*4.4. Sugarcane Plastome Transformation*

The optimized conditions for efficient calli induction and regeneration were used to engineer the *Saccharum* plastome. Among the evaluated genotypes, genotype HSF-240 was selected for transformation owing to its good response to callogenesis and regeneration. Calli were bombarded with SOFM2 plasmid DNA-coated gold particles of 0.6 μm diameter, using a Biolistic gun PDS-1000/He (Bio-Rad, Hercules CA, USA), following the transformation procedures described by Khan and Maliga [11]. The calli were incubated in the dark at 26 ± 1 ◦C for 48 h before transfer to selective medium supplemented with 350 mg/L of streptomycin sulfate (Phytotechnology, Lenexa, KS, USA). After two weeks of incubation on callus induction medium, antibiotic-resistant calli were shifted to selective RMSDBK medium. The streptomycin-resistant shoots were rooted on MS medium containing 4 mg/L IBA.
