Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 123, Issue 1, pp 121–132 | Cite as

Impacts of photoautotrophic and photomixotrophic conditions on in vitro propagated Billbergia zebrina (Bromeliaceae)

  • João Paulo Rodrigues Martins
  • Veerle Verdoodt
  • Moacir Pasqual
  • Maurice De Proft
Original Article


Micro-propagation techniques contribute to the multiplication of several bromeliad species. However, micropropagated plantlets often present low survival rate due to anatomical and physiological disorders induced by in vitro conditions. This study aimed to evaluate the sucrose and gas exchange impact on in vitro propagated Billbergia zebrina plants and to check if there is any residual effect of the in vitro conditions on micropropagated plants after acclimatization. Previously in vitro-established B. zebrina plants were transferred to culture media containing 0.0, 15.0, 30.0, 45.0 or 60.0 g L−1 sucrose. Two different culture container sealing systems were tested: lids with a filter (permitting an excellent gas exchange) and a filter covered with PVC (blocking fluent gas exchange). At 45 days in vitro growth, B. zebrina plantlets were transplanted onto plastic pots containing peat and cultivated for 80 days in greenhouse. At 45 days in vitro and 80 days of acclimatization in the greenhouse, the plants were evaluated. High sucrose levels in the in vitro media resulted in reduced growth. Plantlets exposed to aerated containers presented better rooting, being the sugar-free medium the best in vitro condition (photoautotrophic condition). Limited air exchange resulted in plantlets with anatomical and physiological disorders at the end of the in vitro period. The highest growth rate in the greenhouse was observed in plants previously propagated in unlimited gas exchange system and sugar-free medium. The use of photoautotrophic conditions induces B. zebrina plantlets without anatomical and physiological disorders and it interfere positively on ex vitro growth.


Bromeliad In vitro plant Plant anatomy Photoautotrophic growth Tissue culture 



The authors would like to thank the CAPES (Brazil) for the financial support as a scholarship granted to João P. R. Martins during his sandwich Ph.D. They also thank to Katholieke Universiteit Leuven for the technical support.


  1. Aragón C, Carvalho L, González J, Escalona M, Amancio S (2012) The physiology of ex vitro pineapple (Ananas comosus L. Merr. var MD-2) as CAM or C3 is regulated by the environmental conditions. Plant Cell Rep 31:757–769. doi: 10.1007/s00299-011-1195-7 CrossRefPubMedGoogle Scholar
  2. Bao C, Wang J, Zhang R, Zhang B, Zhang H, Zhou Y, Huang S (2012) Arabidopsis VILLIN2 and VILLIN3 act redundantly in sclerenchyma development via bundling of actin filaments. Plant J 71:962–975. doi: 10.1111/j.1365-313X.2012.05044.x CrossRefPubMedGoogle Scholar
  3. Barboza SBSC, Graciano-Ribeiro D, Teixeira JB, Portes TA, Souza LAC (2006) Anatomia foliar de plantas micropropagadas de abacaxi. Pesqui Agropecu Bras 41:185–194. doi: 10.1590/S0100-204X2006000200002 CrossRefGoogle Scholar
  4. Bresta P, Nikolopoulos D, Economou G, Vahamidis P, Lyra D, Karamanos A, Karabourniotis G (2011) Modification of water entry (xylem vessels) and water exit (stomata) orchestrates long term drought acclimation of wheat leaves. Plant Soil 347:179–193. doi: 10.1007/s11104-011-0837-4 CrossRefGoogle Scholar
  5. Carvalho V, Santos DS, Nievola CC (2014) In vitro storage under slow growth and ex vitro acclimatization of the ornamental bromeliad Acanthostachys strobilacea. S Afr J Bot 92:39–43. doi: 10.1016/j.sajb.2014.01.011 CrossRefGoogle Scholar
  6. Cha-um S, Chanseetis C, Chintakovid W, Pichakum A, Supaibulwatana K (2011) Promoting root induction and growth of in vitro macadamia (Macadamia tetraphylla L. ‘Keaau’) plantlets using CO2-enriched photoautotrophic conditions. Plant Cell Tissue Organ Cult 106:435–444. doi: 10.1007/s11240-011-9940-8 CrossRefGoogle Scholar
  7. Cui XH, Murthy HN, Wu CH, Paek KY (2010) Sucrose-induced osmotic stress affects biomass, metabolite, and antioxidant levels in root suspension cultures of Hypericum perforatum L. Plant Cell Tissue Organ Cult 103:7–14. doi: 10.1007/s11240-010-9747-z CrossRefGoogle Scholar
  8. De Proft MP, Maenen LJ, Debergh PC (1985) Carbon dioxide and ethylene evolution in the culture atmosphere of Magnolia cultured in vitro. Physiol Plant 65:373–379. doi: 10.1111/j.1399-3054.1985.tb08660.x CrossRefGoogle Scholar
  9. Deccetti SFC, Soares AM, Paiva R, Castro EM (2008) Effect of the culture environment on stomatal features, epidermal cells and water loss of micropropagated Annona glabra L. plants. Sci Hortic 117:341–344. doi: 10.1016/j.scienta.2008.05.020 CrossRefGoogle Scholar
  10. Dias MC, Pinto G, Guerra C, Jesus C, Amaral J, Santos C (2013) Effect of irradiance during acclimatization on content of proline and phytohormones in micropropagated Ulmus minor. Biol Plant 57:769–772. doi: 10.1007/s10535-013-0341-1 CrossRefGoogle Scholar
  11. Dias GMG, Soares JDR, Pasqual M, Silva RAL, Rodrigues LCA, Pereira FJ, Castro EM (2014a) Photosynthesis and leaf anatomy of Anthurium cv. Rubi plantlets cultured in vitro under different silicon (Si) concentrations. Aust J Crop Sci 8:1160–1167Google Scholar
  12. Dias MC, Correia C, Moutinho-Pereira J, Oliveira H, Santos C (2014b) Study of the effects of foliar application of ABA during acclimatization. Plant Cell Tissue Organ Cult 117:213–224. doi: 10.1007/s11240-014-0434-3 CrossRefGoogle Scholar
  13. Eckstein A, Zieba P, Gabrys H (2012) Sugar and light effects on the condition of the photosynthetic apparatus of Arabidopsis thaliana cultured in vitro. J Plant Growth Regul 31:90–101. doi: 10.1007/s00344-011-9222-z CrossRefGoogle Scholar
  14. Ferreira WM, Suzuki RM, Pescador R, Figueiredo-Ribeiro RCL, Kerbauy GB (2011) Propagation, growth, and carbohydrates of Dendrobium Second Love (Orchidaceae) in vitro as affected by sucrose, light, and dark. In Vitro Cell Dev Biol Plant 47:420–427. doi: 10.1007/s11627-010-9311-x CrossRefGoogle Scholar
  15. Freschi L, Takahashi CA, Cambui CA, Semprebom TR, Cruz AB, Mioto PT, Versieux LM, Calvente A, Latansio-Aidar SR, Aidar MPM, Mercier H (2010) Specific leaf areas of the tank bromeliad Guzmania monostachia perform distinct functions in response to water shortage. J Plant Physiol 167:526–533. doi: 10.1016/j.jplph.2009.10.011 CrossRefPubMedGoogle Scholar
  16. Fuentes G, Talavera C, Desjardins Y, Santamaria JM (2005a) High irradiance can minimize the negative effect of exogenous sucrose on the photosynthetic capacity of in vitro grown coconut plantlets. Biol Plant 49:7–15. doi: 10.1007/s10535-005-7015-6 CrossRefGoogle Scholar
  17. Fuentes G, Talavera C, Opereza C, Desjardins Y, Santamaria J (2005b) Exogenus sucrose can decrease in vitro photosynthesis but improve field survival and growth of coconut (Cocos nucifera L.) in vitro plantlets. In Vitro Cell Dev Biol Plant 41:69–76. doi: 10.1079/IVP2004597 CrossRefGoogle Scholar
  18. Guerra MP, Vesco LLD (2010) Strategies for the micropropagation of bromeliads. In: Jain SM, Ochatt SJ (eds) Protocols for in vitro propagation of ornamental plants: methods in molecular biology, v.589. Humana Press, New York, pp 47–66. doi: 10.1007/978-1-60327-114-1_6 CrossRefGoogle Scholar
  19. Hameed M, Ashraf M, Naz N, Nawaz T, Batool R, Ahmad MSA, Ahmad F, Hussain M (2013) Anatomical adaptations of Cynodon dactylon (l.) Pers. from the salt range (Pakistan) to salinity stress. II. Leaf anatomy. Pak J Bot 45:133–142Google Scholar
  20. Hazarika BN (2003) Acclimatization of tissue-cultured plants. Curr Sci 85:1704–1712Google Scholar
  21. Hazarika BN (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
  22. Iarema L, Cruz ACF, Saldanha CW, Dias LLC, Vieira RF, Oliveira EJ, Otoni WC (2012) Photoautotrophic propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tissue Organ Cult 110:227–238. doi: 10.1007/s11240-012-0145-6 CrossRefGoogle Scholar
  23. Ivanova M, Staden JV (2010) Natural ventilation effectively reduces hyperhydricity in shoot cultures of Aloe polyphylla Schönland ex Pillans. Plant Growth Regul 60:143–150. doi: 10.1007/s10725-009-9430-8 CrossRefGoogle Scholar
  24. Jo EA, Tewari RK, Hahn EJ, Paek KY (2009) In vitro sucrose concentration affects growth and acclimatization of Alocasia amazonica plantlets. Plant Cell, Tissue Organ Cult 96:307–315. doi: 10.1007/s11240-008-9488-4 CrossRefGoogle Scholar
  25. Johansen DA (1940) Plant microtechnique, 2a edn. Mc Graw-Hill, New York, p 523Google Scholar
  26. Kitaya Y, Ohmura Y, Kubota C, Kozai T (2005) Manipulation of the culture environment on in vitro air movement and its impact on plantlets photosynthesis. Plant Cell Tissue Organ Cult 83:251–257. doi: 10.1007/s11240-005-6839-2 CrossRefGoogle Scholar
  27. Kozai T (2010) Photoautotrophic micropropagation—environmental control for promoting photosynthesis. Prop Ornam Plants 10:188–204Google Scholar
  28. Kozai T, Kubota C (2001) Developing a photoautotrophic micropropagation system for woody plants. J Plant Res 114:525–537. doi: 10.1007/PL00014020 CrossRefGoogle Scholar
  29. Kurita FMK, Tamaki V (2014) In vitro growth of the bromeliad Alcantarea imperialis (Carrière) Harms with different concentrations of nitrogen. Acta Sci Biol Sci 36:279–285. doi: 10.4025/actascibiolsci.v36i3.22933 CrossRefGoogle Scholar
  30. Mantovani A, Venda AKL, Almeida VR, Costa AF, Forzza RC (2012) Leaf anatomy of Quesnelia (Bromeliaceae): implications for the systematics of core bromelioids. Plant Syst Evol 298:787–800. doi: 10.1007/s00606-012-0590-z CrossRefGoogle Scholar
  31. Martins JPR, Schimildt ER, Alexandre RS, Santos BR, Magevski GC (2013) Effect of synthetic auxins on in vitro and ex vitro bromeliad rooting. Pesqui Agropec Trop 43:138–146. doi: 10.1590/S1983-40632013000200009 CrossRefGoogle Scholar
  32. Martins JPR, Schimildt ER, Alexandre RS, Castro EM, Nani TF, Pires MF, Pasqual M (2014) Direct organogenesis and leaf-anatomy modifications in vitro of Neoregelia concentrica (Vellozo) L.B. Smith (Bromeliaceae). Pak J Bot 46:2179–2187Google Scholar
  33. Mengesha A, Ayenew B, Tadesse T (2013) Energy sources affect in vitro propagation and subsequent acclimatization of Ananas comosus, var. smooth cayenne plants. J Microbiol Biotechnol Food Sci 2:2372–2376Google Scholar
  34. Mohamed AA (2008) Promotive effects of a 5-aminolevulinic acid-based fertilizer on growth of tissue culture-derived date palm plants (Phoenix dactylifera L.) during acclimatization. Sci Hortic 118:48–52. doi: 10.1016/j.scienta.2008.05.034 CrossRefGoogle Scholar
  35. Mohamed MA, Alsadon HAA (2010) Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets. Sci Hortic 123:295–300. doi: 10.1016/j.scienta.2009.09.014 CrossRefGoogle Scholar
  36. Murashige T, Skoog F (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
  37. Ola H, Reham AE, Farag E, Eisa SS, Habib SA (2012) Morpho-anatomical changes in salt stressed kallar grass (Leptochloa fusca L. Kunth). Res J Agric Biol Sci 8:158–166Google Scholar
  38. Park SY, Moon HK, Murthy HN, Kim YW (2011) Improved growth and acclimatization of somatic embryo-derived Oplopanax elatus plantlets by ventilated photoautotrophic culture. Biol Plant 55:559–562. doi: 10.1007/s10535-011-0125-4 CrossRefGoogle Scholar
  39. Pedroso ANV, Lazarini RAM, Tamaki V, Nievola C (2010) In vitro culture at low temperature and ex vitro acclimatization of Vriesea inflata an ornamental bromeliad. Braz J Bot 33:407–414. doi: 10.1590/S0100-84042010000300004 CrossRefGoogle Scholar
  40. Pereira TAR, Silva LC, Azevedo AA, Francino DMT, Coser TS, Pereira JD (2013) Leaf morpho-anatomical variations in Billbergia elegans and Neoregelia mucugensis (Bromeliaceae) exposed to low and high solar radiation. Botany 91:327–334. doi: 10.1139/cjb-2012-0276 CrossRefGoogle Scholar
  41. Pospíšilová J, Haisel D, Synková H, Čatský J, Wilhelmová N, Plzáková S, Procházková D, Šrámek F (2000) Photosynthetic pigments and gas exchange during ex vitro acclimation of tobacco plants as affected by CO2 supply and abscisic acid. Plant Cell Tissue Organ Cult 61:125–133. doi: 10.1023/A:1006402719458 CrossRefGoogle Scholar
  42. Rodríguez-Gamir J, Intrigliolo DS, Primo-Millo E, Forner-Giner MA (2010) Relationships between xylem anatomy, root hydraulic conductivity, leaf/root ratio and transpiration in citrus trees on different rootstocks. Physiol Plant 139:159–169. doi: 10.1111/j.1399-3054.2010.01351.x CrossRefPubMedGoogle Scholar
  43. Sáez PL, Bravo LA, Sáez KL, Sánchez-Olate M, Latsague MI, Ríos DG (2012) Photosynthetic and leaf anatomical characteristics of Castanea sativa: a comparison between in vitro and nursery plants. Biol Plant 56:15–24. doi: 10.1007/s10535-012-0010-9 CrossRefGoogle Scholar
  44. Saldanha CW, Otoni CG, Rocha DI, Cavatte PC, Detmann KSC, Tanaka FKO, Dias LLC, DaMatta FM, Otoni WC (2014) CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets. Plant Cell Tissue Organ Cult 118:87–99. doi: 10.1007/s11240-014-0464-x CrossRefGoogle Scholar
  45. Salih AA, Ali IA, Lux A, Luxova M, Cohen Y, Sugimoto Y, Inanaga S (1999) Rooting, water uptake and xylem structure adaptation to drought of two sorghum cultivars. Crop Sci 39:168–173. doi: 10.2135/cropsci1999.0011183X003900010027x CrossRefGoogle Scholar
  46. Shao HB, Chud LY, Jaleelc CH, Zhao CX (2008) Water-deficit stress-induced anatomical changes in higher plants. C R Biol 331:215–225. doi: 10.1016/j.crvi.2008.01.002 CrossRefPubMedGoogle Scholar
  47. Shin KS, Park SY, Paek KY (2013) Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions. In Vitro Cell Dev Biol Plant 49:445–454. doi: 10.1007/s11627-013-9524-x CrossRefGoogle Scholar
  48. Skirycz A, Inzé D (2010) More from less: plant growth under limited water. Curr Opin Biotech 21:197–203. doi: 10.1016/j.copbio.2010.03.002 CrossRefPubMedGoogle Scholar
  49. Trevisan F, Mendes BMJ (2005) Optimization of in vitro organogenesis in passion fruit (Passiflora edulis f. flavicarpa). Sci Agric 62:346–350. doi: 10.1590/S0103-90162005000400007 CrossRefGoogle Scholar
  50. Wang L, Ruan YL (2013) Regulation of cell division and expansion by sugar and auxin signaling. Front Plant Sci 4:1–9. doi: 10.3389/fpls.2013.00163 Google Scholar
  51. Xiao Y, Niu G, Kozai T (2011) Development and application of photoautotrophic micropropagation plant system. Plant Cell Tissue Organ Cult 105:149–158. doi: 10.1007/s11240-010-9863-9 CrossRefGoogle Scholar
  52. Zobayed SMA (2000) In vitro propagation of Lagerstroemia spp. from nodal explants and gaseous composition in the culture headspace. Environ Control Biol 38:1–11CrossRefGoogle Scholar
  53. Zobayed SMA (2005) Ventilation in micropropagation. In: Kozai T, Afreen F, Zobayed SMA (eds) Photoautotrophic (sugar-free medium) micropropagation as a new micropropagation and transplant production system. Springer, Netherlands, pp 147–186. doi:  10.1007/1-4020-3126-2_9

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • João Paulo Rodrigues Martins
    • 1
  • Veerle Verdoodt
    • 2
  • Moacir Pasqual
    • 1
  • Maurice De Proft
    • 2
  1. 1.Tissue Culture Laboratory of the Department of AgricultureFederal University of LavrasLavrasBrazil
  2. 2.Division of Crops Biotechnics, Department of BiosytemsKatholieke Universiteit LeuvenLouvainBelgium

Personalised recommendations