ABSTRACT

A simple expression assay for evaluation of gene constructs for input of traits into cotton cells (Gossypium hirsutum L.) using oncogenic binary Agrobacterium strains is presented. Seedling explants from four commercial cotton varieties, representing diverse genotypes, exhibited tumor or root formation to an equal degree in response to infection by different types of oncogenic Agrobacterium strains. Cotyledon or hypocotyl explants readily developed tumors (100%) within a week and the tumors doubled in fresh weight every two weeks. A. rhizogenes super-rooting mutant strain MT232 was highly infective on cotyledon explants. Experiments with oncogenic strains served as a basis for development of an assay using tumor-inducing binary vectors carrying the gene to be evaluated, an insecticidal Bt protoxin gene. An oncogenic binary vector containing a chimeric neomycin phosphotransferase II and a Bt protoxin gene conferred antibiotic resistance and insect resistance to Lepidopteran larvae in tumorigenic cells from cotyledon explants. The efficacy of the insecticidal protoxin gene towards the control of a Lepidopteran cotton insect pest, tobacco budworm (Heliothis virescens), was demonstrated in this study using oncogenic cotton cells. In a parallel study, the efficiency of this gene construct was also demonstrated using the tobacco model system against another Lepidopteran pest, tobacco hornworm (Manduca sexta). The time needed to conduct the experiment with cotton tumor cells was about three to four months from the time of initiation, same as the time needed for the tobacco model system. The rapidity of this assay is extremely useful in evaluation of gene constructs for input traits such as insect-, disease -, or herbicide resistance in the laboratory, especially in recalcitrant species such as cotton, where more than 15 months are needed for selection and regeneration of transgenic plants.