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

FLOW CYTOMETRY ANALYSIS AND MODIFIED AFLP TECHNIQUE FOR A RAPID CHARACTERIZATION OF INTRASPECIFIC HYBRID CROSSES BETWEEN “FEMMINELLO” LEMON + “VALENCIA” ORANGE CYBRIDS AND A SELECTION OF “FEMMINELLO” LEMON TOLERANT TO “MALSECCO” DISEASE

NARDI L.*, ABBATE L.**, AIELLO M.*, FERRANTE S.**, LUCRETTI S.*, SCARANO M.-T.**, TUSA N.**

* ENEA C.R. Casaccia Divisione Biotecnologia e Agricoltura, Via Anguillarese 301, 00100 Roma

luca.nardi@casaccia.enea.it

** Istituto di Ricerca per la Genetica degli Agrumi – CNR, Viale delle Scienze 11, 90128 Palermo

cybrids, breeding, malsecco, flow cytometry, modified AFLP

Somatic hybridization and cybridization via protoplast fusion has become an integral part of Citrus breeding programs worldwide for obtaining seedless and more healthy new cultivar (Grosser et al, In Vitro Cell Dev. Biol.-Plant 2000, 36:434-449). Our collection of ‘Valencia’ sweet orange [Citrus sinensis (L.) OSbeck] and ‘Femminello’ lemon [C. limon (L.) Burm.] somatic hybrid and cybrids (Grosser et al, Plant Cell Reports 1996, 15:672-676) was tested toward “mal secco” infection caused by Phoma tracheiphila (Petri) Kanc. et Ghik. and it showed an intermediate degree of resistance, with slight differences in disease symptoms, in comparison with resistant 'Monachello' lemon and susceptible 'Femminello' lemon, used as controls (Tusa et al, Hortscience 2000,35:125-127). An attempt to obtain new genotypes of lemon tolerant to “malsecco” has been carried out by intra-specific crossing using 2n and 4n cybrids of ‘Femminello’ + ‘Valencia’ crossed with one of our selections of diploid ‘Femminello’ lemon tolerant to the disease (LRMS). In order to investigate mitochondrial inheritance in the progeny, cybrids have been used as mother-plant and the LRMS clone as pollen donor parent. In the crosses between the tetraploid cybrid and this diploid lemon clone, seedless triploid genotypes tolerant to the “malsecco” could be obtained. The immature small fruits have been collected by embryo-rescue at approximately 105 days after pollination when the zygotic embryos are more numerous than nucellar embryos. Since the two parents are phylogenetically very close, it was almost impossible to distinguish the zygotic embryos from the nucellar ones on the basis of biochemical results such as zymograms. The combined use of flow cytometry for ploidy discrimination and AFLP technique to evaluate genetic recombination proved to be very effective in identifying true crosses and their genetic constitution. Molecular screening was conducted on the basis of a fast but reproducible molecular marker technique as the modified AFLP (Vos et al, Nuc. Acids Res. 1995, 23:4407-4414) that has been recently set on for plants (Ranamukhaarachchi et al, BioTechniques 2000, 29:858-866) and which uses one restriction enzyme, one adapter and one primer; PCR products were analysed by agarose gel electrophoresis followed by ethidium bromide staining and incorporating formamide to generate more intense and uniform bands. This approach turns out to be faster, more user-friendly and less expensive than the classic AFLP, and it does not require the use of radioisotopes. Our investigations confirmed the presence of zygotic hybrids among the screened population which could be of interest in obtaining new valuable resistant lemon cultivars for the italian citriculture.