The plant material comprised of local sugarcane cultivar Co 238 taken from Regional Research Station, Kapurthala during the July-August (formative phase) 2019. The spindle leaf roll segments were prepared from the top apical cuttings. The in vitro experiments were carried out in Tissue culture and Genetic Transformation Laboratory, School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana.

The spindle leaf roll segments excised from fieldgrown healthy sugarcane were used as explant. The explants were thoroughly washed for 10 minutes under running tap water. Then explants were washed with Teepol solution and 1 % Tween-20 for 5 min, then rinsed with sterile distilled water to remove the detergent. Subsequently, they were treated with 1 % Bavistin by shaking on a rotary shaker (New Brunswick Scientific, USA) at 150 rpm for 30-60 minutes and 0.1 % HgCl₂ for 9-10 min to the method described by Sandhu et al. (2016). This was followed by three items of washing with sterile distilled water.

The shoot regeneration was induced on MS media supplemented with 5 mg/l NAA + 0.5 mg/l Kinetin + 30 g/l sucrose + 8 g/l agar designated as M1. Second shoot regeneration media, namely M2 contained MS + 2 mg/L NAA + 0.5 mg/L Kinetin + 30 g/l sucrose + 8 g/l agar. Third media i.e. M3 contained MS supplemented with 7 mg/l NAA + 1 mg/l Kinetin + 30 g/l sucrose + 8 g/l agar. Root induction was carried on two media designated as M1 and M4. MS containing 0.5 mg/L IAA and 0.5 mg/L IBA and 0.5 mg/L BAP and 30 g/l sucrose and 8 g/l agar was designated as M4. Cultures were incubated in a growth room at 25 ± 2 °C under 16/8 h light/dark photoperiod.

The shoot clumps were hardened on moist cotton soaked in water and kept in an incubation room for 3-5 days at 25 ± 2 ºC under 16/8 h light/ dark photoperiod. After hardening, clumps were separated into individual plantlets and were transferred to plastic bags containing soil. The plants were watered regularly. The shoot length, root length, and no. shoots per explant were analyzed using R software (ANOVA test).

The leaf roll segments showed unwhorling after 5 to 10 days of incubation. The unwhorling was observed after the sixth day of incubation on M1 medium, after the ninth day of incubation on M2 medium, and after the seventh day of incubation on M3 medium. The direct shoot regeneration was detected from the cut ends of leaf tissue after the third week of incubation in clusters of 2-3 shoots without the callus phase (Fig. 1A). The shoots were excised from the explants and sub-cultured on the same media for another passage leading to shooting proliferation after 2 weeks of incubation (Fig. 1B). After six weeks of culture initiation, data on the number of shoots per explant, length of shoots were recorded, and after hardening the plantlets were transferred to soil (Fig. 1C, D). The number of shoots per explant was maximum on M1 (7.68 ± 1.26) as compared to M2 (6.04 ± 1.05) and M3 (4.5 ± 0.79). Maximum mean shoot length 8.13 ± 1.87 cm was recorded on M1 medium, followed by 6.19 ± 1.23 cm on M2 and 4.78 ± 0.94 cm on M3 (Fig. 2). Redae and Ambaye (2018) obtained 10.667 number of shoots per explant on MS media containing 2.0 mg/l BAP and 1.0 mg/l NAA. They also recorded a maximum shoot length of 8.4 ± 0.008 cm on 1.5 mg/l BAP and 0.5 mg/l kinetin. Tesfa and Ftwi (2018) observed 10.73 ± 0.35 mean a number of shoots per explant on BAP 2 mg/l and kinetin 1.5 mg/l and maximum shoot length of 3.77 ± 0.40 cm on 2 mg/l BAP and 1.5 mg/l kinetin. In our study, the number of shoots per explants were lesser than these studies. The main differences pertain to the absence of BAP in shoot regeneration media in our study; on the contrary, we used Kinetin. Kinetin is involved in the uninuclear division of cells (Miller et al. 1961), whereas BAP leads to the cell cycle participating in cellular division. In addition, BAP contains NO3− nitrogen and a NO3− to NH4+ that affects the plant growth activity. In contrast, NAA in combination with other hormones, promotes shoot and root regeneration in parallel, as observed in our study. It was interesting to note that maximum mean shoot length was obtained in this study as compared to other studies. It can be concluded that direct shoot regeneration is affected by the nature of genotype and the optimum concentration of growth regulators.

The elongated shoots clumps were then rooted on M1 and M4 media. Roots were observed after two weeks of incubation. The rooting percentage was maximum on M1 (100 %) followed by M4 (96.43%). The maximum root length of 3.89 cm was recorded on M1 and 2.9 cm on M4 (Fig. 3). Tesfa and Ftwi (2018) obtained rooting of 100 % as well as root length of 2.21 cm in half-strength MS with 5 mg/l NAA as compared to 3 and 7 mg/l NAA. In contrast, Redae and Ambaye (2018) obtained a maximum root length of 5.06 cm on half MS with 2 mg/l NAA. The role of NAA for root induction in sugarcane has been highlighted by Tesfa and Ftwi (2018) and Redae and Ambaye (2018). Gill et al. (2006) observed that a lower concentration of NAA (less than 5.0 mg/l) induced rhizogenesis, and a higher concentration of NAA was not conducive for root induction. It can be concluded that NAA alone or in combination plays a vital role in root induction in sugarcane.