Gamma irradiation-induced variability in morpho-agronomic and oil quality traits of Mentha piperita L.

PURPOSE: To develop elite mutant in Mentha piperita having morphotype and oil content of Mentha arvensis, and oil quality of M. piperita. MATERIALS AND METHODS: The dormant runner of menthol rich genotype MPK-5 were subjected to different doses of γ- irradiation (10, 20, 30, 50, 70, 90, and 110 Gy) at a dose rate of 55 Gy/min to induce the genetic variability for herb and oil yield as well as oil quality. A wide spectrum of variability for agro-morphological traits, herb yield, and quality profile was observed among the mutants. RESULTS: The developed and selected superior mutants viz. MPK-5(1) and MPK-5(3) have a mean herb yield potential of 23.923 and 21.503 Kg/9 m2; mean oil yield of 92.953 and 80.047 ml/plot; mean menthol content of 69.012% and 69.160% with mean menthofuran content of 1.554% and 0.531%, respectively. CONCLUSIONS: Mutational breeding through γ- irradiation is considered complementary to the conventional breeding method, to broaden the spectrum of genetic variability. The developed and selected mutants namely viz. MPK-5(1) and MPK-5(3) identified as promising mutants, based on herb yield, oil yield, and essential oil quality, could be used as a parental line for exploitation in hybridization program/recombinant breeding.

Improving Phenolic Total Content and Monoterpene in Mentha x piperita by Using Salicylic Acid or Methyl Jasmonate Combined with Rhizobacteria Inoculation.

The effects of plant inoculation with plant growth-promoting rhizobacteria (PGPR) and those resulting from the exogenous application of salicylic acid (SA) or methyl jasmonte (MeJA) on total phenolic content (TPC) and monoterpenes in Mentha x piperita plants were investigated. Although the PGPR inoculation response has been studied for many plant species, the combination of PGPR and exogenous phytohormones has not been investigated in aromatic plant species. The exogenous application of SA produced an increase in TPC that, in general, was of a similar level when applied alone as when combined with PGPR. This increase in TPC was correlated with an increase in the activity of the enzyme phenylalanine ammonia lyase (PAL). Also, the application of MeJA at different concentrations in combination with inoculation with PGPR produced an increase in TPC, which was more relevant at 4 mM, with a synergism effect being observed. With respect to the main monoterpene concentrations present in peppermint essential oil (EO), it was observed that SA or MeJA application produced a significant increase similar to that of the combination with rhizobacteria. However, when plants were exposed to 2 mM MeJA and inoculated, an important increase was produced in the concentration on menthol, pulegone, linalool, limonene, and menthone concentrations. Rhizobacteria inoculation, the treatment with SA and MeJA, and the combination of both were found to affect the amount of the main monoterpenes present in the EO of M. piperita. For this reason, the expressions of genes related to the biosynthesis of monoterpene were evaluated, with this expression being positively affected by MeJA application and PGPR inoculation, but was not modified by SA application. Our results demonstrate that MeJA or SA application combined with inoculation with PGPR constitutes an advantageous management practice for improving the production of secondary metabolites from M. piperita.

Weed interference with peppermint (Mentha x piperita L.) and spearmint (Mentha spicata L.) crops under different herbicide treatments: effects on biomass and essential oil yield.

BACKGROUND: 'Minor crops' such as spearmint and peppermint are high added value crops, despite the fact that their production area is comparably small worldwide. The main limiting factor in mint commercial cultivation is weed competition. Thus, field experiments were carried out to evaluate the effects of weed interference on growth, biomass and essential oil yield in peppermint and spearmint under different herbicide treatments. RESULTS: The application of pendimethalin and oxyfluorfen provided better control of annual weeds resulting in higher crop yield. Additionally, when treated with herbicides both crops were more competitive against annual weeds in the second year than in the first year. All pre-emergence herbicides increased biomass yield, since pendimethalin, linuron and oxyfluorfen reduced the density of annual weeds by 71-92%, 63-74% and 86-95%, respectively. Weed interference and herbicide application had no effect on essential oil content; however, a relatively strong impact on essential oil production per cultivated area unit was observed, mainly due to the adverse effect of weed interference on plant growth. CONCLUSION: Considering that pendimethalin and oxyfluorfen were effective against annual weeds in both spearmint and peppermint crops, these herbicides should be included in integrated weed management systems for better weed management in mint crops. © 2017 Society of Chemical Industry.

The effect of lunisolar tidal acceleration on stem elongation growth, nutations and leaf movements in peppermint (Mentha × piperita L.).

Orbital movement of the Moon generates a system of gravitational fields that periodically alter the gravitational force on Earth. This lunar tidal acceleration (Etide) is known to act as an external environmental factor affecting many growth and developmental phenomena in plants. Our study focused on the lunar tidal influence on stem elongation growth, nutations and leaf movements of peppermint. Plants were continuously recorded with time-lapse photography under constant illumination as well in constant illumination following 5 days of alternating dark-light cycles. Time courses of shoot movements were correlated with contemporaneous time courses of the Etide estimates. Optical microscopy and SEM were used in anatomical studies. All plant shoot movements were synchronised with changes in the lunisolar acceleration. Using a periodogram, wavelet analysis and local correlation index, a convergence was found between the rhythms of lunisolar acceleration and the rhythms of shoot growth. Also observed were cyclical changes in the direction of rotation of stem apices when gravitational dynamics were at their greatest. After contrasting dark-light cycle experiments, nutational rhythms converged to an identical phase relationship with the Etide and almost immediately their renewed movements commenced. Amplitudes of leaf movements decreased during leaf growth up to the stage when the leaf was fully developed; the periodicity of leaf movements correlated with the Etide rhythms. For the fist time, it was documented that lunisolar acceleration is an independent rhythmic environmental signal capable of influencing the dynamics of plant stem elongation. This phenomenon is synchronised with the known effects of Etide on nutations and leaf movements.

MAPK-mediated regulation of growth and essential oil composition in a salt-tolerant peppermint (Mentha piperita L.) under NaCl stress.

Peppermint (Mentha × piperita L.) is an important and commonly used flavoring agent worldwide, and salinity is a major stress that limits plant growth and reduces crop productivity. This work demonstrated the metabolic responses of essential oil production including the yield and component composition, gene expression, enzyme activity, and protein activation in a salt-tolerant peppermint Keyuan-1 with respect to NaCl stress. Our results showed that Keyuan-1 maintained normal growth and kept higher yield and content of essential oils under NaCl stress than wild-type (WT) peppermint.Gas chromatography-mass spectrometry (GC-MS) and qPCR results showed that compared to WT seedlings, a 150-mM NaCl stress exerted no obvious changes in essential oil composition, transcriptional level of enzymes related to essential oil metabolism, and activity of pulegone reductase (Pr) in Keyuan-1 peppermint which preserved the higher amount of menthol and menthone as well as the lower content of menthofuran upon the 150-mM NaCl stress. Furthermore, it was noticed that a mitogen-activated protein kinase (MAPK) protein exhibited a time-dependent activation in the Keyuan-1 peppermint and primarily involved in the modulation of the essential oil metabolism in the transcript and enzyme levels during the 12-day treatment of 150 mM NaCl. In all, our data elucidated the effect of NaCl on metabolic responses of essential oil production, and demonstrated the MAPK-dependent regulation mechanism of essential oil biosynthesis in the salt-tolerant peppermint, providing scientific basis for the economic and ecological utilization of peppermint in saline land.

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.

Salicylic acid elicitation during cultivation of the peppermint plant improves anti-diabetic effects of its infusions.

Peppermint (Mentha piperita) infusions represent an important source of bioactive compounds with health benefits, which can be enhanced by applying salicylic acid (SA) during plant cultivation. The aim of this study was to evaluate the effect of SA (0, 0.5 and 2 mM) during peppermint cultivation on the chemical profile of saponins and alkaloids, as well as the anti-diabetic properties of the resulting infusions. The results showed that a 2 mM SA treatment significantly improved the chemical profiles of the infusions. Furthermore, the administration of 2 mM SA-treated peppermint infusions for 4 weeks to a high-fat diet/streptozotocin-induced diabetic rats decreased serum glucose levels (up to 25%) and increased serum insulin levels (up to 75%) as compared with the diabetic control. This can be related to the observed protection on pancreatic ß-cells. Furthermore, 0.5 and 2 mM SA-treated peppermint infusions decreased LDL (24 and 47%, respectively) and increased HDL levels (18 and 37%, respectively). In addition, all groups treated with peppermint infusions had lower serum and liver triglyceride contents, where 2 mM SA peppermint infusion showed the highest effect (44% and 56%, respectively). This is probably caused by its higher capacity to inhibit pancreatic lipase activity and lipid absorption. Moreover, SA-treated peppermint infusions improved the steatosis score in diabetic rat liver and decreased serum transaminase levels, probably as a result of the increase in steroidal saponins and alkaloids, such as trigonellin. Therefore, the application of 2 mM SA during cultivation of peppermint could be used to improve the anti-diabetic properties of peppermint infusions.

Metabolite profile, antioxidant capacity, and inhibition of digestive enzymes in infusions of peppermint (Mentha piperita) grown under drought stress.

Peppermint (Mentha piperita) infusions represent an important source of antioxidants, which can be enhanced by inducing abiotic stress in plants. The aim of this study was to evaluate the effect of drought stress on peppermint cultivation as well as the metabolite profile, antioxidant capacity, and inhibition of digestive enzymes of resulting infusions. At 45 days after planting, irrigation was suppressed until 85 (control), 65, 35, 24, and 12% soil moisture (SM) was reached. The results showed that 35, 24, and 12% SM decreased fresh (20%) and dry (5%) weight. The 35 and 24% SM treatments significantly increased total phenolic and flavonoid contents as well as antioxidant capacity. Coumaric acid, quercetin, luteolin, and naringenin were detected only in some drought treatments; however, in these infusions, fewer amino acids and unsaturated fatty acids were identified. The 24 and 12% SM treatments slightly improved inhibition of pancreatic lipase and α-amylase activity. Therefore, induction of moderate water stress in peppermint is recommended to enhance its biological properties.

Reguladores de crescimento vegetal no cultivo in vitro de Mentha x Piperita L / Plant growth regulators on in vitro culture of Mentha x piperita L

A micropropagação é uma técnica muitas vezes indicada para a multiplicação em larga escala de plantas com propriedades medicinais. Dentre elas, destaca-se a hortelã-pimenta (Mentha x Piperita L.), cujo óleo essencial é utilizado no tratamento de transtornos digestivos e respiratórios. Para otimizar o protocolo de micropropagação dessa espécie são necessários estudos, principalmente quanto à suplementação do meio de cultura para garantir a produção massal in vitro e posterior extração do óleo essencial. Nesse contexto, objetivou-se avaliar o efeito de concentrações e combinações de reguladores de crescimento vegetal na morfogênese in vitro de hortelã-pimenta. Segmentos nodais provenientes de plântulas estabelecidas in vitro foram utilizados como fonte de explante e inoculados em meio de cultura MS suplementado com 0; 2,0 e 4,0 mg L-1 de BAP (6-benzilaminopurina), 0; 0,5 e 1,5 mg L-1 de ANA (ácido naftaleno-acético) e 0; 0,5 e 1,0 mg L-1 de GA3 (ácido giberélico). O delineamento experimental adotado foi inteiramente casualizado, com os tratamentos distribuídos em esquema fatorial 3x3x3 com oito repetições. Concluiu-se que o BAP favoreceu a sobrevivência de segmentos nodais de M. x Piperita inoculados in vitro e, quando combinado ao GA3, promoveu a brotação dos explantes. Essas características, no entanto, não foram estimuladas pela adição de ANA ao meio de cultura. Conclui-se que após a multiplicação dos brotos in vitro estes devem ser transferidos para meio sem reguladores para seu desenvolvimento. Apesar dos efeitos benéficos do BAP na organogênese de M. x Piperita, elevadas concentrações deste regulador de crescimento vegetal promoveram a formação de calos.

Micropropagation is a technique used for the large-scale production of medicinal plants. Among them, peppermint (Mentha x piperita L.) may be mentioned because of the pharmacological importance of its essential oil, which is used on the treatment of digestive and respiratory disorders. Studies are needed in order to optimize the micropropagation protocol of this species, especially concerning the culture medium, to ensure the in vitro mass clonal production and to enable the future extraction of the plant essential oil. Therefore, this work aimed to evaluate the effects of concentrations and combinations of different plant growth regulators on the in vitro morphogenesis of peppermint. Nodal segments from plantlets already established in vitro were used as explants and inoculated on MS medium supplemented with 0, 2.0 and 4.0 mg L-1 of BAP (6-benzylaminopurine), 0, 0.5 and 1.5 mg L-1 of NAA (naphthalene acetic acid) and 0; 0.5 and 1.0 mg L-1 of GA3 (gibberellic acid). The experiment was in a completely randomized design, set up as a 3x3x3 factorial design with eight replicates. We concluded that BAP increases the survival rate of in vitro inoculated nodal segments of M. x piperita. In addition, its combination with GA3 stimulates explants shooting. Those aspects, however, are not promoted by the addition of NAA into the culture medium. Also, the results indicate that, after in vitro multiplication, peppermint shoots must be transferred to another medium without plant growth regulators for shoot elongation. High concentrations of BAP promote calli induction, despite having beneficial effects on the organogenesis of M. piperita.

Binomial and enumerative sampling of Tetranychus urticae (Acari: Tetranychidae) on peppermint in California.

The two-spotted spider mite, Tetranychus urticae Koch, is an economic pest on peppermint [Mentha x piperita (L.), 'Black Mitcham'] grown in California. A sampling plan for T. urticae was developed under Pacific Northwest conditions in the early 1980s and has been used by California growers since approximately 1998. This sampling plan, however, is cumbersome and a poor predictor of T. urticae densities in California. Between June and August, the numbers of immature and adult T. urticae were counted on leaves at three commercial peppermint fields (sites) in 2010 and a single field in 2011. In each of seven locations per site, 45 leaves were sampled, that is, 9 leaves per five stems. Leaf samples were stratified by collecting three leaves from the top, middle, and bottom strata per stem. The on-plant distribution of T. urticae did not significantly differ among the stem strata through the growing season. Binomial and enumerative sampling plans were developed using generic Taylor's power law coefficient values. The best fit of our data for binomial sampling occurred using a tally threshold of T = 0. The optimum number of leaves required for T urticae at the critical density of five mites per leaf was 20 for the binomial and 23 for the enumerative sampling plans, respectively. Sampling models were validated using Resampling for Validation of Sampling Plan Software.

Essential oils to control Botrytis cinerea in vitro and in vivo on plum fruits.

BACKGROUND: The consequence of misusing chemical biocides in controlling pests and diseases has drawn the attention of policy makers to the development of methods potentially available in nature for this purpose. In the present study the inhibitory effects of black caraway, fennel and peppermint essential oils against Botrytis cinerea were tested at various concentrations in vitro and in vivo. RESULTS: The in vitro results showed that the growth of B. cinerea was completely inhibited by the application of black caraway and fennel oils at concentrations of 400 and 600 µL L⁻¹ respectively. The in vivo results indicated that black caraway, fennel and peppermint oils at all applied concentrations inhibited B. cinerea growth on plum fruits compared with the control. In addition, all three oils at higher concentrations showed positive effects on fruit quality characteristics such as titrable acidity, total soluble solids, carbohydrate content, pH and weight loss percentage. Thus the oils inhibited the infection of plum fruits by B. cinerea and increased their storage life. CONCLUSION: This research confirms the antifungal effects of black caraway, fennel and peppermint essential oils both in vitro and in vivo on plum fruits postharvest. Therefore these essential oils could be an alternative to chemicals to control postharvest phytopathogenic fungi on plum fruits.

In vitro production of M. × piperita not containing pulegone and menthofuran.

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.

Volatile organic compounds from rhizobacteria increase biosynthesis of essential oils and growth parameters in peppermint (Mentha piperita).

Volatile organic compounds (VOCs), characterized by low molecular weight and high vapor pressure, are produced by all organisms as part of normal metabolism, and play important roles in communication within and between organisms. We examined the effects of VOCs released by three species of plant growth-promoting rhizobacteria (Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense) on growth parameters and composition of essential oils (EO) in the aromatic plant Mentha piperita (peppermint). The bacteria and plants were grown on the same Petri dish, but were separated by a physical barrier such that the plants were exposed only to VOCs but not to solutes from the bacteria. Growth parameters of plants exposed to VOCs of P. fluorescens or B. subtilis were significantly higher than those of controls or A. brasilense-treated plants. Production of EOs (monoterpenes) was increased 2-fold in P. fluorescens-treated plants. Two major EOs, (+)pulegone and (-)menthone, showed increased biosynthesis in P. fluorescens-treated plants. Menthol in A. brasilense-treated plants was the only major EO that showed a significant decrease. These findings suggest that VOCs of rhizobacteria, besides inducing biosynthesis of secondary metabolites, affect pathway flux or specific steps of monoterpene metabolism. Bacterial VOCs are a rich source for new natural compounds that may increase crop productivity and EO yield of this economically important plant species.

Determination of polyphenols in Mentha longifolia and M. piperita field-grown and in vitro plant samples using UPLC-TQ-MS.

Nine polyphenols in the aerial parts of Mentha longifolia have been separated by chromatographic techniques. Their structures have been confirmed by HPLC/electrospray ionization-MS/MS. The compounds identified included rosmarinic acid, salvianolic acid L, dedihydro-salvianolic acid, luteolin-glucuronide, luteolin-diglucuronide, luteolin-glucopyranosyl-rhamnopyranoside, and eriodictyol-glucopyranosyl-rhamnopyranoside. The extracts of M. longifolia and M. piperita field plants, in vitro plants, callus tissues, and cell suspension cultures were profiled, and their polyphenol composition was compared in different tissues and quantified using ultra-performance column liquid chromatography (UPLC)/triple-quadrupole-MS in the selected-ion recording detection mode. Determination of desired compounds was based on calibration curves obtained for standards, which were previously isolated from M. longifolia aerial parts. The UPLC profiles revealed considerable differences in the synthesis of secondary metabolites among samples coming from field plants, in vitro plants, callus tissues, and cell suspension cultures. Plant tissues coming from field cultivation (for both M. piperita and M. longifolia) contained several phenolic compounds (flavonoids and phenolic acids), whereas plants from in vitro conditions, callus tissues, and suspension cultures contained only a few of them. Rosmarinic acid dominated in all of these samples. These results show that under in vitro conditions, the metabolism of phenolics undergoes a fundamental change.

Citocininas na multiplicação in vitro de hortelã-pimenta (Mentha x Piperita L. ) / Cytokinins on in vitro multiplication of peppermint (Mentha x Piperita L. )

O hortelã-pimenta é um hibrido triplo, Mentha x piperita, utilizado de forma medicinal no tratamento de náuseas, cólicas gastrointestinais, flatulências, cálculos biliares, icterícia, ansiedade, expectoração e expulsão de vermes intestinais. Na micropropagação, o BAP (6-benzilaminopurina) e KIN (cinetina) têm sido as fontes de citocininas mais empregadas. O objetivo deste trabalho foi avaliar o efeito de reguladores vegetais na multiplicação in vitro de hortelã-pimenta. Explantes constituídos de segmentos nodais provenientes de plântulas já estabelecidas in vitro com aproximadamente 0,5 cm foram inoculados em meio MS, suplementado com diferentes associações de BAP e KIN, em adição de 30 g L-1 de sacarose. As concentrações de citocininas utilizadas foram 0,0; 1,0 e 2,0 mg L-1 de BAP e 0,0; 0,5 e 1,0 mg L-1 de KIN. O delineamento experimental foi o inteiramente casualizado, com nove tratamentos consistindo de cinco frascos cada um, sendo que cada frasco continha quatro explantes. A utilização de 2,0 mg L-1 de BAP promove a multiplicação in vitro de hortelã-pimenta, porém, diminui o índice de sobrevivência. O uso de citocininas aumenta a massa fresca e seca dos explantes e a ausência destes reguladores propicia o alongamento nesta espécie.

Peppermint is used for medical treatments of nausea, gastrointestinal cramps, flatulence, gallstones, jaundice, anxiety, sputum and expulsion of intestinal worms. In micropropagation, BAP (6-benzylaminopurine) and KIN (kinetin) have been the most widely used sources of cytokinins. The aim of this study was to evaluate the effect of plant growth regulators in multiplication of peppermint. Explants consisting of nodal segments from seedlings already established in vitro with approximately 0,5 cm were inoculated on MS medium supplemented with different combinations of BAP and KIN and 30 g L-1 of sucrose. The concentrations of cytokinins used were 0,0; 1,0 and 2,0 mg L-1 of BAP and 0,0; 0,5 and 1,0 mg L-1 of KIN. The experiment was set up in a completely randomized design with nine treatments consisting of five bottles each, and each vial contained four explants. The use of 2,0 mg L-1 of BAP promotes the in vitro multiplication of peppermint, but decreases the survival rate. Cytokinins increase fresh and dry weight of explants and the absence of these regulators provides the elongation in this specie.

Yield, content, and composition of peppermint and spearmints as a function of harvesting time and drying.

Peppermint ( Mentha × piperita L.) and spearmints ('Scotch' spearmint, M. × gracilis Sole, and 'Native' spearmint, Mentha spicata L.) are widely grown essential oil crops in more northern latitudes; however, there is limited information on how harvest time and drying influence peppermint and spearmint yield, oil composition, and bioactivity, when grown south of the 41st parallel. In this 2-year study, the effects of harvest time and drying on the yield, oil composition, and bioactivity of peppermint ('Black Mitcham' and 'B90-9'), 'Scotch' spearmint, and 'Native' spearmint were evaluated. Peppermint oil from the dried material had higher menthol and eucalyptol concentrations. Menthone in both peppermint cultivars decreased from harvest 1 (late June) to harvest 5 (late August) or 6 (early September), whereas menthol increased. (-)-Carvone in spearmints accumulated early, before flowering, allowing for early harvest. Oil yields from the dried spearmint biomass reached the maximum at harvest 3 (mid-July). The essential oil compositions of the four mint genotypes were similar to that of 11 commercially available oils, suggesting that these genotypes can be grown in the hot, humid environment of the southeastern United States. The antioxidant activities (ORAC(oil) values) of the essential oils were 4372, 1713, 1107, and 471 µmol of TE L(-1) for 'Scotch' spearmint, 'Native' spearmint, peppermint, and Japanese cornmint ( Mentha canadensis ), respectively. The oils of the four mint genotypes did not affect ruminal fermentation in vivo, and did not exhibit antimicrobial, antileishmanial, or antimalarial activity at levels that would warrant bioassay-directed fractionation in a drug-discovery screening program. Specifically, the oils did not show greater than 50% growth inhibition against Leishmania donovani , Plasmodium falciparum clones D6 and W2, Candida albicans , Escherichia coli , Pseudomonas aeruginosa , Cryptococcus neoformans , Mycobacterium intracellulare , or Aspergillus fumigates at 50 µg mL(-1).

An efficient protocol for high-frequency direct multiple shoot regeneration from internodes of peppermint (Mentha x piperita).

A simple, repeatable and efficient protocol for direct multiple shoot regeneration from internodal explants has been defined in peppermint (Mentha x piperita var. Indus). In vitro regenerated shoots of peppermint were excised into 4 to 8 mm long internodes and cultured on Murashige and Skoog's medium supplemented with different cytokinins. In the hormonal assay, 3.0 mg L(-1) zeatin or 6-isopentenyl adenine independently supplemented to half strength MS medium exhibited multiple shoot regeneration, while thiaduzorn (0.1-3.0 mg L(-1)) showed no morphogenetic effect. A maximum of 85% in vitro cultured explants showed multiple shoot formation with an average of 7 shoots per explant on MS medium supplemented with zeatin. Multiple shoots were initiated within three weeks of cultivation. Internodes with regenerated multiple shoots were transferred to half- strength MS medium without supplementing with any plant growth hormone for shoot elongation and rhizogenesis. Rooted plants acclimatized and grew to maturity under glasshouse conditions. The plantlets developed were phenotypically identical to the parent plant and exhibited 96% survival.

Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol.

Accessions of Mentha x piperita, M. x villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant re-growth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25 degree C or in alternating temperature regimes (25/15C or 25/-1C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25/-1C regime than in plants cultivated at 20C or 25C or at 25/15C regime for all nine tested accessions. The mean re-growth levels increased from 36 percent at 20C to 69percent at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89 percent. A pre-culture at alternating temperatures of 25/15C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.

Assessment of wool waste and hair waste as soil amendment and nutrient source.

A field and two container experiments were conducted to assess uncomposted wool and hair wastes as a nutrient source for crops and to evaluate their potential to improve soil biological and chemical properties. Overall, addition of wool or hair waste to soil increased yields of basil (Ocimum basilicum L. 'Trakia'), thorn apple (Datura innoxia Mill. 'Inka'), peppermint (Mentha x piperita L. 'Black Mitchum'), and garden sage (Salvia officinalis L. 'Desislava'), increased NH(4)-N and NO(3)-N in soil, increased total N (and protein) content in plant tissue, stimulated soil microbial biomass, and decreased mycorrhizae colonization of plant roots of thorn apple but not in basil. Wool and hair waste additions to soil altered slightly the content and composition of plant secondary metabolites (essential oils or alkaloids); however, overall the constituents remained within the "typical" range for the respective crops. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis demonstrated that wool and hair wastes decompose slowly under field or greenhouse conditions, and act as a slow release S, N, P, and K fertilizer. These results, along with the measured concentrations of NO(3)-N in soil at harvest, suggest that the addition of wool or hair waste of only 3.3 g kg(-1) of soil may support two to five harvests or crops under greenhouse conditions and two to four field seasons in field production systems, and would improve soil biological and chemical characteristics. Further research is needed to optimize the rate of application of these waste materials to the nutrient requirements of specific crops to avoid nitrate leaching into the ground water. In addition, the effect of wool and hair waste on other environmental end points should also be further investigated before specific recommendations for growers are provided.

Effect of an arbuscular mycorrhizal fungus, Glomus mosseae, and a rock-phosphate-solubilizing fungus, Penicillium thomii, on Mentha piperita growth in a soilless medium.

Rock phosphate effect on English mint (Mentha piperita L.) grown on steamed perlite:vermiculite (1:1, v:v) substrate, with and without rock phosphate, was evaluated in greenhouse experiments. Five treatments were carried out by inoculation with an arbuscular mycorrhizal fungus Glomus mosseae and a phosphorus solubilizing microorganism Penicillium thomii. Plant aerial biomass, phosphorus concentration in plant tissue, and P available in the substrate, were evaluated upon two harvests. After the first harvest, plant aerial biomass did not show significant differences between treatments using rock phosphate as fertilizer, although P content in plants inoculated with P. thomii was higher. The second harvest revealed a higher biomass and plant tissue P content in treatments inoculated with G. mosseae. P. thomii increased P available in the substrate, whereas in the absence of G. mosseae, it did not enhance plant tissue P content. Mycorrhizal colonization was not affected by P. thomii. Microbial inoculation effect on English mint growth was also evaluated. The microbial effect was positive in all treatments when compared with the control without rock phosphate.