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IVES 9 IVES Conference Series 9 OIV 9 OIV 2024 9 Short communications - Viticulture, table grapes, dried grapes and unfermented grape products 9 Enhancing grapevine transformation and regeneration: A novel approach using developmental regulators and BeYDV-mediated expression

Enhancing grapevine transformation and regeneration: A novel approach using developmental regulators and BeYDV-mediated expression

Abstract

Grapevine (Vitis vinifera L.) is a challenging plant species to transform and regenerate due to its complex genome and biological characteristics. This limits the development of cisgenic and gene-edited varieties. One hurdle is selecting the best starting tissue for the transformation process, much like isolating suitable tissue for protoplasts. One promising method involves delivering CRISPR/Cas components to protoplasts isolated from embryogenic calli, which are then induced to regenerate. However, this process is inefficient, time-consuming, and only applicable to a few genotypes. To enhance grapevine regeneration efficiency, the expression of developmental and plant growth regulators shows promise in escaping the recalcitrance encountered in traditional tissue culture methods. A strategy based on the Bean yellow dwarf virus (BeYDV) allows for the temporary expression of regulators while minimizing the risk of obtaining transgenic plants. Additionally, the high copy number replication of BeYDV allows for high CRISPR/CAS levels, thereby improving editing. The goal of this study is to evaluate the effects of individual or combined developmental regulators’ expression on grapevine embryogenic tissues. We conducted preliminary assays using BeYDV-derived vectors for luciferase reporter gene expression to optimize delivery efficiencies. Assays were performed on ‘Chardonnay’ calli and protoplasts using both agrobacterium-mediated transformation and protoplast transfection approaches. The present study seeks to enhance the transformation protocols and regeneration processes, with the ultimate aim of realizing the full potential of editing technologies in grapevine.

Miglioramento dei processi di trasformazione e rigenerazione in vite: un nuovo approccio che utilizza regolatori dello sviluppo ed espressione mediata da BeYDV

La vite (Vitis vinifera L.) è una specie vegetale difficile da trasformare e rigenerare a causa si del suo complesso genoma che delle sue caratteristiche biologiche. Ciò limita lo sviluppo di varietà cisgeniche e di varietà modificate mediante editing genomico. Una delle principali difficoltà è selezionare il tessuto di partenza più adatto per il processo di trasformazione, così come l’isolamento di tessuti adatti per i protoplasti. Un metodo promettente è l’utilizzo del sistema crispr/cas su protoplasti isolati da calli embriogenici, i quali vengono poi indotti a rigenerare. Tuttavia, questo processo è inefficiente, richiede tempo ed è applicabile solo a pochi genotipi. Per migliorare l’efficienza di rigenerazione della vite, una strada promettente sembra l’espressione di regolatori dello sviluppo e della crescita delle piante che potrebbe aiutare a superare i limiti posti dai metodi tradizionali di coltura dei tessuti. Una strategia basata sul bean yellow dwarf virus (beydv) consente l’espressione temporanea dei regolatori riducendo al minimo il rischio di ottenere piante transgeniche. Inoltre, la replicazione ad alto numero di copie di beydv consente di avere livelli elevati di crispr/cas, migliorandone così l’efficienza. L’obiettivo di questo studio è valutare gli effetti dell’espressione singola o combinata di regolatori dello sviluppo su tessuti embriogenici di vite. Abbiamo condotto test preliminari utilizzando vettori derivati da beydv contenente come gene reporter la luciferasi in modo da ottimizzare l’efficienza di trasformazione. Gli esperimenti sono stati eseguiti sui calli e sui protoplasti di ‘chardonnay’ utilizzando sia l’approccio di trasformazione mediato da agrobatterio che la trasfezione di protoplasti. Questo studio mira a migliorare i protocolli di trasformazione e i processi di rigenerazione in modo da sfruttare a pieno il potenziale delle tecnologie di editing anche sulla vite.

Mejora de la transformación y regeneración de la vid: un enfoque novedoso utilizando reguladores del desarrollo y expresión mediada por BeYDV

La vid (Vitis vinifera L.) Es una especie de planta desafiante de transformar y regenerar debido a su complejo genoma y características biológicas. Esto limita el desarrollo de variedades cisgénicas y editadas genéticamente. Un obstáculo es seleccionar el mejor tejido inicial para el proceso de transformación, al igual que aislar tejido adecuado para protoplastos. Un método prometedor implica la entrega de componentes crispr/cas a protoplastos aislados de callos embriogénicos, que luego se inducen a regenerar. Sin embargo, este proceso es ineficiente, consume tiempo y solo es aplicable a unos pocos genotipos. Para mejorar la eficiencia de regeneración de la vid, la expresión de reguladores del desarrollo y crecimiento de plantas muestra promesa para escapar de la recalcitrancia encontrada en los métodos tradicionales de cultivo de tejidos. Una estrategia basada en el bean yellow dwarf virus (beydv) permite la expresión temporal de reguladores mientras se minimiza el riesgo de obtener plantas transgénicas. Además, la alta replicación del número de copias de beydv permite niveles altos de crispr/cas, mejorando así la edición genética. El objetivo de este estudio es evaluar los efectos de la expresión de reguladores del desarrollo individual o combinados en tejidos embriogénicos de la vid. Realizamos ensayos preliminares usando vectores derivados de beydv para la expresión del gen reportero luciferasa para optimizar las eficiencias de entrega. Los ensayos se realizaron en callos y protoplastos de ‘chardonnay’ utilizando tanto la transformación mediada por agrobacterium como enfoques de transfección de protoplastos. El presente estudio busca mejorar los protocolos de transformación y procesos de regeneración, con el objetivo final de realizar todo el potencial de las tecnologías de edición en la vid.

DOI:

Publication date: November 18, 2024

Issue: OIV 2024

Type: Article

Authors

Flavia Angela Maria Maggiolini¹, Margherita D’Amico¹, Lucia Rosaria Forleo¹, Annalisa Prencipe², Bruna Suriano¹, Mario Ventura², Maria Francesca Cardone¹, Riccardo Velasco¹, Carlo Bergamini¹

¹ Council for Agricultural Research and Economics – Research Center for Viticulture and Enology (CREA-VE), Italy
² Department of Biosciences, Biotechnology, and Environment, University of Bari, Italy

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Tags

Full papers OIV 2024 | IVES Conference Series | OIV | OIV 2024

Citation

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