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A new method of in vitro interaction between cell suspension and Cf-elicitor was employed to understand red rot resistance in sugarcane. RT-PCR, differential display (DD)-RT-PCR, RACE-PCR, semi-quantitative RT-PCR and different bioinformatic tools were used to identify and characterize the defense related transcripts. In DD-RT-PCR, many differentially expressed transcripts were identified and functionally grouped. Important candidate genes were identified through RACE-PCR and their functional domains were characterized. Sugarcane chitinase was characterized as a class IV chitinase. Differential accumulation of several transcripts of the flavanoid biosynthetic pathway was established through spatial expression by semi quantitative RT-PCR and this study further confirms the role of sugarcane phytoalexins in red rot resistance. The study has resulted in the generation of large number of TDFs that can be used in future studies in sugarcane-C. falcatum interaction as well as well as other biotic/abiotic stress factors. For the first time, this study established that in vitro interaction between cell suspension and Cf-elicitor mimics molecular interaction in cane tissue and C. falcatum.