Plant regeneration from Citrus protoplasts: Variability in methodological requirements among cultivars and species

Nucellar callus lines were established from two orange cultivars ('Nucellar Shamouti', 'Shamouti Landau'), three mandarin cultivars ('Murcott', 'Dancy', 'Ponkan') one grapefruit cultivar ('Duncan') and sour orange (Citrus aurantium). These callus lines were initiated from in vitro cultured ovules of young fruits and maintained an embryogenic capacity. The plating efficiencies of protoplasts derived from these calli, as well as those of protoplasts from lemon (cv. 'Villafranca') nucellar callus were differentially affected by the maceration enzymes and by the sugars used as osmotic stabilizers. Plants with normal morphological features were regenerated from cultured protoplasts derived from each of the nucellar callus lines. The establishment of eight new protoplast systems in Citrus paves the way for cell genetics studies and for novel breeding approaches in these economically important orchard trees.