Factors Affecting Cell Biomass and Flavonoid Production of Ficus deltoidea var. kunstleri in Cell Suspension Culture System.

A study was conducted to establish in vitro culture conditions for maximum production of biomass and flavonoid content for Ficus deltoidea var. kunstleri, locally named as Mas Cotek, known to have a wide variety of potential beneficial attributes for human health. Size of initial inoculum, cell aggregate and initial pH value have been suggested to influent content of biomass and flavonoid for cell suspension culture in several plant species. In the present study, leaf explants were cultured by cell suspension culture procedures in MSB5 basal medium supplemented with predetermined supplements of 30 g/L sucrose, 2.75 g/L gelrite, 2 mg/L picloram and 1 mg/L kinetin with continuous agitation of 120 rpm in a standard laboratory environment. Establishment of cell suspension culture was accomplished by culturing resulting callus in different initial fresh weight of cells (0.10, 0.25, 0.50, 1.0, and 2.0 g/25 mL of media) using similar basal medium. The results showed that the highest production of biomass (0.65 g/25 mL of media) was recorded from an initial inoculum size of 2.0 g/25 mL media, whereas the highest flavonoid (3.3 mg RE/g DW) was found in 0.5 g/25 mL of media. Cell suspension fractions classified according to their sizes (500-750 µm, 250-500 µm, and <250 µm). Large cell aggregate size (500-750 µm) cultured at pH 5.75 produced the highest cell biomass (0.28 g/25 mL media) and flavonoid content (3.3 mg RE/g DW). The study had established the optimum conditions for the production of total antioxidant and flavonoid content using DPPH and FRAP assays in cell suspension culture of F. deltoidea var. kunstleri.

Cellulases and pectinases act together on the development of articulated laticifers in Ficus montana and Maclura tinctoria (Moraceae).

The presence of articulated laticifers in the Moraceae family was recently discovered, which means that the location of pectinase and cellulase activities must be of great importance for their growth. Thus, the present study aimed to determine the role of these enzymes in the laticifer growth in Ficus montana and Maclura tinctoria. Reproductive meristems were collected and fixed in Karnovsky. Pectinase and cellulase labeling was performed in part of the samples, while another part was processed for usual TEM analyses. Pectinase and cellulase activities were detected in the vacuole and close to the middle lamella in both species. The presence of cellulases in the laticifers supports their articulated origin. Therefore, the occurrence of pectinase and cellulase activity in the laticifers points out that these enzymes could act in the dissolution of the transverse walls and in the processes of intrusive growth (through the dissolution of the middle lamella) and cell elongation (through the partial disassembly of components of the wall making it more plastic). Both enzymes are synthesized in the endoplasmic reticulum and transported to the cell wall by exocytosis or stored in the vacuole. The species studied showed a diverse subcellular composition, which is probably related to the species and not to the laticifer type (they present the same type) and to the composition of the latex (they show similar latex composition). We conclude that the presence of pectinases and cellulases can be used as a diagnostic condition for the laticifer types (articulated vs. non-articulated).

Mineral Deposits in Ficus Leaves: Morphologies and Locations in Relation to Function.

Ficus trees are adapted to diverse environments and have some of the highest rates of photosynthesis among trees. Ficus leaves can deposit one or more of the three major mineral types found in leaves: amorphous calcium carbonate cystoliths, calcium oxalates, and silica phytoliths. In order to better understand the functions of these minerals and the control that the leaf exerts over mineral deposition, we investigated leaves from 10 Ficus species from vastly different environments (Rehovot, Israel; Bologna, Italy; Issa Valley, Tanzania; and Ngogo, Uganda). We identified the mineral locations in the soft tissues, the relative distributions of the minerals, and mineral volume contents using microcomputed tomography. Each Ficus species is characterized by a unique 3D mineral distribution that is preserved in different environments. The mineral distribution patterns are generally different on the adaxial and abaxial sides of the leaf. All species examined have abundant calcium oxalate deposits around the veins. We used micromodulated fluorimetry to examine the effect of cystoliths on photosynthetic efficiency in two species having cystoliths abaxially and adaxially (Ficusmicrocarpa) or only abaxially (Ficuscarica). In F. microcarpa, both adaxial and abaxial cystoliths efficiently contributed to light redistribution inside the leaf and, hence, increased photosynthetic efficiency, whereas in F. carica, the abaxial cystoliths did not increase photosynthetic efficiency.

Pollutant-induced cell death and reactive oxygen species accumulation in the aerial roots of Chinese banyan (Ficus microcarpa).

Industrial pollutants induce the production of toxic reactive oxygen species (ROS) such as O2.-, H2O2, and ·OH in plants, but they have not been well quantified or localized in tissues and cells. This study evaluated the pollutant- (HSO3-, NH4NO3, Al3+, Zn2+, and Fe2+) induced toxic effects of ROS on the aerial roots of Chinese banyan (Ficus microcarpa). Root cell viability was greatly reduced by treatment with 20 mM NaHSO3, 20 mM NH4NO3, 0.2 mM AlCl3, 0.2 mM ZnSO4, or 0.2 mM FeSO4. Biochemical assay and histochemical localization showed that O2.- accumulated in roots in response to pollutants, except that the staining of O2.- under NaHSO3 treatment was not detective. Cytochemical localization further indicated that the generated O2.- was present mainly in the root cortex, and pith cells, especially in NH4NO3- and FeSO4-treated roots. The pollutants also caused greatly accumulated H2O2 and ·OH in aerial roots, which finally resulted in lipid peroxidation as indicated by increased malondialdehyde contents. We conclude that the F. microcarpa aerial roots are sensitive to pollutant-induced ROS and that the histochemical localization of O2.- via nitrotetrazolium blue chloride staining is not effective for detecting the effects of HSO3- treatment because of the treatment's bleaching effect.

Root contraction in Cycas and Zamia (Cycadales) determined by gelatinous fibers.

UNLABELLED: • PREMISE OF THE STUDY: Reaction wood (RW) in seed plants can induce late and usually secondary changes in organ orientation. Conifers produce compression wood (CW), generated by compression tracheids, which generate a push force. Angiosperms produce tension wood (TW), generated by tension wood fibers (TWF) often described as "gelatinous fibers," which exert a pull force. Usually RW is produced eccentrically, but it can occur concentrically, as in aerial roots of Ficus. However, gymnosperms can produce gelatinous fibers (tension fibers, TF), as in cortical and secondary phloem tissues (Gnetum). TFs are therefore limited neither to wood, xylem, nor angiosperms. Here we demonstrate that TFs in secondary phloem are involved in contraction of roots of cycads and compare them with TFs of Ficus.• METHODS: We sectioned root material of cycads at various stages of seedling development using simple staining and histochemical procedures to follow the course of secondary phloem development. Aerial roots of Ficus were compared with the cycad root material.• KEY RESULTS: Tension fibers (gelatinous fibers) occur extensively and continuously in the secondary phloem in roots that undergo contraction. Older tissues, but notably the xylem, become distorted by contraction. TFs in cycads correspond in cell wall features to TFs that occur in Ficus, but do not occur in secondary xylem. The individual fibers visibly contract.• CONCLUSIONS: Tissue contraction in Cycas and Zamia corresponds to that found in angiosperms and Gnetum and further broadens the scope of the activity of tension tissues. This finding possibly indicates that gelatinous fibers originated at a very early period of seed plant evolution.

Relationship between pollination and cell wall properties in common fig fruit.

Most botanical types in fig Ficus carica require pollination to fulfil their development and ensure quality onset of the fruit. Cell wall behaviour and composition was followed in fig fruit in response to pollination during maturity. Figs, when ripe, soften drastically and lose of their firmness and cell wall cohesion. Pollination increased peel thickness, flesh thickness, fresh weight and dry matter content of the fruit. Alcohol insoluble solids (AIS), more concentrated in the flesh tissue, were not influenced by the lack of pollination. Concentrations in uronic acids were higher in the AIS of the peel than that of the flesh and differences were significant between pollinated and non-pollinated fruits. Pectin polymers in figs were high methylated (DM>50). The methylation degree (DM) increased more with pollination affecting textural properties of the fig receptacle. The major neutral sugars from the AIS were glucose (Glc) from cellulose followed by arabinose (Ara). No significant changes in neutral sugars content could be allocated to pollination. Pollination is essential in fruit enlargement and softening. Minor changes were determined in the cell wall composition of the fruit at maturity. Fertile seeds resulting from pollination may possibly take place in hormonal activity stimulating many related enzymes of the wall matrix depolymerisation in particular polygalacturonase (PG) and pectin methylesterase (PME).

Leaf anatomy and histochemistry of three species of Ficus sect. Americanae supported by light and electron microscopy.

In this work the leaf anatomy of three species of Ficus section Americanae (Miq.) Miq. from Brazil, whose leaves and latex are used in folk medicine is reported. The work was carried out using light and scanning electron microscopy in order to characterize these species and to evaluate their taxonomic significance, and also contribute to the quality control of their ethnodrugs. The three species (Ficus cyclophylla, Ficus elliotiana, and Ficus caatingae) showed hypostomatic leaves, anomocytic stomata, straight epidermal cell outlines, and a dorsiventral mesophyll. Some micro-morphological characters such as density and distribution of epicuticular waxes, glandular trichomes, the length and width of stomata, as well as the palisade of mesophyll and petiole outlines proved to be the most useful and distinctive characters for the separation of species. These may contribute as additional support for the taxonomy of the section and for the quality control of their ethnodrugs.

Development of a large-area plasmonic sensor substrate with dielectric subwavelength gratings using nanoimprint lithography.

We demonstrated an enhanced surface plasmon resonance (SPR) detection by incorporating subwavelength SiO2 gratings built on a thin silver film. Large-area SiO2 gratings were fabricated by nanoimprint lithographic technique and dry etching processes and used to sense a surface-limited biomolecular interaction. Numerical results based on rigorous coupled-wave analysis method exhibited that the dielectric gratings can provide a notable sensitivity improvement by 2 times, which is attributed to an increase in surface reaction area. As another possible application of the fabricated plasmonic substrate, experimental data of imaging the cell morphology were also presented. This study was intended to show the feasibility and extend the applicability of a large-area grating-based SPR substrate to diverse optical bioengineering fields.

Fluorescence kinetic parameters and cyclic electron transport in guard cell chloroplasts of chlorophyll-deficient leaf tissues from variegated weeping fig (Ficus benjamina L.).

Residual chlorophyll in chlorophyll-deficient (albino) areas of variegated leaves of Ficus benjamina originates from guard cell chloroplasts. Photosynthetic features of green and albino sectors of F. benjamina were studied by imaging the distribution of the fluorescence decrease ratio Rfd within a leaf calculated from maximum (Fm) and steady-state leaf chlorophyll fluorescence (Fs) at 690 and 740 nm. Local areas of albino sectors demonstrated an abnormally high Rfd(740)/Rfd(690) ratio. Fluorescence transients excited in albino sectors at red (640 and 690 nm) wavelengths showed an abrupt decrease of the Rfd values (0.4 and 0.1, correspondingly) as compared with those excited at blue wavelengths (1.7-2.4). This "Red Drop" was not observed for green sectors. Normal and chlorophyll-deficient leaf sectors of F. benjamina were also tested for linear and cyclic electron transport in thylakoids. The tests have been performed studying fluorescence at a steady-state phase with CO(2)-excess impulse feeding, photoacoustic signal generated by pulse light source at wavelengths selectively exciting PSI, fluorescence kinetics under anaerobiosis and fluorescence changes observed by dual-wavelength excitation method. The data obtained for albino sectors strongly suggest the possibility of a cyclic electron transport simultaneously occurring in guard cell thylakoids around photosystems I and II under blue light, whereas linear electron transport is absent or insufficient.

Presence of triploid cytotypes in the common fig (Ficus carica L.).

Ficus carica (2n = 26) is one of the oldest fruit trees of the Mediterranean basin. Recently there has been increasing interest in this species, in particular for questions related to germplasm such as genetic diversity and cultivar identification. This study was undertaken to gain more knowledge of F. carica cytogenetics and provide data useful for the characterization of its germplasm. Karyomorphological analysis and physical mapping of 18S-25S and 5S rRNA genes by the FISH technique contributed to defining the basic traits of the chromosome complement of F. carica. However, the most interesting result was the discovery of triploid (2n = 39) cytotypes of the cultivated common fig. This result demonstrates the importance of cytogenetic investigations in studies of fig germplasm and emphasizes the role of cross-fertilization as a source of variability not only in wild populations but also in cultivated forms. The results of pollen analysis suggest spontaneous sexual polyploidization as a possible origin of triploid cytotypes. Further studies are necessary to clarify the origin and effective spreading of polyploidy, the presence of other ploidy levels, and their distribution in wild and cultivated forms.

A novel method to identify the Chinese herbal medicine Wuzhimaotao by quantification of laticifers.

Histology and the microscope have been used to identify Chinese herbal medicines for a long time. However, research on using the microscope for quantitative determination of identification characters is limited. A novel method which combines histological and microscopic analysis of laticifers by "blob" analysis is established to identify Wuzhimaotao, which is derived from species of Ficus (primarily F. hirta Vahl, but F. simplicissima Lour., F. hirta Vahl var. imberbis Gagnep., and F. esquiroliana Lévl. are also used). Results indicate that laticifers, which are stained orange-red by Sudan III, are mainly scattered in the phloem of Wuzhimaotao. The blob area of the laticifers is varied according to the species: F. hirta Vahl, F. simplicissima Lour., F. hirta Vahl var. imberbis Gagnep., and F. esquiroliana Lévl. showed 86,609 +/- 3,768 (mean +/- SD, n = 10), 48,582 +/- 2,603 (n = 10), 68,745 +/- 2,179 (n = 5), and 27,966 +/- 2,121 (n = 3) blob area, respectively. By directly measuring the blob area of laticifers in transverse sections, we could distinguish species of Wuzhimaotao in the same genus which were difficult to distinguish by microscopic examination of the dry roots, and we could provide objective data to describe and standardize the characters observed in microscopic images. This method is rapid, accurate, and inexpensive.

[Leaf anatomy of the mosaic ficus benjamina cv. Starlight and interaction of source and sink chimera components].

Leaf anatomy was studied in the mosaic Ficus benjamina cv. Starlight and non-chimeric Ficus benjamina cv. Daniel. The number of chloroplasts in a white, chlorophyll-deficient tissue declines as compared to the green tissue. However, their functional activity is retained. The leaf of the mosaic F. benjamina contains two or, sometimes, three subepidermal layers. Mesophyll forms one layer in the green and white parts of leaf palisade and one white and one green layer in the transitional zone (edge). In the transitional zone, green spongy mesophyll is located between two white spongy layers and the proportion of photosynthesizing cells varies. In cv. Daniel, there are two subepidermal layers and one layer of columnar mesophyll cells. According to the morphometry data, the proportion of white zone in the leaf correlates with the leaf position in the whole shoot: the higher the branch order, the larger the proportion of white zone. The total leaf area depends also on its position in the shoot. No such correlation was found in non-chimeric F. benjamina cv. Daniel. In the mosaic chimera, the source-sink status appears to depend on the leaf position in the shoot. Experiments with individual shoots of the same order and elimination of all lateral shoots have shown that the proportion of white zone in new leaves on the shoot increases with the total area of green zone. Thus, the area of assimilating shoot surface affects the formation of leaves in the meristem. A hypothesis was put forward that the source-sink state affects the ratio of green and white parts in the leaf primordium. Products of photosynthesis (carbohydrates) are a possible metabolic signal affecting the meristem. It cannot be excluded as well that the hormonal state undergoes changes in the chimeric plant.

Ficus rubiginosa 'variegata', a chlorophyll-deficient chimera with mosaic patterns created by cell divisions from the outer meristematic layer.

BACKGROUND AND AIMS: Sections leaves of Ficus rubiginosa 'Variegata' show that it is a chimera with a chlorophyll deficiency in the second layer of the leaf meristem (GWG structure). Like other Ficus species, it has a multiseriate epidermis on the adaxial and abaxial sides of the leaf, formed by periclinal cell divisions as well as anticlinal divisions. The upper and lower laminae of the leaf often exhibit small dark and light green patches of tissue overlying internal leaf tissue. METHODS: The distribution of chlorophyll in transverse sections of typical leaves was determined by fluorescence microscopy. KEY RESULTS: Patches of dark and light green tissue which arise in the otherwise colourless palisade and spongy mesophyll tissue in the entire leaf are due to further cell divisions arising from the bundle sheath which is associated with major vascular bundles or from the green multiseriate epidermis. Leaves produced in winter exhibit more patches of green tissue than leaves which expand in mid-summer. Many leaves produced in summer have no spotting and appear like a typical GWG chimera. There is a strong relationship between the number of patches on the adaxial side of leaves and the number on the abaxial side, showing that the cell division in upper and lower layers of leaves is strongly coordinated. In both winter and summer, there are fewer patches on the abaxial side of leaves compared with the adaxial side, indicating that periclinal and anticlinal cell divisions from the outer meristematic layer are less frequent in the lower layers of leaf tissue. Most of the patches are small (<1 mm in longest dimension) and thus the cell divisions which form them occur late in leaf development. Leaves which exhibit large patches generally have them on both sides of the leaves. CONCLUSION: In this cultivar, the outer meristematic layer appears to form vascular bundle sheaths and associated internal leaf tissue in the entire leaf lamina.