Callus induction

Spindle leaf explants (Figure 1A) of sugarcane cultivars LSC and B36464 formed callus on MS media containing varying concentrations of 2,4-D or picloram by the twelfth day of incubation (Figure 1B, C). For both cultivars, the absence of 2,4-D and picloram in the culture media resulted in no callus production, indicating an auxin requirement for callus formation in sugarcane, similar to other plant species (Ikeuchi et al., 2013; Osman et al., 2016; Zang et al., 2016). Similar observations were made by Ramgareeb et al. (2010) and Alcantara et al. (2014)where various types of sugarcane explants failed to form callus on auxin-free media. Exogenous auxin supplementation is required to reprogram somatic cells to acquire pluripotency as well as initiate cell division leading to the formation of callus (Skoog and Miller, 1957; George et al., 2008; Fehér, 2019). An increase in the number and size of calli was observed after 21 days of incubation on both 2,4-D (Figure 1D, E) and picloram-supplemented media (Figure 1F, G).

Callus induction on 2,4-D supplemented media was significantly higher (p ≤ 0.05) compared to picloram at the same concentrations (Table 1; supplementary Table 1) suggesting that 2,4-D is an effective auxin for callus induction in sugarcane. This is similar to earlier reports (Ho and Vasil, 1983; Gill et al., 2004; Dibax et al., 2011; Alcantara et al., 2014; Arjun Srinath, 2015). However, our findings contradict those of Gallo-Meagher et al. (2012)who reported higher callus induction on picloram- supplemented medium rather than 2,4-D in sugarcane. This may be due to differences in genotypic response to auxins as reported by Gandonou et al. (2005) and Mekonnen et al. (2014).

In general, induction of embryogenic calli improved with increasing auxin concentration (Table 1). For LSC, callus induction was best achieved at the highest concentrations of 3 and 4 mg/L for 2,4-D. Similar observations have been reported by several authors including Badawy et al. (2008), Jahangir et al. (2010) and Arjun Srinath (2015). A similar trend was observed for picloram. However, as picloram concentration increased from 3 to 4 mg/L callus induction rate declined (from 44 to 34%) in LSC. The reduction in callus formation may be attributed to the presence of endogenous hormones in the explant tissues prior to culture initiation (Can et al., 2008). Wernicke et al. (1986), explain that high auxin concentrations may prevent meristematic cell divisions, resulting in decreased reactions such as callus induction. In the genotype B36464, the best concentration for callus formation is between 2- 4 mg/L 2,4-D or 4 mg/L picloram. Nonetheless, the response of the two genotypes in this study to callus induction is beneficial in that, it is a first and crucial step in in vitro mutagenesis as well as genetic transformation protocols for improvement of local sugarcane genotypes. Calli are ideal propagules for reception of genes since they are mostly of single cell origin (Nagmani et al., 1987)and can also be reached by either physical or chemical mutagens in mutagenesis (ref).

Mean production of embryogenic callus is the ratio of callus formed to the number of explants cultured expressed as a percentage. Data represent mean ± SD. Values in a column followed by the same letters are not significantly different from each other at p ≤ 0.05 (Tukey’s pairwise comparison test).

Shoot induction from calli

The addition of cytokinins, particularly BAP, to MS medium has been shown to be effective for inducing shoots from sugarcane callus cultures (Ather et al.. 2009; Dibax et al.. 2011; Arjun Srinath. 2015). Thus, to determine the optimum concentration of BAP for shoot induction from callus for the two cultivars, we transferred friable calli to MS medium containing BAP ranging from 0.1 to 1.5 mg/L BAP. By the second week, green microstructures were observed on the surfaces of calli of both B36464 (Figure 2A, B) and LSC (Figure 2C, D), suggesting the formation of totipotent shoot primordia. The presence of high concentration of cytokinin in the shoot induction medium induces shoot stem cell regulators from callus (Gordon et al., 2007). According to Dibax et al. (2013), the formation of such shoot primordia is triggered from both nodular and friable calli due to the presence of cytokinins which initiate a pathway redirection. The formation of shoot primordia on callus of sugarcane resulted in the development of shoots within 28 days of culture. The mean number of shoots formed per callus explants ranged from 9.69 ± 2.55 to 18.13 ± 3.07 in B36464 genotype and 8.44 ± 4.19 to 16.75 ± 3.55 in LSC genotype (Figure 3; Supplementary Table 2). The shoot induction rates obtained from callus cultures in this study are higher than those obtained from shoot tip or lateral bud culture (Danso et al., 2011; Mekonnen et al., 2014)making this protocol efficient for commercial production of sugarcane planting materials. Callus develops more shoot primordium, hence more shoots than from direct organogenesis in shoot tips. Shoot regeneration from callus is a result of localised cell division leading to differentiation of globular meristemoids which can develop into shoots (Ove?ka et al., 1997). For example, in Nicotiana tabacum, the palisade cells divide to form callus which gives rise to shoot primordia and finally regeneration into shoot (Gupta et al., 1966; Zhi-hong and Gui-Yun, 1980).

The lowest concentrations of BAP (0.1 and 0.5 mg/L) induced the least number of shoots from callus in both genotypes. However, at higher BAP concentrations of 1.0 and 1.5 mg/L, we recorded a significant increase (p ≤ 0.05) in shoot induction in both genotypes (Supplementary Table 2). An increase in shoot production from sugarcane callus cultures with increasing BAP concentration has been reported by Behera and Sahoo (2009) and Hapsoro (2017). A comparison between the two genotypes showed higher shoot induction from callus of variety B36464 compared with LSC, suggesting genotype effect on shoot induction. Similarly, genotypic differences have been observed in shoot regeneration from callus cultures of different sugarcane cultivars (Gandonou et al., 2005; Kaur and Kapoor, 2016; Di Pauli et al., 2021).

Shoot proliferation and root induction

Given the increasing frequency of shoot induction (from callus) with increasing BAP concentration in the previous experiment, we evaluated the proliferation of shoots on MS medium supplemented with a higher concentration of BAP (2.5 mg/L) in combination with low concentrations of NAA (0.1 mg/L) and GA₃ (0.1 mg/L). In sugarcane, as in many plant species, a high cytokinin to auxin ratio in the culture medium has been shown to effectively stimulate shoot regeneration and differentiation (Ikeuchi et al., 2013; Fehér, 2019). In order to increase plantlet production and maximise economic gains, four serial subcultures were used to multiply the shoots in culture.

After subculture cycle 1, the total number of shoots produced in LSC genotype was higher (2902) compared to B36464 (2797) (Table 2 and Figure 4A, B). However, after 8 weeks of culture on SPM (cycle 2), shoot production was higher (7209) in B36464 compared to LSC (6927) and this trend was maintained for the next two cycles (till 16 weeks of culture). The total number of shoots formed in both genotypes increased more than two-fold by the second subculture (at 8 weeks) and four-fold by the fourth subculture (at 16 weeks) (Table 2). This high frequency of plantlet production indicates the efficiency of the callus-mediated technique for sugarcane micro-propagation.

Interestingly, shoot multiplication rates decreased with successive subcultures (second subculture onwards) likely due to the cumulative effects of BAP in succeeding subcultures which might have led to high endogenous concentration of BAP, hence the phytotoxic effect. In general, decline in shoot multiplication rates as a result of repeated in vitro subculture has been reported in several plant species (Norton and Norton, 1986; Hussain et al., 2007; Vujovi? et al., 2012). Although reducing the concentration of hormones in the culture medium can delay the decline in shoot multiplication rates (Vujovi? et al., 2012), regular initiation of new cultures from spindle leaf explants is necessary to maintain high shoot multi-plication rates. The initiation of new cultures is important to reduce the risk of somaclonal variation which is quite common in sugarcane (Khan, 1999; Khan et al., 2008). It also ensures genetic homogeneity of plantlets and the production of true-to-type planting materials for the establishment of commercial plantations (Petolino et al., 2003). However, in this work using LSC and B36464 genotypes, we did not observe any genetic or somaclonal variation in plants transferred to the field at six months old.

With respect to root induction, 25% of shoot clusters of LSC formed well-defined roots spontaneously on BAP-containing medium (SPM) after 4 weeks of culture com-pared to 57% in B36464 shoot clusters (Figure 4C). This phenomenon of spontaneous root formation at the shoot maturation stage on media containing only cytokinins has been reported by Zamir et al. (2012). Nonetheless, to improve root formation from in vitro shoots, we supplemented MS media with 3 mg/L NAA which resulted in marked improvement in root development in both B36464 (88%) and LSC (72%) after 8 weeks of culture.

Plantlet acclimatization

Efficient weaning and hardening of in vitro-generated plantlets are critical for successful transfer of plantlets to field conditions. To determine the efficiency of weaning and hardening, a total of 200 plantlets (100 per genotype) were transferred to the greenhouse for weaning. Survival rates of 85 and 91% were achieved for LSC and B36464, respectively (Figure 5). The rates of acclimatization obtained for plantlets of both sugarcane genotypes in this study are similar to survival rates obtained for other commercial Indian varieties (Kaur and Kapoor, 2016)but higher compared to other reports where up to 87% survival rates were obtained (Mekonnen et al., 2014). Well-developed roots in in vitro plantlets prior to weaning have been shown to enhance survival of plants at the weaning stage (Danso et al., 2011). Other factors such as genotype and the medium used for weaning also play significant role in post-flask acclimatisation: cocopeat: soil: sawdust mixture (2:1:1) could explain the differences in plantlet acclimatization compared with other studies. The weaned plantlets were successfully transferred to field after 8 weeks of hardening for further development.