Morphological,histochemical and ultrastructural indicators of maize and barley leaf senescence

In this study we report on morphological and histochemical indicators of maize and barley leaf senescence. We determined how the traits such as distribution of stomata and hairs, presence of epicuticular wax, staining of tissues with toluidine blue, change with leaf age and within the leaf blade. We identified regions of young, non-mature leaves as exhibiting juvenile phase, regions with features typical for mature and fully differentiated leaves-as an adult phase and regions with traits of age damage as a senescing phase. Ultrastructural analysis of these regions of leaves gives a clear picture of the time development of the senescence process. Appearance of morphological and histological indicators of senescence in certain regions of leaf is correlated with ultrastructural changes of mesophyll cells of the same regions. We have thus found a relatively simple method of estimating the stage of senescence both in maize and barley.     

Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces

The leaf surface properties of 11 cuticular wax mutants of maize were characterized, and this information was used to identify the quantitative relations among distinct leaf surface traits. Compared with the wild‐type maize, these mutants were reduced 3–24% in their leaf surface hydrophobicity, 20–88% in the mass of cuticular waxes on their leaves, and 52–94% in the percentage of planar leaf surface area covered with epicuticular crystalline waxes. They also differed in the presence and abundance of the epicuticular crystalline waxes in each of seven structural classes. With the exception of one mutant, the mass of cuticular waxes produced by these mutants was positively correlated with the number of epicuticular crystalline waxes per unit area on their leaves. Furthermore, an increase of 0·4 mg of cuticular wax per gram of leaf (dry weight) was associated with a 1% increase in leaf surface area covered by epicuticular crystalline waxes, and this 1% increase was associated with a 2° increase in the contact angle of a water droplet on the leaf surface. Linear differences in the leaf surface hydrophobicity were associated with exponential differences in the mass of the cuticular waxes produced. Quantitative knowledge of these leaf surface properties is highly relevant to the interactions of leaves with environmental factors such as microbes, insects, agricultural chemicals, and pollutants.     

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.     

Epidermal conductance in different parts of durum wheat grown under Mediterranean conditions: the role of epicuticular waxes and stomata

Abstract. Epidermal (non-stomatally-controlled) conductance from the fourth leaf, first node leaf, flag leaf and ear of durum wheat (Triticum turgidum var durum L.) grown under Mediterranean field conditions has been measured, along with leaf stomatal frequency and the amount and distribution of epicuticular waxes. Measurements were carried out on varieties and land-races from the Middle East, North Africa, ‘Institut National de la Recherche Agricole’ (INRA) and ‘Centra Internacional de Mejora de Maiz y Trigo’ (CIMMYT). Significant differences were observed among genotypes in the epidermal conductances (g_e) of the four organs. For each of the four organs tested, genotypes from the Middle East and CIMMYT showed higher g_e. values than those from North Africa and INRA. Ears showed epidermal conductances that were more than four times higher than those of leaves when g_e. values were expressed per unit dry weight. The amount of epicuticular waxes was higher in the fourth leaves, intermediate in the first node and flag leaves and lower in the ears. For each organ, g_e differences among genotypes were unrelated with the amount of epicuticular waxes. Removal of epicuticular waxes by dipping the organs into chloroform significantly increased the epidermal conductance for the fourth and first node leaves and the ear. However, this did not occur for the flag leaf. For the fourth leaf, g_e of intact leaves and g_e of leaves in which epicuticular waxes were removed were unrelated (r = -0.265). The regression coefficient of this relation for the first node and flag leaves showed values of 0.666 and 0.650 (P > 0.05), respectively, and values were even higher in the ear (r > m 0.892, P > 0.01). Scanning electron microscope analysis showed that wax bloom decreased from the fourth leaf to the flag leaf, whereas the extent of amorphous wax increased. Wax bloom in leaves consisted mainly of deposits of thin wax plates. In the ears and the adaxial surface of flag leaves, fibrillar waxes predominated. In the first node and flag leaves, the wax deposits on the adaxial side cover the surface of the leaf more densely and uniformly than those on the abaxial side. There was no significant correlation between g_e and total stomatal density, or between g_e and either adaxial or abaxial stomatal density for any sample of the three different leaves. The contribution of epicuticular waxes plus total stomatal frequency only explained 42.4, 11.8, 28.3 and 16% of g_e (per unit leaf area) variations for the fourth leaf, first node leaf, flag leaf and the combined variation of the three leaves together, respectively. From these results, it is concluded that complex interrelationship between different morphophysiological characteristics probably control g_e differences among genotypes and that these interrelationships differ for each different plant part.     

Cabbage waxes affect Trissolcus brochymenae response to short‐range synomones

We show that induced synomones, emitted as a consequence of Murgantia histrionica activity on Brassica oleracea, are adsorbed by the epicuticular waxes of leaves and perceived by the egg parasitoid Trissolcus brochymenae. Leaves were exposed to M. histrionica females placed on the abaxial leaf surface. After 24 h, the leaves were treated mechanically using gum arabic, or chemically using chloroform, on the adaxial surface, and finally the adaxial surface was assayed with T. brochymenae by two‐choice tests in a closed arena. Wasp females responded to mechanically dewaxed cabbage leaf portions with feeding punctures and footprints (Ff) and with feeding punctures, oviposition and footprints (FOf), showing no effect of wax removal. In contrast, the removal of the epicuticular waxes from leaf portions close to FOf, and from leaves with oviposition and footprints (Of), determined the lack of responses by T. brochymenae. Solvent extracts of different treatments were bioassayed, but only FOf triggered parasitoid response. Thus the detection of oviposition‐induced synomones by the parasitoid depends on their adsorption by the epicuticular waxes. Mechanical wax removal from leaf portions contaminated with host footprints (f) also determined a lack of wasp responses, suggesting that the footprints might trigger the induction of a “footprint‐induced synomone” adsorbed onto the epicuticular waxes and exploited by the parasitoid. Leaf portions with the abaxial lamina previously dewaxed and then contaminated by footprints (D+f) of M. histrionica did not affect the parasitoid response, indicating that the abaxial epicuticular waxes are not directly involved in the chemicals induced by M. histrionica footprints.     

Influence of polishing and abrasion on the diffusive conductance of leaf surface of Festuca arundinacea Schreb.

Abstract Leaf surfaces of Festuca arundinacea Schreb. were subjected to controlled polishing and abrasion on a test-bed designed to simulate components of abrasive wind damage. Both treatments substantially increased the leaf surface conductance, particularly the polishing treatment. Scanning electron micrographs of cryo-fixed leaf surfaces showed displacement and smoothing of the epicuticular waxes, damage to the cuticle, collapse of epidermal cells and fracture of trichomes. The importance of the epicuticular waxes in determining leaf diffusivity and permeability is discussed.     

The leaf internal morphology and ultrastructure of Zostera muelleri irmisch ex aschers. (Zosteraceae): a comparative study of the intertidal and subtidal forms

The leaf anatomy, histochemistry and ultrastructure of the intertidal and subtidal seagrass Zostera muelleri Irmish ex Aschers. from Westernport Bay, Victoria were studied. Unusual anatomical and ultrastructural features are compared with other seagrasses and their functional significance is assessed. Subcuticular cavities are present in the young blade, but not observed in the older blade nor young and old leaf sheath. Wall ingrowths occur in the blade epidermal cells particularly on the inner tangential walls and the lower portions of the radial walls. Plasmodesmata are present between adjacent epidermal cells and between the epidermal and mesophyll cells, suggesting that solutes could transfer between these tissues both symplastically and apoplastically. Each leaf has three longitudinally aligned vascular bundles, each of which comprises a single xylem element isolated from the phloem tissue. The phloem consists of nacreous-walled sieve elements accompanied by phloem parenchyma cells which also process wall ingrowths. The xylem walls are completely hydrolysed and the middle lamella borders directly on the xylem lumen. Leaves have prominent air lacunae bisected transversely by septa at regular intervals along their length. Each septum consists of a file of small parenchyma cells with wall protuberances projecting into intercellular space. There are no major structural differences between the subtidal and intertidal plants, but the former have larger leaves and more leaves per shoot than the latter. In addition, a network of unusual reticulated fungal hyphae is present in the leaf intercellular spaces of the subtidal form and this network may facilitate solute transfer in these plants.     

Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

Guard cells emit an alkali-induced, blue fluorescence upon excitationby ultraviolet radiation (emission maximum energy at 365 nm).Fluorescence emission of guard cells was brighter than thatof the neighbouring epidermal cells in a number of wild andcultivated plants including conifers, but the relative fluorescenceintensity and quality was species-dependent. Three representativeplants possessing stomatal complexes which differed morphologicallywere studied: Olea europaea, Vicia faba and Triticum aestivum.Immersing leaves of these plants in chloroform for 30 s (therebyremoving epicuticular waxes) significantly reduced the intensityof the fluorescence emitted by guard cells. This indicates thatguard cell fluorescence could be due to either an increasedconcentration of fluorescing compounds (probably wax-bound phenolics),or a thicker cuticular layer covering the guard cells. Giventhat the alkali-induced blue fluorescence of the guard cellsis a common characteristic of all plants examined, it couldbe used as a rapid and convenient method for in situ measurementsof the number, distribution and size of stomatal complexes.The proposed experimental procedure includes a single coatingof the leaf surface by, or immersion of the whole leaf in, a10% solution of KOH for 2 min, washing with distilled water,and direct observation of the leaf surface under the fluorescencemicroscope. Fluorescence images were suitable for digital imageanalysis and methodology was developed for stomatal countingusing Olea europaea as a model species. Copyright 2001 Annalsof Botany Company Cuticle, epicuticular waxes, fluorescence microscopy, image analysis, phenolics, stomata     

Stomatal behaviour and cuticular properties of maize leaves of different chilling-resistance during cold treatment

Stomatal behaviour and the amount and synthesis of epicuticular waxes were studied in maize leaves of various degrees of chilling resistance in order to investigate the rapid desiccation characteristic of chilling injury. In contrast to the resistant variety (KSC 360), the sensitive line (Lye) failed to close the stomata during chilling treatment. Both, the amount of epicuticular waxes and their degree of labeling by 1-14C-acetate were much higher in the resistant than in the sensitive leaves. Chilling-sensitivity was also related to a suppressed conversion of free fatty acids into free primary alcohols.     

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.     

Epicuticular wax crystals of Wollemia nobilis: morphology and chemical composition

BACKGROUND AND AIMS: The morphology of the epicuticular leaf waxes of Wollemia nobilis (Araucariaceae) was studied with special emphasis on the relationship between the microstructure of epicuticular wax crystals and their chemical composition. Wollemia nobilis is a unique coniferous tree of the family Araucariaceae and is of very high scientific value as it is the sole living representative of an ancient genus, which until 1994 was known only from fossils. METHODS: Scanning electron microscopy (SEM), gas chromatography (GC) combined with mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) were used for characterizing the morphology and the chemical structure of the epicuticular wax layer of W. nobilis needles. KEY RESULTS: The main component of the leaf epicuticular wax of W. nobilis is nonacosan-10-ol. This secondary alcohol together with nonacosane diols is responsible for the tubular habit of the epicuticular wax crystals. Scanning electron micrographs revealed differences in the fine structure of adaxial and abaxial leaf surfaces that could be explained by gas chromatographic studies after selective mechanical removal of the waxes. CONCLUSIONS: SEM investigations established the tubular crystalline microstructure of the epicuticular wax of W. nobilis leaves. GC-MS and NMR experiments showed that nonacosan-10-ol is the major constituent of the epicuticular wax of W. nobilis leaves.     

Water-stressed maize, barley and rice seedlings show species diversity in mechanisms of leaf growth inhibition

The possible occurrence of species diversity in mechanisms underlying leaf-growth inhibition by water stress, was investigated in related cereal plants. Water stress was generated by additions of the osmoticum polyethylene glycol 6000 to the root medium. Effects of external water potentials ranging from 0 to -0.6MPa, on early growth parameters of emerging leaves were measured under controlled environment conditions, using pairs of maize, barley or rice genotypes with differing resistance to water stress under field conditions. Water potentials of -0.4 MPa for 24 h, similarly reduced leaf growth, comparative production rates of leaf epidermal cells and cell size in all genotypes. These reductions did not appear to be caused by reductions in the osmotic potential gradients between the expanding leaf cells and their external water source. However, growth inhibition in maize and barley, was accompanied by significant reductions in comparative leaf and cell wall extensibility. Moreover, regression plots revealed good linear correlations (r=0.83** for maize and r=0.77** for barley) between the reductions in leaf growth induced by a series of water potentials and associated reductions in leaf extensibility. In contrast, the reduction in growth of rice leaves, was not accompanied by any significant changes in leaf or cell wall extensibility. Similarly, regression plots revealed poor correlations between leaf growth and leaf extensibility in both paddy and upland rice (r=0.17 and r=0.07, respectively). Thus, despite numerous inter-species similarities, biophysical changes associated with stress-induced leaf growth inhibition in maize and barley, differed from those in rice.Key words: Cell walls, extensibility, water stress, cereal diversity, leaf growth.      

Glossy15 Controls the Epidermal Juvenile-to-Adult Phase Transition in Maize

Loss-of-function mutations at the maize Glossy15 (Gl15) locus alter the normal transition from juvenile-to-adult growth by conditioning the abbreviated expression of juvenile epidermal cell traits and the coordinate precocious expression of adult epidermal cell features. These include epicuticular wax composition, cell wall characteristics, and the presence or absence of differentiated epidermal cell types (e.g., epidermal macrohairs and bulliform cells). A transposon-induced mutable allele of Glossy15 (gl15-m1) was isolated and employed in both phenotypic and genetic analyses to characterize the role of Gl15 in the maize juvenile-to-adult phase transition. Comparisons between Gl15-active and Gl15-inactive somatic sectors in the leaves of variegated plants demonstrated that the Gl15 gene product acts in a cell-autonomous manner to direct juvenile epidermal differentiation but does not affect factors that regulate the overall process of phase change. Examination of the gl15-m1 phenotype in the Corngrass1, Teopod1, and Teopod2 mutant backgrounds showed that the prolonged expression of juvenile epidermal traits associated with these mutations also required Gl15 activity. These results support a model whereby the cell-autonomous Gl15 gene product responds to a juvenility program that operates throughout the vegetative shoot to condition the juvenile differentiation of maize leaf epidermal cells.     

Effect of Salinity on the Composition, Number and Size of Epidermal Cells along the Mature Blade of Wheat Leaves

Salinity inhibits leaf growth in association with changes in cell size. The objective of this study was to determine the spatial distributions of the composition, number and dimensions of epidermal cells in the mature blades of leaf four of wheat seedlings under saline conditions. Plants were grown in loamy soil either with or without 120 mmol/L NaCl in a growth chamber, and harvested after leaf four was fully developed. The results of the spatial distribution analyses of width along the blade showed that salinity not only reduced the width of the leaf blade, but that it also altered the distribution pattern of blade width along the leaf axis. The reduction in the final size of the leaf blade was associated with a reduction in the total number of epidermal cells and in their widths and lengths. This study also revealed the spatial effects of salinity on the blade and epidermal cell dimensions along the leaf axis. In particular, salinity inhibited the total cell number for interstomatal, sister and elongated cells, implying that cell division in wheat leaves is inhibited by salinity. However, the lengths of interstomatal cells were not affected by salinity （unlike those for the sister and elongated cells）, suggesting the relative contributions of cell length and numbers to the reduction in the final length of the blade under salinity is dependent on cell type.     

Abnormal Stomatal Behaviour and Leaf Anatomy in Capsicum annuum, scabrous diminutive, a Wilty Mutant of Pepper

An attempt is made to explain the tendency to excessive wiltingin scabrous diminutive, a pepper mutant. For this, mutant andnormal plants were compared with respect to leaf anatomy, transpirationof whole plants and detached drying leaves, density and openingof stomata, staining of potassium in epidermal cells and rootpressure. A much greater proportion of intracellular space was found inthe mutant leaf which contains fewer and smaller mesophyll cellsthan the normal plant. The anticlinal walls of the epidermisof the mutant leaf were almost straight whereas those of thenormal were wavy. Transpiration per unit leaf area of wholeplants, percentage of stomata open both day and night, and waterloss from detached drying leaves were all higher in the mutant.Potassium staining in guard cells was similar in both planttypes, slightly less in darkness and marginally higher in light.The subsidiary cells of normal leaves did not stain at all,but those of the mutant leaves stained heavily in both lightand darkness. Root pressure was lower in the mutant. Possible explanations for the tendency of the pepper mutantto wilt are discussed.     

Fused organs in the adherent1 mutation in maize show altered epidermal walls with no perturbations in tissue identities

In the absence of wounding, the epidermis is only rarely involved in cell or organ fusion events; in fact, intact epidermal layers prevent graft unions. In Zea mays L. the mutation adherent1 (ad1) shows abnormal fusions between cells and organs. Fusions involve epidermal cells of vegetative and floral organs and occur early in the ontogeny of organs. Even so, epidermal cell types differentiate normally in the fused regions and internal tissue identities are maintained. In contrast, the extracellular matrix (cell wall and cuticle) of the epidermal cells is perturbed. Epidermal cell walls in adherent leaves are thicker than normal. Epicuticular wax particles appear reduced in size and number and altered in shape in mutant leaves. In addition, the outer epidermal cell walls of adherent leaves fluoresce when stained with aniline blue, a reagent that binds to callose. Immunolocalizations to specific cell wall epitopes suggest that pectins but not arabinogalactans may have a role in the fusion events. Taken together, these results suggest that the ad1 mutation results in cell-wall and epicuticular-wax defects similar to responses seen in wounding, pollination by incompatible pollen, or pathogen attack. Since cell wall components and epicuticular waxes are extracellular secreted products, the ad1 mutation may disrupt normal functioning and/or composition of the secretory pathway and its cargo. Received: 30 January 1998 / Accepted: 5 March 1998     

STRUCTURE OF THE PEAR LEAF CUTICLE WITH SPECIAL REFERENCE TO CUTICULAR PENETRATION

Study of the pear leaf cuticle (Pyrus communis L. ‘Bartlett‘), in both intact and enzymatically isolated forms, has revealed that the cuticular membrane is separated from the underlying epidermal cell wall by a layer of pectic substances which extend into but not through the membrane. A layer of embedded birefringent waxes occurs towards the outer surface of the cuticular membrane. Platelet-like epicuticular waxes are deposited on the outer surface. The upper cuticular membrane is astomatous. The lower epidermis is stomatous, and the outer cuticular membrane is continuous with that lining the substomatal cavity. The lower cuticular membrane is also generally thicker than the upper, and both the upper and lower cuticular membranes are thicker over veinal than over mesophyll tissue. The birefringence frequently is discontinuous over anticlinal walls and over veinal tissue. The lower cuticle appears to contain fewer embedded waxes (as indexed by birefringence) than the upper. Enzymatic isolation of the cuticular membrane from the underlying tissues does not appear to cause any discernible change in structure as viewed with a light microscope. These findings are discussed in light of current knowledge concerning penetration of foliar applied substances into the leaf.     

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.     

Chemical composition of Tipuana tipu,a source for tropical honey bee products

Tipuana tipu (Benth.) Kuntze is a tree from the leguminosae family (Papilionoideae) indigenous in Argentina and extensively used in urbanism, mainly in Southern Brazil. The epicuticular waxes of leaves and branch, and flower surface were studied by high temperature high resolution gas chromatography. Several compounds were characterized, among which the aliphatic alcohols were predominant in branch, leaves and receptacle. Alkanes were predominant only in the petals and the aliphatic acids were predominant in stamen. In branches and leaf epicuticular surfaces, six long chain wax esters series were characterized, as well as lupeol and b-amyrin hexadecanoates.     

Relationship between selected morphological, anatomical and cytological characteristics of leaves and the level of tolerance to herbicides in strawberry cultivars

Foliar application of a mixture of herbicides containing phenmedipham, desmedipham and ethofumesate to the plants of nine strawberry cultivars revealed that there were differences in the level of plant tolerance to the applied chemicals. Light, polarized light and scanning electron microscopy were used to explain differences in tolerance to herbicides. The surface of strawberry leaves and cells was examined for stomata, hairs, trichomes, surface structures, cucticle, vacuole and oxalate crystals. The thicker the cuticle on the adaxial leaf surface, the thicker the layer of epicuticular waxes, greater number of large vacuoles and greater number of calcium oxalate crystals in epidermis cells were characteristic for cultivars with very good tolerance to herbicides. The cracking of epicuticular waxes layer was typical to cultivars with respectively low tolerance to herbicides.