Histological insights in to cryopreservation of Rubia cordifolia L. using encapsulation-dehydration

This study explores the cryopreservation of Rubia cordifolia L. using the encapsulation-dehydration technique. The economic importance of this species, coupled with the depletion of its genetic diversity due to overexploitation and inadequate cultivation, necessitates effective conservation strategies. Cryopreservation offers a viable strategy for long-term conservation of plant genetic resources. Six regeneration media combinations and three explant types (shoot tips, nodal halves, and nodal explants) were evaluated for optimizing a cryopreservation protocol for R. cordifolia. Murashige and Skoog (MS) medium supplemented with gibberellic acid (GA3) and 2-isopentenyl adenine (2-iP) proved optimal, inducing 50% shoot regeneration within 3 days in control explants and demonstrating superior shoot length and node count. Nodal explants (2 mm) were most effective, achieving a mean shoot length of 3.57 ± 0.20 cm. On testing the explants for cryopreservation using encapsulation-dehydration by encapsulating them in calcium alginate beads and air desiccation before liquid nitrogen storage, both control and desiccated explants exhibited 100% survival rates. Post-cryopreservation, predominantly callogenesis was observed, with the highest callus induction (60.00 ± 3.24%) on MS + 2- iP (1.0 mg/L) + GA3 (0.2 mg/L) medium after 6 h of air desiccation. Histological analysis revealed stress-induced cellular changes in desiccated explants. Control explants showed prominent meristematic zones, vascular bundles, and shoot primordia, while explants exposed to liquid nitrogen lacked shoot primordia, suggesting encapsulation-dehydration cryopreservation induced damage in cellular structures critical for shoot regeneration. This study offers valuable insights into effect of low temperature on R. cordifolia and serves as a foundation for further research on its long-term conservation.

The online version contains supplementary material available at 10.1007/s13205-026-04718-x.