Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli

Symplasmic isolation marks cell fate changes during somatic embryogenesis

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis

Quantitative and qualitative characteristics of cell wall components and prenyl lipids in the leaves of Tilia x euchlora trees growing under salt stress

The study was focused on assessing the presence of arabinogalactan proteins (AGPs) and pectins within the cell walls as well as prenyl lipids, sodium and chlorine content in leaves of Tilia x euchlora trees. The leaves that were analyzed were collected from trees with and without signs of damage that were all growing in the same salt stress conditions. The reason for undertaking these investigations was the observations over many years that indicated that there are trees that present a healthy appearance and trees that have visible symptoms of decay in the same habitat. Leaf samples were collected from trees growing in the median strip between roadways that have been intensively salted during the winter season for many years. The sodium content was determined using atomic spectrophotometry, chloride using potentiometric titration and poly-isoprenoids using HPLC/UV. AGPs and pectins were determined using immunohistochemistry methods. The immunohistochemical analysis showed that rhamnogalacturonans I (RG-I) and homogalacturonans were differentially distributed in leaves from healthy trees in contrast to leaves from injured trees. In the case of AGPs, the most visible difference was the presence of the JIM16 epitope. Chemical analyses of sodium and chloride showed that in the leaves from injured trees, the level of these ions was higher than in the leaves from healthy trees. Based on chromatographic analysis, four polyisoprenoid alcohols were identified in the leaves of T. x euchlora. The levels of these lipids were higher in the leaves from healthy trees. The results suggest that the differences that were detected in the apoplast and symplasm may be part of the defensive strategy of T. x euchlora trees to salt stress, which rely on changes in the chemical composition of the cell wall with respect to the pectic and AGP epitopes and an increased synthesis of prenyl lipids

Identifikasi Bakteri Resisten Merkuri Pada Urine, Feses Dan Kalkulus Gigi Pada Individu Di Kecamatan Malalayang, Manado, Sulawesi Utara

: Consumption of shells, fishes and some sea mammals is one of the main track in which people get contaminated by mercury. Mercury is neurotoxin and is very harmful for human body. Mercury accumulation in human body resulted in bacteria adapting and its being resistance to mercury. Objective: To detect the existence of bacteria that can live in the environmental condition contaminated by mercury and to identify mercury resistance bacteria on urine, feces and dental calculus of individual in district Malalayang, Manado, North Sulawesi. Method: This research employs explorative descriptive research design. The sample of the study is the mercury resistance bacteria on urine, feces, and dental calculus in district Malalayang, Manado, North Sulawesi. The study is begun conducted in October and end in December 2013. The study is begun done by isolating mercury resistance bacteria on Broth Nutrient containing HgCl2 and Phenylmercury, continued by morphology testing, physiology testing and biochemistry testing in Biotechnology Laboratory of MIPA faculty of Sam Ratulangi University. Result: There are mercury resistance bacteria on urine, feces and dental calculus taken from individual in district Malalayang. There are 3 genus of Bacteria founded, which are E. Coli, Staphylococcus and Bacillus sp. In this study, the highest level of bacteria resistance on mercury conducted on HgCl2 concentration of 10 ppm, 20 ppm and 40 ppm is on concentration of 40 ppm. On Phenylmercury with concentration of 5 ppm, 10 ppm and 20 ppm is on concentration of 20 ppm

Rays, intrusive growth, and storied cambium in the inflorescence stems of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana is a model plant used in analysis of different aspects of plant growth and development. Under suitable conditions, secondary growth takes place in the hypocotyl of Arabidopsis plants, a finding which helps in understanding many aspects of xylogenesis. However, not all developmental processes of secondary tissue can be studied here, as no secondary rays and intrusive growth have been detected in hypocotyl. However, results presented here concerning the secondary growth in inflorescence stems of Arabidopsis shows that both secondary rays and intrusive growth of cambial cells can be detected, and that, in the interfascicular regions, a storied cambium can be developed

Nuclear genome stability in long-term cultivated callus lines of Fagopyrum tataricum (L.) Gaertn

© 2017 Betekhtin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Long-term cultivated Fagopyrum tataricum (L.) Gaertn. (Tartary buckwheat) morphogenic and non-morphogenic callus lines are interesting systems for gaining a better understanding of the mechanisms that are responsible for the genetic stability and instability of a plant tissue culture. In this work, we used histological sections and transmission electron microscopy to identify and describe the morphology of the nuclei of all of the analysed callus lines. We demonstrated that the embryogenic callus cells had prominent round nuclei that did not contain heterochromatin clumps in contrast to the non-morphogenic callus lines, in which we found nuclei that had multiple lobes. Flow cytometry analysis revealed significant differences in the relative DNA content between the analysed calli. All of the analysed morphogenic callus lines had peaks from 2C to 8C as compared to the nonmorphogenic callus lines, whose peaks did not reflect any regular DNA content and exceeded 8C and 16C for the line 6p1 and 16C and 32C for the callus line 10p2A. The results showed that non-morphogenic calli are of an aneuploid nature. The TUNEL test enabled us to visualise the nuclei that had DNA fragmentation in both the morphogenic and non-morphogenic lines. We revealed significantly higher frequencies of positively labelled nuclei in the non-morphogenic lines than in the morphogenic lines. In the case of the morphogenic lines, the highest observed frequency of TUNEL-positive nuclei was 7.7% for lines 2-3. In the non-morphogenic calli, the highest level of DNA damage (68.5%) was revealed in line 6p1. These results clearly indicate greater genome stability in the morphogenic lines

Histology and symplasmic tracer distribution during development of barley androgenic embryos

The present study concerns three aspects of barley androgenesis: (1) the morphology and histology of the embryos during their development, (2) the time course of fluorescent symplasmic tracers’ distribution, and (3) the correlation between symplasmic communication and cell differentiation. The results indicate that barley embryos, which are developing via an androgenic pathway, resemble their zygotic counterparts with respect to their developmental stages, morphology and histology. Analysis of the distribution of the symplasmic tracers, HPTS, and uncaged fluorescein indicates the symplasmic isolation of (1) the protodermis from the underlying cells of the late globular stage onwards, and (2) the embryonic organs at the mature stage of development