Progress 09/27/03 to 05/15/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This research supports NP 301, Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, primarily component II: Genomic Characterization and Genetic Improvement by characterizing the genetic diversity of selected tropical crop plant germplasm; and contributes to NP 302, Plant Biological and Molecular Processes, primarily component I: Analysis and Modification of Plant Genomes by identifying DNA molecular markers linked to loci encoding important phenotypic traits, isolating and characterizing agronomically important genes, developing technologies for improving crop germplasm by marker- assisted selection and through genetic transformation, improving transgene expression by developing methods that minimize position effects, transgene copy number and integrity, and
creating tissue- specific or other desired patterns of expression in engineered tropical crop plants by developing new gene promoters. Although more than 30,000 of the 250,000 plant species known to man are edible, only nine species, accounting for 75% of human food, are the focus of the majority of crop improvement efforts. Genes found in the remaining largely-untapped wealth of under-developed and undomesticated plants may be critical for crop improvements that are needed to feed the worlds future population. Unfortunately, we have only a very limited knowledge about the genetic makeup of this group of plants, many of which are threatened for survival. There are strong indications that the survival of about a quarter of the worlds flora, much of it from the tropics, may be threatened over the next few decades by population growth, deforestation, habitat loss, destructive development, and agricultural expansion. Further loss of plant diversity is predicted through genetic erosion and
narrowing of the genetic basis of many species. The disappearance of such vital and massive amounts of biodiversity is one of the greatest challenges faced by the world community: to halt the destruction of plant resources that are essential for meeting present and future needs. These challenges are exacerbated by the high costs of plant conservation programs and our lack of knowledge about these genetic resources. The planned research is uniquely positioned to utilize Hawaiis plant germplasm collections, proven scientific expertise, and successful collaborative network to expand current knowledge on genome evaluation and improvement of tropical plant species. Because conservation and utilization of plant genetic resources for food and agriculture are inextricably linked, the goals of our proposed research are to: 1) genetically characterize tropical crop genetic resources; 2) identify agriculturally important traits and the genes controlling their expression in the preceding
material; and 3) develop better methods for manipulating these genes for improving the traits. The planned research will facilitate optimal management of genetic resources and increase use of these resources by characterizing and manipulating genes important for increasing productivity, value, and sustainability of tropical crop species. Better characterization of the genetic diversity of tropical fruits and selected crops will be used to identify which accessions to preserve in the ARS germplasm collections. Genome analysis will identify genes conferring useful agronomic traits for assisting breeders in the improvement of germplasm through both traditional breeding and selection and transgenic approaches. Genetic transformation with candidate genes and proper level of expression of the transgenes can confer desired agronomic traits such as increased resistance to pests and diseases. Increased genetic resistance to pests and diseases will allow farmers to reduce dependence on use of
chemicals and reduce quarantine treatments currently used to allow commodity export. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2003) Map yield, quality, and resistance genes and loci in sugarcane. Clone and characterize constitutive gene promoters from sugarcane. Year 2 (FY 2004) Produce a high density genetic map of the papaya genome. Develop a comparative map of sugarcane and sorghum. Conduct AFLP analysis of the genetic diversity of ginger. Clone and characterize papaya pathogenesis-related (PR) genes. Introgress the ringspot virus coat protein into specific cultivars. Year 3 (FY 2005) Fine map the papaya sex determination locus. Year 4 (FY 2006) Produce genetic and QTL maps of coffee. Conduct AFLP analysis of the genetic diversity of tea. Clone and characterize organ-specific promoters from papaya. Develop methods to regulate transgene expression. Transform papaya with pest and disease resistance genes. Year 5 (FY2007) Map
yield, quality, and resistance genes and loci in papaya. Clone and characterize papaya flower organ determination genes. Clone and characterize the papaya sex determination gene(s). Evaluate new gene promoters and sub-cellular targeting peptides. Develop and evaluate technologies for production of plant produced recombinant proteins. 4a List the single most significant research accomplishment during FY 2006. (NP 302 Component III Plant Biotechnology Risk Assessment (a) Improving and Assessing Genetic Engineering Technology). Developing an efficient transformation/selection system. The plant cell culture systems for selectively growing transformed cells are generally based on antibiotic resistance that has several operational limitations and suffers from perceived health risks. Alternative selection gene technology is needed to advance plant transgenic research and development. A collaborative research project in Hawaii between the Tropical Plant Physiology Production and Disease Unit
of the ARS U. S. Pacific Basin Agricultural Research Center and the Hawaii Agriculture Research Center evaluated a transformation selection protocol based on overcoming mannose (Man) toxicity with a gene encoding the enzyme phospho-mannose isomerase (PMI). The PMI/Man selection system was more efficient in transforming papaya than were methods reported using antibiotic selection or visual markers. Mannose selection increases selection efficiency, facilitates stacking of multiple genes of interest, and avoids issues of antibiotic use. Plant transgenic research and development are advanced with this new system. 4b List other significant research accomplishment(s), if any. (NP 302 Component III Plant Biotechnology Risk Assessment (a) Improving and Assessing Genetic Engineering Technology) Papaya with increased resistance to insect pests. Increased genetic resistance to insect pests is needed to reduce pesticide use, lower production costs, and protect human health and the environment.
Collaborative research between scientists of the Hawaii Agriculture Research Center and ARS in Hawaii produced papaya plants transformed with a tobacco hornworm gene and evaluated plant response to carmine spider mites, a major insect pest of a very wide range of fruit, vegetable, and ornamental plants. In field trials, the transformed papaya plants grew more vigorously and reduced the population of infecting spider mites more than did the non- transformed controls. The transformed papaya lines will be advanced in field trials for performance in fruit production and may prove beneficial to the papaya fruit industry. The same technology holds promise for a wide range of crop species. 5. Describe the major accomplishments to date and their predicted or actual impact. (NP 302, Component 2) Genetic maps, increasingly important for improving the efficiency of breeding and selection of superior cultivars, are few or totally lacking for polyploid plants. This situation is unfortunate since a
majority of crop plant species are polyploid. A collaborative project conducted primarily in Texas but involving researchers from the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center, the Hawaii Agriculture Research Center, Texas A&M University, and University of Georgia produced three informative genetic maps for polyploid sugarcane consisting of a consensus map from two interspecific crosses, a quantitative trait map for parameters of yield, and a quantitative trait map for sugar yield. These findings are allowing sugarcane breeders to use DNA markers to select potentially superior parental lines and will supplement the art of plant breeding with the science of genetics for more rapid, less costly, and more specific phenotype improvements in sugarcane breeding and selection programs. (NP 301, Component 1) Plant scientists need to know the DNA phylogenetic relationship among accessions to assure adequate and efficient
conservation of germplasm and to identify accessions having agronomically desirable genes that can be manipulated to produce useful traits in a crop improvement program. A collaborative tropical plant genomics project in Hawaii between the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center and the Hawaii Agriculture Research Center has developed DNA fingerprints of coffee, papaya and pineapple varieties and accessions. AFLP analyses on 68 coffee varieties, 190 pineapple varieties, and 54 papaya varieties showed sufficient polymorphism to distinguish among lines and revealed unexpected phylogenetic relationships. Information will assist germplasm curators and crop breeders to do their job more efficiently and will lead to discovery of new genes for producing improved crop varieties. (NP 302, Component 2) Few details are known about how sex chromosomes are involved in plant sex determination even though proper expression of plant sex
is the single most important contributor to yield of all fruit and seed producing crops. Collaborative research in Hawaii among the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center, the Hawaii Agriculture Research Center, and the University of Georgia produced a high density DNA linkage map of papaya. The papaya linkage map revealed a primitive sex chromosome having a very small male-specific region (MSY) of 4-5 Mb in size compared to the MSY in humans that is 10 fold larger (60-70 Mb), and 100 fold larger (500-600 Mb) in the most studied plant sex chromosome of white campion. The incipient sex chromosome of papaya is postulated to resemble the ancestor of the human Y chromosome as it existed 240-320 million years ago so that sequencing this chromosome provides a unique opportunity to test hypotheses and theories about sex chromosome evolution near the inception of this process and may reveal genes and their regulatory elements
that can be managed to attain proper sex expression for high yield stability in seed and fruit crops. (NP 302, Component 2) The concept of using short segments of coat protein genes from different strains of PRSV has been validated, and a selected line with segmented genes is being characterized with the aim of deregulating the line in the US. The selected line is a transgenic Kapoho with resistance to PRSV from Hawaii and from Thailand. The approach for using segmented genes to obtain resistance to different viruses and strains of a virus could be a generic method for engineering virus resistance in plants. A patent was obtained for this technology. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Sugarcane ubi9 gene promoter was provided to Dr.
Antonio Figueira at Universidade de Sao Paulo under an ARS MTA. Dr. Figuera intends to use this promoter for transgene expression in garlic. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Food E-News. The pineapple fingerprint. Oct 05, 2005. http://www.rssl. com/OurServices/foodENews http://search.rssl.com/cgi-bin/MsmGo.exe? grab_id=199&page_id=14815488&query=pineapple&hiword=PINEAPPLES+pineapple+ Schoebi, Katharina. No escape for transgenic papaya. Checkbiotech August 23, 2005. http://www.checkbiotech.org/root/index.cfm? fuseaction=search&search=apaya&doc_id=11059&start=1&fullsearch=0 Presentation on our research on SCYLV P0 to Kamehameha Schools summer Biotechnology Class, June 06.
Impacts
(N/A)
Publications
- Jeffrey Lai, C., Yu, Q., Hou, S., Skelton, R.L., Jones, M.L., Lewis, K., Murray, J., Eustice, M., Agbayani, R., Guan, P., Moore, P.H., Ming, R., Presting, G. 2006. Analysis Of Papaya BAC End Sequences Reveals First Insights Into The Organization Of A Tree-Fruit Genome. Plant and Animal Genome XIV International Conference Proceedings. P491, Pg. 224.
- Lai, C.W., Yu, Q., Hou, S., Skelton, R.L., Jones, M.R., Lewis, K.L., Murray, J., Eustice, M., Agbayani, R., Eguan, P., Moore, P.H., Ming, R., Presting, G. 2006. Analysis of papaya BAC end sequences reveals first insights into the organization of a tree-fruit genome. 18th Annual CTAHR Student Research Symposium. (Abstracts) #78. Pg. 50.
- Agbayani, R., Nishijima, W.T., Moore, P.H., Zhu, Y.J. 2006. Characterization and Improvement of Disease Response to Phytophthora Palmivora in Papaya. 18th Annual CTAHR Student Research Symposium [abstract]. Paper No. 104-63.
- Eustice, M., Lai, C.W., Yu, Q., Presting, G., Moore, P.H., Ming, R. 2006. Variation of microsatellite markers among selected papaya varieties. 18th Annual CTAHR Student Research Symposium. (Abstracts)#77, pg. 49.
- Chang, W.P., Combs, G.F., Scanes, C.G., Marsh, J.A. 2005. The effects of dietary vitamin E and selenium deficiencies on plasma thyroid and thymic hormone concentrations in the chicken. Developmental and Comparative Immunology 29:265-273.
- Guan, P., Moore, P.H., Albert, H.H. 2006. RNA-induced gene silencing in papayas. 18th Annual CTAHR Student Research Symposium. (Abstract)#51. Pg. 63.
- Jones, M., Zee, F.T., Moore, P.H., Kim, M.S., Ming, R. 2006. Genetic diversity of litchi germplasm assessed by AFLP marker. Plant Animal and Microbe Genomes Conference XIV. P489, Pg. 223.
- Ming, R., P. H. Moore, K. K. Wu, A. DHont, J. C. Glaszmann, T. L. Tew, T. E. Mirkov, J. da Silva, J. Jifon, M. Rai, R. J. Schnell, S. M. Brumbley, P. Lakshmanan, J. C. Comstock, A. H. Paterson Sugarcane Improvement through Breeding and Biotechnology. In (J Janick ed.) Plant Breeding Reviews vol 27: 15-118. 2006.
- Ostroff, R.L., Yu, Q., Srinivasan, R., Manshardt, R., Moore, P.H., Ming, R. 2006. The Expression Of The Gene For Lycopene B-Cyclase Is Elevated In Leaves And Flowers And Down-Regulated In Both Yellow- And Red- Fleshed Papaya Fruits. Plant and Animal Genome XIV International Conference Proceedings. P479, Pg. 221.
- Paidi, M., Lee, S.E., Li, Q., Moore, P.H., Zhu, Y.J. 2006. Differential Expression of Papaya Protein Profile Using Proteomic Tools. 18th Annual CTAHR Student Research Symposium. (Abstracts)#21, Pg. 21.
- Veatch, O.J., Yu, Q., Eustice, M., Moore, P.H., Paull, R., Ming, R. 2006. Mapping physiological traits in Carica papaya using microsatellite markers. 18th Annual CTAHR Student Research Symposium [abstract]. Paper No. 30-25.
- Yu, Q., Moore, P.H., Alam, M., Jiang, J., Paterson, A.H., Vyskot, B., Ming, R. 2006. The evolution of sex chromosomes in papaya. Plant Animal and Microbe Genomes Conference XIV. W340, Pg. 85.
- Zhu, Y.J., Agbayani, R., Mccafferty, H., Albert, H.H., Moore, P.H. 2005. Effective selection of transgenic papaya plants with the PMI/Man selection system. Plant Cell Reports. 24:426-432.
- Zhu, Y.J., Fitch, M.M., Moore, P.H. 2006. Papaya (Carica papaya L.). Methods of Molecular, vol 344: Agrobacterium Protocols, 2/e, vol. 2:209- 217.
- Fermin, G., Gonsalves, D. 2004. Engineering resistance against papaya ringspot virus by native, chimeric and synthetic transgenes. In: G Lebenstein and G. Thottappilly, editors. Virus and virus-like diseases of major crops in developing countries. The Netherlands: Kluwer Academic Press Publishers. Chapter 20:P 497-518.
- Souza, M., Gonsalves, D. 2005. Sequence similarity between the viral CP gene and the transgene in transgenic papayas. Pesc. Agrotech. Bras., Brasilia. 40(5):479-486.
- Tripathi, S., Suzuki, J., Gonsalves, D. 2006. Development of Genetically Engineered Resistant Papaya for Papaya Ringspot Virus in a Timely Manner-A Comprehensive and Successful Approach. In P. Ronald (ed.) Plant-Pathogen Interactions: Methods and Protocols, The Humana Press, Inc., Totowa, New Jersey. P. 197-239.
- Souza Junior, M.T., Tennant, P., Gonsalves, D. 2005. Influence of Coat Protein Transgene Copy Number on Resistance in Transgenic Line 63-1 Against Papaya Ringspot Virus Isolates. Hortscience. 40(7). P2083-2087.
- Souza, J., Nickel, O., Gonsalves, D. 2005. Development of virus resistant transgenic transgenic papayas expressing the coat protein from a Brazilian isolate of papaya ringspot virus (prsv). Fitopatologia Brasileira. 30:357- 365.
- Shah, D.A., Dillard, H.R., Mazumdar-Leighton, S., Gonsalves, D., Nault, B. A. 2006. Incidence, spacial patterns and association among viruses in snap beans and alfalfa in New York. Plant Disease 90:203-210.
- Kresovich, S., Barbazuk, B., Bedell, J., Borrell, A., Buell, R., Burke, J. J., Clifton, S., Cordonnier-Pratt, M., Cox, S., Dahlberg, J., Erpelding, J. E., Fulton, T.M., Fulton, B., Fulton, L., Gingle, A., Goff, S., Hash, C., Huang, Y., Jordan, D., Klein, P., Klein, R.R., Magalhaes, J., McCombie, R., Moore, P.H., Mullet, J.E., Ozias-Akins, P., Paterson, A.H., Porter, K., Pratt, L., Roe, B., Rooney, W., Schnable, P., Steely, D.M., Tuinstra, M., Ware, D., Warek, U. 2005. Toward sequencing the sorghum genome: A US National Science Foundation-sponsored workshop report. Plant Physiology. 138(4):1898-1902.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Although more than 30,000 of the 250,000 plant species known to man are edible, only nine species, accounting for 75% of human food, are the focus of the majority of crop improvement efforts. Genes found in the remaining largely-untapped wealth of under-developed and undomesticated plants may be critical for crop improvements that are needed to feed the worlds future population. Unfortunately, we have only a very limited knowledge about the genetic makeup of this group of plants, many of which are threatened for survival. There are strong indications that the survival of about a quarter of the worlds flora, much of it from the tropics, may be threatened over the next few decades by population growth, deforestation, habitat loss, destructive development, and agricultural expansion. Further loss of plant
diversity is predicted through genetic erosion and narrowing of the genetic basis of many species. The disappearance of such vital and massive amounts of biodiversity is one of the greatest challenges faced by the world community: to halt the destruction of plant resources that are essential for meeting present and future needs. These challenges are exacerbated by the high costs of plant conservation programs and our lack of knowledge about these genetic resources. The planned research is uniquely positioned to utilize Hawaiis plant germplasm collections, proven scientific expertise, and successful collaborative network to expand current knowledge on genome evaluation and improvement of tropical plant species. Because conservation and utilization of plant genetic resources for food and agriculture are inextricably linked, the goals of our proposed research are to: 1) genetically characterize tropical crop genetic resources; 2) identify agriculturally important traits and the genes
controlling their expression in the preceding material; and 3) develop better methods for manipulating these genes for improving the traits. This research supports NP 301, Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, primarily component II: Genomic Characterization and Genetic Improvement by characterizing the genetic diversity of selected tropical crop plant germplasm; and NP 302, Plant Biological and Molecular Processes, primarily component I: Analysis and Modification of Plant Genomes by identifying DNA molecular markers linked to loci encoding important phenotypic traits, isolating and characterizing agronomically important genes, developing technologies for improving crop germplasm by marker-assisted selection and through genetic transformation, improving transgene expression by developing methods that minimize position effects, transgene copy number and integrity, and creating tissue-specific or other desired patterns of expression in
engineered tropical crop plants by developing new gene promoters. The planned research will facilitate optimal management of genetic resources and increase use of these resources by characterizing and manipulating genes important for increasing productivity, value, and sustainability of tropical crop species. Better characterization of the genetic diversity of tropical fruits and selected crops will be used to identify which accessions to preserve in the ARS germplasm collections. Genome analysis will identify genes conferring useful agronomic traits for assisting breeders in the improvement of germplasm through both traditional breeding and selection and transgenic approaches. Genetic transformation with candidate genes and proper level of expression of the transgenes can confer desired agronomic traits such as increased resistance to pests and diseases. Increased genetic resistance to pests and diseases will allow farmers to reduce dependence on use of chemicals and reduce
quarantine treatments currently used to allow commodity export. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2003) Map yield, quality, and resistance genes and loci in sugarcane Clone and characterize constitutive gene promoters from sugarcane Year 2 (FY 2004) Produce a high density genetic map of the papaya genome Develop a comparative map of sugarcane and sorghum Conduct AFLP analysis of the genetic diversity of ginger Clone and characterize papaya pathogenesis-related (PR) genes Introgress the ringspot virus coat protein into specific cultivars Year 3 (FY 2005) Fine map the papaya sex determination locus Year 4 (FY 2006) Produce genetic and QTL maps of coffee Conduct AFLP analysis of the genetic diversity of tea Clone and characterize organ-specific promoters from papaya Develop methods to regulate transgene expression Transform papaya with pest and disease resistance genes Year 5 (FY2007) Map yield, quality, and resistance genes and loci in
papaya Conduct AFLP analysis of the genetic diversity of cacao Clone and characterize papaya flower organ determination genes Clone and characterize the papaya sex determination gene(s) Evaluate new gene promoters and sub-cellular targeting peptides Develop and evaluate technologies for production of plant produced recombinant proteins 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Fine map the papaya sex determination locus. Milestone Fully Met 2. Produce genetic and QTL maps of coffee. Milestone Fully Met 3. Conduct AFLP analysis of the genetic diversity of tea. Milestone Substantially Met 4. Clone and characterize organ-specific promoters from papaya. Milestone Not Met Other 5. Transform papaya with pest and disease resistance genes. Milestone Substantially Met 6. Develop methods to regulate transgene expression. Milestone Substantially Met 7. Map yield,
quality, and resistance genes and loci in papaya. Milestone Substantially Met 8. Conduct AFLP analysis of the genetic diversity of cacao. Milestone Substantially Met 9. Clone and characterize papaya flower organ determination genes. Milestone Substantially Met 10. Evaluate new gene promoters and sub-cellular targeting of peptides. Milestone Not Met Other 11. develop tissue culture system for efficient transformation of anthurium. Milestone Substantially Met 12. Characterize molecular content of transgenic papaya containing sysnthetic constructs. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The milestones listed below with a description of the anticipated outcomes. The entire project is scheduled to be completed during FY 2007 and a new project will be developed for OSQR review and subsequent implementation
beginning during FY 2007. Year 4 (FY 2006) Produce genetic and QTL maps of coffee: We will conduct linkage mapping using the true F2 population and compare the mapping results with the linkage maps generated from the segregating F1 population. Conduct AFLP analysis of the genetic diversity of tea: Done Clone and characterize organ-specific promoters from papaya: Continue to optimize RNA extraction methods from various papaya tissues/organs. cDNA AFLP analysis, originally targeted to begin FY 2005, will now begin FY 2006 due to delays in obtaining good quality RNA & making cDNA. Develop methods to regulate transgene expression: Continue to study transgene expression in existing sugarcane lines, using ELISA, quantitative RT-PCR, and nuclear run-offs. New cell lines produced using Ac-Ds transposon vectors are undergoing selection and will be analyzed in the same way. Data will allow the analysis of the relationship between transgene expression/silencing and transgene insertion number
and structure. New sugarcane lines transformed with viral suppressors of PTGS will be produced. Deletion analysis to identify functional domains in SCYLV P0 will continue, and point mutations will be made and tested to advance this understanding. Sequence analysis suggests the functional domains of SCYLV P0 represent novel, currently uncharacterized protein structures. Transform papaya with pest and disease resistance genes: A transgenic Kapoho line with good horticultural properties and resistance to PRSV was selected, and will be further characterized with the aim of getting it deregulated and commercialized in the US. Map yield, quality, and resistance genes and loci in papaya: We will collect phenotypic data of fruit weight, flesh color, bearing height, and insect resistance from two locations, and map QTLs controlling these traits. Conduct AFLP analysis of the genetic diversity of cacao: We will study the diversity of cacao germplasm collected in Hawaii using SSR markers. Clone
and characterize papaya flower organ determination genes: We will construct papaya floral cDNA libraries using young flower buds from male, female, and hermaphrodite, and sequence thousands of ESTs. We expect to clone flower organ determination genes through sequence homology with those identified in model species. Clone and characterize the papaya sex determination gene(s): We will sequence the MSY and the corresponding region of the X chromosome. Sequence comparison will reveal a limited number of candidate genes for sex determination, and these candidate genes will be characterized using RT-PCR and in situ hybridization using floral bud tissues. Develop tissue culture system for efficient transformation of anthurium: Transgenic callus lines will be evaluated for the presence of the transgenes. Transformed callus will be generated into plants. Characterize molecular content of transgenic papaya containing synthetic constructs: We have synthetic PRSV resistance constructs that may
impart resistance to a wide range of PRSV strains. The constructs will be transformed into several papaya cultivars that will be tested for resistance to a wide range of PRSV strains. Transgenic callus lines will be identified and regeneration initiated along with molecular characterization by PCR. Transgenic plants with the synthetic genes will be tested for resistance with a range of PRSV strains. Papaya cultivars originating from Bangladesh and Africa will be transformed with the synthetic gene constructs. Year 5 (FY2007) Produce genetic and QTL maps of coffee: We will conduct QTL mapping for bean size and leaf characteristics using the true F2 mapping population. Significant difference in cupping quality was detected between the two parents, but the cost for cupping test by professional cuppers is very high. We will map QTLs for cupping quality if funds permit. Conduct AFLP analysis of the genetic diversity of tea: Done Clone and characterize organ-specific promoters from
papaya: Papaya genes identified to have tissue-specific expression patterns will be cloned, and expression patterns verified by quantitative RT-PCR. Develop methods to regulate transgene expression: Transgenic sugarcane plants harboring SCYLV P0 or other viral suppressors of PTGS will be characterized for growth abnormalities, expression of transgenes, accumulation of siRNAs and miRNAs, and steady state levels of predicted miRNA target mRNAs. Transform papaya with pest and disease resistance genes: Done Map yield, quality, and resistance genes and loci in papaya: Done Conduct AFLP analysis of the genetic diversity of cacao: We will continue to analyze genetic diversity of cacao if it has not been completed in 2006. Clone and characterize papaya flower organ determination genes: We will construct papaya floral cDNA libraries using young flower buds from male, female, and hermaphrodite, and sequence thousands of ESTs. We expect to clone flower organ determination genes through
sequence homology with those identified in model species. Clone and characterize the papaya sex determination gene(s): We will sequence the MSY and the corresponding region of the X chromosome. Sequence comparison will reveal a limited number of candidate genes for sex determination, and these candidate genes will be characterized using RT-PCR and in situ hybridization using floral bud tissues. Develop tissue culture system for efficient transformation of anthurium: Work initiated on developing a transformation system for anthurium has produced several hundred transgenic lines this past year. Transgenes for leaf blight and nematode resistance were obtained and used in preliminary transformation trials. Transgenic callus lines will be evaluated for the presence of the transgenes. Transformed callus will be generated into plants. Characterize molecular content of transgenic papaya containing synthetic constructs: We have synthetic PRSV resistance constructs that may impart resistance
to a wide range of PRSV strains. The constructs will be transformed into several papaya cultivars that will be tested for resistance to a wide range of PRSV strains. Transgenic callus lines will be identified and regeneration initiated along with molecular characterization by PCR. Selected lines that show resistance will be transferred to Bangladesh and Africa and started towards the process of deregulation in the respective countries. 4a What was the single most significant accomplishment this past year? Results from a series of field trials to compare the growth and fruit yields of papaya orchards that had been established from planted seed, branch cuttings, or tissue culture showed the early highest fruit yields from the tissue culture clones. Due to the genetics of papaya sex determination, its seedlings segregate 50:50 for female and hermaphrodite trees but only the hermaphrodites are grown because of their higher productivity and better shipping quality. The traditional way to
establish hermaphrodite orchards by over-planting approximately 10 seed per hole, allowing the seedlings to grow to 3-4 months of age when sex can be determined, and then chopping out the female trees is wasteful of labor, seed, water, and fertilizer, and can leave non-producing gaps in the field. Replacing this practice with planting clonal hermaphrodite propagules has economic potential that needed to be evaluated. Field trials, designed and conducted by US PBARC scientists with field help provided by the Hawaii Agriculture Research Center, were conducted over two years on the two major papaya producing islands, Oahu and Hawaii, of the State of Hawaii. Changing Hawaiis papaya cropping system from one that is seed-based to one that is clone-based will take time but may prove beneficial to the industry as current technologies are improved to produce and establish the cloned materials. 4b List other significant accomplishments, if any. Benzothiadiazole (BTH) was shown to induce
systemic acquired resistance (SAR) in papaya and the response shows many similarities to SAR characterized in arabidopsis. Two dozen papaya genes induced by BTH were identified, including several with known roles in plant defense, but also including several novel genes not previously described in plant defense. Two papers on this work were published in Physiological & Molecular Plant Pathology. Follow up characterization of the new defense genes may reveal new mechanisms and genetic sources for resistance to plant diseases. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Genetic maps, increasingly important for improving the efficiency of breeding and selection of superior cultivars, are few or totally lacking for polyploid plants. This situation is unfortunate since a majority of crop plant species are polyploid. A collaborative project conducted primarily in Texas but involving researchers from the Tropical Plant
Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center, the Hawaii Agriculture Research Center, Texas A&M University, and University of Georgia produced three informative genetic maps for polyploid sugarcane consisting of a consensus map from two interspecific crosses, a quantitative trait map for parameters of yield, and a quantitative trait map for sugar yield. These findings are allowing sugarcane breeders to use DNA markers to select potentially superior parental lines and will supplement the art of plant breeding with the science of genetics for more rapid, less costly, and more specific phenotype improvements in sugarcane breeding and selection programs. Action plans 1.2.5 and 1.2.7. Plant scientists need to know the DNA phylogenetic relationship among accessions to assure adequate and efficient conservation of germplasm and to identify accessions having agronomically desirable genes that can be manipulated to produce useful traits in a crop
improvement program. A collaborative tropical plant genomics project in Hawaii between the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center and the Hawaii Agriculture Research Center has developed DNA fingerprints of coffee, papaya and pineapple varieties and accessions. AFLP analyses on 68 coffee varieties, 190 pineapple varieties, and 54 papaya varieties showed sufficient polymorphism to distinguish among lines and revealed unexpected phylogenetic relationships. Information will assist germplasm curators and crop breeders to do their job more efficiently and will lead to discovery of new genes for producing improved crop varieties. Action plans 1.2.5, 1.2.7 and 1.2.8. Few details are known about how sex chromosomes are involved in plant sex determination even though proper expression of plant sex is the single most important contributor to yield of all fruit and seed producing crops. Collaborative research in Hawaii among the
Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center, the Hawaii Agriculture Research Center, and the University of Georgia produced a high density DNA linkage map of papaya. The papaya linkage map revealed a primitive sex chromosome having a very small male-specific region (MSY) of 4-5 Mb in size compared to the MSY in humans that is 10 fold larger (60-70 Mb), and 100 fold larger (500- 600 Mb) in the most studied plant sex chromosome of white campion. The incipient sex chromosome of papaya is postulated to resemble the ancestor of the human Y chromosome as it existed 240-320 million years ago so that sequencing this chromosome provides a unique opportunity to test hypotheses and theories about sex chromosome evolution near the inception of this process and may reveal genes and their regulatory elements that can be managed to attain proper sex expression for high yield stability in seed and fruit crops. Action plans 1.2.5 and 1.2.6.
The concept of using short segments of coat protein genes from different strains of PRSV has been validated, and a selected line with segmented genes is being characterized with the aim of deregulating the line in the US. The selected line is a transgenic Kapoho with resistance to PRSV from Hawaii and from Thailand. The approach for using segmented genes to obtain resistance to different viruses and strains of a virus could be a generic method for engineering virus resistance in plants. A patent was obtained for this technology. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The segmented gene approach for developing transgenic plants with resistance to a range of viruses or strains of a virus was validated. A patent was obtained for this
technology. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Goldstein, C. Cutting through Biotech Myths. Agriculture Hawaii July-Sept 2004. pp:6-7. Shows photos of ARS and UH researchers and discusses the success of transgenic papaya. Delan Perry. Bringing Papayas to the Market. Agriculture Hawaii July- Sept 2004. p:14. Discusses the marketing of transgenic papaya, and how the development of transgenic lines saved the Hawaii industry. June 7, 2005 Pang, S.-Z., Gonsalves, D., and Jan, F.-J. 2004. DNA constructs and methods to impart resistance to papaya ringspot virus on plants. Patent no. 6,750,382 B2, Issued June 15, 2004 Scorza, R., Ravelonandro, M., and Gonsalves, D. 2004. Plum Tree Named 'Honeysweet'. Patent no. PP 15,154, Issued September 21, 2004 Zhu, H., Ling, K., and Gonsalves, D. 2005. Grapevine leafroll virus (Type 2) proteins and their
uses. Patent no. US 6,858,426 B1, Issued February 22, 2005
Impacts
(N/A)
Publications
- Eustice, M., Moore, P.H., Presting, G., Ming, R. 2005. Frequency and variation of microsatellite markers in the papaya genome. CTAHR Student Research Symposium (Abstracts). P17.
- Ackerrman, C.M., Yu, Q., Moore, P.H., Paull, R.E., Steiger, D.L., Ming, R., Cloning and characterization of a superman ortholog in polygamous papaya. Annual International Plant & Animal Genome XIII Conference. Abstracts P507, pg 197. 2005.
- Ming, R., Van Droogenbroeck, B., Moore, P.H., Zee, F.T., Kyndt, T., Scheldeman, X., Sekioka, T., Gheysen, G. 2005. Molecular diversity of Carica papaya and related species. Plant Genome: Biodiversity and Evolution. A.K Sharma, A. Sharma (eds.). Science Publishers, Enfield, New Hampshire, USA. V. IB: Phanerograms;P. 229-254.
- Qiu, X., Guan, P., Wang, M., Moore, P.H., Zhu, Y.J., Hu, J., Borth, W., Albert, H.H., 2004. Identification and expression analysis of bth induced genes in papaya. Physiological and Molecular Plant Pathology. 65:21-30.
- Wang, M., Goldstein, C., Su, W., Moore, P.H., Albert, H.H. 2005. Production of biologically active gm-csf in sugarcane: a secure biofactory. Transgenic Research. V14:P167-178.
- Tennant, P., Souza Junior, M.T., Gonsalves, D., Fitch, M.M., Manshardt, R. M., Slightom, J.L. 2005. Line 63-1, a new virus-resistant transgenic papaya for Hawaii. Hortscience 40(5):1196-1199.
- Fitch, M.M., Leong, T., Akashi, L., Yeh, A., White, S., Dela Cruz, A., Santo, L., Ferreira, S., Moore, P.H. 2005. Clonaly propagated and seed- derived papaya orchards: 2. comparison of yields. Journal of Horticultural Science and Biotechnology. 40(5)1291-1297.
- Fitch, M.M., Leong, T., Akashi, L., Yeh, A., White, S., Dela Cruz, A., Santo, L., Ferreira, S., Moore, P.H. 2005. Growth and yield of clonally propagated and seedling-derived papayas. i. growth. ii yield.. Journal of Horticultural Science and Biotechnology. 40(5): 1283-1290.
- Ackerman, C.M., Yu, Q., Moore, P.H., Paull, R.E., Ming, R. 2005. Cloning and characterization of apetala3 and pistillata orthologs in papaya. Plant Biology (Abstracts) P728. P237.
- Albert, H.H., Qui, X., Buan, P., Wang, M.L., Moore, P.H., Ahu, Y.J., Hu, J. , Borth, W.,. Establishment of cellular reducing conditions with sar induction in papaya, plant, animal & microbe genomes x11. abstract.. Annual International Plant & Animal Genome XIII Conference. Abstracts W265, P61. 2005.
- Fermin, G., Inglesses, V., Garbozo, C., Rangel, S., Dagert, M., Gonsalves, D. 2004. Engineered resistance against prsv in venezuelan transgenic papayas. Plant Disease 88 (5):516-522.
- Fermin, G., Tennant, P., Gonsalves, C., Lee, D., Gonsalves, D. 2004. Comparative development and impact of transgenic papayas in hawaii, jamaica and venezuela. Transgenic Plants: Methods and Protocols. 286:399- 430.
- Fitch, M.M., Leong, T., Albert, H.H., Mcafferty, H., Zhu, J., Nikolov, K., Megwende, T., Moore, P.H., Gonsalves, D. 2005. Improvement in transformation of anthurium. In Vitro Cellular and Developmental Biology - Plants. 41:p30-A.
- Gonsalves, D. 2004. Chronological summary of transgenic papaya work in Hawaii. In: Gonsalves, D., editor. OECD/USAID/ARS Conference on Virus Resistant Transgenic Papaya in Hawaii: A Case for Technology Transfer to Lesser Developed Countries, October 22-24, 2003, Hilo, Hawaii. p. 5-8.
- Gonsalves, D. 2004. Premier papaya plantations rescued through science and teamwork. Agricultural Research Service Publication 52(1):2.
- Gonsalves, D. 2004. Transgenic papaya in Hawaii and beyond. Agbioforum. Vol. 4:36-40.
- He, X., Miyasaka, S.C., Fitch, M.M., Zhu, J.Y., Moore, P.H. 2004. Toward improved fungal resistance in taro through genetic transformation. Abstracts 3rd International Symposium on Tropical and Subtropical Fruits: Abstract P19, p76.
- Kato, C.Y., Nagai, C., Moore, P.H., Zee, F.T., Kim, M.S., Steiger, D.L., Ming, R. 2004. Intra-specific dna polymorphism in pineapple (ananas comosus (l.) merr.) assessed by aflp markers. Genetic Resources and Crop Evolution. 51:815-825.
- Mangwende, T., Mirkov, E., Albert, H.H. 2005. The P0 protein of sugarcane yellow leaf virus is a suppressor of posttranscriptional gene silencing. Plant and Animal Genome Conference XIII Proceedings. Abstract A161, P38.
- Meng, B., Li, C., Wang, W., Goszczynski, D., Gonsalves, D. 2005. The complete genome sequence of two new variants of Grapevine rupestris stem pitting-associated virus and comparative analyses. Journal of General Virology. 86:1555-1560.
- Zhu, Y.J., Agbayani, R., Tang, C.S., Moore, P.H. 2004. Developing transgenic papaya to improve broad disease resistance against fungal pathogens [abstract]. Proc 3rd International Symposium on Tropical and Subtropical Fruits. p48:O-03.
- Meng, B., Li, C., Goszczynski, D., Gonsalves, D. 2005. Genome sequences and structures of two biologically distinct strains of grapevine leafroll- associated virus 2 and sequence analysis. Virus Genes 31(1):31-41.
- Moore, P.H. 2004. Chapter introduction: breeding and molecular biology to improve sugarcane for the 21st century. Book Chapter, p461-464.
- Moore, P.H. 2005. Integration of sucrose accumulation across hierarchical scales: towards developing an understanding of the gene-to-crop continuum. Field Crop Research. 92:119-135.
- Moore, P.H. A personal view on coordinating international progress in sugarcane improvement and linking biotechnology to application. Sugar Cane International. v107:1273, p27-31. 2005.
- Schenck, S., Pearl, H.M., Liu, Z., Moore, P.H., Ming, R. 2005. Genetic variation of ustilago scitaminea pathotypes in hawaii evaluated by host range and AFLP markers. Sugar Cane International. 23(1):15-19.
- Wang, M., Schenck, S., Albert, H.H. 2005. Antibody to a short peptide sequence detected sugarcane yellow leaf virus isolates from several sources. Sugar Cane International. 23:25-27.
- Zhu, Y.J., Agbayani, R., Jackson, M.C., Tang, C.S., Moore, P.H. 2004. Expression of the grapevine stilbene synthase gene vst1 in papaya provides increased resistance against diseases caused by phytophthora palmivora. Planta 220:241-250.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Although more than 30,000 of the 250,000 plant species known to man are edible, only nine species, accounting for 75% of human food, are the focus of the majority of crop improvement efforts. Genes found in the remaining largely-untapped wealth of under-developed and undomesticated plants may be critical for crop improvements that are needed to feed the world's future population. Unfortunately, we have only a very limited knowledge about the genetic makeup of this group of plants, many of which are threatened for survival. There are strong indications that the survival of about a quarter of the world's flora, much of it from the tropics, may be threatened over the next few decades by population growth, deforestation, habitat loss, destructive development, and agricultural expansion. Further loss of
plant diversity is predicted through genetic erosion and narrowing of the genetic basis of many species. The disappearance of such vital and massive amounts of biodiversity is one of the greatest challenges faced by the world community: to halt the destruction of plant resources that are essential for meeting present and future needs. These challenges are exacerbated by the high costs of plant conservation programs and our lack of knowledge about these genetic resources. The planned research is uniquely positioned to utilize Hawaii's plant germplasm collections, proven scientific expertise, and successful collaborative network to expand current knowledge on genome evaluation and improvement of tropical plant species. Because conservation and utilization of plant genetic resources for food and agriculture are inextricably linked, the goals of our proposed research are to: 1) genetically characterize tropical crop genetic resources; 2) identify agriculturally important traits and the
genes controlling their expression in the preceding material; and 3) develop better methods for manipulating these genes for improving the traits. This research supports NP 301, Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, primarily component II: Genomic Characterization and Genetic Improvement by characterizing the genetic diversity of selected tropical crop plant germplasm; and NP 302, Plant Biological and Molecular Processes, primarily component I: Analysis and Modification of Plant Genomes by identifying DNA molecular markers linked to loci encoding important phenotypic traits, isolating and characterizing agronomically important genes, developing technologies for improving crop germplasm by marker-assisted selection and through genetic transformation, improving transgene expression by developing methods that minimize 'position effects', transgene copy number and integrity, and creating tissue-specific or other desired patterns of expression in
engineered tropical crop plants by developing new gene promoters. The planned research will facilitate optimal management of genetic resources and increase use of these resources by characterizing and manipulating genes important for increasing productivity, value, and sustainability of tropical crop species. Better characterization of the genetic diversity of tropical fruits and selected crops will be used to identify which accessions to preserve in the ARS germplasm collections. Genome analysis will identify genes conferring useful agronomic traits for assisting breeders in the improvement of germplasm through both traditional breeding and selection and transgenic approaches. Genetic transformation with candidate genes and proper level of expression of the transgenes can confer desired agronomic traits such as increased resistance to pests and diseases. Increased genetic resistance to pests and diseases will allow farmers to reduce dependence on use of chemicals and reduce
quarantine treatments currently used to allow commodity export. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2003) Map yield, quality, and resistance genes and loci in sugarcane. Clone and characterize constitutive gene promoters from sugarcane. Year 2 (FY 2004) Produce a high density genetic map of the papaya genome. Develop a comparative map of sugarcane and sorghum. Conduct AFLP analysis of the genetic diversity of ginger. Clone and characterize papaya pathogenesis-related (PR) genes. Introgress the ringspot virus coat protein into specific cultivars. Year 3 (FY 2005) Fine map the papaya sex determination locus. Year 4 (FY 2006) Produce genetic and QTL maps of coffee. Conduct AFLP analysis of the genetic diversity of tea. Clone and characterize organ-specific promoters from papaya. Transform papaya with pest and disease resistance genes. Year 5 (FY2007) Map yield, quality, and resistance genes and loci in papaya. Conduct AFLP analysis of the
genetic diversity of cacao. Clone and characterize papaya flower organ determination genes. Clone and characterize the papaya sex determination gene(s). Evaluate new gene promoters and sub-cellular targeting peptides. Develop and evaluate technologies for production of plant produced recombinant proteins. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones below scheduled to be completed in FY 2004 were all completed. -Produce a high density genetic map of the papaya genome. A high density genetic map of papaya was constructed with 1501 molecular and morphological markers. This map revealed suppression of recombination at the sex determination locus, and was instrumental in the discovery of a primitive Y chromosome in papaya. -Develop a comparative map of
sugarcane and sorghum. Two sugarcane genetic maps were constructed with 442 DNA markers and aligned to sorghum genetic map. These maps were used for QTL analysis of sugarcane yellow leaf virus resistance, and selected markers from the maps were used to fingerprint sugarcane materials used in the breeding program at HARC. -Conduct AFLP analysis of the genetic diversity of ginger. Eighteen ginger varieties from China, Japan, Australia, and Honduras were analyzed with 572 informative AFLP markers. Greater genetic diversity was detected in ginger compared with papaya, coffee, and pineapple; this might be the consequence of ginger's enormous genome of 6,031 Mb that has accumulated large amount of repetitive DNA sequences through the course of evolution. -Clone and characterize papaya pathogenesis-related (PR) genes. Suppression subtractive hybridization (SSH) was used to produce a Carica papaya L. cDNA library enriched for benzothiadiazole (BTH) induced genes. From this library 360 clones
were screened by reverse-northern dot blot. The 360 screened clones produced 24 unique papaya ESTs with expression altered more than or equal to 1.5-fold in at least one assay. These ESTs included homologs of defense-related genes from other species and several genes for which no defense-related function has been previously described. -Introgress the ringspot virus coat protein into specific cultivars. Selected 14 superior clones of papaya to micropropagate for conducting replicated field trials. Improved micropropagation procedures to minimize losses, increase throughput, and decrease costs. Provided propagules sufficient to plant 7 acres for 3 growers. Trials showed that clonally propagated plants yield significantly more fruit than traditionally planted and thinned seedlings. -Begin developing a genetic transformation system for improving anthurium with increased resistance to bacterial blight and burrowing nematodes. Beginning to develop a regeneration capable anthurium target
tissue culture system. Initiated transformation of anthurium with bacterial blight and nematode resistance genes. -Initiate transformation of papaya with synthetic PRSV resistance genes. Initiated and increased tissue cultures needed for the transformation trials. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The milestones listed below with a description of the anticipated outcomes. The entire project is scheduled to be completed during FY 2007 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY 2007. Year 3 (FY 2005) -Fine map the papaya sex determination locus. Since we now know that sex determination in papaya is controlled by a large block (4 - 5 Mb) of male specific region of the Y chromosome (MSY), we will complete physical mapping of the MSY region towards eventual cloning of the sex
determination gene. -Complete experiments characterizing the sugarcane yellow leaf virus P0 gene effect on gene silencing in Nicotiana benthamiana and submit a manuscript for publication. -Begin cDNA AFLP analysis of tissue-specific gene expression in papaya. Extract mRNA to produce cDNA libraries of specific tissues and stages of development. Use AFLPs or SSH to identify differentially expressed genes for promoter isolation. Year 4 (FY 2006) -Produce genetic and QTL maps of coffee. We will continue to collect phenotypic data for two years and complete genetic and QTL mapping in FY 2006. We expect to map coffee leaf and bean characteristics that contribute to the coffee yield. Cupping quality is a tricky trait without reliable quantitative measurement, and it could be very difficult to identify QTLs influencing coffee quality. We will study the trigonelline, caffeine, and sugar biosynthetic pathways in developing coffee cherries of three genotypes having different quality beans. We
will look at gene expression of key enzymes that are involved in these pathways. -Conduct AFLP analysis of the genetic diversity of tea. We will collect leaf samples and conduct DNA extractions for conducting a diversity study based on AFLPs. -Clone and characterize organ-specific promoters from papaya. Continue analysis of temporal and special control of gene expression. -Transform papaya with pest and disease resistance genes. We will continue to produce transgenic lines with antifungal and insecticidal genes and to develop bioassays to detect tolerant lines. -Develop a transient gene silencing system in papaya for functional gene analysis. We will continue to study transgene expression in existing sugarcane lines, using ELISA, quantitative RT-PCR, and nuclear run-offs. New cell lines produced using Cre-lox resolution constructs and Ac-Ds transposon vectors are undergoing selection and will be analyzed in the same way. Data will allow the analysis of the relationship between
transgene expression/silencing and transgene insertion number and structure. Year 5 (FY2007) -Map yield, quality, and resistance genes and loci in papaya. We will collect phenotypic data of fruit weight, flesh color, bearing height, and insect resistance from two locations, and map QTLs controlling these traits. -Conduct AFLP analysis of the genetic diversity of cacao. We initiated a collaborative project with Ray Schnell, ARS in Florida, for molecular analysis of Hawaiian Cacao germplasm using SSR. We reestablished a cacao field to compare SSR profiles of productive versus low-productive trees. -Clone and characterize papaya flower organ determination genes. We will construct papaya floral cDNA libraries using young flower buds from male, female, and hermaphrodite, and sequence thousands of ESTs. We expect to clone flower organ determination genes through sequence homology with those identified in model species. -Clone and characterize the papaya sex determination gene(s). We will
sequence the MSY and the corresponding region of the X chromosome. Sequence comparison will reveal a limited number of candidate genes for sex determination, and these candidate genes will be characterized using RT-PCR and in situ hybridization using floral bud tissues. -Characterize molecular content of transgenic anthurium lines. Anthuriums are susceptible to a bacterial disease, leaf blight, caused by Xanthomonas campestris pv. dieffenbachiae. Work initiated on developing a transformation system for anthurium will result in hundreds of transgenic lines in about 1 year. Transgenes for leaf blight and nematode resistance were obtained and will be transformed into anthurium. The tissue culture system will be developed and transformation of the cultures with the resistance transgenes will be initiated. Transgenic callus lines will be generated and molecular tests like PCR will be accomplished. -Characterize molecular content of transgenic papaya containing synthetic constructs. We have
synthetic PRSV resistance constructs that may impart resistance to a wide range of PRSV strains. The constructs will be transformed into several papaya cultivars that will be tested for resistance to a wide range of PRSV strains. Transgenic callus lines will be identified and regeneration initiated along with molecular characterization by PCR. -Develop and evaluate technologies for production of plant produced recombinant proteins. We will continue collaboration with Erik Mirkov at Texas A&M to study the mechanisms of post-transcriptional gene silencing (PTGS), specifically the P0 protein of Sugarcane Yellow Leaf Virus, which acts to suppress PTGS. This could suggest strategies for suppressing PTGS for the maximum production of transgenic proteins. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY2004. Few details are known about how sex chromosomes are involved in plant sex determination even though proper expression
of plant sex is THE single most important contributor to yield of all fruit and seed producing crops. Collaborative research in Hawaii among the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center, the Hawaii Agriculture Research Center, and the University of Georgia produced a high density DNA linkage map of papaya. The papaya linkage map revealed a primitive sex chromosome having a very small male-specific region (MSY) of 4-5 Mb in size compared to the MSY in humans that is 10 fold larger (60-70 Mb), and 100 fold larger (500- 600 Mb) in the most studied plant sex chromosome of white campion. The incipient sex chromosome of papaya is postulated to resemble the ancestor of the human Y chromosome as it existed 240-320 million years ago so that sequencing this chromosome provides a unique opportunity to test hypotheses and theories about sex chromosome evolution near the inception of this process and may reveal genes and their
regulatory elements that can be managed to attain proper sex expression for high yield stability in seed and fruit crops. B. Other Significant Accomplishment(s): Engineering genetic improvements in grass or cereal crops (monocots) is more difficult than in dicots because the best promoter for expression of transgenes, the cauliflower mosaic virus promoter (35S), is generally not very efficient in monocots. A collaborative project in Hawaii among the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center, the University of Hawaii, and the Hawaii Agriculture Research Center succeeded in isolating, partially characterizing, and patenting three sugarcane polyubiquitin promoters. These promoters proved to be more effective than 35S in driving transgene expression in a number of monocots including sugarcane, pineapple, sorghum, and rice. These promoters are being evaluated by several scientists and they have good potential for advancing
monocot transformation. C. Significant activities that support special target populations. None D. Progress Report. This report includes progress of research conducted under a specific cooperative agreement between ARS and the Hawaiian Agricultural Research Center. Additional details of that research can be found in the Subordinate Project report for project 5320-21000-008-01S, Molecular Approaches for Improvement of Crops Grown in Hawaii. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Genetic maps, increasingly important for improving the efficiency of breeding and selection of superior cultivars, are few or totally lacking for polyploid plants. This situation is unfortunate since a majority of crop plant species are polyploid. A collaborative project conducted primarily in Texas but involving researchers from the Tropical Plant Physiology Production and Disease Unit of the Pacific Basin Agricultural Research Center,
the Hawaii Agriculture Research Center, Texas A&M University, and University of Georgia produced three informative genetic maps for polyploid sugarcane consisting of a consensus map from two interspecific crosses, a quantitative trait map for parameters of yield, and a quantitative trait map for sugar yield. These findings are allowing sugarcane breeders to use DNA markers to select potentially superior parental lines and will supplement the art of plant breeding with the science of genetics for more rapid, less costly, and more specific phenotype improvements in sugarcane breeding and selection programs. Plant scientists need to know the DNA phylogenetic relationship among accessions to assure adequate and efficient conservation of germplasm and to identify accessions having agronomically desirable genes that can be manipulated to produce useful traits in a crop improvement program. A collaborative tropical plant genomics project in Hawaii between the Tropical Plant Physiology
Production and Disease Unit of the Pacific Basin Agricultural Research Center and the Hawaii Agriculture Research Center has developed DNA fingerprints of coffee, papaya and pineapple varieties and accessions. AFLP analyses on 68 coffee varieties, 190 pineapple varieties, and 54 papaya varieties showed sufficient polymorphism to distinguish among lines and revealed unexpected phylogenetic relationships. Information will assist germplasm curators and crop breeders to do their job more efficiently and will lead to discovery of new genes for producing improved crop varieties. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Albert, H.H, Wei, H. Promoter of the sugarcane UBI4 gene. 2003. USDA; Univ. Hawaii US Patent 6,638,766. Albert, H.H., Wei, H.
Sugarcane ubi9 gene promoter and methods of use thereof. 2004. USDA, Univ. Hawaii US Patent 6,706,948. Albert, H.H., Wei, H. Sugarcane UBI9 gene promoter sequence and methods of use thereof. 2004. USDA, Univ. Hawaii US Patent 6,686,513 B1. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Anon, Papaya sex chromosome study illuminates human sex evolution. Pacific Business News, Jan 19, 2004. http://pacific.bizjournals. com/pacific/stories/2004/01/19/daily24.html Anon, Dawn of the Y. Papaya: Glimpse of early sex chromosomes. SCIENCE NEWS This Week. January 24, 2002 v. 165. pp52-53. Anon, New Insights on Papaya Evolution. Crop Biotech Update. Feb. 12, 2004 Easley, Becky. Papayas' evolution might explain human sex chromosomes. The Flat Hat. Feb. 13, 2004. http://flathat.wm.edu/2004-02-13/story.php? type=1&aid=6 Gottlieb, Barry. Women Are from Venus, Men Are Like Papayas. AlterNet. April 27, 2004.
http://www.alternet.org/module/printversion/18528 Hawaii Papaya Industry Association. Papaya research earns award. Agriculture Hawaii 4(1): p19. Holmes, Cat. Scientists: papaya plants have sex chromosomes. University of Georgia College of Agricultural and Environmental Sciences press release Jan 22, 2004. Maugh, Thomas H. Crude Papaya Chromosome Tied t Human Sex Marker. Los Angeles Times Jan 24, 2004 http://www.latimes.com/news/nation/la-sci- papaya24jan24,1,2785009.story Milius, Susan. Dawn of the Y. Papaya: Glimpse of early sex chromosomes. SCIENCE NEWS This Week. January 24, 2002 vol 165. pp52-53. Proffitt, Fiona. Papayas' Sexy Secret. AAAS Science now. www.sciencenow. sciencemag.org/cgi/content/full/2004/121/2 Daily News Coverage 21 January 2004. Selim, Jocelyn. A Fruity Look at the Origin of Man. Discover; May2004, Vol. 25 Issue 5, p.16 Schenck, Susan. Molecular biology Serving Ag. Agriculture Hawaii. April- June 2004: p.15. Thompson, T. Replanting papaya: New tactics may cut
costs. The Produce News. Jan. 26, 2004. p.1. <www.producenews.com/index.html>. Wang, M-L. Producing high-value products. Agriculture Hawaii. 2004. p. 15. Wood, Marcia. Papaya Sex-Chromosome Study Provides New Glimpse of Evolution. Agricultural Research , January 23, 2004. Wood, Marcia. Flavorful Coffees and Creamy Macadamia Nuts. Agricultural Research, June 2004: p.18-20. Wood, Marcia. Papaya! High-tech tactics enhance a tropical favorite. Agricultural Research, January 2004: p.4-7 and cover.
Impacts
(N/A)
Publications
- Yu, Q., Alvarez, M., Moore, P.H., Zee, F.T., Kim, M.S., De Silva, A., Hepperly, P.R., Ming, R. 2003. Molecular diversity of Ralstonia solanacearum ginger strains. Phytopathology 93: 1124-1130. 2003.
- Yu, Q., Ming, R., Moore, P.H. 2003. Cloning and characterization of flower development genes in papaya. 15th Annual CTAHR Student Research Symposium (Abstracts) p.30. 2003.
- Yu, Q., Moore, P.H., Ackerman, C.M., Ming, R. 2004. Cloning and characterization of floral homeotic genes in papaya. Plant Animal and Microbe Genomes Conference XII. P597, p. 220. 2004
- Zhou, F., Wang, M., Albert, H.H., Moore, P.H., Zhu, Y.J. 2003. Production of human GM-CSF protein using PVX vector on N. benthamiana plants. Compiled Abstracts for Annual Meeting of American Society of Plant Biologists in Hawaii July 2003. Plant Biology 2003: P810, pg 171. 2003.
- Zhu, Y.J., Agbayani, R., Moore, P.H. 2004. Green fluorescent protein, as a positive selection marker, improves the efficiency of papaya (carica papaya l.) transformation while avoiding selection for antibiotic resistance. Plant Cell Reports 22: 660-667. 2004
- Zhu, Y.J., Agbayani, R., Moore, P.H. 2003. Green fluorescent protein as a sole selectable marker facilitates genetic transformation of papaya (Carica papaya L.). 7th International Congress of Plant Molecular Biology, Barcelona, Spain. p388, Abstract S26-63. 2003
- Zhu, Y.J., Qiu, X., Moore, P.H., Borth, W., Hu, J., Ferreira, S., Albert, H.H. 2004. Systemic aquired resistance induced by BTH in papaya. Physiological and Molecular Plant Pathology 63(5): 237-248.
- Fitch, M., Leong, T., Saito, N., Yamamoto, G., Dela Cruz, A., Yeh, A., White, S., Maeda, S., Ferreira, S., Moore, P. H. Photoautotrophic rooting and Growth of Papayas In Vitro. Am. Soc. Plant Physiol.(Abs.) p. 572. pg. 131. 2003.
- Fitch, M.M., Leong, T., Saito, N., Yamamoto, G., Dela Cruz, A., Yeh, A., White, S., Maeda, S.H., Moore, P.H. 2004. Photoautotrophic medium and ppm(tm) help to alleviate losses from bacterial contamination in papaya micropropagation. In Vitro Cell Dev Biol (2004) 40:32A.
- Liu, Z., Moore, P.H., Ma, H., Kim, M., Makandar, R., Pearl, H., Charlton, J., Ackerman, C., Yu, Q., Stiles, J.I., Fitch, M.M., Paterson, A.H., Ming, R. 2003. The genetic basis of sex determination in papaya. Am. Soc. Plant Physiol. (Abs.) P501, pg. 118. 2003.
- Fitch, M. M. M., Leong, T., Saito, N., Yamamoto, G., Dela Cruz, A., Yeh, A. , White, S., Maeda, S. H.., Ferreira, S., Moore, P. H. Control of bacterial contamination in large scale papaya micro propagation. In Vitro Cell Develop Biology - Plants 39 (Abs) : 19A. 2003
- He, X., Miyasaka, S.C., Fitch, M.M., Zhu, Y.J., Moore, P.H. 2003. Development of a genetic transformation and regeneration system for taro. American Society of Plant Physiology. (Abs.) P647. p.144. 2003.
- Meng, B., Credi, R., Petrovic, N., Tomazic, I., Gonsalves, D. 2003. Antiserum to recombinanat virus coat protein detects Rupestris stem pitting associated virus in grapevines. Plant Disease. 87:515-522.
- Petrovic, N., Meng, B., Ravnikar, M., Mavric, I., Gonsalves, D. 2003. First detection of Rupestris stem pitting associated virus particles by antibody to a recombinant coat protein. Plant Disease. 87:510-514.
- Wakui, C., Akiyama, H., Watanabe, T., Fitch, M.M., Uchikawa, S., Ki, M., Takahashi, K., Chiba, R., Fujii, .., Hino, A., Maitani, T. 2004. A histochemical method using a substrate of beta-glucuronidase for detection of genetically modified papaya. Journal of Food Hygenics Society of Japan. 45 (1): 19-24.
- Wang, M., Goldstein, C.S., Moore, P.H., Albert, H.H. 2003. Toward introduction of single-copy transgenes in sugarcane. (Abs.). Annual Meeting of American Society of Plant Biologists. pg. 194. 2003.
- He, X., Miyasaka, S., Fitch, M.M., Zhu, Y.J., Moore, P.H. 2004. Transformation of taro (Colocasia esculenta) with a rice chitinase gene. In Vitro Cell and Development Biology. 40:68-A. Abstract p-2104. 2004.
- Steiger, D.L., Moore, P.H., Zee, F.T., Liu, Z., Ming, R. 2003. Assessment of genetic diversity in macadamia by AFPL markers. Euphytica 132/2:269-277. 2003.
- Gonsalves, D., Gonsalves, C., Ferreira, S., Pitz, K., Fitch, M.M., Manshardt, R., Slightom, J. 2004. Transgenic virus resistant papaya: from hope to reality for controlling of papaya ringspot virus in Hawaii. Phytopathology Supplement; APSnet (Plant Pathology Online). APSnet Feature, American Phytopathological Society. Available: http://www.apsnet. org/online/feature/ringspot/.
- Ling, K., Zhu, H., Gonsalves, D. 2004. Complete nucleotide sequence and genome organization of grapevine leafroll-associated virus-3, type member of the genus Ampelovirus. Journal of General Virology. 85:2099-2102.
- Gonsalves, D., Fermin, G. 2004. The use of transgenic papaya to control papaya ringspot virus in Hawaii and technology transfer to other countries, In: Christou, P., Klee, H., editors. Handbook of Plant Biotechnology. London: John Wiley & Sons. p. 1165-1182.
- Gonsalves, D. 2002. Transformation of carica papaya l. with virus coat protein genes for studies on resistance to papaya ringspot virus from jamaica. Tropical Agriculture (Trinidad). 79:105-113.
- Gonsalves, D. 2003. Transgenic papaya: A case for worldwide control of papaya ringspot virus. Crop Protection Council British Proceedings. p. 1029-1034.
- Gonsalves, D. 2003. Commercialization of transgenic papaya: Weighing benefits and potential risks. Proceedings of the OECD Workshop on Dissemination of GMOS in Agro-Ecosystems, September 27-28, 2002, Grossrussbach, Austria. p. 131-137.
- Gonsalves, D. 2002. Transgenic papaya: a case study on the theoretical and practical application of virus resistance. In: Vasil, K. editor. Proceedings of the Tenth International Association for Plant Tissue Culture and Biotechnology Congress, June 23-28, 2002, Orlando, Florida. p. 115-118.
- Meng, B., Gonsalves, D. 2003. Rupestris stem pitting associated virus: genome organization, heterogeneity, rapid detection, and relationships with other plant viruses. Research Trends. Current Topics in Virology. 3:125-135. Research Trends.
- Fuchs, M., Chirco, E.M., Gonsalves, D. 2004. Movement of coat protein genes from a commercial virus-resistant transgenic squash into a wild relative. Environmental Biosafety Research. 3(1):5-16.
- Fuchs, M., Chirco, E.M., McFerson, J.R., Gonsalves, D. 2004. Comparative fitness of a wild squash species and three generations of hybrids between wild x virus-resistant transgenic squash. Environmental Biosafety Research. 3(1):17-28.
- Gonsalves, D. 2003. The papaya story: a special case or can it be generic?. In: Eaglesham, A., Hardy, Ristow, S., editors. Science & Society at a Crossroad, National Agricultural Biotechnology Council Report 15, June 1-3, 2003, Seattle, Washington. p. 223-233.
- Gonsalves, D., Ferreira, S. 2003. Transgenic papaya: A case for managing risks of papaya ringspot virus in Hawaii. Plant Health Progress. doi:10. 1094/PHP-2003-1113-03-RV.
- Zhu, Y.J., Tang, C.S., Ferreira, S.E., Fitch, M.M., Gonsalves, D., Moore, P.H. 2003. Developing transgenic resistance to fungal diseases in papaya (carica papaya l.). International Congress of Plant Pathology Abstracts and Proceedings. v.2, P12.3. p.169.
- Qui, X., Zhu, Y.J., Wang, M.L., Ming, R., Moore, P.H., Gonsalves, D., Albert, H.H. 2003. Isolation and characterisation of genes involved in papaya (Carica papaya l.) systemic acquired resistance. International Congress of Plant Pathology Abstracts and Proceedings. v.2, P12.3. p.169.
- Albert, H.H., Qiu, X., Wang, M., Moore, P.H. 2003. Survey of BTH-induced genes in papaya by suppression subtractive hybridization. West Sect ASPB Oct. 10-11. Plant Genomics 2003: p. 21. 2003.
- Albert, H.H., Wang, M., Goldstein, C., Lemaux, P., Yu, X., Moore, P.H. 2004. Does single-copy transgene introduction reduce the frequency of PTGS in sugarcane? Plant, Animal and Microbe Genome Conference XII Proceedings. W125, p. 36.
- Liu, Z., Moore, P.H., Ma, H., Ackerman, C.M., Ragiba, M., Yu, Q., Pearl, H. M., Kim, M.S., Charlton, J.W., Stiles, J.I., Zee, F.T., Paterson, A.H., Ming, R. 2004. A primitive y chromosome in papaya marks incipient sex chromosomes evolution. Nature 427: 348-352. 2004
- Liu, Z., Moore, P.H., Ackerman, C.M., Yu, Q., Paterson, A.H., Ming, R. 2004. A primitive Y chromosome in papaya reveals that sex chromosomes evolve from autosomes. Plant Animal and Microbe Genomes Conference XI. P598, p.220. 2004
- Ma, H., Moore, P.H., Liu, Z., Kim, M.S., Yu, Q., Fitch, M.M., Sekioka, T., Paterson, A.H., Ming, R. 2003. High-density linkage mapping revealed suppression of recombination at the sex determination locus in papaya. Genetics 166: 419-436. 2004.
- Ma, H., Schulze, S., Lee, S., Mirkov, E., Irvine, J., Moore, P.H., Paterson, A. 2004. An est survey of the sugarcane transcriptome. Theoretical and Applied Genetics 108:851-863. 2004.
- Pearl, H., Nagai, C., Moore, P.H., Steiger, D., Osgood, R., Ming, R. 2003. Construction of a genetic map for Arabica coffee. Theoretical and Applied Genetics 108:829-835. 2004.
- Manshardt, R.M., Moore, P.H. 2003. Natural history of papaya and the caricaceae. Plant Biology 2003: P842, pg 177.2003.
- McCafferty, H.R., Moore, P.H., Zhu, Y.J. 2003. Towards improved insect resistance in papaya. Compiled Abstracts for Annual Meeting of American Society of Plant Biologists in Hawaii July 2003. Plant Biology 2003: P731, pg 158.
- McCafferty, H., Moore, P.H., Zhu, Y.J. 2004. Towards improved insect resistance of papaya by transgenic expression of snowdrop lectin. 2004 World Congress on In Vitro Biology, 40, p52-A, abstract P-2039.
- Ming, R., Moore, P.H., Liu, Z., Ma, H., Yu, Q., Kim, M.S., Fitch, M.M., Sekioka, T. 2003. Papaya genomics. Compiled Abstracts for Annual Meeting of American Society of Plant Biologists in Hawaii July 2003. Plant Biology 2003: P999, pg 203.2003.
- Schenck, S., Crepau, M.W., Wu, K.K., Moore, P.H., Yu, Q., Ming, R. 2004. Genetic diversity and relationships of native hawaiian saccharum officinarum sugarcane. Journal of Heredity 95:327-331. 2004.
- Schenck, S., Crepeau, M.W., Wu, K.K., Moore, P.H., Yu, Q., Ming, R. 2003. Native hawaiian Saccarum officinarum sugarcanes: Genetic diversity and relationship to modern commercial Saccarum hybrid cultivars. 4th International Society of Sugarcane Technologists Molecular Biology Workshop. p.29. 2003.
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