*2.5. Tracking Transgene Integration through PCR Approach*

The putative transgenic plants were analyzed for marker gene(s) integration into the plastid genome using the PCR approach. Two gene-specific primers for *gfp* were used. Clones 3, 5, and 6 were found positive for *gfp* gene integration since a fragment of 721 bp was amplified (Figure 5B). Further, to reconfirm the presence of translationally fused genes (*gfp* and *aadA*), another set of primers where forward primer landed on the *aadA* gene and reverse primer on the *gfp* gene were used. Amplification of a fragment of 1452 bp from clones 3, 5, and 6 confirmed the integration of the transgene into the plant genome. These experiments only verified the presence of transgenes in the plant genome but did not authenticate whether transgene was integrated into the plastome. To verify whether the transgenes are integrated into the chloroplast genome, another pair of primers was designed where one primer lands on native plastome and other on transgene (*aadA*). The amplification of a fragment of 2259 bp confirmed their transplasmicity, thus eliminating the possibility of spontaneous mutants, escapees, or even nuclear transformants.

However, plastid transformation was achieved in sugarcane, but low transformation efficiency and heteroplasmy remained to be resolved. The transformation efficiency resulting in one transformation event per 27 bombarded plates was found to be lower than reported in *Arabidopsis* [25,26], potato [27–29], and tomato [30,31]. However, transformation efficiency was comparable with rice [11] since target tissues in both cases were embryogenic calli. Plastids in the dark-grown calli are normally undeveloped, having a size of ~1 μm (approximately 5 to 10-fold smaller than well-developed chloroplasts in the green leaf tissue, turning out to be tough targets for transformation with 0.6 μm gold/tungsten particles owing to increased physical damage. The use of smaller-sized metal particles (0.4 μm) is reported to improve the transformation efficiency of proplastids by three- to four-fold [32]. Another reason could be the levels of transcription and translation that

is lower in proplastids than mature chloroplasts [33]. The strong constitutive promoter (*Prrn*) was used to drive transgene expression but its activity has been reported to be low in proplastids. Thus, promoters encompassing both the PEP (plastid-encoded RNA polymerase) and NEP (nucleus-encoded RNA polymerase) would be the promoters of choice to increase transgene accumulation in sugarcane proplastids [34]. Another major impediment in achieving homoplasmic clones is the anatomy of the sugarcane plant where mesophyll and bundle sheath cells originate from preprogrammed dividing cells, and these cells are aligned in a regular fashion in the leaves.

**Figure 6.** Tracking green fluorescent protein in sugarcane leaves by a stereomicroscope equipped with a GFP detection system. GFP fluoresces green whereas chlorophyll fluoresces red when exposed to fluorescent illumination. (**A**–**C**) Images of transformed sugarcane leaves in bright or dark field illumination. (**D**–**F**) Images of transformed sugarcane leaves in fluorescent illumination. (**H**–**I**) Images of untransformed sugarcane leaves in fluorescent illumination.

**Figure 7.** Visualizing fluorescent sectors in transformed sugarcane leaves under a laser scanning confocal microscope. The red channel and the green channel were imaged separately. Images were processed to detect chlorophyll (**A**–**C**) and Green Fluorescent Protein (**D**–**F**). Transplastomic plant leaf sectors fluoresced green (**D**,**E**) whereas no fluorescence was detected in untransformed sugarcane leaves (**F**) under the green channel.

**Figure 8.** The phenotypic segregation of the antibiotic-resistant sugarcane transformants. Variegation was observed in leaves of plants growing on streptomycin-containing MS medium. (**A**,**C**,**E**) Variegated leaf segments of putative sugarcane transformants. (**B**,**D**,**F**) Lush green leaf segments of untransformed sugarcane plants growing on streptomycin-free medium in the same conditions.
