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Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

  • Genetics and Genomics
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Abstract

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.

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Abbreviations

ABA:

abscisic acid

BA:

6-benzylaminopurine

DMSO:

dimethyl sulfoxide

LN:

liquid nitrogen

SSR:

simple sequence repeats

VNTR:

variable number of tandem repeats

References

  • Ahuja S, Mandal BB, Dixit S, Srivastava PS (2002) Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips. Plant Sci 163:971–977

    Article  CAS  Google Scholar 

  • Anker L, Gopalakrishna R, Jones K, Law R, Couldwell W (1995) Hypericin in adjuvant brain tumor therapy. Drugs Fut 20:511–517

    Google Scholar 

  • Bajaj YPS (1995) Cryopreservation of plant germplasm I. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 32. Springer-Verlag, Berlin, pp 398–416

    Google Scholar 

  • Čellárová E, Kimáková K, Brutovská R (1992) Multiple shoot formation and phenotypic changes of R0 regenerants in Hypericum perforatum L. Acta Biotechnol 12:445–452

    Article  Google Scholar 

  • Chang YJ, Reed BM (2001) Preculture conditions influence cold hardiness and regrowth of Pyrus cordata shoot tips after cryopreservation. HortScience 36:1329–1333

    CAS  Google Scholar 

  • Channuntapipat C, Sedgley M, Collins G (2003) Changes in methylation and structure of DNA from almond tissues during in vitro culture and cryopreservation. J Am Soc Hort Sci 128:890–897

    CAS  Google Scholar 

  • Chen T, Li PH, Brenner M (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol 71:362–365

    PubMed  CAS  Google Scholar 

  • Chetverikova EP (1999) Role of abscisic acid in frost tolerance of plants and cryopreservation of cultured tissues. Russ J Plant Physiol 46:721–727

    CAS  Google Scholar 

  • D'Amato F (1985) Cytogenetics of plant cell and tissue cultures and their regenerates. In: Conger BV (ed) Critical review of plant science. CRC Press, Inc. Boca Raton, Florida, pp 73–112

    Google Scholar 

  • Diwu Z (1995) Novel therapeutic and diagnostic applications of hypocrellins and hypericins. Photochem Photobiol 61:529–539

    PubMed  CAS  Google Scholar 

  • Dixit S (2001) In vitro regeneration and cryopreservation of Asian yams and sweet potato. PhD thesis, Jamia Hamdard, New Delhi.

  • Engelmann F (1997) In vitro conservation methods. In: Ford-Lloyd BV, Newburry JH, Callow JA (eds) Biotechnology and plant genetic resources: conservation and use. CABI, UK, pp 119–161

    Google Scholar 

  • Gagliardi RF, Pacheco GP, Carniero LA, Valls JFM, Vieira MLC, Mansur E (2003) Cryopreservation of Arachis species by vitrification of in vitro grown shoot apices and genetic stability of recovered plants. Cryo-Lett 24:103–110

    CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  PubMed  CAS  Google Scholar 

  • Haberer G, Fischer TC, Torres-Ruiz RA (1996) Mapping of the nucleolus organizer region on chromosome 4 in Arabidopsis thaliana. Mol Gen Genet 250:123–128

    PubMed  CAS  Google Scholar 

  • Hao YJ, Liu QL, Deng XX (2001) Effect of cryopreservation on apple genetic resources at morphological, chromosomal, and molecular levels. Cryobiology 43:46–53

    Article  PubMed  CAS  Google Scholar 

  • Hao YJ, You CX, Deng XX (2002a) Effects of cryopreservation on developmental competency, cytological and molecular stability of citrus callus. Cryo-Lett 23:27–35

    Google Scholar 

  • Hao YJ, You CX, Deng XX (2002b) Analysis of ploidy and the patterns of amplified fragment lenght polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation. Cryo-Lett 23:37–46

    Google Scholar 

  • Harding K (2004) Genetic integrity of cryopreserved plant cells: A review. Cryo-Lett 25:3–22

    Google Scholar 

  • Harding K, Benson EE (1994) A study of growth, flowering and tuberization in plants derived from cryopreserved potato shoot-tips: implications for in vitro germplasm collections. Cryo-Lett 15:59–66

    Google Scholar 

  • Harding K, Marzalina M, Krishnapillay B, Nashatul-Zaimah NA, Normah MN, Benson EE (2000) Molecular stability assessments of trees regenerated from cryopreserved mahogany (Swietenia macrophylla) seed germplasm using non- radioactive techniques to examine the chromatin structure and DNA methylation status of the ribosomal RNA genes. J Trop For Sci 12:149–163

    Google Scholar 

  • Helliot B, Madur D, Dirlewanger E, De Boucaud MT (2002) Evaluation of genetic stability in cryopreserved Prunus. In vitro Cell Dev Biol-Plant 38:493–500

    Article  CAS  Google Scholar 

  • Jeffreys AJ, Wilson V, Thein SL (1985) Hypervariable “minisatellite” regions in human DNA. Nature 314:67–73

    Article  PubMed  CAS  Google Scholar 

  • Kimáková K (1995) Effect of cryopreservation on cell function changes in callus culture of Matricaria recutita (L.) Rauschert and genetic stability of Hypericum perforatum L. meristems. Dissertation thesis (in Slovak), Košice, pp 117

  • Kim HH, Cho EG, Baek HJ, Kim CY, Keller ERJ, Engelmann F (2004) Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions. Cryo-Lett 25:59–70

    Google Scholar 

  • Kirakosyan A, Sirvent TM, Gibson DM, Kaufman PB (2004) The production of hypericins and hyperforin by in vitro cultures of St. John's wort (Hypericum perforatum). Biotechnol Appl Biochem 39:71–81

    PubMed  CAS  Google Scholar 

  • Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127

    Article  CAS  Google Scholar 

  • Liu YG, Wang XY, Liu LX (2004) Analysis of genetic variation in surviving apple shoots following cryopreservation by vitrification. Plant Sci 166:677–685

    Article  CAS  Google Scholar 

  • Mazur P (1984) Freezing of living cells: mechanisms and implications. Am J Physiol 247:C125–C142

    PubMed  CAS  Google Scholar 

  • Moges AD, Shibli RA, Karam NS (2004) Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips. In vitro Cell Dev Biol-Plant 40: 389–395

    Article  CAS  Google Scholar 

  • Moran M, Cacho M, Fernandez-Tarrago J, Corchete P (1999) A protocol for cryopreservation of Digitalis thapsi L. cell cultures. Cryo-Lett 20:193–198

    Google Scholar 

  • Murín G (1960) Substitution of cellophane for glass covers to facilitate preparation of permanent squashes and smears. Stain Technol 35:351–353

    PubMed  Google Scholar 

  • Murray MJ, Haldeman BA, Grant FJ, O'Hara PJ (1988) Probing the human genome with minisatellite-like sequences from the human coagulation factor-VII gene. Nucl Acids Res 16:4166

    Article  PubMed  CAS  Google Scholar 

  • Na HY, Kondo K (1996) Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Sci 118:195–201

    Article  CAS  Google Scholar 

  • Nakamura Y, Leppert M, O'Connel P, Wolff R, Holm T, Culver M, Martin C, Fujimoto E, Hoff M, Kumlin E, White R (1987) Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235:1616–1622

    Article  PubMed  CAS  Google Scholar 

  • Panis B, Lambardi M (2005) Status of cryopreservation technologies in plants (crops and forest trees). In: Electronic forum on biotechnology in food and agriculture: Conference 13 on the role of biotechnology and conservation of crop, forest, animal and fishery genetic resources in developing countries (http://www.fao.org/biotech/C13doc.htm)

  • Reed BM, Chang Y (1997) Medium and long-term storage of in vitro cultures of temperate fruit and nut crops. In: Razdan MK, Cocking EC (eds) Conservation of Plant Genetic Resources in vitro, vol 1. General aspects Enfield: Science Publishers, pp 67–105

  • Ryynanen L (1998) Effect of abscisic acid, cold hardening, and photoperiod on recovery of cryopreserved in vitro shoot tips of silver birch. Cryobiology 36:32–39

    Article  PubMed  CAS  Google Scholar 

  • Sakai A (1985) Cryopreservation of shoot-tips of fruit trees and herbaceous plants. In: Kartha KK (ed) Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, Florida, pp 135–158

    Google Scholar 

  • Schempp CM, Pelz K, Wittmer A, Schopf E, Simon JC (1999) Antibacterial activity of hyperforin from St. John's wort, against multiresistant Staphylococcus aureus and gram-positive bacteria. Lancet 353:2129

    Article  PubMed  CAS  Google Scholar 

  • Smolenskasym G, Gawronska H, Kacperska A (1995) Modifications of abscisic-acid level in winter oilseed rape leaves during acclimation of plants to freezing temperatures. Plant Growth Reg 17:61–65

    CAS  Google Scholar 

  • Tanaka D, Niino T, Isuzugawa K, Hikage T, Uemura M (2004) Cryopreservation of shoot apices of in vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols. Cryo-Lett 25:167–176

    Google Scholar 

  • Urbanová M, Čellárová E, Kimáková K (2002) Chromosome number stability and mitotic activity of cryopreserved Hypericum perforatum L. meristems. Plant Cell Rep 20:1082–1086

    Article  CAS  Google Scholar 

  • Veisz O, Galiba G, Sutka J (1996) Effect of abscisic acid on the cold hardiness of wheat seedlings. J Plant Physiol 149:439–443

    CAS  Google Scholar 

  • Wang YL, Fan MJ, Liaw SI (2005) Cryopreservation of in vitro-grown shoot tips of papaya (Carica papaya L.) by vitrification. Bot Bull Acad Sin 46:29–34

    Google Scholar 

  • Winberg BC, Zhou Z, Dallas JF, McIntype CL, Gustafson JP (1993) Characterization of minisatellite sequences from Oryza sativa. Genome 36:978–983

    Article  PubMed  CAS  Google Scholar 

  • Yoshimatsu K, Yamaguchi H, Shimomura K (1996) Traits of Panax ginseng hairy roots after cold storage and cryopreservation. Plant Cell Rep 15:555–560

    Article  CAS  Google Scholar 

  • Závodná M, Kraic J, Paglia G, Gregová E, Morgante M (2000) Differentiation between closely related lentil (Lens culinaris MEDIK.) cultivars using DNA markers. Seed Sci Technol 28:217–219

    Google Scholar 

  • Zhai ZY, Wu YJ, Engelmann F, Chen RZ, Zhao YH (2003) Genetic stability assessments of plantlets regenerated from cryopreserved in vitro cultured grape and kiwi shoot-tips using RAPD. Cryo-Lett 24:315–322

    Google Scholar 

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Acknowledgements

This work was supported by grant APVT-20-003704 and VEGA 1/2326/05. Authors would like to thank Bc. Ľudmila Majsniarová, Mrs. Zdenka Lacková and Mrs. Alžbeta Maková for their technical assistance.

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Authors and Affiliations

  1. Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University, Mánesova 23, 04154, Košice, Slovakia

    Martina Urbanová, Ján Košuth & Eva Čellárová

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  1. Martina Urbanová
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  2. Ján Košuth
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  3. Eva Čellárová
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Correspondence to Eva Čellárová.

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Communicated by K. Toriyama

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Urbanová, M., Košuth, J. & Čellárová, E. Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation. Plant Cell Rep 25, 140–147 (2006). https://doi.org/10.1007/s00299-005-0050-0

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  • DOI: https://doi.org/10.1007/s00299-005-0050-0

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