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B S Sangwannorreel - One of the best experts on this subject based on the ideXlab platform.
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improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus Niger by seed water soaking
Scientia Horticulturae, 2005Co-Authors: Rajbir S Sangwan, B S SangwannorreelAbstract:Abstract We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus Niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-( N -morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.
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transformation of pollen embryo derived explants by agrobacterium tumefaciens in Hyoscyamus Niger
Plant Cell Tissue and Organ Culture, 2005Co-Authors: Shanjun Tu, V. Raghavan, Rajbir S Sangwan, D P S Verma, B S SangwannorreelAbstract:Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus Niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient β-glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, β-glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species.
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effect of 2 n morpholino ethanesulfonic acid and myo inositol on somatic embryogenesis and plant regeneration from zygotic embryos of Hyoscyamus Niger l
Plant Science, 1996Co-Authors: Thierry Tetu, Rajbir S Sangwan, B S SangwannorreelAbstract:Abstract A rapid and efficient regeneration system via somatic embryogenesis has been developed from zygotic embryos of Hyoscyamus Niger (black henbane). The effect of 2-(n-morpholino)ethanesulfonic acid (MES), myo-inositol (MI) and different combinations of them with a number of growth regulators on somatic embryogenesis was evaluated. Maximum frequency of direct somatic embryogenesis and germination (30.6%) was achieved after 2–5 weeks by a one-step culture procedure on Murashige and Skoog's (MS) basal medium containing 1 mg/l naphthaleneacetic acid, 2 g/l MI and 0.5 g/l MES. Ploidy level of twenty randomly selected regenerants was determined using flow cytometry. No variation in their ploidy levels was observed. This regeneration system may be of value for application with a microparticle gun and Agrobacterium tumefaciens-mediated genetic transformation of black henbane.
Yasuyuki Yamada - One of the best experts on this subject based on the ideXlab platform.
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structures and expression patterns of two tropinone reductase genes from Hyoscyamus Niger
Bioscience Biotechnology and Biochemistry, 1999Co-Authors: Keiji Nakajima, Yoko Oshita, Masahiko Kaya, Yasuyuki Yamada, Takashi HashimotoAbstract:In the biosynthesis of tropane alkaloids, two tropinone reductases (TRs) catalyze reduction of tropinone to different stereoisomers, tropine and pseudotropine. Two TRs from Hyoscyamus Niger have 64% of identical amino acids and hence a common evolutionary origin. In this study, genomic clones of TRs were isolated from H. Niger. Their sequence comparison showed that although they have the same exon/intron organization, sequence similarity was restricted to the coding regions. In H. Niger transgenic hairy roots, both TR promoters activated transcription of the reporter genes in endodermis and pericycle of the roots. A quantitative reporter assay and a nuclear run-on experiment indicated that the two genes are transcribed at a similar rate. The results indicate that although different activity levels have been observed for the TR enzymes in the H. Niger root, the TR genes per se conserve similar tissue-specific expression pattern and transcriptional rate.
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cdna encoding tropinone reductase ii from Hyoscyamus Niger
Plant Physiology, 1993Co-Authors: K Nakajima, Takashi Hashimoto, Yasuyuki YamadaAbstract:Two stereospecific NADPH-dependent reductases, TR-I and TR-11, constitute a branching point in the biosynthesis of tropane alkaloids. TR-I catalyzes the stereospecific reduction of tropinone to tropine (Koelen and Gross, 1982), whereas TR-I1 reduces tropinone to pseudotropine (Drager et al., 1988). We previously characterized TRs that had been purified from cultured roots of Hyoscyamus Niger (Hashimoto et al., 1992) and showed that the two TRs had both common and different biochemical and kinetic properties. To obtain a better understanding of the structure and evolutionary relationship of these reductases, we isolated cDNA clones coding for TR-I1 from H. Niger (Table I). An intemal amino acid sequence was found in both TR-I and TR-I1 that had been purified from the cultured roots of Datura stramonium and H. Niger, respectively (Nakajima et al., 1993). An oligonucleotide probe corresponding to this sequence was synthesized and used to screen the cDNA library from cultured roots of H. Niger. DNA sequencing analysis revealed that a11 four of the isolated cDNA clones encoded the TR-I1 polypeptide, probably due to the low concentration of the TR-I transcript in this genus. None of the cDNA clones contained a full ORF; some lacked the amino-terminal part and others the carboxy-terminal part and the 3‘ nontranslated region. Because the nucleotide sequence of the overlapping part (0.4-0.8 kb) matched perfectly, we concluded that these clones were derived from a single gene. The combined nucleotide sequence (1049 bp) contained a 783-bp ORF coding for a polypeptide composed of 260 amino acids, and the calculated mo1 wt of 28,436 agreed well with the molecular mass for the TR-I1 subunit (29 kD) that had been purified from H. Niger (Hashimoto et al., 1992). The isolated cDNA was expressed in Escherichia coli as a fusion protein to /3-galactosidase under the control of the Zac promoter. The fusion protein was induced by isopropyl-P-Dthiogalactopyranoside, and the bacterial lysate was assayed for TR activities as described elsewhere (Nakajima et al., 1993). The fusion protein catalyzed the same highly stereospecific reduction of tropinone as the TR-I1 from H. Niger; pseudotropine was the sole reaction product detected. The deduced amino acid sequence of TR-I1 from H. Niger is highly homologous to that from D. stramonium (Nakajima
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two tropinone reductases with distinct stereospecificities from cultured roots of Hyoscyamus Niger
Plant Physiology, 1992Co-Authors: Takashi Hashimoto, Keiji Nakajima, Godelieve Ongena, Yasuyuki YamadaAbstract:Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-II, which, respectively, reduce tropinone to 3α-hydroxytropane (tropine) and 3β-hydroxytropane (ψ-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus Niger and characterized. The TR-I reaction was reversible, whereas the TR-II reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol ψ-tropine. Marked differences were found between the two reductase in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by TR-II but not by TR-I. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2,1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-II. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed.
Takashi Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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structures and expression patterns of two tropinone reductase genes from Hyoscyamus Niger
Bioscience Biotechnology and Biochemistry, 1999Co-Authors: Keiji Nakajima, Yoko Oshita, Masahiko Kaya, Yasuyuki Yamada, Takashi HashimotoAbstract:In the biosynthesis of tropane alkaloids, two tropinone reductases (TRs) catalyze reduction of tropinone to different stereoisomers, tropine and pseudotropine. Two TRs from Hyoscyamus Niger have 64% of identical amino acids and hence a common evolutionary origin. In this study, genomic clones of TRs were isolated from H. Niger. Their sequence comparison showed that although they have the same exon/intron organization, sequence similarity was restricted to the coding regions. In H. Niger transgenic hairy roots, both TR promoters activated transcription of the reporter genes in endodermis and pericycle of the roots. A quantitative reporter assay and a nuclear run-on experiment indicated that the two genes are transcribed at a similar rate. The results indicate that although different activity levels have been observed for the TR enzymes in the H. Niger root, the TR genes per se conserve similar tissue-specific expression pattern and transcriptional rate.
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cdna encoding tropinone reductase ii from Hyoscyamus Niger
Plant Physiology, 1993Co-Authors: K Nakajima, Takashi Hashimoto, Yasuyuki YamadaAbstract:Two stereospecific NADPH-dependent reductases, TR-I and TR-11, constitute a branching point in the biosynthesis of tropane alkaloids. TR-I catalyzes the stereospecific reduction of tropinone to tropine (Koelen and Gross, 1982), whereas TR-I1 reduces tropinone to pseudotropine (Drager et al., 1988). We previously characterized TRs that had been purified from cultured roots of Hyoscyamus Niger (Hashimoto et al., 1992) and showed that the two TRs had both common and different biochemical and kinetic properties. To obtain a better understanding of the structure and evolutionary relationship of these reductases, we isolated cDNA clones coding for TR-I1 from H. Niger (Table I). An intemal amino acid sequence was found in both TR-I and TR-I1 that had been purified from the cultured roots of Datura stramonium and H. Niger, respectively (Nakajima et al., 1993). An oligonucleotide probe corresponding to this sequence was synthesized and used to screen the cDNA library from cultured roots of H. Niger. DNA sequencing analysis revealed that a11 four of the isolated cDNA clones encoded the TR-I1 polypeptide, probably due to the low concentration of the TR-I transcript in this genus. None of the cDNA clones contained a full ORF; some lacked the amino-terminal part and others the carboxy-terminal part and the 3‘ nontranslated region. Because the nucleotide sequence of the overlapping part (0.4-0.8 kb) matched perfectly, we concluded that these clones were derived from a single gene. The combined nucleotide sequence (1049 bp) contained a 783-bp ORF coding for a polypeptide composed of 260 amino acids, and the calculated mo1 wt of 28,436 agreed well with the molecular mass for the TR-I1 subunit (29 kD) that had been purified from H. Niger (Hashimoto et al., 1992). The isolated cDNA was expressed in Escherichia coli as a fusion protein to /3-galactosidase under the control of the Zac promoter. The fusion protein was induced by isopropyl-P-Dthiogalactopyranoside, and the bacterial lysate was assayed for TR activities as described elsewhere (Nakajima et al., 1993). The fusion protein catalyzed the same highly stereospecific reduction of tropinone as the TR-I1 from H. Niger; pseudotropine was the sole reaction product detected. The deduced amino acid sequence of TR-I1 from H. Niger is highly homologous to that from D. stramonium (Nakajima
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two tropinone reductases with distinct stereospecificities from cultured roots of Hyoscyamus Niger
Plant Physiology, 1992Co-Authors: Takashi Hashimoto, Keiji Nakajima, Godelieve Ongena, Yasuyuki YamadaAbstract:Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-II, which, respectively, reduce tropinone to 3α-hydroxytropane (tropine) and 3β-hydroxytropane (ψ-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus Niger and characterized. The TR-I reaction was reversible, whereas the TR-II reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol ψ-tropine. Marked differences were found between the two reductase in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by TR-II but not by TR-I. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2,1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-II. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed.
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molecular cloning of hyoscyamine 6 beta hydroxylase a 2 oxoglutarate dependent dioxygenase from cultured roots of Hyoscyamus Niger
Journal of Biological Chemistry, 1991Co-Authors: J Matsuda, Souichi Okabe, Takashi Hashimoto, Yukio YamadaAbstract:Abstract Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus Niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. Niger.
Rajbir S Sangwan - One of the best experts on this subject based on the ideXlab platform.
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improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus Niger by seed water soaking
Scientia Horticulturae, 2005Co-Authors: Rajbir S Sangwan, B S SangwannorreelAbstract:Abstract We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus Niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-( N -morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.
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transformation of pollen embryo derived explants by agrobacterium tumefaciens in Hyoscyamus Niger
Plant Cell Tissue and Organ Culture, 2005Co-Authors: Shanjun Tu, V. Raghavan, Rajbir S Sangwan, D P S Verma, B S SangwannorreelAbstract:Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus Niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient β-glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, β-glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species.
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effect of 2 n morpholino ethanesulfonic acid and myo inositol on somatic embryogenesis and plant regeneration from zygotic embryos of Hyoscyamus Niger l
Plant Science, 1996Co-Authors: Thierry Tetu, Rajbir S Sangwan, B S SangwannorreelAbstract:Abstract A rapid and efficient regeneration system via somatic embryogenesis has been developed from zygotic embryos of Hyoscyamus Niger (black henbane). The effect of 2-(n-morpholino)ethanesulfonic acid (MES), myo-inositol (MI) and different combinations of them with a number of growth regulators on somatic embryogenesis was evaluated. Maximum frequency of direct somatic embryogenesis and germination (30.6%) was achieved after 2–5 weeks by a one-step culture procedure on Murashige and Skoog's (MS) basal medium containing 1 mg/l naphthaleneacetic acid, 2 g/l MI and 0.5 g/l MES. Ploidy level of twenty randomly selected regenerants was determined using flow cytometry. No variation in their ploidy levels was observed. This regeneration system may be of value for application with a microparticle gun and Agrobacterium tumefaciens-mediated genetic transformation of black henbane.
Cinthia Carolina Abbona - One of the best experts on this subject based on the ideXlab platform.
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the chloroplast genome of Hyoscyamus Niger and a phylogenetic study of the tribe hyoscyameae solanaceae
PLOS ONE, 2014Co-Authors: Virginia M Sanchezpuerta, Cinthia Carolina AbbonaAbstract:The tribe Hyoscyameae (Solanaceae) is restricted to Eurasia and includes the genera ArchiHyoscyamus, Anisodus, Atropa, Atropanthe, Hyoscyamus, Physochlaina, Przewalskia and Scopolia. Even though the monophyly of Hyoscyameae is strongly supported, the relationships of the taxa within the tribe remain unclear. Chloroplast markers have been widely used to elucidate plant relationships at low taxonomic levels. Identification of variable chloroplast intergenic regions has been developed based on comparative genomics of chloroplast genomes, but these regions have a narrow phylogenetic utility. In this study, we present the chloroplast genome sequence of Hyoscyamus Niger and make comparisons to other solanaceous plastid genomes in terms of gene order, gene and intron content, editing sites, origins of replication, repeats, and hypothetical open reading frames. We developed and sequenced three variable plastid markers from eight species to elucidate relationships within the tribe Hyoscyameae. The presence of a horizontally transferred intron in the mitochondrial cox1 gene of some species of the tribe is considered here a likely synapomorphy uniting five genera of the Hyoscyameae. Alternatively, the cox1 intron could be a homoplasious character acquired twice within the tribe. A homoplasious inversion in the intergenic plastid spacer trnC-psbM was recognized as a source of bias and removed from the data set used in the phylogenetic analyses. Almost 12 kb of plastid sequence data were not sufficient to completely resolve relationships among genera of Hyoscyameae but some clades were identified. Two alternative hypotheses of the evolution of the genera within the tribe are proposed.