High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Cichorium intybus L. via removing macronutrients

Abstract

Transformed hairy roots technology provides new opportunities for mass production of pharmaceutical metabolites. Hairy roots culture has been considered as an alternative method for producing medicinal biomolecules in plants. Chicory (Cichorium intybus L.) is a medicinal herb from Asteraceae family. It contains many important metabolites including chicoric acid, inulin, scoline, coumarin, and flavonoids. In this study, for the first time, a reliable gene transfer system via modifying co-cultured medium using different strains of Agrobacterium rhizogenes and explants were established for C. intybus. Different co-culture media were used for this purpose, including MS basal medium and MS medium without either of KNO3, NH4NO3, KH2PO4, CaCl2, and MgSO4. The results showed increases in hairy roots induction percentage, biomass production, and phenolic content of the hairy roots. Maximum hairy root induction percentage obtained by using A4 strain when a KNO3-free MS medium was used. High frequency of hairy root induction percentage with ATCC11325 and ATCC15834 strains was achieved when MS medium had KH2PO4 removed. In all of the experiments, very low hairy root induction was observed when NH4NO3 was removed from co-culture MS medium. Molecular confirmation of transgenic hairy roots was done with PCR using gene-specific primers for rolB gene. Total phenols, flavonoids, anthocyanins and chicoric acid contents of hairy root clones and the control (untransformed) roots were also determined. The results indicated significant increases in total metabolites content of hairy root clones induced by A4, ATCC11325 and ATCC15834 strains with some macronatrient–free co-culture MS media and also directly-regenerated shoots from hairy roots were spontaneous without using any plant growth regulators.