Development Of Genetic Transformation System And Regularation Of Plant Growth By Exogenous Melatonin In Isatis Indigotica Fort

Isatis indigotica Fort. is a biennial plant, belonging to the genus Isatis in the cruciferae family. As a Chinese medicinal herb, its dried root is called ban Ian gen, and dried leave da qing ye.Melatonin (N-acetyl-5-methoxytryptamine) is found in various plant species. Several investigators have studied the physiological roles of melatonin in plants. Melatonin is reported to be active in many physiological functions in plants, functioned as antioxidant protector and plant growth regulator. However, the mechanisms by which melatonin carries out remain unclear.In this study, an efficient system of organogenesis and transformation protocols of Isatis indigotica Fort. was established. The transgenic plants with human AANAT (arylalkylamine N-acetyltransferase) and HIOMT (Hydroxyindole O-methyltransferas) gene were obtained. The contents of melatonin and Indirubin in the transgenic plants were analyzed by HPLC. The effects of exogenous melatonin on the seeds germination under the cold stress and the regeneration activity of wild type of coleoptiles Isatis indigotica Fort. were observed.The results were as follows:(1) The root segments, hypocotyls and coleoptiles of Isatis indigotica Fort. could all be in vitro regenerated. High-frequency directed organogenesis was induced from root segments of the in vitro grown seedlings on MS medium supplemented with 6-BA and NAA, which only required 3-7d of culture for differented and leaded to a 100% differented ratio. Regenerated shoots were converted to plantlets on MS medium supplemented with 0.4mg/L IBA. The protocol could provide a rapid means for high fidelity regeneration and in vitro manipulation of Isatis indigotica Fort. plants via the root segments.(2) The transgenic Isatis indigotica Fort. plants were generated via expression of human AANAT and HIOMT gene using Agrobacterium-mediated gene transformation. Factors affecting the efficiency of transformation were optimized. The explants were first infected by agrobaeterium solution at the concentration about A600=0.5-0.7 for 6 min before being co-cultured for 3d; the explants were then selected by 50 mg/L gentamycin. The resistant transgenic plants were obtained from the above procedure. The integration and expression of the transgene in the transgenic Isatis indigotica Fort. seedlings were confirmed by RNA and RT-PCR analyses.(3) Melatonin and indirubin in Isatis indigotica Fort. were extracted from the leaves of transgenic, non-transgenic and wild type plants in the field. The contents of melatonin and indirubin from the above samples were assayed by HPLC. The results showed that the contents of melatonin in the transgenic plants were 4 fold higher than that in non-transgenic plants, but lower than that in the field-plants.(4) The coleoptiles of Isatis indigotica Fort. could differentiated and rooted on MS medium with MT 0.2 mg/L. The regenerated shoots were rooted on MS medium with MT 0.4 mg/L. The seeds were treated with the 100μM exogenous melatonin, an elevated frequency of seeds germination during cold stress was observed. It suggests that melatonin is involved in physiological function during regeneration and growth of Isatis indigotica Fort., and plays a protective role against cold-stress too.