Abstract
This 4-year research project is proposed to develop gene-stacking technology for the breeding of new Eucalyptus varieties in which multiple genetic characteristics are improved to meet the demand in industry. Preservation technology is also proposed to develop for the establishment of mutant bank containing varied transgenic lines of Eucalyptus that will bring the molecular breeding into the new research area of functional genomics. In this year's project, gene stacking by sequential transformation were developed to raise transgenic E. camaldulensis plants with the antisense Pt4CL1 and the sense CAld5H transgenes. The transgenic E. camaldulensis plants previously transformed with the sense CAld5H transgenes were sequentially transferred with recombinant SiRNA of the CAD gene to produce transgenic callus or shoots with double transgenes. In co-transformation, transgenic Eucalyptus plants with the antisense Euc4CL1 and/or the sense CALd5H transgene were produced. The transgenic E. camaldulensis plants previously transformed with the sense CAld5H transgenes were selected and transferred with recombinant SiRNA of the CAD gene to produce transgenic callus or shoots with multiple transgenes. In addition to these, another 5 cellulose synthase (CesA) genes will be cloned from E. camaldulensis readily for the further co-transformation work in the following year. In controlled pollination of gene stacking, transgenic seedlings with C4H and GUS transgenes derived from pollination between transgenic E. grandis x E. urophylla and E. camaldulensis were raised. Transgenic Eucalyptus previously transformed with either the antisense Euc4CL1 or the sense CAld5H transgene were induced to flower for making more controlled pollinations. For rapidly measuring monomeric lignol of transgenic wood, an analytical technique by using pyrolyzer coupled with GC-MS were developed. We already confirmed that the sense CAld5H transgene can raise S/G ratio and the total amount of cellulose is no difference.