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

Matthew J Szarzanowicz1,2,3, Michael Busche4, Ziyu Dai1,5

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The origin of replication family in binary vectors, not plasmid copy number, controls Agrobacterium-mediated transformation outcomes. This finding enables optimized genetic engineering in plants and fungi.

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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.