| Summary: | Seed potato production often relies on mineral fertilizers. However, biofertilizers offer an eco-friendly, cost-effective means to enhance nutrient uptake, plant growth, yields, and quality while bolstering stress resilience. Two cultivars (‘Spunta’ and ‘Russet’), two <i>in vitro</i> materials as microtubers and plantlets, and four bio-fertilizers were used to produce seed minitubers. These bio-fertilizers included mycorrhiza (T2), microalgae (T3), beneficial bacteria (PGPR) (T4), and vermicompost (T5). Treatment T1, which received 100% mineral nutrients, was used as the control, while the bio-fertilizers were given 40% of the mineral nutrition relative to the control. The study clearly demonstrated the effectiveness of the biofertilizers used in improving plant growth parameters, particularly highlighting the efficacy of vermicompost. The highest seed tuber yield of 173.12 g was obtained from the combination of ‘Spunta’ + microtuber + vermicompost’. In both varieties, <i>in vitro</i> microtubers led to a higher seed yield than <i>in vitro</i> plantlets. In terms of tuber diameter, tuber weight, and tuber number, the performance of the ‘Spunta’ cultivar was significantly higher than that of the ‘Russet’ cultivar. Seed tubers derived from <i>in vitro</i> microtubers had a larger diameter and were heavier than those derived from <i>in vitro</i> plantlets. However, seed tubers produced from <i>in vitro</i> plantlets were of a smaller size but more in number. In <i>in vitro</i> potato seed tuber production, we recommend the use of ‘Spunta’ cultivar and <i>in vitro</i> microtuber, supplementing with vermicompost to enhance yield, size, number curbing costs, and eco-friendliness.
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