Efficient protocol for in vitro mass micropropagation of slash pine

  • Sandra Nunes
  • Diana Sousa
  • Vanessa T. Pereira
  • Sandra Correia
  • Liliana Marum
  • Conceição SantosEmail author
  • Maria C. Dias
Plant Tissue Culture


An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.


Flow cytometry Micropropagation Pinus elliottii Ploidy 



Authors thank technical support of A Costa and J Manso.

Funding information

This work was supported by the support of QREN to Klon-UA collaboration and by FEDER/COMPETE/POCI for Projects POCI-01-0145-FEDER-006958 and POCI/01/0145/FEDER/007265 and National Funds of FCT/MEC FEDER co-funding within Partnership Agreement PT2020 UID/QUI/50006/2013, PEst-OE/BIA/UI4004/2011, UID/QUI/00062/2013. FCT supported M Dias (SFRH/BPD/100865/2014) and S Correia (SFRH/BPD/91461/2012).


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Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  1. 1.KLÓN—Innovative Technologies from Cloning, S.A., Biocant ParkParque Tecnológico de CantanhedeCantanhedePortugal
  2. 2.Centre for Functional Ecology (CEF), Department of Life ScienceUniversity of Coimbra, Calçada Martim de FreitasCoimbraPortugal
  3. 3.Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja)BejaPortugal
  4. 4.Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM)Universidade de ÉvoraÉvoraPortugal
  5. 5.Department of Biology, Faculty of Sciences & LAQV/REQUIMTEUniversity of PortoPortoPortugal
  6. 6.Department of Chemistry and QOPNAUniversity of AveiroAveiroPortugal

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