Flow Cytometry-assisted Screening of Gamma Irradiation-induced Nuclear DNA Content Variations in Cassava (Manihot esculenta Crantz) Genotypes

Abstract

Gamma irradiation is a widely used physical mutagen in plant breeding capable of inducing a variety of DNA variations and chromosomal aberrations that manifest in the genome; therefore, flow cytometry (FCM), the gold-standard technique for estimating nuclear DNA content, provides a rapid, high-resolution method for DNA nuclear content quantification, enabling precise evaluation of irradiation effects on crops such as cassava (Manihot esculenta Crantz) by measuring 2C nuclear DNA content with DNA stains such as propidium iodide (PI). This study assessed the impact of gamma irradiation on the infraspecific diversity of three cassava genotypes (CAS-1, CAS-2, CAS-3) using an incomplete factorial design with doses ranging from 0 to 30 Gy. Nuclear DNA content was estimated relative to Zea mays cv. CE-777 as an internal reference standard, and statistical analyses were performed using Welch ANOVA and Games–Howell post hoc tests. Results revealed clear genotype-specific responses: CAS-1 remained stable across all doses (≤0.5% change); CAS-2 exhibited modest reductions at 15–25 Gy (−0.8 to −1.6%); and CAS-3 showed a significant increase at 15 Gy (+4.5%), accompanied by endoreplication peaks (2C–32C) indicative of irradiation-induced polyploidization. Welch ANOVA confirmed highly significant genotype × irradiation dose interactions (η² = 0.989), and post hoc clustering separated irradiated CAS-3 nuclei into a distinct group. These findings highlight cassava’s genomic plasticity under gamma-ray mutagenic stress, with CAS-1 demonstrating stability, CAS-2 showing vulnerability to DNA loss, and CAS-3 exhibiting expansion and altered cell cycle regulation. The study underscores the utility of FCM in detecting subtle genomic shifts and providing insights into tailored gamma irradiation strategies for cassava improvement, particularly in breeding programs targeting enhanced diversity and disease resistance, i.e., cassava brown streak disease.