OBJECTIVE: Epimedium brevicornu Maxim. is a perennial persistent C3 plant of the genus Epimedium Linn. in the family Berberaceae that exhibits severe physiological and morphological seed dormancy.We placed mature E. brevicornu seeds under nine stratification treatment conditions and explored the mechanisms of influence by combining seed embryo growth status assessment with related metabolic pathways and gene co-expression analysis.
RESULTS: We identified 3.9 °C as the optimum cold-stratification temperature of E. brevicornu seeds via a chilling unit (CU) model. The best treatment was variable-temperature stratification (10/20 °C, 12/12 h) for 4 months followed by low-temperature stratification (4 °C) for 3 months (4-3). A total of 63801 differentially expressed genes were annotated to 2587 transcription factors (TFs) in 17 clusters in nine treatments (0-0, 0-3, 1-3, 2-3, 3-3, 4-3, 4-2, 4-1, 4-0). Genes specifically highly expressed in the dormancy release treatment group were significantly enriched in embryo development ending in seed dormancy and fatty acid degradation, indicating the importance of these two processes. Coexpression analysis implied that the TF GRF had the most reciprocal relationships with genes, and multiple interactions centred on zf-HD and YABBY as well as on MYB, GRF, and TCP were observed.
CONCLUSIONS: In this study, analyses of plant hormone signal pathways and fatty acid degradation pathways revealed changes in key genes during the dormancy release of E. brevicornu seeds, providing evidence for the filtering of E. brevicornu seed dormancy-related genes.

This study reveals striking differences in the content and composition of hydrophilic and lipophilic compounds in blackcurrant buds (Ribes nigrum L., cv. Ben Klibreck) resulting from winter chill or chemical dormancy release following treatment with ERGER, a biostimulant used to promote uniform bud break. Buds exposed to high winter chill exhibited widespread shifts in metabolite profiles relative to buds that experience winter chill by growth under plastic. Specifically, extensive chilling resulted in significant reductions in storage lipids and phospholipids, and increases in galactolipids relative to buds that experienced lower chill. Similarly, buds exposed to greater chill exhibited higher levels of many amino acids and dipeptides, and nucleotides and nucleotide phosphates than those exposed to lower chilling hours. Low chill buds (IN) subjected to ERGER treatment exhibited shifts in metabolite profiles similar to those resembling high chill buds that were evident as soon as 3 days after treatment. We hypothesise that chilling induces a metabolic shift which primes bud outgrowth by mobilising lipophilic energy reserves, enhancing phosphate availability by switching from membrane phospholipids to galactolipids and enhancing the availability of free amino acids for de novo protein synthesis by increasing protein turnover. Our results additionally suggest that ERGER acts at least in part by priming metabolism for bud outgrowth. Finally, the metabolic differences presented highlight the potential for developing biochemical markers for dormancy status providing an alternative to time-consuming forcing experiments.

Cylicomorpha solmsii (Urb.) Urb (Caricaceae) is a wild relative of domesticated Carica papaya native to the humid tropical forest of Cameroon. C. solmsii is becoming extinct due to rapid urbanization of its habitat. There is currently no restoration planning, no available data on seed germination, details on morphological description and fruit phenology. We investigated the effects of light and soil on seed germination, updated its morphological description and provided cues of its fruit phenology. In two series of experiments, a germination test was first conducted under light and dark conditions with three seed pre-treatments (scarification, drying and cold). Secondly, pre-treated seeds were sown in native soils of C. solmsii habitat collected at Eloumden I and II, two ex-situ and mixtures soil with sand. Qualitative and quantitative data were collected on different part of the plant and analyzed using R package version 4.3.2. Our findings showed that C. solmsii seeds can germinate only under light. The seeds manifested a physiological embryonic dormancy. The native soils showed the highest germination percentage and seedling establishment. The dioicy of C. solmsii was clearly described with incomplete staminate and pistillate unisexual flower whorls. C. solmsii was observed to produce fruits throughout the year at varying intensity. This information is a vital cue to species restoration and policy makers towards C. solmsii conservation.

Dormancy in seeds is a key persistence mechanism for many flowering plants. Physically dormant (PY) seeds have water impermeable seed coats, and in fire-prone systems a common mechanism for dormancy release is fire-induced soil heating. However, the thermal thresholds innate to seeds with PY may be influenced by vegetation, climate, and fire regimes, varying substantially between populations of the same species. To investigate intraspecific variation of thermal thresholds in PY seeds, we sampled obligate seeding Acacia pulchella (Fabaceae) which produces PY seeds. Sampling was undertaken from 13 populations across a climate gradient of rainfall and temperature, and between two vegetation communities in fire-prone Mediterranean-type ecosystems of south-west Western Australia. To test a range of weather and fire-induced soil heating dormancy-break scenarios, we conducted dry heat shock experiments between 40 and 140 °C for 10 min and scored germination for 16 weeks. We created population-specific thermal performance curves and extracted the dormancy release temperature at which 50 % of the seeds had germinated (DRT50), the optimum dormancy-breaking temperature to stimulate maximum germination (T0), and the lethal temperature at which 50 % of the seeds were killed (LT50). Generalised linear models were used to examine relationships between thermal thresholds and possible vegetation, climate, and fire regime drivers of intraspecific variation in seed traits. We found that thermal thresholds differed between vegetation communities, with thresholds consistently higher in forest-type ecosystems compared to open woodland, and the influence of climate varied significantly between the two communities. Seeds from Jarrah Forest populations had a DRT50 16.0 °C higher, a T0 9.7 °C higher, and LT50 7.8 °C higher than seeds from Banksia woodlands. A high rate of non-dormancy was identified in one population that had lost fire in its system and displayed significant germination after both summer and fire-related temperatures. The PY thermal thresholds modelled here provide insight into the strong influence of variable soil heating as a function of vegetation and fuel dynamics in fire-prone environments. Our findings highlight the significant intraspecific variation for this species and suggest that fire-induced soil heating generated by vegetation characteristics may be an overlooked element of fire regimes shaping seed traits.

Flowering plants adjust their reproductive period to maximize the success of the offspring. Monocarpic plants, those with a single reproductive cycle that precedes plant senescence and death, tightly regulate both flowering initiation and flowering cessation. The end of the flowering period involves the arrest of the inflorescence meristem activity, known as proliferative arrest, in what has been interpreted as an evolutionary adaptation to maximize the allocation of resources to seed production and the viability of the progeny. Factors influencing proliferative arrest were described for several monocarpic plant species many decades ago, but only in the last few years studies performed in Arabidopsis have allowed to approach proliferative arrest regulation in a comprehensive manner by studying the physiology, hormone dynamics, and genetic factors involved in its regulation. However, these studies remain restricted to Arabidopsis and there is a need to expand our knowledge to other monocarpic species to propose general mechanisms controlling the process. In this work, we have characterized proliferative arrest in Pisum sativum, trying to parallel available studies in Arabidopsis to maximize this comparative framework. We have assessed quantitatively the role of fruits/seeds in the process, the influence of the positional effect of these fruits/seeds in the behavior of the inflorescence meristem, and the transcriptomic changes in the inflorescence associated with the arrested state of the meristem. Our results support a high conservation of the factors triggering arrest in pea and Arabidopsis, but also reveal differences reinforcing the need to perform similar studies in other species.

Dependency on in vitro culture and regeneration limits the ability to use genome editing on elite wheat (Triticum aestivum L.) varieties. We recently developed an in planta particle bombardment (iPB) technique for gene editing in wheat that utilizes shoot apical meristems (SAMs) as a target tissue. Since the method does not require in vitro culture, it can therefore be used on recalcitrant varieties. In this chapter, we describe in detail the steps used in the iPB method. With this protocol, 3% to 5% of T0 plants grown from bombarded SAMs typically carry mutant alleles and approximately 1% to 2% of the T0 plants inherit mutant alleles in the next generation.

Transgenesis technologies, such as overexpression or RNA interference-mediated suppression, have often been used to alter the activity of target genes. More recently developed targeted genome modification methods using customizable endonucleases allow for the regulation or knockout mutation of target genes without the necessity of integrating recombinant DNA. Such approaches make it possible to create novel alleles of target genes, thereby significantly contributing to crop improvement. Among these technologies, the Cas9 endonuclease-based method is widely applied to several crops, including barley (Hordeum vulgare). In this chapter, we describe an Agrobacterium-based approach to the targeted modification of grain dormancy genes in barley using RNA-guided Cas9 nuclease.

Knockout mutants provide definitive information about the functions of genes related to agronomic traits, including seed dormancy. However, it takes many years to produce knockout mutants using conventional techniques in polyploid plants such as hexaploid wheat. Genome editing with sequence-specific nucleases is a promising approach for obtaining knockout mutations in all targeted homoeologs of wheat simultaneously. Here, we describe a procedure to produce a triple recessive mutant in wheat via genome editing. This protocol covers the evaluation of gRNA and Agrobacterium-mediated transformation to obtain edited wheat seedlings.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.

Genome-wide association study (GWAS) is widely used to characterize genes or quantitative trait loci (QTLs) associated with preharvest sprouting and seed dormancy. GWAS can identify both previously discovered and novel QTLs across diverse genetic panels. The high-throughput SNP arrays or next-generation sequencing technologies have facilitated the identification of numerous genetic markers, thereby significantly enhancing the resolution of GWAS. Although various methods have been developed, the fundamental principles underlying these techniques remain constant. Here, we provide a basic technological flow to perform seed dormancy assay, followed by GWAS using population structure control, and compared it with previous identified QTLs and genes.