The 5-azacytidine-induced epimutagenesis of sugarcane (Saccharum spp. hybrids) for aluminium tolerance

A protocol for generating aluminium (Al) tolerant sugarcane using 5-azacytidine (5-azaC)-induced epimutagenesis was developed. Eight (8) plantlets per gram of cultivar N51 calli were generated from the 5-azaC (100 μM) and Al (1.5-50 mM) treatment (+Az + Al) when compared with 104 plantlets from non-mutagenised, no Al control treatment. Following in vitro selection on 60 mM Al, ex vitro stress over four rounds of chimera dissolution during vegetative propagation was applied. In the final stress round, 36% of the Mut 2 epilines survived with plants showing higher relative water content (61.2%) when compared with 47.3% from the stressed control (S N51). The Mut 2 line maintained a higher green leaf area (83.4%) and longer roots (32.4 cm) under stress than S N51 (61.4% and 26.3 cm, respectively). Overall, Mut 2 had a high stress tolerance index of 85.4%, compared with 79.0% from the S N51, nearing that of the non-stressed N51 control (NS N51, 100%) when data were analysed using PCA and clustering analyses of morpho-physiological traits. Analysis of molecular variance (AMOVA) revealed high epigenetic differentiation (ɸst = 0.67) and a variation of 66.6% observed among N51 genotypes. The principal coordinate analysis (PCoA) showed that Mut 2 was epigenetically distinct from S N51. These findings support previous studies that 5-azaC can be used for novel trait creation via epimutagenesis and highlights the necessity for chimera dissolution to achieve stable traits in epibreeding of sugarcane.