The development of a precision breeding toolkit: Towards genome editing in pollen

Abstract

Plant biotechnology has long been a vital tool for crop improvement and is poised to play a crucial role in addressing global food security and environmental challenges. This thesis details the development of an innovative plasmid toolkit for targeted gene expression and precise genome editing in tomato pollen, with the goal of enabling heritable genetic modifications. Initial research optimised tomato pollen germination conditions, establishing a robust protocol for high viability and germination, essential for subsequent gene delivery experiments. A key innovation was the creation of a versatile plasmid toolkit with high-copy-number origins, antibiotic resistance markers, a modular design, tissue-specific and constitutive promoters, and CRISPR/Cas9 compatibility, facilitating both transient and stable expression, validated through sfGFP reporter protein expression. Poly(2- dimethylaminoethyl methacrylate) (PDMAEMA) nanoparticles were explored for efficient biomolecule delivery, with studies on DNA-binding interactions, stability, and cytotoxicity to maintain pollen viability. While CRISPR/Cas9-based genome editing was not demonstrated within this research, the toolkit shows potential for future targeted genetic modifications. This foundational work advances plant biotechnology and offers a platform for precision breeding to support more adaptive agriculture.