Symposium Leiden 29-11-2024

Summaries of the lectures on the SVPW fall symposium, Friday, November 29th, 2024, Leiden

The science of (micro)grafting

Charles Melnyk, Swedish University of Agricultural Sciences, Upsalla

For millennia people have used grafting to improve and propagate plants. Grafting is widely used in horticulture to increase stress tolerance, improve disease resistance and promote yields. However, we have a limited understanding of how two plants successfully fuse tissues and form vascular connections. Here, I will present my group’s latest efforts to develop and use micrografting techniques to understand the molecular basis for how plants successfully graft. I will discuss the process of micrografting in species including Arabidopsis, tomato, monocots and conifers. Incompatibility is a major cause of graft failure and I will also present our work on understanding and overcoming this phenomenon. Altogether, our research sheds light on the mechanism of graft formation and suggests that micrografting young tissues could play an important role in improving graft success rates.

Micrografting: a tool for investigating scion-rootstock interactions in olive tree.

Nourhene Jouini, University of Palermo , Department of Agricultural, Food and Forestry Sciences

The olive tree (Olea europaea L.) is a cornerstone of Mediterranean agriculture, increasingly recognized for its economic and nutritional value worldwide. Consequently, breeding and propagation of this species to attend the demand of the intensive cultivation system have become focal points of numerous research endeavors. However, the recalcitrance of many cultivars to self-rooting remains a significant obstacle in olive propagation. Therefore, the aim of the present work is to investigate the use of micropropagation and specifically in vitro micrografting (IVM), through a morphological, histological and molecular analysis, to address these challenges and select suitable rootstocks based on their vigor and rooting ability.

Decoupling shoot and root physiology using micro-grafting in Arabidopsis and lettuce.

Kiki Spaninks, Leiden University, Institute of Biology

Throughout plant development, the communication between the shoot and root via long-distance signaling molecules is of vital importance. For instance, shoot-root signaling aids plants in adjusting to a range of environmental stresses, nutrient deficiencies, and pathogen or herbivore defense. Although mostly used in horticultural crop production, the micro-grafting technique holds great potential for research on shoot-root signaling. Using this technique, it is possible to decouple shoot-specific factors from root-specific factors when studying a phenotype of interest. For instance, we applied micro-grafting of lettuce seedlings to study tipburn, a calcium deficiency disorder that affects head-forming crops. Aside from studying shoot- and root-specific physiology for one phenotype (tipburn), we are also using the micro-grafting technique in a broad screen to identify which phenotypic traits rely (partially) on shoot-root communication. To achieve this, we are currently screening a large set of graft combinations with different Arabidopsis thaliana ecotypes.

Fern or Foe: Development of a novel genetic transformation technique in ferns

Aaron Ang, Wageningen University

Ferns represent a key phylogenetic lineage in the land plants that hold the key to understanding the evolution of numerous morphological innovations. Nonetheless, ferns have been severely underrepresented in genetic studies due to their cumbersome genomes and lack of amenable model systems. My project aims to explore and optimise an alternative genetic transformation technique for the model fern Ceratopteris richardii using Agrobacterium rhizogenes to overcome the limitations of current techniques. In addition, I aim to investigate the conservation of cellular polarity in ferns. 

Protoplast cell wall regeneration and the establishment of isolated cell culture for biophysical studies

Stéphane Verger, Umeå Plant Science Centre

The plant cell wall is a major hub of plant biomechanics, regulating cell and tissue shape, mechanics and adhesion as well as relaying physical signals back to the cell. In planta, the cell wall presents major heterogeneities ranging from the subcellular to tissue scale, often rendering interpretations difficult when studying whole plants. Thus, our goal is to establish the simplest model possible using isolated single cells (spherical and with homogeneous cell walls) as a model for biophysical studies of the cell wall. This can in principle be achieved through protoplast extraction and cell wall regeneration. However, in our original attempts we encountered difficulties in obtaining cell wall regeneration after protoplasting despite trying most of the published protocols. This led us to develop a novel user-friendly imaging and data analysis pipeline to screen in an unbiased, traceable, accessible and high-throughput manner, ideal in vitro culture conditions for protoplast survival and cell wall regeneration, tailored for our goal. Here I will describe our original challenges as well as the development and implementation of this workflow along with example use cases for upcoming studies.

Impedance Flow Cytometry and Fractional Factorial Design: Identifying critical parameters affecting regeneration of B. napus protoplasts.

Tiago Rodrigues – Ghent University, Department of Plants and Crops, Horticell

Given their nature, protoplasts are sensitive structures, and their isolation and culture methods need to be optimized for the desired objective, specific genotype, and even tissue of interest. This often entails systematic and repetitive testing of many factors reported to be important at this early stage. The time-consuming and labour-intensive endeavor is crucial to ensure a reliable and effective protocol. As such, it cannot be overlooked and often constitutes the first major hurdle for anyone working with protoplasts.

The combination of fractional factorial experimental design and impedance flow cytometry provides a quick, efficient, robust and comprehensive set of tools for screening those important variables. This approach delivers a solid basis for further optimization, not only significantly facilitating early protoplast research, but also ensuring that all critical factors are thoroughly evaluated before progressing to the next stages.

(Gaining) Mechanical control of protoplast regeneration

Rik Froeling/Patricia Schöppl, Wageningen University, Green Mechanobiology

Protoplast regeneration is an elegant tool in plant cell culture and breeding, and yet it remains notoriously difficult. To obtain functional protocols, researchers are optimizing many parameters, ranging from adjustments in isolation protocols, osmolytes, basal salts, and hormones, to variations in temperature and

cell density – a highly empirical and time-consuming approach. However, in planta, cells are not only influenced by chemical signals but are also mechanically constrained by their neighboring cells. And yet, we rarely find that this mechanical dimension is considered in current regeneration strategies. We propose that this negligence of the mechanical dimension contributes largely to the challenges of successful protoplast regeneration. Using examples from both plant and animal cell culture, we will demonstrate how mechanical signaling impacts cellular behavior, explain why integrating this mechanical dimension into our regeneration protocols could be the key to success, and share our approach for gaining mechanical control of protoplast regeneration.

Research pitches by PhD students/Post Docs

Identification of new maternal haploid inducers

Dawit Tekleyohans, Wageningen University, Laboratory of Molecular Biology

Double haploid (DH) technology is an integral part of genetic improvement programs whereby rapid attainment of large number of genetically homozygous inbred lines within reduced time and labor is possible. In vivo development of DH involves the crossing of source germplasm with pollen from maternal haploid inducer (MHI) lines followed by identification of haploid haploids and genome doubling. In dicots, the haploid induction rate of MHIs is still quite low. Here we describe our efforts to identify new MHI mutants in arabidopsis.

Transcriptional regulation of the LEAFY COTYLEDON1 morphogene

Dandan Wang, Wageningen University, Laboratory of Biochemistry

LEAFY COTYLEDON1 (LEC1) is an NF-YB transcription factor that controls many aspects of zygotic embryo development. Ectopic overexpression of LEC1 also promotes 2,4-D mediated somatic embryogenesis and induces spontaneous formation of embryogenic tissue.  We used CRISPR mutagenesis of evolutionarily conserved nucleotide sequences in LEC1 non-protein coding regions to identify cis-regulatory elements that control LEC1 expression in arabidopsis. Using this approach, we identified novel alleles with characteristic LEC1 overexpression phenotypes.

Genetic dissection of mutagenic repair at CRISPR-induced DNA breaks in Arabidopsis thaliana

Lycka Kamoen, Leiden University, Institute of BiologyIy,

practical and powerful approach for genome editing in plants involves the delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. This method utilizes a double-strand break (DSB)-inducing enzyme, expressed from a transferred segment of bacterial DNA (T-DNA). To develop efficient strategies for precise genome editing, it is crucial to understand the mechanisms that repair CRISPR-induced DSBs. In this study, we present a detailed and comprehensive genetic analysis of DSB repair induced by Cas9 and Cas12a in the model plant Arabidopsis thaliana. Our findings reveal that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) both act, sometimes redundantly, on CRISPR-induced DSBs, resulting in distinct mutational outcomes. Additionally, we discovered that DSBs flanked by short repeats stimulate a specific mode of TMEJ, enabling predictable mutagenesis at these sites. Our study supports the development of tailor-made strategies for predictable engineering of crop plants.

Optimization of crop response to climate change with plant hormones and biostimulants

Jing Li, Ghent University,

Stem cell factor induced regeneration
Jana Wittmer, Wageningen University,

In the plant kingdom pluripotent stem cells are maintained in the stem cell niches throughout a plants life cycle. Under suitable in vitro conditions involving the application of phytohormones, pluripotency can be equally induced from differentiated plant cells. However, regenerative recalcitrance to respond to tissue culture manipulation presents a bottleneck that lies in the species, cultivars and explant origin. Here we took an approach similar to the induced pluripotent stem cell concept in animals and overexpress a set of root stem cell specific factors in Arabidopsis thaliana, to induce and study regeneration in the absence of hormones. We find that regeneration induced by these factors goes via the somatic embryogenesis program. We provide evidence that this setup can be translated to crops using the same set of factors by regenerating lettuce on hormone-free media.