Keywords:
n Rhizobiumn rhizogenesn , Hairy roots, n Oroxylumn indicumn , Flavonoid, UV–VIS spectroscopy, FTIR
Oroxylum indicum (L) Vent (O. indicum), a significant endangered medicinal plant, was transformed by the Rhizobium rhizogenes (R. rhizogenes) mediated transformation method using the bacterial strain MTCC 532. For a successful transformation, factors such as explant selection, Co-cultivation time, temperature for induced root development, transformation technique, and antibiotic concentration were optimized. Various methods were employed for enhancing the rate of transformation. A drastic increase in transformation frequency was observed when CaCl2 was used in concentration of 10 mM and 15 mM along with ultrasonication during Co-cultivation. Total phenolic and flavonoid content was determined in various extracts of O. indicum by using Folin–Ciocalteau reagent and Aluminium chloride colorimetric method respectively. Moreover, the antioxidant potential of different extracts of O. indicum were assessed with the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method. Using specific rolA primers, molecular analysis was performed, revealing T-DNA integration in the hairy roots and confirming the expression of hairy root inducible genes. Murashige and Skoog (MS) medium with 3% sucrose was shown to have the maximum induction rate of hairy roots after 28 days of Co-cultivation. TLC as well as spectroscopic methods (UV–VIS and FTIR) were performed to compare the specific flavonoid (baicalein) in transformed roots as well as several non-transformed extracts. Comparing hairy roots to non-transformed roots, the total phenolic, flavonoid contents, and antioxidant activities were greater in hairy roots. The findings indicate that O. indicum hairy root cultures have a greater capacity for producing beneficial chemicals and researching their biological activities. The transformation of O. indicum by R. rhizogenes (MTCC 532) has been reported for the first time.
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Ahad A, Ganai A, Sareer O, Najm M, Kausar A, Mujeeb M, Siddiqui W (2012) Therapeutic potential of Oroxylum indicum: a review. J Pharma Res Opin 2:163–172
Akramian M, Mohammad S, Tabatabaei F, Mirmasoumi M (2008) Virulence of different strains of agrobacterium rhizogenes on genetic transformation of four Hyoscyamus species American–Eurasian. J Agric Environ Sci 3(5):759–763
Algabri A, Pandhure N (2019) Original research paper in vitro micropropagation studies in Spilanthes Acmella Murr. Int J Sci Res 8(2):1–2
Ali A, Naz S, Siddiqui FA, Iqbal J (2008) Rapid clonal multiplication of sugarcane (Saccharum officinarum) through callogenesis and organogenesis. Pak J Bot 40(1):123–138
Alibert B, Lucas O, LeV KJ, Alibert G (1999) Pectolytic enzyme treatment of sunflower explantsprior to wounding and co cultivation with Agrobacterium tumefaciens, enhances efficiency of transient β-glucuronidase expression. Physiol Plant 106:232–237
Altamura M, Capitani F, Gazza L, Capone I, Costantino P (1994) The plant oncogenes rolB stimulates the formation of flower and root meristemoids in tobacco thin cell layers. New Phytol 126:283–293
Amer A, Omar H (2019) In-vitro propagation of the multipurpose Egyptian medicinal plant Pimpinellaanisum. Egypt Pharmac J 18:254–262
Ayadi and Trémouillaux-Guiller (2003) Root formation from transgenic calli of Ginkgobiloba. Tree Physiol 23:713–718
Bansal YK, Gokhale M (2012) Effect of additives on micropropagation of an endangered medicinal tree Oroxylum indicum (L.) Vent. In: Leva A, Rinaldi LMR (eds) Recent advances in plant in vitro culture, Rinaldi Intech Publishers, p 183–196. https://doi.org/10.5772/50743
Begum M, Islam A, Begum R, Uddin MS, Rahman MS, Alam S, Akter W, Das M, Rahman MS, Imon AHMR (2019) Ethnopharmacological inspections of organic extract of Oroxylum indicum in rat models: a promising natural gift. Evid Based Complement Altern Med 2019:1562038. https://doi.org/10.1155/2019/1562038
Brijwal L, Tamta S (2015) Agrobacterium rhizogenes mediated hairy root induction in endangered Berberisaristata DC. Springerplus 4:443. https://doi.org/10.1186/s40064-015-1222-1
Chandra S, Chandra R (2011) Engineering secondary metabolite production in hairy roots. Phytochem Rev 10:371–395
Chandrappa et al (2012) Identification and separation of quercetin from ethanol extract of Carmona retusa by thin layer chromatography and high-performance liquid chromatography with diode array detection. World J Pharmacy Pharmac Sci 3(6):2020–2029
Chang C, Yang M, Wen H, Chern J (2002) Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 10:178–182
Chen L, Cai Y, Liu X, Guo C, Sun S, Wu C, Jiangb B, Hanb T, Hou W (2018) Soybean hairy roots produced in vitro by Agrobacterium rhizogenes mediated transformation. Crop J 6(2):162–171
Colling J, Groenewald J, Makunga N (2010) Genetic alterations for increased coumarin production lead to metabolic changes in the medicinally important Pelargonium sidoides DC (Geraniaceae). Metab Eng 12(6):561–572
Darshan S, Ved DK (2003) A balanced perspective for management of Indian. Medicinal Plants Ind Fort 129:275–288
DiCola A, Costantino P, Spano L (1996) Cell commitment and rolB gene expression in the induction of root differentiation. Plant Cell, Tissue Organ Cult 46:203–209
Doma M, Abhayankar G, Reddy VD, KaviKishor PB (2012) Carbohydrate and elicitor enhanced withanolide (withaferin A and withanolide A) accumulation in hairy root cultures of Withania somnifera (L.). Indian J ExpBiol 50:484–490
Ercan AG, Taskin KM (1999) Agrobacterium rhizogenes-mediated hairy root formation in some Rubiatinctorum L. population growth in Turkey. Turk J Botany 23:373–377
Faraz R, Gokhale M, Gothalwal R (2020) Callus extracts of Oroxylum indicum (L.) vent containing baicalein have in vitro antioxidant and antibacterial activities. Biotecnol Veget 20(1):51–62
Garagounis C, Georgopoulou ME, Beritza K, Papadopoulou KK (2020) An Agrobacterium rhizogenes mediated hairy root transformation protocol for fenugreek. Methods 7:101098
Giri C, Dhingra V, Narasu M (2001) Enhanced podophyllotoxin production from Agrobacterium rhizogenes transformed cultures of Podophyllum hexandrum. Nat Prod Lett 15:229–235
Gokhale M, Bansal YK (2010) Assessment of secondary metabolites in in vitro regenerated plantlets of Oroxylum indicum (L.) Vent. Plant Tissue Cult Biotechnol 20(1):21–28
Gokhale M, Bansal YK, Sandhu S (2016) Optimization of baicalein and chrysin production in cell cultures of Oroxylum indicum (L.) Vent. J Anal Chem Lett 6(6):834–849
Hwang S (2009) Catapol production in Chinese foxglove (Rehmannia glutinosa Libos) hairy roots transformed with Agrobacterium rhizogenes ATCC15834. Methods Mol Biol 547:263–273. https://doi.org/10.1007/978-1-60327-287-2_21
Ismail H, Dilshad E, Waheed MT, Sajid M, Kayani WK, Mirza B (2016) Transformation of Lactuca sativa L. with rol C gene results in increased antioxidant potential and enhanced analgesic, anti-inflammatory and antidepressant activities in vivo. 3 Biotech. 6(2):215
Jasik J, Boggetti G, Caricato S (1997) Characterization of morphology and root formation in the model woody perennial shrub Solanuma viculare Forst. Expressing rolABC genes of Agrobacterium rhizogenes. Plant Sci 124:57–68
Joubert P, Beaupere D, Lelievre P, Wadouachi A, Sangwan RS, Sangwan-Norreel BS (2002) Effect of phenolic compounds on Agrobacterium vir genes and gene transfer induction-a plausible molecular mechanism of phenol binding protein activation. Plant Sci 162:733–743
Karadag A, Ozeelik B, Saner S (2009) Review of methods to determine antioxidant capacities. Food Anal Method 2:41–60
Karthikeyan A, Palanivel S, Parvathy S, Bhakyaraj R (2007) Hairy root induction from hypocotyls segments of groundnut (Arachis hypogaea L.). Afr J Biotechnol 6(15):1817–1820
Kim JS, Lee YS, Park S (2008) Resveratrol production in hairy root culture of peanut, Arachishypogaea L. transformed with different Agrobacterium rhizogenes strains. Afr J Biotechnol 7(20):3788–3790
Kirtikar KR, Basu BD (2001) Indian medicinal plants. Oriental Enterprises, Uttaranchal, 2nd edn, vol 8, p 2604
Kobayashi Y, Fukui H, Tabata M (1989) effect of oxygen-supply on berberine production in cell-suspension cultures and immobilized cells of Thalictrum minus. Plant Cell Rep 8(4):255–258
Kopustinskiene DM, Jakstas V, Savickas A, Bernatoniene J (2020) Flavonoids as anticancer agents. Nutrients 12(2):457. https://doi.org/10.3390/nu12020457
Kumar DRN, George VC, Suresh PK, Kumar RA (2012) Cytotoxicity, apoptosis induction and anti-metastatic potential of Oroxylum indicum in human breast cancer cells. Asian Pac J Cancer Prev 13:2729–2734
Kumar R, Mamrutha H, Kaur A, Grewal A (2017) Synergistic effect of cefotaxime and timentin to suppress the Agrobacterium overgrowth in wheat (Triticum aestivum L.) transformation. Asian J Microbiol Biotechnol Environ Exp Sci 19:961–967
Kumar V, Sharma A, Narasimha P, Gururaj H, Ravishankar G (2006) Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone. Electron J Biotechnol 9(4):349–357
Laupattarakasem P, Houghton P, Hoult J, Itharat A (2003) An evaluation of the activity related to inflammation of four plants used in Thailand to treat arthritis. J Ethnopharmacol 85:207–215. https://doi.org/10.1016/S0378-8741(02)00367-7
Lee MH, Yoon ES, Jeong JH (2004) Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters. Plant Cell Rep 22:822–827
Lister E, Wilson P (2001) Measurement of total phenolics and ABTS assay for antioxidant activity (personal communication). Crop Research Institute Lincoln, New Zealand
Liu SJ, Wei ZM, Huang JQ (2008) The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties. Plant Cell Rep 27:489–498. https://doi.org/10.1007/s00299-007-0475-8
Liu S, Su L, Liu S, Zeng X, Zheng D, Hong L, Li L (2016) Agrobacterium rhizogenes mediated transformation of Arachis hypogaea: an efficient tool for functional study of genes. Biotechnol Biotechnol Equip 30(5):869–878
Mao AA (2002) Oroxylum indicum vent. A potential anticancer medicinal plant. Ind J Trad Know 1:17–21
Mehrotra S, Kukreja A, Khanuja S, Mishra BN (2008) Genetic transformation studies and scale up of hairy root culture of Glycyrrhizaglabra in bioreactor. Electron J Biotechnol 11(2):1–7
Moghadam AY, Piri K, Bahramnejad B, Ghiasvand T (2014) Dopamine production in hairy root cultures of Portulacaoleracea (Purslane) using Agrobacterium rhizogenes. J AgricSci Technol 16:409–420
Nilsson O, Olsson O (1997) Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol Plantarum 100:463–473
Nourozi et al (2016) Influences of various factors on hairy root induction in Agastache foeniculum(Pursh) Kuntze. Acta Agric Sloven 107(1):45–54
Panche AN, Diwan AD, Chandra SR (2016) Flavonoids: an overview. J Nutr. Sci. 5:e47
Panda BM, Mehta UJ, Hazra S (2017) Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L. 3 Biotech 7:21
Park S-U, Facchini PJ (2000) Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures. J Exp Bot 347:1005–1016
Pawar PK, Maheshwari VL (2004) Agrobacterium rhizogenes mediated hairy root induction in two medicinally important members of family Solanaceae. Indian J Biotechnol 3:414–417
Pedroso ANV, Lazarini RAM, Tamaki V, Nievol CC (2010) Invitro culture and ex vitro acclimatization of Vriesea inflate. Rev Bras Bot 33(3):407–414
Peng CX, Gong JS, Zhang XF, Zhang M, Zheng SQ (2008) Production of gastrodin through biotransformation of p-hydroxybenzyl alcohol using hairy root cultures of Daturatatula L. Afr J Biotechnol 7(3):211–216
Phan T, Nguyen VH, Salazar MA, Wong P, Diamond JD, Yim JH, Melstrom LG (2020) Inhibition of autophagy amplifies baicalein-induced apoptosis in human colorectal cancer. Mol Ther Oncolyt 19:1–7
Pistelli L, Giovannini A, Ruffoni B, Bertoli A (2010) Hairy root cultures for secondary metabolites production. Adv Exp Med Biol 698:167–184
Rahnama H, Hasanloo T, Shams MR, Sepehrifar R (2008) Silymarin production by hairy root culture of Silybum marianum (L.) Gaertn. Irn J Biotechnol 6(2):113–118
Rajeev P, Deepa A, Vanathi P, Vidhya D (2017) Screening for phytochemicals and FTIR analysis of Myristica dactyloids fruit extracts. Int J Pharmac Sci 9(1):315–318
Rolfs CH, Schön H, Steffens M, Kindl H (1987) Cell-suspension culture of Arachis hypogaea L.: model system of specific enzyme induction in secondary metabolism. Planta 172:238–244
Romero FR, Delate K, Kraus GA, Solco AK, Murphy PA, Hannapel DJ (2009) Alkamide production from hairy root cultures of Echinacea. In Vitro Cell Dev Biol Plant 45(5):599–609. https://doi.org/10.1007/s11627-008-9187-1.[CrossRef][GoogleScholar]
Rostampour S, Sohi SH, Jourabchi E, Ansari E (2009) Influence of Agrobacterium rhizogenes on induction of hairy roots and benzyliso quinoline alkaloids production in Persian poppy (Papaver bracteatumLindl.): preliminary report. World J Microbiol Biotechnol 25:1807–1814. https://doi.org/10.1007/s11274-009-0081-8.[CrossRef][GoogleScholar]
Rout GR, Samantaray S, Das P (2000) In vitro manipulation and propagation of medicinal plants. Biotechnol Adv 18(2):91–120
Sahu R et al (2014) Ultraviolet-visible and fourier transform infrared spectroscopic studies on non-conventional species of Curcuma. IJACS 2(4):300–302
Samadi A, Carapetian J, Heidari R, Jafari M, Hassanzadehgorttapeh A (2012) Hairy root induction in Linum mucronatum ssp. mucronatum, an anti-tumor lignans producing plant. Not Bot HortiAgrobo 40(1):125–131
Sanders D, Brownlee C, Harper JF (1999) Communicating with calcium. Plant Cell 11(4):691–706
Santhanakrishnan D, Sripriya N, Chandrasekaran B (2014) Studies on the phytochemistry, spectroscopic characterization and antibacterial efficacy of Salicornia Brachiata. Int J Pharm Pharm Sci 6:6430–6432
Schmidt JF, Moore MD, Pelcher LE (2007) Covello PS (2007) High efficiency Agrobacterium rhizogenes mediated transformation of Saponaria vaccaria L. (Caryophyllaceae) using fluorescence selection. Plant Cell Rep 26:1547–1559
Sevón N, Oksman-Caldentey KM (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Med 68:859–868
Showmiya K, Ananthi T (2018) Phytochemical screening and FTIR analysis of Citrus maxima Linn. leaves. Int J Pharma Res Health Sci 6(3):2565–2569
Singh AK (2015) Morphological expressions in seedlings of Oroxylum indicum. Mod Phytomorphol 8:41–48
Spanos GA, Wrolstad RE (1990) Influence of processing and storage on the phenolic composition of Thompson seedless grape juice. J Agric Food Chem 38:1565–1571
Stalikas CD (2007) Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci 30(18):3268–3295
Stojakowska A, Kisiel W (1999) Secondary metabolites from a callus culture of Scutellaria columnae. Fitoterapia 70:324–325
Srivastava S, Srivastava AK (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol 27(1):29–43
Subramanyam K, Subramanyam K, Sailaja KV, SrinivasuluM LK (2011) Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration. Plant Cell Rep 30:425–436
Swain SS, Sahu L, Pal A (2012) Hairy root cultures of butterfly pea (Clitoria ternatea L.): agrobacterium plant factors influencing transformation. World J Micro Biol Biotechnol 28:729–739
Thiruvengadam M, Praveen N, Kim EH, Kim SH, Chung IM (2014a) Production of anthraquinones, phenolic compounds and biological activities from hairy root cultures of Polygonum multiflorum Thunb. Protoplasma 251:555–566
Thiruvengadam M, Praveen N, Maria John KM, Yang YS, Kim SH, Chung IM (2014b) Establishment of Momordica charantia hairy root cultures for the production of phenolic compounds and determination of their biological activities. Plant Cell Tissue Organ Cult 118:545–557
Thwe A, ValanArasu M, Li X, Park CH, Kim SJ, Al-Dhabi NA, Park SU (2016) Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenyl propanoid biosynthesis in Tartary buckwheat (Fagopyrum tataricum Gaertn). Front Microbiol 7:318
Toivonen L, Laakso S, Rosenqvist H (1992) The effect of temperature on hairy root cultures of Catharanthus roseus: Growth, indole alkaloid accumulation and membrane lipid composition. Plant Cell Rep 11(8):395–399
Trick HN, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–336
Trick HN, Finner JJ (1998a) Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue. Plant Cell Rep 17:482–488
Trick HN, Finner JJ (1998b) Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.)Merrill] embryogenic suspension culture tissue. Plant Cell Rep 17:482–488
Valimehr S, Sanjarian F, Sohi HH, Sharafi A, Sabouni F (2014) A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepetapogono sperma. Physiol Mol Biol Plants 20(3):351–356
Visveshwari M, Subbaiyan B, Thangapandian V (2017) Phytochemical analysis, antibacterial activity, FTIR and GCMS analysis of Ceropegia junceaRoxb. Int J Pharmacogn Phytochem Res 9(7):914–920
Vojin T, Snežana M, Aleksandar C (2014) Production of hairy root cultures of lettuce (Lactuca sativa L). Central Eur J Biol 9(12):1196–1205
Weremczuk-Jezyna I, Grzegorczyk-Karolak I, Frydryc B, Kroılicka A, Wysokinıska H (2013) Hairy roots of Dracocephalum moldavica :rosmarinic acid content and antioxidant potential. Acta Physiol Plant 35:2095–2103
White PJ, Broadley MR (2003) Calcium in plants. Ann Bot 92(4):487–511
Xie Z, Lu W, Yang L, Li G, Ye B (2017) A voltammetry sensor platform for baicalein and baicalin simultaneous detection in vivo based on Ta2O5-Nb2O5@CTS composite. Talanta 170:358–368
Yogananth N, Basu MJ (2009) TLC method for the determination of plumbagin in hairy root culture of Plumba gorosea L. Global J Biotech Biochem 4(1):66–69
Zaker A, Abrishamchi P, Asili J, Mousavi S, Rezaee A (2013) Induction of callogenesis and rhizogenesis in Perovskia abrotanoides Karel., a little known medicinal plant. J Med Plants Res 7(46):3385–3392
Zhong JJ, Konstantinov KB, Yoshida T (1994) Computer aided on-line monitoring of physiological variables in suspended cell cultures of Perila frutescens in a biorreator. J Ferment Bioeng 77(4):445–447
Zhong JJ, Bai Y, Wang SJ (1996) Effects of plant growth regulators on cell growth saponin production by suspension cultures of Panax quinque folium. J Biotechnol 45:227–234
We are grateful to the Principal, St. Aloysius(autonomous) College, Jabalpur (MP, India) for supporting this research.