The initiation of callus culture and the stabilization of cell suspensions are important steps in obtaining a callus mass for in vitro secondary metabolite production. The protocol described here could be used for efficient callus induction in a wide range of Passiflora spp. Murashige and Skoog medium supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) and Picloram (PIC) in different concentrations and combinations with Benzylaminopurine (BAP) and Kinetin (KIN) were used for culture initiation. A total of 11 treatments and different plant organs as explant source were tested. Callus proliferation was performed in solid and liquid culture systems. The interaction of factors showed that internodal segments callused at 100% on medium containing 2 mg/l 2,4-D and 0.5 mg/l BAP. On this culture medium, the leaf fragments also performed the highest callusing rate (98.33%). For P. quadrangularis, the liquid culture system proved to be superior in terms of cell mass and dry matter content, which can be explained by the better access to nutrients and water in a liquid medium. This research demonstrates that P. quadrangularis callus culture is strongly influenced by the plant growth regulators combination, the type of explants and culture system. The cell suspension obtained is the first step in the secondary metabolite production system.

Picloram (PIC) is an herbicide widely used in Uruguay to control weeds, especially in forest crops. In the present study, we evaluate the acute toxicity and genotoxicity of formulated 24.9% PIC-based Tordon®24K on Boana pulchella tadpoles under standard laboratory conditions. In the acute toxicity test we use mortality as the endpoint and we obtained a 48-h LC50 of 16.5 mg/L, LOEC 14.5 mg/L, and NOEC 12.5 mg/L. We determined genotoxicity in GS36 tadpoles exposed to sublethal concentrations of 8.5 (NOEC/1.5) and 12.5 mg/L (NOEC) PIC using micronucleus frequency (MN) and occurrence of nuclear abnormalities (AN) as endpoints. We noticed a significant increase in the frequency of MN and AN in all treatments tested at sublethal concentrations. Our findings demonstrate the genotoxic effects of this herbicide, which represent a danger to amphibians that inhabit agroecosystems. Based on the results obtained and considering that Boana pulchella is not a threatened species and has a wide distribution in the Uruguayan territory and the Pampa Biome, it could be considered a reference organism in environmental monitoring programs in the region.

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

Lilium brownii var. viridulum, commonly called Longya lily, is a well-known flower and vegetable plant in China that has poor tolerance to Botrytis fungal disease. The molecularimprovement has mainly been restricted to an efficient regeneration and transformation system. In this study, the highly efficient regeneration of Longya lily was established through the optimization of embryogenic callus, adventitious shoot and rooting induction. The major factors influencing transformation (antibiotics, Agrobacterium concentration, infection time, suspension solution and coculture medium) were examined. The expression responses of PR promoters (ZmPR4 and BjCHI1) to B. cinerea were assessed in transgenic calli. The results showed that Murashige and Skoog (MS) medium with 1.0 mg·L-1 picloram (PIC) and 0.2 mg·L-1 1-naphthaleneacetic acid (NAA) under light conditions and MS with 0.5 mg·L-1 6-benzylaminopurine (6-BA) and 1.0 mg·L-1 NAA under darkness were optimal for embryogenic callus induction (64.67% rate) and proliferation (3.96 coefficient). Callus inoculation into MS containing 2.0 mg·L-1 thidiazuron (TDZ), 0.4 mg·L-1 NAA, 1.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA led to shooting induction (92.22 of rate) and proliferation (3.28 of coefficient) promotion, respectively. The rooting rate reached 99.00% on MS with 0.3 mg·L-1 NAA. Moreover, a transformation rate of 65.56% was achieved by soaking the callus in Agrobacterium at an OD600 of 0.4 for 10 min in modified MS without NH4NO3 as the suspension solution and coculture medium before selecting 75 mg·L-1 hygromycin and 300 mg·L-1 cefotaxime. Only the BjCHI1 promoter was obviously expressed in transgenic calli. These results could facilitate the generation of Longya lily transgenic plants with improved B. cinerea resistance.

Agapanthus praecox has become a burgeoning variety in the flower market due to its high ornamental value with unique large blue-purple inflorescence. For rapid entering into the market, tissue culture technology or organogenesis has an attractive application over the conventional reproduction approach. In this study, a highly efficient protocol based on indirect organogenesis has been successfully established for A. praecox subsp. orientalis ‘Big Blue’. Two types of explants, root tips versus root segments, were compared for callus induction frequency in response to the induction culture media. The induction media contain Murashige and Skoog’s (MS) Basal Salt supplemented with various concentrations of picloram (PIC), 2,4-Dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), kinetin (KT) and naphthalene acetic acid (NAA). Of the two types of explants, root tips were found to be more effective for callus induction than root segments. Among the induction media tested, the highest callus induction rate (100.00%) was achieved when cultured on MS supplemented with 2.0 mg/L PIC, 1.5 mg/L KT and 0.1 mg/L NAA, which was probably accredited to higher endogenous phytohormone contents, especially of 3-indoleacetic (IAA). The optimal medium for callus proliferation was MS + 1.0 mg/L PIC + 1.0 mg/L 6-BA + 0.4 mg/L NAA, and the fresh weight increased by 72.74%. After being transferred onto the adventitious bud induction medium for 25 days, shoots were dedifferentiated from the surface of the flourishing callus, which then developed to the plantlet with roots in 90 days. The plantlets were transplanted in a greenhouse with a survival rate of 92.86%. This study innovatively established an indirect organogenesis tissue culture system of A. praecox with roots as explants, which provided a practical reference in its application.

Abstract The objective of this work was to investigate the effect of plant growth regulators (PGR) on the induction of calli from cotyledons of Enterolobium contortisiliquum grown in vitro, as well as to evaluate the inhibitory activity of trypsin in these calli. Cotyledons of E. contortisiliquum were cultivated in culture medium supplemented with dichlorophenoxyacetic acid (2,4-D), picloram (PIC), kinetin (KIN), and 6-benzylaminopurine (BAP). Callus mass, inhibitory activity of trypsin, morphogenetic and cytochemical observations, and biological activity were measured after 60 days. The Pareto diagram showed that only 0.5 mg L-1 PIC influenced the increase of fresh mass, whereas 0.5 mg L-1 BAP influenced dry mass. The principal component analysis plot showed the formation of two clear clusters. The inhibitory activity of trypsin is achieved at a higher intensity in the treatment with 0.5 mg L-1 2,4-D + 2.0 mg L-1 PIC + 0.5 mg L-1 KIN + 2.0 mg L-1 BAP. The combination of PGR at different rates causes an intense callus formation. Low concentrations of PGR result in a higher fresh mass, dry mass, and biological activity. The use of PGR is efficient in obtaining E. contortisiliquum calli with a satisfactory inhibitory activity of trypsin.

Abstract Medicinal plants are an important therapeutic option for a large share of the world’s population. To establish an in vitro culture system for the production of secondary metabolites from Hovenia dulcis, we studied the effect of auxins, cytokinins, absence of light, and silver nitrate on the development of friable callus. Callus cultures were established for the first time and used to obtain cell suspension cultures. Supplementation with KIN (Kinetin) produced calli with both compact and friable areas, while the addition of TDZ (Thidiazuron) only produced compact callus. The maintenance of cultures in the dark induced a slight enhancement on friability when the auxin PIC (Picloram) was present in the culture medium. The addition of silver nitrate promoted the formation of friable calli. Dry weight analysis showed no significant differences in biomass growth, and, therefore, 2.0 mg.L-1 was considered the most suitable treatment. The presence of silver nitrate was not required for the establishment of cell suspension cultures. Dry weight analysis of cell suspensions showed higher biomass production in the absence of silver nitrate. PIC promoted 100% of cell suspension culture formation in the absence of silver nitrate, and higher biomass production was observed with the lowest concentration (0.625 mg.L-1). No morphological differences were observed among the different concentrations of PIC. Phytochemical screening showed the presence of saponins, flavonoids, flavonols and catechins in the extracts obtained from H. dulcis calli. These results show that the cell cultures herein established are potential sources for the production of H. dulcis secondary metabolites of medicinal interest.

Sulfate radical-based oxidation of the aminopyralid and picloram herbicides: The role of amino group on pyridine ring

In this work, voltammetric methods were applied using a boron-doped diamond electrode (BDD) to detect Picloram (PCR), a very toxic and persistent herbicide. The method was applied to investigate environmental waters in the Amazon. In this context, an environmental-friendly method was developed using a miniaturized BDD electrode, and a compact electrochemical cell manufactured by a 3D printer using a polylactic acid biodegradable polymer to reduce the generated residue, leading to more sustainable research procedures with potential to be applied in field analysis. In addition, studies were carried out to establish the rate-limiting step of the reaction and the number of electrons and protons involved, being three protons and three electrons suggested for PCR electrochemical oxidation. For analytical purposes, the optimization of the square wave voltammetry (SWV) parameters and tests of precision, accuracy and sensitivity were performed. Thus, a low detection limit of 390 nmol L−1 and values for the intra-day and inter-day repeatability tests of 8.65% and 4.64%, respectively, were obtained. Due to its biodiversity abundance, environmental waters in the Amazon should be better monitored and this method can contribute to this task quite efficiently.

Temperature-enabled reversible “On/Off” switch-like hazardous herbicide picloram voltammetric sensor in agricultural and environmental samples based on thermo-responsive PVCL-tethered MWCNT@Au catalyst

Lamprocapnos spectabilis (L.) Fukuhara is a perennial plant species valued in the horticultural, cosmetic, and pharmaceutical markets. To date, however, there were no studies on tissue culture systems in this species when adjusted from non-meristematic explants. The aim of this study is to induce callogenesis, organogenesis, and somatic embryogenesis in non-meristematic explants of Lamprocapnos spectabilis ‘Alba’ cultured in various media and to analyze the chemical diversity of the produced callus. Leaf, petiole, and internode explants were cultured on the modified Murashige and Skoog (MS) medium fortified with various combinations and concentrations of 6-benzyladenine (BA), indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorphenoxyacetic acid (2,4-D), and picloram (PIC). After 10 weeks of culturing, the morphogenetic response of explants was evaluated and the concentration of chlorophylls, carotenoids, anthocyanins, and polyphenols in callus was analyzed. There was no influence of explant type on the callogenesis efficiency (62.1–65.3%). The highest fresh weight of callus was produced on leaf explants in the presence of 2,4-D or PIC. In contrast, the highest share of dry weight was found in internode-derived calli and cultured on IAA-supplemented medium (up to 30.8%). Only 2.5% of all explants regenerated adventitious shoots, while rhizogenesis was reported in 4.5% of explants. Somatic embryos were produced indirectly by 0% to 100% of explants, depending on the culture medium and explant type. The highest mean number of embryos (11.4 per explant) was found on petioles cultured in the MS medium with 0.5 mg·L−1 BA and 1.0 mg·L−1 PIC. Calli cultured in media with NAA usually contained a higher content of primary and secondary metabolites. There was also a significant impact of explant type on the content of anthocyanins, polyphenols, and carotenoids in callus. Further studies should focus on the elicitation of metabolites production in callus culture systems of the bleeding heart.

Somatic embryogenesis (SE) is a unique method of in vitro regeneration for plant reproduction, germplasm resource preservation, and molecular-assisted breeding. Although lily is an internationally valued ornamental plant, its SE technology is still incomplete. Here, the effects of different explants, picloram (PIC) concentrations, and light conditions on somatic embryo induction, development, germination and seedling formation were studied in five Lilium varieties belonging to different hybrids. The embryo histomorphology, development and origins were observed, and a broadly efficient embryogenesis system was firstly established in Lilium. With leaf explants, all varieties tested underwent indirect somatic embryogenesis (SE). With scale explants, the Lilium Asiatic ‘Strawberry and Cream’, Lilium Oriental ‘Double Surprise’, and Lilium longiflorum Thunb. ‘White Heaven’ underwent indirect SE and the Lilium longiflorum × Asiatic hybrid ‘Eyeliner’ and Lilium longiflorum × Oriental ‘Triumphator’ underwent direct SE. Somatic embryogenesis in the above varieties involved four stages: globular, heart-shaped, torpedo-shaped, and cotyledon embryos. Two original SE pathways (endogenous and exogenous) were observed, and their appearance times differed. In indirect SE, exogenous embryos appeared first, followed by endogenous embryos; whereas in direct SE, the order was reversed. The different somatic embryo origins and the proportion of internal and external origins were closely related to the occurrence of somatic embryos by direct or indirect pathways and significantly affected the induction cycle of somatic embryos. Dark culture promoted lily somatic embryo induction, while 16 h/day light culture promoted germination, and the germination rates of all five lily varieties exceeded 90%. Murashige Skoog (MS) medium + (1.5–2.5 mg L−1) PIC + 0.2 mg L−1 1-Naphthaleneacetic acid (NAA) was suitable for SE from lily leaves and scales with an induction efficiency of 88–96%. Our research developed a widely applicable regeneration procedure for different hybrids of lily via somatic embryogenesis. Morphohistological analysis confirmed the origins, developmental stages and dynamic changes of cells during in vitro morphogenesis. This study will provide a valuable reference for the propagation of Lilium.

There is little information on the in vitro tissue culture systems in Lamprocapnos spectabilis (bleeding heart). The aim of this study was to analyze the influence of plant growth regulators (PGRs) on the development, quality, and physiological state of in vitro-grown bleeding heart “Gold Heart” and “White Gold”. Single-node explants were inoculated on the modified MS medium (Murashige and Skoog in Physiol Plant 15:473–497, 1962), fortified with different auxins, which included indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and picloram (PIC), along with cytokinins, which included 6-benzyladenine (BA), kinetin (KIN), and thidiazuron (TDZ) at various concentrations. The morphogenetic response of the explants was cultivar-specific. KIN was preferable for the proliferation and development of shoots in “Gold Heart.” However, none of the auxins or cytokinins improved the development of “White Gold” explants, compared with the PGR-free control medium. NAA was the most effective for stimulating rhizogenesis in both cultivars, although IAA resulted in the regeneration of the longest roots. TDZ, NAA, and PIC suppressed the development of shoots in both cultivars tested and stimulated abundant callus formation. Indirect regeneration of somatic embryos occurred on the NAA- and PIC-fortified media. In particular, the latter media stimulated regeneration of the highest number of somatic embryos per nodal segment. Composition of the culture medium also affected the levels of primary and secondary metabolites in shoots and callus of L. spectabilis. IAA (at 1.0 mg L−1) stimulated the synthesis of chlorophyll a and carotenoids in the “Gold Heart,” while BA and KIN (at 0.5 mg L−1) had a negative impact on the concentration of chlorophyll b in the shoots of this cultivar. None of the PGRs increased the level of the pigments in the shoots of bleeding heart “White Gold.” The concentration of chlorophylls and carotenoids in the callus of both cultivars tested was significantly lower compared with the shoots; however, callus was abundant in flavanols.

Plant cell totipotency is one of the 25 major topics in current scientific research, and somatic embryos are good experimental material for studying cell totipotency. Polar auxin transport plays an important regulatory role in somatic embryogenesis (SE). However, little is known about the auxin transport genes and their regulatory mechanisms in Lilium SE. In this study, we applied single-molecule real-time (SMRT) sequencing to Lilium pumilum DC. Fisch. for the first time and obtained a total of 119,649 transcripts, of which 14 encoded auxin transport genes. Correlation analyses between somatic embryo induction and gene expression under different treatments revealed that auxin transport genes, especially ATP-binding cassette (ABC) transporter B family member 21 (ABCB21) and PIN-FORMED (PIN) LIKES 7 (PILS7), may be key players in SE, and the necessary duration of picloram (PIC) treatment to induce SE is as short as 3 days. Our research provides valuable genetic information on Lilium pumilum, elucidating the candidate auxin transport genes involved in SE and their influencing factors. This study lays a foundation for elucidating the regulatory mechanism of auxin transport in SE.

Cassava (Manihot esculenta Crantz) is a tropical root crop that serves as a food staple and a vital income source to resource deprived farmers in the tropics. Despite its importance, cassava production and consumption is faced by a myriad of biotic/abiotic constraints. Genetic transformation which can be applied to mitigate these challenges however has as a prerequisite the availability of robust regeneration systems. This study evaluated the effect of explant type and hormone regime on somatic embryogenesis and regeneration of Kenyan cassava cultivars. The embryogenic competence of immature leaf lobe and stem explants of three cassava cultivars was determined by culturing them in MS medium supplemented with picloram (4, 6, 8 and 10 mg/l). The optimum media composition for embryo maturation, germination and plant recovery was assessed by culturing embryos in media supplemented with varying concentrations of 6-Benzylaminopurine (BAP), α–Naphthalene acetic acid (NAA) and Gibberellic acid (GA3). Somatic embryo formation frequencies in leaf explants were significantly higher (P≤0.05) than in stem explants. Embryo formation rates were found to increase with increasing concentrations of picloram. Cultivar 08/080 gave the least response to embryogenesis while embryogenesis rates for cultivar 08/274 were similar to the control cultivar TMS 60444. Maturation and plant recovery rates differed significantly with regard to the ratios of BAP, NAA and GA3 in the maturation media. The optimal medium for embryo germination and plant recovery was supplemented with 1 mg/l BAP, 0.02 mg/l NAA and 1.5 mg/l GA3. This optimized regeneration protocol can be coupled with mutation breeding or genetic transformation to improve the cassava germplasm. Key words: Cassava, explant, regeneration, somatic embryogenesis, plant growth regulator.

The picloram (PCM) adsorption on nontronite, illite and kaolinite was studied at pH 3, 5 and 7. The adsorption isotherms had well-fitted to Langmuir and Freundlich models equations. The interactions of PCM with the clay mineral surfaces exhibited an anionic profile adsorption, with a decrease in adsorption when the pH increases. The PCM adsorption capacity increases in the following order: kaolinite < illite < nontronite. The X-ray diffraction (XRD) analysis of PCM-clay samples revealed that the picloram molecule does not enter into the clays basal space. The interaction of PCM with clays surface sites through nitrogen of the pyridine ring was confirmed by X-ray photoelectron spectroscopy (XPS). Due to the anionic form of PCM, the adsorption onto the external and edges surface sites of the clay minerals was proposed.

This study aimed to fabricate a novel TiNT-RuO2 electrocatalyst to produce active chlorine with the capacity to oxidize recalcitrant organic matter. Titanium oxide nanotubes (TiNTs) were first grown via Ti foil anodization in potentiostatic mode in an ethylene glycol solution containing H2O and NH4F and then annealed at 450°C in air. RuO2 nanoparticles were deposited on the TiNTs using a hybrid strategy that included pulsed electrodeposition of the Ru nanoparticles, followed by thermal treatment. X-ray diffraction (XRD) only revealed peaks related to metallic Ti and anatase (TiO2) for the TiNTs, whereas additional peaks associated with the tetragonal RuO2 structure were shown for TiNT-RuO2. Scanning electron microscopy showed a well-organized structure for the TiNTs with an average inner diameter of approximately 185 nm for the NTs, while RuO2 nanoparticles (size <10 nm) were homogenously deposited on the walls and around the openings of the TiNTs. TiNT-RuO2 preferred the active chlorine formation over oxygen evolution owing to the presence of the RuO2 nanoparticles. Rapid degradation testing revealed complete elimination of picloram (PCL) in 2 h when 10 mA cm−2 was imposed on the TiNT-RuO2, whereas 48% of the 2,4-dichlorophenoxyacetic acid (2,4-D) was removed under the same conditions using the electro-generated active chlorine on the TiNTs.

Arachis pintoi is a peanut species native to Brazil, which is cultivated in many countries for animal forage, soil cover, landscaping, and recovery of degraded areas. Tissue culture studies for this species have been focused in plant production, whereas works on in vitro secondary metabolites production are scarce. The goal of the present work was to establish callus cultures from different seed explants of A. pintoi, aiming at evaluating the potential for metabolites production and antioxidant activity. Embryonic axes, leaflets, and cotyledons were cultured on solidified MS medium supplemented with picloram (PIC), 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA), under light or dark conditions. Friable calluses with a high biomass (4.3 ± 0.3 g FW per callus) were obtained from embryonic leaflets cultured on medium supplemented with 17.6 µM BA plus 5.4 µM NAA, in the dark. Cotyledons and embryonic axes cultured in the presence of 4.4 µM BA combined with 10.8 µM NAA formed heterogeneous calluses with a compact base and a large friable surface. Trans-resveratrol and other stilbenes that were not found in seeds were detected in callus extracts, especially those originated from cotyledons, although these materials showed lower total phenolic contents (TPC) when compared with seeds with and without testa, as well as cotyledons. Extracts from seeds with testa and from calluses derived from cotyledons and embryonic axes showed the highest EC50 in DPPH assays. No correlation between TPC, trans-resveratrol and antioxidant activity was observed.

This communication provides a protocol for stable transformation of Opuntia callus cultures. Itis a summary of ten years research from 2006 to 2016 of more than 340 experiments toobtain stable transformation and regeneration of five clones of Opuntia ficus-indica. Althoughregeneration was not achieved, stable transformation was achieved as evidenced byexpression of a fluorescent marker gene six months after inoculation by soaking explantsderived from unopened floral buds in Agrobacterium tumefaciens EHA 101. As cactus hadtoo much auto fluorescence to permit use of even enhanced green fluorescent proteins, thefluorescent marker TdTomato with red/orange emission was used. To avoid consumerobjections from antibiotic selective marker systems, the Phosphomannose Isomerase (PMI)gene that screens for growth on mannose was used. Explants that fluoresced grew well up to10 g L-1 mannose, while explants that did not fluoresce senesced and died when cultured on2 g L-1 mannose. The optimal basal media were found to be either Murashige-Skoog with 30g L-1 sucrose, with either double the standard Ca concentration or Woody Plant media with anadditional 2,000 mg L-1 KNO3. Standard liquid shake, temporary immersion system or solidagar with 3 g L-1 gel rite was examined and the solid gel rite media was used. Previousreports that reported stable transformation of meristems by needle injection could not berepeated, possibly because those experiments were conducted with non-intron GUS thatwould have permitted the Agrobacterium to properly transcribe and splice transcripts for theuidA gene. Two independent reports of somatic embryogenesis Bouamama et al. (2011) andGomez et al. (2006) in Opuntia were intensively examined, but could not be repeated.However smooth, green structures similar to “nodules” described by McGown et al. (1988)that can be induced to produce shoots were obtained but could not be induced to produceshoots. The hormone combinations that resulted in the greatest “structure” from immaturefloral explants were Zeatin (ZA) 0.2 to 0.75 mg L-1 with naphthalene acetic acid (NAA) 0.2 mg&#x0D; L-1, or Thidiazuron (TDZ) 0.75 with ZA 0.5 and NAA 0.4 mg L-1, or metatopolin 1.5 with NAA0.25 mg L-1. Long-term culture on Picloram (PIC) resulted in cultures with a red tinge,thought to be stress-induced betalain production. However, the most promising hormonecombination with (PIC) on floral explants was 0.01 TDZ/0.06 ZA/0.02 mg L-1 PIC. It issuggested that the most promising approaches to obtain shoots from these types ofstructures will come from transient or stable expression of the WUSCHEL and/orBABYBOOM regulatory genes in order to stimulate shoot development.

On the performance of HOCl/Fe2+, HOCl/Fe2+/UVA, and HOCl/UVC processes using in situ electrogenerated active chlorine to mineralize the herbicide picloram