Summary
Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N6-Benzyladenine (BA) alone at 5.0µM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5µM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.
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References
Abdullah, A. A.; Yeoman, M. M.; Grace, J. Micropropagation of mature Calabrian pine (Pinus brutia Ten.) from fascicular buds. Tree Physiol. 3:123–136; 1987.
Aitken, J.; Horgan, K. J.; Thorpe, T. A. Influence of explant selection on the shoot-forming capacity of juvenile tissue ofPinus radiata. Can. J. For. Res. 11:112–117; 1981.
Baxter, R.; Brown, S. N.; England, N. F., et al. Production of clonal plantlets of tropical pine in tissue culture via axillary shoot activation. Can. J. For. Res. 19:1338–1342; 1989.
Bornman, C. H. Possibilities and constraints in the regeneration of trees from cotyledonary needles ofPicea abies in vitro. Physiol. Plant 57:5–16; 1983.
Chang, S.; Sen, S.; McKinley, C. R., et al. Clonal propagation of Virginia pine (Pinus virginiana Mill.) by organogenesis. Plant Cell Rep. 10:131–134; 1991.
Cheah, K. T.; Cheng, T. Y. Histological analysis of adventitious bud formation in cultured Douglas-fir cotyledons. Am. J. Bot. 65:845–849; 1978.
Debergh, P.; Aitken-Christie, J.; Cohen, L. D. et al. Reconsideration of the term “vitrification” as used in micropropagation. Plant Cell Tissue Organ Cult. 30:135–140; 1992.
Gupta, P. K.; Durzan, D. J. Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga manziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep. 4:177–179; 1985.
Harry, I. S.; Thompson, M. R.; Thorpe, T. A. Regeneration of plantlets from mature embryos of Western larch. In Vitro Cell. Dev. Biol. 27P:32–41; 1991.
Hartmann, H. T.; Kester, D. E.; Davies, F. T. Plant propagation, principles and practices. Englewood Cliffs, NJ: Prentice Hall; 1990:283.
Lambardi, M.; Sharma, K. K.; Thorpe, T. A.In vitro regeneration of shoot buds and plantlet formation from mature embryos ofPinus halepensis Mill. Acta Hortic. 289:123–124; 1991.
Marascuilo, L. A.; McSweeney, M. Nonparametric and distribution-free methods for the social sciences. Belmont, CA: Brooks/Cole; 1977:141–147.
Martinez Pulido, C.; Harry, I. S.; Thorpe, T. A.In vitro regeneration of plantlets of Canary Island pine (Pinus canariensis). Can. J. For. Res. 20:1200–1211; 1990.
Minocha, S. C. Callus and adventitious shoot formation in excised embryos of white pine (Pinus strobus). Can. J. Bot. 58:366–370; 1980.
Mohammed, G. H.; Vidaver, W. E. Root production and plantlet development in tissue-cultured conifers. Plant Cell Tissue Organ Cult. 14:137–160; 1988.
Mott, R. L.; Amerson, H. V. Tissue culture plantlets produced fromPinus monticola embryonic materials. For. Sci. 27:299–304; 1981.
Mott, R. L.; Smeltzer, R. H.; Mehra-Palta, A., et al. Production of forest trees by tissue culture. Tappi 60:62–64; 1977.
O’Brien, T. P.; McCully, M. E. The study of plant structure principles and selected methods. Melbourne: Termarcarphi PTY. Ltd; 1981:6.97–98.
Panetsos, K. P. Genetics and breeding in the group halepensis. Foret mediterraneenne, t. VIII:15–21; 1986.
Quoirin, M.; Le Poivre, P. Etudes de milieux adaptes aux cultures in vitro dePrunus. Acta Hortic. 78:437–442; 1977.
Schenk, R. U.; Hildebrandt, A. C. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50:199–204; 1972.
Sharma, K. K.; Bhojwani, S. S.; Thorpe, T. A. The role of cotyledonary tissue in the differentiation of shoots and roots from cotyledon explants ofBrassica juncea (L.) Czern. Plant Cell Tissue Organ Cult. 24:55–59; 1991.
Snedecor, G. W.; Cochran, W. G. Statistical methods. Ames, IA: Iowa University Press; 1980:507.
Thorpe, T. A.; Biondi, S. Conifers. In: Sharp, W. R.; Evans, D. A.; Ammirato, P. A., et al., eds. Handbook of plant cell culture, vol. 2, crop species. New York: Macmillan Publishing Co.; 1984:435–470.
Thorpe, T. A.; Harry, I. S.; Kumar, P. P. Application of micropropagation to forestry. In: Debergh, P. C.; Zimmerman, R. H., eds. Micropropagation. The Netherlands: Kluwer Ac. Publishers; 1991:311–336.
Thorpe, T. A.; Patel, K. R. Clonal propagation: adventitious buds. In: Vasil, I. K., ed. Cell culture and somatic cell genetics of plants, vol. 1. New York: Academic Press; 1984:49–60.
von Arnold, S.; Eriksson, T.In vitro studies on adventitious shoot formation inPinus contorta. Can J. Bot. 59:870–874; 1981.
Yeung, E. C.; Aitken, J.; Biondi, S., et al. Shoot histogenesis in cotyledon explants of Radiata pine. Bot. Gaz. 142:494–501; 1981.
Yeung, E. C.; Law, S. K. Serial sectioning techniques for a modified LKB historesin. Stain Technol. 62:147–153; 1987.
Zel, J.; Gogala, N.; Camloh, M. Micropropagation ofPinus sylvestris. Plant Cell Tissue Organ Cult. 14:169–175; 1988.
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Lambardi, M., Sharma, K.K. & Thorpe, T.A. Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.). In Vitro Cell Dev Biol - Plant 29, 189–199 (1993). https://doi.org/10.1007/BF02632034
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DOI: https://doi.org/10.1007/BF02632034