Plant regeneration of mulberry (<Emphasis Type="Italic">Morus indica</Emphasis>) from mesophyll-derived protoplasts

A protocol is presented for regenerating plants from protoplasts of tropical mulberry. Leaves from seedling node cultures maintained in vitro were used as donor tissue. Optimal cell wall digestion was achieved with a combination of cellulase (2%) and macerozyme (1%). The plant growth regulator (PGR) combination zeatin (2.3 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.3 μM) resulted in the highest number (29%) of cell divisions. First cell divisions were observed at day 4 after plating. Only zeatin (2.3 μM) and 2-methoxy-3,6-dichlorobenzoic acid (dicamba) (13.5 μM) supplemented medium supported subsequent divisions in protoplast cultures. Microcolonies reached a cell number of approximately 50, after 40 to 42 days of culture. The cells of these colonies continued dividing, leading to formation of microcalli. Whole plants were obtained after culture of microcalli on Murashige and Skoog (MS) medium containing thidiazuron (TDZ) (4.5 μM) and indole-3-acetic acid (IAA) (17.1 μM). The regenerated shoots were rooted on MS medium supplemented with 4.9 μM indole butyric acid (IBA). With a low survival rate during acclimation, regenerated plants were established in the greenhouse.