*4.1. Choice of Explant Material and Callus Induction*

Various plant parts, including nodal tissues, apical meristem, the sub-apical region of the shoot apex, young leaf whorls, and root meristematic tissues, were used to develop calli and for proliferation. Release of phenolic compounds from all explants except young leaf whorls were a major problem in developing the calli and regeneration protocol, resulting in browning of tissues. Hence, the protocol for young leaf whorls was developed. Sugarcane tops of 6–8-month-old plants were sterilized using 70% ethanol and then sliced into transverse sections. Young leaf roll discs of 1.5–2.0 mm thickness and 60 to 95 mm<sup>2</sup> area cross-section were used to develop on callus induction medium, having different levels of 2,4-D (1, 2, 3, 4, and 5 mg/L). A suitable level of 2,4-D was selected that was tested in combination with different levels of kinetin (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L).
