Tropical maize transformation through cell suspension and protoplast culture systems
Manipulation of tropical maize in tissue culture is crucial to harnessing the benefits of biotechriology, especially those involving upgrading the genetic base of the maize to resist or tolerate biotic and abiotic constraints of production. The strategies involved are many, having transformed from those involving conventional breeding to the more appropriate molecular plant breeding methods. Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many tropical maize inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the transgene delivery method. Availability of very efficient Agrobacterium-mediated maize inbred line transfonriation protocol will improve future opportunities for maize genetic manipulation and functional genomics. Most molecular and gene function studies in maize requiring tissue culture have been done using cell suspensions mainly among temperate inbred lines of maize in which the protocols have been optimized to suit these lines. In this study, efficient quick generation of transgenic plants and progeny from five maize inbred lines will be investigated using an Agrobacterium-mediated standard binary vector system comparatively with polyethylene glycol (PEG) and calcium phosphate clean gene delivery systems. These systems are• envisaged to target cell suspension and protoplast cultures as explants. The cell suspensions and protoplasts developed from the maize inbred lines will be first pre-screened for regeneration frequency and efficiency on Murashige and Skoog medium. The established cell suspensions and protoplast culture systems will thus fast-track transformation of tropical Kenyan adopted white maize using direct. DNA uptake method. The maize transformation evaluation studies will be done using GUS reporter gene and pmi selectable marker gene. The transformation efficiency of direct DNA uptake method coupled with the use of clean transgenes and the new culture systems will be evaluated through histochemical GUS staining, southern, western blotting and polymerase chain reaction analysis. The established protocols will be useful in generating transgenic maize lines resistant to various biotic and abiotic stresses.