Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.60

gfp as a useful reporter for transient expression and stable transformation in alfalfa (Medicago sativa L.)

M. BELLUCCI*, F. DE MARCHIS*, R. MANNUCCI**, S. ARCIONI*

*) Institute of Plant Genetics, Research Division of Perugia, CNR, Via Madonna Alta, 130, 06128 Perugia, Italy

**) Dept Clin. Exp. Med. Sect. Internal Medicine & Oncology, Perugia University Medical School, Policlinico Monteluce, 06122 Perugia, Italy

transgenic alfalfa, green fluorescent protein, transit peptide, plastid targeting, bombardment

Reporter genes coding for proteins that possess an unique activity, or produce a signal that allows the protein to be easily distinguished from other proteins, are essential tools of molecular biology. The use of jellyfish green fluorescent protein (GFP) as a reporter in plant biology research is extensively documented. Transient expression of two green fluorescent protein (GFP) gene constructions was compared in alfalfa (Medicago sativa L.) leaves: the gfp containing the mutation S65C in an essentially mgfp4 background (Reichel et al. 1996) coding for GFP S65C versus the mgfp5-ER (Haseloff et al. 1997) coding for mGFP5. The fluorescent signal was brighter in cells transiently transformed with the gfp S65C gene than with the mgfp5-ER gene. Based on these results, the gfp S65C gene was used to assemble other chimaeric genes. First, a chloroplast-targeted GFP producing green-fluorescent chloroplasts inside transiently transformed cells was constructed. Then, a chloroplast-targeted fusion protein between a maize protein (beta-zein) and GFP was assembled, and this protein accumulated in small aggregates into the chloroplasts of transiently transformed cells. Furthermore, the gfp S65C gene was used to obtain transformed alfalfa plants by particle bombardment. Strong GFP expression was observed in several kinds of tissues from T0 plants derived from a single genotype.