Optimization of Selection Agent Concentrations and Expanding G418 Utility for Gentamicin Resistance in Marchantia polymorpha DOI Creative Commons
Andisheh Poormassalehgoo,

Elżbieta Kaniecka,

Mohamadreza Mirzaei

et al.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 28, 2024

Abstract Genetic transformation of plants is pivotal for advancing biotechnology, with success depending largely on effective selection methods. Marchantia polymorpha has emerged as a model plant due to its evolutionary importance, ease manipulation, and simple genetic structure. However, inconsistent antibiotic performance limited studies optimal agent concentrations have posed challenges. This study aimed optimize use in M. transformation. We assessed the effects five antibiotics (hygromycin, kanamycin, G418, neomycin, gentamicin) herbicide chlorsulfuron gemmae growth. For each agent, we identified minimum lethal concentration nontransgenic safe thresholds transgenics, balancing false-positive prevention reduced toxicity. Hygromycin, exhibited broad selective ranges, enabling efficient transformant selection. Notably, observed cross-activity gentamicin resistance enzyme phenomenon also seen tobacco. effectively determined agents Additionally, unexpected underscores need careful marker highlights potential strategic use. Our findings enhance protocols possibly other species.

Language: Английский

Pan-phylum genomes of hornworts reveal conserved autosomes but dynamic accessory and sex chromosomes DOI
Peter Schafran,

Duncan A. Hauser,

Jessica Nelson

et al.

Nature Plants, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 3, 2025

Language: Английский

Citations

3
Optimizing Promoters and Subcellular Localization for Constitutive Transgene Expression in Marchantia polymorpha DOI Creative Commons
Sze Wai Tse, Davide Annese, Facundo Romani

et al.

Plant and Cell Physiology, Journal Year: 2024, Volume and Issue: 65(8), P. 1298 - 1309

Published: June 1, 2024

Abstract Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterization of genetic elements would make heterologous gene expression more predictable in this test bed circuit assembly bioproduction. Yet, the toolbox parts includes only a few constitutive promoters that need benchmarking to assess their utility. We compared patterns previously characterized new promoters. found driving with double enhancer version cauliflower mosaic virus 35S promoter (pro35S × 2) provided highest yield proteins, although it also inhibits growth transformants. In contrast, derived from genes ETHYLENE RESPONSE FACTOR 1 CLASS II HOMEODOMAIN-LEUCINE ZIPPER protein drove higher levels across all tissues without penalty can provide intermediate expression. addition, we showed cytosol is best subcellular compartment target proteins significant burden. To demonstrate potential these Marchantia, expressed RUBY, polycistronic betalain synthesis cassette linked by P2A sequences, coordinated metabolic enzymes. A heat-shock-inducible was used further mitigate burdens associated high amounts accumulation. have expanded existing tool kit resources research community.

Language: Английский

Citations

7
Callose in leptoid cell walls of the moss Polytrichum and the evolution of callose synthase across bryophytes DOI Creative Commons
Karen S. Renzaglia,

E. Duran,

Laxmi Sagwan‐Barkdoll

et al.

Frontiers in Plant Science, Journal Year: 2024, Volume and Issue: 15

Published: Feb. 7, 2024

Introduction Leptoids, the food-conducting cells of polytrichaceous mosses, share key structural features with sieve elements in tracheophytes, including an elongated shape oblique end walls containing modified plasmodesmata or pores. In callose is instrumental developing pores that enable efficient photoassimilate transport. Aside from a few studies using aniline blue fluorescence yielded confusing results, little known about moss leptoids. Methods Callose location and abundance during development leptoid cell was investigated Polytrichum commune quantitative immunogold labeling (label density) transmission electron microscope. To evaluate changes abiotic stress, leptoids hydrated plants compared to dried for 14 days under field conditions. A bioinformatic study assess evolution within across bryophytes conducted synthase (CalS) genes 46 (24 15 liverworts, 7 hornworts) one representative each five tracheophyte groups. Results increases around meristematic mature Controlled drying resulted significant increase label density over counts plants. Phylogenetic analysis CalS protein family recovered main clades (A, B, C). Different where greatest diversity homologs found clade A, majority gene duplication B. Discussion This work identifies as crucial wall polymer their inception functioning leptoids, water stress similar tracheophytes. Among bryophytes, mosses exhibit number multiple events, while only two duplications are revealed hornwort none liverworts. The absence essential pore angiosperms, reveals different responsible synthesizing associated mosses.

Language: Английский

Citations

5
Biolistics-mediated transformation of hornworts and its application to study pyrenoid protein localization DOI
Declan Lafferty, Tanner A. Robison, Andika Gunadi

et al.

Journal of Experimental Botany, Journal Year: 2024, Volume and Issue: 75(16), P. 4760 - 4771

Published: May 23, 2024

Hornworts are a deeply diverged lineage of bryophytes and sister to mosses liverworts. have an array unique features that can be leveraged illuminate not only the early evolution land plants, but also alternative paths for nitrogen carbon assimilation via cyanobacterial symbiosis pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts one few plant lineages with limited available genetic tools. Here we report efficient biolistics method generating transient expression stable transgenic lines in model hornwort, Anthoceros agrestis. An average 569 (±268) cells showed per bombardment, green fluorescent protein observed within 48-72 h. A total 81 stably transformed were recovered across three separate experiments, averaging six bombardment. We followed same transiently transform nine additional hornwort species, obtained transformants from one. This was further used verify localization Rubisco activase pyrenoids, which central proteins CCM function. Together, our approach offers key advantages over existing methods as it enables rapid applied widely diverse species.

Language: Английский

Citations

5
Streamlined regulation of chloroplast development in the liverwort Marchantia polymorpha DOI Creative Commons
Nataliya E. Yelina, Eftychios Frangedakis,

Zhemin Wang

et al.

Cell Reports, Journal Year: 2024, Volume and Issue: 43(9), P. 114696 - 114696

Published: Sept. 1, 2024

Language: Английский

Citations

5
What can hornworts teach us? DOI Creative Commons
Eftychios Frangedakis, Alan O. Marron, Manuel Waller

et al.

Frontiers in Plant Science, Journal Year: 2023, Volume and Issue: 14

Published: March 8, 2023

The hornworts are a small group of land plants, consisting only 11 families and approximately 220 species. Despite their size as group, phylogenetic position unique biology great importance. Hornworts, together with mosses liverworts, form the monophyletic bryophytes that is sister to all other plants (Tracheophytes). It recently became amenable experimental investigation establishment Anthoceros agrestis model system. In this perspective, we summarize recent advances in development A. an system compare it plant systems. We also discuss how can help further research comparative developmental studies across solve key questions associated colonization terrestrial environment. Finally, explore significance crop improvement synthetic applications general.

Language: Английский

Citations

12
A Simple and Scalable Chopped-Thallus Transformation Method for Marchantia polymorpha DOI Creative Commons
Rituraj Batth, Andisheh Poormassalehgoo, Kritika Bhardwaj

et al.

Plants, Journal Year: 2025, Volume and Issue: 14(4), P. 582 - 582

Published: Feb. 14, 2025

The liverwort Marchantia polymorpha has emerged as a valuable model for studying fundamental biological processes and the evolutionary history of land plants. Agrobacterium-mediated transformation is widely used genetic modification M. using spores, thalli, gemmae. While spores offer high efficiency, they result in diverse backgrounds due to sexual reproduction. Conversely, thallus- gemma-based methods maintain consistency but are impractical large-scale applications. To address these limitations, we developed novel chopped-thallus method. This technique improves efficiency by generating numerous thallus fragments through chopping optimizing regeneration duration. method demonstrated superior compared traditional approaches achieved sufficient numbers transformants simplified Gamborg's B5 medium, previously considered suboptimal. scalable straightforward enables generation large genetically consistent transformants, facilitating high-throughput experiments, including mutant screening other

Language: Английский

Citations

0
A tRNA-gRNA multiplexing system for CRISPR genome editing inMarchantia polymorpha DOI Creative Commons
Eftychios Frangedakis, Nataliya E. Yelina,

Satish Kumar Eeda

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 20, 2025

Abstract The liverwort Marchantia polymorpha is a widely used model organism for studying land plant biology, which has also proven to be promising testbed bioengineering. CRISPR/Cas9 technology emerged as transformative tool precise genome modifications in M. . However, robust method the simultaneous expression of multiple gRNAs, crucial enhancing efficiency and versatility CRISPR/Cas9-based editing, yet fully developed. In this study, we introduce an adaptation from OpenPlant kit tools, that facilitates gRNAs single transcript through incorporation tRNA sequences. This approach significantly improves scalability editing Additionally, by combining vector system with simplified optimized protocol thallus transformation, further streamline generation mutants resulting gene- offers versatile, time-saving straightforward advancing functional genomics , enabling more comprehensive genetic engineering.

Language: Английский

Citations

0
Streamlined regulation of chloroplast development in the liverwort Marchantia polymorpha DOI Creative Commons
Nataliya E. Yelina, Eftychios Frangedakis,

Zhemin Wang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Jan. 23, 2023

Photosynthesis in eukaryotic cells takes place chloroplasts that develop from undifferentiated plastids response to light. Two families of transcription factors known as Golden2-like (GLK) and GATA regulate plant chloroplast development, the MIR171-targeted SCARECROW-LIKE (SCL) GRAS chlorophyll biosynthesis. Additionally, Elongated Hypocotyl 5 (HY5) factor plays a critical role photomorphogenesis. The extent which these proteins carry out conserved roles non-seed plants such liverworts is not known. Here we determine degree functional conservation GLK, GATA, SCL HY5 controlling development model liverwort Marchantia polymorpha. Our results indicate have minimal or undetectable biogenesis but loss GLK function leads reduced size, underdeveloped thylakoid membranes lower accumulation. ChIP-seq RNA-seq analyses revealed can directly many photosynthetic development-related genes. But there extensive divergence between its M. polymorpha flowering plants. Collectively our findings suggest well more specialised photomorphogenesis, either evolved after vascular bryophytes, were lost polymorpha, redundancy masking their roles. In contrast, consistent with presence algae,

Language: Английский

Citations

9
Insights into U‐to‐C RNA editing from the lycophyte Phylloglossum drummondii DOI Creative Commons
Farley M. Kwok van der Giezen,

Amy Viljoen,

Leni Campbell‐Clause

et al.

The Plant Journal, Journal Year: 2024, Volume and Issue: 119(1), P. 445 - 459

Published: April 23, 2024

The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in Huperzioideae group and one a small minority plants which perform uridine to cytidine RNA editing. We assembled P. chloroplast mitochondrial genomes used sequence data build comprehensive profile organellar editing events. In addition many C-to-U events both organelles, we found just four U-to-C transcripts cob, nad1, nad5 rpl2. These are conserved related lycophytes genera Huperzia Phlegmariurus. De novo transcriptomes for three these were search putative enzymes. Four factors could be matched sites. Due unusually few numbers sites, useful models studying this poorly understood mechanism.

Language: Английский

Citations

1