Effect of Suberoylanilide Hydroxamic Acid and Phytosulfokine-Alpha on Successful Plant Regeneration from Embryogenic Callus-Derived Protoplasts of Garlic (Allium sativum L.)

Garlic's vegetative reproduction limits genetic improvement, necessitating advanced biotechnological tools like protoplast culture. However, efficient protoplast regeneration in monocots such as garlic remains a significant challenge. This study establishes an optimized protocol for embryogenic callus induction and subsequent protoplast-to-plant regeneration in garlic (<i>Allium sativum</i> L.), aiming to overcome current limitations using suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, and phytosulfokine-alpha (PSK). We successfully induced embryogenic callus from four garlic accessions and refined protoplast isolation and culture conditions. Key optimizations included using a specific enzyme mixture (2% cellulase R-10 and 0.2% pectolyase Y23) for high yields (from 0.8 to 2.1 × 10⁶ protoplasts per g FM) of viable (approx. 90%) protoplasts and employing the enriched K8M culture medium. Short exposure of protoplasts to SAHA (0.05 or 0.1 µM) significantly improved microcallus formation and plant regeneration. Notably, only callus derived from SAHA-treated cultures displayed regeneration potential, highlighting its pivotal role in embryo differentiation and development. This optimized protocol achieved a 70% success rate for plant acclimatization to ex vitro conditions, with 97% of regenerated plants retaining the ploidy of the donor accession. We demonstrate that SAHA and PSK application enhances garlic protoplast regeneration efficiency. This reliable system provides the groundwork for advanced biotechnological applications, including gene editing technologies in garlic.