2. Materials and methods
2.1. Plant materials and culture conditions
Seeds of different silybium genotypes collected from different
environmental locations (Kafr-El-Shiekh, Asuiet, El-Menia,
Beni-Sewaf, El-Fayuim and Agricultural road of Alexandria)
were surface sterilized with 70% ethanol for 1 min, followed
by 30% commercial Clorox (contained 5.25% sodium hypochlorite)
for 20 min. After three successive rinses in sterile distilled
water, sterilized seeds were cultured on MS [7] basal
medium supplemented with 30 g L1 sucrose, 100 mgL1
myo-inositol and solidified with 7 gL1 agar.
All media were autoclaved (121 C and a pressure of
1.2 kg cm2 for 20 min) and the pH was adjusted to 5.8 (using
1 M NaOH or HCl). All cultures were incubated in the growth
chamber for 1 month at 25 ± 2 C under a 16-h photoperiod
(irradiance of about 40 mmol m2 s
1 provided by cool white
fluorescent lamps).