GENETIC-TRANSFORMATION OF CITRUS PROTOPLASTS AND REGENERATION OF TRANSGENIC PLANTS

Citrus protoplast-derived embryogenic calli can readily be induced to differentiate embryos which further develop into mature fruit bearing trees. We successfully introduced plasmid pCAP212 DNA, harboring the coding sequences of neomycin phosphotransferase (nptII) and chloramphenicol acetyltransferase (cat) genes into Citrus protoplasts via polyethylene glycol (PEG) treatment resulting in transgenic plants. Transient expression of chloramphenicol acetyltransferase (CAT) activity was detected 3 days after the direct transformation was performed. Microcolonies (0.5 mm in diameter) were selected on agar medium containing paromomycin sulfate (PAR). Twenty one resistant clones, presumably containing the nptII gene, were isolated. To verify the transgenic nature of the isolated clones either neomycin phosphotransferase (nptII) activity was measured or Southern hybridization was performed. Altogether 9 positive stably transformed embryogenic clones were obtained; two of these were regenerated into transgenic rooted plants. © 1990.