Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced inCrocus sativusfrom zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a fromGardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehydein bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium-mediated supertransformation of multi genes and in transiently transformedNicotiana benthamianaleaves. We demonstrate that co-expression ofGjCCD4awithphytoene synthaseandβ-carotene hydroxylasegenes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and usingin vitroassays, we show that GjCCD4a cleaved β-carotene,in planta, and produced crocetin dialdehyde via C30β-apocarotenoid intermediate. GjCCD4a also cleaved C27β-apocarotenoids, providing a new route for C17-dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency inNicotianaleaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activityin plantaand an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.