Exploring somatic embryogenesis in Ajuga multiflora Bunge: Profiling lipophilic metabolites via HPLC, GC-FID, and GCMS– analysis

Abstract

The present study aims to assess the effect of different concentrations of auxins and combinations of plant growth regulators (PGRs), specifically auxin and cytokinin, on somatic embryogenesis in Ajuga multiflora. We also aim to compare the lipophilic metabolite profiles, such as carotenoids (CAs), fatty acids (FAs), phytosterols (PSs), and tocopherols (TPs), in globular somatic embryos (SEs), germinated SEs, and shoots of SEs derived plantlets. This assessment is carried out utilizing high performance liquid chromatography, gas chromatography-flame-ionization detection, and gas chromatography–mass spectrometry techniques. The results reveal that the application of 2,4-dichlorophenoxyacetic acid (2,4-D) had the most significant impact on somatic embryogenesis, inducing both SE initiation (34.4%) and the number of SEs (3.5) per explant. Among the explants used, leaf explants were found to be the most effective for SE induction (SEI). Additionally, the study explores the combined effect of 2,4-D and cytokinins (N6-benzyladenine, N6-BA, or 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, TDZ) on somatic embryogenesis, highlighting optimal conditions for SEI percentages and the number of SEs per explant. The maximum SE induction rate (100%) and the number of SEs (45.9) per leaf explant were obtained on an SEI medium with 30 µM of 2,4-D and 2.3 µM of TDZ. The maturation and conversion of SEs were also examined, emphasizing the role of GA3 in achieving the highest conversion percentage (94.0 ± 2.7 at 0.8 µM GA3). Furthermore, the study details the biochemical composition during various stages of somatic embryogenesis, including CAs, TPs, PSs, and FAs. Globular SEs displayed comparatively lower concentrations of CAs than their germinated counterparts and shoots from SE-derived plantlets, while the total PSs content remained relatively consistent across all stages. Conversely, fatty acids tended to accumulate more in globular SEs, with the exception of α-Linolenic acid. These findings provide valuable insights into the optimization of somatic embryogenesis protocols in A. multiflora, with implications for plant tissue culture, regeneration and lipophilic metabolites production.