High-frequency somatic embryogenesis and artificial seeds for mass production of true-to-type plants in Ledebouria revoluta: an important cardioprotective plant

Abstract

Scaly bulb of Ledebouria revoluta is a natural source of several cardiac glycosides and is traditionally used in Indian and South African ethno-medicinal system from ancient age. High-frequency indirect somatic embryogenesis protocol via callus culture had been developed from bulb scale explant. Optimum embryogenic calli was induced on Murashige and Skoog (MS) medium supplemented with 3.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.75 mg l−1 β-naphthoxyacetic acid. A maximum of 93.3 % of the cultures responded after 6 weeks of culture on MS medium containing 3.0 mg l−1 of thidiazuron, 0.75 mg l−1 α-naphthalene acetic acid and 1.75 mM spermidine forming 46.7 ± 0.58 somatic embryos per 500 mg callus. Individual somatic embryo was encapsulated in calcium alginate beads to produce artificial seeds (ASs). The ASs were stored at 4, 15, and 24 °C temperatures up to 180 days. The ASs showed 57.8 % germinability even after 4 months of storage at 15 °C. Plantlets were acclimatized with a survival rate of 96.0 % and after 13–14 months, 92.4 % of these plants produced flowers. Chromosomal studies revealed cytological stability of the regenerants containing 2n = 30 chromosomes in root tips and n = 15 chromosomes in pollen grains, same as parental plants. The meiotic behaviour of the regenerants is also similar to that of parental plants. Randomly amplified polymorphic DNA analysis revealed that there is no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirm the very reliable method for short-term conservation and large-scale production of true-to-type plantlets of L. revoluta, which can be applied in pharmaceutical industries.