CRISPR-Edited Wheat PPO1 and PPO2 Genes Reduce Browning
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Solution Overview
Problem
Polyphenol oxidases (PPO) in plants cause undesirable browning and discoloration of plant tissues, particularly in wheat, leading to reduced value of flour and end-use products during processing, for which existing methods lack effective solutions beyond reducing flour extraction rates.
Innovation Solution
Modified plants with reduced PPO activity are developed by introducing mutations in the PPO1 or PPO2 genes, specifically in the copper binding domain, using genome editing techniques such as CRISPR/Cas9 to decrease PPO polypeptide expression or activity, which are then bred and propagated to produce grains with lower browning propensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If PPO activity is reduced through genome editing, then browning and discoloration of plant tissues is reduced, but the complexity of the manufacturing process increases
Solution Approach 1:
The patent extracts and removes the harmful PPO enzyme function by targeting and disabling specific PPO genes (PPO1 and PPO2) through genome editing. CRISPR/Cas9 is used to create mutations that prevent PPO polypeptide expression, thereby eliminating the browning reaction source while maintaining the rest of the plant system intact.
Solution Approach 2:
The patent changes the genetic parameters of the plant by introducing mutations in the PPO1 and PPO2 genes. Specifically, it modifies the copper binding domain regions of these genes to reduce or eliminate PPO activity, thereby changing the biochemical parameters that control browning without affecting other plant functions.
2Object-affected harmful factors
If flour extraction rate is reduced during milling, then PPO activity in flour is reduced, but the productivity and yield of flour production decreases
Solution Approach 1:
The patent applies preliminary action by genetically modifying the plant before harvest to reduce PPO activity. The genome editing is performed on the living plant, allowing the plant to grow and produce grain with inherently reduced PPO content, so that when the grain is milled, the flour naturally contains less PPO without requiring reduced extraction rates.
Solution Approach 2:
The patent replaces the mechanical control method (reducing flour extraction rate during milling) with a biological/genetic solution. Instead of physically limiting how much flour is extracted, the approach substitutes genetic modification to reduce PPO activity at its source, thereby maintaining both flour yield and reduced browning potential.
3Object-affected harmful factors
If PPO polypeptide expression is reduced through gene mutation, then browning propensity is reduced, but the difficulty of detecting and measuring PPO activity changes
Solution Approach 1:
The patent employs feedback mechanisms by using guide RNAs that are designed to specifically target and bind to PPO1 and PPO2 gene sequences. This allows for detection and verification of the genetic modifications. The system provides feedback on whether the intended mutations have been successfully introduced, enabling measurement and confirmation of reduced PPO activity through molecular detection methods.
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
The modified plants significantly reduce browning in wheat products, enhancing their quality and value by minimizing discoloration and flavor changes, allowing for the production of commodity plant products with improved stability and appearance.
Implementation Method 1
using genome editing techniques such as CRISPR/Cas9 to decrease PPO polypeptide expression or activity
Implementation Method 2
the mutation is in a region of the endogenous PPO1 or PPO2 gene that encodes a copper binding domain of a PPO polypeptide
Data Source
AI summary
The disclosure relates to compositions and methods for reducing polyphenol oxidase (PPO) activity in plants, including wheat plants. The compositions comprise plants, plant parts, plant cells, and seeds comprising a mutation in at least one endogenous PPO1 or PPO2 gene. Products produced from the grain of plants have reduced browning.