Protoplast isolation from bamboo leaves

Abstract

Protoplasts with a yield greater than 2.0×106 cells per 0.3 g fresh weight were isolated from leaves of four bamboo species: an herbaceous bamboo, Lithachne pauciflora, and three woody bamboos, Phyllostachys meyeri, Sasa jotanii, and Bambusa vulgaris. For aseptic protoplast isolation, folded leaves were sterilized in 70% ethanol, and the internal tissues were drawn out from the leaf sheath. The leaf material was incubated for 4 h in a modified White's medium solution containing 0.8 M mannitol (pH 5.8, 32°C) and an enzyme mixture containing 3% Macerozyme R-10, 2.5% Meicelase, and 2% Cellulase Onozuka for woody bamboos, and 2.5%, 2.5%, and 1% of these enzymes, respectively, for L. pauciflora. Protoplasts were filtered through a double-layered Miracloth funnel and then washed with 0.6 M to 0.4 M mannitolcontaining White's medium (pH 5.8) prior to analysis. Protoplast viability averaged 83% as determined by FDA staining. © 2010 The Japanese Society for Plant Cell and Molecular Biology.