Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 120, Issue 2, pp 571–584 | Cite as

The apoplastic oxidative burst as a key factor of hyperhydricity in garlic plantlet in vitro

  • Jie Tian
  • Fangling Jiang
  • Zhen Wu
Original Paper


The phenomenon of hyperhydricity, a physiological disorder occurring frequently in tissue culture, causes ultrastructural modification and metabolic alteration of shoots. Reactive oxygen species (ROS) accumulation and oxidative stress induction are common features during the development of hyperhydricity, but the relationship between organelle redox homeostasis and hyperhydricity with ultrastructural abnormalities is unclear. To investigate the origin of oxidative stress-induced hyperhydricity, changes in oxygen metabolism in different subcellular compartments of garlic plantlets in vitro were studied. Under exogenous hydrogen peroxide (H2O2) stress, the chloroplastic and mitochondrial ultrastructure was disrupted, which was concomitant with aggravated frequency and severity of hyperhydricity. The addition of H2O2 to the growth medium enhanced superoxide anion generation and H2O2 content in the subcellular compartments. Accumulation of ROS was the highest in apoplasts. Compared with control shoots, in apoplasts exogenous H2O2 stimulated a sharp increase in superoxide dismutase activity within 4 days and a sharp increase in ascorbate peroxidase and glutathione reductase activities and in ascorbic acid and glutathione contents after 8 days of H2O2 treatment. In the other subcellular compartments, dramatic improvement of the antioxidant system occurred after 12 days. Thus, the apoplast was the most sensitive compartment among those investigated. Apoplastic ROS might play a signaling role to participate in the coordination of stress adaptation. The apoplastic oxidative burst in garlic plantlets in vitro is an early response to the development of hyperhydricity.


Garlic Plantlet in vitro Reactive oxygen species Subcellular compartments Hyperhydricity 



Ascorbate peroxidase


Ascorbic acid


Bovine serum albumin




5′,5′-Dithiobis-2-nitrobenzoic acid




Ethylene diamine tetraacetic acid


Ethylene glycol tetraacetic acid


Glutathione reductase




Oxidized glutathione


4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid


Hydrogen peroxide


Infiltrated washing fluid


Potassium iodide


β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt


Nitro blue tetrazolium


Superoxide anion


Singlet oxygen


Hydroxyl radical


Potential of hydrogen


Phenylmethanesulfonyl fluoride




Reactive oxygen species


Superoxide dismutase


Trichloroacetic acid



This work was supported by National Natural Science Foundation of China (31372056) and Doctoral Fund of Ministry of Education of China (200803071012).

Supplementary material

11240_2014_623_MOESM1_ESM.doc (74 kb)
Supplementary material 1 (DOC 74 kb)


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126PubMedCrossRefGoogle Scholar
  2. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  3. Arakawa N, Tsutsumi K, Sanceda NG, Kurata T, Inagaki C (1981) A rapid and sensitive method for the determination of ascorbic acid using 4,7-diphenyl-1,10-phenanthroline. Agric Biol Chem 45:1289–1290CrossRefGoogle Scholar
  4. Ayabe M, Sumi S (1998) Establishment of a novel tissue culture method, stem–disc culture, and its practical application to micropropagation of garlic (Allium sativum L.). Plant Cell Rep 17:773–779CrossRefGoogle Scholar
  5. Ayabe M, Sumi S (2001) A novel and efficient tissue culture method—“stem–disc dome culture”—for producing virus-free garlic (Allium sativum L.). Plant Cell Rep 20:503–507CrossRefGoogle Scholar
  6. Balen B, Tkalec M, Pavoković D, Pevalek-Kozlina B, Krsnik-Rasol M (2009) Growth conditions in in vitro culture can induce oxidative stress in Mammillaria gracilis tissues. J Plant Growth Regul 28:36–45CrossRefGoogle Scholar
  7. Bhattacharjee S (2005) Reactive oxygen species and oxidative burst: roles in stress, senescence and signal transduction in plants. Curr Sci India 89:1113–1121Google Scholar
  8. Blackman LM, Hardham AR (2008) Regulation of catalase activity and gene expression during phytophthroa nicotianae development and infection of tobacco. Mol Plant Pathol 9:495–510PubMedCrossRefGoogle Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  10. Cassells AC, Curry RF (2001) Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. Plant Cell Tiss Organ Cult 64:145–157CrossRefGoogle Scholar
  11. Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2005) Hyperhydricity in apple: ultrastuctural and physiological aspects. Tree Physiol 26:377–388CrossRefGoogle Scholar
  12. Chen J, Ziv M (2001) The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus. Plant Cell Rep 20:22–27CrossRefGoogle Scholar
  13. Dewir YH, Chakrabarty D, Ali MB, Hahn EJ, Paek KY (2006) Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots. Environ Exp Bot 58:93–99CrossRefGoogle Scholar
  14. Diaz-Vivancos P, Rubio M, Mesonero V, Periago PM, Ros Barceló A, Martínez-Gómez P, Hernández JA (2006) The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection. J Exp Bot 57:3813–3824PubMedCrossRefGoogle Scholar
  15. Fernandez-García N, Piqueras A, Olmos E (2008) Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation. Environ Exp Bot 62:168–175CrossRefGoogle Scholar
  16. Foyer CH, Noctor G (2009) Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxid Redox Signal 11:861–905PubMedCrossRefGoogle Scholar
  17. Franck T, Kevers C, Gaspar T (1995) Protective enzymatic systems against activated oxygen species compared in normal and vitrified shoots of Prunus avium L. raised in vitro. Plant Growth Regul 16:253–256CrossRefGoogle Scholar
  18. Franck T, Kevers C, Gaspar T, Dommes J, Deby C, Greimers R, Serteyn D, Deby-Dupont G (2004) Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response. Plant Physiol Biochem 42:519–527PubMedCrossRefGoogle Scholar
  19. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension culture of soybean root cells. Exp Cell Res 50:151–158PubMedCrossRefGoogle Scholar
  20. Giannopolitis CN, Ries SK (1977) Superoxide dismutases: occurrence in higher plants. Plant Physiol 59:309–314PubMedCentralPubMedCrossRefGoogle Scholar
  21. Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212PubMedCrossRefGoogle Scholar
  22. Hassannejad S, Bernard F, Mirzajani F, Gholami M (2012) SA improvement of hyperhydricity reversion in Thymus daenensis shoots culture may be associated with polyamines changes. Plant Physiol Biochem 51:40–46PubMedCrossRefGoogle Scholar
  23. Hu XL, Jiang MY, Zhang A, Lu J (2005) Abscisic acid-induced apoplastic H2O2 accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves. Planta 223:57–68PubMedCrossRefGoogle Scholar
  24. Huang YC, Chiang CH, Li CM, Yu TA (2010) Transgenic watermelon lines expressing the nucleocapsid gene of Watermelon silver mottle virus and the role of thiamine in reducing hyperhydricity in regenerated shoots. Plant Cell Tiss Organ Cult 106:21–29CrossRefGoogle Scholar
  25. Ivanova M, Staden J (2009) Natural ventilation effectively reduces hyperhydricity in shoot cultures of Aloe polyphylla Schönland ex Pillans. Plant Growth Regul 60:143–150CrossRefGoogle Scholar
  26. Ivanova M, Staden JV (2011) Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. Plant Cell Tiss Organ Cult 104:13–21CrossRefGoogle Scholar
  27. Jaspers P, Kangasjärvi J (2010) Reactive oxygen species in abiotic stress signaling. Physiol Plant 138:405–413PubMedCrossRefGoogle Scholar
  28. Kangasjärvi S, Kangasjärvi J (2014) Towards understanding extracellular ROS sensory and signaling systems in plants. Adv Bot 2014Google Scholar
  29. Kevers C, Frank T, Strasser RJ, Dommes J, Gaspar T (2004) Hyperhydricity of micropropagated shoots: a typically stress-induced change of physiological state. Plant Cell Tiss Organ Cult 77:181–191CrossRefGoogle Scholar
  30. Leadsham JE, Gourlay CW (2010) cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation. BMC Cell Biol 11:92PubMedCentralPubMedCrossRefGoogle Scholar
  31. Luciani GF, Mary AK, Pellegrini C, Curvetto NR (2006) Effects of explants and growth regulators in garlic callus formation and plant regeneration. Plant Cell Tiss Organ Cult 87:139–143CrossRefGoogle Scholar
  32. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410PubMedCrossRefGoogle Scholar
  33. Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, Gollery M, Shulaev V, Breusegem FV (2011) ROS signaling: the new wave? Trends Plant Sci 16:300–309PubMedCrossRefGoogle Scholar
  34. Møller IM, Sweetlove LJ (2010) ROS signalling-specificity is required. Trends Plant Sci 15:370–374PubMedCrossRefGoogle Scholar
  35. Munné-Bosch S, Alegre L (2003) Drought-induced changes in the redox state of α-tocopherol, ascorbate, and the diterpene carnosic acid in chloroplasts of Labiatae species differing in carnosic acid contents. Plant Physiol 131:1816–1825PubMedCentralPubMedCrossRefGoogle Scholar
  36. Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1–13PubMedCentralPubMedCrossRefGoogle Scholar
  37. Okada Y, Tanaka K, Fujita I, Sato E, Okajima H (2005) Antioxidant activity of thiosulfinates derived from garlic. Redox Rep 10:96–102PubMedCrossRefGoogle Scholar
  38. Olmos E, Hellín E (1998) Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants. Sci Hort 75:91–101CrossRefGoogle Scholar
  39. Park SW, Jeon JH, Kim HS, Park YM, Aswath C, Joung H (2004) Effect of sealed and vented gaseous microenvironments on the hyperhydricity of potato shoots in vitro. Sci Hort 99:199–205CrossRefGoogle Scholar
  40. Ramírez-Malagón R, Pérez-Moreno L, Borodanenko A, Salinas-González GJ, Ochoa-Alejo N (2006) Differential organ infection studies, potyvirus elimination, and field performance of virus-free garlic plants produced by tissue culture. Plant Cell Tiss Org Cult 86:103–110CrossRefGoogle Scholar
  41. Rojas-Martinez L, Visser RG, de Klerk GJ (2010) The hyperhydricity syndrome: waterlogging of plant tissues as a major cause. Propag Ornam Plants 10:169–175Google Scholar
  42. Saher S, Piqueras A, Hellin E, Olmos E (2005) Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling: implications of oxidative stress. Plant Cell Tiss Organ Cult 81:149–158CrossRefGoogle Scholar
  43. Sayyari M, Babalar M, Kalantari S, Serrano M, Valero D (2009) Effect of salicylic acid treatment on reducing chilling injury in stored pomegranates. Postharvest Biol Technol 53(3):152–154CrossRefGoogle Scholar
  44. Schaedle M, Bassham JA (1977) Chloroplast glutathione reductase. Plant Physiol 59:1011–1012PubMedCentralPubMedCrossRefGoogle Scholar
  45. Schwarzländer M, Finkemeier I (2013) Mitochondrial energy and redox signaling in plants. Antioxid Redox Signal 18:2122–2144PubMedCentralPubMedCrossRefGoogle Scholar
  46. Song XS, Tiao CL, Shi K, Mao WH, Ogweno JO, Zhou YH, Yu JQ (2006) The response of antioxidant enzymes in cellular organelles in cucumber (Cucumis sativus L.) leaves to methyl viologen-induced photo-oxidative stress. Plant Growth Regul 49:85–93CrossRefGoogle Scholar
  47. Sreedhar RV, Venkatachalam L, Neelwarne B (2009) Hyperhydricity-related morphologic and biochemical changes in Vanilla (Vanilla planifolia). J Plant Growth Regul 28:46–57CrossRefGoogle Scholar
  48. Tan MP, Lu J, Zhang AY, Hu B, Zhu XW, Li WB (2011) The distribution and cooperation of antioxidant (iso) enzymes and antioxidants in different subcellular compartments in maize leaves during water stress. J Plant Growth Regul 30:255–271CrossRefGoogle Scholar
  49. Taşkın H, Baktemur G, Kurul M, Büyükalaca S (2013) Use of tissue culture techniques for producing virus-free plant in garlic and their identification through real-time PCR. Sci World J. doi: 10.1155/2013/781282 Google Scholar
  50. Tattelman E (2005) Health effects of garlic. Am Fam Physician 72:103–106PubMedGoogle Scholar
  51. Uchida A, Jagendorf AT, Hibino T, Takabe T, Takabe T (2002) Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice. Plant Sci 163:515–523CrossRefGoogle Scholar
  52. van den Dries N, Giannì S, Czerednik A, Krens FA, de Klerk GJ (2013) Flooding of the apoplast is a key factor in the development of hyperhydricity. J Exp Bot 64:5221–5230PubMedCentralPubMedCrossRefGoogle Scholar
  53. 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 system. Plant Physiol 127:426–435PubMedCentralPubMedCrossRefGoogle Scholar
  54. Wang YL, Wang XD, Zhao B, Wang YC (2007) Reduction of hyperhydricity in the culture of Lepidium meyenii shoots by the addition of rare earth elements. Plant Growth Regul 52:151–159CrossRefGoogle Scholar
  55. Wang CQ, Zhang YF, Zhang YB (2008) Scavenger enzyme activities in subcellular fractions of white clover (Trifolium repens L.) under PEG-induced water stress. J Plant Growth Regul 27:387–393CrossRefGoogle Scholar
  56. Wrzaczek M, Brosché M, Kollist H, Kangasjärvi J (2009) Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS. Proc Natl Acad Sci USA 106:5412–5417PubMedCentralPubMedCrossRefGoogle Scholar
  57. Wrzaczek M, Brosché M, Salojärvi J, Kangasjärvi S, Idänheimo N, Mersmann S, Robatzek S, Karpiński S, Karpińska B, Kangasjärvi J (2010) Transcriptional regulation of the CRK/DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis. BMC Plant Biol 10:95PubMedCentralPubMedCrossRefGoogle Scholar
  58. Wu YX, von Tiedemann A (2002) Impact of fungicides on active oxygen species and antioxidant enzymes in spring barley (Hordeum vulgare L.) exposed to ozone. Environ Pollut 116:37–47PubMedCrossRefGoogle Scholar
  59. Wu Z, Chen LJ, Long YJ (2009) Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoot. In Vitro Cell Dev Biol-Plant 45:483–490CrossRefGoogle Scholar
  60. Zobayed SMA, Armstrong J, Armstrong W (2001) Micropropagation of potato: evaluation of closed, diffusive and forced ventilation on growth and tuberization. Ann Bot 87:53–59CrossRefGoogle Scholar
  61. Zurbriggen MD, Carrillo N, Hajirezaei MR (2010) ROS signaling in the hypersensitive response: when, where and what for? Plant Signal Behav 5:393–396PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.College of Horticulture, Nanjing Agricultural University/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East ChinaMinistry of AgricultureNanjingPeople’s Republic of China

Personalised recommendations