A simple and efficient protocol for transient transformation of sliced grape berries

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Agricultural Science

Background:

• Grape (Vitis spp.) is a vital crop, yet it exhibits recalcitrance to Agrobacterium-mediated genetic transformation and in vitro regeneration.
• Developing efficient transformation protocols is crucial for genetic improvement and functional studies in grape.

Purpose of the Study:

• To develop and optimize a simple and efficient protocol for transient transformation of grape berries.
• To investigate the impact of explant pre-culture and vacuum infiltration duration on transformation efficiency.

Main Methods:

• Explants from "Shine-Muscat" grape berries were pre-cultured or not, then subjected to Agrobacterium infiltration with varying vacuum durations (5, 10, 15 min).
• Transformation efficiency was assessed using a β-glucuronidase (GUS) reporter system (histochemical assay and RT-qPCR).
• The protocol's utility was validated by transiently transforming grape berries with a hydrogen peroxide sensor gene (VvHPCA3).

Main Results:

• Pre-culture of grape berry explants significantly increased susceptibility to Agrobacterium infection and enhanced transient transformation efficiency.
• Transformation efficiency positively correlated with vacuum infiltration duration, with longer durations yielding higher efficiencies.
• Gene expression analysis and GUS activity assays confirmed successful transient transformation and gene function (VvHPCA3).

Conclusions:

• A simple and effective transient transformation protocol for grape berries has been established.
• This protocol facilitates rapid gene function testing and aids in studying key regulatory networks in grapes.
• The optimized method overcomes Agrobacterium-mediated transformation challenges in this important crop.