5-Azacytidine promotes the induction of embryogenic calli and somatic embryos from transverse thin cell layer (tTCL) cultures in coconut

Coconut (Cocos nucifera L.) is highly recalcitrant to in vitro interventions.There is a need to overcome various bottlenecks to standardize a repeatable protocol for in vitro regeneration in coconut to meet the requirements of quality planting materials.Epigenetic processes, especially DNA methylation, are known to assay crucial roles in regulating genes controlling plant growth and development, especially somatic embryogenesis.In this study, we demonstrate that the supplementation of 5-azacytidine (5-azaC), a demethylating agent, in the coconut tissue culture media can enhance the formation of embryogenic calli, somatic embryos, and plantlet regeneration from transverse thin sections of mature zygotic embryos.Transverse thin cell layer (tTCL) sections of zygotic embryos were cultured onto Y3 medium supplemented with different concentrations of 5-azaC (0, 10, 15, and 20 M), auxins (2,4-D, picloram and atrazine; 75 and 100 M) and thidiazuron (TDZ;4.5 M).Explants were exposed to constant 5-azaC and reduced auxin concentration in subsequent sub-culturing.Our results indicated supplementing 15 M 5-azaC, in combination with picloram (75 M) and TDZ (4.54 M), improved the percentage of callusing (95.8%) and formation of embryogenic calli (87.5%), and formation of somatic embryos (4.7) and plantlets (4.0) per explant in comparison with control having 80.8%, 75.0%, 1.6 and 0.67, respectively from tTCL sections of mature zygotic embryos.The results will form the basis for designing more efficient coconut tissue culture protocols.