The different matrix components in enamel contribute to its large

The different matrix components in enamel contribute to its larger and more rigid hydroxyapatite crystal structures than dentin and bone. Enamel matrix proteins are produced at their highest levels by ameloblasts

during the secretory and transition stages of amelogenesis and collectively orchestrate the proper assembly and growth Afatinib of crystals within mineralized enamel. These proteins are nearly completely degraded by specific proteases such as MMP-20, mainly produced during the secretory/transition stage, and KLK4, mainly produced during the transition/maturation stage, resulting in a highly ordered and purposefully designed meshwork of carbonated hydroxyapatite crystals with astonishing mechanical properties [1]. Cell adhesion to the extracellular matrix is of fundamental importance for a wide range of cellular functions, including cell differentiation, proliferation, and survival [2]. Inner and outer enamel epithelial cells interact with the basement membrane, of which major constituents are type IV collagen, laminin, and heparan-sulfate proteoglycan perlecan. For example, laminin α5 (Lama5)-deficient mice show small tooth germs without a cusp [3]. In addition, proliferation and polarization of the dental epithelium

are inhibited in these selleckchem mice, indicating that interactions between the dental epithelium and the basement membrane are important to determine tooth sizes and dental epithelial cell differentiation. Ameloblasts are polarized in the secretory stage and secrete enamel matrix components, including amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM). Furthermore, amelotin (AMTN) and Apin/odontogenic ameloblast-associated (ODAM) are secreted from those

in the maturation stage. These enamel matrix components are considered important for enamel crystal formation. In fact, AMEL and ENAM mutations are identified in patients with amelogenesis imperfecta (AI), a common group of inherited defects of dental enamel formation that exhibit marked genetic and clinical Staurosporine heterogeneity, with at least 14 different sub-types recognized on the basis of their clinical appearance and mode of inheritance [4], [5] and [6]. However, a recent study that used a gene-targeting method and in vitro analysis showed that these matrices also have a large number of biological functions in addition to enamel formation, including bone formation, tumorigenesis, and the regulation of stem cell function. Mineralized tissue formation and maintenance are controlled by matrix proteins such as several secretory calcium-binding phospho-proteins (SCPPs), which are encoded by SCPP genes clustered on human chromosome 4 [7]. Half of these proteins, including bone sialoprotein, osteopontin, dentin sialophosphoprotein, and dentin matrix protein, are associated with bone and dentin formation, while the other half of consists of enamel-associated proteins, including AMBN, ENAM, AMTN, and Apin/ODAM (Table 1).

21) [46] and [47] Mutational studies also indicated that the fir

21) [46] and [47]. Mutational studies also indicated that the first arginine (R1), fourth proline (P4) and fifth asparate

(D5) were important for binding. Because the surface of titanium is covered with an oxide film TGF-beta inhibitor displaying both positively and negatively charged hydroxyl groups under physiological conditions, electrostatic interactions between –O− and R1, and –OH2+ and D5, have been proposed to underlie the interaction between minTBP-1 and titanium. Four kinds of peptide were prepared as summarized in Table 3. Adsorption assay of the synthesized peptides was carried out on crystal quartz sensors coated with Ti using a QCM-D instrument. The results demonstrated that accretion of surfactant reduced nonspecific interactions, dramatically enhancing the selectivity

and specificity of the Ti-binding peptides, and ensuring reversible specific binding (Fig. 22). In addition, the bioactivities GDC-973 of P. gingivalis cells on peptide-modified titanium were evaluated by ATP-bioluminescent assay. The bioactivity test revealed that ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control [45]. These findings indicate that surface modification with conjugated molecules consisting of antimicrobial and titanium-binding peptides is a promising method for reduction of biofilm formation on titanium implants. In conclusion, a cold-plasma surface modification is useful in controlling the physicochemical nature of surfaces, including the surface energy and the surface electrical

charge, leading to immobilize the drugs and peptides, and in developing bio-functional implants (Fig. 4). Considering HSP90 the present technology, it may be possible to produce implants with highly controlled surfaces that maintain homeostasis. The authors have no financial relationship with the organization that sponsored the research. This research was supported by an Oral Health Science Center Grant hrc7 and hrc8 from Tokyo Dental College, a “High-Tech Research Center” Project for Private Universities: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) of Japan, 2006–2010 and 2010–2012, and a Grant-in-Aid for Scientific Research (B:18390524 and 18659581) from the Japan Society for the Promotion of Science. “
“Dental caries is the most common pathological change of dentin. Fusayama’s research demonstrated that carious dentin consists of two distinct layers: an outer layer of bacterially infected dentin, and an inner layer of affected dentin [1]. The outer layer (caries-infected dentin) was characterized as being highly demineralized, physiologically unreminerazable and showing irreversible denatured collagen fibrils with a virtual disappearance of cross-linkages.

However the quercetin standard showed a higher percentage of oxid

However the quercetin standard showed a higher percentage of oxidation inhibition, possibly due to its more hydrophobic nature. The XO inhibitory capacity of hydrolyzed rutin (after 4, 8 and 12 h of hydrolysis with hesperidinase) was not statistically different from rutin, which could be considered a weak inhibitor of XO. Quercetin, on the other hand, exhibited the strongest inhibitory activity, as shown in Table 1. The antiproliferative properties of the samples before and after bioconversion were assessed using nine human cancer cell lines, and the chemotherapeutic drug, doxorubicin, as a positive control (Fig. 4 and Table 2). A horizontal line at 0% was traced to visualize

Total Growth Inhibition (TGI) that represents the concentration required to completely inhibit cell growth (total Enzalutamide molecular weight cytostatic effect) (Table 2). For all cell lines tested, rutin hydrolyzed by hesperidinase displayed a moderate Selleckchem ATM inhibitor antiproliferative activity with selectivity for OVCAR-3 (ovarian, TGI = 1.5 μg/mL), MCF-7 (breast, TGI = 2.3 μg/mL) and U251 (glioma, TGI = 3.6 μg/mL) while quercetin presented a weak activity with selectivity for U251 (glioma, TGI = 31.4 μg/mL), MCF-7 (breast, TGI = 31.9 μg/mL), 786–0 (renal, TGI = 42.7 μg/mL) and NCI-ADR/RES (ovarian expressing

multidrug resistance, TGI = 44.0 μg/mL). Rutin did not inhibit cell proliferation of any of the cancer cell lines tested. Flavonoid glycosides production of by removing rhamnose from rutinosides can be performed through controlled enzymatic catalysis. In the present study, we were able to define a good condition of β-d-glucosidase inactivation for hesperidinase and naringinase, while keeping a high level of α-l-rhamnosidase activity. After 4 h of enzymatic reaction catalyzed by hesperidinase, previously heated at 70 °C for 30 min, significant amounts of quercetin-3-glucoside (approximately

70%) were obtained. Hesperidinase hydrolyzed rutin more efficiently than naringinase. Hydrolyzed rutin produced by bioconversion using hesperidinase was subsequently selected for further Erythromycin investigation. Vila-Real, Alfaia, Bronze, Calado, and Ribeiro (2011) performed a similar procedure to produce flavonoid monoglycosides, including quercetin-3-glucoside, from rutinosides, using naringinase from Penicillium decumbens as biocatalyst. The authors reported that a selective inactivation of β-d-glucosidase activity of naringinase was achieved at 81.5 °C and pH 3.9, keeping a very high residual activity of α-l-rhamnosidase (78%). Similarly, You et al. (2010) reported that β-d-glucosidase activity of crude enzyme extract of Aspergillus niger was completely inactivated by treatment for 30 min at 70 °C while the α-l-rhamnosidase activity was decreased by only 50%. The difference in the Rha/Glu activity ratio between hesperidinase and naringinase (Fig.

e would explain the overall lower concentration of anthocyanins

e. would explain the overall lower concentration of anthocyanins per head. Consistently, it has been established www.selleckchem.com/products/AZD2281(Olaparib).html that inner leaves of lettuce heads have lower concentrations of flavonols than outer leaves- not due to a lack of competence but due to lower incident radiation intensity compared to the situation with outer leaves (Hohl, Neubert, Pforte, Schonhof, & Böhm, 2001). The observation that there was no significant difference anymore between mature heads of warm- and cool-cultivated plants (Fig. 3 and Table 1) may indicate an acclimation of the all the time cool-cultivated plants to the lower temperature.

In these plants the light-harvesting chlorophyll antenna may have been down-scaled and the chlorophyll a/b ratio altered (Havaux & Kloppstech, 2001). Thereby, again, the amount of energy captured and funnelled into the electron transport chain would be reduced and no anthocyanin accumulation would be necessary to encounter an enhanced oxidative load. Regarding quercetin-3-O-(6″-O-malonyl)-glucoside, quercetin-3-O-glucuronide/luteolin-7-O-glucuronide, and quercetin-3-O-glucoside concentration, there were no significant

differences between small heads that were cultivated either cool or warm ( Fig. 3 and Table 1). Furthermore, there were no significant differences concerning these compounds between mature heads cultivated in different temperature regimes ( Fig. 3 and ADP ribosylation factor Table 1). If we compare warm- and cool-cultivated AZD5363 plants after the same number of days, we detect significantly higher

concentrations of quercetin-3-O-(6″-O-malonyl)-glucoside and quercetin-3-O-glucuronide/luteolin-7-O-glucuronide ( Table 2 and Fig. 3). However, the data of Romani et al. (2002) suggest a higher concentration of quercetin glycosides in early growth stage-lettuce compared to later stages. In Section 3.2 we demonstrated that warm- and cool-cultivated plants in our experiment were in different growth stages after 26 days of treatment. Hence, we conclude that the higher concentrations in the cool-cultivated plants were rather due to their growth stages than to the temperature treatment. This is in line with results Løvdal et al. (2010) obtained on leaves of tomato plants (Solanum lycopersicum): Quercetin glycosides were accumulated in response to increasing light intensity and nitrogen depletion rather than to lowered temperature alone. Indeed, quercetin glycoside concentration in red leaf lettuce does respond sensitively to radiation intensity ( Becker et al., 2013). In our experiment, we closely monitored the macro nutrients in the nutrient solution to ensure they are sufficient and the PPFD we applied was constant (247 μmol m−2 s−1). The lowest temperature in our experiment (7 °C) was applied outside of the photoperiod and it, therefore, did not concur with radiation.

On the other hand, 3,7,11,11-tetramethyl-10,15-dioxo-hexadec-2,4,

On the other hand, 3,7,11,11-tetramethyl-10,15-dioxo-hexadec-2,4,6,8-tetra-enal was identified as two different hydrazones: one of them resulting from the reaction between DNPHi and one carbonyl group and the other from the reaction between DNPHi and two carbonyl groups of the CC. This fact shall be related to differences in the reaction rates between DNPHi and each different CC structure. BLU9931 mouse The factors which can interfere with these rates include the electrophilic character of the carbonylic carbons, the steric conditions in the molecules (for example, for aldehydes the carbonyl group is located in the molecule’s extremity, making easier the approximation of a reagent than in ketones), the pH of the media

and the DNPHi concentration Metabolism inhibitor (Bicking and Cooke, 1998). The time elapsed between the sampling and the elution of the cartridge for analysis is also a critical factor, depending on the type of compound reacting with the derivatisation agent (Van Leeuwen, Hendriksen, & Karst, 2004). Some authors (Zwiener, Glauner,

& Frimmel, 2002) have demonstrated that a minimum of 1 h of contact is necessary for the compounds which have high reaction rates with DNPHi, such as monocarbonyl compounds. However, for the dicarbonyl and hydroxicarbonyl compounds, a minimum of 12 h may be necessary to carry out the reaction in the experimental conditions evaluated. In this study, as stated in section 2.5, the total elapsed time between the sampling and the elution of the cartridges was seven hours. Although the formation of some of the compounds highlighted in this work (e.g., 5,6-epoxy-12´-apo-β-carotenal, 7-apo-β-caroten-7-al (β-cyclocitral), 12´-apo-β-carotenal, amongst others) as oxidation products of β-carotene is well documented, there is not a well-defined mechanism in the literature for their formation yet (Rodriguez and Rodriguez-Amaya,

2007, Waché, Bosser-Deratuld, LY, & Belin, 2002). In fact, it is inadequate to propose just one mechanism for all of the products obtained, considering that the precursor molecule is highly unsaturated and offers several possibilities for the initial ozone attack. In addition, the products that are initially formed may also react with ozone themselves, giving rise to new products as described previously. Casein kinase 1 When a double bond is broken, several different compounds may be formed; for example, when the double bond localised inside the ring is broken, epoxyde and secocarotenoids are formed. Fig. 2 shows the mechanism proposed for the formation of 2-methyl-buten-2-dial, beginning with the ozone attack on the double bonds between C12´–C11´ and C8´–C7´. A highly unstable ozonide is then formed, followed by the dienal and two Criegee’s biradicals. Fig. 3 shows, on the other way, the proposed mechanism for the formation of an epoxycarotenoid, which is originally based on that suggested by other authors for the ozonolysis of alkenes (Bailey, Mann, & Maittlis, 1975).

Pregnant women who reported living in the United States for their

Pregnant women who reported living in the United States for their entire lives had 38% (95% CI: − 0.1, 89.3; p = 0.05) and 35% (95% CI: 2.6, 78.0; p = 0.03) higher uncorrected and specific gravity-corrected AZD2281 manufacturer urinary BPA concentrations, respectively, compared with women who reported living in the United States for 5 years or less. Additionally, women who reported drinking at least three sodas per day had approximately 58% (95% CI: 18.0, 112.1; p = 0.002) and 41% (95% CI: 9.9, 80.9; p = 0.01) higher uncorrected and specific gravity-corrected urinary BPA concentrations,

respectively, compared with women who did not consume soda. Compared with women who reported not consuming any hamburgers, women who reported eating hamburgers three times

per week or more had 20% (95% CI: − 0.2, 45.2; p = 0.05) and 17.3% (95% CI: 0.5, 36.9; p = 0.04) higher uncorrected and specific gravity-corrected urinary BPA concentrations, respectively. Lastly, we observed that for every one-hour increase in sample collection time, there was a 3% (95% CI: 0.3, 6.0; p = 0.03 and 95% CI: 0.8, 5.8; p = 0.01 for uncorrected and specific gravity-corrected concentrations, respectively) increase in urinary BPA concentrations. Results were similar when we restricted our analysis to women with no missing covariate data (i.e., no imputed covariates) and when we included collection time as a categorical variable based on potential meal times (i.e., higher BPA concentrations

selleck screening library Amino acid were observed as samples were collected later in the day and associations with other predictor variables were largely unchanged). When we evaluated the relationship between time spent living in the United States and significant dietary predictors, we observed that there was a higher percentage of women who reported consuming sodas (> 1 soda/day vs. no sodas) and hamburgers (≥ 1 time per week vs. ≤ 1–3 times per month) in women who reported living in the United States their entire lives compared with women who had lived less time in the country (Fig. 1). We observed significantly higher BPA concentrations with longer residence in the United States among pregnant women of Mexican descent. Pregnant women who consumed more servings of soda and hamburgers also had higher BPA concentrations. Urinary BPA concentrations from samples collected twice during pregnancy varied greatly, with high within- versus between-woman variability, and seemed to be marginally higher in samples collected in the afternoon/evening hours. The higher BPA concentrations in pregnant women in our study who lived in the United States their entire lives compared with recent immigrants may reflect differences in diet that accompany U.S. acculturation.

01–0 23 cm year−1 Differences between

01–0.23 cm year−1. Differences between Linsitinib purchase observed and predicted values were mostly less than 2 cm. Higher values were found for Moses with Scots pine, for Prognaus with Scots pine in Arnoldstein and spruce in Litschau, and for Silva for both species in Litschau. Although not presented here,

we plotted observed and predicted individual tree values for each plot and growth simulator. For spruce, BWIN and Silva in most cases underestimated the diameters of small trees and overestimated the diameters of large trees. For BWIN in particular, observed and predicted dbh matched quite well except that the very large trees were considerably overestimated. In contrast, Prognaus and Moses overestimated the diameters of small trees and underestimated the diameters of large trees. Similarly for pine, all four growth simulators overestimated the size of small trees and underestimated the size of large trees. Predicted heights deviated 0.3–3.5 m from observed values. This corresponds to 0.01–0.12 m year−1. Observed and predicted height growth matched quite well see more in Arnoldstein, and there was little deviation between observed and predicted values for both mean and maximum values. In Litschau, however there was poor agreement

with observed values, except for Scots pine height growth predicted by Silva. Moses overestimates the mean height but underestimates the maximum values. This seems to indicate that the shape of the height growth curve is inappropriate. Examining the plots of observed and predicted heights, we found that from in Arnoldstein all four growth simulators for both species overestimated the height of small trees and underestimated the height of large trees. Patterns were less homogenous in Litschau. For pine, a pattern similar to that in Arnoldstein was prevalent, with an overestimation of small heights and the underestimation of large heights; for spruce the opposite was true except for Prognaus. In many cases observed and predicted height:diameter

ratios agreed fairly well. Within a plot low height:diameter ratios were overestimated and high height:diameter ratios were underestimated, except for predictions of spruce with the simulator Silva in Litschau. Height:diameter ratios are the result of the predictions of height and diameter increment. There are four different cases for the resulting height:diameter ratio: (1) increment and allometry correct, (2) height or diameter increment wrong, allometry distorted, (3) height and diameter increment wrong, allometry correct and (4) height and diameter increment wrong, allometry distorted. Indeed there were cases where neither model largely deviated, but the resulting height:diameter ratios were biased. Also, there were cases were both models deviated, but the resulting height:diameter ratio agreed fairly well with observed values. Compare, for example, the simulation results for Norway spruce in Litschau using Moses in Table 6, Table 7 and Table 8.

, 2007) The seed production of many agroforestry trees is often

, 2007). The seed production of many agroforestry trees is often informal and very few countries have included these species in their tree improvement programmes. Germplasm of exotic tree species, typically from introductions of unknown provenance and uncharacterised performance, is often collected by smallholders directly for their find more own planting.

Lillesø et al. (2011), for example, identified five sources for farmers’ tree planting material (farmland, natural forest, plantations, seed orchards and vegetative propagules) and indicated heavy reliance on the first source, with natural forest sources being underutilised. Farmers and local seed dealers often prefer to collect seed from previously learn more introduced exotic trees in farmland rather than source externally because the transaction costs are lower, even when better-performing seed sources of the same trees may be available elsewhere (Lengkeek et al., 2005 and Muriuki, 2005). In recent decades, there has been a greater focus on the cultivation of indigenous tree species in agroforestry systems, with the involvement of local people in carrying out genetic selection for tree characteristics of importance

to them. One such approach, known as participatory domestication, has been developed in Africa on indigenous fruit trees (see Dawson et al., 2014, this special issue). The advantage of this approach is that genetic quality as a concept is explicitly considered, and local wild stands provide significant genetic variation that is a pool for selection

(Tchoundjeu et al., 2006). The risk of spreading pests and diseases while transferring reproductive material is often considerable. Pests and diseases travel in different substrates and it is challenging anti-PD-1 antibody inhibitor to monitor the way they spread; for example, to reconstruct the exact pathways of their past movements. In Europe, Santini et al. (2013) reconstructed the most probable pathways of alien invasive forest pathogen spread since 1800. They found that living plants (57% of all pathogen introductions) and wood (10%) were likely major vectors for introductions, while the share of any other pathway, such as bark, seed, soil and cuttings, was less than 10% over the last two centuries. According to the same authors, over the last few decades, the invasion rate of alien forest pathogens has increased exponentially in Europe, with soil recently becoming a major transfer substrate second to living plants. In the USA, a similar study attributed 69% of the introductions of non-native forest insects and pathogens since 1860 to the trade in living plants (Liebhold et al., 2012). These studies confirm the need for phytosanitary regulations and their careful implementation while transferring tree germplasm. However, they also show that the pathogen risk associated with transferring seed is considerably lower than the risk connected with transferring other materials such as living plants or wood.

To further demonstrate concordance as a complete system, the Nati

To further demonstrate concordance as a complete system, the National Institute for Standards and Technology (NIST) performed an initial concordance study comparing genotypes from 652 unrelated individuals using a pre-release PowerPlex® Fusion System to commercially available PowerPlex® 16 HS and PowerPlex® 21 Systems and further compared to AmpFLSTR® NGM™, Identifiler™, YFiler™, Profiler®, Minifiler™ and Sinofiler™ PCR Amplification Kits (Life Technologies™), and Investigator® ESSplex Plus and IDplex Plus systems (Qiagen). At its commercial release a minor change check details was made to the D16S539 primers. A confirmatory concordance study was performed

using a subset of 182 African-American samples. Samples were selleck chemical detected using an Applied Biosystems® 3130 Series Genetic Analyzer with a 1 kV 3 s injection for the original sample set and 2kv 5 s injection for the confirmatory sample set. Three discordant calls out of 39,198 alleles tested were observed at amelogenin, D7S820, and D22S1045. No discordances were observed at D16S539 with the updated primers. One discordant sample generated Y, Y results at amelogenin with the PowerPlex® Fusion System and all other systems except Investigator® ESSplex Plus and IDplex Plus. In the second sample, sequencing confirmed 8 and 11 alleles at D7S820. The 8, 11 genotype was

generated using the PowerPlex® 16 and Minifiler™ systems. However, the PowerPlex® Fusion, Profiler®, Sinofiler™, and PowerPlex® 21 systems produced an 8, 9.3 genotype. A deletion is suspected between the primer binding sites of the two sets of systems. Finally, a previously unknown discordance was observed at D22S1045. Well balanced

14, 17 alleles were amplified using the PowerPlex® ESI and ESX Systems. In contrast, amplification using the PowerPlex® Fusion System yielded a severely imbalanced 14 allele. The PowerPlex® Fusion System is suitable for comparison with science previously gathered profiles from multiple systems, as the observed discordances were rare and unique. Allele calls rely on similar migration between the sample and allelic ladder standard. Therefore, migration and sizing precision must be consistent and within the bin window for accurate allele calls. To demonstrate precision, allelic ladders were detected at five sites on Applied Biosystems® 3130 and 3500 Series Genetic Analyzers and an Applied Biosystems® 3730 DNA Analyzer. This study addressed typical sources of variability such as differences between capillaries and injections. Standard deviations in sizing were calculated for each allele. The maximum standard deviation of an allele was 0.1 bases on the 3130xl and 3500xl Genetic Analyzers ( Fig. 4 and Supplemental Fig. 8).

Based on specific reamplification of this band from multiple test

Based on specific reamplification of this band from multiple tested bands, we concluded that the artifact bands Selleck INCB018424 could be derived

from the formation of heteroduplexes during PAGE analysis when more than two similar bands coexisted in the same PCR product [39]. Therefore, we consider the appearance of the heteroduplex artifact bands as a signature for the mixture of the two species that can be beneficial for authentication or identification of mixtures in large volumes of processed ginseng samples [40]. The InDel-based codominant marker has limitations in high-throughput analysis to detect mixtures of the species because genotyping with the marker depends on high-resolution gel electrophoresis. Even though HRM can detect both individual genotypes without gel electrophoresis, the method has limited application to mixed samples [24], [29], [30] and [31]. To address this, we tested the ability of the species-specific markers to identify mixtures. The Pg-specific marker could reveal the presence of P. ginseng at a 1% level in the American ginseng products ( Fig. 6A). Conversely, the Pq-specific marker could identify down to 1% P. quinquefolius in P. ginseng products ( Fig. 6B). Quantitative PCR with the same primer set was consistent selleck products with the AGE results, and revealed quantitative mixing ratios

down to 1% (Fig. 7). The quantitative PCR method reports the quantitative mixing ratio without requiring gel electrophoresis, which is an advantage for mass and high-throughput analysis for monitoring mislabeling or false trading in commercial ginseng products [41]. These markers will be useful to prevent the illegal distribution or intentional mixing of American and Korean ginseng in the ginseng market. Korean and American ginseng are important herbal medicines and each species has some

unique medicinal functions [42] and [43]. Applying the evaluation system we have developed here will promote and increase the value of Korean ginseng as well as American ginseng in Inositol monophosphatase 1 Korea and worldwide, by allowing consumers to be confident in the contents of commercial ginseng products. All authors declare no conflicts of interest. This study was supported by the Next-Generation BioGreen21 Program (No. PJ008202), Rural Development Administration, Korea. “
“Korean ginseng (Panax ginseng) is a renowned perennial herb that has long been used for medicinal purposes in East Asia [1]. P. ginseng has a large genome estimated to be more than 3 Gbp in size [2] and 2n = 48 chromosomes [3]. Species belonging to the genus Panax have 2n = 24 chromosomes or 48 chromosomes, so that the species with 2n = 48 chromosomes have been regarded as tetraploids [4] and [5].