Somatic embryogenesis and plantlet regeneration from protoplast culture of Muscari neglectum Guss

Abstract

A protocol has been developed for plant regeneration from protoplast culture of Muscari neglectum using regenerable embryogenic calli obtained from bulb culture on Murashige and Skoog medium (MS) plant growth regulators (PGR)-free or containing lower 1-naphthaleneacetic acid (NAA) and 6- benzylaminopurine (BA) concentrations and 30 g/l sucrose. Protoplasts were isolated directly from embryogenic calli, embedded in Ca-alginate beads and cultured with nurse cells in MS medium supplemented with 1 mg/l each of NAA and BA, 100 mg/l ascorbic acid and 0.5 M mannitol at 25°C in darkness. After 4 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads. Growth of microcalli in the medium with nurse cells (33.3%) was much better than those in the medium without nurse cells (6.5%). Transferring beads onto MS medium supplemented with 0.1 mg/l BA increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium PGR-free or with 1 mg/l abscisic acid at 25°C under continuous illumination with fluorescent light. Maturated embryos germinated and then converted to plantlets on half strength MS medium containing 1 mg/l BA after 3 months. The plantlets left in the medium produced bulbs after 5 months.

Key words: Ca-alginate beads, Muscari neglectum, nurse culture, plantlet regeneration, protoplast culture, somatic embryogenesis.