Factors influencing axillary shoot proliferation and ... - Tree Physiology

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484 RENAU-MORATA, OLLERO, ARRILLAGA AND SEGURA exclude a possible ABA influence in our experiments. The use of defoliated microcuttings makes it difficult to compare our results with those reported by Piola and Rohr (1996) for juvenile C. libani microcuttings; nevertheless, we found that an incubation temperature of 30 °C enhanced shoot yield in juvenile microcuttings of both C. atlantica and C. libani. Piola and Rohr (1996) found that a 3-h pulse with 0.1 mM BA induced the highest proliferation rate (4.5 buds per explant) in C. libani microcuttings. We observed similar results for C. libani microcuttings grown in the presence of 2.2 µM BA; however, BA negatively affected bud sprouting in C. atlantica microcuttings. Nested ANOVA attributed most of the variability in shoot yield from cultured microcuttings of juvenile origin to single seedlings (77 and 65% for C. atlantica and C. libani, respectively). This strong genotypic effect was even more evident in cultured explants of bicentennial C. libani trees, where only three out of the six genotypes were successfully established in vitro. A similar genotypic effect on the bud-forming capacity of explants was observed by Tang et al. (2001). In some conifers, variation in morphogenic responses was evident not only among embryos from different seed lots but also among embryos obtained from controlled crossings (von Arnold and Eriksson 1982, 1986, Shen and von Arnold 1982). We observed differences in the initial in vitro performance of the three genotypes, but these differences decreased with increasing number of subcultures. Usually, in vitro manipulation of woody plants is more difficult with mature explants than with juvenile explants, and Cedrus was no exception. A comparison between cedar explants of juvenile origin and those of adult origin showed that microcuttings were the preferred explant for axillary shoot proliferation in juvenile and adult cultures. Although this response did not require the presence of cytokinin in the culture medium, Z increased axillary shoot yield in explants of mature origin. Hormone-free nutrient media have been successfully employed for culture establishment of other mature Pinaceae including Pseudotsuga menziesii (Mirb.) Franco and Pinus lambertiana Dougl. (Gupta and Durzan 1985), although the presence of growth regulators favored axillary bud sprouting in shoot apices and microcuttings isolated from 20-year-old Larix occidentalis Nutt. trees (Chesick et al. 1990). Shoot proliferation rates in cultures of adult C. libani were generally greater on basal MSBN/2 medium than on basal WPM. Although the influence of nutrient medium on in vitro morphogenesis is well documented, its effect remains one of the most empirical aspects of plant cell culture (Preece 1995). Compared with basal MSBN/2 medium, basal WPM has higher concentrations of calcium, sucrose, micronutrients and organic supplements, and contains biotine and folic acid (both absent in MSBN/2 medium). A reduced amount of nutrients stimulates axillary branching in some plants (Karhu 1997) and low calcium availability can affect apical meristem integrity (McCown and Sellmer 1987), thus favoring axillary bud development; however, we are unable to offer an explanation for the differential effect of MSBN/2 and WPM media on the basis of our experimental data. TREE PHYSIOLOGY VOLUME 25, 2005 Adventitious organogenesis offers higher potential for shoot production than axillary bud proliferation, and is the preferred method for coniferous micropropagation because coniferous buds are generally induced directly on the explant (Thorpe et al. 1991). With a few exceptions, however, techniques for adventitious budding of coniferous explants obtained from trees in the adult growth phase are still limited, because the caulogenic potential of isolated organs is affected by the ontogenic age of the tissues (von Aderkas and Bonga 2000, Giri et al. 2004). In our experiments, callus derived from shoot apices differentiated adventitious buds in the presence of cytokinin, but only when explants of juvenile origin were used and we were unable to promote their elongation. In contrast, Hosseyni et al. (1999) reported the induction of adventitious organogenesis (buds and needles) from winter buds of 10–15year-old C. libani cultured on WPM with BA or kinetin. This difference between studies may be associated with tree age, because our trees were older. We successfully generated adventitious shoots from C. atlantica embryos, which opens up the possibility of mass propagation of this species. Our regeneration system included: (1) adventitious budding in the presence of 9.0 µM Z; (2) bud development on medium containing 4.4 µM Z; and (3) shoot elongation on cytokinin-free medium. Zeatine or benzyladenine alone was enough to stimulate both direct and indirect adventitious budding, which is in agreement with previous findings in other conifer species (Thorpe et al. 1991); however, the presence of Z improved the bud-forming capacity of the explants, and the process of bud development required transfer of C. atlantica embryos to a medium with cytokinin. In contrast, in other conifers, bud development depends on transfer of explants to hormone-free medium (Gómez and Segura 1994, Mata et al. 2001, Villalobos-Amador et al. 2002, Schestibratov et al. 2003). Successful micropropagation of many woody species is frequently limited by their reluctance to form adventitious roots, and C. libani and C. atlantica were no exception. In many species, rooting can be achieved by adding auxins to the culture medium or by using different support substrates (Hartmann et al. 1997). Activated charcoal, a high sucrose concentration, variations in environmental temperature conditions and light reduction in the rooting zone improve auxin-induced rooting in many species, including conifers (George 1993). A variety of ancillary compounds such as paclobutrazol, phosphoric acid, calcium, triacontanol, oligosaccharins and coumarin (George 1993, Haissig and Davis 1994, Tantos et al. 2001, Nandi et al. 2002) can improve rooting capacity in recalcitrant species. In some conifer species, infection with A. rhizogenes enhances rhizogenesis (McAfee et al. 1993, Tzfira et al. 1996, Mihaljevic et al. 1998, Villalobos-Amador et al. 2002). In our experiments, all of these variables failed to induce rooting in shoots of axillary or adventitious origin of C. libani and C. atlantica. Nicholson (1984) and Nandi et al. (2002) were able to induce rooting in C. deodara (D. Don) G. Don and rooting has been reported in C. libani and C. atlantica (Dirr and Heuser 1987) cuttings, although in the last two species, rooting percentages were minimal and rooting was observed only when Downloaded from http://treephys.oxfordjournals.org/ by guest on June 5, 2013
cuttings were sampled at the end of the winter season. Hosseyni et al. (1999) also reported rooting (29–33%) from adventitious shoots of C. libani cultured on MS/4 or WPM/2 media with 1.4 µM IAA but not with 1.4 µM IBA; both hormones, however, were ineffective in promoting rooting in shoots generated from winter buds of 10–15-year-old C. libani trees. In conclusion, we have developed an efficient method to promote axillary bud proliferation from cedar microcuttings of juvenile and adult origin. Our regeneration system (bud sprouting in defoliated microcuttings cultured on basal MSBN/2 medium at 26 or 30 °C, and isolation and continuous subculture of sprouted buds onto the same medium) provides a means of obtaining multiple shoots from a single microcutting from cedar trees old enough to have demonstrated their superior characteristics. Our regeneration method may be useful for clonal propagation of elite genotypes provided that a protocol for the successful rooting of proliferating shoots is developed. The phenotypic characteristics of the putative clones were similar to those of seedlings grown from seeds. We also developed a protocol for the successful regeneration of adventitious shoots from mature embryos of C. atlantica. Acknowledgments The authors thank the European Union, Contract Number ERBIC18- CT97-0177, and Generalitat Valenciana (Grupos 03/102) for financial support. References Abourouh, M. and L. Najim. 1990. Culture in vitro de fragments de cotyledons des plantules de Cedrus atlantica Manetti. Saussurea 21:75–80. Altman, A. 2003. From plant tissue culture to biotechnology: scientific revolutions, abiotic stress tolerance and forestry. In Vitro Cell. Dev. Biol. Plant 39:75–84. Benchekroun, F. 1994. The economy of the Moroccan cedar forest and its impact on the development of local collectivities. Ann. Rech. Fort. Maroc 27:713–724. Bhatnagar, S.P., M.N. Singh and N. Kapur. 1983. Preliminary investigations on organ differentiation in tissue cultures of Cedrus deodara and Pinus roxburghii. Indian J. Exp. Biol. 21:524–526. Boulay, M. and A. Franclet. 1977. Recherches sur la propagation végétative du Douglas Pseudotsuga menziesii (Mirb.) Franco. Possibilités d’obtention de plantes viables à partir de la culture in vitro de bourgeons de pieds-mères juvéniles. CR Acad. Sci. 284D: 1405–1407. Bourgin, J.P. and J.P. Nitsch. 1967. Obtention de Nicotiana haploides à partir d’étamines cultivées in vitro. Ann. Physiol. Vég. 9: 337–338. Brunetti, M., E.L. De Capua, N. Macchioni and S. Monachello. 2001. Natural durability, physical and mechanical properties of Atlas cedar (Cedrus atlantica Manetti) wood from Southern Italy. Ann. For. Sci. 58:607–613. Chesick, E.E., D.E. Bilderback and G.M. Blake. 1990. In vitro multiple bud formation by 20-year-old western larch buds and stems. HortScience 25:114–116. Dirr, M.A. and C.W. Heuser, Jr. 1987. The reference manual of woody plant propagation: from seed to tissue culture. Varsity Press, Athens, GA, 239 p. SHOOT PROLIFERATION IN CEDAR CULTURES 485 TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Fenning, T.M. and J. Gershenzon. 2002. Where will the wood come from? Plantation forests and the role of biotechnology. Trends Biotechnol. 20:291–296. George, E.F. 1993. Plant propagation by tissue culture. 2nd Edn. Exegetics, Edington, U.K., 1361 p. Giri, C.C., B. Shyamkumar and C. Anjaneyulu. 2004. Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. Trees 18:115–135. Gómez, M.P. and J. Segura. 1994. Factors controlling adventitious bud induction and plant regeneration in mature Juniperus oxycedrus leaves cultured in vitro. In Vitro Cell. Dev. Biol. Plant 30: 210–218. Gupta, P.K. and D.J. Durzan. 1985. Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep. 4:177–179. Haissig, B.E. and T.D. Davis. 1994. A historical evaluation of adventitious rooting research to 1993. In Biology of Adventitious Root Formation. Eds. T.D. Davis and B.E. Haissig. Plenum Press, New York, pp 275–331. Hapla, F., J.V. Oliver-Villanueva and J.M. González-Molina. 2000. Effect of silvicultural management on wood quality and timber utilisation of Cedrus atlantica in the European mediterranean area. Holz als Roh-Werkstoff 58:1–8. Hartmann, H.T., D.E. Kester, F.T. Davies and R.L. Geneve. 1997. Plant propagation: principles and practices. Prentice Hall, London, 770 p. Hoagland, D.R. and D.I. Arnon. 1950. The water culture method for growing plants without soil. Circular 347, California Agricultural Experiment Station, Berkeley, CA, 32 p. Hosseyni, N., A. Ozan and Z. Kaya. 1999. Basal nutrient and hormonal requirements for direct organogenesis of Cedrus libani A. Rich. In Plant Biotechnology and In Vitro Biology in the 21st Century. Eds. A. Altman, M. Ziv and S. Izhar. IX International Congress on Plant Tissue and Cell Culture, Jerusalem. Kluwer Academic Publishers, Dordrecht, pp 53–56. Karhu, S.T. 1997. Axillary shoot proliferation of blue honeysuckle. Plant Cell Tissue Organ Cult. 48:195–201. Khuri, S., M.R. Shmoury, R. Baalbaki, M. Maunder and S.N. Talhouk. 2000. Conservation of the Cedrus libani populations in Lebanon: history, current status and experimental application of somatic embryogenesis. Biodivers. Conserv. 9:1261–1273. Lloyd, G.B. and B.H. McCown. 1980. Comercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoottip culture. Proc. Int. Plant Propagation Soc. 30: 421–421. Mata, M., V.M. Chávez and R. Bye. 2001. In vitro regeneration of plantlets from immature zygotic embryos of Picea chihuahuana Martínez, an endemic Mexican endangered species. In Vitro Cell. Dev. Plants 37:73–78. McAfee, B.J., E.E. White, L.E. Pelcher and M.S. Lapp. 1993. Root induction in pine (Pinus) and larch (Larix) spp. using Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult. 34:53–62. McCown, B.H. and J.C. Sellmer. 1987. General media and vessels suitable for woody plant culture. In Cell and Tissue Culture in Forestry. Eds. J.M. Bonga and J. Durzan. Vol. 1. Marinus Nijhoff Publishers, Dordrecht, pp 4–16. Merkle, S.A.and J.F.D. Dean. 2000. Forest tree biotechnology. Curr. Opin. Biotechnol. 11:298–302. Mihaljevic, S., V. Katavic and S. Jeleska. 1998. Root formation in micropropagated shoots of Sequoia sempervirens using Agrobacterium. Plant Sci. 141:73–80. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497. 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