Siong Blue Hairy Root And Its Impact Of Exogenous Melatonin

Isatis indigotica Fort. is the main botanical origin of the common herbal medicine, Ban Lan Gen and Da Qing Ye. Isatis indigotica Fort. contains many chemical ingredients, and one component in the plant, indirubin, has depicted extensively anti-tumor activities. As a secondary metabolites production of Isatis indigotica Fort., indirubin has low content in nature, limiting its application in medicine.Hairy roots have been reported to be a potential approach to yield higher amounts of secondary metabolites. In order to obtain the optimal cultural condition of hairy roots of Isatis indigotica Fort., the L-tryptophan precursors, melatonin and IAA are used to enhance the productivity of indirubin, and the growth curve and the indirubin content are tested to assess their effects. The content of related enzyme and the level of secondary metabolites are measured, which would be evidenced on the antioxidant capacity of melatonin and rol gene in hairy root regenration plants of Isatis indigotica Fort., through which the functional roles of melatonin can be acquired and the medical application of melatonin can be expanded.(1) An Agrobacterium rhizogenes-mediated genetic transformation for I. indigotica has been developed. Hairy root cultures were induced from both cotyledon andhypocotyl explants of I. idigotica through transformation with agropine wild-type strains of Agrobaterium rhizogenes A4. The transgenic character of the hairy root cultures was proved by PCR amplification of the rolA, B, C genes and aux genes. The production of mannopine production in positive transgenic lines was detected by high voltage paper electrophoresis as well. The results confirmed the successful transfer of Ri T-DNA (root-inducing transferred DNA) of A. rhizogenes into the I. Indigotica genome. The regeneration plants of hairy root were induced(2) The effect of L-tryptophan precursors on the growth and accumulation of indirubin was assessed, and the impact of IAA and MT in hairy root cultures of I. Indigotica was studiedThe biomass increase was observed after25d of culturing hairy roots in MS medium supplemented with0.2mg/L IAA or0.2mmol/L MT, which was1.20and1.12times higher than that of the control. And supplementation with IAA or MT also resulted in the enhancement of indirubin production from35.7ng/gFW (control untreated culture) to42.4,38.6ng/g FW. The highest yield of indirubin (48.05ng/g FW) was observed when the medium was supplemented with0.20mg/L of the precursor L-tryptophan. Addition of0.20mg/L of the precursor L-tryptophan to the medium also resulted in the greatest IAA accumulation (27.7ng/g FW) and the greatest increase (1.235times) in biomass compared to the untreated hairy root control.(3) Under NaCl stress, the MDA contents of both hairy roots and control were increased gradually, and the activities of the SOD, POD and CAT were reduced, which indicated stress injuries in plants. But the physiological indexes of hairy roots were obviously superior to that of control. The indirubin content of hairy roots was87.2ng/g FW, which was2.52-fold higher than the corresponding value of the control under100mg/L NaCl treatment. Expression of rolB and rolC was significantly increased in the roots treated with NaCl compared to the control roots, which was tightly correlated with increased salt tolerance. Supplementation with IAA or melatonin together with100mg/L NaCl resulted in the enhancement of antioxide enzymes (control NaCl treated alone). The highest yield of indirubin (102.2ng/g FW) was observed when the hairy roots treated with100mg/L NaCl in combination with0.2mmol/L melatonin.