*2.1. Development of a Proficient Regeneration Protocol*

Unfurled leaves from six field-grown elite sugarcane genotypes (CPF-246, US-127, HSF-242, US-778, HSF-240, and US-64) were sacrificed to develop leaf roll discs, and subsequently placed on callus induction medium. Of six genotypes, maximum embryogenic cells were recovered from genotypes US-127, US-778 on MS medium supplemented with 2,4-D (2,4-Dichlorophenoxyacetic acid) ranging from 3 to 5 mg/L. Five-week-old dark proliferated calli were subjected to many combinations of hormones (RMOS, RMSD, RMSDB, and RMSDBK) (Section 4.2) and the maximum number of shoots was observed on RMSDBK medium (Figure 1). However, it was observed that maintenance of 2,4-D in combination with kinetin and BAP (6-Benzylaminopurine) results in healthy and separable mature plants, contrary to earlier studies (Figure 2) [14,15]. BAP, kinetin, and 2,4-D had synergistic effects on shoot regeneration [16,17].

**Figure 1.** Efficient multiple shoot regeneration from embryogenic calli developed from undifferentiated young leaf whorls of different cultivars of sugarcane on the RMSDBK medium. (**A**) US-778 (**B**) HSF-240 (**C**) US-64 (**D**) CPF-246 (**E**) US-127 (**F**) HSF-242.

**Figure 2.** Effect of different types of media on shoot induction from calli derived from young leaf whorls of different genotypes. Most responsive genotype to regeneration was CPF-246 on RMSDBK medium.
