We report a peptide based in vivo plant cell mitochondrial genetic engineering method. Peptide vectors were created by selecting mitochondrial protein sorting signal sequences that contain similar physical and chemical properties as a cell-penetrating peptide (CPP). In the presence of exogenous double-stranded DNA (dsDNA) the peptide vectors called mitochondrial targeting peptides (mTPs) form peptide-nucleic acid nanoparticles. The nanoparticles when incubated with plant cells first cross the cellular membrane and then cross the outer and inner mitochondrial membranes to deliver exogenous DNA into the mitochondria of plant cells. The exogenous DNA integrates into the mitochondrial genome and the transfected plant cells can be cultured into plantlets. A linear double stranded gene construct was delivered into protoplasts and microspores of AC Ultima spring triticale (X. Triticosecale Wittmack) plants using the mTP peptide vector system to study in vivo mitochondrial transfection of plant cells and transient mitochondrial gene expression.