In order to circumvent the constraints in the existing methods for In vitro culture growth, germplasm conservation and large-scale propagation of grape, a study was undertaken to devise and formulate an appropriate growth media for micropropagation of new grape genotypes. Explants of two germplasm accessions viz., Sunder Khani (SK) and 019972 from a recently established field gene bank were used. In total, 12 different media formulations involving macroand micro-nutrients (MS media), Fe-EDTA levels employed in Murashige-Skoog (MS) media were tested for their effects on In vitro growth performance of the two grape germplasm accessions. It was found that 75% levels of macronutrients worked best for the genotypes tested. The protocol fine tuned in this study for In vitro growth of grape cultures and micropropagation process achieved as many as 72 and 62 plants in each cycle of subculturing every 4-6 weeks, in case of Sundar Khani and 019972 accession, respectively. Use of coconut husk produced 80% rooting frequency when double node cuttings were used, without any application of rooting hormone within a period of 6 weeks, whereas, single node cuttings failed to root. On the other hand, In vitro plants showed maximum rooting incidence (1.26 gm per explant) when grown on media containing adenine sulfate (543 uM) in combination with IAA (1.1 uM) and kinetin (4.6 uM). The protocol developed in this study has paved the way to establish In vitro conservation of all grape germplasm accessions of our gene bank as a back up for sustainable plant genetic resources (PGRs) conservation and their utilization.