Skip to main content
SpringerLink
Log in

Study of the antioxidant enzymatic system during shoot development from cultured intercalar meristems of saffron

  • Original paper
  • Published:
Plant Growth Regulation Aims and scope Submit manuscript

Abstract

As preliminary research to develop a clonal in vitro propagation system for saffron, we have evaluated the morphogenic response of intercalary meristems present in shoots from sprouting saffron corms when cultured in media with different cytokinins. The best results in terms of shoot production and quality were achieved in the medium supplemented with 5 mg/l of benzylaminopurine (BAP). During the multiplication of the meristems, several changes in the activities of the antioxidant enzymatic system were detected. Catalase (CAT) activity was reduced in the presence of the assayed cytokinins, whereas peroxidase (POX) activity was stimulated with all the cytokinins assayed. When TDZ (thidizuron) was used, a slight decrease in superoxide dismutase (SOD) activity occurred, whereas BAP and 2iP (2-isopentenyl adenine) treatments produced a significant increase in SOD activity. The ascorbate–glutathione cycle enzymes showed a similar pattern in response to the addition of cytokinins. These results suggest a possible use of antioxidant enzyme activities as a parameter that could be use to evaluate the efficiency of micropropagation protocols for saffron and even in other species.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

2iP:

2-isopentyladenin

APX:

Ascorbate peroxidase (EC 1.11.1.1)

BAP:

6-benzylaminopurine

CAT:

Catalase (EC 1.11.1.6)

DHAR:

Dehydroascorbate reductase (EC 1.8.5.1)

GR:

Glutathione reductase (EC 1.6.4.2)

MDHAR:

Monodehydroascorbate reductase (EC 1.6.5.4)

POX:

Peroxidases (EC 1.11.1.7)

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase (EC 1.15.1.1)

TDZ:

Thidiazuron

References

  • Aebi H (1984) Catalase in vitro. In: Lester P (ed) Methods in enzymology oxygen radicals in biological systems. Academic Press, London, pp 121–126

    Chapter  Google Scholar 

  • Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 53:1331–1341

    Article  PubMed  CAS  Google Scholar 

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Ann Rev Plant Biol 55:373–399

    Article  CAS  Google Scholar 

  • 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 50:601–639

    Article  CAS  Google Scholar 

  • Barba-Espin G, Diaz-Vivancos P, Clemente-Moreno MJ, Albacete A, Faize L, Faize M, Pérez-Alfocea F, Hernández JA (2010) Interplay between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant Cell Environ 33:981–994

    Article  PubMed  CAS  Google Scholar 

  • Batkova P, Pospisilova J, Synkova H (2008) Production of reactive oxygen species and development of antioxidative ayatems during in vitro growth and ex vitro transfer. Biol Plant 52:413–422

    Article  CAS  Google Scholar 

  • Benson EE (2000) Do free radicals have a role in plant tissue culture recalcitrance? In Vitro Cell Dev Biol-Plant 36:163–170

    CAS  Google Scholar 

  • Benson EE, Lynch PT, Jones J (1992) Variation in free-radical damage in rice cell-suspensions with different embryogenic potentials. Planta 188:296–305

    Article  CAS  Google Scholar 

  • Blázquez S, Olmos E, Hernandez JA, Fernandez-Garcia N, Fernandez JA, Piqueras A (2009) Somatic embryogenesis in saffron (Crocus sativus L) histological differentiation and implication of some components of the antioxidant enzymatic system. Plant Cell Tissue Organ Culture 97:49–57

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Cakmak I, Strbac D, Marschner H (1993) Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds. J Exp Bot 44:127–132

    Article  CAS  Google Scholar 

  • Chahota RK, Dhiman KC, Rana SS, Singh M (2003) Efficacy of different propagating methods for higher daughter corm production in saffron (Crocus sativus L). Indian Perfumer 47:155–158

    Google Scholar 

  • Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2006) Hyperhydricity in apple: ultrastructural and physiological aspects. Tree Physiol 26:377–388

    Article  PubMed  CAS  Google Scholar 

  • Chen J, Ziv M (2001) The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus. Plant Cell Rep 20:22–27

    Article  Google Scholar 

  • Decker EL, Frank W, Sarnighausen E, Reski R (2006) Moss systems biology en route: phytohormones in physcomitrella development. Plant Biol 8:397–400

    Article  PubMed  CAS  Google Scholar 

  • Del Rio LA, Pastori GM, Palma JM, Sandalio LM, Sevilla F, Corpas FJ, Jiménez A, Lopez-Huertas E, Hernandez JA (1998) The activated oxygen role of peroxisomes in senescente. Plant Physiol 116:1195–1200

    Article  PubMed  CAS  Google Scholar 

  • Diaz-Vivancos P, Barba-Espin G, Clemente-Moreno MJ, Hernandez JA (2010) Characterization of the antioxidant system during the vegetative development of pea plants. Biol Plant 54:76–82

    Article  CAS  Google Scholar 

  • Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28:1056–1071

    Article  CAS  Google Scholar 

  • Gupta D (2010) Role of free radicals and antioxidants in in vitro morphogenesis. In: Gupta D (ed) Reactive oxygen species and antioxidants in higher plants. CRC Press, pp 230–247

  • Gupta SD, Datta S (2003) Antioxidant enzyme activities during in vitro morphogenesis of gladiolus and the effect of application of antioxidants on plant regeneration. Biol Plant 47:179–183

    Article  CAS  Google Scholar 

  • Hernandez JA, Jiménez A, Mullineaux PM, Sevilla F (2000) Tolerance of pea (Pisum sativum L) to long-term salt stress is associated with induction of antioxidant defenses. Plant Cell Environ 23:853–862

    Article  CAS  Google Scholar 

  • Hernandez JA, Ferrer MA, Jimenez A, Ros-Barcelo A, Sevilla F (2001) Antioxidant systems and O2 −/H2O2 production in the apoplast of Pisum sativum L leaves: its relation with NaCl-induced necrotic lesions in minor veins. Plant Physiol 127:817–831

    Article  PubMed  CAS  Google Scholar 

  • Hernandez JA, Escoba C, Creissen G, Mullineaux PM (2004) Role of hydrogen peroxide and the redox state of ascorbate in the induction of antioxidant enzymes in pea leaves under excess light stress. Func Plant Biol 31:359–368

    Article  CAS  Google Scholar 

  • Imin N, Nizamidin M, Daniher D, Nolan KE, Rose RJ, Rolfe BG (2005) Proteomic analysis of somatic embryogenesis in medicago truncata explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments. Plant Physiol 137:1250–1260

    Article  PubMed  CAS  Google Scholar 

  • Jimenez A, Hernandez JA, del Rio LA, Sevilla F (1997) Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol 114:275–284

    PubMed  CAS  Google Scholar 

  • Jirage D, Ravishankar G, Suvarnlatha G, Venkataraman L (1994) Production of polyamines during sprouting and growth of saffron (Crocus sativus L) corms. Plant Growth Regul 13:69–72

    Article  CAS  Google Scholar 

  • Kataeva NV, Alexandrova IG, Butenko RG, Dragavtceva EV (1991) Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro. Plant Cell Tissue Organ Cul 27:149–154

    Article  CAS  Google Scholar 

  • Kawaoka A, Matsunaga E, Endo S, Kondo S, Yoshida K, Shinmyo A, Ebinuma H (2003) Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen. Plant Physiol 132:1177–1185

    Article  PubMed  CAS  Google Scholar 

  • Kwak JM, Nguyen V, Schroeder JI (2006) The role of reactive oxygen species in hormonal responses. Plant Physiol 141:323–329

    Article  PubMed  CAS  Google Scholar 

  • Lai Q-X, Bao Z-Y, Zhu Z-J, Qian Q-Q, Mao B-Z (2007) Effect of osmotic stress on antioxidant enzymes activities in leaf discs of PSAG12-IPT modified gerbera. J Zhejiang Univ Sci B 8:458–464

    Article  PubMed  CAS  Google Scholar 

  • Majourhat K, Fernandez JA, Martinez-Gomez P, Piqueras A (2007) Enhanced plantlet regeneration from cultured meristems in sprouting buds of saffron corms. Acta Hortic 739:275–278

    Google Scholar 

  • Mathew B (1982) The Crocus, a revision of the genus Crocus (Iridaceae). BT Batsford, London

    Google Scholar 

  • McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymic function for erythrocuprein. J Biol Chem 244:6049–6055

    PubMed  CAS  Google Scholar 

  • Molassiotis AN, Dimassi K, Diamantidis G, Therios I (2004) Changes in peroxidases and catalase activity during in vitro rooting. Biol Plant 48:1–5

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3):473–497

    Article  CAS  Google Scholar 

  • Negbi M, Dagan B, Dror A, Basker D (1989) Growth, flowering, vegetative reproduction, and dormancy in the saffron crocus (Crocus sativus L). Israel J Botany 38:95–113

    Google Scholar 

  • Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279

    Article  CAS  Google Scholar 

  • Obert B, Benson EE, Millam S, Pretovb A, Bremner DH (2005) Moderation of morphogenetic and oxidative stress responses in flax in vitro cultures by hydroxynonenal and desferrioxamine. J Plant Physiol 162:537–547

    Article  PubMed  CAS  Google Scholar 

  • Olmos E, Piqueras A, Martinez-Solano JR, Hellin E (1997) The subcellular localization of peroxidase and the implication of oxidative stress in hyperhydrated leaves of regenerated carnation shoots. Plant Sci 130:97–105

    Article  CAS  Google Scholar 

  • Pasternak TP, Potters G, Caubergs R, Jansen MAK (2005) Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ and cellular level. J Exp Bot 58:1991–2001

    Article  Google Scholar 

  • Piqueras A, Fernandez JA (2004) Phase change from dormancy to continuous shoot proliferation in cultured meristems of saffron corms. In Vitro Cell Dev Biol-Animal 40:74A

    Article  Google Scholar 

  • Piqueras A, Han BH, Van Huylenbroeck JM, Debergh PC (1998) Effect of different environmental conditions in vitro on sucrose metabolism and antioxidant enzymatic activities in cultured shoots of Nicotiana. Plant Growth Regul 25:5–10

    Article  CAS  Google Scholar 

  • Piqueras A, Han BH, Escribano J, Rubio C, Hellin E, Fernandez JA (1999) Development of cormogenic nodules and microcorms by tissue culture, a new tool for the multiplication and genetic improvement of saffron. Agronomie 19:603–610

    Article  Google Scholar 

  • Plessner O, Ziv M, Negbi M (1990) In vitro corm production in the saffron crocus (Crocus sativus L). Plant Cell Tissue Organ Culture 20:89–94

    Article  Google Scholar 

  • Pomar F, Caballero N, Pedreño MA, Ros Barceló A (2002) H2O2 generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis. FEBS Lett 529:198–202

    Article  PubMed  CAS  Google Scholar 

  • Quorin M, Lepoivre P (1977) Etude de milieux adaptes aux cultures in vitro de Prunus. Acta Hort 78:437–442

    Google Scholar 

  • Romano CP, Hein MB, Klee HJ (1991) Inactivation of auxin in tobacco transformed with the indolacetic acid-lysine synthetase gene of Pseudomonas savastoni. Genes Dev 5:446–538

    Article  Google Scholar 

  • Ros Barcelo A (1998) The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme. Planta 207:207–216

    Article  Google Scholar 

  • Ros Barceló A, Gómez-Ros LV, Ferrer MA, Hernández JA (2006) The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees. Trees 20:145–156

    Article  Google Scholar 

  • Saher S, Piqueras A, Hellin E, Olmos E (2004) Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress. Physiol Plant 120:152–161

    Article  PubMed  CAS  Google Scholar 

  • Stephens JM (2003) Saffron-Crocus sativus L IFAS extension. Horticultural Sheets 661:2p

    Google Scholar 

  • Synkova H, Semoradova S, Schnablova R, Witters E, Husak M, Valcke R (2006) Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny. Biol Plant 50:31–41

    Article  CAS  Google Scholar 

  • 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 43:760–769

    Article  PubMed  CAS  Google Scholar 

  • Tang W, Harris LC, Outhavong V, Newton RJ (2004) Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (Pinus virginiana Mill). Plant Cell Rep 22:871–877

    Article  PubMed  CAS  Google Scholar 

  • Vatankhah E, Niknam V, Ebrahimzadeh H (2010) Activity of antioxidant enzyme during in vitro organogenesis in Crocus sativus. Biologia Plantarum 54(3):509–514

    Article  CAS  Google Scholar 

  • Veljovic-Jovanovic SD, Pignocchi C, Noctor G, Foyer CH (2001) Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant. Plant Physiol 127:426–435

    Article  PubMed  CAS  Google Scholar 

  • Werner T, Schmülling T (2009) Citokinin action in plant development. Current Opinion Plant Biol 12:527–538

    Article  CAS  Google Scholar 

  • Yeung EC, Law SK (1987) Serial sectioning techniques for a modified Lkb historesin. Stain Technol 62:147–153

    PubMed  CAS  Google Scholar 

  • Zavaleta-Mancera HA, Lopez-Delgado H, Loza-Tavera H, Mora-Herrera M, Trevilla-Garcia C, Vargas-Suarez M, Ougham H (2007) Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence. J Plant Physiol 164:1572–1582

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research has been mainly supported by the projects PII1I09-0029-9367 (Consejería de Educación y Ciencia, JCCM, Spain), RF2004-0032-C03, PET2007-014-C05-03 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, MEC, Spain), and AGRI GEN RES 018 Action ‘‘Genetic Resources of Saffron and Allies’’ (CROCUSBANK).

Author information

Authors and Affiliations

  1. Department of Plant Breeding, CEBAS-CSIC, PO Box 164, 30100, Espinardo, Murcia, Spain

    Pedro Díaz-Vivancos, José Antonio Hernández & Abel Piqueras

  2. Faculté des Sciences Semlalia, University Cadi-Ayyad of Marrakech, BP 2390, 40000, Marrakech, Morocco

    Khalid Majourhat

  3. Department of Biotechnology, IDR-University of Castilla-La Mancha, Campus Universitario de Albacete s/n, 02071, Albacete, Spain

    José Antonio Fernández

Authors
  1. Pedro Díaz-Vivancos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Khalid Majourhat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José Antonio Fernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. José Antonio Hernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Abel Piqueras
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pedro Díaz-Vivancos.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Díaz-Vivancos, P., Majourhat, K., Fernández, J.A. et al. Study of the antioxidant enzymatic system during shoot development from cultured intercalar meristems of saffron. Plant Growth Regul 65, 119–126 (2011). https://doi.org/10.1007/s10725-011-9581-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10725-011-9581-2

Keywords

Search

Navigation