Gujarat is one of India's top potato-producing regions, making it one of the world's top producers of potatoes. The demand for potatoes is driven by the food processing industry, domestic consumption, and export opportunities. While potato production in India has been growing, there are several issues that affect the industry. The availability of high-quality potato seeds as well as post-harvest losses due to improper handling and storage are major challenges. The purpose of this study was to investigate the effects of various culture systems, and nutrient supplements to establish and optimize a suitable system for in vitro shoot growth, microtuberization, and storage conditions. In vitro cultures of locally adapted six different potato cultivars have been initiated and the shoot multiplication protocol has been standardized. The microtubers protocol was optimized using four-week-old shoots and a mean of 4 microtubers per shoot was observed on the Murashige and Skoog medium supplemented with 6-benzylaminopurine (0.88 µM) and sucrose (8%). Harvested microtubers were used for storage conditions and shoot growth was evaluated from microtubers under in vitro as well as ex vitro conditions. All microtubers developed healthy shoots after 18 days of storage at 4 ℃ both in vitro and ex vitro, and the resulting plantlets showed > 90% survival in the greenhouse. The distribution of high-quality potato seeds in Gujarat, which are in high demand, may benefit from the optimal microtubarization protocol. This study confirms the potential of long-term germplasm preservation and microtuber based cultivation practices in the Gujarat.