Construction of a species-specific vector for improved plastid transformation efficiency in L.

In the present study, we focused on designing a species-specific chloroplast vector for L. and finding out its transformation efficiency compared to a heterologous vector. The plastid transformation vector (CaIA) was designed to target homologous regions and of IR region. A selectable marker gene , whose expression is controlled by promoter and terminator, was cloned between two flanking regions. A heterologous vector pRB95, which targets and of LSC region along with driven by promoter and terminator, was also used for developing plastid transformation in . Cotyledonary explants were bombarded with stabilized biolistic parameters: 900 psi pressure and 9 cm flight distance, and optimized regeneration protocol (0.7 mg/L TDZ + 0.2 mg/L IAA) was used to obtain transplastomic lines on selection medium (300 mg/L spectinomycin). The integration and homoplasmy were confirmed by obtaining 1.2 and 3.7 kb amplicons in CaIA transformants and subsequently verified by Southern blotting, whereas in pRB95 transformants, integration was confirmed by PCR with 1.45 kb and 255 bp amplicons corresponding to integration and flanks, respectively. The transformation efficiencies attained with two plastid vectors were found to be 20%, i.e., 10 transplastomic lines in 50 bombarded plates, with CaIA and 2%, i.e., 1 transplastomic line in 50 bombarded plates, with heterologous pRB95, respectively.