Research Assistant Fort Valley State University Fort Valley, Georgia
Body of Abstract: Hemp (Cannabis sativa L.) is a dioecious crop and has therapeutic potential for human diseases because Phyto- cannabinoids in medical therapy are getting more attention recently. The purpose of this study was to establish a protocol for Agrobacterium-mediated transformation for foreign gene integration. Several factors influencing transformation efficiency including the effect of explant type and age, the concentration of silver nitrate, calcium chloride, bacteria concentration, infection time, acetosyringone concentration, wounding, and different co-culture periods of bacteria were evaluated to optimize the transformation efficiency. The Agrobacterium-mediated transformation was optimized using the binary vector pCAMBIA1304 (1) encoding the GFP and GUS as reporter genes which are driven by the CaMV35S promoter for early detection of transgene expression. Results obtained were based on the average number of blue spots and percentage of transformed GUS expressing leaf tissue which was observed 3 days post-transformation. The results indicated that 14 and 21 days old fully expanded leaves showed increased transient transformation recorded as the number of GUS blue spots and GFP expression at 7-day-old leaf explant. Enhanced transient GUS expression with an average frequency of 79% was noticed at 30 minutes of the co-cultivation period while 10 minutes of the co-cultivation period resulted in 20% transient GUS expression with an average of 132 blue spots. The addition of 150 µM acetosyringone, 60 µM of silver nitrate, and 0.5 µM of calcium chloride in the co-cultivation medium increased the transient transformation frequency. The highest transient gene expression of 74% was obtained from a leaf incubated compared to other explants such as roots, cotyledons, petiole, or leaf-derived calli. The results indicate that the GUS gene is superior to the GFP gene in following transgene expression in transiently transformed materials in different explants. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.