Assistant Professor University of Tennessee Knoxville, Tennessee
Body of Abstract: Plastid genetic engineering in higher plants has relied on homologous recombination (HR) site-specific integrating vectors for nearly 30 years. In a recent investigation, the small-synthetic genome (mini-synplastome) was designed as a valuable alternative tool for engineering plastids of the crop plant Solanum tuberosum (potato). The core sequence of the mini-synplastome is a chloroplast-specific origin of replication (ori) that confers the ability to this vector to persist as extra-chromosomal DNA (episome) without integration in the chloroplast genome (plastome). Many intrinsic characteristics make the mini-synplastome a valuable choice for plastid genetic engineering: i) ability to persist at high-copy number at ~1:1 ratio with the endogenous plastome throughout all plant life cycle; ii) stability throughout multiple vegetative plant generations, including plants originated from tubers; iii) high levels of heterologous protein accumulation, equaling or even exceeding the levels achieved in the state-of-the-art HR vectors; iv) no phenotypic penalties have been observed in synplastomic plants with equal phenotypic parameters compared to wild-type plants. Among potential applications of this novel genetic tool, the mini-synplastome have been redesigned to produce marker-free transplastomic plants. The utilization of these novel vectors allows stable transgene integration at homoplasmy and at the same time complete removal of the selectable-marker gene (aadA) in only one vegetative generation in tissue culture. We anticipate that in the near future the mini-synplastome has the potential to revolutionize agriculture providing novel synthetic biology tools for more flexible, precise, and safe genetic engineering of plastids for improving crop traits.