Efforts to deregulate Rainbow papaya in Japan: Molecular Characterization of Transgene and Vector Inserts

Authors: Suzuki, Jon; TRIPATHI, SAVARNI - UNIV OF HAWAII; FERMIN, GUSTAVO - UNIV DE LOS ANDES; JAN, FUH-JYH - NAT'L CHUNG HSING UNIV; HOU, SHAOBIN - UNIV OF HAWAII; SAW, JIMMY - UNIV OF HAWAII; ACKERMAN, CHRISTINE - HI AGRI RESEARCH CTR; YU, QINGYI - HI AGRI RESEARCH CTR; SCHATZ, MICHAEL - UNIV OF MARYLAND; PITZ, KAREN - UNIV OF HAWAII; YEPES, MARCELA - CORNELL UNIV; FITCH, MAUREEN - HI AGRI RESEARCH CTR; MANSHARDT, RICHARD - UNIV OF HAWAII; SLIGHTOM, JERRY - AUREOGEN BIOSCIENCES; FERREIRA, STEPHEN - UNIV OF HAWAII; SALZBERG, STEVEN - UNIV OF MARYLAND; ALAM, MAQSUDUL - UNIV OF HAWAII; MING, RAY - HI AGRI RESEARCH CTR; Moore, Paul; Gonsalves, Dennis

Submitted to: Acta Horticulturae
Publication Type: Proceedings
Publication Acceptance Date: 1/20/2009
Publication Date: 1/1/2010
Citation: Suzuki, J.Y., Tripathi, S., Fermin, G.A., Jan, F., Hou, S., Saw, J.H., Ackerman, C.M., Yu, Q., Schatz, M.C., Pitz, K.Y., Yepes, M., Fitch, M.M., Manshardt, R.M., Slightom, J.L., Ferreira, S.A., Salzberg, S.L., Alam, M., Ming, R., Moore, P.H., Gonsalves, D. 2010. Efforts to deregulate Rainbow papaya in Japan: Molecular Characterization of Transgene and Vector Inserts. Acta Horticulturae. 851:235-240.

Interpretive Summary: Transgenic papaya, primarily the Rainbow cultivar, is grown in Hawaii and exported to the mainland U.S. and Canada. Japan is a potential market for the transgenic papaya, but the transgenic papaya must first be deregulated in Japan before this market avenue will be available to Hawaii papaya growers. One of the criteria 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 summarizes results on the characterization of the transgene inserts that are in the genome of the transgenic papaya. Three inserts were detected: one is the functional gene cassette that imparts resistance to papaya ringspot virus; the second is a fragment of the tetA transgene; and the third is a fragment of the nptII transgene. This information is being used as part of the petition to deregulate the transgenic papaya in Japan. Deregulation of the transgenic papaya in Japan would provide a significant economic boost to the Hawaiian papaya industry.

Technical Abstract: Transformation plasmid-derived insert number and insert site sequence in 55-1 line papaya derivatives Rainbow and SunUp was determined as part of a larger petition to allow its import into Japan (Suzuki, et al., 2007, 2008). Three insertions were detected by Southern analysis and their corresponding sequences determined by clones (Fermín, 2002) or via the whole genome shotgun (WGS) sequence database of SunUp (Ming, et al., 2008). All functional transgenes including the coat protein (CP) gene that confers resistance to PRSV, and visible and selectable marker genes, uidA encoding glucuronidase (GUS) and nptII encoding neomycin phosphotransferase II were found in a single 9,789 basepair (bp) insert referred to as the functional transgene. The two other insertion sites consisted of a 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. Detection of the same three inserts in Rainbow and in samples of SunUp representing transgenic generations five to eight (R5 to R8) suggests that the inserts are stable. Five out of the six genomic DNA segments flanking the three inserts were nuclear plastid sequences (nupts). 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. These results should support a positive review of the petition to allow the import and consumption of Rainbow and its derivatives in Japan, which is currently in its final stages. Export of Rainbow papaya to Japan will greatly benefit the local papaya industry in Hawaii and will provide a case for testing consumer acceptance of genetically engineered fresh products in Japan.