Submitted to: Tropical Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2008
Publication Date: 12/10/2008
Citation: Suzuki, J.Y., Tripathi, S., Fermin, G., Hou, S., Saw, J., Ackerman, C., Schatz, M., Pitz, K., Yepes, M., Fitch, M., Manshardt, R., Slightom, J.L., Ferreira, S., Salzberg, S.L., Alam, M., Ming, R., Moore, P., Gonsalves, D. 2008. Characterization of insertion sites in Rainbow papaya, the first commercialized transgenic tree-fruit crop. Tropical Plant Biology. 1:293-309. Interpretive Summary: Transgenic Rainbow papaya is the most widely grown papaya in Hawaii, and is sold throughout the U.S. and Canada. However, Rainbow papaya cannot be marketed in Japan because that country has not yet deregulated the transgenic papaya. One of the criterium for getting regulatory approval in Japan is the thorough characterization of the transgenes that were inserted into the papaya during the transformation process. This work provides the thorough analysis required by Japan. It showed that three transgene inserts are present in the Rainbow papaya. The functional insert contains the genes that provides resistance as well as those that enhance our ability to transform papaya. The other inserts represented fragments of the tetA and nptII genes. From the biosafety standpoint, our analyses of the transgene insertion sites and flanking genomic DNA did not predict any allergenic or toxic proteins. This information has been used to file a petition to deregulate the transgenic Rainbow papaya in Japan. Deregulation of the transgenic papaya in Japan would provide a significant economic boost to the Hawaiian papaya industry.
Technical Abstract: Inserts and insert sites in transgenic, commercial papaya line 55-1 derivatives Rainbow and SunUp were characterized as part of a petition to Japan to allow import of fresh fruit of these cultivars from the U.S. and to provide data for a larger study aimed at understanding the global impact of DNA transformation on whole genome structure. Insert number and species were determined by Southern analysis using probes spanning the entire transformation plasmid and their sequences determined from corresponding clones or sequence reads from the whole-genome shotgun (WGS) sequence of SunUp papaya. All functional transgenes were found encoded in a single 9,789 basepair (bp) insert. Only two other insertion sites, one consisting of 290 bp nonfunctional sequence of the nptII gene and a 1,533 bp plasmid-derived fragment containing a nonfunctional 222 bp segment of the tetA gene were detected in Rainbow and SunUp. Detection of the same three inserts in samples representing transgenic generations five to eight (R5 to R8) suggests that the inserts are stable and co-inherited. Five out of the six genomic DNA segments flanking the three inserts were nuclear plastid sequences (nupts). From the biosafety standpoint, no changes to endogenous gene function based on sequence structure of the transformation plasmid DNA insertion sites could be determined and no allergenic or toxic proteins were predicted from analysis of the insertion site and flanking genomic DNA.