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In Vitro rooting of micropropagated micro-shoots of two commercial sugarcane varieties was carried out with the aim of evaluating the root induction responses of the sugarcane varieties (B41-227 and N14) to alpha naphthalene acetic acid (NAA) and Indole -3- butyric acid (IBA). Accordingly, four levels of NAA (0, 1, 2, and 3 mg L-1) and IBA (0, 1, 2 and 3 mg L-1) in a completely randomized design with 4 × 4 × 2 factorial treatment combination arrangements were tested. Data on the number of roots per shoot and average root length (cm) were collected after 30 days of culture on ½ MS root induction medium. Analysis of variance revealed that the interaction effects of NAA, IBA and the sugarcane genotypes on number of roots per shoot and average root length of both sugarcane varieties was very highly significant (P<0.0001). Culture medium containing 2 mg L-1 NAA and 1 mg L-1 IBA for B41-227 and 1 mg L-1 NAA alone for N14 was found to be optimum. On these medium, B41-227 gave 33 ± 0.15 roots per shoot with 2.92 ± 0.18 cm root length and N14 produced 35± 0.20 roots per shoot with 3.2 ± 0.25 cm root length. The rooted plantlets were survived 100% after four weeks of acclimatization in greenhouse on Farmyard manure and soil at 2:8 ratios. The optimized protocol can be used to develop healthy and profuse root system in the sugarcane micro-shoots, an essential stage in sugarcane micropropagation.