Identification of the most suitable reference genes for nanoparticle stress response in Salvia rosmarinus (rosemary) produced under in vitro conditions

Abstract Salvia rosmarinus L. (rosemary) is known to have a wide range of pharmacological effects including antidiabetic, anticarcinogenic, and antitumorigenic properties owing to its secondary metabolites. Studies aiming for the elevation of these metabolites have utilized various elicitors and stresses under in vitro conditions, although underlying molecular mechanisms remain unexplored. Gene expression studies using qRT-PCR might provide valuable information regarding how plant and plant cells interact and perceive various treatments and elicitors. Although, despite being able to calculate accurate fold changes, the accuracy of the qRT-PCR data highly depends on the expression of housekeeping (H.K.) genes. To the best of our knowledge, there is no information available on the stable H.K. genes in rosemary under in vitro conditions. Thus, in this paper, we assessed the stability of seven commonly used H.K. genes with qRT-PCR. Thereafter, the five most commonly used software (comparative ΔCt, BestKeeper, NormFinder, geNorm, and RefFinder) were used to rank the candidates based on their expression stabilities. We recommend using a combination of F1-ATPase and 18S rRNA to normalize the gene expression experiments in rosemary under in vitro conditions. The selected H.K. genes were verified using 4-coumarate-CoA ligase i.e., a pharmacologically important gene, whose expression might alter under nanoparticle treatment. Additionally, reference genes for several plant tissues, elicitors, and stresses are also proposed. The conclusions obtained from this current study will accelerate the future molecular work in S. rosmarinus and other related species.