A procedure to introduce point mutations into the Rubisco large subunit gene in wild?type plants

Photosynthetic inefficiencies limit the productivity and sustainability of crop production, resilience agriculture to future societal environmental challenges. Rubisco is a key target for improvement as it plays central role in carbon fixation during photosynthesis remarkably inefficient. Introduction mutations chloroplast-encoded large subunit rbcL particular interest improve catalytic activity efficiency enzyme. However, manipulation hampered by its location plastome, with many species recalcitrant plastome transformation, plastid’s efficient repair system, which can prevent effective maintenance introduced homologous recombination. Here we present system where introduction number silent into within model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This was used successfully generate range transplastomic lines from wild-type N. stable point 40% transformants, allowing assessment effect these on assembly activity. With further optimization, approach offers viable way forward mutagenic testing function planta tobacco modifying other crops chloroplast transformation feasible. The strategy could also be applied introduce genes.