J.M. Cordts

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene

SummaryPlum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

Plant regeneration from cotyledons of Prunus persica, Prunus domestica, and Prunus cerasus

Shoots were regenerated from the proximal region of immature cotyledons (with the embryonic axis removed) of Prunus persica (peach) and from the same area in mature cotyledons of P. domestica (plum) and P. cerasus (sour cherry) on MS medium containing (in mgl-1) thiamine-HCl, 0.4; nicotinic acid, 0.5; pyridoxine-HCl, 0.5; sucrose, 25 000; and 0.7% agar. The medium was supplemented with 0.0–2.5 μM indole-butyric acid and 5–12.5 μM thidiazuron. Cultures were incubated at 24 °C under 16h photoperiod. Shoots regenerated adventitiously over a broad range of thidiazuron concentrations and 2.5 μM indole-butyric acid in 35 days. The presence of the embryonic axis inhibited the development of shoots. Regenerated shoots of peach and plum were rooted on half-strength MS inorganic semi-solid medium with 2.5–5.0 μM indole-butyric acid. Rooted plants were acclimatized and transferred to the greenhouse.

Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants

SummaryTransgenic grape plants were regenerated from somatic embryos derived from immature zygotic embryos of seedless grape (Vitis vinifera L.) selections. Somatic embryos were bombarded twice with 1 μm gold particles using the Biolistic PDS-1000/He device (Bio-Rad Laboratories) and then exposed to Agrobacterium tumefaciens strain C58/Z707 containing the binary plasmid pGA482GG or pCGN7314. Following cocultivation, secondary embryos were allowed to proliferate on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 20–40 μg/ml kanamycin (kan). Transgenic embryos were identified after 3–5 months under selection and allowed to germinate and develop into rooted plants on Woody Plant Medium containing 1 μM 6-benzylaminopurine (BAP), 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 kan, positive GUS and PCR assays, and Southern analysis.

Effects of carbohydrates and nitrogen on the development of anthocyanins of a red leaf peach (Prunus persica (L.) Batsch) in vitro

Heterozygous red leaf peach (Prunus persica (L.) Batsch) shoots were implanted on media with varying nitrogen and carbohydrate regimes to identify a combination which elicited maximum anthocyanin production in explants. A medium with relatively low nitrogen (5 mM NH4+ and 10 mM NO3-) and high sucrose (234 mM) was most effective in stimulating anthocyanin production. Sucrose was more effective as a carbon source than glucose, fructose, or starch under given nitrogen levels. The major anthocyanin in red leaf peach was tentatively identified as cyanidin 3-glucoside based on PC and HPLC analysis.