Visualisation of calcium oxalate crystal macropatterns in plant leaves using an improved fast preparation method.

Leaves of the majority of plants contain calcium oxalate (CaOx) crystals or druses which often occur in spectacular distribution patterns. Numerous studies on CaOx in plant tissues across many different plant groups have been published, since it can be visualised readily under a light microscope (LM). However, there is surprisingly limited knowledge on the actual, precise distribution of CaOx in the leaves of quite ordinary plants such as common native and exotic trees. Traditional sample preparation for the documentation of the distribution of CaOx crystals in a given sample - including overall distribution - requires time-consuming clearing procedures. Here we present a refined fast preparation method to visualise the overall CaOx complement in a sample: The plant material is ashed and the ash viewed under the polarising microscope. This is a rapid method which overcomes many shortcomings of other methods and permits the visualisation of the entire CaOx content in most leaf samples. Pros and cons in comparison with the conventional clearing technique are discussed. Further aspects for CaOx investigations by micro-CT and scanning electron microscopy are discussed.

Traces of calcium oxalate biomineralization in fossil leaves from late Oligocene maar deposits from Germany.

Calcium oxalate (CaOx) is one of the most common bio-mineral in extant plants and is believed to serve a variety of functions such as calcium storage and herbivore defense. However, traces of CaOx crystals have rarely been identified in fossil plants, and they are primarily known from fossil gymnosperms, where empty cavities of former CaOx crystals or ghost crystals have been reported from leaf cuticles of some Late Cretaceous and Cenozoic conifers. Here we investigate fossil angiosperm leaves from the late Oligocene Rott Fossil Lagerstätte and report ghost crystals of various shapes, sizes and topology (distribution patterns), and cavities. These micromorphological structures of fossil leaves are compared to CaOx deposits in leaves of extant plants: globular structures in fossil leaves resemble CaOx druses (crystal aggregates) in fresh leaves in size and distribution; and angular or brick-shaped structures in the vascular system of fossil leaves closely resemble prismatic CaOx crystals in the vascular system of extant leaves in both size and topology. Chemically, CaOx druses have survived fossilization as cavities only, and were replaced by organic matter and ghost minerals containing Ca, Si, Al, S, and Fe. The identification of former CaOx remains in leaf fossils provides novel insights on the fate of plant bio-minerals during fossilization. More importantly, it provides an additional aspect of the ecophysiology of fossil plants thus improving the accuracy of palaeoecological reconstructions and can provide a broader perspective on the evolution of CaOx and their rule in plant ecology across geological timescales. Alternative interpretations of the fossil microstructures are discussed but ruled out.

Distribution of Biominerals and Mineral-Organic Composites in Plant Trichomes.

Biomineralization is a common phenomenon in plants and has been shown to be chemically, functionally and topologically diverse. Silica and calcium carbonate have long been known as structural plant biominerals and calcium phosphate (apatite)-long known from animals-has recently been reported. Strikingly, up to three different biominerals may occur in a single trichome in, e.g., Urticaceae and Loasaceae, and in combination with organic compounds, can form organic/inorganic composite materials. This article presents an extension of previous studies on the distribution of these biominerals in Loasaceae trichomes with a focus on their spatial (three-dimensional) distribution and co-localization with organic substances. Light microscopy and scanning electron microscopy with high-resolution EDX element analyses of sample surfaces and sections illustrate the differential distribution and composition of the different biomineral phases across cell surfaces and cell walls. Raman spectroscopy additionally permits the identification of organic and inorganic compounds side by side. All three biominerals may be found in a nearly pure inorganic phase, e.g., on the plant surfaces and in the barbs of the glochidiate trichomes, or in combination with a larger proportion of organic compounds (cellulose, pectin). The cell lumen may be additionally filled with amorphous mineral deposits. Water-solubility of the mineral fractions differs considerably. Plant trichomes provide an exciting model system for biomineralization and enable the in-vivo study of the formation of complex composite materials with different biomineral and organic compounds involved.

Distribution, Ecology, Chemistry and Toxicology of Plant Stinging Hairs.

Plant stinging hairs have fascinated humans for time immemorial. True stinging hairs are highly specialized plant structures that are able to inject a physiologically active liquid into the skin and can be differentiated from irritant hairs (causing mechanical damage only). Stinging hairs can be classified into two basic types: Urtica-type stinging hairs with the classical "hypodermic syringe" mechanism expelling only liquid, and Tragia-type stinging hairs expelling a liquid together with a sharp crystal. In total, there are some 650 plant species with stinging hairs across five remotely related plant families (i.e., belonging to different plant orders). The family Urticaceae (order Rosales) includes a total of ca. 150 stinging representatives, amongst them the well-known stinging nettles (genus Urtica). There are also some 200 stinging species in Loasaceae (order Cornales), ca. 250 stinging species in Euphorbiaceae (order Malphigiales), a handful of species in Namaceae (order Boraginales), and one in Caricaceae (order Brassicales). Stinging hairs are commonly found on most aerial parts of the plants, especially the stem and leaves, but sometimes also on flowers and fruits. The ecological role of stinging hairs in plants seems to be essentially defense against mammalian herbivores, while they appear to be essentially inefficient against invertebrate pests. Stinging plants are therefore frequent pasture weeds across different taxa and geographical zones. Stinging hairs are usually combined with additional chemical and/or mechanical defenses in plants and are not a standalone mechanism. The physiological effects of stinging hairs on humans vary widely between stinging plants and range from a slight itch, skin rash (urticaria), and oedema to sharp pain and even serious neurological disorders such as neuropathy. Numerous studies have attempted to elucidate the chemical basis of the physiological effects. Since the middle of the 20th century, neurotransmitters (acetylcholine, histamine, serotonin) have been repeatedly detected in stinging hairs of Urticaceae, but recent analyses of Loasaceae stinging hair fluids revealed high variability in their composition and content of neurotransmitters. These substances can explain some of the physiological effects of stinging hairs, but fail to completely explain neuropathic effects, pointing to some yet unidentified neurotoxin. Inorganic ions (e.g., potassium) are detected in stinging hairs and could have synergistic effects. Very recently, ultrastable miniproteins dubbed "gympietides" have been reported from two species of Dendrocnide, arguably the most violently stinging plant. Gympietides are shown to be highly neurotoxic, providing a convincing explanation for Dendrocnide toxicity. For the roughly 648 remaining stinging plant species, similarly convincing data on toxicity are still lacking.

Trichome Biomineralization and Soil Chemistry in Brassicaceae from Mediterranean Ultramafic and Calcareous Soils.

Trichome biomineralization is widespread in plants but detailed chemical patterns and a possible influence of soil chemistry are poorly known. We explored this issue by investigating trichome biomineralization in 36 species of Mediterranean Brassicaceae from ultramafic and calcareous soils. Our aims were to chemically characterize biomineralization of different taxa, including metallophytes, under natural conditions and to investigate whether divergent Ca, Mg, Si and P-levels in the soil are reflected in trichome biomineralization and whether the elevated heavy metal concentrations lead to their integration into the mineralized cell walls. Forty-two samples were collected in the wild while a total of 6 taxa were brought into cultivation and grown in ultramafic, calcareous and standard potting soils in order to investigate an effect of soil composition on biomineralization. The sampling included numerous known hyperaccumulators of Ni. EDX microanalysis showed CaCO3 to be the dominant biomineral, often associated with considerable proportions of Mg-independent of soil type and wild versus cultivated samples. Across 6 of the 9 genera studied, trichome tips were mineralized with calcium phosphate, in Bornmuellera emarginata the P to Ca-ratio was close to that of pure apatite-calcium phosphate (Ca5(PO4)3OH). A few samples also showed biomineralization with Si, either only at the trichome tips or all over the trichome. Additionally, we found traces of Mn co-localized with calcium phosphate in Bornmuellera emarginata and traces of Ni were detected in trichomes of the Ni-hyperaccumulator Odontarrhena chalcidica. Our data from wild and cultivated plants could not confirm any major effect of soil chemistry on the chemistry of trichome biominerals. Hyperaccumulation of Ni in the plants is not mirrored in high levels of Ni in the trichomes, nor do we find large amounts of Mn. A comparison based on plants from cultivation (normal, calcareous and serpentine soils, Mg:Ca-ratios ca 1:2 to 1:20) shows at best a very weak reflection of different Mg:Ca-ratios in the mineralized trichomes. The plants studied seem to be able to maintain highly conserved biomineralization patterns across a wide range of soil chemistries.

Ethylenediurea (EDU) mitigates the negative effects of ozone in rice: Insights into its mode of action.

Monitoring of ozone damage to crops plays an increasingly important role for the food security of many developing countries. Ethylenediurea (EDU) could be a tool to assess ozone damage to vegetation on field scale, but its physiological mode of action remains unclear. This study investigated mechanisms underlying the ozone-protection effect of EDU in controlled chamber experiments. Ozone sensitive and tolerant rice genotypes were exposed to ozone (108 ppb, 7 hr day-1 ) and control conditions. EDU alleviated ozone effects on plant morphology, foliar symptoms, lipid peroxidation, and photosynthetic parameters in sensitive genotypes. Transcriptome profiling by RNA sequencing revealed that thousands of genes responded to ozone in a sensitive variety, but almost none responded to EDU. Significant interactions between ozone and EDU application occurred mostly in ozone responsive genes, in which up-regulation was mitigated by EDU application. Further experiments documented ozone degrading properties of EDU, as well as EDU deposits on leaf surfaces possibly related to surface protection. EDU application did not mitigate the reaction of plants to other abiotic stresses, including iron toxicity, zinc deficiency, and salinity. This study provided evidence that EDU is a surface protectant that specifically mitigates ozone stress without interfering directly with the plants' stress response systems.

Stinging hair morphology and wall biomineralization across five plant families: Conserved morphology versus divergent cell wall composition.

PREMISE OF THE STUDY: Stinging hairs are striking examples of plant microengineering-the plant equivalent of the hypodermic syringe. The requisite mechanical properties are mostly achieved by cell wall mineralization. Stinging hairs of Urtica dioica (Urticaceae) are known to be mineralized with silica and calcium carbonate and those of Loasaceae also with calcium phosphate, but no comparative study has been provided across different taxa with stinging hairs. METHODS: Light microscopy and scanning electron microscopy (SEM) with cryo-SEM and energy-dispersive x-ray spectroscopy were used to analyze morphology and biomineralization of stinging hairs of 43 species from the families Caricaceae, Euphorbiaceae, Loasaceae, Namaceae, and Urticaceae. KEY RESULTS: Stinging hair morphology is similar across the taxa studied, in striking contrast to the divergent patterns of biomineralization. Trichome bases are mostly calcified, sometimes silicified, the shafts are mostly calcified, and the apices silicified (Urticaceae), and contain calcium phosphate (Caricaceae, Namaceae), both silica and calcium phosphate (some Loasaceae), or no minerals (Cnidoscolus, Euphorbiaceae). Some stinging hairs are superficially thinly coated with silica over a cell wall otherwise mineralized with calcium carbonate or calcium phosphate. CONCLUSIONS: Mineralization patterns are surprisingly diverse and involve three different biominerals deposited in different parts of individual trichomes with calcium phosphate a common component. The physical properties of different wall regions of the stinging trichomes are thus fine-tuned to optimize their function via modulation of wall thickness and differential element deposition. Similar function is apparently achieved through divergent wall compositions.

A new blind snake of the genus Letheobia (Serpentes: Typhlopidae) from Rwanda with redescriptions of L. gracilis (Sternfeld, 1910) and L. graueri (Sternfeld, 1912) and the introduction of a non-invasive preparation procedure for scanning electron microscopy in zoology.

A new species of blind snake in the genus Letheobia is described from Akagera National Park in eastern Rwanda. The new species is most similar to species of the L. gracilis complex, particularly L. gracilis and L. graueri. It differs from all other species of the genus by a unique combination of morphological characters, including the highest number of middorsal scale rows (834) and the most extreme elongation (total-length/midbody-width ratio 131) of all species in the genus and of any species of snake in the world; 22-22-22 longitudinal scale rows; snout in dorsal profile rounded, in lateral profile bluntly rounded with an angular horizontal edge ventrally; rostral broad, posteriorly rounded; eyes invisible; supralabial imbrication pattern T-0; tail short (1.3 percent of total length) with an apical spine; and a pink life colouration. The holotype of the new species was collected in gallery forest at a lake shore surrounded by savanna at 1300 m elevation. We produced scanning electron microscope images of the heads of the investigated specimens applying a liquid-substitution preparation procedure which does not require coating or drying and thus does not irreversibly damage the investigated samples. The obtained images allow an easy and more accurate examination of the scalation.

Mineralized trichomes in Boraginales: complex microscale heterogeneity and simple phylogenetic patterns.

Background and Aims: Boraginales are often characterized by a dense cover of stiff, mineralized trichomes, which may act as a first line of defence against herbivores. Recent studies have demonstrated that the widely reported silica and calcium carbonate in plant trichomes may be replaced by calcium phosphate. The present study investigates mineralization patterns in 42 species from nine families of the order Boraginales to investigate detailed patterns of mineralization and the possible presence of a phylogenetic signal in different mineralization patterns. Methods: The distribution of biominerals was analysed by scanning electron microscopy (SEM) including cryo-SEM and energy-dispersive X-ray analyses with element mapping. The observed distribution of biominerals was plotted onto a published phylogeny of the Boraginales. Three colours were selected to represent the principal elements: Si (red), Ca (green) and P (blue). Key Results: Calcium carbonate was present in the mineralized trichomes of all 42 species investigated, silica in 30 and calcium phosphate in 25; multiple mineralization with calcium carbonate and silica or calcium phosphate was found in all species, and 13 of the species were mineralized with all three biominerals. Trichome tips featured the most regular pattern - nearly all were exclusively mineralized with either silica or calcium phosphate. Biomineralization of the trichome shafts and bases was found to be more variable between species. However, the trichome bases were also frequently mineralized with calcium phosphate or silica, indicating that not only the tip is under functional constraints requiring specific patterns of chemical heterogeneity. The complete absence of either silica or phosphate may be an additional feature with systematic relevance. Conclusions: This study demonstrates that complex, site-specific and differential biomineralization is widespread across the order Boraginales. Calcium phosphate, only recently first reported as a structural plant biomineral, is common and appears to be functionally analogous to silica. A comparison with the phylogeny of Boraginales additionally reveals striking phylogenetic patterns. Most families show characteristic patterns of biomineralization, such as the virtual absence of calcium phosphate in Cordiaceae and Boraginaceae, the triple biomineralization of Heliotropiaceae and Ehretiaceae, or the absence of silica in Namaceae and Codonaceae. The complex chemical and phylogenetic patterns indicate that trichome evolution and functionalities are anything but simple and follow complex functional and phylogenetic constraints.

Calcium phosphate in plant trichomes: the overlooked biomineral.

MAIN CONCLUSION: Calcium phosphate was unknown as a plant biomineral until recently reported in Neotropical Loasaceae. Here, we demonstrate its widespread occurrence in the trichomes of several plant families, including Brassicaceae. Calcium phosphate is the primary biomineral in, e.g., the bones and teeth of higher animals; in plants, it was only recently discovered in the stinging hairs and scabrid-glochidiate trichomes of South American Loasaceae (Ensikat et al. in Sci Rep UK 6:26073, 2016), where it appears to be deposited highly specifically, often replacing the common plant biomineral silica. We initiated a broader survey in a range of different plant orders to investigate a possibly wider distribution of calcium phosphate biomineralization in plants. Scanning electron microscopy with EDX element analysis and mapping was used for the detection of the biominerals: calcium phosphate, calcium carbonate, and silica in the trichomes of several common plant species of different orders. Results were authenticated with Raman spectroscopy. Calcium phosphate was found in the trichomes of several species in the orders Malpighiales, Rosales, Boraginales, and Brassicales. It occurred in trichome tips, replacing the more common silica, or together with silica and calcium carbonate at specific locations in the trichome cell walls. Most surprisingly, it was found in the trichomes of Arabidopsis thaliana, one of the most studied plant species-where it had been overlooked so far. The wide distribution of calcium phosphate as plant biomineral here demonstrated and the striking mineralization patterns with three different biominerals in the walls of single-celled trichomes underscore an unexpected complexity in plant biomineralization.

Ontogeny and the process of biomineralization in the trichomes of Loasaceae.

PREMISE OF THE STUDY: South American Loasaceae have a morphologically complex trichome cover, which is characterized by multiple biomineralization. The current study investigates the ontogeny of these complex trichomes and the process of their biomineralization, since both are very poorly understood. METHODS: The development of stinging trichomes on various parts of the plants and the process of mineralization were studied using scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDX). KEY RESULTS: Trichomes are initiated very early in organ development and the different trichome types begin developing their distinctive morphology at a very early developmental stage. Biomineralization in the stinging trichomes starts with the deposition of silica or calcium phosphate in the apex and then proceeds basipetally, with a more irregular, subsimultaneous mineralization of the base and the shaft. Mineralization of the scabrid-glochidiate trichomes starts on the surface processes and in the apex (silica, calcium phosphate), with a subsequent mineralization of the shaft with calcium carbonate. CONCLUSION: Mineralized trichomes in Loasaceae provide an excellent model for the study of biomineralization. The overall sequence of mineralization is typically from distal and peripheral to proximal and central. Typically, three biominerals-silica, calcium carbonate, and calcium phosphate-are differentially and sequentially deposited in different parts of each unicellular stinging trichome.

Complex patterns of multiple biomineralization in single-celled plant trichomes of the Loasaceae.

PREMISE OF THE STUDY: Plants of the family Loasaceae are characterized by a usually dense indument of various trichome types, including two basically different types of mineralized, unicellular trichomes (stinging hairs or setae and scabrid-glochidiate trichomes). Mineralized trichomes have long been known to have silicified or calcified walls, but recent studies demonstrated that trichomes of Loasaceae may also contain calcium phosphate. The current study investigates the distribution of different biominerals in the mineralized trichomes across several different taxa. METHODS: Plants from cultivation were studied with scanning electron microscopy including energy dispersive x-ray analyses and element mapping. KEY RESULTS: The vast majority of the 31 species investigated had at least two different biominerals in their trichomes, and 22 had three different biominerals in their trichomes. Thirty of the species had calcium phosphate in their trichomes. Loasa was mostly free of silica, but contained calcium phosphate in trichome tips and barbs, whereas calcium phosphate and silica were found in representatives of other genera of the family (Blumenbachia, Caiophora, Nasa). CONCLUSIONS: Biomineralization is remarkably diversified between species, different trichome types and parts of the same trichome. Individual genera largely had different patterns of biomineralization. The presence of three biominerals in the trichomes of the basally branching Eucnide urens indicates either an early evolution and subsequent loss or several independent origins of multiple biomineralization. Differential biomineralization of the parts of individual, unicellular trichomes clearly indicates an extraordinary degree of physiological control over this process.

A first report of hydroxylated apatite as structural biomineral in Loasaceae - plants' teeth against herbivores.

Biomineralization provides living organisms with various materials for the formation of resilient structures. Calcium phosphate is the main component of teeth and bones in vertebrates, whereas especially silica serves for the protection against herbivores on many plant surfaces. Functional calcium phosphate structures are well-known from the animal kingdom, but had not so far been reported from higher plants. Here, we document the occurrence of calcium phosphate biomineralization in the South-American plant group Loasaceae (rock nettle family), which have stinging trichomes similar to those of the well-known stinging nettles (Urtica). Stinging hairs and the smaller, glochidiate trichomes contained nanocrystalline hydroxylated apatite, especially in their distal portions, replacing the silica found in analogous structures of other flowering plants. This could be demonstrated by chemical, spectroscopic, and diffraction analyses. Some species of Loasaceae contained both calcium phosphate and silica in addition to calcium carbonate. The intriguing discovery of structural hydroxylated apatite in plants invites further studies, e.g., on its systematic distribution across the family, the genetic and cellular control of plant biomineralization, the properties and ultrastructure of calcium phosphate. It may prove the starting point for the development of biomimetic calcium phosphate composites based on a cellulose matrix.

Fossil nutlets of Boraginaceae from the continental Eocene of Hamada of Méridja (southwestern Algeria): The first fossil of the Borage family in Africa.

PREMISE OF THE STUDY: The Paleogene deposits of the Hamada of Méridja, southwestern Algeria, are currently dated as lower-to-middle Eocene in age based on fossil gastropods and charophytes. Here we report the presence of fruits that can be assigned to the Boraginaceae s.str., apparently representing the first fossil record for this family in Africa, shedding new light on the historical biogeography of this group. METHODS: Microscopic studies of the fossil nutlets were carried out and compared to extant Boraginaceae nutlets, and to types reported in the literature for this family. KEY RESULTS: The fossils are strikingly similar in general size and morphology, particularly in the finer details of the attachment scar and ornamentation, to nutlets of extant representatives of the Boraginaceae tribe Echiochileae, and especially the genus Ogastemma. We believe that these nutlets represent an extinct member of this lineage. CONCLUSIONS: The Ogastemma-like fossils indicate that the Echiochileae, which are most diverse in northern Africa and southwestern Asia, have a long history in this region, dating back to the Eocene. This tribe corresponds to the basal-most clade in Boraginaceae s.str., and the fossils described here agree well with an assumed African origin of the family and the Boraginales I, providing an important additional calibration point for dating the phylogenies of this clade.

Self-healing of voids in the wax coating on plant surfaces.

The cuticles of plants provide a multifunctional interface between the plants and their environments. The cuticle, with its associated waxes, is a protective layer that minimizes water loss by transpiration and provides several functions, such as hydrophobicity, light reflection and absorption of harmful radiation. The self-healing of voids in the epicuticular wax layer has been studied in 17 living plants by atomic force microscopy (AFM), and the process of wax film formation is described. Two modes of wax film formation, a concentric layer formation and striped layer formation, were found, and the process of multilayer wax film formation is discussed. A new method for the preparation of small pieces of fresh, water-containing plant specimens for AFM investigations is introduced. The technique allows AFM investigations of several hours duration without significant shrinkage or lateral drift of the specimen. This research shows how plants refill voids in their surface wax layers by wax self-assembly and should be useful for the design of self-healing materials.

Superhydrophobicity of biological and technical surfaces under moisture condensation: stability in relation to surface structure.

The stability of superhydrophobic properties of eight plants and four technical surfaces in respect to water condensation has been compared. Contact and sliding angles were measured after application of water drops of ambient temperature (20 degrees C) onto cooled surfaces. Water evaporating from the drops condensed, due to the temperature difference between the drops and the surface, on the cooled samples, forming "satellite droplets" in the vicinity of the drops. Surface cooling to 15, 10, and 5 degrees C showed a gradual decrease of superhydrophobicity. The decrease was dependent on the specific surface architecture of the sample. The least decrease was found on hierarchically structured surfaces with a combination of a coarse microstructure and submicrometer-sized structures, similar to that of the Lotus leaf. Control experiments with glycerol droplets, which show no evaporation, and thus no condensation, were carried out to verify that the effects with water were caused by condensation from the drop (secondary condensation). Furthermore, the superhydrophobic properties after condensation on cooled surfaces from a humid environment for 10 min were examined. After this period, the surfaces were covered with spherical water droplets, but most samples retained their superhydrophobicity. Again, the best stability of the water-repellent properties was found on hierarchically structured surfaces similar to that of the Lotus leaf.

The hydrophobic coatings of plant surfaces: epicuticular wax crystals and their morphologies, crystallinity and molecular self-assembly.

Plant surfaces are the interfaces of the organisms with respect to their environment. In the micro-dimension they show an enormous variety of functional three-dimensional structures. Their materials and structures developed over millions of years by evolutionary processes in which their functionality has been proven and selected by environmental pressures. As a result, nature developed highly functional materials with several amazing properties like superhydrophobicity and superhydrophilicity. These functional structures are built up by a complex biopolymer called cuticle. The cuticle is mainly composed of a three-dimensional network of cutin, and integrated and superimposed lipids called "waxes". Superimposed waxes are also called "epicuticular waxes". Epicuticular waxes often form two- and three-dimensional structures, in dimensions between hundreds of nanometers and some micrometers, which influence the wettability, self-cleaning behaviour and the light reflection at the cuticle interface. This review gives a brief introduction into the functions of the plant epicuticular waxes and summarises the current knowledge about their morphologies, crystal structures, growth by self-assembly and provides an overview about the microscopy and preparation techniques for their analysis.

Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM).

The cuticle of terrestrial vascular plants and some bryophytes is covered with a complex mixture of lipids, usually called epicuticular waxes. Self-assembly processes of wax molecules lead to crystalline three-dimensional micro- and nanostructures that emerge from an underlying wax film. This paper presents the first AFM study on wax regeneration on the surfaces of living plants and the very early stages of wax crystal formation at the molecular level. Wax formation was analysed on the leaves of Euphorbia lathyris, Galanthus nivalis, and Ipheion uniflorum. Immediately after wax removal, regeneration of a wax film began, consisting of individual layers of, typically, 3-5 nm thickness. Subsequently, several different stages of crystal growth could be distinguished, and different patterns of wax regeneration as well as considerable variation in regeneration speed were found.