This one year BARD project was funded to develop methods to monitor promoter activity a gene expression patterns in citrus fruit. To fulfill this goal, we divided the research tasks between both labs so that the Israeli side evaluated the use of microprojectile bombardment ; a tool to evaluate transient gene expression in various citrus fruit tissues, and the US side optimized technical parameters required for Agrobacterium-mediated transformation of various citrus cultivars. Microprojectile bombardment appeared to be a very efficient method for transient gene expression analysis in citrus leaf tissues but was somewhat less applicable in fruit tissues. Nevertheless, we did succeeded to achieve significant levels of 35S-GUS gene expression in young green flavedo tissue. However, only single random spots of 35S-GUS gene expression were detected mature flavedo and in juice sacs and albedo tissue. Overall, we assume that following some more technical improvements particle bombardment could provide a useful technique to rapidly analyze promoter activity at least in the flavedo tissue. For Agrobacterium-mediated transformation, we found that shoot cultures of 'Washington' navel oranges,'Fairchild' mandarins,'Eureca' lemons,'Troyer' citrange and various grapefruits provided a more reliable and consistent source of tissue for transformation than germinated seedlings. Moreover, various growth media's (McCown, Quoirin & Lepoivre, DCR) further improved shoot and root growth relative to MS mineral media, which is commonly used. Also pure white light (using bulbs which do not emit UV or blue light) improved shoot growth in various citrus varieties, and paromomycin appeared to be a more efficient antibiotic for the selection of transgenic plants than Kanamycin. Overall, these optimizations improve transformation efficacy and shoot growth and rooting capacity. In addition to the development of transformation methods, both Israeli and US labs achieved progress in the identification of citrus fruit-specific promoters. In Israel, we isolated a 3.6 kb promoter fragment of the thiamine biosynthesis c-thi gene, which is highly expressed in fruit peel tissue, whereas in the US we isolated a 1.5 kb promoter fragment of the citrus seed-specific cDNA CssH. The identification of more fruit-specific cDNAs and their corresponding promoter regions is currently in progress.
Development, Characterization and Experimental Validation of a Cultivated Sunflower (Helianthus annuus L.) Gene Expression Oligonucleotide Microarray
