Growth and monoterpene production by transformed shoot cultures of Mentha citrata and Mentha piperita in flasks and fermenters

This paper reports studies on the growth and biosynthesis of monoterpenes by transformed shoot cultures of Mentha citrata and Mentha piperita, originally developed 5 years ago and since maintained by regular subculturing. Throughout this time, the M. citrata culture has stably maintained production of an oil closely resembling that of the parent plant in which linalool and linalyl acetate are the predominant components. However, M. piperita, which initially showed a divergence from the parent plant in producing significant amounts of menthofuran in addition to the characteristic oil components menthol and menthone, has now been found to produce pulegone and menthofuran as the major components. The cultures were subjected to different environmental conditions of varying periods of light and temperature in an attempt to restore menthol and menthone production. Increased illumination reduced the yields of pulegone and menthofuran but did not stimulate the production of either menthol or menthone, which remained only at trace levels (below 0.2 g/g fresh weight). Cultures of M. citrata were, however, stimulated by increased illumination, and produced more linalool and linalyl acetate. Shoot cultures of M. citrata and M. piperita were grown in 14–1 fermenters for up to 60 dys during which the biomass increased from approximately 100 g to 2.5 kg and 3.5 kg respectively. Both cultures rapidly consumed sucrose with a concomitant release of glucose, and the uptake of inorganic ions was similar except that M. citrata consumed far less Na+ during the fermentation. The total yields of monoterpenes from the fermentations were 1.16 g (M. piperita) and 0.18 g (M. citrata). *** DIRECT SUPPORT *** AG903062 00005     

Phylogenetic Analysis of Selected Menthol-Producing Species Belonging to the Lamiaceae Family

Menthol is an organic compound with diverse medicinal and commercial applications, and is made either synthetically or through extraction from mint oils. The aim of the present study was to investigate menthol levels in selected menthol-producing species belonging to the Lamiaceae family, and to determine phylogenetic relationships of menthol dehydrogenase gene sequence among these species. Three genus of Lamiaceae, namely Mentha, Salvia, and Micromeria, were selected for phytochemical and phylogenetic analyses. After identification of each species based on menthol dehydrogenase gene in NCBI, BLAST software was used for the sequence alignment. MEGA4 software was used to draw phylogenetic tree for various species. Phytochemical analysis revealed that the highest and lowest amounts of both essential oil and menthol belonged to Mentha spicata and Micromeria hyssopifolia, respectively. The species Mentha spicata and Mentha piperita, which were assigned to one cluster in the dendrogram, contained the highest amounts of essential oil and menthol while Micromeria species, which was in the distinct cluster and placed in the farther evolutionary distance, contained the lowest amount of essential oil and menthol. Phylogenetic and phytochemistry analyses showed that essential oil and menthol contents of menthol-producing species are associated with menthol dehydrogenase gene sequence.     

Influence of ethephon and daminozide on growth and essential oil content of peppermint and sage

Foliar application of Daminozide at 1000 ppm reduces the growth of Salvia officinalis (sage) and decreases essential oil yield, but increases both growth and essential oil yield of Mentha piperita at the same concentration. Ethephon at 250 ppm reduces the growth and essential oil yield of peppermint and slightly increases growth and essential oil content of sage. Both growth regulators markedly reduce the level of menthone and menthol in peppermint oil and increase the level of isomenthone and neoisomenthol. Both growth regulators decrease the level of camphor and increase the level of β-pinene in sage oil. Changes in essential oil composition induced by these growth regulators are most readily explained by alterations in the levels or activities of the relevant biosynthetic enzymes.     

Chemical Diversity of Volatile Compounds of Mints from Southern Part of Pannonian Plain and Balkan Peninsula – New Data

Mints are the most popular economic and traditional herbs. The aim of this article was chemical characterization of volatile compounds from wild populations of Mentha aquatica, M. arvensis, M. longifolia, M. microphylla, M. pulegium, M. spicata, M.×dumetorum, M.×gentillis and M.×verticillata, as well as cultivated samples of M. spicata, M.×piperita ‘Alba’ and M.×piperita ‘Crispa’. Analyses were performed directly from dried aerial parts (herb) of collected samples by headspace gas chromatography‐mass spectrometry. In total 54 compounds were detected, representing from 89.99 % to 99.66 % of volatile fractions of all investigated samples. The recorded volatiles were primarily monoterpene hydrocarbons and oxygenated monoterpenes, while oxygenated aromatic monoterpenes, sesquiterpene hydrocarbons and aliphatic compounds were present in lower concentrations in analyzed samples. The major components were linalool, limonene, 1,8‐cineol, α‐terpinyl acetate, pulegone, β‐pinene and menthol. The cluster analysis revealed five main groups or chemotypes according to qualitative and quantitative content of volatiles, as well as similarities among samples. These results contribute to the knowledge on the mints chemistry in Pannonian Plain and Balkan Peninsula.     

Plant growth‐promoting effects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study

Plant growth‐promoting rhizobacteria (PGPR) affect growth of host plants through various direct and indirect mechanisms. Three native PGPR (Pseudomonas putida) strains isolated from rhizospheric soil of a Mentha piperita (peppermint) crop field near Córdoba, Argentina, were characterised and screened in vitro for plant growth‐promoting characteristics, such as indole‐3‐acetic acid (IAA) production, phosphate solubilisation and siderophore production, effects of direct inoculation on plant growth parameters (shoot fresh weight, root dry weight, leaf number, node number) and accumulation and composition of essential oils. Each of the three native strains was capable of phosphate solubilisation and IAA production. Only strain SJ04 produced siderophores. Plants directly inoculated with the native PGPR strains showed increased shoot fresh weight, glandular trichome number, ramification number and root dry weight in comparison with controls. The inoculated plants had increased essential oil yield (without alteration of essential oil composition) and biosynthesis of major essential oil components. Native strains of P. putida and other PGPR have clear potential as bio‐inoculants for improving productivity of aromatic crop plants. There have been no comparative studies on the role of inoculation with native strains on plant growth and secondary metabolite production (specially monoterpenes). Native bacterial isolates are generally preferable for inoculation of crop plants because they are already adapted to the environment and have a competitive advantage over non‐native strains.     

国产野生薄荷挥发油化学组分变异及其化学型

采用气－质联用的方法,对国产野生薄荷（ＭｅｎｔｈａｈａｐｌｏｃａｌｙｘＢｒｉｑ．）居群的挥发油成分进行了分析,２２个样品可归纳为６个化学型：（１）薄荷酮－胡薄荷酮型（ｍｅｎｔｈｏｎｅ－ｐｕｌｅｇｏｎｅｔｙｐｅ）;（２）胡椒酮型（ｐｉｐｅｒｉｔｏｎｅｔｙｐｅ）;（３）氧化胡椒酮－氧化胡椒烯酮型（ｐｉｐｅｒｉｔｏｎｅｏｘｉｄｅ－ｐｉｐｅｒｉｔｅｎｏｎｅｏｘｉｄｅｔｙｐｅ）;（４）芳樟醇－氧化胡椒酮型（ｌｉｎａｌｏｏｌ－ｐｉｐｅｒｉｔｏｎｅｏｘｉｄｅｔｙｐｅ）;（５）香芹酮型（ｃａｒｖｏｎｅｔｙｐｅ）;（６）薄荷醇－乙酸薄荷酯型（ｍｅｎｔｈｏｌ－ｍｅｎｔｈｙｌａｃｅｔａｔｅｔｙｐｅ）。结合地理分布和薄荷属单萜类成分生物合成途径,对上述主要类型进行了讨论。     

Variations in composition of peppermint oil in relation to production areas

Compositional data obtained for peppermint oil fromMentha piperita L. produced in Tasmania as well as oils produced in other major production areas, were analysed by principal coordinate analysis. The 6 variates included in the analysis were the oil compounds limonene, cineole, menthone, menthofuran, menthyl acetate and menthol. In general, Tasmanian oils were characterised by low menthofuran, low limonene, low menthyl acetate and to a lesser extent high menthone, low cineole and high menthol concentrations relative to most major production areas. When the variation in composition of oil samples from within Tasmania was displayed in 3 dimensions (using the first 3 principal coordinates) it was not possible to achieve any pronounced separation of oils produced at different locations within southern Tasmania. Principal coordinates which were based mainly on cineole and menthol concentrations, respectively, did allow a degree of separation between oils produced in southern and northern Tasmania. Generally, oils produced in northern Tasmania had lower cineole and, in some cases, higher menthol than southern Tasmanian oils. Oil extracted from regrowth herb after commercial harvest was distinguished by having very high menthyl acetate, low menthone, high menthol, high menthofuran, low cineole and low limonene concentrations. The effect of some cultural and environmental factors on oil composition is also discussed.     

Contact Toxicity and Repellency of the Essential Oil from Mentha haplocalyx Briq. against Lasioderma serricorne

The chemical composition of the essential oil obtained by hydrodistillation from the aerial parts of Mentha haplocalyx was investigated by GC‐FID and GC/MS analyses. In sum, 23 components, representing 92.88% of the total oil composition, were identified, and the main compounds were found to be menthol (59.71%), menthyl acetate (7.83%), limonene (6.98%), and menthone (4.44%). By bioassay‐guided fractionation (contact toxicity), three compounds were obtained from the essential oil and identified as menthol, menthyl acetate, and limonene. The essential oil and the three isolated compounds exhibited potent contact toxicity against Lasioderma serricorne adults, with LD₅₀ values of 16.5, 7.91, 5.96, and 13.7 μg/adult, respectively. Moreover, the oil and its isolated compounds also exhibited strong repellency against L. serricorne adults. At the lower concentrations tested and at 2 h after exposure, menthol showed even significantly stronger repellency than the positive control DEET. The study revealed that the bioactivity properties of the essential oil can be attributed to the synergistic effects of its diverse major and minor components, which indicates that the M. haplocalyx oil and its isolated compounds have potential for the development as natural insecticides and/or repellents to control insects in stored grains and traditional Chinese medicinal materials.     

A New Histochemical Method for Localization of the Site of Monoterpene Phenol Accumulation in Plant Secretory Structures

A new method is reported for the histochemical localizationof monoterpene phenols in essential oil secretory structures.The method was adapted from a spot test originally devised forin vitro detection of phenolic compounds in organic analyses.Plant subjects were the Lamiaceae species Thymus vulgaris L.,Oreganum vulgare L. and Mentha x piperita L., which accumulateessential oil in glandular trichomes. A reagent consisting of4-nitrosophenol in conc. H₂SO₄was applied to sample leaves ofeach species. A positive test for phenol was indicated by theproduction of coloured indophenols. Using this method, monoterpenephenols were identified in the trichomes of T. vulgaris(thymol)and O. vulgare(carvacrol), indicated by colour changes to redand green respectively. No phenol was detected in trichomesof M. x piperita. Results were confirmed by GC-MS analysis ofleaf volatile extracts from each species, and in vitro testswith thymol and carvacrol. The method could be used in fieldsurveys for rapid identification of potential medicinal plantsand bioactive compounds. Copyright 2001 Annals of Botany Company Histochemistry, secretory structures, glandular trichomes, Lamiaceae, Thymus vulgaris, Oreganum vulgare, Mentha x piperita, essential oil, aromatic monoterpenes     

A unique interspecific hybrid spearmint clone with growth properties of Menthaarvensis L. and oil qualities of Mentha spicata L.

Crossing blocks of Mentha arvensis cv Kalka (menthol mint without carvone) and Mentha spicata cv Neera (carvone mint without menthol) in alternate rows (2:1::Kalka:Neera), and pollination of florets of Kalka with that from Neera, yielded a carvone-rich variant among the open-pollinated seedlings. The variant possesses the hybrid phenotype, including the vigorous M. arvensis growth habit and the synthesis of rich oil aroma supplemented with a menthol tinge (carvone 64%–76% against 58% for the normal carvone mint cv Neera), and thus a novel combination of the essential oil. Chromosome counts and random amplified polymorphic DNA analysis confirmed that this spearmint variant, designated as the variety Neerkalka, is a unique interspecific hybrid (2n=5x=60) of M. arvensis cv Kalka (2n=8x=96) and M. spicata cv Neera (2n=2x=24). Vegetative multiplication of the hybrid was facilitated by its underground sucker-reproducing ability which is otherwise absent in spearmints. The per cent improvement in the variant ranged from 31–97 for herbage yield and 95–317 for oil yield over the standard spearmint varieties (MSS-5, Arka and Neera), with per hectare oil yields of 125.0 kg, 139.0 kg and 65.0 kg, respectively. Received: 25 March 2000 / Accepted: 3 July 2000     

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.     

Stimulation of menthol production in <Emphasis Type="Italic">Mentha piperita</Emphasis> cell culture

Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid at 15 d interval and used for further studies. Precursor feeding alone, i.e., menthone, at 35 μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60 μM concentration and in combination with menthone feeding at 35 μM increased menthol yield up to 92 and 110 mg l⁻¹ in comparison to 77 mg l⁻¹ of control culture. Synergistic potentiation effect of menthone feeding at 35 μM and γ-cyclodextrin at 60 μM treatment followed by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8 mg l⁻¹ in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol yield up to 278 mg l⁻¹. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Positive correlation between menthol content andin vitro menthol tolerance inMentha arvensis L. cultivars

Menthol is a highly valued monoterpene produced by Japanese mint (Mentha arvensis) as a natural product with wide applications in cosmetics, confectionery, flavours, beverages and therapeutics. Selection of high menthol yielding genotypes is therefore the ultimate objective of all genetic improvement programmes inMentha arvensis. A positive correlation was observed in the present study between menthol content in oils of evaluated genotypes and the level of tolerance to externally supplied menthol of explants of these genotypes in culture medium. The easy use of this relationship as a selectable biochemical marker opens the practical applicability of largescalein vitro screening of the germplasm, clones and breeders' material for selection of elite genotypes.     

Influence of foliar applied cytokinins on growth and essential oil content of several members of the lamiaceae

Foliar application of the cytokinins kinetin, dipbenylurea, benzylaminopurine and zeatin at the 1–10 ppm level has a general growth promoting effect on Mentha piperita, M. spicata and Salvia officinalis, but not on M. suaveolens and Lavandula vera, grown under controlled environmental conditions. The essential oil yield of cytokinintreated plants is also increased up to two-fold on a fr. wt basis relative to untreated controls, with only a minor influence on oil composition in most cases. The increase in oil yield cannot be attributed to alteration in growth or development of the treated plants, or to changes in oil gland populations. In vitro assay of the enzymes catalysing the rate limiting steps of camphor biosynthesis in S. officinalis and of menthone biosynthesis in M. piperita indicated that the increase in oil yield under the influence of cytokinin is a result of increased monoterpene biosynthesis.     

Effect of chemical complexity of essential oils on feeding deterrence in larvae of the cabbage looper

The relationship between chemical composition and feeding deterrent activity of seven essential oils is explored, including those from Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae), Cinnamomum zeylanicum Blume (Lauraceae), Lavendula latifolia (L.) Medicus, Lavendula angustifolia L., Mentha crispa L., Mentha arvensis L. and Mentha piperita L. (Lamiaceae) against an important agricultural pest, the cabbage looper (Trichoplusia ni Hübner, Noctuidae), using a leaf‐disc choice bioassay. Comparison of the deterrent activity of ‘full mixtures' with respective artificial blends missing individual constituents demonstrates that, for most oils, minor constituents in a mixture can be as important as major constituents with respect to the overall feeding deterrent effect. There is a lack of correlation between the feeding deterrent effect of an individual constituent of an essential oil and its contribution to the overall activity of the ‘full mixture’ in some cases. The effect of removing an individual constituent from the mixture of an essential oil depends on the unique properties of the mixture, which in turn may reflect the interaction of its constituents. Understanding the role and contribution of each constituent to the overall activity of the oil can facilitate the creation of artificial blends that optimize their efficacy against different pests.