In Vitro Propagation of Clausena lenis Drake

Clausena lenis Drake, a valuable medicinal plant in the Rutaceae family, faces threats from wildlife predation, overharvesting, and climate change. In the wild, C. lenis primarily propagates through seeds; however, their rapid loss of viability poses challenges for long-term storage and germplasm conservation. Plant tissue culture offers a practical solution for both its conservation and large-scale production. This study examines seed sterilization, callus induction, shoot multiplication, and root induction protocols for C. lenis. Seeds attained a 100% sterilization rate using 0.2% (w/v) HgCl₂ for 20 min without compromising germination. When cultured on MS medium containing 0.5 mg/L 2,4-D, seed, stem-node, and 1-week-old seedling explants produced abundant callus. A 2.0 mg/L BA treatment achieved 100% shoot induction, with stem-node explants yielding the highest shoot proliferation (3.90 ± 0.31 shoots/explant), followed by 1-week-old seedlings (2.30 ± 0.21 shoots/explant) and seed explants (1.60 ± 0.16 shoots/explant). Rooting was most effective on half-strength MS medium supplemented with 20.0 mg/L IBA, producing an average of 4.30 ± 0.83 roots per shoot in shoot-tip-deprived explants. The rooted plantlets successfully acclimatized, attaining a 100% survival rate in a 1:1:1 mixture of sterile soil, cocopeat, and vermiculite. These findings provide a robust platform for the sustainable propagation and conservation of C. lenis in response to its growing vulnerabilities.