An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis x Eucalyptus urophylla).

Regeneration problems are one of the main limitations preventing the wider application of genetic engineering strategies to the genus Eucalyptus. Seedlings from Eucalyptus grandis x Eucalyptus urophylla were selected according to their regeneration (adventitious organogenesis) and transformation capacity. After in vitro cloning, the best genotype of 250 tested was transformed via Agrobacterium tumefaciens. A cinnamyl alcohol dehydrogenase (CAD) antisense cDNA from Eucalyptus gunnii was transferred, under the control of the 35S CaMV promoter with a double enhancer sequence, into a selected genotype. According to kanamycin resistance and PCR verification, 120 transformants were generated. 58% were significantly inhibited for CAD activity, and nine exhibited the highest down-regulation, ranging from 69 to 78% (22% residual activity). Southern blot hybridisation showed a low transgene copy number, ranging from 1 to 4, depending on the transgenic line. Northern analyses on the 5-16 and 3-23 lines (respectively one and two insertion sites) demonstrated the antisense origin of CAD gene inhibition. With respectively 26 and 22% of residual CAD activity, these two lines were considered as the most interesting and transferred to the greenhouse for further analyses.