Abstract
Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47 ± 0.63 mg g−1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS + 5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08 ± 1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3 ± 1.36 roots with an average length of 15.31 ± 2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L−1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.
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References
Ahmed MR, Anis M (2012) Role of TDZ in the quick regeneration of multiple shoots from nodal explant of Vitex trifolia L.—an important medicinal plant. Appl Biochem Biotechnol 168(5):957–966. https://doi.org/10.1007/s12010-012-9799-0
Alvarez SIP, Spollansky TC, Giulietti AM (2000) The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzym Microb Technol 26(2-4):252–258. https://doi.org/10.1016/S0141-0229(99)00137-4
Amali P, Kingsley SJ, Ignacimuthu S (2014) High frequency callus induction and plant regeneration from shoot tip explants of Sorghum bicolor L. Moench. Int J Pharm Pharm Sci 6:213–216
Bhattacharyya P, Kumaria S, Diengdoh R, Tondon P (2014) Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid. Meta Gene 2:489–504. https://doi.org/10.1016/j.mgene.2014.06.003
Bopaiah CP, Pradhan N (2001) Central nervous system stimulatory action from root extract of Plumbago zeylanica in rats. Phytother Res 15(2):153–156. https://doi.org/10.1002/ptr.702
Ceasar SA, Ayyanar M, Ignacimuthu S (2013) An improved micropropagation protocol for Plumbago zeylanica L. An important medicinal plant. Asian J Biol Sci 6(4):214–220
Chaplot BB, Dave AM, Jasrai YT (2006) A valued medicinal plant-Chitrak (Plumbago zeylanica Linn.): successful plant regeneration through various explants and field performance. Plant Tissue Cult Biotechnol 16(2):77–84. https://doi.org/10.3329/ptcb.v16i2.1108
Chauhan M (2014) A review on morphology, phytochemistry and pharmacological activities of medicinal herb Plumbago zeylanica Linn. J Phytochem 3(2):95–118
Checker R, Sharma D, Sandura SK, Khanam S, Poduval TB (2009) Anti-inflammatory effects of plumbagin are mediated by inhibition of NF-kappaB activation in lymphocytes. Int Immunopharmacol 9(7-8):949–958. https://doi.org/10.1016/j.intimp.2009.03.022
Chetri SK, Sardar PR, Agrawal V (2014) Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker. Physiol Mol Biol Plants 20(4):517–526. https://doi.org/10.1007/s12298-014-0257-z
Das G, Rout R (2002) Direct plant regeneration from leaf explants of Plumbago species. Plant Cell Tissue Organ Cult 68(3):311–314. https://doi.org/10.1023/A:1013945513317
Ding Y, Chen ZJ, Liu S, Che D, Vetter M, Chang CH (2005) Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone. J Pharm Pharmacol 57(1):111–116. https://doi.org/10.1211/0022357055119
Durand R, Zenk MH (1971) Biosynthesis of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) via the acetate pathway in higher plants. Tetrahedron Lett 12(32):3009–3012. https://doi.org/10.1016/S0040-4039(01)97077-7
Edwin S, Joshi SB, Jain DC (2009) Antifertility activity of leaves of Plumbago zeylanica Linn. In female albino rats. Eur J Contracept Reprod Health Care 14(3):233–239. https://doi.org/10.1080/13625180902874310
Erisen S, Atalay E, Yorgancilar M (2011) The effect of thidiazuron on the in vitro shoot development of endemic Astragalus cariensis in Turkey. Turk J Bot 35(5):521–526. https://doi.org/10.3906/bot-1009-74
Fesel PH, Zuccaro A (2016) β-glucan: crucial component of the fungal cell wall and elusive MAMP in plants. Fungal Genet Biol 90:53–60. https://doi.org/10.1016/j.fgb.2015.12.004
Franklin G, Sheeba CJ, Sita GL (2004) Regeneration of eggplant (Solanum melongena L.) from root explants. In Vitro Cell Dev Biol Plant 40(2):188–191. https://doi.org/10.1079/IVP2003491
Gangopadhyay M, Dewanjee S, Bhattacharya S (2011) Enhanced plumbagin production in elicited Plumbago indica hairy root cultures. J Biosci Bioeng 111(6):706–710. https://doi.org/10.1016/j.jbiosc.2011.02.003
Gbadamosi IT, Egunyomi A (2010) Micropropagation of Plumbago zeylanica L. (Plumbaginaceae) in Ibadan, southwestern Nigeria. J Med Plant Res 4(4):293–297. https://doi.org/10.5897/JMPR09.263
Goyal AK, Pradhan S, Basistha BC, Sen A (2015) Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) Nees using RAPD and ISSR markers. 3. Biotech 5(4):473–482. https://doi.org/10.1007/s13205-014-0244-7
Guo B, Abbasi BH, Zeb A, Xu LL, Wei YH (2011) Thidiazuron: a multi-dimensional plant growth regulator. Afr J Biotechnol 10:(45)8984–9000. https://doi.org/10.5897/AJB11.636
Heikrujam M, Kumar D, Kumar S, Gupta SC, Agrawal V (2014) High efficiency cyclic production of secondary somatic embryos and ISSR based assessment of genetic fidelity among the emblings in Calliandra tweedii (Benth.) Sci Hortic 177:63–70. https://doi.org/10.1016/j.scienta.2014.07.036
Huetteman CA, Preece JE (1993) Thidiazuran: a potent cytokinins for woody plant tissue culture. Plant Cell Tissue Organ Cult 33(2):105–119. https://doi.org/10.1007/BF01983223
Hutchinson MJ, Murr D, Krishnaraj S, Senaratna T, Saxena PK (1997) Does ethylene play a role in thidiazuron-regulated somatic embryogenesis of Geranium (Pelargonium x Hortorum bailey) hypocotyl cultures? In Vitro Cell Dev Biol Plant 33(2):136–141. https://doi.org/10.1007/s11627-997-0012-z
Jaisi A, Panichayupakaranant P (2016) Increased production of plumbagin in Plumbago indica root cultures by biotic and abiotic elicitors. Biotechnol Lett 38(2):351–355. https://doi.org/10.1007/s10529-015-1969-z
Jones MPA, Cao J, O’Brien R, Murch SJ, Saxena PK (2007) The mode of action of thidiazuron: auxins, indoleamines, and ion channels in the regeneration of Echinacea purpurea L. Plant Cell Rep 26(9):1481–1490. https://doi.org/10.1007/s00299-007-0357-0
Joshi P, Dhawan V (2007) Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biol Plant 51(1):22–26. https://doi.org/10.1007/s10535-007-0005-0
Jumali SS, Said IM, Ismail I, Zamri Z (2011) Genes induced by high concentration of salicylic acid in Mitragyna speciosa. Aust J Crop Sci 5(3):296–303
Kishore N, Mishra BB, Tiwari VK, Tripathi V (2012) An account of phytochemicals from Plumbago zeylanica (Family: Plumbaginaceae): a natural gift to human being. Chron Young Sci 3(3):178–198. https://doi.org/10.4103/2229-5186.99564
Kodja H, Noirot M, Khoyratty SS, Limbada H, Verpoorte H, Palama TL (2015) Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment. Plant Physiol Biochem 96:337–344. https://doi.org/10.1016/j.plaphy.2015.08.017
Komaraiah P, Amrutha RN, Kishor PK, Ramakrishna SV (2002) Elicitor enhanced production of plumbagin in suspension cultures of Plumbago rosea L. Enzym Microb Technol 31(5):634–639. https://doi.org/10.1016/S0141-0229(02)00159-X
Komaraiah P, Ramakrishna SV, Reddanna P, Kishor PBK (2003) Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption. J Biotechnol 101(2):181–187. https://doi.org/10.1016/S0168-1656(02)00338-3
Kumar R, Kumar S, Patra A, Jayalakshmi S (2009) Hepatoprotective activity of aerial parts of Plumbago zeylanica Linn. against carbon tetrachloride-induced hepatotoxicity in rats. Int J Pharm Pharm Sci l:171–175
Kuo PL, Hsu YL, Cho CY (2006) Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther 5(12):3209–3221. https://doi.org/10.1158/1535-7163.MCT-06-0478
Liu Y, Deng F, Liu C, Meng QU, Gao ZW (2006) Determination of plumbagin in different parts of Plumbago zeylanica by RP-HPLC. Zhongguo Zhong Yao Za Zhi 31(20):1684–1686
Mallikadevi T, Senthilkumar P, Paulsamy S (2008) In vitro regeneration of medicinal plant, Plumbago zeylanica L. with reference to a unique population in Maruthmalai, the western Ghats, India. Plant Tissue Cult Biotechnol 18(2):173–179. https://doi.org/10.3329/ptcb.v18i2.3648
Mittal V, Sharma SK, Kaushik D, Khatri M, Tomar K (2010) A comparative study of analgesic activity of Plumbago zeylanica Linn. callus and root extracts in experimental mice. Res J Pharm Biol Chem Sci 1(4):830–836
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Murthy BNS, Murch SJ, Saxena PK (1995) Thidiazuran-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogea): endogenous growth regulator levels and significance of cotyledons. Physiol Plant 94(2):268–276. https://doi.org/10.1111/j.1399-3054.1995.tb05311.x
Paiva SR, Lima LA, Figueiredo MR, Kaplan MA (2011) Chemical composition fluctuations in roots of Plumbago scandens L. in relation to floral development. An Acad Bras Ciênc 83(4):1165–1170. https://doi.org/10.1590/S0001-37652011000400004
Panda S, Kamble A (2016) Genotypic and seasonal variation of plumbagin content from different populations of Plumbago zeylanica L. in India. Proc Natl Acad Sci India Sect B Biol Sci 86(1):165–169. https://doi.org/10.1007/s40011-014-0432-6
Pant M, Lal A, Rana S, Rani A (2012) Plumbago zeylanica L.: a mini review. Int J Pharmaceut Appl 3(3):399–405
Pathak H, Dhawan V (2011) ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793. In Vitro Cell Dev Biol Plant 48(1):137–143. https://doi.org/10.1007/s11627-011-9385-0
Putalun W, Udomsin O, Yusakul G, Juengwatanatrakul T, Sakamoto S, Tanaka H (2010) Enhanced plumbagin production from in vitro cultures of Drosera burmanii using elicitation. Biotechnol Lett 32(5):721–724. https://doi.org/10.1007/s10529-010-0202-3
Rahimi S, Devi BSR, Khorolragchaa A, Kim YK, Kim JH, Jung SK, Yang DC (2014) Effect of salicylic acid and yeast extract on the accumulation of jasmonic acid and sesquiterpenoids in Panax ginseng adventitious roots. Russ J Plant Physiol 61(6):811–817. https://doi.org/10.1134/S1021443714060156
Rahman MS, Anwar MN (2007) Antimicrobial activity of crude extract obtained from the root of Plumbago zeylanica. Bangladesh J Microbiol 24(1):73–75. https://doi.org/10.3329/bjm.v24i1.1244
Rathore NS, Rai MK, Phulwaria M, Rathore N, Shekhawat NS (2014) Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis. Acta Physiol Plant 36(2):555–559. https://doi.org/10.1007/s11738-013-1429-0
Razaq M, Heikrujam M, Chetri SK, Agrawal V (2013) In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker. Physiol Mol Biol Plants 19(2):251–260. https://doi.org/10.1007/s12298-012-0152-4
Ren G, Li L, Hu H, Li Y, Liu C, Wei S (2016) Influence of the environmental factors on the accumulation of the bioactive ingredients in Chinese Rhubarb products. PLoS One 11(5):e0154649. https://doi.org/10.1371/0154649
Rout GR (2002) Direct plant regeneration from leaf explants of Plumbago species and its genetic fidelity through RAPD markers. Ann Appl Biol 140(3):305–313. https://doi.org/10.1111/j.1744-7348.2002.tb00186.x
Rout GR, Saxena C, Samantaray S, Das P (1999) Rapid plant regeneration from callus cultures of Plumbago zeylanica. Plant Cell Tissue Organ Cult 56(1):47–51. https://doi.org/10.1023/A:1006224404734
Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci U S A 81(24):8014–8018. https://doi.org/10.1073/pnas.81.24.8014
Sahoo S, Debata BK (1998) Micropropagation of Plumbago zeylanica Linn. J Herbs Spices Med Plants 5(4):87–93. https://doi.org/10.1300/J044v05n04_10
Sankara AJ, Kumar N, Mokkapati A (2013) In vitro anti-cancer activities of few plant extracts against MCF-7 and HT-29 cell lines. Int J Pharm Sci 3:185–188
Sivanesan I (2007) Shoot regeneration and somaclonal variation from leaf callus cultures of Plumbago zeylanica L. Asian J Plant Sci 6:83–86
Sivanesan I, Jeong BR (2009) Induction and establishment of adventitious and hairy root cultures of Plumbago zeylanica L. Afr J Biotechnol 8(20):5294–5300
Sjahril R, Haring F, Riadi M, Rahim MD, Khan RS, Amir RS, Amir A, Trishawat AR (2016) Performance of NAA, 2iP, BAP and TDZ on callus multiplication, shoots initiation and growth for efficient plant regeneration system in Chrysanthemum (Chrysanthemum morifolium Ramat.) Int J Agric Syst 4(1):52–61. https://doi.org/10.20956/ijas.v4i1.241
Tyagi R, Menghani E (2014) A review on Plumbago zeylanica: a compelling herb. Int J Pharm Sci Res 5(4):119–126
Wang YC, Huang TL (2005) Anti-Helicobacter pylori activity of Plumbago zeylanica L. FEMS Immunol Med Microbiol 43(3):407–412. https://doi.org/10.1016/j.femsim.2004.10.015
Acknowledgments
The authors are grateful to the University of Delhi, India, for providing Research and Development Grant. Prof. VA is thankful to the Science and Engineering Research Board, Government of India, New Delhi, for the sanction of a Major Research Project (SERB/SR/SO/PS/05/2012). US is indebted to University Grants Commission, New Delhi, for the award of JRF and SRF. Financial assistance provided by DST-Purse grant to Department of Botany is gratefully acknowledged.
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Sharma, U., Agrawal, V. In vitro shoot regeneration and enhanced synthesis of plumbagin in root callus of Plumbago zeylanica L.—an important medicinal herb. In Vitro Cell.Dev.Biol.-Plant 54, 423–435 (2018). https://doi.org/10.1007/s11627-018-9889-y
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DOI: https://doi.org/10.1007/s11627-018-9889-y