Genetic Transformation of Commercially Important Mature Citrus Scions

A Mature Citrus Facility (MCF) was established at the University of Florida's Citrus Research and Education Center (CREC) to produce transgenic citrus (Citrus spp.) tolerant to canker and greening bacterial diseases. Plants produced from mature tissue should flower and fruit early. Budsticks of 'Hamlin', 'Valencia', and 'Pineapple' sweet orange [C. sinensis (L.) Osbeck] and 'Ray Ruby' grapefruit (C. paradisi Macfad.) were obtained from the Florida Division of Plant Industry (DPI) and introduced into the facility through shoot-tip grafting (STG). These plants were tested for bacteria, phloem-limited Huanglongbing (HLB), and indexed for viruses to ensure freedom from disease. Mature buds were grafted onto immature rootstock and the first two flushes of mature scion used in transformation feasibility studies with Agrobacterium strain EHA105 harboring pCAM-BIA2301 and strain EHA101 harboring pTLAB21, which carry the beta-glucuronidase (GUS) and green fluorescent protein (GFP) reporters, respectively. beta-glucuronidase assays or GFP fluorescence was used to identify transgenic shoots, which developed through organogenesis from stem explants. Transgenic shoots were micrografted onto immature rootstock, and secondarily micrografted onto 4- to 5-mo-old rootstock. NPTII enzyme expression in older trees was confirmed using the immunostrip and enzyme-linked immunosorbent assays (ELISA). Southern blots of a representative group of transgenics showed that most were low copy number with 1 to 3 copies of the T-DNA integrated into the genome. There were a large number of escapes on kanamycin, and many transgenic shoots were lost in micrografting. Several transgenic trees flowered 18 to 19 mo after cocultivation with Agrobacterium. Transformation of mature citrus with disease resistance genes would be a feasible way to produce citrus tolerant to canker and greening.