Optimization of Plant Growth Regulators for in Vitro Callus Induction and Shoot Proliferation in the Natural Sweetener Stevia Rebaudiana (Bertoni)

Stevia rebaudiana (Bertoni), a perennial herb of the Asteraceae family, is the source of zero-calorie steviol glycosides (notably stevioside and rebaudioside A) that are 250–300 times sweeter than sucrose and possess antidiabetic, antihypertensive, anti-inflammatory and antioxidant properties (Salehi et al., 2023). Because seed germination is poor and the species is highly self-incompatible, in-vitro propagation offers the most reliable route to genetically uniform, large-scale planting material (Abouelela et al., 2025). The present study optimised plant growth regulator (PGR) combinations for callus induction and shoot proliferation from leaf explants of S. rebaudiana on Murashige and Skoog (MS) medium. For callogenesis, 2,4-dichlorophenoxyacetic acid (2,4-D) was combined with either kinetin (Kn) or a-naphthaleneacetic acid (NAA), and for shoot proliferation, 6-benzylaminopurine (BAP) was combined with NAA at eight concentration ratios. The maximum callus-induction frequency (86.2 ± 3.6%), yielding green, organogenic callus, was obtained on MS medium supplemented with 2,4-D + NAA at 1.5 + 3.0 mg L^-1, which outperformed all 2,4-D + Kn combinations. For shoot proliferation, MS medium with BAP + NAA at 0.5 + 1.0 mg L^-1 gave the highest response (88% of explants), the greatest mean shoot length (3.61 ± 0.21 cm after four weeks) and a four-fold multiplication rate, whereas higher cytokinin-auxin ratios were progressively inhibitory. The optimised protocol provides an efficient, reproducible system for rapid clonal multiplication of this commercially important sweetener crop and a platform for downstream steviol-glycoside production and germplasm conservation.