Functional Genome Analysis of a Conditionally Pathogenic Rhizobacterial Strain, Pseudomonas putida AKMP7.

This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.

Agrobacterium-mediated Genetic Transformation of Cotton and Regeneration via Somatic Embryogenesis.

Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide. This protocol was validated in: Nat Biotechnol (2016), DOI: 10.1038/nbt.3665.

Failure of methanol detoxification in pests confers broad spectrum insect resistance in PME overexpressing transgenic cotton.

Methanol is noxious to insect pests, but most plants do not make enough of it to shield themselves from encroaching insects. Methanol emission is known to increase in the instance of herbivory. In the current study, we showed that Aspergillus niger pectin methylesterase over-expression increases methanol emission and confers resistance to polyphagous insect pests on transgenic cotton plants by impeding the possible methanol detoxification pathways. Transgenic plants emitted ∼11 fold higher methanol displaying insect mortality of 96% and 93% in Helicoverpa armigera and Spodoptera litura, respectively. The larvae were unable to survive and finish their life cycle and the surviving larvae exhibited severe growth retardation. Insects try to detoxify methanol via catalase, carboxylesterase and cytochrome P450 monooxygenase enzymes, amongst which cytochrome P450 plays a major role in oxidizing methanol to formaldehyde and formaldehyde to formic acid, which is broken down into carbon dioxide and water. In our study, catalase and esterase enzymes were found to be upregulated, but cytochrome P450 monooxygenase levels were not much affected. Leaf disc assays and In-planta bioassays also showed 50-60% population reduction in the sap sucking pests, such as Bemisia tabaci and Phenacoccus solenopsis. These findings imply that elevated methanol emissions confer resistance in plants against chewing and sap-sucking pests by tampering the methanol detoxification pathways. Such mechanism will be useful in imparting expansive resistance against pests in plants.

Toxicity assessment of metribuzin and its amelioration through plant growth regulators in Vigna radiata (L.) R. Wilczek.

The present experiment was conducted to evaluate the metribuzin-induced stress response in Vigna radiata and to explore the ameliorative role of exogenous application of plant growth regulators (PGRs) against metribuzin toxicity by assessing important biochemical and yield parameters. Prior to the field experiment, dose standardization experiments were performed, and EC50 was calculated for metribuzin. On day 21, field grown V. radiata plants were treated with graded concentrations of metribuzin (0-1000 mg [Formula: see text]). Plants treated with 600 mg [Formula: see text] (EC50) and 1000 mg [Formula: see text] (highest dose) of metribuzin were co-treated individually and simultaneously with gibberellic acid-3 (GA), indole-3 acetic acid (IAA), and salicylic acid (SA). After 7 days of treatment, leaf tissues were analyzed for biochemical parameters, whereas those related to yield were recorded during harvest. The result of this study indicated that metribuzin treatment to V. radiata resulted in increase in lipid peroxidation and reduce chlorophyll and carotenoid contents as well as yield parameters. However, metribuzin-treated plants induced proline accumulation and activity of antioxidant enzymes. Exogenous application of GA, IAA, and SA significantly reduced lipid peroxidation and increased contents of photosynthetic pigments, proline, and antioxidant enzymes thereby increasing yield parameters. It was observed that during metribuzin stress, SA exhibited a better ameliorative response out of the three exogenously applied PGRs, while the combined use of all PGRs exhibited much improved ameliorative response on biochemical and yield parameters of plants.

A systematic approach towards optimization of brackish groundwater treatment using nanofiltration (NF) and reverse osmosis (RO) hybrid membrane filtration system.

This research compares the performance efficiencies of Nanofiltration (NF), Reverse osmosis (RO), and Nanofiltration-Reverse Osmosis (NF-RO) hybrid membrane filtration systems, for treatment of brackish groundwater in Delhi-NCR region. Central composite design (CCD) of response surface methodology (RSM) were applied to formulate predictive models for the optimization and simulation of various responses, viz. Water flux, salt rejection, permeate recovery and specific energy consumption (SEC). Three different input factors (feed concentration, pH and pressure) were evaluated. Significance of RSM model was tested and validated with use of an analysis of variance (ANOVA). The models were also compared graphically for their prediction ability. The optimum conditions were determined by numerical optimization of the NF and RO membrane pilot plants. Finally, hybrid configurations of NF and RO were investigated utilising the optimum conditions to identify the technology suitable for the remediation of brackish groundwater. Despite, RO membrane eliminating over 99% of TDS from groundwater, the lower recovery rate renders it environmentally unfavourable. Result indicates that recovery of the hybrid system (40.35%) was greater than that of RO alone (18.796%) and SEC also decreases (5.090-3.8 kW H/m3). Hence, combining a hybrid membrane arrangement with NF-RO to treat brackish groundwater for improved recovery and lower SEC is a viable alternative.

Biomarker-based evaluation of cytogenotoxic potential of glyphosate in Vigna mungo (L.) Hepper genotypes.

Herbicides have proven to be a boon for agricultural fields. Their inherent property to kill weeds and unwanted vegetation makes them an essential biological tool for farmers and agricultural systems. Besides being capable of destroying weeds, they also exhibit certain effects on non-target crop plants. In the present study, a laboratory experiment was performed to assess the effect of glyphosate on Vigna mungo root meristem cells. Seeds of five different genotypes of V. mungo were treated with a series of concentrations of glyphosate ranging from 1 to 10 mM, and their effects on mitotic cell division were studied. Healthy and uniform-sized seeds were selected and were allowed to grow in Petri plates for 3 days, and all the doses were maintained in triplicates. Roots were fixed at day 3 after treatment (DAT) for cytological microscopic slide preparation. The results obtained indicate the dose-dependent reduction in the mitotic index in all the genotypes and an increase in the percentage of chromosomal aberrations (CAs) and relative abnormality rate (RAR). Most commonly observed chromosome aberrations at lower doses (< 6 mM) were fragments, stickiness, and disoriented metaphase, while at higher doses (6 to 10 mM) bridges, laggards, spindle disorientation, and clumping were obvious. The increase in the percentage of CAs and RAR indicates the inhibitory effect of glyphosate on cell cycle progression at various stages in root tip cells. The present study is a fine example of a biomarker-based genotoxic assessment of mitotic damage caused by glyphosate.

Response surface methodology and artificial neural network modelling for the performance evaluation of pilot-scale hybrid nanofiltration (NF) & reverse osmosis (RO) membrane system for the treatment of brackish ground water.

Artificial neural network (ANN) and response surface methodology (RSM) were employed to develop models for process optimisation of pilot scale nanofiltration (NF) and reverse osmosis (RO) membrane filtration system for the treatment of brackish groundwater. The process variables for this study were feed concentration, temperature, pH and pressure. The performance of NF/RO was assessed in terms of permeate flux, water recovery, salt rejection and specific energy consumption, which were considered as responses. The experimental design was employed to develop both RSM and ANN models. RSM model was validated for the whole range of experimental levels, while the ANN model was considered for the whole range of experimental design. RSM and ANN models were statistically analysed using analysis of variance (ANOVA). Further, the models were graphically compared for its predictive capacity. Numerical optimisation of NF and RO pilot plant to determine the optimum conditions were verified. Finally, using the optimum conditions, three hybrid configurations of NF and RO were studied to determine the best mode for the treatment of brackish groundwater. It was found that parallel NF-RO had a recovery of 57.18% and rejection of 44.89%, for RO-concentrate-NF (RO-C-NF) recovery was 49.55% and rejection of 38.64% & for NF-concentrate-RO (NF-C-RO), the recovery of 39.53% and rejection of 49.66% was obtained. Results obtained also suggested that the mode of configurations and the feed concentration affect the performance of the hybrid system.

Distinct defensive activity of phenolics and phenylpropanoid pathway genes in different cotton varieties toward chewing pests.

Identifying the maximum level of inherent defense against harmful insects in natural variation among wild lineages of crop plants may result in high yield tolerant varieties and reducing use of chemical insecticides. However, knowledge of natural cotton genotypes with high insect-resistance is still indistinguishable at the biochemical or molecular level. In the present study, different cultivated Gossypium hirsutum varieties were evaluated for their inherent insect-tolerance against two major cottons chewing pests. The insect bio-assay identified two tolerant and one susceptible cotton varieties. The study demonstrates difference in phenolic acids, proanthocyanidin and tannin accumulation in tolerant and susceptible varieties. The post-infestation of chewing pests increases transcript level of the phenylpropanoid pathway genes were detected in tolerant varieties as compared to the susceptible varieties. Altogether, chewing pest-tolerance level in cotton varieties is the cumulative effect of enhanced phenylpropanoid pathway genes expression and secondary metabolite leading to defense responses to conventional host plant.

Phyto-genotoxicity of arsenic contaminated soil from Lakhimpur Kheri, India on Vicia faba L.

The present experiment was designed to evaluate physico-chemical characteristics and phyto-genotoxicity of arsenic (As) contaminated soil collected from different sites of Lakhimpur, Uttar Pradesh (UP), India by employing Vicia faba L. The analyses included various biochemical as well as cyto-genotoxicity assays. The results showed that soil pH was slightly acidic to neutral in nature. The bulk density (1.18-1.23 gcm-3), particle density (2.51-2.59 gcm-3) and porosity (44-53%) varied in different places. The level of available nutrients, nitrogen, phosphorus and potassium was found to vary as 124-165 mgkg-1, 173-186 mgkg-1 and 48-98 mgkg-1, respectively. The maximum As levels were found in soil of Fulvareya (27.13 mgkg-1) and Atareya (24.12 mgkg-1), the level of As in water samples of these sites were 0.19 mgl-1and 0.21 mgl-1, respectively. Phytotoxicity of the As present in soils was evident through significant increases in stress metabolites, hydrogen peroxide (H2O2), malondialdehyde (MDA) and carbonyl groups in root and shoot of V. faba. Cyto-genotoxic effects were also seen through reduced mitotic index (MI) and increased mitotic depression (MD), relative abnormality rate (RAR) as well as other chromosomal abnormalities along with micronuclei in root meristematic cells of V. faba. The phytotoxicity and cyto-genotoxicity assessment suggests the harmful soil properties that might affect biota.

Assessment of various genetic components through NCD-I and NCD-III designs of biparental mating in opium poppy.

Creation of genetic variability and development of varieties having higher yield potential depends on information about nature of gene action. The present investigation was undertaken to decipher the nature of gene action and allied genetic parameters involved in the inheritance of yield and yield-related component traits in opium poppy (Papaver somniferum L.). The biparental inbreeding progenies derived from four segregating base populations of crosses NB-1Kr40-3/3×NB-1Kr30+0.2-2/1, NB-5Kr40-7/2×58/1, NB-1Kr30+0.2-2/1×58/1 and NB-Kr40-3/3×NB-5Kr40-7/2 of opium poppy were analysed to study the gene actions involved in the inheritance of yield and component traits. Additive component of variance played a predominant role in North Carolina design (NCD)-I, while both additive and dominance genetic components were found important in NCD-III design. The presence of additive as well as nonadditive components of variance suggested that one or two generations of intermating in further generations followed by selection may lead to development of novel genotypes.

Morphotoxicity and cytogenotoxicity of pendimethalin in the test plant Allium cepa L. - A biomarker based study.

Pesticides have brought tremendous benefits to mankind by increasing food production and controlling various crop diseases. But their prolonged and extensive use has been reported to induce toxicity. Biological markers used for the evaluation of toxic effects of pesticides have increased these days. The aim of this study was to determine the morphotoxic and cytogenotoxic effects of pesticide pendimethalin applied to the soil by using morphological and genotoxic biomarkers in the test plant Allium cepa L. A pot experiment was set up in which pendimethalin was added to soil at the rate of 0, 0.033, 0.044, 0.055 and 0.066 g kg-1 soil. Similar sized onion bulbs were planted in each pot and 3 replicates were maintained for each dose of pendimethalin at 1, 7, 15, 30 and 45 days after treatment. Average root number (ARN) and average length of roots (ALR) of onion bulbs were recorded and on the day 3 of sowing roots were harvested and fixed for cytological analysis. Morphological biomarkers revealed significant concentration and duration dependent inhibition of ARN and ALR as compared to control which shows the morphotoxicity of pendimethalin. The results also showed inhibitory effect on the mitotic index (%) of A. cepa while relative abnormality rate (%) increased. Further, we observed aberrations in both the dividing and non-dividing cells along with spotting of few ring chromosomes. Reduced mitotic index, increased relative abnormality rate; various chromosomal and interphase nuclear aberrations all being mitosis endpoint markers reflect the cytogenotoxicity of pendimethalin, even at lower concentrations.

Cyto-genotoxic consequences of carbendazim treatment monitored by cytogenetical analysis using Allium root tip bioassay.

Environmental pollution is one of the major problems of these days. One of the reasons of environmental pollution is the indiscriminate use of agrochemicals in agriculture. Fungicides are being extensively used in agriculture for enhancing crop yield and growth by controlling fungal growth. Fungicide carbendazim is widely applied to soil and seeds of vegetable/cereal crops in India and is effective against a very broad spectrum of fungi. The present study was designed to monitor the cyto-genotoxic effects of carbendazim directly in treated soils by cytogenetical analysis using Allium cepa root tip bioassay. In a pot experiment, fungicide carbendazim was added to soil at the rates of 2.5, 5, 7.5, and 10 mg kg-1 soil and uniform size onion bulb was planted in each pot, and three replicates were maintained for each dose at 1, 7, 15, 30, and 45 days after application and roots from onion bulbs were fixed for cytogenetical analysis. Findings indicate that carbendazim treatment leads to a significant dose and duration-dependent decrease in percent mitotic index with related increase in mitotic inhibition. Statistical analysis showed a significant effect of carbendazim doses and duration of treatment on the percentage relative abnormality rate of A. cepa. Phase indices of our study showed high numbers of cells in prophase as compared to other phases at some doses of treatment. The different types of chromosomal abnormalities observed in our study serve as indicators of genotoxicity of carbendazim and we report for the first time the effect of its application directly in soil using a plant test system.

Phytotoxicity of pesticides mancozeb and chlorpyrifos: correlation with the antioxidative defence system in Allium cepa.

Pesticides are a group of chemical substances which are widely used to improve agricultural production. However, these substances could be persistent in soil and water, accumulative in sediment or bio-accumulative in biota depending on their solubility, leading to different types of environmental pollution. The present study was done to assess the impact of pesticides-mancozeb and chlorpyrifos, via morphological and physiological parameters using Allium cepa test system. Phytotoxic effects of pesticides were examined via germination percentage, survival percentage, root and shoot length, root shoot length ratio, seedling vigor index, percentage of phytotoxicity and tolerance index. Oxidative stress on Allium seedlings caused by pesticides was also assessed by investigating the activity of antioxidative enzymes viz. catalase, peroxidase and superoxide dismutase. Correlation was worked out between morphological parameters and antioxidative enzymes to bring out the alliance between them. Mancozeb and chlorpyrifos concentrations were significantly and positively correlated with the activity of antioxidative enzymes and negatively correlated with morphological parameters. Significant positive correlation between various morphological parameters showed their interdependency. However, negative correlation was obtained between activity of antioxidative enzymes and morphological parameters. The enzymes however, showed positive correlation with each other. Based on our result we can conclude that all morphological parameters were adversely affected by the two pesticides as reflected by phytotoxicity in Allium. Their negative correlation with activity of antioxidative enzymes indicates that upregulation of antioxidative enzymes is not sufficient to overcome the toxic effect, thereby signifying the threat being caused by the regular use of these pesticides.

Monitoring of morphotoxic, cytotoxic and genotoxic potential of mancozeb using Allium assay.

The present experiment was designed to monitor the morphotoxic, cytotoxic and genotoxic potential of Mancozeb (fungicide) in non-target plants using bulbs of Allium cepa. Mancozeb is classified as a contact fungicide and is registered for use on a variety of crop plants. In the present monitoring, Allium cepa bulbs were exposed to different concentrations of mancozeb viz., 10, 30, 50, 70, 90, 110, 130 and 150 ppm for 24 and 48 h. The potential morphotoxic and cytotoxic effects of mancozeb were examined by determining the average root number, average root length, mitotic index, relative abnormality rate (%) and frequency of abnormalities (%). A progressive significant concentration and time dependent inhibition of the average root number, average root length indicated the morphotoxic nature. The cytotoxic effect was significantly increased for 48 h treatment as compared to 24 h treatment time, by reducing the mitotic index of meristematic cells. The results indicated an indirect genotoxic effect by inducing different types of chromosomal abnormalities, likely sticky, disoriented and fragmented chromosomes. Thus indicating that the investigated fungicide have genotoxic potential due to abnormal DNA condensation and chromosome coiling by spindle inactivation. The observations of cyto and genotoxic effects suggest that the fungicide mancozeb is clastogenic agent. Thus the different concentrations used in the field could be harmful for the end-receptors of food-chain and needs constant monitoring and management for the better development of crop plants.

Cytomorphologic parameters in monitoring cytogenotoxic effects of fertilizer in Allium cepa L.

The present investigation was done to assess cytomorphologic parameters as indicators of genotoxicity as it is a simpler method and could be suggested for rapid screening of the vast range of agrochemicals used all over the world. The excessive and indiscriminate use of agrochemicals is responsible for increasing the level of pollutants in the soil environment resulting in cellular and molecular damage to the plants. The cellular damage caused manifestation of the resulting oxidative stress due to pollutants which can go up to the level of DNA. The roots of Allium cepa were treated with 0.5 mg N ml-1 concentration of ammonium nitrate fertilizer for 3, 6, 9 and 12 h, and in mitotic preparation of their respective root, mitotic index, phase indices and the genotoxic markers viz. chromosomal aberrations and binucleate cells were observed and the data statistically analysed. A significant decrease in mitotic index and increase in abnormality percentage as compared to control was observed which increased with the treatment duration. Chromosomal aberrations like stickiness, fragmentation, precocious movement, bridges and disorientations were observed in varying frequencies. A cytomorphologic study revealed that the interphase cell volume of cells of treated roots and their respective interphase nuclear volume were reduced as compared to control. The ratio between nuclear and cytoplasmic volume has been reported to relate to cell integrity. Both these markers viz. cytomorphologic and genotoxic can be used for assessment of the toxicity of agrochemicals including fertilizer; in our study, they have revealed the cytogenotoxic behaviour of ammonium nitrate fertilizer.

Nitric oxide ameliorates the damaging effects of oxidative stress induced by iron deficiency in cyanobacterium Anabaena 7120.

In cyanobacterium Anabaena 7120, iron deficiency leads to oxidative stress with unavoidable consequences. Nitric oxide reduces pigment damage and supported the growth of Anabaena 7120 in iron-deficient conditions. Elevation in nitric oxide accumulation and reduced superoxide radical production justified the role of nitric oxide in alleviating oxidative stress in iron deficiency. Increased activities of antioxidative enzymes and higher levels of ROS scavengers (ascorbate, glutathione and thiol) in iron deficiency were also observed in the presence of nitric oxide. Nitric oxide also supported the membrane integrity of Anabaena cells and reduces protein and DNA damage caused by oxidative stress induced by iron deficiency. Results suggested that nitric oxide alleviates the damaging effects of oxidative stress induced by iron deficiency in cyanobacterium Anabaena 7120.

Biomonitoring of air pollution using antioxidative enzyme system in two genera of family Pottiaceae (Bryophyta).

Bryophyte particularly mosses, have been found to serve as reliable indicators of air pollution and can serve as bryometers-biological instruments for measuring air pollution. They are remarkable colonizers, as they have the ability to survive in adverse environments and are also particular in their requirement of environmental conditions, which makes them appropriate ecological indicators. The purpose of this study was to evaluate the activity of antioxidative enzymes in two mosses viz., Hyophila rosea R.S. Williams and Semibarbula orientalis (Web.) Wijk. & Marg. and assess their suitability as biomonitors. Three different locations viz., Lucknow University, Residency (contaminated sites) and Dilkusha Garden (reference site) within Lucknow city with different levels of air pollutants were used for comparison. Our results indicate that air pollution caused marked enhancement in activity of antioxidative enzymes viz., catalase, peroxidase and superoxide dismutase. All the three are capable of scavenging reactive oxygen species. In the genus S. orientalis, catalase, peroxidase and superoxide dismutase activity was minimum at the reference site Dilkusha Garden and was significantly higher at the two contaminated sites for catalase and peroxidase, whereas the difference was non significant for superoxide dismutase. In H. rosea the activity of catalase and peroxidase at the three locations was almost similar, however superoxide dismutase activity showed a significant increase in the two contaminated sites when compared to the reference site, the value being highest for Lucknow University site. It was thus observed that the two genera, from the same location, showed difference in the activity of the antioxidative enzymes. Based on our results, we recommend bryophytes as good monitors of air pollution.

Monomerization alters the dynamics of the lid region in Campylobacter jejuni CstII: an MD simulation study.

CstII, a bifunctional (α2,3/8) sialyltransferase from Campylobacter jejuni, is a homotetramer. It has been reported that mutation of the interface residues Phe121 (F121D) or Tyr125 (Y125Q) leads to monomerization and partial loss of enzyme activity, without any change in the secondary or tertiary structures. MD simulations of both tetramer and monomer, with and without bound donor substrate, were performed for the two mutants and WT to understand the reasons for partial loss of activity due to monomerization since the active site is located within each monomer. RMSF values were found to correlate with the crystallographic B-factor values indicating that the simulations are able to capture the flexibility of the molecule effectively. There were no gross changes in either the secondary or tertiary structure of the proteins during MD simulations. However, interface is destabilized by the mutations, and more importantly the flexibility of the lid region (Gly152-Lys190) is affected. The lid region accesses three major conformations named as open, intermediate, and closed conformations. In both Y121Q and F121D mutants, the closed conformation is accessed predominantly. In this conformation, the catalytic base His188 is also displaced. Normal mode analysis also revealed differences in the lid movement in tetramer and monomer. This provides a possible explanation for the partial loss of enzyme activity in both interface mutants. The lid region controls the traffic of substrates and products in and out of the active site, and the dynamics of this region is regulated by tetramerization. Thus, this study provides valuable insights into the role of loop dynamics in enzyme activity of CstII.

Molecular events during the early stages of aggregation of GNNQQNY: An all atom MD simulation study of randomly dispersed peptides.

This study probes the early events during lag phase of aggregation of GNNQQNY using all atom MD simulations in explicit solvent. Simulations were performed by varying system size, temperature and starting configuration. Peptides dispersed randomly in the simulation box come together early on in the simulation and form aggregates. These aggregates are dynamic implying the absence of stabilizing interactions. This facilitates the exploration of alternate arrangements. The constituent peptides sample a variety of conformations, frequently re-orient and re-arrange with respect to each other and dissociate from/re-associate with the aggregate. The size and lifetime of aggregates vary depending upon the number of inter-peptide backbone H-bonds. Most of the aggregates formed are amorphous but crystalline aggregates of smaller size (mainly 2-mers) do appear and sustain for varying durations of time. The peptides in crystalline 2-mers are mostly anti-parallel. The largest crystalline aggregate that appears is a 4-mer in a single sheet and a 4-, 5-, or 6-mer in double layered arrangement. Crystalline aggregates grow either by the sequential addition of peptides, or by the head-on or lateral collision-adhesion of 2-mers. The formation of various smaller aggregates suggests the polymorphic nature of oligomers and heterogeneity in the lag phase.