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 to elevate these metabolites have utilized various elicitors and stresses under in vitro conditions, although underlying molecular mechanisms remain unexplored. Gene expression studies using RT-qPCR might provide valuable information regarding how plant and plant cells interact and perceive various treatments and elicitors. However, despite being able to calculate accurate fold changes, the accuracy of the RT-qPCR data highly depends on the expression of reference genes. To the best of our knowledge, there is no information available on the stable reference genes in rosemary under in vitro conditions. Thus, in this paper, we assessed the stability of seven commonly used reference genes under different elicitor and stress conditions using RT-qPCR. Thereafter, the five most commonly used software and algorithms (comparative ΔCt, BestKeeper, NormFinder, geNorm, and RefFinder) were used to rank the candidates based on their expression stabilities. In conclusion, we recommend using a combination of F1-ATPase, ATP synthase and ACCase to normalize the gene expression experiments in rosemary under in vitro conditions. The selected reference genes were verified using 4-coumarate-CoA ligase, 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.

Department of Agroecology and Crop Production Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 165 00 Prague 6 Czech Republic

Department of Botany and Plant Physiology Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 165 00 Prague 6 Czech Republic

Department of Crop Sciences and Agroforestry The Faculty of Tropical AgriSciences Czech University of Life Sciences Prague Kamýcká 129 165 00 Prague 6 Czech Republic

Department of Plant Protection Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamýcká 129 165 00 Prague 6 Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

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Kompelly A., Kompelly S., Vasudha B., Narender B. Rosmarinus Officinalis L.: An Update Review of Its Phytochemistry and Biological Activity. J. Drug Deliv. Ther. 2019;9:323–330. doi: 10.22270/jddt.v9i1.2218. DOI

Moore J., Yousef M., Tsiani E. Anticancer Effects of Rosemary (Rosmarinus Officinalis L.) Extract and Rosemary Extract Polyphenols. Nutrients. 2016;8:731. doi: 10.3390/nu8110731. PubMed DOI PMC

Villegas-Sánchez E., Macías-Alonso M., Osegueda-Robles S., Herrera-Isidrón L., Nuñez-Palenius H., González-Marrero J. In Vitro Culture of Rosmarinus Officinalis L. in a Temporary Immersion System: Influence of Two Phytohormones on Plant Growth and Carnosol Production. Pharmaceuticals. 2021;14:747. doi: 10.3390/ph14080747. PubMed DOI PMC

Cui W., Yang K., Yang H. Recent Progress in the Drug Development Targeting SARS-CoV-2 Main Protease as Treatment for COVID-19. Front. Mol. Biosci. 2020;7:616341. doi: 10.3389/fmolb.2020.616341. PubMed DOI PMC

de Rivero-Montejo S.J., Vargas-Hernandez M., Torres-Pacheco I. Nanoparticles as Novel Elicitors to Improve Bioactive Compounds in Plants. Agriculture. 2021;11:134. doi: 10.3390/agriculture11020134. DOI

Yao D., Zhang Z., Chen Y., Lin Y., Xu X., Lai Z. Transcriptome Analysis Reveals Differentially Expressed Genes That Regulate Biosynthesis of the Active Compounds with Methyl Jasmonate in Rosemary Suspension Cells. Genes. 2022;13:67. doi: 10.3390/genes13010067. PubMed DOI PMC

Joseph J.T., Poolakkalody N.J., Shah J.M. Plant Reference Genes for Development and Stress Response Studies. J. Biosci. 2018;43:173–187. doi: 10.1007/s12038-017-9728-z. PubMed DOI

Sen M.K., Hamouzová K., Košnarová P., Roy A., Soukup J. Identification of the Most Suitable Reference Gene for Gene Expression Studies with Development and Abiotic Stress Response in Bromus Sterilis. Sci. Rep. 2021;11:13393. doi: 10.1038/s41598-021-92780-1. PubMed DOI PMC

Chapman J.R., Waldenström J. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS ONE. 2015;10:e0141853. doi: 10.1371/journal.pone.0141853. PubMed DOI PMC

Bustin S.A. Quantification of MRNA Using Real-Time Reverse Transcription PCR (RT-PCR): Trends and Problems. J. Mol. Endocrinol. 2002;29:23–39. doi: 10.1677/jme.0.0290023. PubMed DOI

Czechowski T., Stitt M., Altmann T., Udvardi M.K., Scheible W.-R. Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiol. 2005;139:5–17. doi: 10.1104/pp.105.063743. PubMed DOI PMC

Hadi Soltanabad M., Bagherieh-Najjar M.B., Mianabadi M. Carnosic Acid Content Increased by Silver Nanoparticle Treatment in Rosemary (Rosmarinus Officinalis L.) Appl. Biochem. Biotechnol. 2020;191:482–495. doi: 10.1007/s12010-019-03193-w. PubMed DOI

Aminfar Z., Rabiei B., Tohidfar M., Mirjalili M.H. Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Rosmarinus Officinalis L. in Various Tissues and under Elicitation. Biocatal. Agric. Biotechnol. 2019;20:101246. doi: 10.1016/j.bcab.2019.101246. DOI

Kotrade P., Sehr E.M., Wischnitzki E., Brüggemann W. Comparative Transcriptomics-Based Selection of Suitable Reference Genes for Normalization of RT-QPCR Experiments in Drought-Stressed Leaves of Three European Quercus Species. Tree Genet. Genomes. 2019;15:38. doi: 10.1007/s11295-019-1347-4. DOI

Silver N., Best S., Jiang J., Thein S.L. Selection of Housekeeping Genes for Gene Expression Studies in Human Reticulocytes Using Real-Time PCR. BMC Mol. Biol. 2006;7:33. doi: 10.1186/1471-2199-7-33. PubMed DOI PMC

Pfaffl M.W., Tichopad A., Prgomet C., Neuvians T.P. Determination of Stable Housekeeping Genes, Differentially Regulated Target Genes and Sample Integrity: BestKeeper—Excel-Based Tool Using Pair-Wise Correlations. Biotechnol. Lett. 2004;26:509–515. doi: 10.1023/B:BILE.0000019559.84305.47. PubMed DOI

Andersen C.L., Jensen J.L., Ørntoft T.F. Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Res. 2004;64:5245–5250. doi: 10.1158/0008-5472.CAN-04-0496. PubMed DOI

Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F. Accurate Normalization of Real-Time Quantitative RT-PCR Data by Geometric Averaging of Multiple Internal Control Genes. Genome Biol. 2002;3:research0034. doi: 10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

Xie F., Xiao P., Chen D., Xu L., Zhang B. MiRDeepFinder: A MiRNA Analysis Tool for Deep Sequencing of Plant Small RNAs. Plant Mol. Biol. 2012;80:75–84. doi: 10.1007/s11103-012-9885-2. PubMed DOI

Ullah A., Munir S., Badshah S.L., Khan N., Ghani L., Poulson B.G., Emwas A.-H., Jaremko M. Important Flavonoids and Their Role as a Therapeutic Agent. Molecules. 2020;25:5243. doi: 10.3390/molecules25225243. PubMed DOI PMC

Coskun Y., Duran R.E., Kilic S. Striking Effects of Melatonin on Secondary Metabolites Produced by Callus Culture of Rosemary (Rosmarinus Officinalis L.) Plant Cell Tissue Organ Cult. 2019;138:89–95. doi: 10.1007/s11240-019-01605-7. DOI

Mosavat N., Golkar P., Yousefifard M., Javed R. Modulation of Callus Growth and Secondary Metabolites in Different Thymus Species and Zataria Multiflora Micropropagated under ZnO Nanoparticles Stress. Biotechnol. Appl. Biochem. 2019;66:316–322. doi: 10.1002/bab.1727. PubMed DOI

Ali A., Mohammad S., Khan M.A., Raja N.I., Arif M., Kamil A., Mashwani Z.-R. Silver Nanoparticles Elicited in vitro Callus Cultures for Accumulation of Biomass and Secondary Metabolites in Caralluma Tuberculata. Artif. Cells Nanomed. Biotechnol. 2019;47:715–724. doi: 10.1080/21691401.2019.1577884. PubMed DOI

Ashrafi M., Azimi Moqadam M.R., Moradi P., Mohsenifard E., Shekari F. Evaluation and Validation of Housekeeping Genes in Two Contrast Species of Thyme Plant to Drought Stress Using Real-Time PCR. Plant Physiol. Biochem. 2018;132:54–60. doi: 10.1016/j.plaphy.2018.08.007. PubMed DOI

Ling H., Wu Q., Guo J., Xu L., Que Y. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR. PLoS ONE. 2014;9:e97469. doi: 10.1371/journal.pone.0097469. PubMed DOI PMC

Chen M., Wang B., Li Y., Zeng M., Liu J., Ye X., Zhu H., Wen Q. Reference Gene Selection for QRT-PCR Analyses of Luffa (Luffa Cylindrica) Plants under Abiotic Stress Conditions. Sci. Rep. 2021;11:3161. doi: 10.1038/s41598-021-81524-w. PubMed DOI PMC

Long X.-Y., Wang J.-R., Ouellet T., Rocheleau H., Wei Y.-M., Pu Z.-E., Jiang Q.-T., Lan X.-J., Zheng Y.-L. Genome-Wide Identification and Evaluation of Novel Internal Control Genes for Q-PCR Based Transcript Normalization in Wheat. Plant Mol. Biol. 2010;74:307–311. doi: 10.1007/s11103-010-9666-8. PubMed DOI

Mascia T., Santovito E., Gallitelli D., Cillo F. Evaluation of Reference Genes for Quantitative Reverse-Transcription Polymerase Chain Reaction Normalization in Infected Tomato Plants: RT-QPCR Normalization in Infected Tomato. Mol. Plant Pathol. 2010;11:805–816. doi: 10.1111/j.1364-3703.2010.00646.x. PubMed DOI PMC

Reid K.E., Olsson N., Schlosser J., Peng F., Lund S.T. An Optimized Grapevine RNA Isolation Procedure and Statistical Determination of Reference Genes for Real-Time RT-PCR during Berry Development. BMC Plant Biol. 2006;6:27. doi: 10.1186/1471-2229-6-27. PubMed DOI PMC

Gopalam R., Rupwate S.D., Tumaney A.W. Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis in Salvia Hispanica. PLoS ONE. 2017;12:e0186978. doi: 10.1371/journal.pone.0186978. PubMed DOI PMC

Galli V., Borowski J.M., Perin E.C., da Silva Messias R., Labonde J., dos Santos Pereira I., dos Anjos Silva S.D., Rombaldi C.V. Validation of Reference Genes for Accurate Normalization of Gene Expression for Real Time-Quantitative PCR in Strawberry Fruits Using Different Cultivars and Osmotic Stresses. Gene. 2015;554:205–214. doi: 10.1016/j.gene.2014.10.049. PubMed DOI

Oneto C.D., Bossio E., Faccio P., Beznec A., Lewi D. Validation of Housekeeping Genes for QPCR in Maize during Water Deficit Stress Conditions at Flowering Time. Maydica. 2018;62:6.

Roy A., Palli S.R. Epigenetic Modifications Acetylation and Deacetylation Play Important Roles in Juvenile Hormone Action. BMC Genom. 2018;19:934. doi: 10.1186/s12864-018-5323-4. PubMed DOI PMC

Systematic Identification of Suitable Reference Genes for Quantitative Real-Time PCR Analysis in Melissa officinalis L