Efficient protocol for in vitro mass micropropagation of slash pine
- 461 Downloads
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).
- Cézar TM, Higa AR, Koehler HS, Ribas LLF (2015) Influence of culture medium, explant length and genotype on micropropagation of Pinus taeda L. Ciência Florestal 25:13–22Google Scholar
- Coke JE (1996) Basal nutrient medium for in vitro cultures of loblolly pines. Google PatentsGoogle Scholar
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
- Nandwani D, Kumaria S, Tandon P (2001) Micropropagation of Pinus kesiya Royle Ex Gord (khasi pine). Gartenbauwissenschaft 66:68–71Google Scholar
- Newton R, Marek-Swize K, Magallanes-Cedeno M, Dong N, Sen S, Jain S (1995) Somatic embryogenesis in slash pine (Pinus elliottii Engelm.) In: Jain S, Gupta P, Newton R (eds) Somatic embryogenesis in woody plants. Vol 3. Gymnosperms. Kluwer Academic Publishers, The Netherlands, pp 183–195. https://doi.org/10.1007/978-94-011-0960-4_11 CrossRefGoogle Scholar
- Nunes S, Marum L, Farinha N, Pereira VT, Almeida T, Sousa D, Mano N, Figueiredo J, Dias MC, Santos C (2017b) Somatic embryogenesis of hybrid Pinus elliottii Var. Elliottii × P. caribaea Var. Hondurensis and ploidy assessment of somatic plants. Plant Cell Tissue Organ Cult 132(1):71–84. https://doi.org/10.1007/s11240-017-1311-7 CrossRefGoogle Scholar
- Oliveira L, Ribas L, Quoirin M, Koehler H, Amano E, Higa A (2012) Micropropagation of Pinus taeda L. from juvenile material. Tree For Sci Biotech 6:96–101Google Scholar
- Tang W, Newton R (2007) Protocols for micropropagation of woody trees and fruits. In: Jain SM, Häggman H (eds) Protocols for micropropagation of woody trees and fruits, Springer Netherlands, pp 15–22Google Scholar
- Tereso S, Gonçalves S, Marum L, Oliveira M, Maroco J, Miguel C (2006) Improved axillary and adventitious bud regeneration from Portuguese genotypes of Pinus pinaster Ait. Propag Ornam Plants 6:24–33Google Scholar