Sex-Linked Molecular Markers Identify Female Lines in Endosperm-Derived Kiwifruit Callus and in Regenerants.

This is the first report of molecular markers application for the analysis of endosperm-derived callus and nonaploid kiwifruit (Actinidia chinensis var. deliciosa, formerly: Actinidia deliciosa) plants. As a source of explants, fruits of 'Hayward', the most popular cultivar, were used. Additionally, analyses of the nuclear DNA content and sex were conducted on the regenerated plants. Hexaploid seedlings were used as control for the flow cytometric analyses. Most of the plants (about 90%) regenerated via endosperm-derived callus possessed 2C = 9Cx DNA, which confirmed their endosperm origin and nonaploidy. Because Actinidia is a dioecious species, and female plants bearing fruits are desired by breeders, it is crucial to identify the sex of an individual at early stages of development. Analyses were conducted with ex vitro and in vitro samples. Results revealed that specific markers for a Y-chromosome applied at the callus stage allowed us to reliably predict the sex of plants regenerated from it. This is a novel application of sex-linked markers for early selection of female and male callus lines when the sex of the initial explants is still unknown, such as fresh isolated embryos and endosperm. It may have significant importance for breeding kiwifruit programs, which involve tissue culture techniques.

Phylogeny and historical biogeography analysis support Caucasian and Mediterranean centres of origin of key holoparasitic Orobancheae (Orobanchaceae) lineages.

The extensive diversity of the tribe Orobancheae, the most species-rich lineage of holoparasitic Orobanchaceae, is concentrated in the Caucasus and Mediterranean regions of the Old World. This extant diversity has inspired hypotheses that these regions are also centres of origin of its key lineages, however the ability to test hypotheses has been limited by a lack of sampling and phylogenetic information about the species, especially in the Caucasus region. First, we assessed the phylogenetic relationships of several poorly known, problematic, or newly described species and host-races of four genera of Orobancheae occurring in the Caucasus region-Cistanche, Phelypaea, Phelipanche and Orobanche-using nuclear ribosomal (ITS) and plastid (trnL-trnF) sequence data. Then we applied a probablistic dispersal-extinction-cladogenesis model of historical biogeography across a more inclusive clade of holoparasites, to explicitly test hypotheses of Orobancheae diversification and historical biogeography shifts. In sum, we sampled 548 sequences (including 196 newly generated) from 13 genera, 140 species, and 175 taxa across 44 countries. We find that the Western Asia (particularly the Caucasus) and the Mediterranean are the centre of origin for large clades of holoparasitic Orobancheae within the last 6 million years. In the Caucasus, the centres of diversity are composed both of long-branch taxa and shallow, recently diversified clades, while Orobancheae diversity in the Mediterranean appears to represent mainly recent diversification.

The First Report of Orobanche laxissima Parasitizing Pomegranate (Punica granatum) in Georgia.

Orobanche laxissima Uhlich & Rätzel (Orobanchaceae) is a polyphagous root parasitic plant distributed in the Caucasus Mountains and Transcaucasia; especially Russia, Georgia, Armenia, Azerbaijan, N.E. Turkey (Piwowarczyk et al. 2019). It infects many wild or sometimes cultivated trees and shrubs, such as Betulaceae, Oleaceae, Fagaceae, Aceraceae, Cornaceae, usually Fraxinus L., Fagus L., Carpinus L. (Piwowarczyk et al. 2019, 2020). Punica granatum L. (Lythraceae), commonly known as pomegranate, is native to the Caucasus, the Himalayas in North Pakistan and Northern India, and is widely cultivated, e.g. in USA and throughout the Mediterranean. Pomegranate is one of the first domesticated fruits and have been used in folk medicine or as a food for centuries. Fruit, seed, leaves, flower, root, or barks extracts have extensive medicinal properties (Shaygannia et al. 2015). Field surveys conducted in south-eastern Georgia in May 2019 revealed extensive infestations of O. laxissima on the roots of P. granatum in one locality in Kakheti Province, near Sighnaghi (41°37,4 N, 45°56,3 E, 480 m elevation), in roadside or hills scrub and cultivated areas of pomegranate. The infection was confirmed by verifying the attachment of the Orobanche to the Punica root. The population of the parasite consisted of at least a ca. thousand shoots, sometimes in one clump was ca. 100 individuals. A single plant of pomegranate was parasitized by few to c.a. a hundred of broomrape plants, and 10 to 20% of the ca. 1 ha location was infested. The main botanical features of the O. laxissima are: i) stem simple, (10-)25-40(-100) cm high, with haustoria; ii) inflorescence usually long to short cylindrical or lax, usually many-flowered; iii) calyx-segments entire or bidentate, rarely with 4 teeth; iv) corolla (16-)22-24(-31) mm long, tubular-bell-shaped; purple, pink, rarely dirty yellow, light brown; v) stigma purple, orange, or yellow (Piwowarczyk et al. 2019, 2020). For molecular analysis, total genomic DNA was extracted from the sample and the plastid gene rbcL (rubisco large subunit) was sequenced and amplified as described in Piwowarczyk et al. (2015). The sequence (1231 bp) was deposited in GenBank (MN384886). BLAST search found that it was most similar to (Query Cover 100%, Per Ident. 100%) O. laxissima (KR260928). To the best of our knowledge, this is the first report of a O. laxissima parasitizing P. granatum. O. laxissima appearing in large numbers on singles pomegranate shrubs can weaken the plants, and reduce flowering and fruiting. In the Caucasus region, O. laxissima was observed in mesophilic forests and shrubs, but our report suggests the possibility of a potential spread to neighboring cultivated areas, especially fruit trees and shrubs. Until now, only one report of pathogenic plants was documented for P. granatum, included Phelipanche aegyptiaca (Pers.) Pomel and O. crenata Forssk. in Israel (Dor et al. 2014).

Does actually mean chromosome number increase with latitude in vascular plants? An answer from the comparison of Italian, Slovak and Polish floras.

WE COMPARED CHROMOSOME NUMBER (CN) VARIATION AMONG VASCULAR FLORAS OF THREE DIFFERENT COUNTRIES WITH INCREASING LATITUDE IN THE BOREAL HEMISPHERE: Italy, Slovakia, Poland. Aim of the study was to verify whether the patterns of CN variation parallel the differences in latitudinal ranges. The three datasets comprised 3426 (Italy), 3493 (Slovakia) and 1870 (Poland) distinct cytotypes. Standard statistics (ANOVA, Kruskal-Wallis tests) evidenced significant differences among the three countries, mean CN increasing together with latitude. On the contrary, an inverse relation (r = -1) was evidenced among the frequency of odd CNs and latitude. Our results show that the hypothesis of a polyploid increase proportional with distance from the Equator seems to be confirmed, when territories from the same hemisphere are compared.

Are extracellular matrix surface network components involved in signalling and protective function?

Endosperm is an interesting model for in vitro experiments, because of its unique origin, development and ploidy level. Here we used Actinidia deliciosa endosperm-derived callus to investigate morphology, histology and chemistry of extracellular matrix (ECM) structures in morphogenically stable tissue from long-term culture. SEM and TEM analysis showed that ECM is a heterogenous layer which consists of amorphous, dark-staining material, osmiophilic granules and reticulated fibres outside the outer callus cell wall. This structure may serve as a structural marker of morphogenic competence in endosperm-derived callus, because of its presence on the surface of callus forming morphogenic domains and its disappearance during organ growth. Based on immunolabelling, histochemistry, solvent and enzyme treatments, we suggest that pectins and lipids are components of the ECM layer. These results might indicate protective, water retention and/or cell communication functions for this ECM layer.

Multicellular structures developing during maize microspore culture express endosperm and embryo-specific genes and show different embryogenic potentialities.

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.