# Development of an efficient in vitro micropropagation and biochemical profiling of Salvia halophila, an endemic Turkish sage

## TL;DR

This study develops an efficient method to grow an endangered Turkish sage species in the lab, improving its survival and phytochemical production.

## Contribution

A stage-specific in vitro micropropagation protocol for Salvia halophila that enhances germination, shoot regeneration, and secondary metabolite accumulation.

## Key findings

- GA₃-enriched WPM medium achieved 20% germination, with minimal contamination.
- MS medium with meta-topolin and IBA yielded optimal shoot regeneration and rooting.
- Micropropagated plants showed higher phenolic content and antioxidant capacity than seed-derived plants.

## Abstract

Salvia halophila Hedge is an endemic sage species confined to the salt steppe ecosystems of Central Anatolia (Türkiye), particularly around the Lake Tuz Basin, and is classified as Endangered (EN) by the IUCN. Its narrow distribution, low seed germination rates, and habitat pressures threaten its survival and restrict the sustainable exploitation of its valuable phytochemicals, such as rosmarinic acid and other phenolics. In vitro micropropagation represents a strategic approach for producing genetically uniform and phytochemically rich plant material, thereby supporting both ex situ conservation and biotechnological applications.

Three germination strategies were compared. Chemical priming and direct gibberellic acid (GA₃) applications were largely ineffective, whereas in vitro germination on GA₃-enriched media produced viable seedlings. WPM medium supplemented with 0.5–1.0 mg L⁻¹ GA₃ achieved the highest germination rate (20%). The optimized sterilization treatment minimized fungal contamination (4.4%) while sustaining a high proportion of healthy shoots (73%). For shoot induction, MS medium consistently outperformed WPM, achieving 78% induction and 1.51 shoots per explant versus 50% and 0.86 on WPM. Among cytokinins, meta-topolin (mT) provided the most reliable results, ensuring 90–100% shoot induction with high shoot quality at 0.2–1.0 mg L⁻¹. Rooting experiments demonstrated the superiority of IBA over NAA, with 0.5 mg L⁻¹ IBA yielding 66% rooting and 77% acclimatization. Biochemical profiling showed that micropropagated plantlets accumulated higher phenolic contents (TPC = 15.9 mg GAE g⁻¹ DW) and stronger antioxidant capacity (IC₅₀=24.6 µg mL⁻¹) compared with seed-derived plants.

The study successfully established a stage-specific and efficient micropropagation protocol for S. halophila. While WPM medium was more favorable for seed germination, MS medium supplemented with mT and IBA was optimal for shoot regeneration and rooting. The protocol not only enhances propagation efficiency but also increases secondary metabolite accumulation, providing a reliable platform for conservation, sustainable utilization, and potential commercial production of this endangered halophyte.

The online version contains supplementary material available at 10.1186/s12870-026-08152-2.