monkshood GAP bases etc.
Lay a good foundation.
Embodiment：
With reference to embodiment, the present invention is described in further detail.
But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following embodiments.
Inventor (chooses the stem of plant respectively by material of the river monkshood plant selected from Pingwu, sichuan monkshood introduces a collection base
Point, blade, root, stem section, the stem section with axillary bud, petiole and seed for collecting upper one year etc. are explant), using hormon
The culture medium of proportioning, quickly bred by tissue cultures evoked callus, observe the developmental state of different explants, be
Seed selection river monkshood improved seeds provide theoretical and test basis；As described below is which part test situation.
Inductivity and differentiation rate described in each embodiment are calculated by following formula to obtain respectively：
Explant number/inoculation explant number × 100% of inductivity=have callus；
Differentiation rate=budding callus number/inoculation callus number × 100%.
MS minimal mediums used, each mean the MS culture mediums by improvement in following embodiments：Wherein KH2PO4 (phosphorus
Acid dihydride potassium) dosage be 200mg/L；Separately containing sucrose 3%, agar powder 0.4% etc.；PH is 5.8.
Embodiment 1
The stem apex that the present embodiment chooses the river monkshood plant in Pingwu, sichuan monkshood introduces a collection base is explant, by organizing to train
Evoked callus is supported quickly to be bred, including following key steps：
The first step：The selection and sterilization of explant
In Pingwu, sichuan monkshood introduces a collection base, the stem apex that annual healthy and strong rhizome of Chinese monkshood plant is chosen in the March is explant
(as inoculation material), 4 DEG C of low temperature are handled 3 days；Rinsed well with running water, with 75% ethanol postincubation 15~20s, 0.1% liter
Mercury solution soaks 10~12min, removes mercuric chloride liquid, is changed clothes 4~5 times with sterilized water；0.2mm length is cut into after stem apex is peeled off, it is stand-by；
Second step：Inoculation, evoked callus
Using MS as minimal medium, additional NAA 0.1mg/L+6-BA1.0mg/L are cured as induction in minimal medium
The culture medium of injured tissue, the explant that the above-mentioned first step is handled well are inoculated with carry out callus induction according to a conventional method, each
Processing 15 test tubes of inoculation, 1 explant of every test tube, repeat, dark culturing 30 days or so, obtain callus three times；Meter
It is 90.0% to calculate inductivity result；
3rd step：Induce Multiple Buds
NAA0.1mg/L+6-BA2.0mg/L is added in MS minimal mediums and induces Multiple Buds as differential medium, will
The callus that above-mentioned second step obtains is transferred on the differential medium, progress inducing clumping bud, illumination cultivation 30 days or so,
Obtain Multiple Buds；
Differentiation rate is calculated, is as a result 83.3%；
4th step：Root induction
IBA1.0mg/L+AC3.0g/L+PVP3.0g/L is added in MS minimal mediums as root media, will be upper
State the 3rd step-length to 3~5cm Multiple Buds and be transferred to the formation that root is induced on the root media, incubation time is 20 days left sides
The right side, the test tube seedling taken root；Rooting rate reaches 95%；
5th step：Acclimatization and transplantses
Treat that the test tube seedling of above-mentioned 4th step is cultivated on root media to root long 4cm or so, plant 6cm or so, open
Bottle cap, room temperature hardening clean the culture medium on seedling, transplanting to the corruption treated with 0.1% carbendazim with running water after 2~3 days
Matter soil and (detritus soil is 2: 1 with perlite volume ratio) in perlite mixed-matrix, are placed in 22 ± 1 DEG C of temperature, periodicity of illumination
12h/d, in 1500~2000lx of intensity of illumination growth room, film covering, environment moistening is kept, crop field cultivation is transferred to after one week
Training, watering daily is once；Survival rate more than 96%.
Embodiment 2
The river monkshood plant leaf that the present embodiment chooses Pingwu, sichuan monkshood introduces a collection base is explant, passes through tissue cultures
Quickly bred, including following key steps：
The first step：The selection and sterilization of explant
In Pingwu, sichuan monkshood introduces a collection base, the blade that annual healthy and strong rhizome of Chinese monkshood plant is chosen in the March is explant
(as inoculation material), 4 DEG C of low temperature are handled 3 days；Rinsed well with running water, with 70% ethanol postincubation 15~20s, 0.1% liter
Mercury solution soaks 10~12min, removes mercuric chloride liquid, is changed clothes 4~5 times with sterilized water；It is stand-by that blade is cut into 1cm × 1cm squares；
Second step：Evoked callus
Using MS as minimal medium, NAA0.2mg/L+6-BA4.0mg/L is added in minimal medium as callus induction
The culture medium of tissue, the explant that the above-mentioned first step is handled well are inoculated with carry out callus induction according to a conventional method, each place
Reason 15 test tubes of inoculation, 1 explant of every test tube, repeat, dark culturing 32 days or so, obtain callus three times；
Inductivity is calculated, is as a result 90.0%；
3rd step：Inoculation, induction are grown thickly
NAA0.2mg/L+6-BA2.0mg/L is added in MS minimal mediums and induces Multiple Buds as differential medium, will
The callus that above-mentioned second step obtains is transferred on the differential medium, progress inducing clumping bud, illumination cultivation 32 days or so,
Obtain Multiple Buds；Differentiation rate is calculated, is as a result 80.8%；
4th step：Root induction
IBA1.0mg/L+AC3.0g/L+PVP3.0g/L is added in MS minimal mediums as root media, will be upper
State the 3rd step-length to 3~5cm Multiple Buds and be transferred to the formation that root is induced on the root media, incubation time is 20 days or so
The test tube seedling taken root；Rooting rate reaches 95%；
5th step：Acclimatization and transplantses
Treat that the test tube seedling of above-mentioned 4th step is cultivated on root media to root long 4cm or so, plant 6cm or so, open
Bottle cap, room temperature hardening clean the culture medium on seedling, transplanting to the corruption treated with 0.1% carbendazim with running water after 2~3 days
Matter soil and (detritus soil is 2: 1 with perlite volume ratio) in perlite mixed-matrix, are placed in 22 ± 1 DEG C of temperature, periodicity of illumination
12h/d, in 1500~2000lx of intensity of illumination growth room, film covering, environment moistening is kept, crop field cultivation is transferred to after one week
Training, watering daily is once；Survival rate more than 90%.
Embodiment 3
It is outer that the present embodiment, which chooses the stem section of river monkshood plant in Pingwu, sichuan monkshood introduces a collection base and the stem section with axillary bud,
Implant, quickly bred by tissue cultures evoked callus, including following key steps：
The first step：The selection and sterilization of explant
In Pingwu, sichuan monkshood introduces a collection base, the stem section and band of annual healthy and strong river monkshood plant are chosen respectively in the March
The stem section of axillary bud is explant (as inoculation material), and 4 DEG C of low temperature is handled 3 days；Rinsed well with running water, at 70% alcohol
15~20s is managed, 0.1% mercuric chloride liquid soaks 10~12min, removes mercuric chloride liquid, changed clothes 4~5 times with sterilized water；By stem section and with armpit
The stem section of bud is cut into 1.5cm or so, stand-by；
Second step：Inoculation, evoked callus
Using MS as minimal medium, NAA0.2mg/L+6-BA2.0mg/L is added in minimal medium as callus induction
The culture medium of tissue, the explant that the above-mentioned first step is handled well are inoculated with carry out callus induction according to a conventional method, each place
Reason 15 test tubes of inoculation, 1 explant of every test tube, repeat, dark culturing 28 days or so, obtain callus three times；Calculate
Inductivity, it is as a result 90.0%；
3rd step：Induce Multiple Buds
NAA0.1mg/L+6-BA 2.5mg/L are added in MS minimal mediums and induce Multiple Buds as differential medium,
The callus that above-mentioned second step obtains is transferred on the differential medium, carries out inducing clumping bud, 28 days left sides of illumination cultivation
The right side, obtain Multiple Buds；
Differentiation rate is calculated, is as a result 89.0%；
4th step：Root induction
IBA1.0mg/L+AC3.0g/L+PVP3.0g/L is added in MS minimal mediums as root media, will be upper
State the 3rd step-length to 3~5cm Multiple Buds and be transferred to the formation that root is induced on the root media, incubation time is 25 days left sides
The right side, the test tube seedling taken root；Rooting rate reaches 95%；
5th step：Acclimatization and transplantses
Treat that the test tube seedling of above-mentioned 4th step is cultivated on root media to root long 4cm or so, plant 6cm or so, open
Bottle cap, room temperature hardening clean the culture medium on seedling, transplanting to the corruption treated with 0.1% carbendazim with running water after 2~3 days
Matter soil and (detritus soil is 2: 1 with perlite volume ratio) in perlite mixed-matrix, are placed in 22 ± 1 DEG C of temperature, periodicity of illumination
12h/d, in 1500~2000lx of intensity of illumination growth room, film covering, environment moistening is kept, crop field cultivation is transferred to after one week
Training, watering daily is once；Survival rate more than 96%.
It is explant that inventor, which also chooses root, petiole, seed of the river monkshood plant in Pingwu, sichuan monkshood introduces a collection base etc.,
Above-mentioned tissue cultures and the experimental study quickly bred have been carried out, has obtained different degrees of inductivity, differentiation rate and rooting rate
Deng.
Claims (4)
- A kind of 1. scientific and effective river monkshood propagation method, it is characterised in that：The first step：Stem apex, blade, root, the stem of annual healthy and strong river monkshood plant are chosen in the selection of explant with the sterilization March Section, the stem section with axillary bud, petiole or the seed collected upper one year are explant, and 4 DEG C of low temperature is handled 3 days；Rinsed with running water Totally, 10~12min is soaked, mercuric chloride liquid is removed, is changed with sterilized water with 70~75% 15~20s of ethanol postincubation, 0.1% mercuric chloride liquid Wash 4~5 times；0.2mm length is cut into after stem apex is peeled off, root, stem section, the stem section with axillary bud and petiole are cut into 1.3~1.7cm, leaf Piece is cut into 1cm × 1cm sizes, stand-by；Second step：Inoculation, evoked callus add NAA0.1~0.2mg/ using MS as minimal medium in minimal medium Culture mediums of the L+6-BA1.0~4.0mg/L as evoked callus, the explant that the above-mentioned first step is handled well are routinely square Method inoculation carries out callus induction, dark culturing 28~32 days, obtains callus；3rd step：Induce Multiple Buds to add 2.0~2.5mg/L of NAA0.1~0.2mg/L+6-BA in MS minimal mediums to make Multiple Buds are induced for differential medium, the callus that above-mentioned second step obtains is transferred on the differential medium, carry out clump Sprout induction, illumination cultivation 28~32 days, induce Multiple Buds；4th step：Root induction adds IBA1.0mg/L+AC3.0g/L-1 in MS minimal mediums；It is adapted to callus proliferation Culture medium with inducing clumping bud is：MS+NAA0.2mgL+-1PVP3.0g/L is as root media, by above-mentioned 3rd step-length Multiple Buds to 3~5cm or so are transferred to the formation of induction root on the root media, and incubation time is 20~25 days, is obtained The test tube seedling taken root；5th step：Acclimatization and transplantses treat the test tube seedling of above-mentioned 4th step cultivated on root media to 4~5cm of root long, plant 6~ 7cm, select healthy and strong seedling to open bottle cap and carry out hardening, room temperature hardening cleans the culture medium on seedling with running water after 2~3 days, moves Plant into the detritus soil and perlite mixed-matrix treated with 0.1% carbendazim, be placed in 22 ± 1 DEG C of temperature, periodicity of illumination 12h/d, in 1500~2000lx of intensity of illumination growth room, film covering, keep environment to remove film after moistening one week and be transferred to Field production.
- 2. scientific and effective river monkshood propagation method according to claim 1, it is characterised in that：In the described first step Explant is selected from the stem apex of the disease-free river monkshood plant of annual stalwartness, blade, stem section, the stem section with axillary bud, through 4 DEG C of low temperature Processing is used to sterilize, be inoculated with, induce after 3 days.
- 3. scientific and effective river monkshood propagation method according to claim 1, it is characterised in that：Described MS is cultivated substantially Base refers to the MS culture mediums by improvement, wherein KH2PO4 dosage 200mg/L.
- 4. scientific and effective river monkshood propagation method according to claim 1, it is characterised in that：In the 5th described step Detritus soil and perlite mixed-matrix are detritus soil and mixed-matrix of the perlite using volume ratio as 2: 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112106666A (en) * | 2020-10-28 | 2020-12-22 | 中国科学院昆明植物研究所 | Method for regenerating radix aconiti kusnezoffii through organogenesis |
| CN113475397A (en) * | 2021-07-26 | 2021-10-08 | 西藏自治区高原生物研究所 | Tissue culture rapid propagation method of Aconitum carmichaeli and application thereof |
-
2016
- 2016-06-23 CN CN201610458336.1A patent/CN107535354A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112106666A (en) * | 2020-10-28 | 2020-12-22 | 中国科学院昆明植物研究所 | Method for regenerating radix aconiti kusnezoffii through organogenesis |
| CN112106666B (en) * | 2020-10-28 | 2022-04-08 | 中国科学院昆明植物研究所 | Method for regenerating radix aconiti kusnezoffii through organogenesis |
| CN113475397A (en) * | 2021-07-26 | 2021-10-08 | 西藏自治区高原生物研究所 | Tissue culture rapid propagation method of Aconitum carmichaeli and application thereof |
| CN113475397B (en) * | 2021-07-26 | 2022-06-28 | 西藏自治区高原生物研究所 | Tissue culture and rapid propagation method of Aconitum carmichaeli Debx and application thereof |
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