Optimizing Pharmaceutical-Grade Micropropagation of Digitalis purpurea L.: A Two-Stage Protocol Integrating Benzyladenine and Gibberellic Acid for Enhanced Shoot Multiplication and Elongation

Digitalis purpurea L. represents a critical pharmaceutical resource for cardiac glycoside production. Conventional propagation methods suffer from prolonged germination periods, variable secondary metabolite content, and limited scalability for industrial applications. This study establishes an innovative two-stage micropropagation protocol optimizing benzyladenine and gibberellic acid concentrations to achieve pharmaceutical-grade plantlet production with enhanced standardisation. Single-node explants were cultured on Murashige and Skoog medium supplemented with BA (0.0, 0.25, 0.50, 0.75 mg L-1) or GA3 (0.0, 0.25, 0.50, 0.75 mg L-1) in a completely randomized design. After 4 weeks, 0.25 mg L-1 BA significantly enhanced shoot multiplication (2.40 shoots), fresh weight (1.10 g), and dry weight (0.11 g) compared to control (1.00 shoot, 0.36 g, 0.05 g; P < 0.001), representing a 140% increase in propagation efficiency. Conversely, 0.75 mg L-1 GA3 maximized shoot length (8.65±49%) and facilitated robust plantlet development (P < 0.001) without compromising biomass accumulation. The complementary physiological responses BA promoting lateral branching via apical dominance disruption and GA3 stimulating internodal elongation through DELLA protein degradation enable a synergistic two-stage approach: Stage 1 (multiplication: MS + 0.25 mg L-1 BA, 4 weeks) followed by Stage 2 (elongation: MS + 0.50 mg L-1 GA3, 2-3 weeks). This protocol yields approximately 3.1 transplant-ready plantlets per explant within 6-7 weeks, offering a 50-fold annual multiplication rate with superior genetic uniformity and cost-effectiveness for pharmaceutical-grade Digitalis production systems