Normalising intolerance: the efforts of Christian Right groups to block LGBTIQ+ inclusion in South African schools.

Post-apartheid, South Africa has come a long way in making the inclusion of gender and sexuality equality explicit in its Constitution. To make schools more inclusive for lesbian, gay, bisexual, transgender, intersex and queer (LGBTIQ+) identifying learners, the Department of Basic Education has developed what it claims are South Africa's first guidelines regarding sexual orientation, gender identity and sex characteristics (SOGIESC). Despite the emphasis on equality in South Africa's post-apartheid policies, which set out to protect the rights of LGBTIQ+ individuals, there has been a backlash from conservative advocacy groups, many with links to the US Christian Right. This paper argues that contrary to the disinformation being propagated by anti-LGBTIQ + groups, it is queer and transgender individuals who experience extreme levels of violence and marginalisation in schools. The empirical research makes explicit the bullying and exclusion that transgender and gender-diverse youth experience in school; however, this evidence is neglected in conservative claims that SOGIESC (SOGIESC) guidelines and Comprehensive Sexuality Education (CSE) endanger other learners. Notably, and in stark contrast to those advocating for measures to make schools safer for LGBTIQ + learners, far-right advocacy groups have no empirical basis to support their claims.

Cell cycle arrest mediated by Cd-induced DNA damage in Arabidopsis root tips.

Accumulating evidence demonstrates that the aberrant expression of cell cycle regulation and DNA repair genes can result in abnormal cell proliferation and genomic instability in eukaryotic cells under different stresses. Herein, Arabidopsis thaliana (Arabidopsis) seedlings were grown hydroponically on 0.5 × MS media containing cadmium (Cd) at 0-2.5mgL-1 for 5d of treatment. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that expression of DNA damage repair and cell cycle regulation genes, including BRCA1, MRE11, WEE1, CDKA;1 and PCNA1, showed an inverted U-shaped dose-response. In contrast, notably reduced expression was observed for G1-to-S transition-related genes, Histone H4, E2Fa and PCNA2; DSB end processing, GR1; G2-to-M transition-related gene, CYCB1;1; and DNA mismatch repair, MSH2, MSH6 and MLH1 genes in root tips exposed to 0.125-2.5mg/L Cd for 5d. Flow cytometry (FCM) analysis revealed significant increases of cells with a 2C nuclear content and with a 4C and 8C nuclear content under Cd stresses of 0.125 and 1-2.5mgL-1, respectively. Our results suggest that 0.125mgL-1 Cd-induced DNA damage induced the marked G1/S arrest, leading to accelerated growth in root tips, while 1.0-2.5mgL-1 Cd-induced DNA damage caused a notable G2/M arrest in root tips, leading to reduced growth in root tips. This may be a protective mechanism that prevents cells with damaged DNA from dividing under Cd stress.

In tobacco BY-2 cells xyloglucan oligosaccharides alter the expression of genes involved in cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

Xyloglucan oligosaccharides (XGOs) are breakdown products of XGs, the most abundant hemicelluloses of the primary cell walls of non-Poalean species. Treatment of cell cultures or whole plants with XGOs results in accelerated cell elongation and cell division, changes in primary root growth, and a stimulation of defence responses. They may therefore act as signalling molecules regulating plant growth and development. Previous work suggests an interaction with auxins and effects on cell wall loosening, however their mode of action is not fully understood. The effect of an XGO extract from tamarind (Tamarindus indica) on global gene expression was therefore investigated in tobacco BY-2 cells using microarrays. Over 500 genes were differentially regulated with similar numbers and functional classes of genes up- and down-regulated, indicating a complex interaction with the cellular machinery. Up-regulation of a putative XG endotransglycosylase/hydrolase-related (XTH) gene supports the mechanism of XGO action through cell wall loosening. Differential expression of defence-related genes supports a role for XGOs as elicitors. Changes in the expression of genes related to mitotic control and differentiation also support previous work showing that XGOs are mitotic inducers. XGOs also affected expression of several receptor-like kinase genes and transcription factors. Hence, XGOs have significant effects on expression of genes related to cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

Silence, nostalgia, violence, poverty : what does 'culture' mean for South African sexuality educators?

In in-depth interviews with 25 Life Orientation teachers in South Africa, we found that teachers spontaneously drew upon notions of culture to explain and justify people's sexual beliefs and behaviours and their own role as educators. Drawing upon a Bakhtinian understanding of discourse, we apply critical semantic analysis to explore how culture is deployed as a discursive strategy. Teachers draw upon particular understandings of culture available to them in their social contexts. Furthermore, the substitution of the word 'culture' for a series of other phenomena (silence, violence and poverty) affords these phenomena a certain authority that they would otherwise not wield. We argue, first, that systems teacher education and training needs to (re)define culture as dynamic, interactive and responding to, but not determined by, socio-historical realities. Beyond this, teachers need to learn how to critically engage with cultural practices and perceptions and to be provided with some basic tools to do so, including more sophisticated understandings of cultural and training in dialogic methodologies. Teaching sexuality education in multicultural societies such as South Africa will require meaningful engagement in intercultural dialogues that may need to include voices that have traditionally been excluded from school spaces.

A yeast mitotic activator sensitises the shoot apical meristem to become floral in day-neutral tobacco.

True day-neutral (DN) plants flower regardless of day-length and yet they flower at characteristic stages. DN Nicotiana tabacum cv. Samsun, makes about forty nodes before flowering. The question still persists whether flowering starts because leaves become physiologically able to export sufficient floral stimulus or the shoot apical meristem (SAM) acquires developmental competence to interpret its arrival. This question was addressed using tobacco expressing the Schizosaccharomyces pombe cell cycle gene, Spcdc25, as a tool. Spcdc25 expression induces early flowering and we tested a hypothesis that this phenotype arises because of premature floral competence of the SAM. Scions of vegetative Spcdc25 plants were grafted onto stocks of vegetative WT together with converse grafts and flowering onset followed (as the time since sowing and number of leaves formed till flowering). Spcdc25 plants flowered significantly earlier with fewer leaves, and, unlike WT, also formed flowers from axillary buds. Scions from vegetative Spcdc25 plants also flowered precociously when grafted to vegetative WT stocks. However, in a WT scion to Spcdc25 stock, the plants flowered at the same time as WT. SAMs from young vegetative Spcdc25 plants were elongated (increase in SAM convexity determined by tracing a circumference of SAM sections) with a pronounced meristem surface cell layers compared with WT. Presumably, Spcdc25 SAMs were competent for flowering earlier than WT and responded to florigenic signal produced even in young vegetative WT plants. Precocious reproductive competence in Spcdc25 SAMs comprised a pronounced mantle, a trait of prefloral SAMs. Hence, we propose that true DN plants export florigenic signal since early developmental stages but the SAM has to acquire competence to respond to the floral stimulus.

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery.

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 interacting partner 1 (SKIP1). Furthermore, the AtWEE1-green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1-YFP(C) (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1-YFP(N) negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.

Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25.

BACKGROUND: Entry into mitosis is regulated by cyclin dependent kinases that in turn are phosphoregulated. In most eukaryotes, phosphoregulation is through WEE1 kinase and CDC25 phosphatase. In higher plants a homologous CDC25 gene is unconfirmed and hence the mitotic inducer Schizosaccharomyces pombe (Sp) cdc25 has been used as a tool in transgenic plants to probe cell cycle function. Expression of Spcdc25 in tobacco BY-2 cells accelerates entry into mitosis and depletes cytokinins; in whole plants it stimulates lateral root production. Here we show, for the first time, that alterations to cytokinin and ethylene signaling explain the rooting phenotype elicited by Spcdc25 expression in Arabidopsis. RESULTS: Expressing Spcdc25 in Arabidopsis results in increased formation of lateral and adventitious roots, a reduction of primary root width and more isodiametric cells in the root apical meristem (RAM) compared with wild type. Furthermore it stimulates root morphogenesis from hypocotyls when cultured on two way grids of increasing auxin and cytokinin concentrations. Microarray analysis of seedling roots expressing Spcdc25 reveals that expression of 167 genes is changed by > 2-fold. As well as genes related to stress responses and defence, these include 19 genes related to transcriptional regulation and signaling. Amongst these was the up-regulation of genes associated with ethylene synthesis and signaling. Seedlings expressing Spcdc25 produced 2-fold more ethylene than WT and exhibited a significant reduction in hypocotyl length both in darkness or when exposed to 10 ppm ethylene. Furthermore in Spcdc25 expressing plants, the cytokinin receptor AHK3 was down-regulated, and endogenous levels of iPA were reduced whereas endogeous IAA concentrations in the roots increased. CONCLUSIONS: We suggest that the reduction in root width and change to a more isodiametric cell phenotype in the RAM in Spcdc25 expressing plants is a response to ethylene over-production. The increased rooting phenotype in Spcdc25 expressing plants is due to an increase in the ratio of endogenous auxin to cytokinin that is known to stimulate an increased rate of lateral root production. Overall, our data reveal important cross talk between cell division and plant growth regulators leading to developmental changes.

Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants.

BACKGROUND AND AIMS: How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. METHODS: Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1(oe)), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. KEY RESULTS: Quantitative data indicated a repressive effect in WEE1(oe) and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1(oe) seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1(oe) and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1(oe) for all three ground tissues but for wee1-1 only cortical cell size was reduced. CONCLUSIONS: There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Teacher positioning on the teaching of sexual diversity in South African schools.

This paper reports on research with 11 high school teachers in Durban and draws on positioning theory to raise important questions on the teaching of issues relating to homosexuality and bisexuality within sexuality education. Using data collected from classroom observations and in-depth interviews, it demonstrates that issues related to sexual diversity were in most cases ignored or avoided by teachers and when teachers did include aspects of homosexuality, they took up positions that endorsed the idea of 'compulsory heterosexuality'. I conclude by paying attention to ways we can deepen the teaching of homosexual and bisexual issues.

A commentary on the G2/M transition of the plant cell cycle.

BACKGROUND: The complex events of mitosis rely on precise timing and on immaculate preparation for their success, but the G2/M transition in the plant cell cycle is currently steeped in controversy and alternative models. SCOPE: In this brief review, the regulation of the G2/M transition in plants is commented on. The extent to which the G2/M transition is phosphoregulated by WEE1 kinase and CDC25 phosphatase, as exemplified in yeasts and animals, is discussed together with an alternative model that excludes these proteins from this transition. Arabidopsis T-DNA insertional lines for WEE1 and CDC25 that develop normally prompted the latter model. An argument is then presented that environmental stress is the norm for higher plants in temperate conditions. If so, the repressive role that WEE1 has under checkpoint conditions might be part of the normal cell cycle for many proliferative plant cells. Arabidopsis CDC25 can function as either a phosphatase or an arsenate reductase and recent evidence suggests that cdc25 knockouts are hypersensitive to hydroxyurea, a drug that induces the DNA-replication checkpoint. That other data show a null response of these knockouts to hydroxyurea leads to an airing of the controversy surrounding the enigmatic plant CDC25 at the G2/M transition.

Arabidopsis T-DNA insertional lines for CDC25 are hypersensitive to hydroxyurea but not to zeocin or salt stress.

BACKGROUND AND AIMS: In yeasts and animals, cyclin-dependent kinases are key regulators of cell cycle progression and are negatively and positively regulated by WEE1 kinase and CDC25 phosphatase, respectively. In higher plants a full-length orthologue of CDC25 has not been isolated but a shorter gene with homology only to the C-terminal catalytic domain is present. The Arabidopis thaliana;CDC25 can act as a phosphatase in vitro. Since in arabidopsis, WEE1 plays an important role in the DNA damage/DNA replication checkpoints, the role of Arath;CDC25 in conditions that induce these checkpoints or induce abiotic stress was tested. Methods arath;cdc25 T-DNA insertion lines, Arath;CDC25 over-expressing lines and wild type were challenged with hydroxyurea (HU) and zeocin, substances that stall DNA replication and damage DNA, respectively, together with an abiotic stressor, NaCl. A molecular and phenotypic assessment was made of all genotypes Key RESULTS: There was a null phenotypic response to perturbation of Arath;CDC25 expression under control conditions. However, compared with wild type, the arath;cdc25 T-DNA insertion lines were hypersensitive to HU, whereas the Arath;CDC25 over-expressing lines were relatively insensitive. In particular, the over-expressing lines consistently outgrew the T-DNA insertion lines and wild type when challenged with HU. All genotypes were equally sensitive to zeocin and NaCl. CONCLUSIONS: Arath;CDC25 plays a role in overcoming stress imposed by HU, an agent know to induce the DNA replication checkpoint in arabidopsis. However, it could not enhance tolerance to either a zeocin treatment, known to induce DNA damage, or salinity stress.

Differential spatial expression of A- and B-type CDKs, and distribution of auxins and cytokinins in the open transverse root apical meristem of Cucurbita maxima.

BACKGROUND AND AIMS: Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima. METHODS: Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization. KEY RESULTS: The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM. CONCLUSIONS: Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.

Queering the (Ab)Normalization of Gender, (Hetero)Sexuality and Schooling in South Africa.

Schools are places where youth do work on the construction of their sexual identities which is intimately connected with issues around gender. Using one-on-one in-depth interviews, this article addresses how queer youth navigate dominant understandings of gender and sexuality in the context of their identity and practice. Cognizant of how gender remains a significant force in organizing social relations in schools, the youth parody and abnormalize heteronormativity calling into question the fragility of hegemonic heterosexuality. Moreover, the findings demonstrate that despite evidence that associates schooling with social exclusion, the queer youth's accounts highlight, strikingly, that queer identity and inclusion are not necessarily separate storylines. Offering an alternative view of the schooling experiences of queer youth, the paper motivates that within exclusion, in a matter of speaking with all its unduly assemblages, is inclusion.

Molecular Analysis of the E2F/DP Gene Family of Daucus carota and Involvement of the DcE2F1 Factor in Cell Proliferation.

E2F transcription factors are key components of the RB/E2F pathway that, through the action of cyclin-dependent kinases, regulates cell cycle progression in both plants and animals. Moreover, plant and animal E2Fs have also been shown to regulate other cellular functions in addition to cell proliferation. Based on structural and functional features, they can be divided into different classes that have been shown to act as activators or repressors of E2F-dependent genes. Among the first plant E2F factors to be reported, we previously described DcE2F1, an activating E2F which is expressed in cycling carrot (Daucus carota) cells. In this study, we describe the identification of the additional members of the E2F/DP family of D. carota, which includes four typical E2Fs, three atypical E2F/DEL genes, and three related DP genes. Expression analyses of the carrot E2F and DP genes reveal distinctive patterns and suggest that the functions of some of them are not necessarily linked to cell proliferation. DcE2F1 was previously shown to transactivate an E2F-responsive promoter in transient assays but the functional role of this protein in planta was not defined. Sequence comparisons indicate that DcE2F1 could be an ortholog of the AtE2FA factor of Arabidopsis thaliana. Moreover, ectopic expression of the DcE2F1 cDNA in transgenic Arabidopsis plants is able to upregulate AtE2FB and promotes cell proliferation, giving rise to polycotyly with low frequency, effects that are highly similar to those observed when over-expressing AtE2FA. These results indicate that DcE2F1 is involved in the control of cell proliferation and plays important roles in the regulation of embryo and plant development.

Nuclear Migration: An Indicator of Plant Salinity Tolerance in vitro.

In order to understand the mechanisms underlying acquisition of tolerance to salinity, we recently produced callus tissues of tobacco and Medicago truncatula resistant to NaCl-induced salt stress following application of a step-up recurrent selection method. The effects of salinity on cell size are known, but those on cell morphometry including cell and nuclear surface area and position of nuclei within salt stress resistant cells were never studied before. This work fills that gap, using suspension cultured cells of M. truncatula A17 initiated from callus, and Nicotiana tabacum BY-2 cell line resistant to increasing NaCl concentrations up to 150 mM NaCl. The surface area of salinity resistant cells of M. truncatula A17 and N. tabacum BY2 and their nuclei, produced by step-up recurrent selection, were reduced, and cells elongated as NaCl increased, but these parameters proved to be unreliable in explaining cell survival and growth at high NaCl. Conversely, nuclei of resistant cells migrated from the center to the periphery of the cytoplasm close to the walls. Nuclear marginalization was for the first time observed as a result of salt stress in plant cells, and could be a novel helpful morphological marker of acquisition of salinity tolerance.

Expression of Arabidopsis WEE1 in tobacco induces unexpected morphological and developmental changes.

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.

A strong nucleotypic effect on the cell cycle regardless of ploidy level.

BACKGROUND AND AIMS: In published studies, positive relationships between nucleotype and the duration of the mitotic cell cycle in angiosperms have been reported but the highest number of species analyzed was approx. 60. Here an analysis is presented of DNA C-values and cell cycle times in root apical meristems of angiosperms comprising 110 measurements, including monocots and eudicots within a set temperature range, and encompassing an approx. 290-fold variation in DNA C-values. METHODS: Data for 110 published cell cycle times of seedlings grown at temperatures between 20-25 degrees C were compared with DNA C-values (58 values for monocots and 52 for eudicots). Regression analyses were undertaken for all species, and separately for monocots and eudicots, diploids and polyploids, and annuals and perennials. Cell cycle times were plotted against the nuclear DNA C-values. KEY RESULTS: A positive relationship was observed between DNA C-value and cell cycle time for all species and for eudicots and monocots separately, regardless of the presence or absence of polyploid values. In this sample, among 52 eudicots the maximum cell cycle length was 18 h, whereas the 58 monocot values ranged from 8-120 h. There was a striking additional increase in cell cycle duration in perennial monocots with C-values greater than 25 pg. Indeed, the most powerful relationship between DNA C-value and cell cycle time and the widest range of cell cycle times was in perennials regardless of ploidy level. CONCLUSIONS: DNA replication is identified as a rate limiting step in the cell cycle, the flexibility of DNA replication is explored, and we speculate on how the licensing of initiation points of DNA replication may be a responsive component of the positive nucleotypic effect of C-value on the duration of the mitotic cell cycle.

Toward Unravelling the Genetic Determinism of the Acquisition of Salt and Osmotic Stress Tolerance Through In Vitro Selection in Medicago truncatula.

Changes in global climate and the nonstop increase in demographic pressure have provoked a stronger demand for agronomic resources at a time where land suitable for agriculture is becoming a rare commodity. They have also generated a number of abiotic stresses which exacerbate effects of diseases and pests and result in physiological and metabolic disorders that ultimately impact on yield when and where it is most needed. Therefore, a major scientific and agronomic challenge today is that of understanding and countering the impact of stress on yield. In this respect, in vitro biotechnology would be an efficient and feasible breeding alternative, particularly now that the genetic and genomic tools needed to unravel the mechanisms underlying the acquisition of tolerance to stress have become available. Legumes in general play a central role in a sustainable agriculture due to their capacity to symbiotically fix the atmospheric nitrogen, thereby reducing the need for fertilizers. They also produce grains that are rich in protein and thus are important as food and feed. However, they also suffer from abiotic stresses in general and osmotic stress and salinity in particular. This chapter provides a detailed overview of the methods employed for in vitro selection in the model legume Medicago truncatula for the generation of novel germplasm capable of resisting NaCl- and PEG-induced osmotic stress. We also address the understanding of the genetic determinism in the acquisition of stress resistance, which differs between NaCl and PEG. Thus, the expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1) proline synthesis (P5CS), and ploidy level and cell cycle (CCS52 and WEE1) was upregulated under NaCl stress, while under PEG treatment the expression of MtWEE1 and MtCCS52 was significantly increased, but no significant differences were observed in the expression of genes MtSERK1 and MtP5CS, and MtSOS1 was downregulated. A number of morphological and physiological traits relevant to the acquisition of stress resistance were also assessed, and methods used to do so are also detailed.

Environmental impact of estrogens on human, animal and plant life: A critical review.

BACKGROUND: Since the inception of global industrialization, steroidal estrogens have become an emerging and serious concern. Worldwide, steroid estrogens including estrone, estradiol and estriol, pose serious threats to soil, plants, water resources and humans. Indeed, estrogens have gained notable attention in recent years, due to their rapidly increasing concentrations in soil and water all over the world. Concern has been expressed regarding the entry of estrogens into the human food chain which in turn relates to how plants take up and metabolism estrogens. OBJECTIVES: In this review we explore the environmental fate of estrogens highlighting their release through effluent sources, their uptake, partitioning and physiological effects in the ecological system. We draw attention to the potential risk of intensive modern agriculture and waste disposal systems on estrogen release and their effects on human health. We also highlight their uptake and metabolism in plants. METHODS: We use MEDLINE and other search data bases for estrogens in the environment from 2005 to the present, with the majority of our sources spanning the past five years. Published acceptable daily intake of estrogens (µg/L) and predicted no effect concentrations (µg/L) are listed from published sources and used as thresholds to discuss reported levels of estrogens in the aquatic and terrestrial environments. Global levels of estrogens from river sources and from Waste Water Treatment Facilities have been mapped, together with transport pathways of estrogens in plants. RESULTS: Estrogens at polluting levels have been detected at sites close to waste water treatment facilities and in groundwater at various sites globally. Estrogens at pollutant levels have been linked with breast cancer in women and prostate cancer in men. Estrogens also perturb fish physiology and can affect reproductive development in both domestic and wild animals. Treatment of plants with steroid estrogen hormones or their precursors can affect root and shoot development, flowering and germination. However, estrogens can ameliorate the effects of other environmental stresses on the plant. CONCLUSIONS: There is published evidence to establish a causal relationship between estrogens in the environment and breast cancer. However, there are serious gaps in our knowledge about estrogen levels in the environment and a call is required for a world wide effort to provide more data on many more samples sites. Of the data available, the synthetic estrogen, ethinyl estradiol, is more persistent in the environment than natural estrogens and may be a greater cause for environmental concern. Finally, we believe that there is an urgent requirement for inter-disciplinary studies of estrogens in order to better understand their ecological and environmental impact.

PEG Induces High Expression of the Cell Cycle Checkpoint Gene WEE1 in Embryogenic Callus of Medicago truncatula: Potential Link between Cell Cycle Checkpoint Regulation and Osmotic Stress.

Polyethylene glycol (PEG) can be used to mimic osmotic stress in plant tissue cultures to study mechanisms of tolerance. The aim of this experiment was to investigate the effects of PEG (M.W. 6000) on embryogenic callus of Medicago truncatula. Leaf explants were cultured on MS medium with 2 mg L-1 NAA and 0.5 mg L-1 BAP for 5 months. Then, calli were transferred to the same medium further supplemented with 10% (w/v) 6000 PEG for 6 months in order to study physiological and putative molecular markers of water stress. There were no significant differences in growth rate of callus or mitotic index ± PEG although embryogenic potential of PEG treated callus was morphologically enhanced. Cells were rounder on PEG medium and cell size, nuclear size and endoreduplication increased in response to the PEG treatment. Significant increases in soluble sugar and proline accumulation occurred under PEG treatment compared with the control. Significantly, high MtWEE1 and MtCCS52 expression resulted from 6 months of PEG treatment with no significant differences in MtSERK1 or MtP5CS expression but down regulation of MtSOS expression. The results are consistent in showing elevated expression of a cell cycle checkpoint gene, WEE1. It is likely that the cell cycle checkpoint surveillance machinery, that would include WEE1 expression, is ameliorating the effects of the stress imposed by PEG.