Establishment Of A Novel System For Production Of Hybrid Rapeseed Seeds Via Chemical Emasculation

Rapeseed is the most important oil crop in China,and heterosis is one of the important ways to improve its yield and oil content.Establishment of effective and efficient a system of producing hybrid seeds is the prerequisite for use of heterosis.Production of hybrid seeds by chemical emasculation is one of the important ways in use of heterosis.However,chemical emasculation has some defects as well as many advantages,and the main defect is low purity of hybrid seeds.In order to solve this problem,we have proposed a new system of producing hybrid rapeseed seeds via chemical emasculation,which is that mother plants are treated with chemical hybridizing agents and then pollinated with pollens from father plants with herbicide-resistance and killing false hybrid seedlings using herbicides.Following works were carried out in order to test the new system of producing hybrid rapeseed seeds via chemical emasculation:1.Heredity and molecular mechanism of herbicide-resistance characteristics of rapeseed germplasm 12 WH31812WH318 is a novel rapeseed germplasm with imidazolinones-resistance selected by our research group.Hybridization,reciprocal cross and back cross were done between 12WH318 and 4 herbicide-sensitive rapeseed varieties.The results indicate that the herbicide-resistance in 12WH318 is controlled by one pair of a nuclear gene,in a mode of complete dominance without cytoplasmic effect.Because action target of imidazolinones herbicides is acetolactate synthase(ALS),we isolated three members of BnALS gene families,i.e.BnALSl,BnALS2 and BnALS3 from 2WH318 and three other herbicide-sensitive rapeseed germplasms using homologous strategy.Alignment of ribonucleosides of BnALS1,BnALS2 and BnALS3 showed that there are no differences in sequences of both BnALS2 and BnALS3 between 2WH318 and three herbicide-sensitive varieties,but the ribonucleoside of BnALS1 of 2WH318 at position+1913 mutated from G to A,which resulted in a change of the 638th amino-acid reside in protein BnALSl of 2WH318 from serine to asparagine.Gene expression analysis indicated that expression of BnALSl was sharply up-regulated in 2WH318 within 4 days after herbicide treatment.2.Regeneration of doubled haploid plants of rapeseedIn order to improve growth period,comprehensive traits and combining ability of 12WH318,microspores from four back cross generation(BC1)population,(Lisandra×12WH318)xLisandra、(ZS9×2WH318)×ZS9、(ZSlO×12WH318)×ZS10 and(QY14×12WH318)xQY14,were in vitro cultured,but haploid plant regeneration was only found in combination(ZSlO×12WH318)×ZS10.213 regenerated haploid plants were treated with 2.5 g/L colchicine for 5 h and then transplanted in field.Totally,149 plants survived,yielding a survival rate of 70%;of them,57 plants were fertile.These fertile doubled haploid plants can be used for selection of ideal plants with good comprehensive traits and herbicide-resistance.3.Breeding transgenic rapeseed germplasm Z7B10 with herbicide-resistanceHerbicide-resistance gene bar was introduced into cells of herbicide-sensitive rapeseed variety Zhongshuang 7 and transgenic plants were regenerated.Transgenic plants were identified by PCR techniques and the copy numbers of transgenes were identified by Sothern blot.Transgenic plants with single cope of bar gene were selected and self-pollinated.Finally,a homozygous transgenic plant Z7B10 was obtained and herbicide-resistance and tolerance experiments were conducted with it.4.Study on a novel system for production of chemical hybrid seeds of rapeseedAt single nuclear stage of pollens,6 herbicide-sensitive rapeseed varieties,Zhongshuang 7,Zhongshuang 9,Zhongshuang 11,Enyou 4,Qingyou 14 and Mianhangyou 6,were sprayed with tribenuron at 0.00 mg/L,0.05 mg/L,0.075 mg/L and 0.10 mg/L,and pollen abortion,physiological indexes,agronomic traits and oil contents of treated plants were investigated.The results showed that 0.1 mg/L was the optimal level for chemical emasculation of rapeseed when tribenuron was used as chemical hybridizing agent.Three rapeseed cultivars,Zhongshuang 4,Zhongshuang 10 and Zhongshuang 12,were treated with 0.1 mg/L tribenuron and then pollinated with pollens of transgenic Z7B10 plants harboring herbicide-resistance gene bar,and the yields of potential hybrid seeds were 652.8±75.15 kg/hm2,589.8±42.75 kg/hm2 and 469.2±88.5 kg/hm2,respectively.The potential hybrid seeds germinated and developed into seedlings.After herbicide at 0.1 mg/L was applied to the seedlings at stage of 3-4 leaves,the false hybrid seedlings died and the authentic hybrid ones survived well.Based on the death rates of seedlings,the purity of hybrid seeds of combinations of Zhongshuang 4×Z7B10,Zhongshuang lOxZ7B10 and Zhongshuang 12xZ7B10 was 89.64%±11.06%,79.28%±6.58%and 81.29%±2.99%,respectively.These results demonstrated that our novel system of producing hybrid seeds by chemical emasculation can efficiently improve purity of hybrid seeds.