Optimization of protoplast cell division and microcallus formation toward somatic embryogenesis in oil palm

Efficient protoplast regeneration remains a major obstacle to the application of somatic hybridization, genome editing and precision breeding in oil palm (Elaeis guineensis); while early developmental stages, from cell wall regeneration and cell division to microcolony and microcallus formation, could be more important, which could be connected easily with the traditional tissue culture system for succeeding plant regeneration. This study aimed to optimize the protopalst culture conditions for cell division and microcallus formation by identifying the most effective combinations of culture systems, basal media and plant growth regulators.

Protoplasts isolated from different oil palm tissues (embryogenic callus, suspension cultures, and somatic embryos) were investigated by protoplast yield and viability; protoplasts from the embryogenic callus showed the most satisfactory response and was therefore used for subsequent optimization of culture conditions. The protoplasts were cultured using six culture systems (liquid, droplet, agarose pool, well-plate culture, feeder layer, and nurse culture) in combination with eight basal media (BH3, MS, WPM, Y3, KM, B5, N6, and KPR), and the culture effects were evaluated based on protoplast viability, plating efficiency, cell division, microcolony and microcallus formation. Microcallus emerged from the KPR solid medium were transferred to previously established traditional tissue culture system for further somatic embryogenesis and regeneration.

BH3 liquid medium was particularly effective for early protoplast recovery and rapid cell wall regeneration, whereas KPR solid medium combined with the nurse culture system, provided the most favorable conditions for sustained cell division, plating efficiency, microcolony and microcallus formation. Cell division occurred within 3-7 days, followed by microcolony formation after approximately 7 days and visible microcallus formation after 60 days. Microcallus were transferred for further development, somatic embryos and bud regeneration was observed after 90 days and 120 days, respectively.

The present study focused on the early developmental stages of oil palm protoplast culture, and optimized a stage-specific culture strategy for quick microcallus formation; combined with previous oil palm tissue culture system, further somatic embryogenesis and bud regeneration were established successfully. This work not only promoted the protoplast regeneration of oil palm, but also provides a promising strategy for other tropical palm trees such as coconut and betel nut.