Gibberellic Acid (GA3) Detection
The isolates were cultured in 50 ml of sterile Richard’s Solution in triplicate and incubated
under static condition for 10 days (Johnston and Booth 1983). To the control
flasks were supplemented with distilled water treated in the same manner. After ten
days of incubation the medium was filtered through the filter paper with 0.7 μm of
the pore size (Whatman no. 1) and pH of the filtrate was adjusted to 2.5 by using 1N
HCl and extracted following Hasan (2002) with slight modification. The suspended
residue was spotted on a silica gel TLC plate alongside with gibberellic acid standard
(GA3; Sigma) and positioned in vertical direction by using two specific solvent systems
i.e. isopropanol:ammonia:water (10 : 1 : 1), v/v/v and chloroform:ethyl acetate:
formic acid (5 : 4 : 1), v/v/v (Chang and Jacobs 1973; Hasan 2002). Visualization process
was completed according to Hasan (2002) and the Rf values of GA3 was calculated
(Fessenden et al. 2001).