Genetically modified reduced-browning fruit-producing plant and produced fruit thereof, and method of obtaining such
Inventors
Armstrong, John • Lane, William David
Assignees
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Publication Number
US-11359205-B2
Publication Date
2022-06-14
Expiration Date
Abstract
A genetically modified fruit-producing plant, said plant having sufficiently reduced total Polyphenol Oxidase (PPO) activity relative to a wild type of said plant to reduce browning in the fruit of said plant relative to said wild type, wherein the reduced total PPO activity results from a reduction in activity of at least two PPO isoenzymes in said plant relative to said wild type, or a cell, seed, seedling, part, tissue, cell, fruit or progeny of said plant.
Core Innovation
The invention relates to genetically modified fruit-producing plants engineered for reduced browning by lowering total polyphenol oxidase (PPO) activity and/or reducing PPO expression. In embodiments, this reduction is achieved by suppression of at least two PPO isoenzymes, including PPO2 and other PPO isoenzymes such as GPO3 and APO5, thereby targeting total PPO activity rather than a single isoenzyme activity. The described phenotype is a reduced-browning fruit phenotype.
Nucleic acid constructs disclosed in the patent include sense and stem-loop RNA silencing architectures to support suppression of PPO expression/activity. The constructs are described as heterologous nucleic acid molecules comprising nucleotide sequences configured for RNA silencing, including stem-loop dsRNA architectures. Plant-cell and plant embodiments are described, including embodiments relevant to fruit-producing plants such as genetically modified apple plants.
The patent further provides assay concepts and quantitative thresholds for reduced total PPO activity/expression and reduced-browning phenotypes, and it reports experimental examples demonstrating that suppression of at least two PPO isoenzymes provides reduced browning tied to reduced total PPO activity. The examples include constructs and genetically modified plant materials with suppression of multiple PPO isoenzymes, along with evaluation of PPO activity and PPO gene expression and assessment outcomes such as reduced browning linked to reduced total PPO activity.
Claims Coverage
The provided claim set includes independent claims covering genetically modified apple plants and nucleic acid constructs defined by identity relationships to specific reference nucleotide sequences, with four inventive features centered on sequence identity thresholds and contiguous segment requirements.
Genetically modified apple plant with 100% identical contiguous segments from two selected SEQ IDs
A genetically modified apple plant includes at least one heterologous nucleic acid molecule comprising at least 20 contiguous nucleotides 100% identical to at least 20 contiguous nucleotides of a first nucleotide sequence and at least 20 contiguous nucleotides 100% identical to at least 20 contiguous nucleotides of a second nucleotide sequence, where the first and second nucleotide sequences are selected from two different nucleotide sequences selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
Genetically modified apple plant with at least 90% identical different SEQ ID sequences
A genetically modified apple plant includes at least one heterologous nucleic acid molecule comprising at least a first nucleotide sequence and a second nucleotide sequence, where the first and second nucleotide sequences are different from each other and are selected from nucleotide sequences that are at least 90% identical to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
Nucleic acid construct with 100% identical contiguous segments from two selected SEQ IDs
A nucleic acid construct comprises at least 20 contiguous nucleotides 100% identical to at least 20 contiguous nucleotides of a first nucleotide sequence and at least 20 contiguous nucleotides 100% identical to at least 20 contiguous nucleotides of a second nucleotide sequence, where the first and second nucleotide sequences are selected from two different nucleotide sequences selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
Nucleic acid construct with at least 90% identical different SEQ ID sequences
A nucleic acid construct comprises at least a first nucleotide sequence and a second nucleotide sequence, where the first and second nucleotide sequences are selected from two different nucleotide sequences selected from sequences that are 90% identical to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
Across the independent claims, the coverage is defined by genetically modified apple plants and nucleic acid constructs that include heterologous nucleic acid molecules or constructs containing nucleotide sequences selected from specified SEQ ID NOs (30–33), with the central inventive variations being whether the sequences meet 100% identity over at least 20 contiguous nucleotides or meet at least 90% identity.
Stated Advantages
Reduced browning via lowering total polyphenol oxidase (PPO) activity and/or reducing PPO expression.
Suppression of at least two PPO isoenzymes to achieve reduced-browning fruit phenotypes linked to reduced total PPO activity.
Documented Applications
Application to genetically modified fruit-producing plants, including apple plants, to produce a reduced-browning fruit phenotype by lowering total PPO activity and reducing PPO expression.
Use of nucleic acid constructs for RNA silencing to suppress PPO expression/activity in plant embodiments.
Evaluation of PPO activity and gene expression, and assessment of browning outcomes such as controlled bruising/juice browning and field trial events showing reduced browning linked to reduced total PPO activity.
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