Hairy Root Cultures and Plant Regeneration in Solidago nemoralis Transformed with Agrobacterium rhizogenes

Abstract

By screening a native plant extract library we identified Solidago nemoralis as a novel source of agonists for alpha7 nicotinic receptors for acetylcholine with therapeutic potential. The next phase of our drug discovery strategy is to in- crease the yields of active compounds in the plant species by gain of function mutations in hairy root cultures [1]. Here we report a protocol for Agrobacterium rhizogenes-mediated genetic transformation of hairy root cultures of Solidago nemoralis which will enable this. Leaf explants of this species were successfully transformed with a frequency of 30% - 35% using A. rhizogenes strain R1000 harboring the binary vector pCambia 1301. Transformation was confirmed using the β-glucuronidase (GUS) histochemical assay. Transformed hairy roots showed spontaneous regeneration of adventi- tious shoots in media without the addition of cytokines, albeit at very low frequency. However, media supplementation with auxin (α-naphthaleneacetic acid, NAA) increased shoot regeneration frequency to 35% and resulted in viable ad- ventitious shoots. Transformation was confirmed at all phases of plant regeneration by GUS staining. Hairy root trans- formation of Solidago altissima has been previously reported, but this is the first report of genetic transformation of S. nemoralis. The protocol will allow for a large population of activation tagged mutants of S. nemoralis to be generated which will be then screened for the presence of stable mutants which are over-producing metabolites with activity at alpha7 nicotinic receptors. These over-producing mutant cultures will then be regenerated into intact mutant plants.