Author ORCID Identifier

https://orcid.org/0009-0001-3043-9300

Date Available

12-5-2025

Year of Publication

2025

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College

Agriculture, Food and Environment

Department/School/Program

Plant and Soil Sciences

Faculty

Dr. Carlos Rodriguez Lopez

Abstract

Grapevine (Vitis vinifera) faces unprecedented challenges from climate change, with up to 90% of current growing regions potentially becoming unsuitable for production within decades. Traditional breeding approaches require 25-30 years for variety development, creating a time constraint incompatible with the pace of environmental change. This research establishes the approach for a novel mobile RNA-based gene editing platform that addresses longstanding bottlenecks in grapevine improvement while preserving cultivar identity and reducing breeding timelines.

This research developed a three-component system for transgene-free gene editing in grapevine. First, a mobile CRISPR/Cas9 system was established using Agrobacterium rhizogenes transformation and the cut-and-dip method to generate transformed roots and wild-type shoots. Dicistronic transcripts containing tRNA-like structure (TLS) mobility signals successfully transported CRISPR/Cas9 machinery from transgenic roots to non-transformed aerial tissues, achieving targeted gene editing in approximately 7% of treated plants. This tissue culture-free approach eliminates somaclonal variation risks while preserving cell layer chimerism integrity essential to cultivars.

Second, RNA methylation signals were investigated to enhance transcript mobility and editing efficiency. Computational analysis of the 101-bp sequence (S2) from Arabidopsis thaliana mag5 revealed adoption of a cloverleaf structure similar to TLS motifs, suggesting convergent mechanisms for methylation targeting and mobility enhancement. Transgenic Arabidopsis lines expressing S2-enhanced CRISPR constructs were generated, establishing a platform for evaluating methylation-mediated mobility improvements.

Third, comprehensive characterization of native RNA methylation patterns in grapevine tissues using bisulfite sequencing and long read RNA nanopore sequencing revealed 5-methylcytosine across cotyledons, roots, and first true leaves. Principal component analysis demonstrated consistent tissue grouping in methylation patterns, while differential methylation analysis identified key regulatory genes. Critically, the grapevine mag5 homolog was confirmed to be highly methylated, validating its potential as a species-specific mobility enhancer.

The integrated system offers several advantages: a dramatic reduction in breeding timelines by eliminating tissue culture requirements, regulatory benefits by producing plants free from transgenic DNA integration, and applicability to other woody perennials facing similar transformation challenges. Plants edited through this system can be propagated directly from non-transformed tissues, creating varieties that contain desired genetic modifications without foreign DNA with the ability to bypass the bottlenecks associated with gene editing in grapevines.

This work establishes the foundation for a new approach in perennial crop improvement that aligns with natural RNA mobility mechanisms while addressing the urgent time constraints imposed by climate change. By leveraging the plant's endogenous transport systems and methylation patterns, this approach provides a pathway for developing climate-resilient grapevine varieties that maintain their identity while acquiring traits essential for future productivity and sustainability.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.448

Funding Information

California Grape Rootstock Association

Three Years Funded

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Project Report-LZZ01022403-Lifeasible.pdf (1772 kB)
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