CN114532228A - Method for cultivating plukenetia volubilis hexaploid plants - Google Patents
Method for cultivating plukenetia volubilis hexaploid plants Download PDFInfo
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- 241001300674 Plukenetia volubilis Species 0.000 title claims abstract description 36
- 241000196324 Embryophyta Species 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title abstract description 10
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 60
- 210000001938 protoplast Anatomy 0.000 claims abstract description 40
- 208000026487 Triploidy Diseases 0.000 claims abstract description 13
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims abstract description 8
- 238000012364 cultivation method Methods 0.000 claims abstract description 5
- 229960001338 colchicine Drugs 0.000 claims abstract description 4
- 230000001939 inductive effect Effects 0.000 claims abstract description 3
- 238000012216 screening Methods 0.000 claims abstract 2
- 230000006698 induction Effects 0.000 claims description 31
- 239000001963 growth medium Substances 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 12
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- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 6
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 6
- 229930195725 Mannitol Natural products 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000594 mannitol Substances 0.000 claims description 6
- 235000010355 mannitol Nutrition 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 5
- 101710097941 N-acetylmuramoyl-L-alanine amidase CwlA Proteins 0.000 claims description 4
- 238000010494 dissociation reaction Methods 0.000 claims description 4
- 230000005593 dissociations Effects 0.000 claims description 4
- 210000002257 embryonic structure Anatomy 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 claims description 3
- 239000012883 rooting culture medium Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 108010076119 Caseins Proteins 0.000 claims description 2
- 108010059892 Cellulase Proteins 0.000 claims description 2
- 239000007836 KH2PO4 Substances 0.000 claims description 2
- 108010059820 Polygalacturonase Proteins 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 239000005018 casein Substances 0.000 claims description 2
- 108010079058 casein hydrolysate Proteins 0.000 claims description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 229940106157 cellulase Drugs 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 2
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 2
- 210000003608 fece Anatomy 0.000 claims description 2
- 238000005286 illumination Methods 0.000 claims description 2
- 229910052928 kieserite Inorganic materials 0.000 claims description 2
- 239000010871 livestock manure Substances 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 239000003415 peat Substances 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- 239000012882 rooting medium Substances 0.000 claims description 2
- 239000004576 sand Substances 0.000 claims description 2
- 239000002689 soil Substances 0.000 claims description 2
- 238000004043 dyeing Methods 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 5
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- 208000020584 Polyploidy Diseases 0.000 abstract description 3
- 239000003921 oil Substances 0.000 description 10
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
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- 241000221017 Euphorbiaceae Species 0.000 description 2
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 229960004488 linolenic acid Drugs 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 241000257468 Asterias amurensis Species 0.000 description 1
- 241001246270 Calophyllum Species 0.000 description 1
- 241000526900 Camellia oleifera Species 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 241001510071 Pyrrhocoridae Species 0.000 description 1
- 241000133425 Stauntonia Species 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
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- 239000002537 cosmetic Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000004626 essential fatty acids Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 229960004232 linoleic acid Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 229960002969 oleic acid Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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Classifications
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- 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
- 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
-
- 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
- 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
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
-
- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Inorganic Chemistry (AREA)
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- Soil Sciences (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of ploidy breeding of plants, and particularly relates to a cultivation method of a plukenetia volubilis hexaploid plant. The method comprises the steps of inducing callus and free protoplast by using endosperm of plukenetia volubilis seeds under the aseptic culture condition, selecting triploid protoplast, doubling the triploid protoplast by using colchicine to be hexaploid, selecting the hexaploid protoplast by using the free protoplast, and performing induced culture on the hexaploid protoplast to form a hexaploid plant. The method has the advantages that the screening can be carried out at the protoplast stage, the method is rapid and efficient, the inductivity is high, the obtained polyploid material can obtain a large amount of hexaploid test-tube plantlets through enrichment culture, and the breeding process is greatly shortened.
Description
Technical Field
The invention relates to the technical field of ploidy breeding of crops, and particularly discloses a cultivation method of a plukenetia volubilis hexaploid plant.
Background
Plukenetia volubilis L, also called Plukenetia volubilis L, is a perennial woody vine oil plant of Euphorbiaceae (Euphorbiaceae), mainly utilizes seeds and is rich in oil, amino acid, vitamin and the like, wherein the oil mainly comprises polyunsaturated fatty acids such as alpha-linolenic acid, alpha-linoleic acid and oleic acid, the content of the three unsaturated fatty acids is more than 90 percent and is obviously higher than that of oil plants such as peanut, rape, camellia oleifera and the like, and especially the content of the essential fatty acid of human body such as alpha-linolenic acid accounts for 45 to 53 percent of the oil component, so the Plukenetia volubilis L is regarded as high-end plant edible oil and has wide application prospect in the fields of food, medicine, cosmetics and the like.
After the plukenetia volubilis linneo is introduced into China in 2006, introduction and cultivation are carried out in provinces such as Yunnan province and Guizhou province and the success is achieved. The plukenetia volubilis linneo has the characteristics of short period, quick response, good high yield performance, capability of being planted in mountainous areas and hilly areas and the like. In addition, the plukenetia volubilis linneo is planted in the same year, blossoms and fruits in the same year, can continuously gain for many years after being planted once, has high yield value, and is an economic crop with wide development prospect. However, the plukenetia volubilis is a newly introduced species, the germplasm resources are deficient, the improved variety breeding is restricted, and the development of the plukenetia volubilis industry is not facilitated. The reproductive organs such as flowers, fruits and the like of the plant after polyploidization become large, and the content of secondary metabolites is improved, so that the cultivation of polyploid plukenetia volubilis can be one of ways for improving the grease yield of plukenetia volubilis. Therefore, the invention separates a cultivation method of the plukenetia volubilis hexaploid plant, has the characteristics of strong operability, high efficiency, low cost, short period and the like, and can lay a foundation for promoting plukenetia volubilis polyploidy breeding and cultivating a new variety of high-oil plukenetia volubilis.
Disclosure of Invention
The invention aims to provide a cultivation method of a plukenetia volubilis hexaploid plant, which utilizes the characteristic that endosperm is triploid and combines the biological effect that colchicine can double chromosomes to quickly and efficiently obtain the plukenetia volubilis hexaploid plant through tissue culture, thereby realizing the aim of the invention.
The invention relates to a method for cultivating a plukenetia volubilis hexaploid plant, which comprises the following process steps:
(1) endosperm callus induction: under the aseptic condition, after the conventional disinfection treatment of the asterias amurensis seeds, removing seed coats, inoculating embryos and endosperms into a callus induction culture medium, culturing for 30 days under the dark condition, stripping the embryos, and continuously culturing and inducing endosperm callus by the endosperms, wherein the callus induction culture medium is MS + 1.0-3.0 mg/L6-BA + 0.5-2.0 mg/L NAA + 0.5-1.0 g/L casein hydrolysate + 0.5-1.0 g/L morpholine ethanesulfonic acid + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and the pH is 5.4-5.8;
(2) protoplast dissociation and sorting: after endosperm callus is placed in the dark at 4 ℃ for 24h, callus enzymolysis liquid is used for treating free protoplasts for 5-10 h, then living DNA staining agent (5-10 mug/ml Hoechst 33342) is used for staining for 3-5 min, and triploid cells are screened by a flow cytometer under the aseptic condition to be enriched. The callus enzymolysis liquid comprises cell wall hydrolase and a protoplast washing liquid, wherein the cell wall hydrolase is 1.0-2.0% of cellulase, 0.5-1.0% of isolation enzyme and 0.5-1.0% of pectinase, and the protoplast washing liquid is 20-25 mg/L KH2PO4+90～100mg/L KNO3+180～240mg/L MgSO4·H2O+0.1～0.2mg/L KI+0.01～0.02mg/L CuSO4+1.0～1.5g/L CaCl2·H20.5-0.8 mol/L of mannitol and 10-20 mu mol/L of morpholine ethanesulfonic acid, wherein the pH value is 5.4-5.8;
(3) induction and sorting of hexaploid callus: and (3) placing the triploid protoplast obtained by sorting into a hexaploid callus induction culture medium, carrying out soft shaking culture at 25 ℃ under the dark condition until a callus block is formed, transferring the triploid protoplast into the callus induction culture medium for culture to obtain a callus, carrying out protoplast dissociation according to the step (2), and sorting out the hexaploid protoplast. The hexaploid callus induction culture medium is MS + 1.0-3.0 mg/L6-BA + 0.5-2.0 mg/L NAA + 1.0-2.0 g/L colchicine + 0.5-1.0 g/L hydrolyzed casein + 0.5-1.0 g/L morpholine ethanesulfonic acid + 0.5-0.8 mol/L mannitol + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and the pH value is 5.4-5.8;
(4) culturing hexaploid protoplast to form callus: placing the hexaploid protoplast obtained by sorting into a callus induction culture medium containing 0.5-0.8 mol/L mannitol, carrying out soft shaking culture at 25 ℃ under the dark condition until a callus block is formed, transferring the hexaploid protoplast into the callus induction culture medium, and carrying out illumination intensity of 40 mu mol.m at 25 DEG C-2·s-1Culturing under the condition to obtain callus.
(5) Induction of hexaploid callus to form adventitious buds: inoculating the callus into a bud differentiation culture medium for bud differentiation culture to obtain adventitious buds. The bud differentiation culture medium comprises: MS + 0.5-1.0 mg/L6-BA + 0.1-0.5 mg/L NAA + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and pH is 5.4-5.8;
(6) adventitious bud induction rooting to form a hexaploid seedling: inoculating the adventitious bud into a rooting culture medium for rooting culture to obtain the star oil vine hexaploid seedling. The rooting medium is 1/2MS + 0.1-0.5 mg/L IBA + 1.0-2.0 mg/L GGR + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and the pH value is 5.4-5.8;
(7) cultivation: cleaning the root of the seedling of the plukenetia volubilis linneo, and culturing the seedling in a mixed matrix to obtain the plukenetia volubilis linneo seedling. The mixed matrix is formed by mixing peat soil, farmyard manure and river sand according to the ratio of 8:1: 1.
The method comprehensively utilizes endosperm culture and chromosome doubling technologies to culture the plukenetia volubilis hexaploid plant, and has the characteristics of strong operability, high efficiency, low cost, short period and the like.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example one
Star-shaped oil vine endosperm induced callus
Collecting mature Plukenetia volubilis L seeds in late 8 months, removing aril, treating the Plukenetia volubilis L seeds by a conventional disinfection method under aseptic conditions, removing the seed coat, inoculating an embryo and endosperm into a callus induction culture medium, culturing for 30 days under dark conditions, stripping the embryo, continuously culturing endosperm to induce endosperm callus, wherein the callus induction rate can reach over 75.8%.
Example two
Star oil vine callus free protoplast
Selecting pale yellow callus from Plukenetia volubilis L, carrying out dark treatment at 4 ℃ for 24h, weighing 0.5g of the callus under aseptic condition, placing the callus into a 50ml centrifugal tube containing 10ml of enzymolysis liquid, sealing the tube with a sealing film, carrying out dark treatment at 25 ℃ for 5-10 h, and gently inverting and mixing the callus once every 1 h. After the enzyme treatment, the mixture was filtered through a 300-mesh cell sieve, and the filtrate was placed in a new sterile centrifuge tube and sealed with a sealing film, and centrifuged at 100g for 3 min. After the supernatant was aspirated, the protoplast wash was added to resuspend, and the aspiration-resuspension was repeated 3 times. And (3) fixing the suspension to 500ml, taking a little suspension, staining the suspension for 3min by 0.03% trypan blue, then dropwise adding the suspension onto a cell counting plate, and observing and counting the free number and survival rate of the protoplast under a microscope. The results showed that 1g of Stauntonia brachypoides callus could be liberated 2.5 x 106The survival rate of each protoplast reaches more than 90 percent.
EXAMPLE III
Plukenetia volubilis protoplast sorting
Stainers (Plukenetia volubilis L.) protoplasts were stained with 5-10. mu.g/ml of a living DNA stain (Hoechst 33342) for 3-5 min, centrifuged for 3min at 100g, resuspended in a protoplast wash after discarding the staining solution, and the pipetting-resuspension was repeated 3 times. The protoplasts were collected, flow cytometrically sorted under sterile conditions, and triploid or hexaploid protoplasts were sorted according to the instrument instructions.
Example four
Calophyllum hexaploid callus induction
The triploid protoplast of Plukenetia volubilis L is placed in a hexaploid callus induction culture medium, and is subjected to soft shaking culture at 25 ℃ in the dark until callus blocks are formed, and then the triploid protoplast is transferred to callus induction culture to be cultured to obtain callus. Flow cytometric sorting results show that the induction rate of the hexaploid callus can reach 36.3%.
EXAMPLE five
Induction of bud and root differentiation of star oil vine hexaploid callus
Inoculating the hexaploid callus into a bud differentiation culture medium for bud differentiation induction to obtain an adventitious bud, transferring the adventitious bud into a rooting culture medium for rooting induction, and the result shows that the bud differentiation rate and the root differentiation rate of the hexaploid callus of the plukenetia volubilis respectively reach more than 85.5% and 90.3%.
Claims (1)
1. A cultivation method of a plukenetia volubilis hexaploid plant is characterized by comprising the following steps:
(1) endosperm callus induction: under the aseptic condition, after the conventional disinfection treatment of the plukenetia volubilis seeds, removing seed coats, inoculating embryos and endosperms into a callus induction culture medium, culturing for 30 days under the dark condition, stripping the embryos, and continuously culturing and inducing endosperm callus by the endosperms, wherein the callus induction culture medium is MS + 1.0-3.0 mg/L6-BA + 0.5-2.0 mg/L NAA + 0.5-1.0 g/L casein hydrolysate + 0.5-1.0 g/L morpholine ethanesulfonic acid + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and the pH is 5.4-5.8;
(2) protoplast dissociation and sorting: placing endosperm callus in the dark at 4 ℃ for 24h, treating free protoplasts with callus enzymolysis liquid for 5-10 h, then dyeing with living DNA stain (5-10 microgram/ml Hoechst 33342) for 3-5 min, and screening triploid cells by using a flow cytometer under the aseptic condition to enrich the triploid cells; the callus enzymolysis solution comprises cell wall hydrolase and a protoplast washing solution, wherein the cell wall hydrolase is 1.0-2.0% of cellulase, 0.5-1.0% of eductase and 0.5-1.0% of pectinase,the protoplast washing solution is 20-25 mg/L KH2PO4+90～100mg/L KNO3+180～240mg/L MgSO4·H2O+0.1～0.2mg/L KI+0.01～0.02mg/L CuSO4+1.0～1.5g/L CaCl2·H20.5-0.8 mol/L of mannitol and 10-20 mu mol/L of morpholine ethanesulfonic acid, and the pH value is 5.4-5.8;
(3) induction and sorting of hexaploid callus: placing the triploid protoplast obtained by sorting into a hexaploid callus induction culture medium, carrying out soft shaking culture at 25 ℃ under the dark condition until callus blocks are formed, transferring the triploid protoplast into the callus induction culture medium for culture to obtain callus, carrying out protoplast dissociation according to the step (2), and sorting out the hexaploid protoplast; the hexaploid callus induction culture medium is MS + 1.0-3.0 mg/L6-BA + 0.5-2.0 mg/L NAA + 1.0-2.0 g/L colchicine + 0.5-1.0 g/L hydrolyzed casein + 0.5-1.0 g/L morpholine ethanesulfonic acid + 0.5-0.8 mol/L mannitol + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and the pH value is 5.4-5.8;
(4) culturing hexaploid protoplast to form callus: placing the hexaploid protoplast obtained by sorting into a callus induction culture medium containing 0.5-0.8 mol/L mannitol, carrying out soft shaking culture at 25 ℃ under the dark condition until a callus block is formed, transferring the hexaploid protoplast into the callus induction culture medium, and carrying out illumination intensity of 40 mu mol.m at 25 DEG C-2·s-1Culturing under the condition to obtain callus;
(5) induction of hexaploid callus to form adventitious buds: inoculating the callus into a bud differentiation culture medium for bud differentiation culture to obtain adventitious buds; the bud differentiation culture medium comprises: MS + 0.5-1.0 mg/L6-BA + 0.1-0.5 mg/L NAA + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and pH is 5.4-5.8;
(6) adventitious bud induction rooting to form a hexaploid seedling: inoculating the adventitious bud into a rooting culture medium for rooting culture to obtain a plukenetia volubilis hexaploid seedling; the rooting medium is 1/2MS + 0.1-0.5 mg/L IBA + 1.0-2.0 mg/L GGR + 15-30 g/L sucrose + 3.5-6.0 g/L agar, and the pH value is 5.4-5.8;
(7) cultivation: cleaning the root of the seedling of the plukenetia volubilis hexaploid, and culturing in a mixed matrix to obtain the seedling of the plukenetia volubilis hexaploid; the mixed matrix is formed by mixing peat soil, farmyard manure and river sand according to the ratio of 8:1: 1.
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