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A significant part of sugarcane crop is lost each year due to different diseases particularly red rot. As sugarcane is mostly propagated by vegetative method, therefore, plant once infected by pathogen can transfer it generation after generation. Therefore introduction of new disease resistance varieties needs rapid, reliable and efficient methods of multiplication. In this study the, biotechnological methods i.e. Plant Tissue Culture were used for genetic improvement through both physical and chemical mutagenic agents. In this project the major emphasis was focused on in vitro as well field screening of red rot resistant lines using partially purified toxin of fungus Colletotrichum falcatum. The induction of mutation was further supported by using various disease related biochemical markers(Qualitative and quantitative analysis of protein and different enzymes). Finally these selected high yielding red rot resistant lines were multiplied by micropropagation, regeneration through callus to achieve the objectives. Protocols are suitable for induction of mutation and mass scale production of disease resistance, high yielding and premium quality planting material.