Abstract
Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri TL-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.
Similar content being viewed by others
References
Sharafi A, Sohi HH, Mousavi A, Azadi P, Razavi K, Otang Ntui V (2012) A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-012-0246-2
Altamura MM (2004) Agrobacterium rhizogenes rol B and rol D genes: regulation and involvement in plant development. Plant Cell Tiss Org Cult 77:89–101
Banerjee S, Zehra M, Gupta MM, Kumar S (1997) Agrobacterium rhizogenes mediated transformation of Artemisia annua: prodcution of transgenic plants. Planta Med 63:467–469
Benjamin BD, Roja G, Heble MR (1993) Agrobacterium rhizogenes mediated transformation of Rauwolfia serpentina: regeneration and alkaloid synthesis. Plant Cell Tiss Org Cult 35:253–257
Bhattacharjee SK (1998) Handbook of medicinal plants. Pointer Publishers, India
Boulter ME, Croy E, Simpson P, Shields R, Croy RRD, Shirsat AH (1990) Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes—a comparison. Plant Sci 70:91–99
Braun RH, Reader JK, Christey MC (2000) Evaluation of cauliflower transgenic for resistance to Xanthomonas campestris pv. campestris. Acta Hort. 539:137–143
Chandra S (2011) Natural plant genetic engineer Agrobacterium rhizogenes: role of T-DNA in plant secondary metabolism. Biotechnol Lett 34(3):407–415
Chaudhuri KN, Ghosh B, Tepfer D, Jha S (2006) Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation. Plant Cell Rep 25:1059–1066
Choi PS, Kim YD, Choi KP, Chung HJ, Choi DW, Liu JR (2004) Plant regeneration from hairy root cultures transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell Rep 22:828–831
Christey MC (2001) Use of Ri-mediated transformation for production of transgenic plants. In Vitro Cell Dev Biol Plant. 37:687–700
Christey MC, Braun RH, Reader JK (1999) Field performance of transgenic vegetable Brassicas (Brassica oleracea and B. rapa) transformed with Agrobacterium rhizogenes. SABRAO J Breed Genet. 31:93–108
Dunwell JM (2000) Transgenic approaches to crop improvement. J Exp Bot 51:487–496
Falkenhagen H, Stockigt J, Kuzovkina IN, Alterman IE, Kolshorn H (1993) Indole alkaloids from the hairy roots of Rauwolfia serpentina. Can J Chem 71:2201–2203
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158
Gangopadhyay M, Chakraborty D, Bhattacharyya S, Bhattacharya S (2010) Regeneration of transformed plants from hairy roots of Plumbago indica. Plant Cell Tiss Organ Cult. 102:109–114
Goel MK, Mehrotra S, Kukreja AK, Shanker K, Khanuja SPS (2009) In vitro propagation of Rauwolfia serpentina using liquid medium, assessment of genetic fidelity of micropropagated plants, and simultaneous quantitation of reserpine, ajmaline, and ajmalicine. In: Jain SM, Saxena PK (eds) Methods in molecular biology, protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, vol 547. Humana Press, Clifton, pp 17–33
Goel MK, Goel S, Banerjee S, Shanker K, Kukreja AK (2010) Agrobacterium rhizogenes mediated transformed roots of Rauwolfia serpentina for reserpine biosynthesis. Med Aromat Plant Sci Biotech 8–14
Hamill JD, Lidgett AJ (2000) Hairy root cultures—opportunities and key protocols for studies in metabolic engineering. In: Doran PM (ed) Hairy roots: culture and applications. Hardwood Academic, Sydney, pp 1–30
Hamill JD, Rhodes MJC (1988) A spontaneous, light independent and prolific plant regeneration response from hairy roots of Nicotiana hesperis transformed by Agrobacterium rhizogenes. J Plant Physiol 133:506–509
Hatamoto H, Boulter ME, Shirsat AH, Croy EJ, Ellis JR (1990) Recovery of morphologically normal transgenic tobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid. Plant Cell Rep 9:88–92
Hooykass PJJ, Klapwijk PM, Nuti PM, Shilperoot RA, Rorsch A (1977) Transfer of Agrobacterium tumifacience Ti plasmid to a virulent Agrobacterium and Rhizobium explant. J Gen Microbiol 98:477–487
Jaziri M, Yosimatsue K, Homes J, Shimomura K (1994) Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains. Plant Cell Tiss Org Cult 38:257–262
Jouanin L, Tourneur J, Tourneur C, Casse-Delbart F (1986) Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4. Plasmid 16:124–134
Kamble S, Gopalakrishnan R, Eapen S (2011) Production of camptothecin by hairy roots and regenerated transformed shoots of Ophiorrhiza rugosa var. decumbens. Nat Prod Res 25(18):1762–1765
Khanuja SPS, Shasany AK, Darokar MP, Kumar S (1999) Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol Biol Rep 17:74–80
Li XG, Chen SB, Lu ZX, Chang TJ, Zeng QC, Zhu Z (2002) Impact of copy number on transgene expression in tobacco. Acta Bot Sin 44:120–123
Ma Julian KC, Pascal MWD, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nature 4:796–805
Manners JM, Way H (1989) Efficient transformation with regeneration of the tropical pasture legume Stylosanthes humilis using Agrobacterium rhizogenes and a Ti plasmid-binary vector system. Plant Cell Rep 8:341–345
Mehrotra S, Rahman LU, Kukreja AK (2010) An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering. Biotechnol Appl Biochem 56:161–172
Mishra BN, Ranjan R (2008) Growth of hairy-root cultures in various bioreactors for the production of secondary metabolites. Biotechnol Appl Biochem 49:1–10
Noda T, Tanaka N, Mano Y, Nabeshima H, Ohkawa H, Matsui C (1987) Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection. Plant Cell Rep 6:283–286
Ohara A, Akasaka Y, Daimon H, Mii M (2000) Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L. Plant Cell Rep 19:563–568
Ono NN, Tian L (2011) The multiplicity of hairy root cultures: prolific possibilities. Plant Sci 180:439–446
Peres LEP, Morgante PG, Vecchi C, Kraus JE, Sluys MA (2001) Shoot regeneration capacity from roots and transgenic hairy roots of tomato cultivars and wild related species. Plant Cell Tiss Organ Cult 65:37–44
Petit A, Stougaard J, Kühle A, Marcker KA, Tempé J (1987) Transformation and regeneration of the legume Lotus corniculatus: a system for molecular studies of symbiotic nitrogen fixation. Mol Gen Genet 207:245–250
Puddephat IJ, Robinson HT, Fenning TM, Barbara DJ, Morton A, Pink DAC (2001) Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated cotransformation and selection of transformed hairy roots by GUS assay. Mol Breeding 7:229–242
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, New York, pp 47–58
Saxena G, Banerjee S, Rahman LU, Verma PC, Mallavarapu GR, Kumar S (2007) Rose-scented geranium (Pelargonium sp.) generated by Agrobacterium rhizogenes mediated Ri-insertion for improved essential oil quality. Plant Cell Tiss Organ Cult 90:215–223
Shahin EA, Sukhapinda K, Simpson RB, Spivey R (1986) Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA, but no Ri-plasmid T-DNA. Theor Appl Genet 72:770–777
Shi HP, Long YY, Sun TS, Tsang PKE (2011) Induction of hairy roots and plant regeneration from the medicinal plant Pogostemon Cablin. Plant Cell Tiss Organ Cult 107:251–260
Srivastava S, Srivastava AK (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol 27:29–43
Stam M, Joseph NMM, Kooter JM (1997) The silence of genes in transgenic plants. Ann Bot 79:3–12
Tang W, Ronald JN, Douglas AW (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58:545–554
Trivedi KC (1995) Sarpagandha. In: Chadha KL, Gupta R (eds) Advances in horticulture medicinal and aromatic plants, vol 11. Malhotra Publshing House, New Delhi, pp 453–466
Veena V, Taylor CG (2007) Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev Biol Plant 43:383–403
Watase I, Sudo H, Yamazaki M, Saito K (2004) Regeneration of transformed Ophiorrhiza pumila plants producing camptothecin. Plant Biotechnol 21:337–342
Webb KJ, Jones S, Robbins MP, Minchin FR (1990) Characterization of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus. Plant Sci 70:243–254
Wu HJ, Wang XX, Yan L, Zhang DG, Zhang B, Wang XY (2011) Propagation of Gentiana macrophylla (Pall) from hairy root explants via indirect somatic embryogenesis and gentiopicroside content in obtained plants. Acta Physiol Plant 33:2229–2237
Wu J, Yi W, Zhang LX, Zhang XZ, Kong J, Lu J, Han ZH (2012) High efficiency regeneration of Agrobacterium rhizogenes induced hairy roots in apple rootstock Malus baccata. Plant Cell Tiss Org Cult 111:183–189
Yoshimatsu K, Shimomura K, Yamazaki M, Saito K, Kiuchi F (2003) Transformation of Ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes. Planta Med 69:1018–1023
Acknowledgments
Authors are grateful to Director, CIMAP (CSIR) for providing the necessary facilities to carry out work. Post Doctoral Fellowship provided by Department of Science and Technology (DST), Govt. of India to SM is duly acknowledged. Thanks are also due to Dr. K. Shankar for chemical analysis of root samples.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mehrotra, S., Goel, M.K., Rahman, L.U. et al. Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina . Plant Cell Tiss Organ Cult 114, 31–38 (2013). https://doi.org/10.1007/s11240-013-0302-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-013-0302-6