Ectopic co-expression of the SOD and APX genes enhanced callus growth and in vitro regeneration in Arabidopsis

  • Amrina ShafiEmail author
  • Insha Zahoor
  • Tejpal Gill
  • Paramvir Singh Ahuja
  • Sanjay Kumar
  • Anil Kumar SinghEmail author
Original Article


Antioxidant enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), play important role in ROS homeostasis in plants. In the present study, two important antioxidant enzyme-encoding genes, cytosolic Cu/Zn-SOD and APX, were isolated from Potentilla atrosanguinea and Rheum australe plants, which grow at high-altitude regions of Himalaya. Previously, we have reported cytosolic overexpression of both the genes in Arabidopsis, individually and in combination and these transgenic plants exhibit cold and salt stress tolerance. In the present study, wild-type (WT) and transgenic lines (cytosolic Cu/Zn-SOD and APX) were analysed for their regeneration potential and expression profiling of various genes involved in in vitro regeneration was carried out. Among all transgenic lines, dual transgenics showed early callus induction and shoot regeneration. Callus growth rate and in vitro regeneration capacity were significantly higher in transgenic lines compared with control plants. Interestingly, H2O2 accumulation and SOD activity were found to be higher in SOD and dual transgenic lines during callus induction and shoot regeneration stages, indicating a correlation between H2O2 and SOD activity with regeneration process. Whereas APX activity in transgenic lines was found to be decreased in regenerated shoots, cotyledons, it was increased in callus and roots. Further, expression analysis of several genes involved in callus induction and in vitro regeneration using qRT-PCR showed that the majority of genes were significantly up-regulated (two- to fourfold) during different stages of regeneration in transgenic lines. Consequently, our results substantiate that a minimal amount of H2O2 accumulation brought about by overexpression of SOD and APX genes may play an important role in early callus induction and shoot regeneration in transgenic line. The overall results will add knowledge about the role of antioxidant genes in in vitro regeneration of plants.


Antioxidants Growth kinetics In vitro regeneration SOD APX Hydrogen peroxide 



This work was supported by Grants from the Council of Scientific and Industrial Research (CSIR), New Delhi, India, under CSIR Network Projects: SIMPLE (BSC0109) and Pla-Gen (BSC0107) and Indo-German Science and Technology Centre (IGSTC), India. A.S. and T.G. acknowledge fellowships awarded by the CSIR, India.

Author contributions

PSA, SK and AKS designed research; AS and TG performed research; IZ did the statistical analysis; AS, IZ and AKS analysed data; AS, IZ, and AKS participated in writing the whole manuscript. The authors declare that they have no conflict of interest.


This work was supported by Ministry of Science, ICT and Future Planning (Grant no. NRF-2017R1A2B4012820).

Supplementary material

11816_2019_535_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 15 kb)


  1. Abbasi BH, Khan M, Guo B, Bokhari SA, Khan MA (2011) Efficient regeneration and antioxidative enzyme activities in Brassica rapa var. turnip. Plant Cell Tissue Organ Cult 105:337–344CrossRefGoogle Scholar
  2. Asada K (1999) The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Ann Rev Plant Physiol Plant Mol Biol 50:601–639CrossRefGoogle Scholar
  3. Banno H, Ikeda Y, Niu QW, Chua NH (2001) Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell 13:2609–2618CrossRefGoogle Scholar
  4. Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci 316:1194–1199Google Scholar
  5. Benson EE (2000) Do free radicals have a role in plant tissue culture recalcitrance? Vitro Cell Deve Biol-Plant 36:163–170CrossRefGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 72:248–254CrossRefGoogle Scholar
  7. Che P, Gingerich DJ, Lall S, Howell SH (2002) Global and cytokinin related gene expression changes during shoot development in Arabidopsis. Plant Cell 14:2771–2785CrossRefGoogle Scholar
  8. Che P, Lall S, Nettleton D, Howell HS (2006) Gene expression programs during shoot, root, and callus development in Arabidopsis tissue culture. Plant Physiol 141:620–637CrossRefGoogle Scholar
  9. Filipovic BK, Simonovic AD, Trifunovic MM, Dmitrovic SS, Save JM, Jevremovic SB, Subotic AR (2015) Plant regeneration in leaf culture of Centaurium erythraea Rafn. Part 1: the role of antioxidant enzymes. Plant Cell Tissue Organ Cult 121:703–719CrossRefGoogle Scholar
  10. Gantait S, Mandal N, Das PK (2011) In vitro accelerated mass propagation and ex vitro evaluation of Aloe vera L. with aloin content and superoxide dismutase activity. Nat Product Res 25(14):1370–1378CrossRefGoogle Scholar
  11. Gautheret RJ (1966) Factors affecting differentiation of plant tissue grown in vitro. In: Beerman W, Nieuwkoop PD, Wolff E (eds) Cell differentiation and morphogenesis. North-Holland Publishing, Amsterdam, pp 55–95Google Scholar
  12. Gill T, Sreenivasulu Y, Kumar S, Ahuja PS (2010) Over-expression of superoxide dismutase exhibits lignifications of vascular structures in Arabidopsis thaliana. J Plant Physiol 167:757–760CrossRefGoogle Scholar
  13. Hicks GS (1980) Patterns of organ development in plant tissue culture and the problem of organ determination. Bot Rev 46:1–23CrossRefGoogle Scholar
  14. IkedaY Banno H, Niu QW, Howell SH, Chua NH (2006) The ENHANCER OF SHOOT REGENERATION 2 gene in Arabidopsis regulates CUP-SHAPED COTYLEDON 1 at the transcriptional level and controls cotyledon development. Plant Cell Physiol 47:1443–1456CrossRefGoogle Scholar
  15. Ikeuchi M, Sugimoto K, Iwase A (2013) Plant callus: mechanisms of induction and repression. Plant Cell 25:3159–3173CrossRefGoogle Scholar
  16. Jana S, Shekhawat GS (2012) In vitro regeneration of Anethum graveolens, antioxidative enzymes during organogenesis and RAPD analysis for clonal fidelity. Biol Plant 56:9–14CrossRefGoogle Scholar
  17. Kairong C, Gengsheng X, Xinmin L, Gengmei X, Yafu W (1999) Effect of hydrogen peroxide on somatic embryogenesis of Lycium barbarum L. Plant Sci 146:9–16CrossRefGoogle Scholar
  18. Kakimoto T (1996) CKI1, a histidine kinase homolog implicated in cytokinin signal transduction. Science 274:982–985CrossRefGoogle Scholar
  19. Konieczny R, Banas AK, Surowka E, Michalec Z, Miszalski Z, Libik-Konieczny M (2014) Pattern of antioxidant enzyme activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of Mesembryanthemum crystallinum L. Plant Cell Rep 33:165–177CrossRefGoogle Scholar
  20. Libik M, Konieczny R, Pater B, Slésak I, Miszalski Z (2005) Differences in the activities of some antioxidant enzymes and in H2O2 content during rhizogenesis and somatic embryogenesis in callus cultures of the ice plant. Plant Cell Rep 23:834–841CrossRefGoogle Scholar
  21. Meratan AA, Ghaffari SM, Niknam V (2009) In vitro organogenesis and antioxidative enzymes activity in Acanthophyllum sordidum. Biol Plant 53:5–10CrossRefGoogle Scholar
  22. Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33:453–467CrossRefGoogle Scholar
  23. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410CrossRefGoogle Scholar
  24. Molassiotis AN, Dimassi K, Diamantidis G, Therios I (2004) Changes in peroxidases and catalase activity during in vitro rooting. Biol Plant 48:1–5CrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate- specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880Google Scholar
  27. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST@) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl Acids Res 30:e36CrossRefGoogle Scholar
  28. Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha R (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110CrossRefGoogle Scholar
  29. Rout JR, Sahoo SL (2013) In vitro propagation and antioxidant enzymes activities of Elephantopus scaber L. Asia-Pac J Mol Biol Biotechnol 21(2):59–66Google Scholar
  30. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386Google Scholar
  31. Shafi A, Dogra V, Gill T, Ahuja PS, Sreenivasulu Y (2014) Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications. PLoS One 9:e110302CrossRefGoogle Scholar
  32. Shafi A, Chauhan R, Gill T, Swarnkar MK, Sreenivasulu Y, Kumar S, Kumar N, Shankar R, Ahuja PS, Singh AK (2015a) Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress. Plant Mol Biol 87:615–631CrossRefGoogle Scholar
  33. Shafi A, Gill T, Sreenivasulu Y, Kumar S, Ahuja PS, Singh AK (2015b) Improved callus induction, shoot regeneration, and salt stress tolerance in Arabidopsis overexpressing superoxide dismutase from Potentilla atrosanguinea. Protoplasma 252:41–51CrossRefGoogle Scholar
  34. Shi LZ, Wang R, Huang G, Vogel P, Neale G (2011) HIF1a–dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells. J Exp Med 208:1367–1376CrossRefGoogle Scholar
  35. Sonja V, Noctor G, Foyer CH (2002) Are leaf hydrogen peroxide concentrations commonly overestimated? The potential influence of artefactual interference by tissue phenolics and ascorbate. Plant Physiol Biochem 40:501–507CrossRefGoogle Scholar
  36. Tang W, Newton RJ (2005) Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos. Plant Physiol Biochem 43:760–769CrossRefGoogle Scholar
  37. Tian M, Gu Q, Zhu M (2003) The involvement of hydrogen peroxide and antioxidant enzymes in the process of shoot organogenesis of strawberry callus. Plant Sci 165:701–707CrossRefGoogle Scholar
  38. Tubic L, Savic J, Mitic N, Milojevic J, Janosevic D, Budimir S, Zdravkovic-Korac S (2016) Cytokinins differentially affect regeneration, plant growth and antioxidative enzymes activity in chive (Allium schoenoprasum L.). Plant Cell Tissue Organ Cult 124:1–14CrossRefGoogle Scholar
  39. Valvekens D, Van Montagu M, Lijsebettens MV (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540CrossRefGoogle Scholar
  40. Yang HP, Cheng JC, Zhou JY, Li GC, Zhou ZY (1993) Change of superoxide dismutase activities during somatic embryogenesis in Asparagus officinalis L. Acta Bot Sin 35:490–493Google Scholar

Copyright information

© Korean Society for Plant Biotechnology 2019

Authors and Affiliations

  • Amrina Shafi
    • 1
    • 2
    • 6
    Email author
  • Insha Zahoor
    • 3
  • Tejpal Gill
    • 4
  • Paramvir Singh Ahuja
    • 1
    • 2
  • Sanjay Kumar
    • 1
    • 2
  • Anil Kumar Singh
    • 1
    • 2
    • 5
    Email author
  1. 1.Department of BiotechnologyCSIR-Institute of Himalayan Bioresource TechnologyPalampurIndia
  2. 2.Academy of Scientific and Innovative ResearchNew DelhiIndia
  3. 3.Bioinformatics CentreUniversity of KashmirSrinagarIndia
  4. 4.National Institute of Arthritis and Musculoskeletal and Skin DiseasesNational Institute of HealthBethesdaUSA
  5. 5.ICAR-Indian Institute of Agricultural BiotechnologyRanchiIndia
  6. 6.Biotechnology DepartmentUniversity of KashmirSrinagarIndia

Personalised recommendations