Genetic transformation of Micro-Tom tomato with a calcium signal modifying gene and analysis of transgenic plants challenged with ' Candidatus Liberibacter solanacearum'

Candidatus Liberibacter (Ca. L.) spp. infection in citrus and tomatoes causes reduction of productivity and devastating economic losses. Previous results from transgenic citrus plants transformed with a calcium signal modifier (CSM-1) gene showed resistance to various pathogens. Citrus has a long juvenile period, which delays any evaluation of transgene expression, whereas Micro-Tom (MT) tomato has a short life cycle, produces numerous progeny, and is easy to genetically transform; therefore MT can be used as a model organism to evaluate CSM-1 over-expression, possible resistance to Ca. L. solanacearum (Lso), and any alterations to phenotype in short time. The objectives of this study were to develop transgenic MT tomato plants expressing CSM-1, study morphological and physiological changes, observe presence of CSM-1 T1 populations, and screen for resistance to Lso. MT cotyledons were transformed with CSM-1 gene using Agrobacterium strain EHA105 (optical density of 0.2-0.6), putative transformed shoots were identified by kanamycin resistance and histochemical GUS assay. CSM-1 gene expression was verified through reverse transcription PCR for a total of 17 transgenic lines. All transgenic lines had one copy of the CSM-1 gene; copy number was determined through qPCR and verified by Southern blot analysis. Some transgenic MT lines produced seedless fruit; other lines produced low to moderate seeds giving a mean of 2.12 seeds per fruit and a 60% germination rate. Pollen viability was determined by germination on semi-solid medium for seedless lines and observed every 30 minutes. Wild type MT pollen germination was 37.94% while it was 2.19% in one of the transgenic lines; low pollen viability was associated with low seed set. Offspring of transgenic MT tomato plants were infected with Lso via tomato/potato psyllid (Bactericera cockerelli ) feeding and infected plants were confirmed by quantitative PCR (qPCR). A Dunnett's t test determined that transgenic lines were not offer any resistance to Lso compared to the WT Micro-Tom tomato plants. Because the CSM-1 gene was not optimized for expression in tomato this may be the cause of failure to offer resistance to Lso.