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Nicasio Tusa, Sergio Fatta Del Bosco, Franco Nigro, and Antonio Ippolito

The reaction of lemon hybrids obtained by symmetrical and asymmetrical protoplast fusion, toward “mal secco” infection caused by Phoma tracheiphila (Petri) Kanc. et Ghik. was examined. Resistance was tested in `Valencia' sweet orange [Citrus sinensis (L.) Osbeck] and `Femminello' lemon [C. limon (L.) Burm. f.] somatic hybrid and cybrids, by stem and leaf inoculation tests and by analysis of propagule number of P. tracheiphila in the xylem of stem-inoculated plants. In general, the somatic hybrid and the cybrids 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. The lower mortality in the asymmetrical lemon cybrids suggests that specific mechanisms of resistance to the disease could be activated in these genotypes.

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Mirko Siragusa, Fabio De Pasquale, Loredana Abbate, and Nicasio Tusa

A collection of 18 accessions of sour orange (Citrus aurantium L.) coming from Sicily and other countries was investigated by two polymerase chain reaction (PCR)-based DNA marker technologies. Ten inter-simple sequence repeat (ISSR) primers and fifteen randomly amplified polymorphic DNA (RAPD) primers were used to identify and to evaluate the genetic variability and relationship of accessions. A total of 111 ISSR and 145 RAPD amplified fragments were used to estimate the Dice's coefficient of similarity for cluster analysis using a unweighted pair-group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using ISSR and RAPD markers were highly concordant, such that the correlation between ISSR and RAPD genetic distance (GD) estimates was r = 0.93. The ISSR and RAPD analysis of 18 sour orange accessions found a high grade of genetic diversity in foreign accessions, while a low variability was detected in local accessions. Sicilian accessions could be grouped in two distinct clusters, including indistinctly plants from three origin regions. Some markers could be linked to the different growing areas. The ISSR and RAPD molecular reference system seems to be suitable for a fine identification of tightly related plants and the obtained results can form the basis for future setting up of Citrus rootstock genetic improvement projects.

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Mirko Siragusa, Fabio De Pasquale, Loredana Abbate, Letizia Martorana, and Nicasio Tusa

There is a high level of diversity among lemons [Citrus limon (L.) Burm. f. (2n = 2x = 18)] in Sicily, where each growing area has a wide range of landraces mostly derived from bud mutation. Because this variability represents an important resource for future breeding programs and genetic improvement, the relationships among the principal 36 accessions of Sicilian lemon, belonging to three different cultivars (Femminello, Monachello, and Lunario), were examined by intersimple sequence repeat and random amplified polymorphic DNA markers. Three ‘Femminello’ accessions from nearby Italian regions were also examined to study the genetic flow from the continent. The disputed case of the accession ‘Eureka Messina lemon’ was also examined, using ‘Frost Eureka’ as a control. Our results confirmed the extreme polymorphic nature of the three principal Sicilian cultivars and the presence of a wide range of different genotypes. Twenty-two Sicilian genotypes were recognized as unique accessions, reflecting the richness of the lemon germplasm present in Sicily. Each growing area showed the presence of several genetically different landraces, probably preserved by genetic isolation, whereas the continental accessions appeared extremely similar to the island genotypes, showing an exchange of germplasm from the island to the continent.

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Eliezer S. Louzada, Jude W. Grosseti, Frederick G. Gmitter Jr., Beatriz Nielsen, J.L. Chandler, Xiu Xin Deng, and Nicasio Tusa

Protoplast culture following polyethylene glycol-induced fusion resulted in the regeneration of vigorous tetraploid somatic hybrid plants from eight complementary parental rootstock combinations: Citrus reticulata Blanco (Cleopatra mandarin) + C. aurantium L. (sour orange), C. reticulata (Cleopatra mandarin) + C. jambhiri Lush (rough lemon), C. reticulata (Cleopatra mandarin) + C. volkameriana Ten. & Pasq. (Volkamer lemon), C. reticulata (Cleopatra mandarin) + C. limonia Osb. (Rang-pur), C. sinensis (L.) Osb. (Hamlin sweet orange) + C. limonia (Rangpur), C. aurantium (sour orange) + C. volkameriana (Volkamer lemon) zygotic seedling, C. auruntium hybrid (Smooth Flat Seville) + C. jambhiri (rough lemon), and C. sinensis (Valencia sweet orange) + Carrizo citrange [C. paradisi Macf. × Poncirus trifoliata (L.) Raf.]. Diploid plants were regenerated from nonfused callus-derived protoplasts of Valencia sweet orange and Smooth Flat Seville and from nonfused leaf protoplasts of sour orange, Rangpur, rough lemon, and Volkamer lemon. Regenerated plants were classified according to leaf morphology, chromosome number, and leaf isozyme profiles. All somatic hybrid plants were tetraploid (2n = 4× = 36). One autotetraploid plant of the Volkamer lemon zygotic was recovered, apparently resulting from a homokaryotic fusion. These eight new citrus somatic hybrids have been propagated and entered into field trials.