Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 129, Issue 3, pp 457–467 | Cite as

Anatomical and histological features of Ilex paraguariensis leaves under different in vitro shoot culture systems

  • C. V. LunaEmail author
  • A. M. Gonzalez
  • L. A. Mroginski
  • P. A. Sansberro
Original Article


Ilex paraguariensis: (Aquifoliaceae) is an evergreen tree traditionally used to prepare a caffeine-rich infusion that has several medicinal properties. The in vitro propagation of this species has been studied as an alternative to conventional methods such as cuttings and seedlings. The in vitro culture environment consists of closed flasks with high relative humidity, reduced gas exchange and low luminosity conditions, which may induce physiological, anatomical and histological disruption in the cultivated plants. The occurrence of anatomical and histological alterations to the leaves of I. paraguariensis was examined in plants cultivated under greenhouse and different in vitro conditions. The variations in the microclimate inside the culture vessels generated by the different treatments tested affected the anatomy and histology of I. paraguariensis leaves by causing alterations in their architecture, thickness, vascularization and stomatal differentiation. A temporary immersion system was the best treatment for conserving the anatomical and histological features of the leaves. This treatment led to the lowest stomatal index and an extensive system of intercellular spaces that were similar to the characteristics developed under external environment conditions. Consequently, more than 80% of rooted plantlet transferred to pots were successfully rusticated. Plants from temporary immersion had greater photosynthetic rates due to a higher stomatal conductance.


Micropropagation Temporary immersion Leaf anatomy Leaf histology Ilex paraguariensis 



This work was supported by grants from ANPCyT (PICTO 2011 − 0203, PICT 2014 − 1246), SGCyT-UNNE (PI A001/14), and Establecimiento Las Marías S.A.C.I.F.A. We extend our deep appreciation to anonymous reviewers for their critical comments. The authors are members of the Research Council of Argentina (CONICET).


  1. Akdemir H, Süzerer Y, Onay A, Tilkat E, Ersali Y, Ozden Çiftçi Y (2014) Micropropagation of the pistachio and its rootstocks by temporary immersion system. Plant Cell Tissue Org Cult 117:65–72. doi: 10.1007/s11240-013-0421-0 CrossRefGoogle Scholar
  2. Ash A, Ellis B, Hickey L, Johnson K, Wilf P, Wing P (1999) Manual of leaf architecture. Morphological description and categorization of dicotyledonous and net-veined monocotyledonous angiosperms. Smithsonian Institution, Washington, DCGoogle Scholar
  3. Baas P (1975) Vegetative anatomy and the affinities of Aquifoliaceae, Sphenostemon, Phelline, and Oncotheca. Blumea 22:311–407Google Scholar
  4. Caravita Abbade L, Duarte de Oliveira Paiva P, Paiva R, de Castro M, Rabelo Centofante A, de Oliveira C (2009) Anatomia foliar de ipê-branco (Tabebuia roseo alba (Ridl.) Sand.) – Bignoniaceae, proveniente do cultivo ex vitro e in vitro. Acta Sci-Biol Sci 31:307–311. doi: 10.4025/actascibiolsci.v31i3.1937 Google Scholar
  5. Carvalho Pereira D, Franca Barros C, Rubio Scarano F (2009) In situ variation in leaf anatomy and morphology of Andira legalis (Leguminosae) in two neighboring but contrasting light environments in a Brazilian sandy coastal plain. Acta Bot Bras 23:267–273. doi: 10.15446/abc.v21n1.47621 CrossRefGoogle Scholar
  6. Gago J, Martínez-Núñez L, Landín M, Flexas F, Gallego P (2014) Modeling the effects of light and sucrose on in vitro propagated plants: a multiscale system analysis using artificial intelligence technology. PLoS ONE 9:e85989. doi: 10.1371/journal.pone.0085989 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Gambero A, Ribeiro M (2015) The positive effects of yerba maté (Ilex paraguariensis) in obesity. Nutrients 7:730–750. doi: 10.3390/nu7020730 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gonzalez A, Cristóbal C (1997) Anatomía y ontogenia de semillas de Helicteres lhotzkyana (Sterculiaceae). Bonplandia 9:287–294Google Scholar
  9. Gottlieb A, Giberti G, Poggio L (2005) Molecular analyses of the genus Ilex (Aquifoliaceae) in South America, evidence from AFLP and its sequence data. Amer J Bot 92:352–369. doi: 10.3732/ajb.92.2.352.CrossRefGoogle Scholar
  10. Gugliucci A (1996) Antioxidant effects of Ilex paraguariensis: Induction of decreased oxidability of human ldl in vivo. Biochem Biophys Res Commun 224:338–344. doi: 10.1006/bbrc.1996.1030 CrossRefPubMedGoogle Scholar
  11. Hazarika B (2006) Morpho-physiological disorders in in vitro culture of plants. Sci Hortic 108:105–120. doi: 10.1016/j.scienta.2006.01.038 CrossRefGoogle Scholar
  12. Isah T (2015) Adjustments to in vitro culture conditions and associated anomalies in plants. Acta Biol Cracov Bot 57:9–28. doi: 10.1515/abcsb-2015-0026 Google Scholar
  13. Ishida A, Yazaki K, Hoe A (2005) Ontogenetic transition of leaf physiology and anatomy from seedlings to mature trees of a rain forest pioneer tree, Macaranga gigantea Tree Physiol 25:513–522. doi: 10.1093/treephys/25.5.513 CrossRefPubMedGoogle Scholar
  14. Jackson MB (2005) Aereation stress in plant tissue cultures. In: Hvoslef-Eide A, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, p 459–473. doi: 10.1007/1-4020-3200-5_35 CrossRefGoogle Scholar
  15. Khan P, Kozai T, Nguyen Q, Kubota C, Dhawan V (2003) Growth and water relations of Paulownia fortunei under photomixotrophic and photoautotrophic conditions. Biol Plant 46:161–166. doi: 10.1023/A:1022844720795 CrossRefGoogle Scholar
  16. Korn R, Frederick G (1973) Development of D-type stomata in the leaves of Ilex crenata var. crenata. Ann Bot 37:647–656. doi: 10.1093/oxfordjournals.aob.a084731 CrossRefGoogle Scholar
  17. Kozai T, Kubota C (2005) In vitro aerial environments and their effects on growth and development of plants. In: Kozai T, Afreen F, Zobayed S (eds) Photoautotrophic (sugar-free medium) micropropagation as a new micropropagation and transplant production system. Springer, Dordrecht, p 31–52. doi: 10.1007/1-4020-3126-2_4 CrossRefGoogle Scholar
  18. Lorenzo J, González J, Escalona M, Teisson C, Espinosa P, Borroto C (1998) Sugarcane shoot formation in an improved temporary immersion system. Plant Cell Tissue Org Cult 54:197–200CrossRefGoogle Scholar
  19. Luna C, Sansberro P, Mroginski L, Tarragó J (2003) Micropropagation of Ilex dumosa (Aquifoliaceae) from nodal segments in a tissue culture system. BioCell 27:205–212PubMedGoogle Scholar
  20. Luna C, Acevedo M, Gonzalez A, Collavino M, Mroginski L, Sansberro P (2013) Endophytic bacteria in Ilex paraguariensis shoot cultures: localization, characterisation, and control using isothiazolone biocides. In Vitro Cell Dev Biol—Plant 49:326–332. doi: 10.1007/s11627-013-9500-5 CrossRefGoogle Scholar
  21. McAlister B, Finnie J, Watt MP, Blakeway F (2005) Use of the temporary immersion bioreactor system (RITA®) for production of commercial Eucalyptus clones in mondi forests (SA). Plant Cell Tissue Org Cult 81:347–358. doi: 10.1007/s11240-004-6658-x CrossRefGoogle Scholar
  22. Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
  23. Paganini Stein FL, Schmidt B, Furlong EB, Souza-Soares LA, Soares MC, Vaz MR, Muccillo Baisch AL (2005) Vascular responses to extractable fractions of Ilex paraguariensis in rats fed standard and high-cholesterol diets. Biol Res Nurs 7:146–156. doi: 10.1177/1099800405280521 CrossRefPubMedGoogle Scholar
  24. Payne WW (1969) A quick method for clearing leaves. Ward’s Bulletin May, p 4–5Google Scholar
  25. Qi Z, Pic E, Zhanga X, Möllerd M, Jiange B, Lua H (2017) Ontogenesis of D-type stomata and cork-warts on the leaf epidermis of Camellia japonica (Theaceae) and functional assessment. Flora. doi: 10.1016/j.flora.2017.01.010 Google Scholar
  26. Rasband WS (1997–2016) ImageJ, U. S. National Institutes of Health, Bethesda.
  27. Reich P, Wright I, Lusk C (2007) Predicting leaf physiology from simple plant and climate attributes: a global GLOPNET analysis. Ecol Appl 17:1982–1988. doi: 10.1890/06-1803.1 CrossRefPubMedGoogle Scholar
  28. Robinson P, Britto J, Senthilkumar S (2009) Comparative anatomical studies on Emilia zeylanica C. B. Clarke with in vitro regenerated plants. Middle-East J Sci Res 4:140–143Google Scholar
  29. Sansberro PA, Rey HY, Bernardis AC, Luna CV, Collavino MM, Mroginski LA (2000) Plant regeneration of Ilex paraguariensis St. Hil. (Aquifoliaceae) by in vitro culture of nodal segments. BioCell 24:53–63PubMedGoogle Scholar
  30. Sansberro, PA, Rey, HY, Mroginski, LA, Krivenki MA (2001) Plant regeneration from Ilex spp. (Aquifoliaceae) in vitro. BioCell 25:139–146PubMedGoogle Scholar
  31. Spegazzini E, Castro M, Carpano S, Nájera M (2002) Taxonomic determination of therapeutic argentine species of Ilex. Pharm Biol 40:2–15. doi: 10.1076/phbi. CrossRefGoogle Scholar
  32. Tarragó J, Sansberro P, Filip R, López P, Gonzalez A, Luna C, Mroginski L (2005) Effect of leaf retention and flavonoids on rooting of Ilex paraguariensis cuttings. Sci Hortic 103:479–488. doi: 10.1016/j.scienta.2004.07.004 CrossRefGoogle Scholar
  33. Tarragó J, Filip R, Mroginski L, Sansberro P (2012) Influence of the irradiance on phenols content and rooting of Ilex paraguariensis cuttings collected from adult plants. Acta Physiol Plant 34:2419–2424. doi: 10.1007/s11738-012-1009-8 CrossRefGoogle Scholar
  34. Toma I, Toma C, Ghiorghita G (2004) Histo-anatomy and in vitro morphogenesis in Hyssopus officinalis L. Acta Bot Croat 63:59–68Google Scholar
  35. Vasco A, Thadeo M, Conover M, Daly DC (2014) Preparation of samples for leaf architecture studies, a method for mounting cleared leaves. Appl Plant Sci 2:1400038. doi: 10.3732/apps.1400038.CrossRefGoogle Scholar
  36. Vidal N, Blanco B, Cuenca B (2015) A temporary immersion system for micropropagation of axillary shoots of hybrid chestnut. Plant Cell Tissue Org Cult 123:229–243. doi: 10.1007/s11240-015-0827-y CrossRefGoogle Scholar
  37. Yang S, Yeh D (2008) In vitro leaf anatomy, ex vitro photosynthetic behaviors and growth of Calathea orbifolia (Linden) Kennedy plants obtained from semi-solid medium and temporary immersion systems. Plant Cell Tissue Org Cult 93:201–207. doi: 10.1007/s11240-008-9363-3 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • C. V. Luna
    • 1
    Email author
  • A. M. Gonzalez
    • 1
  • L. A. Mroginski
    • 1
  • P. A. Sansberro
    • 1
  1. 1.Facultad de Ciencias Agrarias (UNNE)Instituto de Botánica del Nordeste (CONICET)CorrientesArgentina

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