Progress 09/15/03 to 09/14/05
Outputs
We developed a method for efficient Agrobacterium-mediated genetic transformation and rapid regeneration of papaya plants and transformed papaya cv F65 with four different genetic constructs of the coat protein gene of a Florida strain of PRV. These constructs contained either the sense or antisense PRV coat protein gene or a non-translatable PRV coat protein gene with either a frame-shift or stop-codon mutation. Putatively transformed papaya plants were tested for resistance to PRV. Forty-eight of 257 plants appeared to be immune and attenuated symptoms, as compared to those in control plants, were observed in many of the other transformed plants. All putatively transgenic plants were female. Twenty-one PRV-resistant lines (R0 ) were crossed with the six elite papaya genotypes. Seventeen lines containing either a frame-shift or stop-codon mutation of the PRV coat protein gene were highly fertile. Two lines each with either the sense or anti-sense PRV coat protein gene
constructs produced few viable seeds. Our first field release of the transgenic papaya lines was installed in March 2001 after the seedlings had been screened by spraying with kanamycin for the presence of the NPT II gene. Within one year of planting, 293 (23.3%) of 1258 transgenic progeny from the 50 crosses became naturally infected by PRV. In comparison, 29 (96.7%) of 30 non-transgenic plants in the same planting became infected. Infection varied from 4.6% to 38.9% among the descendants of the six elite male parents, and from 10.4% to 87.9% among the 17 transgenic lines. Thus, although not immune to PRV, the transgenic papaya lines appeared to be less susceptible to natural infection by the virus. The R2 generation, derived from 39 self-pollinated R1 selections, was evaluated in the field in 2002-2003. Successive generations up to the present R5 generation have now been evaluated annually in the field. Usually, 30 or more inbred progeny of each selection were evaluated in the next
generation. Selections have been based on a number of criteria, including PRV resistance, vigor and stature of the tree, fruit and fruit column uniformity, fruit quality and quantity, general resistance to diseases and pests, resistance to environmental stresses, and market preferences. Presently, four of the 17 transgenic lines selected for testing in the field, the X17-2, X26, D6, and D95, lines remain in the breeding program. Currently 20 selections (breeding lines) are in the field and represent nine of the original crosses of the transgenic lines with elite genotypes. Ten or more plants each of 12 transgenic papaya lines and 23 non-transgenic accessions, including named varieties and selections, were planted in a field in May 2003. Within 8 months, all of the non-transgenic papaya plants became naturally infected by PRV and exhibited moderate to high levels of disease severity. In contrast, only a few plants of four of the 12 transgenic lines developed mild symptoms of PRV. Thus,
although not immune to PRV infection, especially when mechanically inoculated, transgenic lines exhibited a high level of resistance to natural infection in the field.
Impacts
In 2004, over 6.7 million metric tons of papaya fruit were produced worldwide; 16,240 metric tons were produced in the USA, and 3,200 metric tons were produced in Puerto Rico. During 2003 in the USA, 101,868 metric tons worth $68.8 million were imported and 7,046 metric tons worth $14.2 million were exported. Production in Hawaii accounts for the vast majority of the total production in the USA. In 1995-96, Florida produced 2,835 metric tons. The Florida industry is worth $2-4 million annually. The potential net income per acre for a three-year planting is estimated to be approximately $26,000, if major pest and disease problems can be controlled, especially PRV. The increase in acreage, production, and economic benefit from overcoming PRV is potentially worth millions of dollars and hundreds of jobs. The same situation applies to much of the Caribbean region
Publications
- Davis, M. J., T. L. White, and J. H. Crane. 2004. Resistance to papaya ringspot virus in transgenic papaya breeding lines. Proc. Fla. Sta. Hort. Soc. 117: 241-245
- Davis, M. J., White, T. L., and Crane, J. H. 2003. Papaya variety development in Florida. Proc. Fla State Hort. Soc. 116:4-6.
- Davis, M. J. and Z. Ying. 2004. Development of papaya breeding lines with transgenic resistance to Papaya ringspot virus. Plant Disease: 88:352-358
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Progress 10/01/03 to 09/30/04
Outputs
The resistance of transgenic papaya breeding lines to Papaya ringspot virus (PRSV) was examined. Resistance was conferred by non-translatable transgenes derived from the coat protein (CP) gene of a PRSV isolate (H1K) from Florida. To render the CP gene non-translatable, either a stop-codon (D6 lines) or frame-shift (X17-2 lines) mutation had been introduced into the CP gene. Although our original transgenic selections all exhibited a high degree of resistance to PRSV following mechanical inoculation with the same isolate used to obtain the CP transgene, some of the progeny of these lines were susceptible to natural infection by PRSV in the field. This susceptibility appeared to vary with both the transgenic line and the parentage of the plants. Thus, selective breeding for a high level of resistance should help to overcome this problem. However, a possibility exists that the transgenic lines might be more susceptible to infection by isolates of PRSV other than the one
originally tested. Investigations reported here were conducted to evaluate the resistance of our transgenic lines to different isolates of PRSV from Florida and to directly compare natural infection with PRSV in 12 of our R3 transgenic lines to that in 23 non-transgenic varieties or selections varying from highly susceptible to tolerant in their reaction to PRSV. Non-transgenic and transgenic papaya lines (R3 generation) were mechanically inoculated with three isolates (H1A, H1C, and H1K) of PRSV representing the genetic diversity of the virus in Florida. The mean severity of symptoms evaluated weekly for 8 weeks post-inoculation was consistently lower in the transgenic lines regardless of the PRSV isolate, and transgenic resistance to the different virus isolates did not differ noticeably. Ten or more plants each of 12 transgenic papaya lines and 23 non-transgenic accessions, including named varieties and selections, were planted in a field in May 2003 and evaluated for the incidence
and severity of PRSV following natural infections. Within 8 months, all of the non-transgenic papaya plants became infected by PRSV and exhibited moderate to high levels of disease severity. In contrast, only a few plants of four of the 12 transgenic lines developed mild symptoms of PRSV. Thus, although not immune to PRSV infection, especially when mechanically inoculated, transgenic lines exhibited a high level of resistance to natural infection in the field.
Impacts
This research should result in the development of new papaya varieties with resistance to papaya ringspot virus. Without the constraints to commercial papaya production imposed by the virus, papaya production should become more profitable and widespread in the Caribbean region.
Publications
- Davis, M. J. and Z. Ying. 2004. Development of papaya breeding lines with transgenic resistance to Papaya ringspot virus. Plant Disease: 88:352-358
- Davis, M. J., T. L. White, and J. H. Crane. 2003. Papaya variety development in Florida. . Proc. Fla. State Hort. Soc. 116:4-6.
- Davis, M. J., T. L. White, and J. H. Crane. 2004. Resistance to Papaya ringspot virus in transgenic papaya breeding lines . Proc. Fla. State Hort. Soc. 117: (in press).
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