Binary vector origin predictably determines Agrobacterium-mediated transformation outcome across eukaryotic kingdoms.

Area of Science:

• Plant science
• Mycology
• Molecular biology
• Genetic engineering

Background:

• Agrobacterium-mediated transformation (AMT) is crucial for genetic engineering in plants and fungi.
• Key transformation outcomes like efficiency, transgene insertion number, and integrity are poorly understood.
• Current AMT methods lack optimization for industrial and academic applications.

Purpose of the Study:

• To systematically analyze how binary vectors impact AMT outcomes.
• To investigate the roles of plasmid origin of replication (ORI) families and copy number variants.
• To compare transformation results across plant and fungal species.

Main Methods:

• Comparative analysis of different plasmid origin of replication (ORI) families.
• Engineering and testing of plasmid copy number variants.
• Systematic evaluation of transformation efficiency, transgene insertion number, and integrity.
• Cross-kingdom comparison of ORI-dependent outcomes in yeast and *Arabidopsis*.

Main Results:

• The ORI family, not plasmid copy number, dictates T-DNA insertion number, backbone inclusion, and transformation efficiency.
• pVS1 ORI-based vectors yield more insertions and higher transgene silencing than pSa ORI vectors.
• pSa ORI vectors promote uniform single insertion events.
• ORI-dependent transformation outcomes in yeast correlate with those in *Arabidopsis*.

Conclusions:

• Origin of replication family is a critical determinant of AMT outcomes.
• Vector design choices significantly influence transgene integration and expression.
• Findings provide a basis for developing optimized binary vectors for predictable transformation across eukaryotes.