Clearing methods allow relatively quick processing of plant material and examination of cellular structures by rendering tissues and organs translucent. They have been adapted for plant embryology, primarily to study ovule development, megasporogenesis, megagametogenesis and embryogenesis. Such clearing methods overcome several disadvantages of the conventional embedding-sectioning techniques that are arduous and time-consuming. Although numerous protocols with different clearing solutions have been described, there have been no reports to date proposing a reliable method to clear the crassinucellate ovules of the sugar beet (Beta vulgaris L.), an economically important crop. Therefore, this study aims to find a suitable approach to improve the tissue transparency of sugar beet ovules at different developmental stages. We established a methyl salicylate-based protocol that significantly improved the transparency of the B. vulgaris ovule structures, which allowed us to observe the megagameto- and embryogenesis of that species. This was achieved by (1) chemical softening of the tissues; (2) vacuum pump-assisted infiltration step; (3) shaking-assisted incubation with clearing mixtures; and (4) manual removal of the chemically softened seed coat. The effectiveness of our method is due to the strategy combining various approaches at different stages of the procedure aiming at increasing the accessibility of the internal ovule structures to the clearing solution. The results of this study may be applied in sugar beet breeding programs, and it will provide a basis for further investigation of numerous aspects of the species’ embryology. Moreover, that unique approach may be easily adapted to other species developing crassinucellate ovules.

Reproductive processes including male and female lines, embryo and endosperm development were studied in\ud Cardaminopsis arenosa (syn. Arabidopsis arenosa) growing on two metalliferous sites (Bukowno and Bolesław,\ud S. Poland), rich in Zn, Pb, Cd and other metals. Disturbances of developmental processes and necroses observed\ud in anthers and ovules influenced plant fertility and seed set of plants from both metal-polluted sites. In anthers,\ud disturbances and necrosis during male meiosis and pollen development occurred at low frequency (4–5%).\ud Pollen grain viability was very high, reaching over 90%. In ovules the frequency of abnormal meiosis, female\ud gametophyte developmental disturbances and necrosis was high, 23.5–28% depending on site. The polluted environment\ud also affected embryo and endosperm. Necrosis of whole generative structures decreased plant fertility.\ud This study indicates that the range of disturbances and necroses in embryological structures and processes (at\ud gametophyte level) gives a set of useful characters to determine plant tolerance to stress, complementary to many\ud tolerance characters at the sporophyte level of plant ontogenesis

Programmed cell death (PCD) is a pivotal biological process that occurs at various stages of plant development, including embryogenesis and seed germination. This study investigated whether the absence of PCD in endosperm cells is connected to the poor germination of Viola odorata seeds. Seeds of poorly germinating V. odorata and well-germinating V. × wittrockiana were either cold-stratified for 10 days or left untreated. Germination tests, tetrazolium viability tests, Western blot analyses for caspase-like proteases, and Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assays for DNA strand break detection were performed. The results revealed that V. odorata seeds did not germinate, regardless of stratification or lack thereof, whereas in V. × wittrockiana, stratification significantly increased their germination capacity (34 ± 6.5% vs. 56.5 ± 9.8% in non-stratified and stratified seeds, respectively). The tetrazolium viability test revealed that V. odorata seeds were nonviable (100% nonviable endosperm and 96% nonviable embryos in total), whereas the seeds of V. × wittrockiana were highly viable (63% and 59% endosperm and embryos in total, respectively). Western blot analysis revealed that in the germinating seeds of V. × wittrockiana, caspase-like activity was detected in the endosperm but not in the embryos, whereas in seeds that failed to germinate, the PCD signal in the endosperm was very weak. In the seeds of V. odorata, caspase-like activity was detected in the embryos and endosperm collected directly after 10 days of stratification, but no signal was detected in the seeds left to germinate for one month after cold stratification. TUNEL assays revealed DNA strand breaks in the peripheral part of the endosperm in V. odorata and in non-germinating V. × wittrockiana, whereas in the germinating seeds of V. × wittrockiana, DNA strand breaks were detected in the endosperm cells adjacent to the embryo. These findings indicate that endosperm-localized PCD facilitates nutrient mobilization to the embryo and seems crucial for successful germination. Overall, these results suggest that PCD contributes to the regulation of seed germination in Viola spp.

Background: Autopolyploids generally exhibit larger phenotypes than their progenitors, suggesting higher biomass and enhanced phytoremediation potential. This study tested this hypothesis using a previously uncharacterized, novel artificial autooctaploid of a Zn- and Cd-tolerant ecotype of Arabidopsis arenosa , derived from a natural tetraploid cytotype. Vegetative development, metal tolerance, and accumulation capacity were compared between the parental tetraploids and the derived autooctaploids under controlled laboratory conditions. Results: During the intermediate growth period (days 56–72), octaploid plants had significantly lower biomass than tetraploids, but this difference was no longer significant by day 84. This transient difference coincided with the earlier entry of octaploids into the generative phase. Despite their autopolyploid origin, the octaploid specimens were fully fertile, enabling sexual reproduction via seeds. They could also be propagated using encapsulated vegetative tissues (artificial seeds). Under extreme heavy metal stress (8 mM Zn and/or 0.2 mM Cd for 9 days), octaploids showed a significantly higher Zn concentration in their roots than tetraploids, while both cytotypes exhibited similar overall resilience to metal toxicity. Conclusions: The autooctaploids demonstrate significant potential for land reclamation due to their ability to tolerate and sequester extreme concentrations of zinc and cadmium without visible signs of toxicity. Under the controlled conditions used here, octaploids showed a > 50% higher Zn concentration in roots than tetraploids, whereas shoot Zn concentrations did not differ, supporting potential for phytostabilisation rather than enhanced phytoextraction. The more rapid life cycle of octaploids may be relevant to future applications, but any advantage under field conditions remains to be tested.