Embryogenic callus induction, proliferation, protoplast isolation, and PEG-induced fusion of Camellia hainanica

Abstract: Tea oil plant is an economically significant woody oil crop in China. Camellia hainanica (C.hainanica) is a high-quality variety of this crop. This study aimed to explore the induction and proliferation of embryogenic callus from C. hainanica ‘Haiyou1’ and ‘Haiyou3’, using different plant growth regulators. Additionally, the study——assessed the impact of various factors on the yield and viability of protoplasts isolated from embryogenic callus, as well as determined the optimal fusion conditions under various PEG concentrations. The optimal medium for inducing embryogenic callus from immature embryos was found to be MS + 2 mg/L 6-BA + 1 mg/L NAA + 30 g/L sucrose + 7 g/L agar, with an induction rate of 56.44%. The optimal medium for proliferating embryogenic callus was MS + 2 mg/L 6-BA + 1 mg/L IBA + 30 g/L sucrose + 7 g/L agar, with a proliferation coefficient of 2.42. Protoplast isolation conditions were determined to be 1.5%(w/v) cellulase R-10, 1% (w/v) Macerozyme R-10 and 0.5% (w/v) pectinase, 0.5 mol/L mannitol, and 7 h of digestion time. The protoplast yield and viability from C. hainanica ‘Haiyou1’ and ‘Haiyou3’ were as follows: 3.70 × 106 protoplasts/g FW, with 88.65% viability and 3.46 × 106 protoplasts/g FW, with 90.01% viability, respectively. The optimal PEG fusion conditions for embryogenic callus protoplasts of C.hainanica and Camellia oleifera Able (C.oleifera) were 40% PEG 6000 for 20 minutes. Fusion rates of ‘Haiyou1’ and ‘Haiyou3’ with C. oleifera were 11.40% and 13.11%, respectively. This study provides a foundation for future research on somatic hybridization in C. hainanica.