Effect of Pollen Genotype, Temperature and Period of Storage on In Vitro Germinability and In Vivo Seed Set in Chrysanthemum—Preliminary Study

Among many challenges in chrysanthemum cross-breeding, the access to viable pollen for hybridization of cultivars distant in location and different in flowering time is required. Low pollen viability along with incompatibility are mainly responsible for low seed set in modern chrysanthemum cultivars. The aim of the study was to test various temperatures and periods of pollen storage of Chrysanthemum × morifolium in order to elaborate the method of chrysanthemum pollen preservation for cross-breeding purposes. In the first experiment, in vitro pollen germination of four cultivars was investigated following storage at 20 °C, 4 °C, −20 °C, and −80 °C, for one, four, and eight weeks. The second experiment focused on in vivo seed set after one week pollen treatment with 20 °C, 4 °C, −20 °C, and −80 °C (three pollen donor cultivars tested). Pollen in vitro germinability, as well as seed set efficiency, was generally low and cultivar dependent. Independent of the period of storage, stored pollen germinability was lower (5.30–6.63%) than fresh pollen (8.15%). Incubation of pollen in −80 °C significantly increased pollen germinability (9.80%), as well as seed set efficiency in comparison to control (19.28% and 10.21%, respectively) provided the cultivars are compatible. Among cultivars, the highest germinability of pollen was found in ‘Brda’ and ‘Donna’ (8.2% and 8.23%, respectively), while ‘Bydgoszczanka’ showed the lowest germinability (2.97%). There were also pollen genotype dependent effects in in vivo seed set efficiency, which was highest in ’Brda’ (17.57%) and much lower in ‘Jutrzenka’ and ‘Polka’ (1.34% and 0.39%, respectively), which contributed to the incompatibility of crossed cultivars rather than pollen viability.

Seed Paternity Analysis Using SSR Markers to Assess Successful Pollen Donors in Mixed Olive Orchards

The olive tree (Olea europaea L.) is a wind-pollinated crop that exhibits an extreme alternate bearing habit. To improve fruit set, several methods have been used to determine the most successful compatible combinations of cultivars. In this study, priority is given to seed paternity analysis based on simple sequence repeats (SSRs), microsatellite markers used for the identification of potential pollen donors of cultivar ‘Oblica’ in a mixed olive orchard during two consecutive years. Seven microsatellite primers were successfully used to examine the paternity of olive embryos from ‘Oblica’ mother trees. Embryos were considered as a product of self-fertilization if only maternal alleles were present, but not a single case of self-fertilization was found among all the embryos analyzed. Two dominant pollen donors were not the closest nor the cultivars with the highest number of trees in the orchard, suggesting that cross-compatibility may have a key role in determining pollen donor success. In our earlier studies, pollen tube growth and fertilization success correlated with fruit set when controlled crosses between cultivars were performed; however, some discrepancy might appear compared to paternity analyses when mother trees have a free choice among different pollen sources from cultivars growing in their surroundings.

Low Outcrossing from an Apple Field Trial Protected with Nets

Regulatory compliance of experimental releases into the environment of not yet approved genetically modified plants often requires implementation of measures to reduce the dispersal of reproductive material. To study the impact of nets on pollen flow in an experimental field site in Switzerland, non-GM apple varieties ‘Ladina’ and ’Nicogreen’ were planted inside and outside a netted plot, respectively. Seeds harvested from mature fruits were germinated and the paternal variety of the seedlings was determined using simple sequence repeat (SSR) molecular markers. We demonstrate that pollination frequency from trees inside the netted plot to trees outside over a two-year (2018 and 2019) study of 4500 seedlings is 0.6% (26 seedlings). Moreover, these outcrossing events decreased with increasing distance from the pollen donor. Over the study period, we found on average 0.9%, 0.5%, 0.4% and 0.09% of the seedlings derived from apples of ‘Nicogreen’ trees at 8 m, 15 m, 72 m and 117 m being generated by fertilisations of ‘Ladina’ pollen, respectively. In comparison, 48.3% (2018 season) and 75.1% (2019 season) of examined ‘Ladina’ seedlings in the netted plot originated from ‘Nicogreen’ tree pollen outside the netted plot. The results suggest that insect netting is effective in minimizing egress of apple pollen from an experimental site and that the likelihood of outcrossing is reduced further (<0.1%) when there are no compatible apple trees within a radius of 100 m of the pollen donor. These data are important for biosafety research/regulation to aid understanding of pollen flow in experimental field sites.

Study of compatibility and determination of the suitable pollinizer for Argane tree (Argania spinosa L.)

During the breeding program studies, the pollen donor parents (pollinizers) were observed to be characterized by a strong flowering rate and very low fruit set, even after hand pollination. Then the notion of pollinizers in the argane species was born, first mentioned, checked, documented and confirmed like other conventional tree species. Since the argane tree is completely self-incompatible, the presence of compatible pollinizers is necessary for the fruit set. Indeed, pollinizers need to be selected accurately based on the synchronization of bloom periods and compatibility to receiver trees (female). The pollinizer is essential in any breeding program and for new orchard plantations. The current study was conducted on 13 argane genotypes including two pollinizers. The flowering period, bloom phenology, floral structure and fruit set of crossed genotypes were monitored, illustrated and a season phenogram was established. The pollen viability and germination were also evaluated. In order to test compatibility, the hand pollination using two selected pollinizer pollens was compared to open pollination. Then, the compatibility system was monitored and evaluated through analysis of crossing diallel programs and through the index of self-incompatibility. The flowering periods are genotype depending and one to three blooms have been observed during the two years study (2018-2019) and the argane tree is a tristylous species (Mesostylous, brevistylous and longistylous flowers). The in vitro tests showed that the pollen originated from crossed genotypes were viable and able to germinate. The cross-compatibility rate depends on cross associations and it varies from 39 to 84 %. In fact, this study showed that the effect of pollen-parent (xenia) occurs in all fruit components of argane tree. It was observed, for the first time, that both compatible pollinizers and xenic effects of pollen on argane fruit have occurred. Artificial pollination is currently feasible for breeding programs and the screening of elite genotypes. Then the selected pollinizer is quite required for the development of argane tree cropping.

Reproductive compatibility of Sorbocotoneaster with mountain ash species and varieties that are promising for breeding in Siberia

In Russia, in the Republic of Yakutia, there is the only point in the world where natural intergeneric hybridization of species of the genera Sorbus and Cotoneaster is observed, resulting in the formation of a new hybridogenic genus ×Sorbocotoneaster with a rich polymorphism of parental genera traits. For the selection of mountain ash in Siberia, it is promising to involve Sorbocotoneaster genotypes in artificial hybridization as sources of a complex of economically valuable traits, including winter hardiness, short stature, and self-fertility. Artificial hybridization of the tetraploid selective form Sorbocotoneaster with selected forms and varieties of Sorbus sibirica, S. aucuparia, S. sambucifolia and their hybrids allows obtaining viable hybrid offspring in combinations where Sorbocotoneaster is used as a pollen donor.

S-Genotyping and Seed Paternity Testing of the Pear Cultivar ‘Celina’

The diploid Celina/QTee® (‘Colorée de Juillet’ × ‘Williams’), one of the most promising pear cultivars developed by the Norwegian breeding program Graminor, was launched in 2010. In Norway, the flowering is medium to late, while the fruits ripen in the beginning of September. The fruits are attractive with an intense red blush (50%) on a green background. Although, ‘Celina’ is cultivated in the most climatically suitable regions for fruit cultivation, present in Norway, unfavorable environmental conditions for pear pollination can have a very negative effect on fruit set and consequent yield. The aim of this study was to determine the S-alleles of ‘Celina’, as well as its frequently used pollinizers, and, through paternity testing of ‘Celina’ seeds, give a recommendation regarding the most important pollinizers of this pear cultivar. In order to accomplish this, ‘Celina’ and its potential pollinizers were all S-genotyped. After harvest, seeds collected from ‘Celina’ fruit in 2017 and 2018 were genotyped using eleven microsatellite markers. Genomic DNA was also extracted from leaf material collected from ‘Celina’, as well as from five pear cultivars used as pollinizers in the three examined orchards, and analyzed using the same marker set. Subsequently a simple sequence repeat (SSR) database was constructed and used for gene assignment analyses with the aim of quantifying pollen donor contribution from individual pollinizers. The obtained results indicate that ‘Anna’, the only examined pollinizer that was fully cross-compatible with ‘Celina’, together with ‘Fritjof’, the genotype which had the highest flowering overlap with ‘Celina’, proved to be the most successful pollinizers across all seasons and orchards. Although both cultivars were ubiquitous in the examined orchards, either as planted trees or as branches introduced during the flowering period, they were the most abundant pollinizers in only one orchard each. It is therefore possible to conclude that pollinizer abundance has a secondary significance in pollinizer success within investigated ‘Celina’ orchards.

The Phylogenetic Position of Vincetoxicum pannonicum (Borhidi) Holub Supports the Species' Allopolyploid Hybrid Origin

The Pannonian endemic species <em>Vincetoxicum pannonicum </em>was described from specimens collected in Hungary and occurs at only few locations. It is considered “vulnerable” according to the International Red List. The chromosome set was reported to be tetraploid, and the species was hypothesized to be an allotetraploid hybrid of the Balkan species <em>V. fuscatum </em>and the Adriatic species <em>hirundinaria </em>subsp. <em>adriaticum. </em>We investigated the origin of <em>V. pannonicum </em>using molecular phylogenetic methods by separately analyzing the multicopy nuclear ribosomal internal transcribed spacer (nrITS) and the plastid-encoded <em>trn</em>H-<em>psb</em>A DNA regions and by evaluating discrepancies between the produced gene trees. Paralogs in the nrITS region clustered in two main groups, one of which was closest to <em>V. fuscatum</em>, and the other included <em>V. hirundinaria </em>subsp. <em>adriaticum</em>. According to <em>trn</em>H-<em>psb</em>A sequences, <em>V. pannonicum </em>and <em>V. hirundinaria </em>subsp. <em>adriaticum </em>formed a single group. Our results show that <em>V. pannonicum </em>diversified because of hybrid speciation, in which <em>V. fuscatum </em>was the pollen donor. We discovered a similar placement of <em>V. maeoticum</em>, which suggests a further hybridization event between <em>V. fuscatum </em>and a species of the <em>V. hirundinaria </em>group. Our genome-size estimate indicates almost sixfold larger genome size in <em>V. pannonicum </em>compared to the maternal diploid parent, suggesting hexaploidy; however, <em>V. pannonicum </em>is tetraploid. This may suggest cytological diploidization in the allopolyploid <em>V. pannonicum</em>. We observed substantial genetic distance between <em>V. hirundinaria </em>subsp. <em>adriaticum </em>and all other subspecies of <em>V. hirundinaria</em>, and we therefore propose that <em>V. adriaticum </em>should be regarded as a separate species.

In-Depth Field Characterization of Teff [Eragrostis tef (Zucc.) Trotter] Variation: From Agronomic to Sensory Traits

Teff is an important food crop that serves to prepare Injera-flat-bread. It is cultivated worldwide and is particularly susceptible to lodging. A diverse collection of teff [Eragrostis tef (Zucc.) Trotter] populations was characterized for a wide range of traits, ranging from agronomic to final Injera sensory parameters, under well-irrigated Mediterranean spring conditions. The populations tested were collected from single plants presenting lodging resistance at the site of collection and their traits were characterized herein. An early type of lodging was observed, which was most likely triggered by a fast and sharp inflorescence weight increase. Other populations were ‘strong’ enough to carry the inflorescence during most of the grain-filling period, up to a point where strong lodging occurred and plants where totally bent to the ground. Three mixed color seed populations were established from a single plant. These were separated into ‘white’ and ‘brown’ seeds and were characterized separately under field conditions. The newly ‘brown’ populations appear to be the result of a rather recent non-self (external) airborne fertilization from a dark pollen donor. Some of these hybrids were found to be promising in terms of Injera sensory traits. The population of these studies might serve as breeding material. Integration between a wide range of parameters and the correlations obtained between agronomic and sensory traits might improve our ability to breed towards a “real world” better end-product.