Abstract
An efficient protocol for the development of genetically uniform clones of a valuable medicinal plant Solanum trilobatum L. has been established. An optimal shoot regeneration response was observed in a modified Murashige and Skoog medium (M-MS) containing 25 mM ammonium nitrate, 2 mg l−1 6-benzyl adenine and 0.1 mg l−1 indole-3-acetic acid using in vitro derived node and shoot tip explants. Consequently, the multiple shoot buds were elongated in MS medium supplemented with 0.5 mg l−1 Gibberellic acid. The in vitro regenerated shoots were rooted best in MS medium containing 1.5 mg l−1 indole-3-butyric acid and successfully acclimatized in the field. The single primer amplification reaction (SPAR) approach, including random amplified polymorphic DNA, inter simple sequence repeats and directed amplification of minisatellite DNA regions markers did not identify any genetic polymorphism among in vitro regenerants. Similarly flow cytometry analysis illustrated that the DNA content and genome size of micropropagated plants were equivalent to that of intact plants from field. In addition, the accumulation of solasodine in micropropagated plants was confirmed by thin layer chromatography and further quantified by high performance liquid chromatography analysis as 2.47 mg g−1 DW which is comparable to field grown plants. Thus the protocol can be effectively exploited for commercial propagation of this species to obtain solasodine and also in genetic transformation studies.
References
Alagumanian SV, Saravana Perumal R, Balachandar K, Rameshkannan M, Rao V (2004) Plant regeneration from leaf and stem explants of Solanum trilobatum L. Curr Sci 86:1478–1480
Anirudhan K, Nair AS (2009) In vitro production of solasodine from Solanum trilobatum. Biol Plant 53(4):719–722
Arockiasamy DI, Muthukumar B, Natarajan E, John Britto S (2002) Plant regeneration from node and internode explants of Solanum trilobatum L. Plant Tissue Cult 12(2):93–97
Balakrishna K, Vasanth S, Sugunthan J, BhimaRao R (1992) Estimation of the total alkaloidal content in various parts of Solanum trilobatum Linn. Ind Drugs 29:509–510
Bhatnagar P, Bhatnagar M, Nath AK, Sharma DR (2004) Production of solasodine by Solanum laciniatum using plant tissue culture technique. Indian J Exp Biol 42:1020–1023
Bradley V, Collins DG, Crabbe PG, Eastwood FW, Irvine MC, Swan JM, Symon DE (1978) A survey of Australian Solanum plants for potentially useful sources of solasodine. Aust J Bot 26:723–754
Breiman AD, Karp RA, Shaskin H (1987) Heritable somaclonal variation in wild barley (Hordeum spontaneum). Theor Appl Genet 71:637–643
Dhavala VNC, Rao DT, Rao YV, Prabavathi K (2009) Effect of explant age, hormones on somatic embryogenesis and production of multiple shoot from cotyledonary leaf explants of Solanum trilobatum L. Afr J Biotechnol 8(4):630–634
Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content of plants using flow cytometry. Nat Protoc 2:2233–2244
Galbraith DW, Lambert G, Macas J, Dolezel J (2002) Analysis of nuclear DNA content and ploidy in higher plants. In: Robinson J, Azmi A, Tutois S (eds) Current protocols in cytometry. Wiley, New York, pp 7.6.1–7.6.15
Ghimire BK, Yu CY, Chung IM (2012) Direct shoot organogenesis and assessment of genetic stability in regenerants of Solanum aculeatissimum Jacq. Plant Cell Tiss Organ Cult 108:455–464
Guo W, Li Y, Gong L, Li F, Dong Y, Liu B (2006) Efficient micropropagation of Robinia ambigua var. idahoensis (Idaho Locust) and detection of genomic variation by ISSR markers. Plant Cell Tiss Organ Cult 84:343–351
Ivanova M, Vav SJ (2009) Nitrogen source, concentration, and NH4+:NO3− ratio influence shoot regeneration and hyperhydricity in tissue cultured Aloe polyphylla. Plant Cell Tiss Organ Cult 99:167–174
Jawahar M, Amalan Rabert G, Jeyaseelan M (2004) Rapid proliferation of multiple shoots in Solanum trilobatum L. Plant Tissue Cult 14(2):107–112
Mallón R, Rodríguez-Oubiña J, González ML (2010) In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tiss Organ Cult 101:31–39
Mann JD (1978) Production of solasodine for the pharmaceutical industry. AdvAgron 30:207–243
Mohanty S, Parida R, Singh S, Joshi RK, Subudhi E, Nayak S (2011) Biochemical and molecular profiling of micropropagated and conventionally grown Kaempferia galangal. Plant Cell Tiss Organ Cult 106:39–46
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Pati R, Chandra R, Chauhan UK, Mishra M, Srivastava N (2008) In vitro clonal propagation of bael (Aegle marmelos Corr.) CV. CISHB1 through enhanced axillary branching. Physiol Mol Biol Plants 14(4):337–346
Poddar K, Vishnoi RK, Kothari SL (1997) Plant regeneration from embryogenic callus of finger millet Eleusine coracana (L.) Gaertn. on higher concentrations of NH4NO3 as a replacement of NAA in the medium. Plant Sci 129:101–106
Purushothaman KK, Saradambal S, Narayanaswamy V (1969) Chemical estimation of Solanum trilobatum L. Aust J Chem 22(7):1569–1570
Rahman MH, Haider SA, Hossain M, Islam R (2011) Effect of potassium and ammonium nitrate media on in vitro growth response of potato (Solanum tuberosum L.). Int J Biosci 1(2):54–57
Shirdel M, Azar AM, Masiha S, Mortazavi N, Matloobi M, Sharafi Y (2011) Effects of inorganic nitrogen source and NH4+:NO3 ratio on proliferation of dog rose (Rosa canina). J Med Plant Res 5(18):4605–4609
Vujović T, Cerović R, Ružic D (2012) Ploidy level stability of adventitious shoots of sour cherry 'Čačanski Rubin' and Gisela 5 cherry rootstock. Plant Cell Tiss Organ Cult 111:323–333
Acknowledgments
J. Shilpha thanks the Council for Scientific and Industrial Research (CSIR) New Delhi, for financial support [CSIR award No. 09/688 (0016) 2011 EMR-I] in the form of Junior Research Fellowship (JRF). The authors sincerely thank Dr. G.S. Selvam, Professor and Head, Department of biochemistry and Dr. A. Mahesh, Project faculty, School of Biological sciences, Madurai Kamaraj University (MKU) for access to the FACS instrumentation facility at UGC-Networking Resource Centre in Biological Sciences, MKU, and the Bioinformatics Infrastructure Facility of Alagappa University (funded by Department of Biotechnology, Government of India: Grant No. BT/BI/25/001/2006) for offering the computational facility to perform statistical analysis of the work.
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Shilpha, J., Silambarasan, T., Largia, M.J.V. et al. Improved in vitro propagation, solasodine accumulation and assessment of clonal fidelity in regenerants of Solanum trilobatum L. by flow cytometry and SPAR methods. Plant Cell Tiss Organ Cult 117, 125–129 (2014). https://doi.org/10.1007/s11240-013-0420-1
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DOI: https://doi.org/10.1007/s11240-013-0420-1