Generation of Transplastomic Tobacco Plants

Plastid transformation was performed as described in previous studies (Nakahira et al., 2013; Kikuchi et al., 2018). Gold particles (0.6 μm) coated with pRGNNV1 plasmid DNA were delivered into young tobacco leaves cultured in vitro using the Biolistic^® PDS1000/He particle delivery system (Bio-Rad, Hercules, CA, United States). The bombarded leaves were kept in the dark at 25°C for 3 days, then cut into small pieces (~0.25 cm²), and placed onto RMOP agar medium [MS medium containing 3% (w/v) sucrose, 0.8% (w/v) agar, 0.1 mg/L 1-naphthaleneacetic acid, 1 mg/L N⁶-benzyladenine, 1 mg/L thiamine, 100 mg/L inositol] supplemented with 200 mg/L spectinomycin dihydrochloride as a selective agent. In tobacco plasmid transformation, it is common to use 500 mg/L spectinomycin dihydrochloride in all selection rounds, but we experienced low efficiency in acquiring resistant shoots or calli when 500 mg/L spectinomycin dihydrochloride was used in primary selection. Therefore, in our research group, the use of 200 mg/L spectinomycin dihydrochloride for primary selection was kept as a standard condition (Nakahira et al., 2013; Kikuchi et al., 2018). Resistant shoots and calli were transferred onto RMOP agar medium containing 500 mg/L spectinomycin dihydrochloride for the second selection. The obtained shoots were subjected to total cellular DNA extraction, as described previously (Kikuchi et al., 2018). To confirm that RGNNV-CPpt was correctly introduced into the specific region of the plastid genome, PCR-based genotyping was performed using the total cellular DNA as a template with the primer pair 1: ADL-F3 (5'-CGGGGGGGACCACCACGGCT-3') and ADL-R3 (5'-AGGGTTGAAGGGAGATAGTGCATCA-3'), and primer pair 2: RGNNV-Fd1 (5'-CCATGGTAAGAAAAGGAGAAAAAAAATTAGC-3') and Sal-TpsbA-Rv (5'-GTCGACCGAATATAGCTCTTCTTTCTTATT-3'). The amplification program using primer pairs 1 and 2 was as follows: 94°C for 1 min, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 2 min. The transgenic lines confirmed by genotyping were subjected to two more rounds of regeneration cycles on RMOP agar medium containing 500 mg/L spectinomycin dihydrochloride to achieve homoplasmy and finally rooted on MS medium supplemented with 3% (w/v) sucrose, 0.8% (w/v) agar, and 500 mg/L spectinomycin dihydrochloride.