A study of T-DNA integration and transgene expression in Pinus radiata
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Pinus radiata D Don. is the key resource for the New Zealand forest industry. Genetic engineering could potentially accelerate its genetic improvement by the introduction of novel traits or modification of existing traits, with both environmental and economical importance for the industry. In this study PCR and Southern hybridisation confirmed stable T-DNA integration in P.radiata plants produced by Agrobacterium-mediated gene transfer. Southern analysis showed a predominance of single T-DNA integration events. However, incomplete transfer leading to truncated copies of the T-DNA appeared to be common. TAIL-PCR was used to identify TDNA/ genomic DNA junctions in five transgenic P.radiata lines. Sequence analysis of these junctions showed attachment of residual vector DNA at the right border for all five lines. We suggest this may reflect the recalcitrance of P.radiata to Agrobacterium infection. Rearrangement of the integrated T-DNA was analysed in detail for two transgenic P.radiata lines and revealed features common to both lines including deletion of T-DNA sequence, presence of plant genomic DNA within the T-DNA loci, and T-DNA rearrangement around the CaMV 355 promoter region. The visual marker genes β-glucuronidase (uidA) and green fluorescent protein (gfp) were evaluated for their potential use in regenerated P.radiata tissue. Detection of uidA by histochemical staining was poor and unpredictable. Factors within the plant cell appeared to either inhibit the B-glucuronidase enzyme or interfere with the chemistry of the histochemical reaction. GFP could be visualised using epi-fluorescence microscopy in all cell types of needles removed from transgenic P.radiata plants, and could be distinguished from background autofluorescence. An image analysis system was used to compare GFP expression between lines. Transgenic lines with high levels of GFP expression were distinguished from transgenic lines with low levesl of GFP expression. Comparison of GFP expression between clonal shoots propagated from single transgenic P.radiata lines showed that the lines analysed were not chimeric in origin. Understanding the predominant characteristics of Agrobacterium-mediated T-DNA integration in P.radiata plants in conjunction with an efficient visual marker-gene system to evaluate transgene expression will improve transformation strategies and thus the stable expression of transgenes.