The effects of plant growth regulators on in vitro seedling development in Verbascum bugulifolium and thidiazuron-induced changes in phenolic substance contents in its callus tissues

Verbascum bugulifolium is a rare and potentially multi-purpose plant species with a narrow natural distribution limited to South?eastern Bulgaria and Northwestern T?rkiye. This study examined the effects of several plant growth regulators on seed germina?tion, organ development, and callogenesis in V. bugulifolium to identify an efficient in vitro culture protocol which would aid in conservation efforts and estimate its medicinal potential. Gibberellic acid (GA3) treatment at 0.5 mg L-1 promoted seed germination and organ development. The efficacy of GA3 treatment in enhancing seed germination and callogenesis increased when combined with thidiazuron (TDZ) at 2.0 mg L-1. The GA3 treatment also increased hypocotyl lengths when combined with other PGRs, except when combined with kinetin (KIN). Leaf production increased in response to GA3 treatment alone or in combination with 6-benzyl?aminopurine (BAP) and TDZ. High concentrations of auxin treatments limited leaf production. Treatment with 1-naphthaleneacetic acid (NAA) at 0.25 mg L-1 increased root production while significantly decreasing its elongation. All cytokinin and auxin treatments except indole-3-butyric acid (IBA) at 0.25 and 0.5 mg L-1 significantly reduced root elongation. GA3 treatments and the combination of GA3 with IBA at 0.5 mg L-1 increased root lengths. TDZ was the most effective callus-inducing growth regulator, and its effect increased when combined with GA3. The calli yielded promising amounts of total phenolic, flavonoid, and tannin contents, which varied in response to different TDZ concentrations. The protocol described here may be used to promote enhanced seedling growth and callogenesis in the species.