Vol. 9, Issue 11, Part E (2025)

This investigation presents a comprehensive in vitro regeneration protocol for Opuntia ficus-indica (L.) Mill., developed through a series of controlled experiments conducted at the Tissue Culture Laboratory, State Level Biotechnology Centre, MPKV, Rahuri (2023-2025). Three explant types were evaluated, and areoles from 2-3-month-old cladodes exhibited the highest regenerative capacity (76.66%), whereas older areoles responded poorly and cladode sections lacking areoles failed to regenerate. A stepwise sterilization protocol- 0.1% Tween-20 for 15 min (86.67% survival), Bavistin (0.1%) + Streptocycline (0.05%) for 20 min (93.33% survival), 0.1% HgCl₂ for 5 min (93.33% survival), 1% NaOCl for 3 min (66.67% survival) and 70% ethanol for 60 s (86.67% survival)- yielded maximum aseptic recovery. Hormonal optimization revealed that MS medium supplemented with 1.0 mg L⁻¹ BAP + 1.0 mg L⁻¹ 2,4-D produced the highest shoot induction (90% areole activation within 30 days). Shoot multiplication peaked on MS + 2.0 mg L⁻¹ BAP + 0.5 mg L⁻¹ NAA, achieving 100% initiation and 10.3 shoots per explant, while combinations of 0.5 mg L⁻¹ NAA + 1.5 mg L⁻¹ KIN and 4.0 mg L⁻¹ KIN + 0.4 mg L⁻¹ IAA also achieved full initiation with 3.3 and 4.3 shoots per explant, respectively. Rooting was maximized on half-strength MS medium containing 0.5 mg L⁻¹ IAA (T₈), resulting in 100% root induction within 28 days. Primary hardening in soil: cocopeat: sand (1:1:1) ensured 100% survival, followed by 100% secondary survival in vermicompost: sand: soil (1:1:2). Issues such as phenolic browning and slow areole activation were resolved through pre-inoculation hydration and reduction of explant size. This fully detailed protocol provides a reliable, reproducible and scalable system for micropropagation, conservation and commercial deployment of O. ficus-indica in arid and semi-arid regions.