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Stefan Gleissberg - One of the best experts on this subject based on the ideXlab platform.
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Duplicated STM-like KNOX I genes act in floral meristem activity in Eschscholzia californica (Papaveraceae)
Development Genes and Evolution, 2013Co-Authors: Angelika Stammler, Annette Becker, Sandra S. Meyer, Alastair R. Plant, Brad T. Townsley, Stefan GleissbergAbstract:In angiosperms, the shoot apical meristem is at the origin of leaves and stems and is eventually transformed into the floral meristem. Class I knotted -like homeobox ( KNOX I ) genes are known as crucial regulators of shoot meristem formation and maintenance. KNOX I genes maintain the undifferentiated state of the apical meristem and are locally downregulated upon leaf initiation. In Arabidopsis , KNOX I genes, especially SHOOTMERISTEMLESS ( STM ), have been shown to regulate flower development and the formation of carpels. We investigated the role of STM -like genes in the reproductive development of Eschscholzia californica , to learn more about the evolution of KNOX I gene function in basal eudicots. We identified two orthologs of STM in Eschscholzia , EcSTM1 and EcSTM2 , which are predominantly expressed in floral tissues. In contrast, a KNAT1 / BP -like and a KNAT2 / 6 -like KNOX I gene are mainly expressed in vegetative organs. Virus-induced gene silencing (VIGS) was used to knockdown gene expression, revealing that both EcSTM genes are required for the formation of reproductive organs. Silencing of EcSTM1 resulted in the loss of the gynoecium and a reduced number of stamens. EcSTM2 -VIGS flowers had reduced and defective gynoecia and a stronger reduction in the number of stamen than observed in EcSTM1 -VIGS. Co-silencing of both genes led to more pronounced phenotypes. In addition, silencing of EcSTM2 alone or together with EcSTM1 resulted in altered patterns of internodal elongation and sometimes in other floral defects. Our data suggest that some aspects of STM function present in Arabidopsis evolved already before the basal eudicots diverged from core eudicots.
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silencing of ecflo a floricaula leafy gene of the california poppy Eschscholzia californica affects flower specification in a perigynous flower context
International Journal of Plant Sciences, 2013Co-Authors: Sara Wreath, Andrea Scholz, Conny Bartholmes, Oriane Hidalgo, Stefan GleissbergAbstract:Flowers are reproductive shoots produced by determinate floral meristems. The role of FLORICAULA/LEAFY-like genes in the specification of flowers varies between lineages and has not been characterized in a basal eudicot species. Here we report the phenotypic effects of virus-induced silencing of EcFLO, a FLORICAULA/LEAFY homologue in the California poppy Eschscholzia californica. EcFLO silencing resulted in repeated sepal and petal whorl formation and internodes between calyxes. A subset of silenced flowers showed reduced petal and stamen numbers and mosaic identities of floral organs. We also provide expression data of other floral regulator genes and conclude that EcFLO contributes to certain aspects of flower development, reflecting both functional conservation and differentiation among basal eudicots, core eudicots, and monocot grasses. EcFLO is involved in stamen and petal initiation, floral organ identity delimitation, suppression of internodes, and limitation of the number of perianth whorls. Furth...
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evolution of the yabby gene family with emphasis on the basal eudicot Eschscholzia californica papaveraceae
Plant Biology, 2011Co-Authors: Conny Bartholmes, Oriane Hidalgo, Stefan GleissbergAbstract:YABBY genes are seed plant-specific transcriptional regulators that are involved in diverse aspects of leaf, shoot and flower development. A series of duplications gave rise to five gene groups found throughout flowering plants. In Arabidopsis and other species, expression of two gene groups, CRABS CLAW and INNER NO OUTER, is restricted to floral organs. In contrast, members of the FILAMENTOUS FLOWER, YABBY2 and YABBY5 gene groups are also expressed in leaves and have been termed 'vegetative YABBYs'. How the five paralogue groups evolved and how their expression and function diversified have remained largely unresolved, precluding a reconstruction of the natural history of this gene family. Here, we report new genes from Eschscholzia californica (Ranunculales, Papaveraceae) that we use together with currently available database sequences in a comprehensive phylogenetic re-evaluation of the YABBY gene family. Multilayered Bayesian analysis covering seed plants allowed us to locate Eschscholzia YABBY sequences within the gene family phylogeny. We established that vegetative YABBYs do not form a monophyletic clade, and that CRABS CLAW and FILAMENTOUS FLOWER arose from a common ancestor gene. INNER NO OUTER genes are sister to that ancestral gene. We identified several conserved motifs outside of known amino acid domains that define all five angiosperm YABBY gene clades. Further, we inferred the evolution of gene expression and provide evidence for release of purifying constraint in certain branches of the gene family tree. Finally, we report expression patterns for five Eschscholzia YABBY genes consistent with functional conservation between early-diverged and core eudicots.
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highly efficient virus induced gene silencing vigs in california poppy Eschscholzia californica an evaluation of vigs as a strategy to obtain functional data from non model plants
Annals of Botany, 2007Co-Authors: Stefanie Wege, Stefan Gleissberg, Andrea Scholz, Annette BeckerAbstract:†Background and Aims Eschscholzia californica (California poppy) is an emerging model plant for ‘evo‐devo’ studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes. †Methods Commercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the Eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower. †Key Results It is shown that VIGS is able to effectively down-regulate the EcPDS gene in Eschscholzia. Various degrees of silencing were observed starting ,2 weeks after infiltration with Agrobacterium tumefaciens in 92 % of the plants. Tissue with silencing symptoms also showed complete or strong reduction of EcPDS transcripts. Strong silencing resulted in almost completely white petals, fruits, shoots and leaves. Plants with a strong degree of silencing will eventually die off; however, others are able to produce EcPDS gene product even after a strong initial silencing and will recover. Silencing was found to be not always systemic, but was often restricted to certain organs or parts of organs. †Conclusions VIGS is an effective, fast and transient method to down-regulate gene expression in Eschscholzia. It serves well to detect prominent phenotypes which may become obvious even if some target gene transcript remains in the plant tissue. However, subtle phenotypes will be more difficult to detect, as extremely strong silencing effects occur in ,10 % of all flowers from infected plants.
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floral and vegetative morphogenesis in california poppy Eschscholzia californica cham
International Journal of Plant Sciences, 2005Co-Authors: Annette Becker, Stefan Gleissberg, David R SmythAbstract:For studies of the evolution of development in angiosperms, early‐diverging eudicot taxa are of particular interest for comparisons with established core eudicot model plants, such as Arabidopsis. Here we provide a detailed description of shoot and floral development of the basal eudicot California poppy (Eschscholzia californica). Rosette formation in the vegetative phase is accompanied by increased leaf complexity and shoot apex size. The flowering phase is characterized by internode elongation, formation of dissected cauline leaves, terminal flowers, and basipetal inflorescence branching. For developing flowers and fruits, we have defined 14 stages according to important landmark events, from inflorescence primordium initiation through seed dispersal. Floral organ initiation, morphogenesis, increase in floral meristem size, and the surface structure of mature floral organs are recorded in detail. The duration of the later floral stages, as well as the path of pollen tube growth in the gynoecium, is doc...
Toni M. Kutchan - One of the best experts on this subject based on the ideXlab platform.
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a concerted mechanism for berberine bridge enzyme
Nature Chemical Biology, 2008Co-Authors: Andreas Winkler, Toni M. Kutchan, Andrzej Franciszek Lyskowski, Sabrina Riedl, Martin Puhl, Peter Macheroux, Karl GruberAbstract:Berberine bridge enzyme catalyzes the conversion of (S)-reticuline to (S)-scoulerine by formation of a carbon-carbon bond between the N-methyl group and the phenolic ring. We elucidated the structure of berberine bridge enzyme from Eschscholzia californica and determined the kinetic rates for three active site protein variants. Here we propose a catalytic mechanism combining base-catalyzed proton abstraction with concerted carbon-carbon coupling accompanied by hydride transfer from the N-methyl group to the N5 atom of the FAD cofactor.
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isolation and analysis of a gene bbe1 encoding the berberine bridge enzyme from the california poppy Eschscholzia californica
Plant Molecular Biology, 1998Co-Authors: Kay Hauschild, Hubert H Pauli, Toni M. KutchanAbstract:A genomic clone, bbe1 was isolated that encodes the methyl jasmonate-inducible berberine bridge enzyme of antimicrobial benzophenanthridine alkaloid biosynthesis in the California poppy Eschscholzia californica. Genomic DNA gel blot analysis indicates that two genes are present in the E. californica genome that code for the berberine bridge enzyme reading frame. Each coding region is apparently preceeded by a unique promoter sequence. The bbe1 gene contains no introns and one transcriptional start site. A 41 nucleotide region between −496 and −455 of the 5′-flanking region appears to be essential for promoter activity in E. californica. The promoter displayed an unexpectedly high species specificity, being active in only E. californica and Thalictrum bulgaricum, out of 28 cell suspension cultures tested.
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barbiturate induced benzophenanthridine alkaloid formation proceeds by gene transcript accumulation in the california poppy
Biochemical and Biophysical Research Communications, 1997Co-Authors: Georg Haider, Thomas Kislinger, Toni M. KutchanAbstract:Abstract Four barbiturates, barbituric acid, butethal, phenobarbital, and 2-thiobarbituric acid, of fourteen tested were found to induce accumulation of benzophenanthridine alkaloids in cell suspension cultures of the California poppy Eschscholzia californica. When the plant cell suspension cultures were treated with 1 mM barbiturate, alkaloids accumulated to 100 mg/l within four days. This is a level comparable to that achieved with 300 μM concentration of the established secondary metabolite inducer methyl jasmonate. In contrast to methyl jasmonate, barbituric acid, and 2-thiobarbituric acid, butethal and phenobarbital treatment resulted in a different alkaloid profile, suggesting that only select cytochrome P-450 genes were activated by these latter two barbiturates. RNA gel blot analysis of barbiturate induced cell cultures confirmed that transcripts of at least two benzophenanthridine alkaloid biosynthetic genes cyp80b1 (encoding the cytochrome P-450-dependent monooxygenase ( S )- N -methylcoclaurine 3′-hydroxylase) and bbe1 (encoding the covalently flavinylated berberine bridge enzyme) increased up to 5- to 7-fold over control values.
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Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture
Phytochemistry, 1994Co-Authors: Toni M. Kutchan, A Bock, H DittrichAbstract:The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvolfia serpentina and the berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes.
Annette Becker - One of the best experts on this subject based on the ideXlab platform.
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Duplicated STM-like KNOX I genes act in floral meristem activity in Eschscholzia californica (Papaveraceae)
Development Genes and Evolution, 2013Co-Authors: Angelika Stammler, Annette Becker, Sandra S. Meyer, Alastair R. Plant, Brad T. Townsley, Stefan GleissbergAbstract:In angiosperms, the shoot apical meristem is at the origin of leaves and stems and is eventually transformed into the floral meristem. Class I knotted -like homeobox ( KNOX I ) genes are known as crucial regulators of shoot meristem formation and maintenance. KNOX I genes maintain the undifferentiated state of the apical meristem and are locally downregulated upon leaf initiation. In Arabidopsis , KNOX I genes, especially SHOOTMERISTEMLESS ( STM ), have been shown to regulate flower development and the formation of carpels. We investigated the role of STM -like genes in the reproductive development of Eschscholzia californica , to learn more about the evolution of KNOX I gene function in basal eudicots. We identified two orthologs of STM in Eschscholzia , EcSTM1 and EcSTM2 , which are predominantly expressed in floral tissues. In contrast, a KNAT1 / BP -like and a KNAT2 / 6 -like KNOX I gene are mainly expressed in vegetative organs. Virus-induced gene silencing (VIGS) was used to knockdown gene expression, revealing that both EcSTM genes are required for the formation of reproductive organs. Silencing of EcSTM1 resulted in the loss of the gynoecium and a reduced number of stamens. EcSTM2 -VIGS flowers had reduced and defective gynoecia and a stronger reduction in the number of stamen than observed in EcSTM1 -VIGS. Co-silencing of both genes led to more pronounced phenotypes. In addition, silencing of EcSTM2 alone or together with EcSTM1 resulted in altered patterns of internodal elongation and sometimes in other floral defects. Our data suggest that some aspects of STM function present in Arabidopsis evolved already before the basal eudicots diverged from core eudicots.
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virus induced gene silencing of the alkaloid producing basal eudicot model plant Eschscholzia californica california poppy
Methods of Molecular Biology, 2013Co-Authors: Dawit G Tekleyohans, Sabrina Lange, Annette BeckerAbstract:Eschscholzia californica (California poppy), a member of the basal eudicot family of the Papaveraceae, is an important species to study alkaloid biosynthesis and the effect of alkaloids on plant metabolism. More recently, it has also been developed as a model system to study the evolution of plant morphogenesis. While progress has been made towards establishing methods for generating genetically modified cell culture lines, transcriptome data and gene expression analysis, the stable transformation and subsequent regeneration of transgenic plants has proven extremely time consuming and difficult. Here, we describe in detail a method to transiently down regulate expression of a target gene by virus-induced gene silencing (VIGS) and the subsequent analysis of the VIGS treated plants. VIGS in E. californica allows for the study of gene function within 2 to 3 weeks after inoculation, and the method proves very efficient, enabling the rapid analysis of gene functions.
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highly efficient virus induced gene silencing vigs in california poppy Eschscholzia californica an evaluation of vigs as a strategy to obtain functional data from non model plants
Annals of Botany, 2007Co-Authors: Stefanie Wege, Stefan Gleissberg, Andrea Scholz, Annette BeckerAbstract:†Background and Aims Eschscholzia californica (California poppy) is an emerging model plant for ‘evo‐devo’ studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes. †Methods Commercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the Eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower. †Key Results It is shown that VIGS is able to effectively down-regulate the EcPDS gene in Eschscholzia. Various degrees of silencing were observed starting ,2 weeks after infiltration with Agrobacterium tumefaciens in 92 % of the plants. Tissue with silencing symptoms also showed complete or strong reduction of EcPDS transcripts. Strong silencing resulted in almost completely white petals, fruits, shoots and leaves. Plants with a strong degree of silencing will eventually die off; however, others are able to produce EcPDS gene product even after a strong initial silencing and will recover. Silencing was found to be not always systemic, but was often restricted to certain organs or parts of organs. †Conclusions VIGS is an effective, fast and transient method to down-regulate gene expression in Eschscholzia. It serves well to detect prominent phenotypes which may become obvious even if some target gene transcript remains in the plant tissue. However, subtle phenotypes will be more difficult to detect, as extremely strong silencing effects occur in ,10 % of all flowers from infected plants.
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floral and vegetative morphogenesis in california poppy Eschscholzia californica cham
International Journal of Plant Sciences, 2005Co-Authors: Annette Becker, Stefan Gleissberg, David R SmythAbstract:For studies of the evolution of development in angiosperms, early‐diverging eudicot taxa are of particular interest for comparisons with established core eudicot model plants, such as Arabidopsis. Here we provide a detailed description of shoot and floral development of the basal eudicot California poppy (Eschscholzia californica). Rosette formation in the vegetative phase is accompanied by increased leaf complexity and shoot apex size. The flowering phase is characterized by internode elongation, formation of dissected cauline leaves, terminal flowers, and basipetal inflorescence branching. For developing flowers and fruits, we have defined 14 stages according to important landmark events, from inflorescence primordium initiation through seed dispersal. Floral organ initiation, morphogenesis, increase in floral meristem size, and the surface structure of mature floral organs are recorded in detail. The duration of the later floral stages, as well as the path of pollen tube growth in the gynoecium, is doc...
Andrea Scholz - One of the best experts on this subject based on the ideXlab platform.
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silencing of ecflo a floricaula leafy gene of the california poppy Eschscholzia californica affects flower specification in a perigynous flower context
International Journal of Plant Sciences, 2013Co-Authors: Sara Wreath, Andrea Scholz, Conny Bartholmes, Oriane Hidalgo, Stefan GleissbergAbstract:Flowers are reproductive shoots produced by determinate floral meristems. The role of FLORICAULA/LEAFY-like genes in the specification of flowers varies between lineages and has not been characterized in a basal eudicot species. Here we report the phenotypic effects of virus-induced silencing of EcFLO, a FLORICAULA/LEAFY homologue in the California poppy Eschscholzia californica. EcFLO silencing resulted in repeated sepal and petal whorl formation and internodes between calyxes. A subset of silenced flowers showed reduced petal and stamen numbers and mosaic identities of floral organs. We also provide expression data of other floral regulator genes and conclude that EcFLO contributes to certain aspects of flower development, reflecting both functional conservation and differentiation among basal eudicots, core eudicots, and monocot grasses. EcFLO is involved in stamen and petal initiation, floral organ identity delimitation, suppression of internodes, and limitation of the number of perianth whorls. Furth...
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funktionelle untersuchungen von floricaula und knox genen in Eschscholzia californica cham
2008Co-Authors: Andrea ScholzAbstract:FLORICAULA (FLO) und KNOTTED1-like Homoobox (KNOX)-Gene ubernehmen neben ihren konservierten Funktionen in der Achsenentwicklung in verschiedenen Eudikotylen eine Funktion in der Fiederblattentwicklung. Zur Klarung der Frage nach dem ursprunglichen Regulationsweg der Fiederblattentwicklung in Hinblick auf FLO und KNOX-Gene innerhalb der Eudikotylen wurde hier die Bedeutung dieser Gene fur die Fiederblattentwicklung von Eschscholzia californica als Modell fur die Ranunculales, die Schwestergruppe aller anderen Eudikotylen untersucht. Es wurde ein Protokoll zur Erzeugung von somatischen Embryonen aus unreifen Samen entwickelt. Wege zur Herstellung von Mutanten durch Agrobacterium-vermittelte Transformation werden vorgeschlagen. Die Bedeutung von Auxin fur die Blattentwicklung und die Untersuchung der Interaktion von Eschscholzia CALIFORNICA FLORICAULA (EcFLO) und des KNOX- Gens Eschscholzia CALIFORNICA SHOOT MERISTEMLESS (EcSTM) mit Auxin wurde durch Hemmung des Auxintransports untersucht. Trotz gravierender Storungen in der Blattpositionierung und -morphologie konnten Expressionsanderungen beider Gene nicht nachgewiesen werden. Ein Funktionsverlust von EcFLO und KNOX-Genen in E. californica wurden mittels Virus induziertem Gen Silencing (VIGS) erzeugt. VIGS von EcFLO rief keinen Phanotypen hervor. VIGS des KNOX-Gens EcSTM erzeugte dagegen in einigen Pflanzen eine Reduktion der Fiederzahl. Auch molekularbiologisch konnte das Silencing von EcSTM, nicht aber das Silencing von EcFLO nachgewiesen werden. Die Ergebnisse belegen die Notwendigkeit des ungestorten Auxintransports fur die Blattentwicklung von E. californica und machen die Beteiligung des KNOX-Gens EcSTM an der Blattentwicklung wahrscheinlich. Die Beteiligung von EcFLO an der Fiederbildung konnte nicht nachgewiesen werden. %%%%Besides their conserved function in the development of the plant axis, FLORICAULA (FLO) and KNOTTED1-like homeobox (KNOX)-genes both play a role in dissected leaf development of some eudicot species. The aim of this work was to elucidate the ancestral genetic pathway of dissected leaf development in eudicots with regard to FLO and KNOX. Eschscholzia californica was used as a model plant for the Ranunculales, the sister group of all other eudicots. A protocol to obtain somatic embryos via callus culture from unripe seeds was established and methods to produce stable mutants via Agrobacterium-mediated transformation are proposed. The importance of auxin for leaf development and the interaction of auxin and Eschscholzia CALIFORNICA FLORICAULA (EcFLO) and the KNOX-gene Eschscholzia CALIFORNICA SHOOT MERISTEMLESS (EcSTM) was investigated by inhibiting auxin transport. The inhibition of auxin transport caused serious defects in leaf positioning and leaf development, but no altered expression of either genes was obvious. Virus induced gene silencing (VIGS) was used to cause a loss of function of EcFLO and EcSTM. While VIGS of EcFLO showed no phenotypic effect, some EcSTM-silenced plants showed a slight reduction of leaflet number. The…
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highly efficient virus induced gene silencing vigs in california poppy Eschscholzia californica an evaluation of vigs as a strategy to obtain functional data from non model plants
Annals of Botany, 2007Co-Authors: Stefanie Wege, Stefan Gleissberg, Andrea Scholz, Annette BeckerAbstract:†Background and Aims Eschscholzia californica (California poppy) is an emerging model plant for ‘evo‐devo’ studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes. †Methods Commercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the Eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower. †Key Results It is shown that VIGS is able to effectively down-regulate the EcPDS gene in Eschscholzia. Various degrees of silencing were observed starting ,2 weeks after infiltration with Agrobacterium tumefaciens in 92 % of the plants. Tissue with silencing symptoms also showed complete or strong reduction of EcPDS transcripts. Strong silencing resulted in almost completely white petals, fruits, shoots and leaves. Plants with a strong degree of silencing will eventually die off; however, others are able to produce EcPDS gene product even after a strong initial silencing and will recover. Silencing was found to be not always systemic, but was often restricted to certain organs or parts of organs. †Conclusions VIGS is an effective, fast and transient method to down-regulate gene expression in Eschscholzia. It serves well to detect prominent phenotypes which may become obvious even if some target gene transcript remains in the plant tissue. However, subtle phenotypes will be more difficult to detect, as extremely strong silencing effects occur in ,10 % of all flowers from infected plants.
S U Park - One of the best experts on this subject based on the ideXlab platform.
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Somatic embryogenesis from embryogenic cell suspension cultures of california poppy, Eschscholzia californica Cham
In Vitro Cellular & Developmental Biology - Plant, 2001Co-Authors: S U ParkAbstract:A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented with 0.5 mg 1^−1 (2.26 μ M ) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition, gyratory shaker speed, various carbon sources, different cytokinins, and AgNO_3 on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog (MS) salts and vitamins supplemented with 40 g 1^−1 (117 m M ) sucrose, 0.05 mg 1^−1 (0.22 μ M ) 6-benzylaminopurine, and 10 mg l^−1 (58.8 μ M ) AgNO_3. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development of somatic embryos was promoted by AgNO_3 at concentrations below 10 mg l^−1 (58.8 μ M ). A semi-solid medium containing 1.5 g l^−1 Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture.
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agrobacterium mediated genetic transformation of california poppy Eschscholzia californica cham via somatic embryogenesis
Plant Cell Reports, 2000Co-Authors: S U ParkAbstract:An efficient Agrobacterium-mediated protocol for the stable genetic transformation of Eschscholzia californica Cham. (California poppy) via somatic embryogenesis is reported. Excised cotyledons were co-cultivated with A. tumefaciens strain GV3101 carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l−1 paromomycin as the selective agent and 200 mg l−1 timentin to eliminate the Agrobacterium. Four to five weeks after infection, paromomycin-resistant calli grew on 80% of explants in the presence of 2.0 mg l−1 1-naphthaleneacetic acid (NAA) and 0.1 mg l−1 6-benzylaminopurine (BAP). Calli were cultured on somatic embryogenesis induction medium containing 1.0 mg l−1 NAA and 0.5 mg l−1 BAP, and somatic embryos were visible on 30% of the paromomycin-resistant calli within 3–4 weeks. Three to four weeks after the somatic embryos were transferred to phytohormone-free plant regeneration medium, 32% converted to paromomycin-resistant plants. Detection of the neomycin phosphotransferase gene and high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all plant tissues confirmed the integrative transformation of the regenerated plants. The normal alkaloid profile of California poppy was unaffected by the transformation process; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of the benzophenanthridine alkaloid pathway.
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high efficiency somatic embryogenesis and plant regeneration in california poppy Eschscholzia californica cham
Plant Cell Reports, 2000Co-Authors: S U ParkAbstract:The development of a rapid protocol for high-efficiency somatic embryogenesis and plant regeneration from seed-derived embryogenic callus cultures of California poppy (Eschscholzia californica Cham.) is reported. The optimized procedure required less than 13 weeks from the initiation of seed cultures to the recovery of plantlets and involved the sequential transfer of cultures onto solid Murashige and Skoog basal medium containing three different combinations of growth regulators. All steps were performed at 25 °C. Friable primary callus was induced from seeds of E. californica cultured on medium supplemented with 1.0 mg l−1 2,4-dichlorophenoxyacetic acid. The primary callus was transferred to medium containing 1.0 mg l−1 1-naphthaleneacetic acid and 0.5 mg l−1 6-benzylaminopurine to establish embryogenic callus and promote somatic embryogenesis. Regenerated plantlets were recovered after the conversion of somatic embryos on medium containing 0.05 mg l−1 6-benzylaminopurine and showed normal development. Embryogenic callus was induced at a frequency of 85%, an average of 45 somatic embryos were produced per callus, 90% of the somatic embryos converted, and about 70% of the plantlets were recovered in soil. The growth rate of somatic embryo-derived shoots could be increased by gibberellic acid treatment, but the resulting plantlets were hyperhydritic.