Somatic embryogenesis and in vitro shoot propagation of Gentiana utriculosa

Study describes protocols for in vitro propagation of Gentiana utriculosa L. via axillary shoot multiplication and indirect somatic embryogenesis. Shoot cultures were established from seedling epicotyl explants cultured on MS medium supplemented with 0.25 mg L-1 BA and 0.1 mg L-1 IAA. Medium containing 2% sucrose and 0.2 mg L-1 BA improved multiple shoot production, providing 2.3 shoots per explant. The highest rooting (29.6%) was obtained on medium with 1/2 MS mineral salts and 0.5 mg L-1 NAA. Somatic embryogenesis was induced using different explants, including immature seeds as well as leaves and roots from shoot cultures. Following auxin treatment with either 1.0 mg L-1 2,4-D (immature seeds and leaves) or 0.1 mg L-1 NAA (roots), explants produced embryogenic calli which upon transfer to plant growth regulator-free medium allowed embryo conversion into plantlets. The best embryogenic response (82%) was obtained in calli derived from leaves cultured with their abaxial surface in contact with medium, whereas the highest embryo conversion rate (68%) was recorded for calli induced on immature seed explants. Histological analysis in all explant types revealed development of proembryogenic cell complexes at callus periphery, giving rise to somatic embryos. The presence of embryos at various stages of development indicated asynchronous somatic embryogenesis in G. utriculosa. Derooted embryo-derived plantlets placed on medium with 0.2 mg L-1 BA multiplied further as shoot cultures.