[Application of strigolactone analogs in storage of Gastrodia elata].

This study explored the preservation effect of strigolactone analogs on Gastrodia elata tubers and screened out the suitable preservation measures of G. elata to provide a safer and more effective method for its storage and preservation. Fresh G. elata tubers were treated with 7FGR24, 2,4-D isooctyl ester, and maleic hydrazide, respectively. The growth of flower buds, the activities of CAT, and MDA, and the content of gastrodin and p-hydroxybenzyl alcohol were measured to compare the effects of different compounds on the storage and preservation of G. elata. The effects of different storage temperatures on the preservation of 7FGR24 were compared and analyzed. The gibberellin signal transduction receptor gene GeGID1 was cloned, and the effect of 7FGR24 on the expression level of GeGID1 was analyzed by quantitative polymerase chain reaction(qPCR). The toxicity of the G. elata preservative 7FGR24 was analyzed by intragastric administration in mice to evaluate its safety. The results showed that compared with 2,4-D isooctyl ester and maleic hydrazide, 7FGR24 treatment had a significant inhibitory effect on the growth of G. elata flower buds, and the CAT enzyme activity of G. elata was the highest, indicating that its preservation effect was stronger. Different storage temperatures had different effects on the preservation of G. elata, and the preservation effect was the strongest at 5 ℃. The open reading frame(ORF) of GeGID1 gene was 936 bp in length, and its expression level was significantly down-regulated after 7FGR24 treatment, indicating that 7FGR24 may inhibit the growth of flower buds by inhibiting the gibberellin signal of G. elata, thereby exerting a fresh-keeping effect. Feeding preservative 7FGR24 had no significant effect on the behavior and physiology of mice, indicating that it had no obvious toxicity. This study explored the application of the strigolactone analog 7FGR24 in the storage and preservation of G. elata and preliminarily established a method for the storage and preservation of G. elata, laying a foundation for the molecular mechanism of 7FGR24 in the storage and preservation of G. elata.

Unraveling the DNA Methylation in the rDNA Foci in Mutagen-Induced Brachypodium distachyon Micronuclei.

Many years have passed since micronuclei were first observed then accepted as an indicator of the effect of mutagens. However, the possible mechanisms of their formation and elimination from the cell are still not fully understood. Various stresses, including mutagens, can alter gene expression through changes in DNA methylation in plants. In this study we demonstrate for the first time DNA methylation in the foci of 5S and 35S rDNA sequences in individual Brachypodium distachyon micronuclei that are induced by mutagenic treatment with maleic acid hydrazide (MH). The impact of MH on global epigenetic modifications in nuclei and micronuclei has been studied in plants before; however, no in situ analyses of DNA methylation in specific DNA sequence sites are known. To address this problem, we used sequential immunodetection of 5-methylcytosine and fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes on the non-dividing cells of B. distachyon. Such investigations into the presence or absence of DNA methylation within specific DNA sequences are extremely important in plant mutagenesis in the light of altering gene expression.

Maleic hydrazide elicits global transcriptomic changes in chemically topped tobacco to influence shoot bud development.

MAIN CONCLUSION: Transcriptomic analysis revealed maleic hydrazide suppresses apical and axillary bud development by altering the expression of genes related to meristem development, cell division, DNA replication, DNA damage and recombination, and phytohormone signaling. Topping (removal of apical buds) is a common agricultural practice for some crop plants including cotton, cannabis, and tobacco. Maleic hydrazide (MH) is a systemic suckercide, a chemical that inhibits shoot bud growth, used to control the growth of apical (ApB) and axillary buds (AxB) following topping. However, the influence of MH on gene expression and the underlying molecular mechanism of controlling meristem development are not well studied. Our RNA sequencing analysis showed that MH significantly influences the transcriptomic landscape in ApB and AxB of chemically topped tobacco. Gene ontology (GO) enrichment analysis revealed that upregulated genes in ApB were enriched for phosphorelay signal transduction, and the regulation of transition timing from vegetative to reproductive phase, whereas downregulated genes were largely associated with meristem maintenance, cytokinin metabolism, cell wall synthesis, photosynthesis, and DNA metabolism. In MH-treated AxB, GO terms related to defense response and oxylipin metabolism were overrepresented in upregulated genes. GO terms associated with cell cycle, DNA metabolism, and cytokinin metabolism were enriched in downregulated genes. Expression of KNOX and MADS transcription factor (TF) family genes, known to be involved in meristem development, were affected in ApB and AxB by MH treatment. The promoters of MH-responsive genes are enriched for several known cis-acting elements, suggesting the involvement of a subset of TF families. Our findings suggest that MH affects shoot bud development in chemically topped tobacco by altering the expression of genes related to meristem development, DNA repair and recombination, cell division, and phytohormone signaling.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

Use of autoclave extraction and liquid chromatography with tandem mass spectrometry for determination of maleic hydrazide residues in tobacco.

Maleic hydrazide has been extensively used as an effective growth regulator in tobacco sucker control. After application, maleic hydrazide distributes itself throughout the tobacco plant where it can exist as free, or forms glucoside conjugates with glucose, or becomes bound with lignin. Among them, free maleic hydrazide and its glucoside conjugates are extractable under conventional solvent extraction, while lignin bound maleic hydrazide is claimed to be non-extractable. Herein, an autoclave extraction method has been developed to extract maleic hydrazide effectively, in which tobacco samples are extracted in an autoclave at 130°C for 1 h using 4 M hydrochloric acid. Under such pressurized hot acidic water conditions, lignin bound maleic hydrazide can be released. Meanwhile, glucoside conjugates are hydrolyzed. Total maleic hydrazide is detected by liquid chromatography coupled with tandem mass spectrometry, and the quantitative results coincide well with that obtained from the international standard method. The proposed autoclave extraction with liquid chromatography and tandem mass spectrometry method exhibits excellent linearity in the range of 5-200 mg/kg (R2  = 0.9998), the matrix matched limit of detection and limit of quantification is 0.68 and 2.27 mg/kg, respectively. This method is simple and improves sample capacity, providing an effective approach to monitoring maleic hydrazide residues in tobacco.

Dissecting the chromosomal composition of mutagen-induced micronuclei in Brachypodium distachyon using multicolour FISH.

Background and Aims: Brachypodium distachyon (Brachypodium) is a model species for temperate cereals and other economically important grasses. Its favourable cytogenetic features and advanced molecular infrastructure make it a good model for understanding the mechanisms of instability of plant genomes after mutagenic treatment. The aim of this study was to qualitatively and quantitatively assess the composition and origin of micronuclei arising from genomic fracture, and to detect possible 'hot spots' for mutagen-induced DNA breaks. Methods: Seeds of Brachypodium were treated with maleic hydrazide (MH) or X-rays. The structure of mutagen-induced micronuclei was analysed in root-tip meristematic cells using multicolour fluorescence in situ hybridization (mcFISH) with various repetitive (5S rDNA, 25S rDNA, telomeric, centromeric) and low-repeat [small and large pools of bacterial artificial chromosome (BAC) clones specific for chromosome Bd1] DNA sequences. Key Results: The majority of micronuclei derive from large, acentric fragments. X-rays caused more interstitial DNA breaks than MH. Double-strand breaks rarely occurred in distal chromosome regions. Bd1 contributed to the formation of more mutagen-induced micronuclei than expected from random chromosome involvement. Conclusions: mcFISH with chromosome-specific BAC clones offers insight into micronuclei composition, in so far as it allows their origin and formation to be determined more specifically. A reliable assay for micronuclei composition is crucial for the development of modern genotoxicity tests using plant cells. The combination of mutagenic treatments and well-developed cytomolecular resources in Brachypodium make this model species very promising for plant mutagenesis research.

Mixture genotoxicity of 2,4-dichlorophenoxyacetic acid, acrylamide, and maleic hydrazide on human Caco-2 cells assessed with comet assay.

Assessment of genotoxic properties of chemicals is mainly conducted only for single chemicals, without taking mixture genotoxic effects into consideration. The current study assessed mixture effects of the three known genotoxic chemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), acrylamide (AA), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0-1.4, 0-20, and 0-37.7 mM for 2,4-D, AA, and MH, respectively. Mixture toxicity was tested with a fixed ratio design at a 10:23:77% ratio for 2.4-D:AA:MH. Results indicated that the three chemicals yielded a synergistic mixture effect. It is not clear which mechanisms are responsible for this interaction. A few possible interactions are discussed, but further investigations including in vivo studies are needed to clarify how important these more-than-additive effects are for risk assessment.

Cytotoxic effects of etephon and maleic hydrazide in Vero, Hep2, HepG2 cells.

The toxicity of etephon and maleic hydrazide, used as plant growth regulators in agriculture, were reported as low in mammals in previous studies. However, in vitro cytotoxicity studies in mammalian cells are currently missing to understand their toxicity at molecular level. In the current study, the cytotoxicity of these compounds, were studied in Vero (African green monkey kidney epithelium), HepG2 (human hepatocellular carcinoma), Hep2 (human epidermoid cancer) cells by MTT ((3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromure) and LDH (lactate dehydrogenase) assays. Maleic hydrazide had lower IC50 values for all cell lines compared to ethephon. Least cytotoxic effect treated by ethephon were observed in Vero, followed by HepG2 and Hep2. Similarly maleic hydrazide also showed least cytotoxicity on Vero cells, followed by Hep2 and HepG2 cells (p < 0.05). IC50 values in general were found to be highest in Vero cells, followed by HepG2 and Hep2 cells (p < 0.05). LDH and MTT assays showed correllation and had close relation except HepG2-maleic hydrazide application with the correlation coefficient for all >0.868 (p < 0.05). This study is expected to be a basis to understand the cytotoxic effects of ethephon and maleic hydrazide in mammal cells to be supplemented by further studies.

Selective extraction of maleic hydrazide in foods using magnetic molecularly imprinted polymers and colorimetric detection via smartphone.

Maleic hydrazide (MH) is a plant growth regulator, herbicide, and sprout inhibitor used to improve the growth and quality of certain vegetables and fruits, unfortunately, MH has genotoxic and carcinogenic effects; thus, MH residues in food need to be analyzed. Herein, magnetic molecularly imprinted polymers (MagMIP) were synthesized by radical polymerization in just 30 min using a microwave for rapid and selective extraction of MH. The colorimetric detection of MH using the immobilized Folin Ciocalteau's reagent (FCR) on 96-well microplate via smartphone sensor exhibits useful sensitivity for MH with a limit of detection (LOD = 0.6 ppm) which is far lower than the maximum residue limits (higher than 5 ppm). The immobilized FCR was stored dry at two different storage conditions at +4 °C and room temperature without losing its performance over six months. The coupling MagMIP-extraction/clean-up and smartphone determination were tested towards food samples (i.e., potatoes, and carrots), obtaining good recovery (79-96 %), high repeatability (RSD 4.5 %; n = 10), and high selectivity for MH determination.

[Determination of maleic hydrazide residues and its influence on the quality of Atractylodes macrocephala].

OBJECTIVE: To determine maleic hydrazide (MH) residues and discuss its influence on the quality of Atractylodes macrocephala. METHODS: At the bud stage, A. macrocephala different concentration of MH. Then MH residues,the contents of sugar and lactone were determined by HPLC and UV. The quality of A. macrocephala was comprehensively evaluated by independent sample t test and principal component analysis. RESULTS: The range of MH residues was 0.3-2.2 mg/kg. The results of independent sample t test revealed that the trend of the contents of lactone was low-high-low with the increase of MH, and the effect of MH on the content of sugar was barely obvious. Meanwhile, principal component analysis showed that comprehensive evaluation on the quality of A. macrocephala was the best when MH with 75 or 100 times water was applied. CONCLUSION: Proper concentration MH is applied to ensure low concentration MH residues and improve yield and quality of A. macrocephala.

[Effect of different plant growth regulators on yield and quality of Angelica dahurica var. formosana development].

OBJECTIVE: To investigate the effect of plant growth regulators on the growth and quality of Angelica dahurica var. formosana. METHOD: Five plant growth regulators: chlormequat chloride (CCC), Mepiquat chloride (PIX), Gibberellic acid (GA3), Paclobutrazol (PP333) and Maleic Hydrazide (MH) were sprayed in rosette stage, the effects of these plant growth regulators (PGRs) on the growth, yield and quality of A. dahurica var. formosanaw were observed. The biological traits were first measured and then imperatorin and isoimperatorin contents in roots were determined by HPLC. RESULT: Low concentration GA3 increased the yield while not influenced the premature bolting rate and the coumarin content. CONCLUSION: Spraying of GA3 (30 mg x L(-1)) could guarantee the growth and development of A. dahurica var. formosana to have a higher yield and maintain the active ingredients content in the root as well.

Real-Time Monitoring of Translocation, Dissipation, and Cumulative Risk of Maleic Hydrazide in Potato Plants and Tubers by Ion Exclusion Chromatography.

In this paper, a high-performance ion exclusion chromatographic (ICE) method was developed and applied for monitoring maleic hydrazide (MH) translocation in complex potato plant tissue and tuber matrices. After middle leaf uptake, most MH was trapped and dissipated in the middle leaf, and the rest was transported to other parts mainly through the phloem. Soil absorption significantly reduced the uptake efficiency of the root system, in which MH was partitioned to dissipate in root protoplasts or transfer through the xylem and persisted in the plant. Tuber uptake enabled MH to remain in the flesh and maintain stable levels under storage conditions, but during germination, MH was translocated from the flesh to the growing buds, where it dissipated through the short-day photoperiodic regime. The results demonstrated successful application of the ICE method and provided necessary insights for real-time monitoring of MH translocation behavior to effectively improve potato edible safety.

Maleic hydrazide prompting growth and delaying senescence of mother frond in S. Polyrriza 7498.

The effect and function mechanism of maleic hydrazide on the growth of mature leaves is unclear. Duckweed is widely used as a model plant to study the effect of compounds on plant growth. The observation of section and ultrastructure of the fronds, the comparation of SOD enzyme activity and related-gene transcriptional expression level showed that 75 µg/mL maleic hydrazide could prompt the growth of the mother fronds in S. Polyrriza 7498. The half-mother fronds (without meristematic tissue, cut from the mother fronds) with little meristematic tissue could repair themselves and delay their senescence by 75 µg/mL MH. The mother fronds turned more greener with 50 µg/mL MH and exogenous 0.1 µmol/L 6-BA (a kind of cytokinin) treatment, as well as with the increasing of fresh and dry weight in S. Polyrriza 7498. RNA-Seq data found that the happy growth of the mother fronds caused by MH, was probably resulted from up-regulating the expression of gene related to the synthesis and signaling transduction of cytokinin in S. Polyrriza 7498. Which are responsible for the maintaining membrane system integrate and transport protein function. The work gives lights to the study of function mechanism of MH prompting mature leaves growth and delaying mature leaves senescence in plant. And it provides a strategy to increase biomass with the application of low concentration MH and 6-BA in the same time in agriculture.

Halloysite nanotube/black phosphorene nanohybrid modified screen-printed carbon electrode as an ultra-portable electrochemical sensing platform for smartphone-capable detection of maleic hydrazide with machine learning assistance.

With the assistance of machine learning (ML), black phosphorene (BP) stabilized by silver nanoparticles (AgNPs) is used to modify halloysite nanotube (HNT) to obtain highly conductive nanomaterials, HNT/BP-AgNPs, which are morphologically characterized and elementally analyzed. Artificial neural network (ANN) and least squares support vector machine (LS-SVM) are adopted for the intelligent and rapid analysis of maleic hydrazide (MH). An ultra-portable electrochemical sensor bases on HNT/BP-AgNPs modifying screen-printed carbon electrode (SPCE), smartphone and mini-palm potentiostat for detection of MH in the linear range 0.7-55 µM with limit of detection (LOD) of 0.3 µM. For comparison, a traditional electrochemical sensor is fabricated by glass carbon electrode (GCE), desktop computer and large electrochemical potentiostat, and the linear range is 0.3-600 µM with low LOD of 0.1 µM. The ultra-portable electrochemical sensor combined with ML for the detection of MH in sweat potato and carrot gain satisfactory recoveries.

The Fate of maleic hydrazide on tobacco during smoking.

Tobacco mainstream smoke (MSS) and sidestream smoke (SSS), butts, and ashes from commercial cigarettes and maleic hydrazide (MH) spiked cigarettes were analyzed for their MH contents. The MH transfer rates obtained for MSS ranged from 1.4% to 3.7%, for SSS ranged from 0.2% to 0.9%, and for butts ranged from 1.1% to 1.9%. And as expected, MH is absent in ashes. The transfer rate of MH into mainstream smoke is the top one during in transfer rate into main-stream, side-stream, ashes, and butts, and higher MH levels lead to more MH in smoke. Further, analysis of total MH in butts and ashes along with that in MSS and SSS indicates that much MH is destructed during the smoking process.

Glyphosate, alachor and maleic hydrazide have genotoxic effect on Trigonella foenum-graecum L.

In the present study effects of herbicides glyphosate (GP), alachlor (AL) and maleic hydrazide (MH) is studied on mitotic cells of Trigonella foenum-graecum L. Seeds of T. foenum-graecum L. treated with a series of concentrations ranging from 0.1%, 0.2%, 0.3%, 0.4% and 0.5% for 1, 2 and 6 h and their effect on mitotic index and chromosomal aberrations was studied. The results indicate that these herbicides reduced mitotic index in dose-dependent manner. In addition, increase in the percentage of abnormal mitotic plates was observed in herbicide treated groups which was both concentration and time dependent. Commonly observed abnormalities were c-mitosis, laggards, bridges, stickiness, c-anaphase, precocious separation, un-equal distribution and fragments. The result of the present investigation indicates that commonly used herbicides GP, AL and MH have significant genotoxic effect on T. foenum-graecum plant.

Maleic hydrazide: sprout suppression of potatoes in the field.

In 2005, the active substance maleic hydrazide was released on the Belgian market. Maleic hydrazide is authorized in potatoes as foliar treatment for instore sprout suppression and control of volunteers. The mode of action is based on blocking cell division whilst cell elongation is not affected. The product must be applied at once during the growing season, only after at least 80% of the tubers have reached 25 mm diameter and not later than 3 weeks before haulm killing. The first 24 h after application, no meaningful precipitation should occur to insure sufficiently uptake of the product by the crop. Field trials were set up for 4 years (2005-2008) and 4 locations per year with application of maleic hydrazide in four different cultivars (Bintje, Fontane, Asterix and Cilena). After application, the cultivar Asterix showed almost every year a temporarily phytotoxicity (bronze discoloration). On the first place yield was determined. When maleic hydrazide was applied too early (80% tubers % 25mm diameter) yield was negatively affected (3 years on 4) except for the cultivar Cilena (fresh market). Internal quality (dry matter and fry quality) was not influenced by the application of maleic hydrazide. Only Fontane had a slightly lower dry matter content. Maleic hydrazide also influenced appearance of secondary growth. However, the results were very variable depending on cultivar, location and time of application. After harvest, the tubers were kept in storage and assessed monthly on germination. Potatoes treated late in the growing season, showed a shorter dormancy period. A part of the tubers was replanted the following spring to verify volunteer control. Additional trials were set up by the Flemish government for two years (2010-2011). The results of previous trials were confirmed. Additional, the influence of maleic hydrazide on internal germination during storage was examined on the cultivar Innovator. The tests clearly showed a positive effect for this parameter.

Intelligent analysis of maleic hydrazide using a simple electrochemical sensor coupled with machine learning.

A simple electrochemical sensing platform based on a low-cost disposable laser-induced porous graphene (LIPG) flexible electrode for the intelligent analysis of maleic hydrazide (MH) in potatoes and peanuts coupled with machine learning (ML) was successfully designed. The LIPG electrode was patterned by a simple one-step laser-induced procedure on commercial polyimide film using a computer-controlled direct laser writing micromachining system and displayed excellent flexibility, 3D porous structure, large specific surface area, and preferable conductivity. A data partitioning technique was proposed for the optimal MH concentration ranges by selecting the size of datasets, including the size of the training set and the size of the test set combined with the performance metrics of ML models. Different algorithms such as artificial neural networks (ANN), random forest (RF), and least squares support vector machine (LS-SVM) were selected to build the ML models. Three ML models were evaluated, and the LS-SVM model displayed unique superiority. Both the recoveries and RSD of practical application were further measured to assess the feasibility of the selected LS-SVM model. This will have important theoretical and practical significance for the intelligent analysis of harmful residuals in agro-product safety using an electrochemical sensing platform.

Herbicide effects on freshwater benthic diatoms: induction of nucleus alterations and silica cell wall abnormalities.

Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5x10(-6), 10(-6), 10(-7)M) for 6h followed by a 24h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per thousand), S.D.=4.23) and the highest concentrations (29.40 per thousand, S.D.=8.49 for 10(-6)M and 35.96 per thousand, S.D.=3.71 for 5x10(-6)M) was statistically significant (Tukey test, P<0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10(-6) and 5x10(-6)M; Tukey test, P<0.05). These two parameters tended to increase together (Pearson correlation=0.702, P<0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic effects of MH. The alterations observed could be related to the effects of MH on the synthesis of the proteins involved in frustule formation or in the regulation of the cytoskeleton of the diatom cells.

Determination of hydrazine in maleic hydrazide technical and pesticide formulations by gas chromatography: collaborative study.

Twelve collaborating laboratories assayed hydrazine in technical maleic hydrazide (MH), 6-hydroxy-2H-pyridazin-3-one, and 2 formulated products, a liquid concentrate and a soluble granule, using gas chromatography (GC) with electron capture detection. The hydrazine content in the samples ranged from 0.03 ppm, in the liquid concentrate, to 0.26 ppm, in MH technical. Hydrazine and MH are dissolved in an aqueous solution. The MH is then precipitated out of solution by acidification. The solution containing hydrazine is treated with excess pentafluorobenzaldehyde (PFB) to form pentafluorobenzaldehyde azine (PFBA). The PFBA is extracted with hexane for analysis by GC using an electron capture detector. Peak area responses of PFBA are measured and quantified by external standardization. Hydrazine concentration is calculated from the PFBA determination. The laboratories weighed each test sample in duplicate with duplicate analysis for each weighing. Data from these laboratories were statistically analyzed. The average relative repeatability was determined to be 5.34% and the average relative reproducibility was 27.99%.