salts, containing MS vitamins and 3% (w/v) sucrose, and solidified
with 0.7% (w/v) agar (Merck). MSM medium formulation is
a modification of MS medium based on the composition of leaves
of field-grown plants of P. edulis f. flavicarpa. It contains reduced
concentrations of nitrogen, potassium, zinc, boron and chloride,
associated to increased concentrations of calcium, magnesium,
sulfur, iron, manganese, copper, sodium and EDTA, and does not
contain iodine. The reduction of chloride is achieved by changing
the calcium source from calcium chloride to calcium nitrate. The
pH for all media was adjusted to 5.8 and growth regulators were
added at different concentrations before autoclaving for 15 min at
121 ◦C. All inorganic chemicals used were P.A. grade, and biochemical
reagents were of the best grade available from Sigma Chemical
Co.
Cultures were maintained in a growth chamber at 25
±
2 ◦C in
the dark or under a 16 h photoperiod, using a total irradiance of
96 mol m−2 s−1 provided by cool-white fluorescent lamps.