**3. Production of terpenes** *via* **plant cell tissue and organ culture technics**

Since terpenes are pharmaceutical compounds with important biological activities, studies on producing these compounds in plants have accelerated in the last 20 years. Plants generally produce low concentrations of terpene in their tissues, with terpene concentration less than 2–3% of a plant's total dry weight. In particular, variation in terpene distribution caused by biotic, abiotic, and seasonal stimuli may vary depending on the plant's chemotype, suggesting that different plants may respond to genetically different stimuli with various terpene syntheses. This situation may differ even between species and individuals [15].

Due to these molecules' complexity and extreme metabolic modifications, their chemical synthesis is inherently tricky, expensive, and relatively low-yield. By 2050, the need for food is expected to double its current level due to overpopulation, and therefore the use of PSMs will increase in the coming decades to meet this demand [24]. With the increase in the world population, modern technologies developed to meet the demand for PSMs and to overcome possible negative situations have begun to be used. Today, many biotechnological techniques such as plant cell and tissue cultures (shoot culture, callus culture, suspension culture, hairy root culture, plant cell immobilization, and bioreactors) and genetic engineering applications are widely used for terpene production [25]. Many other valuable pharmaceutical terpene compounds, such as ginsenosides in Panax ginseng [26, 27], terpenoid indole alkaloids (TIAs) in *Catharanthus roseus* [28, 29], and tanshinones in *Salvia miltiorrhiza* [30, 31], can be produced in high quantities through the shoot, callus, and cell suspension cultures technics. Some studies conducted to increase the production levels of some terpenes and produce them on an industrial scale have focused on bioreactor systems [32]. It has been reported that *Rhizoma zedoariae* cell suspension cultures provide cell proliferation and accumulation of β-element in the bioreactor [33]. Considering that secondary metabolite production may be higher in differentiated tissues, extensive research has also been conducted on hairy root cultures transformed with *Agrobacterium* sp. as an alternative research tool. Extensive research has also been carried out on hairy root cultures transformed with the *Agrobacterium rhizogenes*mediated transformation method, which reduces the risk of somaclonal variation and provides rapid shoot regeneration, has yielded successful results for the production of terpenes. For example, although the concentration of triterpene detected in the leaves of *Centella asiatica* plants was >2 times higher than in the petiole, the amount of triterpene in the petiole-derived hairy root cultures was 1.4 times higher than in the leaf-derived hairy root cultures. In addition, it was determined that the amount of terpene obtained from leaf and petiole root cultures was higher than that of adventitious roots [34]. Advances in immobilization techniques contribute to a significant increase in the production of high value-added pharmaceutical compounds. *Plumbago rosea*, in which cell cultures were immobilized with 10 mM calcium alginate, plumbagin production was doubled compared to control cells [35].

Furthermore, understanding the function of genes involved in terpene production may lead to discovering new compounds or metabolic pathways that can reveal optimal properties. Accordingly, increased terpene emission was observed in *Nicotiana* 

*tabacum* plants' leaves after adding monoterpene synthase genes [36]. In recent years, overproduction, co-overexpression, gene silencing, and genome editing techniques are among the current approaches used for synthesizing different terpene compounds through developments in metabolic engineering.
