Genetic engineering for antibacterial activity in lettuce (Lactuca sativa L.)

Lettuce crop is affected by bacterial diseases (both in the field and in storage) with severe, consequences which can be mitigated through improving disease resistance in lettuce. The numerous constraints associated with sexual crosses in lettuce render novel gene transfer via genetic manipulation a suitable method for lettuce improvement. This study was undertaken to transfer three chimaeric genes encoding antibacterial peptides into lettuce and to ascertain the effectiveness of these peptides in enhancing bacterial disease resistance. Genetic manipulation of plants requires a good regeneration ability of genotypes in tissue culture, to ensure recovery of complete transformed plants. Twenty-two lettuce genotypes belonging to several morphological groups were thus screened for their regeneration response to defined tissue culture conditions and selection of amenable genotypes for genetic manipulation. Genotypic variation was observed in callus, shoot and root production in vitro; two lettuce genotypes Bambino (a crisphead) and Cobham Green (a butterhead) with good tissue culture response were subsequently chosen for Agrobacterium-mediated transformation. Two binary vectors carrying chimaeric genes (with T4 lysozyme, magainin IT coding sequences) were obtained from Crop & Food Research, Lincoln; these vectors had pBINPLUS (a recently constructed binary plasmid) as the backbone. A third binary vector, similar in structure to the two others, with a chimaeric gene encoding Shiva-1 (a cecropin B analogue) was successfully constructed. The three vectors, pBINPLYS, pBINMGN and pBINPLUSH (encoding T4 lysozyme, magainin IT and Shiva-1 respectively) in Agrobacterium strain AGLI were used for the transformation of Bambino and Cobham Green. Two transformation treatments: a delay of 5 days (after co cultivation) before explants were transferred to kanamycin selection medium and an immediate transfer of explants to selection medium were studied for their effect on transformation frequency. Successful regenerated transformed plants of Cobham Green were obtained in this study; however, attempts to transform Bambino proved futile and no regenerated transformed shoots of Bambino were obtained. The specific reasons for the failure to transform Bambino are unknown but crisphead lettuce genotypes are often recorded as being recalcitrant to Agrobacterium-mediated transformation. The delayed selection treatment gave higher transformation frequencies in Cobham Green than the immediate selection treatment. Polymerase Chain Reaction (PCR) with specific npt IT oligonucleotide primers confirmed the presence of the selectable marker gene in all the 22 putative transgenic plants recovered. An inheritance study on the selfed progeny of transformed lines, confirmed transmission of the kanamycin resistance and pointed to a single insertion of the npt II gene in most of the transformed lettuce lines. The presence of the antibacterial genes was established in all the transformed plants, by PCR. The presence and effectiveness of the antibacterial peptides were determined by an in vitro assay and greenhouse evaluation of pathogen resistance in seedlings. Crude leaf extracts from selected transformed lines and control of untransformed Cobham Green were added to cultures of Erwinia carotovora subsp. carotovora and Xanthomonas campestris pv vitians to establish their effect on the growth of the pathogens. Growth of pathogens in cultures with crude extracts from transformed lines was inhibited while extracts from the control showed no such effect on the growth of the pathogens. In the greenhouse, selfed progeny of transformed lines and untransformed control were inoculated with the two lettuce pathogens to assess their response to disease development. Overall, delayed symptoms and reduced disease severity characterised the inoculated transformed lines, compared to the control seedlings. The potential of the antibacterial genes to effectively improve the resistance of bacterial diseases in lettuce was evident in these assays