Introduction of Xa21, a Xanthomonas-resistance gene from rice, into 'Hamlin' sweet orange [Citrus sinensis (L.) Osbeck] using protoplast-GFP co-transformation or single plasmid transformation

Plasmid DNA (pARS108) containing the non-destructive selectable marker Green Fluorescent Protein (GFP) gene, and a plasmid containing a cDNA of the Xa21 gene from rice (pXa21-mtaq) were co-transformed into 'Hamlin' orange protoplasts using polyethylene glycol (PEG). Alternatively, plasmid DNA (pAO3), containing both genes (GFP and Xa21) was directly transformed into 'Hamlin' orange protoplasts. Over 1,000 transgenic plantlets were regenerated from approx. 80 independent transformation events. The transgenic plants showed normal growth and stable GFP expression over more than 2 years in the greenhouse. This is the first report of a large population of transgenic'Hamlin' sweet orange plants containing one or more target gene(s), using a protoplast-GFP transformation system. Polymerase chain reaction (PCR) revealed the presence of the Xa21 cDNA and the GFP genes in all single plasmid transformed plants, and in 35% of the co-transformed plants. Southern blot analysis showed the integration of the cDNA into one-to-five different sites per plant. Western blot analysis showed the accumulation of the rice XA21 protein in the transgenic sweet orange plants. This is the first time that a gene from rice has been stably integrated and expressed in sweet orange plants. Using the protoplast-GFP transformation system, it is possible to avoid the use of Agrobacterium, antibiotic resistance genes, and destructive assay systems.