A Non-Rogue Mutant Line Induced by ENU Mutagenesis in Paramutated Rogue Peas (Pisum sativum L.) Is Still Sensitive to the Rogue Paramutation.

The spontaneously emerging rogue phenotype in peas (Pisum sativum L.), characterized by narrow and pointed leaf stipula and leaflets, was the first identified case of the epigenetic phenomenon paramutation. The crosses of homozygous or heterozygous (e.g., F1) rogue plants with non-rogue (wild type) plants, produce exclusively rogue plants in the first and all subsequent generations. The fact that the wild phenotype disappears forever, is in clear contradiction with the Mendelian rules of inheritance, a situation that impedes the positional cloning of genes involved in this epigenetic phenomenon. One way of overcoming this obstacle is the identification of plant genotypes harboring naturally occurring or artificially induced neutral alleles, non-sensitive to paramutation. So far, such alleles have never been described for the pea rogue paramutation. Here, we report the induction via 1-ethyl-1-nitrosourea (ENU) mutagenesis of a non-rogue revertant mutant in the rogue cv. Progreta, and the completely unusual fixation of the induced non-rogue phenotype through several generations. The reversion of the methylation status of two previously identified differentially methylated genomic sequences in the induced non-rogue mutant, confirms that the rogue paramutation is accompanied by alterations in DNA methylation. Nevertheless, unexpectedly, the induced non-rogue mutant showed to be still sensitive to paramutation.

Acclimation of cadmium-induced genotoxicity and oxidative stress in mung bean seedlings by priming effect of phytohormones and proline.

In this research, eight local mung bean (Vigna radiata) varieties were analyzed for their performance against two levels of CdCl2 solution (0.3 and 0.5 mM) alone and priming with gibberellic acid (GA3) (100 µM), salicylic acid (SA) (50 µM) and proline (5 mM) solution prior to Cd exposure. Mung bean seedlings were analyzed for disturbance in cytological, morphological, biochemical and enzymatic parameters under cadmium stress. For cytological studies, 48 h grown mung bean seedlings root tips were used to prepare slides and studied for percent mitotic index (MI%) and to calculate percent C-mitosis, laggard, sticky and fragmented chromosomes, pictures were captured by a Nikon camera (DS-Fi 1 Japan) attached with a microscope. One-week grown mung seedlings were studied for growth traits, malondialdehyde (MDA), protein, proline and antioxidant enzymes. ANOVA and DMR test of this research revealed that all the tested mung bean varieties and treatments were significantly different regarding mitotic index and number of chromosomal aberrations. Both the Cd treatments exhibited increased total chromosomal aberrations with different types and a maximum decrease in MI%. In pretreated samples, GA3, SA and proline serve as mitigating agents that reduce mutagenic effects of Cd in mung bean by increasing MI% and decreasing chromosomal aberrations as compared to non-pretreated samples. Both the Cd treatments showed a decrease in all growth traits. Total proteins were also found to be significantly reduced in a dose-dependent manner in all genotypes. Cd treatment increased the activities of all antioxidant enzymes tested. Cd caused oxidative damage as indicated by elevated levels of MDA content in treated samples in comparison to control. Proline content levels were also high in Cd treated seedlings indicating stress. Results demonstrated that pretreatment with phytohormones and proline before Cd were found to improve all morphological parameters, by altering antioxidant enzymes activities along with a decrease in MDA and proline contents as well. It was further noticed that the performance of GA3 was better at 0.3 mM Cd treatment while SA was found to be a good mitigating agent at 0.5 mM Cd stress in all tested mung bean varieties. This research concluded less deleterious effects of Cd on AZRI-2006 while more sensitivity to NM-51 towards Cd. Priming with phytohormones and proline is a user-friendly, economical, and simple mitigation strategy to reduce Cd toxicity in plants and get better yield from contaminated lands.

Loss of an ABC transporter in Arabidopsis thaliana confers hypersensitivity to the anti-cancer drug bleomycin.

Bleomycin (BLM) is used as an anti-cancer drug clinically. However, some cancer cells are resistant to BLM, which limits the usage of BLM in chemotherapy. But the underlying mechanism of such resistance is poorly understood. Here we show that the ATP binding cassette (ABC) transporter ABCC3 is required for the BLM-resistance in Arabidopsis. In a genetic screen for ddrm (DNA damage response mutants), we found that loss of ABCC3 confers the hypersensitivity to BLM. In contrast, overexpression of ABCC3 enhances the resistance to BLM. We further found that the expression of ABCC3 is induced by BLM, which is dependent on the protein kinase ATM and the transcription factor SOG1, two master regulators of DNA damage response. Our study revealed that the ABC transporter contributes to BLM-resistance, indicating that the combination of ABC transporter inhibitors and BLM may enhance the efficacy of BLM in cancer therapy.

Comprehensive benchmarking of software for mapping whole genome bisulfite data: from read alignment to DNA methylation analysis.

Whole genome bisulfite sequencing is currently at the forefront of epigenetic analysis, facilitating the nucleotide-level resolution of 5-methylcytosine (5mC) on a genome-wide scale. Specialized software have been developed to accommodate the unique difficulties in aligning such sequencing reads to a given reference, building on the knowledge acquired from model organisms such as human, or Arabidopsis thaliana. As the field of epigenetics expands its purview to non-model plant species, new challenges arise which bring into question the suitability of previously established tools. Herein, nine short-read aligners are evaluated: Bismark, BS-Seeker2, BSMAP, BWA-meth, ERNE-BS5, GEM3, GSNAP, Last and segemehl. Precision-recall of simulated alignments, in comparison to real sequencing data obtained from three natural accessions, reveals on-balance that BWA-meth and BSMAP are able to make the best use of the data during mapping. The influence of difficult-to-map regions, characterized by deviations in sequencing depth over repeat annotations, is evaluated in terms of the mean absolute deviation of the resulting methylation calls in comparison to a realistic methylome. Downstream methylation analysis is responsive to the handling of multi-mapping reads relative to mapping quality (MAPQ), and potentially susceptible to bias arising from the increased sequence complexity of densely methylated reads.

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots.

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.

CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations ('Cu*Ag') acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of 'Cu*Ag', and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis.

Response of the Green Alga Chlamydomonas reinhardtii to the DNA Damaging Agent Zeocin.

DNA damage is a ubiquitous threat endangering DNA integrity in all living organisms. Responses to DNA damage include, among others, induction of DNA repair and blocking of cell cycle progression in order to prevent transmission of damaged DNA to daughter cells. Here, we tested the effect of the antibiotic zeocin, inducing double stranded DNA breaks, on the cell cycle of synchronized cultures of the green alga Chlamydomonas reinhardtii. After zeocin application, DNA replication partially occurred but nuclear and cellular divisions were completely blocked. Application of zeocin combined with caffeine, known to alleviate DNA checkpoints, decreased cell viability significantly. This was probably caused by a partial overcoming of the cell cycle progression block in such cells, leading to aberrant cell divisions. The cell cycle block was accompanied by high steady state levels of mitotic cyclin-dependent kinase activity. The data indicate that DNA damage response in C. reinhardtii is connected to the cell cycle block, accompanied by increased and stabilized mitotic cyclin-dependent kinase activity.

Cadmium stress alters cytosine methylation status and expression of a select set of genes in Nicotiana benthamiana.

In this paper, we evaluated the genotoxicity of cadmium (Cd) in plants by performing a methylation-sensitive amplification polymorphism (MSAP) on the model plant Nicotiana benthamiana. Among 255 loci examined, 14 genes were found to show altered cytosine methylation patterns in response to Cd stress. Four of those genes (NbMORC3, NbHGSNAT, NbMUT, and NbBG) were selected for further analysis due to their predicted roles in plant development. Cd-induced changes of cytosine methylation status in MSAP fragments of selected genes were confirmed using bisulfite sequencing polymerase chain reaction (BSP). In addition, the expression levels of these genes were found to correlate with cadmium dosage, and a knock-down of these four genes via virus-induced genes silencing (VIGS) led to abnormal development and elevated sensitivity to cadmium stress. Silencing of these four genes resulted in altered cadmium accumulation in different parts of the experimental plants. Our data indicate that cadmium exposure causes dramatic changes in the cytosine methylation status of the plant genome, thus affecting the expression of many genes that are vital for plant growth and are involved in cadmium stress response.

Fabrication of iron oxide nanocolloids using metallosurfactant-based microemulsions: antioxidant activity, cellular, and genotoxicity toward Vitis vinifera.

The present work aims at the fabrication of iron oxide nanocolloids using biocompatible microemulsion and their cytotoxic, genotoxic effect on Vitis vinifera plant has been evaluated. The three iron-based metallosurfactant complexes were synthesized. Nanosuspensions (Ns) were prepared using microemulsion technique and for the purpose, the microemulsion was prepared using oleic acid, butanol, tween 80 and as synthesized iron metallosurfactant. In this technique, no additional capping agent and/or reducing agent was added. Tween 80 which is a biocompatible surfactant acted as a reducing agent as well as stabilizing for the iron oxide Ns. Characterization of Ns's was done using TEM, FESEM, EDX, XRD, AFM, and zeta potential. Mixed type of iron oxide nanoparticles i.e. magnetite (Fe3O4), and maghemite (Fe2O3) with a size range of 1-16 nm was found to be present in the nanosuspensions prepared from all the three precursors. The antioxidant activity of the Fe Ns was also confirmed using DPPH assay, with order of activity FeDDA > FeCTAC > FeHEXA. The cellular toxicity of Ns was evaluated by observing the morphological changes on V. vinifera plant (petiole) using a light microscope. Further, the interactions of iron oxide Ns with V. vinifera's DNA (plant-DNA) was assessed using circular dichroism (CD) and gel electrophoresis. For the case of FeCTAC Ns, a decrease in the intensity of bands was observed indicating fragmentation or adduct formation resulting in DNA damage. In the case of FeDDA, a modest decrease in the intensity of bands was observed. However, for FeHEXA Ns, complete neutralization of bands was confirmed implying maximum damage to the plant DNA. CD, gel electrophoresis and antioxidant activity confirmed that FeHEXA Ns were most toxic and FeDDA Ns were safest among the three as-fabricated nanosuspensions.

Roles of CRWN-family proteins in protecting genomic DNA against oxidative damage.

CROWDED NUCLEI (CRWN) family in Arabidopsis consists of four members, CRWN1 to CRWN4. It has been previously reported that the CRWN proteins are involved in the control of nuclear morphology and degradation of ABI5. In this study, however, we discover that CRWN-family proteins are not only involved in attenuating responsiveness to abscisic acid (ABA), but also implicated in inhibiting reactive oxygen species (ROS) production and DNA damage induced by genotoxic agent methyl methanesulfonate (MMS). Our results demonstrate that three crwn double mutants, i.e. crwn1 crwn3, crwn2 crwn3, and crwn2 crwn4, show slightly earlier leaf senescence, enhanced leaf cell death, and obvious overaccumulation of ROS under regular growth conditions. When treated with 0.15 µM ABA or 0.01% MMS, two double mutants, crwn1 crwn3 and crwn2 crwn3, exhibit significant decreased germination rates as well as leaf opening and greening rates. Moreover, subsequent investigations indicate that the MMS treatment strongly inhibits the growth of crwn mutant seedlings, while this inhibition is substantially relieved by imidazole (IMZ); by contrast, DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has no effect on relief of the growth inhibition. Further studies reveal that under 0.01% MMS treatment conditions, crwn mutants, especially the three double mutants, accumulate more ROS compared to Col-0, and their genomic DNA suffers from more severe DNA damage relative to Col-0, which is indicated by significantly higher 8-oxo-7-hydrodeoxyguanosine (8-oxo dG) content as observed in the crwn mutants. Altogether, these data clearly demonstrate that the CRWN-family proteins play important roles in diminishing ROS accumulation and protecting genomic DNA against excessive oxidative damage caused by MMS.

Cytological Effects of Bleaching Agent (Quneex) on Plant Cells and Plant DNA.

BACKGROUND AND OBJECTIVE: There have been a number of reported drawbacks and efficacy issues regarding the use of bleaching agents in the plant industry. This study was conducted to determine the cytological effects of the bleaching agent (Quneex) on the plant cells and plant DNA using the Allium cepa assay. MATERIALS AND METHODS: It was subjected sixteen root meristems of A. cepa to different concentrations of the bleaching agent (0.1, 0.2, 0.3, 0.4 and 0.5%) with different periods of time (6, 12 and 24 h). Recovery was done for 6, 12 and 24 h after exposure. RESULTS: The mitotic index significantly decreased with time and also decreased with increase in the concentration of the bleaching agent. Abnormal chromosomal changes reflecting mutagenesis including stickiness, laggards, bridges, C-metaphase, star-metaphase, binucleation, polyploidy, disturbance and multinucleation were observed in the different concentrations and periods of time. After recovery, a slow increase in the mitotic index was observed. All treatments with or without recovery for 12 and 24 h resulted in reduction in the amount of DNA. CONCLUSION: Bleaching agents similar to Quneex containing sodium hypochlorite have mutagenic properties that can be potentially hazardous to the environment and also to humans. Thus, there is a need to regulate the use and disposal of such chemicals into the environment particularly to the sewers, to prevent contamination of potable water, plant and biodiverse aquatic animals.

Fusarium infection causes genotoxic disorders and antioxidant-based damages in Orobanche spp.

This study aims to evaluate the toxic effects of Fusarium oxysporum on root parasitic weed, Orobanche spp. Comparative genetic and gene expression studies were conducted on uninfected and fungus-infected orobanches. In genetic studies, isolated total DNA was amplified by RAPD PCR. Fragment properties were analysed by GTS test. According to the results, the fragment properties of control and Fusarium infected (experimental) groups varied widely; and it has been observed that Fusarium has genotoxic effects on the DNA of orobanches. In gene expression studies, the expression levels of genes encoding enzymes or proteins were associated with ROS damage and toxic effects, therefore, gene expressions of Mn-superoxide dismutase (SOD), Zn-superoxide dismutase (=SOD2, mitochondrial), glutamine synthetase (GS), heat shock protein gene (HSP70), BAX, Caspase-3 and BCL2 were significantly higher in the experimental group. In the light of obtained data, it was concluded that F. oxysporum (1) caused heavy ROS damage in Orobanche (2) induced significant irrevocable genotoxic effects on the DNA of Orobanche, (3) degraded protein metabolism and synthesis, and finally (4) triggered apoptosis. The results of this study can be a ground for further research on reducing the toxic effects of Fusarium on agricultural products, so that advancements in bio-herbicide technology may provide a sustainable agricultural production.

In planta genotoxicity of nZVI: influence of colloidal stability on uptake, DNA damage, oxidative stress and cell death.

Nanoremediation of soil, ground and surface water using nanoscale zerovalent iron particles (nZVI) has facilitated their direct environmental exposure posing ecotoxicological concerns. Numerous studies elucidate their phytotoxicity in terms of growth and their fate within the plant system. However, their potential genotoxicity and cytotoxicity mechanisms are not known in plants. This study encompasses the physico-chemical characterisation of two forms of nZVI (nZVI-1 and nZVI-2) with different surface chemistries and their influence on uptake, root morphology, DNA damage, oxidative stress and cell death in Allium cepa roots after 24 h. To our knowledge, this is the first report on the cyto-genotoxicity of nZVI in plants. The adsorption of nZVI on root surfaces caused root tip, epidermal and root hair damage as assessed by Scanning Electron Microscopy. nZVI-1, due to its colloidal destabilisation (low zeta potential, conductivity and high polydispersity index), smaller size and high uptake imparted enhanced DNA damage, chromosome/nuclear aberrations (CAs/NAs) and micronuclei formation compared to nZVI-2. Although nZVI-2 exhibited high zeta potential and conductivity, its higher dissolution and substantial uptake induced genotoxicity. nZVI incited the generation of reactive oxygen species (ROS) (hydrogen peroxide, superoxide and hydroxyl radicals) leading to membrane lipid peroxidation, electrolyte leakage and mitochondrial depolarisation. The inactivation of catalase and insignificant glutathione levels marked the onset of oxidative stress. Increased superoxide dismutase and guaiacol peroxidase enzyme activities, and proline content indicated the activation of antioxidant defence machinery to alleviate ROS. Moreover, ROS-mediated apoptotic and necrotic cell death occurred in both nZVI-1 and nZVI-2-treated roots. Our results open up further possibilities in the environmental safety appraisal of bare and modified nZVI in correlation with their physico-chemical characters.

Accumulation efficiency, genotoxicity and antioxidant defense mechanisms in medicinal plant Acalypha indica L. under lead stress.

The present study was designed to assess the physiological and biochemical changes in roots and shoots of the herb Acalypha indica grown under hydroponic conditions during exposure to lead (Pb) (100-500 mg L-1) for 1-12 d. The accumulation of Pb by A. indica plants was found to be 121.6 and 17.5 mg g-1 dry weight (DW) in roots and shoots, respectively, when exposed to a Pb concentration of 500 mg L-1. The presence of Pb ions in stem, root and leaf tissues was confirmed by scanning electron microscope (SEM) and Energy-dispersive X-ray spectroscopy (EDX) analyses. Concerning the activity of antioxidant enzymes, viz., peroxidase (POX) catalase (CAT) and ascorbate peroxidase (APX), they were induced at various regimes during 5, 8 and 12 d of Pb exposure in both the leaves and roots than untreated controls. Lead treatment increased superoxide dismutase (SOD) activity in both the leaf and root tissues over control, irrespective of the duration of exposure. Anew, it was observed that Pb treatments induced variations in the number and intensity of protein bands. Random amplified polymorphic DNA (RAPD) results show that the Pb treatment caused genotoxicity on DNA molecules as evidenced by the amplification of new bands and the absence of normal DNA amplicons in treated plants. Results confirm that A. indica is a Pb accumulator species, and the antioxidants might play a crucial role in the detoxification of Pb-induced toxic effects.

DNA replication after mutagenic treatment in Hordeum vulgare.

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed.

Impact assessment of mercury accumulation and biochemical and molecular response of Mentha arvensis: a potential hyperaccumulator plant.

The present study was focused on examining the effect of Hg oxidative stress induced physiochemical and genetic changes in M. arvensis seedlings. The growth rate of Hg treated seedlings was decreased to 56.1% and 41.5% in roots and shoots, respectively, compared to the control. Accumulation of Hg level in both roots and shoots was increased with increasing the concentration of Hg. Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were found to be increased with increasing the Hg concentration up to 20 mg/L; however, it was decreased at 25 mg/L Hg concentration. The POX enzyme activity was positively correlated with Hg dose. The changes occurring in the random amplification of ploymorphic DNA (RAPD) profiles generated from Hg treated seedlings included variations in band intensity, disappearance of bands, and appearance of new bands compared with the control seedlings. It was concluded that DNA polymorphisms observed with RAPD profile could be used as molecular marker for the evaluation of heavy metal induced genotoxic effects in plant species. The present results strongly suggested that Mentha arvensis could be used as a potential phytoremediator plant in mercury polluted environment.

Utility of RAPD marker for genetic diversity analysis in gamma rays and ethyl methane sulphonate (EMS)-treated Jatropha curcas plants.

The presence of important chemical and physical properties in Jatropha curcas makes it a valuable raw material for numerous industrial applications, including the production of biofuel. Hence, the researcher's interest is diversified to develop more and better varieties with outstanding agronomic characteristics using conventional breeding. Among these, mutation breeding is one of the best approaches to bring genetic changes in plant species. The aim of this study is to evaluate the diversity and genetic relationship among J. curcas mutants, which were obtained from different doses of gamma rays (control, 5 Kr, 10 Kr, 15 Kr, 20 Kr and 25 Kr) and EMS (1%, 2%, 3% and 4%), using RAPD marker. Among the 21 random primers, 20 produced polymorphic bands. The primers, OPM-14 and OPAW-13, produced a minimum number of bands (3) each across the ten mutants, while the primer OPF-13 produced the maximum number of bands (10), followed by the primers OPU-13, OPAM-06, OPAW-09 and OPD-05, which produced 9 bands each. The number of amplicons varied from 3 to 10, with an average of 7 bands, out of which 4.57 were polymorphic. The percentage of polymorphism ranged from 0.00 to 100 with an average of 57%. In the present study, RAPD markers were found most polymorphic, with an average polymorphism information content (PIC) value of 0.347, effective multiplex ratio (EMR) of 35.14, marker index (MI) of 14.19, resolution power (Rp) of 11.19, effective marker index (EMI) of 8.21 and genotype index (GI) of 0.36, indicating that random primers are useful in studies of genetic characterization in J. curcas mutant plants. In a dendrogram constructed based on Jaccard's similarity coefficients, the mutants were grouped into three main clusters viz., (a) control, 10 Kr, 15 Kr, 20 Kr, 2% EMS, and 3% EMS, (b) 5 Kr and 1% EMS, and (c) 25 Kr and 4% EMS mutants. Based on the attributes of the random primers and polymorphism studied, it is concluded that RAPD analysis offers a useful molecular marker for the identification of the mutants in gamma rays and EMS treated plants.

The polar auxin transport inhibitor TIBA inhibits endoreduplication in dark grown spinach hypocotyls.

We addressed the question of whether an additional round of endoreduplication in dark-grown hypocotyls is a common feature in dicotyledonous plants having endopolyploid tissues. Ploidy distributions of hypocotyl tissues derived from in vitro-grown spinach (Spinacia oleracea L. cv. Atlas) seedlings grown under different light conditions were analyzed by flow cytometry. An additional round of endoreduplication (represented by 32C cells) was found in the dark-grown hypocotyl tissues. This response was inhibited by light, the intensity of which is a crucial factor for the inhibition of endoreduplication. The higher ploidy cells in cortical tissues of the dark-grown hypocotyls had larger cell sizes, suggesting that the additional round of endoreduplication contributes to hypocotyl elongation. More importantly, a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), strongly inhibits endoreduplication, not only in spinach but also in Arabidopsis. Because other polar auxin transport inhibitors or an auxin antagonist show no or mild effects, TIBA may have a specific feature that inhibits endoreduplication.

Antioxidant potential and oxidative DNA damage preventive activity of unexplored endemic species of Curcuma.

Free radical scavenging activity, ferrous ion chelating capacity, reducing power and genoprotective effect of the aqueous leaf extracts of four unexplored endemic Curcuma spp. (C. vamana, C. neilgherrensis, C. mutabilis, C. haritha) were found to be dose-dependent and were highest in C. vamana. DNA protection property of the extracts was evaluated against H202/UV-induced oxidative damage. DNA-methyl green displacement assay showed that these extracts were free of DNA intercalating compounds. Further, hemolysis assay also showed that the extracts were non-toxic to human erythrocytes. The results highlight C. vamana as a promising source for herbal preparations possessing high antioxidant potential and genoprotective activity.

Cell-programmed death induced by walnut husk washing waters in three horticultural crops.

Walnut husk washing waters (WHWW), a by-product of walnut production, are indiscriminately used for irrigation without preliminary risk assessment. Basing on previous in vitro results on the toxicity of this by-product, we have followed the morphophysiological development of Zea mays, Lactuca sativa cv. Gentilina and L. sativa cv. Canasta under diluted and undiluted WHWW irrigation. Significant development alterations have been observed in root and shoot elongations for all crops as well as in total biomass and chlorophyll content. The genotoxic potential of WHWW has been concurrently verified; acridine orange/ethidium bromide staining evidenced chromatin modifications and DNA degradation and also was confirmed by DNA laddering. The DNA instability was also assessed through RAPD, thus suggesting the danger of the by-product of walnut processing and focusing the attention on the necessity of an efficient treatment of WHWWs. The findings obtained by PCA of agronomic and physiological traits suggested that establishing guidelines for the administration of WHWW for irrigation is of great importance, and it is necessary to supervise their use in agricultural soils.