Comprehensive Phenotypic, Molecular, and Phenolic Analysis of Kalanchoë daigremontiana Hairy Roots

Hairy root cultures represent a valuable biotechnological tool for the sustainable production of bioactive secondary metabolites. Kalanchoë daigremontiana is known synthesize phenolic compounds and flavonoids associated with antioxidant and health-promoting properties. This study aimed to establish K. daigremontiana hairy root cultures through infection with Rhizobium rhizogenes and to characterize their growth and phenolic profile. Three strains of R. rhizogenes (A4, ATCC 15834 + pTDT, and K599) were evaluated using in vitro-grown plantlets of K. daigremontiana. Root induction and development were monitored, and quantitative image analysis was performed. Next, five hairy root lines were successfully obtained using the ATCC 15,834 + pTDT strain. The rolA, rolB, rolC, rolD, genes and additional T-DNA markers (orf13, orf14 genes), as well as the TDT protein, were detected in all transformed lines. Morphometric evaluation revealed enhanced growth dynamics in lines KdA2, KdB6, and KdB11. Total phenolic and flavonoids contents in hairy roots extracts ranged from 15.60 to 18.37 mg of gallic acid equivalents (GAE)/g extract and 8.77-13.87 mg quercetin equivalents (QE)/g extract, respectively, compared with 278.13 mg GAE/g extract and 32.97 mg QE/g in wild-type roots. Although in hairy root cultures, basal phenolic levels were lower than in wild-type roots, the established cultures provide a genetically stable platform that can be further optimized through elicitation strategies to enhance the production of health-relevant metabolites. These findings underscore the importance of exploring differential accumulation of metabolites in hairy root cultures.