An evaluation of pollen as a vehicle for plant transformation.

This thesis reviews the possibility of pollen grain/tube transformation and defines several problems which have hindered attempts to develop a routine technology for gene transfer. The importance of transforming pollen grains or using the pollen tube as path way for transformation has been discussed. As a prerequisite for transformation, the optimum conditions for pollen germination in vitro and fertilisation system with pre-germinated pollen were established for pea and tobacco. Plasmid DNA (free or liposome-encapsulated) binding to the pollen tube was demonstrated using fluorescein and radioactive labelling techniques. The problems of introducing foreign genes into the pollen grain/tube and obtaining DNA integration and expression have been discussed. Selection techniques allowing potentially the detection of transformed pea and tobacco seedlings were developed. Transformation methods involving using the use of naked DNA in vitro or in combination with PEG, the coating of pollen grain with plasmid, electroporation, microprojectile bombardment or the use of liposome-encapsulated plasmid were attempted. Transformation methods in vivo using a 'pollen tube' pathway were attempted with tobacco, pea and wheat. DNA uptake by injection of Agrobacterium into pea florets was also attempted. Although all of these transformation experiments reported in this thesis were unsuccessful it is still possible that the plant fertilisation system can be perturbed to allow gene transfer and in this respect an outline of possible future work has been given.