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Submitted to: Journal of New Seeds
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/17/2003 Publication Date: 6/1/2003 Citation: Rajasekaran, K. 2003. A rapid assay for gene expression in cotton cells transformed by oncogenic binary Agrobacterium strains. Journal of New Seeds. 5(2-3):179-192. Interpretive Summary: Inserting genes into cotton is a very time consuming procedure and it takes more than 15 months to produce transgenic cotton plants, only to discover that the gene was not suitable or not working properly. For this reason, scientists often use model species such as tobacco, mustard weed, or carrot to understand how the new gene might work in a test plant. Even when the gene is working correctly in a model plant system, the results could not be estimated for crop species such as cotton. This research paper presents a quick and easy method for testing new genes in rapidly growing cotton tumor cells caused by a soil-borne bacterium. The rapidity of the assay bypasses the time-consuming process of transforming (inserting genes) into cotton cells and regenerating cotton plants and allows the researchers to test the gene for input traits such as insect resistance, disease resistance, and herbicide tolerance in a native cotton cell environment. Scientists could test tumor cells expressing the gene of interest for efficacy within a reasonable time frame (3-4 months), similar to the period needed for testing with model plant systems, before using the gene for subsequent transformation and regeneration of fertile cotton plants. The information presented in this report is useful for agricultural biotechnology scientists to test and evaluate new genes in target crop plants such as cotton. Technical 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. Explants from three 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 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 in the laboratory, especially in recalcitrant species such as cotton, where more than 15 months are needed for selection and regeneration of transgenic plants. |
