APPROACHES TO STUDY BIODIVERSITY AND SALINITY TOLERANCE MECHANISMS IN MEDITERRANEAN FRUIT TREE SPECIES

Changes in global climate are going to increase the problem of drought and soil salinity in the Mediterranean area, where increases in temperature, drought and soil and water salinity are expected. Identification of plant genotypes with positive agronomic traits, such as salt tolerance, may reduce the effects of salinity and drought on productivity. Therefore, there is considerable effort being directed toward the development of salt-tolerant genotypes through plant breeding, with the aim to the introgression of salt-tolerance traits into new cultivars. The results presented in this thesis contributed to the develop of new tools to improve knowledge on the biodiversity and salinity tolerance on two minor fruit tree crops grown in Mediterranean countries: Fig (Ficus carica L.) and Pistachio spp. In recent years, the interest for these two tree fruit crops is greater than before because of their nutritional values and economic importance. The Fig tree is one of the most ancient cultivated fruit tree crops in South Italy. To explore biodiversity in the Fig, two different experiments were carried out. The main objective of the first experiment was to characterize and evaluate the genetic diversity among 181 accessions, using SSR markers. A total of 117 alleles were detected with a mean of 6.5 per locus. The average expected (He) and observed heterozygosity (Ho) were 0.56 and 0.66, respectively. The mean polymorphic information content (PIC) was 0.51, from 0.78 (LMFC30) to 0.21 (LMCF26), suggesting a high molecular diversity among the fig accessions. The UPGMA cluster analysis discriminated 174 genotypes and identified to eight groups. The accession ‘Bianca d’Agosto’ from Basilicata and the genotype “FCS138” from Calabria were the most diverse. These findings demonstrate the richness of the fig’s genetic resources in Southern Italy, resolved cases of synonymies and homonymies, and characterized fig accessions. In a second experiment, an analysis into the genetic variability was conducted within the “Dottato” cultivar, which has fruits that are admitted to two EU Protected Designation of Origin (PDO) “Fico Bianco del Cilento” (Campania) and “Fichi di Cosenza” (Calabria). Dottato is grown in large parts of Southern Italy and it is well regarded for its parthenocarpic fruits and their excellent organoleptic characteristics, still maintained after drying and processing. To preserve the high quality standard of the fruits admitted to PDO, it is important to know the potential genetic variation within the Dottato. The morphological and molecular diversity were studied by evaluating 24 morphological traits and by genotyping with 18 microsatellite markers. The microsatellite allelic profiles among the putative clones of ‘Dottato’ indicated a moderate genetic variability. The average expected and observed heterozygosity were 0.42 and 0.62, respectively. The mean polymorphic information content (PIC) was 0.4, varying from 0.08 (LMFC26) to 0.67 (FCUP 38_6), suggesting a low level of genetic diversity. The morphological clustering showed the uniformity of 19 genotypes whereas 5 were different. Part of this PhD project was to develop new methods for studying, at cellular level, the mechanisms of plant salinity resistance by evaluating the uptake, transport and sequestration of the sodium, potassium and chloride ions in roots and in leaves. The research was conducted on “in vitro” plant of pistachio species (P. atlantica, P. integerrima) and their interspecific hybrid (UCB1), that are commonly used as rootstocks. The seedlings obtained from “in vitro” germination were cultivated on a ½ MS medium supplemented with 7gr/L of agar and 15 gr/L sucrose. The salinity treatments (100umM of NaCl) were applied on 2-month-old seedlings for 7 days. Roots and leaves were sectioned with the vibratome, and then incubated in osmolality maintaining buffer to ensure tissue viability. In roots sections, micrographs were recorded on the Confocal Fluorescence Microscopy (ZEISS LSM710/700), the CoroNa-Green AM (Invitrogen), and the Asante Potassium Green were used to detect sodium and potassium respectively. Sections of leaves of UCB-1 and P. integerrima treated and control were incubated in CoroNa-Green AM (Invitrogen) and co-stained with SNARF-1 (carboxylic acid, acetate, succinimidyl ester Molecular Probes, Inc., Eugene, OR), micrographs were recorded on the fluorescent laser scanning confocal microscopy Leica SP8/ SP8 MP Microscope was used. In roots, distinct accumulation patterns of sodium were observed at the subcellular level in UCB-1 and P. integerrima. The potassium signal was stronger in the endodermis of all three genotypes subjected to saline stress. In leaves, a new methodology was developed that removed chlorophyll auto-fluorescence signal, allowing the distinction between different compartmentalization ability of sodium ions between the genotypes: only in UCB1 were vacuoles stained. The higher salt tolerance of UCB1 was also confirmed by a higher survival rate and no damage in the leaves. The inability of P. Integerrima to accumulate sodium in vacuoles can be correlated with a higher sensitivity to salt stress, evidenced by greater damage in the leaves with chlorosis and necrosis, and a lower survival rate. The new methods developed can be transferred to other fruit crops, providing a unique opportunity to assess salt resistance. The results presented in this thesis contributed to develop new tools to improve knowledge on the biodiversity and salinity tolerance. Application of these new approaches in larger genotype screening permits to identify genotypes with salinity tolerance and to have a better understanding of the salinity tolerance mechanisms.