A protocol for efficient transformation and regeneration of Carica papaya L.

A reproducible and effective biolistic method for transforming papaya (Cari ca papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protoc ol included: 1) spreading of young somatic embryo tissue that arose directl y from excised immature zygotic embryos, followed by another spreading of t he actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamyci n-resistant clusters were first isolated from induction media containing ka namycin; 3) transfer of embryos with finger-like extensions to maturation m edium; and 4) transferring explants from germination to the root developmen t medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 tr ansgenic papaya lines expressing the nontranslatable coat protein gene of p apaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. Th is also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated t his procedure in our laboratory by visiting researchers who did four indepe ndent projects to transform seven papaya cultivars with coat protein gene c onstructs of PRSV strains from four different countries. The method is desc ribed in detail and should be useful for the routine transformation and reg eneration of papaya.