Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protoplasts”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1999

Development of an apparatus for monitoring protoplast isolation from plant tissues based on both dielectric and optical methods.

In order to develop a method allowing objective determination of the optimal conditions for the isolation of protoplasts, the process of protoplast isolation from plant tissues was quantitatively evaluated. First, a specialized spectrophotometer cuvette (working volume = 2.0 ml) was designed for the continuous monitoring of protoplast isolation from plant tissues based on the optical method. Homogeneous mixing of tissue sections and the protoplast suspension in the cuvette was accomplished by means of a magnetic bar. The cuvette was divided into upper and lower parts by a nylon mesh. Since tissue sections in the upper part could not pass through the mesh, they did not affect the optical path in the lower part, and only isolated protoplasts were able to move freely between the two parts. At the optimal agitation speed (200 rpm), mechanical damage to protoplasts of Catharanthus roseus did not occur. Increases in the protoplast concentration during their isolation from tissue sections (leaf and petal) could be continuously monitored by measuring the optical density (O.D.), making it possible to estimate the end of protoplast isolation. Degassing treatment of the tissues markedly enhanced protoplast isolation. In order to monitor the viable protoplast concentration, a larger specialized spectrophotometer cuvette (working volume = 25 ml) was developed which enabled simultaneous measurement of the permittivity and O.D. of the suspension to be carried out during protoplast isolation. Permittivity is a measure of the viable protoplast concentration, while the O.D. shows protoplast characteristics such as color. Using this large cuvette, the time courses of protoplast isolation from leaf and petal sections were monitored and large amounts of viable protoplasts were obtained. The protoplast isolation process after degassing treatment was described by a simple first-order reaction model and the viable protoplast isolation rate was quantitatively evaluated from the rate constant (k) on the basis of permittivity changes.

Journal Article↗
PubMed2001

Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts.

We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2*- and H2O2 and 7-fold lower levels of O2*- and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2*-, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2*- and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.

Antioxidants↗
PubMed1993

Visualizing Enzyme Secretion from Individual Barley (Hordeum vulgare) Aleurone Protoplasts.

A method was developed to detect [alpha]-amylase gene expression and [alpha]-amylase secretion from individual barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts. Protoplasts are incubated in liquid media with or without hormones and embedded in a thin film of agarose and starch, where they remain viable for up to 24 h. [alpha]-Amylase secreted by individual protoplasts digests the starch, and starch hydrolysis is visualized after 45 min by staining the preparation with I2KI. After I2KI staining, secreting protoplasts are surrounded by a clear, starch-free halo visible by light microscopy. The formation of starch-free halos is dependent on the synthesis and secretion of [alpha]-amylase and is not caused by carry-over of preformed enzyme from incubation media. Treating protoplasts with inhibitors of protein synthesis or exposing them to anaerobic conditions for 2 h before embedding them in agarose prevents the formation of halos. When [alpha]-amylase secretion is observed by counting the percentage of secreting protoplasts, the data are comparable to that obtained by measuring [alpha]-amylase secretion from a population of cells. The response of individual protoplasts to gibberellic acid (GA3) and abscisic acid measured by the thin-film method is almost identical to the response of populations of protoplasts to these hormones, validating the utility of this method. Although not generally practical for quantifying secretion, the thin-film method is uniquely useful in distinguishing secreting from nonsecreting protoplasts. In none of our experiments did more than 60% of the protoplasts secrete [alpha]-amylase when exposed to GA3, even though more than 95% of the protoplasts in the preparations were viable. Similar results were obtained when the response to GA3 was assayed at the level of gene transcription by visualizing the transient expression of a plasmid containing the promoter from [alpha]-amylase fused to the reporter gene glucuronidase in single protoplasts. The thin-film secretion assay also revealed that the response of a population of protoplasts to GA3 was not uniform with time. The effect of GA3 treatment was to gradually increase the percentage of responding protoplasts up to a maximum of 50 to 60%. Abscisic acid, which inhibits [alpha]-amylase secretion by GA3-treated protoplasts, reduced the proportion of protoplasts that secrete the enzyme.

Journal Article↗
PubMed1987

The Mechanics of Injury to Isolated Protoplasts following Osmotic Contraction and Expansion.

Micro-osmotic manipulation was used to determine the influence of osmotic contraction on the expansion potential of individual protoplasts isolated from rye (Secale cereale L. cv Puma) leaves. For protoplasts isolated from leaves of nonacclimated plants (NA protoplasts), osmotic contraction in sufficiently hypertonic solutions (>1.53 osmolal) predisposed the protoplasts to lysis during osmotic expansion when they were returned to isotonic conditions (0.53 osmolal). In contrast, for protoplasts isolated from leaves of cold acclimated plants (ACC protoplasts), osmotic contraction in either 2.6 or 4.0 osmolal solutions was readily reversible. Following osmotic contraction, the resting tension (gamma(r)) of NA protoplasts was similar to that determined for protoplasts in isotonic solutions (i.e. 110 +/- 22 micronewtons per meter). In contrast, gamma(r) of ACC protoplasts decreased from 164 +/- 27 micronewtons per meter in isotonic solutions to values close to zero in hypertonic solutions. Following expansion in hypotonic solutions, gamma(r)'s of both NA and ACC protoplasts were similar for area expansions over the range of 1.3 to 1.6. Following osmotic contraction and reexpansion of NA protoplasts, hysteresis was observed in the relationship between gamma(r) and surface area-with higher values of gamma(r) at a given surface area. In contrast, no hysteresis was observed in this relationship for ACC protoplasts. Direct measurements of plasma membrane tension (gamma) during osmotic expansion of NA protoplasts from hypertonic solutions (1.53 osmolal) revealed that gamma increased rapidly after small increments in surface area, and lysis occurred over a range of 1.2 to 8 millinewtons per meter. During osmotic expansion of ACC protoplasts from hypertonic solutions (2.6 osmolal), there was little increase in gamma until after the isotonic surface area was exceeded. These results are discussed in relation to the differences in the behavior of the plasma membrane of NA and ACC protoplasts during osmotic contraction (i.e. endocytotic vesiculation versus exocytotic extrusion) and provide a mechanistic interpretation to account for the differential sensitivity of NA and ACC protoplasts to osmotic expansion from hypertonic solutions.

Journal Article↗