Abstract
Plant roots can be manipulated by Agrobacterium rhizogenes to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13) in combination with three co-cultivation media (MS, B5, LS) were examined to establish an efficient and reliable transformation system for chicory (Cichorium intybus L.) using A. rhizogenes. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l-1 in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l-1, respectively). Beta-glucuronidase (GUS) gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 %) was achieved using A13 strain and MS cultivation medium. Detection of GUS and hptII genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.
Highlights
Induction of hairy roots in herbal plants by soilborne bacterium Agrobacterium rhizogenes provides a useful systems for synthesis of valuable pharmaceutical compounds, among them secondary metabolites
In an investigation done by Bais et al (2000) the possible benefits of coumarins production in hairy root cultures of C. intybus was proven; 4.06 and 3.71 fold increase in esculin and esculetin production as derivative of coumarin were achieved in induced hairy roots using fungal elicitors
We realized that the combination of A13 strain and MS medium is the best transformation for C. intybus hairy roots
Summary
Induction of hairy roots in herbal plants by soilborne bacterium Agrobacterium rhizogenes provides a useful systems for synthesis of valuable pharmaceutical compounds, among them secondary metabolites. Sesquiterpene lactones which have high anticancer property were isolated from A. rhizogenes LBA 9402 transformed hairy roots of C. intybus in 2002 (Malarz et al, 2002). It was previously confirmed that optimizing the composition of organic/inorganic nutrients of the media for hairy root cultures is essential to gain high production of secondary metabolites (Sivakumar, et al, 2005). Different concentrations of salts and elicitors in culture media have a major role in hairy root growth and induction of secondary metabolites production (Wang and Wu, 2013). In the present study the effect of different cocultivation media and various A. rhizogenes strains on the induction and growth of hairy root cultures of chicory were studied. The aim of the work presented here was improving a genetic transformation protocol for C. intybus and investigating the potential for genetic manipulation of the important secondary metabolite pathways
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