CN106258870B - Method for obtaining broccoli microspore material in winter - Google Patents
Method for obtaining broccoli microspore material in winter Download PDFInfo
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- CN106258870B CN106258870B CN201510318967.9A CN201510318967A CN106258870B CN 106258870 B CN106258870 B CN 106258870B CN 201510318967 A CN201510318967 A CN 201510318967A CN 106258870 B CN106258870 B CN 106258870B
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- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 title claims abstract description 29
- 240000003259 Brassica oleracea var. botrytis Species 0.000 title claims abstract description 29
- 235000017647 Brassica oleracea var italica Nutrition 0.000 title claims abstract description 28
- 239000000463 material Substances 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 12
- 230000035784 germination Effects 0.000 claims abstract description 14
- 230000007613 environmental effect Effects 0.000 claims abstract description 10
- 238000009331 sowing Methods 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims abstract description 6
- 235000016709 nutrition Nutrition 0.000 claims abstract description 5
- 230000035764 nutrition Effects 0.000 claims abstract description 5
- 230000001954 sterilising effect Effects 0.000 claims abstract description 4
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 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
- 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
- BDOYKFSQFYNPKF-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O BDOYKFSQFYNPKF-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
- 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
- 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
- 239000011790 ferrous sulphate Substances 0.000 claims description 9
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 9
- 238000011049 filling Methods 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
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 9
- 229910000359 iron(II) sulfate Inorganic materials 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
- 239000000203 mixture Substances 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
- 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
- 230000000249 desinfective effect Effects 0.000 claims description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 239000010902 straw Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 230000009105 vegetative growth Effects 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 5
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 235000010333 potassium nitrate Nutrition 0.000 claims description 4
- 239000004323 potassium nitrate Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000001850 reproductive effect Effects 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims 1
- 235000015097 nutrients Nutrition 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000012258 culturing Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 240000007124 Brassica oleracea Species 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica 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
- 241000219193 Brassicaceae Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 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 method for obtaining a broccoli microspore material in winter, which comprises the following steps: a. sowing: sowing broccoli seeds in the hole tray in a shallow layer; b. accelerating germination: placing the plug tray on a hotbed for accelerating germination, wherein the temperature is 20-25 ℃ in the daytime and 10-15 ℃ at night; c. planting: when 2-3 true leaves grow, planting and cultivating in a substrate nutrition cup, and regularly irrigating nutrient solution MB 1; d. and (3) low-temperature vernalization: after 14-16 true leaves grow out, vernalizing at the low temperature of 8-15 ℃ for about 20 days, and pouring nutrient solution MB2 in a matched manner; e. cultivation: after the flower balls are formed, pouring nutrient solution MB3 to continue cultivation; controlling the environmental temperature in the greenhouse to be 20-25 ℃ to obtain normal buds; f. extracting microspores: sterilizing the selected flower buds, and extracting pollen to obtain the microspore material. The soilless culture technology can be used for culturing buds suitable for serving as microspore materials in a greenhouse in winter, and the technical problem that the microspores of the broccoli cannot be normally obtained in winter is solved.
Description
Technical Field
The invention relates to a method for obtaining microspore material, in particular to a method for obtaining the microspore material of broccoli in winter.
Background
The broccoli is also named broccoli, cauliflower and the like, is a first-generation herbaceous plant and a second-generation herbaceous plant which take green bulbs as product organs in brassica cabbage seeds of cruciferae, takes the green bulbs formed at the tops of main stems and side branches as products, is rich in nutrition, has good color, fragrance and taste, and is a famous and special vegetable which is well sold in the international market.
Since Lichter reported that the culture of isolated microspores of rape obtains regeneration plants for the first time in 1982, the culture of microspores of brassica crops, especially rape, has been successful in aspects of embryo yield, embryo culture and regeneration plants. Compared with the crops such as cabbage type rape, common head cabbage and the like, the culture difficulty of the free microspore of the broccoli is higher.
Moreover, in the normal flowering season of broccoli, a large amount of microspores can be obtained; however, when the proper flowering season is over, and after the flowering season enters autumn and winter, since the external cultivation conditions are not allowed, the flower buds which are properly used as microspores cannot be normally extracted, and then the broccoli microspores cannot be obtained in winter, resources are provided for creating DH (double haploid) materials, and more difficulties are brought to the broccoli microspore culture breeding technology.
Disclosure of Invention
In order to overcome the defect that microspore materials cannot be obtained in winter in the prior art, the invention aims to provide a method which can provide microspore materials which normally bloom in winter and provide resources for creating DH.
The technical scheme adopted by the invention for solving the technical problem is as follows: a method for obtaining broccoli microspore material in winter, which mainly comprises the following steps:
a. sowing: preparing a hole tray in a greenhouse, filling a culture medium in the hole tray, thoroughly watering the hole tray with water, and sowing broccoli seeds in the hole tray in a shallow layer;
b. accelerating germination: placing the plug tray with the broccoli seeds in a hotbed for accelerating germination, wherein the accelerating germination temperature is controlled to be 20-25 ℃ in the daytime and 10-15 ℃ at night; watering management according to the dry and wet conditions of the substrate during germination accelerating;
c. planting: when 2-3 true leaves grow out, planting and cultivating the leaves in a matrix nutrition cup, regularly pouring nutrient solution MB1 according to the dry and wet condition of the matrix, and controlling the environmental temperature and humidity so as to meet the conditions required by the vegetative growth;
d. and (3) low-temperature vernalization: after 14-16 pieces of true leaves grow out and the vegetative growth is finished, controlling the environmental temperature at 8-15 ℃, inducing the true leaves to enter reproductive growth by low-temperature vernalization for about 20 days, and pouring nutrient solution MB2 in the period;
e. cultivation: after the flower balls are formed, according to the dry and wet conditions of the matrix, pouring nutrient solution MB3 to continue cultivation; controlling the environmental temperature in the greenhouse to be 20-25 ℃ to obtain normal buds;
f. extracting microspores: selecting a flower bud with proper length, sterilizing the flower bud, and extracting pollen to obtain a microspore material of the broccoli in winter; the extraction method of the microspore specifically comprises the following steps:
(1) selecting buds: selecting main inflorescence buds with good growth length of 2.5-3.5 mm, or selecting lateral branch buds with length of 3.5-4 mm;
(2) and (3) disinfection: disinfecting the selected flower buds with alcohol for 30-40S, then disinfecting with sodium hypochlorite for 10min, and finally cleaning with sterile water for 5min each time for 3 times;
(3) pollen extraction: placing sterilized flower buds into a centrifuge tube, adding 1-2 mLB5 culture solution, grinding with a glass rod to disperse pollen, grinding to paste, adding a half-straw B5 culture solution into the tube, pouring into a filter, filtering, and filling the filtrate into a 10mL centrifuge tube; adding B5 culture solution to flush pollen in the filter, flushing for 2-3 times, combining filtrates, and diluting the pollen suspension in a 10mL centrifuge tube;
centrifuging the pollen suspension at 800 rpm for 5 min; removing supernatant in the centrifugal tube, adding liquid B5 culture solution, dispersing the precipitated pollen with a straw, suspending again, centrifuging, and removing supernatant;
diluting the precipitated pollen with NLN-13 culture solution, filling the diluted suspension into 6cm culture dishes, generally adding about 5mL of suspension into each dish, wherein each mL contains about 1.5-2 buds, and sealing with a sealing film to obtain a required microspore material; the microspore material can be continuously cultured into embryoid in a culture dish.
The specific composition of the culture solution MB1 is as follows: the water comprises 1.6kg of potassium nitrate, 1.4kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.15kg of sodium dihydrogen phosphate, 50g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 6g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.25g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride, wherein the water is counted by one cubic meter.
The specific composition of the culture solution MB2 is as follows: the water comprises 1.05kg of potassium chloride, 1.5kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.5kg of sodium dihydrogen phosphate, 30g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 10g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.35g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride in terms of one cubic meter of water.
The specific composition of the culture solution MB3 is as follows: the water comprises 1kg of potassium chloride, 1.15kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.9kg of sodium dihydrogen phosphate, 10g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 12g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.5g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride in terms of one cubic meter of water.
Further, the mass percent of the alcohol used in the disinfecting step is 75%, and the mass percent of the sodium hypochlorite used in the disinfecting step is 2%.
The invention has the beneficial effects that: compared with the prior art, the method has the advantages that the flower buds suitable for being used as microspore materials are cultivated in the greenhouse in winter through the soilless culture technology, the microspore materials are extracted from the suitable flower buds, and the technical problem that the microspores of the broccoli cannot be normally obtained in winter is solved.
Drawings
FIG. 1 is a schematic diagram of the sowing stage of broccoli seeds according to the present invention.
FIG. 2 is a schematic diagram of the germination accelerating stage of broccoli seeds according to the present invention.
Fig. 3 is a schematic diagram of the colonization phase of the present invention.
FIG. 4 is a schematic view of the low temperature vernalization stage of the present invention.
FIG. 5 is a schematic representation of the ball formation stage of the present invention.
FIG. 6 is a schematic view of a normal curd obtained in the present invention.
FIG. 7 is a diagram of the microspore morphology of the present invention magnified 40 times under an inverted microscope.
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
Examples
A method for obtaining broccoli microspore material in winter, which mainly comprises the following steps:
a. sowing: in a greenhouse, preparing a hole tray, filling a culture medium in the hole tray, thoroughly watering with water, and sowing broccoli seeds in the hole tray in a shallow layer as shown in figure 1;
b. accelerating germination: placing the plug tray with the broccoli seeds in a hotbed for accelerating germination, wherein the accelerating germination temperature is controlled to be 20-25 ℃ in the daytime and 10-15 ℃ at night; watering management during pregermination according to the dry and wet condition of the substrate is shown in FIG. 2;
c. planting: when 2-3 true leaves grow out, planting and cultivating the leaves in a substrate nutrition cup, pouring nutrient solution MB1 regularly according to the dry and wet condition of the substrate, and controlling the environmental temperature and humidity so as to meet the conditions required by the vegetative growth of the leaves, as shown in figure 3;
d. and (3) low-temperature vernalization: after 14-16 pieces of true leaves grow out and the vegetative growth is finished, controlling the environmental temperature at 8-15 ℃, inducing the true leaves to enter reproductive growth by low-temperature vernalization for about 20 days, and pouring nutrient solution MB2 in the period, as shown in figure 4;
e. cultivation: after the flower balls are formed, according to the dry and wet conditions of the matrix, pouring nutrient solution MB3 to continue cultivation; controlling the environmental temperature in the greenhouse to be 20-25 ℃ to obtain normal buds as shown in figure 5;
f. extracting microspores: selecting a bud with a proper length (as shown in figure 6), sterilizing the bud, and extracting pollen to obtain microspore material of broccoli in winter, wherein the shape of the microspore under a microscope is shown in figure 7.
The specific composition of the culture solution MB1 is as follows: the water comprises 1.6kg of potassium nitrate, 1.4kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.15kg of sodium dihydrogen phosphate, 50g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 6g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.25g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride, wherein the water is counted by one cubic meter.
The specific composition of the culture solution MB2 is as follows: the water comprises 1.05kg of potassium chloride, 1.5kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.5kg of sodium dihydrogen phosphate, 30g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 10g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.35g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride in terms of one cubic meter of water.
The specific composition of the culture solution MB3 is as follows: the water comprises 1kg of potassium chloride, 1.15kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.9kg of sodium dihydrogen phosphate, 10g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 12g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.5g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride in terms of one cubic meter of water.
The extraction method of the microspores specifically comprises the following steps:
(1) selecting buds: selecting main inflorescence buds with good growth length of 2.5-3.5 mm, or selecting lateral branch buds with length of 3.5-4 mm;
(2) and (3) disinfection: disinfecting the selected flower buds for 30-40 seconds by using 75% of alcohol by mass percent, then disinfecting for 10min by using 2% of sodium hypochlorite by mass percent, and finally cleaning for 3 times by using sterile water, wherein each time lasts for 5 min;
(3) pollen extraction: placing sterilized flower buds into a centrifuge tube, adding 1-2 mLB5 culture solution, grinding with a glass rod to disperse pollen, grinding to paste, adding a half-straw B5 culture solution into the tube, pouring into a filter, filtering, and filling the filtrate into a 10mL centrifuge tube; adding B5 culture solution to flush pollen in the filter, flushing for 2-3 times, combining filtrates, and diluting the pollen suspension in a 10mL centrifuge tube;
centrifuging the pollen suspension at 800 rpm for 5 min; removing supernatant in the centrifugal tube, adding liquid B5 culture solution, dispersing the precipitated pollen with a straw, suspending again, centrifuging, and removing supernatant;
diluting the precipitated pollen with NLN-13 culture solution, filling the diluted suspension into 6cm culture dishes, generally adding about 5mL of suspension into each dish, wherein each mL contains about 1.5-2 buds, and sealing with a sealing film to obtain a required microspore material;
and (3) carrying out dark culture on the sealed culture dish at the temperature of 25 ℃, beginning to check whether visible granular embryos are formed or not about 10 days, continuing to culture if the visible granular embryos are not formed, and moving to a constant-temperature shaking bed at 50rpm and the temperature of 25 ℃ to continue dark culture if the visible embryos are formed.
The above embodiments are only for illustrating the invention and are not to be construed as limiting the invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention, therefore, all equivalent technical solutions also belong to the scope of the invention, and the scope of the invention is defined by the claims.
Claims (2)
1. A method for obtaining broccoli microspore material in winter is characterized in that: the method mainly comprises the following steps:
a. sowing: preparing a hole tray in a greenhouse, filling a culture medium in the hole tray, thoroughly watering the hole tray with water, and sowing broccoli seeds in the hole tray in a shallow layer;
b. accelerating germination: placing the plug tray with the broccoli seeds in a hotbed for accelerating germination, wherein the accelerating germination temperature is controlled to be 20-25 ℃ in the daytime and 10-15 ℃ at night; watering management according to the dry and wet conditions of the substrate during germination accelerating;
c. planting: when 2-3 true leaves grow out, planting and cultivating the leaves in a matrix nutrition cup, pouring a culture solution MB1 regularly according to the dry and wet condition of the matrix, and controlling the environmental temperature and humidity so as to meet the conditions required by the vegetative growth of the leaves; the specific composition of the culture solution MB1 is as follows: the water comprises 1.6kg of potassium nitrate, 1.4kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.15kg of sodium dihydrogen phosphate, 50g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 6g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.25g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride, wherein the potassium nitrate is counted by one cubic meter of water;
d. and (3) low-temperature vernalization: after 14-16 true leaves grow out and the vegetative growth is finished, controlling the environmental temperature at 8-15 ℃, inducing the true leaves to enter reproductive growth by low-temperature vernalization, and irrigating culture solution MB2 in 20 days; the specific composition of the culture solution MB2 is as follows: the water comprises 1.05kg of potassium chloride, 1.5kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.5kg of sodium dihydrogen phosphate, 30g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 10g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.35g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride, wherein the water is counted by one cubic meter;
e. cultivation: after the flower balls are formed, according to the dry and wet conditions of the matrix, pouring the culture solution MB3 for continuous cultivation; controlling the environmental temperature in the greenhouse to be 20-25 ℃ to obtain normal buds; the specific composition of the culture solution MB3 is as follows: the water comprises 1kg of potassium chloride, 1.15kg of ammonium nitrate, 0.5kg of calcium chloride, 0.37kg of magnesium sulfate, 2.9kg of sodium dihydrogen phosphate, 10g of inositol, 28g of ferrous sulfate, 40g of ethylene diamine tetraacetic acid disodium, 22g of manganese sulfate, 9g of zinc sulfate, 12g of sodium borate, 1g of potassium iodide, 2g of glycine, 0.5g of sodium molybdate, 0.5g of copper sulfate and 0.5g of cobalt chloride, wherein the water is counted by one cubic meter;
f. extracting microspores: selecting flower buds, sterilizing the flower buds, and extracting pollen to obtain microspore material of broccoli in winter; the extraction method of the microspores specifically comprises the following steps:
(1) selecting buds: selecting main inflorescence buds with good growth length of 2.5-3.5 mm, or selecting lateral branch buds with length of 3.5-4 mm;
(2) and (3) disinfection: disinfecting the selected flower buds with alcohol for 30-40S, then disinfecting with sodium hypochlorite for 10min, and finally cleaning with sterile water for 5min each time for 3 times;
(3) pollen extraction: placing sterilized flower buds into a centrifuge tube, adding 1-2 mLB5 culture solution, grinding with a glass rod to disperse pollen, grinding to paste, adding a half-straw B5 culture solution into the tube, pouring into a filter, filtering, and filling the filtrate into a 10mL centrifuge tube; adding B5 culture solution to flush pollen in the filter, flushing for 2-3 times, combining filtrates, and diluting the pollen suspension in a 10mL centrifuge tube;
centrifuging the pollen suspension at 800 rpm for 5 min; removing supernatant in the centrifugal tube, adding liquid B5 culture solution, dispersing the precipitated pollen with a straw, suspending again, centrifuging, and removing supernatant;
diluting the precipitated pollen with NLN-13 culture solution, filling the diluted suspension into 6cm culture dishes, adding 5mL of suspension into each dish, wherein each mL contains 1.5-2 buds, and sealing with a sealing film to obtain a required microspore material; the microspore material can be continuously cultured into embryoid in a culture dish.
2. A method for harvesting broccoli microspore material in winter as claimed in claim 1, wherein: the mass percent of the alcohol adopted in the disinfection step is 75%, and the mass percent of the sodium hypochlorite adopted in the disinfection step is 2%.
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