CN114467747A - Rapid propagation method and culture medium for cymbidium mongolicum - Google Patents
Rapid propagation method and culture medium for cymbidium mongolicum Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 58
- 241000732800 Cymbidium Species 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 16
- 241000233855 Orchidaceae Species 0.000 claims abstract description 47
- 230000035755 proliferation Effects 0.000 claims abstract description 19
- 238000005728 strengthening Methods 0.000 claims abstract description 13
- 238000010899 nucleation Methods 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 5
- 230000035784 germination Effects 0.000 claims description 37
- 239000007640 basal medium Substances 0.000 claims description 21
- 241000196324 Embryophyta Species 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 240000008790 Musa x paradisiaca Species 0.000 claims description 13
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 241000228347 Monascus <ascomycete fungus> Species 0.000 claims description 10
- 244000061456 Solanum tuberosum Species 0.000 claims description 10
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 10
- 239000002775 capsule Substances 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 10
- 230000010152 pollination Effects 0.000 claims description 9
- 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 claims description 8
- 230000007613 environmental effect Effects 0.000 claims description 7
- 238000009331 sowing Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 6
- 244000060011 Cocos nucifera Species 0.000 claims description 5
- 235000013162 Cocos nucifera Nutrition 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000003337 fertilizer Substances 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000012883 rooting culture medium Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
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- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 19
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 18
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 description 15
- 238000011282 treatment Methods 0.000 description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 12
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 12
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 description 9
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 7
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- OVBJJZOQPCKUOR-UHFFFAOYSA-L EDTA disodium salt dihydrate Chemical compound O.O.[Na+].[Na+].[O-]C(=O)C[NH+](CC([O-])=O)CC[NH+](CC([O-])=O)CC([O-])=O OVBJJZOQPCKUOR-UHFFFAOYSA-L 0.000 description 6
- 239000004471 Glycine Substances 0.000 description 6
- 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 6
- 229930006000 Sucrose Natural products 0.000 description 6
- 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 description 6
- 239000008272 agar Substances 0.000 description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 6
- 239000004327 boric acid Substances 0.000 description 6
- 239000001110 calcium chloride Substances 0.000 description 6
- 229910001628 calcium chloride Inorganic materials 0.000 description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 229960000367 inositol Drugs 0.000 description 6
- 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 6
- 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 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 description 6
- 235000001968 nicotinic acid Nutrition 0.000 description 6
- 229960003512 nicotinic acid Drugs 0.000 description 6
- 239000011664 nicotinic acid Substances 0.000 description 6
- 239000004323 potassium nitrate Substances 0.000 description 6
- 235000010333 potassium nitrate Nutrition 0.000 description 6
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 6
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 6
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 6
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 6
- 239000011684 sodium molybdate Substances 0.000 description 6
- 235000015393 sodium molybdate Nutrition 0.000 description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 229960000344 thiamine hydrochloride Drugs 0.000 description 6
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 6
- 239000011747 thiamine hydrochloride Substances 0.000 description 6
- 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 6
- 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 6
- 239000005556 hormone Substances 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 4
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 4
- 230000004075 alteration Effects 0.000 description 4
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 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 2
- -1 0-0.5 mg2 Chemical compound 0.000 description 2
- 241001313857 Bletilla striata Species 0.000 description 2
- 239000001653 FEMA 3120 Substances 0.000 description 2
- 241000218922 Magnoliophyta Species 0.000 description 2
- 244000061661 Orchis Species 0.000 description 2
- 239000005985 Paclobutrazol Substances 0.000 description 2
- 244000295923 Yucca aloifolia Species 0.000 description 2
- 235000004552 Yucca aloifolia Nutrition 0.000 description 2
- 235000012044 Yucca brevifolia Nutrition 0.000 description 2
- 235000017049 Yucca glauca Nutrition 0.000 description 2
- 238000011474 orchiectomy Methods 0.000 description 2
- 229930195732 phytohormone Natural products 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- OVSKIKFHRZPJSS-DOMIDYPGSA-N 2-(2,4-dichlorophenoxy)acetic acid Chemical compound OC(=O)[14CH2]OC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-DOMIDYPGSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241001313855 Bletilla Species 0.000 description 1
- 241001530613 Horminum pyrenaicum Species 0.000 description 1
- 241000030999 Monascus pilosus Species 0.000 description 1
- UCHDWCPVSPXUMX-TZIWLTJVSA-N Montelukast Chemical compound CC(C)(O)C1=CC=CC=C1CC[C@H](C=1C=C(\C=C\C=2N=C3C=C(Cl)C=CC3=CC=2)C=CC=1)SCC1(CC(O)=O)CC1 UCHDWCPVSPXUMX-TZIWLTJVSA-N 0.000 description 1
- 238000012865 aseptic processing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- PYRZPBDTPRQYKG-UHFFFAOYSA-N cyclopentene-1-carboxylic acid Chemical compound OC(=O)C1=CCCC1 PYRZPBDTPRQYKG-UHFFFAOYSA-N 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000003 effect on germination Effects 0.000 description 1
- 230000000459 effect on growth Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960005127 montelukast Drugs 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229940047208 pyridoxine 10 mg Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Cultivation Of Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the field of rapid propagation of orchidaceae plants, and discloses a rapid propagation method and a culture medium for cymbidium mongolicum, which comprise the following steps: s1 preparing materials, S2 aseptic seeding, S3 cluster bud proliferation, S4 rooting and seedling strengthening, and S5 transplanting. The present invention uses the Mongolian orchid seed as explant, adopts different culture mediums and adopts the propagation steps of aseptic seeding, cluster bud multiplication, strong seedling culture and aseptic seedling transplantation to obtain a large number of seedlings. The method has the characteristics of high seed germination rate, quick seedling formation, good seedling quality, low cost and the like, has high seedling transplanting survival rate, and can provide an effective way for the production of the Mongolian orchid seedlings.
Description
Technical Field
The invention relates to the field of rapid propagation of orchidaceae plants, in particular to a rapid propagation method and a culture medium of cymbidium mongolicum.
Background
Mengzia foliosa (King & Pantl.) W.C.Huang, Z.J.Liu & C.Hu, Orchidaceae perennial herbaceous plants, are distributed in southern Yunnan of China and under mountain slopes of Thailand. (note: the original name of bletilla striata, the research result aiming at the phylogenetic development of the family of the orchidaceae, namely, the family of the dragon mouth, indicates that the rhizome of bletilla striata does not belong to the species of the genus bletilla and should be independently established as a new genus-the genus Monascus.
The wild resources of the Mongolian orchid (the original rhizoma bletillae) are rare, and the Endangered plant (Endangered, EN) (the famous book of threatened species of higher plants in China) and the Huashengton Convention (CITES) -appendix II are listed in China. The model specimen was collected from Yunnan Mongolian in 1898 by UK plant, and stored in Qiyuan specimen museum in London, UK. It is found that in about 120 years, there are few reports of wild population discovery, and researchers' understanding of it is limited to 3 specimens collected in a collection. Shanghai Chenshan plant garden researchers found suspected wild species in Yunan Mongolian of its model production area in 2018 in 5 months, transferred to Shanghai Chenshan plant garden, and then flowering, identified and confirmed; subsequently, a series of artificial cultivation and propagation research experiments were carried out. Recently, another field population, the population number of which is not more than 300, was reported to be found in Zhenkang county, Yunnan province, far from the model production place.
The seeds of the orchids are small and the embryos are not fully developed. The germination rate of seeds is very low in the natural state, and nutrients are provided by symbiotic bacteria. Sterile culture media required by different orchid seeds under artificial cultivation conditions are different. The rapid propagation of the seedlings is an effective way for realizing the regeneration and reasonable development and utilization of wild endangered orchidaceae plant resources. The traditional propagation mode is pseudobulb plant division propagation, the method is simple to operate, but the propagation coefficient is low, the propagation period is long, and the large-scale seedling demand is difficult to meet. In order to accelerate the propagation speed and shorten the propagation period, a sterile culture mode is usually adopted in large-scale seedling culture. And the Monagland has no blank in related researches of sterile seeding and rapid propagation due to the scarcity of field material resources. If a set of efficient rapid propagation technical system can be established, the method not only can provide help for conservation biology researches such as population regression and recovery of the species, but also can provide theoretical basis for flowering regulation mechanism research and shortening of breeding cycle of the species, and lays a foundation for further development of Monascus plant resources. Therefore, how to realize the rapid propagation of the Monascus mongolicus and help the large-scale artificial propagation of the species is a problem to be solved urgently in the field.
Disclosure of Invention
The invention provides a fast reproduction method of cymbidium mongolicum, which comprises the following steps:
s1 preparation of material: selecting healthy Mongolian orchid plants, carrying out artificial pollination when the plants bloom, and sowing seeds of capsules formed after pollination;
s2 aseptic seeding: inoculating the seeds on a germination culture medium to germinate, wherein the germination culture medium comprises one of MS basal medium, 1/2MS basal medium and 1/3MS basal medium;
s3 cluster bud proliferation: inoculating the germinated leaf differentiated plantlets to a proliferation culture medium for proliferation, wherein the proliferation culture medium comprises 1/2MS basal medium;
s4 rooting and seedling strengthening: inoculating the proliferated plantlets to a strong seedling culture medium to enable the plantlets to take roots and grow seedlings, wherein the strong seedling culture medium comprises 1/2MS basal medium;
and S5 transplanting: transferring the bottle seedlings of the cymbidium mongolicum after rooting and seedling strengthening into a greenhouse, taking out the aseptic seedlings from a culture bottle after 5-7 days, washing, planting in a medium, placing in a shady place in the greenhouse, keeping the humidity at the room temperature of 28 ℃ or below 60% -70%, and performing water-fertilizer conservation after the plants grow stably.
In a feasible scheme, the maturity of the seeds for germination is 85-115 days in the stages of S1 and S2, wherein the vitality of Mongolian orchid seeds formed 105 days after artificial flower pollination is stronger, and the germination rate is highest under the same conditions.
In one possible embodiment, in step S2, the MS basic medium contains 30g of sucrose, 6.5g of agar, 0.2g of activated carbon, 190mg of potassium nitrate, 165mg of ammonium nitrate, 18mg of magnesium sulfate, 17mg of potassium dihydrogen phosphate, 33mg of calcium chloride, 0.86mg of zinc sulfate heptahydrate, 0.62mg of boric acid, 2.82mg of manganese sulfate tetrahydrate, 0.0025mg of copper sulfate pentahydrate, 0.0212mg of sodium molybdate, 0.0014mg of cobalt chloride, 0.083mg of potassium iodide, 3.73mg of disodium ethylenediaminetetraacetate, 2.78mg of ferrous sulfate heptahydrate, 0.2mg of glycine, 0.05mg of nicotinic acid, 0.01mg of thiamine hydrochloride, 0.05mg of pyridoxine hydrochloride, 10mg of inositol, and the balance of distilled water, and has a pH of 5.8.
In one possible embodiment, in step S2, the 1/2MS basic medium contains 30g of sucrose, 6.5g of agar, 0.2g of activated carbon, 95mg of potassium nitrate, 82.5mg of ammonium nitrate, 9mg of magnesium sulfate, 8.5mg of potassium dihydrogen phosphate, 16.5mg of calcium chloride, 0.43mg of zinc sulfate heptahydrate, 0.31mg of boric acid, 1.41mg of manganese sulfate tetrahydrate, 0.00125mg of copper sulfate pentahydrate, 0.0106mg of sodium molybdate, 0.0007mg of cobalt chloride, 0.0415mg of potassium iodide, 1.865mg of disodium ethylenediaminetetraacetate dihydrate, 1.39mg of ferrous sulfate heptahydrate, 0.1mg of glycine, 0.025mg of nicotinic acid, 0.005mg of thiamine hydrochloride, 0.025mg of pyridoxine hydrochloride, 5mg of inositol, and the balance of distilled water, at pH 5.8.
In one possible implementation, in step S2 above, the 1/3MS basal medium contains 30g sucrose, 6.5g agar, 0.2g activated carbon, 63.3mg potassium nitrate, 55mg ammonium nitrate, 6mg magnesium sulfate, 5.7mg potassium dihydrogen phosphate, 11mg calcium chloride, 0.29mg zinc sulfate heptahydrate, 0.33mg boric acid, 0.94mg manganese sulfate tetrahydrate, 0.0008mg copper sulfate pentahydrate, 0.0071mg sodium molybdate, 0.0005mg cobalt chloride, 0.0277mg potassium iodide, 1.24mg disodium ethylenediaminetetraacetate, 0.93mg ferrous sulfate heptahydrate, 0.07mg glycine, 0.017mg nicotinic acid, 0.003mg thiamine hydrochloride, 0.017mg pyridoxine hydrochloride, 3.33mg inositol per liter, and the balance distilled water, and a pH of 5.8 per liter.
In a possible embodiment, the step S2 specifically includes the following steps of aseptic processing: washing Mengyan capsule under water flow for 20min, soaking in 75% alcohol for 30s on a clean bench, shock sterilizing with 0.1% mercuric chloride solution containing a little Tween 20 for 12min, rinsing with sterile water for 4 times, drying the surface moisture of capsule with filter paper, cutting the capsule, and inoculating the seed to seeding culture medium for germination.
In a possible solution, in the above steps S2-S4, the environmental conditions are specifically:
the temperature is 23-27 ℃,
the illumination intensity is as follows: the concentration of the mixed solution is 2000-2500 lux,
light source: the white light of the LED lamp is,
illumination duration: the reaction time is 12 hours per day,
humidity: 50% -70%.
In a possible solution, in the above steps S2-S4, the environmental conditions are specifically: the temperature is 24-26 ℃, and the illumination intensity is 2000 lx.
In a possible solution, in the step S5, the environmental conditions are specifically:
the temperature is 22-28 ℃,
the natural illumination intensity: 3000-5000 lux of the total weight of the powder,
humidity: 60% -70%.
In one possible solution, in the step S5, the mass ratio of the bark to the peat to the blue stone in the medium is 1:2: 1.
In the research of the growth stages of plants such as a Mongolian orchid seed germination culture medium, a clumpy bud multiplication, rooting and seedling strengthening and the like, basic culture mediums such as MS, KC, Huabao and the like commonly used in Orchidaceae are used, experiments show that the variety of the culture mediums has no significant influence on the germination and growth of the Mongolian orchid seeds, the concentration of the culture mediums has more significant influence on the germination and growth of the Mongolian orchid seeds, and the basic culture medium with the concentration of 1/2MS has better effects on the germination and growth of the seeds when being used as the basic culture medium of the Mongolian orchid. The addition of some phytohormones, plant homogenates (e.g., banana homogenates, potato homogenates, coconut green, etc.) had a significant effect on germination and growth of the Mongolian orchid seeds. For example, 0-1 mg NAA (naphthylacetic acid) and 0-1.5 mg6-BA (6-benzylamino adenine) are added into a germination culture medium of the Monascus mongolicus seeds, so that the germination rate of the Monascus mongolicus seeds can be greatly improved, wherein 1/2MS basal medium +0.6mg NAA per liter +1mg 6-BA per liter is adopted to promote the germination rate of the seeds to be optimal; 0-0.5 mg NAA and 0.5-2.5 mg6-BA per liter are added into the Mongolian orchid clump bud multiplication culture medium to promote the multiplication speed of the Mongolian orchid clump bud, wherein the ratio of 1/2MS basal culture medium plus 1mg6-BA per liter plus 0mg NAA per liter has the best effect; 0-1 mgNAA, 0-100 g banana homogenate, 0-100 g potato homogenate and 0-100 ml coconut green are added into a rooting culture medium for the strong seedlings of the orchidaceae mongolian orchidaceae, and can promote the multiple buds of the orchidaceae mongolian orchidae to quickly grow into small seedlings of the orchidaceae mongolian orchidae which have good appearance and are suitable for transplanting, wherein the proportioning effect of 1/2MS + 1mgNAA per liter and 30g potato homogenate per liter is optimal.
For mature Mongolian orchid plants which are cultured quickly, the ratio of different phytohormones has obvious influence on the promotion of the blooming of the Mongolian orchid and the shape of the flower; therefore, 0-2 mg6-BA, 0-0.5 mg NAA, 0-0.5 mg TDZ (thidiazuron), 0-0.5 mg2,4-D (2, 4-dichlorophenoxyacetic acid), 0-1 mg PP333 (paclobutrazol) and 0-30g banana homogenizing are added into the culture medium of the orchis mongolicaThe pulp can promote the formation of the floral bud of the fast-reproducing orchidaceae plant and the normal flowering rate, wherein 1/2MS culture medium +1mg 6-BA + 0.5mg NAA +1mg PP + per liter333The culture medium can promote the blooming rate of the orchis mongolica to be the highest and reach 84 percent.
Based on the scheme, the method takes the yucca seeds as explants, adopts different culture media, and can obtain a large number of seedlings by the propagation steps of seed sterile treatment, sowing, cluster bud multiplication, strong seedling culture and sterile seedling transplantation by controlling the picking time of the yucca seeds. The method has the characteristics of high seed germination rate, quick seedling formation, good seedling quality, low cost and the like; the seed germination rate is up to 97%, and the seedling transplanting survival rate is up to more than 90%; the normal flowering rate of the fast-propagating orchidaceae plant is more than 84 percent through the culture of the flower forcing culture medium. Therefore, the technical scheme provided by the invention can provide an effective way for the production of the Mongolian orchid seedlings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The implementation scheme is as follows:
s1 preparation of material: selecting healthy Mongolian orchid plants, carrying out artificial pollination during flowering, and sowing seeds in fruits which develop after pollination and do not crack, wherein the days after pollination is the maturity of the seeds.
S2 aseptic seeding: washing Mengyan capsule under water flow for 20min, soaking in 75% ethanol for 30s in clean bench, sterilizing with 0.1% mercuric chloride solution containing Tween 20 under shaking for 12min, rinsing with sterile water for 4 times, and drying the surface water with filter paper. Then the capsule is cut open, and the seeds are inoculated on the prepared germination culture medium.
For better demonstration of the effect, the following two sub-schemes a and b are used for experiments:
a. sowing seeds with different maturity, wherein the maturity is 85 days, 95 days, 105 days and 115 days (the maturity is the days from selfing to sowing) on 1/2MS culture medium, additionally adding 0.5mg Naphthalene Acetic Acid (NAA) per liter, observing seed germination difference, recording germination time and counting germination rate. Wherein, the 1/2MS basal medium contains 30g of sucrose, 6.5g of agar, 0.2g of activated carbon, 95mg of potassium nitrate, 82.5mg of ammonium nitrate, 9mg of magnesium sulfate, 8.5mg of monopotassium phosphate, 16.5mg of calcium chloride, 0.43mg of zinc sulfate heptahydrate, 0.31mg of boric acid, 1.41mg of manganese sulfate tetrahydrate, 0.00125mg of copper sulfate pentahydrate, 0.0106mg of sodium molybdate, 0.0007mg of cobalt chloride, 0.0415mg of potassium iodide, 1.865mg of disodium ethylenediaminetetraacetate dihydrate, 1.39mg of ferrous sulfate heptahydrate, 0.1mg of glycine, 0.025mg of nicotinic acid, 0.005mg of thiamine hydrochloride, 0.025mg of pyridoxine hydrochloride, 5mg of inositol, and the balance of distilled water per liter, and has the pH value of 5.8.
b. Seeds with the best maturity (105 days) are respectively sown on different culture media, wherein the basic culture media comprise MS, 1/2MS and 1/3MS, and growth regulators with different concentrations, namely 0-1 mg of naphthylacetic acid (NAA) and 0-1.5 mg of 6-benzylaminopurine (6-BA), are additionally added. Wherein each liter of MS basic culture medium contains 30g of sucrose, 6.5g of agar, 0.2g of activated carbon, 190mg of potassium nitrate, 165mg of ammonium nitrate, 18mg of magnesium sulfate, 17mg of monopotassium phosphate, 33mg of calcium chloride, 0.86mg of zinc sulfate heptahydrate, 0.62mg of boric acid, 2.82mg of manganese sulfate tetrahydrate, 0.0025mg of copper sulfate pentahydrate, 0.0212mg of sodium molybdate, 0.0014mg of cobalt chloride, 0.083mg of potassium iodide, 3.73mg of disodium ethylenediaminetetraacetate, 2.78mg of ferrous sulfate heptahydrate, 0.2mg of glycine, 0.05mg of nicotinic acid, 0.01mg of thiamine hydrochloride, 0.05mg of pyridoxine hydrochloride and 10mg of inositol, and the balance of distilled water, and the pH value is 5.8. And 1/3MS basal medium contains 30g sucrose, 6.5g agar, 0.2g activated carbon, 63.3mg potassium nitrate, 55mg ammonium nitrate, 6mg magnesium sulfate, 5.7mg potassium dihydrogen phosphate, 11mg calcium chloride, 0.29mg zinc sulfate heptahydrate, 0.33mg boric acid, 0.94mg manganese sulfate tetrahydrate, 0.0008mg copper sulfate pentahydrate, 0.0071mg sodium molybdate, 0.0005mg cobalt chloride, 0.0277mg potassium iodide, 1.24mg disodium ethylenediaminetetraacetate, 0.93mg ferrous sulfate heptahydrate, 0.07mg glycine, 0.017mg nicotinic acid, 0.003mg thiamine hydrochloride, 0.017mg pyridoxine hydrochloride, 3.33mg inositol, and the balance distilled water per liter, and has a pH of 5.8; 1/2MS basal medium is referred to in step a, and culture conditions are referred to in step a. And (5) counting the germination rate of the seeds.
S3 cluster bud proliferation: inoculating the germinated leaf to a proliferation culture medium, wherein the proliferation culture medium comprises 1/2MS basal medium, 0-0.5 mg of naphthylacetic acid (NAA), 0.5-2.5 mg of 6-benzylamino adenine (6-BA) and the balance of distilled water, and the pH value is 5.8. 1/2MS basal medium and culture conditions refer to step a. After 60 days, the proliferation rate was counted.
S4 rooting and seedling strengthening: inoculating the proliferated small seedlings of the cymbidium mongolicum with approximately the same size to different seedling strengthening culture media, wherein the seedling strengthening culture media comprise 1/2MS basal culture media, 0-1 mg of naphthylacetic acid (NAA), 0-100 g of banana homogenate, 0-100 g of potato homogenate, 0-100 ml of coconut green, the balance of distilled water and the pH value of 5.8. 1/2MS basal medium and culture conditions refer to step a. The number of roots, root length and seedling height were measured after 60 days.
And S5 transplanting: transferring the bottle seedlings of the cymbidium mongolicum after rooting and seedling strengthening into a greenhouse, taking out the aseptic seedlings from a culture bottle after 5-7 days, washing out the residual culture medium on the plants, planting the seedlings in a medium with 1:2:1 of bark, turf and orchidine, placing the medium in the greenhouse at a shady place, keeping the humidity at the room temperature of no more than 28 ℃, keeping the humidity at 60-70%, enabling the survival rate of the seedlings to reach more than 90%, and performing conventional water-fertilizer conservation after the plants stably grow.
In addition, the environmental conditions from S2 to S4 are specifically: temperature 23-27 ℃, light intensity: 2000-2500 lux, light source: white light of the LED lamp, illumination time length: 12 hours/day, humidity: 50% -70%. More preferably, the ambient conditions for this step are a temperature of 24-26 ℃ and an illumination intensity of 2000 lx.
In step S5, it is preferable that the environmental conditions are specifically: temperature of 22-28 ℃, natural illumination intensity: 3000-5000 lux, humidity: 60% -70%.
Optionally, before transplanting the cultured artificial seedlings, attempting to promote flowering for the bottle seedlings: after the cluster buds are multiplied, the small seedlings of the orchidaceae with the same size are inoculated on a flower forcing culture medium to be bloomed. The flower forcing culture medium contains 0-2 mg/l6-BA, 0-0.5 mg/l NAA, 0-0.5 mg/l TDZ, 0-0.5 mg/l2,4-D and 0-1 mg/lPP333, 0-30 g/l banana homogenate (wherein tdz is thidiazuron, 2,4-D is 2, 4-dichlorophenoxyacetic acid, PP333 is paclobutrazol).
The above embodiment was performed to obtain the following experimental data:
TABLE 1 germination rates of seeds of different maturity
Of these, 4 groups of treatments A1-A4 were used, each treatment inoculated 10 bottles of about 100 seeds per bottle, and germination time was recorded and germination rate was counted. The germination rate is equal to the number of germinated seeds/total number of inoculated seeds multiplied by 100%. According to the table 1, the optimal sowing time of the seeds can be obtained, and the maturity of the Mongolian orchid seeds has a remarkable influence on the germination rate. The time for seeds 85d to 105d after pollination to germinate is about 30d, and the germination rate is gradually increased along with the increase of days, but the difference is not obvious. The germination rate of the seeds of 105d is 87 percent at most, and is obviously higher than that of the seeds of 115 d.
TABLE 2 Effect of different media on the germination of Mongolian orchid seeds
Note: germination rates in the table are mean values ± standard errors; the letters in the same column indicate significant differences at the level of P-0.05
And (3) setting 20 treatments B1-B20, wherein each treatment inoculates 5 bottles of 100 seeds in each bottle, and counting the germination rate after the seeds germinate. According to the table 2, the optimal culture medium for seed germination can be obtained, the Mongolian orchid seeds with the maturity of 105d are sown on different culture media, and 20 groups of culture media tested can germinate, but the germination rates of all groups are obviously different. The germination rate of the B15 treated medium 1/2MS +0.6mg/l NAA +1mg/l 6-BA was 97% at the highest. 1/2 the germination rate of MS as basal medium is higher than that of MS and 1/3MS as basal medium. Subsequently, the relatively better growth of seedlings on 1/2MS as a basal medium was observed continuously for Mongolian seedlings after seed germination. Therefore, for better comparison, 1/2MS is used as a basic medium for the subsequent comparison experiments of cluster bud proliferation, strong seedling rooting and flower forcing.
TABLE 3 Effect of different hormone concentration ratios on the proliferation of orchidaceae monterey buds
Note: germination rates in the table are mean values ± standard errors; the letters in the same column indicate significant differences at the level of P-0.05
From Table 3, it can be seen that the optimal medium for clump shoot proliferation, Mongolian clumps shoots under 16 treatments all had varying degrees of proliferation, medium C16 without NAA and 6-BA addition was significantly lower than the hormone addition medium, C2 and C4 were significantly higher than the other treatments, and neither treatment was NAA addition. The C4 culture medium is 1/2MS + 2mg/L6-BA, the highest proliferation rate is 47.33 percent, and the concentration of the 6-BA is 2 mg/L; the lowest proliferation rate of C5 was 18.01%, the 6-BA concentration was 2.5mg/l, and NAA was not added in both treatments.
TABLE 4 Effect of different concentrations of NAA and nutrients on the growth of seedlings of Monoalan
Note: germination rate data in the table are mean values ± standard errors; the letters in the same column indicate significant differences at the level of P-0.05
According to the table 4, the best culture medium for rooting and seedling strengthening can be obtained, and the Mongolian orchid clump buds are inoculated to the culture medium D1-D17, wherein the Mongolian orchid growth condition of the D15 culture medium 1/2MS +1mg/l NAA +30g/l potato homogenate is the best, the seedling height, the root length and the number of roots are all obviously higher than those of other treatments, and the next step is D14. The potato with the weight of 30g/l can root optimally, and the potato with the weight of 10g/l can inhibit the rooting when the weight of the potato exceeds 30 g/l; 30ml/l, 50ml/l of coconut green and 100g/l of banana also favour rooting but the effect is poorer than that of potato. The effect of NAA on the height of the seedlings is larger than that of nutrients, 1mg/l of NAA is beneficial to the growth of stems and leaves of the seedlings, and the average height of D14 and D15 seedlings is obviously higher than that of other treatments.
In addition, an experiment of the influence of different hormone concentration ratios on the blooming of the orchidaceae montelukast test tube was also performed, which specifically comprises the following steps:
TABLE 5 Effect of different hormone concentration ratios on the flowering in vitro of Monascus pilosus
Note: germination rate data in the table are mean values ± standard errors; the letters in the same column indicate significant differences at the level of P-0.05
In the rooting and seedling strengthening test of the Mongolian orchid, partial flowering of seedlings is found in the seedling strengthening test of 30g/l banana homogenate, so that the flowering of the seedlings is promoted by adding 30g/l banana homogenate into the test tube flowering test culture medium, and the E17 control group is not added. And (3) inoculating the strong and uniform-seedling-height Mongolian orchid seedlings to a culture medium of E1-E17, observing the influence of different hormones on the flower bud differentiation of the seedlings, carrying out 17 treatments, repeating 25 seedlings in each group for 5, and counting the flowering rate and the normal flowering rate after 90 days. Flowering rate ═ number of open flowers/number of inoculated explants × 100% normal flower rate ═ number of normal flowers/number of open flowers × 100%. As can be seen from Table 5, the flower bud differentiation rate and normal flowering rate of the E2 treatment were significantly higher than those of the other treatments with the addition of PP333, and the medium was the optimal flower forcing medium. Observing the differentiation condition of flower buds, the stems of Mongolian orchids in the culture mediums E1, E2 and E3 are all longer, most flowering plants have 2-4 flowers, and all flowering plants in other culture mediums are 1 stem and 1 flower; e17 vs E1 found that seedlings did not differentiate in flower buds in medium without PP333 added; when the concentration of the added 6-BA is the same, the differentiation rate of the flower buds of the combination of NAA and 6-BA is obviously better than that of the combination of 2,4-D and 6-BA except that E6 does not bloom, and the flower aberration rate is also lower; except for E16, the flower bud differentiation rate of the culture medium containing 2,4-D is obviously lower than that of other treatments, and the flower aberration rate is higher; TDZ also has a promoting effect on the bud differentiation of the cymbidium mongolicum, but the effect is not as good as that of 6-BA, the TDZ effect of 0.5mg/l is obviously higher than that of 0.1mg/l, but the flower aberration rate is higher than that of a culture medium containing 6-BA, so that the addition of TDZ can increase the flower aberration rate of the cymbidium mongolicum, and 2,4-D has a certain inhibiting effect on the bud differentiation of the cymbidium mongolicum. E12 flowering state of seedling on the medium added with 0.5mg/l TDZ and 0.5mg/l NAA, short pedicel and curled petal; e14 added 0.1mg/l TDZ and 0.5mg/l2,4-D medium to bring the seedlings into flowering state, the pedicles short and the flowers closed until withering. In the comparative tables of the experiments of the examples, the absence of banana homogenate shows no flowering, but the absence or addition of banana homogenate is not a determinant of the flowering of Monascus, and the formation of flower buds and flowering rate are only relatively worse in Monascus plants without banana homogenate added to the medium.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A fast reproduction method of Mongolian orchid is characterized by comprising the following steps:
s1 preparation of material: selecting healthy Mongolian orchid plants, carrying out artificial pollination when the plants bloom, and sowing seeds which form capsules after pollination;
s2 aseptic seeding: inoculating the aseptically treated seeds onto a germination medium for germination, the germination medium comprising one of MS basal medium, 1/2MS basal medium, 1/3MS basal medium;
s3 cluster bud proliferation: inoculating the germinated leaf differentiated plantlets to a proliferation culture medium for proliferation, wherein the proliferation culture medium comprises 1/2MS basal medium;
s4 rooting and seedling strengthening: inoculating the proliferated plantlets to a strong seedling culture medium to enable the plantlets to take roots and grow seedlings, wherein the strong seedling culture medium comprises 1/2MS basal medium;
and S5 transplanting: transferring the bottle seedlings of the cymbidium mongolicum after rooting and seedling strengthening into a greenhouse, taking out the aseptic seedlings from a culture bottle after 5-7 days, washing, planting in a medium, placing in a shady place in the greenhouse, keeping the humidity at the room temperature of 28 ℃ or below 60% -70%, and performing water-fertilizer conservation after the plants grow stably.
2. The rapid propagation method of Monascus mongolicus as claimed in claim 1, wherein in step S2, the method specifically comprises the following steps: washing Mengyan capsule under water flow for 20min, soaking in 75% alcohol for 30s on a clean bench, shock sterilizing with 0.1% mercuric chloride solution containing a little Tween 20 for 12min, rinsing with sterile water for 4 times, drying the surface moisture of capsule with filter paper, cutting the capsule, and inoculating the seed to seeding culture medium for germination.
3. The rapid propagation method of Monascus mongolicus as claimed in claim 1, wherein in steps S2-S4, the environmental conditions are specifically:
the temperature is 23-27 ℃,
the illumination intensity is as follows: the concentration of the mixed solution is 2000-2500 lux,
light source: the white light of the LED lamp is,
illumination duration: the reaction time is 12 hours per day,
humidity: 50% -70%.
4. The rapid propagation method of Monascus mongolicus as claimed in claim 1, wherein in step S5, the environmental conditions are specifically:
the temperature is 22-28 ℃,
the natural illumination intensity: 3000-5000 lux of the total weight of the powder,
humidity: 60% -70%;
the mass ratio of the bark to the turf to the blue stone in the medium is 1:2: 1.
5. The rapid propagation method of Monascus purpureus according to claim 1, wherein the seeds obtained at the stage of S1 or S2 for germination have a maturity of 85-115 days.
6. A germination culture medium for Mongolian orchid seeds is suitable for rapid propagation of Mongolian orchid and is characterized in that each liter of the germination culture medium contains 0-1 mg NAA and 0-1.5 mg 6-BA.
7. A cymbidium mongolicum clump bud multiplication culture medium suitable for rapid propagation of cymbidium mongolicum clump bud is characterized in that the multiplication culture medium contains 0-0.5 mg NAA and 0.5-2.5 mg6-BA per liter.
8. A rooting culture medium for strong seedlings of orchidaceae mongolian orchidae is suitable for rapid propagation of orchidaceae mongolian orchidae and is characterized in that each liter of the rooting culture medium for strong seedlings comprises 0-1 mgNAA, 0-100 g of banana homogenate, 0-100 g of potato homogenate and 0-100 ml of coconut green.
9. A cymbidium mongolicum flower forcing culture medium is suitable for flower forcing of cymbidium mongolicum plants and is characterized in that the flower forcing culture medium used in the flower forcing stage of the cymbidium mongolicum plants comprises 0-2 mg6-BA, 0-0.5 mg NAA, 0-0.5 mg TDZ, 0-0.5 mg2,4-D, 0-1 mg PP333 and 0-30g banana homogenate per liter.
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