Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl2, 0.92 mM NaH2PO4 and 6 .25 2-[N-morpholino]ethanesulfonic acid (1.5 mi) mixed with 0.7 M MS(-)8P ( 2.5 mi). MS(-)8P medium consisted of Murashige and Skoog salts without NH4N O3, 1 mg l(-1) thiamine HCl, 100 mg l(-1) myo-inositol, 3.1 g l(-1) glutami ne and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucro se and 0-0.55 M mannitol. Protoplast yields of 3.5x10(6) protoplasts g(-1) were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture mediu m dilution. Under optimum conditions, proembryos developed directly from em bryogenic protoplasts and subsequently into somatic embryos. Optimum condit ions for somatic embryo development included the culture of protoplasts at a density of 0.8-1.6x10(5) ml(-1) in 0.4 M MS(-)8P for 2-3 weeks. followed by subculture in 0.15 M MS(-)8P at a diluted density of 20-40x for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovere d on semisolid medium; however, a low frequency of plantlet recovery (less than or equal to 1%) from protoplast-derived somatic embryos was observed.