Biology subjects

Melzer, R.

Publications and source records attributed to Melzer, R..

13 recordsLinked to original sources

bioRxiv2026

The Viability Gambit: An Optimized Sterilisation Protocol for Industrial Hemp ( Cannabis sativa L.) Seeds

Hemp (Cannabis sativa L.) is an increasingly important crop with applications spanning fibre, seed oil and bioactive cannabinoid production, yet the development of reliable tissue culture systems for this species remains a significant challenge. The establishment of axenic seedling cultures is a prerequisite for hypocotyl-based regeneration and future genetic transformation pipelines, but hemp seeds harbour diverse endophytic microbial communities that frequently overwhelm standard surface sterilisation protocols. Here, we present a systematic comparative evaluation of seed sterilisation strategies across seven industrial hemp accessions, examining the effects of sterilisation chemistry, seed provenance and accession identity on both contamination outcome and the subsequent morphogenic competence of hypocotyl explants. Across all treatments and accessions, in-house glasshouse-harvested seeds achieved higher sterility rates than commercially sourced material regardless of protocol applied. This provenance effect, combined with considerable batch-to-batch variation within seed sources, indicates that contamination load is a primary determinant of successful hemp seed sterilisation. Among the sterilisation treatments evaluated, a baseline of 75% ethanol combined with sequential 1% hydrogen peroxide incubation performed consistently well for low-load seed batches, while supplementation with Plant Preservative Mixture (PPMTM) might be necessary to achieve acceptable rates of non-contaminated seedlings from high contamination load batches. Beyond their effect on contamination, sterilisation treatments influenced the morphogenic fate of hypocotyl explants independently of sterility outcomes. Notably, seedling treatment with the Prochloraz-based fungicide Octave promoted shoot and root co-regeneration in the absence of exogenous plant growth regulators in some cases. Hormone-free organogenesis from hypocotyl explants was achievable across multiple hemp accessions, demonstrating that this developmental capacity is broadly distributed within hemp, though its frequency and consistency varied with accession identity and protocol conditions. Together, these findings provide a practical framework for axenic hemp seed culture that can be used as starting point requiring local adaptation based on seed source, batch history and the intended downstream application.

Plant Biology↗

bioRxiv2026

GENE-FAM: An automated pipeline for mining gene families and its application to MADS-box genes in Cannabis sativa

Understanding how gene families evolve can offer great insight into adaptation at the phenotypic and ecological levels. This is particularly true in plants, where transcription factor gene families are often targeted for breeding programs to improve the agronomic traits of economically important crops. While recent advances in next generation sequencing have accelerated the wealth of genomics data, there remains a lack of accessible and reproducible genome mining pipelines tailored for gene family characterisation. Here, we address this gap by developing GENE-FAM, an automated, scalable and open-source pipeline designed to mine and predict gene families based on conserved domains and motifs. To illustrate its application, we apply GENE-FAM to annotate MADS-box transcription factor genes across multiple Cannabis sativa genomes. A comprehensive set of MADS-box genes was identified across three C. sativa cultivars, including both previously annotated and newly predicted genes. Through phylogenetic analyses, we confirm that all type II MADS-box gene subfamilies represented in flowering plants are present in C. sativa. Comparing our annotations with those of Arabidopsis thaliana and Solanum lycopersicum revealed that while most MADS type II families are highly conserved, SEPALLATA-like genes have undergone diversification in C. sativa. Together, these results demonstrate the application of GENE-FAM for genome-wide identification and characterisation of gene families in non-model species, revealing novel insights into MADS-box gene family evolution in C. sativa.

genomics↗

bioRxiv2025

An ancient X chromosomal region harbours three genes potentially controlling sex determination in Cannabis sativa

O_LISex determination mechanisms in dioecious plants remain poorly understood yet offer an excellent model system to study genetic changes underlying morphological evolution. C_LIO_LIWe investigated the genetic basis of sex determination in Cannabis sativa, combining QTL mapping in a segregating population, comparative transcriptomics between monoecious and dioecious cultivars, and a genomic analysis of X-Y chromosome divergence. C_LIO_LIQTL mapping identified Monoecy1, a locus on the X chromosome putatively controlling the monoecy-dioecy trait. This locus resides in the most ancient and diverged region of the sex chromosomes and contains three genes within 60,000 bp (CsREM16, lncREM16, and CsKAN4) with distinct sex-specific and monoecy-specific expression patterns. C_LIO_LIMonoecy1 harbours genes for male-female as well as monoecious-dioecious sex determination. We propose that the combinatorial interaction of CSREM16, lncREM16 and CsKAN4 provides a unifying genetic framework for understanding male-female and monoecious-dioecious sex determination in Cannabis sativa. C_LI

plant biology↗

bioRxiv2025

A simple and reliable PCR-based method to differentiate between XX and XY sex genotypes in Cannabis sativa

Cannabis sativa is mainly a dioecious plant, which means that female and male flowers develop on separate individuals, which is controlled by an XY sex determination system: females have two X chromosomes and male plants carry an X and a Y chromosome. C. sativa is a crop that has a wide variety of applications, some of which depend on the sex of the plant. Females are, for example, used for the cannabinoid production, as cannabinoids are produced in the female inflorescence. However, while adult C. sativa individuals present high sexual dimorphism, it is not possible to phenotypically distinguish male and female plants before flowering. Here we present the identification of a highly conserved sex marker, CsPDS5, and the development of a robust, reliable and affordable PCR-based method to determine the sex genotype. In contrast to other sex genotyping methods, our approach relies on a gene polymorphic between the X and Y chromosomes and therefore requires only a single PCR with one pair of primers. The method was tested in 14 cultivars and 6 crossings, with different tissues and developmental stages, on a total of 600 samples, with 100% accuracy. Our assay allows early sex identification and hemp selection, which is useful for both research and industrial purposes. Finally, the pipeline presented here to identify genes polymorphic between the X and Y chromosomes can serve to discover new sex markers, not only in C. sativa, but other dioecious plants and other organisms with 2 sexes.

plant biology↗

bioRxiv2024

FT-like genes in Cannabis and hops: sex specific expression and copy-number variation may explain flowering time variation

Cannabis sativa is a fascinating, yet under-researched, species. To facilitate the global expansion of C. sativa cultivation a greater understanding of flowering time control is crucial. The PEBP gene family consists of universal promoters and repressors of flowering, with homologs of FLOWERING LOCUS T (FT) being highly conserved key regulators of flowering. FT encodes florigen, and balancing the florigen and anti-florigen signals is key for fine-tuning a crops flowering to local climatic conditions. Here, we provide an in-depth characterisation of the PEBP gene family in C. sativa and the closely related species H. lupulus. Phylogenetic analysis reveals expansion of FT and TFL1/CEN clades in the Cannabaceae. The retention of the duplicated PEBP genes likely has functional significance with divergent sequences and expression patterns hinting at signatures of sub-functionalisation. We speculate that duplicated PEBP genes have been crucial to the evolution of photoperiod insensitivity and sexually dimorphic flowering in C. sativa and harnessing the available genetic variation for these traits will be key for establishing this future crop.

plant biology↗

bioRxiv2024

Morphological and genetic analysis of inflorescence and flower development in hemp (Cannabis sativa L.)

Hemp (Cannabis sativa) is one of the oldest cultivated crops, and its myriad of uses have fascinated humans for millennia. In contemporary agriculture, the seeds are used for high-quality oil, human food, and nutritional supplements. From female inflorescences, secondary compounds (cannabinoids, terpenes, and flavonoids) are isolated that are implicated to have a wide range of medicinal applications. These elements provide hemp with significant market and research value and emphasise the need to thoroughly understand reproductive development in hemp. Here, we present a detailed morphological and molecular analysis of hemp inflorescence and flower development of the cultivar FINOLA. Hemp is unusual among flowering plants as it is dioecious, i.e. develops male and female plants. We define eight landmark events in male and female flower development and show that developmental differences between male and female plants extend beyond floral morphology, additionally comprising inflorescence structure as well as flowering time. Further, we demonstrate that the time of activation of key reproductive developmental regulators is significantly different in male and female hemp plants. Our comparison of male and female hemp plants shows that developmental pathways diverge very early, already at the two-leaf stage, laying a basis for further exploration into the genetics of sex determination of C. sativa.

plant biology↗

bioRxiv2023

Exploring phenotypic and genetic variability in hemp (Cannabis sativa)

Hemp (Cannabis sativa L.) is a high-value multi-purpose crop with applications ranging from the medicinal use of its secondary metabolites to carbon-sequestering building materials. To fully capitalise on the potential of hemp as a crop for more sustainable agriculture, detailed phenotypic and genotypic characterizations are required in order to inform targeted breeding programmes. Here, we present a detailed morphological and genomic analysis of 10 hemp cultivars. We found high variability in agronomically important traits such as flowering time, plant height, and biomass in most of the hemp cultivars tested. Additionally, genotyping by sequencing demonstrates that genetic differences are pervasive, both between hemp cultivars as well as between individuals of a single cultivar. The significant genetic and phenotypic variability we observe in hemp contrasts with other crops, where cultivars are often phenotypically and genetically relatively uniform. We argue that the variability of hemp is an asset for breeding and increases the potential for further improvement of the crop but is also a challenge for todays highly automated agriculture that relies on phenotypic uniformity. Author summaryHemp (Cannabis sativa L.) stands as one of the earliest domesticated crops. This remarkable plant is a sustainable crop with high carbon sequestration capacity which can be cultivated for soil remediation. Furthermore, hemp oil and fibre are used for many applications ranging from cooking to manufacturing bioplastics, textiles, or building materials of superb characteristics, and its secondary metabolites are sought after because of their medicinal properties. However, in contrast to many modern crops, hemp exhibits extensive variability in key agricultural traits, such as plant height and flowering time. This variability presents a challenge for both farmers and processors. To unravel the fundamentals of hemp diversity we conducted a comprehensive study of phenotypic and genetic characterisation of ten diverse hemp cultivars. We present findings confirming substantial variability not only among individuals of different cultivars but also within the same cultivar. Additionally, we explore heterozygosity in the context of other hemp studies and other crops. Understanding this variability in the context of a single hemp cultivar and across multiple cultivars is paramount for breeding novel, more uniform hemp varieties which will allow us to unlock the full potential of hemp as a crop of the future.

plant biology↗

bioRxiv2023

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is tightly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive ("autoflower") cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. Extensive sequence divergence between alleles of CsFT1 was identified between photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences especially in introns. Genotyping of several mapping populations and a diversity panel confirmed a strong correlation between CsFT1 alleles and photoperiod response as well as affirming that at least two independent loci for this agriculturally important trait, Autoflower1 and Autoflower2, exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

plant biology↗

bioRxiv2022

A protocol for rapid generation cycling (speed breeding) of hemp (Cannabis sativa) for research and agriculture

Hemp (Cannabis sativa) is a highly versatile multi-purpose crop with a multitude of applications, from textiles, biofuel and building material to high-value food products for consumer markets. Furthermore, non-hallucinogenic cannabinoids like cannabidiol (CBD), which can be extracted from female hemp flowers, are potentially valuable pharmacological compounds. In addition, hemp has high carbon sequestration potential due to its rapid growth rate. Therefore, the hemp industry is gaining more traction and breeding hemp cultivars adapted to local climate conditions or bred for specific applications is becoming increasingly important. Here, we present a method for rapid generation cycling (speed breeding) for hemp. The speed breeding protocol makes use of the photoperiod sensitivity of Cannabis. It encompasses vegetative growth of the plants for two weeks under continuous light, followed by flower induction, pollination and seed development for four weeks under short-day conditions and a seed ripening phase under continuous light and water stress. With the protocol introduced here, a generation time of under nine weeks (61 days) from seed to seed can be achieved. Our method furthermore synchronises flowering time of different hemp cultivars, thus facilitating crosses between cultivars. The extremely short generation time will enable hemp researchers and breeders to perform crosses in a time-efficient way and generate new hemp cultivars with defined genetic characteristics in a shorter amount of time.

plant biology↗

bioRxiv2022

Phenotypic Variation from Waterlogging in Multiple Perennial Ryegrass Varieties under Climate Change Conditions

Identifying how various components of climate change will influence ecosystems and vegetation subsistence will be fundamental to mitigate negative effects. Climate change-induced waterlogging is understudied in comparison to temperature and CO2. Grasslands are especially vulnerable through the connection with global food security, with perennial ryegrass dominating many flood-prone pasturelands in North-western Europe. We investigated the effect of long-term waterlogging on phenotypic responses of perennial ryegrass using four varieties grown in atmospherically controlled growth chambers (ambient vs 2050, +2{degrees}C and eCO2) during two months of peak growth. Using image analysis and PCA methodologies, we assess how multiple predictors (phenotypic, environmental, genetic and temporal) influence overall plant performance and productivity. Long-term waterlogging was found to reduce leaf-colour intensity, with younger plants having purple hues indicative of anthocyanins. Plant performance and yield was lower in waterlogged plants, with tetraploid varieties coping better than diploid ones. The climate change treatment was found to reduce colour intensities further. Flooding was found to reduce plant productivity via reductions in colour pigments and root proliferation. These effects will have negative consequences for global food security from facing extreme weather events and flooding. Our approach can be adapted as plant health diagnostics tools via remote sensing and drone-technology.

ecology↗

bioRxiv2019

Genome-wide analysis of MIKC-type MADS-box genes in wheat: pervasive duplications may have facilitated adaptation to different environmental conditions

BackgroundWheat (Triticum aestivum) is one of the most important crops worldwide. Given a growing global population coupled with increasingly challenging climate and cultivation conditions, facilitating wheat breeding by fine-tuning important traits such as stress resistance, yield and plant architecture is of great importance. Since they are involved in virtually all aspects of plant development and stress responses, prime candidates for improving these traits are MIKC-type (type II) MADS-box genes.\n\nResultsWe present a detailed overview of number, phylogeny, and expression of 201 wheat MIKC-type MADS-box genes, which can be assigned to 15 subfamilies. Homoeolog retention is significantly above the average genome-wide retention rate for wheat genes, indicating that many MIKC-type homoeologs are functionally important and not redundant. Gene expression is generally in agreement with the expected subfamily-specific expression pattern, indicating broad conservation of function of MIKC-type genes during wheat evolution.\n\nWe find the extensive expansion of some MIKC-type subfamilies to be correlated with their chromosomal location and propose a link between MADS-box gene duplications and the adaptability of wheat. A number of MIKC-type genes encode for truncated proteins that lack either the DNA-binding or protein-protein interaction domain and occasionally show novel expression patterns, possibly pointing towards neofunctionalization.\n\nConclusionsConserved and neofunctionalized MIKC-type genes may have played an important role in the adaptation of wheat to a diversity of conditions, hence contributing to its importance as a global staple food. Therefore, we propose that MIKC-type MADS-box genes are especially well suited for targeted breeding approaches and phenotypic fine tuning.

plant biology↗

bioRxiv2017

The Floral Homeotic Protein SEPALLATA3 Recognizes Target DNA Sequences By Shape Readout Involving A Conserved Arginine Residue In The MADS-Domain

SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor and a central player in flower development. MADS-domain proteins bind as dimers to AT-rich sequences termed CArG-boxes which share the consensus 5-CC(A/T)6GG-3. Since only a fraction of the abundant CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-box sequences into genuine recognition sites. Here, we investigated to which extent the shape of the DNA contributes to the DNA-binding specificity of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to a variety of DNA probes which all contain the CArG-box motif, but differ in their DNA shape characteristics. We found that binding affinity correlates well with certain DNA shape features associated with A-tracts. Analysis of SEPALLATA3 proteins with single amino acid substitutions in the DNA-binding MADS-domain further revealed that a highly conserved arginine residue, which is expected to contact the DNA minor groove, contributes significantly to the shape readout. Our studies show that the specific recognition of cis-regulatory elements by plant MADS-domain transcription factors heavily depend on shape readout mechanisms and that the absence of a critical arginine residue in the MADS-domain impairs binding specificity.

plant biology↗

bioRxiv2017

Sequence Features Of MADS-Domain Proteins That Act As Hubs In The Protein-Protein Interaction Network Controlling Flower Development

The development of angiosperm flowers is regulated by homeotic MIKC-type MADS-domain transcription factors that activate or repress target genes via the formation of DNA-bound, organ specific tetrameric complexes. The protein-protein interaction (PPI) capabilities differ considerably between different MIKC-type proteins. The floral homeotic protein SEPALLATA3 (SEP3) acts as a hub that incorporates numerous other MADS-domain proteins into tetrameric complexes that would otherwise not form. However, the molecular mechanisms that underlie these promiscuous interactions remain largely unknown. In this study we created a collection of amino acid substitution mutants of SEP3 to quantify the contribution of individual residues on protein tetramerization during DNA-binding, employing methods of molecular biophysics. We show that leucine residues at certain key positions form a leucine zipper structure that is essential for tetramerization of SEP3, whereas the introduction of physicochemically very similar residues at respective sites impedes the formation of DNA-bound tetramers. Comprehensive molecular evolutionary analyses of MADS-domain proteins from a diverse set of flowering plants revealed exceedingly high conservation of the identified leucine residues within SEP3-subfamily proteins throughout angiosperm evolution. In contrast, MADS-domain proteins that are unable to tetramerize among themselves exhibit preferences for other amino acids at homologous sites. Our findings indicate that the subfamily-specific conservation of amino acid residues at just a few key positions account for subfamily-specific interaction capabilities of MADS-domain transcription factors and shaped the present-day structure of the PPI network controlling flower development.

plant biology↗