Androgenesis inphanerogams is a very distinct process. This technique has been proven unique breakthrough to shorten the breeding period of the cultivars for the production and fixing of agronomic traits in the homozygous state in a single generation.Catharanthus roseus(L.) G. Don. (Periwinkle)being an important pharmaceutical plantwas selected for the investigation to study the effect of various pretreatment on callus induction in anther culture at constant concentrations of growth regulators i.e. 2, 4-D, NAA and BAP. Out of the five pretreatments chosen cold pretreatment at 8°C for 10 days was found to be the best66.33 ± 1.74%and least was observed in centrifugation at 2000rpm for 6 hours 28.00 ± 0.57%.While 2,4-D=7mgL-1, NAA=0.5mgL-1 and BAP=1.5mgL-1 were found to be suitable for maximum calli production at all concentrations of 2,4-D.Plantlets regenerated by culturing embryo like structure are all reported to be haploid.