Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from petal explant of Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’. Somatic embryogenesis was induced from petal explants on the Murashige and Skoog (MS) medium supplemented with 1.0 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg l^?1 6-benzyladenine (BA), yielding the highest mean number of embryos (56.3) per explant after 5 weeks of culture. We evaluated the effects of basal medium and various concentrations of sucrose on the proliferation of secondary somatic embryos. MS medium was observed to be more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, 1 % sucrose was also found to be the best in induction of secondary embryogenesis. The highest germination rate (70 %) of the somatic embryos was observed on the MS medium containing 0.2 mg l^?1 α-naphthalene acetic acid and 1 g l^?1 activated charcoal (AC). Shoots elongated rapidly and roots developed well on hormone-free MS medium with 1 g l^?1 AC and successfully acclimated in the greenhouse. Flow cytometric analysis of the primary somatic embryos, secondary somatic embryos, and the somatic embryo-obtained plants along with the parent grown in the greenhouse showed that they all had same identical peaks, indicating that there was no variation of ploidy level during the regeneration process. We expect that our report would be useful for micropropagation and Agrobacterium-mediated genetic transformation studies of this cultivar.     

The Roles of Auxin in Regulating “Shoot Branching” of Cremastra appendiculata

Cremastra appendiculata (D. Don) Makino is a mainly vegetative propagation terrestrial orchid that is a typical representative of the warm-temperate vegetation in China. In this experiment, we investigated the growth and development process of C. appendiculata leaf buds and examined their biochemical components (proteins, auxin, and cytokinin) to gain insight into the “shoot branching” of C. appendiculata pseudobulb string. The results showed that the metabolic activity of C. appendiculata pseudobulbs became lower with the increase of pseudobulb age. However, biennial and triennial pseudobulbs have higher auxin levels than annual pseudobulbs in the intact plant (P < 0.05). After decapitation, the auxin rapidly reduces in biennials. The reduction of auxin level promotes cytokinin biosynthesis, which makes the biennial dormant buds start to germinate 18 days after decapitation. These data and phenomena suggested that auxin plays important roles in regulating shoot branching of C. appendiculata, although further studies are needed to consolidate this viewpoint. Our data indirectly support the classical apical dominance theory whereby biennial pseudobulbs are strongly dependent on reduced auxin to initiate leaf bud outgrowth.

     

Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.The Snf1 protein kinase of Saccharomyces cerevisiae is the yeast ortholog of the AMP-activated protein kinase (AMPK) found in mammals and other eukaryotes. AMPK acts as a nutrient and energy sensor, becoming activated under conditions of nutrient and energy depletion (6). In mammals, AMPK plays a key role in glucose homeostasis and is a target for drugs used to treat metabolic syndrome and type 2 diabetes (34). In yeast, the Snf1 kinase plays an essential role during aerobic growth and fermentative growth on alternative carbon sources. Cells lacking Snf1 kinase activity are viable but display numerous phenotypes including poor or no growth on alternative carbon sources, defects in meiosis and sporulation, defects in response to ion stress, and defects in pseudohyphal growth (7).The Snf1 kinase and all members of the AMPK family function as heterotrimers composed of a catalytic α subunit complexed with regulatory β and γ subunits (2). The γ subunit in mammalian enzymes directly binds three molecules of AMP (26, 33), which stimulates enzyme activity by inhibiting the dephosphorylation of the conserved threonine residue in the kinase activation loop (23). In yeast, there is no evidence that the γ subunit binds AMP; however, similar to mammals, the key glucose-regulated step is the dephosphorylation of the kinase activation loop (22).In this study, we examine the role of the β subunits in the regulation of the Snf1 kinase activity. Yeasts express three isoforms of the Snf1 kinase that differ depending on which of the three distinct β subunits, Sip1, Sip2, and Gal83, is incorporated into the enzyme. Previous studies have shown that the Snf1 isoforms have distinct substrate preferences (24), subcellular localizations (32), and stress response capacities (9). Only the Snf1 isoform containing Gal83 as the β subunit is able to localize to the cell nucleus in a process that requires Sak1, one of the three Snf1-activating protein kinases. Since all three of the Snf1-activating kinases (SAKs) are capable of phosphorylating Snf1 on its activation loop (3), it has remained a mystery as to why the Sak1 kinase is specifically required for Snf1 nuclear localization.The β subunits of Snf1 as well as mammalian AMPK contain a domain that is referred to as either a carbohydrate-binding module (CBM) (11) or a glycogen-binding domain (GBD) (19). The structure of this domain has been solved (20), and it was previously shown that this domain binds most tightly to branched oligosaccharides like glycogen that contain α1→6 branches (12). The binding of glycogen to the β subunit causes an allosteric inhibition of AMPK activity and inhibits phosphorylation by the upstream activating kinase. The β subunits of yeast contain the GBDs, but the importance of binding glycogen is questionable since cells that lack the ability to make glycogen show a normal regulation of Snf1 kinase in response to glucose limitation (15). Nonetheless, the deletion of the GBD from the Gal83 protein caused an increased activity of the Snf1 enzyme and release from glucose repression. Therefore, the GBD acts as a negative regulator of kinase activity in both mammalian and fungal cells.In this study we examine the role of the GBD present in the Sip2 and Sip1 proteins. We also extend the characterization of the Gal83 GBD by determining what connection this domain has with the regulated dephosphorylation of the Snf1 kinase. Finally, we have characterized other N-terminal domains in the β subunits that control accumulation and phosphorylation.     

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Summary Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 μM and 15 μM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 μM and 15 μM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N⁶-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 μM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred. Part of a thesis submitted by M. B. K. in partial fulfillment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

Exogenous Melatonin Alleviates Heat‐Induced Oxidative Damage in Strawberry (Fragaria × ananassa Duch. cv. Ventana) Plant

Journal of Plant Growth Regulation - Melatonin is a signaling molecule that is involved in inducing plants? abiotic stress tolerance. To determine the possible effects of the melatonin...     

Regulation of Involucrin in Psoriatic Epidermal Keratinocytes: The Roles of ERK1/2 and GSK-3β

Psoriasis, a common inflammatory skin disease, is characterized by epidermal hyperplasia, abnormal differentiation, angiogenesis, immune activation, and inflammation. Involucrin is an early terminal differentiation marker of epidermal keratinocytes. In this study, we determined the immunolocalization of involucrin in psoriatic lesions and normal skin of individuals without psoriasis by means of immunofluorescence (IF) assay. Furthermore, the regulation of involucrin by interleukin (IL)-13, IL-17A, endothelin (ET)-1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was investigated by Western blot. Extracellular regulate protein kinases 1/2 (ERK1/2) and glycogen syntheses kinase-3β (GSK-3β) inhibitors were also included to define the roles of these signals in the production of involucrin in both psoriatic and normal keratinocytes. In psoriatic lesional skin, involucrin was detected in the stratum spinosum, but not in the basal or the cornified layer. In normal skin, involucrin was restricted to the granular layer and the upper stratum spinosum. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ up-regulate expression of involucrin in both psoriatic and normal keratinocytes. However, this effect was abolished by ERK1/2 and GSK-3β inhibitors. In conclusion, involucrin is up-regulated in psoriatic keratinocytes. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ could increase involucrin protein levels in psoriatic and normal keratinocytes. The ERK1/2 and GSK-3β signaling pathways may play positive roles in regulating epidermal differentiation as observed in psoriasis.     

The Effect of Rye Chromosomes on the Characteristic Features of Androgenesis in Wheat–Rye Substitution Lines Triticum aestivumL. (cv. Saratovskaya 29)/Secale cerealeL. (cv.Onokhoiskaya) and Triticale

The characteristic features of androgenesis in six wheat–rye substitution lines Triticum aestivumL. (cv. Saratovskaya 29)/Secale cerealeL. (cv. Onokhoiskaya) and triticale (2n= 56) using anther culture at different concentrations of 2,4-D in the growth medium were studied. Under variable cultivation conditions, the significant effect of genotypic diversity on the variability of such androgenesis parameters as the frequency of productive anthers, the frequency of embryoid formation, and the frequency of total regenerated plantlets, was shown. It was demonstrated that chromosomes 1R, 3R, and 7R stimulated the formation of androgenous embryoids, while chromosome 5R produced an opposite effect. In triticale and substitution lines, the regeneration ability of androgenous embryoids induced by elevated 2.4-D concentrations was inhibited. Chromosome 1R of the Onokhoiskaya cultivar was suggested to contain genes suppressing regeneration of green plantlets, while chromosome 3R, conversely, stimulated their formation. Chromosomes 1R, 2R, 3R, and 7R of the Onokhoiskaya cultivar did not inhibit the spontaneous formation of androgenous hexaploids in the substitution lines.     

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for -glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3 flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5 expression control sequences.     

Comparative Analysis of Growth and Photosynthetic Characteristics of (Populus simonii × P. nigra) × (P. nigra × P. simonii) Hybrid Clones of Different Ploidides

To evaluate differences among poplar clones of various ploidies, 12 hybrid poplar clones (P. simonii × P. nigra) × (P. nigra × P. simonii) with different ploidies were used to study phenotypic variation in growth traits and photosynthetic characteristics. Analysis of variance showed remarkable differences for each of the investigated traits among these clones (P < 0.01). Coefficients of phenotypic variation (PCV) ranged from 2.38% to 56.71%, and repeatability ranged from 0.656 to 0.987. The Pn (photosynthetic rate) photosynthetic photon flux density (PPFD) curves of the 12 clones were S-shaped, but the Pn-ambient CO₂ (Ca) curves were shaped like an inverted “V”. The stomatal conductance (Gs)-PPFD and transpiration rate (Tr)-PPFD curves had an upward tendency; however, with increasing PFFD, the intercellular CO₂ concentration (Ci)-PPFD curves had a downward tendency in all of the clones. The Pn-PPFD and Pn-Ca curves followed the pattern of a quadratic equation. The average light saturation point and light compensation point of the triploid clones were the highest and lowest, respectively, among the three types of clones. For Pn-Ca curves, diploid clones had a higher average CO₂ saturation point and average CO₂ compensation point compared with triploid and tetraploid clones. Correlation analyses indicated that all investigated traits were strongly correlated with each other. In future studies, molecular methods should be used to analyze poplar clones of different ploidies to improve our understanding of the growth and development mechanisms of polyploidy.     

Detachment-accelerated ripening and senescence of strawberry (Fragaria × ananassa Duch. cv. Akihime) fruit and the regulation role of multiple phytohormones

To study the detachment stress on the ripeness of strawberry fruit, physiological characteristics of strawberry fruit on and off plant during ripeness and senescence processes were investigated. The results indicated that the ripeness of strawberry fruit upon detachment was accelerated, in terms of firmness, soluble solid content and especially color development. The color of fruit off plant changed rapidly from white to full red in 1–2 days. The respiratory rate in fruit off plant was strengthened, higher than that on plant. Abscisic acid level and ethylene production in fruit off plant were also higher than those on plant and auxin degradation was exacerbated by detachment. Expression levels of FaMYB1, FabHLH3 and FaTTG1 were generally reduced with phenotypes of redder color and more anthocyanin accumulation in fruit off plant. Results also suggested that the detachment initially stimulated ethylene and abscisic acid production and auxin degradation, which modulated ripening-related gene expression and at last enhanced fruit pigmentation.     

Micropropagation of juvenile tissue of Eucalyptus erythronema × eucalyptus stricklandii cv. ‘urrbrae gem’

Summary Micropropagation via enhanced axillary shoot proliferation was investigated in the ornamental Eucalyptus cv. ‘Urrbrae Gem’ using in vitro germinated seedlings and was successfully achieved using woody plant medium (WPM) supplemented with 2.2 μM benzylaminopurine, 1.0 μM α-naphthaleneacetic acid, and 1.5 μM gibberellic acid (GA₃), gelled with 5 g l⁻¹ Phytagel^?. Shoot proliferation was greater on WPM and QL media with GA₃ compared to B5, AP, and TK media with or without GA₃. GA₃ was required for shoot elongation as the internodes were otherwise very short and unsuitable for multiplication or root initiation. Root initiation was improved using (1/2) WPM supplemented with 20 μM indole-3-butyric acid (IBA) over a 7 d pulse, followed by subculture to IBA-free medium, compared to placing shoots on low levels of IBA for 4–6 wk. Plantlets were successfully hardened off to the natural environment via a fogger at 67% relative, humidity at 21°C for 3 d and continued to thrive as potted plants. This is the first report of successful, micropropagation in an ornamental eucalypt (subgenus Symphyomyrtus) from seedling explants.     

Genetic instability in protoclones of potato (Solanum tuberosum L. cv. ‘Bintje’): new types of variation after vegetative propagation

Summary The transmission of variation from protoplast-derived plants of tetraploid potato cultivar Bintje to tuber progeny was examined. The morphological alterations of a majority of the variant protoclones were transmitted to corresponding tuber progeny. Some of the normal and variant protoclones gave new phenotypes, or segregated into parental and new phenotypes after vegetative propagation. The ploidy levels of almost all these clones remained unchanged after propagation. It was concluded that the occurrence of variation after vegetative propagation was due to somatic segregation of chimeras resulting from gene mutations or chromosome structural rearrangements in only part of the regenerated plant. The origin of variation is discussed in the light of these results.     

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2)

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH₄NO₃ and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6–7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GR Glutathione reductase - 2iP Isopentenyl adenine - KT Kinetin - NAA Naphthaleneacetic acid - PFC Perfluorocarbon - PIC Picloram - PO Peroxidase - ROS Reactive oxygen species - SOD Superoxide dismutase - T.HCl Thiamine hydrochloride     

Measurement of selected trace elements in Olea europaea L. cv. ‘Sigoise’

BackgroundOlive-trees (Olea europaea L.) are the dominant rustic trees cultivated in the Mediterranean agricultural zones. Major and micronutrients play an indispensable role in their plant physiological functions although; the effect of trace elements on metabolic processes has not been sufficiently investigated, especially in olive-trees.MethodsIn the current study, we have used X-ray fluorescence (XRF) spectrometry to determine selected major and trace elements (Br, Cu, Fe, K, Mn, P, Rb and Zn) in the main olive cultivar cultivated in Algeria, cv.‘Sigoise’. Certified reference materials viz. IAEA-336 (Lichen) and NIST-1646a (Estuarine sediment) were evaluated simultaneously with the soil and plant samples for quality control of the analytical method.ResultsThe results show that Fe and Mn concentrations were superior in leaves than fruits. However large amounts of K, Cu and Rb were accumulated in the olive-fruits. The contents of all chemical elements were above the threshold limits for possible plant nutrient deficiencies, except for P whose concentration was in borderline requirement of olive trees. High values of a translocation factor index were found for K, Cu and Rb (TFs > 4). Principal component analysis (PCA) indicated that K was highly related with olives-fruits, suggesting that the fruit was the principal organ of K storage. Furthermore, dietary element intake through consuming olives was also estimated and compared to recommended daily intakes (RDIs) and daily permissible limits (DPLs). The estimations of chemical element intakes were below the DPLs set by WHO/FAO guidelines for human nutrition.ConclusionThe present work indicates that the concentrations of macro- and microelements (Cu, Fe, K, Mn and Zn) were above the threshold limits for possible plant deficiencies except for P, and this cultivar can easily accumulate high amount of K in their organs (predominance in olives). These findings will be used to achieve efficient fertilization for O. europaea orchards.     

Cytogenetical studies in the bridge cross Allium cepa× (A. fistulosum×A. roylei)

  Allium fistulosum harbours a number of desirable agronomical traits for the breeding of onions. However exploitation of A. fistulosum for onion breeding via direct sexual hybridization is problematic. Therefore, we examined if a bridge cross, using A. roylei as a bridging species, might provide an alternative. By means of genomic in situ hybridization (GISH) we showed that each of the three parental genomes can be distinguished from the others in interspecific hybrids, suggesting that these genomes contain sufficiently different repetitive DNA families. We succeeded in carrying out multi-colour GISH to metaphase spreads of a first-generation bridge-cross individual [A. cepa× (A. fistulosum×A. roylei], which is composed of three parental genomes. Recombination between the genomes of A. fistulosum and A. roylei took place to a large extent: 7 recombined chromosomes were observed, and it could be shown that the proximal regions of the recombined A. fistulosum/A. roylei chromosomes belonged to the former, whereas the distal parts belonged to the latter. The high percentage of bound bivalent arms in metaphase I of pollen mother cells of a fertile bridge-cross individual suggests the introgression of A. fistulosum genes, mediated by A. roylei, into the genome of A. cepa. However, the presence of univalents reflects decreased pairing and recombination between the three genomes. Pollen fertility and pollen-tube growth of the first-generation bridge-cross individual seem to be sufficient to produce a second generation bridge-cross (A. cepa×first-generation bridge cross) progeny. Received: 27 May 1997 / Accepted: 30 June 1997