0000000000019454
AUTHOR
Wolfgang Wernicke
The apparent loss of tissue culture competence during leaf differentiation in yams (Dioscorea bulbifera L.)
Explants taken from the leaves of yams (Dioscorea bulbifera L.) at different stages of development were cultured in vitro on a checkerboard using various combinations and/or concentrations of auxin (2,4-d) and cytokinin (6-BAP). An addition of cytokinin to the culture media was not essential for callus induction from explants derived from young leaves in the very early stages of expansion. When the leaves expanded further they required cytokinin and the requirement increased considerably during expansion. Explants taken from fully expanded leaves were no longer able to proliferate, even when extremely high concentrations of cytokinins were applied. Callus grown from highly immature leaves w…
Cell Cycle in Potentially Dedifferentiating Cereal Mesophyll Protoplasts Cultured in vitro II. Behaviour of the Cytoskeleton
Summary Following up on the recent finding that mesophyll protoplasts of wheat and oats were able to reenter the cell cycle when cultured in vitro , the behaviour of the cytoskeleton was studied. The aim was to elucidate whether changes in the cytoskeletal patterns correlate with the abnormal progression through the cycle observed previously. Protoplasts freshly isolated from mature mesophyll tissue (type B) exhibited very few, fragmented microtubules, whereas mesophyll protoplasts isolated from immature leaf tissue (type A) showed an abundant network fo microtubules. Type B protoplasts of wheat, which were able to reenter but recalcitrant to complete S-phase, failed to reestablish a microt…
Effect of auxin on the mitotic cell cycle in cultured leaf segments at different stages of development in wheat
Young leaves of Triticum timopheevi Zukh. show a defined gradient of development. One-mm-long sections from such leaves were cultured in vitro. At a low concentration of exogenous auxin, cells in the most basal, highly meristematic explants divided readily in culture, but in the absence of auxin they soon ceased dividing and were arrested in G1 and G2 of the mitotic cell cycle. In the region adjoining the meristem, where most cells were arrested in G1, very high concentrations of auxin had to be applied to reinitiate cell division, i.e. stimulate transitions from G1 to S-phase and from G2 to mitosis. Above this potentially auxin-responsive region, which represented less than 50% of the tota…
Improved Mesophyll Protoplast Culture and Plant Regeneration in Arabidopsis thaliana (L.) Heynh., Genotype Landsberg Erecta
Summary The response of in vitro cultured Arabidopsis thaliana mesophyll protoplasts was investigated with the aim of characterising the crucial factors that affect plating efficiencies and plant regeneration. We selected the genotype Landsberg erecta, which is frequently used for genetic studies, but which is known to respond poorly in protoplast culture. Factors permitting vigorous vegetative growth of the donor plants, i.e. short light period and avoidance of high temperature, were found to be advantageous. Selection of competent leaves and fractionation of protoplast populations indicated that predominantly protoplasts derived from still expanding, yet not highly immature mesophyll cell…
Microtubules and the Establishment of Apparent Cell Wall Invaginations in Mesophyll Cells of Pinus silvestris L.
Summary Ridges of cell wall material protruding into the cellular lumen are characteristic of Pinus silvestris mesophyll cells. These ridges, e.g., appear to increase the inner surface for optimal dispersal of organelles, especially of chloroplasts. We show that the ridges are the result of local, brace-like wall reinforcements deposited during early turgor-driven expansion growth of postmitotic cells. The reinforced sites resist expansion and become the base of deep, narrow folds representing the ridges, while the intervening thin-walled areas evaginate as the cell volume increases. Cell wall material is deposited uniformly after expansion to stabilize the protrusions. There is a distinct …
γ-Tubulin in Barley and Tobacco: Sequence Relationship and RNA Expression Patterns in Developing Leaves during Mitosis and Post-Mitotic Growth
gamma-Tubulin is typically associated with microtubule organising centres, such as the centrosome, and appears to mediate microtubule nucleation. Centrosomes are usually not found in higher plants, but active genes homologous to gamma-tubulin have been identified in the plant kingdom, including the angiosperms Arabidopsis, maize and rice. We have isolated and characterised gamma-tubulin cDNA sequences of two further angiosperm species, barley and tobacco. Sequence comparison revealed a phylogenetic tree with distinct clusters corresponding to the systematic position of the species. Furthermore, domains, thought to be exposed in the folded protein and to be candidates for interaction with as…
The First Cell Division Cycle in Nicotiana Mesophyll Protoplasts Cultured in Vitro. I. Methods to Determine Cycle Kinetics
Summary The progress of freshly isolated and in vitro cultured mesophyll protoplasts through the first mitotic cycle was studied with the aim of determining the frequency and timing of the G2-phase. Putative G1 and G2 phase cells were identified by measuring the relative DNA content through improved cytofluorimetry of DAPI-stained nuclei. S-phase nuclei were identified by labelling with the thymidine-analogue BrdU, which was subsequently immuno-localized with a BrdU-specific monoclonal antibody. The cells entered S-phase after 12–24h and a maximum of G2 cells was observed 24–36h after culture initiation. Most of the cells had divided after 48 to 72 h.
The ambiguous role of 2,4-dichlorophenoxyacetic acid in wheat tissue culture
The very basal, highly immature regions of dissected young leaves of Triticum aestivum L. cv. Kite formed adventitious roots on a nutrient medium supplemented with comparatively low concentrations (0.16 to 0.63 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). Higher concentrations (up to 640 μM) had to be applied to stimulate growth from more mature regions higher up the leaf. Yet, already at 2.5 μM roots were less distinct and more callus-like, and eventually (at 10 to 640 μM) only a subculturable callus of apparently suppressed, slowly proliferating root primordia developed. Furthermore, at the most basal, highly immature regions growth was significantly retarded when the auxin concentratio…
Tubulin gene expression persists during all changes in microtubular arrays related to cell shaping in growing barley leaves
Summary Levels of α-tubulin RNA were determined in consecutive leaf segments taken along young, growing barley leaves by Northern blotting. The leaves exhibited a developmental gradient typical of graminaceous species ranging from a meristem at the base to fully differentiated cells at the tip. The objective was to ascertain whether or not high steady-state tubulin RNA levels accompany characteristic changes in microtubular arrays known to occur during cell division and differentiation. Of particular interest was the differentiation zone comprising a succession of well defined changes in microtubular arrays that appear to be involved in the control of cell shaping. High steady-state levels …
Changes in the density of microtubular networks in mesophyll cells and mesophyll derived protoplasts of Nicotiana and Triticum during leaf development
Changes in the density of microtubular mesh-works were analysed in mesophyll cells and mesophyll derived protoplasts of Nicotiana tabacum L. and Triticum aestivum L. during leaf development. The main purpose of this study was to test whether the low density, if not lack, of microtubular networks recently described in protoplasts that had been isolated from fully differentiated mesophyll cells happened during protoplast isolation or whether the loss of microtubules actually occurred during differentiation of the leaf tissue. Immunofluorescence microscopy showed that the density of the microtubular cytoskeleton in the leaf tissue decreased steadily after cessation of cell growth in both speci…
The First Cell Division Cycle in Nicotiana Mesophyll Protoplasts Cultured in vitro II. Microtubules and the Plane of Division
Summary Reorganization of the microtubular cytoskeleton prior to and during the first cell division cycle of Nicotiana mesophyll protoplasts was investigated using immunofluorescence microscopy. Bands of microtubules reminiscent of PPBs were found. However, their structure was comparatively diffuse. In addition, frequencies were low compared to those in meristem cells of organized tissues. Abnormalities during phragmoplast and cell plate formation were observed. The question is raised whether the formation of abnormal PPBs is related to strategic difficulties in co-ordinating the formation of a coherent plate of cytoplasm (phragmosome) traversing the vacuole to permit smooth bisecting of th…
Changes in tubulin protein expression accompany reorganization of microtubular arrays during cell shaping in barley leaves
Barley (Hordeum vulgare L.) leaves grow from the base and thus exhibit a smooth developmental gradient. Developing mesophyll cells acquire their typical lobed shape synchronously along this gradient. Successive changes in the patterns of cortical microtubules are involved in the shaping process. The changes include formation and dispersal of band-like structures, the establishment of a random network and a dramatic loss of microtubules after completion of cell shaping. When the relative tubulin contents were determined in consecutive segments taken along the leaf, two tubulin maxima were found. They coincided with the establishment of the microtubular bands and the random network, respectiv…
Rates of uptake and metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by cultured leaf segments at different stages of development in wheat
Immature leaf tissue of Triticum timopheevi Zukh. responded to supplied auxin and showed cell division in culture. The rates of uptake and of metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by such tissues were measured and compared with those of mature auxin-unresponsive tissue. The purpose of these experiments was to determine whether or not the concentration of auxin in cultured mature tissue was a factor limiting the cell division response to auxin. The data indicate that neither alterations in rates of uptake nor alterations in rates of metabolism could explain the loss of responsiveness to auxin which apparently occurs during cell differentiation. The results are…
Cell Cycle in Potentially Dedifferentiating Cereal Mesophyll Protoplasts Cultured in vitro. I. Abnormalities in Cycle Kinetics
Summary Protoplasts isolated from immature and mature mesophyll of wheat and oats were cultured in vitro . The potential progression from G1 (G0) to G2, through mitosis and cytokinesis was analysed. The degree and speed of progression through the cell cycle depended on the species and the state of maturity of the tissue from which the protoplasts were derived. In all the protoplast populations that were tested at least initiation of DNA synthesis was detected by labelling nuclei with the thymidine-analogue, bromodeoxyuridine (BrdU). Protoplasts derived from mature leaf tissue of wheat appeared to be recalcitrant to reach G2. Wheat protoplasts derived from immature leaf tissue appeared to pr…