CN106797890B - A kind of culture medium and its method reducing leather leaf winged euonymus tissue culture pollution rate - Google Patents
A kind of culture medium and its method reducing leather leaf winged euonymus tissue culture pollution rate Download PDFInfo
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- CN106797890B CN106797890B CN201710150116.7A CN201710150116A CN106797890B CN 106797890 B CN106797890 B CN 106797890B CN 201710150116 A CN201710150116 A CN 201710150116A CN 106797890 B CN106797890 B CN 106797890B
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- 241000208368 Euonymus alatus Species 0.000 title claims abstract description 44
- 239000001963 growth medium Substances 0.000 title claims abstract description 42
- 240000001371 Chamaedaphne calyculata Species 0.000 title claims abstract description 35
- 235000013691 Chamaedaphne calyculata var. angustifolia Nutrition 0.000 title claims abstract description 34
- 235000013685 Chamaedaphne calyculata var. latifolia Nutrition 0.000 title claims abstract description 34
- 235000013687 Chamaedaphne calyculata var. nana Nutrition 0.000 title claims abstract description 34
- 235000011756 Vitis shuttleworthii Nutrition 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 50
- 238000005406 washing Methods 0.000 claims description 37
- 238000011081 inoculation Methods 0.000 claims description 31
- 238000004659 sterilization and disinfection Methods 0.000 claims description 26
- 230000001954 sterilising effect Effects 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 229930006000 Sucrose Natural products 0.000 claims description 20
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 20
- 239000005720 sucrose Substances 0.000 claims description 20
- 229920001817 Agar Polymers 0.000 claims description 19
- 239000008272 agar Substances 0.000 claims description 19
- 229960002523 mercuric chloride Drugs 0.000 claims description 19
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 19
- 230000008859 change Effects 0.000 claims description 16
- 239000000341 volatile oil Substances 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 9
- 238000005286 illumination Methods 0.000 claims description 7
- 238000002203 pretreatment Methods 0.000 claims description 7
- 238000007654 immersion Methods 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- 230000003442 weekly effect Effects 0.000 claims description 6
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 2
- 238000002255 vaccination Methods 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims 1
- 229910052760 oxygen Inorganic materials 0.000 claims 1
- 239000001301 oxygen Substances 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 12
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 238000012258 culturing Methods 0.000 abstract description 6
- 238000011109 contamination Methods 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 3
- 229920000742 Cotton Polymers 0.000 description 10
- 238000001816 cooling Methods 0.000 description 10
- 238000012545 processing Methods 0.000 description 8
- 241000607479 Yersinia pestis Species 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000008223 sterile water Substances 0.000 description 6
- 102000002322 Egg Proteins Human genes 0.000 description 5
- 108010000912 Egg Proteins Proteins 0.000 description 5
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 5
- 239000003599 detergent Substances 0.000 description 5
- 239000007943 implant Substances 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 210000004681 ovum Anatomy 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 239000000645 desinfectant Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 241000208367 Euonymus Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- PVRBGBGMDLPYKG-UHFFFAOYSA-N 6-benzyl-7h-purine Chemical compound N=1C=NC=2N=CNC=2C=1CC1=CC=CC=C1 PVRBGBGMDLPYKG-UHFFFAOYSA-N 0.000 description 1
- 241000931515 Acer palmatum Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000218195 Lauraceae Species 0.000 description 1
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 240000006909 Tilia x europaea Species 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to field of plant tissue culture technique, in particular to a kind of culture medium and its method for reducing leather leaf winged euonymus tissue culture pollution rate.Culture medium prescription provided by the invention can will remove from office the Contamination rate control of leaf winged euonymus tissue cultures within 10%, guarantee survival rate up to 95% or more simultaneously, leather leaf winged euonymus tissue-culturing rapid propagation production cost is greatlyd save, its tissue-culturing rapid propagation production efficiency is improved, is suitable for large-scale application and popularization.
Description
Technical field
The invention belongs to field of plant tissue culture technique more particularly to a kind of trainings for reducing leather leaf winged euonymus tissue culture pollution rate
Support base and its method.
Background technique
Leaf winged euonymus (Euonymus lecleri L é vl.) shrub or dungarunga are removed from office, it is 1-7 meters high, belong to Celastraceae Euonymus
Plant is Chinese endemic plant.The ground such as Hubei, Hunan, Sichuan, Guizhou are distributed in, are grown on 1,300 meters to 2,900 meters of height above sea level
Area, be grown on that mountainous region woods is shady and limes marginis more.Burning buss in landscape application using more universal, be chiefly used in isolated planting,
Group planting and mass-planting are excellent ornamental plants in garden and green tree species.Leaf winged euonymus is removed from office as a kind of evergreen species, resistance to shade is cold-resistant,
Resistance is strong, has very high ornamental and application value in northern area.With gradualling mature for " Nan Shubei drawing ", leaf winged euonymus is removed from office
Gradually it is widely used in garden landscape planning.
Key areas one of of the plant tissue culture technique as modern biotechnology, in agricultural production and batch production
It is widely used in terms of nursery, the quick breeding etc. of the preservation of plant fine germplasm resources, excellent variety is also played and is focused on
The effect wanted.
Currently, the tissue culture technology of leather leaf winged euonymus is still in conceptual phase.In sterile culture, make to remove from office leaf for various reasons
Winged euonymus tissue-cultured seedling is highly susceptible to pollute, and loses so as to cause some precious materials, this is undoubtedly to cause for tissue culture propagation
Life.And it is not yet had been reported that in relation to reducing the culture technique of leather leaf winged euonymus tissue culture pollution rate at present.Therefore, how to efficiently reduce
Pollution is lost caused by leather leaf winged euonymus tissue-cultured seedling, and building leather leaf winged euonymus Plant Tissue Breeding rapid propagation system is played to pass weight
The effect wanted.
Summary of the invention
In view of this, the present invention provides a kind of culture medium and its method for reducing leather leaf winged euonymus tissue culture pollution rate, the culture
Base can effectively control the pollution rate of leather leaf winged euonymus tissue-cultured seedling, while guarantee higher survival rate.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of culture mediums for reducing leather leaf winged euonymus tissue culture pollution rate, to contain 0.7-1.0mg/L 6-
BA, 0.1-0.4mg/L NAA, 50-80mg/L embrace the MS culture of Piao Xianggui essential oil, 20~30g/L sucrose and 7~8g/L agar
Base, pH value are 5.8~6.0.
It may make the Contamination rate control of leather leaf winged euonymus tissue-cultured seedling within 10% using culture medium prescription provided by the invention,
Guarantee that survival rate up to 95% or more, greatlys save leather leaf winged euonymus tissue-culturing rapid propagation production cost, improves the production of its tissue-culturing rapid propagation simultaneously
Efficiency is suitable for large-scale application and popularization.
Preferably, culture medium provided by the invention are as follows: contain 1.0mg/L 6-BA, 0.1mg/LNAA, 50mg/L, 30g/L
The MS culture medium of sucrose and 7g/L agar, pH value 5.90.
The present invention also provides a kind of methods for reducing leather leaf winged euonymus tissue culture pollution rate, comprising the following steps:
Step 1: leather leaf winged euonymus explant being inoculated on culture medium of any of claims 1 or 2, tissue-cultured seedling is obtained；
Step 2: after 10~15d of tissue-cultured seedling culture that the step 1 obtains, being transferred to containing 0.7-1.0mg/L6-BA, 0.1-
It is cultivated on the MS culture medium of 0.4mg/L NAA, 20~30g/L sucrose and 7~8g/L agar.
The present invention is directed to for the first time to be lowered the fast breeding technique of tissue culture pollution rate of leather leaf winged euonymus and is studied, by culture
That debita spissitudo is added in base embraces Piao Xianggui essential oil, and double induced medium methods, explant disinfection, explant pre-treatment is combined to change
Good operation etc., tissue culture seedling pollution rate is finally controlled within 10% under the premise of guaranteeing tissue culture shoot survival percent, guarantee simultaneously
Survival rate optimizes the production operation process of leather leaf winged euonymus tissue culture rapid propagation system, greatly reduces batch production and educate up to 95% or more
The time of seedling and cost.
Wherein, double induced medium methods refer to and carry out Fiber differentiation using two kinds of culture mediums, firstly, will remove from office outside leaf winged euonymus
Implant be seeded in containing 0.7-1.0mg/L 6-BA, 0.1-0.4mg/L NAA, 50-80mg/L, 20~30g/L sucrose and 7~
The MS culture medium of 8g/L agar, cultivate 10d after, then be transferred to containing 0.7-1.0mg/L 6-BA, 0.1-0.4mg/L NAA, 20~
The MS culture medium of 30g/L sucrose and 7~8g/L agar.
Preferably, further include explant disinfection before the inoculation of explant described in step 1, after explant disinfection again into
Row pre-treatment step.
Preferably, disinfection specifically: take explant to rinse 30-40min using flowing water, then soaked with 0.1% sodium hypochlorite
Steep 10min~15min.
In embodiment provided by the invention, the pre-treatment of explant are as follows: leather leaf winged euonymus explant → alcohol is taken to impregnate 30s
→ aseptic water washing 2 times, the mercuric chloride of each 2min → 0.1% impregnates 8min → aseptic water washing 2 times, and each 2min → change cup →
0.1% mercuric chloride impregnates 8min → aseptic water washing 2 times, each 2min → change cup → 0.1% mercuric chloride immersion 8min → sterile water punching
It washes 2min → change cup → aseptic water washing 3min → and changes cup → aseptic water washing 3min.
Preferably, the inoculation in step 1 specifically: bake two tweezers one before primary vaccination on alcolhol burner knife, one tweezer
Subprocessing explant, one is used to tweezers to be inoculated with；It before inoculation, is baked on alcolhol burner in advance another tweezers and knife, is placed on training
Support spare on ware, finished to the inoculation of this disk, then roasting one tweezers are placed in it is spare on culture dish, and with the one of prior high-temperature sterilization
Tweezers and knife are handled next disk explant, and so on, until inoculation finishes.
In embodiment provided by the invention, inoculation specifically: with being moistened with the absorbent cotton of 75% alcohol for knife, tweezers and connect
Discharge plate wipes one time, and high temperature is roasting once on alcolhol burner, is placed on inoculation disk.After tool is cooling, burning that will handle well
Explant in cup is put into inoculation disk, and explant is trimmed to 1~2cm segment by a 8-10 explant.Access culture medium
Before, tweezers are wiped again with alcohol absorbent cotton, on alcolhol burner after the roasted cooling of high temperature, access preprepared culture medium
In.
In the present invention, the stem section with axillary bud, blade or petiole are chosen as explant.Preferably, explant be selected from 2~
3 years seedling stalwartnesses and the stem section with axillary bud, blade or the petiole of no disease and pests harm.Wherein, the acquisition of explant are as follows: choose 2
~triennial seedling, the explant of acquisition current year raw healthy and strong and no disease and pests harm the stem section with axillary bud, blade or petiole as tissue cultures
Body is removed remaining worm's ovum, spot etc. on explant, is transferred in beaker, with cleaning with the clear water soaking and washing added with detergent
Gauze covers rim of a cup, and 30~50min is rinsed under flowing water.
Preferably, the present invention in, leather leaf winged euonymus tissue cultures cultivation temperature be 24~26 DEG C, light application time be 12~
18h/d, intensity of illumination are 2500~3500lx, and relative humidity is 45%~50%.
In embodiment provided by the invention, the cultivation temperature of leather leaf winged euonymus tissue cultures is 25 DEG C, and light application time is
12h/d, intensity of illumination 3500lx, relative humidity 48%.
It preferably, further include tissue culture room sterilization steps, tissue culture room follpet during removing from office leaf winged euonymus tissue cultures
Body are as follows: every morning opens ultraviolet lamp sterilization 30min at night, opens ozone generator weekly, sterilizes 15min.
Compared to traditional training method, the present invention embraces Piao Xianggui essential oil by add debita spissitudo into culture medium, and
In conjunction with double induced medium methods, explant disinfection and explant pre-treatment evolutionary operation etc., finally to remove from office leaf winged euonymus tissue-cultured seedling
Contamination rate control within 10%, while guaranteeing survival rate up to 95% or more, greatly save leather leaf winged euonymus tissue-culturing rapid propagation production
Cost improves its tissue-culturing rapid propagation production efficiency, is suitable for large-scale application and popularization.
Specific embodiment
The invention discloses a kind of methods and culture medium for reducing leather leaf winged euonymus tissue culture pollution rate, and those skilled in the art can
To use for reference present disclosure, it is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this
It is it will be apparent that they are considered as being included in the present invention for the technical staff of field.Method and application of the invention is
Be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to herein
The methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
To the explanation of the disclosed embodiments, enable those skilled in the art to implement or use the present invention.To this
A variety of modifications of a little embodiments will be readily apparent to those skilled in the art, as defined herein general
Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not
It can be intended to be limited to the embodiments shown herein, and be to fit to consistent with the principles and novel features disclosed in this article
Widest scope.
MS culture medium (unit mg/L) of the present invention includes:
A great number of elements: NH4NO31650, KNO31900, KH2PO41700, MgSO4·7H2O 3700, CaCl2664.4；
Microelement: KI 83, MnSO4H2O 1690, ZnSO4.7H2O 860, H3BO3620, Na2MoO4.2H2O 25,
CuSO4.5H2O 2.5CoCl2.6H2O 2.5；
Molysite: FeSO4.7H2O 2780, Na2.EDTA3730；
Organic principle: inositol 2000, IVB niacin 10, VB610, VB12, glycine 40.
The culture medium need to by high-pressure sterilizing pot sterilize 15-25min, 121 DEG C of high-pressure sterilizing pot operating temperature, pressure
115kPa。
The 6-BA is 6- benzyl purine, and the NAA is methyl α-naphthyl acetate, and embracing Piao Xianggui essential oil is one kind by Henan Acer palmatum ' Atropurpureum' kind
Seedling limited liability company embraces the essential oil with bacteriostasis for extracting and obtaining in Piao Xianggui from Lauraceae bay.The present invention mentions
Used medium component is available on the market in the culture medium and its method of the leather leaf winged euonymus tissue cultures of confession.
Below with reference to embodiment, the present invention is further explained:
The tissue cultures of the leather leaf winged euonymus of embodiment 1
1. growing environment: tissue culture room, light application time 12h/d, intensity of illumination 3500lx, cultivation temperature is 25 DEG C, relatively wet
Degree 48%；Every morning opens ultraviolet lamp sterilization 30min at night, opens ozone generator weekly, sterilizes 15min.
2. the processing of explant
(1) 2~3 years seedlings are chosen, current year acquires the tender stem section with bud of healthy growth no disease and pests harm children, as explant
Body.
(2) after the completion of acquiring, with the clear water soaking and washing added with detergent, remaining worm's ovum, spot on explant are removed
Deng being transferred in beaker, cover rim of a cup with clean gauze.
(3) 30~50min rinses under flowing water in elder generation, then dips the solution containing 0.1%NaClO with banister brush and cleans outside
Implant carries out disinfection to explant.
(4) after the completion of sterilizing, the preceding processing before being inoculated in superclean bench is successively proceeded as follows: being used
75% alcohol impregnates 2 mercuric chloride of 2min → 0.1% immersions 8min → 2 2min of aseptic water washing → of 30s → sterile water and changes cup (in advance
First sterilizing) → 0.1% mercuric chloride impregnates 8min → 2 2min of aseptic water washing → and changes cup → 0.1% mercuric chloride and impregnate 8min → sterile
Water rinses 2min → change cup → aseptic water washing 3min → and changes cup → aseptic water washing 3min.
3. pair induced medium culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, knife, tweezers and inoculation disk are wiped one time with the absorbent cotton for being moistened with 75% alcohol, and
High temperature is roasting once on alcolhol burner, is placed on inoculation disk.After tool is cooling, the explant in the beaker handled well is put into
It is inoculated in disk, a 8-10 explant.Explant is trimmed to 1~2cm.
(4) tweezers are wiped again with alcohol absorbent cotton, on alcolhol burner after the roasted cooling of high temperature, explant that will build
Be inoculated into dispensed containing 1.0mg/L 6-BA, 0.1mg/LNAA, 50mg/L embrace Piao Xianggui essential oil, 30g/L sucrose and 7g/L
In the culture medium of the MS culture medium of agar, 1 bottle of inoculation is to one.
(5) inoculated tissue-cultured seedling is transferred on the sterile tissue culture frame of the tissue culture room of above-mentioned condition of culture immediately and is trained
It supports, clean gown of a doctor and slippers are replaced in disengaging tissue culture room.
(6) after cultivating 10d, by tissue-cultured seedling be transferred to containing 1.0mg/L 6-BA, 0.1mg/LNAA, 30g/L sucrose and 7~
It is cultivated on the MS culture medium of 8g/L agar.
After cultivating 35d, the induction survival rate and pollution rate of tissue-cultured seedling are counted.
The tissue cultures of the leather leaf winged euonymus of embodiment 2
1. growing environment: tissue culture room, light application time 12h/d, intensity of illumination 3500lx, cultivation temperature is 25 DEG C, relatively wet
Degree 48%；Every morning opens ultraviolet lamp sterilization 30min at night, opens ozone generator weekly, sterilizes 15min.
2. the processing of explant
(1) 2~3 years seedlings are chosen, current year acquires the tender stem section with bud of healthy growth no disease and pests harm children, as explant
Body.
(2) after the completion of acquiring, with the clear water soaking and washing added with detergent, remaining worm's ovum, spot on explant are removed
Deng being transferred in beaker, cover rim of a cup with clean gauze.
(3) 30~50min rinses under flowing water in elder generation, then dips the solution containing 0.1%NaClO with banister brush and cleans outside
Implant carries out disinfection to explant.
(4) after the completion of sterilizing, the preceding processing before being inoculated in superclean bench is successively proceeded as follows: being used
75% alcohol impregnate 2 mercuric chloride of 2min → 0.1% of 30s → sterile water impregnate 8min → 2 2min of aseptic water washing → change cup →
0.1% mercuric chloride impregnates 8min → 2 2min of aseptic water washing → and changes cup → 0.1% mercuric chloride immersion 8min → aseptic water washing 2min
→ change cup → aseptic water washing 3min → and change cup → aseptic water washing 3min.
3. pair induced medium culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, knife, tweezers and inoculation disk are wiped one time with the absorbent cotton for being moistened with 75% alcohol, and
High temperature is roasting once on alcolhol burner, is placed on inoculation disk.After tool is cooling, the explant in the beaker handled well is put into
It is inoculated in disk, a 8-10 explant.Explant is trimmed to 1~2cm.
(4) tweezers are wiped again with alcohol absorbent cotton, on alcolhol burner after the roasted cooling of high temperature, explant that will build
It is inoculated into contain 1.0mg/L6-BA, 0.1mg/LNAA, the 60mg/L dispensed and embraces Piao Xianggui essential oil, 30g/L sucrose and 7g/L fine jades
In the MS culture medium of rouge, 1 bottle of inoculation is to one.
(5) inoculated tissue-cultured seedling is transferred on the sterile tissue culture frame of the tissue culture room of above-mentioned condition of culture immediately and is trained
It supports, clean gown of a doctor and slippers are replaced in disengaging tissue culture room.
(6) after cultivating 10d, tissue-cultured seedling is transferred to containing 1.0mg/L 6-BA, 0.1mg/L NAA, 25g/L sucrose and 8g/L
It is cultivated on the MS culture medium of agar.
After cultivating 35d, the induction survival rate and pollution rate of tissue-cultured seedling are counted.
The tissue cultures of the leather leaf winged euonymus of embodiment 3
1. growing environment: tissue culture room, light application time 12h/d, intensity of illumination 3500lx, cultivation temperature is 25 DEG C, relatively wet
Degree 48%；Every morning opens ultraviolet lamp sterilization 30min at night, opens ozone generator weekly, sterilizes 15min.
2. the processing of explant
(1) 2~3 years seedlings are chosen, current year acquires the tender stem section with bud of healthy growth no disease and pests harm children, as explant
Body.
(2) after the completion of acquiring, with the clear water soaking and washing added with detergent, remaining worm's ovum, spot on explant are removed
Deng being transferred in beaker, cover rim of a cup with clean gauze.
(3) 30~50min rinses under flowing water in elder generation, then dips the solution containing 0.1%NaClO with banister brush and cleans outside
Implant carries out disinfection to explant.
(4) after the completion of sterilizing, the preceding processing before being inoculated in superclean bench is successively proceeded as follows: being used
75% alcohol impregnate 2 mercuric chloride of 2min → 0.1% of 30s → sterile water impregnate 8min → 2 2min of aseptic water washing → change cup →
0.1% mercuric chloride impregnates 8min → 2 2min of aseptic water washing → and changes cup → 0.1% mercuric chloride immersion 8min → aseptic water washing 2min
→ change cup → aseptic water washing 3min → and change cup → aseptic water washing 3min.
3. pair induced medium culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, knife, tweezers and inoculation disk are wiped one time with the absorbent cotton for being moistened with 75% alcohol, and
High temperature is roasting once on alcolhol burner, is placed on inoculation disk.After tool is cooling, the explant in the beaker handled well is put into
It is inoculated in disk, a 8-10 explant.Explant is trimmed to 1~2cm.
(4) tweezers are wiped again with alcohol absorbent cotton, on alcolhol burner after the roasted cooling of high temperature, explant that will build
It is inoculated into contain 0.8mg/L 6-BA, 0.2mg/L NAA, the 60mg/L dispensed and embraces Piao Xianggui essential oil, 30g/L sucrose and 7g/L
In the MS culture medium of agar, 1 bottle of inoculation is to one.
(5) inoculated tissue-cultured seedling is transferred on the sterile tissue culture frame of the tissue culture room of above-mentioned condition of culture immediately and is trained
It supports, clean gown of a doctor and slippers are replaced in disengaging tissue culture room.
(6) after cultivating 10d, tissue-cultured seedling is transferred to containing 0.8mg/L 6-BA, 0.2mg/L NAA, 25g/L sucrose and 8g/L
It is cultivated on the MS culture medium of agar.
After cultivating 35d, the induction survival rate and pollution rate of tissue-cultured seedling are counted.
The tissue cultures of the leather leaf winged euonymus of embodiment 4
1. growing environment: tissue culture room, light application time 12h/d, intensity of illumination 3500lx, cultivation temperature is 25 DEG C, relatively wet
Degree 48%；Every morning opens ultraviolet lamp sterilization 30min at night, opens ozone generator weekly, sterilizes 15min.
2. the processing of explant
(1) 2~3 years seedlings are chosen, current year acquires the tender stem section with bud of healthy growth no disease and pests harm children, as explant
Body.
(2) after the completion of acquiring, with the clear water soaking and washing added with detergent, remaining worm's ovum, spot on explant are removed
Deng being transferred in beaker, cover rim of a cup with clean gauze.
(3) 30~50min rinses under flowing water in elder generation, then dips the solution containing 0.1%NaClO with banister brush and cleans outside
Implant carries out disinfection to explant.
(4) after the completion of sterilizing, the preceding processing before being inoculated in superclean bench is successively proceeded as follows: being used
75% alcohol impregnate 2 mercuric chloride of 2min → 0.1% of 30s → sterile water impregnate 8min → 2 2min of aseptic water washing → change cup →
0.1% mercuric chloride impregnates 8min → 2 2min of aseptic water washing → and changes cup → 0.1% mercuric chloride immersion 8min → aseptic water washing 2min
→ change cup → aseptic water washing 3min → and change cup → aseptic water washing 3min.
3. pair induced medium culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, knife, tweezers and inoculation disk are wiped one time with the absorbent cotton for being moistened with 75% alcohol, and
High temperature is roasting once on alcolhol burner, is placed on inoculation disk.After tool is cooling, the explant in the beaker handled well is put into
It is inoculated in disk, a 8-10 explant.Explant is trimmed to 1~2cm.
(4) tweezers are wiped again with alcohol absorbent cotton, on alcolhol burner after the roasted cooling of high temperature, explant that will build
It is inoculated into contain 0.8mg/L6-BA, 0.4mg/L NAA, the 50mg/L dispensed and embraces Piao Xianggui essential oil, 30g/L sucrose and 7g/L
In the MS culture medium of agar, 1 bottle of inoculation is to one.
(5) inoculated tissue-cultured seedling is transferred on the sterile tissue culture frame of the tissue culture room of above-mentioned condition of culture immediately and is trained
It supports, clean gown of a doctor and slippers are replaced in disengaging tissue culture room.
(6) after cultivating 10d, tissue-cultured seedling is transferred to containing 0.8mg/L 6-BA, 0.4mg/L NAA, 25g/L sucrose and 8g/L
It is cultivated on the MS culture medium of agar.
After cultivating 35d, the induction survival rate and pollution rate of tissue-cultured seedling are counted.
Embodiment 5 removes from office leaf winged euonymus method for tissue culture comparative test
Control group 1: the culture medium of explant inoculation is to contain 1.0mg/L 6-BA, 0.1mg/LNAA, 30g/L sucrose and 7
The MS culture medium of~8g/L agar does not add and embraces Piao Xianggui essential oil；Meanwhile pre-treatment is using following operation: 75% alcohol impregnates
30s → aseptic water washing 2 times each mercuric chloride of 2min → 0.1% impregnates 8min → aseptic water washing 5-6 times.In addition to this, other
Condition of culture is same as Example 1.
Control group 2: the culture medium of explant inoculation is to contain 1.0mg/L 6-BA, 0.1mg/LNAA, 30g/L sucrose and 7
The MS culture medium of~8g/L agar does not add and embraces Piao Xianggui essential oil, and in addition to this, other condition of culture are same as Example 1.
Control group 3: explant inoculation culture medium be containing 1.0mg/L 6-BA, 0.1mg/LNAA, 200mg/L Cef,
The MS culture medium of 30g/L sucrose and 7~8g/L agar, in addition to this, other condition of culture are same as Example 1.
Control group 4: explant inoculation culture medium be containing 1.0mg/L 6-BA, 0.1mg/LNAA, 150mg/L Cb,
The MS culture medium of 30g/L sucrose and 7~8g/L agar, in addition to this, other condition of culture are same as Example 1.
Respectively Statistics Implementation example 1~4, control group 1~4 cultural method obtain tissue-cultured seedling pollution rate and induction survive
Rate, the results are shown in Table 1.
Table 1 removes from office leaf winged euonymus pollution rate and induces the statistical analysis of survival rate
| Group | Pollution rate (%) | It induces survival rate (%) |
| Embodiment 1 | 8 | 96 |
| Embodiment 2 | 10 | 95 |
| Embodiment 3 | 9 | 95 |
| Embodiment 4 | 10 | 97 |
| Control group 1 | 69 | 54 |
| Control group 2 | 45 | 40 |
| Control group 3 | 15 | 30 |
| Control group 4 | 15 | 35 |
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of induced medium for reducing leather leaf winged euonymus tissue culture pollution rate, which is characterized in that the culture medium are as follows: contain 0.7-
1.0mg/L 6-BA, 0.1-0.4mg/L NAA, 50-80mg/L embrace Piao Xianggui essential oil, 20 ~ 30g/L sucrose and 7 ~ 8g/L agar
MS culture medium, pH value are 5.86 ~ 5.90.
2. induced medium according to claim 1, which is characterized in that the culture medium are as follows: contain 1.0mg/L 6-BA
, 0.1mg/L NAA, 50mg/L embrace Piao Xianggui essential oil, 30g/L sucrose and 7g/L agar MS culture medium, pH value 5.90.
3. a kind of method for reducing leather leaf winged euonymus tissue culture pollution rate using as claimed in claim 1 or 22 induced mediums, feature exist
In including the following steps:
Step 1: leather leaf winged euonymus explant being inoculated on culture medium of any of claims 1 or 2, tissue-cultured seedling is obtained；
Step 2: after 10 ~ 15d of tissue-cultured seedling culture that the step 1 obtains, being transferred to containing 0.8-1.0mg/L 6-BA, 0.1-
It is cultivated on the MS culture medium of 0.3mg/L NAA, 20 ~ 30g/L sucrose and 7 ~ 8g/L agar.
4. according to the method described in claim 3, it is characterized in that, further including explant before the inoculation of explant described in step 1
Sterilisation step carries out pre-treatment step after the explant disinfection again；
The pre-treatment specifically: take leather leaf winged euonymus explant → alcohol to impregnate 30s → aseptic water washing 2 times, each 2min →
0.1% mercuric chloride impregnates 8min → aseptic water washing 2 times, each 2min → change cup → 0.1% mercuric chloride immersion 8min → aseptic water washing 2
Secondary, each 2min → change cup → 0.1% mercuric chloride, which impregnates 8min → aseptic water washing 2min → and changes cup → aseptic water washing 3min →, to be changed
Cup → aseptic water washing 3min.
5. according to the method described in claim 4, it is characterized in that, the disinfection specifically: explant is taken to rinse using flowing water
30-40min, then 10-15min is impregnated with 0.1% sodium hypochlorite.
6. according to the method described in claim 3, it is characterized in that, being inoculated with described in step 1 specifically:
Two tweezers one are baked before primary vaccination on alcolhol burner, tweezers are handled explant by knife, one, one is used to tweezers to be inoculated with；It connects
Kind before, baked on alcolhol burner in advance it is another tweezers and knife, be placed on it is spare on culture dish, to this disk inoculation finish, then bake one
Tweezers are placed in spare on culture dish, and tweezers and knife are handled next disk explant with the one of prior high-temperature sterilization, with this
Analogize, until inoculation finishes.
7. according to the method described in claim 3, it is characterized in that, the leather leaf winged euonymus explant is the stem section with axillary bud, leaf
Piece or petiole.
8. according to the described in any item methods of claim 3 to 7, which is characterized in that the culture of the leather leaf winged euonymus tissue cultures
Temperature is 24 ~ 26 DEG C, and light application time is 12 ~ 18h/d, and intensity of illumination is 2000 ~ 2500lx, and relative humidity is 45% ~ 50%.
9. further including tissue culture during the leather leaf winged euonymus tissue cultures according to claim 3 to 7 described in any item methods
Room sterilization steps, the tissue culture room sterilizing specifically: every morning opens ultraviolet lamp sterilization 30min at night, opens weekly smelly
Oxygen Generator sterilizes 15min.
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