Seed Dormancy Class and Ecophysiological Features of Veronicastrum sibiricum (L.) Pennell (Scrophulariaceae) Native to the Korea Peninsula

Veronicastrum sibiricum is a perennial species distributed in Korea, Japan, Manchuria, China, and Siberia. This study aimed to determine the requirements for germination and dormancy break of V. sibiricum seeds and to classify the kind of seed dormancy. Additionally, its class of dormancy was compared with other Veronicastrum and Veronica species. V. sibiricum seeds were permeable to water and had a mature embryo during seed dispersal. In field conditions, germination was prevented by physiological dormancy, which was, however, relieved by March of the next year, allowing the start of germination when suitable environmental conditions occurred. In laboratory experiments, the seeds treated with 0, 2, 4, 8, and 12 weeks of cold stratification (4 °C) germinated to 0, 79, 75, 72, and 66%, respectively. After the GA3 treatment (2.887 mM), ≥90% of the seeds germinated during the four incubation weeks at 20/10 °C. Thus, 2.887 mM GA3 and at least two weeks at 4 °C were effective in breaking physiological dormancy and initiating germination. Therefore, the V. sibiricum seeds showed non-deep physiological dormancy (PD). Previous research, which determined seed dormancy classes, revealed that Veronica taxa have PD, morphological (MD), or morphophysiological seed dormancy (MPD). The differences in the seed dormancy classes in the Veronicastrum-Veronica clade suggested that seed dormancy traits had diverged. The results provide important data for the evolutionary ecological studies of seed dormancy and seed-based mass propagation of V. sibiricum.

Resetting of the 24-nt siRNA landscape in rice zygotes

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells - the egg and sperm, which in plants have radically different siRNA transcriptomes from each other, and from multicellular embryos. Due to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Since siRNAs serve as both regulators and outputs of the epigenome, we performed here the successful characterization of small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibited extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation was accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ~70% of the zygote siRNA loci did not overlap any egg cell siRNA loci. Newly-detected siRNA loci in zygote are gene proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci which are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci were associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

EFFECT OF 2,4-D AND NAA ON SOMATIC EMBRYOGENESIS FROM APICAL PORTION OF GROUNDNUT (ARACHIS HYPOGAEA L.)

Somatic embryogenesis was carried out epicotyl portion of the mature embryo/apical portion. The somatic embryo induction medium containing 2,4-D or NAA (10.0 to 50.0 mg/l). Of the two concentrations tested 2,4-D (30.0mg/l) recorded the highest percentage of response followed by NAA (30.0mg/l). But the highest number of somatic embryo were recorded in 30.0mg/l of 2,4-D followed by NAA. The apical portion of the mature embryo formed direct embryos without any intervention of callus. The maximum percentage of embryogenic cultures were noticed in 30.0mg/l of 2,4-D followed by NAA at 30.0mg/l. for the differentiation of somatic embryos, the embryogenic masses were transferred to medium without any growth regulator. The maximum number of somatic embryos per culture was recorded in 30 mg/l of 2,4-D followed by 30.0 mg/l of NAA. Keywords: Arachis hypogaea L.,Somatic Embryogenesis, 2,4-D and NAA

Differential Expression Pattern of Goat Uterine Fluids Extracellular Vesicles miRNAs during Peri-Implantation

Early pregnancy failure occurs when a mature embryo attaches to an unreceptive endometrium. During the formation of a receptive endometrium, extracellular vesicles (EVs) of the uterine fluids (UFs) deliver regulatory molecules such as small RNAs to mediate intrauterine communication between the embryo and the endometrium. However, profiling of small RNAs in goat UFs’ EVs during pregnancy recognition (day 16) has not been carried out. In this study, EVs were isolated from UFs on day 16 of the estrous cycle or gestation. They were isolated by Optiprep™ Density G radient (ODG) and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Immunostaining demonstrated that CD63 was present both in the endometrial epithelium and glandular epithelium, and stain intensity was greater in the pregnant endometrium compared to the non-pregnant endometrium. Small RNA sequencing revealed that UFs’ EVs contained numerous sRNA families and a total of 106 differentially expressed miRNAs (DEMs). Additionally, 1867 target genes of the DEMs were obtained, and miRNA–mRNA interaction networks were constructed. GO and KEGG analysis showed that miRNAs were significantly associated with the formation of a receptive endometrium and embryo implantation. In addition, the fluorescence in situ hybridization assay (FISH) showed that chi-miR-451-5p was mainly expressed in stromal cells of the endometrium and a higher level was detected in the endometrial luminal epithelium in pregnant states. Moreover, the dual-luciferase reporter assay showed that chi-miR-451-5p directly binds to PSMB8 and may play an important role in the formation of a receptive endometrium and embryo implantation. In conclusion, these results reveal that UFs’ EVs contain various small RNAs that may be vital in the formation of a receptive endometrium and embryo implantation.

In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococum) and bread (Triticumaestivum) wheat under boron stress

Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum) wheat were used for callus induction on media containing four (0, 1, 2 and 4 mg L− 1) different doses of 2,4-D and dicamba supplemented with under five (0, 6.2, 12.4, 24.8, and 37.2 mg L− 1) different boron stresses. The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L− 1) different BAP doses with five boron stresses for regeneration. The heaviest callus weight in einkorn wheat was in culture media with 1 mg L− 1 dicamba and 6.2 mg L− 1 (3.71 ± 0.13 g). Bread wheat had the heaviest callus weight on culture media with 4 mg L− 1 dicamba and 12.4 mg L− 1 (3.46 ± 0.40 g). Callus diameters were observed as the highest in culture media with 1 mg L− 1 and 12.4 mg L− 1 (1.10 ± 0.31) for einkorn and 4 mg L− 1 dicamba with 6.2 mg L− 1 boron (1.22 ± 0.27 cm) for bread wheat. Regeneration capacity was highest in control group with 6.2 mg L− 1 for both wheat genotypes (einkorn, 71.33% ± 11.78 and bread, 65.33% ± 10.80). The highest plantlet numbers were in only 2 mg L− 1 BAP (2.92 ± 0.88) for einkorn wheat and 0.5 mg L− 1 BAP supplemented with 6.2 mg L− 1 boron (3.71 ± 1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses may be useful for wheat breeding studies.

The REF6-dependent H3K27 demethylation establishes transcriptional competence to promote germination in Arabidopsis

Seed germination is a critical developmental switch from a dormant state to active growth, which involves extensive changes in metabolism, gene expression and cellular identity. However, our understanding of epigenetic and transcriptional reprogramming during this process is limited. The histone H3 lysine 27 trimethylation (H3K27me3) plays a key role in regulating gene repression and cell fate specification. Here, we profile H3K27me3 dynamics and dissect the function of H3K27 demethylation during germination. Our temporal genome-wide profiling of H3K27me3 and transcription reveal delayed H3K27me3 reprogramming compared with transcriptomic changes during germination, with H3K27me3 changes mainly occurring when the embryo is entering into vegetative development. REF6-mediated H3K27 demethylation promotes germination but does not significantly contribute to H3K27me3 dynamics during germination, but rather stably establishes an H3K27me3-depleted state permissive to transcription. By analyzing REF6 genomic binding, we show that it is absent from mature embryo chromatin and gradually establishes occupancy during the course of germination to counteract increased PRC2 activity. Our study provides key insights into the dynamics of gene expression and H3K27me3 during seed germination and suggests the function of H3K27me3 in facilitating cell fate switch. Furthermore, we reveal the importance of H3K27 demethylation-established transcriptional competence in germination and likely other developmental processes.

Callus Induction and Regeneration from Germinating Mature Embryos of Wheat (Triticum aestivum L.)

A high-performance protocol for callus induction was devised using germinating mature embryos of two local wheat (Triticum aestivum L.) landraces as explant. The results showed that callus development from germinating embryos was rapid starting one day after culture with an induction rate 20 to 25% higher than those of soaked embryos. In addition, the mean rate of growth of callus developed from germinating embryos was 60 to 70% higher than those cultured from soaked embryos. This study also demonstrated a higher frequency of green spots formation(48 to 56%)on callus derived from germinating embryos compared to their soaked counterpart (24 to 28%), suggesting a better differentiation potential of callus cultures derived from germinating embryo. These findings indicate that germinating mature embryo is more suitable explant for wheat callus induction and regeneration than the soaked mature embryo commonly employed for wheat callus culture.

Small Non-Coding RNAs at the Crossroads of Regulatory Pathways Controlling Somatic Embryogenesis in Seed Plants

Small non-coding RNAs (sncRNAs) are molecules with important regulatory functions during development and environmental responses across all groups of terrestrial plants. In seed plants, the development of a mature embryo from the zygote follows a synchronized cell division sequence, and growth and differentiation events regulated by highly regulated gene expression. However, given the distinct features of the initial stages of embryogenesis in gymnosperms and angiosperms, it is relevant to investigate to what extent such differences emerge from differential regulation mediated by sncRNAs. Within these, the microRNAs (miRNAs) are the best characterized class, and while many miRNAs are conserved and significantly represented across angiosperms and other seed plants during embryogenesis, some miRNA families are specific to some plant lineages. Being a model to study zygotic embryogenesis and a relevant biotechnological tool, we systematized the current knowledge on the presence and characterization of miRNAs in somatic embryogenesis (SE) of seed plants, pinpointing the miRNAs that have been reported to be associated with SE in angiosperm and gymnosperm species. We start by conducting an overview of sncRNA expression profiles in the embryonic tissues of seed plants. We then highlight the miRNAs described as being involved in the different stages of the SE process, from its induction to the full maturation of the somatic embryos, adding references to zygotic embryogenesis when relevant, as a contribution towards a better understanding of miRNA-mediated regulation of SE.