Melissa officinalis L., a well-known herb with diverse industrial and ethnopharmacological properties. Although, there has been a significant lack in the breeding attempts of this invaluable herb. This study aimed to enhance the agronomical traits of M. officinalis through in vitro polyploidization. Nodal segments were micropropagated and subjected to oryzalin treatment at concentrations of 20, 40, and 60 mM for 24 and 48 hours. Flow cytometry, chromosome counting, and stomatal characteristics were employed to confirm the ploidy level of the surviving plants. The survival rate of the treated explants decreased exponentially with increasing oryzalin concentration and duration. The highest polyploid induction rate (8%) was achieved with 40 mM oryzalin treatment for 24 hours. The induced tetraploid plants exhibited vigorous growth, characterized by longer shoots, larger leaves, and a higher leaf count. Chlorophyll content and fluorescence parameters elucidated disparities in photosynthetic performance between diploid and tetraploid genotypes. Tetraploid plants demonstrated a 75% increase in average essential oil yield, attributed to the significantly larger size of peltate trichomes. Analysis of essential oil composition in diploid and tetraploid plants indicated the presence of three major components: geranial, neral, and citronellal. While citronellal remained consistent, geranial and neral increased by 11.06% and 9.49%, respectively, in the tetraploid population. This effective methodology, utilizing oryzalin as an anti-mitotic agent for polyploid induction in M. officinalis, resulted in a polyploid genotype with superior morpho-physiological traits. The polyploid lemon balm generated through this method has the potential to meet commercial demands and contribute significantly to the improvement of lemon balm cultivation.

The objective of the current study was to assess the efficiency of oryzalin in inducing polyploids in Callisia fragrans (Lindl.) Woodson by in vitro polyploidization. Shoot tips were subjected to Murashige and Skoog (MS) medium containing oryzalin at concentrations 1, 5, and 10 μM for 4 and 8 weeks. Further, the ploidy levels of the plants were confirmed using flow cytometry and chromosome counting. Among all treatments, six tetraploid plants (2n = 4x = 24) were obtained after 8 weeks in MS medium containing 5 μM oryzalin. Upon ex vitro transfer, tetraploid plants were morphologically distinct compared to diploid plants. The size of the leaf and flower increased significantly and nearly doubled when compared to the mother diploid plant. Further, inductively coupled plasma–optical emission spectrometry showed that tetraploid plants exhibited significantly higher sodium, iron, and calcium content, and the potassium content was increased by 100%. Molecular analysis utilizing iPBS and CDDP markers was tested for the first time in C. fragrans to assess the variation between tetraploid and diploid genotypes. Both the markers generated three major clusters, indicating a clear distinction between diploid, tetraploid, and the mixoploid genotypes. In conclusion, in vitro polyploidization using oryzalin could effectively induce polyploids in this and related species. Additionally, the results obtained in this study will provide a basis for future breeding opportunities in this species.

Melissa officinalis L. is well known for its lemon-scented aroma and various pharmacological properties. Despite these valuable properties, the genes involved in the biosynthetic pathways in M. officinalis are not yet well-explored when compared to other members of the mint family. For that, gene expression studies using quantitative real-time PCR (qRT-PCR) are an excellent tool. Although qRT-PCR can provide accurate results, its accuracy is highly reliant on the expression and stability of the reference gene used for normalization. Hence, selecting a suitable experiment-specific reference gene is very crucial to obtain accurate results. However, to date, there are no reports for experiment-specific reference genes in M. officinalis. Therefore, in the current study, ten commonly used reference genes were assessed for their suitability as optimal reference genes in M. officinalis under various abiotic stress conditions and different plant organs. The candidate genes were ranked based on BestKeeper, comparative ΔCt, geNorm, NormFinder, and RefFinder. Based on the results, we recommend the combination of EF-1α and GAPDH as the best reference genes to normalize gene expression studies in M. officinalis. On the contrary, HLH71 was identified as the least-performing gene. Thereafter, the reliability of the optimal gene combination was assessed by evaluating the relative gene expression of the phenylalanine ammonia lyase (PAL) gene under two elicitor treatments (gibberellic acid and jasmonic acid). PAL is a crucial gene involved directly or indirectly in the production of various economically important secondary metabolites in plants. Suitable reference genes for each experimental condition are also discussed. The findings of the current study form a basis for current and future gene expression studies in M. officinalis and other related species.

Nanotechnology has emerged as a promising tool with virtually unlimited potential to enhance crop and food production. Nanoparticles (NPs) have garnered much attention because of their unique physical and Ferric oxide nano-priming enhances photosynthetic and physicochemical properties of sunflower (Helianthus annuus L.) microgreens