Abstract
This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees.
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References
Abdullah AA, Yeoman MM, Grace J (1987) Micropropagation of mature Calabrian pine (Pinus brutia Ten.) from fascicular buds. Tree Physiol 3:123–136
Ahuja MR (1993) Biotechnology and clonal forestry. In: Ahuja MR, Libby WJ (eds) Clonal forestry I, genetics and biotechnology. Springer, Berlin, pp 135–144
Aitken-Christie J, Singh A, Davies H (1988) Multiplication of meristematic tissue: a new tissue culture system for radiata pine. In: Hanover JW, Keathley DE (eds) Genetic manipulation of woody plants. Plenum Publishing Corporation, New York, pp 413–432
Alonso P, Moncaleán P, Fernández B, Rodríguez A, Centeno ML, Ordás RJ (2006) An improved micropropagation method for Stone Pine (Pinus pinea L.). Ann For Sci 63:879–885. doi:10.1051/forest:2006071
Campbell M, Brunner AM, Jones H, Strauss SH (2003) Forestry’s fertile crescent: the application of biotechnology to forest trees. Plant Biotechnol J 1:141–154. doi:10.1046/j.1467-7652.2003.00020.x
Catalán G (1998) Current situation and prospects of the stonepines as nut producers. FAO-CIHEAM. Nucis Newsl 7:28–32
Chalupa V (1989) Micropropagation of Larix decidua Mill. and Pinus sylvestris L. Biol Plant 31(5):400–407. doi:10.1007/BF02876363
Chang SH, Ho CK, Chen ZZ, Tsay JY (2001) Micropropagation of Taxus mairei from mature trees. Plant Cell Rep 20:496–502. doi:10.1007/s002990100362
Cortizo M, Alonso P, Fernández B, Rodríguez A, Centeno ML, Ordás RJ (2004) Micrografting of mature stone pine (Pinus pinea L.) trees. Ann For Sci 61:843–845. doi:10.1051/forest:2004081
Dumas E, Monteuuis O (1995) In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal. Plant Cell Tissue Organ Cult 40:231–235. doi:10.1007/BF00048128
Ewald D (1998) Advances in tissue culture of adult larch. In Vitro Cell Dev Biol Plant 34:325–330. doi:10.1007/BF02822742
FAO (2004) Preliminary review of biotechnology in forestry, including genetic modification. Forest genetic resources working paper FGR/59E. Forest Resources Development Service, Forest Resources Division, Rome
Greenwood MS (1995) Juvenility and maturation in conifers: current concepts. Tree Physiol 15:433–438
Hackett WP (1988) Donor plant maturation and adventitious root formation. In: Davis TD, Hassing BE, Sankhla N (eds) Aventitious root formation in cuttings. Dioscorides Press, Portland, pp 11–28
Hohtola A (1988) Seasonal changes in explant viability and contamination of tissue cultures from mature Scots pine. Plant Cell Tissue Organ Cult 15:211–222. doi:10.1007/BF00033645
Horgan KJ (1987) Pinus radiata. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry. Martinus Nijhoff, Dordrecht, pp 128–145
Kunze I (1994) Influence of the genotype on growth of Norway spruce (Picea abies L.) in in vitro meristem culture. Silvae Genet 43(1):36–41
Merkle SA, Dean JF (2000) Forest tree biotechnology. Curr Opin Biotechnol 11:298–302. doi:10.1016/S0958-1669(00)00099-9
Murthy B, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol Plant 34:267–275. doi:10.1007/BF02822732
Mutke S, Gordo J, Gil L (2000) The stone pine (Pinus pinea L.) breeding programme in Castile-León (Central Spain). FAO-CIHEAM. Nucis Newsl 9:50–55
Parasharami VA, Poonawala IS, Nadgauda RS (2003) Bud break and plantlet regeneration in vitro from mature trees of Pinus roxburghii Sarg. Curr Sci 84:203–208
Prehn D, Serrano C, Mercado A, Stange C, Barrales L, Arce-Johnson P (2003) Regeneration of whole plants from apical meristems of Pinus radiata. Plant Cell Tissue Organ Cult 73:91–94. doi:10.1023/A:1022615212607
Renau-Morata B, Ollero J, Arrillaga I, Segura J (2005) Factors influencing axillary shoot proliferation and adventitious budding in cedar. Tree Physiol 25:477–486
Spichal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, Strnad M, Schmulling T (2004) Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol 45:1299–1305. doi:10.1093/pcp/pch132
Stange C, Prehn D, Gebauer M, Arce-Johnson P (1999) Optimization of in vitro culture conditions for Pinus radiata embryos and histological characterization of regenerated shoots. Biol Res 32:19–28
Sul IW, Korban SS (2004) Effects of salt formulations, carbon sources, cytokinins, and auxin on shoot organogenesis from cotyledons of Pinus pinea L. Plant Growth Regul 43:197–205. doi:10.1023/B:GROW.0000046013.47892.4f
Tang W, Guo Z (2001) In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regul 33:25–31. doi:10.1023/A:1010764816523
Tang W, Newton RJ (2005) Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos. Plant Physiol Biochem 43:760–769. doi:10.1016/j.plaphy.2005.05.008
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. Propag Ornam Plants 6:24–33
von Aderkas P, Bonga JM (2000) Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol 20:921–928
Acknowledgments
The authors sincerely thank Ronnie Lendrum for English revision, and Patxi Saez de Urturi, Ander Isasmendi and Elena Fernández de Larrinoa (Neiker—Tecnalia) for their technical assistance. This work was supported by “Ministerio de Ciencia y Tecnología de España” (MEC-AGL-2005-08214). Millán Cortizo was supported by an FPU grant from the Spanish Ministry of Education. Nuria de Diego was supported by FT grant from the Basque Government.
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Communicated by D. Treutter.
Paloma Moncaleán and Ricardo Javier Ordás contributed equally.
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Cortizo, M., de Diego, N., Moncaleán, P. et al. Micropropagation of adult Stone Pine (Pinus pinea L.). Trees 23, 835–842 (2009). https://doi.org/10.1007/s00468-009-0325-0
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DOI: https://doi.org/10.1007/s00468-009-0325-0