Re. Witjaksono,"litz et Jw. Grosser, Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts, PL CELL REP, 18(3-4), 1998, pp. 235-242
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.