*2.2. Development of Species-Specific Chloroplast Transformation Vectors*

After the establishment of a proficient regeneration system, a species-specific sugarcane plastid transformation vector was developed. Species-specific flanking sequences play a pivotal role in plastome engineering as a drastic decrease in transformation efficiency was observed when flanking sequences of petunia were used instead of tobacco for *Nicotiana* plastome transformation [18], suggesting that a lack of complete homology of the targeting sequences results in low transformation efficiency [19]. The stable plastid transformation system depends upon the integration of foreign DNA into the plastid genome through homologous recombination [20]. To facilitate homologous recombination, the inverted repeat region of sugarcane was amplified using primers P1 5- -GAT ATC AAA ACC CGT CCT CAG TTC GGA TTG C-3 and P2 5- -GAT ATC CAC GAG TTG GAG ATA AGC GGA-3- . The *trn*I-*trn*A intergenic regions were selected to integrate transgene into the plastome since these regions have successfully been used to develop transgenic chloroplasts [21]. To engineer suitable restriction sites, for further cloning, an adapter sequence carrying restriction sites for *Bcu*I, *Kpn*I, *Apa*I, *Xho*I, *Hinc*II, *Bsu*15I, *Hind*III, *Eco*321, *Eco*RI, *Pst*I, *Sma*I, *Bam*HI, *Bcu*I, *Xba*I, *Not*I, *Bstx*I, and *Sac*I was cloned (Figure 3). The fluorescent selectable marker *FLARE-S* having *aadA* (aminoglycoside-3'-adenyltransferase) gene translationally fused with *gfp* (green fluorescent protein), was used to visually detect transformed cells since this is the most commonly used selection system giving out high transformation efficiency, developed by Khan and Maliga [11].
