Search

Carnation (Dianthus caryophyllus L.) holds great significance as a cut flower, and its vase life plays a crucial role in determining its quality and market appeal. However, the extended transportation distances from the production location to consumer markets can have an unfavourable impact on the vase life and overall quality of cut flowers. The first experiment demonstrated that exposing cut carnation flowers to simulated vibrational stress (SVS) led to a decline in vase life and overall quality. Notably, the application of Nano Silver (NS; 25 and 50 μM) and melatonin (MT; 100 and 200 μM) into the holding solution proved effective in counteracting these negative effects, and extended the vase life by promoting cut flower water relations. In the second experiment, the optimal concentrations of NS and MT into the holding solution (25 and 100 μM, respectively) effectively reduced chlorophyll degradation, electrolyte leakage, and lipid peroxidation by enhancing the activity of catalase and peroxidase enzymes. As a result, it appears that the application of NS and MT in the holding solution has successfully prolonged vase life by both improving water balance and boosting antioxidant activity, while mitigating the negative effects of SVS. [ABSTRACT FROM AUTHOR]

Solanum nigrum (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although Agrobacterium-mediated genetic transformation has been established in S. nigrum, the transformation period is long. Specifically, induction of roots takes approximately five weeks for tetraploid and hexaploid S. nigrum, and 7 weeks for diploid Solanum americanum. In this study, we developed an improved rooting-induced method that requires only about 1 week and avoids the use of tissue culture. After generating the transgenic shoots, they were directly transplanted into the soil to facilitate root formation. Remarkably, 100% of the transgenic shoots developed roots within 6 days. Our improved method is time-saving (saving more than 1 month) and simpler to operate. The improved rooting-induced step can be applied to induce roots in various plants using tissue culture, exemplified by the carnation (Dianthus caryophyllus L.). Furthermore, we applied the improved method to generate S. americanum plants expressing AcMYB110 from kiwifruit (Actinidia chinensis spp.). This method will contribute to speeding up gene functional analysis and trait improvement in S. nigrum and might have potential in fast plant molecular breeding processes in crops and rapid rooting induction in tissue culture. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effects of different types of light and some factors on some vegetative growth and flowering of two cultivars of carnation plant (Dianthus caryophyllus L.). The results showed the maximum leaves number, leaf area per plant, internodes number and intermodal length were recorded under 14h incandescent (41.09 leaves/plant, 363.91 cm2, 18.70 node/plant, and 7.675 cm) respectively, and the increase was significant. While the effect of triple interaction, which showed a significant effect in all characteristics, the largest leaf area per plant (578.66 cm2) was obtained from the moonlight cultivar when cultured on the media containing 60% local compost and exposed to the 14h incandescent, Also, the maximum vase life was found in the Ormea cultivar (17.47 days) when the cultivar was exposed to the 14h incandescent and was cultured on medium that included 60% local compost. The best grade flower was obtained when the Ormea cultivar was cultured on a medium containing 30% and 60% local compost under 14 hours of incandescent light, reaching 3.89. [ABSTRACT FROM AUTHOR]

Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a fower color mutant using the pink-fowered carnation ‘Kane Ainou 1-go’. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of ‘Kane Ainou 1-go’ has not been previously isolated, we initially isolated the ANS gene from ‘Kane Ainou 1-go’ for characterization. Southern hybridization analysis confrmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efciency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplifed polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confrmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology [ABSTRACT FROM AUTHOR]

The intensive cultivation of olive trees and grapevines in the Mediterranean region not only results in large yields but also generate wastes, with high restrictions on their impact on people's well-being and the environment. The current study sought to investigate the potential use of olive-mill waste (OW), grape-mill waste (GW) and their mixtures (OW + GW) at different levels (0%, 5%, 10% and 20% v/v) for partial peat substitution in the production of carnation (Dianthus caryophyllus L.) plants. The presence of OW, GW and OW + GW wastes raised the pH, the electrical conductivity, the content of organic matter and mineral content in substrate mixtures, while they decreased the total porosity and the available free air. The use of OW had more negative impacts than GW, while the OW + GW mixture alleviated, to some extent, the negative OW impacts. The use of high levels of residues decreased plant growth, chlorophyll content and mineral accumulation in plant tissue due to inappropriate growing media properties. The increased OW presence caused oxidative stress to the plants, as verified by the increased malondialdehyde and hydrogen peroxide content. This resulted in an upsurge in the total phenolics. However, GW presence did not impact any oxidative stress. It can be suggested that 10% OW, 10% GW or 20% OW + GW can be used in growing media, as they resulted in suitable plant growth. To ensure sufficient yields, nevertheless, the growing media's characteristics also need to be enhanced. [ABSTRACT FROM AUTHOR]

The Dianthus caryophyllus L. occupies the second position of the best sold cut flowers after rose. The choice of the environment and the cultivation container is crucial for flower production because, besides permitting higher temperature, light, and relative humidity management, it also impacts yield. This study evaluated the cultivation of yellow carnation in different protected environments and pot colours. The experiment was conducted in a factorial 2 × 4 (greenhouse and nursery x blue, red, brown, and black pots; the black pot was considered the control treatment). The photosynthetically active radiation, substrate temperature, and meteorological variables in the environments were monitored during the experiment's conduction as well the growth and productivity variables. The greenhouse significantly reduced solar radiation, generating favourable conditions for the initial growth of carnation plants. The nursery maintained higher levels of solar radiation, stimulating its flowering. The cultivation in brown pots provided higher quality plants regarding both vegetative growth and flowering. Therefore, brown pots are an option to substitute the black pots traditionally used for flower production. The use of the blue pot negatively influenced the development of carnation plants. [ABSTRACT FROM AUTHOR]

To investigate the physiological response and uptake characteristics of three genotypes of Dianthus caryophyllus to Cr stress, we used a heavy metal-contaminated soil sample from a lead-zinc mine in a pot trial to determine several parameters, including plant height, stem thickness, root architecture, biomass, heavy metal accumulation and transport capacity under different levels of Cr contamination. We discovered a dosage effect in the influence of the heavy metal Cr on the growth of Dianthus caryophyllus. Under low-dose conditions, it elicited a protective response favoring plant growth, whereas under high accumulation conditions, it inhibited plant growth. Additionally, chromium stress increased the growth of plant roots. In the range of chromium concentrations between 0-100 mg/kg, the enrichment and transit coefficient of the three species, namely Freedom, Master, and Dallas decrease as the concentration of chromium increases. The enrichment coefficient follows the order Freedom > Dallas > Master, whereas the transit coefficient follows the order Master > Freedom > Dallas. All three genotypes of Dianthus caryophyllus exhibited tolerance, accumulation, and transport capacities when subjected to chromium stress. As a result, they can serve as reference plants for on-site remediation of heavy metals in soils contaminated with tailings in mining regions. [ABSTRACT FROM AUTHOR]

The Dianthus caryophyllus L. occupies the second position of the best sold cut flowers after rose. The choice of the environment and the cultivation container is crucial for flower production because, besides permitting higher temperature, light, and relative humidity management, it also impacts yield. This study evaluated the cultivation of yellow carnation in different protected environments and pot colours. The experiment was conducted in a factorial 2 × 4 (greenhouse and nursery x blue, red, brown, and black pots; the black pot was considered the control treatment). The photosynthetically active radiation, substrate temperature, and meteorological variables in the environments were monitored during the experiment's conduction as well the growth and productivity variables. The greenhouse significantly reduced solar radiation, generating favourable conditions for the initial growth of carnation plants. The nursery maintained higher levels of solar radiation, stimulating its flowering. The cultivation in brown pots provided higher quality plants regarding both vegetative growth and flowering. Therefore, brown pots are an option to substitute the black pots traditionally used for flower production. The use of the blue pot negatively influenced the development of carnation plants.

Colombia is one of the main producers of carnations worldwide. However, its production has been affected by vascular wilting caused by the pathogen Fusarium oxysporum f. sp. dianthi. In previous studies, thiamine has been proposed as a potential resistance inducer for this disease, under greenhouse growth conditions. However, the specific conditions for its application must be studied when applying it under commercial conditions. In this sense, the aim of this study was to evaluate the effect on the disease incidence of the foliar aspersion of thiamine solutions in different concentrations among some susceptible carnation cultivars. For this, two in-vivo trials were carried out, using foliar spraying of thiamine as a treatment before inoculation with the pathogen Fod, then measurements of incidence, severity, and the content of phenolic and flavonoid compounds were made. It was found that thiamine application, in the 4 concentrations evaluated, shows a statistically similar decrease in the incidence and severity of the disease. These results were verified using multivariate statistical analysis. In addition, it was found that the application of thiamine in a concentration of 1mmol*L-1 presents a positive response in reducing the effects of the disease in three susceptible cultivars. The results suggest that thiamine has the potential to be applied as a technique in the integrated management of disease in the productive sector.

Solanum nigrum (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although Agrobacterium-mediated genetic transformation has been established in S. nigrum, the transformation period is long. Specifically, induction of roots takes approximately five weeks for tetraploid and hexaploid S. nigrum, and 7 weeks for diploid Solanum americanum. In this study, we developed an improved rooting-induced method that requires only about 1 week and avoids the use of tissue culture. After generating the transgenic shoots, they were directly transplanted into the soil to facilitate root formation. Remarkably, 100% of the transgenic shoots developed roots within 6 days. Our improved method is time-saving (saving more than 1 month) and simpler to operate. The improved rooting-induced step can be applied to induce roots in various plants using tissue culture, exemplified by the carnation (Dianthus caryophyllus L.). Furthermore, we applied the improved method to generate S. americanum plants expressing AcMYB110 from kiwifruit (Actinidia chinensis spp.). This method will contribute to speeding up gene functional analysis and trait improvement in S. nigrum and might have potential in fast plant molecular breeding processes in crops and rapid rooting induction in tissue culture.

Abstract With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

Dianthus caryophyllus is an economic species often considered excellent cut flowers and is suitable for bouquets and gardens. Here, we assembled the haplotype-resolved genome of D. caryophyllus 'Aili' at the chromosome level for the first time. The total lengths of the two assembled haplotypes of carnation were 584.88 Mb for haplotype genome 1 (hap1) and 578.78 Mb for haplotype genome 2 (hap2), respectively. We predicted a total of 44,098 and 42,425 protein-coding genes, respectively. The remarkable structure variation was identified between two haplotypes. Moreover, we identified 403.80 Mb of transposable elements (TEs) in hap1, which accounted for 69.34% of the genome. In contrast, hap2 had 402.70 Mb of TEs, representing 69.61% of the genome. Long terminal repeats were the predominant transposable elements. Phylogenetic analysis showed that the species differentiation time between carnation and gypsophila was estimated to be ~54.43 MYA. The unique gene families of carnation genomes were identified in 'Aili' and previously published 'Francesco' and 'Scarlet Queen'. The assembled and annotated haplotyperesolved D. caryophyllus genome not only promises to facilitate molecular biology studies but also contributes to genome-level evolutionary studies. [ABSTRACT FROM AUTHOR]

The intensive cultivation of olive trees and grapevines in the Mediterranean region not only results in large yields but also generate wastes, with high restrictions on their impact on people’s well-being and the environment. The current study sought to investigate the potential use of olive-mill waste (OW), grape-mill waste (GW) and their mixtures (OW + GW) at different levels (0%, 5%, 10% and 20% v/v) for partial peat substitution in the production of carnation (Dianthus caryophyllus L.) plants. The presence of OW, GW and OW + GW wastes raised the pH, the electrical conductivity, the content of organic matter and mineral content in substrate mixtures, while they decreased the total porosity and the available free air. The use of OW had more negative impacts than GW, while the OW + GW mixture alleviated, to some extent, the negative OW impacts. The use of high levels of residues decreased plant growth, chlorophyll content and mineral accumulation in plant tissue due to inappropriate growing media properties. The increased OW presence caused oxidative stress to the plants, as verified by the increased malondialdehyde and hydrogen peroxide content. This resulted in an upsurge in the total phenolics. However, GW presence did not impact any oxidative stress. It can be suggested that 10% OW, 10% GW or 20% OW + GW can be used in growing media, as they resulted in suitable plant growth. To ensure sufficient yields, nevertheless, the growing media’s characteristics also need to be enhanced.

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches. Highlights: Melatonin at 2 mg/l substantially increases the Agrobacterium-mediated transformation efficiency of carnation cultivars to 24%. Nitrogen-depleted inoculation and co-cultivation media can greatly enhance carnation transformation. Callus is a potential target explant for Agrobacterium transformation of carnation. Melatonin and lipoic acid can considerably promote the shoot regeneration from transformed callus. The established protocol for carnation transformation is cultivar independent. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Agriculture & Nature / Kahramanmaraş Sütçü İmam Üniversitesi Tarım & Doğa Dergisi is the property of Kahramanmaras Sutcu Imam Universitesi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Nano-selenium (nano-Se) is an eco-friendly and inexpensive compound. The potential of including it in the holding solution to stimulate vase life was investigated by conducting two experiments. In the first one, dose-response curves were generated by applying different nano-Se concentrations (0–1 and 0–1.5 µM for rose and carnation, respectively) in the holding solution of three cultivars per species. Nano-Se in the holding solution improved vase life in all cases, besides one carnation cultivar. The optimal concentration was considerably higher in carnation as compared to rose (1–1.5 and 0.25–0.75 µM, respectively), and was cultivar dependent. Nano-Se improved cut flower water balance by enhancing water absorption more than transpiration. In the second experiment, the optimal holding solution nano-Se concentration was employed to evaluate the underlying processes in the two cultivars per species, where vase life was mostly increased. Physiological and biochemical characteristics were assessed in both leaf and petal tissues. Nano-Se ameliorated chlorophyll degradation, electrolyte leakage and lipid peroxidation owing to up-regulation of both enzymatic and non-enzymatic antioxidants. In conclusion, the nano-Se-induced vase life extension was associated with both upgraded antioxidant status and improved water relations, setting it as a low-cost and environmentally-safe agent of prolonging floral longevity. [ABSTRACT FROM AUTHOR]

In order to investigate the effects of preservative 1-Methylcyclopropene (1-MCP) on storage life, vase life and physiological metabolism during intercalation period of Dianthus caryophyllus 'Sangria' cut flowers, these Dianthus caryophyllus cut flowers were fumigated with 0.64, 1.28 and 1.92 µL/L 1-MCP, respectively. The effects of 1-MCP on the shelf life and bottle life of Dianthus caryophyllus were studied by investigating the states of cut flowers in the processes of cold storage (2~4 °C) and room temperature (25 °C). Results showed that 1-MCP could significantly prolong the vase life of Dianthus caryophyllus cut flowers at room temperature, and the concentration of 1.92 µL/L presented the best effects, the cut flowers vase life was prolonged from 4 days to 8 days. Under cold storage condition, the time of cut flowers opening index reaching to 0.8 with 1.92 µL/L 1-MCP treatment was 101 days, while that in control group was only 18 days, which demonstrated that 1 -MCP treatment greatly prolonged the cold storage time of Dianthus caryophyllus cut flowers. Further investigation of cut flowers vase period after refrigeration found that all the 110-day-refrigerated cut flowers bloomed normally after one day of vase incubation, and 1.92 µL/L 1-MCP treatment significantly promoted the relative water absorption of cut flowers, and retard the relative electrical conductivity of petals. In addition, after 11 days vase incubation, the flower wilting rate in 1.92 µL/L 1-MCP treatment group was only 7.1%, while the flower wilting rate of control group was 78.5%. Therefore, 1.92 µL/L 1 -MCP fumigation treatment can be popularized as a fresh preservation treatment scheme for Dianthus caryop -hylluscut flowers. [ABSTRACT FROM AUTHOR]

Although numerous transcription factors with antagonistic activities have been shown to contribute to growth and development, whether and how they regulate senescence in plants is largely unknown. In this study, we investigated the role of antagonistic transcription factors in petal senescence in carnation (Dianthus caryophyllus), one of the most common types of ethylene-sensitive cut flowers produced worldwide. We identified DcHB30 that encodes a ZF-HD transcription factor that is down-regulated in ethylene-treated petal transcriptomes. We found that silencing DcHB30 accelerated ethylene-induced petal senescence and that DcHB30 physically interacts with DcWRKY75, a positive regulator of ethylene-induced petal senescence. Phenotypic characterization and molecular evidence indicated that DcHB30 and DcWRKY75 competitively regulate the expression of their co-targeted genes DcACS1 , DcACO1 , DcSAG12 , and DcSAG29 by reciprocally inhibiting the DNA-binding activity of each other on the gene promoters. This transcriptional regulation mechanism demonstrates that these transcription factors serve as positive and negative regulators in ethylene-induced petal senescence in carnation. Thus, our study provides insights into how antagonizing transcription factors regulate plant senescence. [ABSTRACT FROM AUTHOR]

Fusarium infection in carnation is the most important limiting factor for carnation production. We isolated 38 fungal isolates from infected carnation plants collected from Mahallat, Iran, and 15 Fusarium species were identified morphologically. Isolates of J14, k5, and k72 were the most pathogenic isolates in pathogenicity test on White Liberty cultivar. Molecular identification of J14, k5, and k72 isolates was done based on Elongation Factor 1-alpha (EF1-α) gene. To investigate the effect of Trichoderma and some of its mutants on reducing the Fusarium infection, six wild type isolates were examined. The species of T. harzianum, T. virense, and T. ghanens showed more inhabitation potential and were chosen for induced mutation via gamma irradiation at 250 Gy. The number of 270 mutants were screened morphologically and 60 mutants were screened using dual culture against J14, k5, and k72. Morphological and molecular identification of J14, k5, and k72 isolates recognized them as F. oxysporum. Three mutants, i.e. ThM7(67.17%), TgM1 (59.45%), and TvM17(57.55%) showed the highest efficacy and were selected. Evaluation test of efficacy in greenhouse by mixture of T. harzianum, T. virense and T. ghanens (TW) and mixture of mutant isolates ThM7, TgM1, and TvM17 (TM) showed that biological method had higher ability to control Fusarium infection on carnation plants in greenhouse condition, and mutation had no adverse effects on plants. The results of this experiment proved that the use of mutation in the Trichoderma genome with the use of gamma radiation could be an effective way to achieve isolates with better performance in this bio-control agent. [ABSTRACT FROM AUTHOR]

Background and Purpose: Candida albicans (C. albicans) is the most common human pathogen owing to the most virulence factors. It seems that extracellular hydrolytic enzymes play a key role in C. albicans pathogenicity. The present study aimed to assess the susceptibility and enzymatic activity of pathogenic C. albicans isolates exposed to the Syzygium aromaticum (S. aromaticum) essential oil.Materials and Methods: S. aromaticum oil was characterized using gas chromatography-mass spectrometry (GC–MS). The broth microdilution technique (CLSI, M27-A3) was used to determine the minimum inhibitory concentration (MIC) of test compounds. Furthermore, before and after treatment with S. aromaticum essential oil, the yeasts were analyzed regarding the proteinase (Prz), hemolysin (Hz), and phospholipase (Phz) production/activity.Results: β-caryophyllene (12.76%) was found to be the major constituent in the essential oil after eugenol (84.64%). Only one isolate of C. albicans showed the antifungal resistance to fluconazole. All isolates were susceptible to S. aromaticum essential oil with MIC of 625-1250 μg/ml. S. aromaticum oil represented the best antifungal effectagainst C. albicans at MIC 1000 μg/ml. The mean±SD enzyme activity of C. albicans not exposed to S. aromaticum essential oil was obtained at 0.55±0.03, 0.73±0.04, and 0.61±0.05 for proteinase, hemolysin, and phospholipase, respectively. The activities of these enzymes were reduced significantly (P

Abstract Background Cut flower longevity is often limited by adverse water relations or oxidative stress. The potential of single- and multi-walled carbon nanotubes (SWCNTs and MWCNTs, respectively) on prolonging vase life was addressed. Dose–response curves were obtained by applying five concentrations (0–80 mg L−1) of SWCNTs or MWCNTs either once as a foliar spray or continuously in the holding solution of three carnation cultivars. Next, the optimal concentration of either SWCNTs or MWCNTs was employed to evaluate several parameters critical for vase life. Results Foliar spray application exerted minor effects on water relations, flower opening and keeping quality. By contrast, including CNTs in the holding solution sustained a positive water balance for a longer period, improved flower opening and prolonged vase life. These effects were similar between SWCNTs and MWCNTs, and were concentration-dependent. The optimal concentration for vase life was higher for MWCNTs as compared to SWCNTs, and for two cultivars as compared to the third one. At optimal concentration, SWCNTs or MWCNTs in the holding solution generally maintained turgidity, and alleviated chlorophyll degradation, electrolyte leakage and lipid peroxidation. These effects were related to increased activation of enzymatic (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) and non-enzymatic (carotenoids, polyphenols, and flavonoids) antioxidants. Conclusion CNTs in the holding solution were very effective in stimulating vase life through improved water relations and enhanced antioxidant machinery stimulation. Graphical Abstract

Summary: Carnation (Dianthus caryophyllus) is one of the most popular ornamental flowers in the world. Although numerous studies on carnations exist, the underlying mechanisms of flower color, fragrance, and the formation of double flowers remain unknown. Here, we employed an integrated multi‐omics approach to elucidate the genetic and biochemical pathways underlying the most important ornamental features of carnation flowers. First, we assembled a high‐quality chromosome‐scale genome (636 Mb with contig N50 as 14.67 Mb) of D. caryophyllus, the 'Scarlet Queen'. Next, a series of metabolomic datasets was generated with a variety of instrumentation types from different parts of the flower at multiple stages of development to assess spatial and temporal differences in the accumulation of pigment and volatile compounds. Finally, transcriptomic data were generated to link genomic, biochemical, and morphological patterns to propose a set of pathways by which ornamental traits such as petal coloration, double flowers, and fragrance production are formed. Among them, the transcription factors bHLHs, MYBs, and a WRKY44 homolog are proposed to be important in controlling petal color patterning and genes such as coniferyl alcohol acetyltransferase and eugenol synthase are involved in the synthesis of eugenol. The integrated dataset of genomics, transcriptomics, and metabolomics presented herein provides an important foundation for understanding the underlying pathways of flower development and coloration, which in turn can be used for selective breeding and gene editing for the development of novel carnation cultivars. [ABSTRACT FROM AUTHOR]

SKP1 gene is the core component of the SCF(Cul1-Rbx1-Skp1-F box)E3 ubiquitin ligase protein complex and is involved in many different biological processes. However, the SKP1 gene of carnation has not been cloned. In this study, a new DcSKP1 gene(the accession number in GenBank was MK931293)was isolated from the flower of Dianthus caryophyllus by using RT-PCR and RACE approaches. The results were as follows:(1)The full length cDNA sequence of DcSKP1 was 962 bp, and a ORF of 567 bp encoded 188 amino acids.(2)Protein sequence alignment showed that DcSKP1 had a highly conserved TPEE motif, Skp1_POZ domain and Skp1 domain, and clustered on a branch with Arabidopsis thaliana Skp1.(3)The DcSKP1 gene expression pattern of Dianthus caryophyllus was studied by qRT-PCR, and it was found that the DcSKP1 gene was expressed in all tissue sites, the expression level in anthers was higher than that in stem and leaf tissues, the expression level was the highest in young anthers, and the expression level decreased with the development of anthers. It is speculated that DcSKP1 gene may play an important role in carnation meiosis in D. caryophllus. [ABSTRACT FROM AUTHOR]

Background: Cut flower longevity is often limited by adverse water relations or oxidative stress. The potential of single- and multi-walled carbon nanotubes (SWCNTs and MWCNTs, respectively) on prolonging vase life was addressed. Dose–response curves were obtained by applying five concentrations (0–80 mg L−1) of SWCNTs or MWCNTs either once as a foliar spray or continuously in the holding solution of three carnation cultivars. Next, the optimal concentration of either SWCNTs or MWCNTs was employed to evaluate several parameters critical for vase life. Results: Foliar spray application exerted minor effects on water relations, flower opening and keeping quality. By contrast, including CNTs in the holding solution sustained a positive water balance for a longer period, improved flower opening and prolonged vase life. These effects were similar between SWCNTs and MWCNTs, and were concentration-dependent. The optimal concentration for vase life was higher for MWCNTs as compared to SWCNTs, and for two cultivars as compared to the third one. At optimal concentration, SWCNTs or MWCNTs in the holding solution generally maintained turgidity, and alleviated chlorophyll degradation, electrolyte leakage and lipid peroxidation. These effects were related to increased activation of enzymatic (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) and non-enzymatic (carotenoids, polyphenols, and flavonoids) antioxidants. Conclusion: CNTs in the holding solution were very effective in stimulating vase life through improved water relations and enhanced antioxidant machinery stimulation. [ABSTRACT FROM AUTHOR]

Fusarium wilt, caused by Fusarium oxysporum f.sp. dianthi (FOD), is one of the most devastating carnation diseases globally, resulting in significant crop loss (40-70%). The purpose of this study was to determine the efficacy of several fungicides against Fusarium wilt of carnation in vitro and in vivo under protected cultivation. The disease infected plant samples were collected, and pathogen isolates were confirmed through morphological and molecular techniques. The rDNA sequences of isolates had 97-100% similarity with other sequences of FOD available in Genbank and the isolates were identified as Fusarium oxysporum f.sp. dianthi. Phylogenetic analysis of rDNA sequences revealed 62-99% sequence similarity among the isolates. Further, we evaluated nine different fungicides against the virulent pathogenic isolate CFODTNAU1 under in vitro and in vivo. Three different concentrations such as 500, 1000 and 1500 ppm were evaluated on mycelial growth of the pathogen under in vitro and percent inhibition over control was calculated. Among the fungicides, three of them namely, tebuconazole 50% + trifloxystrobin 25% WG, tebuconazole 25.9% SC and azoxystrobin 23% EC completely inhibited (100% inhibition) the mycelial growth of the fungus in vitro. Under protected cultivation, application of tebuconazole 50% + trifloxystrobin 25% WG through root dipping and soil drenching @ 1.0 g/L recorded 11.11% of wilt incidence compared to control (29.71% wilt incidence), which was 59.99% reduction over control. Besides, this fungicide also increased the stem length, earliness of flowering and yield than the control and other treatments. Thus, the fungicide tebuconazole 50% + trifloxystrobin 25% WG can be used for amelioration and control of wilt disease in carnation. [ABSTRACT FROM AUTHOR]

Leaf stomata are the main channels for water loss of plants including cut flowers. In this study, we investigated the organographic distribution, morphological characteristics, light–dark response, and water loss contribution of stomata in cut carnations (Dianthus caryophyllus L. ‘Master’), which are prone to typical water deficits despite a few and small leaves. Stomata were observed in the upper and lower leaf epidermis, stem surface, abaxial bract epidermis, and abaxial sepal epidermis. Stomatal density (SD) on the stem surface was the highest and significantly greater than that on the upper and lower leaf and abaxial bract epidermis. The sepal epidermis had the lowest SD and the smallest stomata whereas the upper leaf epidermis had the largest stomata. Changes in the water loss rate increased in the light and decreased in the dark in both intact and leaves-removed cut carnations. The water loss rate of the former was greater than that of the latter. However, the water loss rate for the stem-only cut carnations had weak change rhythms and was much lower than that for the intact and leaves-removed cut carnations. These findings demonstrate the differential contributions of stomata in leaves, stems, and floral organs to water loss, and help to elucidate further the mechanism underlying postharvest water deficit in cut carnations.

In order to investigate the possibility for in in vitro fast regeneration of Dianthus caryophyllus, different concentrations of Kinetin (Kin), 2,4-Dichlorophenoxy Acetic Acid (2,4-D) had been tested for induction of callus by leaves and stems explant using Murashige and Skoog (MS) medium. After two months in culture, response callus in the induction rate, fresh and dry weight, texture and color were evaluated. MS medium that contained 4.5 mg.L-1 2, 4-D was suitable for callus induction with leaf explants. The combination of 0.4 mg.L-1 Kin and 2.0 mg.L-1 2, 4-D also demonstrated a wonderful induction of callus on stem explants. In addition to the culture medium complemented by Kin and 2, 4-D, the influence of Zeatin at various concentrations (0.0, 0.4, 0.8 or 1.0 mg.L-1) was assessed. In MS medium that contained 0.4 mg.L-1 Kin+ 2.0 mg.L-1 2,4-D+ 1.0 mg.L-1 Zeatin, rapid callus induction and also more callus proliferation from stem explants were detected. Results indicate on callus induction that, Zeatin was more successful than Kin or 2, 4-D, but other combinations directly developed a shoot. Callus obtained from stems explant was friable, white and yellow, while callus from leaf explants was green.

In order to investigate the possibility for in in vitro fast regeneration of Dianthus caryophyllus, different concentrations of Kinetin (Kin), 2,4-Dichlorophenoxy Acetic Acid (2,4-D) had been tested for induction of callus by leaves and stems explant using Murashige and Skoog (MS) medium. After two months in culture, response callus in the induction rate, fresh and dry weight, texture and color were evaluated. MS medium that contained 4.5 mg.L-1 2, 4-D was suitable for callus induction with leaf explants. The combination of 0.4 mg.L-1 Kin and 2.0 mg.L-1 2, 4-D also demonstrated a wonderful induction of callus on stem explants. In addition to the culture medium complemented by Kin and 2, 4-D, the influence of Zeatin at various concentrations (0.0, 0.4, 0.8 or 1.0 mg.L-1) was assessed. In MS medium that contained 0.4 mg.L-1 Kin+ 2.0 mg.L-1 2,4-D+ 1.0 mg.L-1 Zeatin, rapid callus induction and also more callus proliferation from stem explants were detected. Results indicate on callus induction that, Zeatin was more successful than Kin or 2, 4-D, but other combinations directly developed a shoot. Callus obtained from stems explant was friable, white and yellow, while callus from leaf explants was green. [ABSTRACT FROM AUTHOR]

• Meta-topolin stimulated carnation shoot formation and reduced vitrification. • SiNPs induced compact shoot structure and developed root system. • SiNPs enhanced activities of CAT, APX and SOD. • SiNPs were negatively correlated with Cu, K and Ca uptake but positively correlated with Mg, Fe and Si uptake. • SiNPs-derived plantlets showed well acclimation and growth under ex vitro conditions. This study is an attempt to explore the potential of meta-topolin (MT) and silica nanoparticles (SiNPs) in carnation micropropagation - an in vitro vitrification sensitive cut flower. Among MT concentrations (0.5 mg/L - 3.0 mg/L), 1.0 mg/L MT treatment showed a significant increase in shoot height, leaf length, chlorophyll content, dry weight and dry matter index (DM). However, MT concentration > 2.5 mg/L resulted in increased number of shoots and vitrification. Beside different concentrations SiNPs (0.1 mg/L - 3.0 mg/L) were tested in shoot multiplication, chlorophyll content and DM increased; in contrast, the number of shoots, shoot height and other growth indicators all decreased significantly. The results indicated that MT improved shoot multiplication and reduced vitrification, while SiNPs were less suitable for this stage due to limited water absorption of shoots. Interestingly, SiNPs induced compact shoot structure and stimulated root system development, as well as induced densely closed stomata. Furthermore, the presence of SiNPs increased the activities of antioxidant enzymes Catalase (CAT), Superoxide dismutase (SOD) and Ascorbate peroxidase (APX); besides, SiNPs showed negative correlation with Cu, K and Ca uptake but significantly improved Mg, Fe and Si uptake. However, the plantlets derived from SiNPs showed 2.1–2.8-fold decrease in shoot height compared to the control but significantly increased chlorophyll content, DM index, primary root length and diameter as well as lateral root formation. At the highly tested concentrations of 2.0 mg/L - 3.0 mg/L SiNPs, a decrease in growth, uptake of Mg, Fe, Si and explosion of CAT, APX and SOD antioxidant activity was found, which presumably express stress-induced SiNPs. Nevertheless, carnation plantlets derived from SiNPs showed almost complete survival and well growth in greenhouse conditions. These findings suggest that MT and SiNPs can be used to stimulate growth, reduce vitrification, and harden carnation plantlet in practical micropropagation. [ABSTRACT FROM AUTHOR]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This research was carried out to evaluate the effects of copper nanoparticles (CuNPs) on the postharvest physiology of carnation and chrysanthemum cut flowers. Synthesized CuNPs were applied at 10 and 20 mg L-1 to both cut flowers by the pulsing method. The physicochemical characteristics and the activity of antioxidant enzymes were measured on 0, 3, 6 and 9 days along the vase life. According to heat map analysis, changes in measured parameters were affected by flower type > vase period > CuNPs concentration. Results also showed that treating the two species of cut flower with CuNPS by the pulse treatment improved the relative fresh weight (RFW), vase solution uptake (VSU), membrane stability index (MSI), flower diameter and total soluble carbohydrate (TSC). Meanwhile, a decrease in both bacterial population of the stem end and the hydrogen peroxide (H2 O2 ) was observed. In comparison with the control, CuNPs at 20 mg L-1 by pulse treatment almost increased the vase life in both cut flowers by 30%.

An experiment was conducted in a green house at the Department of Horticulture and Gardens Engineering, College of Agriculture, University of Baghdad in Jadriya district for the period from 15-8-2017 to 15-8-2018. Plant roots were inoculated with two levels of mycorrhiza (20.0g) subjected to three irrigation intervals (2 days, 4 days, and 6 days) and salicylic acid was sprayed on the vegetative part in three concentrations (150, 100, 50.0 mg l-1. The results showed that the inoculation of carnation roots with mycorrhiza reduced the effectiveness of SOD (Superoxide dismutase) leaves content of proline, which amounted to 168.2 unit mol/12.72 mg l-1 dry weight. The results also indicated a significant increase in the vegetative growth traits in response to salicylic acid spraying at a concentration of 50 mg l-1 by interacting with mycorrhiza and D2 irrigation interval, namely plant height, dry matter, flower diameter, increasing leaf content of nitrogen, phosphorus, potassium, zinc, iron and the mycorrhiza infection percentage and roots surface area that reached 94.3 cm, 62.11% 10.79, 4.69%, 0.570%, 6.143%, 87.06%, 179.26%, 77.79% and 146.4g, respectively. [ABSTRACT FROM AUTHOR]

Soil salinity is one of the major environmental factors limiting plant growth and development; and it is considered a problem in arid and semiarid regions, where rainfall is insufficient to leach salts. The clove pink, Dianthus caryophyllus L. is a major product in Jordan with different irrigation needs and has the capacity to cope with water deficit. Consequently, the current study aimed at investigating the effect of salinityon certain growth, physiological and mineral parameters of two varieties of D. caryophyllus (Bizet Sagr and Grand Slam Hygr). The experiments were conducted under greenhouse conditions at the University of Jordan during the 2015/2016 growing season. The plants were grown in either soil or zeolitic tuff at five salinity levels. The results indicated that the growth parameters of both D. caryophyllus varieties vary significantly among the different salinity levels in both growing media. Increasing salinity caused a significant reduction in plant height, fresh and dry weights, flower length and diameter, and a delayed flowering time. Increasing salinity level caused also a significant reduction in leaf greenness, fluorescence yield and relative water content, and an increased stomata resistance of both plant varieties in both growing media. Increasing salinity level caused a significant increase in Na and Cl, and a decrease in K, P and N concentrations in plants of both tested varieties and media. In conclusion, salinity caused a significant effect on all tested growth, physiological and mineral parameters of D. caryophyllus. An appropriate irrigation regime should be used as a key to success in ornamentals’ growth control. [ABSTRACT FROM AUTHOR]

Developing scale-up system and automation of micropropagation in a bioreactor has been a possible way of cost reduction and intensive manual handling. We report a comparison between the results of experiments aimed at improving carnation micropropagation using new bioreactor according to Temporary Immersion Bioreactor (TIB) and solid culture. By applying different levels of BAP, at the concentration of 3 mg L−1, we observed 14.3 new shoots in TIB, but the number of new shoots on solid medium reached to 5.7 at the same treatment. Our results also showed that with 3 mg L−1 BAP in TIB, the initial fresh weight of plant material increased from 10 g to 450 g after 15 days. It is concluded that TIB showed more than 10 times shoot production of carnation. Shoot elongation and rooting induction was successfully stimulated in TIB by applying 1 mg L−1 IBA. Rooting of proliferated plantlets from TIB and solid culture were successfully happened, and led to highest number of roots (4.6 cm) and highest length of roots (6.87 cm) in TIB. More than 90% of plantlet was acclimatized to ex vitro. Our results suggested that mass production of carnation shoots in our simple TIB, with effective result, can be considered as a critical first step toward large scale production of carnation.

The effect of salinity stress on growth and photosynthesis has been studied in various plants. However, reports on the impacts of salt stress on the growth and physiological factors in carnation are limited. In this study, we measured how photosynthesis and related parameters were affected by salinity stress at the flowering stage of carnation. Salinity reduced the photosynthetic rate and related parameters, including stomatal conductance, intercellular CO2 concentration, and transpiration. Chlorophyll content slightly increased at lower NaCl concentrations, and showed a decreasing tendency at higher NaCl concentrations. ABA content increased with increasing NaCl concentrations. The duration and extent of sap flow significantly decreased at higher NaCl concentrations compared to the untreated control on both sunny and cloudy days. At 200 mM NaCl, stressed leaves appeared to have smaller cell size and larger intercellular spaces. Stomata subsided at lower and upper epiderma, while stomatal size and density decreased at higher NaCl. Epicuticular wax layer was particularly thicker at 200 mM NaCl and some stomata were completely covered with the epicuticular wax. Hence, the results reported here facilitate a better understanding of the potential salt tolerant mechanism adopted by carnation to reduce transpirational water loss from the plant under salt stress. Overall, the reduced photosynthetic capacity under salinity stress could be attributed to a decreased stomatal conductance. [ABSTRACT FROM AUTHOR]

The carnation (Dianthus caryophyllus L.) is a beloved and widely embraced ornamental flower worldwide, celebrated for vibrant hues, delightful fragrance, and prolonged blooming season. Within the petals of carnation blooms, anthocyanins, natural pigments, are responsible for the diverse range of captivating colors they display. However, the intricate molecular processes orchestrating the synthesis of these anthocyanins in carnations remain unclear. This study unveils DcbHLH1 as a pivotal regulator actively participating in anthocyanin biosynthesis within carnation flowers. Y1H and dual-LUC provided evidences that DcbHLH1 exhibited direct binding to the promoters of DcCHS and DcDFR , consequently leading to the activation of their gene expressions. In the earlier stages (S2 and S3) of blooming, DcMYB1 synergistically enhances the transcriptional activation capacity of DcbHLH1 through their collaborative interaction. As the flower matures into the later stages (S4 and S5), DcMYB1 expression exhibited a rapid decline. Concurrently, the repressor gene, DcMYB2 , experienced a swift upsurge in expression, ultimately inhibiting anthocyanin biosynthesis and curtailing the transcriptional activation activity of DcbHLH1 through direct interaction with DcbHLH1. Our results show a narrative of the dynamic interplay between DcbHLH1-DcMYB1 and DcbHLH1-DcMYB2, unveiling their roles in orchestrating the array of hues that adorn carnation petals over various flowering stages. These findings offer novel insights into the regulatory mechanisms influencing anthocyanin biosynthesis in ornamental plants. [Display omitted] • Flower color is of great significance in ecosystems and the economy. • Transcription factors play a crucial role in the regulation of flower color. • DcbHLH1 positively regulates anthocyanins biosynthesis in carnation. • DcbHLH1 interacts with DcMYB1/2 to dynamically regulate petal color in carnation. • Understanding mechanisms of color regulation is beneficial for improving color. [ABSTRACT FROM AUTHOR]

The fungal pathogen Fusarium oxysporum f. sp. dianthi (Fod) is the causal agent of the vascular wilt of carnation (Dianthus caryophyllus L.) and the most prevalent pathogen in the areas where this flower is grown. For this reason, the development of new control strategies against Fod in carnation has been continuously encouraged, in particular those based on the implementation of plant resistance inducers that can trigger defensive responses to reduce the disease incidence, even at lower economical and environmental cost. In the present study, the effect of the soil supplementation of a biotic elicitor (i.e., ultrasound-assisted dispersion obtained from Fod mycelium) on disease severity and phenolic-based profiles of roots over two carnation cultivars was evaluated. Results suggest that the tested biotic elicitor, namely, eFod, substantially reduced the progress of vascular wilting in a susceptible cultivar (i.e., ‘Mizuki’) after two independent in vivo tests. The LC-MS-derived semi-quantitative levels of phenolic compounds in roots were also affected by eFod, since particular anthranilate derivatives, conjugated benzoic acids, and glycosylated flavonols were upregulated by elicitation after 144 and 240 h post eFod addition. Our findings indicate that the soil-applied eFod has an effect as a resistance inducer, promoting a disease severity reduction and accumulation of particular phenolic-like compounds.

Background: The production and secretion of defense proteins are one of the protective mechanisms exploited by plants against pathogens. The production and secretion of defense proteins are one of the protective mechanisms exploited by plants against pathogens. Ribosome-Inactivating Proteins (RIPs), as the main class of these proteins, are considered to facilitate cancer therapy worldwide, because of the potential anticancer activity. Indeed, some of these proteins have cytotoxic and anticancer properties. Extracted from the carnation (Dianthus caryophyllus), Dianthin inhibits protein synthesis in many different cells. Methods: In this research, the Dianthins was isolated and purified from the leaves of D. caryophyllus, using ion-exchange chromatography column (CM-Sephadex G-50). Subsequently, its cytotoxicity effect on MCF-7 cell line was investigated. The cell cytotoxicity assessment was performed, using 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), neutral red uptake, and alkaline comet assays at the concentrations of 1.25μg/ mL to 10μg/mL of the protein applying the MCF-7 cell line. Results: the toxin induces cell death, mostly via necrosis rather than apoptosis, but in the special range of concentrations. Conclusion: because of the severe side effects of chemotherapy drugs, this toxin can undergo more research as a new drug candidate against breast cancer

The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation 'Dreambyul' plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H2O2 level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation 'Dreambyul' plantlets cultured in the TIS. [ABSTRACT FROM AUTHOR]

The postharvest performance of cut spray-type carnations (Dianthus caryophyllus L.) is often limited by the opening, longevity, and coloration of individual florets, and is apparently related to water stress and sugar deficiency. In this study, we found that, compared with deionised water-treated controls, the vase life of cut spray 'Prince' carnations was extended by 2.1–4.6 days when held in vase solutions containing1–5 mg L–1 nano-silver (NS). Moreover, combination treatments with 5 mg L–1 NS and 2%, 3%, or 4% sucrose consistently further prolonged the vase life, prominently promoted floret opening, and increased the bright pink coloration of petals, thereby markedly enhancing quality performance. Overall, the 5 mg L–1 NS plus 3% sucrose treatment provided the longest vase life extension (6.6 days) compared with the control, and significantly increased the anthocyanin content in floret petals. Besides, we also showed that the treatment with 5 mg L–1 NS alone or 5 mg L–1 NS plus 3% sucrose significantly enhanced their vase solution uptake and relative fresh weight. Further, scanning electron microscopy observations revealed that the NS vase treatments evidently alleviated vascular occlusion by inhibiting bacterial colonisation and biofilm formation on their stem-end cut surfaces and in the xylem vessels. [ABSTRACT FROM AUTHOR]

ABSTRACT This report describes two cases of male cats affected by FLUTD (Feline lower urinary tract disease). The first patient had been affected by numerous relapses after passing through various therapeutic treatments and surgical interventions, and was subjected to the alternative protocol after being recommended for euthanasia, the second patient had no history of urinary tract disorders in the past, and was not subjected to any kind of previous treatment. The two patients had a significant improvement in their clinical condition, were not affected by relapses after participation in the alternative design, and were accompanied for about a year after their treatment.