Increased Zygote-Derived Plantlet Formation through In Vitro Rescue of Immature Embryos of Highly Apomictic Opuntia ficus-indica (Cactaceae).

O. ficus-indica (prickly pear cactus) is an important forage and food source in arid and semiarid ecosystems and is the most important cactus species in cultivation globally. The high degree of apomixis in the species is a hindrance in plant breeding programs where genetic segregation is sought for the selection of superior genotypes. To understand if in ovulo embryo rescue could increase the proportion of zygotic seedlings, we compared the mature seed-derived seedlings with those regenerated from in vitro embryo rescue at 20, 25, 30, 35, and 40 post-anthesis days (PADs) in four Italian cultivars. The seedlings were classified as apomictic or zygotic based on molecular marker analysis using inter-sequence single repeat (ISSR) primers. Multiple embryos were recovered from all the cultured immature ovules, and plantlets were regenerated and acclimatized to the field post hardening, with success rates ranging from 62% ('Senza spine') to 83% ('Gialla'). The level of polyembryony differed among cultivars and recovery dates, with the highest being 'Rossa', producing 4.8 embryos/ovule at 35 PADs, and 'Gialla', the lowest, with 2.7 at 40 PADs. The maximum number of embryos observed within a single ovule was 14 in 'Trunzara bianca'. ISSR analysis revealed that ovule culture at 35 PADs produced the highest percentage of zygotic seedlings in all the cultivars, from 51% ('Rossa') to 98% ('Gialla'), with a high genotype effect as well. Mature seeds produced much fewer seedlings per seed, ranging from 1.2 in 'Trunzara bianca' to 2.0 in 'Rossa' and a lower percentage of zygotic seedlings (from 14% in 'Rossa' to 63% in 'Gialla'). Our research opens a pathway to increase the availability of zygotic seedlings in O. ficus-indica breeding programs through in ovulo embryo culture.

Rootstocks with Different Tolerance Grade to Citrus Tristeza Virus Induce Dissimilar Volatile Profile in Citrus sinensis and Avoidance Response in the Vector Aphis gossypii Glover.

The citrus tristeza virus (CTV) is an agent of devastating epidemics of the citrus plant grafted on Citrus aurantium, one of the main rootstocks still used in the Mediterranean area. Consequently, CTV-tolerant alternative citrus rootstocks are considered necessary to manage this disease and/or its vector; that in Mediterranean countries is the aphid Aphis gossypii. In this study, we analyzed the VOCs emitted from Citrus sinensis plants grafted on the CTV-susceptible C. aurantium and on the CTV-tolerant Volkamer lemon, Forner-Alcaide no. 5, and Carrizo citrange. Furthermore, the aphid preference/avoidance response toward these combinations was evaluated in a semi-field experiment. The VOC profiles recorded on the leaves of C. sinensis grafted on the four rootstocks listed above showed significant differences in the abundances and ratios of the compounds emitted. The behavioral experiments indicated that A. gossypii prefers to orient and establish on the C. sinensis plants grafted on C. aurantium rather than on that grafted on the three CTV-tolerant varieties. The possibility that this avoidance mechanism is triggered by the different profile of the VOC emitted by the different combinations and the consequent susceptibility/tolerance shown toward CTV is discussed.

Different Cell Types Affect the Transition from Juvenile to Mature Phase in Citrus Plants Regenerated through Somatic Embryogenesis.

Robust protocols for the regeneration of somatic embryos in vitro are essential for the efficient use of the most modern biotechnologies. Unfortunately, in perennial trees such as Citrus, plants regenerated from juvenile tissues usually exhibit strong, undesirable juvenile characters such as thorny habit and delayed flowering and fruit production. In this work, we tested whether the cell types (nucellar and stigma/style) used to regenerate Citrus plants through somatic embryogenesis affected the transition from the juvenile to mature phase. The results show that regenerants from nucellar cells presented persistent juvenile characters, whereas plants originating from stigma/style explants transited to the mature phase more rapidly. Our observations support the hypothesis that the totipotent cells originated from different cell types are not equivalent, possibly by maintaining memory of their previously differentiated state.

Influence of Culture Conditions on In Vitro Asymbiotic Germination of Anacamptis longicornu and Ophrys panormitana (Orchidaceae).

This study is the first approach to in vitro asymbiotic germination of two species of Sicilian threatened terrestrial orchids, Anacamptis longicornu and Ophrys panormitana. Seeds were collected in the wild and cultured in two different media-Orchimax medium (OM) and Murashige and Skoog (MS)-and exposed to different photoperiods and temperatures to evaluate the best conditions for the specific stages of development. The germination of A. longicornu was very high on OM (95.5%) and lower on MS medium (21.4%), whereas O. panormitana germinated only on OM medium, with significantly lower percentages (12.0%), compared with A. longicornu. This difference is caused by variation in quality and quantity of nutrients used, primarily by nitrogen source. The results show that temperature and photoperiod widely affect seed germination and development. Although further investigations on asymbiotic and symbiotic germination are needed for the improvement of conservation of Mediterranean terrestrial orchids, our results contribute to the conservation of this group of plants.

Autotetraploid Emergence via Somatic Embryogenesis in Vitis vinifera Induces Marked Morphological Changes in Shoots, Mature Leaves, and Stomata.

Polyploidy plays an important role in plant adaptation to biotic and abiotic stresses. Alterations of the ploidy in grapevine plants regenerated via somatic embryogenesis (SE) may provide a source of genetic variability useful for the improvement of agronomic characteristics of crops. In the grapevine, the SE induction process may cause ploidy changes without alterations in DNA profile. In the present research, tetraploid plants were observed for 9.3% of 'Frappato' grapevine somatic embryos regenerated in medium supplemented with the growth regulators ß-naphthoxyacetic acid (10 µM) and N6-benzylaminopurine (4.4 µM). Autotetraploid plants regenerated via SE without detectable changes in the DNA profiles were transferred in field conditions to analyze the effect of polyploidization. Different ploidy levels induced several anatomical and morphological changes of the shoots and mature leaves. Alterations have been also observed in stomata. The length and width of stomata of tetraploid leaves were 39.9 and 18.6% higher than diploids, respectively. The chloroplast number per guard cell pair was higher (5.2%) in tetraploid leaves. On the contrary, the stomatal index was markedly decreased (12%) in tetraploid leaves. The observed morphological alterations might be useful traits for breeding of grapevine varieties in a changing environment.

From glacial refugia to hydrological microrefugia: Factors and processes driving the persistence of the climate relict tree Zelkova sicula.

With only two tiny populations, the climate relict Zelkova sicula (Sicily, Italy) is one of the rarest trees in the world. It also represents the most marginal member of genus Zelkova that was widespread in the broadleaved forests thriving in warm-temperate climates throughout Eurasia until the Last Glacial Age. Occurring at the westernmost range of the genus under typical Mediterranean climate, the micro-topographic settings have always appeared crucial for the survival of this relict. However, the factors and processes actually involved in its persistence in the current refugia, as well as the response of similar relict trees in arid environments, are poorly understood worldwide. In the aim to elucidate these aspects, in the two sites hosting Z. sicula analyses of topographical attributes were combined with investigations on soil moisture dynamics. Additionally, plants' growth and spatial distribution patterns were analyzed to detect fine-scale differences between populations and assess the possible ecological amplitude of the species. Results revealed that convergent topographies are basic determinants of microrefugia in arid environments. Within the investigated sites, underground moisture never decreases below 25%, buffering seasonal rainfall fluctuations. Therefore, hydrological microrefugia play a key role in decoupling from regional climate, supporting the target species in coping with an unsuitable climatic envelope. Additionally, the inter-population variability of biometric attributes showed that individual growth is site-dependent and the species retains a relative ecological plasticity, whereas the strongly clumped spatial patterns confirmed the common clonal growth. On one hand, deeply incised landforms have acted as effective hydrologic microrefugia, on the other clonality coupled with triploidy supposedly improved the resistance of Z. sicula to harsh environments, though entailing inability to reproduce sexually. Most likely, sterility and environmental/physical barriers that have existed for millennia have prevented this relict from leaving the last suitable microrefugia, resulting in the two current rear edge populations.

Water relations of two Sicilian grapevine cultivars in response to potassium availability and drought stress.

We investigated the response of two Sicilian grapevine cultivars, Catarratto and Nero d'Avola, to potassium deficiency and drought stress. Two-year-old plants grafted on 1103 Paulsen were grown in agriperlite, with or without potassium in the fertigation solution for six weeks, and subjected to moderate drought stress by suspending irrigation for one week. Potassium content of leaves, roots and xylem sap were measured with an ion-selective electrode. Changes in stomatal conductance, stem and leaf water potential and hydraulic conductance were compared between genotypes and treatments. Potassium deficiency led to significant decreases in leaf potassium content in both cultivars and under both well-watered and drought stress conditions. Potassium content in xylem sap showed no significant differences between cultivars and was correlated with stem hydraulic conductance, particularly in the drought stress treatments. Under drought stress conditions, potassium availability led to an increase in stomatal conductance, particularly in Nero d'Avola. Both cultivars showed a rather isohydric behavior under these experimental conditions, and the level of isohydry varied with potassium availability. These results can be useful for the development of optimal fertigation practices and the selection of drought tolerant varieties.

Phylogenetic Relationship Among Wild and Cultivated Grapevine in Sicily: A Hotspot in the Middle of the Mediterranean Basin.

Grapevine (Vitis vinifera ssp. sativa) is a perennial crop especially important for wine and fruit production. The species is highly polymorphic with thousands of different varieties selected by farmers and clonally propagated. However, it is still debated whether grapevine domestication from its wild ancestor (V. vinifera ssp. sylvestris) has been a single event or rather it occurred on multiple occasions during the diffusion of its cultivation across the Mediterranean. Located in the center of the Basin, Sicily is its largest island and has served as a hotspot for all civilizations that have crossed the Mediterranean throughout history. Hundreds of unique grapevine cultivars are still cultivated in Sicily and its surrounding minor islands, though most of them are menaced by extinction. Wild grapevine is also present with isolated populations thriving along riverbanks. With the aim to evaluate the phylogenetic relationships among Sicilian varieties, and to assess the possible contribution of indigenous wild populations to the genetic makeup of cultivated grapevine, we analyzed 170 domestic cultivars and 125 wild plants, collected from 10 different populations, with 23 SSR markers. We also compared our data with published dataset from Eurasia. Results show that Sicilian wild populations are related to the cultivated Sicilian and Italian germplasm, suggesting events of introgression and/or domestication of local varieties.

Rootstock and fruit canopy position affect peach [Prunus persica (L.) Batsch] (cv. Rich May) plant productivity and fruit sensorial and nutritional quality.

The right combination of rootstock and training system is important for increased yield and fruit sensorial and nutritional homogeneity and quality with peach [Prunus persica (L.) Batsch]. We investigated the effects of rootstock and training system on these parameters, testing the effect of vigorous GF677 and weaker Penta rootstock on 'Rich May' peach cultivar. Fruit position effects regarding photosynthetically active radiation availability, along the canopy profile using the Y training system, were investigated. The positive relationships between total polyphenols content and antioxidant capacity according to canopy vigour and architecture were determined for the two scion/stock combinations. Changes in fruit epicarp colour and content of bioactive compounds were also determined. Lower-vigour trees from Penta rootstock grafting yielded larger fruit with improved skin overcolour, and greater total polyphenols content and antioxidant capacity. GF677 rootstock produced more vigorous trees with fruit with lower sensorial and nutritional parameters. Canopy position strongly affects fruit sensorial and nutritional qualities. These data define potential for improvements to peach production efficiency and fruit quality, particularly for southern Europe peach cultivation conditions.

Extra virgin olive oil improves learning and memory in SAMP8 mice.

Polyphenols are potent antioxidants found in extra virgin olive oil (EVOO); antioxidants have been shown to reverse age- and disease-related learning and memory deficits. We examined the effects of EVOO on learning and memory in SAMP8 mice, an age-related learning/memory impairment model associated with increased amyloid-ß protein and brain oxidative damage. We administered EVOO, coconut oil, or butter to 11 month old SAMP8 mice for 6 weeks. Mice were tested in T-maze foot shock avoidance and one-trial novel object recognition with a 24 h delay. Mice which received EVOO had improved acquisition in the T-maze and spent more time with the novel object in one-trial novel object recognition versus mice which received coconut oil or butter. Mice that received EVOO had improve T-maze retention compared to the mice that received butter. EVOO increased brain glutathione levels suggesting reduced oxidative stress as a possible mechanism. These effects plus increased glutathione reductase activity, superoxide dismutase activity, and decreased tissue levels of 4-hydroxynoneal and 3-nitrotyrosine were enhanced with enriched EVOO (3 × and 5 × polyphenols concentration). Our findings suggest that EVOO has beneficial effects on learning and memory deficits found in aging and diseases, such as those related to the overproduction of amyloid-ß protein, by reversing oxidative damage in the brain, effects that are augmented with increasing concentrations of polyphenols in EVOO.