Meristematic Region Research Articles

Eco(geno)toxicity of an acaricidal formulation containing chlorpyrifos, cypermethrin, and fenthion on different plant models and Artemia salina L.

The mixture of pesticides is widely employed in cattle farming to combat ectoparasite resistance, such as ticks. The commercial formulation COLOSSO FC30, which contains three active ingredients (Cypermethrin, Chlorpyrifos, and Fenthion), stands out due to its efficiency. However, animals exposed to this product may become vectors of potentially toxic molecules, possibly causing contamination in aquatic and terrestrial ecosystems. In light of this, this study evaluated the eco(geno)toxic potential of the commercial formulation COLOSSO FC30, using plants (Allium cepa L., Lactuca sativa L., Raphanus sativus L., Pennisetum glaucum L., and Triticum aestivum L.) and Artemia salina L. as model organisms. In the phytotoxicity test, the species were ranked in order of sensitivity to the commercial formulation as follows: P. glaucum > L. sativa > T. aestivum > R. sativus. The most sensitive parameters were root length (RL) and shoot length (SL) of seedlings. In the cytogenotoxicity test with A. cepa, cell division was decreased at concentrations from 0.351mL L-1 in the meristematic region and root F1. Chromosomal aberrations and micronucleus were observed at all concentrations. In the test with A. salina, the IC50 after 24h of exposure was 0.01207mL L-1 of the commercial formulation. The results highlight the need for further research and regulations to understand and minimize the potential environmental impacts of COLOSSO FC30.

Effects of SpGSH1 and SpPCS1 overexpression or co-overexpression on cadmium accumulation in yeast and Spirodela polyrhiza

Cadmium (Cd) is one of the most toxic elements to all organisms. Glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is considered an extremely important mechanism in Cd detoxification in plants. However, few studies have focused on the roles of glutamate-cysteine ligase (GSH1) and phytochelatin synthase (PCS1) in Cd accumulation and detoxification in plants. In this study, SpGSH1 and SpPCS1 were identified and cloned from Spirodela polyrhiza and analyzed their functions in yeast and S. polyrhiza via single- or dual-gene (SpGP1) overexpression. The findings of this study showed that SpGSH1, SpPCS1, and SpGP1 could dramatically rescue the growth of the yeast mutant Δycf1. In S. polyrhiza, SpGSH1 was located in the cytoplasm and could promote Mn and Ca accumulation. SpPCS1 was located in the cytoplasm and nucleus, mainly expressed in meristem regions, and promoted Cd, Fe, Mn, and Ca accumulation. SpGSH1 and SpPCS1 co-overexpression increased the Cd, Mn, and Ca contents. Based on the growth data of S. polyrhiza, it was recommended that biomass as the preferable indicator for assessing plant tolerance to Cd stress compared to frond number in duckweeds. Collectively, this study for the first time systematically elaborated the function of SpGSH1 and SpPCS1 for Cd detoxification in S. polyrhiza.

Visualization of metabolite distribution based on matrix-assisted laser desorption/ionization-mass spectrometry imaging of tea seedlings (Camellia sinensis).

Tea seedlings (Camellia sinensis) have a well-developed root system with a strong taproot and lateral roots. Compared with ordinary cuttings, tea has stronger vitality and environmental adaptability, thus facilitating the promotion of good varieties. However, there is less of detailed research on the rooting and germination process of tea seeds. In this study, matrix-assisted laser desorption ionization time-of-flight-mass spectrometry was used to conduct non-targeted spatial mass spectrometry imaging of the main organs during growth of tea seedlings. A total of 1234 compounds were identified, which could be divided into 24 classes. Among them, theanine, as the most prominent nitrogen compound, was synthesized rapidly at the early stage of embryo germination, accounting for >90% of the total free amino acids in the radicle, and it was then transferred to each meristem region through the mesocolumnar sheath, indicating that theanine-based nitrogen flow plays a decisive role in organ formation during the development of tea seedlings. Nutrients stored in the cotyledon were rapidly hydrolyzed to dextrin and 3-phosphoglyceraldehyde at the early stages of germination, and subsequently converted to other forms that provided carbon and energy for development, such as raffinose and d-galactose (glucose), which were mainly distributed in the growing zones of the root apex and the apical meristems of the stem. This study provides a new perspective on the synthesis and metabolism of substances during the development of tea seedlings and contributes to a better understanding of the biological characteristics of tea varieties.

Polyploidization Impact on Plant Architecture of Watermelon (Citrullus lanatus)

Plant architecture includes traits such as plant height, stem diameter, and branching pattern, which have significant impacts on yield and fruit quality. Polyploidization can bring changes in plant architectural traits in different crops along with other agronomic and biochemical attributes; however, the specific physiological and biochemical mechanisms are still unclear. In this study, we utilized five watermelon lines: ‘91E7’, ‘Zhengzhou No. 3’, ‘Fanzu No. 1’, ‘Shenlong’, and ‘Houlv’, along with their corresponding autopolyploid derivatives (diploid, autotriploid, and autotetraploid) to compare plant architecture differences in different polyploidy watermelon plants. The results showed that the growth habits of diploid, triploid, and tetraploid watermelon plants were noticeably different. Triploid and tetraploid watermelon plants had greater stem diameters and larger leaf sizes. The leaf angle was also larger in polyploid watermelons than in their diploid ancestor lines. Although vine length was significantly higher in diploid watermelon, there was no significant difference in node number, indicating that the short vine length was due to the short internodal length. The major differences between diploid and polyploid watermelon plants were found in the branching pattern, as diploid watermelon lines have more branching compared to their polyploid sister lines. Furthermore, we examined the phytohormone content of diploid, triploid, and tetraploid ‘Fanzu No. 1’. The reasons for the selection of this material are its robust growth and profuse branching habit, which cause visible differences among the ploidy levels. Hormone analysis showed distinct variations in abscisic acid in the nodal and stem regions, gibberellin in the auxiliary bud regions, and brassinosteroids in the apical meristematic regions. The correlation coefficient also strongly correlated these hormones with architecture-related traits. Our findings indicated that gibberellin, ABA, and brassinosteroids might be associated with variations in plant architectural traits like branching, vine length, internodal length, stem thickness, and leaf angle among different ploidy levels of watermelon. The exogenous application of GA3 showed a positive effect on branching, whereas ABA showed a negative effect on branching. The application of brassinosteroid at the apical meristem demonstrated its effect on leaf angle, leaf size, and internodal length. The results of this study can provide a theoretical reference and valuable insights into the link between plant architecture and ploidy levels.

In Vitro Multiplication and Ontogeny of Ferns Tectaria zeylanica (Houtt.) (Dryoptridaceae) from South India under Current Climate Conditions

The oak leaf fern, Tectaria zeylanica (Houtt.) Sledge, is an endangered fern in India, native to the Southeast Asian region. For the ex-situ conservation of this endangered fern, spores were collected and cultured aseptically on full-strength KC Medium with sucrose at pH 5.8. The spores are monolete with a prominent winged perispore. The exine ruptures at the laesura, and three or four germ filaments grow out, following a Vittaria-type germination pattern. The prothallial development was of the Aspidium type. Adult gametophytes are cordate and much broader than their length. Antheridia develop between 120 and 140 days. They are characteristically 3-celled, with a basal cell, a median cell, and an opercular cell. The female sex organs, archegonia, develop between 150 and 170 days. They are superficial and arranged on the lower side of the prothallus near the midrib and the meristematic region. Fertilization and subsequent development of the sporophytes were observed after four months. The highest percentage of sporophyte formation was observed on KC medium fortified with Kin 0.5 mg/l. Sporophytes had uniseriate, unicellular, and multicellular hairs, similar to those found on adult prothalli. The highest percentage of root formation was observed in the medium with IBA + NAA (1.0 mg/l). The in vitro raised plants showed a 54% establishment during hardening in the field. The present study has established a reproducible protocol for the ex-situ conservation of this endangered oak leaf fern.

Decoding the leaf apical meristem of Guarea glabra Vahl (Meliaceae): insight into the evolution of indeterminate pinnate leaves

In seed plants, growth of shoots and roots is indeterminate, while leaves are typically determinate organs that cease to grow after a certain developmental stage. This is due to the characteristics of the leaf meristem, where cell proliferation activity is retained only for a limited period. However, several plants exhibit indeterminacy in their leaves, exemplified by the pinnate compound leaves of Guarea and Chisocheton genera in the Meliaceae family. In these plants, the leaf meristem at the tip of the leaf retains meristematic activity and produces leaflets over years, resulting in a single leaf that resembles a twig. The molecular mechanism underlying the indeterminate leaf meristem of these plants has not been examined. In this research, we used Guarea glabra as a model to investigate the development of indeterminate pinnate leaves. Transcriptome analyses revealed that the gene expression profile in leaf apex tissue differed from that in the shoot apex. However, a class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) gene which is lost in Brassicaceae was highly expressed in both tissues. We established an in situ hybridisation system for this species using Technovit 9100 to analyse the spatial expression patterns of genes. We revealed that the leaf meristematic region of G. glabra expresses KNOX1, LEAFY and ANGUSTIFORIA3 simultaneously, suggesting the involvement of these genes in the indeterminacy of the leaf meristem.

Regulation of Flowering Time and Other Developmental Plasticities by 3' Splicing Factor-Mediated Alternative Splicing in Arabidopsis thaliana.

Plants, as sessile organisms, show a high degree of plasticity in their growth and development and have various strategies to cope with these alterations under continuously changing environments and unfavorable stress conditions. In particular, the floral transition from the vegetative and reproductive phases in the shoot apical meristem (SAM) is one of the most important developmental changes in plants. In addition, meristem regions, such as the SAM and root apical meristem (RAM), which continually generate new lateral organs throughout the plant life cycle, are important sites for developmental plasticity. Recent findings have shown that the prevailing type of alternative splicing (AS) in plants is intron retention (IR) unlike in animals; thus, AS is an important regulatory mechanism conferring plasticity for plant growth and development under various environmental conditions. Although eukaryotes exhibit some similarities in the composition and dynamics of their splicing machinery, plants have differences in the 3' splicing characteristics governing AS. Here, we summarize recent findings on the roles of 3' splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities in Arabidopsis thaliana.

Preserving root stem cell functionality under low oxygen stress: the role of nitric oxide and phytoglobins.

The preservation of quiescent center stem cell integrity in hypoxic roots by phytoglobins is exercised through their ability to scavenge nitric oxide and attenuate its effects on auxin transport and cell degradation. Under low oxygen stress, the retention or induction of phytoglobin expression maintains cell viability while loss or lack of induction of phytoglobin leads to cell degradation. Plants have evolved unique attributes to ensure survival in the environment in which they must exist. Common among the attributes is the ability to maintain stem cells in a quiescent (or low proliferation) state in unfriendly environments. From the seed embryo to meristematic regions of the plant, quiescent stem cells exist to regenerate the organism when environmental conditions are suitable to allow plant survival. Frequently, plants dispose of mature cells or organs in the process of acclimating to the stresses to ensure survival of meristems, the stem cells of which are capable of regenerating cells and organs that have been sacrificed, a feature not generally available to mammals. Most of the research on plant stress responses has dealt with how mature cells respond because of the difficulty of specifically examining plant meristem responses to stress. This raises the question as to whether quiescent stem cells behave in a similar fashion to mature cells in their response to stress and what factors within these critical cells determine whether they survive or degrade when exposed to environmental stress. This review attempts to examine this question with respect to the quiescent center (QC) stem cells of the root apical meristem. Emphasis is put on how varying levels of nitric oxide, influenced by the expression of phytoglobins, affect QC response to hypoxic stress.

Worldwide Population Genomics Reveal Long-Term Stability of the Mitochondrial Genome Architecture in a Keystone Marine Plant.

Mitochondrial genomes (mitogenomes) of flowering plants are composed of multiple chromosomes. Recombination within and between the mitochondrial chromosomes may generate diverse DNA molecules termed isoforms. The isoform copy number and composition can be dynamic within and among individual plants due to uneven replication and homologous recombination. Nonetheless, despite their functional importance, the level of mitogenome conservation within species remains understudied. Whether the ontogenetic variation translates to evolution of mitogenome composition over generations is currently unknown. Here we show that the mitogenome composition of the seagrass Zostera marina is conserved among worldwide populations that diverged ca. 350,000 years ago. Using long-read sequencing, we characterized the Z. marina mitochondrial genome and inferred the repertoire of recombination-induced configurations. To characterize the mitochondrial genome architecture worldwide and study its evolution, we examined the mitogenome in Z. marina meristematic region sampled in 16 populations from the Pacific and Atlantic oceans. Our results reveal a striking similarity in the isoform relative copy number, indicating a high conservation of the mitogenome composition among distantly related populations and within the plant germline, despite a notable variability during individual ontogenesis. Our study supplies a link between observations of dynamic mitogenomes at the level of plant individuals and long-term mitochondrial evolution.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

An enhancer trap system to track developmental dynamics in Marchantia polymorpha.

A combination of streamlined genetics, experimental tractability and relative morphological simplicity compared to vascular plants makes the liverwort Marchantia polymorpha an ideal model system for studying many aspects of plant biology. Here we describe a transformation vector combining a constitutive fluorescent membrane marker with a nuclear marker that is regulated by nearby enhancer elements and use this to produce a library of enhancer trap lines for Marchantia. Screening gemmae from these lines allowed the identification and characterization of novel marker lines, including markers for rhizoids and oil cells. The library allowed the identification of a margin tissue running around the thallus edge, highlighted during thallus development. The expression of this marker is correlated with auxin levels. We generated multiple markers for the meristematic apical notch region, which have different spatial expression patterns, reappear at different times during meristem regeneration following apical notch excision and have varying responses to auxin supplementation or inhibition. This reveals that there are proximodistal substructures within the apical notch that could not be observed otherwise. We employed our markers to study Marchantia sporeling development, observing meristem emergence as defining the protonema-to-prothallus stage transition, and subsequent production of margin tissue during the prothallus stage. Exogenous auxin treatment stalls meristem emergence at the protonema stage but does not inhibit cell division, resulting in callus-like sporelings with many rhizoids, whereas pharmacologically inhibiting auxin synthesis and transport does not prevent meristem emergence. This enhancer trap system presents a useful resource for the community and will contribute to future Marchantia research.

Characterization of the Molecular Events Underlying the Establishment of Axillary Meristem Region in Pepper.

Plant architecture is a major motif of plant diversity, and shoot branching patterns primarily determine the aerial architecture of plants. In this study, we identified an inbred pepper line with fewer lateral branches, 20C1734, which was free of lateral branches at the middle and upper nodes of the main stem with smooth and flat leaf axils. Successive leaf axil sections confirmed that in normal pepper plants, for either node n, Pn (Primordium n) < 1 cm and Pn+1 < 1 cm were the critical periods between the identification of axillary meristems and the establishment of the region, whereas Pn+3 < 1 cm was fully developed and formed a completely new organ. In 20C1734, the normal axillary meristematic tissue region establishment and meristematic cell identity confirmation could not be performed on the axils without axillary buds. Comparative transcriptome analysis revealed that "auxin-activated signaling pathway", "response to auxin", "response to abscisic acid", "auxin biosynthetic process", and the biosynthesis of the terms/pathways, such as "secondary metabolites", were differentially enriched in different types of leaf axils at critical periods of axillary meristem development. The accuracy of RNA-seq was verified using RT-PCR for some genes in the pathway. Several differentially expressed genes (DEGs) related to endogenous phytohormones were targeted, including several genes of the PINs family. The endogenous hormone assay showed extremely high levels of IAA and ABA in leaf axils without axillary buds. ABA content in particular was unusually high. At the same time, there is no regular change in IAA level in this type of leaf axils (normal leaf axils will be accompanied by AM formation and IAA content will be low). Based on this, we speculated that the contents of endogenous hormones IAA and ABA in 20C1734 plant increased sharply, which led to the abnormal expression of genes in related pathways, which affected the formation of Ams in leaf axils in the middle and late vegetative growth period, and finally, nodes without axillary buds and side branches appeared.

A network of CLAVATA receptors buffers auxin-dependent meristem maintenance.

Plant body plans are elaborated in response to both environmental and endogenous cues. How these inputs intersect to promote growth and development remains poorly understood. During reproductive development, central zone stem cell proliferation in inflorescence meristems is negatively regulated by the CLAVATA3 (CLV3) peptide signalling pathway. In contrast, floral primordia formation on meristem flanks requires the hormone auxin. Here we show that CLV3 signalling is also necessary for auxin-dependent floral primordia generation and that this function is partially masked by both inflorescence fasciation and heat-induced auxin biosynthesis. Stem cell regulation by CLAVATA signalling is separable from primordia formation but is also sensitized to temperature and auxin levels. In addition, we uncover a novel role for the CLV3 receptor CLAVATA1 in auxin-dependent meristem maintenance in cooler environments. As such, CLV3 signalling buffers multiple auxin-dependent shoot processes across divergent thermal environments, with opposing effects on cell proliferation in different meristem regions.

Analysis of polycomb repressive complex 2 (PRC2) subunits in Picea abies with a focus on embryo development

BackgroundConserved polycomb repressive complex 2 (PRC2) mediates H3K27me3 to direct transcriptional repression and has a key role in cell fate determination and cell differentiation in both animals and plants. PRC2 subunits have undergone independent multiplication and functional divergence in higher plants. However, relevant information is still absent in gymnosperms.ResultsTo launch gymnosperm PRC2 research, we identified and cloned the PRC2 core component genes in the conifer model species Picea abies, including one Esc/FIE homolog PaFIE, two p55/MSI homologs PaMSI1a and PaMSI1b, two E(z) homologs PaKMT6A2 and PaKMT6A4, a Su(z)12 homolog PaEMF2 and a PaEMF2-like fragment. Phylogenetic and protein domain analyses were conducted. The Esc/FIE homologs were highly conserved in the land plant, except the monocots. The other gymnospermous PRC2 subunits underwent independent evolution with angiospermous species to different extents. The relative transcript levels of these genes were measured in endosperm and zygotic and somatic embryos at different developmental stages. The obtained results proposed the involvement of PaMSI1b and PaKMT6A4 in embryogenesis and PaKMT6A2 and PaEMF2 in the transition from embryos to seedlings. The PaEMF2-like fragment was predominantly expressed in the endosperm but not in the embryo. In addition, immunohistochemistry assay showed that H3K27me3 deposits were generally enriched at meristem regions during seed development in P. abies.ConclusionsThis study reports the first characterization of the PRC2 core component genes in the coniferous species P. abies. Our work may enable a deeper understanding of the cell reprogramming process during seed and embryo development and may guide further research on embryonic potential and development in conifers.

Population dynamics of Steneotarsonemus concavuscutum, a neglected mite pest in coconut fruits (Cocos nucifera).

The meristematic region of Cocos nucifera fruits can be colonized by various species of mites, including Steneotarsonemus concavuscutum, Steneotarsonemus furcatus, and Aceria guerreronis. The consequence of this colonization is the development of necrotic lesions on the fruit, and sometimes its abortion. Losses are commonly attributed to A. guerreronis alone, owing to the similarities in the injuries caused and its predominance in coconut plantations. However, S. concavuscutum may be the predominant pest species in some crops. Despite the possible impact of S. concavuscutum, little is known about its bioecological aspects, such as the influence of biotic and abiotic factors on its population dynamics. Our objective was to document macroclimatic abiotic factors (temperature, relative humidity, and precipitation) and biotic factors (interspecific competition and predation) interfere in the population dynamics of S. concavuscutum. We evaluated the diversity and abundance of mites in the perianth of coconut fruit naturally infested by S. concavuscutum for 1 year. The species found in the fruits of bunch 6 of the plant, which is the fruit age at which the mites commonly reach the highest abundance, were counted every 2 weeks. We found mites from nine families and S. concavuscutum was the predominant species, representing about 92% of the individuals collected. Predators represented approximately 2% of the total collection, with Neoseiulus baraki as the predominant species. Steneotarsonemus concavuscutum population density ranged from 60 to 397 mites/fruit. The highest population densities of S. concavuscutum were observed in the hottest and driest periods of the year. The population densities of S. concavuscutum were negatively associated with the presence of N. baraki, suggesting that this predator may have a role in the biological control of this pest.

Toxicity Analysis of Some Frequently Used Food Processing Chemicals Using Allium cepa Biomonitoring System.

Frequent use of various food processing chemical agents sometimes causes damage to our bodies by inducing cytotoxicity, genotoxicity, and mutagenesis. In Bangladesh, among various chemical agents, formalin, saccharin, and urea are vastly used for processing foodstuffs by industry and local people. This study is focused to assess the toxic effects of formalin, saccharin, and urea on the popularly used eukaryotic test model, Allium cepa L. The assay was carried out by exposing different concentrations of test samples to A. cepa at 24, 48, and 72 h, where distilled water and CuSO4·5H2O (0.6 µg/mL) were utilized as the vehicle and positive control, respectively. The root length of the onions was measured in mm, and the results propose that all the chemical agents demonstrated toxicity in onions in a concentration- and exposure-time-dependent manner. The highest root length was examined at the lower concentrations, and with the increase in the concentration of the test sample and exposure time, the RG (root growth) was inhibited due to the deposition of chemicals and hampering of cell division in the root meristematic region of A. cepa. All the chemical agents also revealed a concentration- and time-dependent adaptive effect up to 72 h inspection of 24 h and a depletion of % root growth at 72 h inspection of 48 h. Our study suggests that sufficient precautions should be confirmed during its industrial and traditional usage as a toxicological response to the chemical agents observed in the A. cepa assay.

Control effects and sublethal effects of four pesticides on Atheloca bondari (Lepidoptera: Pyralidae) caterpillars

• The concentration of abamectin used to control A. guerreronis caused 100% mortality of A. bondari caterpillars; • The residues of abamectin, azadirachtin, and fenpyroximate affected the behaviour of A. bondari caterpillars; • Acaricide residues did not interfere with infestation of coconut fruits by A. bondari caterpillars. Atheloca bondari Heinrich is an opportunistic lepidopteran that uses necrosis caused by the mite Aceria guerreronis Keifer, the most important pest of the coconut palm, to access the meristematic region of coconuts. Because of this moth-mite positive association, pesticides used to control A. guerreronis (abamectin, azadirachtin, fenpyroximate, and pyridaben) may impact A. bondari caterpillar behavior and/or survival. Thus, we used three methodologies (toothpick, triangle, and mesocarp fragment) to evaluate mortality caused by abamectin, the most commonly used pesticide to control A. guerreronis . The toothpick method proved to be suitable for toxicity tests of A. bondari caterpillars. Subsequently, the mortality caused by these four pesticides was evaluated, and abamectin caused 100% mortality in A. bondari caterpillars. Abamectin showed higher toxicity than the other pesticides to A. bondari caterpillars, with LC 50 and LC 95 values of 1.35 and 3.64 mg/L, respectively. The preference test showed that A. bondari caterpillars infested pesticide-treated fruits in the same proportion as untreated fruits. In the behavioral test, A. bondari caterpillars showed lower locomotor speed when exposed to surfaces with abamectin, azadirachtin, and fenpyroximate residues. Our study presented that pesticides used to control A. guerreronis affect the behavior and survival of A. bondari caterpillars. We discuss how spraying these pesticides can indirectly control the population of A. bondari , reducing the impact of this moth on coconut production, and modifying the importance of this species as a pest of coconut palm.

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

Evaluation of toxicity and clot lysis activity of thymol.

Thymol (TML) is a highly adaptable compound that has numerous practical uses in fields including medicine, dentistry, veterinary science, food production, and agriculture, among many others. The focus of our study was twofold: firstly, to investigate the toxicity of TML, and secondly, to explore the potential thrombolytic activity of TML. The investigation was carried out to assess the toxicity of TML using the eukaryotic test model Allium cepa (A. cepa). To conduct the experiment, various concentrations of TML were applied to A. cepa for 24, 48, and 72 hours. Distilled water was used as a vehicle while, CuSO4 (0.6 µg/mL) served as the positive control. The length of the roots of the onions was measured in millimeters, and the findings indicated that TML exhibited toxicity in onions in a manner that depended on both the concentration and duration of exposure. At lower concentrations, the longest root length was observed, whereas root growth was hindered with increasing concentrations of TML and exposure time. This inhibition was attributed to the deposition of chemicals and interference with cell division in the root meristematic region of A. cepa. To investigate the thrombolytic ability of TML, an experimental model (in vitro) was used in a laboratory setting. Streptokinase was utilized as a reference or positive control, while water was used as a negative control. The findings of the study revealed that TML exhibited a moderate percentage of clot lysis compared to streptokinase. This effect was dependent on the dosage of TML, and the clot-lytic activity of TML was considerably lower than that of streptokinase.

Chromatin Immunostaining of Plant Nuclei.

Recent evidence has demonstrated that specific epigenetic changes are also related to plant growth and development. Immunostaining enables the detection and characterization of chromatin modification, e.g., histone H4 acetylation (H4K5ac), histone H3 methylation (H3K4me2 and H3K9me2), and DNA methylation (5mC) with unique and specific patterns in plant tissues. Here we describe experimental procedures to determine the histone H3 methylation (H3K4me2 and H3K9me2) patterns in 3D-chromatin in whole roots tissue and 2D-chromatin in single nuclei in rice. To analyze both iron and salinity treatments, we show how to test for changes to the epigenetic chromatin landscape using heterochromatin (H3K9me2) and euchromatin (H3K4me) markers for chromatin immunostaining, especially in the proximal meristem region. To elucidate the epigenetic impact of environmental stress and external plant growth regulators, we demonstrate how to apply a combination of salinity, auxin, and abscisic acid treatments. The results of these experiments provide insights into the epigenetic landscape during rice root growth and development.