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R. Scorza, J.M. Cordts, D.J. Gray, D. Gonsalves, R.L. Emershad, and D.W. Ramming

Transgenic grape plants were regenerated from somatic embryos derived from leaves of in vitro-grown plants of `Thompson Seedless' grape (Vitis vinifera L.) plants. Somatic embryos were either exposed directly to engineered Agrobacterium tumefaciens or they were bombarded twice with 1-μm gold particles and then exposed to A. tumefaciens. Somatic embryos were transformed with either the lytic peptide Shiva-1 gene or the tomato ringspot virus (TomRSV) coat protein (CP) gene. After cocultivation, secondary embryos proliferated on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 40 μg·mL-1 kanamycin (kan). Transgenic embryos were identified after 3 to 5 months under selection and allowed to germinate and develop into rooted plants on woody plant medium containing 1 μm 6-benzylaminopurine, 1.5% sucrose, 0.3% activated charcoal, and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kanamycin (kan), positive β-glucuronidase (GUS) and polymerase chain-reaction (PCR) assays, and Southern analysis.

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S. Lius, R. Manshardt, D. Gonsalves, M. Fitch, J. Slightom, and J. Sanford

Twenty transgenic Carica papaya plants ('Sunset', Roclone 55-l) carrying the coat protein gene (cp) of papaya ringspot virus (PRV) strain HA 5-l have remained symptomless and ELISA-negative for 18 mo. after inoculation with Hawaiian PRV under field conditions. Control plants showed disease symptoms within 1 mo. after manual inoculation or within 4 mo. when aphid populations were the inoculum vectors. Trunk diameter was significantly greater in cp + plants (14.3 cm) than in PRV-infected controls (9.3 cm). Fruit brix, plant morphology, and fertility of cp + plants were all norm al. Segregation analysis in R1 seedlings indicated that 55-1 contains a single transgenic insertion site. PRV resistance in R1 plants was linked with the cp gene, although in some progenies, up to 50% of cp + plants developed mild PRV symptoms more than 3 mo. after inoculation. Preliminary tests suggest that this is not due to genesis of virulent mutant strains of PRV.

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R. Manshardt, S. Lius, D. Gonsalves, M. Fitch, J. Slightom, and J. Sanford

Transgenic papaya lines carrying the coat protein gene (CP) of papaya ringspot virus (PRV) strain HA 5-1 display PRV reactions ranging from complete susceptibility (39-3 & 39-4), to slight delay in onset of symptoms (39-1) and attenuation of symptoms (60-3), to high-level resistance (55-1, 63-1). Normal Mendelian segregation of transgene expression was lost in R1 of 39-3 and 39-4, and inbred R1 60-3 gave an aberrant 1:1 ratio. R0 55-1 plants were resistant in the field (Hawaii) for 2 years following manual and/or aphid inoculation, and the high-level resistance remained stable in the R1 after repeated manual inoculations in the greenhouse and graft inoculation for up to 1 year (Cornell). However, inoculation with PRV HA-Oahu strain produced symptoms in some plants at Cornell (9% after 6 weeks) and in Hawaii (50% after 1 year). Two 55-1 and one 60-3 plant subsequently underwent remission of symptoms and became ELISA-negative (Hawaii). Transmission of PRV isolates from symptomatic 55-1 plants to other CP+ 55-1 bioassay plants was unsuccessful.