Minigrafting was used for rescue of tissue culture regenerants of the following four species of Citrus: sour orange (C. aurantium L. `AA CNR 31'), sweet orange [C. sinensis (L.) Osb. `Valencia Late'], lemon [C. limon (L.) Burm. `Femminello Comune'] and mandarin (C. deliciosa Tenore `Tardivo di Ciaculli'). The grafting was carried out with different scion types including shoots, roots, inverted roots and somatic embryos. This material was obtained in vitro from embryogenic style-derived callus. Seedlings of open-pollinated sour orange (C. aurantium L.), Cleopatra mandarin (C. reshni Hort. ex Tan.) and `Troyer' citrange [C. sinensis Osb. × Poncirus trifoliata (L.) Raf.] were used as rootstocks. Minigrafting of shoots, roots, inverted roots and embryos regenerated in vitro allowed successful rescue of these four species. Percentages of successful minigrafts ranged from 100% (shoots) to 2.5% (inverted roots). The probability of successful graft unions increased with the age of the rootstock. The final mean canopy leaf area (120 days after grafting) ranged from 5.2 cm2 (`Tardivo di Ciaculli' mandarin grafted on 6-month-old Cleopatra mandarin) to 157.9 cm2 (`Valencia Late' sweet orange grafted on 18-month-old Cleopatra mandarin). In this work we examined some of the variables which influenced minigrafting and we determined the efficacy of this method for rescue of in vitro regenerants of Citrus. This method is also suggested as a technique to produce a high percentage of viable plants from in vitro regenerants difficult to root.
Fabio De Pasquale, Salvatore Giuffrida, and Francesco Carimi
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.
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.