Antioxidant and anti-stress compounds improve regrowth of cryopreserved Rubus shoot tips

  • Esther E. Uchendu
  • Magfrat Muminova
  • Sandhya Gupta
  • Barbara M. Reed
Biotechnology

Abstract

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.

Keywords

Blackberry Lipoic acid Germplasm Glutathione Glycine betaine Long-term storage Oxidative stress Polyvinylpyrrolidone 

References

  1. Abdelwahd R.; Hakam N.; Labhilili M.; Udupa S. M. Use of an adsorbent and antioxidants to reduce the effects of leached phenolics in in vitro plantlet regeneration of faba bean. African J Biotech 7: 997–1002; 2008.Google Scholar
  2. Allard F.; Houde M.; Kröl M.; Ivanov A.; Huner N. P. A.; Sarhan F. Betaine improves freezing tolerance in wheat. Plant Cell Physiol 39: 1194–1202; 1998.Google Scholar
  3. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 144: 391–396; 2006.CrossRefGoogle Scholar
  4. Bast A.; Haenen G. R. M. M. Interplay between lipoic acid and glutathione in the protection against microsomal lipid. Biochim Biophys Acta 963: 558–561; 1988.PubMedGoogle Scholar
  5. Batkova P.; Pospisilova J.; Synkova H. Production of reactive oxygen and development of antioxidative systems during in vitro growth and ex vitro transfer. Biol Plant 52: 413–422; 2008.CrossRefGoogle Scholar
  6. Benson E. E. Do free radicals have a role in plant tissue culture recalcitrance? In Vitro Cell Dev Biol-Plant 36: 163–170; 2000.Google Scholar
  7. Biewenga G. P.; Haenen G. R. M. M.; Bast A. The pharmacology of the antioxidant lipoic acid. Gen Pharmacol 29: 315–331; 1997.PubMedGoogle Scholar
  8. Busse E.; Zimmer G.; Schopohl B.; Kornhuber B. Influence of α-lipoic acid on intracellular glutathione in vitro and in vivo, Arzneim. Arznei-Forschung 42: 829–831; 1992.Google Scholar
  9. Chang Y.; Reed B. M. Extended cold acclimation and recovery medium alteration improve regrowth of Rubus shoot tips following cryopreservation. CryoLetters 20: 371–376; 1999.Google Scholar
  10. Chen H. H. T.; Murata N. Glycinebetaine: an effective protectant against abiotic stress in plants. Trends Plant Sci 13: 499–505; 2008.CrossRefPubMedGoogle Scholar
  11. Dan Y.; Armstrong C. L.; Dong J.; Feng X.; Fry J. E.; Keithly G. E.; Roberts M. B. J.; LA GA S.; Tan L. J.; Duncan D. R. Lipoic acid—a unique plant transformation enhancer. In Vitro Cell Dev Biol-Plant 45: 630–638; 2009.CrossRefGoogle Scholar
  12. Day J. G.; Fleck R. A.; Benson E. E. Cryopreservation-recalcitrance in microalgae: novel approaches to identify and avoid cryo-injury. J Appl Phycol 12: 369–377; 2000.CrossRefGoogle Scholar
  13. Elstner EF, Osswald W (1994) Mechanisms of oxygen activation during plant stress. Proc Roy Soc Edinburgh 102Google Scholar
  14. Esterbauer H.; Grill D. Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol 61: 119–121; 1978.CrossRefPubMedGoogle Scholar
  15. Esterbauer H.; Schaur R. J.; Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radical Bio Med 11: 81–128; 1991.CrossRefGoogle Scholar
  16. Figueiredo S. F. L.; Albarello N.; Viana V. R. C. Micropropagation of Rollinia mucosa (Jacq) Baill. In Vitro Cell Dev Biol-Plant 37: 471–475; 2001.Google Scholar
  17. Foyer C. H.; Lopez-Delgado H.; Dat J. F.; Scott I. M. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signaling. Physiol Plant 100: 241–254; 1997.CrossRefGoogle Scholar
  18. Foyer C. H.; Noctor G. The molecular biology and metabolism of glutathione. In: Grill D.; Tausz M.; De Kok L. J. (eds) Significance of glutathione in plant adaptation to the environment. Kluwer Academic Publishers, Dordrecht, pp 27–57; 2001.Google Scholar
  19. Gupta S.; Reed B. M. Cryopreservation of shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification. CryoLetters 27: 29–42; 2006.PubMedGoogle Scholar
  20. Gupta S. D.; Datta S. Antioxidant enzyme activities during in vitro morphogenesis of Gladiolus and the effect of application of antioxidants on plant regeneration. Biol Plant 47: 179–183; 2003.CrossRefGoogle Scholar
  21. Halliwell B. Reactive species and antioxidants: redox biology is a fundamental theme of aerobic life. Plant Physiol 141: 312–322; 2006.CrossRefPubMedGoogle Scholar
  22. Ishitani M.; Takabe T.; Kojima K.; Takabe T. Regulation of glycinebetaine accumulation in the halotolerant cyanobacterium Aphanothece halophytica. Aust J Plant Physiol 20: 693–703; 1993.CrossRefGoogle Scholar
  23. Itai C.; Paleg L. G. Responses of water-stressed Hordeum distichum L. and Cucumis sativus to proline and betaine. Plant Sci Letters 25: 329–335; 1982.CrossRefGoogle Scholar
  24. Jocelyn PC(ed) (1972) Biochemistry of the SH Group the Occurrence, Chemical Properties, Metabolism and Biological Function of Thiols and Disulphides. Academic Press, New York NY, USAGoogle Scholar
  25. Johnson J. W.; Benson E. E.; Harding K. Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm. Plant Physiol Biochem 47: 123–131; 2009.CrossRefGoogle Scholar
  26. Jolivet Y.; Larher F.; Hamelin J. Osmoregulation in halophytic higher plants: the protective effect of glycinebetaine against the heat destabilization of membranes. Plant Sci Letters 25: 193–201; 1982.CrossRefGoogle Scholar
  27. Kagan V. E.; Shvedova A.; Serbinova E.; Khan S.; Swanson R.; Powell R.; Parker L. Dihydrolipoic acid-a universal antioxidant both in the membrane and in the aqueous phase. Biochem Pharmacol 44: 1637–1649; 1992.CrossRefPubMedGoogle Scholar
  28. Kytridis V. P.; Manetas Y. Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source. J Exp Bot 57: 2203–2210; 2006.CrossRefPubMedGoogle Scholar
  29. Lascano H. R.; Antonicelli G. E.; Luna C. M.; Melchiorre M. N.; Gomez L. D.; Racca R. W.; Trippi V. S.; Casano L. M. Antioxidant system response of different wheat cultivars under drought: field and in vitro studies. Aust J Plant Physiol 28: 1095–1102; 2001.Google Scholar
  30. Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 405–410; 2002.CrossRefPubMedGoogle Scholar
  31. Mittler R.; Vanderauwera S.; Gollery M.; van Breusegem F. Reactive oxygen gene network of plants. Trends Plant Sci 9: 490–498; 2004.CrossRefPubMedGoogle Scholar
  32. Møller I. M.; Jensen P. E.; Hansson A. Oxidative modifications of cellular components in plants. Annu Rev Plant Biol 58: 459–481; 2007.CrossRefPubMedGoogle Scholar
  33. Murashige T.; Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497; 1962.CrossRefGoogle Scholar
  34. Navari-Izzo F.; Quartacci M. F.; Sgherri C. Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species. Plant Physiol Biochem 40: 463–470; 2002.CrossRefGoogle Scholar
  35. Nishizawa A.; Yabuta Y.; Shigoeka S. Galactinol and raffinose constitute a novel function to protect plants from oxidative damage. Plant Physiol 147: 1251–1263; 2008.CrossRefPubMedGoogle Scholar
  36. Nomura K.; Matsumoto S.; Masuda K.; Inoue M. Reduced glutathione promotes callus growth and shoot development in a shoot tip culture of apple root stock M26. Plant Cell Rep 17: 597–600; 1998.CrossRefGoogle Scholar
  37. Packer L.; Tritschler H. J. Alpha-lipoic acid: the metabolic antioxidant. Free Radical Bio Med 20: 625–626; 1996.CrossRefGoogle Scholar
  38. Packer L.; Witt E. H.; Tritschler H. J. α-lipoic acid as biological antioxidant. Free Rad Biol Med 19: 227–250; 1995.CrossRefPubMedGoogle Scholar
  39. Packer L.; Witt E. H.; Tritschler H. J. Antioxidant properties and clinical applications of alpha-lipoic acid and dihydrolipoic acid. In: Cadenas E.; Parker L. (eds) Handbook of antioxidants. Dekker Marcel Inc, New York, pp 545–591; 1996.Google Scholar
  40. Papageorgiou G. C.; Murata N. The unusually strong stabilizing effects of glycinebetaine on the structure and function of the oxygen-evolving Photosystem II. Photosynth Res 44: 243–252; 1995.CrossRefGoogle Scholar
  41. Park E. J.; Jeknic Z.; Chen H. H. T. Exogenous application of glycinebetaine increases chilling tolerance in tomato plants. Plant Cell Physiol 47: 706–714; 2006.CrossRefPubMedGoogle Scholar
  42. Prajapati H. A.; Patel D. H.; Mehta S. R.; Subramanian R. B. Direct in vitro regeneration of Curculigo orchioides Gaertn., an endangered anticarcinogenic herb. Curr Sci 84: 747–749; 2003.Google Scholar
  43. Reed B. M. Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. CryoLetters 9: 166–171; 1988.Google Scholar
  44. Reed B. M. Responses to ABA and cold acclimation are genotype dependent for cryopreserved blackberry and raspberry meristems. Cryobiology 30: 179–184; 1993.CrossRefGoogle Scholar
  45. Reed BM (ed) (2008) Plant cryopreservation: a practical guide. Springer, New York NY, USA.Google Scholar
  46. Rhodes D.; Hanson A. D. Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol and Plant Mol Biol 44: 357–384; 1993.CrossRefGoogle Scholar
  47. Roach T.; Ivanova M.; Beckett R. P.; Minibayeva F. V.; Green I.; Pritchard H. W.; Kranner I. An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation. Physiol Plant 133: 131–139; 2008.CrossRefPubMedGoogle Scholar
  48. Sakai A.; Kobayashi S.; Oiyama I. Survival by vitrification of nucellar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) cooled to −196°C. J Plant Physiol 137: 465–470; 1991.Google Scholar
  49. SAS Statistical Software Version 9.1.3 (2007) SAS Institute, Inc. Cary NC, USAGoogle Scholar
  50. Saxena P. K.; Gill R. Removal of browning and growth enhancement by polyvinylpolypyrrolidone in protoplast cultures of Cyamopsis tetragonoloba L. Biologia Plant 28: 313–315; 1986.CrossRefGoogle Scholar
  51. Shalata A.; Neumann P. M. Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation. J Exp Bot 52: 2207–2211; 2001.PubMedGoogle Scholar
  52. Takahama U. Oxidation of vacuolar and apoplastic phenolics substrates by peroxidase: physiological significance of the oxidation reactions. Phytochem Rev 3: 207–219; 2004.CrossRefGoogle Scholar
  53. Tang W.; Harris L.; Outhavong V.; Newton R. J. Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (Pinus virginiana Mill.). Plant Cell Rep 22: 871–877; 2004.CrossRefPubMedGoogle Scholar
  54. Tyagi A. K.; Rashid A.; Maheshwari S. C. Promotive effect of polyvinylpolypyrrolidone on pollen embryogenesis in Datura innoxia. Physiol Plant 53: 405–606; 1981.CrossRefGoogle Scholar
  55. Uchendu E. E.; Leonard S. W.; Traber M. G.; Reed B. M. Vitamin E and C reduce lipid peroxidation and improve regrowth of blackberry shoot tips following cryopreservation. Plant Cell Rep 29: 25–35; 2010.CrossRefPubMedGoogle Scholar
  56. Valenzuela A. The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress. Life Sci 48: 301–309; 1991.CrossRefPubMedGoogle Scholar
  57. Valluru R.; Van den Ende W. Plant fructans in stress environments: emerging concepts and future prospects. J Exp Bot 59: 2905–2916; 2008.CrossRefPubMedGoogle Scholar
  58. Vysotskaya O. N.; Mochammed A. I.; Butenko R. G. Cryopreservation of red raspberry meristems (Rubus idaeus L.) isolated from in vitro plantlets. Biology Bulletin 26: 19–22; 1999.Google Scholar
  59. Wang Q.; Laamanen J.; Uosukainen M.; Valkonen J. P. T. Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation-vitrification and encapsulation-dehydration. Plant Cell Rep 24: 280–288; 2005.CrossRefPubMedGoogle Scholar
  60. Wang Z.; Deng X. Cryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione. CryoLetters 25: 43–50; 2004.PubMedGoogle Scholar
  61. Wu Z.; Chen L. J.; Long Y. J. Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoots. In Vitro Cell Dev Biol--Plant 45: 483–490; 2009.Google Scholar
  62. Zhang J. X.; Kirkham M. B. Lipid peroxidation in sorghum and sunflower seedling as affected by ascorbic acid, benzoic acid and propyl gallate. J Plant Physiol 149: 489–493; 1996.Google Scholar

Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  • Esther E. Uchendu
    • 1
    • 2
  • Magfrat Muminova
    • 3
  • Sandhya Gupta
    • 4
  • Barbara M. Reed
    • 5
  1. 1.Department of HorticultureOregon State UniversityCorvallisUSA
  2. 2.Department of Plant Agriculture, Ontario Agriculture CollegeUniversity of GuelphGuelphCanada
  3. 3.Department of BiotechnologyTashkent Chemical-Technology InstituteTashkentUzbekistan
  4. 4.Tissue Culture and Cryopreservation UnitNational Bureau of Plant Genetic ResourcesNew DelhiIndia
  5. 5.United States Department of Agriculture-Agricultural Research ServiceNational Clonal Germplasm RepositoryCorvallisUSA

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