CN103210839B - Method for improving sclerotiniose resistance of oilseed rapes - Google Patents
Method for improving sclerotiniose resistance of oilseed rapes Download PDFInfo
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- CN103210839B CN103210839B CN201310162015.3A CN201310162015A CN103210839B CN 103210839 B CN103210839 B CN 103210839B CN 201310162015 A CN201310162015 A CN 201310162015A CN 103210839 B CN103210839 B CN 103210839B
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- sclerotinia sclerotiorum
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Abstract
The invention relates to a method for improving the sclerotiniose resistance of oilseed rapes. The sclerotiniose resistance of a cabbage is transferred to a turnip type rape through hybridization and backcross; the sclerotiniose resistance of the cabbage and the sclerotiniose resistance of the turnip type rape are combined through hybridization again; and a cabbage type oilseed rape with high sclerotiniose resistance can be selected and cultivated.
Description
Technical field
The invention belongs to plant new lines breeding technique field, be specifically related to a kind of method of sclerotinia rot of colza resistance improvement.Its international Patent classificating number is A01H1/04.
Background technology
Rape is by turnip type rape and wild cabbage distant hybridization, and dyed body doubles to evolve and next a kind of oil crop.Rape is the main oil crop of China, and its planting area is mainly distributed in the Yangtze river basin.This region is with heavy rainfall spring, and sclerotinia rot of colza is popular serious, causes every year rape because of the stalk break harm underproduction 10% to 30%.Breeding resistant variety is to solve the most economical effective approach of sclerotinia rot of colza harm.
Cabbage type rape and sibling species resource thereof are carried out in the research of resistance to sclerotinia sclerotiorum evaluation, the disease-resistant level of finding Chinese cabbage is poor, cabbage type rape resistance is placed in the middle, and wild cabbage is to the better resistance of stalk break (Mei et al. 2011, Euphytica 134:599-604).
Two parents of rape plant and belong to rape genus together, and their common ancestors kinds were divided into these two species (Rana, et al. 2006, Plant Journal 40:725-733) before 4,000,000 years.Existing autoploidy (Attia and Robbelen 1986, the Can J Genet Cytol 28:323-329 that has height between turnip type rape and the genome of wild cabbage that studies confirm that; Cheung, et al. 2009, Plant Cell 21:1912-1928).By turnip type rape and brassica specie intermolecular hybrid, the genetic constitution of species can be imported in another species.
Summary of the invention
The object of the present invention is to provide a kind of method of sclerotinia rot of colza resistance improvement, improve the resistance to sclerotinia sclerotiorum of rape.
Technical scheme of the present invention is as follows:
A method for sclerotinia rot of colza resistance improvement, first it utilize wild cabbage and turnip type rape distant hybridization that resistance to sclerotinia sclerotiorum is strong, through embryo, saves, and obtains monoploid hybrid; Filial generation and turnip type rape parent are backcrossed and selfing again, the resistance to sclerotinia sclerotiorum of wild cabbage is transferred in turnip type rape, screening obtains the turnip type rape of resistance to sclerotinia sclerotiorum improvement; Finally by hybridization, the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape, the cabbage type rape of seed selection high resistance stalk break.
Wherein, method for the resistance to sclerotinia sclerotiorum that passes through hybridization polymerization wild cabbage and turnip type rape of final step has two kinds, a kind of: with the turnip type rape of resistance to sclerotinia sclerotiorum improvement and the wild cabbage hybridization of anti-stalk break, through embryo, save, chromosome doubling, and offspring is carried out to resistance to sclerotinia sclerotiorum evaluation, screening obtains the cabbage type rape that resistance to sclerotinia sclerotiorum is strong.
Another kind is: first utilize cabbage type rape and wild cabbage hybridization, 6 times of body materials that chromosome doubling obtains are bridge, then hybridizes with the turnip type rape of resistance to sclerotinia sclerotiorum improvement, and offspring carries out resistance to sclerotinia sclerotiorum evaluation, the strong cabbage type rape of screening resistance to sclerotinia sclerotiorum.
Its main feature of method of sclerotinia rot of colza resistance improvement provided by the invention is: by wild cabbage and turnip type rape distant hybridization, and backcross with turnip type rape, selfing, the resistance to sclerotinia sclerotiorum of wild cabbage is transferred in turnip type rape, by hybridization, the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape, the cabbage type rape of seed selection high resistance stalk break.
Adopt the present invention, the resistance to sclerotinia sclerotiorum of synthetic cabbage type rape has obtained remarkable improvement.
Embodiment
The wild cabbage material C 01 of the high resistance stalk break that identified of take is for examination material (Mei; et al. 2011; Euphytica 177; 393-400); the embodiment that improvement sclerotinia rot of colza resistance is the inventive method; but be not the restriction to the inventive method, any conversion not surpassing from flesh and blood of the present invention, must belong to protection scope of the present invention.
embodiment 1: the improvement of turnip type rape resistance to sclerotinia sclerotiorum
Turnip type rape and wild cabbage distant hybridization: take turnip type rape landrace " Xishui white oil dish " as maternal (oil crop research institute of the Chinese Academy of Agricultural Sciences provides), 2 days artificial strip off buds before blooming, award with high resistance stalk break wild cabbage C01(Mei, et al. 2011, Euphytica 177, pollen 393-400), bagging one week.By embryo, save, morphology, pollen fertility and fecundity are observed, and cytological Identification obtains distant hybridization material.
131 buds of pollinating altogether, dissect 26 of ovarys, and embryo is saved and obtained a clone totally 24 strain groups training seedlings, and pollen fertility is observed and found that its pollen can dye rate average out to 35.6%.Hybrid plant seedling stage its blade face there is the soft fine, soft fur similar to wild cabbage parent, leaf look greener, has petiole, it is fairly obvious that its leaf morphology and Chinese cabbage parent compare difference, its strain phase form is similar to cabbage type rape.Hybrid F1 is carried out to somatic chromosome and observe discovery, hybrid F1 plant is 19 chromosomes, and it is 1.53(grain that fecundity is observed average seeds per pod).
Hybrid and turnip type rape backcross, and self progeny selects: 2 days strip off hybrid plant buds before blooming, award the pollen with turnip type rape " Xishui white oil dish ", bagging one week.The embryo of the 8th day after pollinating is carried out to embryo redemption, expand numerously, the plant after taking root is transplanted and moves to land for growing field crops at rape season, adopts general field management.According to offspring's appraisal, backcross after 1 to 2 time with turnip type rape, then selfing.
Backcross and carry out resistance to sclerotinia sclerotiorum evaluation with self progeny, the observation of pollen fertility and fecundity, and cytology detects.Get turnip type rape pollen and authorize hybrid, 976 buds of pollinating altogether, dissect 214 of ovarys, obtained 12 strain BC1, pollen fertility is observed and is found that average pollen can the rate of dying be 51.2%, one of them chromosome number is returned to the BC1 of turnip type rape parental set of chromosome number, parent compares with turnip type rape, the degree of its bacterial plaque expansion, and blade is 0.67, cane is 0.82, and fecundity is that average seeds per pod is 7.53.
It is as follows that this embodiment relates to specific experiment method:
Embryo is saved: latter the 8th day of pollination, win the ovary of growing, and with 75% alcohol surface sterilization, after 15 seconds, sterile water washs 1 minute, then sterilizes 15 minutes with 10% liquor natrii hypochloritis, then with sterile water washing 3 times, each 5 minutes.Under aseptic condition, peeling off ovary taking-up rataria is inoculated in the 1/2MS medium that contains 1.5% sucrose, be placed in 25℃ culturing room and cultivate (2000 lx illumination, 16 hours every days, 8 hours dark), until after rataria sprouting, transfer in the MS medium that contains 3% sucrose, 2.0 mg/L 6-BA and 0.2 mg/L NAA and break up cultivation, be then transferred to and in MS medium, carry out culture of rootage.Plant after taking root is transplanted and moves to land for growing field crops at rape season, adopts general field management.
Pollen fertility is observed: get the stamen of newly opening untainted flower the same day, pollen is shaken off in 10% acetic acid magenta to dye gently, then at optical microphotograph Microscopic observation counting, the flat little and shallow pollen grain that dyes is pollen sterile, and full and Hyperchromatic pollen grain is fertile pollen.What every individual plant was observed pollen adds up to 300.
Plant fecundity is observed: choose 30 pieces of the angle fruits of main inflorescence or two branch open pollinations of topmost, statistics full seed grain number, calculates average Seed number per pod.
Cytological Identification: get the immature bud of initial bloom stage, process 3h through the oxine of 0.002 mol/L at 22 ℃, then use Kano fixer (V
ethanol: V
glacial acetic acid=3:1), fixedly more than 24h, change afterwards to 70% ethanol and preserve at 4 ℃.During observation, get ovary in the HCl of 1 mol/L solution in 60 ℃ of hydrolysis 10 min, afterwards column cap is placed on slide, drip the carbolfuchsin of 10% improvement, after compressing tablet in light microscope (200 times) time observation of cell chromosome number.
Resistance to sclerotinia sclerotiorum is identified: with reference to the method for Mei etc., (Euphytica 2011,177:393-399; Eur J Plant Pathol 2012,34:599-604), carries out Vitro Inoculation Technique for Identification to seedling leaf and strain phase cane.When plant is in 9-12 leaf during the phase, clip falls 4 leaves and carries out stalk break inoculation, and is placed in 22 ℃ of temperature, the transfer room that humidity is 95%.Within after inoculation the 3rd day, measure major diameter and the minor axis of bacterial plaque, according to formula S=π * a*b/4, calculate lesion area.When blue or green pod is during the phase, intercept approximately 45 cm long stem or side shoot, two ends are wound around with preservative film, with the card punch that diameter is 4 mm, locate to create an identical wound on cane, then, at wound inoculation mycelia piece, within after inoculation the 4th day, measure scab length.The calculating of the degree of its bacterial plaque expansion is the relative ratio with plant scab observed value to be measured and turnip type rape parent scab observed value.
embodiment 2: turnip type rape and wild cabbage hybridization Synthetic rapeseed
Turnip type rape and disease-resistant wild cabbage C01(Mei with resistance to sclerotinia sclerotiorum improvement, et al. 2011, Euphytica 177,393-400) carry out interspecific cross, after pollination, hybrid embryo was carried out to embryo redemption according to the method in embodiment 1 in the 8th day, until after rataria sprouts, transfer in the MS medium that contains 3% sucrose, 2.0 mg/L 6-BA and 0.2 mg/L NAA and break up cultivation, be developed to be transferred to after predetermined copy number and in MS medium, carry out culture of rootage.After taking root, adopt 0.5 % colchicine bubble root to process 6 hours induce chromosome redoublements, to develop artificially synthesized Brassica type rape, then plantlet of transplant is arrived to land for growing field crops, carry out general field management.Finally according to the method for Resistance Identification in embodiment 1,20 parts that obtain artificial synthetic rapes are carried out to resistance to sclerotinia sclerotiorum evaluation, screening obtains resistance to sclerotinia sclerotiorum than two No. 9 strong individualities of 1.5 times in cabbage type rape variety.
embodiment 3: the 6 times of body materials of take are bridge, polymerization turnip type rape and wild cabbage resistance
With disease-resistant wild cabbage C01(Mei, et al. 2011, Euphytica 177, 393-400) with cabbage type rape variety in two No. 9 (oil crop research institute of the Chinese Academy of Agricultural Sciences provides) hybridization, chromosome doubling obtains 6 times of body materials that genotype is AACCCC, the turnip type rape hybridization of the resistance to sclerotinia sclerotiorum improvement then obtaining with embodiment 1, offspring carries out resistance to sclerotinia sclerotiorum evaluation, the resistance to sclerotinia sclerotiorum of polymerization turnip type rape and wild cabbage, 200 parts of materials that obtain are carried out to resistance to sclerotinia sclerotiorum evaluation, screening obtains resistance to sclerotinia sclerotiorum than two No. 9 strong individualities of 2 times in cabbage type rape variety.
Claims (3)
1. a method for sclerotinia rot of colza resistance improvement, is characterized in that, first utilizes wild cabbage and the turnip type rape distant hybridization of anti-stalk break, through embryo, saves, and obtains monoploid F1 hybrid; Again filial generation and turnip type rape parent are backcrossed, from backcross progeny, select resistance to sclerotinia sclerotiorum to be significantly better than former turnip type rape parent and chromosome number
recover the due chromosome number of turnip type rapeindividuality carry out selfing, the resistance to sclerotinia sclerotiorum of wild cabbage is transferred in turnip type rape, by Morphological Identification, cytological analysis and resistance to sclerotinia sclerotiorum identify, screening obtains the turnip type rape of resistance to sclerotinia sclerotiorum improvement; Finally by hybridization, the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape, the cabbage type rape of seed selection high resistance stalk break.
2. the method that sclerotinia rot of colza resistance according to claim 1 improves, it is characterized in that described by hybridization, one of method of the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape is: with the turnip type rape of resistance to sclerotinia sclerotiorum improvement and the wild cabbage hybridization of anti-stalk break, through embryo, save, chromosome doubling, and offspring is carried out to resistance to sclerotinia sclerotiorum evaluation, screening obtains the cabbage type rape that resistance to sclerotinia sclerotiorum is strong.
3. the method that sclerotinia rot of colza resistance according to claim 1 improves, it is characterized in that described by hybridization, two of the method for the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape is: first utilize cabbage type rape and wild cabbage hybridization, 6 times of body materials that chromosome doubling obtains are bridge, hybridize with the turnip type rape of resistance to sclerotinia sclerotiorum improvement again, offspring carries out resistance to sclerotinia sclerotiorum evaluation, the strong cabbage type rape of screening resistance to sclerotinia sclerotiorum.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103766212B (en) * | 2014-02-24 | 2015-05-13 | 西南大学 | Method for improving cabbage type rape by use of cabbage |
| 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 |
| 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 |
| CN110367118A (en) * | 2019-08-28 | 2019-10-25 | 安徽省农业科学院作物研究所 | A kind of method and its application based on the anti-clubroot bridge kind of fast period rape breeding |
| CN111149689A (en) * | 2020-03-09 | 2020-05-15 | 汉中市农业科学研究所（陕西省水稻研究所） | Breeding method of cabbage type hybrid rape with antibacterial nuclear disease |
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