, Volume 255, Issue 3, pp 741–750 | Cite as

Histological characterization of Passiflora pohlii Mast. root tips cryopreserved using the V-Cryo-plate technique

  • Mariela J. Simão
  • Myriam Collin
  • Renata O. Garcia
  • Elisabeth Mansur
  • Georgia Pacheco
  • Florent Engelmann
Original Article


Cryopreservation stands out as the main strategy to ensure safe and cost efficient long-term conservation of plant germplasm, especially for biotechnological materials. However, the injuries associated with the procedure may result in structural damage and low recovery rates after cooling. Histological analysis provides useful information on the effects of osmotic dehydration, LN exposure, and recovery conditions on cellular integrity and tissue organization, allowing the determination of the critical steps of the cryopreservation protocol and, thus, the use of optimized treatments. Passiflora pohlii Mast. (Passifloraceae) is a native species from Brazil with potential agronomic interest. Recent studies showed the presence of saponins in its roots, which presented antioxidant activity. The goal of this work was to develop a cryopreservation technique for root tips of in vitro-derived plants of P. pohlii using the V-Cryo-plate technique and to characterize the anatomical alterations that occurred during the successive steps of the protocol. Root tips were excised from in vitro plants and precultured before adhesion to cryo-plates and then treated for different periods with the plant vitrification solutions PVS2 or PVS3. Treatment with PVS2 for 45 min resulted in higher recovery (79%) when compared with PVS3 (43%). The greatest number of adventitious roots per cryopreserved explant was also observed after a 45-min exposure to PVS2. Plasmolysis levels were higher in cortical cells of cryopreserved explants treated with PVS2, while pericycle and central cylinder cells were not damaged after this treatment. Thirty days after rewarming, no plasmolysis could be detected, regardless of the experimental conditions.


Cryopreservation Cytological features Passiflora Plasmolysis 



The authors acknowledge Marc Lartaud and Jean-Luc Verdeil (Histology and Plant Imagery Platform, CIRAD, Montpellier, France) for the instructions on the use of ImageJ software and for discussion of the histology results and to the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—Programa Ciência sem Fronteiras) for the research fellowships and financial support.


  1. Antognoni F, Zheng S, Pagnucco C et al (2007) Induction of flavonoid production by UV-B radiation in Passiflora quadrangularis callus cultures. Fitoterapia 78:345–352. CrossRefPubMedGoogle Scholar
  2. Barraco G, Sylvestre I, Iapichino G, Engelmann F (2013) Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species. Plant Biotechnol Rep 7:141–146. CrossRefGoogle Scholar
  3. Benson EE (1990) Free radical damage in stored plant germplasm. IBPGR, RomeGoogle Scholar
  4. Benson EE, Hamill JD (1991) Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica. Plant Cell Tissue Organ Cult 24:163–172CrossRefGoogle Scholar
  5. Buffard-Morel J, Verdeil JL, Pannetier C (1992) Embryogenèse somatique du cocotier (Cocos nucifera L.) à partir d’explants foliaires: étude histologique. Can J Bot 70:735–741CrossRefGoogle Scholar
  6. Dhawan K, Dhawan S, Sharma A (2004) Passiflora: a review update. J Ethnopharmacol 94:1–23. CrossRefPubMedGoogle Scholar
  7. Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol—Plant 47:5–16. CrossRefGoogle Scholar
  8. Fisher DB (1968) Protein sustaining of ribboned epon sections for light microscopy. For Hist 16:92–96Google Scholar
  9. Hughes ZE, Malajczuk CJ, Mancera RL (2013) The effects of cryosolvents on DOPC−β-sitosterol bilayers determined from molecular dynamics simulations. J Phys Chem B 117:3362–3375. CrossRefPubMedGoogle Scholar
  10. Hughes ZE, Mancera RL (2014) Molecular mechanism of the synergistic effects of vitrification solutions on the stability of phospholipid bilayers. Biophys J 106:2617–2624. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Junqueira NTV, Braga MF, Faleiro FG et al (2005) Potencial de espécies silvestres de maracujazeiro como fonte de resistência a doenças. In: Faleiro FG, Junqueira NTV, Braga MF (eds) Maracujá: Germoplasma e Melhoramento Genético. Embrapa Cerrados, Planaltina, DF, pp 81–108Google Scholar
  12. Kim HH, Lee SC (2012) Personalisation of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects. Cryo-Lett 33:271–279Google Scholar
  13. Kim HH, Popova EV, Shin DJ et al (2012) Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (Nakai) hairy roots using a systematic approach. Cryo-Lett 33:506–517Google Scholar
  14. Kulus D, Zalewska M (2014) Cryopreservation as a tool used in long-term storage of ornamental species—a review. Sci Hortic 168:88–107. CrossRefGoogle Scholar
  15. Monteiro ACBA, Higashi EN, Gonçalves AN, Rodriguez APM (2000) A novel approach for the definition of the inorganic medium components for micropropagation of yellow passionfruit (Passiflora edulis sims. F. Flavicarpa Deg.) In Vitro Cell Dev Biol—Plant 36:527–531. CrossRefGoogle Scholar
  16. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  17. Murch SJ, Peiris SE, Liu C-Z, Saxena PK (2004) In vitro conservation and propagation of medicinal plants. Biodiversitas 5:19–24. CrossRefGoogle Scholar
  18. Nan ON, Hocher V, Verdeil J et al (2008) Cryopreservation by encapsulation -dehydration of plumules of coconut (Cocos nucifera L.) Cryo-Lett 29:339–350Google Scholar
  19. Niino T, Matsumoto T, Yamamoto SI et al (2017) Manual of cryopreservation methods using cryo-plate. Impresso, JaliscoGoogle Scholar
  20. Niino T, Yamamoto SI, Fukui K et al (2013) Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plates. Cryo-Lett 34:549–560Google Scholar
  21. Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci 91:67–73. CrossRefGoogle Scholar
  22. Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55. CrossRefGoogle Scholar
  23. Ramachandra Rao S, Ravishankar G (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153. CrossRefGoogle Scholar
  24. Rahmah S, Mubbarakh SA, Ping KS, Subramaniam S (2015) Effects of droplet-vitrification cryopreservation based on physiological and antioxidant enzyme activities of Brassidium Shooting Star orchid. Sci World J.
  25. Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. Cryo-Lett 28:151–172Google Scholar
  26. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33. CrossRefPubMedGoogle Scholar
  27. Salma M, Engelmann-Sylvestre I, Collin M et al (2013) Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots. Protoplasma 251:649–659. CrossRefPubMedGoogle Scholar
  28. Simão MJ, Fonseca E, Garcia R et al (2016) Effects of auxins and different culture systems on the adventitious root development of Passiflora pohlii Mast. and their ability to produce antioxidant compounds. Plant Cell Tissue Organ Cult 124:419–430. CrossRefGoogle Scholar
  29. Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary metabolites. Phytochem Rev 1:13–25. CrossRefGoogle Scholar
  30. Volk GM, Caspersen AM (2007) Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha × piperita. Protoplasma 231:215–226CrossRefPubMedGoogle Scholar
  31. Yamamoto S, Rafique T, Priyantha WS et al (2011) Development of a cryopreservation procedure using aluminium cryo-plates. Cryo-Lett 32:256–265Google Scholar
  32. Yap LV, Noor NM, Clyde MM, Chin HF (2011) Cryopreservation of Garcinia cowa shoot tips by vitrification: the effects of sucrose preculture and loading treatment on ultrastructural changes in meristematic cells. Cryo-Lett 32:188–196Google Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2017

Authors and Affiliations

  • Mariela J. Simão
    • 1
  • Myriam Collin
    • 2
  • Renata O. Garcia
    • 1
  • Elisabeth Mansur
    • 1
  • Georgia Pacheco
    • 1
  • Florent Engelmann
    • 2
  1. 1.Núcleo de Biotecnologia Vegetal, Instituto de Biologia Roberto Alcantara GomesUniversidade do Estado do Rio de JaneiroRio de JaneiroBrazil
  2. 2.DIADE, IRD, CIRAD, CNRS, Université de MontpellierMontpellierFrance

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