Time-course live imaging of maize and sorghum protoplasts v1
Authors: Baschieri Rachel 
Published: September 2022

A protoplast is a living plant, fungal, or bacterial cell with the cell wall removed. Protoplasts offer a simplified system for studies of gene expression compared to more complex whole-plant systems and are promising for the interrogation of cellular physiology, metabolism and responses to stimuli. Performing live imaging of protoplast experiments in a time-course experiment provides more information about how these cellular processes progress over time. Protoplast time-course live imaging studies have previously explored intracellular auxin localization [1], auxin’s effect on gene expression [2], the cellular localization of a mobile transcription factor, and its dynamic response to chemical endomembrane system inhibitors [3], cell wall regeneration [4], and cell division and micro-callus formation [5]. Though time-course live imaging of protoplasts is a useful tool, it is also a skill that can be difficult to acquire. Practice and troubleshooting of various protoplast isolation, transfection, and immobilization protocols can be time-consuming and may deter many from performing experiments with protoplasts. This protocol combines protoplast isolation, transfection, and immobilization methods to form a cohesive protoplast time-course live imaging protocol with notes and modifications from our troubleshooting. This protocol was developed for time-course live imaging of maize and sorghum protoplasts. The isolation and transfection steps are based on methods published in Meng et al. 2020 with a few modifications. First, 50mM MES was added to the PEG-calcium transfection solution to buffer the transfection solution at pH 5.7 and increase protoplast transfection efficiency . Second, 1% bovine serum albumin(BSA) was added to the incubation solution based on Ohshima et al. 1989 to increase protoplast viability after overnight incubation. Third, 50ug/mL ampicillin is added to the incubation solution to decrease bacterial growth overnight [8]. The alginate immobilization steps were adapted from Middleton et al. 2018 and Dovzhenko et al. 1998. References Middleton, A. M., Dal Bosco, C., Chlap, P., Bensch, R., Harz, H., Ren, F., ... & Dovzhenko, A. (2018). Data-driven modeling of intracellular auxin fluxes indicates a dominant role of the ER in controlling nuclear auxin uptake. Cell reports, 22(11), 3044-3057. Maurel, C., Leblanc, N., Barbier-Brygoo, H., Perrot-Rechenmann, C., Bouvier-Durand, M., & Guern, J. (1994). Alterations of auxin perception in rolB-transformed tobacco protoplasts (time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin). Plant Physiology, 105(4), 1209-1215.. Wu, S., & Gallagher, K. L. (2014). The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on the endomembrane system. The Plant Journal, 80(3), 396-409. Kuki, H., Higaki, T., Yokoyama, R., Kuroha, T., Shinohara, N., Hasezawa, S., & Nishitani, K. (2017). Quantitative confocal imaging method for analyzing cellulose dynamics during cell wall regeneration in Arabidopsis mesophyll protoplasts. Plant Direct, 1(6), e00021. Xu, M., Du, Q., Tian, C., Wang, Y., & Jiao, Y. (2021). Stochastic gene expression drives mesophyll protoplast regeneration. Science Advances, 7(33), eabg8466. Meng, R., Wang, C., Wang, L., Liu, Y., Zhan, Q., Zheng, J., & Li, J. (2020). An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9. PeerJ, 8, e10077. Ohshima, M., & Toyama, S. (1989). Studies on Culture of Cells and Tissues of Crop Plants: I. Survey on enzymatic isolation and culture of rice leaf sheath protoplasts. Japanese Journal of Crop Science, 58(1), 103-110. Yoo, S. D., Cho, Y. H., & Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature protocols, 2(7), 1565-1572. Dovzhenko, A., Bergen, U., & Koop, H. U. (1998). Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma, 204(1), 114-118.