Background

Plant cell suspension cultures provide a feasible alternative for the production of plant secondary metabolites and the study of dedifferentiated tissue (Wu and Ge, 2004; Lee et al., 2010). Interest in their use has been increasing for decades as they can offer continuous production systems for high-value biotechnological products for human use. There is a wide variety of cell cultures producing high amounts of secondary metabolites of commercial and industrial interest such as anthocyanins, betalains, flavourings, steviosides, among others (Rao and Ravishankar, 2002).

The lack of significant success for plant cell cultures in becoming regularly used for commercial purposes could be attributed in part to the failure of the dedifferentiated cell lines to produce the desired product with the same characteristics found in the organs of parent plants. The use of modern molecular biology and synthetic biology techniques opens the possibility to genetically improve cell lines and plants to enhance metabolite production (Davies and Deroles, 2014).

Methyl jasmonate (MeJA), jasmonyl-isoleucine (JA-Ile) and Jasmonic acid (JA) are collectively referred to as jasmonates (JAs), and they are involved in the regulation of many developmental processes and defense mechanisms in vascular plants (Pauwels et al., 2008, Pérez-Salamó et al., 2019). JA signaling involves ubiquitination of specific target proteins by the SCF^COI1 complex and their subsequent degradation by the 26S proteasome (Thines et al., 2007).

Bömer et al. (2018) has reported the stably transformed Arabidopsis thaliana cell suspension cultures with an epitope-tagged COI1-overexpressing construct (Devoto et al., 2002) to analyze the effects of JA signaling on cell growth and the production of cell wall proteins and metabolites. In that study, a stable transformation of Arabidopsis cell suspensions was successfully achieved and validated. Changes in the primary metabolism of the transformed cell suspensions were observed. The overexpression of COI1 reinforced its role in mediating defense responses after increasing levels of Oligogalacturonide oxidase 1, beta-Glucosidase/endoglucanases and Polygalacturonase inhibiting protein 2. Furthermore, it affected the availability of metabolites such as beta-alanine, threonic acid, putrescine, glucose and myo-inositol, providing a connection between JA-Inhibited growth and stress responses (Bömer et al., 2018).

The protocol described here was initially devised by O’Brien et al. (2012) to silence Arabidopsis peroxidases adapting a transient transformation system from Ferrando et al. (2000). The gene insertion was mapped by O’Brien et al. (2012) (two independent lines generated, each with a single insertion, thus homogenous cell cultures were obtained). The construct used in this work was prepared and used by Devoto et al. (2002) to transiently overexpress the CORONATINE INSENSITIVE 1 (COI1) JAs receptor (35S:: COI1::HiA). Stable transformation with the same construct was successfully achieved by Bömer et al. (2018). Stably transformed Arabidopsis cell cultures, generated by using this protocol, have been maintained since O’Brien et al. (2012) and Bömer et al. (2018) were published. The fully detailed protocol is presented here.