Orchid is one of the important ornamental plants in Indonesia this plant generally propagated by seed. Enhancing quality of this plant through breeding technology by various plant tissue culture methods and biotechnology, including doubled haploid technology are necessary. The most efficient method in creating doubled haploids plant is via microspore embryogenesis. We have develop new, innovative doubled haploid technology using the technique of isolated microspore culture. The goals are to obtain data on the male gametophyte development, viable embryogenic microspores, microspores derived embryos and double haploid plants of Orchid.
Development of male gametophytes were analysed by isolation of microspores and pollen at various stages and staining with DAPI. Isolated orchid buds of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata were subjected to cold temperatures (4oC) for 7 days, microspores were then isolated by crushing the pollinia using glass rod and cultured them in embryogenesis A2, NP, MS and VW medium, viability of the microspores were determine by using Flourescein diacetate (FDA). Isolated Orchid pollinia were cultured in starvation medium B at various temperatures and duration of time to evaluate embryogenic response, isolated microspores then were cultured further in the basic embryogenesis medium and incubated at 25 oC in the darkness.
The result showed that floral characteristics for the late-uninucleate stage of the microspores were different for every orchid spesies. Ovulum lenght was used for Vanda, while in Dendrobium, Phalaenopsis, Arachnis, Spathoglottis plicata and Cattleya, varied length of flower bud was used. Isolated microspores of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata at 7th days of culture in different media formulation showing different respond of viability. Medium A2 keeping viability of Dendrobium hybrid 1 microspores better than any other medium, while in Vanda tricolor and Spathoglotis plicata embryogenesis NP medium was superior. Incubation of orchid pollinia at 4 and 25 oC were successfully maintain viability of the microspores during starvation periods but not able to block gametophytic development. In contrast starved pollinia at 33oC were succesfully block gametophytic development, percentage of embryogenic microspores after starvation of isolated pollinia at 33°C for 4 days was superior compare to any other treatments. Symmetrical divisions and some multicellular structures were observed, which were clear indication for the sporophytic development of microspore-derived embryos, they had developed and after a few weeks they degenerated and died.

Keywords: flower bud-pollinia-microspore-stress-embriogenic-embryo-Orchid

Bell, P.R.1992. Apospory and apogamy: Implications for understanding the plant life cycle. Int J Plant Sci 153: 123-136.
Binarova, P., G. Hause, V. Cenklova, J.H.G. Cordewener, and M.M. Van Lookeren Campagne. 1997. A short-severe heat shock is required to induce embryogenesis in late bicellular pollen of Brassica napus L. Sex. Plant Reprod 10:200-208
Cordewener, J.H.G., R. Busink, J.A. Traas, J.B.M Custers, H.J.M. Dons, and M.M. Van Lookeren Campagne. 1994. Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis. Planta 195:50-56
Cordewener, J.H.G., G. Hause, E. Görgen, R. Busink, B. Hause, J.J.M. Dons, A.A.M. Van Lammeren, M.M. Van Lookeren Campagne, and P. Pechan. 1995. Changes in synthesis and localization of the 70 kDa class of heat shock proteins accompany the induction of embryogenesis in Brassica napus L microspores. Planta 196:747-755
Dafni, A., and D. Firmage. 2000.Pollen viability and longevity: Practical, ecological and evolutionary implications. Plant Syst.Evol.222: 113-132
Dickinson, H.G. 1994. The regulation of alternation of generation in flowering plants. Biol Rev 69:419-442
Duncan, E.J., and E. Heberle. 1976. Effect of temperature shock on nuclear phenomena in microspores of Nicotiana tabacum and consequently on plantlet production. Protoplasma 90:173-177
Garrido, D., O. Vicente, E. Heberle-Bors, and M. Isabel Rodriguez-Garcia. 1995. Cellular changes during the acquisition of embryogenic potential in isolated pollen grains of Nicotiana tabacum. Protoplasma 186:220-230.
Guha, S., and S.C. Maheshwari. 1964. In vitro production of embryos from anther of Datura. Nature 204: 497.
Guha, S., and S.C. Maheshwari. 1966. Cell division and differentiation of embryos in the pollen grains of Datura in vitro. Nature 212: 97-98.
Hause, B., G. Hause, G. Pechan, and A.A.M. Van Lammeren. 1993. Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L. Cell Biol Int 17:153-168
Hoekstra, S., M.H. van Zijderveld, J.D. Louwerse, F. Heidekamp, and F. van der Mark. 1992. Anther and microspore culture of Hordeum vulgare L. cv. Igri. Plant Sci 86: 89-96.
Indrianto, A., E. Heberle-Bors, and A. Touraev. 1999. Assessment of various stresses and carbohydrates for their effect on the induction of embryogenesis in isolated wheat microspores. Plant Sci 143: 71-79.
Indrianto, A., I. Barinova, A. Touraev, and E. Heberle-Bors. 2001. Tracking individual wheat microspores in vitro: Identification of embryogenic microspores and body axis formation in the embryo. Planta 212: 163-174
Kyo, M., and H. Harada. 1990a. Specific phosphoproteins in the initial period of tobacco pollen embryogenesis. Planta 182 :58-63.
Yo, M., and H. Harada. 1990b. Phosphorylation of proteins associated with embryogenic dedifferentiation of immature pollen grains of Nicotiana rustica. J. Plant Phsiol 136:716­722
Mc Cormick, S. 1993. Male gametophyte development. The Plant Cell 5: 1265-1275
Morison, R.A., and D.A Evans. 1988. Haploid plants from tissue culture: new plant varieties in a shorthened time frame. Biotechnology 6: 684-690
Ogawa, T., H. Fukuoka, and Y. Ohkawa. 1994. Induction of cell division of isolated pollen grains by sugar starvation in rice. Breed Sci 44: 75-77.
Segui-Simarro, J.M., P.S. Testillano, and M.C. Risueno. 2003. HSP70 and HSP90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L. J Struc Biol 142:379-391
Simmonds, D.H., and W.A. Keller. 1999. Significance of preprophase bands of microtubuls in the induction of microspore embryogenesis of Brassica napus. Planta 208: 383-391.
Telmer, C.A., W. Newcom, and D.H. Simmonds. 1993. Microspore development in Brassica napus and the effect of high temperature on division in vivo and in vitro. Protoplasma 172:154-165
Touraev, A., A. Ilham, O. Vicente, and E. Heberle-Bors. 1996a. Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep 15: 561-565.
Touraev, A., A. Indrianto, I. Wratscho, O. Vicente, and E. Heberle-Bors. 1996b. Efficient microspore embryogenesis in wheat (Triticum aestivum L) induced by starvation at high temperatures. Sex Plant Repr 9: 209-215.