Title : Tissue culture optimization and genetic transformation of Calotropis
Abstract:
Calotropis is a continual (perennial) shrub belonging to Apocynaceae family is a medicinally valuable shrub abundant in arid landscapes, yet its biotechnological utilization remains largely unexplored due to the unavailability of efficient tissue culture and transformation protocols. This study aimed to develop and optimize reproducible methods for seed germination, shoot regeneration via multiple explant systems, and stable genetic transformation in this species. Seeds treated with mercuric chloride and sodium hypochlorite showed markedly improved germination on MS basal medium, yielding vigorous, contamination-free seedlings as explant donors. Three explant types internode segments, petiole sections, and leaf discs isolated from fourweek old in vitro seedlings were screened for organogenic potential on MS medium supplemented with BAP and kinetin at varying concentrations. Internode explants proved most responsive, achieving maximum shoot proliferation on MS containing BAP (2.0 mg/L) and kinetin (0.5 mg/L). Petiole explants showed moderate response, whereas leaf discs were least efficient. Regenerated shoots rooted easily on halfstrength MS with IBA. Indirect regeneration was established through friable embryogenic callus induced from petiole and leaf disc explants on BAP+Kinetin medium, later differentiated into shoot primordia using BAP and kinetin combinations. This callus system was afterward working for Agrobacterium tumefaciens-mediated transformation using strains EHA105 carrying the gus (uidA) reporter and nptII marker genes. Parameters including bacterial density, co-cultivation duration, and acetosyringone concentration were fine-tuned for optimal T-DNA delivery and with the GUS staining the transformed tissue is confirm because the GUS staining producing indigo blue coloration absent in controls with PCR analysis providing molecular confirmation of stable transgene integration. These findings establish the first reliable regeneration and transformation framework for Calotropis, offering a foundation for future gene functional studies and secondary metabolite engineering.
