CN103210839A - Method for improving sclerotiniose resistance of oilseed rapes - Google Patents
Method for improving sclerotiniose resistance of oilseed rapes Download PDFInfo
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- CN103210839A CN103210839A CN2013101620153A CN201310162015A CN103210839A CN 103210839 A CN103210839 A CN 103210839A CN 2013101620153 A CN2013101620153 A CN 2013101620153A CN 201310162015 A CN201310162015 A CN 201310162015A CN 103210839 A CN103210839 A CN 103210839A
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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 mainly is distributed in the Yangtze river basin.This zone 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 sclerotinia rot of colza to endanger most economical valid approach.
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 relatively poor, the cabbage type rape resistance is placed in the middle, and wild cabbage is to the better resistance (Mei et al. 2011, Euphytica 134:599-604) of stalk break.
Two parents of rape plant and belong to the 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.Have the autoploidy (Attia and Robbelen 1986, the Can J Genet Cytol 28:323-329 that there are height between the genome that studies confirm that turnip type rape and wild cabbage now; 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 kind of method of sclerotinia rot of colza resistance improvement, it at first utilizes strong wild cabbage of resistance to sclerotinia sclerotiorum and turnip type rape distant hybridization, saves through embryo, 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 the turnip type rape, screening obtains the turnip type rape of resistance to sclerotinia sclerotiorum improvement; At last by hybridizing the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape, the cabbage type rape of the high anti-stalk break of seed selection.
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 ofly be: the turnip type rape and the wild cabbage of anti-stalk break with the resistance to sclerotinia sclerotiorum improvement are hybridized, save through embryo, chromosome doubling, and the offspring is carried out resistance to sclerotinia sclerotiorum identify that screening obtains the strong cabbage type rape of resistance to sclerotinia sclerotiorum.
Another kind is: utilize the hybridization of cabbage type rape and wild cabbage earlier, 6 times of body materials that chromosome doubling obtains are bridge, hybridize with the turnip type rape of resistance to sclerotinia sclerotiorum improvement again, and the offspring carries out resistance to sclerotinia sclerotiorum and identifies, 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 the turnip type rape, by hybridization, the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape, the cabbage type rape of the high anti-stalk break of seed selection.
Adopt the present invention, the resistance to sclerotinia sclerotiorum of synthetic cabbage type rape has obtained remarkable improvement.
Embodiment
Wild cabbage material C 01 with the high anti-stalk break that identified serves as for examination material (Mei; et al. 2011; Euphytica 177; 393-400); improvement sclerotinia rot of colza resistance is the embodiment of the inventive method; but be not the qualification to the inventive method, any not super conversion 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: with turnip type rape landrace " Xishui white oil dish " is female parent (oil crop research institute of the Chinese Academy of Agricultural Sciences provides), at the preceding 2 days artificial strip off buds of blooming, award with the anti-stalk break wild cabbage of height C01(Mei, et al. 2011, Euphytica 177, pollen 393-400), one week of bagging.Save by embryo, morphology, pollen fertility and fecundity are observed, and cytological Identification obtains the distant hybridization material.
131 buds of pollinating are altogether dissected 26 of ovarys, and embryo is saved and obtained a clone totally 24 strain tissue cultivating seedling, 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 have the soft fine, soft fur similar to the wild cabbage parent, the leaf look greener, and petiole is arranged, it is fairly obvious that its leaf morphology and Chinese cabbage parent compare difference, it becomes strain phase form similar to cabbage type rape.Hybrid F1 is carried out somatic chromosome observe discovery, hybrid F1 plant is 19 chromosomes, and it is the 1.53(grain that fecundity is observed average seeds per pod).
Hybrid and turnip type rape are backcrossed, and the self progeny selects: at the preceding 2 days strip off hybrid plant buds of blooming, award the pollen with turnip type rape " Xishui white oil dish ", one week of bagging.The 8th day the embryo in back of pollinating is carried out embryo save, expands numerously, the plant after taking root moves to the land for growing field crops in the rape transplanting season, adopts general field management.Identify situation according to the offspring, backcross after 1 to 2 time selfing then with turnip type rape.
Backcross and the self progeny carries out resistance to sclerotinia sclerotiorum and identifies that the observation of pollen fertility and fecundity and cytology detect.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, the 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 concrete experimental technique:
Embryo is saved: pollination back the 8th day, win the ovary of growing, and the alcohol surface sterilization with 75% is after 15 seconds, and sterile water washing 1 minute was sterilized 15 minutes with 10% liquor natrii hypochloritis again, followed with sterile water and washed 3 times, each 5 minutes.Peeling off ovary taking-up rataria under aseptic condition is inoculated in the 1/2MS medium that contains 1.5% sucrose, place 25 ℃ of culturing room to cultivate (lx illumination 16 hours 2000 every day, 8 hours dark), after rataria is sprouted, transfer in the MS medium that contains 3% sucrose, 2.0 mg/L 6-BA and 0.2 mg/L NAA and carry out differentiation culture, be transferred to then and carry out culture of rootage in the MS medium.Plant after taking root is transplanted at rape and moves to the land for growing field crops 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% aceto-camine solution to dye gently, observe under light microscope then and counting, the flat little and shallow pollen grain that dyes is a pollen sterile, and full and Hyperchromatic pollen grain is the fertile flower powder.What every individual plant was observed pollen adds up to 300.
The 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 every angle fruit grain number.
Cytological Identification: get the immature bud of initial bloom stage, handle 3h down at 22 ℃, use Kano fixer (V then through the oxine of 0.002 mol/L
Ethanol: V
Glacial acetic acid=3:1) fixedly more than the 24h, change afterwards to 70% ethanol and preserve down in 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 the slide, drip the carbolfuchsin of 10% improvement, behind the compressing tablet in the following observation of cell chromosome number of light microscope (200 times).
Resistance to sclerotinia sclerotiorum is identified: (Euphytica 2011,177:393-399 with reference to the method for Mei etc.; Eur J Plant Pathol 2012,34:599-604), to seedling leaf with become the inoculation of exsomatizing of strain phase cane to identify.When plant is in the 9-12 leaf during phase, clip falls 4 leaves and carries out the stalk break inoculation, and places 22 ℃ of temperature, and humidity is 95% transfer room.Major diameter and minor axis that the inoculation back was measured bacterial plaque on the 3rd day calculate lesion area according to formula S=π * a*b/4.When blue or green pod during the phase, intercept about 45 cm long stem or side shoot, twine with preservative film at two ends, is that the card punch of 4 mm locates to create an identical wound on cane with diameter, then at wound inoculation mycelia piece, inoculation back measurement in the 4th day 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 the synthetic cabbage type rape of wild cabbage hybridization
Turnip type rape and disease-resistant wild cabbage C01(Mei with the resistance to sclerotinia sclerotiorum improvement, et al. 2011, Euphytica 177,393-400) carry out interspecific cross, the pollination back was carried out embryo to the hybrid embryo according to the method among the embodiment 1 on the 8th day and is saved, after rataria is sprouted, transfer in the MS medium that contains 3% sucrose, 2.0 mg/L 6-BA and 0.2 mg/L NAA and carry out differentiation culture, be developed to be transferred to behind the predetermined copy number and carry out culture of rootage in the MS medium.After taking root, adopt 0.5 % colchicine bubble root to handle 6 hours induced chromosomes and double,, then plantlet of transplant is arrived the land for growing field crops, carry out general field management with development artificially synthesized Brassica type rape.The method of identifying according to resistance among the embodiment 1 is carried out the resistance to sclerotinia sclerotiorum evaluation to 20 parts of synthetic rapes that obtain at last, and screening obtains resistance to sclerotinia sclerotiorum than two No. 9 strong individualities of 1.5 times in the cabbage type rape variety.
Embodiment 3: with 6 times of body materials is 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, it is 6 times of body materials of AACCCC that chromosome doubling obtains genotype, the turnip type rape hybridization of the resistance to sclerotinia sclerotiorum improvement that obtains with embodiment 1 then, the offspring carries out resistance to sclerotinia sclerotiorum and identifies, the resistance to sclerotinia sclerotiorum of polymerization turnip type rape and wild cabbage, 200 parts of materials that obtain are carried out resistance to sclerotinia sclerotiorum identify, screening obtains resistance to sclerotinia sclerotiorum than two No. 9 strong individualities of 2 times in the cabbage type rape variety.
Claims (3)
1. the method for a sclerotinia rot of colza resistance improvement is characterized in that, at first utilizes the wild cabbage and the turnip type rape distant hybridization of anti-stalk break, saves through embryo, 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 the turnip type rape, identify by morphology, cytological analysis and resistance to sclerotinia sclerotiorum identify that screening obtains the turnip type rape of resistance to sclerotinia sclerotiorum improvement; At last by hybridizing the resistance to sclerotinia sclerotiorum of polymerization wild cabbage and turnip type rape, the cabbage type rape of the high anti-stalk break of seed selection.
2. the method for sclerotinia rot of colza resistance improvement according to claim 1, 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, save through embryo, chromosome doubling, and the offspring is carried out resistance to sclerotinia sclerotiorum identify that screening obtains the strong cabbage type rape of resistance to sclerotinia sclerotiorum.
3. the method for sclerotinia rot of colza resistance improvement according to claim 1, 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: utilize the hybridization of cabbage type rape and wild cabbage earlier, 6 times of body materials that chromosome doubling obtains are bridge, hybridize with the turnip type rape of resistance to sclerotinia sclerotiorum improvement again, the offspring carries out resistance to sclerotinia sclerotiorum and identifies, the strong cabbage type rape of screening resistance to sclerotinia sclerotiorum.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103766212A (en) * | 2014-02-24 | 2014-05-07 | 西南大学 | Method for improving cabbage type rape by use of cabbage |
| CN106035059A (en) * | 2016-05-26 | 2016-10-26 | 西北农林科技大学 | Method for artificially synthesizing novel anti-clubroot brassica napus material |
| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103766212A (en) * | 2014-02-24 | 2014-05-07 | 西南大学 | Method for improving cabbage type rape by use of cabbage |
| CN103766212B (en) * | 2014-02-24 | 2015-05-13 | 西南大学 | Method for improving cabbage type rape by use of cabbage |
| CN106035059A (en) * | 2016-05-26 | 2016-10-26 | 西北农林科技大学 | Method for artificially synthesizing novel anti-clubroot brassica napus material |
| 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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