An optimized protocol for in vitro regeneration of tropical maize inbred lines through cell suspension and semi-protoplast cultures

Abstract

Maize being an important staple food crop is widely consumed in Kenya but its production remains low due to biotic and abiotic challenges that have not been addressed sufficiently through conventional breeding. This study sought to optimize the establishment of cell suspension and protoplast cultures for 10 tropical inbred maize genotypes in order to identify genotypes that can readily establish in liquid cultures and those whose cell suspensions and protoplasts are capable of in vitro regeneration. Callus were induced using immature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 3 mg/L 2, 4-D, 5 mg/L dicamba or 5 mg/L picloram. Dicamba and picloram induced friable calli that readily dispersed into cell suspensions in liquid MS medium supplemented with 0.1 g/L asparagine and either 0.4 or 0.8 g/L proline. Cell growth was determined by packed cell volume (PCV) every seven days. The highest PCV (240 µl/ml) was recorded in genotype EO4 followed by CML 216 (188 µ/ml). Optimal growth was observed in cells maintained in MS Amended with 0.4 or 0.8 g/L of proline in combination with 0.1 g/L asparagine compared to medium without proline. It was also observed that cells cultured in media with reduced Ammonium Nitrate (12 fold reduction) recorded higher PCV values than controls. Protoplasts were generated from the resulting cells using 2% cellulase and 0.5% pectolyase in an enzyme digestion cocktail containing Mannitol and Calcium Chloride (MaCa) and washed in MS with vitamins containing Mannitol (MSMa). Only cell clusters of genotype EO4 gave rise to plants with a regeneration frequency of 42.51%. In conclusion, success was achieved in callus initiation, formation of cell suspension cultures and their eventual regeneration into whole plants for selected tropical maize genotypes.