Optimization of a high-efficiency single-cell regeneration system in sugarcane (Saccharum officinarum) via hormonal regulation and physiological characterization.

Abstract

Efficient regeneration of plants from single cells is a critical yet challenging step for applying modern biotechnologies to sugarcane (Saccharum spp.), a vital sugar and bioenergy crop. The main obstacles include low embryogenic competence and recalcitrant differentiation. Here, we established a standardized, high-efficiency single-cell regeneration system for the model cultivar ROC22 by systematically optimizing key hormonal and physiological parameters. Embryogenic callus, induced from young leaf sheaths, was used to establish suspension cultures. A homogeneous population of single cells with 58 % viability was isolated via 200-mesh sieve filtration. Dynamic growth analysis identified 2.0 mg L^-1 2,4-dichlorophenoxyacetic acid (2,4-D) as optimal for proliferation, yielding a peak density of 2.4 × 10⁵ cells/mL. The differentiation of compact callus was maximized on medium containing 2.0 mg L^-1 6-benzylaminopurine (6-BA) and 0.5 mg L^-1 kinetin (KT), resulting in minimal browning (17.8 %) and large callus clusters (1.52 cm in diameter). Ultimately, a high green plantlet regeneration efficiency of 81.1 % was achieved on a regeneration medium with half-strength MS macronutrients, 3 mg L^-1 naphthaleneacetic acid (NAA), and 0.5 mg L^-1 6-BA. This reproducible and efficient system provides a robust platform for genetic transformation and single-cell-based studies in sugarcane.