CRISPR/Cas9-induced trehalase knockout for drought resistance in grapevine

Abstract

Climate change is predicted to alter the water availability where grapevine is cultivated, potentially impacting grape yield and quality [1]. Conventional grapevine breeding requires long periods of time and may alter a cultivar’s distinctive characteristics [2]. These factors make CRISPR/Cas9 molecular breeding techniques an attractive tool in limiting the effects of drought [3]. Trehalose is a well-known osmoprotectant sugar, with known functions in reducing reactive oxygen species levels (ROS), preserving membrane stability and accumulating of starch and other soluble sugars in leaves of plants under drought stress [4]. Exogenous application of trehalose has been shown to improve abiotic stress tolerance in many plants, including grapevine, thus increasing its content endogenously should enhance grapevine productivity in water-limited environments [5]. Woody plants generally biosynthesize small quantities of trehalose, which is later degraded by the trehalase enzyme. By utilising CRISPR/Cas9 to induce a knockout mutation, trehalase activity can be inactivated, thus allowing the accumulation of trehalose [6]. Agrobacterium-mediated transformation was used to transform Chardonnay embryogenic callus with a CRISPR construct containing sgRNAs targeting the trehalase gene.

Lines of grapevine were regenerated and confirmed, with next generation sequencing, containing complete knockout edits of trehalase. Gas chromatography-mass spectroscopy (GCMS) analysis performed on leaf tissue of the edited and control plants, showed an accumulation of trehalose and other soluble sugars in the edited lines. A population of the control and edited lines have been hardened off and will undergo more comprehensive phenotyping to evaluate growth and morphology, as well as water stress under greenhouse conditions. To confirm that the edited grapevine lines are less susceptible to water stress, physiological water-related parameters will be monitored throughout the trial, and biochemical tests and RT-qPCR analysis of drought and sugar-metabolism related genes are to be performed on leaves of the plants under water stress conditions.

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