*4.3. Construction of Species-Specific Chloroplast Transformation Vectors*

Sugarcane species-specific plastid transformation vector SOFM2 was developed for targeted insertions into the *trn*I (97663–98685)-*trn*A (98751–99633) inverted repeat region of the plastome (GenBank accession No. NC 006084). The cloned fragment served as flanking borders in the final transformation vector. These flanking sequences (*trn*I-*trn*A) were amplified from the sugarcane plastid genome by PCR, 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- ). P1 primer annealed to nucleotides 97101–97126 and P2 annealed to 99620–99641 of the plastome. A DNA fragment carrying restriction sites for endonucleases: *Bcu*I, *Kpn*I, *Apa*I, *Xho*I, *Hinc*II, *Bsu*15I, *Hind*III, *Eco*321, *EcoR*I, *Pst*I, *Sma*I, *Bam*HI, *Bcu*I, *Xba*I, *Not*I, *Bstx*I, and *Sac*I, was amplified by primers P3 (5- -GGT ACC ACT AGT GGG CCC CCC CTC GA-3- ) and P4 (5- -GAG CTC CAC CGC GGT GGC GGC CGC T-3- ) and cloned in the intergenic region to facilitate further cloning. The selection marker FLARE-S (fluorescent antibiotic resistance enzyme, spectinomycin, and streptomycin) encoding a bifunctional protein obtained by translational fusion of the aminoglycoside 3'-adenyltransferase (*aadA*) gene with the green fluorescent protein (*gfp*) gene was cloned at *EcoR*I/*Hind*III sites.
