BiBAC Modification and Stable Transfer into Maize (<i>Zea mays)</i> Hi‐II Immature Embryos via <i>Agrobacterium‐</i>Mediated Transformation

Cody, Jon P.; Graham, Nathaniel D.; Birchler, James A.

doi:10.1002/cppb.20061

Cited by 6 publications

(4 citation statements)

References 8 publications

(8 reference statements)

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“…To the best of our knowledge, pMIN‐Ri represents the first practical pRi‐based modular cloning vector in the literature; the only other published Golden Gate backbone with the pRi origin is the MoClo toolkit vector pICH89921, a BIBAC vector based on the F‐plasmid origin. This backbone is over 4 kb larger than pMIN‐Ri, and the single‐copy F‐plasmid origin requires specialized cultures to reach appreciable plasmid yields in E. coli preparations (Cody et al., 2017). We have successfully conducted plant transformation with constructs built using both optimized backbones, using common Agrobacterium strains including AGL1, EHA105, GV3101, and LBA4404.…”

Section: Resultsmentioning

confidence: 99%

An extensible vector toolkit and parts library for advanced engineering of plant genomes

et al. 2023

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Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from crosscassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

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Section: Resultsmentioning

confidence: 99%

An extensible vector toolkit and parts library for advanced engineering of plant genomes

et al. 2023

Self Cite

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“…To the best of our knowledge, pMIN-Ri represents the first practical pRi-based modular cloning vector in the literature; the only other published Golden Gate backbone with the pRi origin is the MoClo toolkit vector pICH89921, a BIBAC vector based on the F-plasmid origin. This backbone is over 4kb larger than pMIN-Ri, and the single-copy F-plasmid origin requires specialized cultures to reach appreciable plasmid yields in E. coli preparations 39 . We have successfully conducted plant transformation with constructs built using both optimized backbones, using common Agrobacterium strains including AGL1, EHA105, GV3101, and LBA4404.…”

Section: Resultsmentioning

confidence: 99%

An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes

Chamness

Kumar

Cruz

et al. 2022

Preprint

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Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

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“…The GV3101 Agrobacterium strain was transformed via the freeze-thaw method and delivered to true leaves via leaf infiltration as previously described [ 9 ]. DNA was extracted from infiltrated plants 5 days post infiltration using a plate-based CTAB method [ 26 ].…”

Section: Methodsmentioning

confidence: 99%

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing

Atkins

Gamo

Voytas

2021

IJMS

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The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

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BiBAC Modification and Stable Transfer into Maize (Zea mays) Hi‐II Immature Embryos via Agrobacterium‐Mediated Transformation

Cited by 6 publications

References 8 publications

An extensible vector toolkit and parts library for advanced engineering of plant genomes

An extensible vector toolkit and parts library for advanced engineering of plant genomes

An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing

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