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