Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture

Rapid and efficient in vitro regeneration methods\ud that minimise somaclonal variation are critical for the\ud genetic transformation and mass propagation of commercial\ud varieties. Using a transverse thin cell layer culture\ud system, we have identified some of the developmental and\ud physiological constraints that limit high-frequency regeneration\ud in sugarcane leaf tissue. Tissue polarity and consequently\ud the orientation of the explant in culture, size and\ud developmental phase of explant, and auxin concentration\ud play a significant role in determining the organogenic potential\ud of leaf tissue in culture. Both adventitious shoot\ud production and somatic embryogenesis occurred on the\ud proximal cut surface of the explant, and a regeneration\ud gradient, decreasing gradually from the basal to the distal\ud end, exists in the leaf roll. Importantly, auxin, when\ud added to the culture medium, reduced this spatial developmental\ud constraint, as well as the effect of genotype on\ud plant regeneration. Transverse sections (1–2 mm thick) ob-\ud tained from young leaf spindle rolls and orienting explants\ud with its distal end facing the medium (directly in contact\ud with medium) are critical for maximum regeneration.\ud Shoot regeneration was observed as early as 3 weeks on\ud MS medium supplemented with α-naphthalenencetic acid\ud (NAA) and 6-benzyladenine, while somatic embryogenesis\ud or both adventitious shoot organogenesis and somatic embryogenesis\ud occurred on medium with NAA and chlorophenoxyacetic\ud acid. Twenty shoots or more could be generated\ud from a single transverse section explant. These shoots regenerated\ud roots and successfully established after transplanted\ud to pots. Large numbers of plantlets can be regenerated\ud directly and rapidly using this system. SmartSettr,\ud the registered name for this process and the plants produced,\ud will have significant practical applications for the\ud mass propagation of new cultivars and in genetic modification\ud programs. The SmartSettr system has already\ud been used commercially to produce substantial numbers\ud of plants of orange rust-resistant and new cultivars in\ud Australia