CN101785425B - Method of improved artificially synthesized Brassica napa L. for heterosis utilization - Google Patents
Method of improved artificially synthesized Brassica napa L. for heterosis utilization Download PDFInfo
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- CN101785425B CN101785425B CN 200910251074 CN200910251074A CN101785425B CN 101785425 B CN101785425 B CN 101785425B CN 200910251074 CN200910251074 CN 200910251074 CN 200910251074 A CN200910251074 A CN 200910251074A CN 101785425 B CN101785425 B CN 101785425B
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Abstract
The invention discloses a method of improved artificially synthesized Brassica napa L. for heterosis utilization, belonging to the technical field of breeding plant novel varieties. The method comprises the following steps: transferring the excellent oil economical characters (the content of thioglycoside in seed cake dregs is lower than 30 mu mol/g, and the content of erucic acid in seeds is lower than 1% of double-low Brassica napa L.) of the double-low Brassica napa L. to wild cabbage; culturing a novel oil wild cabbage material, hybridizing the material with a double-low Brassica campestris variety to obtain the artificially synthesized Brassica napa L. with normal fecundity, wherein, the content of thioglycoside in seed cake dregs is lower than 30 mu mol/g, the content of erucic acid in seeds is lower than 1%, and the superior combination prepared by the artificially synthesized Brassica napa L. and the natural Brassica napa L. is screened. In the invention, the good genes of Brassica napa L. are transferred to wild cabbage to culture oil wild cabbage material which is for overcoming the disadvantages of low quality and low fecundity of the existing artificially synthesized Brassica napa L., thereby improving the potential of artificially synthesized Brassica napa L. for heterosis utilization.
Description
Technical field
The invention belongs to plant new lines breeding technique field, be specifically related to a kind of method that artificially synthesized Brassica type rape is used for heterosis utilization that improves.Its international Patent classificating number is A01H1/04.
Background technology
Cabbage type rape (Brassica napus, AACC) is by turnip type rape (B.rapa, AA) and wild cabbage (B.oleracea, CC) natural crossing, and chromosome doubling is evolved and next allotetraploid (U 1935).Compare with parent's kind, cabbage type rape has narrow hereditary basis, and this has limited the genetic improvement of existing cabbage type rape.By wild cabbage and turnip type rape hybridization, obtain the hereditary basis that artificially synthesized Brassica type rape can be used to widen existing cabbage type rape, and heterosis utilization potentiality (Seyis et al.2003, Plant Breeding 122:473-478).
Yet, in the existing wild cabbage resource, there is not the oil wild cabbage, this is embodied in seed grouts sulphur glycosides content and surpasses 30 μ mol/g so that artificial synthetic cabbage type rape has relatively poor oil economical character, and content of erucic acid surpasses 1% in the seed.
Summary of the invention
The object of the invention is to overcome the existing inferior deficiency of artificially synthesized Brassica type rapeseed quality, a kind of method that artificially synthesized Brassica type rape is used for heterosis utilization that improves is provided, thereby improves the potentiality that artificially synthesized Brassica type rape is used for heterosis utilization.
The present invention is achieved through the following technical solutions:
A kind of artificially synthesized Brassica type rape of improveing is for the method for heterosis utilization, (seed grouts sulphur glycosides content is lower than 30 μ mol/g to the method with economical character with the good oil of two low cabbage type rapes, content of erucic acid is lower than 1% in the seed) transformation is in wild cabbage, cultivate a kind of new oil wild cabbage material, with this material and two low turnip type rape inter breed crossings, by selecting, obtain seed grouts sulphur glycosides content and be lower than 30 μ mol/g, content of erucic acid is lower than 1% in the seed, the normal artificially synthesized Brassica type of fecundity rape, and screen the strong advantage combination of itself and natural cabbage type rape preparation.
This method may further comprise the steps:
(1) the new oil seed selection of wild cabbage
Backcross by cabbage type rape and wild cabbage recurrent parent, perhaps cabbage type rape and wild cabbage distant hybrid progeny are carried out microspores culture, use the economical character transformation in wild cabbage the good oil of cabbage type rape, cultivate a kind of new oil wild cabbage material, specific practice is:
Be lower than 30 μ mol/g with seed grouts sulphur glycosides content, content of erucic acid is lower than 1% two low B. napus work female parent in the seed, wild cabbage is made male parent, after artificial emasculation pollination 8-12 days, obtain the species hybrid test-tube plantlet by embryo redemption mode, then backcross with the wild cabbage parent, obtain backcross progeny by embryo redemption mode, perhaps microspores culture is carried out in species hybrid, obtain amphidiploid.Backcross progeny or amphidiploid are carried out the selection of economical character and quality trait, and chromosome number and chromosome behavior analysis filter out seed grouts sulphur glycosides content and are lower than 30 μ mol/g, and content of erucic acid is lower than 1% new oil wild cabbage material in the seed;
(2) improvement artificially synthesized Brassica type rape seed selection
The new oil of step (1) is lower than 30 μ mol/g with wild cabbage and grouts sulphur glycosides content, and content of erucic acid is lower than 1% turnip type rape hybridization in the seed, and its hybrid is saved by embryo and obtains the clone test-tube plantlet.Before transplanting the field, be the root system 6 to 12 hours that the colchicine of 0.5% concentration soaks test-tube plantlet with mass concentration, induce chromosome redoublement, artificially synthesized Brassica type rape, and it is normal to screen pollen fertility, the artificially synthesized Brassica type rape that fecundity is good;
(3) screening of strong advantage combination
With the artificially synthesized Brassica type rape after the improvement and the hybridization of natural cabbage type rape, the preparing hybrid combination, by the field trial of a plurality of environment, the strong advantage combination that the screening performance is good.
Advantage of the present invention is: backcross by cabbage type rape and wild cabbage recurrent parent, use the economical character transformation in wild cabbage the good oil of cabbage type rape, cultivate a kind of new oil wild cabbage material, the deficiency poor quality that is used for the existing artificially synthesized Brassica type rape of improvement, the artificially synthesized Brassica type rape after the improvement can be used to improve the hybrid vigour potentiality of existing cabbage type rape.
Embodiment
Below be a kind of embodiment of the inventive method, but be not the restriction to the inventive method that any not super conversion from flesh and blood of the present invention must belong to protection scope of the present invention.
1, the new oil seed selection of wild cabbage
For maternal, wild cabbage C-1 is male parent take cabbage type rape SWU-1 (seed grouts sulphur glycosides content is as 20 μ mol/g, and content of erucic acid is lower than 0.5% in the seed), female parent is shelled flower bud castrates, sexual hybridization was pollinated after 8-12 days, obtained the species hybrid test-tube plantlet by embryo redemption mode.Then the species hybrid test-tube plantlet is transplanted to the field, during flowering stage, artificial emasculation is also awarded pollen with wild cabbage, obtains BCF by embryo redemption mode
1, perhaps microspores culture is carried out in species hybrid, obtain amphidiploid.
Embryo rescue method concrete steps are: winning 8-12 days ovary after the pollination, is 10% clorox surface sterilization with mass concentration, is inoculated into and contains mass concentration 3% sucrose, in the MS medium of 1.0mg/L 6-BA and 0.2mg/L NAA hormone.Be placed in the illumination cultivation chamber and cultivate, illumination in 16 hours, 8 hours dark place reasons, 20 ℃.Approximately inoculation was peeled off the embryo that expands and is germinateed in the MS medium after 15 days from ovule.
The concrete steps of microspores culture are: 16 of the flower buds of winning diameter and be the 2.5-3.5mm size, be ethanol surface sterilization half a minute of 70% with volumetric concentration, distilled water cleans 2 times, is 10% clorox immersion 15-20 minute with mass concentration, sterile water wash 3-5 time, each 3-5 minute.The bud that disinfects grinds with the B5 culture fluid of 2mL, the nylon net filter in 45 μ m apertures is (all vessel are aseptic) in the 10mL centrifuge tube, add B5 to 5mL, centrifugal 2 minutes of 8000rpm, suck supernatant, the B5 extract that adds 8ml washs, after centrifugal 2 times, suck supernatant, add and to contain mass concentration and be 0.01% colchicin and contain the NLN culture fluid 8mL that mass concentration is 16% sucrose, and the aseptic dark cultivation of the insulating box middle-shallow layer that places 32 ℃ 48 hours, centrifugal, suck supernatant, add 16mL and contain the NLN culture fluid mixing that mass concentration is 13% sucrose, divide and install in the culture dish that diameter is 60mm, 4 flower buds/ware, 4mL culture fluid/ware places 25 ℃ insulating box secretly to cultivate, until the formation embryoid forwards to contain in the MS solid culture medium that mass concentration is 1.2% agarose and cultivates seedling.
To DH, BCF
1With its self progeny BCF
2, BCF
3Carry out the field phenotypic character and select, select flowering stage suitable, branch is many, and the proterties that bears pods is good, the strain that disease resistance is strong; Utilize near-infrared analyzer, the BCF elected to the field
3The individual plant selfing seed carries out erucic acid, and the sulphur glycosides is analyzed, and the not affine individual plant of selfing obtains selfed seed by the selfing of stripping flower bud, selects content of erucic acid to be lower than 1%, and seed grouts sulphur glycosides content is lower than the individuality of 30 μ mol/g; Simultaneously candidate's individual plant is carried out cytological Identification, gather the tender bud of plant children, with Kano solution (V
Alcohol: V
Acetic acid=3: 1) fixing, at optical microphotograph Microscopic observation pollen mother cell chromosomes number, and chromosome pairing behavior during reduction division, each sample is observed 10-30 and is divided phase.Economical character and quality trait are good, and chromosome number reverts to 20, and the normal plant of reduction division is elected.
2, improvement artificially synthesized Brassica type rape seed selection
Elected wild cabbage and grouts sulphur glycosides content are lower than 30 μ mol/g, content of erucic acid is lower than 1% turnip type rape hybridization in the seed, its hybrid is saved by embryo and obtains the clone test-tube plantlet, test-tube plantlet is before transplanting the field, adopting mass concentration is that 0.05% colchicine bubble root was processed 6 to 12 hours, induce chromosome redoublement, artificially synthesized Brassica type rape.
Embryo rescue method concrete steps are: winning 8-12 days ovary after the pollination, is 5% clorox surface sterilization with mass concentration, is inoculated into that to contain mass concentration be 3% sucrose, in the MS medium of 1.0mg/L 6-BA and 0.2mg/L NAA hormone.Be placed in the illumination cultivation chamber and cultivate, illumination in 16 hours, 8 hours dark place reasons, 20 ℃.Approximately inoculation was peeled off the embryo that expands and is germinateed in the MS medium after 15 days from ovule.
Fecundity is good during the open pollination of screening artificially synthesized Brassica type rape, the normal artificially synthesized Brassica type of pollen fertility rape, and observe its chromosome pairing behavior, screening nonhomologous chromosome pair frequency is low individual.
Pollen fertility observation procedure concrete steps are: get and bloomed the same day; it is dyeing about 1 minute in 10% the acetic acid magenta that the flower that does not have to pollute is shaken off in volumetric concentration gently; then at the optical microphotograph Microscopic observation; little and the shallow pollen grain that dyes is undesired pollen; large and Hyperchromatic is the fertile flower powder; each candidate plant is observed 10 flowers, 10 to 20 visuals field of every flower observation, and the ratio of calculating fertile pollen.
3, the screening of strong advantage combination
Artificially synthesized Brassica type rape after the improvement and the hybridization of natural cabbage type rape, the preparing hybrid combination.By the field trial of many environment, screen the combination of strong advantage.
The concrete steps of many environment output comparison field trial are: take natural cabbage type rape and artificially synthesized Brassica type rape as the parent, configuration hybrid combination, plant in different environment, each plantation point arranges repetition and contrast, growing period, growth potential and the resistance of investigation hybrid, when ripe, investigate its seed production, in many environment, the combination of performance high yield is elected.
Claims (4)
1. improve the method that artificially synthesized Brassica type rape is used for heterosis utilization for one kind, it is characterized in that, may further comprise the steps:
(1)
The oil seed selection of wild cabbage
Backcross by cabbage type rape and wild cabbage recurrent parent, use the economical character transformation in wild cabbage the good oil of cabbage type rape, cultivate a kind of oil wild cabbage material, specific practice is:
Be lower than 30 μ mol/g with seed grouts sulphur glycosides content, content of erucic acid is lower than the two low B. napus work female parent of 1 % in the seed, and wild cabbage is made male parent, after artificial emasculation pollination 8-12 days, obtains the species hybrid test-tube plantlet by embryo redemption mode; Then the species hybrid test-tube plantlet is transplanted to the field, during flowering stage, artificial emasculation is also awarded pollen with wild cabbage, obtains BCF by embryo redemption mode
1, perhaps microspores culture is carried out in species hybrid, obtain amphidiploid; Backcross progeny is carried out the selection of economical character and quality trait, and chromosome number and chromosome behavior analysis filter out seed grouts sulphur glycosides content and are lower than 30 μ mol/g, and content of erucic acid is lower than the oil wild cabbage material of 1 % in the seed;
The concrete steps of described embryo rescue method are:
Winning 8-12 days ovary after the pollination, is the clorox surface sterilization of 10 % with mass concentration, is inoculated into that to contain mass concentration be 3 % sucrose, in the MS medium of 1.0 mg/L 6-BA and 0.2 mg/L NAA hormone; Be placed in the illumination cultivation chamber and cultivate, illumination in 16 hours, 8 hours dark places reason, 20 ℃, inoculate about 15 days after, from ovule, peel off the embryo that expands and in the MS medium, germinate;
(2) improvement artificially synthesized Brassica type rape seed selection
The oil of step (1) is lower than 30 μ mol/g with wild cabbage and seed grouts sulphur glycosides content, content of erucic acid is lower than the turnip type rape hybridization of 1 % in the seed, its hybrid is saved by embryo and obtains the clone test-tube plantlet, test-tube plantlet is before transplanting the field, and the employing mass concentration is that the colchicine bubble root of 0.05 % was processed induce chromosome redoublement 6 to 12 hours, artificially synthesized Brassica type rape, and the screening fecundity is good, and pollen fertility is normal, the artificially synthesized Brassica type rape that the nonhomologous chromosome pair frequency is low;
(3)
The screening of strong advantage combination
With the artificially synthesized Brassica type rape after the improvement and the hybridization of natural cabbage type rape, the preparing hybrid combination by the output comparison field trial of many environment, is screened the combination of strong advantage.
2. improvement artificially synthesized Brassica type rape according to claim 1 is used for the method for heterosis utilization, it is characterized in that the microspores culture method concrete steps of described step (1) are:
Winning diameter and be 16 of the flower buds of 2.5-3.5 mm size, is ethanol surface sterilization half a minute of 70 % with volumetric concentration, and distilled water cleans 2 times, and mass concentration is that 10% clorox soaked 15-20 minute, sterile water wash 3-5 time, 3-5 minute at every turn; The bud that the disinfects B5 extract with 2 mL is ground, the nylon net filter in 45 μ m apertures is in 10 mL centrifuge tubes, add B5 to 5 mL, centrifugal 2 minutes of 8000 rpm, suck supernatant, the B5 extract that adds 8 ml washs, after centrifugal 2 times, suck supernatant, adding and containing mass concentration is that 0.01% colchicin and mass concentration are NLN culture fluid 8 mL of 16 % sucrose, and the aseptic dark cultivation of the insulating box middle-shallow layer that places 32 ℃ 48 hours, centrifugal, suck supernatant, add 16 mL and contain the NLN culture fluid mixing that mass concentration is 13 % sucrose, divide and install in the culture dish that diameter is 60 mm, 4 flower buds/ware, 4 mL culture fluid/wares place 25 ℃ insulating box secretly to cultivate, until the formation embryoid forwards to contain in the MS solid culture medium that mass concentration is 1.2 % agaroses and cultivates seedling.
3. improvement artificially synthesized Brassica type rape according to claim 1 is used for the method for heterosis utilization, it is characterized in that the pollen fertility observation procedure concrete steps of step (2) are:
Get and bloomed the same day; it is dyeing about 1 minute in the acetic acid magenta of 10 % that the flower that does not have to pollute is shaken off in volumetric concentration gently; then at the optical microphotograph Microscopic observation; little and the shallow pollen grain that dyes is undesired pollen; large and Hyperchromatic is the fertile flower powder; each candidate plant is observed 10 flowers, 10 to 20 visuals field of every flower observation, and the ratio of calculating fertile pollen.
?
4. improvement artificially synthesized Brassica type rape according to claim 1 is used for the method for heterosis utilization, it is characterized in that the concrete steps of many environment output comparison field trial are:
Take natural cabbage type rape and artificially synthesized Brassica type rape as the parent, configuration hybrid combination, plant in different environment, each plantation point arranges repetition and contrast, growing period, growth potential and the resistance of investigation hybrid, when ripe, investigate its seed production, in many environment, the combination of performance high yield is elected.
?
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102783410A (en) * | 2012-08-21 | 2012-11-21 | 江苏丘陵地区镇江农业科学研究所 | Breeding method of high-quality and double-low rapes suitable for mechanization work and with high yield, and lodging resistance |
| AU2013224677B2 (en) | 2012-09-13 | 2017-05-11 | Plant Bioscience Limited | Brassica oleracea plants with improved nutritional value |
| CN103704131A (en) * | 2013-01-17 | 2014-04-09 | 西南大学 | Method for utilizing heterosis between rape sub-genomes |
| CN104542027A (en) * | 2015-02-02 | 2015-04-29 | 中国农业科学院油料作物研究所 | Method for identifying bird damage of rape by use of thioglycoside content |
| CN106035059B (en) * | 2016-05-26 | 2018-04-24 | 西北农林科技大学 | A kind of method of artificial synthesized anti-clubroot brassica napus |
| CN106718823B (en) * | 2016-11-16 | 2019-04-23 | 西南大学 | A method of high oleic acid rape material is prepared using artificial synthesis Brassica napus |
| CN107907541A (en) * | 2017-12-26 | 2018-04-13 | 青海省农林科学院 | The identification method of heterozygote in a kind of cabbage type rape male and female sterile system fertile plant |
| CN109349099A (en) * | 2018-11-20 | 2019-02-19 | 贵州省油料研究所 | Brassica napus recessive gms line is close, distant hybridization selectively breeding hybrid rape selection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101243776A (en) * | 2008-03-04 | 2008-08-20 | 贵州省生物技术研究所 | Cultivation method for cabbage type rape Isolated microspore plant strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101243776A (en) * | 2008-03-04 | 2008-08-20 | 贵州省生物技术研究所 | Cultivation method for cabbage type rape Isolated microspore plant strain |
Non-Patent Citations (2)
| Title |
|---|
| 张晓伟等.人工合成甘蓝型油菜研究.《河南农业科学》.2001,(第2期),第7-10页. * |
| 星晓蓉等.春性甘蓝型油菜小孢子培养技术研究.《西北农业学报》.2007,第16卷(第1期),第114-118页. * |
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