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In vitro Direct Regeneration and Agrobacterium tumefaciens Mediated in planta Transformation of Ocimum sanctum L.


Affiliations
1 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, Uttar Pradesh, India
2 CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow 226 015, Uttar Pradesh, India
 

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II (npt-II) & β-glucoronidase (uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Keywords

Gus-A, Kanamycin, MAPs, Npt-II, O. tenuiflorum, Petiole.
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  • Darrah H H, The cultivated basils, Independence (MO Buckeye Printing Company) 1980.

  • Gupta S K, Prakash J & Srivastava S, Validation of traditional claim of Tulsi, Ocimum sanctum Linn. as a medicinal plant, Indian J Exp Biol, 40 (2002) 765–73.

  • Singh N & Hoette Y, Tulsi: The mother medicine of nature, Lucknow, India: (International Institute of Herbal Medicine) 2002.

  • Uma Devi P, Radioprotective, anticarcinogenic and antioxidant properties of the Indian holy basil, Ocimum sanctum (Tulasi), Indian J Exp Biol, 39 (2001) 185–190.

  • Shishodia S, Majumdar S, Banerjee S & Aggarwal B B, Urosolic acid inhibits nuclear factor-kappaB activation induced by carcinogenic agents through suppression of Ikappa Balpha kinase and p65 phosphorylation: Correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1, Cancer Res, 63 (2003) 4375–83.

  • Pattanayak P, Behera P, Das D & Panda S K, Ocimum sanctum Linn. A reservoir plant for therapeutic applications: An overview, Pharmacogn Rev, 4 (2010) 95–105.

  • Khan S, Fahim N, Singh P & Rahman L, Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop, Ind Crops Prod, 71 (2015) 138–146.

  • Deschamps C & Simon J E, Agrobacterium tumefaciens-mediated transformation of Ocimum basilicum and O. citriodorum, Plant Cell Rep, 21 (2002) 359–364.

  • Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962) 473–497.

  • Khanuja S P S, Shasany A K, Darokar M P & Kumar S, Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils, Plant Mol Biol Rep, (1999) 1–7

  • Huang D & Dai W, Direct regeneration from in vitro leaf and petiole tissues of Populus tremula ‘Erecta’, PCTOC, 107 (2011) 69–174

  • Gawali P M, Akhare A A & Gahukar S J, In vitro regeneration of pigeonpea from leaf with petiole explants, Asian Sciences, 5 (2010) 34–38.

  • Kumar N & Reddy M P, Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant, Ann Appl Biol, 156 (2010) 367–375.

  • Kumar N, Anand K G V & Reddy M P, In vitro regeneration from petiole explants of non-toxic Jatropha curcas, Ind Crops Prod, 33 (2011) 146–151.

  • Bernula D, Benko P, Kaszler N, Domonkos I, Valkai I, Szollosi R, Ferenc G, Ayaydin F, Feher A, Gemes K, Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots, PCTOC, 140 (2020) 327–339.

  • Catterou M, Dubois F, Smets R, Vaniet S & Kichey T, Onckelen H V, Noreel B S S, Sangwan R S, hoc: an Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity, Plant J, 30 (2002) 273–287.

  • Kakimoto T, Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP Isopentenyltransferases, Plant Cell Physiol, 42 (2001) 677–685.

  • Sun J, Niu Q W, Tarkowski P, Zheng B, Tarkowska D & Sandberg G, Chua N H, Zuo J, The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis, Plant Physiol, 131 (2003) 167–176.

  • Cheng Z J, Wang L & Sun W, Zhang Y, Zhou C, Su Y H, LI W, Sun T T, Zhao X Y, Li X G, Cheng Y, Zhao Y, Xie Q, Zhang S X, Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of cytokinin biosynthesis by auxin response factor3, Plant Physiol, 161 (2013) 240–251.

  • Confalonieri M, Balestrazzi A, Bisoffi S & Carbonera D, In vitro culture and genetic engineering of Populus spp.: synergy for forest tree improvement, PCTOC, 72 (2003) 109–138.

  • Magyar-Tábori K, Dobránszki J, Teixeira da Silva J A, Bulley S M & Hudák I, The role of cytokinins in shoot organogenesis in apple, PCTOC, 101 (2010) 251–267

  • Kurepa J, Shull T E & Smalle J A, Antagonistic activity of auxin and cytokinin in shoot and root organs, Plant Direct, 3(2) (2019) 1–9.

  • Besnard F, Refahi Y, Morin V, Marteaux B, Brunoud G, Chambrier P & Vernoux T, Cytokinin signalling inhibitory fields provide robustness to phyllotaxis, Nature, 505 (2014) 417–421.

  • Chickarmane V S, Gordon S P, Tarr P T, Heisler M G, & Meyerowitz E M, Cytokinin signaling as a positional cue for patterning the apical-basal axis of the growing Arabidopsis shoot meristem, Proc Natl Acad Sci U.S.A, 109 (2012) 4002–4007.

  • Dello Ioio R, Nakamura K, Moubayidin L, Perilli S, Taniguchi M, Morita M T, & Sabatini S, A genetic framework for the control of cell division and differentiation in the root meristem, Science, 322 (2008) 1380–1384.

  • Truskina J & Vernoux T, The growth of a stable stationary structure: Coordinating cell behavior and patterning at the shoot apical meristem, Curr Opin Plant Biol, 41 (2018) 83–88.

  • Chisti N, Shawl A S, Sultan P, Kaloo Z A & Khan F A, In vitro organogenesis in Lavendula officinalis, a multipurpose plant of industrial importance, Proc Natl Semin, 2003, 14

  • Ikeuchi M, Sugimoto K & Iwase A, Plant callus: mechanisms of induction and repression, Plant Cell, 25 (2013) 3159–3173

  • Bustillo-Avendaño E, Ibáñez S, Sanz O, Barros J A S, Gude I, Perianez-Rodriguez J, Micol J L, Pozo J C D, Moreno-Risueno M A & Pérez-Pérez J M, Regulation of hormonal control, cell reprogramming, and patterning during DeNovo root organogenesis, Plant Physiol, 176 (2018) 1709–1727

  • Rahdari P, Khosroabadi M, Delfani K & Hoseini S M, Effect of different concentration of plant hormones (IBA and NAA) on rooting and growth factors in root and stem cuttings of Cordyline terminalis, J Med Bioeng, 3 (2014) 190–194.

  • Liu Y, Lu J, Zhu H, Li L, Shi Y & Yin X, Efficient culture protocol for plant regeneration from cotyledonary petiole explants of Jatropha curcas L., Biotechnol Biotechnol Equip, 30 (2016) 907–914.

  • Saha P, Datta K, Majumder S, Sarkar C, China S P, Sarkar S N, Sarkar D & Datta S K, Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv: JRC 321 using shoot tip explants, PCTOC, 118 (2014) 313–326

  • Jin S, Zhang X, Liang S, Nie Y, Guo X & Huang C, Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens, PCTOC, 81 (2005) 229–237

  • Sheikholeslam S N & Weeks D P, Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens, Plant Mol Biol, 8 (1987) 291–298

  • Janssen B & Gardner R C, Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium, Plant Mol Biol, 14 (1989) 61–72


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  • In vitro Direct Regeneration and Agrobacterium tumefaciens Mediated in planta Transformation of Ocimum sanctum L.

Abstract Views: 101  |  PDF Views: 56

Authors

Sana Khan
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, Uttar Pradesh, India
Zakir Husain
CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow 226 015, Uttar Pradesh, India
Laiq ur Rahman
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, Uttar Pradesh, India

Abstract


An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II (npt-II) & β-glucoronidase (uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Keywords


Gus-A, Kanamycin, MAPs, Npt-II, O. tenuiflorum, Petiole.

References