An alternative transformation method in citrus using cell-penetrating peptides (CPPs)

Citrus transformation is typically Agrobacterium tumefaciens-mediated, in which citrus tissues are cultivated with the bacterium and regenerated on selection media. Part of the bacterial plasmid, with the gene of interest and resistance genes, is then inserted into the citrus genome. Due to the slow growth of citrus, this long process must be optimized for each cultivar and ultimately the transformation efficiency in citrus is substantially less than with other model systems. Commercialization of transgenic citrus is even slower because of the regulations on genetically modified produce worldwide. In order to decrease the dependence upon bacterial transformation and increase transformation efficiency, we propose an alternative method of transformation using cell penetrating peptides (CPPs) that does not involve Agrobacterium. CPPs are positively charged short amino acid sequences able to simultaneously bind proteins and nucleic acids and deliver them across cellular membranes and cell walls. CPPs are used currently in plants in transient expression assays and gene silencing, but have not been used in citrus or in stable transformation experiments. We have developed a standard method for the transient expression of reporter genes (gus and gfp) in citrus. Our data indicate that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have been tested and the efficiency is increased to 100% when CPPs are used in conjunction with a lipid reagent. We have transformed several explant segments which survived kanamycin selection, produced shoots and roots, and were planted. PCR and reporter gene analysis will confirm stable integration. Further experiments with CPPs, comprising RNAi and protein trafficking, will also be performed.