Induction Of Sporophytic Division In Orchids Microspores By Stress

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