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 - 5.59

KANAMYCIN-RESISTANT ALFALFA HAS A POINT MUTATION IN THE 16S PLASTID rRNA

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

*) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Perugia, Italy

**) Center for Applied Genetic Technologies, University of Georgia, Athens, GA 30602, USA

antibiotic resistance, homoplastomy, Medicago sativa L., plastid, Ribosomal RNA

Kanamycin, an aminoglycoside antibiotic, is used frequently to obtain transgenic plants by transforming plant cells with the nptII gene from E. coli as the selectable marker. Aminoglycoside antibiotics are toxic to prokaryotic cells and chloroplasts because they bind to the 30S ribosomal subunit at the decoding site (A-site) which causes the amino acid incorporation error rate to increase and inhibit ribosome translocation. Kanamycin-sensitive prokaryotic ribosomes, including those of the plastid and mitochondrion, have an A at the 1408 position of the 16S rRNA. Eukaryotic, kanamycin-insensitive, cytoplasmic ribosomes, have a G at position 1408 (E. coli base numbering). Spontaneous mutations to kanamycin resistance occur in Chlamidomonas reinhardtii (1, 3) but have not been reported to date in higher plants.

In attempting to transform the plastid genome of alfalfa using a kanamycin resistance gene as the selectable marker (2), somatic embryos were obtained after a minimum of two months of culture of leaf explants in the presence of 50 mg l^-1 kanamycin. Kanamycin resistance was confirmed by 4 to 5 regeneration cycles on kanamycin-containing media using mature somatic embryos to start each cycle. PCR and Southern analyses demonstrated that the kanamycin resistance gene was not present in the resistant embryos. Resistant plants produced 85% less biomass than controls and produced very few flowers. Their leaves were chlorotic as they formed and slowly became green. When leaf explants from resistant plants were used for regeneration, they produced less callus and embryos than control leaves on both kanamycin and kanamycin-free media; kanamycin presence resulted in slower regeneration and further reduced callus and embryo production, indicating that kanamycin resistance was not complete.

Sequencing of the plastid DNA region corresponding to the decoding site of the16S rRNA in 10 independent resistance events revealed a A=>C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A=>G conversion confers kanamycin resistance to C. reinhardtii.

All the progeny of the resistant plants obtained through somatic embryogenesis in the absence of kanamycin had the mutated phenotype, indicating that the mutation was homoplastomic.

We propose that kanamycin resistance in alfalfa requires homoplastomy for the described plastid rDNA mutation, which reduces binding affinity of the plastid ribosome for the antibiotic and adversely affects plastid translation.

REFERENCES

1. Bartlett SG, Harris EH, Grabowy CT, Gillham NW, Boynton JE (1979) Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii. Mol Gen Genet 176: 199-208

2. Bateman JM, Purton S (2000) Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker. Mol Gen Genet 263: 404-410

3. Harris EH, Burkhart BD, Gillham NW, Boynton JE (1989) Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome. Genetics 123: 281-292.