Development and validation of a simple and efficient method for the analysis of commercial formulations containing clopyralid, picloram and aminopyralid as active ingredients.

Liquid chromatography plays a pivotal role in evaluating pesticide formulations as it enables the determination of multiple active substances in plant protection products. An adaptable separation technique has been developed, enabling the qualitative and quantitative analysis of clopyralid, picloram, and aminopyralid within pesticide formulations in line with SANCO/3030/99 rev. 5 guidelines. This article offers an insight into the validation procedure encompassing key aspects such as selectivity, linearity, accuracy, precision, and recovery. It places emphasis on critical stages, including sample preparation, chromatographic separation, detection, quantification, and data analysis. The active ingredients are separated using chromatography with isocratic elution, utilizing a mobile phase consisting of a mixture of water, acetonitrile, and acetic acid in a specific ratio (83:15:2 v/v/v). This separation is carried out on a YMC-Pack ODS-AQ column (250 mm x 4.6 mm, 5 µm) at a flow rate of 1.5 mL/min. The method's validation parameters have produced satisfactory outcomes. The recovery rates for each individual compound were found to be in the range of 98.6% to 101.0%. Precision, as indicated by the relative standard deviation (%RSD), was lower than the values predicted by the modified Horwitz equation. Furthermore, the correlation coefficients assessing the linearity of the response exceeded 0.99.

Long-term organogenic callus cultivation of Ranunculus illyricus L.: a blueprint for sustainable ex situ conservation of the species in urban greenery.

The growing trend of introducing wild plant species into urban environments necessitates the identification of novel species adapted to prevailing conditions. A promising reservoir of such species may be xerothermic communities where Ranunculus illyricus occurs. This study aimed to establish a micropropagation protocol for R. illyricus using indirect organogenesis. The protocol includes initiation of culture from various explants, callus proliferation, shoot regeneration, multiplication, and concurrent rooting. Callus appeared on most types of vegetative explants tested, but stolons were considered the best due to their good availability, high disinfection (85%), and robust callus production (maximum increase - 363.1%). The growth rate of the callus fresh matter (CFM) obtained from stolons was calculated. Greater CFM was obtained on the medium with the supplemented picloram 8.0 mg L- 1 with kinetin 5.0 mg L- 1 and in second part of experiment on medium with the addition of 2,4-D (2,4-dichlorophenoxyacetic acid) 2.0 mg L- 1 alone or picloram 6.0 mg L- 1 with kinetin 8.0 mg L- 1. Shoot organogenesis was observed on macronutrients B5 (Gamborg medium), micronutrients MS (Murashige and Skoog) medium with the addition of 2.0 mg L- 1 IBA (indole-3-butyric acid) and 4.0 mg L- 1 BAP (6-benzylaminopurine). To document the process of callus differentiation, microscopic preparations were prepared. Subsequently, the regenerated plants underwent acclimatisation and their growth in an ex situ collection was monitored over three growing seasons. In particular, in vitro-origin plants exhibited developmental patterns similar to those of their seed-origin counterparts. The incorporation of R. illyricus into urban landscapes not only increases aesthetic appeal, but also ensures the preservation of valuable genetic resources for this rare species, potentially contributing to effective ex situ conservation in the future. This marks the first scientific report on in vitro cultures of R. illyricus.

Study on Design, Synthesis and Herbicidal Activity of Novel 6-Indazolyl-2-picolinic Acids.

Thirty-eight new 4-amino-3,5-dicholo-6-(1H-indazolyl)-2-picolinic acids and 4-amino-3,5-dicholo-6-(2H-indazolyl)-2-picolinic acids were designed by scaffold hopping and synthesized to discover potential herbicidal molecules. All the new compounds were tested to determine their inhibitory activities against Arabidopsis thaliana and the root growth of five weeds. In general, the synthesized compounds exhibited excellent inhibition properties and showed good inhibitory effects on weed root growth. In particular, compound 5a showed significantly greater root inhibitory activity than picloram in Brassica napus and Abutilon theophrasti Medicus at the concentration of 10 µM. The majority of compounds exhibited a 100% post-emergence herbicidal effect at 250 g/ha against Amaranthus retroflexus and Chenopodium album. We also found that 6-indazolyl-2-picolinic acids could induce the up-regulation of auxin genes ACS7 and NCED3, while auxin influx, efflux and auxin response factor were down-regulated, indicating that 6-indazolyl-2-picolinic acids promoted ethylene release and ABA production to cause plant death in a short period, which is different in mode from other picolinic acids.

Chemically engineered unzipped multiwalled carbon nanotube and rGO nanohybrid for ultrasensitive picloram detection in rice water and soil samples.

Picloram (4-Amino-3,5,6-trichloro pyridine-2-carboxylic acid) is a chlorinated herbicide that has been discovered to be tenacious and relatively durable in both soil and water. It is known to have adverse and unpleasant effects on humans causing several health complications. Therefore, the determination of picloram is profoundly effective because of its bio-accumulative and persistent nature. Because of this, a sensitive, rapid, and robust detection system is essential to detect traces of this molecule. In this study, we have constructed a novel nanohybrid system comprising of an UZMWCNT and rGO decorated on AuNPs modified glassy carbon electrode (UZMWCNT + rGO/AuNPs/GCE). The synthesized nanomaterials and the developed system were characterized using techniques such as SEM, XRD, SWV, LSV, EIS, and chronoamperometry. The engineered sensor surface showed a broad linear range of 5 × 10-2 nM to 6 × 105 nM , a low limit of detection (LOD) of 2.31 ± 0.02 (RSD < 4.1%) pM and a limit of quantification (LOQ) of 7.63 ± 0.03 pM. The response time was recorded to be 0.2 s, and the efficacy of the proposed sensor system was studied using rice water and soil samples collected from the agricultural field post filtration. The calculated recovery % for picloram in rice water was found to be 88.58%-96.70% (RSD < 3.5%, n = 3) and for soil it was found to be 89.57%-93.24% (RSD < 3.5%, n = 3). In addition, the SWV responses of both the real samples have been performed and a linear plot have been obtained with a correlation coefficient of 0.97 and 0.96 for rice and soil samples, respectively. The interference studies due to the coexisting molecules that may be present in the samples have been found to be negligible. Also, the designed sensor has been evaluated for stability and found to be highly reproducible and stable towards picloram detection.

Picloram binds to the h1 and h4 helices of HSA domain IIIA at drug binding site 2.

Picloram (PC) is a systemic herbicide that controls herbaceous weeds and woody plants. HSA, the most abundant protein in human physiology, binds to all exogenic and endogenic ligands. PC is a stable molecule (t1/2∼157-513 days) and a potential threat to human health via the food chain. HSA and PC binding study has been done to decipher the location and thermodynamics of binding. It has been studied with prediction tools like autodocking and MD simulation and then confirmed with fluorescence spectroscopy. HSA fluorescence was quenched by PC at pH 7.4 (N state), pH 3.5 (F state), and pH 7.4 with 4.5 M urea (I state) at temperatures 283 K, 297 K, and 303 K. The location of binding was found to be interdomain between II and III which overlaps with drug binding site 2. The binding was spontaneous, and entropy-driven that show a noticeable increase in binding with the increase in temperature. No secondary structure change at the native state has been observed due to binding. The binding results are important to understand the physiological assimilation of PC. In silico predictions and the results of spectroscopic studies unambiguously indicate the locus and nature of the binding.

Design, Synthesis, Herbicidal Activity, and Structure-Activity Relationship Study of Novel 6-(5-Aryl-Substituted-1-Pyrazolyl)-2-Picolinic Acid as Potential Herbicides.

Picolinic acid and picolinate compounds are a remarkable class of synthetic auxin herbicides. In recent years, two new picolinate compounds, halauxifen-methyl (ArylexTM active) and florpyrauxifen-benzyl (RinskorTM active), have been launched as novel herbicides. Using their structural skeleton as a template, 33 4-amino-3,5-dicholor-6-(5-aryl-substituted-1-pytazolyl)-2-picolinic acid compounds were designed and synthesized for the discovery of compounds with potent herbicidal activity. The compounds were tested for inhibitory activity against the growth of Arabidopsis thaliana roots, and the results demonstrated that the IC50 value of compound V-7 was 45 times lower than that of the halauxifen-methyl commercial herbicide. Molecular docking analyses revealed that compound V-7 docked with the receptor auxin-signaling F-box protein 5 (AFB5) more intensively than picloram. An adaptive three-dimensional quantitative structure-activity relationship model was constructed from these IC50 values to guide the next step of the synthetic strategy. Herbicidal tests of the new compounds indicated that compound V-8 exhibited better post-emergence herbicidal activity than picloram at a dosage of 300 gha-1, and it was also safe for corn, wheat, and sorghum at this dosage. These results demonstrated that 6-(5-aryl-substituted-1-pyrazolyl)-2-picolinic acid compounds could be used as potential lead structures in the discovery of novel synthetic auxin herbicides.

Herbicides widely used in the world: an investigation of toxic effects on Caenorhabditis elegans.

Dicamba, paraquat, picloram, clopyralid and linuron are herbicides widely used in agriculture. The aim of the present study is to evaluate the toxicity effects of the herbicides used on survival, fertility and length of Caenorhabditis elegans. Kaplan-Meier Survival Analysis method was used to identify the toxicity effect of herbicides on survival, and ANOVA and Post Hoc tests were used to determine the toxicity effects on fertility and length. In the study, C. elegans was exposed to 5 different concentrations (62.5, 125, 250, 500, 1000 µM) of each herbicide. When the results were evaluated, it was observed that survival (life span) and length (physical growth) were more affected, respectively, by paraquat, dicamba, linuron, picloram and clopyralid herbicides, fertility (egg productivity) were more affected, respectively, by paraquat, linuron, dicamba, picloram and clopyralid herbicides. As a result, it was determined that increasing the dose amounts of herbicides caused many toxic reactions on C. elegans, affecting survival, egg productivity and length.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Determination of genotoxic damages of picloram and dicamba with comet assay in Allium cepa rooted in tissue culture and distilled water.

BACKGROUND: Many genotoxicity tests allow us to understand the mechanism of damages on genetic material occurring in living organisms against various physical and chemical agents. One of them is the Comet test. The current study aimed to evaluate genotoxic caused by picloram and dicamba to root meristems of Allium cepa utilizing comet assay. METHODS: Two different protocols were used for rooting and auxin/pesticide application. (i) A. cepa bulbs were rooted in MS medium and then treated with Murashige and Skoog (MS) medium (control) and 0.67, 1.34, 2.01, 2.68, 3.35, 4.02, and 8.04 mg/L of picloram and dicamba using aseptic tissue culture techniques. (ii) A. cepa bulbs were then rooted in bidistilled water and treated with 0 (control), 0.67, 1.34, 2.01, 2.68, 3.35, 4.02, and 8.04 mg/L of picloram and dicamba in distilled water. The A. cepa root tip cells in both treatment groups were examined using comet test to find the possible DNA damaging effects of picloram and dicamba. RESULTS: The results obtained at all the concentrations were statistically compared with their control groups. Almost at all the concentrations of Picloram and dicamba increased comet tail intensity (%) and tail moment in roots treated in MS medium. Two highest concentrations revealed toxic effect. On the other hand, DNA damaging effect of both auxins was only noted on the highest (> 4.02 mg/L) in roots treated in distilled water. CONCLUSIONS: This study approve and confirm genotoxic effects of how growth regulators on plants. These findings give an evidence of DNA damage in A. cepa. Therefore, both picloram and dicamba should only be used in appropriate and recommended concentrations in agriculture to conserve ecosystem and to pose minimum threat to life.

Study of feces of neotropical otters (Lontra longicaudis) in the Ayuquila-Armería basin, Mexico as biomonitors of the spatiotemporal distribution of pesticides.

The pesticides used have contributed to increasing food production; it has also caused them to be found in most ecosystems and have negative effects on biota. The neotropical otter (Lontra longicaudis) is vulnerable to pesticide accumulation and is characterized by being elusive, so it is necessary to address the use of indirect techniques that evaluate its populations' state in an efficient, logistically simple, and non-invasive way. This study aimed to determine the concentration of 20 pesticides in neotropical otter feces in the Ayuquila-Armería basin and to describe the spatiotemporal variation of these pesticides. The presence of 11 pesticides was determined. Imazalil, picloram, and malathion the pesticides with the highest concentrations; emamectin, λ-cyhalothrin, methomyl, and picloram were present in all samples. Emamectin was the only pesticide that presented significant differences concerning the temporality of the samplings, presenting higher concentrations in the wet season. Molinate concentrations showed significant differences concerning the location of the sampling sections in the basin; the lower part of the basin presented higher concentrations. The distribution of the populations of L. longicaudis in the Ayuquila-Armería basin does not respond to the degree of contamination by pesticides in surface waters or to the proximity to agricultural activities, and this in places with evident chemical and organic contamination and human presence. The use of otter feces for pesticide monitoring is an accepted non-invasive method to assess the degree of exposure and can be used to determine sites with pollution problems.

Mabea fistulifera and Zeyheria tuberculosa can be indicated for phytoremediation programs of soils contaminated with hormonal herbicides.

Hormone-like herbicides, used for large crops, can contaminate non-target areas with their waste. The objective of this study was to evaluate the tolerance of Mabea fistulifera and Zeyheria tuberculosa to 2,4-D + picloram herbicides by means of morphological and anatomical evaluations. The experiment was performed in a greenhouse in a 4 × 2 factorial scheme. The first factor was the control (without herbicide) and three doses (0.166; 0.333, and 0.666 L ha-1) of the herbicide Tordon® (402 g L-1 2,4-D + 103.6 g L-1 picloram) and the second factor, the species Mabea fistulifera and Zeyheria tuberculosa. The number of M. fistulifera leaves was lower after treatment with the highest dose of the 2,4-D + picloram mixture. The herbicide rates did not influence the number of Z. tuberculosa leaves. The higher dose of 2,4-D + picloram caused a more than 50% reduction in leaf area. Toxicity increased linearly as a function of the doses of the 2,4-D + picloram mixture. Changes in the leaf anatomy of the two species treated with herbicides were observed; however, the roots did not show any changes. Mabea fistulifera and Zeyheria tuberculosa can be recommended for phytoremediation programs in areas contaminated by the herbicides 2,4-D + picloram.

Phytoremediation of soils contaminated with herbicides is a recent and viable tool for environmental decontamination and for the protection of water resources. Mabea fistulifera and Zeyheria tuberculosa can be used to compose riparian forests and retain the arrival of herbicides in the water. Plant anatomy and morphological characteristics are viable tools to assess the tolerance and phytoremediation potential of plant species. Mabea fistulifera and Zeyheria tuberculosa are tolerant to the presence of hormonal herbicides. In this way, they can be used to recover natural areas close to the cultivation areas where the herbicides 2,4-D and picloram are used.

Two Auxinic Herbicides Affect Brassica napus Plant Hormone Levels and Induce Molecular Changes in Transcription.

With the introduction of the new auxinic herbicide halauxifen-methyl into the oilseed rape (Brassica napus) market, there is a need to understand how this new molecule interacts with indigenous plant hormones (e.g., IAA) in terms of crop response. The aim of this study was to investigate the molecular background by using different growth conditions under which three different auxinic herbicides were administered. These were halauxifen-methyl (Hal), alone and together with aminopyralid (AP) as well as picloram (Pic). Three different hormone classes were determined, free and conjugated indole-3-acetic acid (IAA), aminocyclopropane carboxylic acid (ACC) as a precursor for ethylene, and abscisic acid (ABA) at two different temperatures and growth stages as well as over time (2-168 h after treatment). At 15 °C growth temperature, the effect was more pronounced than at 9 °C, and generally, the younger leaves independent of the developmental stage showed a larger effect on the alterations of hormones. IAA and ACC showed reproducible alterations after auxinic herbicide treatments over time, while ABA did not. Finally, a transcriptome analysis after treatment with two auxinic herbicides, Hal and Pic, showed different expression patterns. Hal treatment leads to the upregulation of auxin and hormone responses at 48 h and 96 h. Pic treatment induced the hormone/auxin response already after 2 h, and this continued for the other time points. The more detailed analysis of the auxin response in the datasets indicate a role for GH3 genes and genes encoding auxin efflux proteins. The upregulation of the GH3 genes correlates with the increase in conjugated IAA at the same time points and treatments. Also, genes for were found that confirm the upregulation of the ethylene pathway.

Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant.

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.

Aqueous picloram degradation by hydroxyl radicals: Unveiling mechanism, kinetics, and ecotoxicity through experimental and theoretical approaches.

Pesticides are chemical compounds widely used to combat pests in crops, and they thus play a key role in agricultural production. However, due to their persistence in aquatic environments, even at low concentrations, their use has been considered an environmental problem and caused concern regarding the adverse effects on human health. This paper reports, for the first time, the mechanisms, kinetics, and an evaluation of the toxicity of picloram degradation initiated by OH radicals in the aqueous environment using quantum chemistry and computational toxicology calculations. The rate constants are calculated using a combination of formulations derived from the Transition State Theory in a realistic temperature range (250-310 K). The results indicate that the two favorable pathways (R1 and R5) of OH -based reactions occur by addition to the pyridine ring. The calculated rate constant at 298 K is compared with the overall second-order reaction rate constant, quantified herein experimentally via the competition kinetics method and data available in the literature showing an excellent agreement. The toxicity assessment and a photolysis study provide important information: i) picloram and the majority of degradation products are estimated as harmful; however, ii) these compounds can suffer photolysis in sunlight. The results of the present study can help understand the mechanism of picloram, also providing important clues regarding risk assessment in aquatic environments as well as novel experimental information.

The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Searching an auxinic herbicide to use as positive control in toxicity assays.

Due to rising concerns for environmental and human health, many toxic compounds, such as auxin-based herbicides, have been tested in relation their toxicity effect. Especially cyto- and phytotoxic assays have been performed on a number monocot and eudicot plant species. In these approaches the toxicity level of the auxin is compared to a positive control - usually a commercial compound with known effects and chemical similarity to the target compound. However, many target compounds still lack an indication of an adequate positive control. Here, we evaluate the phytotoxic and cytotoxic effect of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram in order test their potential use as positive controls. All tested auxinic herbicides showed clastogenic and aneugenic effect mechanisms. The results indicate 2,4-dichlorophenoxyacetic acid as the most phyto- and cytotoxic in the discontinuous method in Lactuca sativa L. and Allium cepa L., and also in the continuous method in A. cepa. Thus, we suggest 2,4-dichlorophenoxyacetic acid as a positive control for future mutagenesis studies involving new auxins. For studies with L. sativa in continuous method, we recommend the auxin picloram as positive control as this one was the only one which allowed the development of roots.

Somatic embryogenesis from leaf tissues of macaw palm [Acrocomia aculeata (Jacq.) Lodd. ex Mart.].

A somatic embryogenesis protocol was developed from the immature leaves of adult plants of the macaw palm. Leaf explants from different regions of the palm heart were used for callus initiation in a modified Y3 medium, supplemented with 2,4-D or Picloram at 450 µM. Calli were separated from the leaf explants at 6-, 9- and 12-month periods and transferred to a fresh culture medium of the same composition. They were multiplied for up to 120 days. Reduced concentrations of 2,4-D and Picloram were used to differentiate somatic embryos. They were then germinated in a medium without plant growth regulators. Morphological and anatomical analyses were conducted at different stages of the embryogenic process. The best results for callus induction were achieved by Picloram, when explants were maintained for up to 9 months on culture medium (64.9%). The farthest portions of the apical meristem were those that provided the biggest calli formation. The formation of the somatic embryos was observed from the calli multiplication phase. Reduction in concentrations of growth regulators failed to promote the formation of complete plants. Picloram at 450 µM promotes high callogenesis in leaf tissues of macaw palm, with a potential for somatic embryo formation.

Different Roles of Auxins in Somatic Embryogenesis Efficiency in Two Picea Species.

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.

Exogenous Auxin Induces Transverse Microtubule Arrays Through TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX Receptors.

Auxin plays a central role in controlling plant cell growth and morphogenesis. Application of auxin to light-grown seedlings elicits both axial growth and transverse patterning of the cortical microtubule cytoskeleton in hypocotyl cells. Microtubules respond to exogenous auxin within 5 min, although repatterning of the array does not initiate until 30 min after application and is complete by 2 h. To examine the requirements for auxin-induced microtubule array patterning, we used an Arabidopsis (Arabidopsis thaliana) double auxin f-box (afb) receptor mutant, afb4-8 afb5-5, that responds to conventional auxin (indole-3-acetic acid) but has a strongly diminished response to the auxin analog, picloram. We show that 5 µm picloram induces immediate changes to microtubule density and later transverse microtubule patterning in wild-type plants, but does not cause microtubule array reorganization in the afb4-8 afb5-5 mutant. Additionally, a dominant mutant (axr2-1) for the auxin coreceptor AUXIN RESPONSIVE2 (AXR2) was strongly suppressed for auxin-induced microtubule array reorganization, providing additional evidence that auxin functions through a transcriptional pathway for transverse patterning. We observed that brassinosteroid application mimicked the auxin response, showing both early and late microtubule array effects, and induced transverse patterning in the axr2-1 mutant. Application of auxin to the brassinosteroid synthesis mutant, diminuto1, induced transverse array patterning but did not produce significant axial growth. Thus, exogenous auxin induces transverse microtubule patterning through the TRANSPORT INHIBITOR 1/AUXIN F-BOX (TIR1/AFB) transcriptional pathway and can act independently of brassinosteroids.

The pesticide picloram affects biomembrane models made with Langmuir monolayers.

Cell membrane models are useful to obtain molecular-level information on the interaction of biologically-relevant molecules such as pesticides whose activity is believed to depend on its effects on the membrane. In this study, we investigated the interaction between the widely used pesticide picloram with Langmuir monolayers of binary and ternary mixtures comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM) and cholesterol (Chol), which could be taken as representative of ocular membranes in humans. Picloram expanded the molecular area of DOPC/SM and DOPC/SM/Chol monolayers as the pesticide penetrated the hydrophobic region of the mixtures. A clear correlation was also found between the compressibility modulus (Cs-1) and the presence of cholesterol in the ternary monolayer. Data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) confirmed that picloram interacts with both the acyl chains and headgroups. Spectral shifts and band broadening were induced by picloram, particularly for the phosphate and choline groups, probably owing to its H-bonding ability. The effects reported here on the lipid monolayers may be evidence of the possible activity of picloram on mammalian cell membranes, which highlights the importance of strict control of the level of exposure of humans dealing with pesticides.