Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still ...Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.展开更多
文摘Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.
