The Study Of Nodule And Nitrogen Fixation Of Hybrid Progenies From Caucasian Clover Crossed With White Clover

In the previous studies,a series of hybrid offsprings were obtained by distant hybridization and embryo rescue techniques with Caucasian clover originated in the Caucasian region as the female parent and usant white clover in the Greater Khingan Mountains as the male parent.Over the years,after systematically exploring the adaptive conditions for embryo isolation and culture,through in-vitro organogenesis of stem segments and embryo germination of the hybrid F₁generation,the F₁generation lines with stable traits were obtained,and successfully cultivated and propagated in greenhouse and field to form lines with excellent traits.However,as with the female parents,the hybrid lines could not spontaneously produce nodules for nitrogen fixation.After inoculating the F₁generation of hybrid strains of Trifolium with Rhizobium,the strains gained the ability of nodulation and nitrogen fixation,showing high biomass and strong adaptability.Therefore,it is very important to analyze the nitrogen fixation mechanism of F₁strain.Five strains of Rhizobia isolated from the experimental field of Qinghai University’s Mengnong clover No.1 were selected and inoculated with the hybrid progeny strains after numbering,having no Rhizobia inoculation as the control,repeating for 10 trials.After 60days of inoculation,the growth phenotype,root configuration and nitrogenase activity of nodules of the plants in each treatment were measured,and the microstructure of the dominant strains was observed.Transcriptome analysis was performed on select dominant strains.Specifically,the plants were sampled three,six,and nine days after inoculation and compared with the uninoculated plants as the control.Each treatment was repeated three times to form 18 analysis samples.The main results were as follows:（1）The comprehensive performance of plants inoculated with No.5 Rhizobia was the best.Its growth phenotype and root index were significantly better than those of other plants;the acid detergent fiber（ADF）and neutral detergent fiber（NDF）of dry grass decreased by 45.34%and 54.11%respectively compared with the control.Observation of the microstructure revealed that Rhizobia colonized additional Rhizobia cells,and there were a large number of infected Rhizobia cells.Inspection of nitrogenase activity of Rhizobia showed that the plants with the strongest nitrogen fixation ability were inoculated with No.3 Rhizobia,followed by No.5 Rhizobia.（2）The plant samples inoculated with the dominant strain（No.5）were used for transcriptome analysis,and the differences in gene expression of these groups when compared to the control group were as follows:CK-Z-3 vs Z-3,44 genes were up-regulated and 41 genes were down-regulated;CK-Z-6 vs Z-6,904 genes were up-regulated and 182 genes were down-regulated;CK-Z-9 vs Z-9,558 genes were up-regulated and 26genes were down-regulated.（3）GO and KEGG enrichment analysis showed that the signal exchange between Rhizobia and plants were more frequent 3 days after inoculation,and the root system began to release flavonoid compounds.6 days after inoculation,Rhizobia began to invade plant cells,which resulted in immediate plant response.9 days after inoculation,the Rhizobia successfully colonized plant cells,and the nitrogen fixation activity of nitrogenase was activated,and biological nitrogen fixation began.