Endosperm Fluorescence Zygosity Detection in Maize Seeds

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for determining the zygosity of transgenes in seeds are time-consuming and labor-intensive, particularly in angiosperm plants like maize, where double fertilization complicates fluorescence measurement in the embryo, leading to potential errors in identifying homozygous, heterozygous, and wild-type seeds.

Innovation Solution

Genetically linking a fluorescent protein (FP) gene to the transgene under the control of an endosperm-specific promoter, allowing for the measurement of fluorescence in the endosperm, which indicates the zygosity of the transgene, using a device that emits an exciting wavelength and measures fluorescence intensity, enabling classification into wild-type, heterozygous, or homozygous categories.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If fluorescence measurement is performed in the embryo to determine zygosity, then direct detection of transgene copy number is possible, but measurement errors occur due to double fertilization in angiosperms

Engineering Contradiction:

Improvezygosity detection accuracyVSAvoiddetection reliability

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent extracts the fluorescence measurement from the embryo and relocates it to the endosperm. By using an endosperm-specific promoter (such as the Zea mays 27-kD albumin promoter) to drive FP expression, the measurement is performed in a tissue that is easier to access and less affected by the complications of double fertilization, thereby improving reliability while maintaining measurement precision

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces an intermediary approach by using the endosperm as a proxy for determining embryonic zygosity. Since the endosperm's genotype reflects the parental contributions, measuring FP expression in the endosperm provides indirect but reliable information about the transgene copy number in the embryo, resolving the reliability issue

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If seeds are germinated and qPCR is performed to identify zygosity, then accurate classification into homozygous, heterozygous, and wild-type is achieved, but the process is time-consuming and labor-intensive

Engineering Contradiction:

Improvezygosity classification accuracyVSAvoidtime for zygosity determination

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent performs preliminary action by genetically linking the FP gene to the transgene and establishing endosperm-specific expression before the actual zygosity determination is needed. This allows seeds to be screened directly after harvest without requiring germination and molecular analysis, dramatically reducing the time and labor required while maintaining accurate classification

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent replaces the mechanical/biochemical process of germination and qPCR with an optical detection system. By using fluorescence microscopy or flow cytometry to detect FP expression in the endosperm, the method substitutes complex laboratory procedures with a simpler, faster optical measurement that can be performed on dry seeds

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Adaptability or versatility

If fluorescent protein is used as a selectable marker, then transgenic events can be tracked, but fluorescence measurement in the embryo is complicated by double fertilization in angiosperms

Engineering Contradiction:

Improvetransgene tracking capabilityVSAvoidmeasurement system complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent applies local quality by restricting FP expression to a specific tissue (the endosperm) through the use of an endosperm-specific promoter. This localized expression simplifies the measurement system because the endosperm is larger, more accessible, and its fluorescence can be detected without the complications affecting embryonic tissue, thereby reducing device complexity while maintaining tracking versatility

Inventive Principle:

Principle #3Local quality

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

This method allows for rapid and accurate determination of transgene zygosity in seeds without the need for germination, reducing time and cost, and provides a reliable means to select seeds for further breeding, with high success rates in identifying homozygous plants.

Implementation Method 1

Genetically linking a fluorescent protein (FP) gene to the transgene under the control of an endosperm-specific promoter, allowing for the measurement of fluorescence in the endosperm

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS11668703B2Method for determining the level of zygosity of a seed

Publication Date: 2023.06.06 LIMAGRAIN EURO SA

AI summary

The invention relates to a method for determining the level of transgene zygosity in a poaceae seed, the transgene being genetically linked to a gene coding for a fluorescent protein (FP protein) under the control of a promoter operative in the endosperm, comprising the step of exposing the endosperm of the seed to a wavelength exciting the FP protein, and measuring the intensity of the emitted fluorescence.