Induction of somatic hybrid by protoplast fusion between Populus koreana X Populus nigra var. italica and Populus euramericana cv. Guardi

Park, Y.-Goo; Kim, J.-Hee; Son, S.-Ho

Journal of Korean Forestry Society 81(3): 273-279


Accession: 008859437

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Protoplasts isolated from leaf mesophyll tissues of Populus koreana X P. nigra var. italica were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana times P. nigra var. italica and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase; w/v), respectively. The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of Ca-2+ in eluting solution at pH 10.5. Dividing cells and/or mini-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, 0.45-mu-M, 2,4-D, and 0.5-mu-M BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing 5.0-mu-M zeatin. To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein and patterns compared with those of the original source plants.