The Caricaceae family consists of six genera, including Vasconcellea, which contains 21 of the 35 Caricaceae species, and Carica papaya L., which is the most economically important species attributable largely to its cultivation in the tropics for fruit production (Van Droogenbroeck et al., 2004). Breeding and selection of C. papaya for cultivation has resulted in the development of numerous C. papaya varieties and this comprises the majority of accessions at the USDA, ARS, PBARC, Tropical Plant Genetic Resources and Disease Research (TPGRDR) unit in Hilo, HI. The TPGRDR is part of the National Plant Germplasm System and the designated location for Caricaceae germplasm.
Papaya ringspot virus (PRSV) is a devastating disease that has a detrimental impact in Hawaii on both commercial production and germplasm conservation. Although tolerance but not genetic resistance to PRSV has been reported in the Carica germplasm, PRSV-resistant plants have been achieved by genetic engineering with the commercial release of transgenic papaya cultivars SunUp and Rainbow. In 2009, the Hawaii papaya industry is based on ‘Rainbow’ (77%), ‘Kapoho’ (9%), ‘Sunrise’ (9%), and other (5%) (USDA, National Agricultural Statistics Service and State of Hawaii Department of Agriculture Agricultural Development Division, 2009). The ‘SunUp’ variety is the homozygous version of the original transgenic line 55-1 and ‘Rainbow’ is the F1 hybrid cross between ‘SunUp’ and ‘Kapoho’ (reviewed in Gonsalves et al., 2006). The plasmid used in the generation of the transgenic line 55-1 consisted of the Agrobacterium transformation vector pGA482GG/cpPRV4, which contained 70 bases of the 5′ Cucumber mosaic virus untranslated region fused to the 16 amino acid sequence of the N-terminal end of the Cauliflower mosaic virus coat protein that is translationally fused to the coat protein of PRSV HA 5-1. Expression is controlled by the 35S promoter and selection was based on the NPTII (neomycinphosphotransferase for kanamycin resistance) and uidA [β-glucoronidase (GUS)] reporter genes NCBI accession FJ467933 (Fitch et al., 1992; Ling et al., 1991).
The presence of transgenic papaya plants in Hawaii presents a potential increased risk of contamination of nontransgenic seed sources by the transgenic material. Although there is an ongoing effort to obtain regulatory clearance in Japan for the importation of the transgenic 55-1 papaya, genetically engineered papayas cannot be shipped into Japan or the European Union (Ohmori et al., 2008). Contamination of transgenic material or any other unintentional genetic outcrossing of the germplasm material is of great concern to the USDA TPGRDR because we distribute Caricaceae germplasm to locations throughout the world. Therefore, to optimize our production, we developed a polymerase chain reaction (PCR) protocol to test our material for the presence of the transgene and to determine the sex of the seedlings planted into the field to reduce the labor required to remove the undesired female plants after flowering.
Standard operating procedures at the TPGRDR unit for Caricaceae seed preservation include regeneration of seeds every 4 to 6 years depending on the storage viability of each accession. Papaya seedlings are germinated in the same area that they are planted to reduce the chance of introducing PRSV and other diseases into a new growing area. An average of three to five seedlings (depending on the cultivar) are planted per planting hole to ensure the presence of at least one hermaphrodite plant for gynodioecious accessions or one male or one female plant for dioecious accessions. Once flowering occurs, the remaining female or male plants are removed. Because the hermaphrodite papayas are in-bred, seed production is achieved by bagging flowers in glassine envelopes before anthesis to prevent outcrossing. For highly heterozygous, dioecious papaya lines, it is necessary to gather pollen from all male trees within the same genetic line to pollinate all the female plants to ensure preservation of the genetic integrity of the accession. During the regeneration cycle, observations on plant stature and growth characteristics are monitored to ensure the consistent phenotypic traits for the genetic line. In addition, papaya fruit morphological data are obtained, submitted, and stored in the national plant database system known as the Germplasm Resources Information Network (GRIN). Morphological data on papaya fruit can be obtained at http://www.ars-grin.gov/cgi-bin/npgs/html/desclist.pl?126.
Because genetically engineered plants are indistinguishable from the original genetic source with the exception of the transgene, it is almost impossible to distinguish the transgenic and nontransgenic material based only on morphological characteristics. Thus, various researchers have used PCR techniques to detect the presence of the transgene insert in 55-1 papaya lines in seeds, seedlings, and fruits (Ohmori et al., 2008; Wall et al., 2004). Identification of the transgene inserts from seeds was based on a homogenous population and not a mixture of seeds from transgenic and nontransgenic sources (Wall et al., 2004). Numerous methods have been developed to screen seeds such as maize, canola, soybeans, safflower, and rice for the adventitious presence of transgenes through various PCR techniques for selectable makers, promoters, or genes of interest (Christianson et al., 2008; Demeke et al., 2006; Freese et al., 2007). Statistical methods (i.e., SeedCalc) have been developed to assist in designing and implementing seed testing procedures to detect the adventitious presence of transgenes and analyze risk considerations for seed producers and seed consumers (Remund et al., 2001). We describe a procedure that has a higher than normal producer risk resulting from the potential of large numbers of seeds not meeting the stringent criteria. We believe this is necessary for our role in germplasm conservation and distribution. However, others can still use the same protocols outlined in this article by lowering the stringency of the rejection criteria. To the best of our knowledge, this is the first publication on the detection of the adventitious presence of transgenes in large-scale nonhomogeneous papaya seeds.
Christianson, J. , McPherson, M. , Topinka, D. , Hall, L. & Good, A.G. 2008 Detecting and quantifying the adventitious presence of transgenic seeds in safflower, Carthamus tinctorius L J. Agr. Food Chem. 56 5506 5513
Demeke, T. , Perry, D.J. & Scowcroft, W.R. 2006 Adventitious presence of GMOs: Scientific overview for Canadian grains Can. J. Plant Sci. 86 1 23
Deputy, J.C. , Ming, R. , Ma, H. , Liu, Z. , Fitch, M.M.M. , Wang, M. , Manshardt, R. & Stiles, J.I. 2002 Molecular markers for sex determination in papaya (Carica papaya L.) Theor. Appl. Genet. 106 107 111
Fitch, M.M.M. , Manshardt, R.M. , Gonsalves, D. , Slightom, J.L. & Sanford, J.C. 1992 Virus resistant papaya plants derived from tissues bombarded with the coat protein gene of papaya ringspot virus BioTechnology 10 1466 1472
Freese, L. , Scholdberg, T.A. , Burton, D.D. , Norden, T.D. , Shokere, L.A. & Jenkins, G.R. 2007 Evaluating homogeneity of LL601 rice in commercial lots using quantitative real-time PCR J. Agr. Food Chem. 55 6060 6066
Gonsalves, D. , Vegas, A. , Prasartsee, V. , Drew, R. , Suzuki, J.Y. & Tripathi, S. 2006 Developing papaya to control papaya ringspot virus by transgenic resistance, intergeneric hybridization, and tolerance breeding Plant Breed. Rev. 26 35 78
Ling, K. , Namba, S. , Gonsalves, C. , Slightom, J.L. & Gonsalves, D. 1991 Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene BioTechnology 9 752 758
Ohmori, K. , Tsuchiya, J. , Watanabe, T. , Akiyama, H. , Maitani, T. , Yamada, T. , Hirayama, K. & Satoh, S. 2008 A DNA extraction method using silica-base resin type kit for the detection of genetically modified papaya J. Food Hyg. Soc. Jpn. 49 63 69
Remund, K.M. , Dixon, D.A. , Wright, D.L. & Holden, L.R. 2001 Statistical considerations in seed purity testing for transgenic traits Seed Sci. Res. 11 101 119
USDA, ARS, National Genetic Resources Program Germplasm Resources Information Network (GRIN) [online database] National Germplasm Resources Laboratory Beltsville, MD 16 Sept. 2008 <http://www.ars-grin.gov/cgi-bin/npgs/html/desclist.pl?126>.
USDA, National Agricultural Statistics Service and State of Hawaii Department of Agricutlure Agricultural Development Division Hawaii papayas 27 Oct. 2009 <http://www.nass.usda.gov/Statistics_by_State/Hawaii/Publications/Fruits_and_Nuts/papaya.pdf>.
Van Droogenbroeck, B. , Kybdt, T. , Maertens, I. , Romeijn-Peeters, E. , Scheldman, X. , Romero-Motochi, J.P. , Van Damme, P. , Goetghebeur, P. & Gheysen, G. 2004 Phylogenic analysis of the highland (Vasconcellea) and allied genera (Caricaceae) using PCR-RFLP Theor. Appl. Genet. 108 1473 1486
- Search Google Scholar
- Export Citation
Van Droogenbroeck, B. Kybdt, T. Maertens, I. Romeijn-Peeters, E. Scheldman, X. Romero-Motochi, J.P. Van Damme, P. Goetghebeur, P. Gheysen, G. 2004 Phylogenic analysis of the highland (Theor. Appl. Genet. Vasconcellea) and allied genera (Caricaceae) using PCR-RFLP 108 1473 1486
Wall, E.M. , Lawrence, T.S. , Green, M.J. & Rott, M.E. 2004 Detection and identification of transgenic virus resistant papaya and squash by multiplex PCR Eur. Food Res. Technol. 219 90 96