*2.3. Plastid Transformation and Recovery of Transplastomic Plants*

Selectable markers are essential tools for chloroplast transformation [11,22] and the only available dominant marker is *aadA*, conferring resistance to streptomycin and spectinomycin. Similar to other monocotyledonous cereals, sugarcane is naturally resistant to spectinomycin. Hence, streptomycin was used to select transformation events. After developing a kill curve, 300–350 mg/L streptomycin was determined as optimal for selection [23]. Three-week-old calli were bombarded using the PDS-1000/He biolistic gun (Bio-Rad, Hercules, CA, USA) following the established protocol [24]. The bombarded calli maintained on MS1.5 medium for 2–3 weeks were transferred onto streptomycin (350 mg/L)-containing RMSDBK medium where transformed cells proliferated and regenerated into shoots. However, wild-type calli turned brown and ultimately became dead

(Figure 4). GFP facilitated the selection and screening of the transformants. Calli chunks fluorescing green under UV light were separated from non-fluorescent cells and were regenerated into shoots. The regenerated shoots were shifted to the rooting medium. The antibiotic-resistant primary regenerants showed a great degree of phenotypic segregation during selection. Variegation was also observed in proliferating leaves along with green shoots. The green shoots appeared to be positive for marker gene presence and integration into plastome (Figure 5), inferring that the variegated shoots were highly heteroplasmic and transgenic plastids were segregating at the cell as well as tissue levels.

**Figure 3.** Schematic diagram showing the cloning strategy for the development of sugarcane plastid transformation vector SOFM2. (**A**) Sugarcane flanking sequences used for site-specific integration of transgene in the plastome inverted repeats. (**B**) Adapter sequence was cloned in between the flanking sequences to engineer multiple cloning sites. (**C**) Final sugarcane plastid transformation vector (SOFM2) with *aadA* and *gfp* genes.
