Protocols for Micropropagation of Woody Trees and Fruits

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B. PINTOS ET AL.
3. CONCLUSION
We present here a new protocol to produce an isolated microspore culture that leads
to cell divisions and proembryo formation in Olea europaea L., cv. Arbequina. The
morphology of floral buds containing responsive anthers and microspores (open buds
with yellow-greenish anthers) from trees grown in the field has also been described.
Moreover, also the optimum microspore stage for the induction of pollen division
has been determined (from late uninucleate microspore to binucleate pollen stage) as
well as the culture conditions for the production of proembryoids from isolated olive
microspores have been defined (33°C for 24 h or 3°C during 96 h). In this work, the
obtained multicellular olive microspores (proembryos) represent the first step
towards haploid olive embryo production.
Acknowledgements. This research was supported by project granted by Spanish Ministry of
Education and Science (MEC) RTA2005-00118-C02-02 and CAO00-018 from the Consejería
de Agricultura y Pesca of the Junta de Andalucía. The collaboration of Drs. J.M. Caballero
and C. del Rio, from the CIFA of Córdoba, Spain, is very much appreciated. The authors
acknowledge the bilateral agreement between INIA-CSIC Joint Collaboration Program
(Convenio CC03-023). Dr. Pintos was recipient of a PhD grant of the National Institute of
Agronomic Research (INIA).
4. REFERENCES
Barro, F. & Martin, A. (1999) Response of different genotypes of Brassica carinata to microspore
culture. Plant Breeding 118, 79–81.
Bayliss, K.L., Wroth, J.M. & Cowling, W.A. (2004) Pro-embryos of Lupinus spp. produced from isolated
microspore culture. Austr. J. Agricult. Res. 55, 589–593.
Bueno, M.A., Gómez, A., Boscaiu, M., Manzanera, J.A. & Vicente, O. (1997) Stress-induced formation
of haploid plants through anther culture in cork-oak (Quercus suber L.). Physiol. Plant. 99, 335–341.
Bueno, M.A., Gómez, A. & Manzanera, J.A. (2000) Somatic and gametic Embryogenesis in Quercus
suber L. In Jain, S.M., Gupta, P.K. & Newton, R.J. (Eds). Somatic Embryogenesis in Woody Plants.
Forestry Sciences. Vol 6. Kluwer Academic Publishers. Dordrecht/Boston/London. ISBN 0-7923-
6419-8, pp. 479–508.
Bueno, M.A. & Manzanera, J.A. (2003) Oak anther culture. In Doubled Haploid Production in Crop
Plants. A Manual. Maluszynski, M., Kasha, K.J., Foster, B.P. & Szarejko, I. (Eds). Kluwer Academic
Publishers, Dordrecht. ISBN 1-4020-1544-5, pp. 297–301.
Bueno, M.A., Pintos, B., Gomez, A. & Martin, A. (2004) Monitoring of in vitro cultured microspores
switched to embryogenesis in Olea europaea L. Polen: Proc. XI International Palynological Congress,
14, pp. 26.
Bueno, M.A., Pintos, B., Höfer, M. & Martin, A. (2005) Pro-embryos induction from Olea europaea L.
isolated microspore culture. Acta Physiol. Plant. 27, 695–701.
Chupeau, Y., Caboche, M. & Henry, Y. (1998) Androgenesis and haploid plants., Springer-Verlag,
Berlin, Heidelberg.
Germana, M.A. & Chiancone, B. (2003) Improvement of the anther culture protocol in Citrus clementina
Hort. ex Tan. Plant Cell Rep. 22, 181–187.
Höfer, M. (2004) In vitro androgenesis in apple – improvement of the induction phase. Plant Cell Rep. 22,
365–370.
Höfer, M., Touraev, A. & Heberle-Bors, E. (1999) Induction of embryogenesis from isolated apple
microspores. Plant Cell Rep. 18, 1012–1017.
Kyo, M. & Harada, H. (1986) Control of the developmental pathway of tobacco pollen in vitro. Planta
168, 427–432.