Valentina Scariot

Development and evaluation of microsatellite markers in Phoenix dactylifera L. and their transferability to other Phoenix species

Forty one simple sequence repeats were isolated from two microsatellite enriched libraries of date palm (Phoenix dactylifera L.). After screening, 17 selected microsatellite loci were characterized and evaluated on a set of 31 cultivars and clones from Algerian and Californian germplasm. All primer pairs produced an amplification product of the expected size and detected high polymorphism among the analysed samples. These nuclear simple sequence repeat (SSR) markers are expected to be a very effective tool for evaluating genetic diversity in date palm germplasm. Acrosstaxa amplification showed the usefulness of most SSR markers in 14 other species across the genus Phoenix.

Application of laser microdissection to identify the mycorrhizal fungi that establish arbuscules inside root cells

Obligate symbiotic fungi that form arbuscular mycorrhizae (AMF; belonging to the Glomeromycota phylum) are some of the most important soil microorganisms. AMFs facilitate mineral nutrient uptake from the soil, in exchange for plant-assimilated carbon, and promote water-stress tolerance and resistance to certain diseases. AMFs colonize the root by producing inter- and intra-cellular hyphae. When the fungus penetrates the inner cortical cells, it produces a complex ramified structure called arbuscule, which is considered the preferential site for nutrient exchange. Direct DNA extraction from the whole root and sequencing of ribosomal gene regions are commonly carried out to investigate intraradical AMF communities. Nevertheless, this protocol cannot discriminate between the AMFs that actively produce arbuscules and those that do not. To solve this issue, the authors have characterized the AMF community of arbusculated cells (AC) through a laser microdissection (LMD) approach, combined with sequencing-based taxa identification. The results were then compared with the AMF community that was found from whole root DNA extraction. The AMF communities originating from the LMD samples and the whole root samples differed remarkably. Five taxa were involved in the production of arbuscules, while two taxa were retrieved inside the root but not in the AC. Unexpectedly, one taxon was found in the AC, but its detection was not possible when extracting from the whole root. Thus, the LMD technique can be considered a powerful tool to obtain more precise knowledge on the symbiotically active intraradical AMF community.

A contribution to the classification of evergreen azalea cultivars located in the Lake Maggiore area (Italy) by means of AFLP markers

The amplified fragment length polymorphism (AFLP) technique was employed to assess the genetic conformity within a gene pool of evergreen azaleas. Ninety-three genotypes grown in historical gardens and nurseries of the Lake Maggiore area (Northern Italy), locally classified in the groups Indica, Japonica and Amoena, 44 reference cultivars of the four more or less universally recognized groups Belgian, Hirado, Kurume and Satsuki, and 40 species and species accessions that most concurred in the origin of the reference groups were included. Aims of the study were to define the genetic profiles of the Italian accessions and to provide useful information for their classification and pedigree reconstruction. Ordination analyses were used to evaluate if the AFLP technique can be applied to establish phylogenetic relationships and to investigate the DNA-based relatedness within the Italian gene pool and between the latter and the reference one. The results regarding the Italian accessions were compared with morphological data by means of Mantel’s test. Assignment tests both on the level of the groups and on individual plant level for the reference and Italian genotypes were applied to further evaluate the relatedness of the groups or individual accessions. Results demonstrated that the AFLP technique together with morphological characterization can contribute to depict the phylogeny of the evergreen azalea. Comparing the reference and Italian genotypes, conclusions about the classification of the evergreen azalea cultivars located in the Lake Maggiore area were drawn.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 μg/g, 0.00015 and 0.00030 μg/g, and 0.0089 and 0.018 μg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.

In vitro seed germination and seedling propagation in Campanula spp.

Abstract The present research investigated in vitro seed germination and seedling multiplication methods to preserve and introduce new naturalized valuable Campanula species to the floriculture markets. Populations of Campanula barbata L., Campanula latifolia L., Campanula rapunculoides L., Campanula spicata L., and Campanula trachelium L. from various sites in northern Italy were considered in this study. Seeds were sown on half-strength Murashige and Skoog (MS) basal medium with 1-naphthaleneacetic acid (NAA), 6-benzyladenine (BAP) and kinetin, added or withheld, after which final germination percentage (FGP), mean germination time and time of 50% germination (T 50) indices were measured. C. rapunculoides showed the highest FGP after 2 weeks in both the presence (53%) and absence (56%) of plant growth regulators. Among the other species, no statistical differences were detected in the presence of growth regulators, except for C. barbata, in which FGP was nearly 0%. In the absence of phytohormones, seed germination in C. spicata was statistically higher (34%), compared to C. barbata (0%), and both C. latifolia and C. trachelium (about 19%). After 4 months, the seedlings were transferred to full-strength MS basal medium with or without NAA and BAP. In all Campanula species tested, the best shoot-forming and rooting response was observed on substrates that contained plant growth regulators.

Application of nonspecific commercial AMF inocula results in poor mycorrhization in Camellia japonica L.

Camellia japonica L. (Theaceae, Theales) is an acidophilic evergreen flowering shrub and is traded worldwide. Symbiotic associations between the roots of this plant species and arbuscular mycorrhizal fungi (AMF), commonly recognized as natural biofertilizers and biocontrol agents, have been poorly studied so far. The aim of our study was to verify whether the application of nonspecific commercial AMF-based inocula could succeed and improve the growth of C. japonica. An experiment was conducted concerning the application of commercial inocula constituted by a specific AMF isolate (Funneliformis mosseae) or a consortium of different fungi and bacteria as alternatives to fertilization in pot cultivated C. japonica ‘Dr. Burnside’. Several growth parameters, plant nutrition and mycorrhization levels were monitored at the end of plant cultivation. Generally, increases in some macroelements (Ca, Mg, K) and microelements (Cu, Mn, Fe, Zn) were detected in the root system of inoculated camellias, while only Cu and Mn accumulation was increased in the leaves. Plants inoculated with the consortium inoculum exhibited increased chlorophyll content and foliage diameter while plants inoculated with F. mosseae exhibited increased number of flowers. Based on the polymorphism of an 18S rDNA region, we assessed the inoculated AMF that colonized C. japonica roots. The experiments showed that the applied AMF poorly colonized the root system of C. japonica. We suggest that commercial AMF formulations should be more targeted and host-specific in order to successfully colonize the host root and, potentially, fully express their benefits.

Azalea phylogeny reconstructed by means of molecular techniques.

Plants belonging to the Rhododendron subgenera Pentanthera (deciduous) and Tsutsusi and Azaleastrum (evergreen) are called azaleas. Concerning their mutual phylogenetic positions, the Pentanthera subgenus is closer to evergreen rhododendrons (subgenera Rhododendron and Hymenanthes) than to the Tsutsusi subgenus. Both azalea types are important ornamentals with a long breeding tradition. Different hybrid groups are often named after the supposed principal ancestor species. Molecular techniques for phylogenetic and kinship research have been evaluated to a great extent. First, some studies using comparative gene sequencing are presented; this approach was then widened to the use of molecular markers to reveal more detailed genetic relationships. Finally, the use of candidate genes as functional markers for the assessment of genetic diversity is presented. This opens new research lines to the genetic mapping of plant traits and azalea genomics.

The influence of water stress on growth, ecophysiology and ornamental quality of potted Primula vulgaris ‘Heidy’ plants. New insights to increase water use efficiency in plant production.

Efficient irrigation practices are required to reduce the amount of water used. In this study, the effects of different irrigation regimes on changes in growth, ecophysiology and ornamental traits of potted Primula vulgaris ‘Heidy’ plants were investigated. Three experiments were carried out. In the first, the plants were either fully irrigated (100% of container capacity) or not. In the second, plants were watered to full irrigation (control), to 50% of the control (moderate water stress), to 25% of the control (severe water stress), or not irrigated and followed by a rehydration phase. Both experiments were conducted under controlled growth conditions. The third experiment was performed under common nursery conditions in an unheated and shaded greenhouse where plants were either irrigated with common irrigation practices (control), or with 66% of the control amount (moderate water stress), or with 33% of the control (severe water stress). In general, the percentage of senescent plants, the growth index, the number of leaves, and the aerial fresh and the dry weight were not affected by moderate water stress treatments. As expected, increasing water stress resulted in a general decrease in all studied gas exchange parameters. However, stressed plants were more efficient in using water than control plants, suggesting that stomata closed to cope with drought conditions without damaging photosynthesis events. The number of fully opened flowers during the growing season was highest in both control and moderately water stressed plants. In conclusion, moderate, but not severe, water stress could be imposed in P. vulgaris ‘Heidy’ pot production to reduce the water consumption, still maintaining plant ecophysiological performances and ornamental quality.

Pollen Grain Preservation and Fertility in Valuable Commercial Rose Cultivars

In the cut flower market, traditional breeding is still the best way to achieve new rose cultivars. The geographical delocalization of cultivar constitution (generally made in Europe and North America) and plant cultivation (large areas in Africa and South America) represents a limit point for crossing and selection. Rose breeders often need to overcome geographical distances, resulting in asynchrony in flowering among crossing parents, by storing and sending pollen. Hence, a key aspect in breeding programs is linked to pollen availability and conservation, jointly with the identification of parameters related to pollen fertility. In this study we present the results of three different trials. In the first, pollen diameter and pollen viability were chosen as fertility predictors of 10 Rosa hybrida commercial cultivars. In the second trial, aliquots of dried pollen grains of six R. hybrida cultivar were stored under two different temperatures (freezer at T = −20 °C and deep freezer at T = −80 °C) and after a wide range of conservation period, their viability was measured. In the third trial, the effective fertilization capacity of frozen pollen of 19 pollen donor cultivars was evaluated during 2015 crossing breeding plan, performing 44 hybridizations and correlating the number of seeds and the ratio seeds/crossing, obtained by each cultivar, with in vitro pollen germination ability.

Cold Treatment Breaks Dormancy but Jeopardizes Flower Quality in Camellia japonica L.

Camellia japonica L. is an evergreen shrub whose cultivars are of great ornamental value. In autumn, after flower bud differentiation, dormancy is initiated. As in many other spring flowering woody ornamentals, winter low temperatures promote dormancy release of both flower and vegetative buds. However, warm spells during late autumn and winter can lead to unfulfilled chilling requirements leading to erratic and delayed flowering. We hypothesized that storing plants at no light and low temperature could favor dormancy breaking and lead to early and synchronized flowering in response to forcing conditions in C. japonica ‘Nuccio’s Pearl’. Plants with fully developed floral primordia were stored at dark, 7°C, and RH > 90% for up to 8 weeks. To monitor endodormancy release during the storage, we evaluated the content of abscisic acid (ABA) in flower buds and the expression profiles of five putative genes related to dormancy and cold acclimation metabolism in leaves and flower buds. In addition, the expression of four anthocyanin biosynthesis pathway genes was profiled in flower buds to assess the effect of the treatment on flower pigment biosynthesis. At 0, 4, 6, and 8 weeks of cold treatment, 10 plants were transferred to the greenhouse and forced to flower. Forced plant flower qualities and growth were observed. The ABA content and the expression profiles of two dormancy-related genes (CjARP and CjDEH) suggested that dormancy breaking occurred after 6–8 weeks of cold treatment. Overall, plants treated for 6–8 weeks showed earlier vegetative sprouting, enhanced, and homogeneous flowering with reduced forcing time. Prolonged cold treatments also reduced flower size and longevity, anthocyanin content, and pigment biosynthesis-related gene transcripts. In conclusion, the cold treatment had a promotive effect on dormancy breaking but caused severe drawbacks on flower quality.