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

## TL;DR

This study improves garlic plant regeneration from protoplasts using SAHA and PSK, enabling better genetic research and biotechnology applications.

## Contribution

A novel protocol using SAHA and PSK to enhance garlic protoplast regeneration and plant acclimatization is developed.

## Key findings

- SAHA treatment significantly improves microcallus formation and plant regeneration from garlic protoplasts.
- The optimized protocol achieves a 70% success rate in plant acclimatization with 97% ploidy retention.
- Protoplast isolation using cellulase R-10 and pectolyase Y23 yields high viability and yield.

## Abstract

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 (Allium sativum 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.