• Journal of Korean Society of Forest Science
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• v.74 no.1
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• pp.29-36
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• 1986

A method isolating Populus euramericana cv. I-214 mesophyll protoplasts was developed to facilitate application of genetic engineering techniques to this species. The suitable medium for shoot multiplication in vitro was MS basal medium with $0.1mg/{\ell}$ BAP. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf in vitro and known volumes of maceration and washing media. The best yields of mesophyll protoplasts were obtained using leaves in vitro in 2.0% Cellulase R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, 0.05% Pectolyase Y-23, and O.6M Mannitol in addition to DTT and MES buffer adjusted to pH 5.6. Over $2.4{\times}10^6$ protoplasts per gram of leaf were produced using these conditions. For protoplast purification, the most favorable sucrose concentration of floating solution was 0.6M after washing them with CPW solution. This method of screening factors affecting protoplast isolation could be applicable to other species.