Agrobacterium tumefaciens-mediated transformation of Eucalyptus urophylla clone BRS07-01

Abstract

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 µM 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.

Keywords

Eucalyptus / Genetic transformation / In vitro regeneration / Plant growth regulators / Somaclonal variation

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