CN116530382A - Breeding method for improving survival rate of radish seeds - Google Patents
Breeding method for improving survival rate of radish seeds Download PDFInfo
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- 241000220259 Raphanus Species 0.000 title claims abstract description 58
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 title claims abstract description 58
- 238000009395 breeding Methods 0.000 title claims abstract description 45
- 230000004083 survival effect Effects 0.000 title claims abstract description 19
- 239000002689 soil Substances 0.000 claims abstract description 97
- 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 30
- 238000009331 sowing Methods 0.000 claims abstract description 30
- 230000001488 breeding effect Effects 0.000 claims abstract description 20
- 229960001338 colchicine Drugs 0.000 claims abstract description 15
- 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 abstract description 12
- 229930195725 Mannitol Natural products 0.000 claims abstract description 12
- 235000010355 mannitol Nutrition 0.000 claims abstract description 12
- 239000000594 mannitol Substances 0.000 claims abstract description 12
- 241000167854 Bourreria succulenta Species 0.000 claims abstract description 11
- 235000019693 cherries Nutrition 0.000 claims abstract description 11
- 239000003895 organic fertilizer Substances 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000003337 fertilizer Substances 0.000 claims description 53
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 49
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- 235000013399 edible fruits Nutrition 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 22
- 241000196324 Embryophyta Species 0.000 claims description 21
- 210000001161 mammalian embryo Anatomy 0.000 claims description 17
- 239000000618 nitrogen fertilizer Substances 0.000 claims description 14
- 238000007789 sealing Methods 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 14
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 claims description 9
- 238000005507 spraying Methods 0.000 claims description 9
- 244000068988 Glycine max Species 0.000 claims description 8
- 235000010469 Glycine max Nutrition 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 235000007164 Oryza sativa Nutrition 0.000 claims description 7
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 7
- 238000009835 boiling Methods 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 238000011049 filling Methods 0.000 claims description 7
- 238000011010 flushing procedure Methods 0.000 claims description 7
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- 239000003755 preservative agent Substances 0.000 claims description 7
- 230000002335 preservative effect Effects 0.000 claims description 7
- 235000009566 rice Nutrition 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 7
- 239000010902 straw Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 7
- 239000000052 vinegar Substances 0.000 claims description 7
- 235000021419 vinegar Nutrition 0.000 claims description 7
- 238000002635 electroconvulsive therapy Methods 0.000 claims description 6
- 210000002257 embryonic structure Anatomy 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 12
- 238000011161 development Methods 0.000 abstract description 5
- 230000018109 developmental process Effects 0.000 abstract description 5
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- 230000013020 embryo development Effects 0.000 abstract description 4
- 230000006543 gametophyte development Effects 0.000 abstract description 4
- 235000003642 hunger Nutrition 0.000 abstract description 4
- 230000003204 osmotic effect Effects 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 230000037351 starvation Effects 0.000 abstract description 4
- 230000023409 microsporogenesis Effects 0.000 abstract description 3
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 8
- 241000209094 Oryza Species 0.000 description 6
- 238000007596 consolidation process Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 229910001961 silver nitrate Inorganic materials 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
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- 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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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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
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
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- 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
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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Abstract
The invention discloses a breeding method for improving the survival rate of radish seeds, and belongs to the technical field of radish breeding. A breeding method for improving the survival rate of radish seeds comprises the following steps of mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing the soil pits at intervals of not less than 5cm, and sowing cherry radish seeds in the soil. In order to solve the problem that the number of finished buds is reduced due to relatively weak spore activity of cherry radishes in the breeding process influenced by self variety factors, mannitol can be used for maintaining proper osmotic pressure, improving the activity of microspores, simultaneously, the method can cause short-time nutrition starvation, promote microspore development, colchicine can improve the activity of microspore groups after separation and purification, so that more microspores can deviate from the original gametophyte development path to the sporophyte development path, and the use of colchicine can generate tissue stress to induce microspore division, thereby promoting embryogenesis.
Description
Technical Field
The invention relates to the technical field of radish breeding, in particular to a breeding method for improving the survival rate of radish seeds.
Background
Radish is a vegetable crop planted in a large area in China, is also a vegetable widely eaten by people, the breeding work of the radish is always one of the key points of scientific research, and the conventional radish breeding method adopts conventional methods such as selective breeding, sexual hybridization and the like.
In the breeding link of the conventional cherry radishes, the spore activity for breeding is relatively weak under the influence of the variety factors of the cherry radishes, so that the quantity of finished buds is reduced; therefore, the existing requirements are not met, and a breeding method for improving the survival rate of radish seeds is provided.
Disclosure of Invention
The invention aims to provide a breeding method for improving the survival rate of radish seeds, which can maintain proper osmotic pressure by using mannitol, improve the activity of microspores, simultaneously can cause short-time nutrition starvation, promote the development of microspores, and can improve the activity of microspore groups after separation and purification by colchicine, so that more microspores can deviate from the original gametophyte development path to be turned to a sporophyte development path, and the use of colchicine can generate tissue stress to induce the microspore division, promote embryogenesis and solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a breeding method for improving the survival rate of radish seeds comprises the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
step two: when 3-4 leaves grow out, thinning, picking off weak and small seedlings, keeping strong seedlings for continuous cultivation, filling soil and consolidating roots of the rest seedlings after thinning, and spraying rice vinegar water on leaf surfaces after the leaves grow completely;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: mannitol and colchicine are added into the culture medium, microspores are transferred to a 25 ℃ condition for dark culture after heat shock treatment, embryo yield is calculated, and cotyledon type embryos are transferred to an incubator body for bud embryo growth.
Preferably, in the first step, the soil for sowing needs to be disinfected by adopting plant ash, the straw and the stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clean water in a ratio of 1:5, and the supernatant is taken and irrigated in the soil.
Preferably, in the second step, if the seedling grows weakly in the early growth stage, 1-2 times of self-made organic liquid nitrogen fertilizer can be applied, the diluted liquid fertilizer is sucked by an injector and injected into soil, wherein the preparation method of the organic liquid nitrogen fertilizer comprises the following steps:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer needs to be diluted by adding water and then applied, and the dilution ratio of the fertilizer to the water is 1:8.
Preferably, in the fourth step, the seeds in the buds are taken out, suitable seed objects are screened out, and hypocotyls of the seeds are cut into segments.
Preferably, the hypocotyl is placed in a medium and nitrate-free and thidiazuron are added.
Preferably, in the seventh step, A, B, C, D groups are further cultured at 4 ℃, and anthers are taken for free microspores for culture respectively, and the anthers are subjected to pretreatment with a culture medium to obtain free microspores as a control.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, mannitol can maintain proper osmotic pressure, improve the activity of microspores, simultaneously can cause short-time nutrition starvation, promote microspore development, and colchicine can improve the activity of microspore groups after separation and purification, so that more microspores can deviate from the original gametophyte development path to be turned to a sporophyte development path, and the use of colchicine can generate tissue stress to induce microspore division, and promote embryogenesis;
2. according to the invention, the exogenous addition of silver nitrate and thidiazuron can increase the regeneration coefficient of adventitious buds, increase the number of buds generated by the regenerated buds, the change trend of enzyme activity and soluble protein is more obvious in the process of in-vitro cultivation of hypocotyls, and the browning rate can be effectively reduced by both the exogenous addition of silver nitrate, especially the browning rate is greatly reduced, the browning rate is kept at about 5%, and the rooting of the regenerated buds of radishes is simpler and faster;
3. according to the invention, soybean is utilized to make the organic fertilizer, when the organic liquid fertilizer is made to be finished, some orange peels are put in to ferment together with the fertilizer, a large amount of essential oil contained in the orange peels can effectively neutralize peculiar smell, and the orange peels are a good vegetable-cultivating fertilizer after fermentation, so that peculiar smell generated by the traditional fertilizer can be effectively avoided.
Detailed Description
The described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A breeding method for improving the survival rate of radish seeds comprises the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
the soil for sowing needs to be disinfected by adopting plant ash, straw and stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clear water in a ratio of 1:5, and supernatant liquid is taken and irrigated in the soil;
step two: when 3-4 leaves are grown out successively, thinning is carried out, weak and small seedlings are picked off, robust seedlings are left for continuous cultivation, soil filling consolidation is needed to be carried out on the roots of the rest seedlings after thinning, rice vinegar water is sprayed on leaf surfaces after the leaves are grown completely, and peculiar smell generated by the traditional fertilizer can be effectively avoided by matching with self-made organic liquid nitrogen fertilizer;
the method for preparing the organic liquid nitrogen fertilizer comprises the following steps of:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer is diluted by adding water and then fertilized, and the dilution ratio of the fertilizer to the water is 1:8;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
taking out seeds in the buds, screening out proper seed objects, taking hypocotyl sections of the seeds, placing the hypocotyl sections in a culture medium, and adding 5 mg.L-1 nitrate-free and 0.7 mg.L-1 thidiazuron;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: adding 15g/L mannitol and 10mg/L colchicine into the culture medium, transferring microspores to a condition of 25 ℃ for dark culture after heat shock treatment, calculating embryo yield, and finally transferring cotyledon type embryos into an incubator for bud embryo growth.
Example 2
A breeding method for improving the survival rate of radish seeds comprises the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
the soil for sowing needs to be disinfected by adopting plant ash, straw and stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clear water in a ratio of 1:5, and supernatant liquid is taken and irrigated in the soil;
step two: when 3-4 leaves are grown out successively, thinning is carried out, weak and small seedlings are picked off, robust seedlings are left for continuous cultivation, soil filling consolidation is needed to be carried out on the roots of the rest seedlings after thinning, rice vinegar water is sprayed on leaf surfaces after the leaves are grown completely, and peculiar smell generated by the traditional fertilizer can be effectively avoided by matching with self-made organic liquid nitrogen fertilizer;
the method for preparing the organic liquid nitrogen fertilizer comprises the following steps of:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer is diluted by adding water and then fertilized, and the dilution ratio of the fertilizer to the water is 1:8;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
taking out seeds in the buds, screening out proper seed objects, taking hypocotyl sections of the seeds, placing the hypocotyl sections in a culture medium, and adding 5 mg.L-1 nitrate-free and 0.7 mg.L-1 thidiazuron;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: and adding 15g/L mannitol into the culture medium, transferring microspores to a 25 ℃ condition for dark culture after heat shock treatment, calculating embryo yield, and finally transferring cotyledon type embryos to an incubator body for bud embryo growth.
Example 3
A breeding method for improving the survival rate of radish seeds comprises the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
the soil for sowing needs to be disinfected by adopting plant ash, straw and stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clear water in a ratio of 1:5, and supernatant liquid is taken and irrigated in the soil;
step two: when 3-4 leaves are grown out successively, thinning is carried out, weak and small seedlings are picked off, robust seedlings are left for continuous cultivation, soil filling consolidation is needed to be carried out on the roots of the rest seedlings after thinning, rice vinegar water is sprayed on leaf surfaces after the leaves are grown completely, and peculiar smell generated by the traditional fertilizer can be effectively avoided by matching with self-made organic liquid nitrogen fertilizer;
the method for preparing the organic liquid nitrogen fertilizer comprises the following steps of:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer is diluted by adding water and then fertilized, and the dilution ratio of the fertilizer to the water is 1:8;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
taking out seeds in the buds, screening out proper seed objects, taking hypocotyl sections of the seeds, placing the hypocotyl sections in a culture medium, and adding 5 mg.L-1 nitrate-free and 0.7 mg.L-1 thidiazuron;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: adding 10mg/L colchicine into the culture medium, transferring microspores to a condition of 25 ℃ for dark culture after heat shock treatment, calculating embryo yield, and finally transferring cotyledon embryo into a culture box for bud embryo growth.
Example 4
A breeding method for improving the survival rate of radish seeds comprises the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
the soil for sowing needs to be disinfected by adopting plant ash, straw and stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clear water in a ratio of 1:5, and supernatant liquid is taken and irrigated in the soil;
step two: when 3-4 leaves are grown out successively, thinning is carried out, weak and small seedlings are picked off, robust seedlings are left for continuous cultivation, soil filling consolidation is needed to be carried out on the roots of the rest seedlings after thinning, rice vinegar water is sprayed on leaf surfaces after the leaves are grown completely, and peculiar smell generated by the traditional fertilizer can be effectively avoided by matching with self-made organic liquid nitrogen fertilizer;
the method for preparing the organic liquid nitrogen fertilizer comprises the following steps of:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer is diluted by adding water and then fertilized, and the dilution ratio of the fertilizer to the water is 1:8;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
taking out seeds in the buds, screening out proper seed objects, taking hypocotyl sections of the seeds, placing the hypocotyl sections in a culture medium, and adding 5 mg.L-1 nitrate-free and 0.7 mg.L-1 thidiazuron;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: 7.5g/L mannitol and 5mg/L colchicine are added into the culture medium, microspores are transferred to a condition of 25 ℃ for dark culture after heat shock treatment, embryo yield is calculated, and cotyledon embryos are transferred to an incubator for embryo growth.
Example 5
A breeding method for improving the survival rate of radish seeds comprises the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
the soil for sowing needs to be disinfected by adopting plant ash, straw and stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clear water in a ratio of 1:5, and supernatant liquid is taken and irrigated in the soil;
step two: when 3-4 leaves are grown out successively, thinning is carried out, weak and small seedlings are picked off, robust seedlings are left for continuous cultivation, soil filling consolidation is needed to be carried out on the roots of the rest seedlings after thinning, rice vinegar water is sprayed on leaf surfaces after the leaves are grown completely, and peculiar smell generated by the traditional fertilizer can be effectively avoided by matching with self-made organic liquid nitrogen fertilizer;
the method for preparing the organic liquid nitrogen fertilizer comprises the following steps of:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer is diluted by adding water and then fertilized, and the dilution ratio of the fertilizer to the water is 1:8;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
taking out seeds in the buds, screening out proper seed objects, taking hypocotyl sections of the seeds, placing the hypocotyl sections in a culture medium, and adding 5 mg.L-1 nitrate-free and 0.7 mg.L-1 thidiazuron;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: adding 20g/L mannitol and 15mg/L colchicine into the culture medium, heat shock treating, transferring microspores to 25deg.C for dark culture, calculating embryo yield, and transferring cotyledon embryo into incubator for bud embryo growth
Comparing examples 1-5, the following table was prepared:
| mannitol (mannitol) | Colchicine | Number of embryo produced | Regeneration rate | |
| Example 1 | 15g/L | 10mg/L | 13.0*10 5 | 75％ |
| Example 2 | 15g/L | 0mg/L | 7.33.0*10 5 | 54.22％ |
| Example 3 | 0g/L | 10mg/L | 3*10 5 | 36％ |
| Example 4 | 7.5g/L | 5mg/L | 6.7.0*10 5 | 66.7％ |
| Example 5 | 20g/L | 15mg/L | 9.67*10 5 | 100％ |
As is known from the comparison of the above examples, mannitol can maintain proper osmotic pressure, improve the activity of microspores, cause short-time nutrient starvation, promote microspore development, and colchicine can improve the activity of microspore populations after separation and purification, so that more microspores can deviate from the original gametophyte development path to be turned to a sporophyte development path, and the use of colchicine can generate tissue stress to induce microspore division, thereby promoting embryogenesis.
The exogenous addition of silver nitrate and thidiazuron can promote the regeneration coefficient of adventitious buds, increase the number of buds generated by the regenerated buds, ensure that the change of enzyme activity and soluble protein tends to be more obvious in the process of in-vitro cultivation of hypocotyls, and can effectively reduce the browning rate, especially the addition of silver nitrate, greatly reduce the browning rate, ensure that the browning rate is kept at about 5 percent, and ensure that the rooting of the regenerated buds of radishes is simpler and faster.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A breeding method for improving the survival rate of radish seeds is characterized by comprising the following steps:
step one: mixing organic fertilizer and soil in soil pits together, watering thoroughly in the pits, sowing cherry radish seeds in the soil at a soil pit interval of not less than 5cm, scattering 3 seeds in each sowing pit, covering a layer of thin soil, and slightly spraying the soil with a soil layer thickness of 1-2 cm;
step two: when 3-4 leaves grow out, thinning, picking off weak and small seedlings, keeping strong seedlings for continuous cultivation, filling soil and consolidating roots of the rest seedlings after thinning, and spraying rice vinegar water on leaf surfaces after the leaves grow completely;
step three: after sowing for 2-3 months, pulling out the radish and the stem and leaf from the soil, cleaning, measuring the sizes of the cleaned fruits, classifying the fruits with consistent sizes into a group, measuring the fruits with the same size for the second time, and selecting fruits with weight within a certain range as breeding objects;
step four: storing a breeding object for a period of time, waiting for water loss in stems and leaves, and then planting the radishes into soil of an incubator again, watering the soil and applying fertilizer appropriately every other week at the temperature of 20-22 ℃, wherein the radishes grow out of the leaves again after a period of time, and the tops of the leaves grow out of the branches and bloom;
step five: placing flower branches in B-13 liquid culture medium without organic components, treating at 4deg.C, and culturing free microspores of flower buds 2.0-4.0mm long;
step six: taking flower buds with the length of 2.0-4.0mm at the flowering period, sterilizing for 30s by using 70% alcohol, sterilizing for 15min by using sodium hypochlorite solution containing 2% of effective chloride ions, flushing for 3 times by using sterile water for 5min each time, dissecting the flower buds under the sterile condition, and separating anthers;
step seven: mannitol and colchicine are added into the culture medium, microspores are transferred to a 25 ℃ condition for dark culture after heat shock treatment, embryo yield is calculated, and cotyledon type embryos are transferred to an incubator body for bud embryo growth.
2. The breeding method for improving the survival rate of radish seeds according to claim 1, wherein the breeding method comprises the following steps: in the first step, the soil for sowing needs to be disinfected by adopting plant ash, straw and stalk are combusted to obtain the plant ash, and then the plant ash is diluted by clear water in a ratio of 1:5, and supernatant liquid is taken and irrigated in the soil.
3. The breeding method for improving the survival rate of radish seeds according to claim 1, wherein the breeding method comprises the following steps: in the second step, if the seedling grows weakly in the early growth stage, 1-2 times of self-made organic liquid nitrogen fertilizer can be applied, the diluted liquid fertilizer is sucked by adopting a syringe and is injected into soil, wherein the preparation method of the organic liquid nitrogen fertilizer comprises the following steps:
s1: soaking soybean for 6-8h, boiling, pouring into a bottle, adding equal amount of clear water, sealing with preservative film, and sealing for two to three weeks;
s2: when the organic liquid fertilizer is manufactured to be finished, putting some orange peels into the organic liquid fertilizer to ferment together with the fertilizer;
s3: the prepared liquid fertilizer needs to be diluted by adding water and then applied, and the dilution ratio of the fertilizer to the water is 1:8.
4. The breeding method for improving the survival rate of radish seeds according to claim 1, wherein the breeding method comprises the following steps: in the fourth step, the seeds in the buds are taken out, proper seed objects are screened out, and hypocotyls of the seeds are cut into sections.
5. The breeding method for improving the survival rate of radish seeds according to claim 4, wherein the breeding method comprises the following steps: the hypocotyl is placed in a culture medium, and nitrate-free and thidiazuron are added.
6. The breeding method for improving the survival rate of radish seeds according to claim 1, wherein the breeding method comprises the following steps: in the seventh step, A, B, C, D groups are cultivated again at the temperature of 4 ℃, anthers are respectively taken for free microspores for cultivation, and the anthers are used as a control with the anther free microspores pretreated by the culture medium.
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