Rosemary (Salvia rosmarinus L.), belonging to the lamiaceae family, is an aromatic plant with well-known pharmacological effects. The wide range of pharmacological effects of this perennial herb is mainly due to its ample secondary metabolites which are known to have antioxidant, antibacterial, anti-inflammatory, antidiabetic, anticarcinogenic, and antitumorigenic activities1, 2. In addition to these, recent studies have also shown that carnosic acid, carnosol, and rosmanol (phytochemicals present in rosemary) have inhibitory effects against the coronavirus’s main protease (SARS-CoV-2 Mpro)3, 4. SARS-CoV-2 Mpro contains important catalytic site residues, which play an essential role in virus proliferation. Consequently, interest has been raised in increasing these pharmacologically important secondary metabolites of this species among the industries. In this scenario, plant tissue culture technology can be an excellent option for large-scale disease-free plant and tissue production with elevated quantities of secondary metabolites from rosemary and several other medicinal plants, irrespective of their growing seasons. Furthermore, treatment of various elicitors such as nanoparticles and others in culture systems can also boost or change the secondary metabolite level5. Even though studies aiming to increase the secondary metabolites through plant tissue culture in rosemary are constantly in process, many more will be needed in the future to accomplish its demands in industrial production. In order to effectively manipulate the synthesis of these metabolites under in vitro conditions, underlying molecular mechanisms needs to be explored. Currently, based on the previous literature studies, there is limited information available about the genes and metabolic pathways in rosemary6. Hence gene expression analysis using quantitative real-time PCR (qRT-PCR) can be an essential tool to get insights into the metabolic pathways of secondary metabolites in specific tissues or under different conditions.
Owing to its high specificity, rapidity, and sensitivity, qRT-PCR has gained an edge over the traditional polymerase chain reaction (PCR) for comparative expression studies. Although qRT-PCR can calculate accurate fold changes, its accuracy is extremely reliant on the expression of a suitable housekeeping gene7. The steps of qRT-PCR are prone to technical noises and variations in the sample preparation. Hence, to nullify these variations, appropriate normalization methods are necessary8. The target gene transcription levels must be normalized with suitable reference gene transcription levels. Any inaccuracies in selecting a suitable reference gene may lead to deceptive results9, 10. The most commonly used references genes in plant gene expression studies include ubiquitin (UBQ), eukaryotic elongation factor (eEF), α-tubulin (α-TUB), β-tubulin (β-TUB), actin (ACT), ribosomal RNA genes (rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Acetyl CoA Carboxylase (ACCase), etc.8. Although it can be assumed that these genes will have a stable expression in any given condition, however, numerous studies have provided evidence of their variability in their expression level between species of plants or different stress conditions or developmental stages7, 11 (Joseph et al., 2018; Czechowski et al., 2005).
Interest in plant tissue culture or in vitro plant culture has grown mainly due to its promising ability to produce improved crop varieties and high yield of crucial secondary metabolites. Several efforts have been made to enhance the production of important secondary metabolites using different biotic and abiotic factors. Currently, the addition of elicitors such as nanoparticles has gained worldwide interest owing to their success in enhancing secondary metabolites in many species. Soltanabad et al., 2020 showed that silver nanoparticle treatment could boost the carnosic acid content in Rosmarinus officinalis L.12 The rising interest in using nanoparticles in commercially important medicinal plants (such as rosemary) will increase the need for gene expression studies in these species. To the best of our knowledge, there are no reports on the identification of the most suitable reference genes for gene expression studies under nanoparticle stress in rosemary produced under in vitro conditions. Hence, this study aims to identify a suitable reference gene for gene expression studies in in vitro S. rosmarinus. We had selected and identified seven common candidate reference genes (18S rRNA, 25S rRNA, 28S rRNA, ACCase, GAPDH, ATP-synthase, and F1-ATPase) in S. rosmarinus and assessed their gene expression stability in three different plant tissues/organs (callus, stem, and leaf), temperature stress (heat stress and cold stress), two elicitor stress (casein hydrolysate and jasmonic acid), osmotic stress (sorbitol) and salt stress (NaCl). Five different widely used statistical software for reference gene analysis (comparative ΔCt13, BestKeeper14, NormFinder15, geNorm16, and RefFinder17) were used to identify the best candidate. Thereafter, the most suitable reference gene was used to validate using the 4-coumarate-CoA ligase (4CL) gene under a nanoparticle (NP)-stress experiment. 4CL catalyzes the enzymatic reactions in the general phenylpropanoid pathway and participates in the synthesis of flavonoids. All flavonoids are produced from one molecule of p-coumaroyl CoA and three molecules of malonyl CoA. Flavonoids are pharmacologically important compounds with numerous probable medicinal properties, including anticancer, antioxidant, anti-inflammatory, and anti-inflammatory activities18. Additionally, suitable candidates under each mentioned experimental condition were also identified. Our study will provide a basis for current and future gene expression studies in S. rosmarinus and its related species and will assist in forthcoming molecular studies aiming for a better understanding of the metabolic pathways associated with secondary metabolites.