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-10227602-B2
Publication Date
2019-03-12
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, including apple, in which fruit browning is reduced relative to wild type. The reduction is achieved by reducing total Polyphenol Oxidase (PPO) activity through reduced expression of multiple PPO isoenzymes in the apple plant.
The genetically modified apple plants are generated by transformation using vectors and nucleic acid constructs comprising heterologous polynucleotide sequences corresponding to PPO isoenzymes. The document targets at least two PPO isoenzymes and, in other embodiments, reduction of expression of at least four PPO isoenzymes by providing multiple heterologous nucleic acid molecules corresponding to specified SEQ ID NOs.
The constructs include promoter-linked heterologous nucleic acid molecules, including sense orientation and optionally antisense orientation, and may be arranged so that an mRNA stem-loop structure can be formed. Genetically modified apple plants and related apple materials can include cells comprising two or more heterologous sequences, and reduced browning is associated with reduced PPO isoenzyme expression relative to wild type.
Claims Coverage
The independent claims cover genetically modified apple plants and nucleic acid constructs that reduce fruit browning potential by reducing expression of multiple PPO isoenzymes. Across the independent claims, the coverage centers on transformation vectors containing defined SEQ ID NO polynucleotides, multi-isoenzyme PPO suppression using multiple heterologous nucleic acid molecules, and nucleic acid constructs that can encode an mRNA capable of forming a stem-loop structure.
Multi-heterologous PPO sequence transformation for apple plants
A genetically modified apple plant generated by transformation of apple plant cells with a vector comprising the heterologous polynucleotide sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, wherein the modified apple plant comprises at least one cell further comprising two or more of the heterologous sequences.
Single defined PPO isoenzyme-sequence transformation for apple plants
A genetically modified apple plant generated by transformation of apple plant cells with a vector comprising the polynucleotide sequence of SEQ ID NO:58, wherein the modified apple plant comprises at least one cell further comprising said sequence.
Reduced-browning potential via reduction of four PPO isoenzyme gene expression
A genetically modified apple plant, or genetically modified cell, seed, seedling, part, tissue, fruit or progeny, having reduced browning potential relative to wild type, wherein the reduced browning potential results from the reduction in expression of genes encoding at least four polyphenol oxidase (PPO) isoenzymes, comprising first, second, third, and fourth heterologous nucleic acid molecules with defined identity to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:24, respectively.
Promoter-linked four-part PPO isoenzyme expression-reducing construct in sense orientation
A nucleic acid construct comprising a promoter and first, second, third, and fourth heterologous nucleic acid molecules having 100% sequence identity to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, respectively, operably linked to the promoter in a sense orientation.
Stem-loop mRNA encoding nucleic acid construct using antisense-oriented PPO-targeting sequences with spacer
A nucleic acid construct encoding an mRNA capable of forming a stem loop structure, comprising a promoter, a first set of nucleic acid sequences, a spacer, and a second set of nucleic acid sequences, wherein the second set is operably linked to the promoter in an antisense orientation and the first and second sets are separated by a spacer.
Overall, the independent claims require genetically modified apple plants generated by transformation with vectors or constructs containing defined PPO-targeting sequences. Several claims focus specifically on reducing expression of multiple PPO isoenzymes, including embodiments using four PPO isoenzymes via multiple heterologous nucleic acid molecules, and on construct architectures that can include sense/antisense promoter linkage and an mRNA stem-loop capable of forming.
Stated Advantages
Reduced fruit browning potential relative to wild type.
Reduction in PPO expression of PPO isoenzymes relative to wild type, associated with reduced browning.
Barely visible or reduced browning in bruised fruit and juice in reported examples.
Reduced PPO activity in reported apple events and varieties.
Documented Applications
Reduced-browning apple varieties and events in bruised fruit and juice, including Golden Delicious, Granny Smith, and Fuji, as reported in field/phenotyping examples.
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