Transient gene expression following DNA transfer to plant cells: The phenomenon; its causes and some applications
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
A preliminary investigation into the practicality of using Ds elements as vectors for onion transformation was undertaken. Transient transposase expression was used to mediate Ds excision following co-bombardment of a transposase expression vector and a Ds element into onion callus tissue. Ds transposition in onion was demonstrated. Further development of this transformation system was not undertaken because, at the time, the low frequency of stable transformation made further investigation impractical. Transient transposase expression as a means to mobilise Ds e1ements for gene tagging and to study transient expression was also investigated. A T-DNA construct carrying the Aetivator (Ae) transposase gene was transferred to leaf discs taken from an Hieracium aurantiaeum plant containing a chromosomal Ds element. Shoots were regenerated under selection for antibiotic resistance resulting from Ds excision. Molecular analysis suggested that regenerants carried unique transposition events. Of 84 regenerated plants, 21 (25%) did not express the T-DNA marker gene and 7 (8.3%) also lacked transposase DNA sequences. These results are consistent with the theory that expression is lost due to loss ofT-DNA sequences. Potential advantages of this gene-tagging method over conventional methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; gene-tagging in clonal or apomictic tissue; and the transi'ent nature ofiransposase expression should facilitate the stabilisation of the transposed element. Different factors involved in the transient nature ofT-DNA expression shortly after co-cultivation were also studied by using the green fluorescent protein reporter gene m-gfp5-ER in Nieotiana plumbaginifolia suspension cell transformation experiments. It was confirmed that transiently expressed T-DNAs can be lost during growth of somatic cells. However, cell death (64% of transient expressers died) and gene silencing (21 % of transient expressers retained T-DNA sequences) were more important barriers to the recovery of "stably" expressing transformants than lack ofT-DNA integration (15% of transient expressers lost all T-DNA sequences). Loss of transgene expression significantly limited the efficiency of plant transformation. Understanding the causes ofloss of trans gene expression should lead to improved transformation strategies.