New gynoecious bitter gourd line, KAU-MCGy-101 identified from Kerala Agricultural University, India, was morphologically characterized. Silver thiosulphate (STS) was used for altering the sex expression of gynoecious line. STS induced hermaphrodite flowers in gynoecious plants and maximum number of hermaphrodite flowers was in plants receiving single spray at 200 ppm after emergence of first female flower. F1 was generated by crossing with monoecious variety Preethi and all the hybrids were monoecious in nature. Expression of gynoecious character in the sib-mated gynoecious inbred and monoecy in F1 hybrids, indicate the recessive nature of gynoecy in bitter gourd. Gynoecious inbred was also maintained through micropropagation. The shoot tip explants cultured on MS medium supplemented with 2.0 mgL−1 BA followed by 1.5 mgL−1 BA have given the highest shoot initiation response in 20.75 days. Multiple shoots, up to 5.50 per explant, were induced on MS medium with 2.0 mgL−1 BA and microshoots elongated best with 0.5 mgL−1 IAA + 0.5 mgL−1 NAA. Best in vitro rooting was on half strength MS medium with 3.0 gL−1 activated charcoal and 1.0 mgL−1 IBA. The gynoecious expression was found to be stable in tissue culture regenerated progenies.