CN106258870A - A kind of method obtaining broccoli sporidiole material in the winter time - Google Patents
A kind of method obtaining broccoli sporidiole material in the winter time Download PDFInfo
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- CN106258870A CN106258870A CN201510318967.9A CN201510318967A CN106258870A CN 106258870 A CN106258870 A CN 106258870A CN 201510318967 A CN201510318967 A CN 201510318967A CN 106258870 A CN106258870 A CN 106258870A
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- Prior art keywords
- sporidiole
- alabastrum
- broccoli
- winter time
- sulfate
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Links
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 title claims abstract description 31
- 235000017647 Brassica oleracea var italica Nutrition 0.000 title claims abstract description 31
- 240000003259 Brassica oleracea var. botrytis Species 0.000 title claims abstract description 29
- 239000000463 material Substances 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000035784 germination Effects 0.000 claims abstract description 15
- 235000015097 nutrients Nutrition 0.000 claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 239000011159 matrix material Substances 0.000 claims abstract description 7
- 235000016709 nutrition Nutrition 0.000 claims abstract description 7
- 230000035764 nutrition Effects 0.000 claims abstract description 7
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 6
- 239000012531 culture fluid Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 15
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- 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 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 9
- 239000004471 Glycine Substances 0.000 claims description 9
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 9
- 229910021538 borax Inorganic materials 0.000 claims description 9
- 239000001110 calcium chloride Substances 0.000 claims description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 9
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 9
- 229940097267 cobaltous chloride Drugs 0.000 claims description 9
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 9
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 9
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 9
- 229960000367 inositol Drugs 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 229940099596 manganese sulfate Drugs 0.000 claims description 9
- 239000011702 manganese sulphate Substances 0.000 claims description 9
- 235000007079 manganese sulphate Nutrition 0.000 claims description 9
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 9
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 9
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 9
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 9
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 9
- 239000011684 sodium molybdate Substances 0.000 claims description 9
- 235000015393 sodium molybdate Nutrition 0.000 claims description 9
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 9
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 9
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 9
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 9
- 229960001763 zinc sulfate Drugs 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000009331 sowing Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 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
- 238000001556 precipitation Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 235000011837 pasties Nutrition 0.000 claims description 3
- 235000010333 potassium nitrate Nutrition 0.000 claims description 3
- 239000004323 potassium nitrate Substances 0.000 claims description 3
- 230000001850 reproductive effect Effects 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 239000002574 poison Substances 0.000 claims description 2
- 231100000614 poison Toxicity 0.000 claims description 2
- 230000004888 barrier function Effects 0.000 abstract description 2
- 239000002689 soil Substances 0.000 abstract description 2
- 238000010586 diagram Methods 0.000 description 7
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 239000011593 sulfur Substances 0.000 description 4
- 229910052717 sulfur Inorganic materials 0.000 description 4
- 241000219198 Brassica Species 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 235000005637 Brassica campestris Nutrition 0.000 description 2
- 244000308180 Brassica oleracea var. italica Species 0.000 description 2
- 241001301148 Brassica rapa subsp. oleifera Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- JLKDVMWYMMLWTI-UHFFFAOYSA-M potassium iodate Chemical compound [K+].[O-]I(=O)=O JLKDVMWYMMLWTI-UHFFFAOYSA-M 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000011331 Brassica Nutrition 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 244000178937 Brassica oleracea var. capitata Species 0.000 description 1
- 244000025254 Cannabis sativa Species 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
- 230000007812 deficiency Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
-
- 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
- A01G31/00—Soilless cultivation, e.g. hydroponics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Abstract
The invention discloses a kind of method obtaining broccoli sporidiole material in the winter time, step is as follows: a. sows: be seeded in hole tray by broccoli seeds shallow-layer；B. accelerating germination: hole tray is placed accelerating germination on hotbed, daytime 20～25 DEG C, night 10～15 DEG C；C. field planting: during until growing 2～3 true leaves, field planting is cultivated in matrix nutrition cup, regular nutrient solution MB1；D. low temperature vernalization: until after true leaf grows 14～16, low temperature vernalization about 20 days at 8～15 DEG C, and coordinate nutrient solution MB2；E. cultivation: until after Curd formation, coordinating nutrient solution MB3 to continue cultivation；The ambient temperature controlled in booth is 20～25 DEG C, it is thus achieved that normal alabastrum；F. sporidiole is extracted: by the alabastrum chosen through sterilizing, extracting pollen, sporidiole material can be obtained.Above-mentioned cultivation technique without soil can cultivate, in warmhouse booth in the winter time, the alabastrum being suitable for doing sporidiole material, solves cannot normally obtain broccoli sporidiole technical barrier winter.
Description
Technical field
The present invention relates to the acquisition methods of a kind of sporidiole material, obtain green grass or young crops more specifically to one in the winter time
The method of Herba Astragali Sinici sporidiole material.
Background technology
Broccoli has another name called Caulis et Folium Brassicae capitatae, broccoli, stem Broccoli etc., is with green in Cruciferae Brassica genus brassica specie
Bouquet is the one of product organ, two raw herbaceous plant, and its green bouquet formed with stem and side shoot top is
Product, nutritious, color tool is good, is international market a kind of name spy vegetable in very great demand.
Since nineteen eighty-two Lichter reported first Brassica campestris L Plantlet Regeneration by Isolated Microspore Culture, Brassica genus
The microspores culture of crop, especially Brassica campestris L, either at germ extraction rate, go out embryo amount, or embryo culture and again
Raw plant aspect is all successful.Relative to the crop such as cabbage type rape, common head cabbage, blue and white
The cultivation difficulty of dish Isolated microspore is bigger.
And, at the normal season of flowers of broccoli, sporidiole can be obtained in a large number；But when suitable season at florescence
Save a mistake, after entering autumn and winter, owing to extraneous cultivation condition does not allows, it is impossible to little spore is properly done in normal extraction
The alabastrum of son, and then broccoli sporidiole cannot be obtained in the winter time, carry for initiative DH (dihaploid) material
For resource, this brings more difficulty to broccoli microspores culture breeding technique.
Summary of the invention
The deficiency of sporidiole material can not be obtained to make up prior art in the winter time, it is an object of the present invention to provide one
The method of kind, the season that the method can cannot normally be bloomed in the winter time, it is provided that the sporidiole material normally bloomed,
Resource is provided for initiative DH.
The present invention solves the technical scheme is that one of technical problem and obtains broccoli sporidiole in the winter time
The method of material, described method mainly comprises the steps that
A. sowing: in warmhouse booth, get out hole tray, fill cultivation matrix in hole tray, and water with water
Thoroughly, broccoli seeds shallow-layer is seeded in hole tray；
B. accelerating germination: above-mentioned sowing has the hole tray of broccoli seeds place accelerating germination on hotbed, and germination temperature controls
20～25 DEG C by day, night 10～15 DEG C；During accelerating germination, water management according to substrate dry and wet situation；
C. field planting: during until growing 2～3 true leaves, field planting is cultivated in matrix nutrition cup, and according to substrate
Dry and wet situation, regular nutrient solution MB1, control ambient temperature and humidity, thus reach to meet its nutrition
The required condition of growth；
D. low temperature vernalization: until after true leaf grows 14～16 end of nourishing and growing, control ambient temperature 8～
15 DEG C, low temperature vernalization induces it to enter reproductive growth, vernalization about 20 days, and period also coordinates pouring battalion
Nutrient solution MB2；
E. cultivation: until after Curd formation, according to substrate dry and wet situation, coordinating nutrient solution MB3 to continue
Continuous cultivation；The ambient temperature controlled in booth is 20～25 DEG C, it is thus achieved that normal alabastrum；
F. sporidiole is extracted: choose the alabastrum being of convenient length, by alabastrum through sterilization, extracting pollen,
Obtain the sporidiole material of broccoli in the winter time.
The particular make-up of described culture fluid MB1 is as follows: in terms of a cubic meter of water, wherein contain potassium nitrate 1.6kg,
Ammonium nitrate 1.4kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.15kg, inositol 50g, sulfur
Acid ferrous 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate 6g, iodate
Potassium 1g, glycine 2g, sodium molybdate 0.25g, copper sulfate 0.5g, cobaltous chloride 0.5g.
The particular make-up of described culture fluid MB2 is as follows: in terms of a cubic meter of water, wherein contains potassium chloride
1.05kg, ammonium nitrate 1.5kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.5kg, inositol
30g, ferrous sulfate 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate
10g, potassium iodide 1g, glycine 2g, sodium molybdate 0.35g, copper sulfate 0.5g, cobaltous chloride 0.5g.
The particular make-up of described culture fluid MB3 is as follows: in terms of a cubic meter of water, wherein contain potassium chloride 1kg,
Ammonium nitrate 1.15kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.9kg, inositol 10g, sulfur
Acid ferrous 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate 12g, iodine
Change potassium 1g, glycine 2g, sodium molybdate 0.5g, copper sulfate 0.5g, cobaltous chloride 0.5g.
The extracting method of above-mentioned sporidiole specifically includes herein below:
(1) flower bud is selected: choose the main inflorescence alabastrum of well-grown a length of 2.5～3.5mm, or choose
The side shoot alabastrum of a length of 3.5～4mm；
(2) sterilization: disinfect the above-mentioned alabastrum chosen in alcohol 30～40S, then disappear with sodium hypochlorite
Poison 10min, finally uses sterile water wash 3 times, each 5min；
(3) extracting pollen: be positioned in centrifuge tube by sterilized alabastrum, adds 1～2mL B5 culture fluid,
Make pollen shed by Glass rod grinding, be ground to pasty state, pipe adds half suction pipe B5 culture fluid, poured into
Filter filters, and filtrate is contained in the centrifuge tube of 10mL；Add the pollen in B5 culture fluid flush filter,
After rushing 2～3 times, merging filtrate, in 10mL centrifuge tube, pollen suspension is diluted；
By centrifugal treating 5min under 800 turns/min of pollen suspension；The supernatant in centrifuge tube is removed,
Add liquid B5 culture fluid again, and the pollen suction pipe of precipitation is broken up, suspended centrifugal again, remove supernatant；
Being diluted with NLN-13 culture fluid by the pollen of precipitation, the suspension after dilution is distributed into 6cm culture dish,
General every ware adds suspension about 5mL, every mL and contains about 1.5～2, alabastrum, seals with sealed membrane,
Just available required sporidiole material；This sporidiole material can continue to be trained embryoid in culture dish.
Further, the mass percent of the ethanol used in described sterilisation step is 75%, described sterilization step
The mass percent of the sodium hypochlorite used in rapid is 2%.
The invention has the beneficial effects as follows: compared with prior art, the present invention is by cultivation technique without soil in the winter time
Warmhouse booth in cultivate the alabastrum being suitable for doing sporidiole material, choose suitable alabastrum extract sporidiole material
Material, solves and cannot normally obtain the technical barrier of broccoli sporidiole winter.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of broccoli seeds sowing stage of the present invention.
Fig. 2 is the schematic diagram in broccoli seeds accelerating germination stage of the present invention.
Fig. 3 is the schematic diagram in field planting stage of the present invention.
Fig. 4 is the schematic diagram in the low temperature of the present invention period of vernalization.
Fig. 5 is the schematic diagram in Curd formation stage of the present invention.
Fig. 6 is the schematic diagram of the normal bouquet obtained in the present invention.
Fig. 7 is the schematic diagram after amplifying 40 times under the present invention medium and small spore shape inverted microscope.
Detailed description of the invention
Below in conjunction with the accompanying drawings and detailed description of the invention, it is further elucidated with the present invention, it should be understood that following be embodied as
Mode is merely to illustrate the present invention rather than limits the scope of the present invention.
Embodiment
A kind of method obtaining broccoli sporidiole material in the winter time, described method mainly comprises the steps that
A. sowing: in warmhouse booth, get out hole tray, fill cultivation matrix in hole tray, and water with water
Thoroughly, broccoli seeds shallow-layer is seeded in hole tray, as shown in Figure 1；
B. accelerating germination: above-mentioned sowing has the hole tray of broccoli seeds place accelerating germination on hotbed, and germination temperature controls
20～25 DEG C by day, night 10～15 DEG C；During accelerating germination, water management such as according to substrate dry and wet situation
Shown in Fig. 2；
C. field planting: during until growing 2～3 true leaves, field planting is cultivated in matrix nutrition cup, and according to substrate
Dry and wet situation, regular nutrient solution MB1, control ambient temperature and humidity, thus reach to meet its nutrition
The required condition of growth, as shown in Figure 3；
D. low temperature vernalization: until after true leaf grows 14～16 end of nourishing and growing, control ambient temperature 8～
15 DEG C, low temperature vernalization induces it to enter reproductive growth, vernalization about 20 days, and period also coordinates pouring battalion
Nutrient solution MB2, as shown in Figure 4；
E. cultivation: until after Curd formation, according to substrate dry and wet situation, coordinating nutrient solution MB3 to continue
Continuous cultivation；The ambient temperature controlled in booth is 20～25 DEG C, it is thus achieved that normal alabastrum, as shown in Figure 5；
F. sporidiole is extracted: choose the alabastrum (as shown in Figure 6) being of convenient length, by alabastrum through sterilizing, taking out
Jacquard weave powder, can obtain the sporidiole material of broccoli, sporidiole such as Fig. 7 of form under the microscope in the winter time
Shown in.
The particular make-up of described culture fluid MB1 is as follows: in terms of a cubic meter of water, wherein contain potassium nitrate 1.6kg,
Ammonium nitrate 1.4kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.15kg, inositol 50g, sulfur
Acid ferrous 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate 6g, iodate
Potassium 1g, glycine 2g, sodium molybdate 0.25g, copper sulfate 0.5g, cobaltous chloride 0.5g.
The particular make-up of described culture fluid MB2 is as follows: in terms of a cubic meter of water, wherein contains potassium chloride
1.05kg, ammonium nitrate 1.5kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.5kg, inositol
30g, ferrous sulfate 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate
10g, potassium iodide 1g, glycine 2g, sodium molybdate 0.35g, copper sulfate 0.5g, cobaltous chloride 0.5g.
The particular make-up of described culture fluid MB3 is as follows: in terms of a cubic meter of water, wherein contain potassium chloride 1kg,
Ammonium nitrate 1.15kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.9kg, inositol 10g, sulfur
Acid ferrous 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate 12g, iodine
Change potassium 1g, glycine 2g, sodium molybdate 0.5g, copper sulfate 0.5g, cobaltous chloride 0.5g.
The extracting method of above-mentioned sporidiole specifically includes herein below:
(1) flower bud is selected: choose the main inflorescence alabastrum of well-grown a length of 2.5～3.5mm, or choose
The side shoot alabastrum of a length of 3.5～4mm；
(2) sterilization: by the above-mentioned alcohol disinfecting that alabastrum mass percent is 75% 30～40S chosen,
Then with the hypochlorite disinfectant 10min that mass percent is 2%, finally sterile water wash is used 3 times, every time
5min；
(3) extracting pollen: be positioned in centrifuge tube by sterilized alabastrum, adds 1～2mL B5 culture fluid,
Make pollen shed by Glass rod grinding, be ground to pasty state, pipe adds half suction pipe B5 culture fluid, poured into
Filter filters, and filtrate is contained in the centrifuge tube of 10mL；Add the pollen in B5 culture fluid flush filter,
After rushing 2～3 times, merging filtrate, in 10mL centrifuge tube, pollen suspension is diluted；
By centrifugal treating 5min under 800 turns/min of pollen suspension；The supernatant in centrifuge tube is removed,
Add liquid B5 culture fluid again, and the pollen suction pipe of precipitation is broken up, suspended centrifugal again, remove supernatant；
Being diluted with NLN-13 culture fluid by the pollen of precipitation, the suspension after dilution is distributed into 6cm culture dish,
General every ware adds suspension about 5mL, every mL and contains about 1.5～2, alabastrum, seals with sealed membrane,
Just available required sporidiole material；
The culture dish of sealing carries out under conditions of 25 DEG C light culture, and general about 10 days start to check whether
There is the formation of visible graininess embryo, without then continuing to cultivate, if there being visible embryo to occur, then moving
Light culture is proceeded to 50rpm, 25 DEG C of constant-temperature tables.
Embodiment of above is merely to illustrate the present invention, and not limitation of the present invention, relevant technical field
Those of ordinary skill, without departing from the spirit and scope of the present invention, it is also possible to make various change
Changing and modification, the technical scheme of the most all equivalents falls within scope of the invention, the patent protection of the present invention
Scope should be defined by the claims.
Claims (6)
1. the method obtaining broccoli sporidiole material in the winter time, it is characterised in that: described method is main
Comprise the following steps:
A. sowing: in warmhouse booth, get out hole tray, fill cultivation matrix in hole tray, and water with water
Thoroughly, broccoli seeds shallow-layer is seeded in hole tray；
B. accelerating germination: above-mentioned sowing has the hole tray of broccoli seeds place accelerating germination on hotbed, and germination temperature controls
20～25 DEG C by day, night 10～15 DEG C；During accelerating germination, water management according to substrate dry and wet situation；
C. field planting: during until growing 2～3 true leaves, field planting is cultivated in matrix nutrition cup, and according to substrate
Dry and wet situation, regular nutrient solution MB1, control ambient temperature and humidity, thus reach to meet its nutrition
The required condition of growth；
D. low temperature vernalization: until after true leaf grows 14～16 end of nourishing and growing, control ambient temperature 8～
15 DEG C, low temperature vernalization induces it to enter reproductive growth, vernalization about 20 days, and period also coordinates pouring battalion
Nutrient solution MB2；
E. cultivation: until after Curd formation, according to substrate dry and wet situation, coordinating nutrient solution MB3 to continue
Continuous cultivation；The ambient temperature controlled in booth is 20～25 DEG C, it is thus achieved that normal alabastrum；
F. sporidiole is extracted: choose the alabastrum being of convenient length, by alabastrum through sterilization, extracting pollen,
Obtain the sporidiole material of broccoli in the winter time.
A kind of method obtaining broccoli sporidiole material in the winter time the most according to claim 1, it is special
Levying and be, the particular make-up of described culture fluid MB1 is as follows: in terms of a cubic meter of water, wherein contains potassium nitrate
1.6kg, ammonium nitrate 1.4kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.15kg, inositol
50g, ferrous sulfate 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate
6g, potassium iodide 1g, glycine 2g, sodium molybdate 0.25g, copper sulfate 0.5g, cobaltous chloride 0.5g.
A kind of method obtaining broccoli sporidiole material in the winter time the most according to claim 1, it is special
Levying and be, the particular make-up of described culture fluid MB2 is as follows: in terms of a cubic meter of water, wherein contains potassium chloride
1.05kg, ammonium nitrate 1.5kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.5kg, inositol
30g, ferrous sulfate 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate
10g, potassium iodide 1g, glycine 2g, sodium molybdate 0.35g, copper sulfate 0.5g, cobaltous chloride 0.5g.
A kind of method obtaining broccoli sporidiole material in the winter time the most according to claim 1, it is special
Levying and be, the particular make-up of described culture fluid MB3 is as follows: in terms of a cubic meter of water, wherein contains potassium chloride
1kg, ammonium nitrate 1.15kg, calcium chloride 0.5kg, magnesium sulfate 0.37kg, sodium dihydrogen phosphate 2.9kg, inositol 10g,
Ferrous sulfate 28g, disodiumedetate 40g, manganese sulfate 22g, zinc sulfate 9g, sodium borate 12g,
Potassium iodide 1g, glycine 2g, sodium molybdate 0.5g, copper sulfate 0.5g, cobaltous chloride 0.5g.
A kind of method obtaining broccoli sporidiole material in the winter time the most according to claim 1, it is special
Levying and be, the extracting method of described sporidiole specifically includes herein below:
(1) flower bud is selected: choose the main inflorescence alabastrum of well-grown a length of 2.5～3.5mm, or choose
The side shoot alabastrum of a length of 3.5～4mm；
(2) sterilization: disinfect the above-mentioned alabastrum chosen in alcohol 30～40S, then disappear with sodium hypochlorite
Poison 10min, finally uses sterile water wash 3 times, each 5min；
(3) extracting pollen: be positioned in centrifuge tube by sterilized alabastrum, adds 1～2mL B5 culture fluid,
Make pollen shed by Glass rod grinding, be ground to pasty state, pipe adds half suction pipe B5 culture fluid, poured into
Filter filters, and filtrate is contained in the centrifuge tube of 10mL；Add the pollen in B5 culture fluid flush filter,
After rushing 2～3 times, merging filtrate, in 10mL centrifuge tube, pollen suspension is diluted；
By centrifugal treating 5min under 800 turns/min of pollen suspension；The supernatant in centrifuge tube is removed,
Add liquid B5 culture fluid again, and the pollen suction pipe of precipitation is broken up, suspended centrifugal again, remove supernatant；
Being diluted with NLN-13 culture fluid by the pollen of precipitation, the suspension after dilution is distributed into 6cm culture dish,
General every ware adds suspension about 5mL, every mL and contains about 1.5～2, alabastrum, seals with sealed membrane,
Just available required sporidiole material；This sporidiole material can continue to be trained embryoid in culture dish.
A kind of method obtaining broccoli sporidiole material in the winter time the most according to claim 5, it is special
Levy and be: the mass percent of the ethanol used in described sterilisation step is 75%, adopts in described sterilisation step
The mass percent of sodium hypochlorite be 2%.
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