Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

Biomedical potential of silver nanoparticles synthesized from calli cells of Citrullus colocynthis (L.) Schrad

Background

Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.

Results

The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m²/cm³. The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.

Conclusion

The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.     

Plant regeneration and Agrobacterium-mediated transformation of cotyledon explants of Citrullus colocynthis (L.) Schrad.

The effect of 6-benzylaminopurine (6-BA) alone or in combination with naphthaleneacetic acid or indoleacetic acid on the morphogenetic response of cotyledon explants of Citrullus colocynthis (L.) Schrad. was tested. The best results were obtained with a medium containing 25 μm 6-BA, which yielded organogenic calli at a frequency of 81.8%. When these organogenic calli were transferred to elongation medium (basal medium supplemented with 0.5 μm 6-BA), 80% produced well-developed shoots. These shoots rooted normally when cultured on rooting medium containing indolebutyric acid at 2.5 or 5.0 μm. Plants grew to maturity under greenhouse conditions and gave normal fruits. Cotyledon explants were transformed by cocultivation with Agrobacterium tumefaciens LBA4404 carrying the binary vector pBI121 which bears the reporter gene β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII). Transformants were selected for growth capacity on medium with 100 mgl^–1 of kanamycin. On the basis of β-glucuronidase expression, the transformation frequency was 14.2%. Molecular characterization by polymerase chain reaction confirmed the presence of the two genes transferred (gus, nptII) in the transgenic plants. Sexual transmission of both genes was also confirmed by studying their expression in progenies from several transgenic plants. Received: 9 May 1996 / Revision received: 3 December 1996 / Accepted: 20 January 1997     

Purification and Properties of Cytosolic Copper, Zinc Superoxide Dismutase from Watermelon (Citrullus vulgaris Schrad.) Cotyledons

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3, 540 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2-4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric points of 4.63 and 4.66.     

Purification and Properties of Cytosolic Copper,Zinc Superoxide Dismutase from Watermelon (Citrullus vulgaris Schrad.) Cotyledons

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3, 540 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2–4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric points of 4.63 and 4.66.     

Peroxisomal membrane manganese superoxide dismutase: characterization of the isozyme from watermelon (Citrullus lanatus Schrad.) cotyledons

In this work the manganese superoxide dismutase (Mn-SOD) bound to peroxisomal membranes of watermelon cotyledons (Citrullus lanatus Schrad.) was purified to homogeneity and some of its molecular properties were determined. The stepwise purification procedure consisted of ammonium sulphate fractionation, batch anion-exchange chromatography, and anion-exchange and gel-filtration column chromatography using a fast protein liquid chromatography system. Peroxisomal membrane Mn-SOD (perMn-SOD; EC 1.15.1.1) was purified 5600-fold with a yield of 2.6 mug of enzyme g(-1) of cotyledons, and had a specific activity of 480 U mg(-1) of protein. The native molecular mass determined for perMn-SOD was 108 000 Da, and it was composed of four equal subunits of 27 kDa, which indicates that perMn-SOD is a homotetramer. Ultraviolet and visible absorption spectra of the enzyme showed a shoulder at 275 nm and two absorption maxima at 448 nm and 555 nm, respectively. By isoelectric focusing, a pI of 5.75 was determined for perMn-SOD. In immunoblot assays, purified perMn-SOD was recognized by a polyclonal antibody against Mn-SOD from pea leaves, and the peroxisomal enzyme rapidly dissociated in the presence of dithiothreitol and SDS. The potential binding of the Mn-SOD isozyme to the peroxisomal membrane was confirmed by immunoelectron microscopy analysis. The properties of perMn-SOD and the mitMn-SOD are compared and the possible function in peroxisomal membranes of the peripheral protein Mn-SOD is discussed.     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski)

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.Abbreviations BAP 6 Benzylaminopurine - NAA -Naphthalene acetic acid - MS Murashige and Skoog's medium - IAA Indole acetic acid - KN Kinetin     

Peroxidases as taxonomic characters in Lophozia incisa (Schrad.) Dum. and Lophozia opacifolia (Culm.) Meyl.

Abstract

Peroxidases in 21 samples of Lophozia incisa from the Tatra Mountains and 14 samples of L. opacifolia from the Tatra Mountains and the Swiss Alps were electrophoretically assayed. Although both taxa proved to be polymorphic, consistent differences were found between them. This indicates that electrophoretic peroxidase phenotypes provide an additional taxonomic character supporting the morphological and ecological differences, a summary of which is provided.     

High efficient somatic embryogenesis development from leaf cultures of <Emphasis Type="Italic">Citrullus colocynthis</Emphasis> (L.) Schrad for generating true type clones

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L^?1 BAP + 1.0 mg L^?1 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L^?1 BAP + 0.5 mg L^?1 NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.     

Microbiology of controlled rotting of egusi (Colocynthis citrullus L.) fruits for the harvesting of the seeds

Microorganisms isolated from naturally rotting egusi fruits which are used as a source of dietary seeds, were mainly bacteria: Bacillus subtilis, B. licheniformis, B. polymyxa, B. megaterium, B. pumilus, Lactobacillus plantarum, La. brevis, Leuconostoc mesenteroides, Enterobacter aerogenes, E. cloacae, Klebsiella aerogenes, K. pneumoniae, Staphylococcus epidermidis, S. aureus, Micrococcus and Candida spp. The rotting period of egusi fruits could be decreased from 120 to 72 h by using a mixture of B. subtilis and B. licheniformis.     

Karyologische Untersuchungen an keimenden Ascosporen und Hyphenzellen von Lophodermium pinastri (Schrad.) Chev.

Zusammenfassung Die vorliegenden Untersuchungen sollen die karyologischen Verhältnisse in keimenden Ascosporen und Hyphenzellen von Lophodermium pinastri klären. Die Kernuntersuchungen erfolgten an fixiertem und gefärbtem Material (HCl-Giemsa).Bevor sich bei den Ascosporen Keimhyphen bilden, setzen Kernteilungen sowie die Bildung von Querwänden ein. 2 hauptsächliche Keimformen können unterschieden werden. L. pinastri ist ein Organismus mit mehrkernigen Hyphenzellen (im Mittel 3–6 Kerne/Zelle). Die Terminalzelle enthält im Mittel 7–30 Zellkerne. Zwischen den Kernzahlen der Terminal-und der ersten Folgezelle besteht eine schwache positive Korrelation.Die 4 untersuchten Stämme unterscheiden sich in ihren Kernzahlen hoch signifikant. Die mittlere Kernzahl je Hyphenzelle kann durch veränderte Umweltbedingungen (z. B. Nährboden) variieren.Die Ergebnisse stellen eine cytologische Bestätigung der zu beobachtenden morphologischen und physiologischen Variabilität von L. pinastri dar.

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Karyological studies of germinating ascospores and hyphal cells in Lophodermium pinastri (Schrad.) Chev.

Summary The studies clarify the karyological constitution in germinating ascopores and hyphal cells of Lophodermium pinastri. For the nuclear studies fixed and stained material (HCl-Giemsa) was used. Before the development of ascospore germ tubes, nuclear divisions occur and septa are formed. 2 main types of germinating ascospores could be distinguished. L. pinastri is an organism with multinucleate hyphal cells (average of 3–6 nuclei/cell). The hyphal tip cell contains an average number of 7–30 nuclei. There is a weak positive correlation between the number of nuclei in the tip cell and in the second cell following.The 4 strains studied differ highly significantly in their numbers of nuclei. The average number of nuclei/cell can be influenced by changed environmental conditions.The results confirm cytologically the morphological and physiological variability observed in L. pinastri.