Biolistic transformation of Eucalyptus grandis x E. urophylla callus

Abstract

A procedure for genetic transformation of the hybrid Eucalyptus grandis × E. urophylla using particle bombardment is described. Cotyledon- and hypocotyl-derived calli growing on SP medium supplemented with 2 m thidiazuron or on MS modified (MSM) medium supplemented with 10 m 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5 m 6-benzylaminopurine (BAP), were used as target material for bombardment assays. Multiple preincubation and bombardment conditions were tested. Tungsten particles were coated with the plasmid pBI426 harbouring a β-glucuronidase (gus) and neomycin phosphotransferase II (npt II) gene fusion controlled by a double 35S cauliflower mosaic virus (CaMV) promoter. Four days after bombardment, the transient transformation efficiency was determined by expression of the gus gene. Fully GUS-positive calli were then obtained after 105 d in MSM medium supplemented with 2,4-D, BAP, and the selective agent kanamycin at 200 mg L^-1. The presence of the gus gene in these kanamycin-resistant calli was confirmed by polymerase chain reaction analysis. Extensive experiments were performed aiming to identify conditions for the regeneration of these GUS-expressing calli. However, they were unable to regenerate transgenic shoots, suggesting that conditions suitable for regeneration are unsuitable for transformation and vice versa.