Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts

Abstract

The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species. These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating wood properties, a number of physical and biochemical limitations to microscopical and molecular experimentation. The use of in vitro wood formation systems for molecular studies and Agrobacterium-mediated introduction of transgenes overcomes many of these obstacles. Using a commercially relevant Eucalyptus species as model organism, we demonstrate here that in vitro wood formation systems can be readily employed to introduce transgenes into growing wood-producing tissue, initially leading to frequent transient gene expression in a range of cell types. Stable transformation events were observed as sectors of transformed tissue derived from primary transformation events in individual cells. The usefulness of such systems for the analysis of gene function during the process of wood formation and wood quality determination, as well as for constructing developmental fate maps of cambial derivatives, is discussed.

Organogenesis and transient genetic transformation of the hybrid Eucalyptus grandis × Eucalyptus urophylla

“…In a study with E. globulus, apical segments were transformed and co-cultivated for periods that varied from 2 to 7 d, with the best results of callogenesis and total number of transformed events being obtained after a 6 d co-culture (Spokevicius et al, 2005). For hypocotyl segments of E. camaldulensis, 2 d were sufficient (Ho et al, 1998).…”

Genetic transformation of eucalyptus

“…Apart from generating transgenic eucalyptus plants, different explants are used to study the role of promoters and genes involved in wood formation. They include in vitro transformation of apical stem segments (Spokevicius et al 2005), in vivo transformation of exposed cambial cells within mature stems Van Beveren et al 2006) and in vivo transformation of developing stems after wounding of dormant lateral buds ).…”

“…Using Agrobacterium-mediated methods, large numbers of independent transient transformation events can be obtained in situ within any cell type. This system holds promise towards becoming a standard for functional analysis of genes involved at various stages in the wood formation process directly in the tissue of interest and within a reasonable timeframe (Spokevicius et al 2005). In vitro systems in general require the establishment and maintenance of tissues in aseptic conditions.…”

“…In vitro systems in general require the establishment and maintenance of tissues in aseptic conditions. This is technically demanding where time consuming where often low efficiency is achieved (Spokevicius et al 2005).…”

“…Efficient use of these systems with Agrobacterium-mediated transformation has been proven to be difficult due to continued contamination following bacterial inoculation which is difficult to control. This results in a low recovery of stably transformed sectors and limited sector size due to the reduced growth periods (Spokevicius et al 2005). Phenotypic characterization of candidate gene function in these systems has not been attempted, till date.…”