Callus induction, suspension culture and protoplast isolation in Camellia oleifera

Abstract

Camellia oleifera is a woody edible oil crop with economical importance. This study established an efficient protocol for the induction of callus, the multiplication of a suspension cell line, and the isolation and purification of protoplasts in Camellia oleifera. It is shown that the callus induction was best when anthers were treated with the hormone of 0.5 mg/L NAA (Naphthaleneacetic acid), 2.0 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) and 0.5 mg/L 6-BA (6-Benzylaminopurine) at 4 °C for 15 days. Callus was further multiplied on MS (Murashige and Skoog) medium augmented with 5% coconut water, 2 mg/L 2,4-D, 0.5 mg/L 6-BA, pH 5.8. Though three types of induced callus transferred to the same liquid medium with the ratio of 1 g callus inoculated into 30 ml liquid medium, it was found that the suspension culture effect of loose particles callus was the best. The maximum yield (11.7 × 106/g·FW) and highest viability (95.1%) of protoplast were reached when cell suspension (cultured for 6 days) was inoculated for 14 h in enzyme solution made of 0.4 mol/L mannitol mixture solution, 1.0% (w/v) Cellulase R-10 and 1.0% (w/v) Macerozyme R-10. The study lays a foundation for future research in cell fusion and transient gene expression in Camellia oleifera.