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The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches.
Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance.
Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi.
Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.
As in other agricultural species, trait introgression from 'wild' species of the genus Nicotiana has been used to improve the cropped species, and characters from at least 13 different species have been transferred into tobacco (Lewis, 2011). As well as smoking tobacco from N. tabacum, several species in the genus are popular ornamental plants. The high concentrations of nicotine, a poison of acetocholine receptors that is toxic to all species with neuromuscular junctions (Baldwin, 2001), provide excellent resistance to herbivorous pests. However, many Nicotiana species are susceptible to fungal and virus diseases as seedlings or adult plants. Economic and environmental considerations require reductions in the use of agrochemicals, increasing interest in exploiting genetic resistance in both field and horticultural crops.
The genus Nicotiana is complex with some 75 species currently recognized (Chase et al., 2003; Kapp et al., 2004). Of these, 35 are allotetraploids (Clarkson et al., 2010). Kovarik et al. (2008) and Lim et al. (2007) investigated the repetitive DNA (rDNA) evolution and chromosome constitution of various hybrid species using genomic in situ hybridization (GISH). These results showed that not only are rDNA gene arrays silenced and lost in the hybrids, but that there is homogenization of rDNA between ancestral genomes, which can be identified by the increasing similarity of the GISH signal with the ancestral genomes used as a probe as age of the polyploids increases. Anssour et al. (2009) examined both allo- and auto-tetraploid Nicotiana species, and showed that there were substantial changes both in morphology and in the genome which are found in the hybrid species compared with their ancestral diploids. Some of these changes have adaptive significance.
Nicotiana × sanderae (section: Alatae), a sexual hybrid between N. alata and N. forgetiana [both 2n = 2x = 18; with the internal transcriber spacer (ITS) of the nuclear rDNA matching the maternal parent, N. alata (Chase et al., 2003)], is cultivated for its attractive flowers that range from white, through green to pink and red, lines of uniform colour being selected by breeders for commercial production. Pigmentation in N. sanderae is derived from N. forgetiana. Nicotiana × sanderae and its parental species are not resistant to blue mould (blue mold), caused by Peronospora tabacina (Clayton, 1945), a fungal disease that devastates ornamental tobaccos in bedding schemes and under glass. This disease is now prevalent in many parts of the world; plants readily succumb to infection and are lost from floral displays. However, Nicotiana germplasm includes species with agronomically important traits, such as tolerance to abiotic stress, including salt and drought (Komari et al., 2000) and resistance to P. tabacina. Thus, N. debneyi Domin (section Suaveolentes), a wild Australian species indigenous to Rockingham Bay in Queensland, was identified as a potential source of resistance to P. tabacina (Wark, 1970; Milla et al., 2005) for transfer to recipient species. Nicotiana debneyi is an allotetraploid (2n = 4x = 48, XXYY genome designation) that has two genomes distinct from those of tobacco (2n = 4x = 48, SSTT) (Goodspeed, 1954; Bai et al., 1995). The Germplasm Resources Information Network (GRIN) database (USDA, 2010) recognizes the species as a gene source for disease resistance and a tertiary genetic relative of tobacco. Resistance to black root rot (Chalara elegans), a common fungal disease of Canadian tobacco, has been transferred from N. debneyi to tobacco by sexual hybridization and backcrossing (Bai et al., 1995, 1996); resistance from this species is likely to be present in some Canadian flue-cured tobacco lines and US burley tobacco lines (R. Lewis, North Carolina State University, Raleigh, NC, USA, pers. comm.). Despite contrary evidence from pedigrees, marker genotyping indicates that blue mould resistance in various commercial tobacco lines has originated from N. debneyi (Milla et al., 2005).
Many horticultural crops do not have the resistances to biotic and abiotic stresses that are present in their wild relatives, the pool of uncultivated germplasm. Sexual hybridization is often impossible because of pre- or post-fertilization barriers (Tezuka et al., 2010). The genetic basis of the resistance and the genes involved are unknown, preventing any transgenic approach. Sexual incompatibility limits the genetic combinations achievable by conventional hybridization. Somatic hybridization, involving protoplast fusion, can be exploited to circumvent sexual incompatibility at both the pre- and post-zygotic stages of development, providing an approach to exploit naturally occurring biodiversity. Additionally, the extensive nuclear and cytoplasmic combinations resulting from protoplast fusion, with potential incorporation of plastids and mitochondria from both parents, generate novel germplasm for incorporation into breeding programmes (Horsman et al., 2001; Davey et al., 2005; Pati et al., 2005). Somatic hybrids are rare that are symmetric at the nuclear level with the complete nuclear complements of both parents, since culture of fusion-treated protoplasts and shoot regeneration from heterokaryon-derived tissues is often accompanied by the loss of chromosomes of one or both fusion partners to produce asymmetric nuclear hybrids (Liu et al., 2005).
Since the pioneering publication describing the first somatic hybrid plants between N. tabacum and N. glauca, which was also the first proof-of-principle of somatic hybridization (Carlson et al., 1972), several somatic hybrid and cybrid plants have been reported in the genus Nicotiana, most of which are cited by the authors listed here in more recent publications (IIcheva et al., 2000). Fitter et al. (2005) fused protoplasts of N. tabacum with those of N. suaveolens to generate three male sterile cybrid plants with recombinant mitochondrial DNA, the DNA sequences correlating with floral morphology. The flowers resembled those of N. tabacum. Chloroplasts were either of the N. tabacum or the N. suaveolans type. Sun et al. (2005) also fused mesophyll protoplasts of N. tabacum with those of N. repanda, generating cybrids with the nuclei of N. tabacum, but mitochondria from N. repanda. These experiments, like those of Fitter et al. (2005), demonstrated the possibility of introgressing cytoplasmic male sterility, carried by mitochondrial DNA, from a wild species into the cultivated crop. The same research group also generated a novel fertile symmetric somatic hybrid between N. tabacum and N. glauca with, unusually, a chromosome number equal to the summation of the complements of the parents. Morphologically, the flowers of the somatic hybrid were similar to those of N. tabacum, but petals were comparable to those of N. glauca (Sun et al., 2007). In other investigations, Sytnik et al. (2005) exploited somatic hybridization to transfer transformed chloroplasts from a transplastomic plant of N. tabacum into Lycium barbarum, demonstrating that organelles can be transferred to remote species by protoplast fusion.
Although N. debneyi can be crossed with N. tabacum, the species is sexually incompatible with N. × sanderae making it impossible to transfer disease resistance into the ornamental N. × sanderae by conventional breeding. As in several other Nicotiana species, plant regeneration from protoplasts has been achieved in N. debneyi (Scowcroft and Larkin, 1980; Kumashiro and Kubo, 1986). The present investigation aimed to generate somatic hybrid plants by protoplast fusion between N. × sanderae and N. debneyi, circumventing sexual incompatibility, and characterizing somatic hybrid plants at the morphological and molecular levels. This study also aimed to examine the fertility of the somatic hybrids, to confirm their chromosomal compositions, which included four genomes from the ancestors present in the hybrid originating from the two tetraploids, N. × sanderae and N. debneyi, and to evaluate seed progeny for their resistance to P. tabacina.
Seeds of Nicotiana × sanderae Hort. ex W.Watson 'Avalon Red Improved' (2n = 2x = 18) and N. debneyi (2n = 4x = 48) were supplied by Floranova, Foxley, Dereham, UK, sown on Levington M3 soil-less compost (Scotts Ltd, Ipswich, UK) and maintained to flowering under glasshouse conditions. Natural daylight in the glasshouse was supplemented with 16 h of fluorescent illumination (195 µmol m−2 s−1; TLD/58 W 35 V 'Daylight' fluorescent tubes; Phillips, Croydon, UK) with day and night temperatures of 25 ± 1 °C.
Three plants of each parent for sexual hybridization were grown in isolation. Buds of recipient plants were opened before anthesis and emasculated. Pollen from dehisced anthers was applied manually to receptive stigmas; reciprocal crosses were performed. Two plants of each parent were also self-pollinated. All pollinated flowers were labelled.
Seeds were immersed in 10 % (v/v) 'Domestos' bleach solution (John Diversey Lever, Northampton, UK) for 5–8 min, followed by three washes in autoclaved reverse osmosis water. Plants were established in vitro by germinating seeds on Murashige and Skoog (MS)-based culture medium (Murashige and Skoog, 1962) with 30 g L−1 sucrose, but lacking growth regulators, and semi-solidified with 8·0 g L−1 agar (Type IV; Sigma-Aldrich). Culture medium was contained in 9-cm-diameter Petri dishes (Bibby Sterilin, Stone, UK) under a 16-h photoperiod (90 µmol m−2 s−1; 'Daylight' fluorescent illumination) at 25 ± 2 °C. Seedlings were transferred to clip-lid axenic plastic vessels (Ashwood, London, UK; one seedling per vessel) each vessel containing 200 mL of the same MS-based medium without growth regulators, to establish axenic shoot cultures. The latter were subcultured every 28 d by excising proliferating shoot apices (each 2 cm in height) and inserting the apices into new medium of the same composition (five shoots per vessel).
Protoplasts were isolated from expanded leaves excised three to six nodes from the apex of axenic shoots. Excised leaves were cut transversely into strips (each 0·5–1·0 mm wide) after removing the main veins. Leaf tissues were plasmolysed by immersion in CPW salts solution (Frearson et al., 1973) containing 9 % (w/v) mannitol (CPW9M) for 1 h in 9-cm Petri dishes (1 g f.wt. tissues/10 mL solution). The plasmolysing solution was replaced with an enzyme mixture consisting of 0·5 % (w/v) Cellulase R-10 and 0·5 % Macerozyme R-10 for N. debneyi, and 1 % (w/v) Cellulase R-10 and 0·5 % Macerozyme R-10 for N. × sanderae (enzymes from Duchefa Biochimie B.V., BH-Haarlem, The Netherlands) and dissolved in CPW9M solution, pH 5·8. Incubation was in the dark at 25 ± 2 °C on a horizontal rotary shaker (40 rpm) for 12–16 h. Mesophyll protoplasts were filtered through a nylon mesh (64 µm pore size) and washed twice by gentle centrifugation (700 rpm for 5 min in 16-mL screw-capped tubes; MSE Centaur bench centrifuge) in CPW9M solution overlaying CPW solution containing 25 % (w/v) sucrose. Protoplasts were removed using a Pasteur pipette from the interface of the two solutions and were resuspended in EF9M electrofusion solution [9 % (w/v) mannitol, 0·5 mm CaCl2] at 1·0 × 105 protoplasts mL−1. Protoplast viability was determined by staining with fluorescein diacetate (Widholm, 1972).
The electrofusion apparatus was described by Jones et al. (1994). Isolated protoplasts of the two parental species were mixed in equal numbers. Aliquots (1 mL) of the protoplast mixture were dispensed into the inner nine wells of a 5 × 5 square grid dish (Bibby Sterilin Ltd), and the protoplasts allowed to settle for 5–10 min. A multi-electrode block of seven brass plates, each separated by 2·7-mm Perspex spacers, was inserted sequentially into each well. Protoplasts were aligned into pearl chains in an AC field (70 V cm−1, 1·0 MHz, 4 s) which was increased to 400 V cm−1 (0·5 s). Protoplasts were fused by 2 DC pulses (each 890 V cm−1) of 1·5-ms and 500-μs duration, separated by a period of 1 s. Parental protoplasts were self-fused. Other parental protoplast preparations were not electrofused, but mixed or cultured separately as controls.
Protoplasts were cultured in liquid MS-based medium containing 9 % (w/v) mannitol, 2·0 mg L−1 α-naphthaleneacetic acid (NAA) and 0·5 mg L−1 6-benzylaminopurine (benzyladenine; BAP) at a density of 1·0 × 105 protoplasts mL−1 (8 mL/9-cm Petri dish). After 21 d, the medium was replaced with the same MS-based medium, but with mannitol reduced to 4·5 % (w/v). Individual protoplast-derived cell colonies were selected randomly and transferred manually when 2–4 mm in size to 5·5-cm-diameter Petri dishes, the latter each containing 4 mL of MS-based medium with 1·0 mg L−1 BAP, 2·0 mg L−1 indole acetic acid (IAA), 4·5 % (w/v) mannitol and 0·8 % (w/v) agar (Type IV; Sigma-Aldrich), pH 5·8 (ten tissues/dish). Green, nodular tissues were sub-cultured when approx. 1 cm2 in size to the same medium, but lacking mannitol (5 tissues/9 cm Petri dish; 8 mL medium). Regenerated shoots were excised and transferred to semi-solid MS-based medium lacking growth regulators for root induction.
Regenerated, rooted plants (R0 generation) were potted in Levington M3 soil-less compost when 6–8 cm in height and maintained under high humidity for 10–14 d by covering the plants and pots with plastic bags. The bags were opened gradually before removal and exposing the plants to growth room conditions (25 ± 1 °C; 'Daylight' fluorescent illumination; 90 µmol m−2 s−1; 16-h photoperiod). Regenerated plants were grown to flowering; any plants that resembled N. debneyi were discarded.
Five putative somatic hybrid plants were retained, selfed manually with flowers from the same plant, and the flowers bagged after selfing. Seeds were collected from these five putative somatic hybrid plants, ten seeds were selected at random from each seed batch and germinated under glasshouse conditions to give R1 generation plants. R2 and R3 generation plants were established in the same way. Morphological characteristics of five R1, R2 and R3 generation protoplast-derived plants were recorded 75 d after seed sowing (Table 1). The same characteristics of five seed-derived plants of both N. × sanderae and N. debneyi were also recorded, but 90 d after seed sowing. Statistical analysis employed Minitab 15® software.
Root tips, each 0·5 cm in length, were excised from R0 generation protoplast-derived plants and from parental plants maintained in the growth room, pre-treated in 2 mm 8-hydroxyquinoline solution for 2–4 h in the dark, and fixed in 3 : 1 (v/v) ethanol : glacial acetic acid for 24 h. Preparations were hydrolysed in 1 m HCl (60 °C, 10 min), and stained with aqueous 1 % (w/v) aceto-orcein. Cells were examined by light microscopy for metaphase chromosomes. Ten preparations were examined per plant; chromosome numbers were recorded.
Root tips for in situ hybridization were excised from glasshouse-grown plants (R0 generation protoplast-derived), pre-treated with water-saturated α-bromonaphthalene for 24 h at 4 °C, fixed in freshly prepared 3 : 1 (v/v) ethanol : glacial acetic acid (24 h), and stored at 4 °C until use. Root tips were digested by incubating in a mixture of 2 % (w/v) Cellulase Onozuka RS and 2 % (w/v) Pectolyase Y23 (Duchefa) in citrate buffer at 37 °C for 30–45 min, transferred to glass slides and the meristematic tissue macerated in 45 % or 60 % (v/v) acetic acid (Schwarzacher et al., 1989). Slides were screened for cells with visible chromosomes prior to GISH.
Total genomic DNA of parental Nicotiana species, used as probes, was labelled with biotin-11-dUTP (GIBCO-BRL) and/or digoxigenin-11-dUTP (Boehringer Mannheim) using a nick translation kit (Bioprime CGH; Invitrogen). The hybridization mixture (30–40 µL per slide) contained 50 % (v/v) deionized formamide, 10 % (w/v) dextran sulphate, 0·125 % (w/v) sodium dodecyl sulphate, 1·0 µg mL−1 salmon sperm DNA and 2× SSC (Schwarzacher and Heslop-Harrison, 2000). The denaturation of chromosomes, hybridization conditions, stringency washing, amount of probe DNA, blocking DNA and hybridization mixture, were as described (Schwarzacher and Heslop-Harrison, 2000), with the most stringent wash being carried out in 0·2× SSC at 42 °C. Digoxigenin-labelled probe hybridization sites were detected using fluorescein isothiocyanate (FITC; Sigma-Aldrich) conjugated to sheep anti-digoxigenin antibody (Boehringer Mannheim) simultaneously to biotin-labelled sites with Cy3-streptavidin (Sigma-Aldrich). Chromosomes were observed using a Zeiss epifluorescence microscope, and images overlayed and processed, using only functions affecting the whole image equally (except drawing identification lines, cropping and filling edges outside crops) with Photoshop CS3.
Total DNA was isolated from leaves using a GenElute plant genomic DNA miniprep kit (Sigma-Aldrich) following the manufacturer's instructions. InterRetroelement Amplified Polymorphism (IRAP) and Retroelement-Microsatellite Amplified Polymorphism (REMAP) analyses were carried out following Saeidi et al. (2008) and Kalendar et al. (1999) using the primer sequences Nikita (5′-CGCATTTGTTCAAGCCTAAACC-3′) with the microsatellite sequence REMAP-GA, (GA)9C and Sukkula (5′-GATAGGGTCGCATCTTGGGCGTGAC-3′) with LTR6149 (5′-CTCGCTCGCCCACTACATCAACCGCGTTTATT-3′). Briefly, 50-ng genomic DNA samples were amplified in a 20-μL PCR mixture containing 1× PCR buffer (Kapa Biosystems), 2 mm MgCl2, 5 pmol of each primer, 200 mm dNTP mix and 1 U Kapa Taq polymerase. The annealing temperatures were optimized using gradients and the PCR programme consisted of 95 °C (2 min); 30 cycles of 95 °C (60 s), annealing at the optimized temperature for 60 s, ramp of 0·5 °C s−1 to 72 °C, and 72 °C for 2 min, adding 3 s per cycle, with a final extension at 72 °C for 10 min. PCR products were analysed by electrophoresis on 2 % (w/v) agarose gels and detected by ethidium bromide staining.
For RAPD analysis, PCR was carried out using the primer B07 (5′-GGTGACGCA-3′; Operon Technologies Inc.) in a programmed thermal cycler (Techne PHC-3) using 40 cycles of 94 °C (3 min), 94 °C (1 min), 35 °C (1 min), 72 °C (1 min), 72 °C (7 min), followed by 4 °C. PCR products were separated by 1·5 % (w/v) agarose gel electrophoresis and detected by ethidium bromide staining.
For cpDNA analysis, the trnL-ndhD fragment of the chloroplast genome was amplified by PCR (Sytnik et al., 2005) using the primers 5′-GTAGACACGCTGCTCTTAGG-3′ (specific to the trnL gene) and 5′ -CGCCAGATGTTCTATGGATAC- 3′ (specific to the ndhD gene; MWG-Biotech AG) to amplify a product of 1536 bp. The PCR programme was 35 cycles of 94 °C (3 min), 94 °C (20 s), 60 °C (20 s), 72 °C (1·5 min), 72 °C (5 min) and 4 °C. The amplified fragment was digested with AluI restriction endonuclease and the products analysed as above.
For mtDNA analysis, the forward primer 5′-CACGGGTCGCCCTCGTTCCG-3′ (rps14) and the reverse primer 5′-GTGTGGAGGATATAGGTTGT-3′ (cob; MWG – Biotech AG), were used to amplify mitochondrial DNA sequences (Demesure et al., 1995). PCR involved 40 cycles of 95 °C (5 min), 94 °C (30 s), 53 °C (30 s), 72 °C (2 min), 72 °C (7 min) and 4 °C. Reaction products were analysed as above.
Seeds harvested from the R1 generation of the five somatic hybrid plants SH28, SH31, SH354, SH411 and SH447 were sown on Levington M3 soil-less compost and individual R2 generation seedlings transferred 14 d after seed sowing to 13-cm-diameter plastic pots of the same compost. Plants were given a liquid feed (N : P : K, 3 : 2 : 3, v/v/v; Vitax) at every watering. Twelve plants of each somatic hybrid were evaluated for their fungal resistance against the same number of plants of N. debneyi, N. × sanderae 'Avalon Red Improved' and two other lines of N. × sanderae, namely 'Avalon Formula Mixed' and 'Perfume Formula Mixed' (Floranova). Plants were randomized in the glasshouse (natural daylight; maximum day temperature 25–30 °C; minimum night temperature 10–15 °C) and sprayed to run off on all their surfaces when 49 and 63 d old, with an aqueous suspension of spores prepared from leaves harvested from field-grown plants of N. × sanderae exhibiting extensive infestation by P. tabacina. Disease symptoms were recorded on glasshouse-grown plants when the latter were at the early flowering stage, 12 d after the second spraying with the spore suspension.
Of 20 flowers on two plants of N. × sanderae and the same number on N. debneyi that were selfed, all except one flower of N. × sanderae produced pods. Each pod had 510·0 ± 5·6 and 220·0 ± 11·6 (mean ± s.d.) seeds for N. × sanderae and N. debneyi, respectively. Pollination of 20 flowers of N. × sanderae with pollen from N. debneyi and the same number of flowers in the reciprocal cross, failed to produce pods, confirming the unpublished but known sexual incompatibility of the parental species (L. Garland and N. Belfield-Smith, Floranova, http://floranova.co.uk, pers. com.).
Protoplast yields (mean ± standard deviation; n = 5) of 2·0 ± 0·5 × 105 and 4·0 ± 1·0 × 106 g−1 f. wt., were obtained from leaves of cultured shoots of N. × sanderae and N. debneyi, respectively. Protoplasts in all preparations, including those subjected to electrofusion treatment, had a viability of approx. 80 % and protoplasts commenced division within 7 d of culture. Protoplasts developed into cell colonies, each approx. 1 mm diameter, within 35 d in liquid MS-based medium containing 9 % (w/v) mannitol, 2·0 mg L−1 NAA and 0·5 mg L−1 BAP. Protoplasts of N. × sanderae that were electrofused alone produced tissues but, importantly, the latter failed to regenerate shoots, providing a means of eliminating the N. × sanderae parental material. In contrast, tissues derived from protoplasts of N. debneyi electrofused alone and those not exposed to electrofusion, regenerated shoots following transfer to MS-based medium of the same composition, but with mannitol reduced to 4·5 % (w/v) and, subsequently, to MS-based medium with 1·0 mg L−1 BAP, 2·0 mg L−1 IAA and lacking mannitol.
Tissues from the electrofusion of protoplasts of N. debneyi with those of N. × sanderae also regenerated shoots. Of 1570 protoplast-derived cell colonies selected manually using forceps and transferred to MS-based medium with 1·0 mg L−1 BAP, 2·0 mg L−1 IAA, 4·5 % (w/v) mannitol and 0·8 % (w/v) agar, 580 developed into tissues. Eighty-nine tissues regenerated shoots when cultured on the same medium without mannitol. All the plants regenerated from the electrofusion of protoplasts of N. debneyi electrofused with those of N. × sanderae were screened for their phenotypic characteristics at flowering. Five plants that exhibited mainly intermediate characteristics compared with seed-derived parental plants, were retained, but the remaining 84 plants resembled N. debneyi and were discarded. The five somatic hybrids (designated SH28, SH31, SH354, SH411 and SH447; R0 generation) were taller than plants of N. × sanderae, but with a spreading habit comparable to that of N. debneyi. Their dominant flower colour, 64 A/B, was similar to that of N. × sanderae (61 A/B; Fig. 1). Importantly, plants were self-fertile. As in N. × sanderae, trichomes were present on both surfaces of the leaves of putative somatic hybrid plants. In contrast, trichomes were present only on the midrib at the base of the laminae of the leaves of N. debneyi.
The parameters evaluated for the subsequent R1, R2 and R3 seed generations, and for seed-derived parental plants, are summarized in Table 1. The mean leaf index and pollen viability of parental and somatic hybrid plants were similar in three seed generations. In contrast, flower length and flower width of all somatic hybrid plants were intermediate in size compared with those of parental plants; these parameters were consistent over the three generations assessed. Two somatic hybrid plants, SH411 and SH447, were closer to the N. debneyi parent in the R1, R2 and R3 generations in terms of plant height and internode length. In contrast, in somatic hybrid plants SH28, SH31 and SH354, such characters were closer to N. × sanderae in the R1 generation, but this stability was not maintained in the R2 and R3 generations when these parameters were closer to those of the N. debneyi parent. The flower colour of the somatic hybrids was stable in the R1, R2 and R3 generations and was consistent with that of the R0 generation plants. The number of seeds per pod was less for somatic hybrids compared with parental plants, with the lowest seed germination in SH31.
The chromosome number of each of the five somatic hybrids was 60 (SH31) or 62 (SH28, SH354, SH411 and SH447), fewer than the 66 expected from addition of 18 from N. × sanderae and 48 from N. debneyi (Fig. 2A). GISH analysis using labelled total genomic DNA as a probe to chromosomes of the R0 generation somatic hybrids (Fig. 2A–D), enabled the parental chromosomes to be distinguished in root tip cells at mitotic metaphase by probing simultaneously with total genomic DNA from N. × sanderae (labelled with biotin) and N. debneyi (labelled with digoxigenin). All metaphases showed 18 chromosomes of N. × sanderae origin. Somatic hybrids SH28, SH411 and SH447 had 44 chromosomes from N. debneyi. In situ hybridization also differentiated chromosome sets from the two parents, themselves both of sexual hybrid origin; the differentiation was particularly clear in SH447 (Fig. 2A, B). Among the 44 chromosomes of N. debneyi origin, there were 24 with more label from one ancestor and 20 with less label from the other ancestor, indicating that all four chromosomes that were missing in the somatic hybrid plant SH447 came from the same ancestral genome. The chromosome complements of the original somatic hybrid plants were retained in their R1, R2 and R3 seed progeny.
In metaphases of N. debneyi hybridized with its own DNA, half of the chromosomes were labelled more strongly than the remaining chromosomes, and some of these had terminal sites which were brighter, thus confirming the amphipolyploid hybrid origin of N. debneyi (Fig. 2E, F), with substantial differentiation of the two genomes, although there remains speculation about their origin (Khan and Narayan, 2007; Clarkson et al., 2010).
The REMAP and IRAP analysis with genomic DNA extracted from the parental species and the five somatic hybrids (R0 generation) showed that the hybrids had a pattern generally representing the sum of both parents (Fig. 3A, B). Although four or six of the chromosomes of N. debneyi origin were missing in the somatic hybrids, all but one band seen in N. debneyi were also detected in the hybrids (Fig. 3B); primer competition may have reduced amplification of this and another band which is less clear in SH447 and SH411 which showed weaker amplification. Two other primer pairs were also used. REMAP-GA with the primer Sukkula showed shared bands and one specific to N. × sanderae that was present in all hybrids; primer Nikita with LTR6149 showed little amplification with N. × sanderae DNA, but four bands in N. debneyi and the hybrids (not shown).
Primer OPB07 used for RAPD analysis generated distinct bands for the parents (Fig. 4, lanes 2, 3 and 6) with somatic hybrids having a summation of the parental bands (lanes 4, 5 and 7–9). The bands obtained for the cpDNA of N. × sanderae (Fig. 5A, lane 2) differed from those of N. debneyi (lane 8) and the bands of the somatic hybrids (lanes 3–7). The cpDNA banding pattern of somatic hybrid plants resembled the pattern of N. debneyi, indicating that plastids in the somatic hybrids were derived from the wild N. debneyi parent. mtDNA analysis revealed a band of 1396 bp present in the somatic hybrid plants (Fig. 5B, lanes 3–7) and in N. debneyi (lane 8). This band was absent from N. × sanderae (lane 2), suggesting that the mitochondrial genomes of the five somatic hybrid plants were probably derived from N. debneyi.
All plants of N. × sanderae 'Avalon Red Improved', 'Avalon Formula Mixed' and 'Avalon Perfume Formula Mixed' (n = 12 for each cultivar) showed characteristic and extensive infection when evaluated 12 d after the second spraying with a spore suspension of P. tabacina. Infection was accompanied by yellowing and twisting of infected leaves (Fig. 6A, B), with thick mats of sporulating mycelium on the lower surfaces of infected leaves (Fig. 6C) and subsequent death of the plants. In contrast, leaves of all plants of N. debneyi (Fig. 6D, E; n = 12) and 12 R2 generation seedlings of each of the somatic hybrids N. × sanderae (+) N. debneyi (60 plants in total) were all completely resistant to P. tabacina and did not exhibit any signs of fungal infection (Fig. 6F, G).
Wild germplasm frequently has novel characteristics that are valuable for breeding, including resistances to biotic or abiotic stress and quality characters. Interspecific sexual hybridization has been used extensively and, increasingly, transgenic approaches have been exploited for gene transfer. However, there are formidable regulatory obstacles relating to transformation technology and the products generated, adding to the laboratory costs to this latter strategy. In the genus Nicotiana, despite the presence of many hybrid species, it was impossible to obtain seed from sexual crosses between N. × sanderae (Nicotiana section Alatae), an ornamental tobacco affected by blue mould, and N. debneyi (section Suaveolentes), a wild species which has been used in breeding fungal resistance into tobacco by sexual hybridization. Tezuka et al. (2010) examined sexual crosses within the section Suaveolentes and found both prezygotic barriers to fertilization and post-zygotic failures during seed development, some of which could be overcome by ovule culture. Hybrid lethality, where seedlings die soon after germination, was also frequent in the progeny of the successful sexual crosses of Tezuka et al. (2010). In contrast, the somatic hybrids between N. × sanderae and N. debneyi flowered (Fig. 1) and produced seed, enabling assessments of subsequent generations. It would be interesting to determine whether hybrid lethality occurs in seedlings from somatic hybrids of those species combinations in the successful sexual crosses reported by Tezuka et al. (2010), provided such somatic hybrids can be generated by the fusion of diploid parental cells.
The shoot regeneration potential of cultured tissues in the genus Nicotiana was evaluated by Li et al. (2003), who showed that organogenesis was poor in N. × sanderae. The present investigation confirmed that although cells derived from electrofusion-treated protoplasts of N. × sanderae commenced division during the first 7 d of culture, they failed to regenerate shoots. This inability of protoplast-derived tissues of N. × sanderae to regenerate was exploited as a half selection procedure to eliminate the N. × sanderae parent from electrofusion-treated mixtures of parental protoplasts. In contrast, and in agreement with the results of Scowcroft and Larkin (1980), tissues that developed from electrofusion-treated protoplasts of N. debneyi regenerated shoots under the same conditions. Thus, any hybrid cells that developed from heterokaryons following electrofusion of protoplasts of N. × sanderae with those of N. debneyi would also be expected to develop through heterosis and to regenerate shoots under the same conditions; the conspicuous morphology of the somatic hybrids compared with that of N. debneyi allowed the somatic hybrids to be identified.
Somatic hybrids and their seed progeny retained a flower colour similar to that of the N. × sanderae parent (Fig. 1), making them attractive ornamentals in their own right. Other morphological characteristics of the five somatic hybrid plants and their seed-derived progeny were mainly intermediate compared with those of the parental plants. Plastids and mitochondria in the somatic hybrids were probably from the wild N. debneyi parent (Fig. 5). Loss of cytoplasmic genomes from one partner is not uncommon in somatic hybrids, with organelles from the other fusion partner becoming dominant (Davey et al., 2005). For example, in Solanum bulbocastanum (+) S. tuberosum plastids and mitochondria were from the cultivated parent (S. tuberosum) in most of the hybrid plants (Lovene et al., 2007).
DNA and GISH analysis of metaphase chromosomes revealed that the somatic hybrids of N. × sanderae (+) N. debneyi included nuclear genomes from both parents (Figs 2, ,33 and and4),4), although four to six chromosomes from N. debneyi were absent from these hybrids (Fig. 2). Loss of parental chromosomes in somatic hybrids is not uncommon (Liu et al., 2005). In fact, most somatic hybrid plants generated between N. tabacum and wild Nicotiana species have been asymmetric at the nuclear level with only a limited number being symmetric hybrids (Sun et al., 2007). Yang et al. (2007) also reported a range of chromosome numbers from 54 to 74 in somatic hybrids between Gossypium hirsutum and G. klozschianum. Despite a loss of chromosomes, the somatic hybrid plants of N. sanderae (+) N. debneyi were morphologically stable as demonstrated by the retention of characteristics that were scored for the original individual somatic hybrid plants (R0 generation) and their seed-derived progeny over the R1, R2 and R3 generations. The five somatic hybrids that were generated were self-fertile, an extremely important attribute, enabling characterization of seed progeny. Whilst characteristics of the somatic hybrids were generally intermediate between those of the parental plants, some of the seed-derived progeny exhibited characteristics that were more similar to those of one parent, as in the case of plant height and internode length. Somatic hybrid plants and their seed progeny also exhibited novel floral morphology and a consistent flower colour, the latter being slightly more intense than that of N. × sanderae as judged using the universally accepted colour charts of The Royal Horticultural Society of London. Segregation of characteristics was not observed in the R1 to R3 seed generations, with 60–80 % seed germination, except in SH31 in which seed germination was approx. 40 %.
Seed-derived progeny were resistant to P. tabacina, this resistance being introgressed from the wild species, N. debneyi. These experiments emphasize the relevance of somatic hybridization through protoplast fusion to transfer complex genetic traits from wild species (Fig. 6) into sexually incompatible recipients without the requirement for identification of the genes involved and recombinant DNA technology. Indeed, N. debneyi may be a useful protoplast fusion partner in attempts to introgress fungal resistance into other sexually incompatible Nicotiana species of value as ornamentals, or for the production of secondary plant products, including pharmaceuticals, where complex disease resistance traits could be combined with simple monogenic traits carried by transgenes. This approach may be exploited more in the future, especially as P. tabacina appears to be more widespread and, for example, has become endemic in the UK in the last 20 years. The fertility and stability of somatic hybrid plants is crucial for the incorporation of this germplasm into breeding programmes. Future experiments will also evaluate transfer of fungal resistance from somatic hybrids of N. × sanderae (+) N. debneyi by sexual backcrossing to lines of N. × sanderae and, possibly, to other ornamental species of Nicotiana. Whilst the semi-dwarf stature of N. × sanderae may have been lost in the somatic hybrid plants generated in these experiments, in their seed progeny and in any future back-cross progeny, additional genetic manipulation of these somatic hybrids and back-cross progeny by the introduction and expression of genes controlling gibberellin metabolism and, hence, stature (Dijkstra et al., 2008) could enable restoration of the dwarf habit characteristic of N. sanderae.
The authors thank L. Garland and N. Belfield-Smith (Floranova) for seeds of N. × sanderae and N. debneyi, for financial assistance to D.P., and for experiments relating to assessments of fungal resistance. Dr K. Pyke (Plant and Crop Sciences Division, University of Nottingham) assisted with the formatting of Figs 1 and 4–6.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Boswell: General Manager Mike Rizzo gives Nats an offseason makeover
Article | Working with a smaller budget than he should have to, General Manager Mike Rizzo has made the Nationals a deeper all-around team in less than a year's time.
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Nationals' Dunn dedicated to playing first base
Article | VIERA, FLA. -- Adam Dunn can't really explain his aversion to becoming a designated hitter. He thought about it for a moment Tuesday, shrugged, and said, "I don't know." Stubbornness plays a part. He thinks playing in the field is fun, and he is not going to play in the American League no matter how...
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Article | The Washington Nationals' Dominican Republic operation is making progress one year after the Smiley Gonzalez scandal.
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Yoga Teacher Training
Yoga Teacher Training Courses
We are taking applications for all three, 12 month CYS courses which will begin in September 2016, one weekend each month –
September 2nd/3rd – alternate months in Edinburgh and Perthshire
September 9th/10th – at Merchant City Yoga in Glasgow
September 17th/18th – September in Weybridge, Surrey.
Each year we invite prospective students to join the tutors, assistants and current students for a light lunch and an informal chat about the course and their interest in moving into teaching. The next dates for this are –
- Saturday, 2 July at David Lloyd Newhaven, Edinburgh
- Saturday, 9 July at Merchant City Yoga, Glasgow
- Saturda, 16 July at Weybridge Hall, Weybridge, Surrey
If you would like to come along to one of the above, please contact June Mitchell, the Course Director, at email@example.com or Judi Farrell at Merchant City Yoga for the Glasgow course – email@example.com
All dates for the Sept 2016 – August 2017 courses are available to click on below and by emailing June as above.
About CYS Yoga Teacher Training
Course Director June Mitchell has been teaching yoga full time since the early 1980s, running three very successful yoga centres in Perth, Scotland, the current one being Perth Yoga Studio www.perthyogastudio.com She is qualified in both Sivananda Yoga through the Scottish Yoga Centre 1976/77 and Ashtanga Vinyasa yoga with Paul Dallaghan's Centred Yoga on Koh Samui in Thailand in 2003; had the benefit in the 1980s and 90s of many trainings with Iyengar yoga teachers and in 2004 was a direct student of Sri K. Pattabhi Jois in Mysore, India. June is dedicated to the traditional teachings of yoga and has been one of the UK's leading yoga teachers for very many years. She has taught and inspired a large number of highly successful yoga teachers who are now taking classes and running their own yoga centres throughout Scotland and the South of England.
In 2003 she co-founded and was Course Director of CYS Scotland, then called the Chi Yoga School, as an independent yoga school offering the highest professional standards in yoga teacher training.
In 2006 CYS expanded into the South of England and both courses have continued to thrive since 2010 when June took over as sole owner and Course Director of CYS, now known as the Classical Yoga School. Since September 2015, three courses are now running – Edinburgh/Perthshire, Weybridge Surrey and with Judi Farrell, Lead CYS trainer, at her Merchant City Yoga in Glasgow. By the middle of August 2016 close to 400 students will have successfully graduated from the school over the years and are teaching throughout the UK with others also in Europe, Australia and the States.
Under her guidance, and with the support of an outstanding team of tutors, the school has become known as a Centre of Excellence in the UK, training enthusiastic yoga students to become inspiring yoga teachers in their own right.


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Below are top resources on SparkPeople for Eating Right 4 Your Type.
We hear it all the time—you changed your habits and lost weight steadily, but after awhile, that progress halted. You've been stuck at the same weight for days, weeks, or even months. There's nothing more frustrating t... Read more
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Having recently discovered that I'm a type A blood, I read up on the book entitled, "Eat right for your Type."
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OC Deputies Hunt Career Criminal Who Escaped From Irvine Jail
WANTED: Fernandez
[Update at end of post] Orange County Sheriff's Department (OCSD) deputies this morning are hunting a career criminal who escaped Friday from the James A. Musick Jail in Irvine, according to an OCSD special bulletin by Lieutenant Jeff Hallock.
Officials discovered Max Edward Fernandez missing during a 4 p.m. security check at the facility and are asking citizens to report any related information, especially the inmate's whereabouts, by calling OCSD Investigator D. McKenzie at 714-647-7000 or calling anonymously to OC Crime Stoppers at 885-847-6227.
The 5-foot-8 and 170-pound Fernandez has brown hair, brown eyes and several tattoos, including a tribal band on his upper right arm, skull on his upper right back and USMC with a hammer shark head on his left tricep.
Hallock reports the criminal was last seen wearing a gray t-shirt and red shorts under gray sweat pants.
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Officials also believe the inmate may have injured himself during the escape.
Fernandez was in custody for possession of stolen property and a narcotics violations.
He'd previously compiled a rap sheet that includes thefts, burglaries, narcotics violations, auto theft and resisting arrest, according to Hallock's press statement.
UPDATE, 3:30 p.m., Sept. 21: Authorities found an unarmed Fernandez hiding in an Oceanside Wendy's restaurant; the inmate is now being housed inside the main Orange County Jail in Santa Ana.
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Arts & Life
Movie Studios Reverse Ban on Screener Videos
Hollywood studios back away from a ban on sending promotional videos to Oscar voters. The ban was meant to stop piracy, but it angered many who said it would have given studio films an advantage over smaller, independent features. NPR's Rick Karr reports.
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Tim McGraw has two singles currently climbing the charts—"Right Back Atcha Babe" and "Truck Yeah." Which do you like better?
"Right Back Atcha Babe"
Votes: 31%
"Truck Yeah"
Votes: 69%
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New ink promotes new world tour.
The Gambler's Last Deal begins May 13 in Mahnomen, Minn.
Video features his Wife Alex and Dog Writer as the main characters.
Sam Hunt leads the field with nods in three categories.
New single, "Outskirts of Heaven," has youngsters thinking.
The faith-fueled tune is part of Blake's new album, "If I'm Honest."


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idk I'm not really impressed.
I've always thought video games will eventually evolve into something where a person actually has to interact physically. Like holograms or VR helmets or whatever...you know, you're whole mind is immersed instead of just holding a controller and staring at a screen.
Now, do I think Google has nefarious motives and is probably up to something? Google? The company who's slogan is "don't be evil" or whatever? I couldn't believe that. YEAH RIGHT.
If anything, they're probably testing how popular this new "interactive" gaming idea will work...and also track you and spy on you and whatever else.
Ultimately, it sounds like advancing sci-fi and nothing more. I don't doubt the story line is true. My mind is open to a lot of things, but I doubt a video game is going to open a portal to hell in real life.
Although, video games opening up portals to hell in real life could certainly happen, through channels of degeneracy. But I guess I'm talking physical fire and brimstone type stuff. lol.
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What the fuck are you idiots talking about? Google and Apple are already spying on you and tracking you! They are probably even listening to you right now.
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Robots, drones fill police ranks
Companies around globe packing earth orbit with satellites to meet communications demands
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Report: Mr. Robot's one-time only VR experience will return next week
'Kill More Cops' Graffiti Mural Appears on Freeway
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Should the Postal Service Handle Your Loans as Well as Your Mail? Elizabeth Warren Thinks So.
HERE IT IS=> Detailed List of Findings in Wikileaks DNC Document Dump
BREAKING: That Was Fast! Hillary Immediately Brings Disgraced DNC Chair Onto Her Campaign! Do you think that Hillary has something to hide?
According to Soros, there's about to be an all-out class war riots, police state and civil war for America
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LOL Of The Day - "Hillary for Prison" banner plane spotted over Philadelphia
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What a year for movies, right??? Friggen The Hunger Games! Friggen Avengers! Friggen The Dark Knight Rises! Friggen 2 Days In New York! Wait... you didn't get a chance to see 2 Days In New York?!?! It's okay, it's okay! It was an indie flick, so-- unlike TDKR-- a lot of people missed it. But that's ...
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Translation of a poem
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As stated, somehow certain different pages, perhaps they are called virtual pages as they are created to hold plugin content, now display Blog Archive content. One is Forums, from bbPress where all forums are to be displayed. The other two are from directory and classified plugins which also are virtual, but used to display generated content.
The day's BU got overwritten before I noticed the problem.
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Put your hands together for an Applause.
The Ovation Applause Series AE35 is a 12-string acoustic-electric guitar that's a Roundback. The AE35 guitar incorporates the revolutionary Ovation Lyrachord composite body - the revolutionary acoustic guitar design that has made Ovation a favorite with pro players for over 40 years. The dovetail heel/neck joint allows the neck angle to be accurately and permanently set at the factory. The low neck profile design on the Ovation Applause 12-string guitar is not only comfortable, but super-stable as well.
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Order today and experience a guitar that sounds great acoustic or electric and that can travel without trouble.


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Historical society hosts picnicAugust 07,2014Mendon summer
picnic is Aug. 23
MENDON The Mendon Historical Society and Mendon Seniors will host a summer picnic from 4 to 7 p.m. Aug. 23 at the Mendon Recreational Field on South Mendon Road.
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There will also be musical entertainment, games, activities and a raffle.
For further information, call 773-4854.MORE IN This Just InNORTH BENNINGTON Two public meetings, in North Bennington and Shaftsbury, have been set for... Full StoryThe Board of Aldermen has endorsed a major jump in city pension funding. Full StoryHUBBARDTON The annual celebration of Hubbardton has been moved to Sunday due to the weather... Full Story
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Alberta.ca > Agriculture and Forestry
"This year's event is being offered jointly by Alberta Farm Fresh Producers Association and Organic Alberta. As a result, there will be sessions for organic grain producers and all sessions will be appropriate for both conventional and organic producers," he says.
The school features a range of streams for different audiences over its two days including horticulture, marketing/transition, livestock and grains. Additionally, there will be a trade show, as well as a business/conversation room for specific businesses and topics.
"New Venture Coaches will be on hand to have one-on-one discussions about business plans and business expansion ideas. There are lots of opportunities to take home information, share ideas, and get insightful tips and advice," Spencer says.
There is a full social evening, including a dinner and awards presentation (with cash bar available), followed by a separate mixer on Thursday evening. The AGMs of the Alberta Farm Fresh Producers Association (AFFPA) and Organic Alberta (OA) will be held at noon on Day 1.
The Alberta Farm Fresh School 2016 takes place February 25-26 at the Pomeroy Inn & Suites in Olds, AB (on Olds College campus).
Visit the Alberta Farm Fresh Producers Association or Organic Alberta for registration information and tentative agendas.
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Phone the Ag-Info Centre, toll-free in Alberta at 310-FARM (3276), for agricultural information.
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I had one of the bluff cabins which were very large, however, it was dated and very dark (not enough lighting). My cabin was at the end, and there was a bad smell in that area, which may have been from the septic tank. Considering the price, they do not supply any toiletries, like shampoo, only bar soap and plastic glasses. I also had to ask them to clean the bath tub because it was very dirty and to get rid of a pop can on the balcony that was covered with ants.
The bluff cabins are a relativity long walk down a somewhat steep winding path from the parking lot and have a large balcony, which the other cabins do not have. The prairie cabins are a very short flat walk from the parking lot. (They should really be called parking lot cabins.)
- Also Known As:
- Giant City Hotel Makanda


text:
Two candidates are running for office in the state senate. During a debate, the candidates say the following: Candidate A: "People are responsible for one another. We will all benefit if everyone gives their fair share. My opponent, Candidate B, doesnt really care ...
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American Government
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Monday, May 25, 2015 by Joshua
the south african government is not providing enough welfare or subsidies to the poor and underpriviledged
Monday, May 25, 2015 by nkazimlo
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What are welfare payments and we're South African government providing and to whom
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Check PLZZ History
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What is the south african government providing and to whom
Saturday, May 23, 2015 by siyabonga
American Government
Congrss controls the Executive branch by issuing what?
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Ultimately, Reagan just like Nixon desired less federal government involvement. True or False I think it's false
Thursday, May 21, 2015 by Gaby
The south african government is not providing enough welfare or subsidies to the poor and the underpriviledge. To whom are these welfare or subsidies provided and how?
Tuesday, May 19, 2015 by Zimkhitha
englis :(((
C). Change these active sentences to passive. 1. People speak Portuguese in Brazil. 2. The Government is planning a new road near my house. 3. My grandfather built this house in 1943. 4. Picasso was painting Guernica at that time. 5. The Cleaner has cleaned the office. 6. He ...
Tuesday, May 19, 2015 by rudy
Life Orientation
What did the community members do before the protest? Measure taken by the local government to address the issue
Tuesday, May 19, 2015 by Israel
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Monday, May 18, 2015 by Amy
pleasse check social studies 2/2
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Monday, May 18, 2015 by XenaGonzalez
SOCIAL STDIES HELP! 8th
Ellis Arnall was elected to the office of governor in 1942 by promising to (1 point) reform Georgias government. continue Talmadges policies. fight in World War II.*** support disfranchisement for African Americans.
Monday, May 18, 2015 by XenaGonzalez
American Goverment
The authority of agencies in the Executive Branch of government comes from (Points : 1) the Constitution. Congressional delegations of authority. executive prerogative. executive Branch membership.
Sunday, May 17, 2015 by christopher
Ms. Sue PLZ HELP!!!(Civics)
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Ms. Sue PLZ HELP!!!(Civics)
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Language Arts
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answer lol social studies 1 question
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What is South African government providing and to whom?
Saturday, May 9, 2015 by Slindile
Social Studies
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The 13-member math club needs to choose a student government representative. They decide that the representative, who will be chosen at random, CANNOT be any of the 3 officers of the club. What is the probability that Samara, who is a member of the club but NOT an officer will...
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American Government
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Business Economics
What subsidies and welfare payments is the south african government providing and to whom ?
Monday, May 4, 2015 by Simran
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Thursday, April 30, 2015 by Shane
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Wednesday, April 29, 2015 by reina bonilla
History/ Civics
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Wednesday, April 29, 2015 by Jane
What type of society & government is Brave New World by Aldous Huxley? And what aspect from the novel is relatable to history or modern society? I need to find something to challenge(a topic/idea/argument Huxley made in his novel) and write a research paper to back up my ...
Wednesday, April 29, 2015 by Beth
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Tuesday, April 28, 2015 by TayB
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Tuesday, April 28, 2015 by Student
Leaders from several states of a nation meet once a year to settle common issues. They govern their own states independently, but they work together on issues that affect them all like trade and defense. They do not have a central government that handles these matters or ...
Tuesday, April 28, 2015 by Student
Which of the following statements would you place in the section labeled F? F is under disadvantages of democracy. A. Very little holds the nation together, and local governments can leave anytime. B. The central and local governments may have conflicts and power struggles. C...
Tuesday, April 28, 2015 by Student
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Tuesday, April 28, 2015 by erica
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Tuesday, April 28, 2015 by Emma
Social studies
Which is an event of the progressive era enacted by the local government in south carolina
Monday, April 27, 2015 by ZaKirrah
In which type of government would people elect officials to make government decisions? Monarchy Direct Democracy Representative Democracy*** Autocracy
Monday, April 27, 2015 by Anonymous
Which order lists government types from the least power for the people to the most? Oligarchy, autocracy, republic Autocracy, oligarchy, democracy Democracy, unitary, parliamentary Federation, republic, unitary***
Monday, April 27, 2015 by Anonymous
Which explains why the Compromise of 1850 intensified the argument about slavery? A- People for and against slavery migrated to Western stats to influence laws. B~Free states in the West added more antislavery votes to Congress. C~The equal division of slave and free ...
Monday, April 27, 2015 by Emma
What is the south African government providing and to whom?
Monday, April 27, 2015 by hopisi abulele
parliament democracy is the form of democracy where citizens have the most power of their government right?
Sunday, April 26, 2015 by reina bonilla
10. Which of these statements about the Cuban Missile Crisis is true? A. President Kennedy removed nuclear missiles from Turkey to resolve the crisis. B. Fidel Castro's government had manufactured guided missiles. C. President Kennedy called Nikita Khrushchev on the ...
Saturday, April 25, 2015 by Sarah
In federalism, the national government (Points : 1) is subordinate to the states. shares power and authority with the states. can force the states to do what it wants. can choose the states governing officials.
Saturday, April 25, 2015 by Kiara
unemployment rate is a measure of
Saturday, April 25, 2015 by reina bonilla
government LSN
what is the difference between urban sprawl and smart growth??
Saturday, April 25, 2015 by anonymous
government LSN
how do politicians and interest groups use the media to persuade Americans
Saturday, April 25, 2015 by anonymous
what do candidates do in an electoral campaign
Saturday, April 25, 2015 by anonymous
most important item a candidate needs to wage a succesful campaign
Saturday, April 25, 2015 by Reina Bonilla
I think it is A & C, but am not sure.. In a perfectly competitive market in which no market failure occurs and no government policy interferes with the equilibrium price and quantity: a. deadweight loss is zero b. the sum of producer and consumer surplus is maximized c. ...
Saturday, April 25, 2015 by Joe
Social studies
1. Which word best describes modern life for Japanese teenagers such as Asuka? relaxed crowded isolated traditional 2. Tropical storms often hit Japan during the ________. summer winter spring fall 3. Which of Japan's islands has the largest level area? Hokkaido Honshu ...
Friday, April 24, 2015 by Shawn
the south african government is not providing welfare or suubsidies to the poor and underpriveledged
Thursday, April 23, 2015 by zipho madlala
um ok so what are the words that describe the precedent created by the cases i know judicial review may be one what else can i put??
Tuesday, April 21, 2015 by Reina Bonilla
SOCIAL STDIES HELP! 8th
2. Which was a part of the Populist Partys platform? (1 point) tax breaks for farmers*** civil rights for all Americans higher tariffs on foreign goods increased industrial development 3. Whom did Rebecca Latimer Felton replace when she was appointed a senator in the ...
Tuesday, April 21, 2015 by XenaGonzalez
Personal Finance
Most consumer complaints are resolved by: A. legal action. B. assistance from a government agency. C. contacting a company's headquarters. D. returning to the place of purchase. I believe it's C
Tuesday, April 21, 2015 by Jackie
can you explain this to me government
First decision by the Supreme Court to declare a law unconstitutional. At the very end of his term, President John Adams had made many federal appointments, including William Marbury as justice of the peace in the District of Columbia. Thomas Jefferson, the new president, ...
Tuesday, April 21, 2015 by Reina Bonilla
government LSN
whats Marbury v. Madison can u explain it to me in the simplest way as possible?
Tuesday, April 21, 2015 by Reina Bonilla
In america, our government is based on the principles of society and the rights of man. do you agree or disagree? List at list 3 example.
Tuesday, April 21, 2015 by Allen
Social Studies
How would the Philippine government respond to an insurgency? A. the government would try to work with their members. B. the government would want to support it. C. the government would ignore it. D. the government would fight it fiercely. D?
Tuesday, April 21, 2015 by Samantha
How were William Lloyd Garrison and the Rev. Elijah Lovejoy alike? Both lived in Boston and were Underground Railroad conductors. Both printed newspapers with antislavery messages in the North. Both used their houses as Underground Railroad stations and risked their lives for ...
Monday, April 20, 2015 by Emma
non profit Accounting
The mayor of a large city approaches a group of citizens and suggests that they form an organization to provide social, educational, and recreational programs for local youth. The group agrees and forms an entity called the Community Youth Organization (CYO). The group also ...
Sunday, April 19, 2015 by mary


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Three better than two
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oh ja heiss schaut mal auf die seite kohle oli , voll geil
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Nearly came in my pants watching the moptop wag his dick around while riding the skinhead. Totally HOT!!! :P
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prostitution in jonesboro?
Posted in the Jonesboro Forum
#1 Feb 1, 2012
Does anyone know if this is really a problem in the area?
Since: Jan 09
#2 Feb 1, 2012
I don't think they have a retirement plan or paid sick leave if you consider that a problem.
#3 Feb 2, 2012
Are you concerned or interested?
#4 Feb 2, 2012
What do you consider a problem? I know a lot of them aren't real pretty and since they are illegal they aren't regulated and some will scam you, but other than that, they generally do their job well.
"truth is a three edged sword"
Since: Jan 11
Babylon 5, Epsilon Eridani
#5 Feb 2, 2012
it is so big of a problem that kait8 gave it a two part series a few years ago. The good neighbor station also gave the locations, times, prices, etc...
#6 Feb 2, 2012
Prostitution is a live and well in Jonesboro, AR. It may just take over Trumanns' spot as the VD capital of the world.(per capita) And China Doll keeps losing her babies to DHS. But wait, there's more- coming! LOL
#7 Feb 2, 2012
it's alive and well everywhere. Man goes out with woman, spends money (no big deal). Man and woman continue to talk and go out and things seem normal (simple dating, no problem money wise).
Then woman starts asking for things or expecting things to continue to go out/talk and flirts/talks sexy/dirty, etc. If man doesn't produce, she ignores him for a few days and little does he know she has other guys she is doing this to as well.
Then, if the man spends the right amount or provides certain things she wants, for instance, i don't know maybe a trip somewhere, clothes, helping with the bills, etc..., then he gets intimacy (snuggling, cuddling, and who knows maybe even touching or sex). If he doesn't, she has other options that might do this, so he is left hanging around.
So, isn't this a type of prostitution, buy me this or do this for me and I will see you/be interested in you/if i like you enough have sex with you?
Seriously, a man is paying in one way or another to be noticed by a woman or for her to act interested in him, with or without sex, but in the end he is hoping he impresses her or spends enough to get laid.
At least prostitution and even loose women keep it real, men pay to go out and have a firm grasp that they know what is real. With prostitution, they know they are getting laid. With a s1ut/hoe, they know that at least their money is leading to an opportunity to get laid. NO BS, no Drama, no empty heartache or let down.
#8 Feb 2, 2012
wow, I feel sorry for you if those are the only types of women you date. Women like that are called "gold diggers". And you should be able to spot them before the first date even happens, so why bother dating them if you know you have to buy stuff for intimacy? Why don't you try dating normal women? They do exist, I promise.
#9 Feb 3, 2012
Most women are gold diggers... They want spoiled and want what they see on the Real Housewives of whatever.
Normal women in Arkansas, well, if you're religious, then maybe you'll get lucky at Church. If you're not religious, there is a very small window for normal women. And I don't consider people who worship invisible sky people to be normal.
#10 Feb 3, 2012
Women in church are no different. Women are women.
United States
#11 Feb 3, 2012
Where r they located
#12 Feb 3, 2012
Look if a man is to stupid to realize he is with a dumbcunt, then he gets what he deserves
#13 Feb 3, 2012
United States
#14 Feb 3, 2012
what about the men are there any men who are prostitue in jonesboro i am interested between the age of 19 and 25
#15 Feb 3, 2012
I've never seen them....
#16 Feb 4, 2012
#17 Feb 4, 2012
AMEN BROTHER! Been in the middle of one like that and wanted to kill somebody over it. Then i stopped to think, and realized, the woman ain't worth it.
Since: Nov 11
#18 Feb 4, 2012
why is there even laws regarding such as this...it's 'capitalism' at its purest form...
#19 Feb 4, 2012
There isn't anything pure about prostitution. Why is hooking better that selling crack? After u sell crack it's gone. But she can always wash hers and sell it again.
I do think it's probably a rare thing for the cracks to get washed between uses
" phd in clinical ignorance"
Since: Apr 09
Location hidden
#20 Feb 4, 2012
actually, under obama's stimulant plan, all eligible ho's get paid time off for qualifying illness, up to and including booby bites, std's, and pregnancy when ended with abortion.
as for retirement, the stimulant plan offers them a choice of food stamps or welfare, or both.
either way they win, they get to claim to be poor while living off of those who work for their living and pay taxes to support them.
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First clip of Jeremy Clarkson's The Grand Tour released
It looks and feels exactly the same as old Top Gear
It looks and feels exactly the same as old Top Gear
Evans has announced he will be stepping down as presenter of Top Gear
If Harris, Reid, and Schmitz are given more screen time, next season could be great
Chris Evans has resigned. FACT.
Fewer than 2 million people tuned in to watch the finale
The show has now lost more than half of its audience since its series premiere, which attracted 4.3m viewers
'Our version is much, much better and will ultimately be more successful'
'It is a lot of fun. I was a big fan of it before and then I went on it to promote Episodes and did a good time in the car. I set the record'
Host Chris Evans has defended the show and argued that overnight viewing figures 'have never been less relevant'
'Overnight television viewing figures for Top Gear have never been less relevant'
The episode was down a third on last week's launch
Evans' co-host Matt LeBlanc has so far proved more popular with viewers
One audience member claimed: 'It sounded as though we were in fits of hysterics'
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'In many respects it was a pale imitation of the previous hosts' popular show, and in others it was a very enjoyable programme about cars'
The most surprisingly common complaint amongst viewers? Things got just a little too loud to be bearable
Do they really think there is anything novel about taking the mickey out of Reliant three-wheelers?