CN118000096A - Tissue culture detoxification rapid propagation method of waterlogging-resistant and saline-alkali-resistant large cherry stock seedlings - Google Patents
Tissue culture detoxification rapid propagation method of waterlogging-resistant and saline-alkali-resistant large cherry stock seedlings Download PDFInfo
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- 241000167854 Bourreria succulenta Species 0.000 title claims abstract description 35
- 235000019693 cherries Nutrition 0.000 title claims abstract description 35
- 238000001784 detoxification Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000003513 alkali Substances 0.000 title claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 46
- 238000012360 testing method Methods 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 238000012258 culturing Methods 0.000 claims description 24
- 238000005520 cutting process Methods 0.000 claims description 22
- 239000000463 material Substances 0.000 claims description 21
- 235000016709 nutrition Nutrition 0.000 claims description 16
- 230000035764 nutrition Effects 0.000 claims description 16
- 238000005286 illumination Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 10
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- 239000010455 vermiculite Substances 0.000 claims description 9
- 235000019354 vermiculite Nutrition 0.000 claims description 9
- 229910052902 vermiculite Inorganic materials 0.000 claims description 9
- 239000002689 soil Substances 0.000 claims description 8
- 241000700605 Viruses Species 0.000 claims description 7
- 239000003599 detergent Substances 0.000 claims description 7
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- 229920001817 Agar Polymers 0.000 claims description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 235000021552 granulated sugar Nutrition 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000012452 mother liquor Substances 0.000 claims description 6
- 239000003895 organic fertilizer Substances 0.000 claims description 6
- 239000002985 plastic film Substances 0.000 claims description 6
- 229920006255 plastic film Polymers 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 6
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 5
- 239000000645 desinfectant Substances 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 4
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- 239000000758 substrate Substances 0.000 claims description 4
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 3
- ZHJGWYRLJUCMRT-UHFFFAOYSA-N 5-[6-[(4-methylpiperazin-1-yl)methyl]benzimidazol-1-yl]-3-[1-[2-(trifluoromethyl)phenyl]ethoxy]thiophene-2-carboxamide Chemical compound C=1C=CC=C(C(F)(F)F)C=1C(C)OC(=C(S1)C(N)=O)C=C1N(C1=C2)C=NC1=CC=C2CN1CCN(C)CC1 ZHJGWYRLJUCMRT-UHFFFAOYSA-N 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229930191978 Gibberellin Natural products 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 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 3
- 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 description 3
- 239000003448 gibberellin Substances 0.000 claims description 3
- 150000004687 hexahydrates Chemical class 0.000 claims description 3
- 229940064880 inositol 100 mg Drugs 0.000 claims description 3
- XNCMOUSLNOHBKY-UHFFFAOYSA-H iron(3+);trisulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XNCMOUSLNOHBKY-UHFFFAOYSA-H 0.000 claims description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000004323 potassium nitrate Substances 0.000 claims description 3
- 235000010333 potassium nitrate Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- 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 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 2
- 230000002745 absorbent Effects 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 2
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 2
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 2
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 2
- 230000000630 rising effect Effects 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 2
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 2
- 239000012466 permeate Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 229910001051 Magnalium Inorganic materials 0.000 description 5
- 241000218378 Magnolia Species 0.000 description 4
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
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- 238000011161 development Methods 0.000 description 3
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- 238000009472 formulation Methods 0.000 description 3
- 229960002523 mercuric chloride Drugs 0.000 description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 3
- 230000035613 defoliation Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000006870 ms-medium Substances 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- XQMVBICWFFHDNN-UHFFFAOYSA-N 5-amino-4-chloro-2-phenylpyridazin-3-one;(2-ethoxy-3,3-dimethyl-2h-1-benzofuran-5-yl) methanesulfonate Chemical compound O=C1C(Cl)=C(N)C=NN1C1=CC=CC=C1.C1=C(OS(C)(=O)=O)C=C2C(C)(C)C(OCC)OC2=C1 XQMVBICWFFHDNN-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001506304 Kadsura japonica Species 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000004883 flower formation Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940098524 pyridoxine 1 mg Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
- A01G2/10—Vegetative propagation by means of cuttings
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Soil Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a tissue culture detoxification rapid propagation method of a waterlogging-resistant and saline-alkali-resistant large cherry stock seedling, which comprises four steps of primary culture, propagation and subculture, detoxification and cuttage rooting. The invention adopts the improved F14 culture medium, so that the buds germinate fast, the leaves are light green, the propagation effect is good, the consistency of large cherry stock seedlings is higher, the growth vigor is good, a large number of detoxified test tube seedlings can be obtained in a short period, and the large cherry stock seedlings can be supplied to the market after grafting, thereby effectively solving the demands of planting large cherries in river beach lands and saline-alkali lands.
Description
Technical Field
The invention belongs to the technical field of plant artificial propagation, and particularly relates to a tissue culture detoxification rapid propagation method of a waterlogging-resistant and saline-alkali-resistant large cherry stock seedling.
Background
The large cherry stock magnalium No. 2 is obtained by the remote hybridization of woods and fruits of the academy of agricultural and forestry sciences in Beijing city. The 2014 application is approved and passed, compared with other stocks, the method has the following advantages: the tree vigor grows vigorously, the root system is deep and developed, the number of the tillers is small, and the soil fixation is good; the soil-logging resistance is high, and the soil-logging resistance is high compared with saline-alkali resistance, brown spot resistance and root nodule resistance; the grafting affinity is good, the big and small foot phenomenon is not obvious, the grafted tree is opened, the young tree is early in flower formation, good in early fruiting, high in yield and stable in yield, strong in tree vigor and semicircular in tree crown, and the grafting tree is a stock with wider and excellent application range.
The large cherry stock magnalium No. 2 is widely applied in Hebei, is relatively lack of water in Gansu days, is relatively less in application, and needs to be reserved in advance and transported for a long distance, so that the cost of seedlings is increased in an intangible way, and the cost of the seedlings is relatively high. In recent years, with the rapid development of science and technology, plant tissue culture technology has entered a production application stage, and is widely applied to large-scale in vitro rapid propagation of flower and fruit tree seedlings. The plant tissue culture detoxification rapid propagation is produced all the year round under the manual control condition, and a large number of tissue culture detoxification seedlings can be obtained in a short time. Therefore, the development of the tissue culture detoxification rapid propagation related experimental study of the large cherry stock seedlings has important significance.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art, and provides a tissue culture detoxification rapid propagation method of large cherry stock seedlings with waterlogging resistance and saline-alkali resistance, which can obtain a large number of large cherry stock magnolia D No.2 detoxification seedlings in a short period by applying the plant detoxification rapid propagation method, and supply the large cherry stock magnolia D No.2 detoxification seedlings to the market after grafting, so that the requirements of large cherry planting in river beach and saline-alkali lands can be met, and the large cherry planting range is wider.
In order to solve the technical problems, the invention adopts the following technical scheme: a tissue culture detoxification rapid propagation method of waterlogging-resistant and saline-alkali-resistant large cherry stock seedlings comprises the following steps:
S1, primary culture
After natural defoliation of No. 2 of a 2-5 year old large cherry stock magnolia, shearing an annual branch, carrying out surface cleaning by running water, then inserting into a glass culture bottle, giving a temperature of 22 ℃ in an artificial incubator, carrying out water culture until a new bud grows to 2cm, collecting a top bud and a bud stem section of a first side bud below, placing into a clean culture bottle, placing into a tissue culture chamber, soaking the bud stem section in 84 disinfectant with a volume concentration of 1 per mill for 5-10min, then washing for 2-6min in running water, then placing into a detergent diluent, washing for 10-15min, then placing into degreasing gauze, carrying out water washing for 1-2h, soaking for 10-25ms in alcohol with a volume concentration of 75% on an ultra-clean workbench, then carrying out treatment for 1.5-3min in a mercuric rising solution with a volume concentration of 0.1%, transferring the bud stem section into the sterile water culture bottle for 4-6 times, then cutting the bud stem section into 84 disinfectant with a volume concentration of 1 per mill, placing into a sterile culture bottle with a humidity of 1.5-0 cm to 7%, transferring the bud stem section into a sterile culture bottle with a humidity of 20-20% for 1-7 days, and transferring the sterile culture bottle culture medium to a sterile water culture bottle with a humidity of the bud culture bottle for 20-20 days;
the formula of the primary culture medium is an improved F14 culture medium, 0.2-0.6 mg/L6-benzyladenine (6-BA), 0.02-0.1mg/L indolebutyric acid (IBA), 10-20g/L active carbon, 20g/L white granulated sugar and 5g/L agar, and the pH value is 5.6;
The improved F14 culture medium consists of major elements, trace elements and organic matters, wherein the major elements are 480mg/L of ammonium nitrate, 220mg/L of potassium nitrate, 360mg/L of magnesium sulfate heptahydrate, 240mg/L of potassium dihydrogen phosphate and 1000mg/L of calcium nitrate tetrahydrate; the microelements are manganese sulfate 0.67mg/L, zinc sulfate heptahydrate 0.86mg/L, sodium molybdate dihydrate 0.25mg/L, boric acid 12.4mg/L, potassium iodide 0.08mg/L, copper sulfate pentahydrate 0.025mg/L, chloridizing drill hexahydrate 0.025mg/L, EDTA-Na 2 27.8.8 mg/L, ferric sulfate heptahydrate 37.3mg/L; the organic matters are glycine 2mg/L, pyridoxine 1mg/L, nicotinic acid 1mg/L, thiamine 0.8mg/L and inositol 100mg/L;
S2, expanding propagation and subculture
Expanding propagation of sterile line seedlings obtained by primary culture of S1, selecting buds with strong growth and consistent integrity, transferring the buds into an expanding propagation culture medium, transferring the buds into a culture chamber, performing dark culture for 7-10 days at the culture temperature of 21-23 ℃ and the humidity of 60-70%, and performing culture for 20-35 days under the condition of illumination of 2000-2400LX to obtain clustered bud sterile line materials;
The propagation medium comprises an improved F14 medium, 0.60 mg/L6-benzyladenine (6-BA), 0.06-0.12mg/L naphthylacetic acid (NAA), 0.06-0.12mg/L gibberellin (GA 3), 20g/L white granulated sugar and 5g/L agar, and the pH value is 5.6;
S3, detoxification
Cutting the sterile series material of the cluster buds subjected to S2 propagation and subculture, cutting 0.4-0.6cm of new buds, carrying out secondary propagation and subculture transfer, transferring to the propagation culture medium in S2, and inoculating 4 strains per bottle, wherein the method specifically comprises the following steps: culturing at 22-25deg.C for 15-20 days, transferring to artificial climate box, respectively culturing under light for 14 hr and dark culturing for 10 hr, and culturing at 26deg.C for 10-15 days while raising temperature to 32deg.C and keeping 32deg.C; the illumination culture is started from 26 ℃, the temperature is increased to 1 ℃ every day, the temperature is kept unchanged after the temperature is increased to 36-38 ℃, then the illumination culture is carried out in a 20-day artificial climate box at 35-38 ℃, the dark culture is carried out at 32 ℃, the stem tips of 0.4-0.5mm are peeled from the tender tips of the materials after the culture is completed, the transfer culture is carried out for 14-21 days, then the stem tips are peeled for the second time, when the stem tips of 0.4-0.5mm are peeled and cultured for 35-45 days, buds of 2.0-4.0cm are cut, the virus detection is carried out, and the virus-free test tube seedlings are obtained for propagation and subculture of the virus-free test tube seedlings according to the method of S2;
S4, cutting rooting
S401, taking out the virus-free test-tube plantlet propagation material for 35-45 days from a culture flask by forceps, cutting off basal callus, cutting off individual stem segments with the length of 2.0-3.0cm, picking off basal 2-5 lobules, filling into a container, spraying water and sealing for standby;
s402, preparing a nutrition pot, putting a substrate, and adding rooting nutrient solution;
S403, inserting the cut test tube seedlings into matrixes in the nutrition pot according to a spacing of 2X 2cm, wherein the humidity of the matrixes is 60-80%, and covering the whole nutrition pot with a white plastic film after the cut test tube seedlings are fully inserted, so that the seedlings are placed in a relatively closed space in the nutrition pot for rooting culture;
s404, after culturing for 15-20 days, observing rooting condition, when the root of the seedling grows into cluster root, removing the plastic film, transferring the nutrition pot to the ground for shading culture, and maintaining the humidity above 85 percent for management;
S405, after culturing for 40-60 days, the leaves of the seedlings in the nutrition pot become green and thick, when the root system accelerates growth, the individual seedlings are transplanted into vermiculite and decomposed organic fertilizer, water is poured in time, the seedlings are gradually adapted to the open field growth environment, when the seedlings grow to 5-10cm, the seedlings with soil are transplanted into the field, and management is carried out according to a field seedling raising method.
Preferably, the detergent diluent in the step S1 is obtained by adding 2-3mL of detergent into 1000mL of water and uniformly mixing.
Preferably, the step S1 is carried out in a mercuric chloride solution with the volume concentration of 0.1 percent for 1.5-3min, namely, the bud-bearing stem is soaked in the mercuric chloride solution with the volume concentration of 0.1 percent, so that the liquid medicine is soaked for 1-2cm through the material, and the liquid medicine is stirred continuously by forceps, so that the liquid medicine uniformly reaches the surface of the material, and various fungi on the surface of the material are killed fully.
Preferably, in S402, the rooting matrix for cutting is vermiculite and perlite, and the mass ratio is 8:2.
Preferably, the rooting nutrient solution for rooting in the cutting in S402 is: adding 25g of rooting powder into 10kg of water, and then adding 100mL of a large amount of component mother liquor of the improved F14 culture medium; the major component mother liquor of the modified F14 culture medium is 50 times of the concentration of the modified F14 culture medium formula.
Preferably, the mass ratio of the vermiculite to the decomposed organic fertilizer in S405 is 1:0.5.
Compared with the prior art, the invention has the following outstanding advantages:
1. The invention provides a tissue culture detoxification rapid propagation method of large cherry stock seedlings with waterlogging resistance and salt and alkali resistance, which adopts an improved F14 culture medium, and has the advantages of rapid germination of new buds, almost continuous growth, light green leaves, good propagation expansion effect, high consistency of large cherry stock seedlings, good growth vigor, capability of obtaining a large number of large cherry stock magnolia D No. 2 detoxification test tube seedlings in a short period, capability of effectively solving the requirements of large cherry planting in river beach and saline-alkali soil after grafting and supplying to market.
2. When the detoxification temperature is too low, incomplete detoxification can be caused, and the virus detoxification rate is low; the detoxification temperature and time of the invention can ensure that the number of stem tips without viruses is reserved while viruses are removed, thereby being beneficial to the later propagation and the development of virus detection tests. In addition, the cutting matrix is not suitable for easily causing seedling mildew and has low rooting rate; the matrix formula and the nutrient solution adopted by the invention can promote the growth vigor of the large cherry stock seedling, have high rooting rate, improve the production efficiency and achieve the purposes of saving cost and enhancing efficiency.
The present invention will be described in further detail with reference to examples.
Detailed Description
Example 1
The embodiment is a tissue culture detoxification rapid propagation method of a waterlogging-resistant and saline-alkali-resistant large cherry stock seedling, which comprises the following steps:
S1, primary culture
Preparing materials, cutting annual branches of 2-5 years old after natural defoliation of 11 months large cherry stock magnum No. 2, cleaning the surfaces of the branches with flowing water, inserting the branches into a glass culture bottle, putting the branches into an artificial incubator at the temperature of 22 ℃, lighting 2000-2400LX, performing water culture to promote new buds, generally producing the new buds within 35-45 days, putting the new buds into a clean culture bottle after about one worship grows to 2cm, collecting terminal buds and bud-bearing stem sections of the first side buds below, respectively marking the dates, putting the bud-bearing stem sections into a tissue culture chamber, soaking the tissue culture chamber for 5-10min in 84 disinfectant with the volume concentration of 1 per mill, washing the tissue culture chamber for 2-6min in flowing water, then putting the tissue culture chamber into detergent diluent with the volume fraction of 2-3 per mill, washing for 10-15min, then placing into absorbent gauze, washing for 1-2h with running water, soaking in 75% alcohol for 10-25ms on an ultra-clean workbench, washing with sterile water for 2-3 times, soaking in 0.1% mercuric chloride solution for 1.5-3min, stirring continuously with forceps, transferring into sterile water, washing for 4-6 times, cutting into 0.4-0.5cm bud-bearing stems, absorbing water with sterile filter paper, transferring into culture bottles filled with primary culture medium, inoculating 2 bud-bearing stems into each bottle, transferring into a culture chamber, culturing at 21-23 ℃ and 60-70% humidity, dark culturing for 7-10 days, and culturing for 15-20 days under the condition of 2000-2400LX to obtain sterile bottle seedlings;
the formula of the primary culture medium is an improved F14 culture medium, 0.2-0.6 mg/L6-benzyladenine (6-BA), 0.02-0.1mg/L indolebutyric acid (IBA), 10-20g/L active carbon, 20g/L white granulated sugar and 5g/L agar, and the pH value is 5.6;
The improved F14 culture medium consists of major elements, trace elements and organic matters, wherein the major elements are 480mg/L of ammonium nitrate, 220mg/L of potassium nitrate, 360mg/L of magnesium sulfate heptahydrate, 240mg/L of potassium dihydrogen phosphate and 1000mg/L of calcium nitrate tetrahydrate; the microelements are manganese sulfate 0.67mg/L, zinc sulfate heptahydrate 0.86mg/L, sodium molybdate dihydrate 0.25mg/L, boric acid 12.4mg/L, potassium iodide 0.08mg/L, copper sulfate pentahydrate 0.025mg/L, chloridizing drill hexahydrate 0.025mg/L, EDTA-Na 2 27.8.8 mg/L, ferric sulfate heptahydrate 37.3mg/L; the organic matters are glycine 2mg/L, pyridoxine hydrochloride 1mg/L, nicotinic acid 1mg/L, thiamine hydrochloride 0.8mg/L and inositol 100mg/L;
S2, expanding propagation and subculture
Expanding propagation of sterile line seedlings which survive primary culture, selecting buds which grow robustly and have consistent integrity, transferring the buds into an expanding propagation culture medium, inoculating 4 buds into a culture chamber, culturing at 21-23 ℃ and 60-70% humidity, culturing in dark for 7-10 days, culturing for 20-35 days under 2000-2400LX of illumination, and culturing a large number of cluster buds for 30-40 days to obtain a cluster bud sterile line material;
The propagation medium comprises an improved F14 medium, 0.60 mg/L6-benzyladenine (6-BA), 0.06-0.12mg/L naphthylacetic acid (NAA), 0.06-0.12mg/L gibberellin (GA 3), 20g/L white granulated sugar and 5g/L agar, and the pH value is 5.6;
S3, detoxification
Cutting the sterile series material of the cluster buds subjected to propagation and subculture, cutting 0.4-0.6cm of new buds, carrying out secondary propagation and subculture transfer, transferring to a propagation culture medium (same as S2), and inoculating 4 strains per bottle, wherein the method specifically comprises the following steps: culturing at 22-25deg.C for 15-20 days, transferring to artificial climate box, respectively culturing under light for 14 hr and dark culturing for 10 hr, and culturing at 26deg.C for 10-15 days while raising temperature to 32deg.C and keeping 32deg.C; the illumination culture is started from 26 ℃, the temperature is increased to 1 ℃ every day, the temperature is kept unchanged after the temperature is increased to 36-38 ℃, then the illumination culture is carried out in a 20-day artificial climate box at 35-38 ℃, the dark culture is carried out at 32 ℃, the stem tips of 0.4-0.5mm are peeled from the tender tips of the materials after the culture is completed, the transfer culture is carried out for 14-21 days, then the stem tips are peeled for the second time, when the stem tips of 0.4-0.5mm are peeled and cultured for 35-45 days, buds of 2.0-4.0cm are cut, the virus detection is carried out, and the virus-free test tube seedlings are obtained for propagation and subculture of the virus-free test tube seedlings according to the method of S2;
S4, cutting rooting
S401, taking out the virus-free test-tube plantlet propagation material for 35-45 days from a culture flask by forceps, cutting off basal callus, cutting off individual stem segments with the length of 2.0-3.0cm, picking off basal 2-5 lobules, filling into a container, spraying water and sealing for standby;
S402, preparing a nutrition pot, putting a substrate (the substrate is vermiculite and perlite, the mass ratio is 8:2), and adding rooting nutrient solution, wherein the rooting nutrient solution is as follows: adding 25g of rooting powder of the Kadsura brand of Shanxi Jiale fertilizer company Limited into 10kg of water per bag, and then adding 100mL of a large amount of component mother liquor of an improved F14 culture medium; the major component mother liquor of the improved F14 culture medium is 50 times of the concentration of the formula of the improved F14 culture medium;
S403, inserting the cut test tube seedlings into matrixes in the nutrition pot according to a spacing of 2X 2cm, wherein the humidity of the matrixes is 60-80%, and covering the whole nutrition pot with a white plastic film after the cut test tube seedlings are fully inserted, so that the seedlings are placed in a relatively closed space in the nutrition pot for rooting culture;
S404, after culturing for 15-20 days, observing rooting conditions, when a large number of white cluster roots are generated at the base of the young seedling, removing the plastic film, transferring the nutrition pot to the ground for shading culture, and maintaining the humidity above 85 percent for management;
s405, after culturing for 40-60 days, the leaves of the young seedlings in the nutrition pot become green and thick, when the root system accelerates growth, the young seedling single plants are moved into vermiculite and decomposed organic fertilizer, wherein the mass ratio of the vermiculite to the decomposed organic fertilizer is 1:0.5, watering thoroughly in time to enable the seedlings to gradually adapt to the open field growth environment, and when the seedlings grow to 5-10cm, moving the seedlings into a field with soil, and managing the seedlings by referring to a field seedling method.
Comparative example 1
A tissue culture detoxification rapid propagation method of waterlogging-resistant and saline-alkali-resistant large cherry stock seedlings, which has the steps similar to those of example 1, is characterized in that the used culture medium is an MS culture medium, and the comparison of the formulas of the MS culture medium and an improved F14 culture medium is shown in Table 1.
Table 1 comparison of MS medium and modified F14 medium formulations
Comparative example 2
A tissue culture detoxification rapid propagation method of a waterlogging-resistant and saline-alkali-resistant large cherry stock seedling, which is the same as that of the embodiment 1, is characterized in that the used culture medium is an improved MS culture medium, the specific composition of the culture medium is shown in a patent CN 112293255A, and the formula comparison of the improved MS culture medium and an improved F14 culture medium is shown in a table 2.
Table 2 comparison of modified MS medium and modified F14 medium formulations
Comparative example 3
A tissue culture detoxification rapid propagation method of waterlogging-resistant and saline-alkali-resistant large cherry stock seedlings, which has the steps similar to those of example 1, is characterized in that the used culture medium is F14 culture medium, and the comparison of the formulas of the F14 culture medium and the modified F14 culture medium is shown in Table 3.
TABLE 3 comparison of F14 Medium and F14 modified Medium formulations
The test material was Landing No. 2 primary culture sterile line seedlings, 50 bottles of each medium of example 1 and comparative examples 1 to 3 were prepared in total, 4 shoots were inoculated per bottle, and propagation coefficients were investigated for 25 days. 9 bottles were randomly withdrawn and the results of the investigation are shown in Table 4.
TABLE 4 comparison of propagation effects of different media
The data analysis result shows that the culture medium rate is low by adopting MS and improving MS, and the phenomena of dry tip, aging and dry-up appear in the new buds; the F14 culture medium is adopted, the new buds are yellow, the stems become crisp, the survival rate of primary culture is low, the seedling growth vigor is weak, and the leaves curl; the improved F14 culture medium is adopted, the germination of the new buds is quick, the new buds are almost continuous and long, the leaves are light green, the propagation effect is best, the consistency of stock seedlings is higher, the growth vigor is good, and a large number of detoxified test tube seedlings can be obtained in a short period.
The large cherry stock magnalium No. 2 has deep and developed root system, less tillering, good soil fixation, strong waterlogging resistance, salt and alkali resistance, brown spot resistance, root nodule resistance and strong soil adaptability. The tissue culture detoxification rapid propagation method of the waterlogging-resistant and saline-alkali-resistant large cherry stock seedling can obtain a large number of large cherry stock magnalium No. 2 detoxification test tube seedlings in a short time, and the large cherry stock magnalium No. 2 detoxification test tube seedlings are supplied to the market after grafting, so that the requirements of large cherry planting in the river beach land and the saline-alkali soil can be effectively met.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention. Any simple modification, variation and equivalent variation of the above embodiments according to the technical substance of the invention still fall within the scope of the technical solution of the invention.
Claims (6)
1. A tissue culture detoxification rapid propagation method of waterlogging-resistant and saline-alkali-resistant large cherry stock seedlings is characterized by comprising the following steps:
S1, primary culture
After natural fallen leaves of 2 # 2 large cherry stock and 2 # 5 large cherry stock are cut for 11 months, an annual branch is cut, surface cleaning is carried out in running water, water culture is carried out under the condition that the temperature is 22 ℃ and the illumination is 2000-2400LX, when new buds grow to 2cm, terminal buds and bud stem sections of the first lateral buds below are collected, the bud stem sections are soaked in 84 disinfectant with the volume concentration of 1 millfor 5-10min and then washed under the running water for 2-6min, then the bud stem sections are placed in a liquid detergent diluent for cleaning for 10-15min, then the bud stem sections are placed in absorbent gauze for washing for 1-2h, sterile water is used for washing for 2-3 times after the soaking in alcohol with the volume concentration of 75% for 10-25ms, then the bud stem sections are treated in a mercuric rising solution with the volume concentration of 0.1% for 1.5-3min, the bud stem sections are transferred to a sterile water bottle for 4-6 times after the treatment is completed, then the bud stem sections with the volume concentration of 0.4-0.5cm to 0.5cm are soaked in 84 disinfectant with the volume concentration of 1 millfor 5-10min, then the bud sections are placed in a sterile water bottle with the sterile water culture medium for transferring to the sterile water culture medium for 1-20-15 h under the conditions that the culture stems are placed in a dark bottle with the water for 20-2000-15 days, and the culture humidity is placed under conditions of 20-20 days, and the conditions of 1-20% for culturing seedlings are obtained;
The formula of the primary culture medium is an improved F14 culture medium, 0.2-0.6 mg/L6-benzyladenine, 0.02-0.1mg/L indolebutyric acid, 10-20g/L active carbon, 20g/L white granulated sugar and 5g/L agar, and the pH value is 5.6;
The improved F14 culture medium consists of major elements, trace elements and organic matters, wherein the major elements are 480mg/L of ammonium nitrate, 220mg/L of potassium nitrate, 360mg/L of magnesium sulfate heptahydrate, 240mg/L of potassium dihydrogen phosphate and 1000mg/L of calcium nitrate tetrahydrate; the microelements are manganese sulfate 0.67mg/L, zinc sulfate heptahydrate 0.86mg/L, sodium molybdate dihydrate 0.25mg/L, boric acid 12.4mg/L, potassium iodide 0.08mg/L, copper sulfate pentahydrate 0.025mg/L, chloridizing drill hexahydrate 0.025mg/L, EDTA-Na 2 27.8.8 mg/L, ferric sulfate heptahydrate 37.3mg/L; the organic matters are glycine 2mg/L, pyridoxine hydrochloride 1mg/L, nicotinic acid 1mg/L, thiamine hydrochloride 0.8mg/L and inositol 100mg/L;
S2, expanding propagation and subculture
Expanding propagation of sterile line seedlings which survive primary culture in the step S1, selecting buds which grow robustly and have consistent integrity, transferring the buds into an expanding propagation culture medium, transferring the buds into a culture chamber, performing dark culture for 7-10 days at the culture temperature of 21-23 ℃ and the humidity of 60-70%, and performing culture for 20-35 days under the condition of 2000-2400LX illumination to obtain cluster bud sterile line materials;
The formula of the propagation medium comprises an improved F14 medium, 0.60 mg/L6-benzyl adenine, 0.06-0.12mg/L naphthylacetic acid, 0.06-0.12mg/L gibberellin, 20g/L white granulated sugar and 5g/L agar, and the pH value is 5.6;
S3, detoxification
Cutting the sterile series material of the cluster buds subjected to S2 propagation and subculture, cutting 0.4-0.6cm of new buds, carrying out secondary propagation and subculture transfer, transferring to the propagation culture medium in S2, and inoculating 4 strains per bottle, wherein the method specifically comprises the following steps: culturing for 15-20 days at 22-25 ℃, transferring to a climatic chamber, then respectively carrying out illumination culture for 14h and dark culture for 10h each day, starting the dark culture of the climatic chamber at 26 ℃ for 10-15 days, raising the temperature to 1 ℃ each day, raising the temperature to 32 ℃ and then not raising the temperature again, keeping the temperature unchanged at 32 ℃, starting the illumination culture at 26 ℃, raising the temperature to 1 ℃ each day, raising the temperature to 36-38 ℃ and then keeping the temperature unchanged, carrying out illumination culture of the climatic chamber at 35-38 ℃ for 20 days, carrying out dark culture at 32 ℃, peeling stem tips of 0.4-0.5mm from tender tips of materials after the culture is completed, carrying out transfer culture for 14-21 days, then carrying out secondary stem tip peeling, peeling stem tips of 0.4-0.5mm, culturing for 35-45 days, shearing buds of 2.0-4.0cm, carrying out virus detection, and obtaining virus-free test tube seedlings, and carrying out virus-free test tube seedling expansion and secondary culture according to the method of S2;
S4, cutting rooting
S401, cutting off basal callus from a virus-free test-tube plantlet propagation material for 35-45 days, cutting off a single stem section with the length of 2.0-3.0cm, picking off 2-5 lobules of the basal part, and filling the single stem section into a container for water spraying and sealing for standby;
s402, preparing a nutrition pot, putting a substrate, and adding rooting nutrient solution;
s403, inserting the cut test tube seedlings into matrixes in the nutrition pot according to a spacing of 2X 2cm, wherein the humidity of the matrixes is 60-80%, and covering the whole nutrition pot with a white plastic film after the cut test tube seedlings are fully inserted, so as to perform rooting culture;
S404, after culturing for 15-20 days, observing rooting condition, when the root of the seedling grows into cluster root, removing the plastic film, transferring the nutrition pot to the ground for shading culture, and keeping the humidity above 85%;
and S405, after culturing for 40-60 days, transferring the individual plantlets into vermiculite and decomposed organic fertilizer, and watering thoroughly on time to enable the plantlets to gradually adapt to the open field growth environment, and transferring the plantlets into a field with soil when the plantlets grow to 5-10cm, and managing by referring to a field seedling method.
2. The method according to claim 1, wherein the detergent diluent of S1 is prepared by adding 2-3mL of detergent into 1000mL of water and mixing uniformly.
3. The method according to claim 1, wherein the step S1 of treating in the 0.1% by volume mercuric solution is to soak the material in the 0.1% by volume mercuric solution to allow the liquid medicine to permeate 1-2cm of the material, and stirring the material with forceps to allow the liquid medicine to uniformly reach the surface of the material.
4. The method according to claim 1, wherein the rooting matrix in S402 is vermiculite and perlite with a mass ratio of 8:2.
5. The method of claim 1, wherein the rooting nutrient for cutting rooting in S402 is: adding 25g of rooting powder into 10kg of water, and then adding 100mL of a large amount of component mother liquor of the improved F14 culture medium; the major component mother liquor of the modified F14 culture medium is 50 times of the concentration of the modified F14 culture medium formula.
6. The method of claim 1, wherein the mass ratio of vermiculite to decomposed organic fertilizer in S405 is 1:0.5.
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