Optimization of a regeneration and transformation protocol for selected tropical inbred maize genotypes through cell suspension and semi-protoplast cultures
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This study sought to optimize establishment of cell suspension and protoplast cultures for ten tropical inbred maize genotypes in order to identify genotypes that can readily establish in liquid cultures and those whose cell suspensions and protoplasts are competent to Agrobacterium mediated transformability and regeneration. Callus tissues were first induced using immature zygotic embryos cultured on Murashige and Skoog (MS) medium supplemented with 2 mgIL 2, 4-D, or 3 mgIL dicamba and 1 mgIL kinetin. Among the hormones used, 3 mg/L dicamba and 1 mg/L kinetin induced appropriate friable embryogenic callus (FEe) that readily dispersed into cell suspensions. Induction of friable embryogenic callus was found to be genotype dependent. The liquid MS medium was then supplemented with 0.1 g/L asparagine and either 0.4 or 0.8 gIL proline, and the cell growth determined by packed cell volume (PCV %) every seven days. The highest PCV (240 Ill/mn was recorded in genotype E04 followed by CML 216 (188 l1/ml).Cells maintained in medium with 0.4 gIL or 0.8 g/L of proline in combination with 0.1 g/L asparagine supported the most optimal growth compared to medium without proline. The growth rate of the established cells indicated an exponential increase of PCV only up to the 8th day of culture. It was also observed that cells cultured in media with reduced ammonium nitrate (12 fold) recorded higher PCV values than controls. Protoplasts were generated from the resulting cells using 2% cellulase and 0.5% pectolyasc in an enzyme digestion cocktail. Only cell clusters of genotype E04 gave rise to plants with optimal regeneration frequency of 28.95% in media containing 0.4 gIL of proline, indicating that regeneration from tropical maize liquid cultures is possible for' genotypes that initiate such clusters. Upon infection of cells with Agrobacterium tumefaciens. harboring a GUS reporter gene, only cells in clusters showed a blue staining (characteristic of transformation), while cells that completely dispersed in liquid media did not transform. In conclusion, success was achieved in initiation of friable callus, the subsequent formation of cell suspension cultures and their eventual regeneration into whole plants as well as showing transformability of cell clusters.