Ochrobactrum-mediated plant transformation via VirD2-dependent T-DNA transfer

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Solution Overview

Problem

Current methods for plant transformation, particularly using Agrobacterium, face challenges such as genotype-independent transformation inefficiencies and unpredictable transgene expression, necessitating the exploration of alternative bacterial strains for improved transformation efficiencies.

Innovation Solution

The use of Ochrobactrum strains, specifically Ochrobactrum haywardense H1, for plant transformation, which involves transferring polynucleotides using VirD2-dependent methods, incorporating virulence genes and T-DNA border sequences, and employing selectable markers for efficient gene introduction and stable integration.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If Agrobacterium-mediated transformation is used, then transformation efficiency is improved, but genotype-independent transformation efficiency deteriorates and transgene expression stability deteriorates

Engineering Contradiction:

Improvetransformation efficiencyVSAvoidtransgene expression stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent uses Ochrobactrum as an intermediary bacterial system to mediate plant transformation, replacing Agrobacterium. The Ochrobactrum strain is engineered with specific virulence genes (virD2, virE2, virB1-virB11) and T-DNA border sequences to enable efficient and stable transgene delivery into plant genomes, resolving the contradiction between transformation efficiency and expression stability.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the bacterial species parameter from Agrobacterium to Ochrobactrum, and modifies the genetic parameters by introducing specific virulence genes and T-DNA border sequences. This parameter change enables both high transformation efficiency and stable transgene expression across different plant genotypes.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If Agrobacterium-mediated transformation is used, then transformation efficiency is improved, but adaptability to different plant genotypes deteriorates

Engineering Contradiction:

Improvetransformation efficiencyVSAvoidgenotype-independent transformation

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The engineered Ochrobactrum system is designed with universal functionality to transform diverse plant genotypes including monocots and dicots. The combination of virulence genes and T-DNA border sequences creates a multi-functional system that can efficiently deliver transgenes across different plant species and genetic backgrounds, overcoming the genotype-specific limitations of Agrobacterium.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Adaptability or versatility

If non-Agrobacterium bacterial strains are used, then adaptability to different plants is improved, but transformation efficiency deteriorates

Engineering Contradiction:

Improveplant species rangeVSAvoidtransformation efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent creates a composite bacterial system by combining Ochrobactrum with engineered virulence genes and T-DNA border sequences from Agrobacterium. This composite approach integrates the adaptability of non-Agrobacterium strains with the transformation efficiency mechanisms of Agrobacterium, achieving both broad plant species range and high transformation efficiency.

Inventive Principle:

Principle #40Composite materials

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach enhances transformation efficiencies and stability, allowing for the successful integration of desired sequences into plant genomes, including monocots and dicots, with potential applications in various crop species.

Implementation Method 1

using an Ochrobactrum strain to transfer a polynucleotide of interest to a plant cell. These methods include VirD2-dependent methods

Methodology Applied

Scientific EffectVirD2-dependent transformation:

Implementation Method 2

Many, if not most of genetically modified crops were generated using Agrobacterium-mediated transformation to integrate the trait(s) of interest

Methodology Applied

Scientific EffectBacterial-mediated transformation:

Data Source

PatentUS11236347B2Ochrobactrum-mediated transformation of plants

Publication Date: 2022.02.01 CORTEVA AGRISCIENCE LLC

AI summary

Methods and compositions for Ochrobactrum-mediated transformation of plants are provided. Methods include but are not limited to using an Ochrobactrum strain to transfer a polynucleotide of interest to a plant cell. These include VirD2-dependent methods. Compositions include an Ochrobactrum strain, transfer DNAs, constructs and/or plasmids. These include Ochrobactrum strains having a plasmid comprising one or more virulence gene(s), border region, and/or origin of replication. Plant cells, tissues, plants, and seeds comprising a polynucleotide of interest produced by the methods are also provided.