Polystyrene nanoparticles induce concerted response of plant defense mechanisms in plant cells.

Recent advances in knowledge suggest that micro- and nanoplastics pose a threat to plant health, however, the responses of plants to this stressor are not well-known. Here we examined the response of plant cell defence mechanisms to nanoparticles of commonly used plastic, polystyrene. We used plant cell cultures of widely cultivated plants, the monocots wheat and barley (Triticum aestivum L., Hordeum vulgare L.) and the dicots carrot and tomato (Daucus carota L., Solanum lycopersicum L.). We measured the activities of enzymes involved in the scavenging of reactive oxygen species and nonenzymatic antioxidants and we estimated potential damages in plant cell structures and functioning via lipid peroxidation and DNA methylation levels. Our results demonstrate that the mode of action of polystyrene nanoparticles on plant cells involves oxidative stress. However, the changes in plant defence mechanisms are dependent on plant species, exposure time and nanoplastic concentrations. In general, both monocots showed similar responses to nanoplastics, but the carrot followed more the response of monocots than a second dicot, a tomato. Higher H2O2, lipid peroxidation and lower enzyme activities scavenging H2O2 suggest that tomato cells may be more susceptible to polystyrene-induced stress. In conclusion, polystyrene nanoplastics induce oxidative stress and the response of the plant defense mechanisms involving several chain reactions leading to oxidoreductive homeostasis.

Structural Equation Modeling (SEM) Analysis of Sequence Variation and Green Plant Regeneration via Anther Culture in Barley.

The process of anther culture involves numerous abiotic stresses required for cellular reprogramming, microspore developmental switch, and plant regeneration. These stresses affect DNA methylation patterns, sequence variation, and the number of green plants regenerated. Recently, in barley (Hordeum vulgare L.), mediation analysis linked DNA methylation changes, copper (Cu2+) and silver (Ag+) ion concentrations, sequence variation, ß-glucans, green plants, and duration of anther culture (Time). Although several models were used to explain particular aspects of the relationships between these factors, a generalized complex model employing all these types of data was not established. In this study, we combined the previously described partial models into a single complex model using the structural equation modeling approach. Based on the evaluated model, we demonstrated that stress conditions (such as starvation and darkness) influence ß-glucans employed by cells for glycolysis and the tricarboxylic acid cycle. Additionally, Cu2+ and Ag+ ions affect DNA methylation and induce sequence variation. Moreover, these ions link DNA methylation with green plants. The structural equation model also showed the role of time in relationships between parameters included in the model and influencing plant regeneration via anther culture. Utilization of structural equation modeling may have both scientific and practical implications, as it demonstrates links between biological phenomena (e.g., culture-induced variation, green plant regeneration and biochemical pathways), and provides opportunities for regulating these phenomena for particular biotechnological purposes.

Heritability of meiotic restitution and fertility restoration in haploid triticale.

KEY MESSAGE: A single division meiosis mechanism of meiotic restitution is incompletely penetrant but significantly associated with restored fertility in triticale haploids (n = 21, genome formula ABR). Meiotic restitution, or failure of meiosis to produce gametes with a reduced chromosome number, can lead to the restoration of fertility in allohaploids. Meiotic restitution is of major interest for producing doubled haploids, as haploid plants undergoing meiotic restitution can often form seeds without the need to apply mitosis inhibitors to double chromosome number. We aimed to characterize meiotic restitution in a population of 183 haploids (n = 21, genome formula ABR) derived from an F1 wheat-rye hybrid where one parent was known to carry factors responsible for restoration of fertility in wide-cross haploids. Based on cytological analysis, approximately half of the plants analyzed were characterized by normal meiosis, while half showed at least some cytological evidence of meiotic restitution. However, this mechanism was incompletely penetrant in the population, with no individual plant showing 100% unreduced gamete formation: restitution occurred sectorially within each anther and was not observed in all the anthers of a given plant. Hence, the absence of meiotic restitution could not be confirmed conclusively for any individual plant, confounding this analysis. However, cytological observation of meiotic restitution was significantly associated with seed set, further confirming the role of meiotic restitution in fertility restoration. Our results provide insight into this mechanism of unreduced gamete formation, and provide a basis for future work identifying the genetic factors responsible for this trait.

DNA methylation changes and TE activity induced in tissue cultures of barley (Hordeum vulgare L.).

BACKGROUND: In vitro plant regeneration via androgenesis or somatic embryogenesis is capable of inducing (epi)mutations that may affect sexual progenies. While epimutations are associated with DNA methylation, mutations could be due to the movement of transposons. The common notion is that both processes are linked. It is being assumed that demethylation activates transposable elements (TEs). Analysis of methylation changes and their relation with TEs activation in tissue cultures requires uniquely derived donor plants (Ds), their regenerants (Rs) and respective progeny (Ps) that would allow discrimination of processes not related to changes introduced via in vitro cultures. Moreover, a set of methods (RP-HPLC, SSAP, and MSTD) is needed to study whether different TEs families are being activated during in vitro tissue culture plant regeneration and whether their activity could be linked to DNA methylation changes or alternative explanations should be considered. RESULTS: The in vitro tissue culture plant regeneration in barley was responsible for the induction of DNA methylation in regenerants and conservation of the methylation level in the progeny as shown by the RP-HPLC approach. No difference between andro- and embryo-derived Rs and Ps was observed. The SSAP and MSTD approach revealed that Ds and Rs were more polymorphic than Ps. Moreover, Rs individuals exhibited more polymorphisms with the MSTD than SSAP approach. The differences between Ds, Rs and Ps were also evaluated via ANOVA and AMOVA. CONCLUSIONS: Stressful conditions during plant regeneration via in vitro tissue cultures affect regenerants and their sexual progeny leading to an increase in global DNA methylation of Rs and Ps compared to Ds in barley. The increased methylation level noted among regenerants remains unchanged in the Ps as indicated via RP-HPLC data. Marker-based experiments suggest that TEs are activated via in vitro tissue cultures and that, independently of the increased methylation, their activity in Rs is greater than in Ps. Thus, the increased methylation level may not correspond to the stabilization of TEs movement at least at the level of regenerants. The presence of TEs variation among Ds that were genetically and epigenetically uniform may suggest that at least some mobile elements may be active, and they may mask variation related to tissue cultures. Thus, tissue cultures may activate some TEs whereas the others remain intact, or their level of movement is changed. Finally, we suggest that sexual reproduction may be responsible for the stabilization of TEs.

The origin of unusual chromosome constitutions among newly formed allopolyploids.

PREMISE OF THE STUDY: Wide hybridization followed by spontaneous chromosome doubling of the resulting hybrids plays an important role in plant speciation. Such chromosome doubling is usually accomplished via unreduced gametes produced by altered meiosis, the so-called 'meiotic restitution'. Unreduced gametes are expected to carry somatic chromosome numbers and constitutions. However, it has been shown recently that new allopolyploids often carry unusual chromosome constitutions which include compensating and noncompensating nulli-tetrasomies and monotrisomies, and translocations of homoeologues. METHODS: We have reanalyzed meiotic divisions in a wheat-rye hybrid by in situ probing with labeled DNA focusing on deviations from the standard pattern of meiotic restitution. KEY RESULTS: In a typical first division restitution in a wide hybrid, there is no chromosome pairing, univalents separate sister chromatids in anaphase I, and there is no meiosis II. Here we illustrate that occasional pairing of homoeologous chromosomes in metaphase I, combined with separation of sister chromatids of univalents, generates diads with compensating nulli-disomies and associated translocations of homoeologues. Similarly, precocious metaphase I migration to the poles of some undivided univalents generates a wide range of noncompensating simple and complex nulli-disomies in the gametes. CONCLUSIONS: Both alterations to the standard pattern of meiotic restitution tend to maintain the somatic chromosome numbers in the gametes; chromosome constitutions are variable but mostly genetically balanced. This source of variation among progeny may be an important factor contributing to greater success of natural allopolyploids.

Albinism in barley androgenesis.

Androgenesis is highly useful for plant breeding, significantly reducing breeding cycle times, as well as in a wide range of biological research. However, for widespread use this process must be efficient. Despite several decades of research on the phenomenon of androgenesis, many processes involved are obscure and there is much to be understood about androgenesis. One of the problems inherent in androgenesis, and reducing its efficiency, is albinism. This article reviews albinism in barley anthers and microspores in vitro cultures. Of special interest is the fate of plastids throughout androgenesis, which is important at several levels, including the genes responsible for driving the green-to-albino ratios. We also summarize the external factors that reduce the incidence of albino plants that are regenerated via androgenesis.

Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack).

BACKGROUND: While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. RESULTS: Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. CONCLUSIONS: The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.

Aneuploidy among androgenic progeny of hexaploid triticale (XTriticosecale Wittmack).

Doubled haploids are an established tool in plant breeding and research. Of several methods for their production, androgenesis is technically simple and can efficiently produce substantial numbers of lines. It is well suited to such crops as hexaploid triticale. Owing to meiotic irregularities of triticale hybrids, aneuploidy may affect the efficiency of androgenesis more severely than in meiotically stable crops. This study addresses the issue of aneuploidy among androgenic regenerants of triticale. Plant morphology, seed set and seed quality were better predictors of aneuploidy, as determined cytologically, than flow cytometry. Most aneuploids were hypoploids and these included nullisomics, telosomics, and translocation lines; among 42 chromosome plants were nulli-tetrasomics. Rye chromosomes involved in aneuploidy greatly outnumbered wheat chromosomes; in C(0) rye chromosomes 2R and 5R were most frequently involved. While the frequency of nullisomy 2R was fairly constant in most cross combinations, nullisomy 5R was more frequent in the most recalcitrant combination, and its frequency increased with time spent in culture with up to 70% of green plants recovered late being nullisomic 5R. Given that 5R was not involved in meiotic aberrations with an above-average frequency, it is possible that its absence promotes androgenesis or green plant regeneration. Overall, aneuploidy among tested combinations reduced the average efficiency of double haploid production by 35% and by 69% in one recalcitrant combination, seriously reducing the yield of useful lines.

Agrobacterium-mediated gene transfer to cereal crop plants: current protocols for barley, wheat, triticale, and maize.

The development of powerful "omics" technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.

The application of the AFLP method to determine the purity of homozygous lines of barley (Horedum vulgare L.).

Amplified fragment length polymorphism of DNA has been used to analyse the equality of plants obtained from isolated microspores. Although the control parental material was regarded as being highly homozygous, the analysis of the banding patterns of single plants showed a certain level of polymorphism. The analysis of regenerants with a doubled chromosome number did not show any diversity within the progeny of a single line. The differences in banding patterns coming from single plants were only observed in microspore donor lines. These results have proven the high purity of homozygous lines obtained via androgenesis from isolated microspores.