An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (Ecs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.
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