CN116671437A - Acer truncatum culture medium and application thereof - Google Patents
Acer truncatum culture medium and application thereof Download PDFInfo
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- CN116671437A CN116671437A CN202310565288.6A CN202310565288A CN116671437A CN 116671437 A CN116671437 A CN 116671437A CN 202310565288 A CN202310565288 A CN 202310565288A CN 116671437 A CN116671437 A CN 116671437A
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- China
- Prior art keywords
- acer truncatum
- medium
- callus
- acer
- embryogenic callus
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- 241000219226 Acer truncatum Species 0.000 title claims abstract description 136
- 239000001963 growth medium Substances 0.000 title claims abstract description 21
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 138
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims abstract description 93
- 230000000408 embryogenic effect Effects 0.000 claims abstract description 92
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000002609 medium Substances 0.000 claims abstract description 43
- 229930191978 Gibberellin Natural products 0.000 claims abstract description 9
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000003448 gibberellin Substances 0.000 claims abstract description 9
- 230000001939 inductive effect Effects 0.000 claims abstract description 7
- 239000007640 basal medium Substances 0.000 claims description 24
- 239000005556 hormone Substances 0.000 claims description 14
- 229940088597 hormone Drugs 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 11
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 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 9
- 239000005720 sucrose Substances 0.000 claims description 9
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- 238000009395 breeding Methods 0.000 claims description 7
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 7
- 235000021240 caseins Nutrition 0.000 claims description 7
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 6
- 230000006698 induction Effects 0.000 abstract description 65
- 238000000034 method Methods 0.000 abstract description 39
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 13
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- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 abstract 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 abstract 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 18
- 230000012010 growth Effects 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
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- 238000012360 testing method Methods 0.000 description 10
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- 239000004062 cytokinin Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 241000208140 Acer Species 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 210000001082 somatic cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
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- 239000008223 sterile water Substances 0.000 description 5
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- 229930192334 Auxin Natural products 0.000 description 4
- 239000002363 auxin Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 4
- 238000004161 plant tissue culture Methods 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000005708 Sodium hypochlorite Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- 230000000392 somatic effect Effects 0.000 description 3
- 230000030118 somatic embryogenesis Effects 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
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- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 2
- 239000012883 rooting culture medium Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- PCRHXLPZZNPXMQ-UHFFFAOYSA-N 2-n-benzyl-7h-purine-2,6-diamine Chemical compound N=1C=2NC=NC=2C(N)=NC=1NCC1=CC=CC=C1 PCRHXLPZZNPXMQ-UHFFFAOYSA-N 0.000 description 1
- 241000931515 Acer palmatum Species 0.000 description 1
- 240000004144 Acer rubrum Species 0.000 description 1
- 235000004476 Acer rubrum Nutrition 0.000 description 1
- 241001143500 Aceraceae Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 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
- 241000238631 Hexapoda Species 0.000 description 1
- -1 IAA Chemical compound 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- LXNHXLLTXMVWPM-UHFFFAOYSA-N Vitamin B6 Natural products CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002242 embryoid body Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 229960000367 inositol Drugs 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
- 159000000014 iron salts Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000000877 morphologic effect Effects 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
- 210000000056 organ Anatomy 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 150000003839 salts Chemical class 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
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 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 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
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- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 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
- A01H4/002—Culture media 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Acer truncatum is taken as a arbor with great ornamental value and economic value, and scientific researchers have conducted a great deal of research on leaf color, unsaturated fatty acid in seeds and the like. In order to clarify the action mechanism of candidate genes in Acer truncatum, researchers need to establish an Acer truncatum genetic transformation system first. The invention relates to an induction method of acer truncatum embryo callus, which comprises the following steps: the embryogenic callus induction culture medium provided by the invention is used for culturing Acer truncatum, so that the embryogenic callus induction probability of Acer truncatum can be improved. The embryogenic callus induction medium is selected from the group consisting of: 1/2MS+0.5mg/L indolebutyric acid+1.0 mg/L thidiazuron+3.0 mg/L6-benzylaminopurine+0.1 mg/L gibberellin; 1/2MS+3.0 mg/L6-benzylaminopurine+2.0 mg/L thidiazuron+0.5 mg/L indolebutyric acid+0.1 mg/L gibberellin. The method for inducing the embryo callus of the acer truncatum is not only suitable for researching an acer truncatum genetic transformation system, but also suitable for rapid propagation of acer truncatum.
Description
The invention relates to a Acer truncatum culture medium and a divisional application applied for the Acer truncatum culture medium, wherein the application number is 2022108950651, the application date is 2022, 07 and 27, the application type is the invention, and the application name is Acer truncatum culture medium and application of the Acer truncatum culture medium.
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to an induction method of acer truncatum embryo callus.
Background
Acer truncatum is also called Acer truncatum, and is a deciduous tree of Acer of Aceraceae. Acer truncatum is a plant with strong ornamental value and strong viability. Acer truncatum has special positions in landscaping due to the characteristics of barren resistance, drought resistance, low temperature resistance, strong adaptability and the like.
The crown of the Acer truncatum Bunge is wide and round, the bark is grey yellow to grey, has longitudinal crack stripes, and the small branches have no hair on smooth and smooth, and grow branches for 1 year. Acer truncatum is resistant to half shadow, cold and wind, and is not resistant to dry heat and strong in sun.
Acer truncatum leaves are light reddish brown or green and have redness, and then are gray. The single leaves of the acer truncatum are grown oppositely, palm-shaped is 5-split, the length is 5-10 cm, the width is 6-15 cm, the whole edge is full, the tip is pointed, and the fruits are turned into the shape of a samara. The Acer truncatum Bunge crown is dense, the tree appearance is beautiful, the leaf shape is beautiful, and the leaf color is gorgeous. The leaf color in autumn is changed into bright redness or golden yellow, and is a famous autumn leaf tree species in northern China.
In the prior art, a new acer truncatum single plant is mostly obtained by means of cuttage or grafting. However, the new superior variety of Acer truncatum is difficult to cut, and the main reason is that the rooting is difficult, so far, the rooting rate of cutting seedlings is still very low, and the requirement of large-scale propagation cannot be met.
With the maturation of tissue culture technology, tissue culture of Acer truncatum becomes a means for breeding Acer truncatum. The Chinese patent application with publication number of CN 111657151A discloses a rapid seedling method of Acer truncatum, which comprises the following steps: collecting current annual branches of Acer truncatum, cutting stem segments with buds as explants, sterilizing, inoculating to an explant germination culture medium, and expanding the buds to obtain aseptic seedlings; and then placing the new stem segment of the aseptic seedling on a rooting culture medium, directly rooting to form the seedling, and then transplanting. The Chinese patent with publication number of CN102726295A discloses a tissue culture propagation method of Acer truncatum, which comprises the following steps: collecting current annual branch buds of Acer truncatum Bunge parent tree with 4-6 years of age in the last 3 months, sterilizing, cutting, inoculating into MS culture medium containing benzylaminoadenine and naphthylacetic acid, performing tissue induction culture in the conditions of pH 5.6-5.8, 20-24 deg.C and illumination intensity 1500lux for 18 hr each day, and lighting for 6 hr darkness. The above method allows each of the explants to differentiate into a single plant by inducing directed differentiation of the explants.
Up to now, although many researchers have conducted studies on a acer truncatum regenerating system and a genetic transformation system, a truly effective acer truncatum genetic transformation system has not been established. The invention relates to an induction method of acer truncatum embryo callus based on cultivation of embryogenic callus to realize the redifferentiation of acer truncatum.
Furthermore, there are differences in one aspect due to understanding to those skilled in the art; on the other hand, since the applicant has studied a lot of documents and patents while making the present invention, the text is not limited to details and contents of all but it is by no means the present invention does not have these prior art features, but the present invention has all the prior art features, and the applicant remains in the background art to which the right of the related prior art is added.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an induction method of embryogenic callus of Acer truncatum and an embryogenic callus induction culture medium. The culture medium and the induction method can obviously improve the stem section somatic embryogenesis rate of the acer truncatum, and lay a technical foundation for cultivating acer truncatum with low germination rate and establishing an acer truncatum genetic transformation system.
The invention relates to an induction method of acer truncatum embryo callus, which comprises the following steps:
collecting Acer truncatum stems, sterilizing the Acer truncatum stems to obtain explants for callus culture, and placing the explants on the surface of a primary callus induction culture medium to obtain primary callus of Acer truncatum; selecting healthy primary callus, transferring the healthy primary callus to embryogenic callus induction medium, the healthy primary callus being white, green or reddish, wherein the embryogenic callus induction medium is selected from the group consisting of:
1/2MS+0.5mg/L indolebutyric acid (IBA) +1.0mg/L Thidiazuron (TDZ) +3.0 mg/L6-benzylaminopurine (6-BA) +0.1mg/L Gibberellin (GA);
1/2MS+3.0 mg/L6-benzylaminopurine+2.0 mg/L thidiazuron+0.5 mg/L indolebutyric acid+0.1 mg/L gibberellin.
According to a preferred embodiment, the primary callus induction medium comprises: 1/2MS, 2 mg/L2, 4-dichlorophenoxyacetic acid (2, 4-D), 0.3 mg/L6-BA, 0.5mg/L Naphthalene Acetic Acid (NAA), 30g/L Sucrose (Sucrose), 7g/L Agar (Agar) and 0.5g/L Acid Hydrolyzed Casein (AHC).
According to a preferred embodiment, the method for sterilizing the stem segments of Acer truncatum comprises the steps of:
washing the detergent with water after soaking; sterilizing the surface of the alcohol and cleaning with sterile water; immersing in sodium hypochlorite, and washing with aseptic water.
According to a preferred embodiment, the method of subculturing embryogenic callus comprises the steps of:
selecting a new embryogenic callus for culture in an embryogenic callus induction medium, wherein the embryogenic callus induction medium is selected from the group consisting of:
1/2MS+0.5mg/L IBA+1.0mg/L TDZ+3.0mg/L 6-BA；
1/2MS+3.0mg/L 6-BA+2.0mg/L TDZ+0.5mg/L IBA。
according to a preferred embodiment, the embryogenic callus induction medium for the secondary culture comprises 1/2MS, 0.5mg/L IBA, 2.0mg/L TDZ, 3.0 mg/L6-BA, 0.1mg/L GA.
According to a preferred embodiment, the embryogenic callus induction medium for the secondary culture comprises 1/2MS, 0.5mg/L IBA, 1.0mg/L TDZ, 3.0 mg/L6-BA, 0.1mg/L GA.
According to a preferred embodiment, the embryogenic callus can be green granular or white granular.
According to a preferred embodiment, the stem segments after sterilization can be divided into growth point free segments between 1 and 2cm in length, which can be cultivated as explants in the primary callus induction medium.
According to a preferred embodiment, embryogenic callus can be subcultured every 2 to 4 weeks to maintain stable growth and proliferation.
An embryogenic callus induction medium for acer truncatum, wherein the embryogenic callus induction medium comprises: 1/2MS basal medium consisting of 1/2MS, 30g/L sucrose, 0.5g/L acid hydrolyzed casein and 8g/L agar and hormone composition, wherein the hormone composition consists of the following components in mass concentration:
IBA 0.1-1.0 mg/L, TDZ 0.1-4.0 mg/L, 6-BA 1.0-5.0 mg/L, GA 0.1mg/L. Preferably, the gibberellin can be GA 3 。
The invention provides a method for cultivating acer truncatum, or application of the method for cultivating acer truncatum in embryogenic callus induction of acer truncatum based on acer truncatum embryo seedling cultivation.
The invention provides a method for cultivating acer truncatum, or application of the method for cultivating acer truncatum in embryogenic callus induction of acer truncatum to improvement of embryogenic transformation rate of acer truncatum somatic cells.
According to a preferred embodiment, the MS medium of the present invention comprises macroelements, microelements, iron salts and organic substances. Macroelements comprising 1650mg/L NH 4 N0 3 、1900mg/L KN0 3 、440mg/L CaCl 2 ·2H 2 O、370mg/L MgSO 4 ·7H 2 O、170mg/L KH 2 PO 4 . The trace elements comprise 0.83mg/L KI, 6.3mg/L H 3 BO 3 、22.3mg/L MnSO 4 ·4H 2 O、8.6mg/L ZnSO 4 ·7H 2 O、0.25mg/L Na 2 MoO 4 ·2H 2 O、0.025mg/L CuSO 4 ·5H 2 O、0.025mg/L CoCl 2 ·6H 2 O. The ferric salt comprises 27.8mg/L FeSO 4 ·7H 2 O、37.3mg/L EDTA-FeNa 2 ·3H 2 O. The organic material comprises 100mg/L inositol,0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride (vitamin B6), 0.1mg/L thiamine hydrochloride (vitamin B1), 2.0mg/L glycine.
According to a preferred embodiment, 1/2MS medium in the present invention means reducing the major elements in MS medium to 1/2 of the original.
Drawings
FIG. 1 is a comparison of the callus yields of embryogenic callus cultured on embryogenic callus induction medium containing IAA and IBA at different concentrations provided by the present invention;
FIG. 2 is a comparison of the callus yields of embryogenic callus cultured on embryogenic callus induction medium containing different concentrations of TDZ provided by the present invention;
FIG. 3 is a comparison of the callus yields of embryogenic callus cultured on embryogenic callus induction medium containing different concentrations of 6-BA provided by the present invention;
FIG. 4 is a different type of callus provided by the present invention;
FIG. 5 shows the structure of cell sections of the embryogenic callus and non-embryogenic callus of Acer truncatum under a 40 Xobjective lens.
Detailed Description
The following detailed description refers to the accompanying drawings.
In the prior art, the traditional asexual propagation mode of Acer truncatum mainly comprises two modes of cuttage and grafting. The invention provides an induction method of embryogenic callus of Acer truncatum. The induction method is different from the traditional propagation method, and the acer truncatum single plant is obtained by converting acer truncatum somatic cells into embryogenic callus and then differentiating. In the invention, the acer truncatum explant can be converted into embryogenic callus, and then the embryogenic callus can be differentiated into complete acer truncatum individuals. The induction method provided by the invention can be used for establishing a regeneration system and a genetic transformation system of Acer truncatum.
Preferably, acer truncatum callus is transferred to a differentiation medium to differentiate into different organ primordia or form embryoid bodies. And transferring the Acer truncatum bud seedlings differentiated into a rooting culture medium to promote the Acer truncatum bud seedlings to root. The rooting treatment can improve the robustness of the Acer truncatum bud seedlings.
The acer truncatum is bred by adopting a short branch cutting or explant bud differentiation mode at present, but the induction method of the embryogenic callus of the acer truncatum provided by the invention is suitable for a tissue culture method of somatic cell transformation into embryoid and then differentiation.
The method for cultivating the acer truncatum by using tissue culture has the following advantages:
the propagation speed is high, and the propagation coefficient is large: the test tube rapid propagation technology is used for propagation, propagation materials can be saved, a large number of embryogenic callus can be produced in a short period by taking a small tissue on the stem section of the Acer truncatum, each embryogenic callus has the possibility of differentiating into a complete Acer truncatum individual, and the method can not damage the original plant of the Acer truncatum but also obtain higher economic benefit;
the propagation offspring are orderly and consistent, and the excellent characters of the original variety can be maintained: the test tube propagation is a miniature asexual propagation, which is obtained from somatic cells of the same acer truncatum rather than sexual cells, so that hereditary properties of offspring are very consistent, the excellent properties of original varieties of acer truncatum can be maintained, special effects on quality guarantee and purity preservation are achieved, a large number of acer truncatum with uniform specification and high quality can be obtained by a tissue culture propagation method, and the commercialization of the acer truncatum is good;
can be used for industrial production: the tissue culture rapid breeding is the intensive production under the condition of manual control, and is not influenced by seasons and bad weather in natural environment. The plant tissue culture can be continuously produced throughout the year without being limited by seasons, and the production efficiency is high. The process can realize industrial production, has special effect on out-of-season production, such as cultivation of high temperature intolerant Acer truncatum in summer, production of tissue culture seedlings in a culture room in summer, and transplanting of Acer truncatum in autumn;
the economic benefit is high: the tissue culture rapid propagation of Acer truncatum Bunge can exert manpower, material resources and financial resources to the maximum extent due to the growth of the seedling in the culture flask, the three-dimensional arrangement, small space, land saving, strict execution of production according to a certain program, miniaturized and precise production process, and high production efficiency, such as millions of test tube seedlings can be produced in a 200 square meter culture room for one year.
The plant embryogenic callus makes the explant expand at the incision of the explant under the induction of external hormone and the like to form a random cell mass with the potential of generating complete plants in a dividing way. Along with the development of plant tissue culture technology, somatic embryogenesis has become a main mode of large-scale plant propagation, and the method has high propagation coefficient and higher genetic stability. High-quality embryogenic callus is a precondition for embryo formation, is a basis for large-scale plant regeneration, and is an important material for genetic transformation. Along with the continuous development of genetic engineering, plant body is introduced with exogenous genes and forms new character varieties with stable genetic expression, which become hot directions, and plant body regeneration becomes a great difficulty in restricting plant transgenic technology.
Acer deciduous tree of Acer, and tissue culture of Acer plant has less research, and only several kinds of tree species such as Acer palmatum, acer americanum, acer rubrum, acer aculeatus, etc. can be referred to. Most of researches related to tissue culture of Acer truncatum are stem tip culture direct seedling technology. The method for inducing and differentiating embryogenic callus can be used for individual breeding and can also be used for breeding new varieties of Acer truncatum. The genetic engineering technology is applied to the breeding of new varieties of Acer truncatum, so that the cultivation of the new varieties of Acer truncatum with stable ornamental property, insect resistance and disease resistance is simpler, more convenient and faster.
Auxins and cytokinins, which control the direction of cell division and differentiation, are involved in plant tissue culture. The invention adopts IAA, IBA, 6-BA, TDZ and GA to induce the callus. Cytokinin and auxin can effectively promote the formation and proliferation of embryogenic callus. 6-BA is the most commonly used cytokinin, can promote cell division and is beneficial to the morphogenesis of callus; TDZ belongs to an artificially synthesized heterocyclic urea cytokinin, which can directly act on cytokinin receptors to activate enzymes in downstream signal networks.
According to a preferred embodiment, the embryogenic callus can be green granular or white granular.
The invention adds biological hormone on the basis of MS culture medium. The bio-hormone can be IBA, IAA, TDZ, GA or 6-BA. The concentration of IBA is 0.1-1.0 mg/L. Preferably, IBA is selected from IAA and IBA. IBA concentration is 0.5mg/L. The concentration of TDZ is 0.1-4.0 mg/L. Preferably, the concentration of TDZ is 2mg/L. The concentration of 6-BA is 1.0-5.0 mg/L. Preferably, the concentration of 6-BA is 3.0mg/L. Preferably, the concentration of GA3 is 0.1mg/L.
Example 1
The test steps comprise collection of test materials, disinfection treatment of explants, induction of primary calli of Acer truncatum, induction of embryogenic calli of Acer truncatum, subculture proliferation of embryogenic calli of Acer truncatum, cytological observation and identification of embryogenic calli of Acer truncatum and data statistical analysis.
1. Acquisition of test materials
The young stem segments of Acer truncatum 'Lihong' are collected in the last ten days of 4 to 5 months each year as test materials. The collection time is 8 to 10 am, and the weather is good when the collection is carried out.
2. Disinfection treatment of explants
The collected stem segments of Acer truncatum Bunge are washed for 10 minutes by running water, then washed for 10 minutes by adding 1/1000 of a detergent, and then repeatedly rinsed for 4 hours by running water. After washing the explant, placing the explant in a sterile culture flask for surface sterilization. The surface sterilization treatment comprises the following steps: firstly, cleaning with sterile water for 2 times and 3 minutes each time; adding 75% alcohol for sterilization for 1 minute, and rinsing with sterile water for 3 times each for 3 minutes; 10% sodium hypochlorite was added to sterilize for 10 minutes, and finally rinsed 3 times with sterile water for 3 minutes each. The surface of the stem section is sucked by sterile filter paper and cut into small sections with 1cm length and no growing point.
3. Induction of Acer Truncatum Bunge primary callus
Culturing stem in callus induction culture medium with formulation of Acer Truncatum Bunge callus induction culture medium of 1/2MS, 2 mg/L2, 4-D, 0.3 mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose, 0.5g/L acid hydrolyzed casein, and 7g/L agar, and inducing for 2 weeks to obtain Acer Truncatum Bunge primary callus.
4. Induction of embryogenic callus of Acer truncatum
Healthy primary calli of white, green or reddish colour were selected from the primary calli obtained and transferred to embryogenic callus induction medium. Embryogenic callus induction was performed using 1/2MS basal medium: 1/2MS, 30g/L sucrose, 0.5g/L AHC and 8g/L agar, and performing control experiments on auxin (IAA) and indole-3-butyric acid (IBA) under the conditions of the same concentration of different species and different concentrations of the same species, respectively setting cytokinin (TDZ) and 6-benzyl amino purine (6-BA) with different concentration gradients, and observing the influence of the hormone proportion on embryogenic callus formation. The invention explores the influence of different hormones on the induction of the embryogenic callus of Acer truncatum based on a controlled variable method.
5. Subculture of acer truncatum embryo callus
The new-born callus with compact structure, hard texture and granular shape is selected once every 2 weeks for subculture, and the growth state, texture and color of the callus are summarized and recorded. The inventors found that embryogenic callus can be subcultured every 2 weeks, and that stable growth and proliferation of the callus can be maintained.
6. Acer truncatum embryo callus cytology observation and identification
And (3) fixing a small amount of subculture callus by using FAA fixing solution, and embedding resin to prepare ultrathin slices after vacuum pumping. And (5) placing the prepared ultrathin slice under a microscope for observation and photographing, and determining the type of embryogenic callus.
7. Statistical analysis of data
Embryogenic callus induction rate (%) = number of explants forming embryogenic callus/number of inoculated explants x 100%;
experimental data was analyzed using Excel 2013.
In this example, the main objective was to explore the effect of IAA and IBA on embryogenic callus induction.
In this example, IAA of 0.1 to 1.0mg/L and IBA of 0.1 to 1.0mg/L were used for the control test. The present example provides ten sets of media, each numbered corresponding to the following composition:
1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.1mg/L IAA;
b.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.2mg/L IAA;
c.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.4mg/L IAA;
d.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IAA;
e.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+1.0mg/L IAA;
f.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.1mg/L IBA;
1.0mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.2mg/L IBA of the 1/2MS basal medium;
h.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.4mg/L IBA;
1.2 MS basal medium +1.0mg/L TDZ +3.0 mg/L6-BA +0.1mg/L GA +0.5mg/L IBA;
j.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+1.0mg/L IBA.
As shown in Table 1, the growth conditions and tissue properties of embryogenic callus on IAA and IBA media at different concentrations were greatly different. As shown in FIG. 1, calli in IAA medium at all concentration gradients grew slowly and continued to brown during subculture, and overall performed less than IBA-supplemented medium. However, when IBA concentration is too high or too low, the callus structure starts to become dense, the texture is harder, the color is relatively deepened, and eventually the brown death occurs. The conditions of callus growth in the dishes with IAA concentrations of 0.1, 0.2, 0.4, 0.5 and 1.0mg/L in FIG. 1, respectively; f-j are the growth conditions of the calli in the culture dishes with the IBA concentration of 0.1, 0.2, 0.4, 0.5 and 1.0mg/L respectively. Thus, it was determined that 1/2MS, 0.5mg/L IBA, 1.0mg/L TDZ, 3.0 mg/L6-BA and 0.1mg/L GA were more desirable to induce embryogenic callus.
TABLE 1
Example 2
The test method used in this embodiment is the same as that used in embodiment 1, and the repeated description is omitted.
In this example, the main objective was to explore the effect of TDZ on embryogenic callus induction.
In this example, a control test was conducted using a TDZ of 0.1 to 4.0mg/L. The present example provides five sets of media, each numbered corresponding to the following composition:
1/2MS basal medium+0.1 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
b.1/2MS basal medium+0.5 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
c.1/2MS basal medium+1.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
d.1/2MS basal medium+2.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
e.1/2MS basal medium +4.0mg/L TDZ +3.0 mg/L6-BA +0.1mg/L GA +0.5mg/L IBA.
As is clear from Table 2 and FIG. 2, the adjustment of TDZ concentration was performed well on the premise of unchanged IBA, 6-BA and GA concentrations, and the embryogenic callus induction rate was largely expanded in the whole explant, with little morphological difference between treatments. However, as the TDZ concentration increases, the callus gradually changes from a loose state to a dense state. The conditions of callus growth in the dishes at TDZ concentrations of 0.1, 0.5, 1.0, 2.0 and 4.0mg/L are shown in FIGS. 2A-e, respectively. After 4 weeks observation, no regeneration shoot differentiation occurred, indicating that TDZ has a positive effect on embryogenic callus induction, which may be related to other hormone concentrations in the formulation.
TABLE 2
Example 3
The test methods used in this embodiment are the same as those used in embodiment 1 and embodiment 2, and the repeated descriptions are omitted.
In this example, the main objective was to explore the effect of 6-BA on embryogenic callus induction.
In this example, a control test was conducted using 1.0 to 5.0mg/L of 6-BA. The present example provides five sets of media, each numbered corresponding to the following composition:
1/2MS basal medium +2.0mg/L TDZ +1.0 mg/L6-BA +0.1mg/L GA +0.5mg/L IBA;
b.1/2MS basal medium+2.0 mg/L TDZ+2.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
c.1/2MS basal medium+2.0 mg/L TDZ+3.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
d.1/2MS basal medium+2.0 mg/L TDZ+4.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA;
e.1/2MS basal medium+2.0 mg/L TDZ+5.0 mg/L6-BA+0.1 mg/L GA+0.5mg/L IBA.
As can be seen from Table 3 and FIG. 3, the embryogenic callus was induced by different concentrations of 6-BA, and the induction rate increased with increasing hormone concentration, but peaked at 3mg/L, indicating that 1/2MS, 3.0 mg/L6-BA, 2.0mg/L TDZ, 0.5mg/L IBA and 0.1mg/L GA were the optimal medium for embryogenic callus induction of Acer truncatum. However, no differentiation buds are generated after two subcultures, which means that the induction conditions of the embryogenic callus of Acer truncatum have been initially established, but the embryogenesis process of the somatic cells of Acer truncatum needs to be further explored. The calli growth conditions in the dishes at 1.0, 2.0, 3.0, 4.0, 5.0 mg/L6-BA concentration were shown in FIGS. 3A-e.
TABLE 3 Table 3
The key point of embryogenic callus induction and maintenance is the concentration and proportion of plant growth regulator in the culture medium used.
The results shown in example 1 show that auxin IBA performs better overall than IAA. The results shown in example 2 demonstrate that cytokinin TDZ has a positive effect on the induction of acer truncatum embryogenic callus. The results shown in example 3 demonstrate that different concentrations of 6-BA can induce embryogenic callus and that the induction rate increases with increasing hormone concentration.
Based on the results in the above examples, the method for inducing embryogenic callus of Acer truncatum involves the following steps:
collecting Acer truncatum stems, sterilizing the Acer truncatum stems to obtain explants for callus culture, and placing the explants on the surface of a primary callus induction culture medium to obtain primary callus of Acer truncatum; healthy primary calli are selected, and transferred to embryogenic callus induction medium, which can be white, green or reddish.
The primary callus induction medium comprises: 1/2MS, 2 mg/L2, 4-D, 0.3 mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose, 7g/L agar and 0.5g/L acid hydrolyzed casein.
The disinfection method of the acer truncatum stem comprises the following steps:
washing the detergent with water after soaking; sterilizing the surface of the alcohol and cleaning with sterile water; immersing in sodium hypochlorite, and washing with aseptic water.
The subculture method of embryogenic callus comprises the following steps:
selecting a new embryogenic callus to culture in an embryogenic callus induction medium, wherein the embryogenic callus induction medium is: 1/2MS+0.5mg/L IBA+2.0mg/L TDZ+3.0 mg/L6-BA.
The stem segments after sterilization can be divided into growth point free segments between 1 and 2cm in length, which can be cultivated as explants in the primary callus induction medium.
An embryogenic callus induction medium for acer truncatum, wherein the embryogenic callus induction medium comprises: 1/2MS basal medium consisting of 1/2MS, 30g/L sucrose, 0.5g/L acid hydrolyzed casein and 8g/L agar and hormone composition, wherein the hormone composition consists of the following components in mass concentration:
IBA at 0.5mg/L, TDZ at 2.0mg/L, 6-BA at 1.0mg/L, GA at 0.1mg/L.
Example 4
The test method used in this embodiment is substantially the same as that used in embodiment 1, and the repeated description is omitted.
The embodiment is used for exploring different types of calli of embryogenic calli after multiple subculture of Acer truncatum.
The callus which is newly induced by the stem is mostly white or green, but if subculture is not performed for a long time, the callus gradually turns yellow and finally becomes soft and round until death after browning.
Primary calli of Acer truncatum are inoculated onto embryogenic callus culture medium, subculture is carried out once every 14d, and 6 types of calli can be seen after multiple subcultures: the type I is white soft callus (figure 4A), which grows very fast, presents a water stain shape as a whole, has no browning basically after subculture and has no transformation; the type II is white granular callus (figure 4B), and the white granular callus grows vigorously and can be converted into type III and type V callus after multiple subcultures; class III is green soft callus (FIG. 4C), grows faster, and has loose texture; the class IV calli were green compact calli (fig. 4D), growing slowly, gradually browning and dying in subsequent subcultures; class V is green granular callus (fig. 4E), growing vigorously, remaining viable after multiple subcultures; class VI is red callus (fig. 4F), growing slowly, and turning into class IV callus in subsequent subcultures.
Callus with good growth condition and basically consistent growth state is selected to be inoculated on a culture medium of 1/2MS, 2mg/L TDZ, 0.5mg/L IBA and 3 mg/L6-BA, and subcultured every 14 d. 6 calli were visible after multiple subcultures: white soft callus, white granular callus, green loose callus, green compact callus, green granular callus, red callus.
Example 5
The invention adopts a tissue section method to observe the cell morphology of different types of calli at different periods.
Microscopic examination of the prepared six calli showed that the green granular calli could be embryogenic cell mass with compact cell arrangement and small volume, and the nucleus was located in the center of the cells (fig. 5 e); white granular callus had both embryogenic cell masses and irregular and large-sized coreless cells (FIG. 5 b), indicating that such callus might be in an intermediate state between embryogenic and non-embryogenic callus; the remaining four types of calli were large in cell volume, loosely arranged, without distinct nuclei (FIG. 5a, c, d, f), and identified as non-embryogenic calli.
The green granular callus obtained by ultra-thin section observation of various calli can be embryogenic cell mass, the white granular callus can be in the intermediate state of embryogenic and non-embryogenic calli, and the other four calli are non-embryogenic calli.
Example 6
The embryogenic callus induction method of Acer truncatum provided by the invention obviously improves the somatic embryogenesis rate of Acer truncatum.
The invention provides a method for cultivating acer truncatum, or application of the method for cultivating acer truncatum in embryogenic callus induction of acer truncatum based on acer truncatum embryo seedling cultivation.
The invention provides a method for cultivating acer truncatum, or application of the method for cultivating acer truncatum to embryogenic callus induction of acer truncatum to improvement of embryogenic transformation rate of acer truncatum somatic cells.
The embryogenic callus of Acer truncatum Bunge can be used for overcoming the abortion of hybrid embryo to obtain rare hybrid, quickly breeding special varieties and reducing the formation of chimera so as to improve the genetic transformation efficiency.
The acer truncatum embryogenic callus obtained by the induction method provided by the invention can be transferred to a somatic embryo differentiation medium for differentiation culture to obtain somatic embryos. The somatic embryo differentiation medium can be a conventional differentiation medium.
And selecting rooting seedlings which grow well and strong for outdoor seedling hardening, and transplanting the rooting seedlings into a loose and breathable matrix after the seedlings adapt to the external environment. And transplanting the Acer truncatum tissue culture seedlings to a field after the Acer truncatum tissue culture seedlings survive completely and grow.
It should be noted that the above-described embodiments are exemplary, and that a person skilled in the art, in light of the present disclosure, may devise various solutions that fall within the scope of the present disclosure and fall within the scope of the present disclosure. It should be understood by those skilled in the art that the present description and drawings are illustrative and not limiting to the claims. The scope of the invention is defined by the claims and their equivalents. The description of the invention encompasses multiple inventive concepts, such as "preferably," "according to a preferred embodiment," or "optionally," all means that the corresponding paragraph discloses a separate concept, and that the applicant reserves the right to filed a divisional application according to each inventive concept. Throughout this document, the word "preferably" is used in a generic sense to mean only one alternative, and not to be construed as necessarily required, so that the applicant reserves the right to forego or delete the relevant preferred feature at any time.
Claims (10)
1. A acer truncatum medium, wherein the medium comprises: 1/2MS, 2 mg/L2, 4-dichlorophenoxyacetic acid, 0.3 mg/L6-benzylaminopurine, 0.5mg/L naphthylacetic acid, 30g/L sucrose, 7g/L agar and 0.5g/L acid hydrolyzed casein.
2. Use of the acer truncatum medium of claim 1 for inducing primary calli of acer truncatum.
3. A acer truncatum medium, which is characterized by comprising the following components in mass concentration:
0.1-1.0 mg/L indolebutyric acid, 0.1-4.0 mg/L thidiazuron, 1.0-5.0 mg/L6-benzylaminopurine and 0.1mg/L gibberellin.
4. A acer truncatum medium, wherein the medium comprises: 1/2MS, 0.1-1.0 mg/L indolebutyric acid, 1.0mg/L thidiazuron, 3.0 mg/L6-benzylaminopurine and 0.1mg/L gibberellin.
5. A acer truncatum medium, wherein the medium comprises: 1/2MS, 3.0 mg/L6-benzylaminopurine, 0.1-4.0 mg/L thidiazuron, O.5mg/L indolebutyric acid and 0.1mg/L gibberellin.
6. A acer truncatum medium, wherein the medium comprises: 1/2MS, 1.0-5.0 mg/L6-benzylaminopurine, 2.0mg/L thidiazuron, 0.5mg/L indolebutyric acid and 0.1mg/L gibberellin.
7. A acer truncatum medium, wherein the medium comprises: 1/2MS basal medium and hormone composition consisting of 1/2MS, 30g/L sucrose, 0.5g/L acid hydrolyzed casein, 8g/L agar.
8. The culture medium according to claim 7, wherein the hormone composition consists of the following components in mass concentration:
0.5mg/L indolebutyric acid, 2.0mg/L thidiazuron, 1.0 mg/L6-benzylaminopurine, 0.1mg/L gibberellin.
9. Use of the acer truncatum medium according to any of claims 3 to 8 for inducing embryogenic callus of acer truncatum.
10. Use of the acer truncatum culture medium according to claim 1 or any of claims 3-8 in obtaining rare hybrids of acer truncatum and breeding special varieties of acer truncatum.
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