Enhanced embryogenesis and plant regeneration from cucumber (Cucumis sativus L.) callus by activated charcoal in solid/liquid double-layer cultures

Enhanced embryogenesis and plant regeneration methods were established in cucumber (Cucumis sativus L. cv. 'Delilah') using hypocotyl segments as explants. Callus formation, followed by pro-embryogenic aggregates and globular embryoids required liquid shake cultures. In liquid medium, however, many of the embryoids developed into abnormal structures - 'neomorphs' or succulent plantlets. Embryoids subcultured to stationary liquid or agar cultures dedifferentiated and underwent secondary embryogenesis. Neither increased osmolarity nor adding abscisic acid (ABA), zeatin or activated charcoal to the liquid medium inhibited abnormal morphogenesis. The use of double layer cultures containing activated charcoal in the lower agar layer and ABA with elevated calcium in the upper liquid phase prevented dedifferentiation and secondary embryogenesis and allowed normal organized growth of the embryoids. Hardening in vitro by partial desiccation with CaSO₄ under aseptic conditions improved the cucumber plantlet's leaf growth and their survival after transplanting to soil.