Camelina [Camelina sativa (L.) Crantz] has gained extensive attention in Europe and North America as a potential dietary oil and biofuel feedstock. It is a relatively new crop in Asia (e.g., China, Korea). There is great potential for the cropping of camelina in eastern China on marginal lands where the climatic conditions (e.g., cooler temperature) may be suitable for cultivating this crop. However, little has been done to evaluate its agronomic performance in eastern China. To address this, a three-year (2019-2021) field study was conducted to evaluate the effect of fall and spring seeding dates on seed yield and quality of sixteen spring camelina genotypes across the three different growing environments in eastern China and to select potentially high-yielding genotypes for fall or spring seeding with the suitable seeding dates for each growing environment. The study showed that fall seeding camelina between late Oct. and the third week of Nov. in eastern China, including Anyang, Qingdao, and Yangzhou, produced a sustainable and satisfactory seed and oil yield (mean across genotypes, locations, and years: 2372 and 921 kg ha-1, respectively). While spring seeding between mid- and the end of April at Qingdao showed a lower productive performance (mean seed and oil yield across genotypes: 1081 and 373 kg ha-1, respectively), it still provides an alternative option for the production of high-quality edible oil compared to other oilseed crops such as soybean [Glycine max (L.) Merr.]. Although the strong genotype x environment interactions showed, among the tested camelina genotypes, fall seeding camelina accessions of CamK9, CamC2, and CamC4 at the suitable seeding dates showed a consistently greater mean seed yield (range: 1648-3170 kg ha-1) and oil yield (747-1368 kg ha-1) in all test locations compared to other genotypes. At the suitable fall seeding dates, mean seed oil content and yield across the tested genotypes and locations were 43.5% (range: 39.0-48.9%) and 856 kg ha-1 (range: 161-1489 ha-1), respectively, with the highest mean oil content of 45.9% determined at Yangzhou (range: 43.6-48.9%) and the highest mean seed yield of 2539 kg ha-1 at Qingdao (range: 1365-3501 kg ha-1). The camelina genotypes indicated would be good candidates for large-scale cropping in eastern China and other parts of the world with similar climatic conditions.

Pre-release risk assessment of genetically modified (GM) Camelina sativa (L.) Crantz requires a careful evaluation of the reproductive compatibility with its closely-related Camelina species. Camelina rumelica Velen. is a naturalized weed occurring in C. sativa production region in the northwestern China. In this study, a large number of reciprocal crosses was conducted between the tetraploid C. rumelica and hexaploid C. sativa. The F1 hybrids were produced by the tetraploid C. rumelica (♀) × hexaploid C. sativa (♂) at the rate of one hybrid for 217 ovules pollinated, and one hybrid for 220 ovules pollinated in the reciprocal direction. All F1 hybrids required vernalization treatment to induce timely flowering and showed the significant lower pollen viability (< 2%) compared to the parental lines. Despite that the F1 failed to backcross with the parental lines, selfed seeds (F2) were obtained from F1 hybrids plants. The F2 hybrids showed some degree of restored pollen viability (about 20%) and successfully produced seeds by both backcrossing and self-pollination. A similar pattern was observed in the field, with F1 hybrids showing self-compatibility and reduced seed production potential. It is worthwhile to mention that all F1 and F2 plants grew well both in the greenhouse and field conditions, but with the significant lower seed production ability. A portion of selfed F2 seeds from naturally shattering persisted in the soil seedbank during summer period and subsequently germinated in late fall. By contrast, other F2 seeds may have been dormant in the soil seedbank and germinated the following spring. These altered life-cycle related traits in hybrids generates the novel ecological concerns on the persistence and population dynamics of transient hybrids in the seedbank. In summary, this study provided the evidence that the tetraploid C. rumelica and hexaploid C. sativa, which have the sympatric distributions and overlapped flowering periods, gene flow between them probably could occur. Although the rate was relatively low (about 0.5%), the introgression of life-cycle related traits into C. sativa population could alter its key life-cycle traits and raise the concerns on the soil seedbank persistence and invasiveness of transient hybrid in C. sativa production region in the northwestern China.

Camelina [Camelina sativa (L.) Crantz], a member of Brassicaceae family, is a relatively new oilseed crop in China. It is a highly adaptable cool season crop species that can be grown in a wide range of environment with low input, making it potentially suitable for growing in northern China. A five-year (2010-2014) field experiment was conducted to evaluate the seed yield and quality of two camelina cultivars across three different locations in China. The study showed camelina can be cultivated successfully in a relatively short growth period of 1308-1920 degrees C d (base temperature: 4 degrees C) across a wide range of environmental conditions. Overall, camelina cv. 'Xiaoguo' displayed a satisfactory high seed yield dry matter (d.m.) (mean: 1946 kg ha(-1), range: 1274-2650 kg ha(-1)) and oil yield (mean: 598 kg ha(-1), range: 387-778 kg ha(-1)) across three different locations over the 5-year field trial, indicating the potential commercial production of camelina in northern China. The average contents of saturated, monounsaturated, and polyunsaturated fatty acids in camelina oil across locations and years varied from 10-21% (mean: 13 %), 24-38% (mean: 33 %), and 47-63% (mean: 56 %), respectively. In the present study, a variety of polyunsaturated fatty acids, particularly C22:6 (DHA) which is commonly presented in aquatic ecosystem were detected in camelina oil, making camelina an attractive oil crop for its peculiar fatty acid composition. This is the first study evaluating the agronomic performance of camelina in different growing environments in China and the relatively short growing cycle and high seed yield make camelina as a suitable oilseed crop in northern China.

Echinochloa species is one of the most problematic weed species due to its high competitiveness and increasing herbicide resistance. Florpyrauxifen-benzyl, a new auxin herbicide, was recently introduced for Echinochloa management; however, the potential risk for the development of herbicide resistance in Echinochloa species has not been well-investigated. Thus, this study was conducted to evaluate the baseline sensitivity of Echinochloa species to florpyrauxifen-benzyl to estimate the risk of future resistance development. A total of 70 and 71 accessions of Echinochloa crus-galli and Echinochloa oryzicola were collected from paddy fields in Korea, respectively. These two Echinochloa species were grown in plastic pots up to the 5-leaf stage, and treated with florpyrauxifen-benzyl at a range of doses from 2.2 g to 70.0 g a.i. ha–1. Nonlinear regression analyses revealed that GR50 values for E. oryzicola ranged from 4.54 g to 29.66 g a.i. ha–1, giving a baseline sensitivity index (BSI) of 6.53, while those for E. crus-galli ranged from 6.15 g to 16.06 g a.i. ha–1, giving a BSI of 2.61. Our findings suggest that E. oryzicola has a greater potential risk than E. crus-galli for the development of metabolism-based resistance to florpyrauxifen-benzyl.

Crops during their early growth stages are vulnerable to a wide range of environmental stressors; thus, earlier seed invigoration and seedling establishment are essential in crop production. As an alternative to synthetic chemical treatments, plasma technology could be one of the emerging technologies to enhance seed germination and seedling vigor by managing environmental stressors. Recent studies have shown its beneficial effects in various stress conditions, suggesting that plasma treatment can be used for early crop stress management. This paper reviewed the effects of different types of plasma treatments on plant responses in terms of the seed surface environment (seed scarification and pathogen inactivation) and physiological processes (an enhanced antioxidant system and activated defense response) during the early growth stages of plants. As a result, plasma treatment can enhance seed invigoration and seedling establishment by alleviating the adverse effects of environmental stressors such as drought, salinity, and pathogen infection. More information on plasma applications and their mechanisms against a broad range of stressors is required to establish a better plasma technology for early crop stress management.

The potential for wide-scale adoption of genetically engineered (GE) Camelina sativa (L.) Crantz in North America has raised ecological concerns of gene flow to its weedy relatives. The objectives of this study were to determine the pollen longevity, flowering phenology and seedbank persistence of C. sativa and congenic species. Percent pollen germination of all Camelina species was reduced with increasing temperature and exposure time. Pollen germination was not observed for all taxa after 72 h between 20-40 degrees C. Weather conditions had a significant effect on pollen longevity. Under cloudy conditions, all taxa pollen had a longer period of longevity (half-life: 0.6-0.8 h) than sunny conditions (0.3-0.4 h). Under both conditions, C. sativa, Camelina alyssum (Mill.) Thell., and Camelina microcarpa Andrz. Ex DC. pollen had a longer period of longevity than Camelina hispida var. grandiflora (Boiss.) Hedge, Camelina laxa C. A. Mey, and Camelina rumelica Velen., but pollen germination was not observed after 5 h exposure. The flowering phenology of C. sativa, C. microcarpa, and C. alyssum behaved similarly, which was different from C. hispida, C. laxa, and C. rumelica. Flowering synchrony among taxa was observed, facilitating pollen-mediated gene flow among taxa. Seeds of C. sativa, C. alyssum, and C. microcarpa did not exhibit dormancy in the soil, but the germination ability of C. sativa throughout the growing season raises the ecological concerns of gene flow from C. sativa volunteers. This new information on pollen longevity, flowering phenology, and seedbank persistence will be helpful for creating containment and coexistence strategies between GE and Non-GE C. sativa, isolating field trials, and managing GE C. sativa volunteers associated weed problems.

Crops during their early growth stages are vulnerable to a wide range of environmental stressors; thus, earlier seed invigoration and seedling establishment are essential in crop production. As an alternative to synthetic chemical treatments, plasma technology could be one of the emerging technologies to enhance seed germination and seedling vigor by managing environmental stressors. Recent studies have shown its beneficial effects in various stress conditions, suggesting that plasma treatment can be used for early crop stress management. This paper reviewed the effects of different types of plasma treatments on plant responses in terms of the seed surface environment (seed scarification and pathogen inactivation) and physiological processes (an enhanced antioxidant system and activated defense response) during the early growth stages of plants. As a result, plasma treatment can enhance seed invigoration and seedling establishment by alleviating the adverse effects of environmental stressors such as drought, salinity, and pathogen infection. More information on plasma applications and their mechanisms against a broad range of stressors is required to establish a better plasma technology for early crop stress management.

AbstractParaquat was the most successful nonselective herbicide in Korea due to its rapid herbicidal activity. However, its high mammalian toxicity, frequent self-poisoning incidents, and a lack of effective antidotes led to a paraquat ban in Korea in 2012. Therefore, this review was conducted to revisit the toxicological profile of paraquat and to investigate the impacts of the paraquat ban on human health and agriculture in Korea. A review of toxicological information reconfirmed that paraquat is highly acutely toxic to humans, and ingestion, inhalation, or dermal administration of the herbicide can cause severe clinical signs and inevitably lead to death by respiratory failure. In Korea, the paraquat ban immediately decreased the suicide rate due to pesticides (mainly paraquat) by 46.1%, resulting in a 10% decrease of the total suicide rate. However, this also led to an increase in suicide attempts with other poisons such as carbon monoxide, suggesting that suicide attempts and rates of suicide by poisoning depend on not only the toxicity of the poison but also the accessibility of the poisoning agents. In agriculture, paraquat was quickly replaced by other nonselective herbicides such as glufosinate and glyphosate. Thus, the paraquat ban did not have a significant impact on agricultural practices but influenced the nonselective herbicide market; the use of glufosinate was higher than use of glyphosate due to glufosinate’s rapid herbicidal activity, which is similar to that of paraquat. Though the paraquat ban can be considered as a national strategy to lower suicide rates, the increase in suicide attempts with other poisons suggests that multilateral efforts are required for not only keeping suicidal agents away from people but also minimizing motives for suicide.

The cultivation of genetically modified (GM) crops has raised many questions regarding their environmental risks, particularly about their ecological impact on non-target organisms, such as their closely-related relative species. Although evaluations of transgene flow from GM crops to their conventional crops has been conducted under large-scale farming system worldwide, in particular in North America and Australia, few studies have been conducted under smallholder farming systems in Asia with diverse crops in co-existence. A two-year field study was conducted to assess the potential environmental risks of gene flow from glufosinate-ammonium resistant (GR) Brassica napus to its conventional relatives, B. napus, B. juncea, and Raphanus sativus under simulated smallholder field conditions in Korea. Herbicide resistance and simple sequence repeat (SSR) markers were used to identify the hybrids. Hybridization frequency of B. napus × GR B. napus was 2.33% at a 2 m distance, which decreased to 0.007% at 75 m. For B. juncea, it was 0.076% at 2 m and decreased to 0.025% at 16 m. No gene flow was observed to R. sativus. The log-logistic model described hybridization frequency with increasing distance from GR B. napus to B. napus and B. juncea and predicted that the effective isolation distances for 0.01% gene flow from GR B. napus to B. napus and B. juncea were 122.5 and 23.7 m, respectively. Results suggest that long-distance gene flow from GR B. napus to B. napus and B. juncea is unlikely, but gene flow can potentially occur between adjacent fields where the smallholder farming systems exist.

Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output-input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. germplasm collections, the efforts to date to phenotype and genotype the diversity available for breeding, and on the scale of breeding work as indicated by number of attempted deliberate crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis, and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic datasets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed-based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass-scale deployment of PBCs.

Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output–input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed‐based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass‐scale deployment of PBCs.

1-Methylcyclopropene (1-MCP) is a cyclopropene compound that is widely used as an ethylene antagonist to extend the storage life of fresh fruit but has a difficulty in its use due to its chemically unstable and physically volatile properties. A cyclopropene compound with a long chain substituent instead of the 1-mehtyl in 1-MCP is expected to be non-volatile and easy to formulate for applications in open working spaces due to its higher molecular weight and lipid-like chemical structure. Therefore, this study investigated the effect of 1-(3-phenyl-propyl) cyclopropene (PPCP) versus 1-MCP in the gaseous state on the quality and storage life of tomato fruit. The application of PPCP maintained a lower ethylene production and respiration rate of the tomatoes and delayed the color change and softening of the tomatoes up to 12 d when compared to the untreated controls. Thus, 16 mu L L-1 of PPCP reduced the fruit ethylene production and respiration rate by 42% and 25% of the untreated control, respectively, at 6 d after storage and showed equivalent effects on the delay in color change and softening of the tomato fruit to those of 1 mu L L-1 of 1-MCP. Therefore, our results suggest that PPCP can be used as an alternative ethylene antagonist for the postharvest storage of tomatoes and other climacteric fruit.

Pollen-mediated gene flow (PMGF) from genetically modified (GM) Brassica napus to its wild relatives by wind and insects is a major ecological concern in agricultural ecosystems. This study conducted is to estimate maximum potential gene flow and differentiate between wind- and bee-mediated gene flows from herbicide resistant (HR) B. napus to its closely-related male sterile (MS) relatives, B. napus, B. juncea and Raphanus sativus. Various markers, including pods formation in MS plants, herbicide resistance, and SSR markers, were used to identify the hybrids. Our results revealed the following: 1) maximum potential gene flow (a maximum % of the progeny of pollen recipient confirmed hybrid) to MS B. napus ranged from 32.48 to 0.30% and from 14.69 to 0.26% at 2-128m from HR B. napus under open and wind pollination conditions, respectively, and to MS B. juncea ranged from 21.95 to 0.24% and from 6.16 to 0.16%, respectively; 2) estimates of honeybee-mediated gene flow decreased with increasing distance from HR B. napus and ranged from 17.78 to 0.03% at 2-128 m for MS B. napus and from 15.33 to 0.08% for MS B. juncea; 3) a small-scale donor plots would strongly favour insect overwind pollination; 4) no gene flow occurred from HR B. napus to MS R. sativus. Our approach and findings are helpful in understanding the relative contribution of wind and bees to gene flow and useful for estimating maximum potential gene flow and managing environmental risks associated with gene flow. (C) 2018 Published by Elsevier B.V.

This study was conducted to evaluate the cross-resistance of acetolactate synthase (ALS) inhibitors with different chemistries, specifically azimsulfuron (sulfonylurea), penoxsulam (triazolopyrimidine sulfonanilide) and bispyribac-sodium (pyrimidinyl thio benzoate), in Echinochloa oryzicola and Echinochloa crus-galli that had been collected in South Korea and to investigate their herbicide resistance mechanism. Both Echinochloa spp. showed cross-resistance to the ALS inhibitors belonging to the above three different chemistries. In a whole plant assay with herbicides alone, the resistant/susceptible ratios for azimsulfuron, penoxsulam and bispyribac-sodium were 12.6, 28.1 and 1.9 in E. oryzicola and 21.1, 13.7 and 1.8 in E. crus-galli, respectively. An in vitroALS enzyme assay with herbicides showed that the I (50)-values of the resistant accessions were approximately two-to-three times higher than the susceptible accessions, with no statistical difference, suggesting that the difference in ALS sensitivity cannot explain ALS inhibitor resistance in Echinochloa spp. for azimsulfuron, penoxsulam and bispyribac-sodium. A whole plant assay with fenitrothion showed that the GR (50)-values significantly decreased in both the resistant E. oryzicola and E. crus-galli accessions when azimsulfuron, penoxsulam and bispyribac-sodium were applied with the P450 inhibitor, while no significant decrease was observed in the susceptible accessions when the P450 inhibitor was used. Thus, these results suggest that ALS inhibitor cross-resistance for azimsulfuron, penoxsulam and bispyribac-sodium is related to enhanced herbicide metabolism.

Barnyardgrass (Echinochloa crus-galli) is a pernicious weed in agricultural fields worldwide. The molecular mechanisms underlying its success in the absence of human intervention are presently unknown. Here we report a draft genome sequence of the hexaploid species E. crus-galli, i.e., a 1.27 Gb assembly representing 90.7% of the predicted genome size. An extremely large repertoire of genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with detoxification are found. Two gene clusters involved in the biosynthesis of an allelochemical 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and a phytoalexin momilactone A are found in the E. crus-galli genome, respectively. The allelochemical DIMBOA gene cluster is activated in response to co-cultivation with rice, while the phytoalexin momilactone A gene cluster specifically to infection by pathogenic Pyricularia oryzae. Our results provide a new understanding of the molecular mechanisms underlying the extreme adaptation of the weed.

The chlorophyll fluorescence measurement to diagnose herbicide resistant Echinochloa species at the reproductive (late) growth stage was applied. The significant correlation between F-v/F-m (chlorophyll fluorescence measurements) and fresh weight (whole plant test) and the statistical similarity of R/S ratios between the two tests demonstrated that the chlorophyll fluorescence test could be reliably used to diagnose herbicide resistant Echinochloa spp. at the reproductive growth stage in a shorter period of time (within 10 days) compared with the conventional whole plant test.

Lack of understanding the effects of single- and multiple-weed interference on soybean yield has led to inadequate weed management in Primorsky Krai, resulting in much lower average yield than neighboring regions. A 2 yr field experiment was conducted in a soybean field located in Bogatyrka (43.82°N, 131.6°E), Primorsky Krai, Russia, in 2013 and 2014 to investigate the effects of single and multiple interference caused by naturally established weeds on soybean yield and to model these effects. Aboveground dry weight was negatively affected the most by weed interference, followed by number of pods and seeds. Soybean yield under single-weed interference was best demonstrated by a rectangular hyperbolic model, showing that common ragweed and barnyardgrass were the most competitive weed species, followed by annual sowthistle, American sloughgrass, and common lambsquarters. In the case of multiple-weed interference, soybean yield loss was accurately described by a multivariate rectangular hyperbolic model, with total density equivalent as the independent variable. Parameter estimates indicated that weed-free soybean yields were similar in 2013 and 2014, i.e., estimated as 1.72 t and 1.75 t ha−1, respectively, and competitiveness of each weed species was not significantly different between the two years. Economic thresholds for single-weed interference were 0.74, 0.66, 1.15, 1.23, and 1.45 plants m−2for common ragweed, barnyardgrass, annual sowthistle, American sloughgrass, and common lambsquarters, respectively. The economic threshold for multiple-weed interference was 0.70 density equivalent m−2. These results, including the model, thus can be applied to a decision support system for weed management in soybean cultivation under single and multiple-weed interference in Primorsky Krai and its neighboring regions of Russia.

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation.

Three Echinochloa species inhabit various crop fields with different soil moisture conditions. Therefore, a growth pouch test was conducted to investigate adaptive diversity of six Echinochloa species, three from Korea and three from USA, to osmotic stress by assessing shoot and root growths. Echinochloa crus-galli var. praticola showed the greatest tolerance to osmotic stress in both root (GR 50 =1316.3 g PEG L −1 ) and shoot (GR 50 =212.2 g PEG L −1 ) growths, while Korean E. oryzicola was most sensitive to osmotic stress in both root (GR 50 =116 g PEG L −1 ) and shoot (GR 50 =126.2 g PEG L −1 ) growths. Root to shoot (R/S) ratio of Echinochloa crus-galli var. praticola increased with increasing osmotic stress, while that of Korean E. oryzicola decreased, suggesting that R/S ratio is closely related to osmotic stress tolerance in Echinochloa species. Our results clearly demonstrate that E. crus-galli var. praticola maintains high R/S ratio even under high osmotic stress, which enables this species to well adapt to dry upland condition. In contrast, while E. oryzicola fails to maintain sufficiently high R/S ratio, resulting in poor adaptability to dry upland condition.