In Vitro Propagation of Rhododendron tomentosum – an Endangered Essential Oil Bearing Plant from Peatland

Open access

Abstract

Rhododendron tomentosum Harmaja (formerly Ledum palustre L.) is a medicinal peat bog plant native to northern Europe, Asia and North America. This plant has a distinctive aroma thanks to the presence of essential oil, to which it also owes its anti-inflammatory, analgesic, antimicrobial and insecticidal properties. However, in Europe R. tomentosum is classified as an endangered species, mainly due to degradation of peatlands. In the present work, the micropropagation protocol for R. tomentosum was established for the first time, providing both an ex situ conservation tool and a means of continuous production of in vivo and in vitro plant material for further studies. R. tomentosum microshoots were initiated from leaf explants and further multiplied using Schenk-Hildebrandt (SH) medium supplemented with 9.84 μM 2iP and 1.00 μM TDZ. The shoots were elongated on the SH medium supplemented with 24.6 μM 2iP and subsequently rooted using the perlite substrate saturated with half-strength Woody Plant medium supplemented with 1.0% sucrose and 4.92 μM IBA. The regenerated plants were hardened on the phytohormone-free SH medium and acclimatized using 3:1:1 deacidified peat:perlite:gravel substrate. The identity of the mother plant was confirmed at morphological and molecular levels and Random Amplified Polymorphic DNA (RAPD) method was implemented to assess the genetic fidelity of the regenerants. The essential oil content of the maternal plant, in vitro shoots and the regenerants was determined by steam-distillation, and the obtained volatile fractions were analyzed by GC/MS.

Anderson WC. 1978. Tissue culture propagation of rhododendrons. In Vitro 14: 334.

Anderson WC. 1984. A revised tissue culture medium for shoot multiplication of Rhododendron. Journal of the American Society for Horticultural Science 109: 343–347.

Bojarczuk K. 1989. Micropropagation of rhododendrons. Acta Horticulturae 251: 241–248.

Briggs BA, McCulloch SM, and Caton LA. 1994. In vitro propagation of Rhododendron. Acta Horticulturae 364: 21–26.

Butkiene R, Šakociute V, Latvenaite D, and Mockute D. 2008. Composition of young and aged shoot essential oils of the wild Ledum palustre L. Chemija 19: 19–24.

Chapman S, Buttler A, Francez A-J, Laggoun-Defarge F, Vasander H, Schloter M, Combe J, Grosvernier P, Harms H, Epron D, Gilbert D, and Mitchell E. 2003. Exploitation of northern peatlands and biodiversity maintenance: a conflict between economy and ecology. Frontiers in Ecology and the Environment 10: 525–532.

Cites Plants Comitee. 2011. Convention on international trade in endangered species of wild fauna and flora. Global strategy for plant conservation of the convention on biological diversity (decision 15.19).

Čizkova H, Kvet J, Comin FA, Laiho R, Pokorny J, and Pithart D. 2013. Actual state of European wetlands and their possible future in the context of global climate change. Aquatic Sciences 75: 3–26.

Dampc A, and Luczkiewicz M. 2013. Rhododendron tomentosum (Ledum palustre). A review of traditional use based on current research. Fitoterapia 85: 130–143.

Debnath SC. 2007. Propagation of Vaccinium in vitro. International Journal of Fruit Science 6: 47–71.

Devarumath R, Nandy S, Rani V, Matimuthu S, Muraleedharan N, and Raina S. 2002. RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam – India type). Plant Cell Reports 21: 166–173.

Doyle JJ, and Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.

Dubova I, Šmite D, Klavina D, and Rila R. 2010. First results of ex situ conservation of endangered wild plants of Latvia in the National Botanic Garden. Environmental and Experimental Biology 8: 75–80.

Eeckhaut T, Janssens K, De Keyser E, and De Riek J. 2010. Micropropagation of Rhododendron. In: Jain SM, Ochatt SJ (eds) Protocols for in vitro propagation of ornamental plants, 141–152. Humana Press, New York, USA.

Ejankowski W. 2010. Demographic variation of dwarf birch (Betula nana) in communities dominated by Ledum palustre and Vaccinium uliginosum. Biologia 65: 248–253.

Fay MF. 1992. Conservation of rare and endangered plants using in vitro methods. In Vitro Cellular and Developmental Biology 28: 1–4.

Gibbs D, Chamberlain D, and Argent G. 2011. The red list of rhododendrons. Botanic Gardens Conversation International, Richmond, UK.

Goetsch L, Eckert AJ, and Hall BD. 2005. The molecular systematics of Rhododendron (Ericaceae): A phylogeny based upon RPB2 gene sequences. Systematic Botany 30: 616–626.

Guo WL, Gong L, Ding ZF, Li YD, Li FX, Zhao SP, and Liu B. 2006. Genomic instability in phenotypically normal regenerants of medicinal plant Codonopsis lanceolata Benth. et Hook. f., as revealed by ISSR and RAPD markers. Plant Cell Reports 25: 896–906.

Harmaja H. 1991. Taxonomic notes on Rhododendron subsection Ledum (Ledum, Ericaceae), with a key to its species. Annales Botanici Fennici 28: 171–173.

Hilton MG, Jay A, Rhodes MJC, and Wilson PDG. 1995. Growth and monoterpene production by transformed shoot cultures of Mentha citrata and Mentha piperita in flasks and fermenters. Applied Microbiology and Biotechnology 43: 452–459.

Kokotkiewicz A, Luczkiewicz M, Hering A, Ochocka R, Gorynski K, Bucinski A, and Sowinski P. 2012. Micropropagation of Cyclopia genistoides, an endemic South African plant of economic importance. Zeitschrift für Naturforschung 67c: 65–76.

Lloyd G, and McCown BH. 1981. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by shoot tip culture. Proceedings of the International Plant Propagators Society 30: 421–427.

Luczkiewicz M, Jesionek A, Kokotkiewicz A, Migas P, Mardarowicz M, Szreniawa-Sztajnert A, Zabiegala B, and Bucinski A. 2015. Production of essential oils from in vitro cultures of Caryopteris species and comparison of their concentrations with in vivo plants. Acta Physiologiae Plantarum 37: 58.

Luczkiewicz M, and Piotrowski A. 2005. Two-stage system for micropropagation of several Genista plants producing large amounts of phytoestrogens. Zeitschrift für Naturforschung 60c: 557–566.

Maddison WP, and Maddison DR. 2015. Mesquite: A Modular System for Evolutionary Analysis. Version 2.73. http://mesquiteproject.org. Accessed 7 August 2015.

Matysiak B, and Nowak J. 2008. Coir substrates for rooting of ornamental ericaceous plants. Propagation of Ornamental Plants 8: 76–80.

McCown BH. 2013. Woody shrubs and trees. In: Smith RH (ed), Plant tissue culture, techniques and experiments, third edition, 93-101. Elsevier, San Diego, USA.

Mulder-Krieger TH, Verpoorte R, Baerheim-Svendsen A, and Scheffer JJC. 1988. Production of essential oils and flavours in plant cell and tissue cultures. A review. Plant Cell, Tissue and Organ Culture 13: 85–154.

Polish Pharmacopoeia VI. 2002. The determination of essential oil, 151. Polish Pharmaceutical Society, Warsaw, Poland.

Racine CH, Johnson LA, and Viereck LA. 1987. Patterns of vegetation recovery after tundra fires in northwestern Alaska, USA. Arctic and Alpine Research 19: 461–469.

Schenk RU, and Hildebrandt AC. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian Journal of Botany 50: 199–204.

Schluter PM, and Harris SA. 2006. Analysis of multilocus fingerprinting data sets containing missing data. Molecular Ecology Notes 6: 569–572.

Spencer A, Hamill JD, and Rhodes MJC. 1990. Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37. Plant Cell Reports 10: 601–604.

Sugii N, and Lamoureux C. 2004. Tissue culture as a conservation method: An empirical view from Hawaii. In: Guerrant EO, Kavens K, Maunder M (eds), Ex situ plant conservation supporting species survival in the wild, 189–205. Island Press, Washington, USA.

Thiem B, Kikowska M, Kurowska A, and Kalemba D. 2011. Essential oil composition of the different parts and in vitro shoot culture of Eryngium planum L. Molecules 16: 7115–7124.

Acta Biologica Cracoviensia s. Botanica

The Journal of Polish Academy of Sciences

Journal Information


IMPACT FACTOR 2016: 0.491
5-year IMPACT FACTOR: 0.787

CiteScore 2016: 0.51

SCImago Journal Rank (SJR) 2016: 0.242
Source Normalized Impact per Paper (SNIP) 2016: 0.264

Ministry of Science and Higher Education: 20 points

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 423 350 21
PDF Downloads 179 168 12