# SeedSeg: image-based transgenic seed counting for segregation analysis of T-DNA loci

## TL;DR

SeedSeg is an automated tool that counts transgenic and wild-type seeds using images, helping researchers analyze T-DNA insertion patterns more efficiently.

## Contribution

SeedSeg introduces an image-based, automated method for T-DNA segregation analysis, reducing manual labor in transgenic plant research.

## Key findings

- SeedSeg uses a segmentation algorithm to count transformed and wild-type seeds in images.
- The tool performs a chi-squared test to determine the number of T-DNA loci.
- SeedSeg is adaptable to different seed sizes and visual markers, making it versatile for plant research.

## Abstract

Transgenic plants are essential for both basic and applied plant biology. Recently, fluorescent and colorimetric markers were developed to enable nondestructive identification of transformed seeds and accelerate the generation of transgenic plant lines. Yet, transformation often results in the integration of multiple copies of transgenes in the plant genome. Multiple transgene copies can lead to transgene silencing and complicate the analysis of transgenic plants by requiring researcher to track multiple T-DNA loci in future generations. Thus, to simplify analysis of transgenic lines, plant researchers typically screen transformed plants for lines where the T-DNA inserted in a single locus — an analysis that involves laborious manual counting of fluorescent and non-fluorescent seeds for screenable markers.

To expedite T-DNA segregation analysis, we developed SeedSeg, an image analysis tool that uses a segmentation algorithm to count the number of transformed and wild-type seeds in an image. SeedSeg runs a chi-squared test to determine the number of T-DNA loci. Parameters can be adjusted to optimize for different brightness intensities and seed sizes.

By automating the seed counting process, SeedSeg reduces the manual labor associated with identifying transgenic lines containing a single T-DNA locus. SeedSeg is adaptable to different seed sizes and visual transgene markers, making it a versatile tool for accelerating plant research.

The online version contains supplementary material available at 10.1186/s13007-025-01406-4.