Making the Genotypic Variation Visible: Hyperspectral Phenotyping in Scots Pine Seedlings.

Hyperspectral reflectance contains valuable information about leaf functional traits, which can indicate a plant's physiological status. Therefore, using hyperspectral reflectance for high-throughput phenotyping of foliar traits could be a powerful tool for tree breeders and nursery practitioners to distinguish and select seedlings with desired adaptation potential to local environments. We evaluated the use of 2 nondestructive methods (i.e., leaf and proximal/canopy) measuring hyperspectral reflectance in the 350- to 2,500-nm range for phenotyping on 1,788 individual Scots pine seedlings belonging to lowland and upland ecotypes of 3 different local populations from the Czech Republic. Leaf-level measurements were collected using a spectroradiometer and a contact probe with an internal light source to measure the biconical reflectance factor of a sample of needles placed on a black background in the contact probe field of view. The proximal canopy measurements were collected under natural solar light, using the same spectroradiometer with fiber optical cable to collect data on individual seedlings' hemispherical conical reflectance factor. The latter method was highly susceptible to changes in incoming radiation. Both spectral datasets showed statistically significant differences among Scots pine populations in the whole spectral range. Moreover, using random forest and support vector machine learning algorithms, the proximal data obtained from the top of the seedlings offered up to 83% accuracy in predicting 3 different Scots pine populations. We conclude that both approaches are viable for hyperspectral phenotyping to disentangle the phenotypic and the underlying genetic variation within Scots pine seedlings.

Leaf Functional Traits in Relation to Species Composition in an Arctic-Alpine Tundra Grassland.

The relict arctic-alpine tundra provides a natural laboratory to study the potential impacts of climate change and anthropogenic disturbance on tundra vegetation. The Nardus stricta-dominated relict tundra grasslands in the Krkonose Mountains have experienced shifting species dynamics over the past few decades. Changes in species cover of the four competing grasses-Nardus stricta, Calamagrostis villosa, Molinia caerulea, and Deschampsia cespitosa-were successfully detected using orthophotos. Leaf functional traits (anatomy/morphology, element accumulation, leaf pigments, and phenolic compound profiles), were examined in combination with in situ chlorophyll fluorescence in order to shed light on their respective spatial expansions and retreats. Our results suggest a diverse phenolic profile in combination with early leaf expansion and pigment accumulation has aided the expansion of C. villosa, while microhabitats may drive the expansion and decline of D. cespitosa in different areas of the grassland. N. stricta-the dominant species-is retreating, while M. caerulea did not demonstrate significant changes in territory between 2012 and 2018. We propose that the seasonal dynamics of pigment accumulation and canopy formation are important factors when assessing potential "spreader" species and recommend that phenology be taken into account when monitoring grass species using remote sensing.

Barley Genotypes Vary in Stomatal Responsiveness to Light and CO2 Conditions.

Changes in stomatal conductance and density allow plants to acclimate to changing environmental conditions. In the present paper, the influence of atmospheric CO2 concentration and light intensity on stomata were investigated for two barley genotypes-Barke and Bojos, differing in their sensitivity to oxidative stress and phenolic acid profiles. A novel approach for stomatal density analysis was used-a pair of convolution neural networks were developed to automatically identify and count stomata on epidermal micrographs. Stomatal density in barley was influenced by genotype, as well as by light and CO2 conditions. Low CO2 conditions resulted in increased stomatal density, although differences between ambient and elevated CO2 were not significant. High light intensity increased stomatal density compared to low light intensity in both barley varieties and all CO2 treatments. Changes in stomatal conductance were also measured alongside the accumulation of pentoses, hexoses, disaccharides, and abscisic acid detected by liquid chromatography coupled with mass spectrometry. High light increased the accumulation of all sugars and reduced abscisic acid levels. Abscisic acid was influenced by all factors-light, CO2, and genotype-in combination. Differences were discovered between the two barley varieties: oxidative stress sensitive Barke demonstrated higher stomatal density, but lower conductance and better water use efficiency (WUE) than oxidative stress resistant Bojos at saturating light intensity. Barke also showed greater variability between treatments in measurements of stomatal density, sugar accumulation, and abscisic levels, implying that it may be more responsive to environmental drivers influencing water relations in the plant.

Light and CO2 Modulate the Accumulation and Localization of Phenolic Compounds in Barley Leaves.

Barley (Hordeum vulgare) accumulates phenolic compounds (PhCs), which play a key role in plant defense against environmental stressors as antioxidants or UV screening compounds. The influence of light and atmospheric CO2 concentration ([CO2]) on the accumulation and localization of PhCs in barley leaves was examined for two varieties with different tolerances to oxidative stress. PhC localization was visualized in vivo using fluorescence microscopy. Close relationships were found between fluorescence-determined localization of PhCs in barley leaves and PhC content estimated using liquid chromatography coupled with mass spectroscopy detection. Light intensity had the strongest effect on the accumulation of PhCs, but the total PhC content was similar at elevated [CO2], minimizing the differences between high and low light. PhCs localized preferentially near the surfaces of leaves, but under low light, an increasing allocation of PhCs in deeper mesophyll layers was observed. The PhC profile was significantly different between barley varieties. The relatively tolerant variety accumulated significantly more hydroxycinnamic acids, indicating that these PhCs may play a more prominent role in oxidative stress prevention. Our research presents novel evidence that [CO2] modulates the accumulation of PhCs in barley leaves. Mesophyll cells, rather than epidermal cells, were most responsive to environmental stimuli in terms of PhC accumulation.

Forest Microhabitat Affects Succession of Fungal Communities on Decomposing Fine Tree Roots.

Belowground litter derived from tree roots has been shown as a principal source of soil organic matter in coniferous forests. Fate of tree root necromass depends on fungal communities developing on the decaying roots. Local environmental conditions which affect composition of tree root mycobiome may also influence fungal communities developing on decaying tree roots. Here, we assessed fungal communities associated with decaying roots of Picea abies decomposing in three microhabitats: soil with no vegetation, soil with ericoid shrubs cover, and P. abies deadwood, for a 2-year period. Forest microhabitat showed stronger effect on structuring fungal communities associated with decaying roots compared to living roots. Some ericoid mycorrhizal fungi showed higher relative abundance on decaying roots in soils under ericoid shrub cover, while saprotrophic fungi had higher relative abundance in roots decomposing inside deadwood. Regardless of the studied microhabitat, we observed decline of ectomycorrhizal fungi and increase of endophytic fungi during root decomposition. Interestingly, we found substantially more fungal taxa with unknown ecology in late stages of root decomposition, indicating that highly decomposed roots may represent so far overlooked niche for soil fungi. Our study shows the importance of microhabitats on the fate of the decomposing spruce roots.

Alien ectomycorrhizal plants differ in their ability to interact with co-introduced and native ectomycorrhizal fungi in novel sites.

Alien plants represent a potential threat to environment and society. Understanding the process of alien plants naturalization is therefore of primary importance. In alien plants, successful establishment can be constrained by the absence of suitable fungal partners. Here, we used 42 independent datasets of ectomycorrhizal fungal (EcMF) communities associated with alien Pinaceae and Eucalyptus spp., as the most commonly introduced tree species worldwide, to explore the strategies these plant groups utilize to establish symbioses with EcMF in the areas of introduction. We have also determined the differences in composition of EcMF communities associated with alien ectomycorrhizal plants in different regions. While alien Pinaceae introduced to new regions rely upon association with co-introduced EcMF, alien Eucalyptus often form novel interactions with EcMF species native to the region where the plant was introduced. The region of origin primarily determines species composition of EcMF communities associated with alien Pinaceae in new areas, which may largely affect invasion potential of the alien plants. Our study shows that alien ectomycorrhizal plants largely differ in their ability to interact with co-introduced and native ectomycorrhizal fungi in sites of introduction, which may potentially affect their invasive potential.

Image Analysis: Basic Procedures for Description of Plant Structures.

This chapter gives examples of basic procedures of quantification of plant structures with use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified "rules of thumb" are presented. The basic procedures mentioned in the text are: (1) sampling, (2) calibration, (3) manual length measurement, (4) leaf surface area measurement, (5) estimation of particle density demonstrated on an example of stomatal density, and (6) analysis of epidermal cell shape.

New Insight into Sperm Capacitation: A Novel Mechanism of 17ß-Estradiol Signalling.

17ß-estradiol (estradiol) is a natural estrogen regulating reproduction including sperm and egg development, sperm maturation-called capacitation-and sperm⁻egg communication. High doses can increase germ cell apoptosis and decrease sperm count. Our aim was to answer the biological relevance of estradiol in sperm capacitation and its effect on motility and acrosome reaction to quantify its interaction with estrogen receptors and propose a model of estradiol action during capacitation using kinetic analysis. Estradiol increased protein tyrosine phosphorylation, elevated rate of spontaneous acrosome reaction, and altered motility parameters measured Hamilton-Thorne Computer Assisted Semen Analyzer (CASA) in capacitating sperm. To monitor time and concentration dependent binding dynamics of extracellular estradiol, high-performance liquid chromatography with tandem mass spectrometry was used to measure sperm response and data was subjected to kinetic analysis. The kinetic model of estradiol action during sperm maturation shows that estradiol adsorption onto a plasma membrane surface is controlled by Langmuir isotherm. After, when estradiol passes into the cytoplasm, it forms an unstable adduct with cytoplasmic receptors, which display a signalling autocatalytic pattern. This autocatalytic reaction suggests crosstalk between receptor and non-receptor pathways utilized by sperm prior to fertilization.

Rough wave-like heaped overburden promotes establishment of woody vegetation while leveling promotes grasses during unassisted post mining site development.

Geodiversity plays an important role in species establishment during spontaneous succession. At post-mining sites in the Czech Republic in 2003, we established plots in which the surface of the heaped overburden was either kept wave-like or leveled. Based on surveys conducted from 2006 to 2015, leveled plots were increasingly dominated by grasses and herbs (and especially by the grass Calamagrostis epigejos) while the wave-like plots were increasingly dominated by the trees Salix caprea and Betula pendula. In 2015, a detailed survey was conducted of the dominant species. Both S. caprea and B. pendula occurred more often in wave-like plots than in leveled plots; this was particularly true for trees taller than 1 m, which were absent in leveled plots. In wave-like plots, leaf and root biomasses of both woody species were higher on the wave slopes than on the wave depressions. Nitrogen content was higher but content stress indicating proline in leaves of S. caprea was lower in wave-like plots than in leveled plots. In wave-like plots, both woody species occurred mainly on wave slopes but C. epigejos occurred mainly in the depressions. We speculate that trees were more abundant in wave-like plots than in leveled plots because the waves trapped tree seeds and snow and because the soil porosity was greater in wave-like than in leveled plots. Grasses may have preferred the leveled plots because soil porosity was lower and clay content was higher in leveled than in wave-like plots.

A sex-chromosome inversion causes strong overdominance for sperm traits that affect siring success.

Male reproductive success depends on the competitive ability of sperm to fertilize the ova, which should lead to strong selection on sperm characteristics. This raises the question of how heritable variation in sperm traits is maintained. Here we show that in zebra finches (Taeniopygia guttata) nearly half of the variance in sperm morphology is explained by an inversion on the Z chromosome with a 40% allele frequency in the wild. The sperm of males that are heterozygous for the inversion had the longest midpieces and the highest velocity. Furthermore, such males achieved the highest fertility and the highest siring success, both within-pair and extra-pair. Males homozygous for the derived allele show detrimental sperm characteristics and the lowest siring success. Our results suggest heterozygote advantage as the mechanism that maintains the inversion polymorphism and hence variance in sperm design and in fitness.

The Effect of Leaf Stacking on Leaf Reflectance and Vegetation Indices Measured by Contact Probe during the Season.

The aims of the study were: (i) to compare leaf reflectance in visible (VIS) (400-700 nm), near-infrared (NIR) (740-1140 nm) and short-wave infrared (SWIR) (2000-2400 nm) spectral ranges measured monthly by a contact probe on a single leaf and a stack of five leaves (measurement setup (MS)) of two broadleaved tree species during the vegetative season; and (ii) to test if and how selected vegetation indices differ under these two MS. In VIS, the pigment-related spectral region, the effect of MS on reflectance was negligible. The major influence of MS on reflectance was detected in NIR (up to 25%), the structure-related spectral range; and weaker effect in SWIR, the water-related spectral range. Vegetation indices involving VIS wavelengths were independent of MS while indices combining wavelengths from both VIS and NIR were MS-affected throughout the season. The effect of leaf stacking contributed to weakening the correlation between the leaf chlorophyll content and selected vegetation indices due to a higher leaf mass per area of the leaf sample. The majority of MS-affected indices were better correlated with chlorophyll content in both species in comparison with MS-unaffected indices. Therefore, in terms of monitoring leaf chlorophyll content using the contact probe reflectance measurement, these MS-affected indices should be used with caution, as discussed in the paper. If the vegetation indices are used for assessment of plant physiological status in various times of the vegetative season, then it is essential to take into consideration their possible changes induced by the particular contact probe measurement setup regarding the leaf stacking.

Trade-off between carotenoid-based sexual ornamentation and sperm resistance to oxidative challenge.

It has been hypothesized that carotenoid-based sexual ornamentation signals male fertility and sperm competitive ability as both ornamentation and sperm traits may be co-affected by oxidative stress, resulting in positive covariation (the 'redox-based phenotype-linked fertility hypothesis'; redox-based PLFH). On the other hand, the 'sperm competition theory' (SCT) predicts a trade-off between precopulatory and postcopulatory traits. Here, we manipulate oxidative status (using diquat dibromide) and carotenoid availability in adult zebra finch (Taeniopygia guttata) males in order to test whether carotenoid-based beak ornamentation signals, or is traded off against, sperm resistance to oxidative challenge. Initial beak colouration, but not its change during the experiment, was associated with effect of oxidative challenge on sperm velocity, such that more intense colouration predicted an increase in sperm velocity under control conditions but a decline under oxidative challenge. This suggests a long-term trade-off between ornament expression and sperm resistance to oxidative challenge. Shortening of the sperm midpiece following oxidative challenge further suggests that redox homeostasis may constrain sperm morphometry. Carotenoid supplementation resulted in fewer sperm abnormalities but had no effect on other sperm traits. Overall, our data challenge the redox-based PLFH, partially support the SCT and highlight the importance of carotenoids for normal sperm morphology.

Comparison of Reflectance Measurements Acquired with a Contact Probe and an Integration Sphere: Implications for the Spectral Properties of Vegetation at a Leaf Level.

Laboratory spectroscopy in visible and infrared regions is an important tool for studies dealing with plant ecophysiology and early recognition of plant stress due to changing environmental conditions. Leaf optical properties are typically acquired with a spectroradiometer coupled with an integration sphere (IS) in a laboratory or with a contact probe (CP), which has the advantage of operating flexibility and the provision of repetitive in-situ reflectance measurements. Experiments comparing reflectance spectra measured with different devices and device settings are rarely reported in literature. Thus, in our study we focused on a comparison of spectra collected with two ISs on identical samples ranging from a Spectralon and coloured papers as reference standards to vegetation samples with broadleaved (Nicotiana Rustica L.) and coniferous (Picea abies L. Karst.) leaf types. First, statistical measures such as mean absolute difference, median of differences, standard deviation and paired-sample t-test were applied in order to evaluate differences between collected reflectance values. The possibility of linear transformation between spectra was also tested. Moreover, correlation between normalised differential indexes (NDI) derived for each device and all combinations of wavelengths between 450 nm and 1800 nm were assessed. Finally, relationships between laboratory measured leaf compounds (total chlorophyll, carotenoids and water content), NDI and selected spectral indices often used in remote sensing were studied. The results showed differences between spectra acquired with different devices. While differences were negligible in the case of the Spectralon and they were possible to be modelled with a linear transformation in the case of coloured papers, the spectra collected with the CP and the ISs differed significantly in the case of vegetation samples. Regarding the spectral indices calculated from the reflectance data collected with the three devices, their mean values were in the range of the corresponding standard deviations in the case of broadleaved leaf type. Larger differences in optical leaf properties of spruce needles collected with the CP and ISs are implicated from the different measurement procedure due to needle-like leaf where shoots with spatially oriented needles were measured with the CP and individual needles with the IS. The study shows that a direct comparison between the spectra collected with two devices is not advisable as spectrally dependent offsets may likely exist. We propose that the future studies shall focus on standardisation of measurement procedures so that open access spectral libraries could serve as a reliable input for modelling of optical properties on a leaf level.

Genetic variability and heritability of chlorophyll a fluorescence parameters in Scots pine (Pinus sylvestris L.).

Current knowledge of the genetic mechanisms underlying the inheritance of photosynthetic activity in forest trees is generally limited, yet it is essential both for various practical forestry purposes and for better understanding of broader evolutionary mechanisms. In this study, we investigated genetic variation underlying selected chlorophyll a fluorescence (ChlF) parameters in structured populations of Scots pine (Pinus sylvestris L.) grown on two sites under non-stress conditions. These parameters were derived from the OJIP part of the ChlF kinetics curve and characterize individual parts of primary photosynthetic processes associated, for example, with the exciton trapping by light-harvesting antennae, energy utilization in photosystem II (PSII) reaction centers (RCs) and its transfer further down the photosynthetic electron-transport chain. An additive relationship matrix was estimated based on pedigree reconstruction, utilizing a set of highly polymorphic single sequence repeat markers. Variance decomposition was conducted using the animal genetic evaluation mixed-linear model. The majority of ChlF parameters in the analyzed pine populations showed significant additive genetic variation. Statistically significant heritability estimates were obtained for most ChlF indices, with the exception of DI0/RC, φD0 and φP0 (Fv/Fm) parameters. Estimated heritabilities varied around the value of 0.15 with the maximal value of 0.23 in the ET0/RC parameter, which indicates electron-transport flux from QA to QB per PSII RC. No significant correlation was found between these indices and selected growth traits. Moreover, no genotype × environment interaction (G × E) was detected, i.e., no differences in genotypes' performance between sites. The absence of significant G × E in our study is interesting, given the relatively low heritability found for the majority of parameters analyzed. Therefore, we infer that polygenic variability of these indices is selectively neutral.

Inbreeding depression of sperm traits in the zebra finch Taeniopygia guttata.

Inbreeding depression, or the reduction in fitness due to mating between close relatives, is a key issue in biology today. Inbreeding negatively affects many fitness-related traits, including survival and reproductive success. Despite this, very few studies have quantified the effects of inbreeding on vertebrate gamete traits under controlled breeding conditions using a full-sib mating approach. Here, we provide comprehensive evidence for the negative effect of inbreeding on sperm traits in a bird, the zebra finch Taeniopygia guttata. We compared sperm characteristics of both inbred (pedigree F = 0.25) and outbred (pedigree F = 0) individuals from two captive populations, one domesticated and one recently wild-derived, raised under standardized conditions. As normal spermatozoa morphology did not differ consistently between inbred and outbred individuals, our study confirms the hypothesis that sperm morphology is not particularly susceptible to inbreeding depression. Inbreeding did, however, lead to significantly lower sperm motility and a substantially higher percentage of abnormal spermatozoa in ejaculate. These results were consistent across both study populations, confirming the generality and reliability of our findings.

Murine cytomegalovirus is not restricted to the house mouse Mus musculus domesticus: prevalence and genetic diversity in the European house mouse hybrid zone.

UNLABELLED: Murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus. It is a common infectious agent of wild mice and a highly studied pathogen of the laboratory mouse. Betaherpesviruses are specific to their hosts, and it is not known if other Mus taxa carry MCMV or if it is restricted to M. m. domesticus. We sampled mice over a 145-km transect of Bavaria-Bohemia crossing a hybrid zone between M. m. domesticus and Mus musculus musculus in order to investigate the occurrence of MCMV in two Mus subspecies and to test the limits of the specificity of the virus for its host. We hypothesized that if the two subspecies carry MCMV and if the virus is highly specific to its host, divergent MCMV lineages would have codiverged with their hosts and would have a geographical distribution constrained by the host genetic background. A total of 520 mice were tested by enzyme-linked immunosorbent assay (ELISA) and/or nested PCR targeting the M94 gene. Seropositive and PCR-positive individuals were found in both Mus subspecies. Seroprevalence was high, at 79.4%, but viral DNA was detected in only 41.7% of mice. Sequencing revealed 20 haplotypes clustering in 3 clades that match the host genetic structure in the hybrid zone, showing 1 and 2 MCMV lineages in M. m. domesticus and M. m. musculus, respectively. The estimated time to the most recent common ancestor (1.1 million years ago [Mya]) of the MCMVs matches that of their hosts. In conclusion, MCMV has coevolved with these hosts, suggesting that its diversity in nature may be underappreciated, since other members of the subgenus Mus likely carry different MCMVs. IMPORTANCE: Murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus, an important lab model for human cytomegalovirus (HCMV) infection. The majority of lab studies are based on only two strains of MCMVs isolated from M. m. domesticus, Smith and K181, the latter derived from repeated passage of Smith in mouse submaxillary glands. The presence of MCMV in other members of the Mus subgenus had not even been investigated. By screening mouse samples collected in the European house mouse hybrid zone between M. m. domesticus and M. m. musculus, we show that MCMV is not restricted to the M. m. domesticus subspecies and that MCMVs likely codiverged with their Mus hosts. Thus, the diversity of MCMV in nature may be seriously underappreciated, since other members of the subgenus Mus likely carry their own MCMV lineages.

Sperm morphology in two house mouse subspecies: do wild-derived strains and wild mice tell the same story?

Being subject to intense post-copulatory selection, sperm size is a principal determining component of male fitness. Although previous studies have presented comparative sperm size data at higher taxonomic levels, information on the evolution of sperm size within species is generally lacking. Here, we studied two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus, which undergo incipient speciation. We measured four sperm dimensions from cauda epididymis smears of 28 wild-caught mice of both subspecies. As inbred mouse strains are frequently used as proxies for exploring evolutionary processes, we further studied four wild-derived inbred strains from each subspecies. The subspecies differed significantly in terms of sperm head length and midpiece length, and these differences were consistent for wild mice and wild-derived strains pooled over genomes. When the inbred strains were analyzed individually, however, their strain-specific values were in some cases significantly shifted from subspecies-specific values derived from wild mice. We conclude that: (1) the size of sperm components differ in the two house mouse subspecies studied, and that (2) wild-derived strains reflect this natural polymorphism, serving as a potential tool to identify the genetic variation driving these evolutionary processes. Nevertheless, we suggest that more strains should be used in future experiments to account for natural variation and to avoid confounding results due to reduced variability and/or founder effect in the individual strains.

MtDNA segregation in heteroplasmic tissues is common in vivo and modulated by haplotype differences and developmental stage.

The dynamics by which mitochondrial DNA (mtDNA) evolves within organisms are still poorly understood, despite the fact that inheritance and proliferation of mutated mtDNA cause fatal and incurable diseases. When two mtDNA haplotypes are present in a cell, it is usually assumed that segregation (the proliferation of one haplotype over another) is negligible. We challenge this assumption by showing that segregation depends on the genetic distance between haplotypes. We provide evidence by creating four mouse models containing mtDNA haplotype pairs of varying diversity. We find tissue-specific segregation in all models over a wide range of tissues. Key findings are segregation in postmitotic tissues (important for disease models) and segregation covering all developmental stages from prenatal to old age. We identify four dynamic regimes of mtDNA segregation. Our findings suggest potential complications for therapies in human populations: we propose "haplotype matching" as an approach to avoid these issues.

Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach.

Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use.