ALISHER TOURAEV
Institute of Microbiology and Genetics, Vienna Biocenter, Vienna University, Dr. Bohrgasse 9, A-1030 Vienna, Austria
The natural destination of microspore development is to differentiate into mature pollen and accomplish fertilisation. Isolated and in vitro cultured microspores or young pollen grains can either differentiate into mature, fertile pollen (the male gametophytes) by culture in a rich medium without stress, or divide repeatedly and develop into embryos (sporophytes) after a stress treatment.
As experimental systems, microspore cultures are used to investigate pollen development and pollination, embryogenesis, totipotency, cytodifferentiation, cell cycle, and the role of stress in development. As a tool in genetic engineering, they can be used to produce doubled haploids (recombinant inbreds) for plant breeding and gene mapping, to induce and select for mutants and to create transgenic plants.
The development of isolated, defined microspores undergoing in vitro embryogenesis has been followed by cell tracking. A modified suppression subtractive hybridization method was used to isolate genes involved in the formation of embryogenic tobacco microspores. One of isolated genes, called NtSM10 (Nicotiana tabacum Stressed Microspore N10) was subjected to more detail analyses of expression pattern using stable transgenic plants harbouring the NtSM10 promoter region fused to the GUS reporter gene. The expression of the GUS reporter gene in transgenic plants was found in embryogenic microspores and microspore derived embryos, only. The function of the NtSM10 gene was studied using RNAi and over-expression strategies. Transgenic tobacco plants, which contained the NtSM10 cDNA over-expressed under the DC3 promoter were completely male sterile. The inhibition of transcription by RNAi in transgenic plants led to the normal pollen development, but all microspores died when cultured under the stress condition to deviate the gametophytic development towards the sporophytic pathway.
Taken together these experiments strongly suggest that the NtSM10 gene is responsible for stress induced microspore embryogenesis and causes male sterility when over-expressed.