Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0-5.0×10⁶ were obtained following 4-6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10⁵ protoplasts/ml was optimal for subsequent division. An initial division frequency of 12-15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μM), and zeatin (2.3 μM). Colonies consisting of 100-200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1-2 mm in diameter) were formed after an additional 4-6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μM) and benzylaminopurine (2.2 μM). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μM gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.