Journal of Advances in Biology & Biotechnology

Secondary hardening plays a crucial role in acclimatizing tissue-cultured plantlets to natural environmental conditions by gradually exposing them to variations in humidity, light, and temperature. Therefore, the present study aims to optimize secondary hardening conditions by evaluating different substrate compositions, humidity regimes, and light intensities. A laboratory experiment was conducted at the Department of Horticulture, College of Agriculture, UAS, Raichur, during 2023–2024. The study was laid out in a Completely Randomized Design (CRD) with seven treatments : T₁: Cocopeat, T₂: Vermiculite, T₃: Vermicompost, T₄: Cocopeat: Vermiculite (1:1), T₅: Cocopeat: Vermicompost (1:1), T₆: Vermiculite: Vermicompost (1:1), and T₇: Cocopeat: Vermiculite: Vermicompost (1:1:1)  to evaluate suitable secondary hardening media for 30-day-old embryo-rescued plantlets derived from the cross UARChH 42 (Capsicum annuum) × PBC 80 (Capsicum baccatum).Among the treatments, cocopeat (T₁) recorded significantly superior performance with maximum shoot length (11.41 cm), root length (13.18 cm), and higher chlorophyll content, leaf number, and biomass accumulation, along with the highest survivability (>90%). It also showed a 47.15% increase in shoot growth over other treatments. In contrast, vermicompost alone (T₃) resulted in complete mortality of plantlets. The findings indicate that cocopeat is an ideal medium for secondary hardening of embryo-rescued chilli plantlets. The standardized protocol developed can enhance plantlet survival and establishment, thereby supporting interspecific breeding programs and facilitating future research on large-scale propagation and development of stress-resilient chilli hybrids.