Breakthrough of wild tomato protoplast regeneration and DNA-free CRISPR-Cas9 genome editing.The cell wall free, single-cell plant protoplast provides a versatile system for conducting gene functional characterization by taking advantage of the low incidence of chimerism. It is also an ideal system for genome editing since all mutated alleles in the T0 generation can be transmitted to the next generation. Currently, the main bottleneck of this strategy is the difficulty of performing protoplast regeneration and concerns of genome instability. Wild tomato (Solanum peruvianum) is an important genetic source of introducing stress-resistant genes for the tomato breeding. However, the progress has been hampered due to the self-incompatibility and highly heterozygous genome. We developed a DNA-free CRISPR-Cas9 genome editing tool based on an optimized protoplast regeneration protocol. Cytogenetic analysis, whole genome sequencing and genome analysis confirms the stable and inheritable diploid and tetraploid transgene-free, homozygous edited tomatoes. Most of the economically important crops are polyploid and a genome editing platform for polyploid crops would accelerate the development of designer crops for a range of climatic conditions.
Co-researchers:LIN, YAO-CHENG, Choun-Sea Lin, Chen-Tran Hsu, Yu-Hsuan Yuan, Po-Xing Zheng, Fu-Hui Wu, Qiao-Wei Cheng, Yu-Lin Wu, Ting-Li Wu, Steven Lin, Jin-Jun Yue, Ying-Huey Cheng, Shu-I Lin, Ming-Che Shih, Jen Sheen