Flower buds were collected as donor material when most
microspores were at the late-uninucleate to early-binucleate stage
of development. Buds were placed under running tap water for
1 h, then surface-sterilized by immersion in 70% (v/v) ethanol
for 30 s followed by 2% (v/v) sodium hypochlorite (NaClO) for
8–10 min. The buds were then rinsed three times in sterile distilled
water. Under aseptic conditions, anthers were isolated from
buds by excision from the filaments, then 20 anthers were placed
immediately onto a single plate of induction medium. Different
media were tested using a completely randomized design, with
30 replicates for each type of induction medium. Anther cultures
were incubated for 20 d at 25 ± 1 ◦C in darkness, then incubated
under fluorescent 20W daylight lamps (30–40 mol/m2/s) for
14/10 h (light/dark) conditions at 25 ± 1 ◦C until callus formed.
Callus induction rates were determined on day 60 after anthers
were placed into culture. Data were analyzed using the Statistical
Package for Social Science (SPSS 17.0; Chicago, IL, USA). Analysis
of variance (one-way ANOVA) was used to determine which variables
were significant (P = 0.05 level). Duncan’s multiple range test
was used to determine significant differences between treatment
means.