J.H.N. Schel

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10−3 M were transferred to hormone-free medium containing 10−2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10−4 M calcium were transferred to hormone-free medium with 10−3 M calcium. At calcium concentrations between 6·10−3 and 10−2 M globular stage somatic embryos were found in cultures supplemented with 2·10−6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.

Distribution of membrane-bound calcium and activated calmodulin during somatic embryogenesis of carrot (Daucus carota L.)

SummaryThe distribution of membrane-bound calcium and activated calmodulin was visualized during carrot somatic embryogenesis by chlorotetracycline (CTC) and fluphenazine fluorescence respectively. Somatic embryos of all stages possessed a higher CTC fluorescence in comparison with the signal from their precursors, the proembryogenic masses. The CTC fluorescence was evenly distributed in the somatic embryos. In contrast, fluphenazine was observed in some regions of the proembryogenic masses only. In the globular, heart-shaped and early torpedo-shaped stage its fluorescence was restricted to the basal part of the embryo. In the older torpedo-shaped embryos also the shoot apex showed fluphenazine fluorescence. It is concluded that during carrot somatic embryogenesis a polarity in the distribution of the activated calmodulin already exists before this polarity is morphologically expressed.

In vitro development of embryoids from punched leaf disc of Coffea canephora

SummaryThe development of embryoids on punched leaf expiants fromCoffea canephora was studied. Both culture conditions as well as structural changes were investigated. The expiants were placed on a modified Murashige and Skoog medium with different concentrations of KNO3. It was found that an increased amount of KNO3 did not raise the rate of embryoid formation.After an initial callus phase embryoids developed on the edges of the punched leaf discs. First embryoids appeared after 37 days in culture. Microscopical examination showed that they arised from clusters of small, plasma-rich cells, situated at the periphery of the callus mass. Because of the small width of the suspensors the embryoids most likely originated from single cells which, however, may be part of a larger proembryonal cell complex.

Golgi Body Motility in the Plant Cell Cortex Correlates with Actin Cytoskeleton Organization

The actin cytoskeleton is involved in the transport and positioning of Golgi bodies, but the actin-based processes that determine the positioning and motility behavior of Golgi bodies are not well understood. In this work, we have studied the relationship between Golgi body motility behavior and actin organization in intercalary growing root epidermal cells during different developmental stages. We show that in these cells two distinct actin configurations are present, depending on the developmental stage. In small cells of the early root elongation zone, fine filamentous actin (F-actin) occupies the whole cell, including the cortex. In larger cells in the late elongation zone that have almost completed cell elongation, actin filament bundles are interspersed with areas containing this fine F-actin and areas without F-actin. Golgi bodies in areas with the fine F-actin exhibit a non-directional, wiggling type of motility. Golgi bodies in areas containing actin filament bundles move up to 7 μm s⁻¹. Since the motility of Golgi bodies changes when they enter an area with a different actin configuration, we conclude that the type of movement depends on the actin organization and not on the individual organelle. Our results show that the positioning of Golgi bodies depends on the local actin organization.

Isozymes as biochemical and cytochemical markers in embryogenic callus cultures of maize (Zea mays L.).

Isozyme analyses were carried out on protein extracts of non-embryogenic and embryogenic callus fromZea mays L., using polyacrylamide gel electrophoresis. We examined the isozyme patterns of glutamate dehydrogenase, peroxidase and acid phosphatase for their utility as biochemical markers of maize embryogenic callus cultures. These isozyme systems were also used to examine possible correlations between isozymes and different stages of regeneration. The zymograms of peroxidase and glutamate dehydrogenase differed for non-embryogenic and embryogenic callus. Further, some isozymes were correlated with the morphological appearance of the tissue while others seemed to be involved with the duration of the culture period. Using the same enzyme assays on fresh tissue samples we were able to test the three enzymes as cytochemical markers in embryogenic cultures. Glutamate dehydrogenase proved to be most successful to discriminate embryogenic from non-embryogenic cells.

AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana

The Arabidopsis thaliana primordia timing (pt) mutant was transformed with an AtSERK1::GUS construct. Liquid cultures of this line were used to study the relationship between somatic embryogenesis and the expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (AtSERK1) as a marker for cells competent to form embryos. In order to search for the expression of AtSERK1::GUS during early stages of somatic embryogenesis, histochemical as well as immunochemical approaches were used for the detection of beta-glucuronidase (GUS). Four sites of AtSERK1 expression were found in the embryogenic cultures: in embryogenic callus, where primary somatic embryos developed; in the basal parts of primary somatic embryos; in the outer layers of cotyledons of primary somatic embryos where secondary embryos were formed; and in provascular and vascular strands of developing somatic embryos. The in vitro expression of AtSERK1::GUS coincides with embryogenic development up to the heart-shaped stage. Prior to the expression in embryos, AtSERK1 was expressed in single cells and small cell clusters, indicating that AtSERK1 indeed marks embryogenic competence. Its expression in (pro)vascular strands, suggests that embryogenic cells in tissue culture retain at least in part their original identity.

Structural observations during androgenic microspore culture of the 4c1 genotype of Zea mays L.

SummaryThe capacity of the maize genotype 4c1 to regenerate microcalli and embryos from cultured microspores has been examined by comparing various cold pretreatments and culture media, using microspores and pollen at different stages of development. Viability of cultured cells was tested with FDA and their development was traced with light and fluorescence microscopy using DAPI as a nuclear dye.It was found that a pre-incubation of dissected flowers floating in a liquid nutrient medium at 8°C during 10–14 days was most successful for the induction of cell division. Among the developmental stages tested only the microspores appeared to regenerate. Subculture at 25°C in the same liquid medium, supplemented with 0.1 mg/l TIBA, gave highest rates of microspore division, i.e. up to 70% at 4 to 6 days of culture.All pathways described earlier for maize androgenic embryogenesis were observed within the 4c1 genotype. Symmetric divisions occurred in cultured microspores but most frequently asymmetric divisions lead to the formation of microcalli within 12 days of culture. In at least 60% of all dividing microspores cells were derived from the generative nucleus. Microcalli further developed either into loose or compact calli. Compact calli formed embryo-like structures.

SPECTRIN-LIKE PROTEINS IN PLANT NUCLEI

We analysed the presence and localization of spectrin‐like proteins in nuclei of various plant tissues, using several anti‐erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross‐reactive pair of bands at 220–240kDa, typical for human erythrocyte spectrin, and a prominent 60kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin‐like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti‐spectrin antibodies used. In some nuclei tracks of spectrin‐like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin‐like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin‐like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

Distribution of splicing proteins and putative coiled bodies during pollen development and androgenesis in Brassica napus L.

SummarySmall nuclear ribonucleoprotein particles (snRNPs) are subunits of splicing complexes, which show a transcriptiondependent localization pattern. We have analyzed the labelling pattern of snRNPs during pollen development and microspore and pollen embryogenesis inBrassica napus with an antibody which recognizes protein D of Ul, U2, U4, U5, and U6 snRNPs. It was found that nuclei were labelled almost uniformly for snRNPs in microspores and young bicellular pollen. In the generative nuclei of late-bicellular pollen and in the vegetative nuclei and sperm nuclei of mature pollen no snRNPs could be detected. The snRNP-positive nuclei contained mostly one or two brightly labelled nuclear bodies, most likely coiled bodies, often closely related to the nucleolus. These nuclear bodies increased in size from 0.5 um in nuclei of young microspores up to 2 urn in nuclei of late microspores and the vegetative nucleus of early-bicellular pollen. Also their number increased during these developmental stages. After induction of embryogenesis the size of the coiled bodies decreased to about 0.5 μm and in several occasions the coiled body was found free in the nucleoplasm, away from the nucleolus. The results support the idea that the size and number of coiled bodies coincide with changes in general nuclear activity. They also indicate that, in nuclei ofBrassica napus, at least assembly and disassembly of coiled bodies takes place in the nucleoplasm, whereas mature coiled bodies are located adjacent to the nucleolus.

Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation.

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non‐uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the%TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1 mg/l each of benzylaminopurine (BA) and α‐naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.