High-efficiency somatic embryogenesis of a broad range of Brazilian Saccharum spp. hybrids (sugarcane) varieties using explants from previously established in vitro plants

Abstract

The current study assessed the embryogenic potential of different Brazilian Saccharum spp. hybrid (sugarcane) varieties, using explants from previously established in vitro plants, and determined the morphogenic capacity of calluses to regenerate plants during consecutive subcultures. After examining various explant lengths (1 to 15 mm) for callus initiation, an optimal length of 12 mm was determined, and 14 sugarcane varieties were studied. Callus induction occurred on Murashige and Skoog medium with 3.0 mg L−1 2,4-dichlorophenoxyacetic acid, in the dark. After 40 d, calluses were divided according to their predominant type: the mucilaginous part remained in the dark on fresh induction medium, whereas the compact nodular callus fraction was transferred to a regeneration medium containing 1.86 mg L−1 1-naphthaleneacetic acid and 0.09 mg L−1 6-benzylaminopurine and was cultivated in light. This callus selection based on morphological type took place over three consecutive subcultures, spanning 22 wk. The embryogenic process was asynchronous and the formation of the first plants was registered at 6 wk of cultivation. The embryogenic callus regeneration rate was kept at over 60% until the third subculture in 13 of the 14 varieties examined. However, delayed regeneration was observed, likely due to the aging of calluses. The protocol reported here utilized the embryogenic potential of sugarcane varieties in combination with efficient and quick regeneration and should thus provide an attractive alternative source material for the production of transgenics and/or large-scale clonal multiplication of sugarcane.