Monitoring the fate of GFP-targeted organelles during pollen development in wheat

摘要

Genetic modification of the plastid genome complements and has several advantages over conventional nuclear transformation. The predominance of homologous recombination in plastids allows precise gene targeting and the elimination of unwanted marker gene sequences. Additionally, the predominantly maternal pattern of plastid inheritance prevents trait gene transmission via pollen and results in enhanced transgene containment. However, the mechanisms that control maternal inheritance of plastids are far from clear. We are using a transformation-based approach to generate stable nuclear transgenic wheat plants containing plastid targeted-GFP to monitor the fate of 'GFP-tagged' plastids during pollen development. GFP is an excellent in vivo marker for gene expression and protein localisation studies. Transgenic cereals transformed with pACTlIsGFP express GFP in the cytosol. To target GFP to plastids, the maize ferredoxin presequence was fused in frame to GFP coding sequences. Particle bombardment was used to transform these constructs into immature wheat embryos. Transient expression studies in bombarded wheat scutella revealed localisation of plastid targeted GFP to plastids whilst cytosolic targeted GFP was distributed uniformly within cells. Observations of pollen from transgenic lines revealed that plastid targeted GFP was strongly expressed in late uni-nucleate microspores and subsequent pollen developmental stages. GFP-tagged plastids will enable the fate of plastids to be followed during pollen maturation.