Epidermal Cell Density Research Articles

Effects of different light conditions on morphological, anatomical, photosynthetic and biochemical parameters of Cypripedium macranthos Sw.

In this study, the morphological (plant height, leaf length and width, stem diameter and leaf number), anatomical (epidermal cell density and thickness, Stomatal length and width), photosynthetic (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, relative humidity, leaf temperature and chlorophyll fluorescence parameters) and biochemical parameters (the content of soluble sugar, soluble protein, proline, malondialdehyde and electrical conductivity) of Cypripedium macranthos Sw. in Changbai Mountain were determined under different light conditions (L10, L30, L50, L100). The results showed that morphological values including plant height, leaf area, stem diameter and leaf number of C. macranthos were smaller under the condition of full light at L100. The epidermal cell density and epidermal thickness of C. macranthos were the highest under L30 and L50 treatments, respectively. It had the highest net photosynthetic rate (Pn) and chlorophyll content under L50 treatment. Meanwhile, correlation analysis indicated that photosynthetically active radiation (PAR) and water use efficiency (WUE) were the main factors influencing Pn. C. macranthos accumulated more soluble sugars and soluble proteins under L100 treatment, while the degree of membrane peroxidation was the highest and the plant was severely damaged. In summary, the adaptability of C. macranthos to light conditions is ranked as follows L50 > L30 > L10 > L100. Appropriate light conditions for C. macranthos are 30%-50% of full light, which should be taken into account in protection and cultivation.

Ecological implications of stomatal density and stomatal index in the adult stage of Mimosa L. (Leguminosae, Caesalpinioideae).

Differences in stomatal density (SD) and stomatal index (SI) are associated with the conditions of the environment in which they are distributed. Mimosa species are important elements in different plant communities, yet knowledge of the ecological implications of its stomatal characteristics is scarce. For this reason, SD and SI were determined in seven Mimosa species from different environments in this study. Five individuals per species were selected, and a sample of leaflets was obtained from each. Fifteen mature leaflets per individual were then extracted and observed by optical microscopy. SD, SI, epidermal cell density (ECD), and guard cell length (GCL) values were obtained. Differences between species were analyzed through a balanced analysis of variance test, and the correspondence between the stomatal characteristics and 21 climate variables was determined by canonical correspondence analysis. The species differed in all evaluated characteristics. It should be noted that only M. affinis showed differences between the leaflet surfaces. Both DE and ECD were negatively associated with altitude and solar radiation and positively with temperature and precipitation. SI was explained by temperature and seasonality of precipitation, and GCL by temperature oscillation and seasonality of precipitation. The results suggest that the stomatal characteristics of the leaflets confer resistance in the species to alterations in environmental conditions.

Improving drought tolerance in soybean by classical breeding leads to physiological adjustments of photosynthesis and stomata functioning

Soybean (Glycine max) is one of the main crops cultivated worldwide and drought is the main abiotic stress affecting its production. However, breeding towards drought tolerance is often set aside due to the difficulties in phenotyping. The aim of this study was to design an effective and simple screening technique that allows to discriminate genotypes according to their drought response and to identify physiological traits responsible for a superior tolerance. This rapid screening evaluated morphological (shoot and root length and weight) and developmental traits (days to germination and plant establishment) of 604 genotypes under control and drought conditions. Two genotypes with contrasting responses (Sensitive Genotype and Tolerant Genotype, SG and TG, respectively) were selected according to their performance in the screening, and were subjected to further testing in order to physiologically characterize the drought response of the chosen cultivars. Wilting, normalized transpiration ratio, leaf gas exchange parameters, water use efficiency (WUE) and stomatal characterizations were performed. TG plants exhibited slow wilting and higher WUE when subjected to drought treatments, product of a reduced water loss due to an anticipated stomatal closure which allowed to preserve soil moisture. Besides, under control conditions, the TG genotype presented a higher stomatal aperture index and a bigger density of epidermal cells and stomata than the drought-sensitive genotypes analyzed. In summary, the analysis of easily quantifiable traits, as the growth of the plant aerial organs and root development, together with the plant development timing, allowed us to select two soybean cultivars with contrasting drought responses. In our opinion, this selection method might be efficiently applied for the selection of drought tolerant cultivars in plant species of agronomic interest.

Nicotiana benthamiana as a model plant host for Fusarium verticillioides to investigate RNA interference, cross-kingdom RNA exchange, and competitive endogenous RNA (ceRNA) network.

Fusarium verticillioides is among the most devastating fungal pathogen of cereals. Therefore, it is crucial to employ effective and long-term strategies for managing F. verticillioides for sustainable agriculture. RNA interference (RNAi) being a natural defense mechanism of plants via regulation of gene expression, has emerged as a promising tool for eradicating pathogens. RNAi also operates between the host and pathogen through RNA exchange. RNAi interacts with competitive endogenous RNAs (ceRNAs) including long non-coding RNA (lncRNA), microRNA (miRNA), and mRNA. Due to the lack of an elaborate model to investigate all these mechanisms, this study aimed to establish a Nicotiana benthamiana (Nb)-F. verticillioides (Fv) phyto-pathosystem as an experimental model. Nb seedlings were inoculated with Fv, and the pathogenicity was monitored morphologically, microscopically, biochemically, and transcriptionally. To observe the role of RNAi and RNA-exchange in pathogenicity, Nb-miR172 and Nb-miR399 targeting Nb-lncRNA-IPS (Induced by Phosphate Starvation1) and Nb-AP2 (Apetala2) and Nb-PHO2 (phosphate over-accumulator) ceRNA network and Fv-V-ATPase (Vesicle-fusing ATPase) targeted by Nb-miR172 were investigated. As a result, epidermal cell density, leaf area, petiole length, and chlorophyll content were reduced while the density of stomata and trichome and phenolic content and the activity of ascorbate peroxidase (APX) and glutathione reductase (GR) were increased in response to Fv infection in Nb. The expression of AP2 and PHO2 were downregulated against Fv but no significant changes were detected in IPS, miR172, and miR399 expression. These findings suggested the Fv-Nb phyto-pathosystem as a useful experimental model to reveal genetic regulations.

Growth and Morpho-Stomatal Response of Kenaf (Hibiscus cannabinus) to Varying Water, Light, and Soil Conditions

This study investigated the stomatal responses of kenaf (Hibiscus cannabinus) to varying water, light, and soil conditions to explain how the species acclimatize to the changes in growing conditions. Seedlings were subjected to different watering regimes (daily – CON, 2 days interval – W2, 3 days interval – W3), light intensities (high, moderate, low), and soil conditions (garden soil – GS, Mt Makiling soil – MAK, UP Land Grant soil – UPL). The biomass, stomatal density (SD), epidermal cell density (ECD), stomatal index (SI), stomatal aperture length (SAL), guard cell length (GCL), stomatal area (SA), and potential conductance index (PCI) were measured across treatments. Water and light treatments had significant effects, but soil treatment did not affect most of the parameters measured. CON and/or W2 and high light intensity resulted in a higher SD, SI, SAL, and GCL, which resulted in a higher PCI, compared with the other water and light treatments. Contrarily, W3-treated seedlings had lower SD but significantly lower SAL, GCL, PCI, and aboveground biomass, compared to CON and W2-treated ones. Biomass allocation to root was also significantly higher in W3-treated seedlings. Therefore, kenaf seedlings exhibited a degree of stomatal plasticity in response to contrasting water, light, and soil conditions. Keywords: biomass allocation, potential conductance index, shade tolerance, stomatal index, water stress

Optimal protocol for in vitro polyploid induction of Cymbidium aloifolium (L.) Sw

BackgroundCymbidium aloifolium is a popular ornamental flower in Thailand with both economic and medical values. Polyploid induction techniques are used to improve plant quality. This study identified polyploidy levels of C. aloifolium induction by colchicine. Protocorms of C. aloifolium were treated on solid New Dogashima Medium (NDM) with various concentrations of colchicine (0, 0.01, 0.02, 0.03, 0.04 and 0.05% w/v) for 2, 4 and 8 weeks.ResultsColchicine effectively induced plant polyploidy. Tetraploid plants were found after treatment with 0.03% and 0.04% colchicine for 8 weeks, while at increased concentration and duration, survival, response and growth performance decreased. Tetraploid plants showed the lowest growth performance but highest size of stomatal and epidermal cells. Growth performance and leaf surface anatomy data were analyzed by Pearson’s correlation and PCA. Results showed that stomatal and epidermal cell sizes had strongly negative correlations with other variables, while HCA revealed that stomatal and epidermal cell sizes of tetraploid plantlets were larger but stomatal and epidermal cell densities decreased when compared with the diploids.ConclusionColchicine at suitable concentrations and duration produced polyploid plants with alteration of morphological and anatomical traits. This study provides potential information to support orchid quality production as ornamental plants and a source of pharmaceutical raw materials.

Modelling Climate Using Leaves of Nothofagus cunninghamii—Overcoming Confounding Factors

Fossil leaf anatomy has previously been used as a proxy for paleoclimate. However, the exposure of leaves to sun or shade during their growth can lead to morphotype differences that confound the interpretation of fossil leaf anatomy in relation to climate and prevent reliable paleoclimate reconstruction. This work aims to model the differences in leaf anatomy that are due to various climatic drivers and differences attributable to sun or shade positions, using Nothofagus cunninghamii as the model species. Leaves from the sun and shade parts of three trees have been sampled from each of 11 sites in Victoria and Tasmania, Australia. The gross morphological and cuticular features have been scored and modelled with climate data from the sites. Random forest models can accurately predict Nothofagus cunninghamii contemporary climatic conditions of the spring temperature and summer rainfall based on leaf anatomical measurements. Leaf area, stomatal density and epidermal cell density are the most accurate predictors of whether a leaf grew in the sun or shade. Leaf area is also the strongest predictor of the maximum and minimum spring temperatures and rainfall. The models have implications for the use of fossilised leaves in paleoclimate reconstruction. The models we have built can be used to effectively predict whether a fossil leaf was from a sun or shade position on the tree and thus enable more reliable inference of paleoclimate by removing the confounding issues of variable leaf anatomy due to sun exposure during growth. Finally, these models could conceivably be used to make predictions of past paleoclimatic conditions provided suitable training and validation data on climatic conditions are available.

Microscopic and metabolic investigations disclose the factors that lead to skin cracking in chili-type pepper fruit varieties.

The hydrophobic cuticle encasing the fruit skin surface plays critical roles during fruit development and post-harvest. Skin failure often results in the fruit surface cracking and forming a wound-periderm tissue made of suberin and lignin. The factors that make the fruit skin susceptible to cracking have yet to be fully understood. Herein, we investigated two varieties of chili peppers (Capsicum annuum L.), Numex Garnet, whose fruit has intact skin, and Vezena Slatka, whose fruit has cracked skin. Microscopical observations, gas chromatography-mass spectrometry, biochemical and gene expression assays revealed that Vezena Slatka fruit form a thicker cuticle with greater levels of cutin monomers and hydroxycinnamic acids, and highly express key cutin-related genes. The skin of these fruit also had a lower epidermal cell density due to cells with very large perimeters, and highly express genes involved in epidermal cell differentiation. We demonstrate that skin cracking in the Vezena Slatka fruit is accompanied by a spatial accumulation of lignin-like polyphenolic compounds, without the formation of a typical wound-periderm tissues made of suberized cells. Lastly, we establish that skin cracking in chili-type pepper significantly affects fruit quality during post-harvest storage in a temperature-dependent manner. In conclusion, our data highlight cuticle thickness and epidermal cell density as two critical factors determining fruit skin susceptibility to cracking in chili-type pepper fruit.

Ignorance is not bliss: evolutionary naiveté in an endangered desert fish and implications for conservation.

Predator naiveté has been invoked to explain the impacts of non-native predators on isolated populations that evolved with limited predation. Such impacts have been repeatedly observed for the endangered Pahrump poolfish, Empetrichthys latos, a desert fish species that evolved in isolation since the end of the Pleistocene. We tested Pahrump poolfish anti-predator responses to conspecific chemical alarm cues released from damaged epidermal tissue in terms of fish activity and water column position. Pahrump poolfish behavioural responses to conspecific alarm cues did not differ from responses to a dechlorinated tap water control. As a positive control, the well-studied fathead minnow, Pimephales promelas, showed significant alarm cue responses in terms of reduced activity and lowered water column position. The density of epidermal club cells, the presumptive source of alarm cues, was significantly lower in Pahrump poolfish relative to fathead minnows. Therefore, anti-predator competence mediated by conspecific alarm cues does not seem to be a component of the ecology of Pahrump poolfish. These findings provide a proximate mechanism for the vulnerability of Pahrump poolfish to non-native predators, with implications for the conservation and management of insular species.

Comparative leaf epidermis study in Habenaria spp. (Orchidaceae) from Thailand

Abstract. Muangsan N, Saensouk P, Chanokkhun T, Watthana S. 2022. Comparative leaf epidermis study in Habenaria spp. (Orchidaceae) from Thailand. Biodiversitas 23: 4159-4168. The variation of epidermal and stomatal characteristics could be used for plant classification, including orchids. This study aimed to investigate leaf epidermis characters of Habenaria from Thailand and focused on the H. rhodocheila group and related species. Fourteen accessions belonging to 10 species were studied and compared. Epidermal peels were taken from the upper and lower surfaces of the leaves and were observed under a light microscope. The stomata of all accessions studied were distributed only in the lower epidermis, where they were hypostomatic and anomocytic. The epidermal cell density showed significant differences between the two surface sides. The stomatal size is significantly different among all samples. The highest stomatal index was presented in H. rostrata and the lowest on H. rhodocheila (red). There is no gap variation of epidermis characteristics in H. rhodocheila and its closely related species. It is indicated that the leaf epidermal characters are the shared characters of the genus Habenaria from Thailand. H. rostrata has tentatively separated from other species based on epidermal anatomical characteristics.

The Topical Effect of rhGDF-5 Embedded in a Collagen–Gelatin Scaffold for Accelerated Wound Healing

The application of exogenous growth factors such as the recombinant human growth and differentiation factor 5 (rhGDF-5) represents a major research topic with great potential for the treatment of complex wounds. In a randomized, controlled minipig study, the topical effect of rhGDF-5 on full-thickness skin defects was evaluated. A total of 60 deep dermal wounds were either treated with rhGDF-5 embedded in an innovative collagen scaffold or another commonly used collagen matrix or left untreated. Wound healing was analyzed by planimetric analysis to determine wound closure over time. After 21 days, the areas of the initial wounds were excised, and the newly formed tissue was examined histologically. In comparison to untreated wounds, all examined matrices accelerated dermal wound healing. The largest acceleration of wound healing was seen with the high-dose rhGDF-5-treated wounds, which, compared to the untreated wounds, accelerated wound healing by 2.58 days, improved the neoepidermal thickness by 32.40 µm, and increased the epidermal cell density by 44.88 cells. The innovative collagen scaffold delivered rhGDF-5 adequately, served as a template to guide proliferating and restructuring cells, and accelerated wound healing. Thus, this composite product offers a novel tool for developing effective wound dressings in regenerative medicine.

Induction of polyploidy in saffron (Crocus sativus L.) using colchicine

ABSTRACT Saffron (Crocus sativus L.) is triploid (2 n = 3x = 24, x = 8), which limits its improvement through crop breeding programs. This study was initiated to induce hexaploidy with the intent of restoring sexual stability in saffron. Corms were treated with colchicine concentrations of 0.0%, 0.025%, 0.05%, and 0.1% for 3-h, 6-h, and 9-h. A total of 16 in-vitro shoots showed signs of polyploidization. These colchiploid shoots showed slow but robust growth and produced large-sized cormlets (6.0 g) compared to the control (2.8 g). Leaf epidermal cell morphology, stomatal size and density showed evidence of polyploidization. Hexaploidy (2 n = 6x = 48) was confirmed by root-tip cytology. The hexaploid corms germinated but could not establish in the field. We were able to develop a protocol for in-vitro induction of hexaploidy in C. sativus, but the subsequent growth of hexaploid corms in the field remains a major challenge in restoring the sexual stability in saffron.

Early signalling processes in roots play a crucial role in the differential salt tolerance in contrasting Chenopodium quinoa accessions.

Significant variation in epidermal bladder cell (EBC) density and salt tolerance (ST) exists amongst quinoa accessions, suggesting that salt sequestration in EBCs is not the only mechanism conferring ST in this halophyte. In order to reveal other traits that may operate in tandem with salt sequestration in EBCs and whether these additional tolerance mechanisms acted mainly at the root or shoot level, two quinoa (Chenopodium quinoa) accessions with contrasting ST and EBC densities (Q30, low ST with high EBC density versus Q68, with high ST and low EBC density) were studied. The results indicate that responses in roots, rather than in shoots, contributed to the greater ST in the accession with low EBC density. In particular, the tolerant accession had improved root plasma membrane integrity and K+ retention in the mature root zone in response to salt. Furthermore, superior ST in the tolerant Q68 was associated with faster and root-specific H2O2 accumulation and reactive oxygen species-induced K+ and Ca2+ fluxes in the root apex within 30 min after NaCl application. This was found to be associated with the constitutive up-regulation of the membrane-localized receptor kinases regulatory protein FERONIA in the tolerant accession. Taken together, this study shows that differential root signalling events upon salt exposure are essential for the halophytic quinoa; the failure to do this limits quinoa adaptation to salinity, independently of salt sequestration in EBCs.

Interaction of elevated CO2 and form of nitrogen nutrition alters leaf abaxial and adaxial epidermal and stomatal anatomy of wheat seedlings.

Plant's stomatal physiology and anatomical features are highly plastic and are influenced by diverse environmental signals including the concentration of atmospheric CO2 and nutrient availability. Recent reports suggest that the form of nitrogen (N) is a determinant of plant growth and nutrient nitrogen use efficiency (NUE) under elevated CO2 (EC). Previously, we found that high nitrate availability resulted in early senescence, enhanced reactive oxygen species (ROS), and reactive nitrogen species (RNS) production and also that mixed nutrition of nitrate and ammonium ions were beneficial than sole nitrate nutrition in wheat. In this study, the interactive effects of different N forms (nitrate, ammonium, mixed nutrition of nitrate, and ammonium) and EC on epidermal and stomatal morphology were analyzed. Wheat seedlings were grown at two different CO2 levels and supplied with media devoid of N (N0) or with nitrate-N (NN), mixed nutrition of ammonium and nitrate (MN), or only ammonium-N (AN). The stoma length increased significantly in nitrate nutrition with a consistent reduction in stoma width. Guard cell length was higher in EC treatment as compared to AC. The guard cell width was maximum in AN-grown plants at EC. Epidermal cell density and stomatal density were lower at EC. Nitrate nutrition increased the stomatal area at EC while the reverse was true for MN and AN. Wheat plants fertilized with AN showed a higher accumulation of superoxide radical (SOR) at EC, while in NN treatment, the accumulation of hydrogen peroxide (H2O2) was higher at EC. Reactive oxygen species, particularly H2O2, can trigger mitogen-activated protein kinase (MAPK) mediated signaling and its crosstalk with abscisic acid (ABA) signaling to regulate stomatal anatomy in nitrate-fed plants. The SOR accumulation in ammonium- and ammonium nitrate-fed plants and H2O2 in NN-fed plants might finely regulate the sensitivity of stomata to alter water/nutrient use efficiency and productivity under EC. The data reveals that the variation in anatomical attributes viz. cell length, number of cells, etc. affected the leaf growth responses to EC and forms of N nutrition. These attributes are fine targets for effective manipulation of growth responses to EC.

Diversity in Surface Microstructures of Trichomes, Epidermal Cells, and Stomata in Lentil Germplasm.

To develop crops capable of withstanding challenges posed by climate change, breeding strategies must focus on addressing multiple stresses occurring concurrently in plants. Leaf epidermal structures such as trichomes, stomata, and epidermal cells play an important role in mediating plant defense and could be essential traits that impart wide-ranging tolerance to biotic and abiotic stresses. Consequently, it is important to inform on the underlying diversity in these traits in lentil germplasm (Lens spp.). In this study, we characterized foliar microstructures of 12 genotypes belonging to seven wild and cultivated Lens species. We performed scanning electron microscopy on leaflet and pod surfaces for their qualitative characterization. For quantitative characterization, we observed surface imprints via light microscopy and quantified trichome density (TD), trichome length (TL), stomatal density (SD), epidermal cell density (ECD), and stomatal index (SI) on adaxial and abaxial leaflet surfaces for each genotype. We also assessed the heritability of trichome traits by evaluating interspecific recombinant inbred lines (RILs) derived from the cross Lens culinaris CDC Redberry × Lens tomentosus IG 72805. Comparing foliar microstructures, we found that TD and TL varied widely among cultivated and wild lentil genotypes. However, in most lentil genotypes, the adaxial leaflet surface had lower TD and longer trichomes compared to the abaxial surface. Pubescence on pods comprised five major phenotypes: no trichomes or glabrous pods, very short trichomes at low density, short trichomes at high density, medium-length trichomes at high density, and long trichomes at high density. Leaves of all species were amphistomatous, and SI, SD, and ECD were all higher on the adaxial compared to the abaxial surface. Adaxial surfaces had slightly sunken stomata, which might be an adaptive trait to conserve water. Quantifying TD and TL on the leaflets of interspecific RILs revealed transgressive segregation of these traits, suggesting that TD and TL are quantitative in nature. While taxonomic implications of this study are limited, a detailed description of agronomically relevant morphophysiological traits presented in this paper along with the mode of inheritance of trichomes may serve as a resource for scientists developing lentil adapted to concurrent biotic and abiotic stresses of the future.

Nitrate/ammonium ratio effect on the growth, yield and foliar anatomy of grafted tomato plants

The vigorous behaviour of a rootstock modifies the growth and yield of a plant variety or hybrid, altering the plant nutritional requirements. The purpose of this work was to study four ratios of NO3–/NH4+ (100/0, 92/8, 85/15 and 80/20%) over the growth, leaf anatomy and yield of grafted and ungrafted tomato plants. We used a fully randomised experimental block design with factorial arrangement of 2 × 4 (grafted and ungrafted plants and four ratios of NO3–/NH4+), on eight treatments in total with four replicates each, using Tukey’s mean comparison test (P ≤ 0.05). The rootstock was ‘Silex’ by Fito Seeds, with the ‘El Arrojado’ graft (variety) by Gene Seeds. The graft produced an increase in growth, dry biomass production, stomatal density, trichome number, epidermal cell density, fruits per plant, average fruit weight, fruit size and total soluble solids, in comparison with the ungrafted tomato plants. In most of the assessed variables, the grafted tomato plants gave their best response at an 85/15% NO3–/NH4+ ratio; while the ungrafted plants performed better at a 92/8% NO3–/NH4+ ratio. The response of the grafted plants to the different NO3–/NH4+ ratios suggests that grafting induces tolerance to NH4+.

Divergent Leaf Morpho-Physiological and Anatomical Adaptations of Four Lettuce Cultivars in Response to Different Greenhouse Irradiance Levels in Early Summer Season.

Lettuce (Lactuca sativa L.) is a winter-spring leafy vegetable, but the high demand for fresh products available year-round requires off-season production. However, the warm climate of the Mediterranean areas can impair the summer production of lettuce, thus requiring the adoption of genotypes tolerant to high irradiance as well as useful agronomic strategies like shading net installations. The aim of our research was to assess the leaf morpho-physiological and anatomical changes, in addition to productive responses, of four lettuce cultivars (‘Ballerina’, ‘Maravilla De Verano Canasta’, ‘Opalix’, and ‘Integral’) grown under shading and non-shading conditions to unveil the adaptive mechanisms of this crop in response to sub-optimal microclimate (high irradiance and temperature) in a protected environment. Growth and yield parameters, leaf gas exchanges, chlorophyll fluorescence and morpho-anatomical leaf traits (i.e., leaf mass area, stomatal density and epidermal cell density) were determined. Under shading conditions, the fresh yields of the cultivars ‘Ballerina’, ‘Opalix’ (‘Oak leaf’) and ‘Integral’ (‘Romaine’) increased by 16.0%, 26.9% and 13.2% respectively, compared to non-shading conditions while both abaxial and adaxial stomatal density decreased. In contrast, ‘Canasta’ under non-shading conditions increased fresh yield, dry biomass and instantaneous water use efficiency by 9.6%, 18.0% and 15.7%, respectively, while reduced abaxial stomatal density by 30.4%, compared to shading conditions. Regardless of cultivar, the unshaded treatment increased the leaf mass area by 19.5%. Even though high light intensity and high temperature are critical limiting factors for summer lettuce cultivation in a protected environment, ‘Canasta’ showed the most effective adaptive mechanisms and had the best production performance under sub-optimal microclimatic conditions. However, greenhouse coverage with a white shading net (49% screening) proved to be a suitable agricultural practice that ensured an adequate microclimate for the off-season growth of more sensitive cultivars ‘Ballerina’, ‘Oak leaf’ and ‘Romaine’.

Diversity of Leaf Stomatal Traits among Coffea canephora Pierre ex A. Froehner Genotypes

Leaf morpho-anatomical characteristics directly reflect photosynthetic performance and the ability to adapt to different environmental conditions. The study of biometric traits is essential for the selection of promising plant materials for breeding purposes. To identify new varieties of coffee plants with desirable traits for genetic improvement programs, this study investigated the variability of leaf morpho-anatomical traits in 43 genotypes of Coffea canephora (as the species under study is hypostomatous). Seven leaf characteristics were used: epidermal cell density (ECD), stomatal length (SL), stomatal width (SW), stomatal density (SD), stomatal size (SS), stomatal index (SI), and stomatal length/width. Morphological traits (plant height, internodal distance, and leaf area) and grain production were also assessed. The data analyzed multivariate analysis of variance grouped by the unweighted pair group the arithmetic mean hierarchical method, and data were also subjected to a Pearson linear correlation and principal component analyses (PCAs). The results showed wide morphological variability reflecting six morphological groups, which is relevant for the genetic divergence analysis and for breeding purposes, as the results have the potential to identify superior genotypes. Within the groups, genotypes were mainly separated by the number of epidermal cells and the number and size of the stomata, reflecting a high genetic heterogeneity within genotypes. Positive and negative correlations were found, with levels of significance ranging from weak to strong among the analyzed traits. The highest correlation levels were found for SL × SS, SW × SS, and SI × SD. In addition, the PCA indicated that plant height, distance between nodes, and leaf area were positively correlated and associated. The greater the number and width of stomata, the higher the rate of gas exchange. Both characteristics are favorable for the development and production of coffee plants, explaining the positive correlation observed in this study. These results emphasize the usefulness of trait evaluations for the identification and breeding of genotypes to compose new C. canephora cultivars suitable for changing environments.

Colchicine-induced tetraploidy in Dendrobium cariniferum and its effect on plantlet morphology, anatomy and genome size

Dendrobium cariniferum Rchb. f. is an important crossing parent that is primarily used to produce aromatic Dendrobium orchid cultivars due to its beautiful flowers and pleasant orange fragrance. However, there are no offspring within the limited progenies that fully inherit this odor trait. Thus, converting diploid D. cariniferum into tetraploids for interploid hybridization is another promising approach to creating a new orange aroma germplasm. This study reports a novel in vitro colchicine-induced chromosome doubling protocol that would effectively convert diploid D. cariniferum protocorms into tetraploids. A total of 120 tetraploid genotypes were identified by flow cytometry. The tetraploid induction rate was significantly affected by interaction between the colchicine concentration and exposure time. Treatment with 0.05% colchicine for 24 h produced the optimal results, yielding 54% surviving regenerated plantlets with a 33% tetraploid induction rate. The tetraploid plantlets displayed obvious morphological variations (broader, thicker leaves and greater stem and root diameters) than the diploid plantlets. Tetraploidization also caused a significant change in the leaf anatomical structure, including larger stomata with lower densities, more chloroplasts per stomata, thicker spongy tissues, larger leaf veins and decreased adaxial epidermal cell and trichome densities. We were the first to observe that there are one to five branched trichomes in the leaf sheath in both diploids and tetraploids, and we propose that the leaf adaxial epidermal cell density can be used to identify tetraploids. Moreover, tetraploids exhibited a 1.75-fold greater genome size than diploids. Our study lays the foundation for breeding triploid aromatic Dendrobium orchids. Autotetraploidy causes morphological, anatomical and genome size variations. This is the first report of trichome types in the leaf sheath. Leaf adaxial epidermal cell density is useful for identifying tetraploids.

Engraftment of bioengineered three-dimensional scaffold from human amniotic membrane-derived extracellular matrix accelerates ischemic diabetic wound healing.

Human amniotic membrane (HAM) is traditionally used for the treatment of non-healing wounds. However, high density of HAM-matrix (HAM-M) diminishes cellular contribution for successful tissue regeneration. Herein we investigated whether a bioengineered micro-porous three-dimensional (3D) HAM-scaffold (HAM-S) could promote healing in ischemic wounds in diabetic type 1 rat. HAM-S was prepared from freshly decellularized HAM. Then, 30days after inducing diabetes, an ischemic circular excision was generated on rats' skin. The diabetic animals were randomly divided into untreated (Diabetic group), engrafted with HAM-M (D-HAM-M group) and HAM-S (D-HAM-S group). Also, non-diabeticuntreated rats (Healthy group) were considered as control. Stereological, molecular, and tensiometrical assessments were performed on post-surgical days 7, 14, and 21. We found that the volumes of new epidermis and dermis, the numerical density of epidermal basal cells and fibroblasts, the length density of blood vessels, the numbers of proliferating cells and collagen deposition as well as biomechanical properties of healed wound were significantly higher in D-HAM-S group in most cases compared those of the diabetic group, or even in some cases compared to D-HAM-M group. Furthermore, in D-HAM-S group, the transcripts for genes contributing to regeneration (Tgf-β, bFgf and Vegf) upregulated more than those of D-HAM-M group, when compared to diabetic ones. Overall, the HAM-S had more impact on delayed wound healing process compared to traditional use of intact HAM. It is therefore suggested that the bioengineered three dimensional micro-porous HAM-S is more suitable for cells adhesion, penetration, and migration for contributing to wounded tissue regeneration.