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 increase 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 adventitious 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 adventitious shoots. Transformation was confirmed at all phases of plant regeneration by GUS staining. Hairy root transformation 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.
Highlights
Plants have complex biosynthetic machineries that have allowed them to evolve bioactive, complex and multifunctional secondary metabolites as protection against stressors
S. nemoralis stem explants, leaf explants and seedlings were tested for their ability to be transformed by A. rhizogenes and produce hairy roots after infection
Further experiments on regeneration of transformed plants were undertaken with A. rhizogenes transformed leaf explants
Summary
Plants have complex biosynthetic machineries that have allowed them to evolve bioactive, complex and multifunctional secondary metabolites as protection against stressors. In addition to being a high affinity ligand for the insect nAChR, MLA is a highly selective ligand for the alpha7-subtype of human nAChR [3]. Since this receptor is an emerging target for the treatment of neurodegenerative disorders [4] other plant metabolites with this selectivity would be of considerable therapeutic interest. Cological activity using a “differential screening” approach [1] which identified Solidago nemoralis (“gray goldenrod”) as a prime candidate which has not previously been investigated for this activity. Stable mutants over-producing the active metabolites are identified by pharmacological screening, and are regenerated into intact mutant plants.
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