CN105660411B - The tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum - Google Patents
The tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum Download PDFInfo
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- 235000007516 Chrysanthemum Nutrition 0.000 title claims abstract description 24
- 238000012258 culturing Methods 0.000 title claims abstract description 23
- 244000144725 Amygdalus communis Species 0.000 title claims abstract description 22
- 235000011437 Amygdalus communis Nutrition 0.000 title claims abstract description 22
- 235000020224 almond Nutrition 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 19
- 244000189548 Chrysanthemum x morifolium Species 0.000 title 1
- 241000723353 Chrysanthemum Species 0.000 claims abstract description 23
- 230000001939 inductive effect Effects 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000012879 subculture medium Substances 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 6
- 238000005520 cutting process Methods 0.000 claims abstract description 5
- 230000010496 root system development Effects 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 238000002791 soaking Methods 0.000 claims abstract description 3
- 241000196324 Embryophyta Species 0.000 claims description 16
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 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 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 5
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 3
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 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 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 241000607479 Yersinia pestis Species 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 3
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 3
- 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 claims description 3
- 229960000367 inositol Drugs 0.000 claims description 3
- 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 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- BZDIAFGKSAYYFC-UHFFFAOYSA-N manganese;hydrate Chemical compound O.[Mn] BZDIAFGKSAYYFC-UHFFFAOYSA-N 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 239000005416 organic matter Substances 0.000 claims description 3
- 239000004323 potassium nitrate Substances 0.000 claims description 3
- 235000010333 potassium nitrate Nutrition 0.000 claims description 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 3
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 3
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 3
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 3
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 235000009529 zinc sulphate Nutrition 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 230000001932 seasonal effect Effects 0.000 abstract description 3
- 229940088597 hormone Drugs 0.000 description 6
- 239000005556 hormone Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000005286 illumination Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- VGKONPUVOVVNSU-UHFFFAOYSA-N naphthalen-1-yl acetate Chemical compound C1=CC=C2C(OC(=O)C)=CC=CC2=C1 VGKONPUVOVVNSU-UHFFFAOYSA-N 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000003415 peat Substances 0.000 description 4
- 239000010451 perlite Substances 0.000 description 4
- 235000019362 perlite Nutrition 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 238000004017 vitrification Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 3
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 2
- 235000004109 Gymnanthemum amygdalinum Nutrition 0.000 description 2
- 241001635503 Gymnanthemum amygdalinum Species 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 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 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000012286 potassium permanganate Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 238000012090 tissue culture technique Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- RMOGWMIKYWRTKW-UONOGXRCSA-N (S,S)-paclobutrazol Chemical compound C([C@@H]([C@@H](O)C(C)(C)C)N1N=CN=C1)C1=CC=C(Cl)C=C1 RMOGWMIKYWRTKW-UONOGXRCSA-N 0.000 description 1
- 244000144730 Amygdalus persica Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000409198 Packera aurea Species 0.000 description 1
- 239000005985 Paclobutrazol Substances 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 244000145469 Vernonia anthelmintica Species 0.000 description 1
- 235000013018 Vernonia anthelmintica Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- -1 iginate Species 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 235000013311 vegetables Nutrition 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
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum, comprise the following steps：1) explant is gathered：Tender stem section is gathered, blade is removed, clear water is standby as explant after rinsing；2) explant is sterilized：To step 1) obtained explant carries out soaking disinfection, and use aseptic water washing；3) Initial culture：By step 2) the obtained explant disinfected is inoculated in inducing culture and cultivated；4) squamous subculture：By step 3) the adventitious bud separation cutting that is formed in inducing culture, cultivated in subculture medium of transferring；5) culture of rootage：Selecting step 4) the obtained propagation seedling of robust growth is transferred in root media and cultivated；6) transplant：Treat that root system development is completed, wash away base portion culture medium, hardening is transplanted into Light media after 10 days.This method has the advantages that sapling multiplication speed is fast, it is not good by seasonal effect, transplanting survival rate height, seedling uniformity to produce, and process is provided for the scale and standardized production of almond ringdove chrysanthemum.
Description
Technical field
The present invention relates to the tissue-culturing rapid propagation nursery side of field of plant tissue culture technique, more particularly to almond ringdove chrysanthemum
Method.
Background technology
Almond ringdove chrysanthemum (Vernonia amygdalina Del.) also known as peach leaf ringdove chrysanthemum, apricot leaf ringdove chrysanthemum, magical tree,
Bitter tree, Nan Feishu, South Africa leaf, are ironweed plant, originate in Africa.Almond ringdove chrysanthemum leaf can with safe edible,
A kind of vegetables are taken as on Nigeria and other places.Its blade has unique smell and bitterness sense, thus is commonly referred to as bitter leaf, can
It is used as medicine, the great potential in terms for the treatment of tumour especially prevents and treats breast cancer, hypotensive.
Almond ringdove chrysanthemum is more in the ground such as Southeast Asia and Taiwan Popular Utilization, and China's Mainland is then relatively strange, closely
Guangdong and Guangxi Provinces area has introduction successively over year, but maternal quantity and related application research are less.Its nothing is set up using tissue culture technique
Property fast breeding technique system, its breeding coefficient, the quality realized whole year production, ensure seedling can be improved, and be conducive to germplasm
Resource conservation, accelerates to promote its industrialization and standardized production, also provides technology platform for deeper research.
The content of the invention
It is an object of the invention to provide a kind of tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum, this method has sapling multiplication
Speed is fast, produce not by seasonal effect, transplanting survival rate is high, seedling uniformity is good the advantages of, be the scale of almond ringdove chrysanthemum
Process is provided with standardized production.
The tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum of the present invention, comprises the following steps：
1) explant is gathered：Tender stem section is gathered, blade is removed, clear water is standby as explant after rinsing；
2) explant is sterilized：To step 1) obtained explant carries out soaking disinfection, and use aseptic water washing；
3) Initial culture：By step 2) the obtained explant disinfected is inoculated in inducing culture and trained
Support；
4) squamous subculture：By step 3) the adventitious bud separation cutting that is formed in inducing culture, transfer into subculture medium
It is middle to be cultivated；
5) culture of rootage：Selecting step 4) the obtained propagation seedling of robust growth is transferred in root media and cultivated；
6) transplant：Completed after root system development and after hardening, wash away base portion culture medium, be transplanted into Light media.
Step 1 of the present invention) described in the tender stem section of collection, refer to choose robust growth, the elite plant of no disease and pests harm,
The lighter spray of giving birth to then of clip degree of lignification is explant.
Step 2) described in explant sterilization, preferably by step 1) obtained explant is cut into long 1.2-1.5cm belt segment
Segment, cuts off blade, only retains petiole base (being about 0.5cm), it is standby that flowing water rinses 20min；On superclean bench, use
75% (v/v) alcohol-pickled 10s, 0.1% mercuric chloride solution vibration sterilization 8min, then wash away residual liquor 5 times with aseptic water washing,
After aseptic filter paper suck dry moisture, cut stem section two ends and petiole top and (retain length about 0.2cm) a little, it is standby.
Step 3) described in Initial culture, that is, sterile bud is induced, by step 2 on superclean bench) obtained explant
Stem section is inoculated on inducing culture, and inducing culture is additional 6- benzyls aminoadenine (6-BA) 0.5mg/ of MS minimal mediums
L and methyl α-naphthyl acetate (NAA) 0.05mg/L, i.e. MS+6-BA0.5mg/L+NAA0.05mg/L, sucrose and agar powder addition are respectively
Per L culture medium 30g, 6g, medium pH is adjusted to 6.0 before sterilizing, and culture room temperature is (23 ± 2) DEG C, intensity of illumination 1500-
2500lx, light application time is 12h/d.
In inducing culture, lateral bud is easy to induce and grown rapidly, and inductivity is up to 70%, with spring and summer Disinfection Effect
More preferably；During Initial culture 3 weeks, lateral bud may be up to 3-4cm, and tool 3 can be transferred to subculture medium and be carried out subculture training with blade
Support.
Step 4) described in squamous subculture, when sterile bud grows to 3-4cm, be segmented cutting, subculture be transferred to paragraph by paragraph
Culture medium is cultivated, and subculture switching in every 3 weeks or so is once；Culturing room's light application time is 16h/d, the same primary of other condition of culture
Culture；
Subculture medium is MS-z+6-BA0.2mg/L+IBA0.05mg/L+PP333 (paclobutrazol) 0.05mg/L, described
Contained component and concentration (mg/L) are as follows in every liter of MS-z culture mediums：
A great number of elements：Potassium nitrate 1900, ammonium nitrate 412.5, calcium chloride dihydrate 880, epsom salt 370, biphosphate
Potassium 170, ferrous sulfate heptahydrate 27.8, disodium ethylene diamine tetraacetate 37.3；
Trace element：Four water manganese sulfates 22.3, white vitriol 8.6, boric acid 3.1, Sodium Molybdate Dihydrate 0.25, KI
0.83rd, cupric sulfate pentahydrate 0.025, CoCL2 6H2O 0.025；
Organic matter：Inositol 100.0, glycine 2.0, puridoxine hydrochloride (VB6) 0.5, nicotinic acid 0.5, thiamine hydrochloride (VB1)
0.1；
It is other：Agar 8000, sucrose 40000, pH 6.0；
The each subculture cycle growth coefficient of almond ringdove chrysanthemum is up to 4.0, and test tube seedling growth is very fast, and incubation time is more than 4
Easily there is yellow leaf and the dead situation of budlet, influence propagation efficiency week.Test tube seedling is more sensitive to hormone, and 6-BA concentration exceedes
Easily there is vitrification phenomenon during 0.3mg/L, when concentration reaches 0.5mg/L, vitrification phenomenon is particularly acute, therefore is cultivated
Cheng Zhongxu strictly controls hormone concentration.When intensity of illumination is 1500-2500lx, the preferred 16h of light application time.
Step 5) described in culture of rootage, preferably choose the number of blade more than 4, more than height 2cm propagation seedling and be transferred to life
Root culture medium is cultivated, and root media is used as minimal medium (only a great number of elements halves), additional indoles fourth from 1/2MS
Sour IBA0.1mg/L, i.e. 1/2MS+IBA0.1mg/L, light application time are daily 16h, to promote robust plant；Culture of rootage 2 weeks
When, rooting rate is up to 100%, and plant height 5cm or so, plant strain growth is healthy and strong, well developed root system；Medium supplemented hormone concentration IBA
More than 0.15mg/L, plant excessive growth can be caused, internode is long；Easily there is callus more than 0.05mg/L in NAA concentration, influence
Quality of rooting and transplanting survival rate.
Step 6) described in transplanting, preferred culture of rootage 2 weeks and after hardening canopy hardening 10 days is transplanted into Light media container
In, Light media is the mixed-matrix (peat soil of peat soil and perlite：Perlite=3:1) the previous day, is transplanted first with 0.5%
(w/w) disinfecting solution of potassium permanganate is standby, is strengthened management after transplanting, is available for field production or hillside to make when height of seedling 30cm
Woods, transplanting survival rate is up to more than 95%.
Compared with prior art, the invention has the advantages that：
1st, the sapling multiplication of almond ringdove chrysanthemum is carried out using tissue culture technique first, a set of efficient tissue culture is established fast
Propagating technology system, this method energy rapid, high volume breeding test tube seedling, is not subject to seasonal restrictions, is also beneficial to the preservation of germ plasm resource.
2nd, the test tube seedling breeding of the entirely appropriate almond ringdove chrysanthemum of technical matters system in the present invention.Almond ringdove chrysanthemum test tube
Seedling is more sensitive to hormone, very sensitive to 6-BA and NAA concentration, and vitrifying easily occurs in squamous subculture, and 6-BA concentration surpasses
Easily there is vitrification phenomenon when crossing 0.3mg/L, when concentration reaches 0.5mg/L, vitrification phenomenon is particularly acute, therefore culture
During need strictly to control hormone concentration.Additional hormone concentration IBA can cause plant apprentice more than 0.15mg/L in root media
Long, internode is long, and NAA concentration callus, influence quality of rooting and transplanting survival rate easily occurs more than 0.05mg/L.The present invention exists
On the basis of lot of experiments is groped, squamous subculture based formulas, prescription of rooting medium and each link condition of culture have been done greatly
The experiment of amount, the method for obtaining the present invention.
3rd, by the techniqueflow in the present invention, obtained almond ringdove chrysanthemum tissue-culture container seedling and transplant bag seedling robust plant,
Survival rate is high, uniformity is good, is convenient for standardized production, is easy to implement industrialization.During culture of rootage 2 weeks, rooting rate is reachable
100%, plant height 5cm or so, plant strain growth are healthy and strong, well developed root system；Transplanting survival rate is up to more than 95%.
Embodiment
With embodiment, the invention will be further described below, but the invention is not limited in these are implemented
Example.Embodiment：
The tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum, comprises the following steps：
1) explant is gathered：Robust growth, the elite plant of no disease and pests harm are chosen, clip degree of lignification is lighter then
Raw spray is explant；
2) explant is sterilized：The stem section adopted back is cut into long 1.2-1.5cm belt segment segment, blade is cut off, only retains leaf
Handle base portion (is about 0.5cm), and it is standby that flowing water rinses 20min；On superclean bench, with 75% alcohol-pickled 10s, 0.1% liter
Mercury vibration sterilization 8min, then wash away residual liquor 5 times with aseptic water washing, after aseptic filter paper suck dry moisture, cut stem section two
End and petiole top are a little (retaining length about 0.2cm), standby；
3) Initial culture, that is, induce sterile bud, by step 2 on superclean bench) obtained explant stem section is inoculated in
On inducing culture, inducing culture is additional 6- benzyls aminoadenine (6-BA) 0.5mg/L of MS minimal mediums and methyl α-naphthyl acetate
(NAA) 0.05mg/L, i.e. MS+6-BA0.5mg/L+NAA0.05mg/L, sucrose and agar powder addition are respectively per L culture mediums
30g, 6g, medium pH is adjusted to 6.0 before sterilizing, and culture room temperature is (23 ± 2) DEG C, intensity of illumination 1500-2500lx, illumination
Time is 12h/d；
In inducing culture, lateral bud is easy to induce and grown rapidly, and inductivity is up to 70%, with spring and summer Disinfection Effect
More preferably；During Initial culture 3 weeks, lateral bud may be up to 3-4cm, and tool 3 can be transferred to subculture medium and be carried out subculture training with blade
Support；
4) squamous subculture, when sterile bud grows to 3-4cm, is segmented cutting, and subculture medium progress is transferred to paragraph by paragraph
Culture, subculture switching in every 3 weeks or so is once；Culturing room's light application time is 16h/d, the same Initial culture of other condition of culture；Illumination
When intensity is 1500-2500lx, light application time 16h；
Subculture medium is MS-z+6-BA0.2mg/L+IBA0.05mg/L+PP3330.05mg/L, described MS-z cultures
Contained component and concentration (mg/L) are as follows in every liter of base：
A great number of elements：Potassium nitrate 1900, ammonium nitrate 412.5, calcium chloride dihydrate 880, epsom salt 370, biphosphate
Potassium 170, ferrous sulfate heptahydrate 27.8, disodium ethylene diamine tetraacetate 37.3；
Trace element：Four water manganese sulfates 22.3, white vitriol 8.6, boric acid 3.1, Sodium Molybdate Dihydrate 0.25, KI
0.83rd, cupric sulfate pentahydrate 0.025, CoCL2 6H2O 0.025；
Organic matter：Inositol 100.0, glycine 2.0, puridoxine hydrochloride (VB6) 0.5, nicotinic acid 0.5, thiamine hydrochloride (VB1)
0.1；
It is other：Agar 8000, sucrose 40000, pH 6.0；
5) culture of rootage：The number of blade more than 4, more than height 2cm test tube seedling is chosen to be transferred to root media and trained
Support.Root media from 1/2MS as minimal medium (only a great number of elements halves), additional indolebutyric acid IBA0.1mg/L,
That is 1/2MS+IBA0.1.Light application time is daily 16h, to promote robust plant.During culture of rootage 2 weeks, rooting rate is reachable
100%, plant height 5cm or so, plant strain growth are healthy and strong, well developed root system.
6) transplant：After culture of rootage 2 weeks, root system development is completed, and in hardening canopy hardening 10 days, is washed away culture medium, is transplanted into
In Light media container.Light media is the mixed-matrix (peat soil of peat soil and perlite：Perlite=3:1) the previous day, is transplanted
It is first standby with 0.5% (w/w) disinfecting solution of potassium permanganate, strengthened management after transplanting, field production is available for when height of seedling 30cm
Or hillside afforestation, transplanting survival rate is up to more than 95%.
The influence that the different culture media formula of table 1 grows to almond ringdove chrysanthemum Regenerated plant
Claims (4)
1. the tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum, it is characterised in that comprise the following steps：
1) explant is gathered：Tender stem section is gathered, blade is removed, clear water is standby as explant after rinsing；
2) explant is sterilized：To step 1) obtained explant carries out soaking disinfection, and use aseptic water washing；
3) Initial culture：By step 2) the obtained explant disinfected is inoculated in inducing culture and cultivated；
4) squamous subculture：By step 3) the adventitious bud separation cutting that is formed in inducing culture, enter in subculture medium of transferring
Row culture；
5) culture of rootage：Selecting step 4) the obtained propagation seedling of robust growth is transferred in root media and cultivated；
6) transplant：Completed after root system development and after hardening, wash away base portion culture medium, be transplanted into Light media；
Step 3) described in inducing culture be MS+6-BA0.5mg/L+NAA0.05mg/L；
Step 4) described in subculture medium be MS-z+6-BA0.2mg/L+IBA0.05mg/L+PP3330.05mg/L, it is described
Contained component and concentration mg/L are as follows in every liter of MS-z culture mediums：
A great number of elements：Potassium nitrate 1900, ammonium nitrate 412.5, calcium chloride dihydrate 880, epsom salt 370, potassium dihydrogen phosphate
170th, ferrous sulfate heptahydrate 27.8, disodium ethylene diamine tetraacetate 37.3；
Trace element：Four water manganese sulfates 22.3, white vitriol 8.6, boric acid 3.1, Sodium Molybdate Dihydrate 0.25, KI 0.83,
Cupric sulfate pentahydrate 0.025, CoCL2 6H2O 0.025；
Organic matter：Inositol 100.0, glycine 2.0, puridoxine hydrochloride 0.5, nicotinic acid 0.5, thiamine hydrochloride 0.1；
It is other：Agar 8000, sucrose 40000；pH 6.0；
Step 5) described in root media be 1/2MS+IBA0.1mg/L.
2. the tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum according to claim 1, it is characterised in that：Step 1) described in
Gather tender stem section, refer to choose robust growth, the elite plant of no disease and pests harm, clip degree of lignification it is light give birth to tender then
Branch is explant.
3. the tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum according to claim 1, it is characterised in that：Step 2) described in
Explant is sterilized, and is by step 1) obtained explant is cut into long 1.2-1.5cm belt segment segment, and blade is cut off, only retains leaf
Handle base portion, it is standby that flowing water rinses 20min；On superclean bench, with 75% alcohol-pickled 10s, the vibration of 0.1% mercuric chloride solution disappears
Malicious 8min, then wash away residual liquor 5 times with aseptic water washing, after aseptic filter paper suck dry moisture, cut stem section two ends and petiole
Top, it is standby.
4. the tissue-culturing rapid propagation method for culturing seedlings of almond ringdove chrysanthemum according to claim 1, it is characterised in that：Step 6) described in
Transplant, be culture of rootage 2 weeks, root system development is completed and hardening is after 10 days, is transplanted into Light media container.
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| CN105191792A (en) * | 2015-09-08 | 2015-12-30 | 深圳市铁汉生态环境股份有限公司 | Intermediate propagation method of vernonia amygdalina |
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| CN105191792A (en) * | 2015-09-08 | 2015-12-30 | 深圳市铁汉生态环境股份有限公司 | Intermediate propagation method of vernonia amygdalina |
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| 驱虫斑鸠菊的组织培养与快速繁殖;胡石开等;《植物生理学通讯》;20080430;第44卷(第2期);第310页 * |
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