Progress 01/15/09 to 09/30/12
OUTPUTS: With the release of Rainbow and SunUp varieties, a high level of PRSV resistance was successfully used in Hawaii to control PRSV which nearly destroyed the Hawaiian Papaya Industry. The Rainbow and SunUp varieties have been widely deployed and currently nearly 90% of the papaya acreage is comprised of transgenic varieties. While the transgenic resistance was highly effective, it was not useful against divergent strains of PRSV from foreign areas. Thus the industry was at risk to the evolution of new strains in Hawaii or the introduction of exotic PRSV strains. The major goal of this project was to mitigate this threat by broadening the transgenic resistance using a segmented and a synthetic coat protein gene construct that would contain increased similarity to foreign or new strains of PRSV. Two major approaches were employed, namely the segment gene approach and the synthetic gene approach. The segmented gene approach proved to be the most useful and practical. With the segmented gene approach, 200 bp segments of the coat protein gene of PRSV from Taiwan, Thailand and Hawaii were incorporated into a single transgene construct that was used to produce new transgenic papaya lines. With the synthetic gene approach, a consensus sequence derived from published coat protein gene sequences was synthesized and use to create transgenic plants with 96% similarity to all published CP sequences of PRSV. The segmented gene approached worked wonderfully to confer resistance to strains of PRSV from Hawaii, Taiwan and Thailand. This resistance has been incorporated into both Sunrise and Kapoho papaya varieties, and has been crossed with Rainbow to produce a Super-Rainbow, the next generation transgenic papaya variety with broader transgenic resistance. We were not successful in using the synthetic gene approach to broaden PRSV transgenic resistance. Possible reasons for this failure might have been due to the relatively small number of transgenic lines that were produced and evaluated for resistance. Future work will address this deficiency. The other major accomplishment of this project was the successful deregulation of the transgenic Rainbow variety in Japan. Research to develop some of the necessary laboratory information was conducted in this laboratory and Dr. Gonsalves' laboratory in Hilo, that was ultimately used to support the formal deregulation and commercialization of Rainbow papaya for sale in Japan. First shipments of commercial Rainbow papaya were initiated in December, 2011. Extension Component. The overall extension component of this project focused on extending the research results at the Papaya Industry meetings. This occurred at least annually, and additionally once or twice a year depending on results. These secondary meetings occurred the HPIA board meetings, typically convened each month. In addition, to support the deregulation effort in Japan, previous research results were summarized and made available for the HPIA deregulation effort that they spearheaded. Important issues centered on the process of producing the transgenic papayas, and issues on food safety and potential environmental and genetic contamination via pollen drift. PARTICIPANTS: Dennis Gonsalves (Ph.D), Center Director, Pacific Basin Agricultural Research Center, Hilo, HI Savarni Tripathi (Ph.D), Assistant Researcher, Plant & Environmental Protection Sciences, CTAHR, Univ of Hawaii at Manoa, Honolulu, HI Karen Pitz (MS), Jr Researcher, Plant & Environmental Protection Sciences, CTAHR, Univ of Hawaii at Manoa, Honolulu, HI Richard Manshardt (Ph.D), Professor, Tropical Plant & Soil Sciences, CTAHR, Univ of Hawaii at Manoa, Honolulu, HI TARGET AUDIENCES: Members of the Hawaii Papaya Industry Association and other International Papaya Growers interested in managing PRSV or avoiding its' introduction; regulators and governmental agencies in Hawaii, the US and Japan having to make decisions about the use of biotechnology; groups and individuals interested in GMO work and interpretation of research in this area; colleagues and peers in institutions of higher learning in Hawaii, the US and the world. PROJECT MODIFICATIONS: Not relevant to this project.
We have confirmed the validity of using the segmented gene approach to broaden transgenic PRSV resistance in papaya. This resistance will be combined with the first transgenic resistance in Rainbow by backcrossing our new lines to Rainbow, thereby producing a Super-Rainbow. This line will have to be deregulated prior to its' commercial release. The other major impact of this project was to assist with the effort to deregulate Rainbow and SunUp (the original transgenic papaya lines deregulated and commercialized in Hawaii) for commercial sale in Japan. This was completed after nearly a 10 year long process with MAFF and MHLW agencies in Japan. First shipments of commercial Rainbow occurred in early December, 2011. We participated and supported the HPIA effort to deregulate transgenic papayas in Japan. Brochures and several video highlighting the processes and various issues on food safety and environmental concerns were addressed.
- Gonsalves, D., Gonsalves, C., Carr, J., Tripathi, S., Matsumoto, T., Suzuki, J., Ferreira, S., Pitz, K. 2012. Assaying for pollen drift from transgenic Rainbow to nontransgenic Kapoho papaya under commercial and experimental field conditions in Hawaii. Tropical Plant Biol. 5(2):153-160. DOI:10.1007/s12042-011-9090-5.
- Fermin, G., Keith, R.C., Suzuki, J.Y., Ferreira, S.A., Gaskill, D.A., Pitz, K.Y., Manshardt, R.M., Gonsalves, D., Tripathi, S. 2011. Allergenicity assessment of the papaya ringspot virus coat protein expressed in transgenic Rainbow papaya. Journal of Agricultural and Food Chemistry. 59(18):10006-10012. DOI: 10.1021/jf201194r.
- 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.
Progress 10/01/10 to 09/30/11
OUTPUTS: Using a synthetic gene to broaden Papaya Ringspot Virus (PRSV) resistance. We on the field evaluation of the synthetic gene construct, which uses a 96% consensus sequence of the coat protein (CP) gene from published PRSV sequences was continued. This year, we obtained a series of R1 generation lines which should be hemizygous and homozygous for the synthetic gene construct. Six of these lines were selected and are being propagated. Homozygous and hemizygous lines will be evaluated for PRSV resistance to determine if transgene dosage of the synthetic construct will confer a high level of PRSV resistance. This work is being conducted since our previous work indicated that in the hemizygous situation, the synthetic construct did not confer useful PRSV resistance. Incorporating segmented transgenic resistance into Papaya. A back cross program to incorporate the next or third backcross generation of the segmented CP transgene (segmented CP gene contruct with segments of PRSV from Thailand and Taiwan) for both Kapoho and Sunrise was completed. These backcross lines will be compared to the parental Kapoho and Sunrise to confirm horticultural similarity and will be used to develop the next generation of Rainbow which should be more broadly resistant to foreign strains of PRSV. These pyramided or super-lines will then be assessed for broader transgenic resistance. Presentations: 8/6/11: American Phytopathological Society Annual Meeting; Field Trip; Papaya and Banana Diseases and Development of Rainbow, the first commercialized transgenic fruit crop in the US. 8/15/11: Field Day; Keaau, HI; Practical tips for successful field control of papaya diseases. 9/22/11: Hawaii Seed Industry Tour. Intent of this visit was to formalize a relationship between Seed Industry personnel and CTAHR, focusing on issues around which we in CTAHR could be of assistance. This included, educational programs for students they would hire, disease and insect management issues, developing publications of varous types for use by the industry. 9/23/11: HPIA 48th Conference, Hilo, HI; History of funding papaya industry research in the College of Tropical Agriculture and Human Resources: Thoughts on alternative funding options. PARTICIPANTS: The work on synthetic and segmented transgenic approaches to broadening PRSV resistance was a collaborative effort with Dr. Dennis Gonsalves, Center Director, Pacific Basin Agricultural Research Center, Hilo, HI. Dr. Gonsalves was produced the initial transgenic lines, and S. Ferreira conducted the field and crossing work to produce next generation materials and to make the field assessments for PRSV resistance. Ms. Karen Pitz provided technical assistance and labor for the project. TARGET AUDIENCES: This work directly benefits Hawaii papaya growers, most of whom constantly battle the ravages of PRSV, which is still the most serious disease challenge to economic viability of papaya growing activities. Other fungal diseases, such as Black Spot and Powdery Mildew, are also a challenge for growers. In addition, growers in other papaya growing regions of the world benefit indirectly through the proof of concept that this project attempts to resolve to develop transgenic papaya varieties with wide or broad resistance to diverse strains of PRSV that exist around the world. This in turn benefits all papaya growers, over 90% of which continue to battle with PRSV. PROJECT MODIFICATIONS: Not relevant to this project.
While the synthetic gene approach to broadening transgenic PRSV resistance in papaya does not appear promising, we plan to evaluate homozygous lines before concluding that synthetic constructs are not useful for PRSV resistance. The segmented gene approach continues to look promising. We now have a third generation backcross lines of both Kapoho and Sunrise in the field which will be used to develop the next generation or super-Rainbow, which in effect pyramid both the original transgene and a segmented gene construct in orde to broaden Rainbow resistance in order to mitigate the threat from foreign or exotic PRSV virus strains. Thus, we are in a position to now commercialize a second generation or super-Rainbow with broader resistance to PRSV, thus mitigating the potential threat of exotic or foreign strains of PRSV.
- Tripathi, S., Suzuki, J. Y., Carr, J. B., McQuate, G. T., Ferreira, S. A., Manshardt, R. M., Pitz, K. Y., Wall, M. M., and Gonsalves, D. (2011). Nutritional composition of Rainbow papaya, the first commercialized transgenic fruit crop. Jour Food Composition & Analysis 24:140-147.
Progress 10/01/09 to 09/30/10
OUTPUTS: Broadening Transgenic Papaya Ringspot Virus (PRSV) Resistance in Papaya: The thrust of this project centers on a multiyear ongoing effort to broaden the transgenic or genetically engineered Papaya ringspot virus (PRSV) resistance in papaya, with the ultimate goal of protecting Hawaii papaya growers to the threat of new or foreign strains of the virus. Two approaches are being evaluated: (1) Synthetic transgene, and (2) Segmented transgene approach. Synthetic Gene Approach. This past year, we focused on completing the initial field evaluation of the synthetic gene approach (which uses a 96% consensus sequence of the coat protein (CP) gene for the published sequences of PRSV isolates from around the world). Previously we reported preliminary results of field trials for 6 transgenic-synthetic gene lines, and this year we confirmed and concluded that the synthetic gene lines are not resistant to PRSV. These R0 lines (hemizygous for the transgene) were selfed and are now being selected for transgene homozygosity for retesting to determine if transgene dosage, or homozygosity, affects transgenic resistance conferred by the synthetic CP gene. Putative homozygous lines were identified and are being propagated for evaluation broad PRSV resistance next year. Segmented Gene Approach. A back cross program to incorporate a second-generation CP transgene (segmented CP gene construct with segments of PRSV from Thailand and Taiwan) in order to broaden PRSV resistance was continued. A second round of backcrossing was completed this year. When seed is available in about 6 months, lines will be evaluated for similarity to Kapoho and Sunrise (the intended horticultural phenotypes) which will be used to pyramid segmented resistance with the original CP transgene from an Hawaiian PRSV isolate. These pyramided lines, referred to as super-lines, will be assessed for broader transgenic resistance. PARTICIPANTS: Dr. Dennis Gonsalves was a co-PI/PD; research and extension goals, objectives, and experimental designs were reviewed by the Co-PI/PD. Dr. Gonsalves is also the Center Director of PBARC (Pacific Basin Agricultural Research Center) in Hilo, HI. Dr. Savarni Tripathi and Dr. John Suzuki are post-doctoral researchers responsible for the biotechnology aspects of the project. Ms. Karen Pitz, Jr. Researcher is responsible for installing laboratory and field trials, obtaining and summarizing data collected, and participated in project discussions with co-PI/PDs and post-doctorates regarding the work to be completed. TARGET AUDIENCES: In addition, growers in other papaya growing regions of the world benefit indirectly through the proof-of-concept this project attempts to resolve regarding developing transgenic papaya varieties with wide or broad resistance to diverse strains of PRSV that exist around the world. If successful, risk of PRSV strains evolving to overcome resistance would be greatly reduced. This work directly benefits Hawaii papaya growers, most of whom constantly battle the ravages of PRSV, which is still the most serious disease challenge to the economic viability of papaya growing activities. In addition, growers in other papaya growing regions of the world benefit indirectly through the proof-of-concept this project attempts to resolve regarding developing transgenic papaya varieties with wide or broad resistance to diverse strains of PRSV that exist around the world. If successful, risk of PRSV strains evolving to overcome resistance would be greatly reduced. In turn papaya growers worldwide would ultimately benefit since PRSV is important in all growing regions. Following activities were conducted: 04/23/2010: Papaya Disease Control Field Day, Hilo, HI. Attended by 27 growers and potential growers. 09/24/2010: Hawaii Papaya Industry Association 47th Conference. Presentation on Practical Control of Papaya Diseases in the Field. Attended by 63 growers and industry associated personnel. PROJECT MODIFICATIONS: We propose to eliminate the objective on Citrus Tristeza Virus work and replace that objective with one on screening Protea and Leucospermum hybrid varieties for resistance to important protea diseases in Hawaii. This change is proposed because of limited success with developing CTV transgenic lines of citrus, while at the same time, recognizing the need to screen several hundred protea cultivars developed in the last 10 years which lack any evaluation for resistance to the 3 most important disease of protea in Hawaii: Elsinoe or Scab disease, Drechslera dieback, and Root rot caused by Phytophthora nicotianae and P. cinnamomi.
While the synthetic gene approach to broadening transgenic PRSV resistance in papaya does not appear to be useful, the segmented gene approach continues to look promising. We now have second generation backcross lines in the field and will have seed populations from which lines homozygous for the segmented gene construct can be selected in both Kapoho and Sunrise backgrounds. These lines should be important in developing a new super-Rainbow papaya variety with broader PRSV resistance.
- Suzuki, J.Y., S. Tripathi, J.B. Carr, S.A. Ferreira, D. Gonsalves. (2009). Transgene-derived small RNA in papaya ringspot virus-resistant, Hawaiian Rainbow papaya. Plant Biology 2009 by American Society of Plant Biologists/ Phycological Society of America. July 18 to 22, 2009. Honolulu, USA.
- Tripathi, Savarni, Jon Suzuki, Stephen A. Ferreira, Richard Manshardt, Karen Pitz, James Carr, Marisa Wall and Dennis Gonsalves (2009). Food safety assessment of first commercialized genetic engineered fruit crop: Rainbow papaya for protection from Papaya ringspot virus. Journal of Food Composition and Analysis (submitted).
Progress 10/01/08 to 09/30/09
OUTPUTS: Japan Deregulation: After more than an 8 year effort, involving experimental work and literature reviews of numerous aspects of the efficacy and food-safety assessment of transgenic papayas, a final application package to the Ministry of Health, Labor and Welfare (MHLW) was accepted for final review. Drs. D. Gonsalves and R. Manshardt were closely involved with this effort. Broadening Transgenic PRSV Resistance in Papaya: One of major goals of this project focuses on developing papayas with broader transgenic resistance to Papaya rinsgpot virus (PRSV) utilizing a segmented gene [coat protein (CP) gene segments from diverse strains of PRSV] and a synthetic gene [consensus CP sequences based on published sequences of diverse PRSV strains] approach. A field trial to assess 32 R2 and R3 transgenic lines of segmented-transgene lines was completed. In addition, 6 synthetic-gene R0 lines were evaluated in both greenhouse and field trials for resistance. None of these lines showed any degree of resistance to PRSV. They were selfed to produce a segregating R1 population in which about 25% of the individuals should be homozygous for the synthetic transgene, and those of which may show resistance to PRSV. These experiments are in the process of being installed and initial evaluations will made in the next year. Finally, 12 transgenic lines (R1 and R2 selections) with delayed fruit ripening transgenes were also evaluated in the field. All of these lines displayed at least some susceptibility to PRSV, and selected lines confirmed for the presence of the delayed ripening transgene will be assessed for ripening characteristics. Biotechnology Education Program: Initial plans were made to conduct an educational program on transgenic technology targeting state legislators and key agricultural leaders in Hawaii were made. PARTICIPANTS: Dr. Dennis Gonsalves was a co-PI/PD; research goals, objectives, and experimental designs were reviewed by the co-PI/PD. Dr. Gonsalves is also the Center Director of PBARC (Pacific Basin Agricultural Research Center) in Hilo, HI. Ms. Karen Pitz, Jr. Researcher, was responsible for installing trials, obtaining and summarizing data collected, and participated in project discussions with co-PI/PD's regarding the work to be completed. TARGET AUDIENCES: Target audiences: This work most directly benefits papaya growers in Hawaii and in other papaya growing regions of the world, followed by food production scientists and technologists (eg. plant pathologists) and extension personnel with an interest in the use of transgenic resistance to control or manage important virus diseases of papaya and other crops, as our transgenic approach can be widely adapted to numerous viral diseases. Because we also are attempting to broaden this approach to govern traits in addition to virus resistance, other growers, scientists, and extension professionals concerned with modifying other traits such as fruit ripening characteristics are also likely to be interested in our research approaches and findings. Ultimately, policy makers who may decide on whether or not plant transgenic technology is ultimately used will also find this working useful. Of special interest with the deregulation of transgenic papayas for sale in Japan, papayas represent the first whole food (non-processed food product) deregulated in Japan. Because of Japan's requirement for GM labeling, their markets will in effect function as a laboratory for assessing the public acceptance and use of transgenic food product as compared to non-transgenic alternatives from both Hawaii and the Philippines. This market data should provide the most objective data about the acceptability and desirability of transgenic vs non-transgenic food alternatives. PROJECT MODIFICATIONS: Because of funding limitations, the scope of this work may be narrowed to focus on papaya applications only. If additional funding comes available, work on transgenic citrus will be included.
Japan Deregulation: At the end of May 2009, we were notified by MHLW in Japan approved transgenic papayas, Rainbow and SunUp, for sale in Japan, pending the successful completion of labeling protocol requirements and a marketing plan. Co-PI/PD's of this project will support and provide all of the technical information required for labeling and marketing efforts. Broadening Transgenic PRSV Resistance in Papaya: Two segmented gene lines were identified for further evaluation horticulturally (to demonstrate that the transformation process did not affect horticultural performance) and for back-crossing to Kapoho, in order to develop a second generation, "Super Rainbow" line that would combine the first generation transgene, 55-1, and the new segmented CP gene construct by hybridizing Kapoho (with segmented resistance) and SunUp (original 55-1 CP construct) thus pyramiding 2 different transgene constructs in a single papaya line. This should give rise to more effective resistance against most if not all strains of PRSV.
- Gonsalves, Dennis, Stephen A. Ferreira, Jon Y. Suzuki and Savarni Tripathi (2008). PAPAYA. A compendium of Transgenic Crop plants (eds. C. Kole and Timothy C. Hall). Blackwell Publishing, Oxford UK.
- Gonsalves, Jon Y. Suzuki, Savarni Tripathi and Stephen A. Ferreira (2008). Papaya ringspot virus (Potyviridae). Encyclopedia of Virology (B.W.J. Mahy and M.H.V. van Regenmortel, Editors), pp. 1-8. Elsevier, Oxford UK.
- Tripathi, S, J Suzuki, SA Ferreira, and D Gonsalves. 2008. Pathogen profile of Papaya ringspot virus: characteristics, pathogenicity, sequence variability and control. Molecular Plant Pathology 9 (3) 269-280.
- Suzuki, Jon Y., Savarni Tripathi, Gustavo A. Fermin, Fuh-Jyh Jan, Shaobin Hou, Jimmy H. Saw, Christine M. Ackerman, Qingyi Yu, Michael C. Schatz, Karen Y. Pitz, Marcela Yepes, Maureen M. M. Fitch, Richard M. Manshardt, Jerry L. Slightom, Stephen A. Ferreira, Steven L. Salzberg, Maqsudul Alam, Ray Ming, Paul H. Moore and Dennis Gonsalves (2008). Characterization of insertion sites in Rainbow papaya, the first commercialized transgenic fruit crop. Tropical Plant Biology 1:293-309.