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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 3.46

STABLE EXPRESSION OF GREEN FLUORESCENT PROTEIN IN MEDICAGO SATIVA L. USING THE BIOLISTIC TECHNIQUE

Bellucci M., De Marchis F., Arcioni S.

Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere, Via Madonna Alta, 130, 06128 Perugia, Italia

plant transformation, genetic marker, Medicago sativa, green fluorescent protein, biolistic technique

The green fluorescent protein gene (gfp) is a widely used reporter in both animals and plants. A gene construct between the cauliflower mosaic virus 35S promoter and the gfp coding region has been delivered to Medico sativa L. (alfalfa) genome using gold microprojectiles. Alfalfa leaves were bombarded and cultured on a callus-induction medium with kanamycin selection. After forty days GFP-positive calli were selected and transferred to a regeneration-medium. GFP was detected using a conventional fluorescence microscope. Regenerated plants were analysed for gfp expression and all the tissues of the leaves revealed the green fluorescence both in the nucleus and in the cytoplasm of the cells. Nuclear gene introgression using A.tumefaciens with the same plasmid is in progress in order to compare the efficiency of this method for alfalfa transformation with that of biolistic bombardment. The development of an efficient particle bombardment protocol in alfalfa, in addition to existing Agrobacterium technologies, would allow the investigator to be able to choose the most suitable technology in relation to the objectives. For example, when the new traits to be introduced require the co-ordinated expression of several genes, particle bombardment could facilitate and speed up the necessary work because the efficiency of co-integrating plasmids using this system has been well documented in the literature. Moreover, particle bombardment offers the advantage of engineering the chloroplast genome (plastome). Furthermore, the stable expression of gfp in alfalfa demonstrates that GFP can be used as a reporter even in this fodder crop species.