CN114027181A - Radish microspore culture method - Google Patents
Radish microspore culture method Download PDFInfo
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- CN114027181A CN114027181A CN202111104209.9A CN202111104209A CN114027181A CN 114027181 A CN114027181 A CN 114027181A CN 202111104209 A CN202111104209 A CN 202111104209A CN 114027181 A CN114027181 A CN 114027181A
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- buds
- microspores
- radish
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- 235000006140 Raphanus sativus var sativus Nutrition 0.000 title claims abstract description 41
- 240000001970 Raphanus sativus var. sativus Species 0.000 title description 5
- 238000012136 culture method Methods 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 17
- 241000220259 Raphanus Species 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 9
- 230000000408 embryogenic effect Effects 0.000 claims abstract description 6
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 6
- 238000002635 electroconvulsive therapy Methods 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims abstract description 3
- 238000005516 engineering process Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims 1
- 230000035939 shock Effects 0.000 claims 1
- 210000002257 embryonic structure Anatomy 0.000 abstract description 10
- 244000088415 Raphanus sativus Species 0.000 description 27
- 238000009395 breeding Methods 0.000 description 8
- 230000001488 breeding effect Effects 0.000 description 8
- 235000013311 vegetables Nutrition 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 235000005733 Raphanus sativus var niger Nutrition 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000219193 Brassicaceae Species 0.000 description 2
- 235000011380 Raphanus sativus Nutrition 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000003911 Arachis Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 235000019057 Raphanus caudatus Nutrition 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 231100000732 tissue residue Toxicity 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for culturing radish microspores, which is specially used for improving the embryogenic rate of radish isolated microspores, and specifically comprises the following steps: (1) selecting buds with proper sizes according to the difference of the lowest temperature, and pretreating the selected buds at 4 ℃ for 8-10 h, and then at 6 ℃ for 12-14 h; (2) sterilizing bud, and purifying with B5 extractive solution with pH of 5.8 to obtain purified microspore; (3) suspending the purified microspores in 1/2NLN-13 (added with 0.25mg/L6-BA) culture medium with pH of 5.8, carrying out heat shock treatment at 32.5 ℃ for 6-8 h, and then standing and dark culturing at room temperature to obtain embryoids visible to naked eyes; (4) and carrying out shake culture on the embryoid to obtain a cotyledon embryo. The invention respectively improves the average incidence rate of the microspore embryos of the full-length red radish and the piercing red radish from 8 embryos/buds and 3 embryos/buds to 15 embryos/buds and 8 embryos/buds. The invention solves the problems in the prior art.
Description
Technical Field
The invention relates to a method for culturing radish microspores, belongs to a method for culturing plant microspores, and is specially used for improving the embryogenic rate of radish isolated microspores.
Background
Radish (Raphanus sativus L) is a plant belonging to the genus Raphanus of the family Brassicaceae and belonging to the genus Arachis, and the cultivated radish is mainly divided into two subspecies of Chinese radish and four-season radish, wherein Chinese radish is large radish and four-season radish is small radish. Chinese radish is an important root vegetable crop originally produced in China, has expanded fleshy straight roots as a main product organ, is one of the vegetables of the public in China, and has a year planting area of 130 kilohm2And the method plays an important role in the production and supply of vegetables.
The conventional breeding technology is mostly adopted for breeding new radish varieties in China, the selection efficiency is low, the breeding year is long, and the problems related to the sexual degradation such as reduction and deterioration of radish roots caused by multi-generation self-transaction because radishes are cross-pollinated crops occur, so that the development of the radish industry is greatly restricted.
The haploid has a single set of chromosome group, is applied to dominant breeding after being doubled into doubled haploid by chromosomes, and can remarkably accelerate the breeding process of a variety. The culture of the free microspores is one of effective ways for generating the haploid of crops and is an important means for breeding the haploid of plants. The free microspore culture/anther culture technology can directly obtain fresh microspore groups from flower buds or anthers, obtain genotype pure plants on the single cell level, further obtain Double Haploid (DH) homozygotes with stable characters by utilizing a haploid doubling technology, greatly shorten the parental genotype homozygous time, accelerate the breeding process and improve the breeding efficiency of varieties.
Radish is a vegetable crop which is least easy to culture free microspores in cruciferae, has low microspore embryogenic rate and is a problem to be solved urgently in radish microspore culture.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for improving the embryo generation rate of radish microspores, which is used for solving the technical problem of low embryo generation rate of radish microspores and meeting the actual use requirement.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a method for improving the embryogenic rate of radish microspore, which comprises the following steps:
(1) selecting proper buds according to the corresponding height of the lowest temperature and carrying out low-temperature (variable-temperature) pretreatment for 20-24 hours;
radish buds with most microspores in the edge-near period of the single nucleus have the length of 2.5-3.5 mm generally, but the sizes (length, development progress and the like) of the buds are greatly influenced by temperature; selecting buds with the length of 3.0-3.5 mm (the ratio of microspores at the near-edge stage of the mononuclear is the most) when the lowest temperature is lower than 10 ℃, and selecting buds with the length of 2.5-3.0 mm (the ratio of microspores at the near-edge stage of the mononuclear is the most) when the lowest temperature is higher than 10 ℃.
And (3) pretreating the selected flower buds at 4 ℃ for 8-10 h, and then pretreating at 6 ℃ for 12-14 h.
(2) Sterilizing the flower buds obtained in the step (1), and then adding the sterilized flower buds into B5 extracting solution with pH of 5.8 for purification to obtain purified microspores;
(3) suspending the purified microspores obtained in the step (2) in 1/2NLN-13 (added with 0.25mg/L6-BA) culture medium with pH of 5.8, carrying out heat shock treatment at 32.5 ℃ for 6-8 h, and then standing and carrying out dark culture at room temperature to obtain embryoids visible to naked eyes;
(4) and (4) carrying out shake culture on the embryoid in the step (3) to obtain a cotyledon embryo.
Detailed Description
The present invention will be described with reference to specific examples.
Examples
1. Experimental materials:
1) and (3) preparing radish seed strains of flower buds: in autumn, the full-bodied red radish and the thoroughfare red radish are respectively selected and planted in the vegetable seed reserving greenhouse in the Liuhe base of the academy of agricultural sciences of Jiangsu province, and the field management is the same as the conventional method.
2) And B5 medium.
3)1/2NLN-13 preparation:
adding 13g of sucrose into every 100ml of liquid NLN to obtain NLN-13, and mixing the NLN-13 with water according to the volume ratio of 1: 1, mixing and diluting to obtain 1/2NLN-13 culture medium with halved macroelements.
2. The method comprises the following specific steps:
1) collecting flower buds from the full-length red radish and the straight-through red radish seeds for free microspore culture from the first ten days of 3 months to the middle ten days of 4 months;
radish buds with most microspores in the edge-near period of the single nucleus have the length of 2.5-3.5 mm generally, but the sizes (length, development progress and the like) of the buds are greatly influenced by temperature; selecting buds with the length of 3.0-3.5 mm (the ratio of microspores at the near-edge stage of the mononuclear is the most) when the lowest temperature is lower than 10 ℃, and selecting buds with the length of 2.5-3.0 mm (the ratio of microspores at the near-edge stage of the mononuclear is the most) when the lowest temperature is higher than 10 ℃.
And (3) pretreating the selected flower buds at 4 ℃ for 8-10 h, and then pretreating at 6 ℃ for 12-14 h.
2) Microspore separation: 25-30 flower buds are used as a group, the flower buds are washed by running water and drained for standby, the selected flower buds are placed in a sterilized beaker on an aseptic workbench, sterilized by 75% alcohol for 20s, surface sterilized by 2% sodium hypochlorite for 15min, washed by sterile water for 3 times, transferred to a crusher for 2000rmp and 30s, added with a small amount of B5 extracting solution sterilized at high temperature, and extruded to form microspores; filtering the microspore-containing suspension with 400 mesh nylon net, removing larger tissue residue, collecting the suspension in a centrifuge tube, centrifuging for 3min at 1000rmp, discarding the supernatant, adding 4mlB5 extractive solution, centrifuging, repeating for 3 times, discarding the supernatant, and collecting the precipitate as pure microspore.
3) Culturing free microspores: the purified microspore was diluted with 1/2NLN-13 culture medium (0.25 mg/L6-BA added) to maintain pollen density at 1.0X 105Bud/ml, 3ml of microspore suspension per dish is subpackaged into a sterile glass culture dish with the diameter of 30mm, and a sealing film is used for sealing; and (3) carrying out heat shock treatment in a constant-temperature incubator at 32.5 ℃ for 6-8 h, and then, standing and dark culturing at 25 ℃.
4) And after the embryoid appears, transferring the embryoid to a shaking table with the rotating speed of 55rpm, and performing shake culture for 21-27 d to obtain the cotyledon embryo, and counting the embryogenesis rate.
By using the method, the average incidence rate of the microspore embryos of the full-length red radish and the piercing red radish is respectively increased from 8 embryos/buds and 3 embryos/buds to 15 embryos/buds and 8 embryos/buds.
Claims (4)
1. A method for culturing radish microspores is characterized by comprising the following steps: the problem of how to improve the embryogenic rate of the radish isolated microspore; the specific method comprises the following steps:
1) selecting proper buds according to the corresponding height of the lowest temperature and carrying out low-temperature (variable-temperature) pretreatment for 20-24 hours; radish buds with most microspores in the edge-near period of the single nucleus have the length of 2.5-3.5 mm generally, but the sizes (length, development progress and the like) of the buds are greatly influenced by temperature; selecting buds with the length of 3.0-3.5 mm (the ratio of microspores at the single-nucleus side period is the most) when the lowest temperature is lower than 10 ℃, and selecting buds with the length of 2.5-3.0 mm (the ratio of microspores at the single-nucleus side period is the most) when the lowest temperature is higher than 10 ℃; pretreating the selected flower buds at 4 ℃ for 8-10 h, and then pretreating at 6 ℃ for 12-14 h;
2) sterilizing the buds obtained in the step 1), adding the sterilized buds into B5 extracting solution with pH of 5.8, and purifying to obtain purified microspores;
3) suspending the purified microspores obtained in the step 2) in 1/2NLN-13 (added with 0.25mg/L6-BA) culture medium with pH of 5.8, carrying out heat shock treatment at 32.5 ℃ for 6-8 h, and then standing and carrying out dark culture at room temperature to obtain embryoids visible to naked eyes;
4) and (3) carrying out shake culture on the embryoid in the step 3) to obtain a cotyledon embryo.
2. The method for culturing radish microspores according to claim 1, wherein the method comprises the following steps: the bud length with the highest ratio of the microspores at the mononuclear border stage in the step 1) is greatly influenced by the lowest temperature of the environment, so the standard (length) of the collected buds needs to be properly fine-tuned according to the difference of the lowest temperature; meanwhile, the buds are pretreated at low temperature and variable temperature, so that the activity of the microspores is improved.
3. The method for culturing radish microspores according to claim 1, wherein the method comprises the following steps: the step 3)1/2NLN-13 (adding 0.25mg/L6-BA) culture medium (NLN-13 culture medium with halved macroelement) is the optimal culture medium, and 32.5 ℃ is the optimal heat shock temperature.
4. The method for culturing radish microspores according to claim 1, wherein the 1/2NLN-13 (0.25 mg/L6-BA) culture medium added in the step 3) obviously improves the embryogenic rate of radish isolated microspores by adding 0.25mg/L6-BA, and increases the practicability of the technology.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111104209.9A CN114027181A (en) | 2021-09-23 | 2021-09-23 | Radish microspore culture method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111104209.9A CN114027181A (en) | 2021-09-23 | 2021-09-23 | Radish microspore culture method |
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| CN114027181A true CN114027181A (en) | 2022-02-11 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543780A (en) * | 2003-11-21 | 2004-11-10 | 北京市农林科学院 | Method for culturing radish dissociative spore |
| US20040226059A1 (en) * | 1999-12-10 | 2004-11-11 | Kasha Kenneth J. | Embryogenesis and plant regeneration from microspores |
| CN101283674A (en) * | 2008-06-11 | 2008-10-15 | 南京农业大学 | Breeding process of obtaining radish haploidy |
| CN101343619A (en) * | 2007-07-11 | 2009-01-14 | 北京市农林科学院 | Method for improving embryos ratio of radish dissociation sporidiolum cultivation |
| CN102349443A (en) * | 2011-07-19 | 2012-02-15 | 南京农业大学 | Method for increasing incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis |
| CN104186309A (en) * | 2014-07-30 | 2014-12-10 | 镇江瑞繁农艺有限公司 | Method for improving embryogenesis rate of Raphanus sativus L. sinoruber makino |
-
2021
- 2021-09-23 CN CN202111104209.9A patent/CN114027181A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040226059A1 (en) * | 1999-12-10 | 2004-11-11 | Kasha Kenneth J. | Embryogenesis and plant regeneration from microspores |
| CN1543780A (en) * | 2003-11-21 | 2004-11-10 | 北京市农林科学院 | Method for culturing radish dissociative spore |
| CN101343619A (en) * | 2007-07-11 | 2009-01-14 | 北京市农林科学院 | Method for improving embryos ratio of radish dissociation sporidiolum cultivation |
| CN101283674A (en) * | 2008-06-11 | 2008-10-15 | 南京农业大学 | Breeding process of obtaining radish haploidy |
| CN102349443A (en) * | 2011-07-19 | 2012-02-15 | 南京农业大学 | Method for increasing incidence rate of isolated microspore embryos of Brassica campestris var. peruviridis |
| CN104186309A (en) * | 2014-07-30 | 2014-12-10 | 镇江瑞繁农艺有限公司 | Method for improving embryogenesis rate of Raphanus sativus L. sinoruber makino |
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| 周志国等: "不同萝卜品种游离小孢子的诱导及培养体系优化研究", 《西北植物学报》 * |
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