The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell’s chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.

In plants, male reproductive development is extremely sensitive to adverse climatic environments and (a)biotic stress. Upon exposure to stress, male gametophytic organs often show morphological, structural and metabolic alterations that typically lead to meiotic defects or premature spore abortion and male reproductive sterility. Depending on the type of stress involved (e.g. heat, cold, drought) and the duration of stress exposure, the underlying cellular defect is highly variable and either involves cytoskeletal alterations, tapetal irregularities, altered sugar utilization, aberrations in auxin metabolism, accumulation of reactive oxygen species (ROS; oxidative stress) or the ectopic induction of programmed cell death (PCD). In this review, we present the critically stress‐sensitive stages of male sporogenesis (meiosis) and male gametogenesis (microspore development), and discuss the corresponding biological processes involved and the resulting alterations in male reproduction. In addition, this review also provides insights into the molecular and/or hormonal regulation of the environmental stress sensitivity of male reproduction and outlines putative interaction(s) between the different processes involved.

In modern agriculture, seeking eco-friendly ways to promote plant growth and enhance crop productivity is of priority. Biostimulants are a group of substances from natural origin that contribute to boosting plant yield and nutrient uptake, while reducing the dependency on chemical fertilizers. Developing biostimulants from by-products paves the path to waste recycling and reduction, generating benefits for growers, food industry, registration and distribution companies, as well as consumers. The criteria to select designated by-products for valorizing as biostimulant are: absence of pesticide residue, low cost of collection and storage, sufficient supply and synergy with other valorization paths. Over the years, projects on national and international levels such as NOSHAN, SUNNIVA, and Bio2Bio have been initiated (i) to explore valorization of by-products for food and agriculture industries; (ii) to investigate mode of action of biostimulants from organic waste streams. Several classes of waste-derived biostimulants or raw organic material with biostimulant components were shown to be effective in agriculture and horticulture, including vermicompost, composted urban waste, sewage sludge, protein hydrolysate, and chitin/chitosan derivatives. As the global market for biostimulants continues to rise, it is expected that more research and development will expand the list of biostimulants from by-products. Global nutrient imbalance also requires biostimulant to be developed for targeted market. Here, we review examples of biostimulants derived from agricultural by-products and discuss why agricultural biomass is a particularly valuable source for the development of new agrochemical products.

Today's agriculture faces many concerns in maintaining crop yield while adapting to climate change and transitioning to more sustainable cultivation practices. The application of plant biostimulants (PBs) is one of the methods that step forward to address these challenges. The advantages of PBs have been reported numerous times. Yet, there is a general lack of quantitative assessment of the overall impact of PBs on crop production. Here we report a comprehensive meta-analysis on biostimulants (focus on non-microbial PBs) of over one thousand pairs of open-field data in a total of 180 qualified studies worldwide. Yield gains in open-field cultivation upon biostimulant application were compared across different parameters: biostimulant category, application method, crop species, climate condition, and soil property. The overall results showed that (1) the add-on yield benefit among all biostimulant categories is on average 17.9% and reached the highest potential via soil treatment; (2) biostimulant applied in arid climates and vegetable cultivation had the highest impact on crop yield; and (3) biostimulants were more efficient in low soil organic matter content, non-neutral, saline, nutrient-insufficient, and sandy soils. This systematic review provides general biostimulant application guidelines and gives consultants and growers insights into achieving an optimal benefit from biostimulant application.

Polyploidization has played a key role in plant breeding and crop improvement. Although its potential to increase biomass yield is well described, the effect of polyploidization on biomass composition has largely remained unexplored. Here, we generated a series of Arabidopsis (Arabidopsis thaliana) plants with different somatic ploidy levels (2n, 4n, 6n, and 8n) and performed rigorous phenotypic characterization. Kinematic analysis showed that polyploids developed slower compared to diploids; however, tetra- and hexaploids, but not octaploids, generated larger rosettes due to delayed flowering. In addition, morphometric analysis of leaves showed that polyploidy affected epidermal pavement cells, with increased cell size and reduced cell number per leaf blade with incrementing ploidy. However, the inflorescence stem dry weight was highest in tetraploids. Cell wall characterization revealed that the basic somatic ploidy level negatively correlated with lignin and cellulose content, and positively correlated with matrix polysaccharide content (i.e. hemicellulose and pectin) in the stem tissue. In addition, higher ploidy plants displayed altered sugar composition. Such effects were linked to the delayed development of polyploids. Moreover, the changes in polyploid cell wall composition promoted saccharification yield. The results of this study indicate that induction of polyploidy is a promising breeding strategy to further tailor crops for biomass production.

Summary: In the plant kingdom, events of whole genome duplication or polyploidization are generally believed to occur via alterations of the sexual reproduction process. Thereby, diploid pollen and eggs are formed that contain the somatic number of chromosomes rather than the gametophytic number. By participating in fertilization, these so‐called 2n gametes generate polyploid offspring and therefore constitute the basis for the establishment of polyploidy in plants. In addition, diplogamete formation, through meiotic restitution, is an essential component of apomixis and also serves as an important mechanism for the restoration of F1 hybrid fertility. Characterization of the cytological mechanisms and molecular factors underlying 2n gamete formation is therefore not only relevant for basic plant biology and evolution, but may also provide valuable cues for agricultural and biotechnological applications (e.g. reverse breeding, clonal seeds). Recent data have provided novel insights into the process of 2n pollen and egg formation and have revealed multiple means to the same end. Here, we summarize the cytological mechanisms and molecular regulatory networks underlying 2n gamete formation, and outline important mitotic and meiotic processes involved in the ectopic induction of sexual polyploidization. Contents Summary 670 I. Introduction 670 II. General mechanisms of 2n gamete formation 671 III. Cytological processes leading to meiotic restitution 672 IV. Genetic control of 2n gamete formation 675 V. Conclusions and future directions 680 Acknowledgements 680 References 680