Novel In Planta Transformation of Nipponbare Rice (Oryza Sativa) Tissues using Electroporation type Plasma Jet

Abstract

Common plant transformation techniques, such as Agrobacterium-mediated and biolistic transformation, still rely on time-consuming and intensive tissue culture steps, including callus induction and regeneration. Eliminating these steps using in planta transformation may service to further improve advances in plant molecular biology. Moreover, CRISPR-Cas delivery using ribonucleoprotein (RNP) transformations may further improve the efficiency of transformation using a transient approach that does not require integration into the genome. Similar to electroporation-protoplast transformation, non-thermal atmospheric-pressure dielectric barrier discharge jet (DBDJ), can generate an electric field near cells, inducing pore openings in the cellular membrane and cell wall, and allowing the insertion of exogenous genetic material through the cell wall and plasma membrane to be expressed in living plant cells. The ability of DBDJ to cause electrostatic disruptions of cell wall structures has already been reported in gram-negative bacteria. ( To utilize this technology for plant genome editing, improvements to the electroporation-like plasma jet transformation must include temporary disruption of both the plant cell wall and membrane, without irreparable damage to plant tissues or function. Here we demonstrate the transformation potential of DBDJ by insertion of an eYFP expression plasmid into mature rice embryos (ME), and a variety of GFP binary and non-binary vectors into root tissues of rice seedlings. By exposing excised MEs and seedlings to the DBDJ electric field after treatment in a liquid osmotic medium, we allow for the successful insertion of eYFP and GFP plasmids. The application of this technology may not be limited to genome editing with the potential to be utilized for many other molecular biological processes.