Use of the growth retardant tetcyclacis for potato tuber formation in vitro

The effects of the plant growth retardant tetcyclacis on in vitro tuber formation in potatoes was studied, using two different approaches: 1. tuber formation in various lines that did not or hardly form tubers under control conditions, and 2. tuber formation by the variety Bintje, which readily forms tubers. The ABA-deficient (droopy) lines of S. phureja hardly formed tubers without the addition of tetcyclacis. In the presence of this growth retardant tuberization was nearly 100%, within three weeks of in vitro culture, even in the absence of cytokinin. A series of somatic hybrids between S. tuberosum and S. brevidens, that did not form tubers in field and pot experiments, were tested. They all formed tubers in vitro in the presence of tetcyclacis. Stoloniferous shoots formed on single-node cuttings from in vitro grown Solanum tuberosum var Bintje plantlets were transferred to media containing a high level of sucrose. In the presence of tetcyclacis, tuber formation started after 4 days, reaching a maximum level of 80% at day 7. Tubers formed in the presence of tetcyclacis, accumulated starch and expressed several tuber-specific genes. These effects were fully antagonized by gibberellic acid. It is concluded that the growth retardant tetcyclacis is a potent tool in the study of tuber formation in potatoes.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid - STS silver thiosulphate - TET tetcyclacis     

Optimal rates of 2,4‐dichlophenoxyacetic acid foliar application for control of common scab in potato

Applications of the synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) to the foliage of potato plants can reduce common scab, a tuber disease. However, in prior research effective applications at 200 mg L^?1 2,4‐D resulted in phytotoxic side effects with reduced tuber yield and quality. This study showed that minimal significant threshold rates from 8.3 to 23.6 mg L^?1 2,4‐D reduced disease incidence in pot trials, and from 10.8 to 41.0 mg L^?1 minimised disease severity in both pot and field trials. In only one pot trial, significant phytotoxicity was found with rates of 100 mg L^?1 or greater, reducing mean total tuber mass per plot and 38 mg L^?1 or greater, reducing mean mass per tuber. Notably, within the field trial, a more reliable plant growth system for estimation of yield, no significant impacts were observed. Disease control was associated with decreased sensitivity of tubers to thaxtomin A, the phytotoxin produced by the common scab pathogen essential for disease induction. The amount of residual 2,4‐D in tubers at harvest varied with cultivar, Russet Burbank accumulating more 2,4‐D than Desiree. Application rates less than 100 mg L^?1 resulted in levels of 2,4‐D below the Australian standard maximum residue limit. These data suggest that applications of 2,4‐D at low rates could provide a commercially suitable control strategy for common scab.     

Enhanced fructose levels in field grown potato tubers expressing a thermostable glucose isomerase

A thermostable glucose isomerase was expressed in Solanum tuberosum Desirée using the tuber-specific granular-bound starch synthase promoter. The fructose content was substantially increased in microtubers, greenhouse grown tubers as well as tubers produced in field trials as compared with the controls. Furthermore, the tuber yield of field grown potatoes was enhanced by 30% in the transgenic lines (from 1.04 kg/plant in the wild type to 1.36 kg/plant in the transgenic lines).     

Field evaluation of tetraploid somatic potato hybrids

Tetraploid somatic hybrids, derived from ten different fusion combinations, were grown in the field to study the inheritance of quantitatively-controlled agronomic traits. The data from two seasons showed that the heterosis in yield varied between 70% and 230% compared to the mid-parent value of the dihaploid fusion clones. The relative yield of the hybrids compared to two standard varieties (= 100%) ranged from 47% to 106%. Despite large differences in yield between the single hybrids of the same fusion combination, no significant differences could be detected between the hybrids. The analysis of the yield components revealed that the heterosis was mainly due to a highly increased tuber weight, whereas the tuber number was in general intermediate to that of the parent clones. In three combinations, the starch content of the hybrids was 15–20% higher than the midparent value and in the others it was intermediate. Fusion of clones with long oval tubers and with round or round-oval ones showed the round/round-oval form was dominant. Fusion combinations of tuber flesh colour indicated that in most cases yellow was dominant to white, while red skin colour was dominant over yellow.     

Growth and yield of potato plants inoculated with rhizosphere bacteria

Pot and field experiments on small plots were carried out to study the effect of isolates ofPseudomonas fluorescens-putida bacteria, applied to potato tubers, on the growth response of potato plants. Inoculation of tuber pieces with the isolates in pot experiments caused a better growth of young potato plant (111 % increase). The effect depended on the viable count in the bacterial suspension and on growth conditions of the plants. Inoculation of potato seed pieces before planting in field experiments caused a 4 –30 % improvement in plant growth and tuber yield. The complex action of inoculation with rhizobacteria apparently caused changes in the microflora colonizing the roots and stolons, which in turn brought about a better growth and yield.     

Investigating the in vivo calcium transport path to developing potato tuber using 45Ca: a new concept in potato tuber calcium nutrition

Calcium is believed to be transported with water in the xylem. Consistent with this proposal, low‐transpiring organs such as potato Solanum tuberosum tubers are known to suffer from calcium deficiency. Although roots on tubers and stolons have been shown to supply water to tubers, there is no direct evidence for the calcium transport pathway to tubers. Both a xylem and a phloem transport pathway have been suggested. We investigated in vivo calcium transport to developing potato, cv. Dark Red Norland and cv. Russet Burbank, tubers using ⁴⁵Ca in a controlled environment facility. Whole plant split pot experiments allowed the placement of ⁴⁵Ca either in the main (basal) root or the tuber and stolon areas of the pot. The results showed that ⁴⁵Ca was transported to the shoot with the transpiration stream from both areas but was not re‐translocated to tubers or the main (basal) root system even 57 days after ⁴⁵Ca application. Radioactivity could only be detected in the tuber when ⁴⁵Ca was fed to the stolon and tuber area. When ⁴⁵Ca was fed to specific tubers, radioactivity was detected in the aerial shoot; however, no activity was detected in other tubers or the main (basal) roots. In another set of experiments, roots on a stolon near a tuber were precisely fed ⁴⁵Ca and Safranin O. The radioactive signal exactly overlapped the water transport pathway in the tuber marked with Safranin O dye, suggesting that water and calcium can be simultaneously transported from stolon roots to the tuber. No transport of ⁴⁵Ca across the tuber periderm was detected 8 days after ⁴⁵Ca was applied to the tuber periderm. This indicated that no significant transport of calcium occurs from the soil across the periderm. Our results provide evidence that: (1) calcium is not re‐translocated via the phloem from the aerial shoot tubers and main (basal) roots; (2) the main root system does not supply calcium to the tuber; (3) calcium is not transported across the periderm to the interior tuber tissue; (4) calcium is transported to the tuber via the xylem along with water, and the roots on the stolon associated with the tuber supply water and calcium to the developing tuber; and (5) transpirational demand is a significant determinant of calcium distribution within the plant.     

Healthier seed potatoes. I. Effects of inoculating stem cutting stocks with Polyscytalum pustulans and Rhizoctonia solani on growth, yield and disease

Potato seed tubers of seven cultivars derived from stem cuttings in 1965 (healthier seed) were grown in 1969–72 at two sites, one clay with flints soil (Rothamsted) and the other sandy loam soil (Woburn). Inoculating sprouted tubers at planting with Polyscytalum pustulans did not affect the number of stems/plant or total yield but increased stem base and tuber infection. The yields of large tubers (57–83 cm) were increased and small tubers (>57 cm) decreased, indicating a decrease in tuber numbers. Rhizoctonia solani inoculated at planting decreased numbers of stems/plant and yield by up to 14% at Woburn but not at Rothamsted. At both sites, yields of large tubers were increased with cvs Majestic and Record and decreased with Pentland Crown. Stem canker and tuber infection were increased but infection was also prevalent on tubers from non-inoculated seed at Woburn. When both pathogens were inoculated together yields of large tubers were increased in cvs King Edward, Majestic and Record and decreased in Pentland Crown. Infection of stem bases and tubers was sometimes less than when either pathogen was inoculated singly.     

Soil Patches of Inorganic Nitrogen in Subtropical Brazilian Plant Communities with Araucaria angustifolia

One-year old tubers of two hybrid calla lily (calla) cultivars (Zantedeschia ‘Pot of Gold’ and ‘Majestic Red’) were inoculated with the arbuscular mycorrhizal fungus (AMF), Glomus intraradices, or not, and grown at three different rates of phosphorus (P) supply to asses the effects of AMF-inoculation on plant development (time of shoot emergence and flowering), flowering (number, length and rate of flowering), and tuber biomass and composition over two growing cycles (2002, 2003). Tubers and flowers of calla responded differently to AMF inoculation. Differences in mycorrhizal responsiveness between cultivars was related to differences in P requirements for flower and tuber production, and the influence of P supply on resource allocation to different reproductive strategies. Inoculation increased shoot production and promoted early flowering, particularly in 2003. Inoculated plants also produced larger tubers than non-inoculated plants, but only increased the number of flowers per plant in 2003. High P supply also increased tuber biomass, but decreased the number of flowers per plant in 2002. Plants grown at a moderate P-rate, produced the most flowers in 2003. For ‘Majestic Red’, benefits from AMF were primarily in terms of tuber yield and composition, and AMF effects on marketable flower production could potentially have negative impact on production strategies for growers. Inoculation of ‘Pot of Gold’ primarily influenced flower production and aspects of tuber quality that caused detectable enhancement of tuber yield and flowering in the second growing cycle following inoculation (2003). The results of this study show that the responses of calla to AMF are partially a function of how nutrient supply alters resource allocation to sexual and vegetative reproduction. Whether AMF-induced changes in resource allocation to flowering and tubers significantly alters commercial productivity and quality of calla depends on the crop production goals (e.g. tubers, cut flowers or potted plants). The U.S. Government’s right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

The effects of soil moisture at the time potato tubers are forming on the incidence of common scab (Streptomyces scabies)

Two complementary field experiments at Rothamsted in 1965, with the potato variety Majestic, related the incidence of common scab (Streptomyces scabies) to rainfall, soil moisture and time of tuber formation. In plots where the soil was maintained at field capacity (less than 10 cm. Hg moisture tension) by watering, tubers had little or no scab; infection increased in amount on plots allowed to dry to 30 cm., 50 cm. or more Hg moisture tension during late June. The time tubers formed was varied by planting sprouted (chitted) and non-sprouted seed tubers, and tuber development and scab incidence were observed on sample plants lifted at frequent intervals from unwatered plots. Scab lesions were first seen on 12 July when the distribution of lesions on the surface of tubers was affected by size of tuber both within and between the different seed-tuber treatments. These differences were correlated with estimates of tuber size on 28 June, the beginning of the first dry period. Tubers 1.0 cm. or more in diameter on 28 June had few or no lesions at the stolon attachment (heel) end of tubers on 12 July, whereas tubers smaller than this on 28 June had many lesions in this region on 12 July. The larger the tuber was on 28 June, the greater was the area free from scab lesions on 12 July.     

Early generation selection for agronomic characters in a potato breeding programme

Summary A random sample of seedlings representing high, medium and poor vigour was studied for tuber colour, tuber shape, eye depth, tuber cracking, tuber yield per plant, average tuber weight and number of tubers per plant in four successive generations (F₁, F₁, F₁C₂, and F₁C₃). Based on the performance of vigour groups in various generations and inter-generation correlation coefficients, we propose a procedure for the elimination of unproductive genotypes early in the breeding programme. The data indicates that seedlings of poor vigour can be discarded at the seedling stage prior to transplantation in the field. The rejection of clones on the basis of tuber colour, tuber shape, eye depth and tuber cracking can also be initiated at the seedling stage. For tuber yield and average tuber weight a negative selection (rejection of poor phenotypes) is suggested from the first clonal generation and for number of tubers, from second clonal generation, until statistically sound replicated trials can be conducted for carrying out positive selection.     

Effect of mound size and fertilizer on white Guinea yam (Dioscorea rotundata) in Southern Nigeria

Summary Field trials were conducted in the forest zone of southern Nigeria on three soil series, gravelly loamy sand Ibadan soil (Oxic paleustalf), gravelly sandy loam Egbeda soil (Oxic paleustalf) and sandy loam Alagba soil (Oxic paleustalf). The trials were carried out to study the effects of planting on flatversus various mound sizes and NPK fertilizer on performance of white Guinea yam (Dioscorea rotundata) cultivar Laoko.Mound size appeared to have a more pronounced effect on tuber yield than fertilizer even on land which was in the second and third year of cropping after bush fallow. The average tuber yield for the three locations without fertilizers was 7.83 tons/ha on the flat compared with 9.44 tons/ha on large mound (about 30 cm height). With fertilizer application, tuber yields were 7.43 tons/ha on the flat and 11.30 tons/ha on large mound respectively. Total yield reduction on flat may in part be related to physical soil impedence. Planting on large mounds also resulted in longer tubers and shorter harvesting time.     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

Screening of cassava (Manihot esculenta Crantz) accessions against Phytophthora palmivora induced tuber rot of cassava

Cassava (Manihot esculenta Crantz), is an important tropical tuber crop with global importance and plays a significant role in the food, nutritional and livelihood security of around 500 million people. In India, the low productivity of cassava attributes to the soil borne disease, particularly tuber rot caused by Phytophthora palmivora (Butl.) which is destructive and the attack is spreading in alarming rate in all the cassava growing regions causing heavy yield loss of more than 50%. Introduction of disease resistant varieties may alleviate the problem to a certain extent. This paper describes the screening procedures and findings on the disease resistant variety of cassava accession against tuber rot. Variety Sree Padmanabha imparted high resistance against tuber rot, while Sree Sahya was moderately resistant and all other accessions studied were found to be susceptible in in vitro and in field trials. In screening studies, a reproducible positive correlation was obtained between attached tubers in live plant with detached tubers which showed that detached tuber part can be used for the prediction of resistance in attached live plants of cassava for cultivar resistance. The procedure described here could be used as a simple, rapid and efficient method for screening of cassava accessions against tuber rot of cassava.     

Susceptibility of potato tubers to infection by Phytophthora infestans

Phytophthora infestans infects King Edward potato tubers more readily through inoculated eyes than through lenticels, but more lenticels than eyes became infected when whole tubers were sprayed with inoculum. The resistance of lenticels but not of eyes increased as tubers aged. The spores did not infect through intact periderm. The likelihood of tubers on plants grown in pots becoming infected by sporangial suspension poured on to the soil increased the nearer the tubers were to the soil surface, the stem, or the side of the pot. Naturally infected tubers, and those sprayed with sporangial suspension, had most eyes infected at the rose end, and most lenticels infected on the middle region of the tuber. Of naturally infected tubers, on which the site of infection could be identified, most were infected through eyes at the rose end.     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.     

Effects of rotation length, fungicide treatment of seed tubers and nematicide on diseases and the quality of potato tubers

In 1982 – 88, potatoes were grown in 2-, 4- and 6-course rotations with spring barley on a field infested with Globodera rostochiensis. Severity of stem canker and black scurf increased with increasing frequency of previous potato crops, and seed tuber treatment with tolclofos-methyl became less effective in controlling diseases. This suggested that previous crops had increased the amounts of soil-borne inoculum of Rhizoctonia solani. Oxamyl soil treatment increased stem canker in one year and decreased black scurf in four years. Seed tuber treatment with imazalil or prochloraz decreased stem base infection by Polyscytalum pustulans and skin spot and silver scurf on tubers. Black dot was prevalent on tubers in all years and was not affected by seed tuber treatment or previous cropping. Oxamyl increased black dot and common scab in five years and decreased % tuber dry matter in six years. Cysts of G. rostochiensis were found attached to Désirée but not to Maris Piper tubers in August. At harvest tubers of both cultivars were affected by superficial pitting and its severity was related to soil populations of G. rostochiensis at planting. This damage was controlled by oxamyl. It is suggested that the pitting developed from holes made in the tuber skin at larval invasion. In 1989, Désirée seed tubers and healthy mini tubers were planted in all plots and severity of stem canker and black scurf increased with increasing proximity of previous potato crops and with the number of previous crops. Black dot on stems and tubers was not affected by previous cropping but was much less severe in a plot that had not grown potatoes during the seven years of the experiment. The severity of common scab generally decreased as the number of preceding potato crops increased.     

The phenology of skin spot (Oospora pustulans Owen &Wakef.) and other fungal diseases of potato tubers

Underground stems and roots of the potato varieties King Edward and Majestic became more severely infected by Oospora pustulans (skin spot) as the growing season advanced. Tubers became infected at their initiation in June and July but the proportion of infected eyes usually increased during both the growth of the crop and bulk storage. Some buds on tubers in bulk stores died in December, but few were killed until after January, when attacks increased until by March about a quarter of Majestic eyes were usually dead. Boxing (chitting) not only prevented most eyes from dying but also prevented much skin spotting on tubers. At High Mowthorpe, Majestic seed unboxed or boxed in March yielded, respectively, 3 and 1 ton/acre less than seed boxed by January. Early boxing increased the yield of seed–sized tubers but did not affect the infection of progeny tubers. Dipping Majestic seed tubers in organo–mercurial fungicide and then boxing soon after lifting decreased their infection by O. pustulans; it also lessened infection on plants they produced and increased the yield of progeny seed–sized tubers. However, dipping, even in successive years, had little effect on the infection of progeny tubers by O. pustulans. Rhizoctonia solani and Helminihosporum atrovirens became increasingly prevalent on tubers during growth after July and during bulk storage. H. atrovirens was decreased by early boxing or dipping tubers in fungicide. Verticillate conidiophores often grew on incubated tuber plugs but their prevalence was affected by past storage. The results suggest that some of the benefit for chitting may be from disease control, it was not possible to measure the extent to which benefits from chitting depend on effects on disease or on the physiology of the tuber.     

Induction and development of potato tubers in a jar fermentor

Potato (Solanum tuberosum L.) tubers were mass propagated in a jar fermentor using a two-step method consisting of a shoot multiplication step (phase 1) followed by a tuber induction and development step (phase 2). Tuberization was observed within 2 weeks of phase 2 and the number of tubers did not increase after this culture period. In contrast, total tuber weight continuously increased for at least 10 weeks. Although the number of tubers and the total tuber weight clearly decreased under the lower temperature (17°C), this weight decrease was partially prevented by changing the temperature from 17°C to 25°C after 2 weeks of phase 2. This result indicates that tuber development can be controlled independently of induction. Lower temperatures influenced the localization and size distribution of tubers in the jar fermentor.     

Yield components of potato microtubers: in vitro production and field performance

Genetic parameters, character association and path analysis for yield components of microtuber production in vitro and their field performance were studied in 37 potato (Solanum tuberosum L.) genotypes. Among the microtuber yield components, average microtuber weight had maximum genotypic (or phenotypic) coefficient of variation, heritability and predicted genetic advance; however, the estimated values of these genetic parameters were maximum for tuber yield among the field yield components. The heritability estimates of field yield components were higher than that of their corresponding in vitro yield components of microtuber production. The highest correlation coefficients between average microtuber weight and microtuber yield suggested that microtuber weight was more important than microtuber number in determining microtuber yield potential in vitro. However, tuber number was found to be more important than tuber weight in determining tuber yield potential under field conditions. Average microtuber weight had maximum direct effect on microtuber yield, whereas tuber number had maximum direct effect on tuber yield under field conditions. The study showed that the relative importance of the components of microtuber production in vitro differed from that of corresponding field yield components. The expression of a genotype for microtuber production in vitro is different from that of tuber production under field situation i.e. the performance of a genotype in vitro is not a measure of its field performance.