Efficient protocol for in vitro mass micropropagation of slash pine
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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.
KeywordsFlow cytometry Micropropagation Pinus elliottii Ploidy
Authors thank technical support of A Costa and J Manso.
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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