Optimization of plant growth regulators and media compositions to enhance in vitro gametic embryogenesis through anther culture of soybean [Glycine max (L.) Merr.].

Soybean is a major oilseed crop of global importance. Although haploid and doubled haploid (DH) production has been attempted in soybean, androgenesis success remains very low (approximately 2%). The study aims to standardize a protocol for haploid production through anther culture. Under aseptic conditions, anthers were excised from immature buds (2.9-3.1 mm), pre-treated at 4 °C for 24 h and cultured in different induction media. The highest callus induction (100%) was observed in the genotype SKAF148 on BNN medium supplemented with 5 mg/L 2,4-D and 1.5 mg/L BAP. Cultures were initially incubated at 7 °C under dark conditions for two weeks, followed by a 16/8 h light/dark photoperiod at 26 ± 2 °C. For callus proliferation, anther-derived embryoids were transferred to MS medium containing 1.5 mg/L BAP and maintained under a 16/8 h photoperiod at 26 ± 2 °C. Molecular marker analysis confirmed genetic fidelity, while ploidy levels of calli and embryos were assessed using flow cytometry. Scanning and transmission electron microscopy revealed metabolically active embryos (20%) with intact ultrastructural (100%) organization. The in vitro protocol is rapid (40-45 days), suitable for gametic embryogenesis and haploid induction in soybean breeding. The protocol enabled rapid haploid production (within 40-45 days), and the methodology provides a practical framework for accelerating soybean breeding programs through haploid and doubled haploid technology.