Acta Scientiarum Polonorum: Hortorum Cultus (Aug 2014)
TRANSGENIC CALLUS CULTURE ESTABLISHMENT, A TOOL FOR METABOLIC ENGINEERING OF Rhodiola rosea L.
Abstract
Agrobacterium tumefaciens EHA101 (pTd33) strain carrying uidA (GUS) reporter gene was used in model experiments on roseroot callus transformation. The T-DNA of pTd33 binary vector plasmid harbors nptII gene conferring resistance to kanamycin, and a uidA reporter gene, encodes the ß-glucuronidase enzyme. Roseroot seeds were sterilized and germinated on half strength MS media of which 70% germinated without any pretreatment. Calli were obtained from leaf segments of the in vitro grown seedlings. Calli was grown on solid MS medium supplemented with 1 mg l-1 NAA and 0.5 mg l-1 BAP. Different types of calli were obtained of which the green and compact type was chosen for transformation experiments. After co-cultivation with agrobacteria, calli were transferred to the same medium supplemented with 20 mg l-1 kanamycin, 200 mg l-1 carbenicillin and 300 mg l-1 claforan with antioxidants (Polyclar and DTE) for selection. GUS test using a titron buffer was applied for monitoring the transformation of the calli. DNAs of 20 individual samples was extracted and subjected for PCR analysis proved the stable transformation in all of the taken samples by amplifying the nptII gene fragment. The method introduced here can be a tool for inserting and over-expressing the genes encoding for hypothesized enzymes to be involved in the biosynthesis of pharmaceutically important bioactive molecules of roseroot and therefore facilitating the applications for callus culture of roseroot in different bioreactor systems for pharmaceutical productions.
