Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

The combined and single effect of salinity and copper stress on growth and quality of Mentha spicata plants.

Copper is essential for plant growth, but in excess may cause adverse effects on plant physiology. Harmful effects are also caused by plant exposure to salinity (NaCl) due to the excessive use of fertilizers, soil degradation and/or the quality of the water used for irrigation. The impact of single and combined salinity (Sal) and copper (Cu) stress on spearmint metabolism were studied in hydroponics. Spearmint plants (Mentha spicata L.) were subjected to salinity stress (150 mM NaCl) and/or excessive Cu concentration (60 µM Cu) via the nutrient solution. Not only Sal and Cu, but also their combination suppressed plant growth by decreasing plant biomass, root fresh weight and plant height. Chlorophyll content decreased mainly for the combined stress treatment (Sal + Cu). Polyphenols and antioxidants (FRAP, DPPH, ABTS) increased in single stress treatments (Sal or Cu), but decreased in the combined stress (Sal + Cu). The application of Sal or Cu stress decreased Zn, N and K (leaves), K, Ca, P and Mg (roots) content. Copper application increased Ca and Mg in leaves. In conclusion, salinity stress and Cu exposure may change the primary metabolic pathways in favor of major volatile oil components biosynthesis, resulting in significant changes of essential oil yield and composition.

Structural re-arrangement of depolymerized sodium alginate enriches peltate glandular trichomes and essential oil production of spearmint.

Over the past decade, radiation-degraded polysaccharides have been used as regulators of growth and development in several crop plants. In quest of the possible reasons of previously established growth-promotion activity of irradiated sodium alginate (ISA), structural parameters of irradiated and un-irradiated sodium alginate were analysed using Ultraviolet-visible spectroscopy (UV-vis) and Fourier Transform Infrared spectroscopic (FT-IR) studies to develop an understanding of structure-property relationship. Using foliar application, response to graded concentrations of ISA was tested in terms of yield and quality attributes of spearmint (Mentha spicata L.). Among different concentrations of ISA [0 (control), 40, 80, 120 and 160mgL-1], 80mgL-1 proved to be the optimum foliar-spray treatment for most of the parameters studied including peltate glandular-trichomes density, which was increased from 20 to 44mm-2. Measurements made at 150days after planting revealed that foliar application of ISA at 80mgL-1 increased the content and yield of spearmint essential oil (EO) by 36.0 and 122.6%, respectively, in comparison to the control. Compared to the control, gas chromatography mass spectrometry (GC-MS) analysis revealed an increase of 18.7% in the carvone content and a decrease of 15.7% in limonene content of the spearmint EO.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Metal absorption properties of Mentha spicata grown under tannery sludge amended soil-its effect on antioxidant system and oil quality.

Tannery sludge (TS) is hazardous to environment and its disposal in an ecofriendly manner is a major challenge. An experiment was conducted to investigate the metal absorption properties of Mentha spicata grown under different levels of TS amended soil (soil: sludge in 100:0, 75:25, 50:50, 25:75 and 0:100 ratio) and its effect on the antioxidant system and oil quality. At 75:25 ratio of sludge and soil, metal translocation factor was ≥0.5 for Cr, Cd, and Co and for Ni and for Pb ≥ 1. Carvone, limonene, dihydrocarvone and other oil constituents along with biomass were maximum in 75:25 ratio of sludge and soil. Superoxide dismutase (SOD), CAT (Catalases), POD (Peroxidases), MDA (Malondialdehyde) and proline play a major role in detoxification of reactive oxygen species generated due to TS (heavy metal stress). Antioxidant (SOD, CAT and POD), MDA and proline showed an increasing trend as the concentration of TS increased with the treatments. To test the relationship between 23 character principal component analysis (PCA) was performed. PC-I contributed 56% of total variance while PC-II contributed 37% of total variance. The results concluded that M. spicata performed well in terms of oil yield and multiple metal translocations in 75:25 sludge and soil ratio.

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco.

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.