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 ml) mixed with 0.7 M MS^-8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^-1 thiamine HCl, 100 mg l^-1 myo-inositol, 3.1 g l^-1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0-0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^-1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8-1.6×10⁵ 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 20-40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed.