Hom-kaek onion (Allium cepa var. viviparum) has a high potential to serve as a functional food. However, to date, there has been no commercial cultivation of Hom-kaek onion in Thailand, and the demands for this plant has been fulfilled mostly by importation. This study aimed to developed a propagation system of Hom-kaek onion using tissue culture technique. Here, we examined various factors during the tissue culture process for the production of Hom-kaek plantlets. During the initiation phase, onion bulbs were sterilized using 25% Clorox?, 0.1% mercuric chloride, 1% silver nitrate, or 6% hydrogen peroxide for 10, 15, and 20 min. Overall, low rates of microbial contaminations (20.00-25.00%) and shoot induction (83.33-85.00%) were observed. Nonetheless, silver nitrate exposure caused the explanted onion to darken and bruised. Comparing the effects of cutting the onion bulbs (4, 6, and 8 sections per bulb), comparable shoot induction rates were ranged from 61.67% to 67.50%. By cutting the onion bulb into 8 sections, the averages of 1.31 shoots per explant and 6.4 shoots per initial bulb were observed. During the multiplication phase, the initiated plants were cut longitudinally and each section capable of shoot formation was found. This suggested that without re-division, 1 initial section gave rise to 1 shoot while with a longitudinal re-division, 1 initial section gave rise to 2 onion shoots. When the effects of cytokinin supplementations were compared, shoot formation rates of the onion explants grown on modified semi-solid MS medium with 1.0-2.0 mg/L BAP and 0.25-0.5 mg/L TDZ, and without supplementation (control) were evaluated. In general, we found TDZ was more effective than BAP supplementation. Compared to the non-supplemented control (1.0 shoot per piece), an addition of 0.5 mg/L TDZ led to the formation of 3.9 shoots per piece on the average. During the root induction phase, the effects of auxins with varying concentrations (0.5 and 1.0 mg/L of either IBA or NAA) were examined. Interestingly, root formation rates in both the auxin-induced and the non-induced control were observed. It was found that 1.0 mg/L IBA was the most efficient for root induction as it promoted 87.67% of root induction, yielding the highest average numbers of roots per shoot (16.75 roots per shoot). Subsequently, in the adjustment period before the plantlet transfer to greenhouse, root induction periods were varied and plantlet survival rates were determined. The onion shoots were grown on the modified semi-solid MS medium supplemented with 1.0 mg/L IBA and allowed root formation for 0, 1, 2, and 4 weeks prior to the greenhouse transfer. Two weeks of root induction period prior to the plantlet transfer was the most optimal time, yielding an 85.71% survival rate. Additionally, the different planting materialswere compared, which included loam soil: sand (1:1), loam soil: sand: rice husk ash (1:1:3), loam soil: sand: coconut coir fiber (1:1:3), and loam soil: sand: rice husk ash: peat moss (1:1:3:3). It was found that the latter formula (1:1:3:3 loam: sand: rice husk ash: peat moss) led to the highest survival rates of the onion plantlets (80.00%) and the highest growth in terms of shoot height, leaf number, root length, and root number (17.07 cm, 5.13 leaves/shoot, 8.35 cm, 25.49 roots/shoot, respectively). The results from this study will aid in designing and developing the micropropagation system for large-scale production of Hom-kaek plantlets to enhance the domestic cultivation of Hom-kaek onion and reduce the import costs in the future