Establishment Of Plant Regeneration System And TSRF1 Gene Transformation Of Eucalyptus Urophylla

Eucalyptus grew in Australia at first.It belongs to myrtle family,which is an ever-green and fast-growing arbor with broad-leaves.It is both one of the three fast-growing species and an excellent raw economical plant for forestry,which is mainly used for industrial pulp,artificial board and refining eucalyptus oil.Urophylla U6 was the fine line breeded from Urophylla seedling stand,it had been planted in most area of the Guangdong,Guangxi and Hainan provinces.It has several advantages:fast growing and high yield,strong sprout capacity in the second and the third generations.As the area of eucalyptus plantations increased steadily in South China,the pests and diseases of eucalyptus s have been becoming more and more serious,which not only affected the eucalyptus growth,but also led to poor wood output and quality.Genetic improving of eucalyptus us using the genetic engineering was one of the hot regions of eucalyptus us breeding,which can overcome sexual-crossing barriers and share all genes existing in the plant,animals,and microbe. Especialy,the genetic engineering can improve the given characters and significantly shorten the breeding periods.However,many hinder still exist in eucalyptus biotechnology.The main reason is that cultivars of transgenic eucalyptus,which requires transformation of appropriate tissue followed by regeneration,remains extremely difficult in embryogenesis and regeneration.Recalcitrant eucalyptus cultivars,long tissue culture duration,the unpredictability of tissue culture are more troublesome in regeneration of eucalyptus.In this study,The hypocotyls germinated from the seeds of Urophylla U6 was extracted as explants.Firstly,an efficient invitro propagation and plant regeneration system was developed for Eucalyptus urophylla.Factors that affecting shoots regeneration via direct organogenesis,including cultivation conditions and plant growth regulators etc,were also investigated in details.Subsequently,parameters that affecting the efficiency of Agrobacterium tumefaciens-mediate transformation of Eucalyptus urophylla were comprehensively studied and optimized by accessing NPTII expression,and a transformation protocol was established.Finally,on the basis of the established transformation protocol,the regeneration of transgenic plants with TSRF1 gene was developed through transformation mediated by Agrobacterium tumefaciens.The main results of this study were introduced separately as following:1.High efficient regeneration system of Eucalyptus urophylla were established(1) The best medium for inducing callus:MS+2,4-D 0.1mg/L+6-BA 0.6 mg/L +Vc 100 mg/L+sugar 30 g/L+agar 7 g/L(pH 5.8～5.9),the callus induction rate was 96.7%(2) The differentiation medium of adventitious buds:1/2MS+LC 0.8 mg/L+ IBA 0.2 mg/L+TDZ 3μmol/L+put 500μmol/L+spd 50μmol/L+Vc 100 mg/L+ sugar 30 g/L+agar 7 g/L(pH 5.8～5.9),the differentiation rate of adventitious buds was 53.3%,the rate of superior buds was 48.3%(3) The optimum medium for bud elongation:BS+NAA 0.05 mg/L+ LC0.8mg/L+Vc 100 mg/L+sugar 30 g/L+agar 7 g/L(pH 5.8～5.9),the buds above 2.0cm accounted for 82.3%(4) The best medium for buds proliferation:1/2MS+6-BA 0.4 mg/L+NAA 0.05 mg/L+sugar 30 g/L+agar 7 g/L(pH 5.8～5.9),the plantlets increased 4.4 times every month,the average height of buds was 2.7 cm(5) Rooting medium of regenerated plant:1/4 MS+NAA 0.05 mg/L+IAA 0.2 mg/L+sugar 30 g/L+agar 7 g/L(pH 5.8～5.9)2.Construction of Eucalyptus urophylla transformation systemEfficient system of Agrobacterium-mediated Eucalyptus urophylla transformation was also established.The results showed that precultured explants for 7 days,immerged for 3h by need bacterium concentration which was diluted during phase of logarithm OD=0.4～0.6,cocultured for 5 days,and delayed selection for a week could increase the transformation frequency.3.Transgenic Eucalyptus urophylla young plants were obtainedThe transgenic plants were validated with PCR assay.The experiment was conducted by using non-transgenic plantlets as negative control group,plasmid DNA as positive control group,groups including TSRF1 gene as promoters,and PCR results showed:Extended strip extended in non-transgenic plantlets.The results of electrophoresis confirmed that the TSRF1 gene had been introduced into Eucalyptus urophylla.