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


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.

Share and Cite:

S. Gunjan, J. Lutz, A. Bushong, D. Rogers and J. Littleton, "Hairy Root Cultures and Plant Regeneration in Solidago nemoralis Transformed with Agrobacterium rhizogenes," American Journal of Plant Sciences, Vol. 4 No. 8, 2013, pp. 1675-1678. doi: 10.4236/ajps.2013.48203.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] J. Littleton, T. Rogers, et al., “Novel Approaches to Plant Drug Discovery Based on High Throughput Pharmacological Screening and Genetic Manipulation,” Life Sciences, Vol. 78, No. 5, 2005, pp. 467-475. doi:10.1016/j.lfs.2005.09.013
[2] H. Baylis, D. Sattelle, et al., “Genetic Analysis of Cholinergic Nerve Terminal Function in Invertebrates,” Journal of Neurocytology, Vol. 25, No. 1, 1996, pp. 747-762. doi:10.1007/BF02284839
[3] A. Drasdo, M. Caulfield, et al., “Methyl Lycaconitine: A Novel Nicotinic Antagonist,” Molecular and Cellular Neuroscience, Vol. 3, No. 3, 1992, pp. 237-243. doi:10.1016/1044-7431(92)90043-2
[4] M. R. Picciotto and M. Zoli, “Neuroprotection via nAChRs: The Role of nAChRs in Neurodegenerative Disorders Such as Alzheimer’s and Parkinson’s Disease,” Frontiers in Bioscience, Vol. 13, 2008, pp. 492-504. doi:10.2741/2695
[5] L. B. Pickens, Y. Tang, et al., “Metabolic Engineering for the Production of Natural Products,” Annual Review of Chemical and Biomolecular Engineering, Vol. 2, No. 1, 2011, pp. 211-236. doi:10.1146/annurev-chembioeng-061010-114209
[6] M. Inoguchi, S. Ogawa, et al., “Production of an Allelopathic Polyacetylene in Hairy Root Cultures of Goldenrod (Solidago altissima L.),” Bioscience, Biotechnology, and Biochemistry, Vol. 67, No. 4, 2003, pp. 863-868. doi:10.1271/bbb.67.863
[7] S. Ohta, S. Mita, T. Hattori and K. Nakamura, “Construction and Expression in Tobacco of a β-Glucuronidase (GUS) Reporter Gene Containing an Intron within the Coding Sequence,” Plant and Cell Physiology, Vol. 31, No. 6, 1990, pp. 805-813.
[8] M. Faiss, M. Strnad, et al., “Chemically Induced Expression of the rolC-Encoded β-Glucosidase in Transgenic Tobacco Plants and Analysis of Cytokinin Metabolism: rolC Does Not Hydrolyze Endogenous Cytokinin Glucosides in Planta,” The Plant Journal, Vol. 10, No. 1, 1996, pp. 33-46. doi:10.1046/j.1365-313X.1996.10010033.x
[9] J. Palazón, R. M. Cusidó, et al., “Expression of the rolC Gene and Nicotine Production in Transgenic Roots and Their Regenerated Plants,” Plant Cell Reports, Vol. 17, No. 5, 1998, pp. 384-390. doi:10.1007/s002990050411
[10] M. Christey, “Use of ri-Mediated Transformation for Production of Transgenic Plants,” In Vitro Cellular & Developmental Biology—Plant, Vol. 37, No. 6, 2001, pp. 687-700.
[11] E. Casanova, A. Zuker, et al., “The rolC Gene in Carnation Exhibits Cytokinin- and Auxin-Like Activities,” Scientia Horticulturae, Vol. 97, No. 3-4, 2003, pp. 321-331. doi:10.1016/S0304-4238(02)00155-3
[12] T. Saitou, H. Kamada, et al., “Involvement of Phytohormones in Light-Induced Adventitious Shoot Formation of Horseradish Hairy Roots,” Plant Science, Vol. 86, No. 2, 1992, pp. 161-166. doi:10.1016/0168-9452(92)90162-F
[13] A. Jacob, and N. Malpathak, “Plantlet Regeneration Enhances Solasodine Productivity in Hairy Root Cultures of Solanum khasianum Clarke,” In Vitro Cellular & Developmental Biology—Plant, Vol. 41, No. 3, 2005, pp. 291-295. doi:10.1079/IVP2005637
[14] B. Vinterhalter, S. Ninkovic, et al., “Shoot and Root Culture of Hypericum perforatum L. Transformed with Agrobacterium rhizogenes A4M70GUS,” Biologia Plantarum, Vol. 50, No. 4, 2006, pp. 767-770. doi:10.1007/s10535-006-0127-9

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.