Dose-response relationship of gamma irradiation on seed germination and early seedling vigor in selected tomato genotypes

Introduction

The deliberate induction of genetic variation through physical and chemical mutagenesis has been a cornerstone of crop improvement since Muller's demonstration of X-ray-induced mutations in Drosophila in 1927 and Stadler's parallel discovery of radiation effects on barley and maize in 1928. Since those foundational observations, gamma irradiation from Cobalt-60 and Caesium-137 sources has emerged as the most widely utilized physical mutagen in plant breeding, contributing to the release of over 3,400 mutant crop varieties registered in the FAO/IAEA Mutant Variety Database as of 2023. The effectiveness of any mutagenesis program depends fundamentally upon the selection of an appropriate irradiation dose that generates sufficient genetic variation while maintaining adequate plant survival and fertility. The dose-response relationship between gamma irradiation and biological endpoints follows a characteristic sigmoid function, where low doses may produce stimulatory hormetic effects, intermediate doses cause progressive growth reduction, and high doses result in lethality. The critical parameters of this doseresponse curve, including the optimal dose for mutagenic efficiency, the LD50 (dose producing 50% lethality), and the dose reduction factor, are essential for the rational design of irradiation protocols. Seed germination and early seedling development represent the first biological filters through which irradiation-induced genetic changes are expressed. The sensitivity of these early developmental stages to radiation damage is influenced by seed moisture content, genome size, ploidy level, DNA repair capacity, and the efficiency of antioxidant defense systems. In tomato (Solanum lycopersicum L.), published reports on irradiation effects on germination have yielded inconsistent dose recommendations, with reported optimal doses ranging from 10 to 100 Gy depending on the cultivar and experimental conditions employed. The present research was therefore conducted to establish precise dose-response relationships for seed germination and early seedling vigor in three tomato genotypes exposed to gamma irradiation at 25, 50, 75, and 100 Gy. The objectives included determination of LD50 values, characterization of dose reduction factors, evaluation of genotypic differences in radiosensitivity, and identification of the threshold dose for germination stimulation under controlled laboratory conditions at Daejeon, South Korea.

Material and Methods Material

Three tomato genotypes designated GT-1 (early maturing, compact), GT-2 (mid-season, semi-vigorous), and GT-3 (late maturing, vigorous) were used in this investigation. Seeds were obtained from the seed bank of the Daejeon Institute of Plant Biotechnology, Daejeon, South Korea. Seeds were pre-conditioned at 25°C and 45% relative humidity for 48 hours to achieve uniform moisture content (approximately 8%). Gamma irradiation was administered using a Cobalt-60 panoramic source (activity: 12,000 Ci) at the Korea Atomic Energy Research Institute at a dose rate of 1.5 Gy/minute. Four irradiation treatments (25, 50, 75, and 100 Gy) plus a non-irradiated control were applied, with dosimetry confirmed using Fricke dosimeters. The experiment was conducted from March to May 2023 in the controlled environment laboratories of the Daejeon Institute.

Methods

The experiment followed a completely randomized design with factorial treatment structure (3 genotypes × 5 irradiation doses) replicated five times. Each experimental unit consisted of 50 seeds placed on three layers of moistened Whatman No. 1 filter paper in 15-cm Petri dishes and incubated in growth chambers at 25±1°C under a 16/8 hour light/dark photoperiod (light intensity: 40 µmol/m²/s). Filter papers were kept consistently moist with distilled water throughout the germination period. Germination was assessed daily for 21 days, with a seed considered germinated upon radicle emergence of at least 2 mm. Parameters recorded included germination percentage (GP), germination rate index (GRI), mean germination time (MGT), root length, shoot length, seedling fresh weight, seedling dry weight (after oven-drying at 70°C for 48 hours), seedling vigor index (SVI = GP × seedling length), and root-to-shoot ratio. LD50 values were estimated by probit analysis using dose-germination response curves. Dose reduction factors (DRF) were calculated as the ratio of germination at dose n+1 to germination at dose n for each 25 Gy dose increment. Data were analyzed by two-way ANOVA using SAS software (version 9.4), and means were compared using LSD test at p≤0.05.

Results

A concise overview of the germination and vigor data revealed consistent dose-dependent responses across all three tomato genotypes, with significant (p<0.01) effects of dose, genotype, and their interaction on most parameters measured. The probit analysis yielded LD50 values of 118 Gy for GT-1, 112 Gy for GT-2, and 128 Gy for GT-3, confirming GT-3 as the most radiation-tolerant genotype. Dose reduction factors decreased progressively from 0.97-0.98 for the 0→25 Gy increment to 0.79-0.81 for the 75→100 Gy increment, indicating accelerating damage rates at higher doses.

Root-to-shoot ratio increased consistently with irradiation dose in all genotypes, from 0.39-0.44 in controls to 0.64-0.71 at 100 Gy. This shift reflects the differential sensitivity of shoot and root meristems to radiation damage, with shoot apical meristems exhibiting greater susceptibility due to their higher mitotic indices and more rapid cell division rates.

Discussion

The dose-response relationships established in this research provide quantitative parameters essential for the rational design of gamma irradiation protocols in tomato mutation breeding. The progressive decline in germination percentage with increasing dose follows the classic sigmoidal doseresponse model, while the variation in LD50 values across genotypes confirms the necessity of genotype-specific dose calibration in mutagenesis programs. The LD50 values of 112-128 Gy determined in this investigation fall within the range reported for tomato by several previous researchers, though direct comparisons are complicated by differences in seed moisture content, dose rate, and environmental conditions during germination assessment. The finding that GT-3, characterized by late maturity and vigorous growth, exhibited the highest LD50 and greatest radiation tolerance is consistent with the observation that genotypes with larger genome sizes or more efficient DNA repair mechanisms tend to display greater resistance to ionizing radiation. The increasing root-to-shoot ratio with irradiation dose represents a consistent and reproducible phenomenon that has mechanistic implications. Root apical meristems typically maintain lower mitotic indices than shoot meristems and possess more quiescent center cells that can serve as replacement initials following radiation damage. Additionally, the root meristem has been shown to possess higher concentrations of antioxidant enzymes including superoxide dismutase and catalase, which may confer cellular radioprotective mechanisms. GT-3, the most tolerant genotype, exhibited characteristics consistent with enhanced DNA damage repair capacity, including higher baseline seedling vigor, larger seed size, and greater seed dry weight. Previous molecular investigations have linked differential radiosensitivity to polymorphisms in DNA repair genes, particularly those involved in non-homologous end joining and base excision repair pathways. Characterization of these molecular determinants in the genotypes used in this research would provide deeper mechanistic understanding of genotype-dependent radiation responses and potentially identify molecular markers for radiosensitivity prediction.

Conclusion

The present research was designed to establish precise doseresponse relationships for gamma irradiation effects on seed germination and early seedling vigor in three tomato genotypes. The investigation accomplished its primary objective by generating comprehensive dose-response curves and derived parameters including LD50 values (112-128 Gy), dose reduction factors (mean 0.88-0.90 per 25 Gy increment), and GR50 values (88-102 Gy) that provide a quantitative foundation for irradiation protocol design. The practical significance of these findings extends to the recommended dose range of 0.3-0.5 × LD50, which translates to approximately 34-64 Gy for the most sensitive genotype and 38-64 Gy for the most tolerant genotype.

The key findings included the progressive and predictable decline in germination percentage with increasing irradiation dose, the consistent increase in mean germination time reflecting radiation-induced damage to germination machinery, the differential sensitivity of shoot and root growth as evidenced by increasing root-to-shoot ratios, and the significant genotypic variation in radiosensitivity with GT-3 displaying superior radiation tolerance. These doseresponse parameters provide direct practical value for mutation breeding programs by enabling evidence-based dose selection tailored to specific genotypic backgrounds. Future research directions should include molecular characterization of the genetic determinants underlying differential radiosensitivity, evaluation of whether radiosensitivity at the germination stage correlates with mutation frequency at maturity, extension of dose-response analysis to M2 populations to assess heritability of radiation-induced damage, and integration of dose-response data with genomic selection approaches for enhanced efficiency in radiation-based tomato improvement.

Acknowledgement

AcknowledgementsThe Korea Atomic Energy Research Institute is gratefully acknowledged for providing irradiation facilities and dosimetric services essential for the accurate implementation of radiation treatments. The Daejeon Institute of Plant Biotechnology is recognized for laboratory infrastructure and institutional support throughout the research period. The contributions of all laboratory personnel who assisted in seed processing, germination monitoring, and data recording are sincerely valued.

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