Indexed on: 01 Sep '03Published on: 01 Sep '03Published in: Molecular Breeding
A procedure for A. tumefaciens-mediated genetic transformation of a juvenile E. camaldulensis clone is presented. CAD antisense full-length cDNAs from Eucalyptus gunnii or Nicotiana tabacum was introduced under the control of the CaMV 35S DE promoter. From 44 individual transgenic shoots selected by PCR analysis, 32% exhibited a significant reduction of CAD activity, up to 83%. The use of the heterologous tobacco CAD cDNA construct was less efficient (up to 65% reduction). Transcript levels in 3 lines obtained using the homologous eucalyptus cDNA confirmed the under-expression of the CAD gene, and Southern blot data indicated a low transgene copy number ranging between 1 and 3. The most down-regulated plant contained a single transgene copy. Therefore, for the first time in eucalyptus, genetically modified plantlets exhibiting a strong inhibition of CAD activity associated with decreased transcription were recovered. Five transgenic lines, transferred to the greenhouse for 10 months, went through a wood chemical analysis that showed no differences in lignin quantity (through Fourier transform infrared spectroscopy), composition (through analytical pyrolysis) or pulp yield (through Kraft pulping) compared to control trees. Despite the down-regulation of the CAD gene in this Eucalyptus species of economic interest, the lack of significant changes in lignin profiles indicates that probably the trees were not sufficiently suppressed in CAD throughout development to exhibit obvious modifications in lignin and pulping. This raises the problem of the requirements for an efficient modulation of lignification in trees such as eucalyptus.