Development of CE-C4D Method for Determination Tropane Alkaloids.

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C4D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) ß-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters-precision (CV): 1.07-2.08%, accuracy of the assay (recovery, RE): 101.0-102.7% and matrix effect (ME): 92.99-94.23%. Moreover, the optimized CE-C4D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.

Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours.

In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a broad-spectrum monoclonal antibody for tropane alkaloids (TAs) was established for the rapid screening of atropine, scopolamine, homatropine, apoatropine, anisodamine, anisodine and L-hyoscyamine residues in pig urine, pork and cereal flour samples through a simple sample preparation procedure. The half inhibitory concentrations of atropine, homatropine, L-hyoscyamine, apoatropine, scopolamine, anisodamine and anisodine were 0.05, 0.07, 0.14, 0.14, 0.24, 5.30 and 10.15 ng mL-1, respectivelyThe detection and quantitative limits of this method for TAs in samples were 0.18-73.18 and 0.44-74.77 µg kg-1. The spiked recoveries ranged from 69.88% to 147.93%, and the coefficient of variations were less than 14%. Good correlation (R2 = 0.9929) between the results of the ic-ELISA and the high performance liquid chromatography-tandem mass spectrometry support the reliability of the developed ic-ELISA method.

Rapid screening of glycoalkaloids in Solanum scabrum and S. nigrum berries using ultra-high-performance liquid chromatography with pathway-specified in-source fragmentation tandem mass spectrometry.

RATIONALE: The safe consumption of Solanum scabrum and S. nigrum berries (SNBs) depends on a reliable and rapid chemical screen for the testing of the fruit and/or final food and industrial products for the presence and level of toxic glycoalkaloids. Such a rapid and sensitive screen could also be used by those involved in food safety and forensics, industry, research labs and those in agriculture production, breeding and food processing. Significant variation in the content and composition of glycoalkaloids across SNBs has been reported. To facilitate high-throughput targeted analysis, this work overcame the slow scan speed of a traditional triple quadruple mass spectrometry (QqQ) method by development of a pseudo-MS3 method. METHODS: In-source fragmentation functioned as a pseudo-MS or pseudo-hydrolysis to trim down the structurally diverse and complex glycosides into five types of aglycone ions, which were then analyzed using multiple reaction monitoring (MRM). Characteristic product ions were selected based on the aglycone skeleton and substitution pattern and associated fragmentation pathway. RESULTS: A compact method with only 15 MRM transitions were developed for high-throughput screening of very diverse glycoalkaloids. Glycosides of the same aglycone type were readily identified in the same transition window without the need for mass spectra interpretation. Validated using solamargine, the sole available standard, the accuracy was 99.7-101.3%, the intra- and inter-day precision were, respectively, 2.5-5.0% and 8.0-9.2%, and the lower limit of detection and quantification were, respectively, 3.1 and 10.2 ng/mL (with 1 µL injection volume). CONCLUSIONS: The peudo-MS3 method allowed for high-throughput targeted analysis with compact MRM transitions to address a large number of glycoalkaloids with diverse structures. This method could serve to meet the most heavy-duty demand for rapid inspection of glycoalkaloids in SNBs. This method can be adopted and used by those involved in food safety and forensics, in developing food and industrial products and in genetics and breeding.

The application of an aqueous two-phase system combined with ultrasonic cell disruption extraction and HPLC in the simultaneous separation and analysis of solanine and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit.

An aqueous two-phase system was used in conjunction with ultrasonic cell disruption to extract and separate solanine (mainly solasonine and solamargine) and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit. The optimized conditions of the present study were determined by a single-factor experiment and a multifactor experiment. The concentration of ethanol was set at 60% and the duration of the ultrasonic cell disruption extraction was 50 min. In the ethanol-K2CO3 aqueous two-phase separation system, the concentration of ethanol was 36%, the concentration of K2CO3 was 0.21 mg·mL-1, and the temperature was 15 °C. The solasonine and solamargine were determined by high-performance liquid chromatography, and the Solanum nigrum polysaccharide was determined by an ultraviolet-visible spectrophotometer in accordance with the phenol-sulfuric acid method. xUnder optimized conditions, the average extraction efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 95.86%, 95.95% and 96.95%, respectively, and the average separation efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 2.07 mg·g-1, 2.05 mg·g-1 and 8.15 mg·g-1, respectively.

Comparison of Profiling of Hairy Root of Two Tibetan Medicinal Plants Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim.

BACKGROUND: Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry. METHODS: To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions. RESULTS: The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings. CONCLUSION: Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.

Analysis of calystegines in tomato-based products by liquid chromatography-Orbitrap mass spectrometry.

Taking into account the high consumption of tomato and tomato-based products, a study of the occurrence of 7 nortropane alkaloids (calystegines A3, A5, B1, B2, B3, B4 and C1) in this type of matrix has been performed. For that purpose, a simple and fast solid-liquid extraction with methanol/water (50/50, v/v) has been developed, and then liquid chromatography, using a HILIC-A as stationary phase, coupled to high resolution mass spectrometry (LC-HRMS-Orbitrap) has been used for their determination. The developed method was validated and recoveries ranged from 96 to 121%, and relative standard deviations lower than or equal to 16% were obtained. Limits of quantification (LOQ) were established at 0.1 (B4), 0.25 (B3) and 0.5 (A3, A5, B1, B2 and C1) mg/kg. Twelve different samples were analyzed, comprising 4 crushed tomatoes, 4 fried tomatoes and 4 jam tomatoes. Calystegines A3 and B2 were identified in all the analyzed samples, while calystegine B3 was determined in all the fried tomatoes as well as in two crushed tomatoes and one jam tomato at concentrations ranging from 0.4 mg/kg (calystegine B2) to 19.0 mg/kg (calystegine A3). Although calystegine A5 was also detected in all the samples, only 3 of them gave concentrations higher than LOQ. Additionally, calystegine B1 was found in one jam tomato at 1.9 mg/kg.

Fragmentation study of major spirosolane-type glycoalkaloids by collision-induced dissociation linear ion trap and infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: Glycoalkaloids play a key role in the plant protection system against phytopathogens including fungi, viruses, bacteria, insects and worms. They can be toxic to humans if consumed in high concentrations causing gastrointestinal disturbances. METHODS: The structural characterization of the major spirosolane glycoalkaloids, solasonine, solamargine, α-tomatine and dehydrotomatine, were investigated by positive electrospray ionization (ESI) coupled with a hybrid linear ion trap (LIT) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Tandem mass spectrometric analysis of spirosolane glycoalkaloids was performed by both collision-induced dissociation (CID) within the LIT and infrared multiphoton dissociation (IRMPD) in conjunction with the FTICR cell. RESULTS: Several common product ions were observed, generated by losses of the sugar moiety or aglycone fragmentation in the B- or E-ring, that can provide information on the accurate mass of aglycone and the primary sequence and branching of the oligosaccharide chains. Thanks to the multistage CID it was possible to understand the fragmentation pathways and thanks to the high resolution of IRMPD-FTICR the elemental compositions of product ions were obtained. CONCLUSIONS: Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical compositions for the majority of detected fragment ions were feasible. From these data, generalized fragmentation pathways were proposed, providing guidance for the characterization of unknown glycoalkaloids in plants. Copyright © John Wiley & Sons, Ltd.

Maternal Ingestion of Ipomoea carnea: Effects on Goat-Kid Bonding and Behavior.

Ipomoea carnea is a toxic plant found in Brazil and other tropical and subtropical countries and often causes poisoning of livestock. The plant contains the alkaloids swainsonine and calystegines, which inhibit key cellular enzymes and cause systematic cell death. This study evaluated the behavioral effects of prenatal ingestion of this plant on dams and their kids. Twenty-four pregnant goats were randomly allocated into four treatment groups and received the following doses (g/kg BW) of fresh I. carnea: 0 (control group), 1.0 (IC1), 3.0 (IC3), and 5.0 (IC5) from day 27 of gestation until parturition. Dam and kid bonding and behavior were evaluated by several tests, immediately after birth until six weeks of age. Dams from IC3 and IC5 groups spent less time paying attention to the newborn. There was a lack of maternal-infant bonding due to I. carnea intoxication. Kids from treated dams had difficulty in standing, suckling, and in recognizing their mother hours after birth. I. carnea can also compromise the kids' ability to learn and to retain spatial memory. We suggest that kids from pregnant goats given I. carnea during gestation have significant behavioral alterations and developmental delays that may compromise their survival.

Tropane alkaloids and calystegines as chemotaxonomic markers in the Solanaceae.

This study assessed the occurrence and distribution of tropane alkaloids and calystegines in genera of the family Solanaceae to identify patterns of distribution and make evolutionary inferences. A database of tropane alkaloids and calystegines occurrences was constructed from the results of a search of scientific websites and a hand search of periodicals. The terms "Solanaceae", "tropane alkaloids", and "calystegines" were used as index terms for a full-text article search unrestricted by date of publications. The number of occurrence and chemical diversity indices were calculated and cluster analysis and principal components analysis were performed. Overall, 996 occurrences were reported, 879 of tropane alkaloids (88.3%) and 117 of calystegines (11.7%). The calystegines were significantly more relevant than tropane alkaloids for characterization of distinct groups of genera on both analyses performed here. This corroborates the trend toward a chemical dichotomy observed on database analysis and somewhat reinforces the correlation between geographic distribution and occurrence of secondary metabolites, as the presence of calystegines alone (without tropane alkaloids) was only reported in genera that have South America as their center of diversity.

Protein and alkaloid patterns of the floral nectar in some solanaceous species.

The family Solanaceae includes several melliferous plants, which tend to produce copious amounts of nectar. Floral nectar is a chemically complex aqueous solution, dominated by sugars, but minor components such as amino acids, proteins, flavonoids and alkaloids are present as well. This study aimed at analysing the protein and alkaloid profile of the nectar in seven solanaceous species. Proteins were examined with SDS-PAGE and alkaloids were analyzed with HPLC. The investigation of protein profile revealed significant differences in nectar-protein patterns not only between different plant genera, but also between the three Nicotiana species investigated. SDS-PAGE suggested the presence of several Nectarin proteins with antimicrobial activity in Nicotiana species. The nectar of all tobacco species contained the alkaloid nicotine, N. tabacum having the highest nicotine content. The nectar of Brugmansia suaveolens, Datura stramonium, Hyoscyamus niger and Lycium barbarum contained scopolamine, the highest content of which was measured in B. suaveolens. The alkaloid concentrations in the nectars of most solanaceous species investigated can cause deterrence in honeybees, and the nectar of N. rustica and N. tabacum can be considered toxic for honeybees.

Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method.

An electrophoretic method for fast separation of three tropane alkaloids (hyoscyamine, anisodamine and scopolamine) was presented. The substances were complete resolved in less than one minute due to utilization of relatively short capillary (20.2cm effective length) and high voltage (25kV). Detector probing frequency was found among the parameters that significantly affected the detection sensitivity. The performed experiments showed insufficient available probing frequency of used commercial spectrophotometric detector according to capillary electrophoresis (CE) separation potential. Under the optimized conditions the background electrolyte (BGE) was composed of 20mM Tris, 6mM HCl and 20mM NaCl (pH 8.50). All analyses were carried out in fused silica capillaries of 50µm (inner diameter) and 31.2cm (total capillary length). Samples were injected hydrodynamically (5s; 3.45kPa) without any sample preparation step and separation was performed at 25kV. The elaborated method was applied in plant cultures growth media analysis after incubation with hairy roots of selected Solanaceae species. The performed experiments proved the usefulness of CE in quality control of biotechnological processes.

Optimisation and validation of ultra-high performance liquid chromatographic-tandem mass spectrometry method for qualitative and quantitative analysis of potato steroidal alkaloids.

An ultra-high performance liquid chromatographic-tandem mass spectrometry (UHPLC-MS/MS) method for quantification of potato steroidal alkaloids, namely α-solanine, α-chaconine, solanidine and demissidine was developed and validated. Three different column chemistries, i.e. ethylene bridged hybrid (BEH) C18, hydrophilic lipophilic interaction and amide columns, were assessed. The BEH C18 column showed best separation and sensitivity for the alkaloids. Validation data (inter-day and intra-day combined) for accuracy and recovery ranged from 94.3 to 107.7% and 97.0 to 103.5%, respectively. The accuracy data were within the acceptable range of 15% as outlined in the United States Food and Drug Administration (USFDA) guidelines. The recovery data were consistent and reproducible with a coefficient of variation (CV) ranging from 6.2 to 9.7%. In addition, precision of the method also met the criteria of the USFDA with CV values lower than 15% even at lower limit of quantification (LLOQ), while the permissible variation is considered acceptable below 20%. The limit of detection and LLOQ of the four alkaloids were in the range of 0.001-0.004µg/mL whereas the linearities of the standard curves were between 0.980 and 0.995.

Modifying glycoalkaloid content in transgenic potato--Metabolome impacts.

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desirée) tubers, which have been genetically modified (GM) to reduce glycoalkaloid content, via the independent down-regulation of three genes SGT1, SGT2 and SGT3 known to be involved in glycoalkaloid biosynthesis. Differences between the three groups of antisense lines and control lines were assessed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS, and data analysed using principal component analysis and analysis of variance. Compared with the wild-type (WT) control, LC-MS revealed not only the expected changes in specific glycoalkaloid levels in the GM lines, but also significant changes in several other metabolites, some of which were explicable in terms of known pathways. Analysis of polar and non-polar metabolites by GC-MS revealed other significant (unintended) differences between SGT lines and the WT, but also between the WT control and other control lines used.

Identification of anisodamine tablets by Raman and near-infrared spectroscopy with chemometrics.

Vibrational spectroscopy including Raman and near-infrared (NIR) spectroscopy has become an attractive tool for pharmaceutical analysis. In this study, effective calibration models for the identification of anisodamine tablet and its counterfeit and the distinguishment of manufacturing plants, based on Raman and NIR spectroscopy, were built, respectively. Anisodamine counterfeit tablets were identified by Raman spectroscopy with correlation coefficient method, and the results showed that the predictive accuracy was 100%. The genuine anisodamine tablets from 5 different manufacturing plants were distinguished by NIR spectroscopy using partial least squares discriminant analysis (PLS-DA) models based on interval principal component analysis (iPCA) method. And the results showed the recognition rate and rejection rate were 100% respectively. In conclusion, Raman spectroscopy and NIR spectroscopy combined with chemometrics are feasible and potential tools for rapid pharmaceutical tablet discrimination.

Multi-development-HPTLC method for quantitation of hyoscyamine, scopolamine and their biosynthetic precursors in selected solanaceae plants grown in natural conditions and as in vitro cultures.

INTRODUCTION: Hyoscyamine and scopolamine, anti-cholinergic agents widely used in medicine, are typically obtained from plants grown under natural conditions. Since field cultivation entails certain difficulties (changeable weather, pests, etc.), attempts have been made to develop a plant in vitro culture system as an alternative source for the production of these compounds. During experiments to locate the limiting steps in the biotechnological procedure, it is important to monitor not only the levels of the final products but also the changes in the concentration of their precursors. OBJECTIVE: To develop a HPTLC method for the separation and quantitation of the main tropane alkaloids hyoscyamine and scopolamine, their respective direct precursors littorine and anisodamine, and cuscohygrine, a product of a parallel biosynthetic pathway that shares a common precursor (N-methyl-∆(1) -pyrrolium cation) with tropane alkaloids. METHODS: Using alkaloid extracts from Atropa baetica hairy roots, different TLC chromatographic systems and developing procedures were investigated. RESULTS: Full separation of all compounds was obtained on HPTLC Si60 F254 plates preconditioned with mobile phase vapours (chloroform:methanol:acetone:25% ammonia ratios of 75:15:10:1.8, v/v/v/v). The chromatograms were developed twice (at distances of 4.0 and 3.0 cm) in a Camag twin trough chamber and visualised with Dragendorff's reagent. Densitometric detection (λ = 190 and 520 nm) was used for quantitative analyses of the different plant samples. CONCLUSION: This method can be recommended for quantitation of hyoscyamine, scopolamine, anisodamine, littorine and cuscohygrine in different plant material (field grown vs. in vitro cultures).

A two-electrode system-based electrochemiluminescence detection for microfluidic capillary electrophoresis and its application in pharmaceutical analysis.

A two-electrode configuration powered by batteries was designed for a microchip capillary electrophoresis-electrochemiluminescence system. A home-made working electrode for end-column mode detection and wall-jet configuration was made up of a platinum wire (0.3 mm diameter) and a quartz capillary (320 µm internal diameter). The platinum wire served as a pseudoreference electrode. The configuration of the detection power supply comprised two D-size batteries (connected in series), a switch, and an adjustable resistor. The microchip consisted of two layers: the bottom layer was a glass sheet containing injection and separation channels; the upper layer was polydimethylsiloxane block. In order to reduce the loss of electrochemiluminescence signal, a coverslip (0.17 mm thickness) was used as the floor of the detection reservoir. The performance of the system was demonstrated by separation and detection of atropine, anisodamine and proline. The linear response for proline ranged from 5 µM to 100 µM (r = 0.9968), and the limit of detection was 1.0 µM (S/N = 3). The system was further applied to the measurement of atropine in atropine sulfate injection solutions with the limit of detection 2.3 µM. This new system is a potential tool in pharmaceutical analysis.

Glycoalkaloid and calystegine levels in table potato cultivars subjected to wounding, light, and heat treatments.

Potato tubers naturally contain a number of defense substances, some of which are of major concern for food safety. Among these substances are the glycoalkaloids and calystegines. We have here analyzed levels of glycoalkaloids (α-chaconine and α-solanine) and calystegines (A3, B2, and B4) in potato tubers subjected to mechanical wounding, light exposure, or elevated temperature: stress treatments that are known or anticipated to induce glycoalkaloid levels. Basal glycoalkaloid levels in tubers varied between potato cultivars. Wounding and light exposure, but not heat, increased tuber glycoalkaloid levels, and the relative response differed among the cultivars. Also, calystegine levels varied between cultivars, with calystegine B4 showing the most marked variation. However, the total calystegine level was not affected by wounding or light exposure. The results demonstrate a strong variation among potato cultivars with regard to postharvest glycoalkaloid increases, and they suggest that the biosynthesis of glycoalkaloids and calystegines occurs independently of each other.

Mn-doped ZnS quantum dots for the room-temperature phosphorescence detection of raceanisodamine hydrochloride and atropine sulfate in biological fluids.

Now, the development of quantum dots (QDs)-based fluorescence sensors become very quickly, but as phosphorescence compared to fluorescent has many advantages, like longer shine time and emission wavelength. Therefore, the phosphorescence properties of QDs and their potential for phosphorescence detection have raised great concerns. In this paper, a novel room-temperature phosphorescence (RTP) quenching method was developed by Mn-doped ZnS quantum dots (QDs). The developed method is employed for detection of the raceanisodamine hydrochloride and atropine sulfate in biological fluids. The results showed a high selectivity of the Mn-doped ZnS QDs toward these medicines by phosphorescence quenching. Under the optimized experimental conditions, the detection limits (3s) for raceanisodamine hydrochloride and atropine sulfate were 0.11 µM, 0.09 µM, respectively. The relative standard deviations for eleven replicate detections of 2.0 µM were 0.92-1.6%. The recovery of spiked solutions in human urine and serum samples ranged from 95% to 104%.

Steroidal glycoalkaloids in Solanum chacoense.

Potato (Solanum tuberosum L.), a domesticated species that is the fourth most important world agricultural commodity, requires significant management to minimize the effects of herbivore and pathogen damage on crop yield. A wild relative, Solanum chacoense Bitt., has been of interest to plant breeders because it produces an abundance of novel steroidal glycoalkaloid compounds, leptines and leptinines, which are particularly effective deterrents of herbivory by the Colorado potato beetle (Leptinotarsa decemlineata Say). Biochemical approaches were used in this study to investigate the formation and accumulation of SGAs in S. chacoense. SGA contents were determined in various organs at different stages of organ maturity during a time course of plant development. Leptines and leptinines were the main contributors to the increased levels in SGA concentration measured in the aerial versus the subterranean organs of S. chacoense accession 8380-1. Leptines were not detected in aboveground stolons until the stage where shoots had formed mature chlorophyllous leaves. To gain insights into SGA biosynthesis, the abundance of SGAs and steady-state transcripts of genes coding for enzymes of the central terpene and SGA-specific pathways in various plant organs at anthesis were compared. For two genes of primary terpene metabolism, transcript and SGA abundances were correlated, although with some discrepancies. For genes associated with SGA biosynthesis, transcripts were not detected in some tissues containing SGAs; however these transcripts were detected in the progenitor tissues, indicating the possibility that under our standard growth conditions, SGA biosynthesis is largely limited to highly proliferative tissues such as shoot, root and floral meristems.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.