*2.4. Tracking Transgenic Plastids Using Green Fluorescence Protein (GFP)*

The antibiotic-resistant calli and regenerated shoots were initially inspected for fluorescence using a hand-held long-wave UV lamp. The proliferating calli and leaves of the putative transplastomic sugarcane plants were subjected to a stereomicroscope, SZX (Olympus America, Melville, NY, USA) equipped with a GFP detection system. The greenfluorescing calli were selected and proliferated on streptomycin-containing MS medium supplemented with MS vitamins, 3 mg/L 2,4-D and were regenerated into shoots upon transfer to RMSDBK medium. Green-fluorescing sectors were observed in the leaves of antibiotic-resistant transgenic plants, confirming that the *gfp* gene was integrated into sugarcane plastome (Figure 6). These fluorescing sectors varied in size in leaves and even in different plants, depending upon the expression of the transgene as well as segregation of transplastomic and wild-type plastids (Figure 7). Such chimerism necessitates another cycle of regeneration on the selective medium. Accumulation of green fluorescent protein (GFP) was also assessed in the leaves of PCR-positive plants by confocal laser scanning microscopy. The FLARE-S expression confirmed the segregation of transplastomic and wild-type plastids in streptomycin-resistant sugarcane (Figure 8). It was observed that cells

surrounding the veins are fluorescent, indicating chloroplasts in bundle sheath cells are only transformed, and regular files of these cell clones are extended in an aligned fashion.

**Figure 4.** Purification of shoots carrying transformed plastomes on streptomycin-containing regeneration medium. (**A**) Calli proliferated under dark growth conditions were used to transform plastomes (**B**) Selection of cells, after the bombardment, on streptomycin-containing regeneration medium where resistant cells are regenerated into shoots while sensitive are bleached. (**C**) Close up of the resistant shoots as shown at B. (**D**) Root initiation on streptomycin-containing rooting medium. (**E**,**F**) Multiplication of regenerated shoots on MS medium in jars and glass tubes with and without IBA, respectively.

**Figure 5.** Confirmation of integration of FLARE-S cassette into the plastid genome of sugarcane. (**A**) Physical map of sugarcane plastid transformation vector showing the position of primers flanking various genes (**B**) PCR amplification of marker gene (*gfp*) with primer sets P5/P6: lanes M 1 kb DNA ladder, lane +ve shows amplification from plasmid DNA, lanes S1, S2, and S3 are transformed sugarcane plant DNA, lane WT untransformed sugarcane plant DNA (**C**) PCR amplification of FLARE-S with primer sets P6/P7: lanes M represent 1 kb DNA ladder, lane +ve represents plasmid DNA as template, lanes S1, S2, and S3 are transformed sugarcane plant DNA, lane WT untransformed sugarcane plant DNA (**D**) PCR amplification of left border sequence along with marker gene (*aadA*) with primer sets P1/P8: lanes S1, S2, and S3 are transformed sugarcane plant DNA, lane WT untransformed sugarcane plant DNA, lanes M 1 kb DNA ladder.
