CN106561450A - Method for inducing adventitious buds by adopting Lycoris radiate rachis as explant - Google Patents
Method for inducing adventitious buds by adopting Lycoris radiate rachis as explant Download PDFInfo
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- CN106561450A CN106561450A CN201610914403.6A CN201610914403A CN106561450A CN 106561450 A CN106561450 A CN 106561450A CN 201610914403 A CN201610914403 A CN 201610914403A CN 106561450 A CN106561450 A CN 106561450A
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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
- A01H4/001—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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Abstract
The invention provides a method for inducing adventitious buds by adopting Lycoris radiate rachis as explant in order to obtain regenerated plants. The method comprises the following steps: selection and disinfection of the explant, induction of the adventitious buds, subculture and propagation, root induction, and transplantation of tissue culture plants. The method allows the induction germination rate to reach 96%, 4.9 times or above adventitious buds to be proliferated from a per plant and the rooting rate to reach 94%, and can effectively solve the problem of in vitro rapid propagation of Lycoris radiate.
Description
Technical field
The present invention relates to Lycoris radiata（Lycoris radiata （L’Her.）Herb.）Tissue culture propagation, category
In the technical field of herbaceous ornamental sapling multiplication.
Background technology
Lycoris radiata (Lycoris radiata （L’Her.）Herb.) be Amaryllidaceae Lycoris one kind.Perennial grass
This plant.Subterraneous stem is plump.Tip of a leaf shape, from base portion pumping after the florescence.Scape is high 30-60 centimetre, umbel basidixed, spends fresh
Red, cylindrical fireworks are shorter, the narrow lanceolar of tapel, outside turnup, and stamen and style stretch out, and attitude is beautiful, July at florescence ~ September, contains
July is opened in, summer is grown on.Property the dark and damp environment of happiness, be afraid of high light direct projection, be preferably grown on loose fertile sandy loam.In originating in
State and Japan, widely cultivate all over the world.It is excellent perennial root herbage flower, shady spot's greening is commonly used in gardens, can be spent
Altar or flower footpath material, are also beautiful cut-flowers.Bulb is poisonous, and being used as medicine has the effect of emetic, eliminating the phlegm, detumescence, pain relieving.
The kind seedling quantity is considerably less at home at present, and correlational study shows lycoris plants bulb separation in its natural state
Breeding coefficient is very low, and seminal propagation cannot ensure the hereditary character of pattern, therefore Lycoris radiata resource scarcity, and its application is subject to
Limit.
About Lycoris radiata and its tissue culture of congener, it is rarely reported both at home and abroad.Dan Zhuo with bulb as explant,
The research of fragrant Bulbus Lycoridis Radiatae tissue-culturing rapid propagation is carried out（The group culturation rapid propagating technology research of fragrant Bulbus Lycoridis Radiatae, gardens science and technology, the 3rd phase in 2012,8-11
Page）.The differentiation of the clove with the bulb of Bulbus Lycoridis Radiatae as investigation of materials such as Zhu Jin and propagation（The research of Bulbus Lycoridis Radiatae tissue culture propagation technology,
Zhejiang Forest science and technology, the 4th phase in 2002,45-48 page）.In the past research obtained the proliferated culture medium of Lycoris other kinds and
Root media, more with bulb as explant, the relatively low problem that often occurs that pollution rate is high and the rate of increase compares.And positive controls for high proliferation rates
The lycoris plants quick breeding method for tissue culture of low stain rate is not yet set up.
In sum, take tissue culture propagating Lycoris radiata be solve Lycoris radiata shortage of resources important channel it
One, the exploitation and utilization and extention to Lycoris radiata all has very important meaning.
The content of the invention
The invention mainly solves the technical problem of the open side with Lycoris radiata rachises as explant evoking adventive bud
Method.
Adopt the following technical scheme that to solve the technology：
A）Explant select with sterilization, choose Lycoris radiata initial bloom stage floral axis be optimal explant, sterilization method detergent
Shaking 15min, after flowing water rinses 30min, uses 75% ethanol disinfection 30s on the aseptic operating platform, then with 0.2% mercuric chloride sterilization
15min, finally with aseptic water washing 5 times, washes 1min every time；
B）The induction of adventitious bud：Cancel the position centered on floral axis summit of the explant after poison and respectively cut reservation 0.5- up and down
1.0cm it is long（Several branch lengths are equal）, explant is inoculated in into inducing culture MS+6-BA 2mg/L+zeatin 0.3mg/
L + GA3Induce aerosor is carried out in 0.8mg/L, control cultivation temperature cultivates 24h in the dark at 24-26 DEG C, first, so
After carry out normal culture 30 days, the normal culture is：Light application time 10-14h/ days, intensity of illumination are 1600-1800lx；
C）Successive transfer culture and propagation：By step B）Subculture medium MS+6-BA is accessed after middle gained Lycoris radiata seedling cutting
Successive transfer culture is carried out 25 days in 2mg/L+NAA0.2 mg/L, cultivation temperature 24-26 DEG C, light application time 10-14h/ days, illumination are strong
Spend for 1600-1800lx；
D）The induction of root：By step C）Gained Lycoris radiata test tube seedling is entered in accessing root media 1/2MS+IBA1.0 mg/L
Row root culture 10-15 days, cultivation temperature 20-26 DEG C, light application time 10-14h/ days, intensity of illumination are 1600-1800lx;
E）Culture bottle is removed into tissue culture room, bottle cap is opened, the root of tissue cultured seedling is immersed in composite microbial by training tissue culture seedling 2 days
In thing preparation, 10min is transplanted in substrate.
The complex microorganism preparations are：According to Strepiomyces lavendulae fermentation liquid：Trichoderma aureoviride fermentation liquid：Candidiasis
Fermentation liquid：Bacillus foecalis alkaligeness fermentation liquid：Turf：Disodium hydrogen phosphate weight ratio=2：3：4：3：5：2
The Strepiomyces lavendulae (Streptomyces lavendulae) is CGMCC NO.2130；
The trichoderma aureoviride is trichoderma aureoviride（Trichoderma aureoviride）ACCC32248
The candidiasis are specially candidiasis（Candida utilis）ATCC No.22023;
The Bacillus foecalis alkaligeness are Bacillus foecalis alkaligeness（Alcaligenes faecalis）ATCC 31555.
First Strepiomyces lavendulae, trichoderma aureoviride, candidiasis, Bacillus foecalis alkaligeness are lived respectively in a conventional manner
Change, then cultivate into bacterium solution viable count and reach 107Individual/gram acquisition fermentation liquid, by above-mentioned fermentation liquid according to mass ratio 2：3：
4：3 mixing, obtain final product according to weight proportion addition turf, disodium hydrogen phosphate.
Hinge structure of the present invention has the beneficial effect that：Lycoris radiata breeding generally plants bulb breeding, 3-4 using point
Divide and plant once, the rate of increase is very low.And Lycoris radiata is bred using the method for tissue culture of the present invention, can be effective
Substantial amounts of Lycoris radiata aseptic seedling is obtained, solves the problems, such as that Lycoris radiata is quickly bred；By to optimal explant, inducing culture
The selection of base, subculture medium and root media, enables to the induction rate of sprouting and reaches 96%, 4.9 times of clonal expansion adventitious bud
More than, rooting rate reaches 94%, and the rate of increase and transplanting survival rate are high, can efficiently obtain Lycoris radiata aseptic seedling.
Specific embodiment
Embodiment 1
Explant is screened
With Lycoris radiata plateau, 3 positions of rachises and petal base portion as explant, compare the pollution of different parts explant
Rate and the impact to Lycoris radiata evoking adventive bud.
Sterilization method is adopted " to be washed and shakes 15min with detergent, after flowing water rinses 30min, use on aseptic operating platform
75% ethanol disinfection 30s, then use0.2%Mercuric chloride is sterilized 15min, finally with aseptic water washing 5 times, washes 1min every time；”.Culture
In inducing culture MS+6-BA 2mg/L+zeatin 0.3mg/L+GA30.8mg/L.Often process and connect explant 100,
Pollution rate, inductivity and fold induction are counted after 30d.
Result of the test is shown in Table 1, and optimal explant is rachises as can be seen from Table 1, and not only pollution rate is low, inductivity is high,
Adventitious bud multiple is also highest.
Impact of the 1 explant body region of table to Lycoris radiata evoking adventive bud
| Explant body region | Pollution rate % | Inductivity % | Adventitious bud multiple |
| Plateau | 89.41 | 80.09 | 2.1 |
| Rachises | 23.66 | 95.86 | 4.9 |
| Petal base portion | 46.92 | 31.22 | 1.5 |
Embodiment 2
Inducing culture is screened
With rachises as explant, after disinfecting, it is inoculated on the inducing culture containing hormon proportioning, the different trainings of comparison
Impact of the foster base to evoking adventive bud.Observation explant developmental state, counts adventitious bud induction frequency and fold induction after 30d.
As can be seen from Table 2, in MS+6-BA 2mg/L+zeatin 0.3mg/L+GA3In 0.8mg/L culture medium, explant
Body development is best, and inductivity highest, evoking adventive bud number are most.
Impact of 2 inducing culture of table to Lycoris radiata evoking adventive bud
| Culture medium prescription | Inductivity % | Adventitious bud multiple |
| MS+ zeatin 2mg/L+NAA0.2 mg/L | 88 | 2.4 |
| MS+KT 2mg/L +NAA0.2 mg/L | 74 | 1.1 |
| MS+6-BA 2mg/L+zeatin 0.3mg/L+GA3 0.8mg/L | 96 | 4.9 |
| MS+6-BA1mg/L +NAA0.2 mg/L | 80 | 1.7 |
Embodiment 3
Sterilization method is screened
Lycoris radiata is perennial root class flowers, and taken explant is under ground portion, and Contamination rate control is more difficult.It is dirty to reduce explant
Dye rate, the different sterilization methods of comparison combine the impact of external implant body pollution rate.Compare pretreatment sterilization method first, to explant
The mixed liquor that 84 disinfectant solution, Amway disinfectant solution, penicillin and streptomycin is respectively adopted processes 3 kinds of sterilizations of 20min and cross-reference
The each 10min of liquid carries out disinfection pretreatment.Then, aseptic operating platform is gone to, using 75% alcohol-pickled 30s, the process of 0.2% mercuric chloride
10min is to explant disinfection.Secondly, compare mercuric chloride disinfecting time, using Amway cleaning mixture concussion immersion 15min,
Go on aseptic operating platform, with 75% alcohol-pickled 30s.12min, 15min and 18min are processed respectively with 0.2% mercuric chloride again, to red
Flower Bulbus Lycoridis Radiatae explant disinfection.All explants disinfected access adventitious bud induction culture base.Specific experiment sets
Meter is shown in Table 3.
Impact of the pretreatment to Environmental capacity of sterilizing as can be seen from the test results be not notable, and mercuric chloride disinfecting time is to dirt
Dye control affects notable.Optimal sterilization method is Amway cleaning mixture concussion immersion 15min, goes on aseptic operating platform, uses 75%
Alcohol-pickled 30s.15min is processed respectively with 0.2% mercuric chloride again.
3 different impacts of the sterilization method to Lycoris radiata Environmental capacity of table
| Processing method | Mercuric chloride process time | Inoculation number/ | Pollution rate/% |
| ‘84’ 20min | 10min | 100 | 80 |
| ‘Amway’ 20min | 10min | 100 | 76 |
| PG/SM（Penicillin/streptomycin mixed liquor）20min | 10min | 100 | 90 |
| ‘84’ 10min ,‘Amway’ 10min | 10min | 100 | 80 |
| PG/SM 10min, ' Amway ' 10min | 10min | 100 | 84 |
| PG/SM 10min, ' 84 ' 10min | 10min | 100 | 82 |
| ‘Amway’ 15min | 12min | 100 | 75 |
| ‘Amway’ 15min | 15min | 100 | 50 |
| ‘Amway’ 15min | 18min | 100 | 24 |
During transplanting, Strepiomyces lavendulae, trichoderma aureoviride, candidiasis, Bacillus foecalis alkaligeness form a good microecosystem
System, reasonable compatibility between each strain, symbiosis are coordinated, mutually not antagonism, and microorganism secretion root system regulator and ablastins can be takeed good care of
Seedlings root infects rotten mould root rot, phytophthora root rot, bacillary root rot and the seedling stage such as base rot disease and damping-off, damping off
Disease, so as to rotten phenomenon of the dead seedling in effective control seedling stage, disodium hydrogen phosphate continually supply nutrition to root system, maintains Seedling strong
Seedling strengthens the impetus, greatly improves the adaptability of plant, improves whole survival rate, and Jing single factor experiments show, tissue cultured seedling root Jing
After crossing the accelerator immersion, transplanting survival rate improves 29.5% than without any process.
Embodiment 4
1st, explant is selected and sterilization：In by the end of August, 21 floral axis summits for choosing Lycoris radiata initial bloom stage are explant, are sterilized
Method detergent solution shakes 15min, after flowing water rinses 30min, with 75% ethanol disinfection 30s on the aseptic operating platform, then uses
0.2% mercuric chloride sterilization 15min, finally with aseptic water washing 5 times, washes 1min, 100 explants of coprocessing every time；
2nd, the induction of adventitious bud：Cancel the position centered on floral axis summit of the explant after poison and respectively cut reservation 1cm length up and down（It is several
Individual branch length is equal）, explant is inoculated in into inducing culture MS+6-BA 2mg/L+zeatin 0.3mg/L+GA3
Induce aerosor is carried out in 0.8mg/L, control cultivation temperature is cultivated 24h in the dark, just then carried out at 24-26 DEG C, first
Often cultivate 30 days, the normal culture is：Light application time 10-14h/ days, intensity of illumination is 1600-1800lx, explant after 30 days
Body pollution rate is 24%, and inductivity is 96%, and average each explant breeds 4.9；
3rd, successive transfer culture and propagation：Will Lycoris radiata adventitious shoot cut after access subculture medium MS+6-BA 2mg/L+
In NAA0.2 mg/L, per bottle is inoculated with 1, carries out successive transfer culture 25 days, cultivation temperature 24-26 DEG C, light application time 10-14h/ days,
Intensity of illumination is 1600-1800lx, and after cultivating 25 days, subculture multiplication multiple is 3.5 times, can obtain 1251 plants of aseptic seedling, then carry out
After subculture 1 time 2489 plants of aseptic seedling；
4th, the induction of root：Lycoris radiata test tube seedling obtained by successive transfer culture is accessed in 1.0 mg/L of root media 1/2MS+IBA
Carry out root culture 15 days, cultivation temperature 24-26 DEG C, light application time 10-14h/ days, intensity of illumination are 1600-1800lx, are taken root
Rate so far there are 2058 plants up to 94%.
5th, culture bottle is removed into tissue culture room, bottle cap is opened, the root of tissue cultured seedling is immersed in compound by training tissue culture seedling 2 days
In microorganism formulation, 10min is transplanted in substrate.
The complex microorganism preparations are：According to Strepiomyces lavendulae fermentation liquid：Trichoderma aureoviride fermentation liquid：Candidiasis
Fermentation liquid：Bacillus foecalis alkaligeness fermentation liquid：Turf：Disodium hydrogen phosphate weight ratio=2：3：4：3：5：2
The Strepiomyces lavendulae (Streptomyces lavendulae) is CGMCC NO.2130；
The trichoderma aureoviride is trichoderma aureoviride（Trichoderma aureoviride）ACCC32248
The candidiasis are specially candidiasis（Candida utilis）ATCC No.22023;
The Bacillus foecalis alkaligeness are Bacillus foecalis alkaligeness（Alcaligenes faecalis）ATCC 31555.
First Strepiomyces lavendulae, trichoderma aureoviride, candidiasis, Bacillus foecalis alkaligeness are lived respectively in a conventional manner
Change, then cultivate into bacterium solution viable count and reach 107Individual/gram acquisition fermentation liquid, by above-mentioned fermentation liquid according to mass ratio 2：3：
4：3 mixing, obtain final product according to weight proportion addition turf, disodium hydrogen phosphate.
Transplanting survival rate is 91.5%.
1 cultivation cycle 75d or so, can be bred by 21 rachises summits and 1883 plants of plant, can fill up significantly city
Field is blank.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (5)
1. it is a kind of with Lycoris radiata rachises as explant evoking adventive bud obtain regeneration plant method, it is characterised in that bag
Include following step：The selection of explant and sterilization, Induce aerosor, successive transfer culture and propagation, the induction of root, tissue culture transplantation of seedlings.
2. method according to claim 1, it is characterised in that specifically include following step：
Step A）Explant is selected and sterilization：The floral axis for choosing Lycoris radiata initial bloom stage carries out disinfection for explant；
Step B）The induction of adventitious bud：Explant after sterilization cuts into 0.5-1.0cm length, is inoculated in inducing culture (group
It is divided into：MS+6-BA 2mg/L+zeatin 0.3mg/L+GA3Induce aerosor, light application time 10- are carried out in 0.8mg/L)
14h/ days, intensity of illumination was 1600-1800lx；
Step C）Successive transfer culture and propagation：By step B）Subculture medium is accessed after middle gained Lycoris radiata adventitious bud cutting（Group
It is divided into：MS+6-BA 2mg/L +NAA0.2 mg/L）In carry out successive transfer culture, intensity of illumination is 1600-1800lx；
Step D）The induction of root：Will be through step C）Lycoris radiata test tube seedling after culture accesses root media（Component is 1/
2MS+IBA1.0 mg/L）In carry out root culture, obtain tissue cultured seedling;
Step E）Tissue culture transplantation of seedlings：Bottle cap is opened, the root of tissue cultured seedling is immersed in complex microorganism system by training tissue culture seedling 2 days
10min in agent, is transplanted in substrate.
3. method according to claim 2, it is characterised in that the sterilization method is：15min, stream are shaken with detergent
After water rinses 30min, 75% ethanol disinfection 30s is used on the aseptic operating platform, then with mercuric chloride sterilization 15min, finally uses aseptic washing
Wash 5 times, wash 1min every time.
4. the method according to claim 2-3, it is characterised in that the complex microorganism preparations are：According to lilac grey chain
Mold fermentation liquid：Trichoderma aureoviride fermentation liquid：Candida mycoderma fermented liquid：Bacillus foecalis alkaligeness fermentation liquid：Turf：Disodium hydrogen phosphate
Weight ratio=2：3：4：3：5：2 mixings are obtained final product.
5. the Lycoris radiata explant material for being obtained according to claim 1-4 methods described.
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| CN108184678A (en) * | 2018-04-04 | 2018-06-22 | 蒋建华 | A kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent |
| CN108575746A (en) * | 2018-04-04 | 2018-09-28 | 蒋建华 | A kind of Chinese herbaceous peony vitro Regeneration System method for building up |
| CN109329059A (en) * | 2018-11-21 | 2019-02-15 | 江苏省中国科学院植物研究所 | A kind of method of straw short-tube lycoris tissue cultures |
| CN109329060A (en) * | 2018-11-21 | 2019-02-15 | 江苏省中国科学院植物研究所 | The method for carrying out tissue-culturing rapid propagation as explant to change brocade flower short-tube lycoris plateau |
| CN110122327A (en) * | 2018-02-02 | 2019-08-16 | 江苏省中国科学院植物研究所 | A kind of method that alum root rachis vitro Regeneration System is established |
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| CN110122327B (en) * | 2018-02-02 | 2021-05-18 | 江苏省中国科学院植物研究所 | Method for establishing alum root rachis in-vitro regeneration system |
| CN108184678A (en) * | 2018-04-04 | 2018-06-22 | 蒋建华 | A kind of method for promoting paenoiae alba tissue culture expanding propagation using growth promoting bacteria agent |
| CN108575746A (en) * | 2018-04-04 | 2018-09-28 | 蒋建华 | A kind of Chinese herbaceous peony vitro Regeneration System method for building up |
| CN109329059A (en) * | 2018-11-21 | 2019-02-15 | 江苏省中国科学院植物研究所 | A kind of method of straw short-tube lycoris tissue cultures |
| CN109329060A (en) * | 2018-11-21 | 2019-02-15 | 江苏省中国科学院植物研究所 | The method for carrying out tissue-culturing rapid propagation as explant to change brocade flower short-tube lycoris plateau |
| CN109329059B (en) * | 2018-11-21 | 2021-08-06 | 江苏省中国科学院植物研究所 | Tissue culture method for lycoris radiata |
| CN111109081A (en) * | 2020-01-03 | 2020-05-08 | 上海市农业科学院 | Lycoris radiata rootless tissue culture method and lycoris radiata cultivation method |
| CN111109081B (en) * | 2020-01-03 | 2022-03-22 | 上海市农业科学院 | Lycoris radiata rootless tissue culture method and lycoris radiata cultivation method |
| CN112385547A (en) * | 2020-12-18 | 2021-02-23 | 江苏省中国科学院植物研究所 | Method for establishing lycoris longituba regeneration system through callus approach |
| CN112385547B (en) * | 2020-12-18 | 2022-04-01 | 江苏省中国科学院植物研究所 | Method for establishing long-tube lycoris regeneration system |
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