High frequency in vitro plantlet regeneration in Solanum trilobatum L., an important ethno-medicinal plant and confirmation of genetic fidelity of R1 plantlets by using ISSR and RAPD markers


Research Articles | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email: contact@vegetosindia.org
Doi: 10.1007/s42535-019-00069-6
First Page: 508
Last Page: 520
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Keywords: Solanum trilobatum L. , Thidiazuron, Organogenesis, Acclimatization, ISSR, Genetic fidelity


An efficient and reproducible protocol was developed for in vitro clonal propagation of Solanum trilobatum L., an ethno-medicinally important plant. Leaf, stem and cotyledon explants were used for callus induction and shoot regeneration via indirect organogenesis. Initially, maximum amounts (mg) of green friable callus (312.86 ± 0.50, 285.3 ± 0.40 and 305.13 ± 0.62) was induced from leaf, stem and cotyledon explants, respectively, on Murashige and Skoog (MS) medium supplemented with 13.57 μM L−1 of 2,4-dichloro phenoxy acetic acid (2,4-D) and 2.21 μM L−1 of 6-benzylaminopurine (BAP), after 4 weeks of culture. Highest mean number of shoots (69.2 ± 0.73, 34.1 ± 0.62 and 57.1 ± 0.62) were differentiated de novo from leaf, stem and cotyledon calluses, respectively, when cultured on MS medium amended with 2.27 μM L−1 of Thidiazuron (TDZ) and 2.68 μM L−1 of Naphthaleneacetic acid (NAA), after 4 weeks of culture. Maximum rooting response (97%) with mean number of roots (31.7 ± 0.61 roots per shoot) was observed from shoots when cultured on half strength MS medium supplemented with 4.90 μM L−1 of indole-3-butyric acid (IBA), after 4 weeks of culture. In vitro raised plantlets (R1) of S. trilobatum were hardened in plastic pots, acclimatized in green house and successfully transferred to field conditions with 82% survivability. ISSR and RAPD based PCR banding profile of the acclimated R1 plantlets was confirmed as synonymous to the mother plant.

                Solanum trilobatum L.
              , Thidiazuron, Organogenesis, Acclimatization, ISSR, Genetic fidelity

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  1. Alagumanian SV, Saravana PR, Balachandar K, Rameshkannan M, Rao V (2004) Plant regeneration from leaf and stem explants of Solanum trilobatum L. Curr Sci 86:1478–1480. http://www.jstor.org/stable/24108692

  2. Amir M, Kumar S (2004) Possible industrial application of genus Solanum in twenty first century—a review. J Sci Ind Res 63:116–124

  3. Arockiasamy DI, Muthukumar B, Natarajan E, John Britto S (2002) Plant regeneration from node and inter node explants of Solanum trilobatum L. Plant Tissue Cult 12(2):93–97

  4. Arokiadasan PX, Leelaram S, Sayamsiddha P, Meenakshi SM, Brindha P (2013) Antitumor potential of ethanolic extract of Solanum trilobatum against ehrlich’s ascites carcinoma. Int J Pharm Tech Res 5(3):1119–1125

  5. Balakrishnan K, Vasanth S, Sugunthan J, BhimaRao R (1992) Estimation of the total alkaloidal content in various parts of Solanum trilobatum L. Plant Tissue Cult 12(2):93–97

  6. Barbosa-Filho JM, Agra MF, Oliveira RAG, Paulo MQ, Troling G, Cunha EVL, Ataide JR, Bhattacharyya J (1991) Chemical and pharmacological investigation of Solanum species of Brazil—a search for solasodine and other potentially useful therapeutic agents. Mem I Oswaldo Cruz 86:189–191

  7. Bhatia R, Singh KP, Jhang T, Sharma TR (2009) Assessment of clonal fidelity of micropropagated gerbera plants by ISSR markers. Sci Horti 119:208–211. https://doi.org/10.1016/j.scienta.2008.07.024

  8. Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, rev edn. Elsevier Science Publishers, Amsterdam, p 105

  9. Blakesley D (1991) Uptake and metabolism of 6-benzyladenine in shoot proliferation of Musa and Rhododendron. Plant Cell Tiss Org Cult 25:69–74

  10. Bylla P, Gulab KR, Radha T, Ravi Ch, Venkataiah P, Subhash K, Christopher RT (2013) DNA profiling of commercial chilli pepper (Capsicum annuum L.) varieties using random amplified polymorphic DNA (RAPD) markers. Afr J Biotechnol 12:4730–4735. https://doi.org/10.5897/AJB2012.3017

  11. Chavan JJ, Gaikwad NB, Kshirsagar PR, Umdale SD, Bhat KV, Dixit GB, Yadav SR (2015) Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegia evansii by RAPD and ISSR markers: a critically endangered plant of Western Ghats. Plant Biosyst 149(2):442–450. https://doi.org/10.1080/11263504.2013.853700

  12. Chinthana P, Ananthi T (2012) Protective effect of Solanum nigrum and Solanum trilobatum aqueous leaf extract on lead induced neurotoxicity in albino mice. J Chem Pharma Res 4(1):72–74

  13. Desingu K, Devarajan N (2013) Indirect propagation of Solanum trilobatum L. using leaf explants. J Nat Prod Plant Res 3(6):24–28

  14. 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

  15. Doss A, Anand SP (2012) Free radical scavenging activity of Solanum trilobatum Linn., on alloxan—induced diabetic rats. Biochem Anal Biochem 1:6. https://doi.org/10.4172/2161-1009.1000115

  16. Doss A, Rangasamy D (2008) Preliminary phytochemical screening and antibacterial studies of leaf extract of Solanum trilobatum Linn. Ethnobot Leafl 12:638–642

  17. Doss A, Palaniswamy M, Angayarkanni J, Dhanabalan R (2009) Antidiabetic activity of water extract of Solanum trilobatum Linn. in alloxan-induced diabetes in rats. Afr J Biotechnol 8(20):5551–5553

  18. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

  19. Edmonds JM, Chweya JA (1997) Black nightshades, Solanum nigrum L. and related species. IPGRI, Italy

  20. Faisal M, Anis M (2002) Rapid in vitro propagation of Rauvolfia tetraphylla L.—an endangered medicinal plant. Physiol Mol Biol Plant 8:295–299

  21. Govindan S, Viswanathan S, Vijayasekaran V, Alagappan R (1999) A pilot study on the clinical efficacy of Solanum xanthocarpum and Solanum trilobatum in bronchial asthma. J Ethnopharmcol 66(2):205–210. https://doi.org/10.1016/S0378-8741(98)00160-3

  22. Govindan S, Viswanathan S, Vijayasekaran V, Alagappan R (2004) Further studies on the clinical efficacy of Solanum xanthocarpum and Solanum trilobatum in bronchial asthma. Phytother Res 18:805–809. https://doi.org/10.1002/ptr.1555

  23. Hare PD, Van Staden J (1994) Cytokinin oxidase: biochemical features and physiological significance. Physiol Plant 91:128–136. https://doi.org/10.1111/j.1399-3054.1994.tb00668.x

  24. Hulya A, Veysel S, Engin T, Ahmet O, Yelda OC (2016) Detection of variation in long-term micropropagated mature Pistachio via DNA-based molecular markers. Appli Biochem Biotechnol 180(7):1301–1312. https://doi.org/10.1007/s12010-016-2168-7

  25. Joshi P, Dhawan V (2007) Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biol Plant 51:22–26. https://doi.org/10.1007/s10535-007-0005-0

  26. Kaminek M (1992) Progress in cytokinin research. Trends Biotechnol 10:159–162

  27. Kanchana A, Balakrishnan M (2011) Anti-cancer effect of saponins isolated from Solanum trilobatum leaf extract and induction of apoptosis in human larynx cancer cell lines. Int J Pharm Sci 3(4):356–364

  28. Kirtikar KR, Basu BD (1953) Indian medicinal plants. Lalit Mohan Publication, Allahabad, p 220

  29. Kirtikar KR, Basu BD (1975) Indian medicinal plants. Lalit Mohan Publication, Allahabad, p 220

  30. Korra R, Bylla P, Rohela GK, Pendli S, Reuben TC (2017) In vitro micro propagation and confirmation of genetic fidelity using RAPD marker in ethno medicinal plant Stachytarpheta jamaicensis L. Vahl. Intern J Adv Res 5:1494–1502. https://doi.org/10.21474/IJAR01/4862

  31. Kour B, Kour G, Kaul S, Dhar MK (2014) In vitro mass multiplication and assessment of genetic stability of in vitro raised Artemisia absinthium L. Plants using ISSR and SSAP molecular markers. Adv Bot 1–7. https://doi.org/10.1155/2014/727020

  32. Kumar SR, Sakthivel KM, Karthik L, Mythili S, Sathiavelu A (2011) In vitro micropropagation and antimicrobial activity of Solanum trilobatum. Asian J Plant Sci Res 1(1):48–56

  33. Kuznetsova OI, Ash OA, Khartina GA, Gostimski SA (2005) RAPD and ISSR analyses of regenerated pea Pisum sativum L. plants. Genetika 41(1):60–65 PMID: 15771253

  34. Lakshmanan V, Sreedhar RV, Bhagyalahshmi N (2007) Genetic analysis of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers. In Vitro Cell Dev Biol Plant 43:267–274. https://doi.org/10.1007/s11627-007-9028-7

  35. Mann JD (1978) Production of solasodine for the pharmaceutical industry. Adv Agron 30:207–243

  36. Mariappan SK (2018) Evaluation of bioactive compounds of Solanum trilobatum L.: a native medicinal plant. Int J Bot Stud 3(2):21–28

  37. Martins M, Sarmento D, Oliveiria MM (2004) Genetic stability of micropropagated almond plantlets as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496. https://doi.org/10.1007/s00299-004-0870-3PMID: 1537219

  38. Mazri MA, Reda M (2013) An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.). J Plant Biochem Biotechnol 22(2):176–184. https://doi.org/10.1007/s13562-012-0147-9

  39. Meziani R, Jaiti FJ, Mouaad AM, Mohamed A, Mustapha AC, Jamal EF, Chakib A (2015) Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L.) cv. Mejhoul. Crop Sci Biotechnol 18:325. https://doi.org/10.1007/s12892-015-0062-4

  40. Mohanan PV, Devi KS (1996) Cytotoxic potential of the preparation from Solanum trilobatum and the effect sobatum on tumour reduction in mice. Cancer Lett 110:71–76. https://doi.org/10.1016/S0304-3835(96)04463-1

  41. Mohanan PV, Rao JM, Kutty MAS, Devi KS (1998) Cytotoxicity and anti-carcinogenic activity of sobatum. Biomedicine 18:106–111

  42. Murashige T, Skoog F (1962) A revised medium for rapid grown and bioassays with tobacco tissue culture. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052

  43. Muthoni J, Shimelis H, Melis R, Kabira J (2012) Reproductive biology and early generation’s selection in conventional potato breeding. Austral J Crop Sci 6(3):488–497

  44. Nadkarni KM (1976) Indian material medica, 3rd edn. Popular Prakasan Pvt. Ltd, Mumbai, pp 1153–1154

  45. Nee M (1999) Synopsis of solanum in the new world. In: Nee M, Symon DE, Laster RN, Jessop JP (eds) Solanaceae IV: advances in biology and utilization. Royal Botanical Gardens, Kew, pp 285–333

  46. Nirmala Devi N, Ramachandramurthy B (2014) Priliminary pytochemical screening of Solanum trilobatum young leaves. Int Res J Pharm 5(2):80–82

  47. Palombi MA, Damiano C (2002) Comparison between RAPD and SSR molecular markers in detecting variation in kiwifruit (Actinidia deliciosa A. Chev). Plant Cell Rep 20:1061–1066

  48. Pandurangan A, Khosa RL, Hemalatha S (2008) Evaluation of anti-inflammatory activity of the Solanum trilobatum roots. Orient Pharm Exper Med 8(4):416–422. https://doi.org/10.3742/OPEM.2008.8.4.416

  49. Pandurangana A, Khosaa RL, Hemalatha S (2009) Evaluation of anti-inflammatory activity of the leaf extracts of Solanum trilobatum Linn. J Pharm Sci Res 1(1):16–21

  50. Pant G, Nitesh N, Gyana P (2013) Enhancement of antidandruff activity of shampoo by biosynthesized silver nanoparticles from Solanum trilobatum plant leaf. Appli Nanosci 3(5):431–439. https://doi.org/10.1007/s13204-012-0164-y

  51. Pereira TM, Silva VCB, Ribeiro Neto JA, Alves SN, Lima LARS (2014) Larvicidal activity of the methanol extract and fractions of the green fruits of Solanum lycocarpum (Solanaceae) against the vector Culex quinquefasciatus (Diptera: Culicidae). Rev Soc Bras Med Trop 47(5):646–648

  52. Pratheeba M, Rajalakshmi G, Ramesh B (2013) Hepatoprotective and antibacterial activity of leaf extract of Solanum trilobatum. Int J Pharma Res Bio Sci 2:17–28

  53. Premalatha S, Kuppusamy E, Alagarmalai J (2013) Mosquitocidal properties of Solanum trilobatum L. (Solanaceae) leaf extracts against three important human vector mosquitoes (Diptera:Culicidae). Asian Pac J Trop Med 6(11):854–858. https://doi.org/10.1016/s1995-7645(13)60152-2PMID: 24083579

  54. Purushothaman KK, Saradambal S, Narayanaswamy V (1969) Chemical estimation of Solanum trilobatum L. Aust J Chem 22(7):1569–1570

  55. Purushothaman KK, Balakrishana K, Sarada A, Bhema Rao R (1987) Extraction of β-solamarine from Solanum trilobatum Linn. Ind Drugs 24:214–215

  56. Raja DH, Senthilarasu K, Arockiasamy DI (2015) Micropropagation of Solanum trilobatum from shoot tip explants. World J Pharm Pharm Sci 4(9):1730–1734

  57. Rajathi M, Anandan R, Sindhu R, Logeshwari MN (2015) Screening of Solanum nigrum for its phytochemical and antimicrobial activity against respiratory tract pathogen. Int J Pure Appl Zool 3(3):210–215

  58. Rajkumar S, Jebanesan A (2004) Ovicidal activity of Solanum trilobatum Linn (Solanaceae) leaf extract against Culex quinquefasciatus Say and Culex triaeniorhynchus Gile (Diptera: Culicidae). Intl J Trop Sci 24:340–342. https://doi.org/10.1079/IJT200442

  59. Rajkumar S, Jebanesan A (2005) Oviposition deterrent and skin repellent activities of Solanum trilobatum leaf extract against the malarial vector Anopheles stephensi. J Ins Sci 5:1–3. https://doi.org/10.1673/031.005.1501PMCID: PMC1307576

  60. Ravi CH, Avinash KS, Gulab KR, Prasad B, Christopher RT (2012) High frequency in vitro clonal propagation of Solanum surattense burm. F. Int J Pharm Bio Sci 3(4):147–151

  61. Rohela GK, Prasad B, Srinivas K, Sadanandam A, Ravi CH, Christopher RT (2013) In vitro plantlet regeneration from leaf and stem calluses of Rauwolfia tetraphylla (R. canescens) and confirmation of genetic fidelity of plantlets using the ISSR-PCR method. J Herbs Spices Med P 19(1):66–75. https://doi.org/10.1080/10496475.2012.741056

  62. Rohela GK, Prasad B, Ravi CH, Rajender K, Christopher RT (2015) In vitro clonal propagation of Rauwolfia tetraphylla, a relative of Indian snake root plant. Res J Biotechnol 10(11):23–31

  63. Rohela GK, Santhosh D, Prasad B, Rajender K, Sreenu P, Christopher TC (2016a) Somatic embryogenesis and indirect regeneration in Mirabilis jalapa Linn. Mater Today 3:3882–3891. https://doi.org/10.1016/j.matpr.2016.11.045

  64. Rohela GK, Bylla P, Korra R, Reuben C (2016b) Phytochemical screening and antimicrobial activity of leaf, stem, root and their callus extracts in Rauwolfia tetraphylla. Int J Agric Biol 18:521–528. https://doi.org/10.17957/IJAB/15.0120

  65. Rohela GK, Bylla P, Pendli S, Rajender K, Reuben TC (2018a) ISSR marker based DNA profiling studies in Rauwolfia species. Ann Plant Sci 75:2289–2295. https://doi.org/10.21746/aps.2018.7.5.13

  66. Rohela GK, Phanikanth J, Aftab AA, Pawan S, Sadanandam A, Mrinal KG (2018b) In vitro regeneration and assessment of genetic fidelity of acclimated plantlets by using ISSR markers in PPR-1 (Morus sp.): an economically important plant. Sci Hortic 241:313–321. https://doi.org/10.1016/j.scienta.2018.07.012

  67. Rohela GK, Aftab AS, Pawan S, Ravindra A, Mudasir G, Srinivasulu Y, Sharma SP (2018c) In vitro clonal propagation of PPR-1, a superior temperate mulberry variety. Indian J Biot 17(4):619–625

  68. Rohela GK, Shabnam AA, Shukla P, Kamili AN, Ghosh MK (2018d) Rapid one step protocol for the in vitro micro propagation of Morus multicaulis var. Goshoerami, an elite mulberry variety of temperate region. J Exper Biol Agric Sci 6(6):936–946. https://doi.org/10.18006/2018.6(6).936.946

  69. Rohela GK, Phanikanth J, Prasad B, Christopher R (2019) Indirect regeneration and assessment of genetic fidelity of acclimated plantlets by SCoT, ISSR, and RAPD markers in Rauwolfia tetraphylla L.: an endangered medicinal plant. Bio Med Res Int 19:1–14. https://doi.org/10.1155/2019/3698742

  70. Sanatombi K, Sen-Mandi S, Sharma GJ (2010) DNA profiling of Capsicum landraces of Manipur. Sci Hortic 124:405–408

  71. Shahjahan M, Sabitha KE, Mallika J, Shyamala Devi CS (2004) Effect of Solanum trilobatum against carbon tetrachloride induced hepatic damage in albino rats. Indian J Med Res 120:194–198

  72. Shahjahan M, Vani G, Shyamaladevi CS (2005) Effect of Solanum trilobatum on the antioxidant status during diethyl nitrosamine induced and phenobarbital promoted hepato carcinogenesis in rat. Chem Biol Interact 156(2–3):113–123. https://doi.org/10.1016/j.cbi.2005.08.003

  73. Shilpha J, Tamilarasan S, Muthiah Joe VL, Manikandan R (2014) 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. https://doi.org/10.1007/s11240-013-0420-1

  74. Sini H, Devi KS (2004) Antioxidant activities of the chloroform extract of Solanum trilobatum. J Pharml Bio 42(6):462–466. https://doi.org/10.1080/13880200490886238

  75. Sini H, Devi KS, Nevin KG (2016) Chloroform extract of Solanum trilobatum inhibits the progress of ehrlich ascites carcinoma in mice. Int J Cancer Res 12(1):17–28. https://doi.org/10.3923/ijcr.2016.17.28

  76. Smita S, Joseph KS, Jyothi RJ, Manohar SH, Hosakatte NM (2016) Efficient in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood) and assessment of genetic fidelity of micropropagated plants. Physiol Mol Biol Plants 22(4):595–603. https://doi.org/10.1007/s12298-016-0379-6

  77. Subramanian SV, Madhavan VR (1983) Heritage of the tamil siddha medicine. International Institute of Tamil Studies, Madras

  78. Sujatha D, Ravi CH, Raghuvardhan L, Prasad B, Gulab Khan R, Sadanandam A, Christopher RT (2013) In vitro plantlet regeneration and genetic transformation of sponge gourd (Luffa cylindrica L.). Afr J Plant Sci 7(6):244–252. https://doi.org/10.5897/ajps12.196

  79. Swapna LP, Kannabiran K (2006) Antimicrobial activity and phytochemicals of Solanum trilobatum Linn. Afr J Biotechnol 5(23):2402–2404. https://doi.org/10.5897/AJB06.480

  80. Thul ST, Darokar MP, Shaseny AK, Khanuja SPS (2011) Molecular profiling of genetic variability in Capsicum species based on ISSR and RAPD markers. Mol Biotechnol 51(2):137–147. https://doi.org/10.1007/s12033-011-9446-y



The first author is grateful to the UGC New Delhi for award of BSR fellowship. The authors are grateful to Prof. Sadanandam Abbagani, CSIR-Emeritus Scientist for his valuable suggestions. The authors gratefully acknowledge the Head, Department of Botany and Department of Biotechnology for providing research facilities. The authors grateful to UGC New Delhi for funds provided under SAP-DRS-III to Botany and SAP-DRS-II to Biotechnology Departments. The authors also grateful to DST-New Delhi for funds provided under DST-FIST to Botany and Biotechnology Departments. The authors thank Dr. Md. Mustafa for authenticating the plant material.

Author Information

Pendli Sreenu
Department of Botany, Kakatiya University, Warangal, India

Rohela Gulab Khan
Central Sericultural Research and Training Institute, Central Silk Board, Ministry of Textiles, Govt. of India, Pampore, India