Optimization of Somatic Embryogenesis and Plant Regeneration in Areca Palm (Areca catechu L.)

Somatic embryogenesis (SE)-the reprogramming of somatic cells to follow an embryogenic pathway-remains inefficient in the tropical monocot areca palm (Areca catechu L.) due to poorly understood constraints on cell fate transition. Using single-factor and orthogonal experiments, we investigated how plant growth regulators (PGRs), nitrogen source, and carbon supply regulate the acquisition of embryogenic competence. The optimal condition (Y3 medium + 6-BA + TDZ + 30 g/L sucrose) yielded a somatic embryo induction rate of 48.62%, more than six-fold higher than that of the unoptimized control. Cytologically, embryogenic callus consisted of small, densely cytoplasmic cells with large, centrally positioned nuclei with decondensed chromatin (i.e., loosely packed, transcriptionally active chromatin) and minimal vacuolation-hallmarks of meristematic, embryogenic-competent cells-whereas non-embryogenic callus contained large, highly vacuolated irregular cells. Histological tracking revealed that areca somatic embryos faithfully recapitulate the zygotic developmental program, including bipolarity establishment and procambium differentiation. Mechanistically, we show that the 6-BA + TDZ combination likely acts by inducing endogenous auxin synthesis; moderate ammonium supply (Y3 medium, NO₃^-/NH₄⁺ ratio 3.6:1) minimizes browning; and 30 g/L sucrose provides optimal energy supply and osmotic regulation. This optimized SE system establishes a tractable model for studying somatic cell reprogramming in a woody monocot.