5-Azacytidine as a tool to induce somaclonal variants with useful traits in sugarcane (Saccharum spp.)

A possible strategy to produce variant sugarcane plants with beneficial traits was tested by promoting somaclonal variation in vitro through the action of the hypomethylation and mutagenic agent 5-Azacytidine (Azac). Treatment of calli in liquid medium caused high levels of necrosis. Consequently, 6- to 8-week-old calli of cultivar NCo376 were exposed to 50 and $100{\mu}M$ Azac in semi-solid callus induction medium (CIM) (MS salts and vitamins, sucrose, casein hydrolysate, agar, with or without $3mgl^{-1}$ 2,4-D) for 1 week. They were then transferred to fresh CIM with 2,4-D and to CIM without 2,4-D, for 2 and 8-10 weeks, respectively. The highest callus necrosis ([60 %) and reduced recovery (\40 %) were recorded for calli treated with $100{\mu}M$ Azac without 2,4-D, which also resulted in lower plant yield (12 plantlets/0.2 g calli) than the control (18 plantlets/0.2 g calli). From methylation-sensitive amplified fragment length polymorphism analyses, the highest polymorphisms (4.2 %) were also obtained from plants derived from the $100{\mu}M$ Azac treatment without 2,4-D. After 9 months of field growth, Azac-derived plants exhibited phenotypic differences compared with the controls. Ex vitro screening resulted in the identification of one plant from the $100{\mu}M$ Azac with 2,4-D treatment putatively tolerant to smut, and three plants from the $100{\mu}M$ Azac with 2,4-D and one from the $50{\mu}M$ Azac with 2,4-D treatments, potentially tolerant to the herbicide imazapyr.