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NON TECHNICAL SUMMARY:
This project uses molecular and conventional breeding methods to develop broader transgenic PRSV-resistance in papaya and to confirm the transgenic approach for CTV-resistance in citrus. Both of these viruses seriously constrain production in papaya and citrus. Broader resistance to PRSV removes the threat of foreign or new strains that would threaten survival. Broader PRSV resistance would also be useful in other papaya growing areas of the world, and the use of this technology could benefit growers beyond Hawaii. With citrus, CTV resistance would provide an opportunity for Hawaii to resurrect commercial citrus production in Hawaii. Further, because of the diverse nature of the CTV population in Hawaii (probably due to the long association of CTV and its aphid vector), CTAHR could become the world center for CTV screening for breeders around the world for both transgenic and conventional citrus lines. For this to happen, proof of concept is needed, and is one of the basic goals of this project. This project offers the opportunity to integrate the basic research with extension activities supporting the transfer and practical utilization of the research. In the case of papaya, completing the deregulation process for Rainbow and SunUp in Japan is critical to assuring Hawaii's most important market niche in Japan. In addition, given the heightened level of activism opposing GM technology, using the success of transgenic papaya as a spring board for discussions with targeted clients should be very useful in assisting with acceptance of GM. Finally, CTAHR has not released a new papaya variety since 1977. Part of the proposed research and extension efforts will center on evaluating and releasing a new solo papaya with a distinct taste and color profile from the two important export varieties, Kapoho and Sunrise.
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| APPROACH:
Research: 1. To develop broadly resistant transgenic papayas, Kapoho (Kp) and Sunrise (Sr) will be transformed with the CP gene segments of PRSV from Thailand, Taiwan, and Keaau, HI. Transgenic plants will be evaluated for resistance to the Hawaii strain of PRSV both in the greenhouse and field. Selections will be made for horticultural characteristics and for broad virus resistance. Note that all field work must be conducted with necessary biotech release permits from APHIS/BRS. 2. Papaya lines of Kp and Sr transformed with both PRSV CP gene and with 3 isozymes of beta-galactosidase genes originally obtained from papaya (this should result in resistant transgenic lines with down-regulated fruit softening gene expression) to develop new varieties with extended shelf-life. Deregulate these enhanced transgenic papaya lines. 3. In a field trial, evaluate transgenic citrus lines for resistance to CTV. Characterize selected lines molecularly to confirm presence of transgenes and to determine gene expression levels in selected lines. Mexican lime transformants from Spain and Hawaii, and sweet orange transformants from California will be evaluated for CTV resistance.
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CRIS NUMBER: 0217250
SUBFILE: CRIS
PROJECT NUMBER: HAW00975-H
SPONSOR AGENCY: NIFA
PROJECT TYPE: HATCH
PROJECT STATUS: EXTENDED
MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jan 15, 2009
TERMINATION DATE: Sep 30, 2012
GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)
CLASSIFICATION
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| 201 | 0999 | 1101 | 2.2 | 10% |
| 201 | 1030 | 1101 | 2.2 | 40% |
| 212 | 0999 | 1101 | 4.2 | 10% |
| 212 | 1030 | 1101 | 4.2 | 40% |
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CLASSIFICATION HEADINGS
KA201 - Plant Genome, Genetics, and Genetic Mechanisms
KA212 - Pathogens and Nematodes Affecting Plants
S1030 - Papaya
S0999 - Citrus, general/other
F1101 - Virology
G2.2 - Increase Efficiency of Production and Marketing Systems
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks
RESEARCH EFFORT CATEGORIES
| BASIC |
30% |
| APPLIED |
40% |
| DEVELOPMENTAL |
30% |
KEYWORDS: papaya~transgenic~prsv~resistance~virus-resistance~citrus~ctv~extension~education
PROGRESS: Oct 1, 2009 TO Sep 30, 2010
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.
IMPACT: 2009-10-01 TO 2010-09-30
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.
PUBLICATION INFORMATION: 2009-10-01 TO 2010-09-30
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.
PROJECT CONTACT INFORMATION
| NAME: |
Ferreira, S. A. |
| PHONE: |
808-956-7854 |
| FAX: |
808-956-2832 |
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