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

Abstract

Polypogon australis Brong. is a native Chilean grass frequently found colonizing metal-rich mine tailings, yet it lacks an established in vitro regeneration system to support controlled physiological and biotechnological studies. Here, we report a reproducible protocol for seed germination, callus induction, and plant regeneration using coleoptile–mesocotyl explants. Surface-sterilized seeds were germinated on Murashige and Skoog (MS) medium supplemented with sucrose, achieving a cumulative germination percentage of 47.67 ± 3.15% after 15 days. The coleoptile–mesocotyl explant proved highly responsive to culture on callus induction medium (CIM) supplemented with dicamba, resulting in a callus induction frequency of 30.55 ± 11.96% after 3–5 weeks. Induced calli were predominantly embryogenic, with embryogenic calli representing 65.42 ± 8.61% of the total callus population. Embryogenic calli regenerated complete plantlets with a regeneration efficiency of 45.0 ± 23.3%. Organogenic structures, including primary shoots and roots, developed directly from embryogenic calli maintained on callus induction medium (CIM) supplemented with dicamba, without transfer to a specialized regeneration medium containing organogenesis-promoting growth regulators. After the initiation of organogenesis, cultures were exposed to a 16 h light/8 h dark photoperiod while remaining on CIM, and regenerated plantlets were subsequently transferred to MS+10 S medium for further growth and elongation. This study establishes the first complete in vitro regeneration system for P. australis, providing a practical framework for future physiological studies, large-scale propagation, genetic transformation, and genome engineering applications in this ecologically relevant native Chilean grass.