An efficient protoplast-based genome editing protocol for Vitis species DOI Creative Commons
David M. Tricoli, Juan M. Debernardi

Horticulture Research, Journal Year: 2023, Volume and Issue: 11(1)

Published: Dec. 13, 2023

Abstract CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These depend on the delivery of editing components into cells regeneration fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one best avenues producing non-chimeric transgene-free genome-edited However, poor plants from protoplasts has hindered their implementation genome editing. Here, we report an efficient protocol regenerating multiple grape varieties. By encapsulating calcium alginate beads co-culturing them with feeder cultures, divide form callus colonies that regenerate embryos ultimately This worked successfully wine table (Vitis vinifera) varieties, well rootstocks grapevine wild relative Vitis arizonica. Moreover, by transfecting CRISPR-plasmid or ribonucleoprotein (RNP) complexes, regenerated albino edits VvPHYTOENE DESATURASE gene three varieties V. The results reveal potential this platform facilitate species.

Language: Английский

Advances in genomics and genome editing for improving strawberry (Fragaria ×ananassa) DOI Creative Commons

Kaitlyn Vondracek,

Fredy Altpeter, Tie Liu

et al.

Frontiers in Genetics, Journal Year: 2024, Volume and Issue: 15

Published: April 19, 2024

The cultivated strawberry, Fragaria ×ananassa , is a recently domesticated fruit species of economic interest worldwide. As such, there significant in continuous varietal improvement. Genomics-assisted improvement, including the use DNA markers and genomic selection have facilitated improvements numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations precision nucleotide substitutions target genome, revolutionizing functional genomics crop Genome beginning to gain traction more challenging polyploid crops, allo-octoploid strawberry. release high-quality reference genomes comprehensive subgenome-specific genotyping gene expression profiling data octoploid will lead surge trait discovery modification by using CRISPR/Cas. has already been successfully applied for several genes, anthocyanin content, firmness tolerance post-harvest disease. However, reports on many other important breeding characteristics associated with quality production are still lacking, indicating need streamlined approaches tools . In this review, we present an overview latest advancements knowledge efforts involving CRISPR/Cas enhancement varieties. Furthermore, explore potential applications technology improving Rosaceous plant species.

Language: Английский

Citations

4
Transgene-free Genome Editing in Grapevine DOI Creative Commons
Edoardo Bertini, Erica D’Incà,

Stefania Zattoni

et al.

BIO-PROTOCOL, Journal Year: 2025, Volume and Issue: 15(1365)

Published: Jan. 1, 2025

CRISPR/Cas9 genome editing technology has revolutionized plant breeding by offering precise and rapid modifications. Traditional methods are often slow imprecise, whereas allows for targeted genetic improvements. Previously, direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts been demonstrated, but successful regeneration edited into whole plants not achieved. Here, we describe an efficient protocol obtaining transgene/DNA-free transfecting isolated from embryogenic callus subsequently regenerating them. The regenerated were comparable in morphology growth habit wild-type controls. This provides a highly method DNA-free grapevine, addressing regulatory concerns potentially facilitating the improvement other woody crop plants. Key features • Protoplasts one most commonly used systems application new technologies, including editing. accessible platform CRISPR-Cas9 ribonucleoparticles through chemical or physical transfection. avoid use both Agrobacterium tumefaciens plasmids; no stable integration exogenous DNA occurs. background remains unchanged identical original plant.

Language: Английский

Citations

0
DNA-free CRISPR Genome Editing in Raspberry (Rubus idaeus) through RNP-mediated Protoplast Transfection and Comparison of Indel Analysis Techniques DOI Creative Commons
Ryan Creeth, Andrew J. Thompson, Zoltán Kevei

et al.

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

Published: Jan. 15, 2025

Abstract Protoplast-based systems have been utilised in a wide variety of plant species to enable genome editing without chromosomal introgression foreign DNA into genomes. This allows elite cultivars be edited further genetic segregation, preserving their unique composition and regulatory status as non-transgenic. can achieved by DNA-free protoplasts, followed regeneration. However, protoplast isolation presents barrier the development advanced breeding technologies raspberry no protocol has published for species. Pre-assembled ribonucleoprotein complexes (RNPs) do not require cellular processing commercial availability Cas9 proteins synthetic guide RNAs streamlined protocols. study novel high-yielding from stem cultures RNP-mediated transfection with CRISPR-Cas9. Targeted mutagenesis phytoene desaturase gene at two intragenic loci resulted an efficiency 19%, though estimated varied depending on indel analysis technique. Only amplicon sequencing was sensitive enough confirm low sample. To our knowledge, this constitutes first use raspberry. provides valuable platform understanding function facilitates precision important soft fruit crop.

Language: Английский

Citations

0
Advancing plant protoplasts: innovative techniques and future prospects DOI Creative Commons
Nidhi S. Mukundan, Kapaettu Satyamoorthy, Vidhu Sankar Babu

et al.

Plant Biotechnology Reports, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 29, 2025

Language: Английский

Citations

0
Early peak of tannin content and gene expression related to tannin biosynthesis in table grape skin during berry development DOI

Yurim Kang,

Sun Mi Huh, Jeong‐Hwan Mun

et al.

Horticulture Environment and Biotechnology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 27, 2025

Language: Английский

Citations

0
CRISPR-Cas in Woody Perennial Plants: Methods, Efficiency, Applications, and Challenges to Creating Commercial Varieties with High Ecological and Economic Value DOI
Arturo Redondo-López, Nahuel González‐Schain, Mariano Perales

et al.

Springer protocols handbooks/Springer protocols, Journal Year: 2025, Volume and Issue: unknown, P. 227 - 243

Published: Jan. 1, 2025

Language: Английский

Citations

0
Application and Prospects of DNA-free Genome Modifications in Crop Plants through Genome Editing DOI
Ambreen Gul, Muhammad Naeem Sattar

CABI eBooks, Journal Year: 2025, Volume and Issue: unknown, P. 146 - 165

Published: March 21, 2025

Language: Английский

Citations

0
CRISPR plants for studying functional genomics: Methods and applications DOI
Mireia Uranga, Ana Montserrat Martín‐Hernández, Fabio Pasin

et al.

Elsevier eBooks, Journal Year: 2025, Volume and Issue: unknown, P. 27 - 59

Published: Jan. 1, 2025

Language: Английский

Citations

0
Establishing a reliable protoplast system for grapevine: isolation, transformation, and callus induction DOI Creative Commons

Gulsen Kolasinliler,

Cengiz Akkale, Hilal Betül Kaya

et al.

PROTOPLASMA, Journal Year: 2025, Volume and Issue: unknown

Published: April 25, 2025

Abstract Protoplasts are single cells enclosed by the plasma membrane after cell wall removal. They widely used in various biotechnological applications, including gene functional analysis, verification of genome editing reagents, and plant regeneration. Recent advances have enabled production non-chimeric transgene-free genome-edited plants using protoplasts. This process involves protoplast isolation, transformation, regeneration, requiring advanced technical skills. Challenges isolation regeneration limited their use editing. In grapevines, however, very few studies reported protoplasts isolated from leaves. Efficient transformation protocols for Chardonnay remain lacking require cultivar-specific optimization. this study, we established a reliable efficient system optimizing conditions PEG-mediated cultivar. The yield viable was approximately 75 × 10 6 per gram leaf material, with viability 91%. A efficiency 87% achieved under optimized conditions. To evaluate ability mesophyll protoplast, transformed untransformed were cultured on solid liquid MS media supplemented 2 mg/L 2,4-D 0.5 BA to facilitate microcalli formation. Microcalli formed feeder layer developed into calli when transferred culture BA. However, unable regenerate roots or shoots. These findings provide foundation further optimization protoplast-based systems potential enhance applications species.

Language: Английский

Citations

0
Boosting grapevine breeding for climate-smart viticulture: from genetic resources to predictive genomics DOI Creative Commons
Gabriele Magon, Valeria De Rosa, Matteo Martina

et al.

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

Published: Dec. 11, 2023

The multifaceted nature of climate change is increasing the urgency to select resilient grapevine varieties, or generate new, fitter cultivars, withstand a multitude new challenging conditions. attainment this goal hindered by limiting pace traditional breeding approaches, which require decades result in selections. On other hand, marker-assisted has proved useful when it comes traits governed one few genes with great effects on phenotype, but its efficacy still restricted for complex controlled many loci. these premises, innovative strategies are emerging could help guide selection, taking advantage genetic diversity within Vitis genus entirety. Multiple germplasm collections also available as source material introgression alleles interest via adapted and pioneering transformation protocols, present themselves promising tools future applications notably recalcitrant species such grapevine. Genome editing intersects both strategies, not only being an alternative obtain focused changes relatively rapid way, supporting fine-tuning genotypes developed methods. A review state art concerning resources possibilities use techniques aid selection presented here support production climate-smart genotypes.

Language: Английский

Citations

10