In this protocol, the transformation of moss (Physcomitrella patens) protoplasts is performed via Agrobacterium-mediated transfer of T-DNA. Protoplasts are incubated with Agrobacterium and acetoseringone in regeneration medium. They are then washed and plated on antibiotic-containing medium to select for T-DNA insertion in stable transformants. The transformation rate for this protocol is typic...

Intact nuclei were isolated from the protoplasts of the filamentous fungus Aspergillus nidulans. The large amounts of protoplasts required for such nuclear preparations were produced from young mycelia grown in liquid culture. For final purification of the crude nuclear fraction, a Nycodenz density-gradient centrifugation was applied. The resulting nuclei were of good purity and morphological s...

Protoplasts from spinach leaves exhibit a light-induced Ca2+ influx. The half maximum rate of Ca2+ influx is achieved at ~ 5 Wm"2. The action spectrum of this influx is similar to that of photosynthesis. Furthermore, light-induced Ca2+ influx is abolished by DCM U (2* 0.5 îm) and is sensitive to the uncoupler FCCP. Vanadate up to 3 enhances light-induced Ca2+ influx. These data indicate that ph...

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of t...

In vacuoles isolated from petal upper epidermis o f Petunia hybrida through polybase-induced lysis of protoplasts the vacuolar localization o f x-mannosidase and x-galactosidase was dem on­ strated. No association was observed between the tonoplast and these hydrolases. Protoplasts from different mutant lines blocked in anthocyanin biosynthesis were treated with DEAE-dextran to release vacuoles...

A method for the preparation of protoplasts from Candida tropicalis cultivated on n-tetradecane is described. This essentially consists of replacing the mannitolsorbitol solution of the classical helicase technique by 1 M magnesium sulfate and lowering the pH to 4.1 during incubation in the presence of helicase. The protoplasts thus prepared behave like intact cells and are capable of consuming...

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K(m) of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve....

Ion channels in the plasma membrane of protoplasts isolated from cultured cells of Arabidopsis thaliana were studied by means of the patch-clamp technique applied in the whole-cell configuration. In some protoplasts, depolarizing pulses and, in other protoplasts, hyperpolarizing pulses elicited time-dependent currents; both kinds of current were only rarely observed in the same protoplast. The ...

The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functiona...

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 ...