Embryogenic callus induction, proliferation, protoplast isolation, and PEG induced fusion in Camellia oleifera

Artículo

Camellia oleifera (C. oleifera) is one of the most important economic oil species in China. This study established an efficient protocol for the induction and multiplication of embryogenic callus, and the isolation, purification and Polyethylene glycol (PEG) induced fusion of protoplasts in C. oleifera. It is shown that the embryogenic callus induction was best when nodular embryogenic structures were treated with the hormone of 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.5 mg/L 6-Benzylaminopurine (6-BA) on Murashige and Skoog (MS) medium, and the highest induction rate of embryogenic callus was 18.82% after 25 days of culture. Embryogenic callus was further multiplied on woody plant medium (WPM) with 0.75 mg/L 2,4-D, pH 5.8. The Proliferating rate of embryogenic callus reached 1.8 after 20 days of culture. The maximum yield (3.16 × 106 protoplasts per gram fresh tissue) and viability (93%) of embryogenic callus protoplast were reached when embryogenic callus was treated in dark for 24 h, vacuumed at -0.03 MPa for 10 min, digested of embryogenic callus with 0.75% (w/v) Cellulase R-10 and 0.5% (w/v) Macerozyme R-10 for 6 hours at 28ºC. The purification effect was the best when using W buffer as a cleaning agent centrifuged 400 revolutions per minute (rpm) for 4 min by precipitation method. Moreover, the maximum fusion efficiency (11.18%) of mesophyll protoplasts and embryogenic callus protoplasts was obtained with 30% PEG 6000 for 15 min. The study lays a foundation for future research in somatic hybridization in C. oleifera.