Establishment of an in vitro culture and regeneration protocol for the native grass Polypogon australis Brong

Polypogon australis Brong. is a native Chilean grass frequently found colonizing metal-rich tailings, yet it lacks an established in vitro regeneration system to support controlled experimentation. Here, we report an efficient and reproducible protocol for seed germination, callus induction, and plant regeneration using coleoptile–mesocotyl explants. Seeds were surface-sterilized and germinated on MS medium supplemented with sucrose, achieving 44–51% germination. The coleoptile–mesocotyl junction was identified as the most responsive explant type, showing a callus induction rate of 30.55% after 3–5 weeks. Embryogenic calli predominated under Dicamba treatment and regenerated multiple plantlets without the need for exogenous organogenesis-promoting hormones. Regeneration frequency reached 45.5%, producing uniform and totipotent plantlets suitable for downstream analyses. This protocol overcomes a major bottleneck in P. australis research and provides a reliable framework for future physiological and molecular studies involving this ecologically relevant native species.