In Vitro Propagation of the Threatened Atacama Cactus Copiapoa cinerea subsp. cinerea: Explant Position, Cytokinin Dose, Rooting, and Ex Vitro Establishment

Copiapoa cinerea (Phil.) Britton & Rose subsp. cinerea is a slow-growing cactus endemic to the coastal Atacama Desert and threatened by habitat loss, climate change, and poaching. We developed a seed-based in vitro propagation protocol and tested seed disinfection, explant position, cytokinin dose, auxin dose, phenolic responses, and acclimatization substrate, using the culture vessel or acclimatization box as the experimental unit. Seeds disinfected with 1, 3, or 5% (v/v) commercial bleach (0.1, 0.3, and 0.5% available chlorine) reached 76 to 84% germination after 45 days, with no effect of disinfectant concentration on final germination or germination timing. In a factorial experiment with basal and medial seedling segments and 0, 2.22, 4.44, or 6.66 µM 6-benzylaminopurine (BAP), explant position affected all growth variables (shoot number, responsive explants, shoot length, and biomass): medial segments outperformed basal segments by producing 1.85 ± 0.15 shoots per explant (versus 1.12 ± 0.10 in basal segments) with 50% of explants forming new shoots (versus 3% in basal segments). Adding BAP to the culture medium increased the percentage of responsive medial explants from 20% in the control to 86.7% at 2.22 µM BAP, which also yielded the highest proliferation rate (2.33 ± 0.18 shoots per explant), without increasing shoot numbers at higher doses. Apical segments rooted in 47% of cases without growth regulators, and indole-3-acetic acid (1.14 to 5.71 µM) showed a non-significant inductive trend up to 93% rooting. Total phenolics of explant pools ranged from 8.5 to 27.0 mg gallic acid equivalents g−1 dry matter, with the cell-wall-bound fraction accounting for 45 to 86%; phenolic pools responded to explant position and growth regulator dose in a fraction-specific manner, whereas tissue browning was minor. Ex vitro survival reached 98%, and a perlite–organic–granite mixture increased plant height and biomass relative to decomposed granite alone. The protocol supplies plant material for the ex situ conservation of this threatened taxon.