Development of Genetic Transformation Systems for Papaya

We have developed transformation systems for papaya {Carica papaya L.) with the intention of genetically engineering virus resistance into the crop. Papayas in Hawaii and throughout the world are susceptible to papaya ringspot virus (PRV), and resistance is nearly nonexistent in advanced germplasm. Transformation of plants with viral coat protein genes has been shown to confer virus resistance in other crops. The coat protein gene of PRV, along with a kanamycin selection gene (kan) and a B-glucuronidase reporter gene (gush), were targeted into papaya. Papaya transformation has been achieved following the development of two methods for high frequency somatic embryogenesis and the subsequent delivery of genes into somatic embryos via the particle gun and Agrobacterium tumefaciens. Embryogenesis occurred after culturing meristematic tissues on Murashige and Skoog media containing 2.3 to 113.1 /uM 2,4-dichlorophenoxyacetic acid. The meristematic tissues consisted of immature zygotic embryos explanted about 100 days post-anthesis and hypocotyl sections from seedlings about ten days old. Embryogenic cultures regenerated normal-looking plants on media devoid of growth regulators. Evidence is presented for the insertion and/or expression of a chimeric gene for the coat protein of papaya ringspot virus (cpPRV), for kanamycin resistance, and for 6-glucuronidase (GUS). There was 100% correspondence between selective growth of embryos, enzyme assays, and polymerase chain reaction (PCR) amplification of kan sequences. Expression of GUS was detected in about a third of the particle-bombarded samples, in contrast, all of the A. tumefaciens-transformed isolates expressed the reporter gene. PCR amplification of cpPRV sequences and Southern hybridization of genomic DNA to a cpPRV probe were observed in nearly all GUS-positive samples tested to date. A PCRamplified sequence for kan from plants transformed with A. tumefaciens, was larger than the sequence amplified from the plasmid control and from plants transformed with the particle gun. The highest yield of transgenic isolates was 1.38% of the bombarded zygotic embryos. About 70% (23) of the isolates from zygotic embryos regenerated normal-looking plants while only 20% of the isolates from embryogenic calli produced plants