Genetic Transformation of Papaya (Carica Papaya, L.) Cultivar Kapoho by Particle Bombardment

Abstract

Papaya transformation systems were developed by Fitch et al. (1991) at the University of Hawaii, and transgenic 'Sunset' papayas with papaya ringspot virus (PRV) coat protein (cp) gene showed complete resistance to papaya ringspot virus (PRV) in the field tests (Manshardt et al, 1994). In our studies, we transformed 'Kapoho' papaya, the major crop on the Big Island, Hawaii, based on Fitch’s (1991) papaya transformation systems, and obtained transgenic 'Kapoho' papaya plants. The coat protein (cp) gene of PRV, along with a kanamycin selective marker gene (neomycin phosphotransferase, NPTII) and a Pglucuronidase (GUS) reporter gene, were constructed into the same plasmid vector by our collaborators at Cornell University and transformed into papaya tissue by particle bombardment. Transgenic 'Kapoho' papaya plants were obtained following somatic embryogenesis from hypocotyl callus on kanamycin selective medium and showed GUS positive expression. Immature zygotic embryos were excised and bombarded with gold particles. Following different treatments of indole-3-butyric acid (IBA), chimeric hypocotyls were harvested on germination medium 20 days after bombardment. Somatic embryogenesis from sections of chimeric transgenic hypocotyls occurred on induction medium and the transgenic embryos were cultured on selective induction medium or maturation medium with different concentrations of kanamycin for eight months. Then, the embryos were regenerated on germination medium without kanamycin. GUS was assayed in all experimental steps, and different GUS positive results were observed at different developmental stages. ELISA assays of coat protein and NPTII in chimeric transgenic hypocotyls showed positive expression and a high efficiency of transformation.