PMC:4307275 / 28611-29981 JSONTXT

{"project":"2_test","denotations":[{"id":"25561231-21625436-50799924","span":{"begin":735,"end":737},"obj":"21625436"},{"id":"25561231-8580770-50799925","span":{"begin":738,"end":740},"obj":"8580770"},{"id":"25561231-17189339-50799926","span":{"begin":854,"end":856},"obj":"17189339"},{"id":"25561231-21625436-50799927","span":{"begin":994,"end":996},"obj":"21625436"},{"id":"T6193","span":{"begin":735,"end":737},"obj":"21625436"},{"id":"T99663","span":{"begin":738,"end":740},"obj":"8580770"},{"id":"T81459","span":{"begin":854,"end":856},"obj":"17189339"},{"id":"T85412","span":{"begin":994,"end":996},"obj":"21625436"}],"text":"4.2. Plant Materials\nThe dehulled seeds of rice (Oryza sativa L. Japonica cv. “Ilmi”) were sterilized by treatment once in 70% ethanol for 1 min and twice in 2% sodium hypochlorite for 20 min and then planted on Murashige and Skoog (MS) agar medium after washing five times with distilled water. The rice seedlings were grown in the dark for 10 days, and then illuminated with white light for 20 h prior to protoplast isolation.\nThe restricted exposure of the plants to light (20 h) was used to reduce the autofluorescence background resulting from the presence of chlorophyll. The formation of RuBisCo and the biosynthesis of chlorophyll in rice leaves have been reported to be the greatest during greening of etiolated leaf tissues [33,34]. During this process, photosynthesis-related proteins are detected within 2 h of illumination with white light [35], the stromal structures of chloroplast are evident following illumination for 16 h [36], and chlorophyll formation begins subsequently [33,36]. In several preliminary experiments, we established that the optimum illumination time was 16–20 h, which resulted in chlorophyll formation but not to a level where autofluorescence was evident in the images using a fluorescein isothiocyanate (FITC) filter (data not shown). Consequently, 20 h of illumination was used for the FCA analysis of protoplasts in this study."}