Wheat Protoplasts Research Articles

The involvement of a G-protein in phytochrome-regulated, Ca2+-dependent swelling of etiolated wheat protoplasts

The involvement of a G-protein in phytochrome-regulated, Ca2+-dependent swelling of etiolated wheat protoplasts

Regeneration of Plants from Embryogenic Suspension Culture Protoplasts of Wheat (Triticum aestivum L.)

We describe the regeneration of plants from protoplasts of wheat isolated from a regenerable embryogenic suspension culture that was initiated from immature embryo-derived ‘aged’ compact callus. Proto-colonies were also obtained from a non-regenerable cell line established from friable callus that arose spontaneously on the aged callus, but did not give rise to any plants. Plating efficiencies well in excess of twenty percent were routinely obtained from protoplasts isolated from the regenerable as well as the non-regenerable cell lines in Kao and Michyaluk, as well as Murashige and Skoog based, nutrient media. Four to five week old protocolonies, derived from regenerable suspension culture protoplasts, formed distinct somatic embryos and shoots upon transfer to various regeneration media. Shoots were rooted on transfer to rooting media. Successive transfer of the plantlets to MS or half-strength MS medium, with or without activated charcoal, produced healthy plants that were successfully transferred to soil and grown in the greenhouse.

Photosynthesis in chloroplasts rapidly isolated from primary leaves of oat (Avena sativa L.): effects of orthophosphate, metal ion and chelators

Abstract A simple mechanical method for the rapid isolation of chloroplasts with high rates of photosynthesis from young leaves of oat (Avena sativa L.) was described. The photosynthetic activity of these chloroplasts was stable for at least 2 h with rates of CO2‐dependent O2 evolution of 30–40 μmol g 1 Chl s 1. The photosynthetic properties of these chloroplasts were similar to those reported for spinach and pea chloroplasts isolated by mechanical disruption. The pH optimum for photosynthetic O2 evolution was pH 7.6. The induction time was 0.5–2 min. Maximal rates of photosynthetic O2 evolution in these chloroplast preparations were obtained in the absence of both divalent cations and EDTA. Addition of divilent cations strongly inhibited photosynthesis which could be partially restored by the subsequent addition of EDTA. But when these cations were not present in the assay medium the addition of EDTA greater than 1 mol m 3 decreased photosynthetic activity. The optimal orthophosphate concentration required for photosynthesis in these chloroplast preparations was 0.2–0.3 mol m 3. In contrast, the addition of pyrophosphate either in the light or dark inhibited photosynthesis. In a comparative study, chloroplasts were also isolated from oat and wheat (Triticum aestivum L., cultivar Hybrid C306) protoplasts. These chloroplast preparations were found to have properties similar to those determined for oat chloroplasts isolated by the mechanical method reported above.

The effect of a calcium‐channel antagonist, nifedipine and agonist, Bay K‐8644, on the phytochrome‐controlled swelling of etiolated wheat protoplasts

The effect of external Ca2+ and Ca2+‐channel modulators on the phytochrome‐controlled swelling of etiolated wheat (Triticum aestivum L. cv. Arminda) mesophyll protoplasts has been studied. The red light (R)‐stimulated swelling of the protoplasts requires Ca2+ in the surrounding medium and maximum response was observed in a medium containing I mM CaCI2. Far‐red light (FR) irradiation of protoplasts in the presence or absence of Ca2+ does not influence the protoplast volume. The Ca2+‐channel antagonist nifedipine prevents R‐induced protoplast swelling at very low concentrations (0.1 μM). The Ca2+ ‐channel agonist Bay K‐8644 stimulates the swelling of protoplasts incubated in darkness or irradiated with FR. Action of nifedipine depends on whether it is applied before or after the R pulse. The results are compatible with the hypothesis that phytochrome controls the activity of dihydropyridine‐sensitive L‐type Ca2+ channels.

The effect of a calcium-channel antagonist, nifedipine, and agonist; Bay K-8644, on the phytochrome-controlled swelling of etiolated wheat protoplasts

The effect of a calcium-channel antagonist, nifedipine, and agonist; Bay K-8644, on the phytochrome-controlled swelling of etiolated wheat protoplasts

Metabolism of Choline Chloride and Its Analogs in Wheat Seedlings

To clarify the mechanisms of the biological activities of choline and its analogs, the uptake and metabolism of these compounds were studied in wheat protoplasts and wheat seedlings

Compartmentation and transport of 1‐aminocyclopropane‐1‐carboxylic acid and N‐malonyl‐1‐aminocyclopropane‐1‐carboxylic acid in barley and wheat mesophyll cells and protoplasts

Intracellular compartmentation of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and N‐malonyl‐1‐aminocyclopropane‐1‐carboxylic acid (MACC) in wheat (Triticum aestivum L. cv. Kanzler) and barley (Hordeum vulgare L. cv. Gerbel) leaves was studied using different methods: first, the isolation of intact vacuoles from protoplasts and, second, a non‐aqueous fractionation procedure. The two methods gave similar results. ACC concentrations were similar in the extravacuolar space and in the vacuole, whereas MACC was accumulated in the vacuolar space. Transport studies revealed that no specific carrier for ACC exists at the tonoplast. MACC transfer across the tonoplast was enhanced by 120% in the presence of ATP. MACC competitively inhibited malate transport into the vacuole indicating that the same transfer system catalyzes the transfer of the two dicarboxylates.It is concluded that malonylation of ACC is not a prerequisite for the transport of ACC through the tonoplast.

Wheat protoplast culture: embryogenic colony formation from protoplasts

Wheat (Triticum aestivum L. cv Chinese Spring) protoplasts were isolated from immature embryos or embryogenic calli (3-4 weeks of culture on MS medium with 32 mg/1 dicamba) and cultured in R2 medium containing 2 mg/1 2,4-D by the nurse culture methods originally developed for rice protoplasts (Kyozuka et al. 1987). Protoplasts isolated from embryogenic calli started to divide within 3-5 days and formed colonies at frequencies up to 2% after 3-4 weeks of culture, while protoplasts isolated from immature embryos formed colonies at much lower frequency (less than 0.1%). Some of these colonies were "embryogenic", and they appeared at a frequency of approximately 0.5% of colonies formed when callus-derived protoplasts were used. From two of those "embryogenic" colonies, calli were regenerated and albino shoots and roots were obtained.

Effects of N-substituted glycine analogues on photosynthesis in wheat protoplasts.

Effects of N-substituted glycine analogues on photosynthesis in wheat protoplasts.

Isolation, culture and morphogenesis from wheat protoplasts, and study of expression of DNA constructs by direct gene transfer

The regeneration of plants from protoplasts of some monocots has been achieved, but so far the list excludes wheat. We report here on the culture of protoplasts obtained from an inflorescence-derived suspension cell line, which when cultured gives rise to green callus showing limited morphogenesis.

Flow cytometry as a method for assaying the biological activity of phytotoxins

A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat ( Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca(2+) ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg(2+), Ba(2+) or K(+) could not replace this requirement for Ca(2+). The presence of K(+) did not enhance the Ca(2+)-dependent swelling response. When the Ca(2+)-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca(2+)-channelblocker Verapamil and La(3+) inhibited R-induced swelling. It is proposed that R causes the opening of Ca(2+)-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.

Chromosome variation in dividing protoplasts and cell suspensions of wheat

The cytology of bread wheat (Triticum aestivum) suspension lines, recycled lines (selected for high division frequency) and their dividing protoplasts, have been examined. Extensive numerical and structural chromosome variation was present in all the lines. The most frequently observed chromosome numbers were around 2n=32, indicating that considerable chromosome loss from the normal wheat complement (2n=6x=42) had occurred during selection of the lines. Chromosome aberrations also indicated loss of chromosome arms and chromosome segments. The implications of this variation for studies on transformation and for the potential regeneration of whole plants from protoplasts of bread wheat are discussed.

Effects of Choline Chloride and Its Analogues on Photosynthesis in Wheat Protoplasts

Effects of Choline Chloride and Its Analogues on Photosynthesis in Wheat Protoplasts

Effects of choline chloride and its analogues on photosynthesis in wheat protoplasts.

(1987). Effects of Choline Chloride and Its Analogues on Photosynthesis in Wheat Protoplasts. Agricultural and Biological Chemistry: Vol. 51, No. 3, pp. 917-919.

Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum

The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in protoplasts of three important graminaceous plant species after introduction of the gene by electroporation. Gene transfer occurred when high-voltage electric pulses were applied either directly or indirectly (without anode contact) to a solution containing plasmid DNA and protoplasts of rice, wheat, or sorghum. The indirect method was more rapid, resulted in higher protoplast viability, and was less subject to contamination than the direct-contact method. Gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the 35S promoter of cauliflower mosaic virus or the copia long terminal repeat promoter of Drosophila. Together with recent advances in regeneration of callus and whole plants from protoplasts, this system makes it possible to study inheritance and expression of genes introduced into graminaceous monocotyledonous plants.

Subcellular fractionation of wheat leaf protoplasts by centrifugal elutriation

A new method using centrifugal elutriation for subcellular fractionation of plant cells has been developed. This method takes advantage of the fact that particles sedimenting in a gravitational field can be eluted by flow against the field. A wheat protoplast homogenate was fed into an elutriation rotor spinning at high speed and the flow rate into the rotor was gradually increased. The smaller and less dense materials such as mitochondria, microbodies, endoplasmic reticulum, and cytoplasm were elutriated earlier than the larger and denser nuclei and chloroplasts. The intact chloroplasts, free of mitochondria, microbodies, endoplasmic reticulum, and cytoplasm, could be obtained within 40 min following the rupture of protoplasts. The chlorophyll-free mitochondria could be obtained within 80 min.

Suppression of the Hypersensitive Response in Wheat Stem Rust Interaction by Reagents with Affinity for Wheat Plasma Membrane Galactoconjugates

The growth of the wheat stem rust (Puccinia graminis f.sp. tritici, race 32) on the resistant wheat cultivar Feldkrone is restricted by cell necrosis. Those epidermal cells first invaded react hypersensitively as is shown by the appearance of autofluorescence and the formation of lignin in the invaded cells. Cell necrosis was inhibited by soy-bean agglutinin (SBA), a lectin, which reacts specifically with galactose and N-acetyl-galactosamine, by Erythrina corallodendron lectin (ECO), specific for galactose, as well as by galactose oxidase, an enzyme which acts on the non-reducing terminal D-galactosyl and N-acetyl-D-galactosaminyl residues of surface galactoconjugates. As a result of this inhibition of cell necrosis, the size of the rust colony on treated leaves in-creased, i.e. the resistance of the wheat was lowered. Since previous results had shown that SBA binds to galactolipids on the surface of wheat protoplasts, we now postulate that SBA, ECO, and galactose oxidase interfere with the interaction of host plasmalemma with the fungal haustorium wall during the initial stages of infection.

Surface Galactolipids of Wheat Protoplasts as Receptors for Soybean Agglutinin and Their Possible Relevance to Host-Parasite Interaction

Soybean agglutinin, a lectin specific for N-acetyl-d-galactosamine and d-galactose, was previously shown to agglutinate wheat leaf protoplasts (Larkin 1978 Plant Physiol 61: 626-629). We investigated the receptors for soybean agglutinin on the plasma membrane of these protoplasts. After treatment of the protoplasts with galactose oxidase, they were no longer agglutinated by the lectin, whereas upon reduction of the galactose oxidase-treated protoplasts with sodium borohydride the susceptibility to agglutination was restored. Analysis of the glycolipids of protoplasts surface labeled by the galactose oxidase-borotritide method, revealed that the radioactivity was mainly present in monogalactosyldiglyceride and digalactosyldiglyceride. The same galactolipids were identified as the only receptors for soybean agglutinin by direct binding of the (125)I-labeled lectin to a thin layer chromatogram of the glycolipids of wheat leaf protoplasts.

Ion Channels in Plasmalemma of Wheat Protoplasts

The patch-clamp technique was used to study passive movements of ions through the plasmalemma of wheat leaf protoplasts. This method overcomes the problems inherent in conventional electrophysiological study of plant cells. Changes in conductance were recorded in patches excised from the plasmalemma. Two types of patches were observed: (i) regions of low channel density, where discrete single-channel currents could be resolved and conductance ranged from 10 to 200 picosiemens and (ii) regions of high channel density, where single-channel currents could not be resolved and conductance was on the order of a few nanosiemens. The results indicate a striking similarity between animal and plant cell membranes in the basic phenomena of transport. Moreover, the approach used constitutes a new degree of refinement in the study of processes of regulation, pathology, and toxicity in plants.