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BACKGROUND: Plant meristems are structured organs consisting of distinct layers of stem cells, which differentiate into new plant tissue. Mutations in meristematic layers can propagate into large sectors of the plant. However, the characteristics of meristematic mutations remain unclear, limiting our understanding of the genetic basis of somaclonal phenotypic variation. RESULTS: Here, we analyse the frequency and distribution of somatic mutations in an apricot tree. We separately sequence the epidermis (developing from meristem layer 1) and the flesh (developing from meristem layer 2) of several fruits sampled across the entire tree. We find that most somatic mutations (> 90%) are specific to individual layers. Interestingly, layer 1 shows a higher mutation load than layer 2, implying different mutational dynamics between the layers. The distribution of somatic mutations follows the branching of the tree. This suggests that somatic mutations are propagated to developing branches through axillary meristems. In turn, this leads us to the unexpected observation that the genomes of layer 1 of distant branches are more similar to each other than to the genomes of layer 2 of the same branches. Finally, using single-cell RNA sequencing, we demonstrate that layer-specific mutations were only transcribed in the cells of the respective layers and can form the genetic basis of somaclonal phenotypic variation. CONCLUSIONS: Here, we analyse the frequency and distribution of somatic mutations with meristematic origin. Our observations on the layer specificity of somatic mutations outline how they are distributed, how they propagate, and how they can impact clonally propagated crops.

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The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically uniform products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid multiplication of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to study a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

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Arctic alpine species experience extended periods of cold and unpredictable conditions during flowering. Thus, often, alpine plants use both sexual and asexual means of reproduction to maximize fitness and ensure reproductive success. We used the arctic alpine perennial Arabis alpina to explore the role of prolonged cold exposure on adventitious rooting. We exposed plants to 4°C for different durations and scored the presence of adventitious roots on the main stem and axillary branches. Our physiological studies demonstrated the presence of adventitious roots after 21 weeks at 4°C saturating the effect of cold on this process. Notably, adventitious roots on the main stem developing in specific internodes allowed us to identify the gene regulatory network involved in the formation of adventitious roots in cold using transcriptomics. These data and histological studies indicated that adventitious roots in A. alpina stems initiate during cold exposure and emerge after plants experience growth promoting conditions. While the initiation of adventitious root was not associated with changes of DR5 auxin response and free endogenous auxin level in the stems, the emergence of the adventitious root primordia was. Using the transcriptomic data, we discerned the sequential hormone responses occurring in various stages of adventitious root formation and identified supplementary pathways putatively involved in adventitious root emergence, such as glucosinolate metabolism. Together, our results highlight the role of low temperature during clonal growth in alpine plants and provide insights on the molecular mechanisms involved at distinct stages of adventitious rooting.

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BACKGROUND: Bud sports occur spontaneously in plants when new growth exhibits a distinct phenotype from the rest of the parent plant. The Witch's Broom bud sport occurs occasionally in various grapevine (Vitis vinifera) varieties and displays a suite of developmental defects, including dwarf features and reduced fertility. While it is highly detrimental for grapevine growers, it also serves as a useful tool for studying grapevine development. We used the Witch's Broom bud sport in grapevine to understand the developmental trajectories of the bud sports, as well as the potential genetic basis. We analyzed the phenotypes of two independent cases of the Witch's Broom bud sport, in the Dakapo and Merlot varieties of grapevine, alongside wild type counterparts. To do so, we quantified various shoot traits, performed 3D X-ray Computed Tomography on dormant buds, and landmarked leaves from the samples. We also performed Illumina and Oxford Nanopore sequencing on the samples and called genetic variants using these sequencing datasets. RESULTS: The Dakapo and Merlot cases of Witch's Broom displayed severe developmental defects, with no fruit/clusters formed and dwarf vegetative features. However, the Dakapo and Merlot cases of Witch's Broom studied were also phenotypically different from one another, with distinct differences in bud and leaf development. We identified 968-974 unique genetic mutations in our two Witch's Broom cases that are potential causal variants of the bud sports. Examining gene function and validating these genetic candidates through PCR and Sanger-sequencing revealed one strong candidate mutation in Merlot Witch's Broom impacting the gene GSVIVG01008260001. CONCLUSIONS: The Witch's Broom bud sports in both varieties studied had dwarf phenotypes, but the two instances studied were also vastly different from one another and likely have distinct genetic bases. Future work on Witch's Broom bud sports in grapevine could provide more insight into development and the genetic pathways involved in grapevine.

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Induced mutations have been an important tool for plant breeding and functional genomics for more than 80 years. Novel mutations can be induced by treating seed or other plant cells with chemical mutagens or ionizing radiation. The majority of released mutant crop varieties were developed using ionizing radiation. This has been shown to create a variety of different DNA lesions including large (e.g., >=10,000 bps) copy number variations (CNV). Detection of induced DNA lesions from whole genome sequence data is useful for choosing a mutagen dosage prior to committing resources to develop a large mutant population for forward or reverse-genetic screening. Here I provide a method for detecting large induced CNV from mutant plants that utilizes a new tool to streamline the process of obtaining read coverage directly from BAM files, comparing non-mutagenized controls and mutagenized samples, and plotting the results for visual evaluation. Example data is provided from low coverage sequence data from gamma-irradiated vegetatively propagated triploid banana.

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Conversion of potato from a tetraploid, heterozygous, vegetatively propagated crop to a diploid F1 hybrid, propagated via botanical seed, would constitute a considerable advance for global agriculture, but faces multiple challenges. One such challenge is the difficulty in inbreeding potato, which involves purging deleterious alleles from its genome. This commentary discusses possible reasons for this difficulty and highlights a recent sequence-based effort to classify SNP variation, in potato germplasm, according to its deleterious potential. Tools and strategies connected to this database may facilitate development of F1 hybrids.

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The genus Eucalyptus is a globally captivated source of hardwood and is well known for its medicinal uses. The hybrid and wild species of Eucalyptus are widely used as exotic plantations due to their renowned potential of adapting to various systems and sites, and rapid large-scale propagation of genetically similar plantlets, which further leads to the extensive propagation of this species. Tissue culture plays a crucial role in the preservation, propagation, and genetic improvement of Eucalyptus species. Despite unquestionable progression in biotechnological and tissue culture approaches, the productivity of plantations is still limited, often due to the low efficiency of clonal propagation from cuttings. The obtained F1 hybrids yield high biomass and high-quality low-cost raw material for large-scale production; however, the development of hybrid, clonal multiplication, proliferation, and post-developmental studies are still major concerns. This riveting review describes the problems concerning the in vitro and clonal propagation of Eucalyptus plantation and recent advances in biotechnological and tissue culture practices for massive and rapid micropropagation of Eucalyptus, and it highlights the Eucalyptus germplasm preservation techniques.

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PREMISE: The central Oaxaca Basin has a century-long history of agave cultivation and is hypothesized to be the region of origin of other cultivated crops. Widely cultivated for mezcal production, the perennial crop known as "espadín" is putatively derived from wild Agave angustifolia. Nevertheless, little is known about its genetic relationship to the wild A. angustifolia or how the decades-long clonal propagation has affected its genetics. METHODS: Using restriction-site-associated DNA sequencing and over 8000 single-nucleotide polymorphisms, we studied aspects of the population genomics of wild and cultivated A. angustifolia in Puebla and Oaxaca, Mexico. We assessed patterns of genetic diversity, inbreeding, distribution of genetic variation, and differentiation among and within wild populations and plantations. RESULTS: Genetic differentiation between wild and cultivated plants was strong, and both gene pools harbored multiple unique alleles. Nevertheless, we found several cultivated individuals with high genetic affinity with wild samples. Higher heterozygosity was observed in the cultivated individuals, while in total, they harbored considerably fewer alleles and presented higher linkage disequilibrium compared to the wild plants. Independently of geographic distance among sampled plantations, the genetic relatedness of the cultivated plants was high, suggesting a common origin and prevalent role of clonal propagation. CONCLUSIONS: The considerable heterozygosity found in espadín is contained within a network of highly related individuals, displaying high linkage disequilibrium generated by decades of clonal propagation and possibly by the accumulation of somatic mutations. Wild A. angustifolia, on the other hand, represents a significant genetic diversity reservoir that should be carefully studied and conserved.

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Silver nanoparticles (AgNPs) are novel compounds used as antimicrobial and antiviral agents. In addition, AgNPs have been used to improve the growth of different plants, as well as the in vitro multiplication of plant material. In this work the effect of AgNPs on in vitro growth of 'Canino' and 'Mirlo Rojo' cultivars, as well as the leaf ion composition, are studied. Different concentrations of AgNPs (0, 25, 50, 75 and 100 mg L-1) were added to two culture systems: semisolid medium with agar (SSM) in jars and liquid medium in temporary immersion system (TIS). Proliferation (number of shoots), shoot length, productivity (number of shoot × average length), leaf surface, fresh and dry weight were measured. Additionally, the silver and other ion accumulation in the leaves were evaluated by inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis. The productivity of 'Canino' and 'Mirlo Rojo' decreased when increasing the concentration of AgNPs in the semisolid medium. However, the use of AgNPs in the TIS improved the proliferation and productivity of 'Canino' and Mirlo Rojo', increasing biomass production, and the concentration of nutrients in the plants, although these effects are genotype-dependent. TISs are the best system for introducing silver into shoots, the optimum concentration being 50 mg L-1 for 'Canino' and 75 mg L-1 for 'Mirlo Rojo'. Principal component analysis, considering all the analyzed ions along the treatments, separates samples in two clear groups related to the culture system used. The use of bioreactors with a liquid medium has improved the productivity of 'Canino' and 'Mirlo Rojo' in the proliferation stage, avoiding hyperhydration and other disorders. The amount of metallic silver that penetrates apricot plant tissues depends on the culture system, cultivar and concentration of AgNPs added to the culture medium. Silver ion accumulation measured in the shoots grown in the TIS was higher than in shoots micropropagated in a semisolid medium, where it is barely detectable. Furthermore, AgNPs had a beneficial effect on plants grown in TIS. However, AgNPs had a detrimental effect when added to a semisolid medium.

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Introducción: Los arrecifes de coral son ecosistemas altamente degradados, por lo que ha sido necesario implementar acciones de restauración activa para recuperar su estructura y funcionamiento. Se ha implementado la propagación clonal para obtener fragmentos pequeños (~ 10 cm) de las ramas distales de colonias donadoras de corales de la especie Acropora palmata, para posteriormente fijarlos en el sustrato arrecifal, simulando el efecto de dispersión que ocurre de manera natural en esta especie, a lo que en este trabajo se denomina ''dispersión asistida". Sin embargo, es necesario evaluar los efectos de esta técnica como son: la cantidad de fragmentos que se puede obtener de cada colonia, el periodo de recuperación de tejido de las colonias donadoras y los fragmentos sembrados. Objetivo: Evaluar el efecto de poda en las colonias donadoras estimando el porcentaje de tejido podado de colonias donadoras de A. palmata y su tasa de recuperación 30 meses después. Métodos: Se realizaron cuatro monitoreos: antes, inmediatamente después de la poda, un mes después de la siembra, y 30 meses después, en cuatro colonias de A. palmata localizadas en el Parque Nacional Costa Occidental de Isla Mujeres, Punta Cancún y Punta Nizuc en el Caribe mexicano. La modelación 3D basada en fotogrametría se realizó con el software Agisoft Metashape Pro, mientras que las métricas de área de superficie de tejido, extensión radial y apical se obtuvieron mediante el software CloudCompare. Resultados: Posterior a la colecta de fragmentos de las colonias, se observó que el material utilizado en la dispersión asistida representa menos del 12% del tejido vivo. Después de un mes, las colonias donadoras presentaban una recuperación del 5% con tejido nuevo recubriendo las áreas de corte. Las colonias donadoras perdieron, en promedio, 65% de tejido vivo tras el impacto de cuatro huracanes, y en un caso la colonia fue totalmente eliminada, pero con los fragmentos sembrados se pudo conservar el genotipo. Conclusiones: La dispersión asistida podría incrementar el tejido vivo de corales ramificados en intervalos de tiempo relativamente cortos, sin comprometer la integridad de la colonia donadora, si se poda menos del 12%.

Introduction: Coral reefs are highly degraded ecosystems, for which it has been necessary to implement active restoration actions to recover their structure and functioning. Asexual propagation has been implemented to obtain small fragments (~10 cm) from the distal branches of donor colonies of corals of the species Acropora palmata, to subsequently relocate them in the reef substrate, simulating the dispersion effect that occurs naturally in the species, which in this work is called assisted propagation. However, it is necessary to evaluate the effects of this technique, such as the number of fragments that can be obtained from each colony, the tissue recovery period of the donor colonies and fragments. Objective: To address the effect of pruning on donor colonies by estimating the percentage of live tissue removed from donor colonies of A. palmata and their recovery rate after 30-months. Methods: Four surveys were carried out: before, immediately after pruning, one month after outplanting, and 30 months after pruning on four colonies of A. palmata located in the Parque Nacional Costa Occidental de Isla Mujeres, Punta Cancún and Punta Nizuc in the Mexican Caribbean. Photogrammetry-based 3D modeling was performed using Agisoft Metashape Pro software, while tissue surface area, radial and apical growth were obtained using CloudCompare software. Results: After fragment collection, the material used in the assisted propagation represents less than 12% of the living tissue. After one month, the donor colonies showed a recovery of 5%, with new tissue covering the cut areas. The donor colonies lost on average 65 % of living tissue after four hurricanes, and in one case the colony was lost all together, but with the outplanted fragments the genotype could be preserved. Conclusions: Assisted propagation could increase living tissue of branching corals in relatively short intervals of time, without serious damage to the donor colony if less than 12 % is removed.

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Many eukaryotic organisms reproduce by sexual and asexual reproduction. Genetic diversity in populations can be strongly dependent on the relative importance of these two reproductive modes. Here, we compare the amounts and patterns of genetic diversity in related water hyacinths that differ in their propensity for clonal propagation - highly clonal Eichhornia crassipes and moderately clonal E. azurea (Pontederiaceae). Our comparisons involved genotype-by-sequencing (GBS) of 137 E. crassipes ramets from 60 locations (193,495 nucleotide sites) and 118 E. azurea ramets from 53 locations (198,343 nucleotide sites) among six hydrological basins in central South America, the native range of both species. We predicted that because of more prolific clonal propagation, E. crassipes would exhibit lower clonal diversity than E. azurea. This prediction was supported by all measures of clonal diversity that we examined. Eichhornia crassipes also had a larger excess of heterozygotes at variant sites, another signature of clonality. However, genome-wide heterozygosity was not significantly different between the species. Eichhornia crassipes had weaker spatial genetic structure and lower levels of differentiation among hydrological basins than E. azurea, probably because of higher clonality and more extensive dispersal of its free-floating life form. Our findings for E. crassipes contrast with earlier studies from the invasive range which have reported very low levels of clonal diversity and extensive geographic areas of genetic uniformity.

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Somatic embryogenesis is the process by which embryos are formed from a single or small group of somatic cells in response to specific stimuli. Somatic embryogenesis has been applied to achieve mass clonal propagation on an industrial scale and to increase the agronomic performance of species of economic interest, including sugarcane. The use of somatic embryogenesis in sugarcane stands out as a biotechnological tool with a high potential for application in the clonal propagation of disease-free elite varieties, as an essential part of genetic transformation protocols, and in the production of synthetic seeds. A better understanding of each phase of somatic embryogenesis can help to optimize the process to enhance yields and produce high-quality emblings. In this chapter, we describe a detailed protocol for somatic embryogenesis in sugarcane (Saccharum sp.) to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment of sugarcane emblings.

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Emerald ash borer (Agrilus planipennis; EAB) has devastated populations of ash (Fraxinus spp.) trees in dozens of U.S. states and Canada over the past few decades. The continued survival of scattered ash trees known as "lingering ash" in heavily infested natural stands, however, offers evidence of genetic resistance or tolerance to EAB. These surviving or "lingering" ash individuals may form the basis for reforestation programs in EAB-impacted areas, and clonal mass-propagation of these genotypes can help accelerate these efforts. Between 2013 and 2018, we initiated embryogenic cultures by culturing immature zygotic embryos from open-pollinated (OP) seeds collected from several surviving white ash and green ash trees in Michigan and Pennsylvania. In addition, in 2018, we initiated cultures from crosses made between lingering green ash parents from the USDA Forest Service ash breeding program in Ohio. Somatic embryos were produced by growing cultures in liquid suspension, followed by fractionation and plating on semisolid medium to produce developmentally synchronous populations of somatic embryos. Somatic embryo germination and conversion were enhanced by a combination of pre-germination cold treatment and inclusion of activated charcoal and gibberellic acid in the germination medium. Ash somatic seedlings derived from OP explants grew rapidly following transfer to potting mix and somatic seedlings representing nine ash clones were acclimatized, grown in the greenhouse and planted in a preliminary field test, along with EAB-resistant Manchurian ash (F. mandshurica) and EAB-susceptible control seedlings. Somatic seedlings have now been produced from cultures that originated from seeds derived from the progeny of lingering green ash parents and an ex vitro germination protocol has shown some promise for accelerating early somatic seedling growth. Results of this research could provide the basis for scaled-up production of EAB-resistant ash varieties for seed orchard production for forest restoration and cultivar development for urban tree restoration.

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Sugi (Japanese cedar, Cryptomeria japonica) is the most important forestry tree species in Japan, covering 44% of the total artificial forest area. Large amounts of pollen released from these forests each spring cause allergic reactions in approximately 40% of the population, which are a serious social and public health problem in Japan. As a countermeasure, there is an urgent need to reforest using male-sterile plants (MSPs; pollen-free plants); however, the production of MSPs via conventional methods is inefficient, time consuming, and requires considerable resources in terms of labor and space. In the present paper, we described an improved and simplified methodology for the efficient propagation of pollen-free Japanese cedar, combining the use of genetic markers (marker-assisted selection or marker-aided selection) for the early selection of male-sterile genotypes and the use of somatic embryogenesis (SE) for the clonal mass propagation of seedlings. We describe all the stages involved in the production process of somatic seedlings. Our results demonstrated that this methodology easily and efficiently produces MSPs with a discrimination rate of 100% in a short period of time. Production of 243.6 ± 163.6 cotyledonary embryos per plate, somatic embryo germination, and plantlet conversion frequencies of 87.1 ± 11.9% and 84.8 ± 12.6%, respectively, and a 77.6 ± 12.1% survival rate after ex vitro acclimatization was achieved. Moreover, we also describe an easy method for the collection of somatic embryos prior to germination, as well as an efficient and practical method for their storage at 5°C. Finally, a representative schedule for the propagation of pollen-free sugi somatic seedlings is presented as a reference for practical uses. This methodology will definitively help to accelerate the production of C. japonica MSPs across Japan.

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Pollen allergy caused by sugi (Japanese cedar, Cryptomeria japonica) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of MS1 itself, selecting individuals with ms1-1 and ms1-2 male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of MS1 male-sterile sugi seedlings.

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Pathogens cause significant challenges to global food security. On annual crops, pathogens must re-infect from environmental sources in every growing season. Fungal pathogens have evolved mixed reproductive strategies to cope with the distinct challenges of colonizing growing plants. However, how pathogen diversity evolves during growing seasons remains largely unknown. Here, we performed a deep hierarchical sampling in a single experimental wheat field infected by the major fungal pathogen Zymoseptoria tritici. We analysed whole genome sequences of 177 isolates collected from 12 distinct cultivars replicated in space at three time points of the growing season to maximize capture of genetic diversity. The field population was highly diverse with 37 SNPs per kilobase, a linkage disequilibrium decay within 200-700 bp and a high effective population size. Using experimental infections, we tested a subset of the collected isolates on the dominant cultivar planted in the field. However, we found no significant difference in virulence of isolates collected from the same cultivar compared to isolates collected on other cultivars. About 20 % of the isolate genotypes were grouped into 15 clonal groups. Pairs of clones were disproportionally found at short distances (<5 m), consistent with experimental estimates for per-generation dispersal distances performed in the same field. This confirms predominant leaf-to-leaf transmission during the growing season. Surprisingly, levels of clonality did not increase over time in the field although reproduction is thought to be exclusively asexual during the growing season. Our study shows that the pathogen establishes vast and stable gene pools in single fields. Monitoring short-term evolutionary changes in crop pathogens will inform more durable strategies to contain diseases.

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In order to investigate the intraspecific diversity of wild Humulus lupulus (hop) in Central Italy, 12 populations were evaluated for their genetic polymorphism by means of 13 SSR loci together with six commercial cultivars as a reference. High levels of polymorphism were found across the populations, being 140 the number of multilocus genotypes over 159 samples analyzed. Moreover, the observed heterozygosity was higher than expected in most of the populations. High levels of gene flow were thus envisaged to occur within and among wild populations, and our sampling strategy allowed us to gain insights on the propagation modes of this species, i.e. clonal versus sexual propagation. Nevertheless, a genetic structure of populations with at least five genetically different clusters was disclosed. Private alleles were observed in both wild and cultivated hops. Chemical analysis of bittering and aromatic quality of female flowers from a subset of 8 wild populations revealed a high variability among plants, especially for essential oil components. Overall, the high variability of wild accessions here examined represent a valid source to be exploited in future breeding programs for new or improved hop cultivars development.

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BACKGROUND AND AIMS: Inferring the diffusion history of many human-dispersed species is still not straightforward due to unresolved past human migrations. The centre of diversification and routes of migration of the autopolyploid and clonally propagated greater yam, Dioscorea alata, one of the oldest edible tubers, remain unclear. Here, we address yam demographic and dispersal history using a worldwide sample. METHODS: We characterized genome-wide patterns of genetic variation using genotyping by sequencing 643 greater yam accessions spanning four continents. First, we disentangled the polyploid and clonal components of yam diversity using allele frequency distribution and identity by descent approaches. We then addressed yam geographical origin and diffusion history with a model-based coalescent inferential approach. KEY RESULTS: Diploid genotypes were more frequent than triploids and tetraploids worldwide. Genetic diversity was generally low and clonality appeared to be a main factor of diversification. The most likely evolutionary scenario supported an early divergence of mainland Southeast Asian and Pacific gene pools with continuous migration between them. The genetic make-up of triploids and tetraploids suggests that they have originated from these two regions before westward yam migration. The Indian Peninsula gene pool gave origin to the African gene pool, which was later introduced to the Caribbean region. CONCLUSIONS: Our results are congruent with the hypothesis of independent domestication origins of the two main Asian and Pacific gene pools. The low genetic diversity and high clonality observed suggest a strong domestication bottleneck followed by thousands of years of widespread vegetative propagation and polyploidization. Both processes reduced the extent of diversity available for breeding, and this is likely to threaten future adaptation.

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