Impacts of inter-annual climate variability on reproductive phenology and postnatal development of morphological features of three sympatric bat species.

Inter-annual variation in weather conditions has been shown to affect the reproductive phenological patterns of many organisms. Because of their relatively small body size and dependence on ectothermic prey, temperate-zone insectivorous bats are particularly sensitive to adverse spring environmental conditions that affect the duration of gestation and timing of parturition in these animals. This study aimed to compare phenological recruitment, birth seasonality and synchrony and morphological changes during postnatal growth in Rhinolophus euryale, Rhinolophus ferrumequinum and Myotis emarginatus in two consecutive years representing a typical dry (2015) and an extremely wet climatic event (2016) in a nursing colony in Kerend cave, western Iran. Females of these three bat species arrived from their wintering cave to the nursing colony in late April to mid-May each year. Synchrony of parturition as defined by amount clustering of births within a year assessed by circular statistics showed that for R. euryale and R. ferrumequinum the angular variance in dry year were significantly (P < 0.05) lower than in wet year, indicating a low level of synchrony in 2016. Similar comparison showed that births from M. emarginatus were highly synchrony, and there were no significant differences in timing of births among years (P > 0.05). Generalized estimating equation (GEE) for R. euryale indicated that for body mass and forearm length tests of parallelism (interaction term or growth rate) and tests for equal intercepts (y-intercepts or group term) were significant (P < 0.001). In R. ferrumequinum, the initial (y-intercepts) forearm length and body mass were not significantly (P > 0.05) different between the 2 years, but the tests for parallelism showed a significant decrease in growth rates of body mass and forearm length in the wet year (P < 0.05). Similar comparison in M. emarginatus indicated that for body mass, tests of parallelism were significantly different (P = 0.004), while tests for equal intercepts were not (P = 0.23). Our results suggest that climate changes may have unequal effects on different bat species due to differences in foraging habitat, niche partitioning, reproductive requirements and foraging strategies.

Vocalization Development in Geoffroy's bat, Myotis emarginatus (Chiroptera: Vespertilionidae).

Postnatal development of vocalization has been studied in a small number of bats, not including the Geoffroy's bat, Myotis emarginatus. In the present study, we investigated vocalization development at a maternity roost of M. emarginatus in Kerend Cave in western Iran by sequential measurement of acoustic parameters in known-age neonates using mark-recapture sampling. Newborn pups of M. emarginatus produced both short and isolation calls. Duration of short calls of 1-day-old pups was on average 7.56 ± 0.05 ms and decreased during postnatal development to reach an adult-like duration of 2.78 ± 0.13 ms in the third week. Myotis emarginatus emitted various types of isolation calls, including classic, UP-tail-FM and CF-tail during postnatal growth. As bat pups grew, both short and isolation calls changed in their spectro-temporal structure. Discriminant function analyses showed that each bat pup has a vocal signature that facilitates mother-infant communication. The current study reveals that M. emarginatus pups are highly vocal at birth, but gradually elaborate their sounds with an increase in peak, start and end frequencies as well as with a decrease in call duration throughout the postnatal growth period.

Mitochondrial DNA sequence analysis reveals multiple Pleistocene glacial refugia for the Yellow-spotted mountain newt, Neurergus derjugini (Caudata: Salamandridae) in the mid-Zagros range in Iran and Iraq.

Phylogeography is often used to investigate the effects of glacial cycles on current genetic structure of various plant and animal species. This approach can also identify the number and location of glacial refugia as well as the recolonization routes from those refugia to the current locations. To identify the location of glacial refugia of the Yellow-spotted mountain newt, Neurergus derjugini, we employed phylogeography patterns and genetic variability of this species by analyzing partial ND4 sequences (867 bp) of 67 specimens from 15 sampling localities from the whole species range in Iran and Iraq. Phylogenetic trees concordant with haplotype networks showed a clear genetic structure among populations as three groups corresponding to the populations in the north, center, and south. Evolutionary ages of clades north and south ranging from 0.15 to 0.17 Myr, while the oldest clade is the central clade, corresponding to 0.32 Myr. Bayesian skyline plots of population size change through time show a relatively slight increase until about 25 kyr (around the last glacial maximum) and a decline of population size about 2.5 kyr. The presence of geographically structured clades in north, center, and south sections of the species range signifies the disjunct populations that have emerged in three different refugium. This study illustrates the importance of the effect of previous glacial cycles in shaping the genetic structure of mountain species in the Zagros range. These areas are important in terms of long-term species persistence and therefore valuable areas for conservation of biodiversity.

Population genetic structure and phylogeography of the greater horseshoe bat (Rhinolophus ferrumequinum) along Alborz and Zagros Mts. in Iran.

In this paper, we investigate the genetic structure and phylogeography of Rhinolophus ferrumequinum, using the mitochondrial cytochrome b gene (1017 bp) in Iran and adjacent regions. The total haplotype and nucleotide diversity are 0.63 ± 0.055 and 0.0021 ± 0.00017, respectively which suggest that R. ferrumequinum exhibits low genetic diversity. AMOVA analysis shows that more variation of genetic differentiation is present among populations of phylogenetic groupings than within populations. Our phylogenetic results support the monophyly of R. ferrumequinum and suggest this taxon comprises three allopatric/parapatric phylogroups that are distributed in Europe-western Turkey, eastern Turkey-northern Iran, and southern Iran. The Europe-western Turkey lineage (clade 2) split from the eastern Turkey-Iran lineage (clade 1) during the middle Pleistocene (0.8534 (ca.I)-0.6454 (ca.II) Ma). The divergence time among subclades A and B occurred during the mid-Pleistocene (0.4849 (ca.I)-0.369 (ca.II) Ma). All phylogenetic analyses also indicate that the Iranian and eastern Turkey R. ferrumequinum diverged from Europe and western Turkey R. ferrumequinum, with the mean percentage sequence differences ranging from 0.92%-0.75% between them. We infer that long-term isolation of R. ferrumequinum in spatially distinct refugia in parts of southwestern and northeastern Iran has promoted distinct phylogeographic lineages during the Pleistocene.

Historical explanation of genetic variation in the Mediterranean horseshoe bat Rhinolophus euryale (Chiroptera: Rhinolophidae) inferred from mitochondrial cytochrome-b and D-loop genes in Iran.

Molecular phylogeography and species distribution modelling (SDM) suggest that late Quaternary glacial cycles have portrayed a significant role in structuring current population genetic structure and diversity. Based on phylogenetic relationships using Bayesian inference and maximum likelihood of 535 bp mtDNA (D-loop) and 745 bp mtDNA (Cytb) in 62 individuals of the Mediterranean Horseshoe Bat, Rhinolophus euryale, from 13 different localities in Iran we identified two subspecific populations with differing population genetic structure distributed in southern Zagros Mts. and northern Elburz Mts. Analysis of molecular variance (AMOVA) obtained from D-loop sequences indicates that 21.18% of sequence variation is distributed among populations and 10.84% within them. Moreover, a degree of genetic subdivision, mainly attributable to the existence of significant variance among the two regions is shown (θCT = 0.68, p = .005). The positive and significant correlation between geographic and genetic distances (R2 = 0.28, r = 0.529, p = .000) is obtained following controlling for environmental distance. Spatial distribution of haplotypes indicates that marginal population of the species in southern part of the species range have occupied this section as a glacial refugia. However, this genetic variation, in conjunction with results of the SDM shows a massive postglacial range expansion for R. euryale towards higher latitudes in Iran.

Mitochondrial DNA marker (D-loop) reveals high genetic diversity but low population structure in the pale bent-wing bat (Miniopterus pallidus) in Iran.

This study investigates the population genetic structure of Miniopterus pallidus using the D-loop region of mitochondrial DNA (331 bp) from 13 localities in Iran. We found a high number of haplotypes (N = 43) and high nucleotide diversity (π = 0.02). The average K2P genetic distance was high within (2.59%) and between (2.54%) the Iranian populations of M. pallidus and analysis of molecular variance revealed that most genetic variability was related to differences within populations. The positive and significant association between pairwise genetic and environmental distances (r = 0.443, p = .01) after accounting for the effect of geographical distances suggests that substantial influence of the climatic factors among different sites have shaped the genetic variation in this species. Neutrality tests showed historical demographic events when applying Fu's Fs, but Tajima's statistic was nonsignificant, indicating a deviation from the theoretical model of expansion. The hypothesis of sudden-expansion was not rejected by analyses of mismatch distribution as the Raggedness and SSD were insignificant (p(r)= .39 and p(SSD) =  .38). Similarly, BSP results approximately revealed a major historical expansion around 81,000 years ago and a recent population decline around 13,000 years ago. Phylogenetic trees illustrated that all Iranian M. pallidus populations, as well as those from eastern Turkey and Azerbaijan, form a monophyletic clade, whereas the samples from western Turkey, Georgia and Greece belong to M. schreibersii clade. The haplotype network was consistent with these findings for M. pallidus populations in Iran and showed a high level of reticulation.

Postnatal Growth and Vocalization Development in the Long-fingered Bat, Myotis capaccinii (Chiroptera, Vespertilionidae).

Robab Mehdizadeh, Hojjat Eghbali, and Mozafar Sharifi (2018) We simultaneously monitored postnatal growth and vocalization development in a free living population of the long-fingered bat, Myotis capaccinii, in a maternity colony in the Mahidasht cave, western Iran. Length of forearm and body mass of 1-day-old neonates averaged 19.59 ± 1.23 mm and 3.59 ± 0.23 g and followed linear pattern of growth at mean growth rates of 0.74 mm/day and 0.15 g/day until 28 days of age, after which is increased slowly. A similar nonlinear growth pattern was found for ten wing characteristics (wingspan, wing area, handwing length, handwing area, armwing length, armwing area, aspect ratio, wing loading, tip length ratio, and tip area ratio). However, postnatal growth followed a two phase linear pattern of increase-decrease for the epiphyseal-phalangeal gap and decrease- increase for wing loading. At birth, infant bats produced low, short frequency sonar and oral calls in isolation before they could open their eyes. Isolation calls obtained at age 1-4 days old from M. capaccinii pups were frequency modulated shallow calls with longer duration (7.54 ± 1.83 ms) and lower peak frequency (20.07 ± 0.89 kHz) compared to adult female calls (2.35 ± 0.75 ms and 54.02 ± 4.34 kHz). During days 12-16, calls began to resemble adult echolocation calls. Duration of calls increased slightly before 7 days old and then gradually decreased. The peak, start and end frequencies of spontaneous calls increased with age. When the juvenile bats started foraging outside the cave, they only omitted nasal FM calls with 1-2 harmonics. Comparing postnatal changes in body and wing characteristics with those of vocalization, we found that the most positive correlation was between body (forearm length and body mass) and wing features (wing area, wingspan, handwing area, armwing area and aspect ratio) and call parameters (peak, start and end frequency), while wing loading had a significant negative correlation with the call parameters.

Taxonomic Evaluation of the Greater Horseshoe Bat Rhinolophus ferrumequinum (Chiroptera: Rhinolophidae) in Iran Inferred from the Mitochondrial D-Loop Gene.

To examine the level of genetic differentiation in the sequences of the mitochondrial D-loop gene of Rhinolophus ferrumequinum, and to evaluate the current taxonomic status of this species, 50 tissue samples of greater horseshoe bats were collected in 2011-2015 from 21 different localities in northwest, northeast, west, central, and south regions of Iran. Twenty-two published D-loop sequences from Europe (Switzerland, United Kingdom, Bulgaria, and Tunisia), and Anatolia (south, west, and east Turkey) were downloaded from GenBank. Molecular genetic analyses revealed remarkable variation among populations of R. ferrumequinum. Two major clades with strong support were identified within the greater horseshoe bat. One of these clades consists of individuals of R. ferrumequinum from Iran and eastern Turkey, and is further subdivided into two subclades. A second clade includes samples from western Turkey and Europe. The two subclades from Iran and Turkey and the second clade from western Turkey and Europe represent three diagnosable categories, which most probably warrant three subspecies for the species. Thus, based on genetic differences, it is clear that two subspecific populations are found in Iran: R. f. irani (southern Iran) and R. f. proximus (northern Iran).

Variation in food availability mediate the impact of density on cannibalism, growth, and survival in larval yellow spotted mountain newts (Neurergus microspilotus): Implications for captive breeding programs.

In this study, we examined cannibalistic behavior, growth, metamorphosis, and survival in larval and post-metamorph endangered yellow spotted mountain newts Neurergus microspilotus hatched and reared in a captive breeding facility. We designed a 2 × 2 factorial experiment, crossing two levels of food with two levels of density including high food/high density, high food/low density, low food/high density, and low food/low density. The level of cannibalistic behavior (including the loss of fore and hind limbs, missing toes, tail, gills, body damage, and whole body consumption) changed as the larvae grew, from a low level during the first 4 weeks, peaking from weeks 7 to 12, and then dropped during weeks 14-52. Both food level and density had a significant effect on cannibalism. The highest frequency of cannibalism was recorded for larvae reared in the low food/high density and lowest in high food/low density treatments. Growth, percent of larval metamorphosed, and survival were all highest in the high food/low density and lowest in low food/high density treatment. Food level had a significant effect on growth, metamorphosis, and survival. However, the two levels of density did not influence growth and metamorphosis but showed a significant effect on survival. Similarly, combined effects of food level and density showed significant effects on growth, metamorphosis, and survival over time. Information obtained from current experiment could improve productivity of captive breeding facilities to ensure the release of adequate numbers of individuals for reintroduction programs. Zoo Biol. 35:513-521, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

Population Genetic Structure of the Endangered Kaiser's Mountain Newt, Neurergus kaiseri (Amphibia: Salamandridae).

Species often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in an endemic and critically endangered stream breeding mountain newt, Neurergus kaiseri, within its entire range in southwestern Iran. We identified two geographic regions based on phylogenetic relationships using Bayesian inference and maximum likelihood of 779 bp mtDNA (D-loop) in 111 individuals from ten of twelve known breeding populations. This analysis revealed a clear divergence between northern populations, located in more humid habitats at higher elevation, and southern populations, from drier habitats at lower elevations regions. From seven haplotypes found in these populations none was shared between the two regions. Analysis of molecular variance (AMOVA) of N. kaiseri indicates that 94.03% of sequence variation is distributed among newt populations and 5.97% within them. Moreover, a high degree of genetic subdivision, mainly attributable to the existence of significant variance among the two regions is shown (θCT = 0.94, P = 0.002). The positive and significant correlation between geographic and genetic distances (r = 0.61, P = 0.002) following controlling for environmental distance suggests an important influence of geographic divergence of the sites in shaping the genetic variation and may provide tools for a possible conservation based prioritization policy for the endangered species.

Phylogeny, Diversity, Distribution, and Host Specificity of Haemoproteus spp. (Apicomplexa: Haemosporida: Haemoproteidae) of Palaearctic Tortoises.

A complex wide-range study on the haemoproteid parasites of chelonians was carried out for the first time. Altogether, 811 samples from four tortoise species from an extensive area between western Morocco and eastern Afghanistan and between Romania and southern Syria were studied by a combination of microscopic and molecular-genetic methods. Altogether 160 Haemoproteus-positive samples were gathered in the area between central Anatolia and eastern Afghanistan. According to variability in the cytochrome b gene, two monophyletic evolutionary lineages were distinguished; by means of microscopic analysis it was revealed that they corresponded to two previously described species-Haemoproteus anatolicum and Haemoproteus caucasica. Their distribution areas overlap only in a narrow strip along the Zagros Mts. range in Iran. This fact suggests the involvement of two different vector species with separated distribution. Nevertheless, no vectors were confirmed. According to phylogenetic analyses, H. caucasica represented a sister group to H. anatolicum, and both of them were most closely related to H. pacayae and H. peltocephali, described from South American river turtles. Four unique haplotypes were revealed in the population of H. caucasica, compared with seven haplotypes in H. anatolicum. Furthermore, H. caucasica was detected in two tortoise species, Testudo graeca and Testudo horsfieldii, providing evidence that Haemoproteus is not strictly host-specific to the tortoise host species.

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production.

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 µg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17-20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 µg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.

Variation in ectoparasite load in the Mehely's horseshoe bat, Rhinolophus mehelyi (Chiroptera: Rhinolophidae) in a nursery colony in western Iran.

We studied variation of ectoparasite load in a free ranging populations of Mehely's horseshoe bat (Rhinolophus mehelyi) on five successive occasions in a nursery roost in western Iran. In total, 87 Rhinolophus mehelyi were captured. The patterns of abundance differed greatly among parasite species but total parasite load was markedly higher in pregnant females in spring and early summer and lower in solitary males. On average, 90% of bats were infested by Eyndhovenia sp. with a mean intensity of 13.79 individuals per bat. Penicillidia sp. and one species from Streblidae were found in 66.7% and 11.49% of bats with parasite load of 2.31 and 1.8 parasite per bat, respectively. Using ratio of forearm length to body mass as an indication of bat health the correlation coefficient between parasite load and the health indicator was 0.002 for males and 0.06 for females indicating that parasite load has no apparent impact on bat's health.

Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad.

Carvacrol is a major component of Satureja khuzistanica Jamzad (≤90%) that has significant antimicrobial and antioxidant properties. Considering the specific capabilities of S. khuzistanica to produce highly pure carvacrol, this plant is an important potential source of carvacrol that could address the abundant consumption and increasing demand for this monoterpene in current world markets. This research was performed to better understand the process of biosynthesis and accumulation of carvacrol in S. khuzistanica. Tests were performed on shoot cultures of S. khuzistanica in Linsmaier-Skoog (LS) medium treated with different concentrations of fosmidomycin (an inhibitor of the non-mevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway) for 21 days at the following concentrations: 0, 10, 25, 50, 75 and 100 µM. The present study demonstrated that the MEP pathway is the major pathway that provides IPP for the biosynthesis of carvacrol, and the expression and activity levels of the DXR enzyme have a critical effect on carvacrol biosynthesis. Surprisingly, Mevinolin at concentrations of 75 and 100 µM increased the carvacrol content and the DXR activity and gene expression in S. khuzistanica plantlets.

In vitro Cytotoxic and Antimicrobial Activity of Essential Oil From Satureja Intermedia.

BACKGROUND: Many members of the genus Satureja have aromatic and medicinal characteristics. Objectives. OBJECTIVES: The purpose of the present work was to determine cytotoxic activity of the essential oil of S. intermedia CA Mey (Lamiaceae) on two human cancerous cell lines and its in vitro inhibitory effects against 11 pathogenic bacteria and fungi as well. MATERIALS AND METHODS: The essential oil was isolated by hydrodistillation and analyzed by combination of capillary GC-FID and GC-MS. The in vitro toxicological study was based on the MTT cytotoxicity assay and antimicrobial activity of the essential oil was studied according to the disc diffusion method and MIC value. RESULTS: Thymol (34.5%), γ-terpinene (18.2%) and ρ-cymene (10.5%) were the main components of the essential oil. The toxicological study on 5637 and KYSE cell lines showed IC50 values of 156 µg/ml. The essential oil exhibited considerable antimicrobial activity on tested bacteria and fungi. CONCLUSIONS: From the results of the present study, it may be concluded that the essential oil of S. intermedia and its major constitutes are interesting in antibacterial and anticancer applications.

The effects of root endophyte and arbuscular mycorrhizal fungi on growth and cadmium accumulation in wheat under cadmium toxicity.

The effects of Piriformospora indica and Glomus mosseae on some growth and physiological parameters, and cadmium (Cd) amounts in Triticum aestivum cv. Sardari39 plants under the toxic levels of Cd were investigated. The experiment was carried out including four treatments (P. indica inoculation, G. mosseae inoculation, co-inoculation of G. mosseae and P. indica, and non-inoculation), each having four Cd concentrations (0, 0.3, 0.6 and 0.9 mM Cd). In non-inoculated plants, by increasing Cd concentration in the soil, growth parameters, chlorophyll content, performance index (PI) and Fv/Fm were reduced, whereas root and shoot Cd accumulation were increased. The colonized plants with P. indica and G. mosseae (in solitary or together) had higher growth, chlorophyll content and PI compared to control. In the presence of P. indica, the Cd concentration was increased in roots, whereas it was decreased in shoots under higher soil Cd concentrations. The presence of G. mosseae decreased root Cd concentration under lower Cd concentration in the soil. Results from this study indicate that P. indica inoculation yielded greater readings of growth parameters, chlorophyll content, PI, root colonization and also reduced shoot Cd content compared to G. mosseae inoculation.

The effect of light on gene expression and podophyllotoxin biosynthesis in Linum album cell culture.

Podophyllotoxin (PTOX) is a naturally occurring phenolic compound isolated as an active anti-tumor agent. The stimulatory influence of light on the formation of phenolic compounds has been reported, but the molecular mechanism underlying the effect of light on the expression of genes involved in phenolic biosynthesis, especially of lignans, is still not fully understood. A series of experiments was carried out using ordinary fluorescent lamps to study the influence of light irradiation on growth and PTOX accumulation in Linum album cell cultures by varying the type of light and periods of exposure. The biosynthesis of PTOX was variably affected according to the quality of light. The enhancing effects of red light on PTOX production was correlated with increased activities of the enzyme phenylalanine ammonia-lyase (PAL), and the expression of some key genes involved in the biosynthesis of this compound, including the PAL gene itself and the cinnamoyl-CoA reductase (CCR) gene. Blue light was found to have similar effects but mainly on the expression level of CCR and pinoresinol lariciresinol reductase (PLR) genes.

Chromium-induced tropane alkaloid production and H6H gene expression in Atropa belladonna L. (Solanaceae) in vitro-propagated plantlets.

Hyoscyamine and scopolamine tropane alkaloids found in several solanaceous plants are anticholinergic drugs. Hyoscyamine 6ß-hydroxylase (H6H) catalyzes two consecutive oxidation reactions. The first reaction is the hydroxylation of hyoscyamine to 6ß-hydroxyhyoscyamine and the second is epoxidation of 6ß-hydroxyhyoscyamine yielding scopolamine that is the final metabolite in the tropane alkaloid biosynthetic pathway. The effects of trivalent chromium as KCr (SO4)(2) on the production of tropane alkaloids and the expression of hyoscyamine 6ß-hydroxylase gene (h6h) were studied in micro-propagated Atropa belladonna L. plantlets. The results showed that chromium treatment decreased the growth parameters (weights and lengths of the plantlets) and chlorophyll contents and increased proline contents. Moreover, semiquantitave RT-PCR analysis showed that the transcript level of H6H increased under chromium treatment. This treatment also increased hyoscyamine and scopolamine contents as shown by HPLC analysis. Changes of scopolamine contents correlate with the expression levels of h6h gene under different concentrations of chromium.

Time-course changes in fungal elicitor-induced lignan synthesis and expression of the relevant genes in cell cultures of Linum album.

Linum album has been shown to accumulate anti-tumor podophyllotoxin (PTOX) and its related lignans. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [140µgg(-1) dry weight (DW) of the L. album cell culture] which is seven-fold greater than the untreated control, while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol, instead of PTOX, up to 365µgg(-1) DW, which was 8.8-fold greater than the control. Quantitative PCR analyses showed that expression of the enzyme genes responsible for the PTOX biosynthesis cascade, such as pinoresinol-lariciresinol reductase (PLR), phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) genes, were also up-regulated in a fungal extract-selective fashion. These results provide evidence that the fungal extracts used in this study differentially increase the production of PTOX or larisiresinol via the up-regulation of the genes in lignan biosynthesis in L. album cell cultures, and suggest that such selective actions of fungal elicitors on the lignan synthesis will lead to more efficient metabolic engineering-based production of PTOX and other beneficial lignans using L. album cell cultures.

Optimization of podophyllotoxin extraction method from Linum album cell cultures.

CONTEXT: Suspension cultures of Linum album Kotschy ex Boiss. (Linaceae) accumulate podophyllotoxin (an anticancer agent) and could therefore serve as an alternative source of this important aryltetralin lactone lignan. OBJECTIVE: The present work compared four podophyllotoxin extraction methods and optimization of the best one by using single factor experiments. MATERIALS AND METHODS: Linum album cell cultures were established from in vitro plantlets and subcultured in MS medium with hormones every 7-8 days. Four podophyllotoxin extraction methods were assayed and the best one was optimized by single factor experiments, studying the effect of methanol concentration, extraction time, and sonication time. RESULTS: Cell cultures accumulated enough podophyllotoxin to be analyzed by HPLC. The methanol/dichloromethane and buffer extraction methods were found to be the best. Methanol alone and hot ethanol were not effective for extracting podophyllotoxin. DISCUSSION AND CONCLUSION: The optimized method based on methanol/dichloromethane extraction combined with HPLC quantification was able to determine small amounts of podophyllotoxin in Linum album cell cultures, showing that this system could constitute a possible alternative source of podophyllotoxin to Podophyllum (Berberidaceae).