Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

In vitro regeneration via caulogenesis and brassin-induced shoot conversion of dormant buds from plumular explants of peanut (Arachis hypogaea L. cv `Okrun')

Shoot buds were induced from plumular explants of peanut (Arachis hypogaea L., cv `Okrun') preconditioned on medium containing 2,4-dichlorophenoxyacetic acid and kinetin and then transferred to regeneration medium containing benzylaminopurine and β-naphthoxyacetic acid. Buds differentiated 25 days following transfer to regeneration medium. Each explant produced 30 to 40 buds, but only 4 shoots. The remaining buds were dormant and did not produce shoots when maintained on regeneration medium. Shoots were regenerated continuously, however, when explants were subsequently transferred to shoot conversion medium containing 1 μM brassin, benzylaminopurine and β-naphthoxyacetic acid, respectively. Approximately 5 shoots were harvested every 30 days after transfer to shoot conversion medium for up to 7 months. No further shoot production was observed from explants maintained on regeneration medium without brassin. Regenerated shoots could be rooted and produced viable seeds. This procedure provides an efficient and reliable system for regeneration and transformation studies using cv `Okrun'. Received: 9 April 1997 / Revision received: 27 August 1997 / Accepted: 20 September 1997     

Direct shoot formation and plant regeneration from cotyledon explants of rapid-cycling Brassica rapa

Summary An in vitro culture system for direct shoot regeneration from cotyledon explants of rapid-cycling Brassica rapa was developed. Cotyledons from 3-d-old seedlings, when cultured on Murashige and Skoog (MS) medium supplemented with 20 μM N⁶-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA), regenerated shoots directly at a frequency of 20%. The addition of 2 μM aminoethoxyvinylglycine (AVG) to this medium increased shoot regeneration to 33%, but silver nitrate drastically inhibited shoot regeneration. Shoot regeneration occurred directly, at the petiolar cut ends of cotyledonary explants, between 10 to 17 d in culture. The highest percentage of regeneration (33%) was obtained from 3-d-old seedlings. NAA was the most effective auxin for root induction and development, with 49% of shoots producing roots after 2 wk on medium containing 1.0 μM NAA. Regenerated plantlets were grown to maturity in pots containing peat moss and vermiculite (1:1). These plants were morphologically normal and fertile. With this protocol, over 100 independently derived, flowering R₀ plants were obtained from 40 regenerating cotyledonary explants within 40 d after culture initiation.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants

Summary An efficient regeneration system was developed by culturing immature cotyledons and embryo axes of Arachis hypogaea L. cv. Georgia Green on Murashige and Skoog basal medium (MS) supplemented with various concentrations of thidiazuron (TDZ; 1, 5, 10, and 15 μM). Highly morphogenic callus was produced from 100% of the explants comprising the cotyledon with attached embryo axis when cultured in the dark on 10 μM TDZ. Upon excision and continued culture in the dark on 10 μM TDZ, morphogenic callus grew repetitively during monthly subcultures and retained its regeneration potential. For organogenesis, a gradual reduction in TDZ concentration and exposure to light were necessary before transfer to MS basal medium. Inclusion of indole-3-butyric acid in liquid MS medium favored rooting of recovered shoots. A distinct feature of this investigation is the induction of highly morphogenic callus by TDZ and regeneration of morphologically normal, fertile peanut plants after 8 months of callus subculture.     

In vitro regeneration of long spur barrenwort (Epimedium grandiflorum Morr.) from rachis explants

Summary A system for micropropagation of Epimedium grandiflorum Morr. from rachis explants was developed. Explants were cultured onto Murashige and Skoog (MS) basal salts medium supplemented with (per L) 100 mg myo-inositol, 2 mg pyridoxine-HCl, 2 mg nicotinic acid, 0.40 mg thiamine-HCl, 30 g sucrose, and 2 g Phytagel. The medium also contained 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.2, or 0.25 mg/L (0.5, 0.9, or 1.1 μM) combined with either N⁶-benzyladenine (BA) or 2-isopentenyl adenine (2ip) at 2.5, 5, or 10 mg/L (11.1, 22.2, or 44.4 μM BA or 12.3, 24.6, or 49.2 μM 2iP). Cultures were maintained at a 16-h photoperiod (40 μmol/m²/s) and 23±2° C. Callogenesis preceded shoot regeneration. Callus formation increased with higher 2,4-D concentrations. The highest percent regeneration, 83% of explants, was obtained on 10 mg BA per L (44.4 μM) combined with 0.25 mg 2,4-D per L (1.1 μM). The maximum number of shoots, 15 per explant, was obtained from explants cultured on a medium containing 0.1 mg 2,4-D per L (0.45 μM) combined with 2.5 mg BA per L (11.1 μM). Maximum shoot length, 0.4 cm, was obtained on 5 mg BA per L (22.2 μM) combined with 0.2 mg 2,4-D per L (0.9 μM). To produce whole plants, shoots were separated and rooted on hormone-free medium containing 1 g activated charcoal per L. Rachises provided an excellent source of explants for Epimedium micropropagation and proved suitable for callus production.     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

Callus induction and plant regeneration from hypocotyl explants of the forage legume Astragalus adsurgens

An efficient procedure was developed for inducing callus and plant regeneration using hypocotyl segments of Astragalus adsurgens. The combinations and concentrations of different growth regulators were shown to be critical factors for both the frequency and the type of callus formation as well as for the potential of callus differentiation. Of the four morphologically distinct types of calli that were induced, a friable, yellow callus, i.e. type I, induced on MS medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid and 2.2 μM N⁶-benzylaminopurine (BA), and then transferred to MS medium containing 0.5 μM α-naphthaleneacetic acid and 8.9 μM BA, exhibited the maximum frequency of shoot regeneration (75%). After regenerated shoots were transferred onto half-strength MS medium without growth regulators, they rooted and complete plants were obtained. Plantlet regeneration from callus cultures required 7–8 weeks. Received: 26 February 1997 / Revision received: 28 August 1997 / Accepted: 13 September 1997     

Genotypic response of cowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants

Summary A plant regeneration system applicable to 17 cowpea genotypes was developed. Cotyledons were initiated on 1/3 MS medium containing 15 to 35 mg N⁶-benzyladenine (BA) per 1 (66.6 to 155.3 μM) for 5 to 15 d. For shoot regeneration, the explants were transferred to a medium containing 1 mg BA per 1 (4.4 μM). Within 1 wk, shoot formation was visible at the proximal end of the cotyledons. Regeneration percentages (1% to 11%) and the numbers of shoots (4 to 12 per explant) were significantly influenced by genotype. Culture duration and BA concentration in the initiation stage significantly affected regeneration capacity. Explants initiated on media containing 15 mg BA per 1 for 5 d resulted in the highest percentage of explants capable of regeneration. Conversely, the highest number of shoots was obtained from explants initiated on media supplemented with 35 mg BA per 1. Whole plants were obtained on a plant growth regulator-free medium. To our knowledge, this is the first report of plant regeneration from U.S. commercial cowpea cultivars and breeding lines. This system is adaptable to diverse cowpea genotypes and will facilitate cowpea genetic transformation. Published with the approval of the Director of the Arkansas Agricultural Experiment Station.     

Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.     

Induction of somatic embryogenesis and genetic transformation of ohio buckeye (Aesculus glabra willd.)

Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment, four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred to fresh medium for germination.     

Genetic transformation of gentian using wild-type Agrobacterium rhizogenes

Leaf sections of greenhouse-grown Miscanthus x ogiformis Honda 'Giganteus' plants and leaf sections or shoot apices of in vitro shoot cultures were grown on Murashige and Skoog medium containing various concentrations of benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid. On leaf sections, the callus induction decreased with increasing BA concentration. The percentage of embryogenic callus was increased, the percentage of root-forming callus decreased, and a new shoot-forming callus type was formed by inclusion of BA during callus induction. A higher percentage of shoot-forming callus was formed on shoot apices compared with leaf sections of in vitro-grown shoots when cultured on 0.4 μM BA. The largest number of plants per callus piece was regenerated from shoot-forming callus, but maintenance of the high regeneration capacity proved difficult. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of an anticarcinogenic herb, Curculigo orchioides (Gaertn.)

Summary Curculigo orchioides is an endangered anticarcinogenic herb. It is available only during the monsoon season, which lasts approximately 4 mo. each year. In vitro culture of the plant can ensure its availability throughout the year. Leaf explants of Curculigo orchioides cultured on a Murashige and Skoog (MS) medium without cytokinins produced a limited number of plantlets that originated directly from the cut end of the midrib. 6-Benzyladenine (BA) (0.44–6.66 μM) was needed to produce plantlets from rhizome explants. A higher concentration of BA (2.22–4.44 μM) resulted in nodular callus that when transferred to cytokinin-free medium formed shoots. The shoots were rooted on media supplemented with either (0.54–5.37 μM) of 1-naphthaleneacetic acid (NAA) (0.57–5.71 μM) of indole-3-acetic acid (IAA), or (0.49–4.90 μM) indole-3-butyric acid (IBA). Plantlets were kept in sterile sand for 3–4 d and then transferred to soil.     

Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L.

A two-step protocol for the induction of shoots from Alstroemeria leaf explants has been developed. Leaf explants with stem node tissue attached were incubated on shoot induction medium for 10 days, and then transferred to regeneration medium. Shoots from the area adjacent to the region between the leaf base and node tissue regenerated within 3 weeks after transfer to the regeneration medium, without a callus phase. The best induction was obtained with Murashige and Skoog medium containing 10 μm thidiazuron and 0.5 μm indole butyric acid. The regeneration medium contained 2.2 μm 6-benzylaminopurine. After several subcultures of the leaf explants with induced shoots, normal plantlets with rhizome were formed. In Alstroemeria, the percentage of responding leaf explants is more important than the number of shoots regenerated per leaf explant, because rhizome formation is the most important factor for micropropagation. The effect of other compounds in the induction medium, including glucose, sucrose, silver nitrate, and ancymidol, on regeneration was also investigated. Received: 14 June 1996 / Revision received: 27 September 1996 / Accepted: 20 October 1996     

The influence of osmotic pretreatment and inoculum age on the initiation and regenerability of switchgrass suspension cultures

Summary Experiments were conducted to study the influence of osmotic pretreatment and inoculum (callus) age on the initiation and induction of somatic embryogenesis from suspension cultures of ‘Alamo’ switchgrass (Panicum virgatum L.). Embryogenic 10-, 20-, or 30-d-old calluses (hereafter referred to as inocula), produced from in vitro-cultured inflorescences, were used as explants to initiate the suspensions. Inocula were cultured for 30 h on MS solid medium containing 0.1, 0.2 or 0.3 M each of sorbitol and mannitol as an osmotic pretreatment. They were then transferred to liquid MS medium with 5 μM 6-benzylaminopurine and 9 μM 2,4-dichlorophenoxyacetic acid and cultured for 28 d in 2-ml Multiwell™ plantes. Individual multiwell contents were transferred to 125-ml Erlenmeyer flasks containing 20 ml of the above liquid medium and cultured for an additional 2 wk. Cultures initiated from the 10-d-old inoculum produced a higher embryogenic response than those initiated from 20- or 30-d-old inocula. Cultures initiated with 30-d-old inocula produced nonembryogenic clusters and the number of embryogenic cells was low. More embryos and a higher regeneration frequency were produced by 0.3 M than by 0.1 or 0.2 M of each of the osmostica.     

Anther culture and haploid plant regeneration in purple coneflower (Echinacea purpurea L.)

Anthers containing microspores at the uninucleate stage were excised from capitula of field grown plants of purple coneflower, Echinacea purpurea, and cultured on medium conducive to callus growth. In callus induction cultures, N6 basal medium was more effective than Murashige and Skoog (MS), and a combination of benzyladenine (BA) at 2.22 μM with naphthaleneacetic acid (NAA) at 0.054 μM was more effective than 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.52, 9.05 and 13.57 μM. Callus induction rate as high as 85.8% was achieved, and no statistically significant differences were found among cultures with various densities of 20, 40, 60, 80 and 100 anthers per bottle each filled with 40-ml medium. Shoots were regenerated from calluses on MS medium containing 2.22 μM BA and various concentrations of NAA, with the highest regeneration rate of 95.24% obtained when 0.27 μM NAA was applied. Although a large portion of the␣regenerated shoots had prominent symptom of vitrification, some normal shoots could be easily rooted on MS medium containing 0.054 μM NAA. Thirty regenerated plants were randomly selected and 19 of them were confirmed to be haploid by observation of chromosome number of root-tip cells.     

Improvement of somatic embryogenesis inFeijoa sellowiana berg (Myrtaceae) by manipulation of culture media composition

Summary Somatic embryos could be induced from the cotyledons of zygotic embryos from immature fruits ofFeijoa sellowiana Berg (Feijoa) in the presence of a wide range of concentrations of fructose, glucose, maltose, and sucrose. Mannitol or sorbitol alone were ineffective. The highest frequencies of induction (99%) and the greatest number of somatic embryos per explant (134) were obtained with 0.4M fructose and 0.3M sucrose, respectively. This sucrose concentration also showed greater induction capacity than equimolar combinations of its monosaccharide constituents combined. Somatic embryo development was arrested at the globular stage at concentrations higher than 0.5M of all the sugars tested. When transferred to solid germination medium containing 2.0 mg/liter (5.77μM) gibberellic acid, 0.5 mg/liter (2.32μM) kinetin, and 0.029M sucrose, somatic embryos formed under 0.3 or 0.4M sucrose had better germination capacity than those induced under lower (0.1 and 0.2M) concentrations, as assessed by the frequency of explants presenting germinated embryos and by the number of plants obtained from those explants. On liquid media of similar composition somatic embryos did not germinate. Our data suggest that high (0.3 to 0.4M) carbohydrate levels improve somatic embryogenesis by acting both as carbon source and as osmotic regulator.     

Adventitious shoot formation and plant regeneration from Pinus pinaster Sol. ex Aiton

Summary Pinus pinaster plants were regenerated from cotyledons excised from in vitro germinated seeds and axenically cultured on induction medium (GMD). 6-Benzyladenine (2.2 μM) induced the highest frequency of direct bud formation from cotyledons. An average of 13.1 ± 2.1 elongated shoots per cotyledon was obtained. Germination time influenced shoot induction, and the organogenic potential decreased with explant age. Cotyledons remained for 21 d on induction medium, and in order to promote adventitious shoot elongation, they were transferred to Gupta and Durzan’s DCR medium without growth regulators, containing 0.5% (wt/vol) activated charcoal and 3% (wt/vol) sucrose. Rooting was achieved by application of an indole-3-butyric acid, (396.6 μM) pulse (24 h at 4° C), followed by transfer to a sterile mixture of peat plus perlite (1:1 vol/vol). Ninety-eight to 100% of the regenerated plants were successfully acclimatized. All plants have survived after transfer to the field.     

Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed)

Summary Basal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 μM 2,4-dichlorophenoxyacetic acid giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 μM naphthaleneacetic acid, 10 μM gibberellic acid, and 2 μM benzyladenine with either 1 or 20 μM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond’s meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 μM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures.