Promoting plant protoplast fusion via membrane decoration with cell-penetrating peptide-conjugated lipids

Protoplast fusion is a powerful plant cell engineering approach that enables the formation of new hybrids and facilitates genetic exchange across species barriers. However, conventional fusion techniques, such as polyethylene glycol (PEG)-induced fusion and electrofusion, often have low efficiency and poor reproducibility. In this study, we used Tat peptide-conjugated PEG-lipids (Tat-PEG-lipids) with different alkyl chain lengths (C9, C12, and C14) to examine the effect of PEG-lipid conjugates on fusion efficiency in rice (Oryza sativa L. cv. Nipponbare) protoplasts. Initially, we used rhodamine-labeled PEG-lipids to evaluate the efficiency of membrane incorporation, revealing that PEG-lipids bearing longer alkyl chains (C12 and C14) were effectively modified to the protoplast membrane. From the measurement of the zeta potential, surface charge of protoplast modified with Tat-PEG-lipid (C12 and C14) (C12; -4.6 mV, C14; -2.4 mV) showed the positive shift from no treatment surface (-6.7 mV). Moreover, confocal microscopy and flow cytometry-based fusion assays revealed that Tat-PEG-lipid (C12) contributed to promoting a significant fusion of rice protoplast membranes, with a fusion efficiency of 9.1 %. In addition, when we combined this method with electrofusion for protoplasts derived from Eucalyptus species, we observed a slight improvement in fusion efficiency from 5.6 % to 8.2 %. These results emphasize the importance of alkyl chain length in optimizing membrane insertion and fusion activity and enabled us to identify Tat-PEG-lipid (C12) as a promising material for efficient and controllable protoplast fusion in plant biotechnological applications.