Abstract
Agrobacterium rhizogenes mediated transformation has been experimented in leaf explants of the memory herb Bacopa monnieri in order to assess the regeneration potential of hairy roots (HR) followed by the elicitation of transformed plants for increased Bacoside A production. Out of the four strains tested, A4 and MTCC 532 derived HR exhibited regrowth in MS basal medium while MTCC 2364 derived HR showed regeneration in MS medium supplemented with suitable phyto hormones. R1000 derived HR possessed no regeneration potential. Comparable to A4, MTCC 532 derived HR displayed maximum regrowth frequency of about 85.71 ± 1.84 % with an increase in biomass to threefold. Therefore, five HR plant lines (MTCC 532 derived) were generated and maintained in MS basal liquid medium in which HR3 topped the others in producing a huge biomass of about 67.09 ± 0.66 g FW. PCR amplification and southern hybridization analysis of rol A gene (280 bp) has been performed in order to confirm the transformation process. Moreover, HR3 plant line has accumulated highest total phenolic content of about 165.68 ± 0.82 mg GAE/g DW and highest total flavonoid content of about 497.78 ± 0.57 mg QRE/g DW when compared to other lines and untransformed controls. In addition, HR3 plant extract showed 85.58 ± 0.14 % of DPPH (2, 2-diphenyl-1-picryl hydrazyl) inhibition displaying its reliable anti oxidant potential. Further on elicitation with 10 mg/L chitosan for 2 weeks, HR3 has produced 5.83 % of Bacoside A which is fivefold and threefold increased production when compared to untransformed and transformed unelicited controls respectively. This is the first report on eliciting HR plants for increased metabolite accumulation in B. monnieri.
Similar content being viewed by others
References
Bansal M, Kumar A, Reddy MS (2014) Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst. Acta Physiol Plant 36:2793–2801
Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181:1199–1200
Bonhomme V, Matter LD, Lacoux J, Fliniaux M, Dubreuil JA (2000) Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only. J Biotechnol 81:151–158
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 by Agrobacterium rhizogenes. Plant Cell Rep 25:1059–1066
Cui L, Wang Z, Zhou X (2012) Optimization of elicitors and precursors to enhance valtrate production in adventitious roots of Valeriana amurensis Smir. ex Kom. Plant Cell Tissue Organ Cult 108:411–420
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
Jacob A, Malpathak N (2005) Plantlet regeneration enhances solasodine productivity in hairy root cultures of Solanum khasianum Clarke. In Vitro Cell Dev Biol Plant 41:291–295
Jeong GT, Park DK (2005) Enhancement of growth and secondary metabolite biosynthesis: effect of elicitors derived from plants and insects. Biotechnol Bioprocess Eng 10:73–77
Jezyna IW, Karolak IG, Frydrych B, Krolicka A, Wysokin H (2013) Hairy roots of Dracocephalum moldavica: rosmarinic acid content and antioxidant potential. Acta Physiol Plant 35:2095–2103
Lee MH, Yoon ES, Jeong JH, Choi YE (2004) Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters. Plant Cell Rep 22:822–827
Lin YL, Juan IM, Chen YL, Liang YC, Lin JK (1999) Composition of polyphenols in fresh tea leaves and associations of their oxygen-radical-absorbing capacity with anti proliferative actions in fibroblast cells. J Agric Food Chem 44:1387–1394
Majumdar S, Garai S, Jha S (2011) Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants. Plant Cell Rep 30:941–954
Marinova D, Ribarova F, Atanassova M (2005) Total phenolics and total flavonoids in Bulgarian fruits and vegetables. J Univ Chem Technol Metall 40:225–260
Mehrotra S, Goel MK, Rahman LU, Kukreja AK (2013) Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina. Plant Cell Tissue Org Cult 114:31–38
Murthy PBS, Raju VR, Ramakrishna T, Chakravarthy MS, Kumar KV, Kannababu S, Subbaraju GV (2006) Estimation of twelve bacopa saponins in Bacopa monnieri extracts and formulations by High performance liquid chromatography. Chem Pharm Bull 54:907–911
Naik PM, Manohar SH, Praveen N, Murthy HN (2010) Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of Bacopa monnieri and accumulation of bacoside A in regenerated shoots. Plant Cell Tiss Organ Cult 100:235–239
Nilsson O, Olsson O (1997) Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol Plant 100:463–473
Paul P, Sarkar S, Jha S (2015) Effects associated with insertion of cryptogein gene utilizing Ri and Ti plasmids on morphology and secondary metabolites are stable in Bacopa monnieri-transformed plants grown in vitro and ex vitro. Plant Biotechnol Rep 9:231–245
Piatczak E, Kuzma L, Skała E, Zebrowska M, Balcerczak E, Wysokinska H (2015) Iridoid and phenylethanoid glycoside production and phenotypical changes in plants regenerated from hairy roots of Rehmannia glutinosa Libosch. Plant Cell Tissue Org Cult 122:259–266
Rahman LU, Verma PC, Singh D, Gupta MM, Banerjee S (2002) Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell. Biotechnol Lett 24:1427–1429
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory Press, New York (Plain view)
Satheeshkumar K, Jose B, Soniya EV, Seeni S (2009) Isolation of morpho variants through plant regeneration in Agrobacterium rhizogenes induced hairy root cultures of Plumbago rosea L. Indian J Biotechnol 8:435–441
Sharafi A, Sohi HH, Mousavi A, Azadi P, Dehsara B, Khalifani BH (2013) Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation. World J Microbiol Biotechnol 29:2125–2131
Shilpha J, Satish L, Kavikkuil M, Largia MJV, Ramesh M (2015) Methyl jasmonate elicits the solasodine production and anti-oxidant activity in hairy root cultures of Solanum trilobatum L. Indus Crops Prod 71:54–64
Singh HK, Dhawan BN (1997) Neurophychopharmacological effects of the ayurvedic nootropic Bacopa monniera Linn (Brahmi). Ind J Pharmacol 29:8359–8365
Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathi A (2012) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Indus Crops Prod 37:124–129
Srivastava S, Srivastava AK (2007) Hairy root culture for mass production of high-value secondary metabolites. Crit Rev Biotechnol 27:29–43
Tepfer D (1990) Genetic transformation using Agrobacterium rhizogenes. Physiol Plant 79:140–146
Udomsuk L, Jarukamjorn K, Tanaka H, Putalun W (2011) Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation. Biotechnol Lett 33:369–374
Veena V, Taylor CG (2007) Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev Biol Plant 43:383–403
Vinterhalter B, Milosevic DK, Jankovic T, Pljevljakusic D, Ninkovic S, Smigocki A, Vinterhalter D (2015) Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production. Plant Cell Tissue Org Cult 121:667–679
Wang QJ, Zheng LP, Yuan HY, Wang JW (2013) Propagation of Salvia miltiorrhiza from hairy root explants via somatic embryogenesis and tanshinone content in obtained plants. Indus Crops Prod 50:648–653
Wu HJ, Wang XX, Li Y, Zhang DG, Zhang B, Wang YX (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
Xu C, Zhao B, Ou Y, Wang X, Yuan X, Wang Y (2007) Elicitor-enhanced syringing production in suspension cultures of Saussurea medusa. Word J Microbiol Biotechnol 23:965–970
Zehra M, Banerjee S, Sharma S, Kumar S (1999) Influence of Agrobacterium rhizogenes strains on biomass and alkaloid productivity in hairy root lines of Hyoscyamus muticus and H. albus. Planta Med 65:60–63
Acknowledgments
The first and the second author greatly acknowledge the University Grants Commission (UGC) for awarding financial support in the form of Basic Science Research Fellowship (UGC order no. F.4-1/2006 (BSR)/7-326/2011(BSR), dated 25.02.2013). We sincerely thank Prof. A. Ganapathy, Department of biotechnology, Bharathidasan University, Tiruchirapalli for providing us the A. rhizogenes strains used in this study. All authors thank the Department of Biotechnology (DBT), Government of India (Grant No. BT/BI/25/001/2006) for the Computational and Bioinformatics Facility provided by the Alagappa University Bioinformatics Infrastructure Facility.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Largia, M.J.V., Satish, L., Johnsi, R. et al. Analysis of propagation of Bacopa monnieri (L.) from hairy roots, elicitation and Bacoside A contents of Ri transformed plants. World J Microbiol Biotechnol 32, 131 (2016). https://doi.org/10.1007/s11274-016-2083-7
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-016-2083-7