The production of crocin, an unusual and valuable apocarotenoid with strong biological activity, was obtained in a cell suspension culture of saffron (L. day of the cell growth cycle and 24 h harvest time was optimized to exploit these cells for the highest increase of metabolite production in saffron cells. L., cell suspension, crocin, salicylic acid, subcellular compartmentalization, antioxidant activity 1. Introduction Saffron, L., representing probably the most costly spice within the global globe [1], is mostly found in medicine due to its wide pharmaceutical properties such as CDDO-Im for example anti-depressive, antitussive, antigenototoxic, anticonvulsant, antihypertensive, anti-Alzheimers, antioxidant, anti-diabetic, anti-Parkinsons, antinociceptive, and anti-inflammatory actions [2]. Saffron is certainly valorized by crocin (crocetin ester), picrocrocin, and safranal bioactive substances, the three main bioactive substances of saffrons [3] stigmas. Crocin, the very first most abundant substance of saffron, is in charge of the red colorization and may be the highest in support of water-soluble carotenoid, offering as an all natural meals colorant and an antioxidant more powerful than -tocopherol [4 also,5]. Lately, crocin effects are actually responsible for a lot of the saffron therapeutic results [6]. Among various other effects, it got anti-depressive results on topics with metabolic symptoms [7]; its program TP15 on rat hippocampus improved storage function and learning [8] also. Unfortunately, the way to obtain crocin remains costly. Plant supplementary metabolite creation by cell civilizations is gaining interest as a trusted, safe, and constant supply strategy option to chemical substance synthesis [9]. The organic variability taking place in seed in-vitro cultures, often unwanted although, could possibly be exploited for determining interesting biosynthetic variations [10]. Furthermore, the creation of bioactive metabolites through cell civilizations may be beneficial once the metabolites are biosynthesized in particular plant tissue [11]. Elicitor program to seed cell civilizations continues to be proved to improve the biosynthesis of the required bioactive substances successfully. Specifically, it had been proven with tocopherol and phytosterol in mung coffee beans, safflower, and sunflower [12,13], CDDO-Im artemisinin in [14] or taxol in suspension system cultures [15]. Building this kind of bioproduction technique for crocin, utilizing a cell suspension system lifestyle and applying an elicited induction of biosynthesis, can be considered as a relevant improvement in satisfying the great demand for this valuable pigment in the food industry. Salicylic acid (SA), known to be a plant defense signaling compound, has a pivotal role in alleviating injuries due to abiotic and biotic stress in plants [16,17]. SA was also shown to act as an elicitor to induce secondary metabolites accumulation. The effects of SA around the biosynthesis of several metabolites have been extensively clarified in many plants [18]. Phenolic compounds (PC) were elicited in cell suspension cultures of [19] and [20]; similarly, alkanes and fatty acids were elicited in cell cultures of [21]. In our recent work, callus cultures were established from two different corm and style explants of saffron [22]. Applying different herb growth regulators (PGR), we optimized crocin production in style-derived calli, using thidiazuron in combination with naphthalene acetic acid on MS medium. In this study, style-derived calli with the best crocin production were used to establish a saffron cell suspension culture. The influence of different concentrations of SA was studied on cell growth, and on the accumulation of crocin and PC content in saffron cell extracts or their corresponding spent media. The extracts were further tested for their antioxidant activity and potential effect on cancer cells. To better interpret the effects of SA on cells, their compartmentalization was investigated microscopically in vivo using saffron protoplasts transiently transformed with different vacuolar markers such as RFP-SYP51, GFPgl133Chi, and AleuRFP. Altogether, our results provide a wide characterization to support a simple but effective optimization of the crocin bioproduction process. 2. Results 2.1. Cell Suspension Lifestyle and SA Results on Cell Development The saffron cell suspension system was extracted from optimized friable calli [22] along with a 10-time subculture routine was optimized in several a few months. Methanol-dissolved SA at different concentrations (0.1, 0.5, and 1 mM) was implemented to saffron cells. The same quantity of methanol useful for the planning of every SA focus was put on saffron control cells and known as 0.1, 0.5, and 1 mock, while cells without SA or methanol represented handles. The effects of the remedies on cell development had been evaluated by calculating the new weight and proven in Body 1. CDDO-Im Three times after subculturing, the fresh weight.