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PubMed2000

Application of solid phase micro extraction for the rapid analysis of chlorinated organics in breast milk.

The method presented here allows the monitoring of persistent organochlorine compounds in breast milk using the solid phase microextraction technique (SPME) and gas chromatography with electron capture detection (GC-ECD). It describes the determination of hexachlorobenzene (HCB), alpha-, beta-, and gamma-hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT) and its derivatives, and some important congeners of the polychlorinated biphenyls (PCB). Also included are more polar substances such as tri-, tetra- and penta-chlorophenols, which can be analyzed simultaneously with the afore-mentioned less polar compounds without the need of a derivatization for the determination of the phenolic compounds. The reproducibility of the results is very good down to the lower microg/L-region. The method is very fast and of low cost compared to the classic extraction and determination procedures.

Chromatography, Gas↗
PubMed2001

Development of an element-selective monitoring system for adsorbable organic halogens (AOX) with plasma emission spectrometric detection for quasi-continuous waste-water analysis.

An automated quasi-continuously-operating monitor has been developed for element-selective analysis of adsorbable organic halogens (AOX) in water. After extensive optimization the automatic method was applied to the analysis of standard solutions and real waste water samples to prove its analytical applicability. The new instrument is based on the element-selective analysis of halogens by means of a spectroscopic detection system consisting of a microwave-induced helium plasma excitation source (TM010-type; developed in this laboratory) and the plasma emission detector (PED) which operates with oscillating narrow-band interference filters. After enriching the organic components on activated charcoal and pyrolysis in an oxygen stream at 950 degrees C, in accordance with DIN/EN 38409,H14/1485, interfering CO2 and H2O gas generated during combustion is removed from the analytes in the so-called ELSA-system (element-selective AOX-analyzer). For focused injection into the plasma excitation source the analytes (hydrogen halides) are trapped in a deactivated fused silica capillary at -180 degrees C; this is followed by identification and quantification on the basis of element-specific emission of radiation in the VIS and NIR-region (chlorine 837.6 nm, fluorine 685.6 nm). Bromine and iodine could not be detected with satisfactory inter-element selectivity, because of spectral interferences caused by matrix elements, and so results from the respective single-element investigations for determination of AOBr and AOI are not presented. The procedure has been validated and the analytical performance has been examined by calibration with p-chlorophenol and p-fluorophenol. The limit of detection was 1.1 microg (absolute) for chlorine and 6.6 microg (absolute) for fluorine.

Flow Injection Analysis↗
PubMed1998

Contaminants in ospreys from the Pacific Northwest: I. Trends and patterns in polychlorinated dibenzo-p-dioxins and -dibenzofurans in eggs and plasma.

Osprey (Pandion haliaetus) eggs were collected from 1991 to 1997 at nests (n = 121) upstream and downstream of bleached kraft pulp mills and at reference sites in the Fraser and Columbia River drainage systems of British Columbia, Washington, and Oregon. Blood samples were collected from nestling ospreys during the 1992 breeding season on the Thompson River. Samples were analyzed for polychlorinated dibenzo-p-dioxins (PCDDs) and -dibenzofurans (PCDFs). Mean concentrations of 2,3,7,8-TCDD were significantly higher in eggs collected in 1991 at downstream compared to upstream nests near pulp mills at Kamloops and Castlegar, British Columbia. There were no significant temporal trends in 2,3,7,8-TCDD, -TCDF or other measured compounds at a sample of nests monitored between 1991 and 1994 downstream of the Castlegar pulp mill, despite changes in bleaching technology (CIO2 substitution). However, by 1997 concentrations of 2, 3,7,8-TCDD and -TCDF were significantly lower than previous years in nests sampled downstream at both Castlegar and Kamloops. An unusual pattern of higher chlorinated PCDDs and PCDFs was found in many of the osprey eggs collected in this study, and considerable individual variation in the pattern existed among eggs from the same site. For example, eggs from four different nests at one study area (Quesnel) on the Fraser River had concentrations of 1,2,3,4,6,7,8-HpCDD ranging from <1 to 1,100 ng/kg and OCDD from <1 to 7,000 ng/kg wet weight. Higher mean concentrations of HpCDD and OCDD were found in eggs from the Thompson River, a tributary of the Fraser, compared to the Columbia River, and concentrations were generally higher at nests upstream of pulp mills. In plasma samples, 1,2,3,4,6,7,8-HpCDD and OCDD were the main compounds detected, with no significant differences measured between samples upstream versus downstream or earlier versus later in the breeding season. Use of chlorophenolic wood preservatives by lumber processors was considered the main source of higher chlorinated PCDD/Fs throughout the systems, based on patterns of trace PCDFs in eggs and significant correlations between egg concentrations of pentachlorophenol and both HpCDD (r = 0.891, p < 0.01) and OCDD (r = 0.870, p < 0.01).

Animals↗
PubMed2002

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8.

The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.

Blotting, Southern↗
PubMed2002

Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2.

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.

Amino Acid Sequence↗
PubMed2005

Isolation and transcriptional analysis of novel tetrachloroethene reductive dehalogenase gene from Desulfitobacterium sp. strain KBC1.

Strain KBC1, an anaerobic bacterium, that dechlorinates tetrachloroethene (PCE) to trichloroethene was isolated. This strain also dechlorinated high concentrations of PCE at a temperature range of 10 to 40 degrees C and showed high oxygen tolerance. Based on the 16S rRNA gene sequence analysis, this microorganism was identified as a species of the genus Desulfitobacterium. Several species of this genus have been reported to be potent ortho-chlorophenol and PCE dechlorinators; however, the gene coding PCE-specific dehalogenase had not been cloned thus far. In this report, we identified a novel PCE reductive dehalogenase (PrdA) gene from the Desulfitobacterium sp. strain KBC1. These prd genes, including putative membrane anchor protein, were classified as novel type of PCE reductive dehalogenase (approximately 40% homology with the general PCE dehalogenase). It was revealed that the two open reading frames had been transcribed as identical mRNA and were induced strictly in the presence of PCE. This transcriptional regulation appeared to be controlled by the transcriptional activator located downstream of prdAB operon. According to the substrate utility of the strain KBC1 and phylogenetic analysis of PrdA, this microorganism may be expected to play the role of a primary dechlorinator of PCE in the environment.

Amino Acid Motifs↗
PubMed2006

Biodegradation of a phenolic mixture in a solid-liquid two-phase partitioning bioreactor.

A solid-liquid two-phase partitioning bioreactor (TPPB) in which the non-aqueous phase consisted of polymer (HYTREL) beads was used to degrade a model mixture of phenols [phenol, o-cresol, and 4-chlorophenol (4CP)] by a microbial consortium. In one set of experiments, high concentrations (850 mg l(-1) of each of the three substrates) were reduced to sub-inhibitory levels within 45 min by the addition of the polymer beads, followed by inoculation and rapid (8 h) consumption of the total phenolics loading. In a second set of experiments, the beneficial effect of using polymer beads to launch a fermentation inhibited by high substrate concentrations was demonstrated by adding 1,300 and 2,000 mg l(-1) total substrates (equal concentrations of each phenolic) to a pre-inoculated bioreactor. At these levels, no cell growth and no degradation were observed; however, after adding polymer beads to the systems, the ensuing reduced substrate concentrations permitted complete destruction of the target molecules, demonstrating the essential role played by the polymer sequestering phase when applied to systems facing inhibitory substrate concentrations. In addition to establishing alternative modes of TPPB operation, the present work has demonstrated the differential partitioning of phenols in a mixture between the aqueous and polymeric phases. The polymeric phase was also observed to absorb a degradation intermediate (arising from the incomplete biodegradation of 4CP), which opens the possibility of using solid-liquid TPPBs during biosynthetic transformation to sequester metabolic byproducts.

Biodegradation, Environmental↗
PubMed2006

Plasmid-mediated degradation of 4-chloronitrobenzene by newly isolated Pseudomonas putida strain ZWL73.

A strain of Pseudomonas putida ZWL73 was isolated from soil contaminated with chloronitrobenzenes and identified by 16S rDNA sequencing. This bacterium released chloride and ammonia into the medium when grown on 4-chloronitrobenzene (4CNB) as the sole source of carbon, nitrogen and energy. A plasmid designated pZWL73 of approximately 100 kb in this strain was found to be responsible for 4CNB degradation. This was based on the fact that the plasmid-cured strains showed 4CNB- phenotype and the 4CNB+ phenotype could be conjugally transferred. The cell-free extracts of strain ZWL73 exhibited chloronitrobenzene nitroreductase and 2-amino-5-chlorophenol 1, 6-dioxygenase (2A5CPDO) activities, but neither activity was found from that of the plasmid-cured strain. We have also cloned a 4.9-kb EcoRI fragment exhibiting 2A5CPDO activity. Sequencing results revealed beta-subunit (cnbCa) and alpha subunit (cnbCb) of a meta-cleavage dioxygenase, which were subsequently expressed in E. coli with 2A5CPDO activity. The phylogenetic analysis suggested that 2A5CPDO may form a new subgroup in class III meta-cleavage dioxygenase with its close homologs.

Biodegradation, Environmental↗
PubMed1997

Pentachlorophenol biodegradation kinetics of an oligotrophic fluidized-bed enrichment culture.

A fluidized-bed reactor (FBR) was used to enrich an aerobic chlorophenol-degrading microbial culture. Long-term continuous-flow operation with low effluent concentrations selected oligotrophic microorganisms producing good-quality effluent for pentachlorophenol(PCP)-contaminated water. PCP biodegradation kinetics was studied using this FBR enrichment culture. The results from FBR batch experiments were modeled using a modified Haldane equation, which resulted in the following kinetic constants: qmax = 0.41 mg PCP mg protein-1 day-1, Ks = 16 micrograms l-1, Ki = 5.3 mg l-1, and n = 3.5. These results show that the culture has a high affinity for PCP but is also inhibited by relatively low PCP concentrations (above 1.1 mg PCP l-1). This enrichment culture was maintained over 1 year of continuous-flow operation with PCP as the sole source of carbon and energy. During continuous-flow operation, effluent concentrations below 2 micrograms l-1 were achieved at 268 min hydraulic retention time (tHR) and 2.5 mg PCP l-1 feed concentration. An increase in loading rate by decreasing tHR did not significantly deteriorate the effluent quality until a tHR decrease from 30 min to 21 min resulted in process failure. Recovery from process failure was slow. Decreasing the feed PCP concentration and increasing tHR resulted in an improved process recovery.

Biodegradation, Environmental↗
PubMed2000

Evidence for diverse oxidations in the catabolism of toluene by Rhodococcus rhodochrous strain OFS.

Rhodococcus rhodochrous strain OFS grew on toluene as a sole source of carbon and energy with a maximum growth rate of 0.011 h(-1). Initial reaction products were extracted, derivatized and identified by GC-MS. Oxygen consumption studies indicated that OFS grown on an aliphatic substrate required an induction period before oxidizing toluene. OFS grown on toluene transformed an array of aromatic ground water pollutants including styrene, ethylbenzene and chlorobenzene. Products of these transformations were identified. The sole product of chlorobenzene biotransformation was 4-chlorophenol. Products from toluene oxidation included 3- and 4-methylcatechol as well as benzyl alcohol, p-cresol and cis-toluene dihydrodiol. The identification of these and the products of other aromatic substrate conversions affirm that oxidation occurred on the functional group as well as directly on the aromatic nucleus.

Biodegradation, Environmental↗
PubMed2003

Fertile transgenic pearl millet [ Pennisetum glaucum (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues.

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.

Acetyltransferases↗
PubMed2003

The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck).

A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.

Citrus sinensis↗
PubMed2003

Development of a non-lethal selection system by using the aadA marker gene for efficient recovery of transgenic rice (Oryza sativa L.).

The application of aminoglycoside-3"-adenyltransferase ( aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator-Chlorophenol Red-was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.

Aminobutyrates↗
PubMed2003

Ploidy variation among herbicide-resistant bermudagrass plants of cv. TifEagle transformed with the bar gene.

A protocol was developed for biolistic transformation of hybrid bermudagrass cv. TifEagle using the bar gene. TifEagle is an ultradwarf used exclusively on golf greens. Herbicide resistance should serve as a useful management tool, especially if methyl-bromide is unavailable for fumigation prior to plant establishment. Hybrid bermudagrass is completely sterile, which should limit the chance of gene escape via out-crossing. Sliced nodes were used to initiate embryogenic tissue cultures on MS medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01 mg/l 6-benzylaminopurine (BA). Embryogenic tissue was bombarded with the bar gene, and herbicide-resistant tissue was selected in the dark on medium supplemented with 0.75 mg/l 2,4-D, 0.01 mg/l BA and 5-15 mg/l phosphinothricin (PPT). Resistant somatic embryos were induced to germinate in the light on MS medium supplemented with 0.13 mg/l 2,4-D and 0.5 mg/l BA. Plants were transferred to the greenhouse after rooting in the presence of 10-15 mg/l PPT and testing positive in a chlorophenol red assay. A total of 89 herbicide-resistant plants were recovered from at least nine independent events from six separate bombardments, although the number of independent transformation events was not confirmed for the entire group. Flow cytometry indicated that most of the plants (82/89) were hexaploid and that the remaining seven plants were triploid. The hexaploid plants were a darker green than the triploids or TifEagle control. Other variation, present only in the hexaploids, included an increased leaf width and length. Southern blot hybridization confirmed genomic integration of the bar gene in triploid and a subset of hexaploid herbicide-resistant plants. AFLP analysis did not indicate changes in DNA profiles using [33P] and a sample of 32 hexaploid plants recovered from a single bombardment. DNA profiles were very similar to that of the TifEagle control with a semi-automated fluorescence-based AFLP.

Culture Techniques↗
PubMed2004

Regeneration of herbicide-tolerant black locust transgenic plants by SAAT.

A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants. Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes). The effects of bacterial concentration (OD550 of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient beta-glucuronidase expression 3 days after infection. SAAT increases transient expression efficiency especially at an OD550 of 0.6. After determining bacterial concentration and infection method, other factors affecting transformation efficiency, such as explant preconditioning and period of time before applying selection, were tested. From these experiments, the preferred protocol for black locust cotyledon transformation should include sonication of preconditioned cotyledons in AGL1 suspension, coculture for 3 days with 100 microM acetosyringone and transfer to selection medium with 4 mg/l phosphinothricin and 150 mg/l timentin. Of the initial explants, 2% produced at least one transgenic shoot. Genetic transformation was confirmed by Southern hybridization, chlorophenol red assay and herbicide tolerance of the regenerated plants.

Aminobutyrates↗
PubMed2006

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants.

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a beta-glucuronidase (GUS) gene was transferred into apple cv. 'Holsteiner Cox' via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1-2 g l(-1) mannose in combination with 30 g l(-1) sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l(-1) and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.

Glucuronidase↗
PubMed2006

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker.

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and alpha-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris alpha-amylase inhibitor-1 (alphaAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris alpha-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T(1) lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T(1) plants. Transgenic plants could be recovered after 8-10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.

Agrobacterium tumefaciens↗
PubMed2006

Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants.

For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based "positive" selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 microM 2,4-D, 20 g l(-1) sucrose and 3 g l(-1) mannose. Plant regeneration was obtained on MS basal medium with 2.5 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM IBA, 20 g l(-1) sucrose, and 1.5 g l(-1) mannose. An increase in mannose concentration from permissive (1.5 g l(-1)) to selective (3 g l(-1)) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.

Blotting, Southern↗