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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

BIOLISTIC TRANSFORMATION OF MEDICAGO SATIVA L.

BELLUCCI M., DE MARCHIS F.

Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere (IRMGPF), CNR, Via della Madonna Alta 130, 06128 Perugia, Italia

s.arcioni@irmgpf.pg.cnr.it

alfalfa, transgenic plants, chloroplast transformation

A range of different transformation procedures have been developed for alfalfa which are based either on direct gene transfer into protoplasts or on the use of Agrobacterium. Agrobacterium is the most diffuse method for alfalfa genetic transformation and two Agrobacterium species are used, A.tumefaciens and A.rhizogenes. Conversely, there are only few reports on stable transformation of alfalfa by particle bombardment and considerable optimisation of the protocol remains to be done if transformation efficiency by this method is to become comparable with Agrobacterium systems. Indeed, the development of efficient particle bombardment protocols, 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 introduce would 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). Chloroplast genetic engineering usually ensures high levels of gene expression and methods to eliminate the marker genes by using this technology are already available. Several gene constructs, either for nuclear transformation or for plastome transformation, incorporating the green fluorescent protein (GFP) are in preparation. Nuclear gene introgression will be achieved by using A.tumefaciens and the biolistic bombardment in order to compare the efficiency of this two methods for alfalfa transformation. Transient expression of the GFP protein will be utilised to set up a reliable and reproducible protocol for particle bombardment of different tissues. As regards plastome transformation, isolation of gene sequences belonging to the alfalfa plastome has been attempted and these sequences will be used to assemble plasmids for alfalfa chloroplast transformation.