Direct Somatic Embryogenesis for Rapid , Cost Effective Production of Transgenic Sugarcane ( Saccharum Spp . Hybrids )

During the last decade, considerable effort has been expended in developing an efficient transformation system for sugarcane. Most protocols employ microprojectile bombardment of gene constructs into embryogenic sugarcane callus. The numbers of transgenic plants produced per bombardment range from 0.1-20 globally, with genotype response and selection regime appearing to be the most important factors influencing efficiency. The time taken to regenerate transgenic plants from callus via this route of indirect somatic embryogenesis is typically 24-36 weeks. We report a novel approach using a direct somatic embryogenic route of regeneration. The technique involves gene delivery to transverse explants derived from immature leaf roll pre-cultured for only 2 weeks on medium containing low levels (0.3 mg/l) of the auxin 2,4-D. Plantlets are ready for hardening off after 13-22 weeks. In a comparative study, PCR analysis showed that transformation efficiencies for the direct and indirect morphogenic routes were similar. Due to faster regeneration times and fewer subcultures, the cost of production via direct somatic embryo-genesis can be reduced from R215 to R52 per transgenic plant. Rationale The use of embryogenic callus as target material for microprojectile bombardment has been used by sugar industries world wide in genetic engineering programmes for the production of transgenic sugarcane. Embryos have been induced to develop indirectly via an undifferentiated cell mass or callus stage from leaf discs or floral parts The choice of cultivar, genotypic responses to hormone treatment and selection regime may account for the differences in efficiency reported. Aside from widely varying levels in transgenic production efficiency , the use of embryogenic callus as target material for bombardment has other limitations. Establishing, developing and maintaining callus cultures is labour intensive and the recovery of transgenic plants ready for glasshouse planting may take as long as 36 weeks (Bower et al., 1996). To minimise the time spent generating embryogenic callus, one approach would be to employ a route of morphogenesis from leaf discs which is faster than the indirect morphogenic route. In a study of cell suspension-derived protoplast regeneration, Aftab and Iqbal (1999) reported the formation of somatic embryos directly from the cut edges of young sugarcane leaf discs with very little intervening callus, so this route was seen to have the potential to speed up a transformation programme. The aim of this study was to compare the efficiency of production of transgenic sugarcane regenerated via direct somatic embryogenesis from leaf roll discs with the conventionally …