CN110447537A - A kind of tissue culture method obtaining regeneration plant using hippeastrum bulb disk as explant - Google Patents
A kind of tissue culture method obtaining regeneration plant using hippeastrum bulb disk as explant Download PDFInfo
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- CN110447537A CN110447537A CN201910781638.6A CN201910781638A CN110447537A CN 110447537 A CN110447537 A CN 110447537A CN 201910781638 A CN201910781638 A CN 201910781638A CN 110447537 A CN110447537 A CN 110447537A
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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 present invention provides a kind of tissue culture methods that regeneration plant is obtained using hippeastrum bulb disk as explant, the following steps are included: carrying out explant selection first, then it carries out disinfection, bulb is regarded into size cutting as 8-12 block after disinfection, it is inoculated in induced medium and carries out adventitious bud inducing, cultivation temperature is controlled at 24-26 DEG C, then at light application time 10-14h/ days, intensity of illumination is that normal culture 45 days is carried out under 2000-2200lx, the clove being proliferated out cuts leaf and root, then the seedling cutting of gained uncertain buds growth is transferred in subculture medium and carries out culture 25-35 days, finally aseptic seedling is accessed in root media and is carried out culture of rootage 15-25 days.The problem of can be effectively solved Hipeastrum vittalum's new varieties Vitro Quick Reproduction by this method.
Description
Technical field
The present invention relates to Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait (Hippeastrum rutilum) tissue culture propagation, and in particular to it is a kind of with
Hippeastrum bulb disk is the tissue culture method that explant obtains regeneration plant, belongs to the technology neck of herbaceous ornamental sapling multiplication
Domain.
Background technique
Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait (Hippeastrum rutilum) it is one kind that Amaryllidaceae Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait belongs to.Herbaceos perennial.Bulb
It is subsphaeroidal, it 6-8 pieces of leaf, is extracted out after spending, emerald green, scape is hollow, and it is slightly flat, there is white powder；Perianth tube green, cylindric, perianth
Sliver oblong, top point, color is different because of kind, and there is ramentum in throat.
Hipeastrum vittalum's kind is very rich at present, and market is occupied based on imported from Holland bulb.Dutch Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is characterized in spending
Big color is gorgeous, and the bud green cleaning of blade is popular potting ornamental flower and cut-flower material.But it is excellent because of reproduction technique problem
The quantity that kind bulb is cultivated at home is considerably less, and correlative study shows Hipeastrum vittalum's platymiscium kind, and bulb separation is numerous in its natural state
Grow that coefficient is very low, and seminal propagation not can guarantee the inhereditary feature of pattern, therefore as excellent home gardening flowers, Dutch Zhu
Red resource scarcity is pushed up, popularization and application are restricted.
Tissue cultures in relation to Dutch Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait and its congener, the country is rarely reported.Zou Shui equality is with double Long Zhuding
Red clove is that explant carries out rapid propagation in vitro, and breeding coefficient reaches 1.2 times, and (the cutting induction of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait tissue-cultured seedling clove is new
Plant technical research, Agriculture in Hunan science, the 4th phase in 2012,5-7 pages).Liu Duo is using dancing queen's kind plateau as explant
Body carries out tissue culture propagation by adventitious bud induction culture base of MS+6-BA 2mg/L+NAA1.0 mg/L, obtains 2.4 times numerous
Grow coefficient (Hipeastrum vittalum's research that numerous sterile system is established fastly, modern horticultural, the 3rd phase in 2018,23-25 pages).It opens intelligent equal with celestial being
Female's kind tissue-cultured seedling plateau is explant, evoked callus, and then progress adventitious buds differentiation induction, to obtain proliferation rate
Breeding system (foundation and optimization of Hipeastrum vittalum's callus induction and proliferation system, the Shandong forestry section of 7.83 plants/bottle
Skill, the 6th phase in 2018,17-20 pages).Previous research has obtained the proliferated culture medium and root media of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, mostly with squama
Stem disk be explant, but proliferation rate compare it is lower；And the adventitious buds differentiation efficiency of callus proliferating way is in the upper easily appearance of production
Wild effect.By groping hormone kind, proportion formula and cutting method, tissue-cultured seedling breeding coefficient can be increased, in short cycle
Obtain efficient sapling multiplication.
It is to solve bulb imported from Holland Hipeastrum vittalum's kind in conclusion taking the tissue culture propagation of high breeding coefficient
One of the important channel of ball shortage, develops it and utilization and extention all has very important meaning.
Summary of the invention
In order to overcome the drawbacks of the prior art, it is obtained and is regenerated as explant using hippeastrum bulb disk the present invention provides one kind
The tissue culture method of plant.
In order to achieve the above object, the present invention adopts the following technical scheme that:
A kind of tissue culture method obtaining regeneration plant using hippeastrum bulb disk as explant comprising following steps:
The selection of step 1) explant and disinfection；
The induction of step 2 adventitious bud: the explant after cancelling poison, cutting are 8-12 block, retain 0.5-1.0cm length bulb, will
Explant, which is inoculated in induced medium, carries out adventitious bud induction culture, controls cultivation temperature at 24-26 DEG C；
Step 3) squamous subculture and proliferation: in Hipeastrum vittalum's adventitious shoot access subculture medium that step 2 is induced, into
Row squamous subculture 25-35 days, 24-26 DEG C of cultivation temperature, light application time 10-14h/ days, intensity of illumination 1600-1800lx；Appoint
Selection of land, then carry out 1-2 squamous subculture；
The induction of step 4) root: Hipeastrum vittalum's test tube seedling obtained by squamous subculture is cut into leaf and fibrous root, is accessed in root media
Progress culture of rootage 15 days, 24-26 DEG C of cultivation temperature, light application time 10-14h/ days, intensity of illumination 1600-1800lx.
Further,
Explant in the step 1) chooses the plateau in Hipeastrum vittalum's Growing season 5-6 month.
Further,
Disinfecting process in the step 1) are as follows: 15min is shaken with dish washing liquid solution, after flowing water rinses 30min, in aseptic operating platform
Upper 75% ethanol disinfection 60s, then 1min is washed every time finally with sterile water washing 6 times with 0.1% mercuric chloride disinfection 22min.
Further,
The induced medium is MS+6-BA 2mg/L+NAA0.1 mg/L.
Preferably,
The adventitious bud induction culture process are as follows: cultivate in the dark first for 24 hours, then carry out normal culture 45 days.
Preferably,
The subculture medium is MS+6-BA 2mg/L+NAA 0.1mg/L.
Preferably,
The root media is MS+IBA 1.0mg/L+ atlapulgite 1g/L.
Preferably, the condition normally cultivated are as follows: light application time 10-14h/ days, intensity of illumination 2000-2200lx.
The beneficial effect of the present invention compared with the prior art mainly includes but is not limited to the following aspects:
Import ocean Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait breeding generallys use a point bulb and breeds, and per fraction of the year plants once, and proliferation rate is very low.And tissue is used to train
Feeding method is bred, and a large amount of Hipeastrum vittalum's new varieties aseptic seedling can be effectively obtained, and solves the problems, such as quickly to breed；It is logical
The selection and optimization to best explant, induced medium, subculture medium and root media are crossed, plateau is enabled to lure
It leads the rate of sprouting and reaches 75%, 3.5 times of clonal expansion adventitious bud or more, rooting rate reaches 100%, 1 cultivation cycle 135d proliferation rate
Reach 150 times or more, can efficiently obtain import ocean Hipeastrum vittalum's aseptic seedling, have a extensive future.
Specific embodiment
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
Body embodiment more clearly and completely describes the present invention, it is clear that described embodiment is only the application one
Divide embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not making
Every other embodiment obtained, should fall within the scope of the present invention under the premise of creative work.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Raw material as used in the following examples, is commercially available unless otherwise specified.
Embodiment 1
Explant is in 4 positions at the top of plateau using Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait without bulb, the plateau with bulb, rachis base portion and rachis
Body compares the pollution rate of different parts explant and the influence to Hipeastrum vittalum's evoking adventive bud.
Sterilization method, which is all made of, " shakes 15min with dish washing liquid solution, after flowing water rinses 30min, in using on aseptic operating platform
75% ethanol disinfection 60s, then 1min " is washed every time finally with sterile water washing 6 times with 0.1% mercuric chloride disinfection 22min.It is incubated at
Induced medium MS+6-BA 2mg/L+NAA0.1 mg/L.Every processing connects explant 100,3 repetitions, counts dirty after 45d
Dye rate, inductivity and fold induction.
Test result is shown in Table 1, and rachis is low as explant pollution rate as can be seen from Table 1 but inductivity is not high, and squama
Stem disk is higher than rachis position pollution rate as explant, but adventitious bud number is more, and breeding coefficient is high.
Influence of the 1 explant body region of table to Lycoris radiata evoking adventive bud
Embodiment 2
Using band bulb plateau as explant, after disinfection treatment, it is inoculated in containing hormon (CPPU, KT, BA and NAA) proportion
In induced medium, compare influence of the different culture medium to evoking adventive bud.Explant developmental state is observed, is counted not after 45d
Normal bud inductivity and fold induction.
It can be seen that from the following table 2, on MS+6-BA 2mg/L+NAA0.1 mg/L culture medium, explant development is best, lures
Conductance highest, evoking adventive bud number are most.
Influence of 2 induced medium of table to Lycoris radiata evoking adventive bud
Embodiment 3
Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is perennial root class flowers, and taken explant is under ground portion, and Contamination rate control is more difficult.To reduce explant body pollution
Rate, more different sterilization methods combine the influence of external implant body pollution rate.Compare pretreatment sterilization method first, to explant point
Not Cai Yong 84 thimerosals, Amway dish washing liquid, penicillin and streptomysin mixed liquor handle 3 kinds of thimerosals of 20min and cross-reference
Each 10min carries out disinfection pretreatment.Then, aseptic operating platform is gone to, impregnates 60s, the processing of 0.1% mercuric chloride using 75% alcohol
20min carries out disinfection processing to explant.Secondly, comparing mercuric chloride disinfecting time, is shaken and impregnated using Amway dish washing liquid solution
15min is gone on aseptic operating platform, impregnates 60s with 75% alcohol.Handle 18min respectively with 0.1% mercuric chloride again, 22min and
26min carries out disinfection processing to Hipeastrum vittalum's explant.The explant access MS+6-BA 2mg/L of all disinfection treatments+
NAA0.1mg/L culture medium.Specific experiment design is shown in Table 3.
Influence of the disinfection pretreatment to contamination control be not significant as can be seen from the test results, and mercuric chloride disinfecting time is to dirt
Dye control influences significant.Optimal sterilization method is that Amway washs liquor concussion immersion 15min, is gone on aseptic operating platform,
60s is impregnated with 75% alcohol.22min is handled respectively with 0.1% mercuric chloride again.
Influence of the different sterilization methods of table 3 to Hipeastrum vittalum's contamination control
Note: ' 84 ': 84 thimerosals；' Amway ': Amway dish washing liquid；PG/SM: penicillin/streptomycin mixed liquor；Pollution rate is dirt
Contaminate number/inoculation number；The death rate is death toll/inoculation number；Inductivity is induction number/pollution-free number.
Embodiment 4
By gained Hipeastrum vittalum's tissue-cultured seedling cutting be single plant after, access hormon proportion root media in carry out culture of rootage
20 days, detect average root long, mean elements and rooting rate.Specifically it is shown in Table 4.
Influence of 4 root media of table to rooting inhibitors
| Culture medium prescription | Rooting rate % |
| MS+NAA 1.0 mg/L | 80.6 |
| 1.0 mg/+ atlapulgite 1g/L of MS+NAA | 91.7 |
| MS+ IBA 1.0mg/L | 95.4 |
| MS+ IBA 1.0mg/L+atlapulgite 1g/L | 100 |
| MS | 56.2 |
As can be seen from Table 3, on MS+ IBA1.0mg/L+atlapulgite 1g/L root media, rooting rate highest reaches
100%, it is higher than remaining culture medium group；IBA ratio NAA is more conducive to take root, and atlapulgite can adsorb the quinones object of tissue-cultured seedling secretion
Matter avoids root browning, moreover it is possible to provide dark situation for root, promote the stimulation of root exogenous growth hormone, improve rooting rate.
Embodiment 5
A kind of tissue culture method obtaining regeneration plant using hippeastrum bulb disk as explant comprising following steps:
1, explant selection and disinfection: at the end of May, the plateau for choosing Hipeastrum vittalum's Growing season is explant, and sterilization method is with washing
Clean essence solution (5ml Amway dish washing liquid constant volume is in 1L aqueous solution) shakes 15min, after flowing water rinses 30min, in aseptic operating platform
Upper 75% ethanol disinfection 60s, then 1min is washed every time finally with sterile water washing 6 times with 0.1% mercuric chloride disinfection 22min, altogether
Handle 100 explants；
2, the induction of adventitious bud: the explant after cancelling poison regards size cutting as 8-12 block, retains 0.5-1.0cm length bulb,
Explant is inoculated in induced medium MS+6-BA 2mg/L+NAA0.1 mg/L and carries out adventitious bud inducing, control culture temperature
Degree is cultivated for 24 hours in the dark first at 24-26 DEG C, then carries out normal culture 45 days, the normal culture are as follows: light application time
10-14h/ days, intensity of illumination 2000-2200lx, explant pollution rate was 20% after 45 days, inductivity 75%, i.e., 60 outer
2-5 number not equal adventitious bud is grown in implant, average each explant is proliferated 3.6；
3, squamous subculture and proliferation: the clove of Hipeastrum vittalum's adventitious shoot is cut into leaf and root, accesses subculture medium MS+6-
In BA 2mg/L+NAA 0.1mg/L, every bottle is inoculated with 1, connects 216 bottles altogether, carries out squamous subculture 25 days, cultivation temperature 24-26
DEG C, after light application time 10-14h/ days, intensity of illumination 1600-1800lx, culture 25 days, shoot proliferation multiple is 3.5 times, can
756 plants of aseptic seedling, then (every minor tick 25 days, 3.5 times of average proliferation) can obtain 9261 plants of aseptic seedling after carrying out subculture 2 times；
4, the induction of root: Hipeastrum vittalum's test tube seedling obtained by squamous subculture is cut into leaf and fibrous root, accesses root media MS+IBA
Carry out culture of rootage 15 days in 1.0mg/L+ atlapulgite 1g/L, 24-26 DEG C of cultivation temperature, light application time 10-14h/ days, illumination
Intensity is 1600-1800lx, and rooting rate so far there are 9261 plants up to 100%, all healthy and strong main roots of new root.
1 cultivation cycle 135d can be proliferated out 9261 plants of plant by 60 plateau fritters, and proliferation rate is up to 154 times.
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, or the tree species except case study on implementation implement this method, this is to this
It is obvious for the technical staff of field.Therefore, these modifications made without departing from theon the basis of the spirit of the present invention, change
Into or range expansion, fall within the scope of the claimed invention.
Claims (8)
1. a kind of tissue culture method for obtaining regeneration plant using hippeastrum bulb disk as explant comprising following steps:
The selection of step 1) explant and disinfection；
The induction of step 2 adventitious bud: the explant after cancelling poison, cutting are 8-12 block, retain 0.5-1.0cm length bulb, will
Explant, which is inoculated in induced medium, carries out adventitious bud induction culture, controls cultivation temperature at 24-26 DEG C；
Step 3) squamous subculture: in Hipeastrum vittalum's adventitious shoot access subculture medium that step 2 is induced, subculture is carried out
Culture 25-35 days, 24-26 DEG C of cultivation temperature, light application time 10-14h/ days, intensity of illumination 1600-1800lx；Optionally, then
Carry out 1-2 squamous subculture；
The induction of step 4) root: Hipeastrum vittalum's test tube seedling obtained by squamous subculture is cut into leaf and fibrous root, is accessed in root media
Progress culture of rootage 15 days, 24-26 DEG C of cultivation temperature, light application time 10-14h/ days, intensity of illumination 1600-1800lx.
2. tissue culture method according to claim 1, which is characterized in that it is raw that the explant in the step 1) chooses Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait
The plateau in long season 5-6 month.
3. tissue culture method according to claim 1 or 2, which is characterized in that disinfecting process in the step 1) are as follows: clean with washing
Smart solution shakes 15min, after flowing water rinses 30min, on aseptic operating platform with 75% ethanol disinfection 60s, then with 0.1% mercuric chloride
It sterilizes 22min and washes 1min every time finally with sterile water washing 6 times.
4. tissue culture method according to claim 1 or 2, which is characterized in that the induced medium is MS+6-BA 2mg/L
+NAA0.1 mg/L。
5. tissue culture method according to claim 1 or 2, which is characterized in that the adventitious bud induction culture are as follows: first black
It cultivates in the dark for 24 hours, then carries out normal culture 45 days.
6. tissue culture method according to claim 1 or 2, which is characterized in that the subculture medium is MS+6-BA 2mg/L
+NAA 0.1mg/L。
7. tissue culture method according to claim 1 or 2, which is characterized in that the root media is MS+IBA 1.0mg/
L+ atlapulgite 1.0g/L.
8. tissue culture method according to claim 5, which is characterized in that the condition normally cultivated are as follows: light application time 10-
14h/ days, intensity of illumination 2000-2200lx.
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Cited By (4)
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| CN112690213A (en) * | 2021-01-14 | 2021-04-23 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality seedlings of hippeastrum rutilum |
| CN113243295A (en) * | 2021-05-25 | 2021-08-13 | 广东省林业科学研究院 | Hippeastrum rutilum tissue culture breeding method |
| CN116267612A (en) * | 2023-03-16 | 2023-06-23 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116369208A (en) * | 2023-06-07 | 2023-07-04 | 包头市农牧科学技术研究所 | Tissue culture and rapid propagation method for hippeastrum henryi and bulb pretreatment device thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112690213A (en) * | 2021-01-14 | 2021-04-23 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality seedlings of hippeastrum rutilum |
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| CN116267612A (en) * | 2023-03-16 | 2023-06-23 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116267612B (en) * | 2023-03-16 | 2024-01-09 | 中国热带农业科学院热带作物品种资源研究所 | Tissue culture propagation method of hippeastrum |
| CN116369208A (en) * | 2023-06-07 | 2023-07-04 | 包头市农牧科学技术研究所 | Tissue culture and rapid propagation method for hippeastrum henryi and bulb pretreatment device thereof |
| CN116369208B (en) * | 2023-06-07 | 2024-04-26 | 包头市农牧科学技术研究所 | Tissue culture and rapid propagation method for hippeastrum henryi and bulb pretreatment device thereof |
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