3.1. Induction of embryogenic callus
Four to 6 weeks after culturing of ovules of Washington Navel and Clementine mandarin, calli that have either a loose or a compact, small, spherical nodules of about 0.1–1.0 mm in diameter, which varied between white and cream-coloured, were induced.
Four to 6 weeks after culturing the ovules of Zagara Bianca (Fig. 1a and b) and Kutdiken lemon varieties on MT + ME medium developed small embryos, that have cotyledons (Fig. 1c). Within 4 weeks, embryogenic calli were induced from the basement of these embryos on the MT medium containing 2.26 mM of 2,4-D. These calli were the same size as other initial calli from Washington Navel orange and Clementine mandarin, but circular, friable and whitish (Fig. 1c). For stabilization, calli have been sub-cultured on semi-solid MT medium including 5% sucrose for 1 year (Fig. 1d). After a year, calli from all cultivars were very friable and smaller in size than previous calli. After subculturing the nucellar calli with intervals of 1–2 months for about 5 years on the same MT medium, those callus lines maintained embryogenic potential.