Studies of homologous recombination between plasmid and chromosomal DNA in plant protoplasts
A vector, called pWreckl, was constructed for the direct DNA transformation of N. tabacum protoplasts. This vector was shown to confer kanamycin resistance to transformed callus. Using derivatives of this plasmid containing various reporter genes, a protocol for direct DNA transformation by PEG-mediated uptake was established. However, all attempts to obtain transformed callus by electroporation were unsuccessful. The CAB6 gene from N. tabacum was identified as a suitable candidate for gene targeting and was further manipulated to produce constructs in pWreckl which were used in gene targeting experiments. Using a derivative of pWreckl, called BinsupF, transformed tobacco plants were generated and used as a model system for the rescue of integrated sequences by supF selection. This system was found to be capable of rescuing integrated pWreckl sequences, but at an efficiency below that which would be required for the rescue of rare gene targeting events. Therefore a second protocol was developed which used the technique of polymerase chain amplification to amplify specific integration events. Following direct DNA transformation with a pWreckl.CAB6 construct, a number of possible gene targeting events were identified and subcloned into pUC13. Partial sequencing of these clones revealed that they were not the result of homologous recombination, but arose through a combination of plasmid degradation and rearrangement which appeared to precede the actual integration event.