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.
Language
eng
File Type
pdf
File Size
1514539 Bytes
Date Available
July 3, 2014
LC Number
T378.24 L916g
Rights
The right to download or print any of the pages of this dissertation (Material) is granted by the copyright owner only for personal or classroom use. The author retains all proprietary rights, including copyright ownership. Any reproduction or editing or other use of this Material by any means requires the express written permission of the copyright owner. Except as provided above, or any use beyond what is allowed by fair use (Title 17 Section 107 U.S.C.), you may not reproduce, republish, post, transmit or distribute any Material from this web site in any physical or digital form without the permission of the copyright owner of the Material. Inquiries regarding any further use of these materials should be addressed to Administration, Jernigan Library, Texas A&M University-Kingsville, 700 University Blvd. Kingsville, Texas 78363-8202, (361)593-3416.
