For Southern blotting, 2 μg total genomic DNA from untransformed WT, T1 and T2 generation marker‐free plants integrated with PG1‐Smdex, lipY and T0 generation integrated with mut (co) were digested by suitable restriction enzymes and separated in 0.8% agarose gel, transferred onto the nylon membranes (Nytran, GE Healthcare), and probed as described previously (Kumari et al., 2019 (link); Kwon et al., 2018 (link)). Seeds from untransformed WT and previously developed three independent T0 transplastomic lipase (Kumari et al., 2019 (link)) expressing plants were germinated on ½ MS medium without any antibiotics. The germinated seedlings were transferred and grown in magenta box. The genomic DNA from leaves of two different T1 plants in each of the three independent events (in total six plants) and WT was isolated, and digested with SmaI restriction enzyme (New England Biolabs, Hertfordshire, UK).