PMC:4307275 / 30026-31631
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4307275","sourcedb":"PMC","sourceid":"4307275","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4307275","text":"With minor modifications, protoplasts were isolated as described by Bart [37]. Strips (0.5 mm) were cut with new razor blades from 0.5 g leaf and stem tissue sections and then immediately immersed in 15 mL of enzyme solution (Cellulase R-10 and Macerozyme R-10; Yakult Honsha, Japan) for the digestion of the cell walls. The plate was put into a vacuum desiccator and the vacuum infiltration for 10 min was applied to increase digestion efficiency. Following the incubation with gentle shaking of 50 rpm at RT in the dark for 4 h, the enzyme reaction was stopped by the addition of 15 mL W5 solution [37]. Cell debris were filtered twice through 70 μm and 40 μm BD Falcon™ cell strainers (BD, Franklin Lakes, NJ, USA); furthermore, the protoplasts were pelleted and suspended in Mmg buffer solution [37] at 107 protoplasts·mL−1 for PEG-transfection. The number of protoplasts was analyzed using a Marienfeld hemocytometer counting system (Marienfeld-Superior, Berlin, Germany). For transfection, an equal volume of 40% PEG-3350 (Sigma, St. Louis, MO, USA) solution [37] in 0.6 M mannitol and 100 mM CaCl2 was added to 110 µL of the protoplasts (around 106 cells) and DNA solution; the mixture was incubated for 15 min. The protoplasts were washed twice with each of the two volumes of W5 and 1 mL of incubation solution and were finally resuspended in 1 mL of incubation solution and incubated at 28 °C in the dark overnight. All plasticware used for protoplasts was coated with 5% calf serum by swirling for 10 s and all buffers were filtered using 0.45 µm syringe filter (Sartorius, Gottingen, Germany).","tracks":[{"project":"2_test","denotations":[{"id":"25561231-16808845-50799928","span":{"begin":74,"end":76},"obj":"16808845"},{"id":"25561231-16808845-50799929","span":{"begin":601,"end":603},"obj":"16808845"},{"id":"25561231-16808845-50799930","span":{"begin":800,"end":802},"obj":"16808845"},{"id":"25561231-16808845-50799931","span":{"begin":1066,"end":1068},"obj":"16808845"},{"id":"T14631","span":{"begin":74,"end":76},"obj":"16808845"},{"id":"T91854","span":{"begin":601,"end":603},"obj":"16808845"},{"id":"T26326","span":{"begin":800,"end":802},"obj":"16808845"},{"id":"T36228","span":{"begin":1066,"end":1068},"obj":"16808845"}],"attributes":[{"subj":"25561231-16808845-50799928","pred":"source","obj":"2_test"},{"subj":"25561231-16808845-50799929","pred":"source","obj":"2_test"},{"subj":"25561231-16808845-50799930","pred":"source","obj":"2_test"},{"subj":"25561231-16808845-50799931","pred":"source","obj":"2_test"},{"subj":"T14631","pred":"source","obj":"2_test"},{"subj":"T91854","pred":"source","obj":"2_test"},{"subj":"T26326","pred":"source","obj":"2_test"},{"subj":"T36228","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#eceb93","default":true}]}]}}