In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.
When tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed® and Synchrony® both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed.
A prerequisite for most current plant genetic engineering procedures is the ability to produce transgenic plants. The process of producing transgenic plants often requires effective means for identifying and selecting transgenic cells and tissues. An important method of soybean regeneration is somatic embryogenesis. Through somatic embryogenesis, genetic engineering of soybean has proved to be a powerful technique for improving seed compositions including the oil for enhanced edible and industrial purposes [1–5]. Somatic embryogenesis (SE) is the process whereby embryos develop from either microspores or somatic tissues. In contrast to the cotyledonary node or other adventitious (non-embryogenic) regeneration systems, the somatic embryo based system appears to be mostly derived from single cells in the epidermal layers of the primary somatic embryos which increases the chances of obtaining non-chimeric regenerated plants[6, 7]. Another potential advantage of the somatic embryos (SE) system is that they are good targets in many cases of seed specific traits since they can be analyzed at the mature soybean somatic embryo stage prior to the zygotic embryonic stage, thus saving labor and time. Embryogenic tissues can be proliferated by subculture on solid proliferation (MSD20) medium or liquid suspension culture medium[8, 9]. One of the well-established soybean transformation procedures is the particle delivery system (gene gun) bombardment of somatic embryos [10–12]. Christou et al.  were the pioneers in the area of biolistic transformation of soybeans using immature seed meristems. The bombarded SEs are selected with molecules that can be inactivated by genes encoded on the introduced DNA. A selection agent that has been used successfully is the antibiotic hygromycin and this has become the standard for selection of soybean SE. However, the presence of antibiotic resistance genes in food may not be desirable due to the potential incorporation of such genes by human pathogens. There would not be selective pressure for horizontal transfer  of genes such as the bacterial DHPS reported here to human pathogens. Herbicide resistance genes may be of less health concern although they can be transferred to wild plant relatives including weeds potentially reducing the efficacy of weed control by the herbicides. Few studies have been reported on using herbicide selectable agents/markers like glyphosate, glufosinate, and ALS herbicides for selecting soybean somatic embryos. However, herbicides such as glyphosate and glufosinate have been extensively used in the transformation and selection of several crops such as maize, tobacco, wheat, rice, alfalfa, etc. and soybeans with the cotyledonary node adventitious regeneration system . Aragão, Rech and co-workers [16, 17] report on use of a modified acetohydroxy acid synthase gene to bombard embryonic axes and select soybeans transformed by a cotyledonary node system with the ALS inhibiting imidazolinone herbicide, Imazapyr.
Glyphosate is a herbicide that has been evaluated in this study as a potential selection agent. Glyphosate has been proven to be a potent broad-spectrum herbicide that interferes with the biosynthesis of aromatic amino acids by specifically inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (the sixth enzyme) of the shikimate pathway.
Herbicidal compounds that inhibit acetolactate synthase (ALS) which is also called acetohydroxy acid synthase (AHAS) the first enzyme involved in the biosynthesis of the branch-chain amino acids thereby resulting in the death of the selected plant tissues [18–20] have also been evaluated in this study.
Phosphinothricin (PPT) ammonium or L-glufosinate ammonium, the active component of the herbicide Liberty® also called "basta", is a structural analogue of glutamate. As a consequence, PPT acts as a glutamate analogue and inhibits glutamine synthetase leading to plant cell death [21–23]. The bar gene has been used as the selectable marker coupled with glufosinate as a selection agent with the Agrobacterium tumefaciens-cotyledonary node transformation system for soybeans  succeeded in germ-line transformation events at frequencies up to 3% using a selection regime of 5 mg/L glufosinate during the shoot initiation stage and 2 mg/L during shoot elongation with soybean cotyledonary nodes transformed with Agrobacterium. Simmonds and Donaldson  report on the selection of two transgenic soybean lines by selecting somatic embryo cultures expressing a phosphinothricin N-acetyltransferase gene with glufosinate.
Some alternate selectable markers can also be chosen from the pool of information on other amino acid biosynthetic pathways that are available. One such example is the regulatory enzyme aspartate kinase (AK) of the aspartate family pathway that leads to the synthesis of lysine, threonine, methionine and isoleucine and that can be a point of feedback regulation (Figure 1). This pathway is regulated by several feedback inhibition loops . AK consists of several isozymes that are feedback inhibited by lysine and threonine [25, 26]. Treatment with millimolar concentrations of lysine plus threonine (LT) strongly inhibit the growth of a wide range of plant species [25, 27–29] due to inhibition of AK activity causing starvation for methionine [25, 27–29] Mutants selected for resistance to LT possess AK genes encoding desensitized enzymes.
Dihydrodipicolinate synthase (DHPS) is another important regulatory enzyme of this pathway that is extremely sensitive to feedback inhibition by lysine and is the major limiting factor for the synthesis of this amino acid. The relatively poor efficiency of lysine synthesis makes plants generally highly sensitive to the toxic lysine analog, S-2 aminoethyl L-cysteine (AEC), which competes with lysine for incorporation into proteins [28, 30, 31]. Only lysine overproducing plants, or plants with defective uptake of this analog, are resistant to AEC. The use of desensitized E. coli AK and E. coli DHPS insensitive to lysine (I50 being 1 mM lysine) as a plant selection agent has been reported for potatoes [25, 32].
A diagram of the aspartate-family biosynthesis pathway. All the key enzymes and their products are indicated. Curved arrows represent feedback inhibition by the end product amino acids. AK: aspartate kinase; DHPS: dihydrodipicolinate synthase; HSD: homoserine dehydrogenase; TDH: threonine dehydrogenase.
The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin. Given the concerns regarding glyphosate tolerance being developed by weeds Behrens et al.  alternate selection systems which can be developed into potential herbicides is a future necessity Our results indicate that glufosinate, (isopropylamine-glyphosate) (Roundup®), AEC and the ALS inhibitors Exceed® and Synchrony® could be useful as selection agents for soybean somatic embryos. However, lysine and threonine were found to be poor selection agents. We were successful in developing a novel non-antibiotic system to select transgenic soybean somatic embryos using the E. coli dapA gene. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup®), AEC and the ALS inhibitors Exceed® and Synchrony® against different tissues of soybean.
Soybeans [Glycine max (L.) Merrill cv. 'Jack'] were grown in the greenhouse at University of Kentucky, Lexington under a 16 hr photoperiod. Pods with immature seeds were surface sterilized by immersing for 30 sec in 70% 2-iso-propyl alcohol followed by a 10 min immersion in 25% of 10% commercial bleach (1.5% hypochlorite) with a few drops of Liquinox (detergent) per L. The pods were then rinsed three times in sterile water for 5 min each time. Immature seeds 3-6 mm in length were removed from the pods. The hilum-side containing the embryonic axis was cut off and discarded. Then the two cotyledons were pushed out from the seed coat, separated and placed with the abaxial side (round side) down on MSD40 medium [12, 34]. Cultures were then incubated at 25°C at a 23 hr photoperiod and low light intensity, 5-10 μEm-2s-1. Globular staged somatic embryo (SS embryos) clusters were harvested from the explant tissues 4 to 6 weeks after induction and then placed on MSD20 solid medium  for a period of one month for proliferation. Embryogenic tissues were then transferred from MSD20 to FNL (Finer and Nagasawa "lite") "liquid medium"  for further proliferation. Suspension cultures were agitated at 100 rpm and maintained with a 2 week subculture period at 25°C with a 23 hr photoperiod. For each of these experiments conducted, 200 mg of globular stage soybean somatic embryos were used as starting materials in 125 mL Erlenmeyer flasks containing 30 mL of FNL (Finer Lite) culture medium.
The growth of the somatic embryos was tested at different media concentration levels to determine the concentration that reduced the growth of the somatic embryos most. Normal FNL medium was used either at normal full concentration (where indicated) or at 1/5 concentration with or without asparagine. 1/5 FNL medium with asparagine will be referred to as 1/5 normal FNL medium (Figure 2). About 200 mg of globular somatic embryo cultures (in triplicate) were used as starting materials in 125 mL Erlenmeyer flasks containing 30 mL of culture media (full concentration, 1/5 concentration with asparagine and 1/5 concentration without asparagine). No difference in growth was observed in full-strength medium with and without asparagine so for further studies were conducted in full-strength medium without asparagine. For each type of medium, a sample in triplicate (as control) was set and transferred twice (every 15 days) into fresh medium. At the end of this period, the somatic embryos were drained and excess moisture removed and weighed to determine the rate of growth in each culture medium. The average weight of soybean somatic embryos from each culture medium was then recorded after 45 days.
Along with the liquid medium (FNL) selection we also tried the solid (D20) medium selection for soybean somatic embryos for LT and AEC. It has been a common observation among the soybean somatic embryo transformation scientists that while solid medium selection is relatively consistent liquid medium based selection is more rapid (personal communication with John Finer). To select the soybean somatic embryos (SSE) on solid medium, non-transformed globular stage SSE were plated on D20 medium  with different concentrations of AEC and LT. The concentration at which the SS embryos turned brown and stopped any further proliferation was determined.
The coding sequence of E. coli dapA (; Genbank accession number M12844) was amplified from DH5α cells using the primers 5'GGC GCC ATG TTC ACG GGA AGT3'and 5'TCT AGA TTA CAG CAA ACC GGC ATG3' for the sense and antisense strands. A Kas I restriction site was added to the forward primer while the reverse primer was designed with an Xba I site. A pea (Pisum sativum L.) rbcs (Rubisco small subunit) chloroplast transit peptide (TP)  was amplified from pBluescript vector containing TP using the forward 5'AAG CTT ATG GCT TCT ATG ATA TCC3' primer and a reverse 5'GGC GCC GCA CTT TAC TCT TCC ACC3' primer with Hind III and Kas I restriction sites. A Hind III/Kas I digested pea rbcS transit peptide was ligated to Xba I/Kas I digested dapA DNA. The ligated product was amplified using the transit peptide forward and dapA reverse primers and cloned into the PGEM T-vector. The product was sequenced using T7 and SP6 sequencing primers to confirm the ligated product. A 1.1 kbp Hind III/Xba I digested fragment was cloned into a pKYLX71 vector  digested with the same enzymes to insert the transit peptide fused dapA under the CaMV 35S promoter. The whole cassette was digested with EcoR I/Pst I and ligated to pCambia 1201 (Genbank accession number AF234293) digested with the same restriction enzymes. Prior to this ligation the hpt gene (conferring hygromycin resistance) from pCambia 1201 was removed by digesting with Xho I and self ligating the plasmid.
Slightly mashed green embryo clumps were placed in the center of a moist filter paper in sterile petri plates (approximately 100 to 150 mg of somatic embryos per plate) and partly dried in a laminar flow hood for 15 min prior to bombardment. Transformation was carried out via particle bombardment with a gene gun (DuPont PDS1000; Bio-Rad Laboratories, Hercules, and CA) by gold/DNA micro projectile preparations as described by ., for nine shots, 25 μg of DNA was used to coat 7.5 mg of 0.6 μm gold spheres. Cultures were bombarded at 10,687 kPa (1,550 psi) helium gas pressure under a 91 kPa (27 in of Hg) vacuum, at a shooting distance of 11 cm from the rupture disk to target tissue. Immediately after bombardment, embryogenic cultures were placed on D20 proliferation medium without any selection agent for seven days.
Bombarded globular soybean somatic embryos (SSE) that were cultured on D20 were transferred (approximately 100 clumps of 0.3-0.4 cm diameter per plate) onto selection medium containing 5 mM AEC. After 3-4 weeks, visibly growing clumps were moved to fresh selection medium. After approximately 12 weeks, AEC resistant SSE clumps (masses) that appear green in color were transferred to FN lite  liquid proliferation medium. For maturation, the AEC resistant embryogenic clusters were transferred from FN Lite into 500-mL Erlenmeyer flasks (5 clusters per flask) containing 100 mL of liquid MS0M3  medium. Prior to transfer into a flask, each embryogenic cluster was gently pressed with a spatula to partially separate the individual globular-stage embryos. At four weeks, embryos were checked with glucuronidase (GUS) stain (Jefferson et al., 1987) Some of the embryos were desiccated as described by . Ten to 12 matured embryos were placed in a 100 × 15 mm Petri dish and sealed with Nescofilm. To allow gradual desiccation of embryos over a period of 5-7 days, a small piece (approximately 1 cm3) of MS0 medium was placed in the middle of the plate away from the embryos. Desiccated embryos were germinated on 1/5th MS medium.
Leaves and flowers of transgenic plants were placed overnight in vials containing the GUS assay buffer [50 mM Na-phosphate, 500 mM potassium ferrocyanide, 10 mM EDTA, 1 mM X-gluc (5-bromo-4chloro-indolyl-glucuronide) and 0.05% Triton X-100, pH 7.0] and incubated overnight at 37°C . The assay buffer was decanted and the tissues were bleached in 95% EtOH and photographed.
Genomic DNA was isolated from matured soybean somatic embryos  by homogenizing 8-10 embryos in 1 ml of extraction buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.5 M NaCl, 0.5% [wt/vol] sodium dodecyl sulfate [SDS], and 0.5% [vol/vol] β-mercaptoethanol). The homogenate was extracted with 500 μl of a phenol/chloroform/isoamyl alcohol mixture (25:24:1) and centrifuged at 12,000 × g for 5 min. The aqueous phase was collected, mixed with 1 μl of 10 mg/ml of RNase A, and incubated at room temperature for 20 min. The samples were re-extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), followed by two re-extractions with chloroform/isoamyl alcohol (24:1). DNA was precipitated by centrifugation after adding 2.5 volumes of ethanol. Precipitated DNA was washed with 70% ethanol, dried, and resuspended in 100 μl of sterile water.
100 ng of genomic DNA was used to amplify the dapA and lysC genes from transgenic tissues of soybean in a polymerase chain reaction. The primers used for dapA amplification were as follow: 5'GGC GCC ATG TTC ACG GGA AGT3' for the forward primer and 5'TCT AGA TTA CAG CAA ACC GGC ATG3' for the reverse primer. The PCR conditions were 94°C for 2 minutes; 30 cycles at 94°C, 30 seconds; 55°C, 30 seconds; 68°C, 1:30 minutes and a final extension at 68°C for 8 minutes.
RNA was isolated using the Trizol Reagent (Life Technologies, GibcoBrl). Soybean somatic embryos of each individual line that were grown for 3 weeks in maturation medium were homogenized in 1 mL Trizol reagent, transferred into microcentifuge tubes, and incubated for 5 minutes at room temperature. 0.2 mL chloroform per 1 mL of Trizol was added; tubes were vigorously shaken for 15 seconds and incubated at room temperature for 3 minutes. Samples were centrifuged at 12,000 × g for 15 minutes at 2 to 8°C. The upper aqueous phase containing RNA was collected in fresh tubes; RNA was precipitated with 0.5 mL of isopropyl alcohol and samples incubated at room temperature for 10 minutes. Samples were centrifuged at 12,000 × g for 10 minutes, and the RNA pellet was washed once with 1 mL of 75% ethanol. Samples were vortexed and centrifuged at 7,500 × g for 5 minutes. The RNA pellet was air-dried, then dissolved in RNase-free water and incubated for 10 minutes at 60°C. The concentration of RNA was estimated by reading the absorbance at 260 nm. The 260/280 ratio of the RNA was 1.9 to 2.0. The quality of RNA was checked using 1.2% agarose gels. Samples were stored at -80°C until used.
Total RNA extracted from the leaves of the AEC resistant soybean somatic embryos was used to amplify dapA cDNA. Reverse transcription was performed for the synthesis of the first-strand cDNA using oligo dT as prescribed by manufacturers of the Kit (Sigma-Aldrich Corporation, St. Louis, MO, USA) in a 20 μL reaction at 48°C for 45 minutes. A 2 μL aliquot of the RT reaction was used for PCR with the following profile: 94°C for 2 minutes; 30 cycles at 94°C, 30 seconds; 55°C, 30 seconds; 68°C, 1:30 minutes and a final extension at 68°C for 8 minutes. The primers used were the same as used in PCR reaction described for transgene amplification.
Exceed or 3-[4,5-bis (difluoromethoxy)-pyrimidin-2-yl]-1-(2-methoxycarbonyl-phenylsulfonyl) urea is a combination of two different compounds (Primisulfuron methyl 2-[[4,6-bis (difluoromethoxy)-2-pyrimidinyl]amino]sulfonyl]benzoate and Prosulfuron 1-(4-methoxy-6-methyl-triazin-2-yl)-3-[2-(3,3,3-trifluoropropyl)-phenylsulfonyl]-urea) that are blended together; and Synchrony is also a combination of two different chemicals or compounds (Thifensulfuron methyl 3-[[[(4-methoxy-6-methyl-1,3-5-triazin-2-yl) amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate and Chlorimuron ethyl 2-[[[(4-chloro-6-methoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] benzoate) that are blended together .
Roundup® which contains N-phosphonomethyl-glycine predominantly in the form of the isopropyl amine salt at 1.92% is a herbicide known to affect EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) pathway was tested for selection.
For total lysine analysis, 100 mg of ground samples were homogenized in 2.0 mL Milli-Q (Millipore, Bedford, MA) water in a 16 × 100 mm glass tube with Brinkman (Westbury, NJ) Polytron homogenizer equipped with a 7-mm generator for 1 min at a speed setting of 5. Five μL of homogenates were transferred to 6 × 50 mm glass tubes that had been acid-washed with 6 N HCl for 24 hr. Samples were then lyophilized. Gas-phase hydrolysis was performed according to  with modifications. The hydrolyses were done in duplicate. Lyophilized samples were placed in each evacuated glass container with 200 μL of 6 N HCl and 10% phenol in the bottom. Evacuation in 25-30 in-Hg pressure alternated with nitrogen flushing was performed at least three times. Samples were hydrolyzed at 110°C in vacuo for 24 hrs. After hydrolysis, samples were dried under a vacuum and stored in a freezer until derivatization and HPLC analysis. For free lysine analysis, 100 mg of ground samples were extracted in 1.0 mL of 50 mM phosphate buffer, pH 8.0, for 1 hr. Samples were centrifuged at 15,000 × g for 10 min. Aliquots of 5 μL of the supernatants were lyophilized and used for derivatization and HPLC analysis in the same manner as for total lysine analyses. This also was done in duplicate.
All samples including amino acid standards were neutralized by adding 20 μL of a 2:2:1 mixture of ethanol:water: Triethylamine (TEA) (v/v), and mixing well with a vortex. They were dried under vacuum. Derivatization was performed by adding 20 μL of mixture of 7:1:1:1 ethanol:water:TEA: Phenylisothiocyanate (PITC) (v/v), and mixing well by vortexing. The reaction between PITC and the hydrolysate to produce phenylthiocarbamyl (PTC) amino acids was allowed to complete for 20 min at room temperature. Samples were then completely dried under vacuum. PTC amino acids in each sample and standards were dissolved by vortex mixing with 500 μL of 5 mM Na2HPO4 buffer, pH 7.4 containing 5% acetonitrile. It was then filtered through 0.2 μm membrane. Reconstitution of samples was done one at a time due to the PTC amino acid sensitivity to light and ambient temperature. Ten μL of samples were injected and analyzed with a Hewlett-Packard (Agilent Technologies, Wilmington, DE) series 1050 HPLC system equipped with column heater, autosampler, variable wavelength detector series 1100, and ChemStation data acquisition software controller. The reverse-phase column used was a Pico-Tag (Waters Corp., Milford, MA) with an in-line column filter. The column temperature was maintained at 38°C. The PTC amino acids were separated and eluted by a gradient resulting from mixing buffers A and B. Buffer A consisted of 150 mM CH3COONa.3H2O, 0.05% TEA, and 6% acetonitrile, pH 6.4. Buffer B consisted of 6:4 acetonitrile:Milli-Q water (v/v). Both buffers were sparged with helium for 10 min before use. The flow rate was 1 mL/min throughout, and the gradient consisted of the following profiles: 100% A at start, 80% A and 20% B at 5.5 min, 54% A and 46% B at 10 min, 100% B at 10.5 to 12.5 min, 100% A at 13 min. The PTC amino acids eluted from column was detected at 254 nm and recorded. The column was regenerated and equilibrated with buffer A for 10 min. A new and freshly reconstituted sample was injected and analyzed every 23 min.
Two different types of ALS inhibitors, Exceed and Synchrony were tested. Both herbicides were found to be heat labile and work better in the lower nutrient medium rather than in the normal FNL medium (Figure 3).
Roundup® was found to be effective at the concentration level of around 730 μM (123 mg/L) in the full medium with or without asparagine. It works even better at a concentration as low as 245 μM (41.4 mg/L) in the 1/5 medium minus asparagine and at the concentration level of 400 μM (or 68 mg/L) in the 1/5 normal FNL medium. The active ingredient of Roundup (N- (phosphonomethyl)-glycine) from Sigma (St. Louis, MO), needed to be used at a concentration of at least 980 μM (165.6 mg/L) and a longer time was required to inhibit the growth of the tissue.
This selection was first carried out in normal FNL medium. The following concentrations (5, 10, 15, 20, 30, and 35 μg/mL) or (25.3, 50.5; 75.8; 101; 151.5 and 176.7 μM) were used with and without asparagine (Figure 4). One week after incubation, between 45 - 50% of the initial somatic embryo tissues had lost their green color and developed a brownish color. The growth of more than 95% of the somatic embryo tissues was inhibited three weeks later and the growth of the remaining 5% was also inhibited after an additional two weeks. The selection in 1/5 concentration FNL medium without asparagine was then carried out with the following concentration: 0.16, 0.33, and 0.5 μg/mL or (0.81; 1.67; and 2.5 μM) (Figure 4). After two transfers lasting an average of 15 days (between each), the growth of all the somatic embryo tissues was found inhibited. The only difference was the growth inhibition of the embryos started earlier (during the first 7 days after selection), while in the normal FNL medium, the inhibition started to be noticeable only during the second week.
Selection with hygromycin was first conducted in full medium concentrations of 5 to 40 μg/mL or (10 to 76 μM). The treatments with 20 μg/mL (40 μM) and above were found to be effective after two transfers of 15 days each (100% growth inhibition) (Figure 4). The selection was also done in 1/5 normal FNL medium and 1/5 FNL medium without asparagine. In the selection medium without asparagine, the growth of 95% of the materials was inhibited during the first week of selection at all the treatment levels. In comparison with the 1/5 full selection medium somatic embryo tissue growth was inhibited at treatment levels 20 μg/mL (38 μM) and higher (Figure 4). Selection in 1/5 medium without asparagine was complete in less than two weeks while with the full medium it took more than three weeks.
The selection of soybean somatic embryos with lysine and threonine was initiated in normal FNL medium without asparagine at concentrations of 2.5, 5 and 10 mM (0.75, 1.6 and 3.2 mg/mL). This experiment lasted for a period of more than five months with transfers to fresh medium every 15 days. At the end of the second transfer (i.e. after a month), signs of growth inhibition were observed with 10 - 20% of the embryos.
After a period of 120 days, about 70% of the embryos growth was inhibited and the remaining were still green (Figure 3). The preliminary experiments with LT in regular FNL medium never resulted in the growth inhibition of 100% tissues (30-40% of the tissues remained green even after 4 months). The failure of the LT to inhibit the growth of the SSE led us to check whether these compounds will be more effective in reduced strength FNL medium. However, the same concentration of 2,4-D as in the normal FNL medium was kept constant to help maintain embryo proliferation. The 1/5 FNL medium without asparagine was selected to carry out subsequent experiments (Figure 3). Thus, the LT experiment when done in this new environment with respect to the same conditions (surfactant and without asparagine) appeared to be more effective than when it was conducted in the normal FNL medium. However, even in this new environment, we observed that after a 4 months period, only about 70-80% of the embryos stopped growing and the other 20-30% remained green.
At first, the selection was conducted with the lysine analog, AEC, on FNL medium without asparagine but with the addition of surfactant (methylated soybean oil). All the materials stopped growing after seventeen weeks (~4 months) and with a total of 8 transfers. The requirement of a high amount (5 mM) and long duration led to the analysis to determine whether this chemical is more effective in reduced strength 1/5 normal FNL medium. In comparison to LT, AEC appeared to be a better candidate as an alternative selection agent. With the 1/5 medium, all of the somatic embryos stopped growing after 13 weeks at the 5, 7.5 and 10 mM or (1, 1.5, and 2 mg/mL) treatments (Figure 3) and growth was more than 95% inhibited at the 2.5 mM treatment which was not effective in the full concentration medium.
We examined the relationship between the dose of LT or AEC administered and growth inhibition to test whether soybean can be selected with LT and AEC on plates. Seed germination results showed that these compounds can inhibit germination at certain concentrations. Germination of soybean seeds on MS plates containing 0-5 mM AEC, showed that 1.5 mM AEC completely inhibited germination (Figure 5). Similarly, 10 mM LT completely inhibited soybean seed germination (Figure 6). Interestingly, only 250 μM AEC was required to inhibit germination of matured soybean somatic embryos while 5 mM LT was required for the same purpose (Figure 7 &8). 5 mM AEC totally prevented the proliferation of globular stage somatic embryos compared to control, which are the target for biolistic transformation (Figure 9) on D20 plates. Since LT, even at 10 mM did not inhibit somatic embryo proliferation, soybean transformation with lysine insensitive AK was not performed, instead only the dapA construct was used for soybean somatic embryo transformation. The lysine insensitive AK was used by Perl et al.  as a selection marker to select potato. The potato shoot and root regeneration was successfully inhibited by LT in the selection medium. The over expression of a lysine insensitive AK prevented the cell death normally caused by the deprivation of methionine in such cases. Since soybean somatic embryos are never totally inhibited by LT the initial idea of using lysine insensitive AK as a SSE selection marker was shelved. The dapA transformed AEC resistant putative transgenic somatic embryos on D20 medium are shown in (data not shown). However, we never obtained any positive transformants in liquid medium selection. These embryos were further proliferated, matured, and analyzed by GUS staining. All of the lines analyzed were GUS positive. These lines have also been found to have the introduced dapA gene. To test whether the introduced gene was being transcribed total RNA from soybean somatic embryos was subjected to RT-PCR with dapA Figure 10). Soybean dapA transgenic lines were obtained from three different independent experiment using both AEC and hygromycin selection systems. Approximately 100 masses (200-250 mg) of 0.3-0.4 cm diameter per plate were used for one shot and a total of 9 shots per experiment. 5-7 transgenic events were obtained per experiment with the AEC system and these results are also similar to results obtained with solid medium hygromycin selection system. The transgenic T1 and vector control plants were taken to the greenhouse and grown under the same condition as the parent Jack. Different tissues of transgenic plants were analyzed for GUS activity (Figure 11). The PCR results of dapA transgenics showed a 3:1 segregation of the introduced dapA gene in the T2 progeny. No difference in the growth pattern and seed development was seen between transgenic and wild type plants.
Inhibition of soybean seed germination at different AEC concentrations; A. 2.5 mM, B. 5 mM, C. 0 mM, D. 500 μM, E. 1.5 mM.
Inhibition of soybean seed germination at different lysine + threonine (LT) concentrations; A. 10 mM, B. 0 mM, C. 5 mM.
Inhibition of soybean mature somatic embryo germination at different AEC (S-(2aminoethyl)-L-cysteine); A. 0 μM, B. 100 μM, C. 250 μM, D. 500 μM AEC.
Inhibition of soybean mature somatic embryo germination at different LT (Lysine + Threonine) concentrations, Control (A), 5 mM (B) and 10 mM (C).
Proliferation inhibition of non transformed soybean globular embryos at different AEC (S-(2aminoethyl)-L-cysteine) concentrations. Concentrations by row from top to bottom of AEC are (in mM) 0, 0.5, 1, 1.5, 2.5 and 5 are represented by A, B, C, D, E and F respectively.
RT-PCR of different soybean somatic embryo transformants expressing E. coli DHPS. A, B, C, D, E, and F are different transgenic lines.
GUS staining of control (A) and transgenic soybean leave discs (B).
Dap A transgenic, AEC resistant soybean plants had normal phenotypes (data not shown). No abnormalities in the flower development and seed production were noticed in the transgenic plants. When analyzed for their free and total lysine contents, no significant difference was found in the seed of T2 progeny when compared to the control and the parent Jack (Table 1).
Free and total lysine levels of Dap A transgenic, vector control (VC) and parental soybean seeds (Jack).
Cellular selection is often necessary in tissue culture and molecular biology work. Most systems use a specific dominant selectable marker to enable the recovery of transgenic tissues once a selection agent is proven to be efficient. Hygromycin is the standard selection agent for soybean somatic embryo tissues. However, possibilities exist in using alternative selection agents and markers in place of hygromycin. A potential group of herbicides, amino acids, and their analogs have been tested as possible selection agents to further improve transgenic soybean somatic embryo selection. Most herbicides act by affecting biochemical processes including photosynthesis, essential amino acid biosynthesis, and other biochemical processes that are unique to plants or distinctly different from other organisms. Resistance to herbicides or feed-back inhibition by amino acids which results from one or more mutations of the genes involved in these pathways can provide alternative means of selection.
The selection medium salts were diluted and tested at different concentration levels of nutrients with and without asparagine: from full strength to 1/2, 1/3, 1/4, 1/5 and 1/10 concentrations (data was not shown). The concentration of 2,4-D was kept constant in all these different selection media. At the 1/10 medium concentration, the soybean SE stopped growing completely and growth inhibition occurred within the first week presumably from starvation. The minimum selection medium concentration that sustains an acceptable growth of the SSE (increasing by almost 50% relative to full-strength media) was found to be at 1/5 of its normal concentration (Figure 2).
Most of the selection agents tested in this study were found to be more effective in the 1/5 culture medium compared to the full-strength medium. Of the different compounds tested, in either normal FNL or 1/5 normal FNL medium, Liberty® (glufosinate) was found to be effective at the lowest concentration level 2.5 μg/mL. Using the cotyledonary node method of soybean transformation Zhang et al.  observed 3-5 μg/ml glufosinate was sufficient to prevent shoot initiation. These amounts are similar to the concentrations of glufosinate required to inhibit SSE proliferation.
In the case of glyphosate, our experiments showed that the salt form of the chemical works better than the acid form. Glyphosate, in the salt form, was found to work even better in 1/5 selection medium with and without asparagine and at a concentration as low as 400 and 240 μM when compared to the full concentration medium (730 μM). This could be due to the relative insolubility of the compound in water as well as the lack of surfactant (s) found with the Roundup Ultra formulation (data not shown).
Four classes of ALS-inhibiting herbicides including sulfonylureas, imidazolinones, triazolopyrimidines, and pyrimidinyl thiobenzoates are found to inhibit the first enzyme involved in the biosynthesis of valine, leucine, and isoleucine. In our experiments, two types of sulfonylurea herbicides were used: Exceed and Synchrony. Although formulated for two different crops, Exceed® for corn and Synchrony® for soybean , we found that both ALS-inhibiting herbicides tested inhibited soybean somatic embryo growth after a period of one month. However, Exceed® was found to be more effective than Synchrony®. Furthermore, the sequence of phytotoxic mechanisms with these chemicals remains unclear . The growth inhibition of somatic cells by these compounds (Exceed® and Synchrony®) could be a result of any of the following factors: direct depletion of the end products, depletion of intermediates of the pathway for some critical processes, or from buildup of a toxic intermediate . In our experiments we found that the two ALS -inhibiting herbicides (Exceed and Synchrony) were found to be less effective in normal FNL medium than in the reduced concentration medium.
The lysine analog AEC and LT have been used as agents for crop improvement since the 1980's . These chemicals were used to select tissues and plants that overproduce either threonine or lysine. Compared to the other lysine analogues AEC was found to be more effective in inhibiting plant growth [31, 44]. The effect of lysine on the two key enzymes, AK and DHDPS, can be mimicked by AEC in higher plants [31, 44–46].
In our study, AEC proved to be effective in 1/5 normal FNL medium and in a shorter period of time. However, it was found to be as effective when used in 1/5 D20 or full strength D20 solid media (Figure 9). In this experiment, lysine-threonine was observed to be less effective with the soybean somatic embryos.
Our results showed that different concentrations of LT and AEC inhibit different tissues from soybean to differing degrees (Figures 5, 6, 7, 8, 9 and 11). These results are more pronounced with AEC. Our selection results also showed that concentrations of AEC needed for inhibiting soybean tissue growth varied greatly. 2.5 mM AEC was required for the inhibition of soybean seed germination while 5 mM was best for inhibiting somatic embryo proliferation (Figures 5 &9). Interestingly, only 250 μM was needed for the inhibition of soybean somatic embryo germination (Figure 7). This shows that different soybean tissues responded to different amounts of AEC. Our results with soybean were in conformity with some of the AEC concentrations reported in mutagenic studies. Depending on the plant species, prior studies indicate that the AEC concentration for growth inhibition varied: 1.5 mM and 1 mM for rice calli and rice anther-derived cell lines and 1 mM for sweet potato shoot inhibition; 0.25 mM for seeds of Arabidopsis thaliana ; and 125 μM for potato shoot formation . Growth inhibition of all of these tissues at different concentrations is an indication of possible existence of a differential metabolism mechanism of AEC depending on their metabolic state. Similar results were reported by  for the mutagenic agent sodium azide. They concluded that sodium azide was differentially metabolized by maize callus and germinating maize embryos using different detoxification and repair mechanisms. The higher sensitivity shown by some tissues like germinating soybean somatic embryos may be the result of more than one factor. It is known that AEC inhibits AK and DHPS . AEC was also found to inhibit cell growth irreversibly by competing with lysine for incorporation into protein, leading to an altered protein processing and folding, and may also account for its cytotoxic effects [50, 51].
All soybean somatic embryos selected with AEC were GUS and PCR positive. Our selection results suggest that AEC and the dapA gene can make a good selection system for proliferating soybean somatic embryos. Efficiency of transformation with the dapA gene and selection with AEC in soybeans is similar to that of hygromycin (data not shown), where 3-5 clumps is positively transformed for every 9 shots. Due to the use of solid selection medium, the growth of the embryos with the AEC selection system was a little slower compared to hygromycin liquid selection system. This is consistent with Samoylov et al. . On solid D20 selection medium 8-10 weeks led to the identification of AEC resistant green transgenic clumps, whereas 6-8 weeks were required for hygromycin selection. However, it is important to point out that AEC selection of transgenic soybean somatic embryos in liquid medium is not satisfactory compared to solid medium although the kill curve studies clearly showed lesser amounts of AEC are needed for growth inhibition in liquid medium (Figure 3). The opposite is seen with hygromycin selection in which selection on solid medium is much slower (~5 months) than with liquid medium (10-12 weeks) (at least from our observations). To our knowledge, the only plant species previously selected using this dapA selection system was potato . Perl et al.  have shown that shoot and root generation of control potato tubers were completely inhibited at 0.1 and 0.15 mM concentrations of AEC. The dapA transgenic plants showed resistance at these concentrations. However, at higher concentrations of AEC (0.3 mM) transgenic plants were also susceptible. No AEC selection is reported for any other plants expressing dapA.
For biolistic experiments on an average for a single shot 25 globular masses were used. When slightly mashed and matured each clump can give rise to ~50 individual matured embryos. The total number of individual matured embryos obtained from 25 clumps will be ~1000. Each experiment generally consists of 9 such shots by gene gun totaling ~9000 embryos. With AEC selection on the solid medium ~5 transgenic clumps for 9000 embryos were recovered ≈0.05% transformation efficiency. As mentioned earlier a similar number of transgenic plants were obtained with hygromycin as the selection agent. Zhang et al.  using basta for selection for Agrobacterium-mediated soybean cotyledonary node transformation observed a 0 to 3% transformation efficiency based on GUS expression. With the same method using hygromycin as a selection marker Olhoft et al.  reported a 0.7% efficiency. They improved the transformation efficiency to 16.4% by using a super virulent strain of Agrobacterium in presence of thiol compounds in the medium. Compared to the Agrobacterium-based cotyledon node method the biolistic transformation of somatic embryos is low. However, advantages including early testing of seed-specific traits and proliferation of large number of transformed embryos that can ensure the establishment of plants with more surety can balance the drawbacks. Arago et al.  reported a transformation efficiency of 0.7% when Phaseolus vulgaris embryonic axes were bombarded.
When chimeric dapA and lysC genes were introduced into tobacco under a CaMV 35S promoter an increase in free lysine content was reported in leaf tissues. The authors speculated the use of tissue specific promoters to increase lysine content in the seeds [54, 55]. In the later studies Karchi et al., 1994 showed the role of lysine catabolism in the prevention of lysine accumulation tobacco seeds when dapA was expressed under a seed-specific promoter. Falco et al.  increased the soybean and canola seed free lysine and total lysine content by expressing Corynebacterium dapA gene under a seed-specific promoter. The unaltered seed lysine levels in the present study might be the result of the promoter used to express the dapA gene. Since the primary objective of this study was to develop dapA as a selection marker for soybean somatic embryos a 35S promoter was used.
The inhibition of plant growth by LT is reported to be due to poor methionine biosynthesis . It was reported in the literature that 2-3 mM LT is inhibitory to seed germination . Our soybean seed germination results with LT confirm this observation. However, for inhibition of soybean seeds, a little higher concentration of LT is needed. However, the growth of soybean somatic embryos was never totally inhibited even at 10 mM LT.
One of the primary concerns of genetically modified (GM) crops is that antibiotic resistant genes could be transferred to pathogenic microbes in the gastrointestinal tract or soil rendering them resistant to treatment with such antibiotics . With biolistics, the DHPS can be shot with the genes of interest, thus eliminating the need for antibiotics in the transgenic soybeans. In conclusion genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. Using this strategy one can exclude antibiotic selection by introducing the expression cassette of lysine resistant DHPS through biolistics with a cassette expressing a gene of desired trait into plants.
This work was supported by the United Soybean Board, the Kentucky Soybean Promotion Board and the Kentucky Agricultural Experiment Station. Erin Yost provided very helpful technical support.
SR conceived the idea of AEC selection of soybean somatic embryos, made the vector constructs, did the biolistics, selection of the transgenic tissue, and established T1 generation plants. LM did the kill curves, GUS analysis of transgenic material, seed chip analysis of the T3 generation to find out the dapA segregation ratio. RP did the RT-PCR and PCR of the transgenic plants to establish transgenic nature of the material. MM did the tobacco transformation that was used as control and helped with the photographs. Free and total lysine analysis was done by PK. The work was done in DH's lab at the University of Kentucky under his overall guidance and supervision. All authors read and approved the final manuscript.


Manasota Key is neither posh or sophisticated nor is it unstylish or simple. So, what is Manasota Key? It is an enchanting barrier island off the coast of Englewood, and the natural beauty is what makes the island so unique.
Manasota Key is known for its laid back attitude, or "Old Florida Charm." Of course, there are multi million homes on the key, but they are not exposed. They are hidden behind lush, tropical landscaping. Natural and Florida like. You won't see manicured gardens like you see on most of the other Keys.
By following the main road, which runs along the center of the island from north to south, you will see what we mean. Compared to Casey Key or Siesta Key this is a low- key affair. No ritzy entrances; everything is hidden behind a lush landscaping.
If you are particularly looking for that type of "Old Florida Charm" - there are not many places left where you can find it. Islands like Don Pedro, Little Gasparilla Island, or Useppa still breath that "Florida Charm", while Keys like Casey Key, Lido Key or Longboat Key have lost it longtime ago.
However, Manasota Key is one island you may want to consider. This is one last resort, if you are looking for a place with that laid back-Old Florida Charm.
But don't expect to get something for "nothing"'. When you buy real estate on an island, where land is scarce, you will pay according to the scarcity.
The northern and central parts of the island, located in Sarasota County, are exclusively residential. There is no commercial activity permitted. No restaurants, no shops - nothing.
The southern section of the island, which belongs to Charlotte County has some commercial activities. Quit a few low-rise condos, apartments, restaurants, bars, and small shops, like boat and jet ski rentals, are lined up along the beach. However, none of the activities really distracts from the beauty of the island. All the action appears to be in total harmony with the rest of the island.
There are 4 beaches on the key: Manasota Beach at the northern end of the island, Middle Beach in the center of the island, Englewood beach (almost at the south end of the island), and the State Park Beach at the very southern end of the island.
Manasota Key is not very wide. In certain areas the island is only a few hundred yards away from the Gulf of Mexico to the west and the Lemon Bay/Intracoastal Waterway to the east. However, that doesn't bother anybody at all.
Although Manasota Key is a popular tourist destination, residents are still able to enjoy an undisturbed way of life. As a result, the island is not nearly as touristy as its cousins, Siesta Key or Anna Maria Island, further north.
Because there is a total of four beaches on Manasota Key, which includes Englewood Beach, Stump Pass Beach, Blind Pass Beach and Manasota Beach, the crowd is spread pretty evenly. Each of these beaches has something special to offer: fishing, boating, diving, swimming, jet skiing, para-sailing, shelling, snorkeling, canoeing, kayaking, sunning, picnicking, and volleyball playing are some of the favorite activities on those beaches and the list gets longer every year. The kind of activity depends depends on where you go. The best variety of activities you will find on Englewood Beach.
Englewood Beach went through an amazing transformation. It was not like that in the past because it used to be a very "simple" beach. When we moved to Englewood in 1995 the only activity was getting a sun burn and swimming in the Gulf of Mexico. We were still happy because we didn't know better. There were not even restrooms during that time and one of the 5 parking lots was always available.
That has changed dramatically. Of all the beaches, Englewood Beach belongs to the most popular among Florida's visitors (besides the beaches of Siesta Key, of course). Now the visitors can enjoy completely remodeled boardwalks, restrooms, showers and covered barbecue spaces.
The only disadvantage is that parking is not free anymore. One hour of sunburn on the beach will cost you $.75. But what is that cost in comparison to the dermatologist's bill who treats your red skin.
If you spend weeks or months in Englewood you may want to buy a parking pass, which gives you access to all of Charlotte County beaches and boat launches, and saves you some beer money.
There are still homes and lots for sale on Manasota Key, however, expect the prices to be a lot higher compared to homes for sale on the mainland. Properties on barrier islands are always special, and when prime locations are scarce the prices are higher. Very few lots are left on the island and they come with a "prime" price. Look for yourself or call us. We can look for you.


text:
Journey to the East / Fully Booked
Journey to the East 2 / Fully Booked
Orient Express / Paris to Istanbul
Pub Ride / Dublin to Copenhagen
The Odyssey / Athens to Amsterdam
Trans-Europa / St. Petersburg to Gibraltar
Viva Italia / Rome to Rome
Bamboo Road / Taipei to Singapore
Hippie Trail / Agra to Goa
Magical Madagascar / The 8th Continent
The Road to Shangri-La / Saigon to Shangri-La
Trans-Himalaya / Srinagar to Kathmandu
Trans-Oceania / Australia & New Zealand
North American Epic / Tuktoyaktuk to Panama City
Silk Route / Beijing to Istanbul
South American Epic / Cartagena to Ushuaia
Tour d'Afrique / Cairo to Cape Town
West Africa en Vélo / Casablanca to Cape Coast
HOTEL-TO-HOTEL
TDA STARTER KIT
7 EPICS
GUEST RIDERS
TDA FOUNDATION
TOUR BLOGS
WHY TDA?
RIDER FUNDRAISING
Guest Rider Days
Einfo@tdaglobalcycling.com
T+1 416 364 8255
THE TDA EXPERIENCE /
What to Expect FAQ Ratings TDA Starter Kit 7 Epics Guest Riders Reviews
Never cycled with us before?
Try our Guest Riders Days and see what it's all about!
Have you ever considered joining one of our cycle tours but couldn't find the time or felt intimidated by the scale of our trips?
We have cycling tours in over 80 countries around the world. On each of these tours, we pass through a variety of cities and towns on six continents – so there's a pretty good chance that we have a cycle tour passing near to you.
That's why we created Guest Rider Days, so you can come and experience our cycle tours without having to commit to a longer expedition. Just a few nights, close to where you live, to get a feel for the daily routine, and more importantly, what type of people take part in these grand cycling adventures.
>>Email us to work out the details
Pick a tour
Take a look at our calendar, and our tour finder and see where our tours come nearest to where you live.
Send us an email, and tell us which tour and which dates you're interested in.
As the date of the Guest Rider Days draws near, we'll send you a few forms, and let you know where and when to meet with the group.
Then you set off with the group! Headed for new adventures with some new cycling friends.
What We Cover, What You Cover
We will carry your bags in our support vehicles and feed you most meals (breakfast, lunch, dinner) during the ride. We will also cover your camping fees and staff will be available to answer your questions and make you feel at home… on the road.
Transportation to meet us (and to return home) and hotel arrangements (and costs) are at your own expense. You will also need to come with your own bike and camping gear (where necessary). If you are staying offsite from the rest of the group, you will also need to find your own way to join the group each morning.
Guest riders may join ongoing tours for up to 3 days. Spaces are limited to 3 guest riders in any one location, so make sure to contact us well in advance. Preference will be given to those who have never been on a cycle tour with us before.
What Riders Said About Guest Rider Days…
"It was such a pleasure to join your ride from Napier to Wellington as a guest. Wonderful company superb support and a privilege to enjoy the TDA family for a bit. You have quite a package I have to say. Once again thanks for you wonderful generosity in adding me to your trip. I hope I will get to do more with you."
-Veronica, from Wellington, New Zealand
"It was a physically challenging three days but very rewarding…I have always been tempted by the rides offered by TDA and I appreciated the opportunity to get a taste of them. Am I prepared to sign up for a two month ride? Probably not, but a 1-2 week section could be in the cards."
-Ray, from Ottawa, Canada
"I very much enjoyed my 3 day trip…The organization and food for the few days I had was great and of a high standard. Obviously difficult to know what it is like to be on the full tour ( with camping ) for over 90 days but most seemed to have settled in to a good routine and were enjoying their adventure.
I will most definitely join you on a trip or section thereof soon – hopefully next year. Must just decide which one!"
-Charl, from Windhoek, Namibia
"I would like to express my gratitude to all of you that you made this possible. I had high expectations but they were exceeded: the crew was great, the food tasty, the participants very welcoming (it felt like I'd ridden with them since Athens), the beautiful scenery, it was awesome and I loved it from start to finish.
I also learned an awful lot for my future TDA trip from the conversations I had. So yes, I can say from every aspect the Guest Rider Day was very very useful."
-Hans, from the Netherlands (Guest rider on The Odyssey 2019)
We would love to introduce you to what we do and prove to you that this is not only for uber-athletes or experienced adventurers. It's for anyone willing to try something a little different, to take on a real challenge and be rewarded with tired legs and a deep sense of accomplishment. Do you think you might enjoy a couple days on the road with us?
The 2020 Tour d'Afrique
Tomorrow, a group of 23 riders, from various countries including the Netherlands, England, Canada, the United States, Brazil, New…
UPDATED January 15, 2020 BY Sarah Fonger
African Literature 301: Reading for the Tour d'Afrique - Fiction
A few days ago we published a list of our top 10 non-fiction books about Africa that we recommended…
UPDATED January 14, 2020 BY Michael Coo
The Origins of TDA Global Cycling
18. What does that number signify? At the age of 18 a person comes of age in most parts of…
UPDATED January 11, 2020 BY Henry Gold
TDA GLOBAL CYCLING
© 2020 TDA Global Cycling
205-1179 King St West
Toronto, Ontario, Canada M6K 3C5
info@tdaglobalcycling.com
TICO registration # 50023724
SHOW PRICES IN MY OWN CURRENCY
All tours are priced in either USD or EUR. This tool will show you the current equivalent cost in your own currency. Please note that if paying in a different currency, payment must be made using the official daily exchange rate at time of payment.
Sign up for our monthly newsletter and
Get free access to 101 tips
101 Tips for First Time Cycle Tourists
Thank you for signing up for our monthly newsletter
An email has been sent to you. Kindly click on the link to confirm your email address and we will immediately share our 101 Tips wth you.

redpajama_set_name: RedPajamaCommonCrawl


text:
'Almost Wholly Negative': The Ford Foundation's Appraisal of the Virginia School
Sandra J. Peart
University of Richmond - Jepson School of Leadership Studies
In 1960 the Thomas Jefferson Center [TJC] of the University of Virginia applied for a "massive" grant from the Ford Foundation. Although James Buchanan, Warren Nutter and Ronald Coase had all received grants from Ford, Ford turned down their proposal because of the Center's unified "point of view." We report on correspondence and private discussions of the events. Following the submission of their proposal, Buchanan, Nutter and then President of UVA, Edgar Shannon met with representatives of the Ford Foundation, Tom Carroll and Kermit Gordon. Buchanan concluded that the "reaction of the Ford representatives must be considered to have been almost wholly negative." The crux of the matter, in Gordon's assessment was the TJC reflected "a single 'point of view'." As the conversation unfolded, it became clear to the UVA representatives that by this the Foundation officials meant a narrow ideological perspective, one in line with Chicago-style economics. Buchanan attempted to dispel this conclusion, arguing that the program was "sufficiently broad" to "encompass wide and divergent points of view." Coase was particularly incensed by allegation of ideological narrowness since, as he explained, he had close ties to the Fabian Society Despite the attempts of both Coase and Buchanan to defend their proposal, Ford officials turned down the application and the TJC never fully recovered.
Levy, David M. and Peart, Sandra J., 'Almost Wholly Negative': The Ford Foundation's Appraisal of the Virginia School (August 22, 2014). Available at SSRN: https://ssrn.com/abstract=2485695 or http://dx.doi.org/10.2139/ssrn.2485695
University of Richmond - Jepson School of Leadership Studies ( email )
Jepson Hall


The set of 3 info panels, produced by the project for use at national and international climate change events, are available in two language editions: English/ Arabic and French/Arabic.
The content of the info panels may be updated and info panel N.3 can be customised, according to the requirements of national partners.
Each info panel is designed to be printed and assembled as a 200cm x 300cm roll-up, self-supporting panel (see demo).


text:
GAMING TREND
Steady hands required – Doctor! Doctor! Review
A frustrating kind of genius — So Many Me Extended Edition Review
Reimu, Miku, Kizuna Ai, oh my! — Groove Coaster Wai Wai Party!!!! review
Get away from her, you Switch! Alien: Isolation review
Dash for cash – Asphalt 9: Legends review
Not to toot our own horn, but Ride the Rails pre-orders are available this week, will ship early
Check out some new screenshots for Stoneshard, out next month
Celebrate the Lunar New Year in Overwatch with new rewards for a limited time
Pro VR is now even more affordable with HTC VIVE Pro price drop
The Age of Artisans is upon us! New expansion for Architects of the West Kingdom coming soon
Old school dungeon diving in New Tokyo — Operation Abyss: New Tokyo Legacy review
By Keith Schleicher
At one time to get a full RPG experience you needed to be sitting in front of a TV with a console in front of you. As technology moved forward and systems got smaller, some classic RPGs made their way to portable systems. While these portable devices have become more powerful, their adventures have become more robust as well. The RPG genre has made a bit of a renaissance on the 3DS and Vita. Experience Inc, the company behind the Vita RPG Demon Gaze, has now come out with another RPG, Operation Abyss: New Tokyo Legacy.
When first starting out Operation Abyss, I noticed the city was a bright futuristic land that could put most '80's neon lighting to shame. The characters you encounter have on bright outfits and illustrated with a level of detail that you'd find in several anime series. However, most of the interaction is with stills of the characters, and the initial interactions can become very long for the amount of exposition to the plot they actually give.
The members of your team are partially determined if you choose the Basic mode, which comes with a party of pre-determined characters, and the Classic mode which allows you to customize your entire party of six. I often just like to go with the characters created by the system, but it's nice to know that you can try to improve the party yourself by giving them tweaks suited to your play style.
If you are looking for a story other than the typical, "you wake up with no memory, meet a stranger who tells you to follow him, and later explains that you need to fight with them to save the world," then you'll be disappointed. Then again, the giant attacking crocodile being demolished in front of you in one critical blow isn't your standard fare. You'll find out that you are a part of the Code Physics Agency in charge of taking care of Variants, the monsters that populate the dungeons you'll be forced to wander through.
If you thought that the entire world would be full of Hello Kitty pink and "Happy Happy Joy Joy" explosions of wonderful happiness, then the tone in the dungeons will shock you. The dungeons themselves are dark, and the textures on them won't win any graphical achievement awards unless there's one for most retro Wolfenstein 3D look. However, this is a remake of a game from 2008 that never made its way to the states. The Variants contrast this with their hand-drawn look and a darker color palate. The forms used are representative of the Shin Megami Tensei series, having a creep factor that could cause nightmares in young children and sensitive adults.
Movement in the dungeons is similar to the old Might and Magic games. Everything is on a 90-degree angle, so maps could have been designed on graph paper. If you didn't mind travelling this way while playing M&M or the Eye of the Beholder series, then this won't be as jarring to you as people who are expecting an Elder Scrolls experience.
The combat system feels like the Wizardry games. Your party consists of six members, with the three in front typically having strong physical attacks, while the back row consists of those with talents in magical powers. When you choose to attack, you attack a group of enemies. While this speeds up the pace of going through the dungeon, it can be infuriating when you want to hit character A in the group you attack, but B or C get hit instead, leaving a fuller compliment of enemies to attack you. If you are used to being able to control all of your tactical moves, you might get frustrated.
Persona 3 had a mechanic where the party got tired the further it went into the dungeon. Operation Abyss uses a similar system, but instead of making the party tired, the enemies get stronger the further the party delves down. However, you can make a large amount of bank and EXP when the Variant levels jump higher, so a risk-reward factor needs to be factored into every move. This might not be so bad if you gained levels while inside the dungeons, but you are forced to go back to the headquarters and level up in the Medical Center and resting. This can slow down the pace of the game since you will do a lot of backtracking. While at headquarters you can reassign the team's equipment, and even try to forge new weapons. Getting the knack of customization will take some time though, as you'll encounter enemies with different strengths and weaknesses that need to be accounted for.
The design of the dungeons is quite confusing, even with the AutoMap. Some of these dungeons end up becoming combobulated into each other because there isn't much to differentiate them. The same monsters combat you repeatedly, making it feel like you are grinding instead of exploring new areas.
It's been a while since I played a sci-fi RPG like this, and it reminded me of Shin Megami Tensei: Strange Journey. I appreciated the colorful characters and unique enemies, but the level design leaves something to be desired. Worse options could be chosen these days, and while it's not for everyone, fans of the genre looking for something new on their Vita should check it out.
—Keith Schleicher
Wide use of color palate
Uses sci-fi setting instead of typical fantasy
Can jump into the action or customize your character at the start
Extra challenge and reward for delving deeper into dungeon
Long-winded text conversations
Dungeons could be designed better
Typical amnesiac storyline
Lots of backtracking
While not working as a Database Administrator, Keith Schleicher has been associated with Gaming Trend since 2003. While his love of video games started with the Telestar Alpha (a pong console with four different games), he trule started playing video games when he received the ill-fated TI-99/4A. While the Speech Synthesizer seemed to be the height of gaming, eventually a 286 AT computer running at 8/12 Hz and a CGA monitor would be his outlet for a while. Eventually he'd graduate to 386, 486, Pentium, and Athlon systems, building some of those systems while doing some hardware reviews and attending Comdex. With the release of the Dreamcast that started his conversion to the console world. Since then he has acquired an NES, SNES, PS2, PS3, PSP, GBA-SP, DS, Xbox, Xbox 360, Gamecube, and Wii. While not playing video games he enjoys bowling, reading, playing board games, listening to music, and watching movies and TV. He originally hails from Wisconsin but is now living in Michigan with his wife and son.
Related Articles: Operation Abyss: New Tokyo Legacy
Are you destined for a spotlight in the The American Journal of Surgery? Do you dream of assisting McDreamy and not just...
Have you ever played a game that you can tell is great, but you just don't enjoy it for whatever reason? I've...
Ever since a Round 1 opened up near me a couple years ago, I've been fixated on playing Groove Coaster. Like most...
Today, Capstone Games announced that it will open pre-orders for the upcoming railroad game...
Get ready for a lush turn-based RPG experience as Ink Stains Games and Hypetrain...
Blizzard has announced the celebration for the Year of the Rat in Overwatch is...
HTC Vive has announced that they would be permanently lowering their prices for the...
Renegade Game Studios has announced that their newest expansion, Architects of the West Kingdom:...
Return to Dark Tower is taking the eighties classic to new heights
When Restoration Games announced the remake/sequel/spiritual successor to the classic "high tech" board game...
A retro arena shooter for Nintendo Switch – Demon Pit Review
"I'll just try one more run" is what I told myself about 15 attempts...
Sneaking Through Too Many Bugs — Espire 1: VR Operative Review
We first got a glimpse of what to expect from this stealth-oriented VR title...
Sweet dreams are made of this — AI: The Somnium Files review
I'm impressed with how far visual novels have developed in the past few years....
Hell will freeze over before it stops these Horsemen — Darksiders Genesis review
I want to be clear from the start, I really enjoyed this game. So...
One of the longest running independent gaming news, reviews and culture sites on the web.
Gaming unleashed — Razer Phone 2 review
I have a love/hate relationship with my Google devices. On one hand, they get...
Immortal blade — Sekiro: Shadows Die Twice review
Ever since it was announced, Sekiro: Shadows Die Twice has been the subject of...
RE-Masterpiece — Resident Evil 2 Review
There's an art to remaking a classic. The trick is to stay true to...
Running on fumes — Days Gone review
Though I didn't know it at the time, the opening hour of Days Gone...
A hell of a ride — Devil May Cry 5 review
It's been more than a decade since Devil May Cry 4, and it's hard...
Dragon Age: Inquisition Destiny Uncharted 4: A Thief's End Super Smash Bros. for Wii U Street Fighter V The Witcher 3: Wild Hunt Overwatch Final Fantasy XV Grand Theft Auto V Borderlands 2 Fallout 4 Halo 5: Guardians The Last of Us The Order: 1886 Rise of the Tomb Raider Metal Gear Solid V: The Phantom Pain Diablo III Star Wars: The Old Republic Bioshock: Infinite Batman: Arkham Knight
Privacy Policy / Terms
Copyright © 2017 Gaming Trend


text:
RBNY News
New York Red Bulls sign Daniel Royer from FC Midtjylland
As rumored, RBNY has found some attacking help.
By Austin Fido@canetop Aug 3, 2016, 9:57am EDT
Share All sharing options for: New York Red Bulls sign Daniel Royer from FC Midtjylland
William Hauser-USA TODAY Sports
The New York Red Bulls have announced the signing of Austrian winger Daniel Royer. The 26-year-old arrives at RBNY from FC Midtjylland, a top-flight Danish club he joined for the 2015-16 season.
Royer has been capped by his country and played in both UEFA's Champions and Europa Leagues. In the club's official announcement of the new signing, RBNY Sporting Director Ali Curtis described a player he considers a versatile and seasoned addition to the squad:
He's a talented player that is in the prime of his career. His ability to play different positions in the midfield gives us both quality and depth. Daniel is a very intelligent and mature young man, and we look forward to him joining the club.
For his part, Royer is quoted relating his frustration at what had become limited opportunities at FC Midtjylland. Per FCM's official site:
...the last six months have not been satisfactory for me. I have an age now where I need to play. I feel good about the decision and the new challenges that await. This is something very different compared to what I have experienced so far. I've only been in Europe, where I have played in Austria, Germany and Denmark, so it is a new step and a new country. I have considered it carefully before I signed, and I think it is a good solution for me. I'm happy for the change.
FCM Sporting Director Claus Steinlein wishes Royer well:
Daniel started well in FC Midtjylland, but he has since New Year's found it difficult to play on the team. We are therefore pleased that we have agreed with New York Red Bulls on a sale
And Royer wishes his old club well:
I've enjoyed being in Denmark, and I love the people. I've never seen a club like FC Midtjylland. It is without doubt the best people I've met in my career. I felt welcome and comfortable in Denmark and FC Midtjylland. It is a shame that the failure on the field, for I have enjoyed being here and will miss it
He arrives at RBNY with a point to prove to himself and his old team.


text:
What happens if there is a settlement in court?
When is a comparison ineffective?
What to do if a comparison is not complied with?
Can a comparison be reversed?
Is it possible to object to a settlement?
Who bears the costs of a court settlement?
Is a settlement a contract?
When is a settlement final?
When is a comparison made?
How high does a comparison have to be?
How long does it take for debts to expire?
A settlement in court (also called a court settlement) is, simply put, an alternative to a judicial judgment. The parties concerned agree on a solution. A comparison is therefore based on mutual concession between the plaintiff and the defendant; it represents a compromise.
The settlement is ineffective if the facts on which the content of the contract is based do not correspond to reality and the dispute or the uncertainty would not have arisen if the facts were known (779 para. 1 BGB @).
Out of court settlements This will usually be a withdrawal of the action. However, it is also possible for the parties to unanimously declare the legal dispute to be settled. If one party does not meet its obligations arising from the out-of-court settlement, the other must sue for the settlement to be fulfilled.
If a court settlement is revoked after an agreed period, the settlement remains effective. This applies even if the person who missed the deadline is not at all to blame for the delay.
There are no legal remedies in the event of a court settlement. If no reservation of revocation has been made, the settlement is effective and irrevocable. The settlement can then only be challenged because of deception or threats.
According to the basic decision of the legislature, the costs of "the legal dispute" do not include the costs of the court settlement. As a rule, this therefore means that the agreement that one party should bear the costs of the legal dispute also includes the costs of the settlement.
A comparison can also be found in substantive law. In Section 779 of the German Civil Code, the settlement is defined as a contract through which a dispute is resolved through mutual indulgence. This also applies to the process comparison. According to hM, the process comparison has a dual nature.
The process comparison is only effective if both the substantive legal requirements for a comparison and the procedural requirements that are to be placed on an effective process action are met.
According to Section 278, Paragraph 6, Clause 1 of the German Code of Civil Procedure, a settlement is concluded – also – by the fact that the parties accept a written settlement proposal from the court. This is by the pleadings of the defendant from 06.10. The comparison came about.
Basically, the amount depends on the individual case. As a rule of thumb, however, it can be assumed that the offer for the comparison should be higher than the amount that the obligee would receive if the debtor were to go bankrupt.
1 BGB, the three-year limitation period begins at the end of the year in which the claim arose. If, for example, a claim arose from a sales contract on May 16, 2017, the statute of limitations begins on Until the claim is not statute-barred.
← Is Obsessive Compulsive Disorder a Disability?
What happens to the children after the abortion What happens to the children after the abortion? →
How To Make A Master List On Tumblr
Why do you learn English in school?
What does high school mean?


text:
Category: Heart of Malta
October 24, 2020 August 24, 2022 webmaster Heart of Malta, Honors
Two Volunteers Win 2020 "Heart of Malta" Awards
Longtime volunteers Kathy Corbalis and Bradford Wilkinson, MD, have been named this year's "Heart of Malta" honorees, in recognition of their extraordinary efforts as non-medical and medical volunteers, respectively. It is the highest honor a Malta supporter can receive.
Corbalis, a resident of West Hartford, has served as a non-medical volunteer for Malta since 2011, following her retirement as a project manager at The Hartford.
"Kathy has poured her heart and soul into Malta, so it was no surprise that she was a unanimous choice for this award," said Malta Executive Director Michelle M. Murphy. "She volunteers an average of 20 hours per week, helping with all the big and little office tasks that make non-profits hum – and she does it all with a million-dollar smile. We would be lost without her!"
"To say that I am shocked and thrilled (by this award) is indeed an understatement," Corbalis said. "Malta House of Care is a very special place, and I am pleased to work alongside such talented and caring staff and volunteers."
Dr. Wilkinson began volunteering for Malta one day per week in 2006, while he was still running a family practice in Durham; after he retired in 2012, he stepped that up to three days per week. And now, two years after he moved to Wellesley, MA, he still comes back to Hartford two days per month to volunteer for Malta.
"Working at the Malta House of Care Mobile Medical Clinic is the most rewarding thing I have done in 35+ years of practicing medicine," Dr, Wilkinson said. "Seeing the collective happiness, relief, and gratitude on the faces of thousands of patients I have attended when they realize that finally they have found a place where they are not scorned or ignored but rather treated with dignity, warmth, and concern is a magical experience.
"And, to work shoulder-to-shoulder with, and be inspired by, dozens of like-minded volunteer professionals who so freely give of their time, expertise, and humanity is an inexpressible joy," he added.
"Brad exudes a commitment to Malta in every single thing he says and does," said Peter G. Kelly, Sr., Chairman of Malta's Development Committee. "Whether he is treating patients, mentoring medical students and residents, or encouraging other Malta volunteers, he does so with compassion and respect and an utter joy in the beauty of each person he meets.
"We can't begin to count the number of hours he's given to our people and our mission," Kelly continued. "He is a treasure."
The "Heart of Malta" awards were established three years ago to honor members of the Malta family who embody the values at the heart of the Malta mission and passionately demonstrate a commitment to its goals.
Bob Voight, Malta's Board Chair and de facto CEO, received the inaugural Heart of Malta award in 2018.
In 2019, the award was given to the parishioners of St. Catherine of Siena Church in West Simsbury and their charismatic pastor, Rev. Michael Whyte, for their constant support and boundless collective generosity to Malta.
In a departure from the past, the 2020 awards were presented at an extremely small, COVID-safe gathering outside the Malta van during its regular Tuesday Clinic hours at the Cathedral of St. Joseph in Hartford. Previously, the awards were given during the annual "Heart of Malta Benefactor Reception" held in the late Fall.


text:
Das Bildnis der Doriana Gray (1975)
Franco was notorious for shooting back-to-back in his heyday, sometimes even simultaneously, and sometimes without the knowledge of those paying his bills. The most frequently cited example of this is Women Behind Bars, which appears to have been shot alongside Barbed Wire Dolls (both 1975) and thus funded unwittingly by Erwin C. Dietrich. Whatever we make of this practice – whether we consider it to be morally dubious, ingenious or evidence of his compulsion to keep filming – it goes some way towards explaining the director's vast filmography. It may also provide an insight into the unevenness of many of the films, which can oscillate between scenes of obvious inspiration and apparent indifference, as though his mind was elsewhere. Either way, this practice of shooting on a very small scale, of working from a bare outline and shooting with whoever happened to be around, sometimes bore remarkable fruit.
"This picture of blossoming youth will crumble."
Lady Doriana Gray (Lina Romay) lives a life of relative seclusion, alone but for her mute man-servant Ziros (Ramon Ardid). A visit from an American journalist (Monica Swinn) brings to light the presence of a sister, a Siamese-twin sister from whom she was separated at birth. Sadly the operation came at a price, and the nameless sister is now confined to an institution, psychically still tethered to her sister and condemned to feel the sexual sensations which Doriana – despite her craving desires – cannot.
Laying out the film's premise like this, it occurs to me that a first-time viewer might expect a romp in which a horny heroine explores every avenue in order to scratch her sexual itch, and Das Bildnis der Doriana Gray (1975) is nothing of the sort. In fact, what we have here is a languidly-paced, rather artistically-shot tragedy with elements of horror and hardcore sex thrown in – a very strange brew.
In narrative terms, the film actually resembles the director's own Female Vampire (1973) as much as the Wilde story from which it takes its name. There is talk of the sister's beauty being more lasting than Doriana's – making her a very physical equivalent of the portrait in the attic – although it's never made clear why that would be. Actually the fate of Lady Gray is much closer to that of Irina Karlstein's, in that she is compelled to take sexual partners and to kill them in so doing. Like much of the director's best work, this is more about mood than message though, with Dietrich himself commenting that the actual content is largely inconsequential.
Doriana Gray – or Die Marquisse Von Sade, as it's also known – was shot primarily in the South of France towards the beginning of Franco's association with Dietrich, and the aesthetic differences between it and, say, the aforementioned Barbed Wire Dolls are striking from the outset. Franco's cinematography is far more considered here, indeed the film is frequently beautiful to behold. Focus is manipulated for effect this time around, to the point that the kiss between the journalist and Ms Gray begins to resemble Edvard Munch's painting of the same name. Ambient light leaves characters partially silhouetted in front of enormous windows, with a rainbow arcing across the sky in one particularly striking shot. Mirrors are also used throughout, providing frames within frames, leaving us gazing at reflections of reflections. The cumulative effect is at times astonishing.
The film's principal location – the Villa Kerylos, just outside of Nice – also contributes significantly to the atmosphere. It's a remarkable space modelled on the villas of Ancient Greece, quite unlike a functioning, twentieth century home. Doriana's world is one of marble floors, mosaics and sunken baths, a coastal palace out of time which only serves to emphasise her isolation and otherness.
"You radiate the charm of youth and the melancholy of experience."
Franco's decision to shoot hardcore sex scenes presented Dietrich with a distribution problem at the time and will remain a barrier for some viewers to this day. His work had become increasingly explicit through the first half of the seventies, so this might have been a natural progression from the likes of Plaisir a Trois, La Comtesse Perverse and the aforementioned Female Vampire / La Comtesse Noir (all 1973). Of course, the mid-seventies was also the era of 'porno-chic', and Franco would have been aware of the success of films like Deep Throat and Behind the Green Door (both 1972) in the US, not to mention Le Sexe Qui Parle (1975) in France.
Whatever the rationale, the sex scenes herein resembe the director's previous work more than contemporary hardcore, with tender flesh rendered indistinct through extreme close-ups, with obsessive zooms in the nether regions of the female. A number of the encounters are also distinctly carnivorous, pornographically vampiric, in particular two pivotal scenes which occur around the halfway point. In the first, Ziros holds down his beautiful, screaming girlfriend (Martine Stedil) as Doriana gnaws at her genitals, and in the second, he is extinguished in similar fashion, seemingly fellated to death by his mistress. These are powerful scenes, not for the faint hearted, with the former particularly frenzied.
"It's the destiny of all beauty to be destroyed."
As we said at the outset, this is more an exercise is style than storytelling. I do need to acknowledge some narrative shortcomings though. For instance, the transference of Doriana's feelings to her sister is never really explored, and perhaps more problematic, neither is the means by which she kills her partners / victims. None of this really impacts my enjoyment of the film – we are deep in the realms of the fantastique here – but there are also structural problems as it progresses.
The sex scenes really pile up in the final third, at the expense of the more contemplative scenes which contribute considerably to the first hour. The eventual coming together of the sisters – pun very much intended – is also a bit of a let down, and there are some jarring 'meat' shots during the one scene of heterosexual intercourse which look for all the world like inserts and serve only to break the spell of doomed eroticism. The film also ends very abruptly, a frequent Franco failing, especially during this period.
I've said before that I'm not really a fan of the films Franco made with Dietrich, feeling the directorial signature was weekend by the scripts and / or crews assembled by the producer. That's not an issue here though, given the spontaneous nature of the shoot. A review on the IMDB described the film as like watching pornography in slow motion while stoned, which is actually a pretty good description. It's a genuinely haunting example of the erotic horror sub-genre, unmistakably Franco, could only be Franco, thank God for Franco.
Credit must also go to Romay for the way she commits to the dual roles of Doriana and her similarly doomed sister. It's hard to imagine another actress then or now giving so much of herself, particularly to the latter role, writhing around wide-eyed, positively convulsing, pulling at her sex.
WordPress does not lend itself to formal footnotes:
Franco was actually slated to direct a version of Wilde's novel during his association with Harry Alan Towers. The film was ultimately directed by Massimo Dallamano, with Helmut Berger playing the lead.
Dietrich's thoughts on the film can be heard during an interview on the Ascot Elite Blu-ray release from 2013. I believe the interview was actually conducted about ten years earlier, around the time of the first DVD release.
Dietrich prepared a soft cut of Doriana Gray, apparently shooting footage himself to replace that which was excised. It's not a version I would recommend though, even to those who might be squeamish about the hardcore. Dietrich's work stands out like a sore thumb, with Romay wearing an unconvincing wig and occupying locations that are clearly at odds with the surrounding footage.
The sequence in which Romay performs cunnilingus on Stedil ends with liquid dripping from her mouth, which is the kind of thing we might expect at the conclusion of a heterosexual oral scene and might give us a clue as to how Doriana is killing her victims, draining their essence as her predecessor Countess Irina Karlstein did in Female Vampire. The effect is echoed during the scene is which she fellates Ardid – her husband at the time – and which provides the films only 'pop shot'.
Stephen Thrower's enormous Flowers of Perversion (Strange Attractor Press, 2018) was helpful in situating the film in Franco's labyrinthine filmography and revealing its shooting location(s).


Working under the direction of the Lifetime Sports Academy Director, incumbent creates and maintains a fun, safe, and learning environment for the participants of the Lifetime Sports Academy. The incumbent will offer a variety of programs, activities, and learning experiences to academy participants.
• Handles discipline as needed.
Associate's degree (A.A.) or equivalent from two-year college or technical school; or six months to one year related experience and/or training; or equivalent combination of education and experience.
A valid Indiana Driver's License will be required if City vehicle is used.
While performing the duties of this job, the employee is regularly required to stand and walk. The employee frequently is required to use hands to finger, handle, or feel; reach with hands and arms; stoop, kneel, crouch, or crawl; and talk or hear; and taste or smell. The employee must occasionally lift and/or move up to 20 pounds. Specific vision abilities required by this job include close vision, distance vision, color vision, peripheral vision, depth perception, and ability to adjust focus.