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-9580723-B2
Publication Date
2017-02-28
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 apple plants and related biological materials in which total polyphenol oxidase (PPO) activity is sufficiently reduced to reduce browning in fruit relative to a wild type. The reduction in total PPO activity is produced by reducing expression of at least four PPO genes, where each PPO gene encodes a PPO isoenzyme defined by specific amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25.
The reduction in gene expression is obtained by altering or disrupting a coding sequence or regulatory element, or by reducing levels of mRNA transcripts of each of the at least four PPO genes. Reducing levels of mRNA transcripts is carried out using co-suppression, antisense expression, small hairpin (shRNA) expression, interfering RNA (RNAi) expression, double stranded (dsRNA) expression, inverted repeat dsRNA expression, micro interfering RNA (miRNA) expression, or simultaneous expression of sense and antisense sequences, applied to the at least four PPO genes or a combination thereof.
The disclosure further describes genetically modified fruit-producing plants, especially apple, with reduced PPO activity and reduced browning or juice browning phenotypes across field trial events. It includes engineered transgenes targeting multiple PPO genes and reported reductions in PPO activity and browning compared to wild type.
Claims Coverage
The document includes two independent claims, one directed to a genetically modified apple plant and related biological materials and one directed to a method for producing such a genetically modified apple plant. Each independent claim is anchored on the same core inventive concept: sufficiently reducing total PPO activity to reduce fruit browning by reducing expression of at least four PPO genes encoding PPO isoenzymes defined by specific SEQ ID NOs, using genetic alteration/disruption or mRNA transcript reduction via specified RNA-silencing approaches.
Sufficiently reduced total PPO activity reduces fruit browning
A genetically modified apple plant, or a cell, seed, seedling, part, tissue, fruit or progeny thereof, having sufficiently reduced total Polyphenol Oxidase (PPO) activity relative to a wild type to reduce browning in the fruit, where the reduced total PPO activity results from a reduction in expression of at least four PPO genes relative to said wild type.
Four PPO genes defined by SEQ ID NO: 19/21/23/25
The at least four PPO genes comprise a first PPO gene encoding a PPO isoenzyme comprising the amino acid sequence set forth as SEQ ID NO: 19, a second PPO gene encoding a PPO isoenzyme comprising SEQ ID NO: 21, a third PPO gene encoding a PPO isoenzyme comprising SEQ ID NO: 23, and a fourth PPO gene encoding a PPO isoenzyme comprising SEQ ID NO: 25.
Gene expression reduced by coding/regulatory alteration or mRNA transcript reduction
Reduction of gene expression is obtained by altering or disrupting a coding sequence or regulatory element, or by reducing levels of mRNA transcripts of each of said at least four PPO genes.
Reducing mRNA transcripts via specified RNA silencing modalities
Reducing levels of mRNA transcripts is by co-suppression, antisense expression, small hairpin (shRNA) expression, interfering RNA (RNAi) expression, double stranded (dsRNA) expression, inverted repeat dsRNA expression, micro interfering RNA (miRNA), or simultaneous expression of sense and antisense sequences, of said at least four PPO genes or a combination thereof.
Producing a genetically modified apple plant by reducing expression of four PPO genes
A method for producing a genetically modified apple plant having sufficiently reduced total Polyphenol Oxidase (PPO) activity relative to a wild type to reduce browning in the fruit, comprising reducing expression of at least four PPO genes.
Reducing four PPO genes using coding/regulatory alteration or mRNA transcript reduction
Reducing expression of said at least four PPO genes comprises altering or disrupting a coding sequence or regulatory element of, or reducing levels of mRNA transcripts of each of said at least four PPO genes.
mRNA transcript reduction via co-suppression/antisense/RNAi modalities
Reducing levels of mRNA transcripts is by co-suppression, antisense expression, small hairpin (shRNA) expression, interfering RNA (RNAi) expression, double stranded (dsRNA) expression, inverted repeat dsRNA expression, micro interfering RNA (miRNA), or simultaneous expression of sense and antisense sequences, of said at least four PPO genes or a combination thereof.
Across the two independent claims, the inventive coverage centers on genetically modified apple fruit biology where total PPO activity is sufficiently reduced to reduce fruit browning by simultaneously reducing expression of at least four PPO genes encoding PPO isoenzymes specified by SEQ ID NO: 19, 21, 23, and 25, with expression reduction achieved via altering or disrupting coding or regulatory elements or via mRNA transcript reduction using specified co-suppression, antisense, and RNAi-type approaches.
Stated Advantages
Reduction of browning in fruit relative to a wild type fruit
Reduction of total Polyphenol Oxidase (PPO) activity relative to a wild type
Reduction in PPO gene expression using co-suppression, antisense expression, shRNA expression, RNAi expression, dsRNA expression, inverted repeat dsRNA expression, miRNA expression, or simultaneous sense and antisense sequences
Documented Applications
Genetically modified apple plants with reduced-browning fruit phenotypes, including reduced browning and juice browning and reduced PPO activity in field trial events.
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