Productive method for commercial propagation through direct embryogenesis and organogenesis in Ledebouria revoluta—an underutilized medicinal plant with cardioprotective properties

Abstract

The bulbaceous herb Ledebouria revoluta (family Asparagaceae) is an important cardioprotective plant. Two different pathways of in vitro direct regeneration system consisting of somatic embryogenesis and shoot organogenesis have been established for high-frequency micropropagation. A rectangular piece (6 × 10 mm2) of leaf lamina was used as an explant to study the morphogenetic regulation by exogenously supplied auxins, cytokinins, and polyamine within controlled in vitro environment. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose and various concentrations and combinations of plant growth regulators (PGRs), as per experimental needs. Both β-naphthoxyacetic acid and α-naphthalene acetic acid (NAA) auxins individually induce somatic embryogenesis. Combined application of auxins and cytokinins further improves the embryonic response. The synergistic effect of NAA (2.0 mg L–1), kinetin (1.0 mg L–1), and spermidine (SDN, 217.88 mg L–1) provided the best result with 96.7 ± 1.8 % response and highest 63.4 ± 0.8 embryos per explant. The highest (94.5 ± 2.5 %) conversion of embryos to complete plantlets was noted in presence of 1.0 mg L–1 kinetin. The morphogenic path altered from embryogenic to organogenic when auxin was totally withdrawn, and a higher concentration of cytokinin was applied. The synergism between 6-benzylaminopurine (3.0 mg L–1) and SDN (217.88 mg L–1) proved the best PGR combination for the direct shoot (or bulblet) organogenesis with maximum 96.6 ± 2.2 % response and highest 48.6 ± 0.5 bulblets per explant. Total withdrawal of auxin was essential for direct bulblet organogenesis because the presence of any auxin always shifts the morphogenic path from organogenic to embryogenic. Histological study confirms the bipolar structure of somatic embryo and unipolar bulblets. Highest 12.7 ± 0.54 roots per bulblet were induced on ½ strength MS medium containing 2.0 mg L–1 indole-3-butyric acid. Finally, the plantlets were acclimatized with 96.7 % survival rate and all of the survived plants produced bulbs and 92.5 % of them flowered normally in field conditions. This in vitro system permits a high-frequency regeneration through embryogenic and organogenic paths, which can be used for large-scale commercial micropropagation for round-the-year supply of this important cardioprotective medicinal plant.