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Clausena harmandiana (Pierre) Guillaumin is the Thai medicinal plant that possessed several pharmacological activities. The main active constituents of this plant are the carbazole alkaloids, isolated from wild plants. However, the in vitro culture for production of carbazole alkaloids from this plant has never been reported. Therefore, we aimed to develop callus culture of C. harmandiana elicited with two biotic elicitors, Trichoderma harzianum and Bacillus subtilis, as a sustainable source of carbazole alkaloids. The callus treated with living B. subtilis (BL) at 0.1 and 1% (v/v) for 3 days accumulated 5-fold increased level of clausine K. The highest level reached 309.37 ± 34.84 μg/g DW. This treatment also showed a significant increase in both total phenolic content and antioxidant capacity, which support the optimum usage of this elicitor. Moreover, only callus treated with 1% (v/v) Trichoderma culture filtrate (CF) showed a significant increase in the total phenolic contents and antioxidant capacity. The correlation analysis also revealed the significant correlation between antioxidant capacity and total phenolic level, total flavonoids, and clausine K but not 7-methoxymukonal. The results from our study suggested the use of C. harmandiana callus with the Bacillus elicitors for high-level production of clausine K.

Morus alba L. (Moraceae) has been used in traditional medicine for the treatment of several illnesses. Recent research also revealed several pharmacological activities from many groups of secondary metabolites, including the stilbenoids mulberroside A, oxyresveratrol, and resveratrol, which are promising compounds for cosmetic and herbal supplement products. In our previous study, cell cultures of M. alba showed high productivity of these compounds. In this study, we attempted to develop immobilized cell cultures of M. alba and to test the effect of elicitors and precursors on the production of stilbenoids. The immobilization of the M. alba cells significantly promoted the secretion of mulberroside A into the extracellular matrix and culture media to 60%, while enhancing the level of oxyresveratrol and resveratrol by 12- and 27-fold, respectively. The elicitation of immobilized cells with a combination of 50 µM methyl jasmonate and 0.5 mg/mL yeast extract for 24 h promoted a twofold increase in the production of all three stilbenoids. Furthermore, the addition of 0.05 mM l-phenylalanine, 0.03 mM l-tyrosine, or a combination resulted in the enhancement of mulberroside A production for up to twofold. The addition of l-tyrosine significantly enhanced the production of oxyresveratrol and resveratrol. This is the first report of stilbenoid production using immobilized cell cultures of M. alba. The cultures have benefits over normal cell suspension cultures by promoting the secretion of mulberroside A and enhancing the levels of oxyresveratrol and resveratrol. Thus, it could be a candidate method for the production of these stilbenoids.

Using several explants of Pueraria candollei Grah. ex Benth. var. candollei and two strains of Agrobacterium rhizogenes (ATCC 15834 and 43057), hairy root cultures were established. Including 100 μM acetosyringone in the culture medium enhanced frequency of hairy root induction by up to 58 %. Subsequently, effects of inoculum size (IS) and temperature on growth and production of isoflavonoids in hairy roots were determined. Conditions of 1 % IS and 32 °C promoted the highest accumulation of total isoflavonoid content, up to 31.0 ± 22.6 mg/g dry weight (DW), in hairy roots. Moreover, culture of hairy roots at 32 °C decreased browning of hairy roots. Furthermore, this temperature promoted accumulation of the secondary metabolite daidzein; whereas, hairy root cultures at the stationary phase accumulated higher amounts of the isoflavonoid puerarin rather than daidzein.

We established cell suspension cultures derived from leaf, stem, and root calli of Pueraria candollei var. candollei and P. candollei var. mirifica using liquid Murashige and Skoog (MS) medium supplemented with 0.56 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Growth of the cell suspension cultures progressed to the stationary phase within 15-24 days. Methanolic extracts of cell suspension cultures of both varieties of P. candollei were analyzed using a validated HPLC protocol. All cell lines derived from leaf, stem, and root explants produced four major isoflavonoids: daidzein, daidzin, genistein, and genistin; these isoflavonoids were detected only in the roots of intact plants. Furthermore, the isoflavonoid contents of the cell suspension cultures were higher than those of intact plants. Thus, cell suspension culture of both varieties of P. candollei may be an effective tool for isoflavonoid production.