Transformation of an Australian variety of Carica papaya using microprojectile bombardment

Abstract

We have developed a method for the stable transformation and regeneration of a dioecious Australian cultivar of Carica papaya (papaw or papaya) by microprojectile bombardment. This method was developed after investigation of both zygotic and somatic embryos as target tissue and optimisation of a number of parameters using transient expression of the uidA reporter gene. The tissue culture regime prior to bombardment was critical in optimisation of transformation. Factors such as age of embryos and various treatments prior to bombardment, increased transient expression by up to 22- fold. Highest uidA transient expression results were obtained when somatic embryos 3 weeks since last subculture on solid medium were given a 3 day treatment in liquid medium, and a 2 h osmotic treatment pre- and post- bombardment. Stably transformed plants were obtained 6 months after bombardment using this system. Transformation efficiency was high with two experiments yielding 45% and 37.5% of bombarded plates regenerating plantlets on media containing kanamycin. The presence of both the uidA and aphA genes, (selectable marker) which code for the enzymes β-glucuronidase (GUS), and neomycin phosphotransferaseII (NPTII) respectively, was confirmed in regenerated plantlets by Southern hybridisation.