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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

VECTORS FOR CHLOROPLAST TRANSFORMATION OF MEDICAGO SATIVA L.

ROSELLINI D.*, LAFAYETTE P. R.**, VERONESI F.*, PARROTT W. A.**

* Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Sezione Genetica e Miglioramento Genetico, Borgo XX Giugno 74, 06121 Perugia

roselli@unipg.it

** Department of Crop and Soil Sciences, University of Georgia-Athens, USA

Alfalfa, genetic engineering, homologous recombination, plastome

The transformation of the chloroplast genome (plastome) shows promises for genetic engineering of crop plants because of the potential for a high expression level of the introduced gene, absence of position effects, and facilitated gene containment in some crops.

For plastome transformation, the gene of interest must be flanked by plastome sequences of the recipient plant, to allow for homologous recombination. These sequences should target the insertion such that no indispensable gene is mutated.

A 2828 bp plastome sequence encompassing part of the psbA gene, the trnH gene and part of the ndhF gene was amplified from genomic DNA of a selected, highly regenerating plant of the alfalfa cultivar Regen-SY (RSY1). The primers were designed based on the published sequences of alfalfa psbA and tobacco ndhF, and on the putative position of ndhF in an alfalfa plastome restriction map. The psbA-trnH intergenic region has been described as highly polymorphic in plants, and potentially suitable for genetic engineering. Sequencing proved that no stop codon or frame shift mutations were introduced, and that the predicted amino acid sequence of the unpublished ndhF gene does not diverge significantly from those of other plants. The enzymes BstYI and AclI were used to trim the sequence, which was then cloned into the vector pMECA.

A Bstz17I restriction site in the psbA-trnH intergenic region was used for cloning two selection cassettes into the homologous recombination region, resulting in two alfalfa plastome transformation vectors. In the first vector pMScp-aadA, the selection cassette includes the tobacco chloroplast 16S rRNA promoter, the E.coli aminoglycoside 3'-adenyltransferase gene (aadA), conferring spectinomycin and streptomycin resistance, and the 3’ untranslated region of the Chlamydomonas reinhardtii rbcL gene. The susceptibility of alfalfa to spectinomycin and streptomycin was tested, showing that alfalfa does not regenerate in the presence of spectinomycin or streptomycin at concentrations greater than 50 mgl^-1.

In the second vector pMScp-aphA, the selection cassette is formed by the 5’ untranslated region of the C. reinhardtii psbA gene, the Acinetobacter baumannii aminoglycoside phosphotransferase gene (aphA-6), conferring kanamycin resistance to C. reinhardtii, and the 3’ untranslated region of the C. reinhardtii rbcL gene. The aphA-6 has not been previously tested in higher plants. Alfalfa does not regenerate in the presence of kanamycin, at concentration greater than 50 mgl^-1.

In both vectors, the selection cassettes are flanked 5’ by 971 and 3’ by 1431 bp of targeting sequence. The selection cassettes were tested by expressing the antibiotic resistance in E. coli. Both vectors are currently in use for biolistic transformation of RSY1 to develop a system for plastome engineering in alfalfa.