Explants were cultured on MS solid (6 g/L plant agar) medium [47] under three different plant growth regulator (PGR) combinations: (1) VV-4 medium, 5 µM N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU) + 5 µM 2,4-dichlorophenoxy acetic acid (2,4-D); (2) VV-5 medium, 20 µM β-naphthoxyacetic acid (NOA) + 4 µM N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ); and (3) VV-16 medium, 10 µM NOA + 4.4 µM N6-benzylaminopurine (BA) [48]. Media were supplemented with 88 mM sucrose. The pH of the media was adjusted to 5.7–5.8 with 1 N NaOH before autoclaving. All chemicals were purchased from Duchefa Biochemie, Netherland. Explants were incubated in a climatic chamber at 26 °C with a 16 h photoperiod (40 μmol m−2s−1 at shelf level, provided by Osram Cool White 18 W fluorescent lamps) and subcultured in the same culture medium at 60 d intervals. The explants showing embryogenic responses were transferred to basal MS-medium-deprived PGRs, supplemented with 88 mM sucrose and cultured for four more weeks to allow embryo proliferation and development. Then, germinated somatic embryos were collected and individually transferred to Magenta™ vessels containing 100 mL of basal solid MS medium under the same light and temperature conditions as described above, to allow further growth. After rooting, four plants produced from each somatic embryo (10–15 cm) were transferred to autoclaved Jiffy-7 peat pellets and moved onto a heating bench at 25 °C and high relative humidity (95–98%). After 4–5 weeks, plants were transferred into 2 L pots containing sterilized soil under natural daylight at 22/27 °C (night/day). After acclimatization, plantlets were transferred to the greenhouse located in Collesano district, grown in 20 L pots on a composite substrate of 2/3 peat and 1/3 agriperlite and fertilized with full strength nutrient solution as described by Oddo et al. [49]. After a 30–40-day period of acclimatization in the greenhouse the plants were transferred to field conditions for assessment of ampelographic traits.

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