Agrobacterium rhizogenes and methods of transforming cells
AssigneePairwise Plants Services, Inc.
InventorsFang-Ming Lai, Eric Dean, Ming Cheng, Joel Reiner, Qingchun Shi, David Schwark, Sharon Leigh Guffy, Aaron Hummel
Life of this patent
Estimating the term…
Abstract
Agrobacterium rhizogenes are described herein along with methods of transforming cells and methods of using Agrobacterium rhizogenes and/or transformed cells. Also described herein are plants, such as plants in the Rubus family, including a modified nucleic acid, optionally wherein the plants are transgene-free and include a modified nucleic acid.
Claims82
A method of transforming a plant cell, the method comprising:
- introducing a polynucleotide from an Agrobacterium rhizogenes strain into the plant cell to provide a transformed plant cell, thereby transforming the plant cell.
The method of any one of the preceding claims, wherein the introducing step comprises contacting the Agrobacterium rhizogenes strain and an explant (e.g., a shoot stem nodal explant, shoot tip explant, node explant, etc.) comprising the plant cell, optionally wherein the plant cell is present in a callus of the explant and the Agrobacterium rhizogenes strain is contacted to the callus to provide a transformed callus.
The method of any one of the preceding claims, wherein introducing the polynucleotide from the Agrobacterium rhizogenes strain into the plant cell comprises concurrently introducing transfer DNA (T-DNA) from a tumor-inducing (Ti) plasmid and hairy root-inducing (Ri) plasmid of the Agrobacterium rhizogenes strain, optionally wherein the T-DNA from the Ti plasmid (e.g., a Ti plasmid comprising a non-Agrobacterium rhizogenes origin of replication) and the T-DNA from the Ri plasmid are concurrently integrated into the genome of the plant cell to provide a transgenic plant cell.
The method of any one of the preceding claims, further comprising forming a callus (e.g., a transgenic callus) from the transformed plant cell or culturing the transformed callus.
The method of any one of the preceding claims, wherein the plant cell is stably transformed with the polynucleotide.
The method of any one of the preceding claims, wherein the polynucleotide is stably expressed in the transformed plant cell and wherein the method further comprises modifying a target nucleic acid in the transformed plant cell to provide a modified nucleic acid.
The method of any one of the preceding claims, further comprising producing a root (e.g., a hairy root) from the transformed plant cell or edited plant cell, optionally wherein the root comprises cells including a modified nucleic acid.
The method of any one of the preceding claims, further comprising producing a transgenic root from the transformed plant cell, optionally wherein the transgenic root is a transgenic hairy root.
The method of any one of the preceding claims, further comprising evaluating the efficacy of the polynucleotide, optionally wherein evaluating the efficacy of the polynucleotide comprises evaluating the transformation efficiency and/or genome editing efficiency of the polynucleotide.
The method of any one of the preceding claims, wherein the Agrobacterium rhizogenes strain is a wild-type Agrobacterium rhizogenes strain.
The method of any one of the preceding claims, wherein the Agrobacterium rhizogenes strain is selected from the group consisting of Agrobacterium rhizogenes strain 1193, Agrobacterium rhizogenes strain A4;
- Agrobacterium rhizogenes strain Qual, Agrobacterium rhizogenes strain K599 and Agrobacterium rhizogenes strain MSU440.
The method of any one of the preceding claims, further comprising screening for the polynucleotide, a modified nucleic acid, or a given phenotype, optionally wherein the screening comprises screening the callus and/or root for the polynucleotide, the modified nucleic acid, or the given phenotype.
The method of any one of the preceding claims, further comprising performing molecular screening for the polynucleotide, a modified nucleic acid, or a given phenotype, optionally wherein performing molecular screening comprises performing molecular screening on the root for the polynucleotide, the modified nucleic acid, or the given phenotype.
The method of any one of the preceding claims, wherein the plant cell is a dicot plant cell, optionally wherein the plant cell is a blackberry, raspberry (e.g., red raspberry), artic bramble, or cherry plant cell.
The method of any one of the preceding claims, wherein the plant cell is from a plant in the Rubus family, optionally wherein the plant is Rubus allegheniensis, Rubus occidentalis, Rubus odoratus, or Rubus fruticosus.
The method of any one of the preceding claims, wherein the method is genotype-independent.
The method of any one of the preceding claims, wherein the method has a transformation efficiency of at least about 4%.
A method of producing an edited plant, the method comprising:
- introducing a polynucleotide from an Agrobacterium rhizogenes strain into a plant cell of an explant to provide an edited cell, wherein the polynucleotide encodes or comprises at least a portion of an editing system that is capable of modifying a target nucleic acid in the plant cell to provide a modified nucleic acid and thereby an edited cell;
- producing a root from the edited cell;
- and
- producing an edited plant from the root.
The method of 37, wherein the explant is a shoot node explant (e.g., a shoot stem nodal explant).
The method of 37 or 38, wherein the root is a transgenic root and the edited plant is produced from the transgenic root.
The method of 37 or 38, wherein the root is transgene free and comprises cells including the modified nucleic acid, and wherein the edited plant is transgene free and comprises cells including the modified nucleic acid.
A method of transforming a plant cell, the method comprising:
- introducing a polynucleotide from a disarmed Agrobacterium rhizogenes strain MSU440 (e.g., Agrobacterium rhizogenes strain DS101.1 and/or Agrobacterium rhizogenes strain comprising a sequence of SEQ ID NO:70) into the plant cell to provide a transformed plant cell, thereby transforming the plant cell.
The method of 44 or 45, wherein the polynucleotide encodes or comprises at least a portion of an editing system that is capable of modifying a target nucleic acid in the plant cell to provide a modified nucleic acid, optionally wherein the polynucleotide encodes or forms a ribonucleoprotein.
A method for evaluating transgene function in a plant cell, the method comprising:
- introducing a polynucleotide from an Agrobacterium rhizogenes strain into the plant cell to provide a transformed plant cell comprising the transgene, optionally wherein the polynucleotide encodes or comprises at least a portion of an editing system that is capable of modifying the target nucleic acid in the plant cell to provide a modified nucleic acid;
- and
- evaluating the transformed plant cell to determine transgene function.
A disarmed Agrobacterium rhizogenes strain comprising a deletion in the Ri plasmid, wherein the deletion is in a region of the MSU440 Ri plasmid from base pair 70,000 to base pair 130,000, wherein the base pair number is from the start of the origin of replication of the MSU440 Ri plasmid, or an optimally aligned region thereto for a different Ri plasmid.
A disarmed Agrobacterium rhizogenes strain prepared from Agrobacterium rhizogenes strain MSU440 by deleting about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of SEQ ID NO:67 in the Ri plasmid of the Agrobacterium rhizogenes strain MSU440, wherein the Ri plasmid of the Agrobacterium rhizogenes strain MSU440 has a sequence of SEQ ID NO:69.
Dependencies are inferred from the claim text.