Molecular fingerprinting of some Mentha species by sequencing and RFLP analysis of the 5S-rRNA non-transcribed spacer region

The genus Mentha is of particular economic importance. The development of new methods for the characterisation of Mentha species is crucial for their unequivocal identification. Amplification of the non-transcribed spacer (NTS) of the 5S-rRNA gene was used to characterise some Mentha species, which revealed a high-specific variability. Cloning and sequencing of all amplified NTS fragments enabled the discrimination among almost all species. In silico and experimental analyses identified specific restriction sites on the amplified 5S-NTS regions, facilitating the rapid and unambiguous discrimination of all the different species by polymerase chain reaction–restriction fragment length polymorphism. A direct comparison between essential oil composition and DNA fingerprinting confirmed a relationship between chemical and molecular data.     

Comparative Analysis of Antioxidant Activities of Fourteen Mentha Essential Oils and Their Components

The essential oils of 14 species and hybrids, respectively, of the genus Mentha were examined for their antioxidant capacity in the ABTS (2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid)) assay and in a lipid‐peroxidation (LPO) assay. The ABTS^.+‐scavenging capacity of pure essential‐oil components and mixtures of them was also tested. In both assays, Mentha×dumetorum (classification not fully confirmed), Mentha suaveolens, and Mentha×villosa (classification not fully confirmed) showed the highest antioxidant capacity, which was ascribed to the components germacrene D, piperitone oxide, and piperitenone oxide. The high antioxidant activity in the LPO assay of the two hybrids Mentha×gracilis and, to a lower degree, of Mentha×dalmatica (classification not fully confirmed) was ascribed to their high contents of cis‐ocimene and β‐caryophyllene. Of the pure components tested (germacrene D, piperitone oxide, and piperitenone oxide were not tested, as not commercially available), only cis‐ocimene showed a distinct antioxidant effect, whereas dihydrocarvone and linalool had pro‐oxidant effects in the ABTS assay.     

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.     

Natural hybrids in Senecio (Asteraceae): new records from Argentina

Abstract

Hybridisation is very common within Senecio. It is considered a fundamental mechanism of speciation and also a major feature causing systematic complexity. Morphological features are the preliminary characteristics used to identify hybrids; however this approach is usually not sufficient when this is the case chromosome studies represent a valuable tool for reinforcing the observations. Two natural homoploid interspecific hybrids are reported here: Senecio × lulioi M. G. López et Xifreda (S. brasiliensis (Spreng.) Less. × S. grisebachii Baker) and Senecio × piepaloensis M. G. López et Xifreda (S. ragonesei Cabrera × S. sectilis Griseb.). Classic cytogenetic analysis and pollen stainability were performed to evaluate hybrid origins and to designate the putative parents. S. × lulioi presented a low frequency of quadrivalents and heteromorphic bivalents, both strong evidences of hybridisation between closely related species. S. × piepaloensis exhibited a high frequency of meiotic irregularities, and reduced pollen stainability, certain characteristics of hybrids. This analysis is discussed in relation to the role of hybridisation in the evolution of the genus, and its systematic significance.     

Identification of hybrids in broad-leaved Potamogeton species (Potamogetonaceae) in China using nuclear and chloroplast DNA sequence data

Hybridization is relatively frequent in the pondweed genus Potamogeton. A total of five putative hybrids of broad-leaved Potamogeton in China were collected in our recent investigations. We used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to confirm the origins of the putative hybrids. Using ITS sequence additivity, we confirmed that the five putative hybrids were P. × anguillanus Koidzumi (P. wrightii × P. perfoliatus), P. × malainoides Miki (P. distinctus × P. wrightii), P. distinctus × P. nodosus, P. nodosus × P. wrightii, and P. distinctus × P. gramineus. The latter four hybrids are new records for China, and P. distinctus × P. gramineus is a new hybrid combination in Potamogeton. We found a new genotype of P. perfoliatus in northeast China. Hybrids between the new and a common genotype of P. perfoliatus were found in Central China. The maternal parents of the six hybrids were confirmed by chloroplast rbcL gene sequence data. The hybrids P. × anguillanus and P. distinctus × P. gramineus are reciprocal hybrids. P. × anguillanus has multiple origins from different populations. P. distinctus × P. gramineus has multiple origins within a single population.     

Transformation of menthane monoterpenes by Mentha piperita cell culture

(+)-Isopiperitenone (100 mg l^–1) was converted into (4S,6R)-6-hydroxy- and (4S,8R)-8,9-epoxyisopiperitenone, aside from the already known (+)-7-hydroxyisopiperitenone, by suspension cell culture of Mentha piperita. As (–)-isopiperitenone was hydroxylated similarly, this implies that the hydroxylating enzyme(s) have a broad substrate stereospecificity in regards to the stereochemistry at C4. (–)-(4R)-Carvone was reduced by the Mentha cells both at carbonyl and C1-C6 double bond to give (1R,2S,4R)-neodihydrocarveol and (1R,2R,4R)-dihydrocarveol with the former being the major product. (+)-(4S)-Carvone had a similar reduction pattern, producing (1S,2R,4S)-neodihydrocarveol and (1S,4S)-dihydrocarvone. Formation of these compounds indicates that the peppermint cell culture cannot only hydroxylate the allylic position but also reduce the ,-unsaturated carbonyl system.     

Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants

Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and –pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods. Received: 22 November 1998 / Accepted: 4 Januar 1999     

Infectivity of Steinernema mushtaqi (Rhabditida: Steinernematidae) against insect pests and their mass production

The infectivity of entomopathogenic nematode (EPN), Steinernema mushtaqi was tested against legume pod borer, Maruca vitrata, tobacco caterpillar, Spodoptera litura, blue butterfly, Lampides boeticus, red hairy caterpillar, Amsacta moorei, brown bug, Clavigralla gibbosa, mealy bug, Centrococcus somatics, fruit borer, Earias vittella and green bug, Nezara viridula larvae and in vivo mass production of the above-tested species of EPN have been carried out during 2008. S. mushtaqi was found to be more pathogenic to A. moorei, as it brought about 100% mortality within 48 h, than to S. litura, L. boeticus, N. viriduala and E. vittella, as mortality occurred within 72 h; whereas this level of mortality was recorded in C. somatis, C. gibbosa and M. vitrata within 144 h. No mortality was observed in the control treatment. Multiplication of S. mushtaqi and the yield of infective juveniles (IJs) on these insects was the highest (0.94 × 10⁵ IJs/cadaver) from N. viriduala, followed by S. litura (0.76 × 10⁵ IJs/cadaver), L. boeticus as also C. gibbosa (0.31 × 10⁵ IJs/cadaver) and M. vitrata (0.20 × 10⁵ IJs/cadaver). Very poor populations of IJs were found from A. moorei (0.15 × 10⁵ IJs/cadaver) and C. somatics (0.01 × 10⁵ IJs/cadaver). No multiplication of IJs was found from the cadaver of E. vittella. This opens a new hope of utilising S. mushtaqi in the insect pests management programme.     

Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue (p < 0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl₂ as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl₂ domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation.     

Artificial and natural hybrids in pycnanthemum (Labiatae)

Further chromosome counts, principally from the southeastern United States, are reported for 38 accessions of 11 species, together with new or corrected counts forP. nudum (2n = 40),P. montanum (2n = 40),P. curvipes (2n = 40), andP. torrei (2n = 80). Four species have now been shown to have intraspecific polyploidy, five are known only as diploids, and nine occur only at the tetraploid level. Artificially produced interspecific hybrids range from vigorous to dwarfed or nonflowering, and from semifertile to sterile as measured by anther development and stainable pollen. Naturally occurring hybrids are described for the following species combinations:P. clinopodioides ×P. incanum,P. pilosum ×P. virginianum, P. flexuosum ×P. nudum, andP. albescens ×P. loomisii.

     

Effect of Eucalyptus and Mentha leaves and Curcuma rhizomes on Tyrophagus putrescentiae (Schrank) (Acarina: Acaridae) in wheat

Varying concentrations of powdered leaves of Eucalyptus and Mentha and rhizomes of Curcuma were evaluated in controlling Tyrophagus putrescentiae in wheat flour. At higher concentrations, all these plant materials significantly decreased the population build-up of the mite. Eucalyptus and Mentha leaf powders were effective at concentrations ranging from 100 to 5% in bringing about a decrease in the fecundity of the mite and reducing the egg numbers to 51.66 and 25.49 per female, respectively, as compared to 98.16 eggs per female in the controls. Rhizomes of Curcuma were effective even at a concentration of 0.1% reducing the egg laying to 7.66 eggs per female in wheat flour. These materials were more deleterious to immature stages (ova and larvae) than to the mature stages of T. putrescentiae. When tested on whole grains of wheat these plant materials showed more pronounced effects in controlling the mite population compared to that in wheat flour. Curcuma rhizomes were the most promising for possible use against T. putrescentiae.     

Three new hybrids of Ophioglossum (Ophioglossaceae) from Monte Pisano,Tuscany (Central Italy)

Based on general morphology, spore measurements and ornamentation (scanning electron microscope), genome size estimation, and molecular systematics (trnL-trnF IGS), we show the extreme systematic complexity within the European representatives of the genus Ophioglossum. In particular, three hybrids from Tuscany are described: the tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane, seen as the hybrid between diploid O. lusitanicum L. and hexaploid O. azoricum C.Presl; the tetraploid O. × giovanninii Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the homoploid hybrid between tetraploid O. vulgatum L. and tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane; the pentaploid O. × pseudoazoricum Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the hybrid between hexaploid O. azoricum C.Presl and tetraploid O. vulgatum L. All the three new taxa grow in different localities in the Monte Pisano mountain range.     

Recurrent hybridisation events between Primula vulgaris,P. veris and P. elatior (Primulaceae,Ericales) challenge the species boundaries: using molecular markers to re‐evaluate morphological identifications

Three Primula species, Primula vulgaris, P. veris and P. elatior, have been objects of fascination for gardeners and botanists over several centuries. The species are able to hybridise, and where they co‐occur, hybrids are commonly found. In Denmark, Møns Klint on the island of Møn and Købelev Skov on Lolland are examples of localities where all three species occur and where the hybrids P. × digenea, the hybrid between P. vulgaris and P. elatior, and P. × polyantha, the hybrid between P. veris and P. vulgaris, can also be found. To investigate relationships between the species and their hybrids, we sampled 168 specimens from 10 geographical locations and subjected them to genetic analysis. The samples were also identified based on morphological traits: primarily inflorescense structure, the size, shape, colour and markings of corolla and leaf basis, leaf blade texture and hairiness. After identifying species‐specific SNPs in the Internal Transcribed Spacer sequence, these were used to resolve species and hybrid boundaries and status through a cleaved amplified polymorphic sequence assay. Polymorphisms in the chloroplast trnL sequence were used as a high‐throughput marker and used to determine the maternal parent of hybrids. Ten simple sequence repeat markers were applied to obtain further insight into the genetic makeup of the accessions using structure and Introgress, providing information of genetic variability within and between populations. Our results indicated that backcrossing of P. × digenea hybrids with parental species has occurred, and that many of the P. × digenea sampled were later‐generation hybrids rather than F₁s. Analyses of P. × polyantha specimens show mostly the expected pattern for primary hybrids but indications of P. veris ancestry of a P. vulgaris plant was discovered. Our results further indicate that some of the specimens initially identified as P. elatior include P. vulgaris among their progenitors and thus challenge currently accepted species boundaries.     

Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat

To evaluate the pathogenicity of Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) a bioassay was designed under laboratory conditions against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. The fungus was applied at the dose rates of 8 × 10³, 8 × 10⁵, 8 × 10⁷ and 8 × 10⁹ conidia/kg of wheat and the bioassay was conducted at 25°C with 60% relative humidity. The data regarding the mortality was recorded after 7 and 14 days exposure intervals. All the treatments gave the significant mortality of R. dominica and M. anisopliae of 8 × 10⁹ conidia/kg was found to be the most effective after a 14-day exposure interval. There was greater production of progeny when the low rate of M. anisopliae was applied to wheat. Overall, our study showed that M. anisopliae is vigorous when applied at a high dose rate which revealed an effective control of R. dominica and also played a pivotal role in the integrated pest management program (IPM) of stored wheat insect pests.     

Two new spontaneous hybrids of American hard pines from Pinus sect. Trifoliae (Pinaceae) found in the unique Russian Sochi Arboretum

In the 1990s, an extraordinarily abundant live collection of the genus Pinus, including 49 species, 4 subspecies, and 7 interspecific hybrids, found in the Sochi Arboretum in Russia on the northeast coast of the Black Sea, was taxonomically revised. Among these taxa, two previously unknown interspecific hybrids of American hard pine species from P. sect. Trifoliae had arisen spontaneously in culture. These are described here as the new hybrid species Pinus × transamericana (P. muricata × P. rigida) and Pinus × critchfieldii (P. patula × P. taeda). All known natural or artificial interspecific hybrids within P. sect. Trifoliae are listed, and hybridization between American hard pine species is discussed. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Dehydrodimers of Caffeic Acid in the Cell Walls of Suspension-Cultured Mentha

Dehydrodicaffeic acid derivatives were found in the cell walls of suspension-cultured cells of Mentha. Using gas chromatography/mass spectrometry (GC-MS) in a single ion chromatography at m/z 790 and m/z 718, eleven peaks of trimethylsilylated dehydrodimers of caffeic acid were detected in the extracts from the cell walls of suspension-cultured cells of Mentha using sodium hydroxide. The result suggests that dehydrodicaffeates are formed in the cell walls from two molecules of caffeate, probably formed through C-C, and C-O-C coupling processes.     

The Administration of Levocabastine,a NTS2 Receptor Antagonist,Modifies Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Properties

Neurotensin behaves as a neuromodulator or as a neurotransmitter interacting with NTS1 and NTS2 receptors. Neurotensin in vitro inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. This effect is prevented by administration of SR 48692 (antagonist for NTS1 receptor). The administration of levocabastine (antagonist for NTS2 receptor) does not prevent Na⁺, K⁺-ATPase inhibition by neurotensin when the enzyme is assayed with ATP as substrate. Herein levocabastine effect on Na⁺, K⁺-ATPase K⁺ site was explored. For this purpose, levocabastine was administered to rats and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in synaptosomal membranes and [³H]-ouabain binding to cerebral cortex membranes were assayed in the absence (basal) and in the presence of neurotensin. Male Wistar rats were administered with levocabastine (50 μg/kg, i.p., 30 min) or the vehicle (saline solution). Synaptosomal membranes were obtained from cerebral cortex by differential and gradient centrifugation. The activity of K⁺-p-NPPase was determined in media laking or containing ATP plus NaCl. In such phosphorylating condition enzyme behaviour resembles that observed when ATP hydrolyses is recorded. In the absence of ATP plus NaCl, K⁺-p-NPPase activity was similar for levocabastine or vehicle injected (roughly 11 μmole hydrolyzed substrate per mg protein per hour). Such value remained unaltered by the presence of 3.5 × 10^?6 M neurotensin. In the phosphorylating medium, neurotensin decreased (32 %) the enzyme activity in membranes obtained from rats injected with the vehicle but failed to alter those obtained from rats injected with levocabastine. Levocabastine administration enhanced (50 %) basal [³H]-ouabain binding to cerebral cortex membranes but failed to modify neurotensin inhibitory effect on this ligand binding. It is concluded that NTS2 receptor blockade modifies the properties of neuronal Na⁺, K⁺-ATPase and that neurotensin effect on Na⁺, K⁺-ATPase involves NTS1 receptor and -at least partially- NTS2 receptor.     

Using DNA‐based techniques to identify hybrids among linear‐leaved Potamogeton plants collected in China

Abstract It is well known that interspecific hybrids occur in the genus Potamogeton. The linear‐leaved Potamogeton species commonly have highly variable morphological characteristics. Their hybrids often show similar vegetative characters to their parental species and their identification based solely on morphology is not always conclusive. In order to clarify whether there are any hybrids from the linear‐leaved Potamogeton plants collected in China, we used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to identify the hybrids. Using ITS sequence additivity, we identified four hybrids, namely P. orientalis (P. pusillus×P. oxyphyllus), P. pusillus×P. berchtoldii, P. foliosus×P. octandrus, and P. cristatus×P. octandrus. The latter three hybrids should be considered as new hybrids in Potamogeton. The maternal parents of the four hybrids were confirmed using chloroplast rbcL gene sequences.     

Chloroplast DNA variation and evolution in the genus Lens Mill

 Chloroplast DNA (cpDNA) restriction site diversity was assessed by 21 enzyme/probe combinations in 30 accessions of six Lens species, including the recently recognized L. lamottei and L. tomentosus. A total of 118 fragments were scored and 26 restriction site mutations were identified. The cpDNA restriction pattern supports circumscribing L. lamottei and L. tomentosus as independent species. The value of the data for reconstructing phylogeny in the genus is discussed. The cpDNA of all 13 accessions of the lentil’s wild progenitor, L. culinaris subsp. orientalis, differed from that of the single lentil cultivars used in this study. This diversity indicates that other populations of this subspecies from Turkey and Syria examined by Mayer and Soltis (1994) are potentially the founder members of lentil. Examination of L. lamottei×L. nigricans hybrids between accessions having different restriction patterns showed paternal plastid inheritance in L. nigricans. Received: 2 July 1996 / Accepted: 19 July 1996