Progress 10/01/13 to 09/30/17
Outputs
Target Audience:Target Audience 1) Professional colleagues and students in Dept. of Tropical Plant & Soil Sciences, UH Manoa, via MS thesis defense presentation by graduate student Greg Hoover in December 2016. 2) Professional colleagues and students in Dept. of Molecular Biosciences and Bioengineering, UH Manoa, MS thesis defense presentation by graduate student Yosuke Kawai in April 2017. 3) Professional colleagues and student members of the American Society for Horticultural Science at the annual meeting via poster presentation of "Intergeneric Hybridization of Carica Papaya L. with Vasconcellea Wild Relatives" in September 2017 at Waikoloa, HI. 4) Professional colleagues and student members at the annual meeting of the American Society for Horticultural Science via Gabriel Sachter-Smith's oral presentation of "Naturally Occurring Host Resistance to Banana Bunchy Top Virus" in September 2017 at Waikoloa, HI. 5) Members of the Hawaii Tropical Fruit Growers Association at their annual meeting in Hilo, HI, in September 2017, where I presented a poster "Intergeneric Hybridization of Carica Papaya L. with Vasconcellea Wild Relatives" and provide banana germplasm for taste trial. Changes/Problems:Objective 2. The overall goal of Obj. 2 remains to improve economically important characteristics of papaya through introduction of new germplasm using conventional breeding methods. However, the approach has changed. With the award of Supplemental Hatch funding in 2014, the project refocused on germplasm that offers a greater range of new traits, including better sources of resistance to papaya ringspot virus (PRSV), the former main goal. Wild relatives of papaya can provide genes for fruit exocarp color, flavors and fragrances, as well as disease resistances, and these make a more attractive breeding target than relying on the single conspecific variety, 'Cariflora', which offers only moderate levels of multigenic resistance to PRSV. The major drawback to the new approach is hybrid sterility, which the project will seek to remedy by chromosome doubling to create fertile allotetraploids. Flow cytometry confirmed that chromosome-doubling treatment of apical meristems with oryzalin was effective in producing tetraploid sectors in hybrid plants, but the conversion was transient, with subsequent testing showing a resumption of diploid growth. These results indicate that the treatments did not sufficiently convert the entirety of the apical meristem and point to the need to develop a more effective treatment procedure. What opportunities for training and professional development has the project provided?The project has provided research objectives, mentoring, and financial support for three MS graduate students in the Department of Tropical Plant and Soil Sciences (TPSS) and one MS student in the Department of Molecular Biosciences and Bioengineering (MBBE) at UH Manoa. TPSS student Gabriel Sachter-Smith completed his MS thesis, "In Search of Naturally Occurring Host Resistance to Banana Bunchy Top Virus," and graduated in August 2015. He screened a banana germplasm collection consisting of 60 commercial clones and wild species for naturally occurring resistance to banana bunchy top virus (BBTV). He has received training in field maintenance and evaluation of horticultural crops, virus inoculation with insect vectors, disease diagnosis by field symptoms and molecular assay, and experimental design. Another TPSS student, David Shepard, managed the Pic-a-Papaya cell phone application and compiled and analyzed survey data to determine the distributions of papaya ringspot virus (PRSV) and virus-resistant transgenic papayas in Honolulu. He was trained in survey methods, diagnosis of PRSV symptoms, and assay methods to detect transgenic papaya plants. He completed his MS Plan B program and delivered his research defense seminar, "Relating PRSV Incidence to Presence of GE Papayas in Spontaneous Papaya Populations in Honolulu," in December 2015. A third TPSS student, Gregory Hoover, produced interspecific and intergeneric hybrids in the Caricaceae (papaya family) and investigated the effect of chromosome doubling and allotetraploidy on restoration of reproductive fertility. He received training in plant breeding methods, embryo culture, ploidy manipulation, cytology, flow cytometry, and isozyme electrophoresis. He defended his MS thesis "Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives" and graduated in December 2016. The project also funded research and provided oversight for Yosuke Kawai, an MS student in the MBBE Department, who developed qPCR probes and DNA extraction methods to determine the proportion of male-pollinated versus hermaphrodite-pollinated embryos in bulked papaya seed batches. Yosuke received training in DNA extraction from large tissue volumes, fluorophore-labeled DNA probe design, and qPCR assay methodology. He completed his MS thesis "Developing a Methodology to Determine the Relative Effectiveness of Male and Hermaphrodite Papayas in Cross-Pollination " and graduated in May 2017. In addition to supporting graduate student education and training, the program maintained a banana germplasm collection at the Waimanalo Experiment Station that served as a teaching resource for instruction of undergraduate students enrolled in TPSS403 Tropical Fruit Production during spring 2015 and a papaya breeding project, also at the Waimanalo Station, that provided field laboratory instruction for undergraduate students enrolled in TPSS453 Plant Breeding and Genetics during fall 2016. How have the results been disseminated to communities of interest?Our professional colleagues in Plant Sciences were targeted by the poster and oral presentations made by two MS graduate students involved in the project at the CTAHR Student Symposium in April, 2014. Gabriel Sachter-Smith presented an oral summary of progress in evaluating banana germplasm for resistance to BBTV under field conditions at Waimanalo Experiment Station. Gregory Hoover's poster summarized his progress in creating Vasconcellea interspecific hybrids and evaluating their fertility. The targeted audience consisted of over 250 CTAHR faculty and students. The goals and results of Objective 1 were presented in May 2014 to the 30 members of the professional working group WERA-20: Virus and Virus-Like Diseases of Fruit Trees, Small Fruits, and Grapevines, using the banana germplasm collection at the Waimanalo Experiment Station as a virus disease demonstration plot for the visitors. The goals and results of Objective 1 were also presented on August 26, 2014, at a Banana Mini-Conference sponsored by the Cooperative Extension Service for commercial banana growers on Oahu. Approximately 250 professional colleagues and students attending the CTAHR Student Research Symposium, April 10-11, 2015, were again informed of the project's progress through presentations by MS graduate students Gregory Hoover, Gabriel Sachter-Smith, and David Shepard. A smaller audience of 35 faculty and students attended Gabriel Sachter-Smith's MS thesis defense, "In Search of Naturally Occurring Resistance to BBTV," on June 25, 2015, in the Tropical Plant & Soil Sciences Dept., University of Hawaii at Manoa. The objectives and results from the BBTV screening program, as well as a descriptive tour of the project's banana germplasm collection was presented to 15 UH Manoa undergraduate students enrolled in TPSS403 Tropical Fruit Production at a field trip presentation by MS graduate student Gabriel Sachter-Smith on Feb. 13, 2015. Ten local home gardeners, primarily in urban Honolulu, participated as "citizen scientists" in our PRSV survey via the Pic-a-Papaya phone app and associated internet links during the reporting period. The project objectives and results were presented to small audiences of about 20 faculty and students in the Dept. of Tropical Plant and Soil Sciences who attended David Shepard's MS Plan B defense seminar "Relating PRSV Incidence to Presence of GE Papayas in Spontaneous Papaya Populations in Honolulu" on Dec. 9, 2015, and Gregory Hoover's MS Plan A research proposal seminar, "Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives" on January 15, 2016. Approximately 250 professional colleagues and students attending the CTAHR Student Research Symposium, University of Hawaii at Manoa, April 8-9, 2016, were again informed of the project's progress with Objective 2 through MS graduate student Gregory Hoover's oral presentation, "Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives." Project objectives and progress were presented to the following audiences during the most recent (2016-17) reporting period: 1) Professional colleagues and students in Dept. of Tropical Plant & Soil Sciences, UH Manoa, via MS thesis defense presentation, "Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives" by graduate student Greg Hoover in December 2016; 2) Professional colleagues and students in Dept. of Molecular Biosciences and Bioengineering, UH Manoa, MS thesis defense presentation by graduate student Yosuke Kawai in April 2017; 3) Professional colleagues and student members of the American Society for Horticultural Science at the annual meeting via poster presentation of "Intergeneric Hybridization of Carica Papaya L. with Vasconcellea Wild Relatives" in September 2017 at Waikoloa, HI; 4) Professional colleagues and student members at the annual meeting of the American Society for Horticultural Science via Gabriel Sachter-Smith's oral presentation of "Naturally Occurring Host Resistance to Banana Bunchy Top Virus" in September 2017 at Waikoloa, HI; and 5) Members of the HawaiiTropical Fruit Growers Association at their annual meeting in Hilo, HI, in September 2017, where I presented a poster "Intergeneric Hybridization of Carica Papaya L. with Vasconcellea Wild Relatives" and provided banana germplasm for taste trial. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: The high quality banana and papaya industries of Hawaii are threatened by viral diseases. Banana bunchy top virus (BBTV) has eliminated production of susceptible bananas on Oahu, and papaya ringspot virus (PRSV) necessitates growing papaya varieties with GMO resistance. GMOs are safe and effective, but unpopular with consumers. We screened 60 exotic banana varieties to find 4 that combine BBTV resistance with good commercial qualities. We also crossed papaya with 11 distantly related wild relatives and rescued hybrid embryos from 5 species to take advantage of naturally occurring PRSV resistance. The selected BBTV-resistant banana varieties are currently being propagated for demonstration plots and chefs' evaluations prior to making them available to commercial growers. The papaya x wild relative hybrids represent new genetic potential for papaya improvement, and they are currently being treated with chromosome-doubling agents to increase their fertility. Objective 1. Identify exotic banana clones with superior fruit quality and resistance to banana bunchy top virus. 1) Major activities The banana collection, consisting of 6 replications of 41 clones, was planted at Waimanalo Experiment Station in March 2012 and exposed continuously to natural populations of virus-vectoring aphids and, starting in August 2012, to viruliferous aphids taken from BBTV-infected plants. At the end of the 31-month field trial, at least 3 replicates of 5 promising clones were moved into a greenhouse for more stringent BBTV screening to detect any field escapes. These (and 19 new clones) were inoculated by manually transferring 10 viruliferous aphids to each replicate, allowing aphids to feed for 3 days, and enclosing them within insect-proof exclusion cages. BBTV symptom development was evaluated for 3 months using Dwarf Cavendish as a susceptible control. 2) Data collected BBTV symptom data were collected for 5 or 6 reps of 41 clones in the field trial. Almost all clones had at least one rep showing BBTV symptoms, but the rate and extent of disease development varied importantly between clones. Cavendish clones were among the most susceptible. Clones with few symptomatic reps and slow systemic involvement included varieties of Gros Michel (Cocos and Highgate) and Pisang Awak (Kayinja and an unidentified clone ITC0459). Suckers collected from apparently healthy plants of the Pisang Awak clones after 4.5 years of BBTV exposure at Waimanalo were 90% free of BBTV by PCR assay. Several locally sourced wild species remained completely symptomless, including Musa acuminata ssp. malaccensis. Greenhouse trials confirmed field results, with ITC0459 and Kayinja being most resistant, Cocos and Highgate being intermediate, and the Dwarf Cavendish control being least resistant. 3) Discussion of results Four banana clones were identified in our field trial as having the most promising combination of horticultural qualities and BBTV resistance. Bunch weights ranged from 36.5 kg (Cocos) to 21.6 kg (Kayinja) and the disease-free production window between first bunch harvest and first BBTV symptoms ranged from 555 days (Kayinja) to 294 days (ITC0459), compared with current Hawaii commercial varieties Dwarf Cavendish (23.7 kg and 115 days) and Dwarf Brazilian (13 kg and 237 days). Longer disease-free production periods result in greater profit margins. 4) Key outcomes Field trials identified useful levels of naturally occurring resistance to BBTV in 4 varieties of banana with good horticultural characteristics, and greenhouse experiments supported the field results. Objective 2. The goal remains to improve papaya through introduction of new germplasm, but the approach has changed. See Changes Problems section. 1) Major activities Papaya is the only Carica species, so our new germplasm consisted of wild relatives in other genera of the papaya family Caricaceae. Eleven species were obtained as seeds or vegetative cuttings from the USDA National Clonal Germplasm Repository, Hilo, HI. The wild species were used to cross-pollinate papaya, and rare hybrid embryos were rescued by tissue culture from ovules that always lacked endosperm. Six wild species produced viable hybrids, 5 of which were established at least temporarily in greenhouse conditions. Isozyme analyses confirmed the hybrid origin of all putative intergeneric plants. Chromosome doubling technology using the mitotic inhibitor oryzalin was applied to intergeneric hybrids, and the effect of treatment was assessed by flow cytometry. 2) Data collected Six of the 11 wild species produced intergeneric hybrids by embryo rescue in crosses with papaya. Isozyme systems MDH and PGM generated clear and distinctive markers that helped confirm the authenticity of interspecific and intergeneric hybrids. Only C. papaya x V. goudotiana produced flowers with anthers, and acetocarmine staining revealed pollen sterility. In oryzalin-treated meristems of intergeneric hybrids, polyploidy was only transiently induced, and 4n sectors reverted to 2n. Despite the incomplete meristem conversion, one oryzalin-treated V. quercifolia x V. parviflora male plant produced flowers in a transient 4n sector that were visibly larger and shed pollen in amounts visible to the unaided eye, unlike the original F1. Microscopic examination revealed pollen grains of increased size consistent with 2n pollen. However, the pollen did not display an increased percentage of stainable grains (still ~20%). 3) Discussion of results The apparent genome compatibility of individual wild species with the "solo" type papayas used in our crosses was reflected in the viability and vigor of the resulting hybrids. Crosses between papaya and Vasconcellea glandulosa, V. horovitziana, V. pulchra, or Horovitzia cnidoscoloides were unsuccessful. Embryos of C. papaya x V. parviflora did not develop beyond polyembryonic clusters in tissue culture. Only 2 plants each of C. papaya x V. monoica and C. papaya x V. stipulata made the transition from embryo culture to greenhouse, and these were similar in their weak growth. Embryos of C. papaya with V. goudotiana, V. pubescens, V. quercifolia, or V. x heilbornii were no easier to produce than the previously mentioned crosses, but they developed nearly normally in tissue culture and, with the exception of quercifolia hybrids that were lost due to contamination, the other crosses resulted in vigorous hybrids in the greenhouse. Flow cytometry confirmed that oryzalin treatment of apical meristems was effective in producing 4n sectors in hybrid plants. However, their transient nature indicated incomplete conversion of the apical meristem and the need to develop a more effective treatment procedure. 4) Key outcomes Carica x Vasconcellea genomes were merged in 6 intergeneric hybrids produced by embryo rescue. The 4 surviving hybrids offer new genes for papaya improvement, if sterility barriers can be overcome. Objective 3. Introduce new banana clones at demonstration plot field days for chefs and growers 1) Major activities An extension demonstration plot displaying new banana germplasm was planted at the Kona Experiment Station in September 2013. The demonstration clones included the Gros Michael clones Cocos and Highgate, the Pisang Awak clone Kayinja, and the FHIA clones 2, 17, 21 and 23. Distributions of virus-free banana germplasm: 1) Kayinja plants to Hawaii Agriculture Research Center to initiate tissue cultures for a private client (April 2017); 2) Kayinja and ITC0459 to UH colleagues in Clean Plant Network for mericloning for eventual distribution to growers; 3) Wild M. acuminate ssp. malaccensis clone, identified as BBTV-resistant in our research, to world Musa germplasm collection (Bioversity International, Leuven, Belgium) for preservation and potential use in banana breeding programs. 2) Key outcomes Tissue cultures of BBTV-resistant Kayinja and ITC0459 have been initiated locally to accelerate propagation and distribution to growers.
Publications
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2016
Citation:
Hoover, G. 2016. MS thesis. Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives. Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Manshardt, R. 2017. Intergeneric Hybridization of Carica papaya L. with Vasconcellea Wild Relatives. American Society for Horticultural Science annual meeting, Waikoloa, HI, Sept. 2017. (oral presentation).
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2016
Citation:
Kawai, Y. 2017. MS thesis. Developing a Methodolgy to Determine the Relative Effectivness of Male and Hermaphrodite Papayas in Cross-Pollination. Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Sachter-Smith, G. 2017. Naturally Occurring Host Resistance to Banana Bunchy Top Virus. American Society for Horticultural Science annual meeting, Waikoloa, HI. (oral presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Manshardt, R. 2017. Intergeneric Hybridization of Carica papaya L. with Vasconcellea Wild Relatives. Hawaii Tropical Fruit Growers Association annual meeting, Hilo, HI, Sept. 2017. (poster).
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:1) Local home gardeners, primarily in urban Honolulu, via Pic-a-Papaya phone app and associated internet links by MS graduate student David Shepard from October through December 2015. 2) Professional colleagues and students in Dept. of Tropical Plant & Soil Sciences, UH Manoa, via MS defense presentation by graduate student David Shepard in December 2015, and MS research proposal seminar by graduate student Gregory Hoover in January 2016. Greg also made an oral presentation of his research progress at the CTAHR Student Research Symposium, April 8-9, 2016. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, the project has provided research objectives and financial support for two MS graduate students in the Department of Tropical Plant and Soil Sciences at UH Manoa. One student completed his MS thesis during the reporting period, employing the Pic-a-Papaya cell phone application to compile and analyze survey data to determine the incidence of papaya ringspot virus (PRSV) and virus-resistant transgenic papayas in Honolulu. He was trained in survey methods, diagnosis of PRSV symptoms, and assay methods to detect transgenic papaya plants. A second student is making interspecific and intergeneric hybrids in the Caricaceae (papaya family) and investigating the effect of chromosome doubling and allotetraploidy on restoration of reproductive fertility. He is receiving training in plant breeding methods, embryo culture, ploidy manipulation, cytology, flow cytometry, and isozyme electrophoresis. In addition to supporting graduate student education and training, the program maintains a papaya breeding project at the Waimanalo Experiment Station that provided field laboratory instruction for undergraduate students enrolled in TPSS453 Plant Breeding and Genetics during September 2016. How have the results been disseminated to communities of interest?The project objectives and results were presented to small audiences of about 20 faculty and students in the Dept. of Tropical Plant and Soil Sciences who attended David Shepard's MS Plan B defense seminar "Relating PRSV Incidence to Presence of GE Papayas in Spontaneous Papaya Populations in Honolulu" on Dec. 9, 2015, and Gregory Hoover's MS Plan A research proposal seminar, "Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives" on January 15, 2016. Approximately 250 professional colleagues and students attending the CTAHR Student Research Symposium, University of Hawaii at Manoa, April 8-9, 2016, were informed of the project's progress through MS graduate student Gregory Hoover's oral presentation, "Broadening the Genetic Base of Papaya via Intergeneric Hybridization with Wild Relatives." A small collection of wild Vasconcellea species at the UH Waimanalo Experiment Station was used to explain the breeding objectives of the project to students in TPSS453 Plant Breeding and Genetics during a September 2016 field lab. What do you plan to do during the next reporting period to accomplish the goals?Research Objective 1: ABB Pisang Awak clones 'Kayinja' ITC 0087 and unnamed ITC0459, two of the most promising clones from the standpoint of horticultural quality and BBTV-resistance, are being maintained in a glasshouse at the UH Magoon Horticulture Facility. The AAA Gros Michel mutants 'Cocos' ITC 0451 and 'Highgate' ITC 0263, as well as a locally sourced wild banana (M. acuminata ssp. malaccensis), which may have potential for banana breeding programs, need to be similarly collected and maintained. All clones need to be PCR-assayed to assure that they are BBTV free. Research Objective 2: With the graduation of MS assistant Gregory Hoover at the beginning of the next reporting period, emphasis will shift from making crosses and rescuing embryos to establishing and vegetatively cloning hybrids in the greenhouse, where they can be characterized morphologically and treated to restore fertility by chromosome doubling. Extension Objective 3: There has been no progress in this objective since last reporting period, so our plans for the next reporting period remain the same. We will produce online extension summaries of results from screening the banana germplasm collection for BBTV resistance and for the Pic-a-Papaya phone app survey of PRSV and GMO papayas in Honolulu. For growers on Oahu, a small demonstration planting of the four selected banana clones with BBTV resistance will be installed at Waimanalo Experiment Station, along with standard commercial clones 'Williams' and 'Dwarf Brazilian.'
Impacts
What was accomplished under these goals?
Banana and papaya production are mature industries in Hawaii that offer high quality, high value fruits to local and international markets. Both crops are negatively affected by virus diseases. In the case of banana, banana bunchy top virus (BBTV) threatens fruit production. Our approach to improve the banana situation has been to screen new crop resources for naturally occurring resistance to BBTV. In previous reporting periods, a banana collection of 77 genotypes was screened in glasshouse conditions for new sources of resistance to BBTV and under field conditions for tolerance of infection and horticultural quality. Four clones were identified with good quality and significantly better BBTV resistance than standard 'Williams' and 'Dwarf Brazilian' commercial clones. The best selections will be made available in demonstration plots for growers and chefs to evaluate. In papaya, effective control of papaya ringspot virus (PRSV) has necessitated GMO resistance, a benign technology that nonetheless has undermined consumer demand. Our approach to improve the papaya situation is to employ resistance genes in related wild species in the genus Vasconcellea. Major hurdles to utilizing Vasconcellea species in papaya improvement involve overcoming breeding barriers with the papaya genus Carica. Our project has used 1) standard tissue culture methods to rescue and grow rare hybrid embryos on artificial nutrient medium, and 2) the chemical oryzalin for treating sexually sterile hybrids to induce chromosome doubling and restore fertility. In the current reporting period, four new Carica x Vasconcellea hybrids, three involving PRSV-resistant Vasconcellea species, have been produced. If chromosome-doubling can restore fertility, a seed-based breeding program for the doubled-chromosome papaya hybrids will provide the long term means to recombine the best traits of both genera. Research Objective 1. Identify exotic banana clones with superior fruit quality and resistance to banana bunchy top virus: Field and glasshouse surveys of BBTV resistance in banana germplasm have been completed and results were provided in previous reporting periods. No new information in this reporting period. Research Objective 2. Use papaya cultivar 'Cariflora' in a breeding program to improve non-transgenic tolerance to papaya ringspot virus in high quality papayas for export and local consumption: The overall goal of Obj. 2 remains the same, but the approach has changed to utilize wild relatives of papaya, primarily from the genus Vasconcellea. Two previously reported C. papaya x V. monoica hybrids were established in the greenhouse, but both of these were weak, ceased apical growth, and eventually died before flowering. To date, about a dozen C. papaya x V. goudotiana hybrids have been established in the greenhouse and six have flowered. Two reciprocal V. goudotiana x C. papaya hybrids were weaker plants and have subsequently died. During the present reporting period, four more Carica x Vasconcellea intergeneric hybrids have been produced, including crosses with V. cundinamarcensis, V. x heilbornii, V. quercifolia and V. stipulata. All of these wild species except V. x heilbornii, a polymorphic taxon comprising clonal 'babaco' and semi-sterile cultigens, are reported to have resistance to PRSV, making the hybrids particularly important. All hybrids except C. papaya x V. quercifolia are currently established in the greenhouse, where C. papaya x V. cundinamarcensis and C. papaya x V. x heilbornii are displaying normal development and vigorous growth. The C. papaya x V. stipulata hybrids are less vigorous and after a period of normal vegetative growth, exhibit an apical die-back similar to that observed in C. papaya x V. monoica hybrids. In preliminary experiments treating C. papaya seedling meristems with oryzalin, flow cytometry analysis confirmed a success rate of about 25% (12/52 seedlings) in creating tetraploid or mixoploid vegetative growth. This chromosome doubling methodology has been applied to all of the intergeneric hybrids during the current reporting period, and the outcome is under investigation. Extension Objective 3. Introduce new banana clones at demonstration plot field days for chefs and growers: No progress during the current reporting period.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Hoover, G. and Manshardt, R. 2016. Broadening the genetic base of papaya via intergeneric hybridization with wild relatives. 28TH ANNUAL CTAHR and COE STUDENT RESEARCH SYMPOSIUM, University of Hawaii at Manoa.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:1) Local home gardeners, primarily in urban Honolulu, via Pic-a-Papaya phone app and associated internet links, by Richard Manshardt from October 2014 through January 2015, and by David Shepard from Feb. through Sept 2015. 2) Professional colleagues in CTAHR via presentations by MS graduate students Gregory Hoover, Gabriel Sachter-Smith, and David Shepard at the CTAHR Student Research Symposium, April 10-11, 2015, and at Gabriel Sachter-Smith's MS thesis defense on June 25, 2015. 3) UH Manoa undergraduate students enrolled in TPSS403 Tropical Fruit Production through a field trip presentation on banana diversity and disease resistance, presented by MS graduate student Gabriel Sachter-Smith on Feb. 13, 2015. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, the project has provided research objectives and financial support for three MS graduate students in the Department of Tropical Plant and Soil Sciences at UH Manoa. One student completed his MS thesis during the reporting period, screening a banana germplasm collection consisting of 75 commercial clones and wild species for naturally occurring resistance to banana bunchy top virus (BBTV). He has received training in field maintenance and evaluation of horticultural crops, virus inoculation with insect vectors and disease diagnosis, and experimental design. A second student is making interspecific and intergeneric hybrids in the Caricaceae (papaya family) and investigating the effect of chromosome doubling and allotetraploidy on restoration of reproductive fertility. He is receiving training in plant breeding methods, embryo culture, ploidy manipulation, cytology, and flow cytometry. The third student is managing the Pic-a-Papaya cell phone application and compiling and analyzing survey data to determine the incidence of papaya ringspot virus (PRSV) and virus-resistant transgenic papayas in Honolulu. He was trained in survey methods, diagnosis of PRSV symptoms, and assay methods to detect transgenic papaya plants. In addition to supporting graduate student education and training, the program maintains a banana germplasm collection at the Waimanalo Experiment Station that served as a teaching resource for instruction of undergraduate students enrolled in TPSS403 Tropical Fruit Production during spring 2015. How have the results been disseminated to communities of interest?Approximately 250 professional colleagues and students attending the CTAHR Student Research Symposium, April 10-11, 2015, were informed of the project's progress through presentations by MS graduate students Gregory Hoover, Gabriel Sachter-Smith, and David Shepard. A smaller audience of 35 faculty and students attended Gabriel Sachter-Smith's MS thesis defense, "In Search of Naturally Occurring Resistance to BBTV," on June 25, 2015, in the Tropical Plant & Soil Sciences Dept., University of Hawaii at Manoa. The objectives and results from the BBTV screening program, as well as a descriptive tour of the project's banana germplasm collection was presented to 15 UH Manoa undergraduate students enrolled in TPSS403 Tropical Fruit Production at a field trip presentation by MS graduate student Gabriel Sachter-Smith on Feb. 13, 2015. Ten local home gardeners, primarily in urban Honolulu, participated as "citizen scientists" in our PRSV survey via the Pic-a-Papaya phone app and associated internet links during the reporting period. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Completion of the greenhouse screening experiments during the reporting period concludes this research objective. Clean propagative plants of the clones most interesting from a combination of horticultural and BBTV-resistance standpoints will be maintained in greenhouse facilities at UH Manoa. These clones include the AAA Gros Michel mutants 'Cocos' ITC 0451 and 'Highgate' ITC 0263 and the ABB Pisang Awak clones 'Kayinja' ITC 0087 and unnamed ITC0459, as well as a locally sourced wild banana (M. acuminata ssp. malaccensis), which may have potential for banana breeding programs. Objective 2: Our future plans for the papaya intergeneric crossing program remain the same. We will continue to concentrate on producing intergeneric F1 hybrids of Carica papaya with the PRSV-resistant wild relatives Vasconcellea cundinamarcensis, V. quercifolia, and V. stipulata, with which we have thus far been unsuccessful. Papaya seedlings and young V. stipulata plants will be planted for this purpose in a field facility near the UH Manoa campus in fall 2015, and we have made arrangements with the USDA National Clonal Germplasm Repository (NCGR) in Hilo, Hawaii, to obtain young V. quercifolia plants for the same field. Vasconcellea cundinamarcensis is poorly adapted to Manoa or Hilo conditions, but we will make crosses with pollen obtained from NCGR plants being grown at higher altitude at Lalamilo, Hawaii. We hope that use of these improved crossing materials will lead to greater success. Vegetative meristems of existing C. papaya x V. goudotiana hybrids, as well as any rescued embryogenic masses obtained from new crosses, will be treated with chromosome-doubling mutagens colchicine or oryzalin to produce allotetraploid plants for fertility comparison with diploids of the same gametic origin. Objective 3: The Extension aspect of our program will produce a summary of the results of the banana germplasm screening for BBTV resistance, and we plan to make it available online through UH CTAHR's Communications Services. In addition to the banana demonstration plot at the Kona Experiment Station on the Big Island, a small demonstration plot containing the BBTV-resistant AAA Gros Michel mutants 'Cocos' ITC 0451 and 'Highgate' ITC 0263 and the ABB Pisang Awak clones 'Kayinja' ITC 0087 and unnamed ITC0459, as well as BBTV-susceptible AAA 'Williams' Cavendish and AAB 'Dwarf Brazilian', will be established at Waimanalo Experiment Station to allow growers on Oahu to evaluate the results of the project. The Pic-a-Papaya phone app survey of PRSV incidence and the extent of transgenic papaya plants in feral populations in Honolulu will be completed in December 2015 when the MS student conducting that project graduates. We will produce a summary of the results and make it available online through UH CTAHR's Communications Services.
Impacts
What was accomplished under these goals?
Objective 1: At the end of the 31-month field trial, suckers of 8 promising clones were moved into a greenhouse environment for more stringent BBTV resistance screening to detect any "escapes" that might have missed infection in the field. After confirming by PCR that the suckers were free of BBTV, corm axilary buds were used to macropropagate young plants. Three plants each of the AAA Gros Michel mutants 'Cocos' ITC 0451 and 'Highgate' ITC 0263 and six plants each of the ABB Pisang Awak clones 'Kayinja' ITC 0087 and an unnamed ITC0459, as well as 4 plants of a locally sourced wild banana (M. acuminata ssp. malaccensis), were inoculated by manually transferring 10 viruliferous aphids to each plant, allowing them to feed for 3 days, and enclosing them within insect-proof exclusion cages. Similar BBTV screens were performed on 3 replicates each of 6 wild species and 13 banana clones obtained directly as tissue cultured plants from the international Musa germplasm collection. BBTV symptom development was evaluated for 3 months in the greenhouse cages, using AAA Dwarf Cavendish as a susceptible control. Among the greenhouse trial of clones that had performed best in the field, the 'Dwarf Cavendish' positive controls were the most sensitive, with all three reps developing severe BBTV symptoms within the first month after inoculation. All three 'Highgate' plants also became infected (PCR positive), but only one developed clear symptoms, and more slowly than in 'Dwarf Cavendish'. Two of three 'Cocos' plants became infected, with both showing clear symptoms after 1 month, while the remaining plant stayed symptomless and PCR-negative throughout the test period. One of six 'Kayinja' plants developed weak symptoms after three months, and the remaining plants stayed symptomless and PCR-negative. All six plants of the unnamed Pisang Awak clone ITC0459 remained symptomless and PCR-negative throughout the test period. Only one of four M. acuminata subsp. malaccensis plants tested positive for BBTV by PCR, but it never showed symptoms. Objective 2: Four dioecious Vasconcellea quercifolia x V. parviflora interspecific F1 hybrids, created in 2012, yielded no progeny from numerous reciprocal backcrosses to V. parviflora. Acetocarmine pollen stainability of the male F1 parent was about 20%. Numerous F1 sib crosses produced only a single F2 that was rescued by embryo culture on MS medium. The F2 hybrid is developing normally in the greenhouse, but has not yet flowered. Another Vasconcellea interspecific mating, V. glandulosa x V. pulchra, produced a mature fruit with seeds containing well developed endosperm, as well as embryos. These mostly failed to germinate normally, but four F1 hybrids were obtained by culturing the ovule contents on MS medium. The F1 hybrids are now established in the greenhouse and are segregating for different leaf morphologies, but only one female has yet flowered. Intergeneric matings of Carica papaya with V. cundinamarcensis, V. quercifolia, V. stipulata, or Horovitzia cnidoscoloides were attempted numerous times, but none of these produced F1 embryos for rescue on artificial culture medium. C. papaya x V. parviflora crosses yielded well formed ovules from which tiny embryo masses developed on MS medium containing 10% coconut water, but none of these developed further. Crosses of C. papaya with V. monoica or V. goudotiana produced a few well formed ovules from which embryo masses were cultured on MS medium with 10% coconut water. Two weak C. papaya x V. monoica F1 hybrids have survived and become established in non-sterile potting medium, but they lack vigor and have not flowered. In contrast, six C. papaya x V. goudotiana (male) F1 hybrids and two reciprocal V. goudotiana x C. papaya (hermaphrodite) F1 hybrids were recovered via embryo rescue, and all are relatively vigorous plants. The intergeneric nature of one of the C. papaya x V. goudotiana F1's was confirmed by electrophoretic analysis of malate dehydrogenase isozymes. Four of the C. papaya x V. goudotiana F1 hybrids have flowered. Three are female with solitary pistillate flowers in the leaf axils, while the fourth has an unusual combination of long, male-like cymose inflorescences bearing predominantly hermaphrodite flowers. It appears that the dioecious sex system of V. goudotiana shifts to a gynodioecious system in intergeneric hybrids with papaya. Acetocarmine staining of the mature anthers revealed only clumps of poorly stained reproductive tissue, rather than well differentiated microspores. The reciprocal V. goudotiana x C. papaya (hermaphrodite) F1 hybrids have not flowered, but PCR analysis detected the presence of the C. papaya hermaphrodite allele in one of them. Objective 3: During the reporting period, the "Pic-a-Papaya" cell phone application was accessed by "citizen scientists" only 10 times, providing minimal information on papaya plants in the Honolulu area. None of the client-provided photos revealed plants with papaya ringspot virus (PRSV) symptoms, although a range of mite, scale, fungal and nutritional problems was diagnosed. Only one client provided leaf samples for GUS assays, which confirmed that 1) the plants in question were transgenic and virus resistant, and 2) client demand for information about GMO status is low. Although Pic-a-Papaya was minimally utilized by the public, it was employed by our program as a field tool to survey feral papaya populations in Honolulu to determine incidence of PRSV and transgenic genotypes. An August 2015 tally of Pic-a-Papaya survey data indicated that 242 plants had been evaluated for PRSV and assayed for GUS expression. Transgenic plants constituted 61% of our survey, 29% were healthy non-transgenic plants, and 10% were PRSV-infected (all non-transgenic).
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Hoover, G. and Manshardt, R. 2015. Broadening the genetic base of papaya through inter-generic hybridization. 27TH ANNUAL CTAHR and COE STUDENT RESEARCH SYMPOSIUM, University of Hawaii at Manoa.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Sachter-Smith, G. and Manshardt, R. 2015. Naturally occurring host resistance to Banana bunchy top virus. 27TH ANNUAL CTAHR and COE STUDENT RESEARCH SYMPOSIUM, University of Hawaii at Manoa.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Sachter-Smith, G., 2015. IN SEARCH OF NATURALLY OCCURRING HOST RESISTANCE TO BANANA BUNCHY TOP VIRUS. MS Thesis, University of Hawaii at Manoa, Honolulu.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Shepard, D., Nelson, S., and Manshardt, R. 2015. Relating PRSV prevalence to distribution of GE and non-GE papayas in Honolulu. 27TH ANNUAL CTAHR and COE STUDENT RESEARCH SYMPOSIUM, University of Hawaii at Manoa.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Our professional colleagues in Plant Sciences were targeted by the poster and oral presentations made by graduate students at the CTAHR Student Symposium in April, 2014. Gabriel Sachter-Smith presented an oral summary of progress in evaluating banana germplasm for resistance to BBTV under field conditions at Waimanalo Experiment Station. Gregory Hoover's poster summarized his progress in creating Vasconcellea interspecific hybrids and evaluating their fertility. The targeted audience consisted of over 250 CTAHR faculty and students who participated in evaluating the quality of research and the effectiveness of communicating results. Gabriel was awarded the prize for best oral presentation of research results by a MS student in the Department of Tropical Plant & Soil Sciences (TPSS). Greg was awarded the prize for best poster presentation by an MS student in TPSS. Changes/Problems: The award of Supplemental Hatch funding at the beginning of the reporting period has refocused Objective 2 on germplasm that offers a greater range of new traits than those of a single cultivar 'Cariflora'. Wild relatives of papaya can provide genes for fruit exocarp color, flavors and fragrances, as well as better sources of resistance to papaya ringspot virus (PRSV) than 'Cariflora', which offers only moderate levels of multigenic resistance to PRSV. The change to utilization of wild species in the papaya improvement project is attractive, because it offers the possibility of more dramatic enhancements. The other change as a result of receiving the Supplemental Hatch funding is the addition of the Pic-a-Papaya phone application project as an extension component. What opportunities for training and professional development has the project provided? The project has provided research objectives and financial support for two MS graduate students in the Department of Tropical Plant and Soil Sciences at UH Manoa. One student is making interspecific and intergeneric hybrids in the Caricaceae and investigating the effect of chromosome doubling and allotetraploidy on restoration of reproductive fertility. The other student is managing the Pic-a-Papaya cell phone application and compiling the data to determine how the incidence of transgenic papayas in Honolulu impacts incidence of PRSV. The banana germplasm collection at the Waimanalo Experiment Station has served as a teaching resource for instruction of undergraduate students enrolled in TPSS403 Tropical Fruit Production. How have the results been disseminated to communities of interest? Our professional colleagues in Plant Sciences were targeted by the poster and oral presentations made by two MS graduate students involved in the project at the CTAHR Student Symposium in April, 2014. Gabriel Sachter-Smith presented an oral summary of progress in evaluating banana germplasm for resistance to BBTV under field conditions at Waimanalo Experiment Station. Gregory Hoover 's poster summarized his progress in creating Vasconcellea interspecific hybrids and evaluating their fertility. The targeted audience consisted of over 250 CTAHR faculty and students. The goals and results of Objective 1 were presented in May 2014 to the 30 members of the professional working group WERA-20: Virus and Virus-Like Diseases of Fruit Trees, Small Fruits, and Grapevines, using the banana germplasm collection at the Waimanalo Experiment Station as a virus disease demonstration plot for the visitors. The goals and results of Objective 1 were also presented on August 26, 2014, at a Banana Mini-Conference sponsored by the Cooperative Extension Service for commercial banana growers on Oahu. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. The completed field evaluation of BBTV resistance/susceptibility in the germplasm collection will be confirmed and extended by two methods. PCR analysis will be performed on all individual mats to verify the disease diagnosis based on visual symptoms and to identify any possibility of symptomless carriers. Secondly, clones that appear resistant or slow to develop symptoms in the field will be macropropagated to produce at least three suckers per clone that are symptomless visually and by PCR. These and susceptible control 'Williams Cavendish' will be established in the greenhouse and inoculated by transferring viruliferous aphids manually to ensure uniform and stringent conditions for BBTV infection. The rate and severity of symptom development will be compared with controls to determine degrees of resistance. Objective 2. Rescued embryos of interspecific and intergeneric hybrids currently in tissue culture will be regenerated and established in the greenhouse. Their hybrid nature will be confirmed using DNA markers or isozyme analysis. A new generation of young vigorous female and hermaphrodite papaya plants will be established in a field near the UH campus (Magoon Horticulture Facility) to allow production of more intergeneric hybrids, concentrating on crosses with PRSV-resistant V. cundinamarcensis, V. quercifolia, and V. stipulata. To the extent made possible by embryogenic proliferation of hybrid cultures, subcultures of single genotypes will be treated in vitro with chromosome-doubling mutagens colchicine or oryzalin to produce allotetraploid plants for comparison with diploids of the same gametic origin. Fertility in any diploid and allotetraploid hybrids that reach reproductive maturity will be evaluated and compared. Objective 3. The banana demonstration planting in Kona will be the site of a field day for local growers. We will seek to popularize the Pic-a-Papaya phone application by working with local Master Gardener programs and other plant-oriented groups to stimulate client interest. We will use the Pic-a-Papaya client list to solicit permission to collect leaf samples for determining transgenic status of papaya plants already in the data base using the GUS histochemical assay.
Impacts
What was accomplished under these goals?
Objective 1. Project work during the reporting period addressed 1) completing the evaluation of a field-planted banana germplasm collection for resistance to banana bunchy top virus (BBTV) and 2) propagation of promising clones identified in the field trial for more rigorous inoculation tests under greenhouse conditions. The banana collection, consisting of six replications of 44 clones, was planted at Waimanalo Experiment Station in March 2012 and has been exposed to natural populations of virus-vectoring aphids since it was installed. Starting in August 2012, viruliferous aphids taken from BBTV-infected bananas kept in a growth chamber on the UH Manoa campus were intentionally introduced into the field. By the end of the reporting period, BBTV symptomology data had been collected for 5 or 6 reps of 41 clones. Almost all of the clones had at least one rep showing BBTV symptoms, but the rate and extent of disease development varied importantly between different clones. Cavendish clones were among the most susceptible. Clones with few symptomatic reps and with apparently slow systemic involvement included varieties of Gros Michel (Cocos ITC0451 and Highgate ITC0263) and Pisang Awak (Kayinja ITC0087 and an unidentified clone ITC0459). Several locally sourced wild species remained completely symptomless, including Musa acuminata ssp. malaccensis, M. balbisiana ssp. liukiuensis, M. laterita (Rhodochlamys), and M. ornata (Rhodochlamys). At least three suckers from symptomless individuals of these promising BBTV-resistant clones were macropropagated and established in the greenhouse for more intensive inoculations during the next reporting period. Objective 2. The overall goal of Obj. 2 remains to improve economically important characteristics of papaya through introduction of new germplasm using conventional breeding methods. However, the approach has changed. With the award of Supplemental Hatch funding at the beginning of the reporting period, the project refocused on germplasm that offers a greater range of new traits, including better sources of resistance to papaya ringspot virus (PRSV), the former main goal. Wild relatives of papaya can provide genes for fruit exocarp color, flavors and fragrances, as well as disease resistances, and these make a more attractive breeding target than relying on the single conspecific variety, 'Cariflora', which offers only moderate levels of multigenic resistance to PRSV. Since papaya is the only species in the genus Carica, our new germplasm consists of papaya wild relatives from other genera of the papaya family Caricaceae. These were obtained as seeds and vegetative cuttings from the USDA National Clonal Germplasm Repository, Hilo, and included Horovitzia cnidosculoides, Vasconcellea cundinamarcensis, V. glandulosa, V. goudotiana, V. horovitziana, V. monoica, V. parviflora, V. pulchra, V. quercifolia, V. stipulata, and V. x heilbornii. Previous (2012) crosses of V. quercifolia x V. parviflora yielded four F1 interspecific hybrids (3 females and one male), which proved sterile (20% stainable pollen with acetocarmine) and have produced no progeny via ovule rescue when sibbed or backcrossed with V. parviflora. Ovules from Carica papaya x V. cundinamarcensis or V. quercifolia intergeneric crosses were too poorly developed to extract embryos and failed to develop embryogenic growth on our initial MS medium lacking coconut water. Ovules from Carica papaya x V. parviflora intergeneric crosses were too poorly developed to extract embryos, but developed embryogenic callus on MS medium containing 10% coconut water. Ovules from Carica papaya x V. goudotiana or V. monoica intergeneric crosses produced large ovules from which polyembryonic masses could be extracted and cultured successfully on MS medium augmented with 10% coconut water. Objective 3. A extension demonstration plot displaying new banana germplasm was planted at the Kona Experiment Station in September 2013. The demonstration clones include the Gros Michael clone 'Highgate', two Pisang Awak clones including 'Kayinja', and the FHIA clones 3, 17, and 21. A papaya extension project centered on the development of the "Pic-a-Papaya" cell phone application was undertaken from January to April 2014. The application facilitates information exchange between the PI's and "citizen scientists." It combines PRSV diagnosis, GMO testing, and plant replacement services for clients' diseased or GMO plants, as a means of incentivizing exchange of information about incidence of PRSV and GMO plants in client neighborhoods. In exchange for plant tissue samples, digital photos, and GPS coordinates of their papayas, clients are provided (free of charge) with results of virus disease diagnosis and GMO tests, and their choice of seeds of resistant GMO or modestly tolerant non-GMO papayas, if replacement is desired. Data on 119 papaya plants, volunteered by Pic-a-Papaya clients mostly within Honolulu city limits, indicated that 12% were infected with PRSV. Surprisingly, only two clients provided tissue samples for GMO analysis, indicating little interest among users in this aspect of the extension program.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Hoover, G.* and Manshardt, R. 2014. Hybridization of papaya with wild relatives for PRSV resistance. 26TH ANNUAL CTAHR and COE STUDENT RESEARCH SYMPOSIUM, University of Hawaii at Manoa.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Sachter-Smith, G.* and Manshardt, R. 2014. Naturally occurring host resistance to Banana bunchy top virus. 26TH ANNUAL CTAHR and COE STUDENT RESEARCH SYMPOSIUM, University of Hawaii at Manoa.
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