TITLE:
Hairy Root Cultures and Plant Regeneration in Solidago nemoralis Transformed with Agrobacterium rhizogenes
AUTHORS:
Samir Kumar Gunjan, Joseph Lutz, Ashton Bushong, Dennis Trent Rogers, John Littleton
KEYWORDS:
Agrobacterium rhizogenes; Hairy Root; Regeneration; Solidago nemoralis
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.4 No.8,
August
5,
2013
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.