Ploidy Analysis And Regeneration System Establishment Of Orchardgrass (Dactylis Glomerata L.) Germplasm Resources

Orchardgrass(Dactylis glomerata L.)is one of the most widely distributed perennial grasses in the world.It grows well in temperate and high altitude areas and has the advantages of shade tolerance,cold resistance and overwintering ability.It is an important grass species for grassland ecological restoration and livestock production.Traditional breeding methods are mainly used in orchardgrass breeding with low efficiency and long cycle.In order to improve this situation,it is urgent to apply modern biotechnology to transfer fine genes directional.Therefore,it is of great significance to construct a high-frequency regeneration system of orchardgrass and carry out related research on genetic transformation technology.In this study,flow cytometry was used to identify the ploidy of orchardgrass,which was combined with cytological ploidy analysis to supply the method and basis for the ploidy identification and to provide a source for the diploid and tetraploid materials of orchardgrass.Through screening the callus induction of mature seeds of different genotypes of orchardgrass,the effects of different seed pretreatment methods,media and exogenous additives on callus induction of orchardgrass were clarified.In this process,good quality callus with high callus induction rate,strong differentiation ability and good seedling formation rate were screened out and high frequency regeneration system of orchardgrass was established,which laid a foundation for the study of genetic transformation of it.The hairy roots of orchardgrass were induced by screening the best conditions for transformation of Agrobacterium tumefaciens.The main results were as follows:1.A rapid orchardgrass ploidy identification method was established.In this study,4kinds of nuclear DNA lysis buffer were screened and 2 flow cytometry methods were compared by using flow cytometric.Based on cytological identification,flow cytometry was used to study the germplasm resources of orchardgrass.The results showed that DNA contents varied with different lysis buffer.The better effect of nuclear suspension with complete peaks,small percentage of fragments and clear fluorescent signal was obtained by using Otto lysis buffer,the coefficient of variation was about 5%.Both internal and external methods had advantages,external standard method was faster and simpler while internal standard method has higher accuracy in plant ploidy detection.There were 22 diploids and 36 tetraploids in 46 detected accessions and 12 varieties mainly using external standard method,among which all cultivated varieties are tetraploid.The results provided rich materials for genetic breeding of orchardgrass.2.To induce callus and establish an efficient regeneration system of orchardgrass.Mature seeds were used to study the effects of seed pretreatment,variety selection,medium selection and hormone ratio in the process of callus induction.The results showed that the cultivar ‘Anba’ has the strongest callus induction ability among the ten cultivars.The optimum pretreatment conditions for mature seeds as follows: after soaking at 4 °C and cold storaging for 24 hours,the seeds of the ‘Anba’ were sterilized with 75% ethanol for 5 minutes and 1.1% Na Cl O for 30 minutes.The optimum medium for callus induction was SH+2.0 mg/L CPA+0.1 mg/L 6-KT+30 μmol/L dicamba+0.5 mg/L 2,4-D.With this medium and hormone combination,the callus induction efficiency was high,which can reach about 70%,and embryonic callus would be induced easier.3.Hairy root induction system construction of orchardgrass.In this experiment,Agrobacterium rhizogenes A4 was used to infect 3 kinds of explants including leaf segments,root segments and living seedlings of orchardgrass.By comparing the effects of bacterial solution concentration on hairy roots induction,we obtained the results as follows:The hairy roots could not be induced by infecting the leaves,root segments and soil culture seedlings of ‘Anba’,however,it could be produced within a week by injecting sterile seedings with needling method.The hairy roots could not die in long-term subculture,accompanied by branches and biomass increasing.It was found that hairy roots induction efficiency was higher when the concentration of AS was 200umol/L and OD value were0.5,0.6,0.7.The induction rate would decrease when OD value was 0.8 and it was easy to contaminate for long-term subculture.A single stable band of Ri plasmids was detected by PCR,which was proved to be hairy roots.