Universidad Zamorano Agricultural Science and Production B.S. in Agricultural Sciences Special Graduation Project Evaluation of Freezing Tolerance in Solanum sitiens I.M. Johnst. Introgression Lines Student Pablo Josue Betancourth Mejia Advisors Deissy Katherine Juyo Rojas. Ph. D. Gregory Martin Vogel. Ph. D. Honduras, september 2025 2 Authorities KEITH L. ANDREWS President a. i. ANA M. MAIER ACOSTA Vice President and Academic Dean CELIA O. TREJO RAMOS Director of Agricultural Science and Production JULIO NAVARRO Secretary General 3 Acknowledgement I want to express my gratitude to Cornell University, particularly to the Vogel Lab, for their ongoing support and for providing me with the opportunity to conduct this research. Their guidance and trust have been essential throughout this process. I would also like to extend my sincere thanks to the collaborators at the Guterman Bioclimatic Lab for their valuable support and willingness to assist during the development of this work. 4 Content Acknowledgement ........................................................................................................................................ 3 List of Tables .................................................................................................................................................. 6 List of Figures ................................................................................................................................................ 7 Appendix Index ............................................................................................................................................. 9 Abstract ....................................................................................................................................................... 10 Resumen ..................................................................................................................................................... 11 Introduction ................................................................................................................................................ 12 Materials and Methods ............................................................................................................................... 16 Experimental Site ........................................................................................................................................ 16 Experimental Units ...................................................................................................................................... 16 Material Preparation ................................................................................................................................... 18 Treatments .................................................................................................................................................. 20 Variables ...................................................................................................................................................... 20 Freezing tolerance ....................................................................................................................................... 20 Experimental Design ................................................................................................................................... 22 Experiment 1. Freezing tolerance in S. sitiens. ........................................................................................... 22 Experiment 2. Validation of freezing tolerance in S. sitiens at early developmental stages ....................... 23 Experiment 3. Evaluation of freezing tolerance in S. sitiens introgression lines (SILs) ............................... 23 Data Analysis ............................................................................................................................................... 26 Results and Discussion ................................................................................................................................ 28 Experiment 1. Freezing tolerance in S. sitiens. ............................................................................................ 28 5 Experiment 2 Validation of Freezing Tolerance in S. sitiens at Early Developmental Stages ...................... 30 Experiment 3 Evaluation of freezing tolerance in S. sitiens introgression lines .......................................... 31 Conclusions ................................................................................................................................................. 39 Recommendations ...................................................................................................................................... 40 References ................................................................................................................................................... 41 Appendixes .................................................................................................................................................. 44 6 List of Tables Table 1 Number of seeds sown per genotype for 26 accessions: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) 18 Table 2 Treatments used in preliminary experiment #1: Evaluation of freezing tolerance in Solanum sitiens at Cornell University, 2025. ......................................................................................................................... 20 Table 3 Treatments used to evaluate freezing tolerance of 2 genotypes: the commercial accession of tomato (Solanum lycopersicum L.) LA4354 and the wild species Solanum sitiens, conducted at Cornell University, 2025 .......................................................................................................................................... 29 Table 4 Average temperature by block during –2 °C for 6-hour freezing treatment in experiment 2: Validation of freezing tolerance in Solanum sitiens at early stages, Cornell University, 2025 .................... 31 Table 5 Average recorded temperatures during –4 °C for 6-hour freezing treatment over two days in experiment 2: validation of freezing tolerance in Solanum sitiens at early stages, Cornell University, 2025 .................................................................................................................................................................... 32 Table 6 Summary statistics of the linear model assessing freezing damage in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), using the predictors line, day, block, and the day × block interaction, fitted in R ..................................................................................................................................................... 33 Table 7 Analysis of variance (ANOVA) to assess significance of predictors (line, day, block, and day × block interaction) in the linear model assessing freezing damage in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) ............................................................................................................................ 34 Table 8 Estimated differences in freezing damage response of the 26 genotypes included in experiment 3: evaluation of freezing tolerance in 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), Cornell University, 2025 .. 35 7 List of Figures Figure 1 Guterman Bioclimatic Laboratory, Cornell University, Ithaca; NY, 2025 ...................................... 16 Figure 2 Developmental timeline of Solanum sitiens over an 8-week growth period following pre-sowing 4.2% chlorine seed treatment, conducted at Cornell University in 2025 ................................................... 17 Figure 3 Comparison of the experimental units left 7-week Solanum sitiens vs. right 5-week remaining 25 genotypes .................................................................................................................................................... 18 Figure 4 Ordinal scale developed through the study used to evaluate freezing tolerance in a subset of 26 genotypes: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) ..................................................................... 21 Figure 5 Comparison of freezing damage after treatments at two timeframes: 10 minutes and 1 day, in the reference tomato line LA4354 at approximately -4°C for 6 hours at Cornell University, 2025 ................... 22 Figure 6 Block arrangement used in experiment 3 to evaluate freezing tolerance in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), conducted at Cornell University in 2025 in chamber #62 .......... 24 Figure 7 Plot arrangement in potholders used to evaluate freezing tolerance in 26 genotypes of tomato: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), with one replication per block, conducted in chamber #62 at Cornell University, 2025 .............................................................................................................................. 24 Figure 8 Block distribution used in treatment 2 to evaluate freezing tolerance in 26 genotypes of tomato: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), with one replication per block, conducted in chamber #62 at Cornell University, 2025 .............................................................................................................................. 26 8 Figure 9 Temperature tracking in the 0°C, -2°C, and -4°C treatments during experiment 1 on freezing tolerance in Solanum sitiens at Cornell University, 2025 ............................................................................ 28 Figure 10 Temperature tracking -2°C for 6 hours treatment: Validation of freezing tolerance in Solanum sitiens at early developmental stages (Experiment 2), Cornell University, 2025 ........................................ 30 Figure 11 Boxplots comparing freezing tolerance among 26 genotypes in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), under the -4 °C for 6 hours treatment (actual average temperature: - 2 °C) ............................................................................................................................................................. 36 Figure 12 Heat map of adjusted p-values for pairwise comparisons in freezing tolerance in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) ..................................................................... 37 Figure 13 Q–Q plot of residuals from the linear model assessing freezing damage in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) ..................................................................................................... 38 9 Appendix Index Appendix A Chamber #62.......................................................................................................................... 44 Appendix B Chlorine treatment for S. sitiens ............................................................................................ 45 Appendix C Experimental units for experiment 3 ..................................................................................... 46 Appendix D S. Sitiens ................................................................................................................................. 47 10 Abstract Tomato (Solanum lycopersicum L.) is susceptible to thermal stress, both at low and high temperatures, which negatively impacts growth, reproduction, and yield. To improve tolerance to abiotic stresses, breeding programs often incorporate traits from wild relatives. In this context, Solanum sitiens is considered a potential source of freezing tolerance. This study evaluated freezing tolerance in S. sitiens introgression lines (SILs). Preliminary experiments compared the response of S. sitiens with the tomato commercial accession LA4354. Young plants of both genotypes were exposed to 0, –2, and –4 °C for 4 and 6 hours. S. sitiens survived all treatments, showing a consistent level of tolerance. Subsequently, 26 genotypes were evaluated: 24 SILs, S. sitiens as tolerant control, and LA4354 as a susceptible control. Four replicates per genotype were used. Experimental units consisted of 5-week-old SIL and LA4354 plants, and 7-week-old S. sitiens plants. The trial was conducted using an incomplete block design with two treatments: –2 °C for 6 hours and –4 °C for 6 hours. A scoring scale was developed to assess tolerance within the study population, and data were analyzed using a linear model. The overall analysis detected significant differences among genotypes, with SIL LA5287 exhibiting the highest tolerance at –2 °C. However, pairwise comparisons adjusted with the Benjamini–Hochberg procedure did not reveal statistically significant differences. Keywords: freezing tolerance, introgression lines (ILs), Solanum sitiens, wild relatives. 11 Resumen El tomate (Solanum lycopersicum L.) es altamente sensible al estrés térmico, tanto a bajas como a altas temperaturas, lo que afecta de manera negativa su crecimiento, reproducción y rendimiento. Con el fin de incrementar su tolerancia a estreses abióticos, los programas de mejoramiento genético incorporan rasgos provenientes de especies silvestres emparentadas. En este contexto, Solanum sitiens se considera una fuente potencial de tolerancia al congelamiento. Este estudio evaluó la tolerancia al congelamiento en líneas de introgresión de Solanum sitiens (LIs). Inicialmente, se realizaron experimentos preliminares comparando la respuesta de S. sitiens con la accesión comercial LA4354. Plantas jóvenes de ambos genotipos fueron expuestas a temperaturas de 0, –2 y –4 °C durante 4 y 6 horas. S. sitiens sobrevivió a todos los tratamientos, mostrando un nivel consistente de tolerancia. Posteriormente, se evaluaron 26 genotipos: 24 LIs, S. sitiens como control tolerante y LA4354 como control susceptible. Se emplearon cuatro repeticiones por genotipo. Las unidades experimentales estuvieron conformadas por plantas jóvenes de LIs y LA4354 de 5 semanas de edad, y S. sitiens de 7 semanas. El ensayo se estableció bajo un diseño de bloques incompletos, aplicando dos tratamientos: –2 °C por 6 horas y –4 °C por 6 horas. Se desarrolló una escala de evaluación para clasificar la tolerancia en la población estudiada, y los datos fueron analizados mediante un modelo lineal. El análisis general detectó diferencias significativas entre genotipos, destacando la línea SIL LA5287 como la más tolerante al congelamiento a –2 °C. Sin embargo, las comparaciones por pares ajustadas con el método de Benjamini–Hochberg no revelaron diferencias estadísticamente significativas. Palabras clave: especies silvestres, líneas de introgresión (LIs), Solanum sitiens, tolerancia al congelamiento 12 Introduction In 2015, tomato (Solanum lycopersicum L.) production generated around $2.6 billion in farm gate value in the United States (Guan et al., 2018). Over the past decade, it has consistently been the most extensively cultivated and consumed vegetable crop worldwide (Kasimatis et al., 2025). Given its substantial economic significance and its critical contribution to global food and nutritional security, tomato remains a strategic model species in plant science and breeding programs, with considerable potential for genetic improvement. Several factors influence the performance of crops. Both biotic and abiotic factors represent significant limitations in the achievable potential for many crops. Tomatoes are sensitive to various abiotic stresses, such as heat, cold, and drought (Zhou et al., 2023). Tomatoes have an optimal temperature range between 18 °C and 29 °C; temperatures above or below this threshold negatively affect their performance and overall productivity (Arshad et al., 2024). Since tomatoes have a subtropical origin, their production is suboptimal in many growing areas due to unfavorable environmental conditions caused by abiotic factors, such as high and low temperatures, drought, among others (Bai & Lindhout, 2007). Low temperatures affect different aspects of plants; they cause cold-induced tissue damage, which impairs cell survival, cell division, photosynthesis, and water transport, resulting in negative impacts on plant growth and reduced yields (Hussain et al., 2023). Cold stress has adverse effects on the growth and development of plants, affecting their spatial distribution and agricultural productivity (Lv et al., 2018). Low-temperature conditions, whether chilling (0–15 °C) or freezing (<0 °C), severely affect plant growth and development, impacting plant physiology, biochemistry, and molecular processes (Raza et al., 2023). Frost-related losses are not limited to specific regions or species; instead, they are a global phenomenon (Jahed et al., 2023). However, this challenge is more prevalent in regions with harsh winters, leading to shorter market windows and increased competition. 13 Many temperate crops, including tobacco, tomatoes, potatoes, corn, as well as certain ornamentals, are considered “very tender” or “half-hardy” and suffer from freezing damage between 0 and -4°C (Bredow & Walker, 2017). These crops face significant challenges when exposed to suboptimal low temperature conditions due to their limited or nonexistent ability to tolerate cold temperatures. Membranes are very sensitive to environmental changes (Zheng et al., 2016). Exposure to subzero temperatures causes extracellular ice formation in plants, drawing water out of cells and leading to cell dehydration, and contraction this results in membrane damage, electrolyte leakage, and lipid phase changes, intensifying the deleterious effects of freezing stress (Ritonga & Chen, 2020). Similarly, cold or freezing conditions cause a metabolic imbalance in the plasma membrane, leading to premature oxygen reactions and excessive accumulation of reactive oxygen species (ROS), which results in oxidative stress (Satyakam et al., 2022). After thawing events, water returns to its original position in the symplast, causing a sudden expansion of the cell, which leads to induced lysis, lamellar-to-hexagonal II phase transitions, and fracture jump lesions (Ruelland et al., 2009). Natural evolution has led to a reduction in cold tolerance in cultivated tomatoes (Solanum lycopersicum L.) (Guo et al., 2024). It is generally believed that domestication and artificial selection have led to genetic erosion in tomatoes, resulting in a decreased tolerance to both biotic and abiotic stresses (Schouten et al., 2019). It is estimated that the genomes of tomato cultivars contain <5 % of the genetic variation found in their wild relatives (Nakazato et al., 2012). Different wild tomato species exhibit varying levels of cold tolerance (Camalle et al., 2024). Wild or native tomato species often exhibit high tolerance to various biotic and abiotic stresses, adapting to harsh environmental conditions (Kapazoglou et al., 2023). This ability is usually explained by the presence of specific physiological and morphological adaptations, as well as certain genes that allow them to thrive under adverse conditions, Solanum sitiens have been observed to grow and develop in 14 unfavorable environmental settings, suggesting the ability of wild species to tolerate conditions of high salinity, drought and low temperatures (Albrecht et al., 2010). S. sitiens is a self-incompatible wild relative of tomato, characterized by salt and drought- resistance traits, with the potential to contribute through breeding programs to crop improvement in cultivated tomatoes (Molitor et al., 2021). S. sitiens grows in some of the driest parts of the hyper-arid Atacama Desert of northern Chile, at elevations of ~2 500–3 500 m, on slopes of the Cordillera Domeyko and minor cordilleras between the coastal range and the main Andes (Albrecht et al., 2010). The Atacama along the Pacific Coast of Chile and Peru is one of the driest deserts in the world, with reported maximum temperatures of 37.9 °C and minimum temperatures dropping as low as −5.7 °C (McKay et al., 2003). Conditions that, for most Solanaceae species, represent an unfavorable environment for their optimal development. Considering the climatic conditions present in the region where S. sitiens were found, it is expected that the species possesses genes and adaptations that allow it to tolerate freezing stress, suggesting a genetic resource of high importance for its relatives, to develop freezing-tolerant varieties. Cold tolerance refers to the ability of plants to endure seasonal low but non-freezing temperatures (0–15 °C), while freezing tolerance indicates the ability of plants to survive at subzero (< 0 °C) temperatures (Adhikari et al., 2022). Freezing tolerance in plants is achieved through the regulation of ice formation; specifically, by promoting extracellular ice accumulation in the apoplast and xylem while simultaneously preventing lethal intracellular ice formation (Ambroise et al., 2020). S. sitiens are allogamous and self-incompatible, which makes them likely to be highly heterozygous, making genome assembly more challenging than for inbred species (Molitor et al., 2021). Due to the genetic distance between the wild relative S. sitiens and S. lycopersicum, reproductive barriers prevent their hybridization. Specialized techniques such as embryo rescue and the use of bridging lines have enabled the development of introgression lines (ILs) between S. sitiens and S. lycopersicum L. (SIL). The SIL library consists of 56 overlapping introgressions, together representing approximately 93% of S. 15 sitiens genome: 65% in homozygous ILs and 28% in heterozygous SILs. Each IL contains a single S. sitiens chromosome segment, in the genetic background of cv. NC 84173, a fresh market inbred line (Chetelat et al., 2019). The objective of the study was to evaluate freezing tolerance in a subset of S. sitiens introgression lines (SILs). A total of 24 SILs were assessed under subzero temperatures to determine their performance under freezing stress and to identify the most promising lines for future experiments, guiding the development of freezing-tolerant tomatoes. 16 Materials and Methods Experimental Site All experiments presented were conducted at Cornell University, in the Guterman Bioclimatic Laboratory, located at 105 Caldwell Rd, Ithaca, NY 14850, USA (Figure 1). Experiment #1 was conducted in chamber #61, later, due to operating problems with the freezer, we changed to chamber #62. Experimental Units The study consisted of 208 experimental units, comprising 26 different genotypes. It included 24 introgression lines of S. sitiens (SILs), corresponding to accessions LA5243, LA5246, LA5247, LA5252, LA5254, LA5256, LA5257, LA5258, LA5259, LA5263, LA5264, LA5265, LA5270, LA5272, LA5275, LA5276, LA5277, LA5282, LA5287, LA5289, LA5290, LA5295, LA5297, and LA5298. In addition, S. sitiens were included as tolerant control and LA4354 as susceptible control. The experimental units used corresponded to young plants of the 26 genotypes. Seven-week-old plants of the wild species S. sitiens were used; in contrast, five-week-old plants were used for the remaining 25 genotypes. These age differences were established based on what was previously reported by the Vogel Lab team at Cornell University, which indicated that S. sitiens seeds had low germination Figure 1 Guterman Bioclimatic Laboratory, Cornell University, Ithaca; NY, 2025 17 percentages. In addition, seeds and seedlings took more time to germinate and develop compared to other accessions. Considering what was previously reported by the Vogel Lab and the lack of information about wild species S. sitiens and its development cycle, an initial timeline was created to show the development and approximate timing of these events (Figure 2). S. sitiens indeed showed slower development. Wild species tend to grow more slowly than domesticated ones because they assign more resources to survival rather than rapid development. (Tellier et al., 2011). During the monitoring, it was observed that this characteristic was more pronounced in the first weeks of development (1–3 weeks), corresponding to the initial stages (germination, emergence, and cotyledon development). The seeds took about 1–2 weeks to germinate and subsequently emerge from the substrate. Indicating slow development compared to the other accessions, which took about 1 week to germinate and emerge. In slender nightshades such as Solanum nigrescens, the hardness of the seed testa acts as a physical barrier to imbibition, delaying germination and serving as a survival strategy in wild ecosystems (Ramírez-Olvera & Sandoval-Villa, 2023). It was observed that once the plants reached stages of 6–8 weeks, it elongated and produced many shoots at a faster rate. experimental units with similar conditions were obtained, showing comparable height and foliage or plant biomass according to observations (Figure 3). Figure 2 Developmental timeline of Solanum sitiens over an 8-week growth period following pre-sowing 4.2% chlorine seed treatment, conducted at Cornell University in 2025 18 Figure 3 Comparison of the experimental units left 7-week Solanum sitiens vs. right 5-week remaining 25 genotypes Material Preparation Seeds of LA4354 and the 24 SILs were sown in 72-cell (6x12) trays using Cornell Mix substrate. For S. sitiens, seeds were pretreated with a 4.2% chlorine solution for 30 minutes to improve germination. Since it has been reported that S. cheesmaniae, S. galapagense, S. ochranthum, S. juglandifolium, S. lycopersicoides and S. sitiens, will not germinate at appreciable rates without bleach treatment (final concentration ~2.7% sodium hypochlorite) for 30 minutes, or 30 mins at full strength (Tomato Genetics Resource Center [University of California, Davis], 2023). The number of seeds sown per genotype was adjusted based on their observed germination rate recorded by Cornell and by adding 1 more seed (Table 1). Table 1 Number of seeds sown per genotype for 26 accessions: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) Lot Accessio n Germination Rate plants Needed Adjusted GR Seeds Sown +1 Plants Obtained 24-2-009-1 LA5243 50% 8 16 17 8 24-2-010-1 LA5246 50% 8 16 17 6 SS24_2_48 LA5247 33% 8 24 25 8 24-2-012-1 LA5252 83% 8 10 11 8 19 Lot Accessio n Germination Rate plants Needed Adjusted GR Seeds Sown +1 Plants Obtained SS24_2_43 LA5254 8% 8 96 97 6 SS24_2_59 LA5256 50% 8 16 17 7 Lot Accessio n Germination Rate plants Needed Adjusted GR Seeds Sown +1 Plants Obtained SS24_2_52 LA5257 83% 8 10 11 6 SIL24_34 LA5258 92% 8 9 10 8 24-2-018-1 LA5259 58% 8 14 15 8 SIL24_31 LA5263 75% 8 11 12 8 24-2-021-1 LA5264 92% 8 9 10 8 24-2-023-1 LA5265 75% 8 11 12 6 24-2-025-1 LA5270 67% 8 12 13 8 24-2-026-1 LA5272 92% 8 9 10 8 24-2-028-1 LA5275 33% 8 24 25 8 SIL24_48 LA5276 67% 8 12 13 8 24-2-030-1 LA5277 100% 8 8 9 7 24-2-031-1 LA5282 13% 8 64 65 8 24-2-033-1 LA5287 67% 8 12 13 8 24-2-034-1 LA5289 50% 8 16 17 8 24-2-035-1 LA5290 75% 8 11 12 7 SS24_2_02 LA5295 50% 8 16 17 6 24-2-037-1 LA5297 67% 8 12 13 7 SS24_2_46 LA5298 28% 8 29 30 8 2-004-15 LA4354 90% 8 9 10 8 24-2-001- 25 S. sitiens - 8 - 17 8 Note. Lot = indicates the source from which the accessions were obtained; Germination Rate = germination percentages estimated by Cornell University based on prior viability testing ; Adjusted GR = number of seeds to sow in the experiment after adjusting for germination rate to ensure the target number of viable plants is reached; Seeds Sown + 1 = number of seeds sown after applying the Adjusted GR plus one extra seed as a buffer. All plants were grown in the "137" greenhouse at the Guterman Bioclimatic Lab, Cornell University. Greenhouse temperature controls targeted 24 °C during the day and 19 °C at night. Supplemental lighting was used to provide a 13-hour photoperiod, and Irrigation was carried out as needed, including the application of Jack Mix 15-15-15. Subsequently, all genotypes were planted in 4.5-inch pots and filled with Cornell mix substrate. 20 Treatments Two treatments were applied to the experimental units. The treatments involved two factors: temperature and exposure time. The first treatment consisted of −2 °C for 6 hours, and the second of −4 °C for 6 hours. The plants of the different genotypes were exposed to the approximate average temperatures for a set period. The treatments were established taking as reference the ones used in experiment 1 (Table 2). Table 2 Treatments used in preliminary experiment #1: Evaluation of freezing tolerance in Solanum sitiens at Cornell University, 2025. Treatment Genotype Experimental units 0 °C / 4 h S. sitiens 1 0 °C / 4 h LA4354 5 0 °C / 6 h S. sitiens 1 0 °C / 6 h LA4354 5 –2 °C / 4 h S. sitiens 1 –2 °C / 4 h LA4354 5 –2 °C / 6 h S. sitiens 1 –2 °C / 6 h LA4354 5 –4 °C / 4 h S. sitiens 1 –4 °C / 4 h LA4354 5 –4 °C / 6 h S. sitiens 1 –4 °C / 6 h LA4354 5 Variables Freezing tolerance To assess freezing tolerance, an ordinal damage scale was developed based on phenotypic characterization throughout the study. The scale ranged from 0 (indicating the highest level of tolerance) to 5 (indicating little or no tolerance) (Figure 4). After the freezing treatments, plants were monitored, and visible damage was assessed one day later, when freezing stress symptoms were most apparent (Figure 5). Observations were then categorized according to damage severity, forming the basis of the scale. 21 The evaluated genotypes showed different responses according to their freezing tolerance. Common morphological symptoms include stem cracking, poor or no germination, lack of vigor, metabolite leakage, leaf wilting, yellowing, necrosis, and delay in regeneration (Adhikari et al., 2021). Categories were defined based on the observed damage: 0, 1, 2, 3, 4, and 5. Plants with higher levels of freezing tolerance were mainly classified into the first two categories (0 and 1), where category zero (0) was characterized by no visible symptoms, and category 1 showed small, localized areas of affected tissue, with leaf edge curling as the primary symptom, present in only a few leaves. Categories 2 and 3 corresponded to genotypes with moderate freezing tolerance. The observed damage was characterized by sections of leaves being wholly or partially curled, along with leaf wilting. Categories 4 and 5 corresponded to genotypes with low tolerance. Category 4 was characterized by evident wilting in most Figure 4 Ordinal scale developed through the study used to evaluate freezing tolerance in a subset of 26 genotypes: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) 22 of the foliage, along with symptoms curling. Category 5 damage was identified by tissue collapse, with stems totally or partially fallen due to dehydration. Figure 5 Comparison of freezing damage after treatments at two timeframes: 10 minutes and 1 day, in the reference tomato line LA4354 at approximately -4°C for 6 hours at Cornell University, 2025 } Experimental Design Experiment 1. Freezing tolerance in S. sitiens. The experiment was conducted in growth chamber #61 to evaluate freezing tolerance in S. sitiens. A completely randomized design was used, involving two factors: temperature and exposure time. The temperature factor included three levels (0, -2, and -4 °C), while the exposure factor had two levels (4 and 6 hours). Two genotypes were evaluated: S. sitiens and LA4354. For each treatment, five replicates of LA4354 and one replicate of S. sitiens were used. The limited availability of S. sitiens seeds restricted the number of replicates for this genotype. The S. sitiens used in the experiment were 14 weeks old, whereas the LA4354 genotypes were 5 weeks old. This discrepancy in the age of the experimental units was primarily due to the limited availability of S. sitiens seeds and resources. Experimental units were randomly 23 assigned to positions within the chamber. The response of the genotypes to freezing stress was assessed at two-time intervals (10 minutes and 1 day), as the effects of freezing became more apparent several minutes after removal from the chamber. In this preliminary experiment, plant survival was determined based on visual estimated evaluation and expert assessment. Experiment 2. Validation of freezing tolerance in S. sitiens at early developmental stages Experiment 2 was conducted to evaluate whether the younger developmental stages of S. sitiens also exhibit freezing tolerance, as experiment 1 involved mature S. sitiens plants, in contrast to the young plants of tomato accession LA4354. This decision was motivated by findings in Arabidopsis thaliana, where freezing tolerance was shown to increase with plant age during the vegetative stage, suggesting that developmental stage plays a key role in the cold stress response (Zhao et al., 2022). The two genotypes previously evaluated, S. sitiens and the line LA4354, were again assessed. Five replicates of LA4354 and three S. sitiens were used. The treatment applied was -2°C for 6 hours. The experiment was conducted in chamber #62, which was pre-set the day prior to the experiment to reach and stabilize the target temperature. Experimental units were randomly distributed (CRD) within the chamber. Once the exposure period ended, the plants were returned to the greenhouse for evaluation. The evaluation conducted was purely visual, with no quantitative data recorded. Observational insights were gathered, with the purpose of monitoring the behavior of the experimental units prior to the main experiment. The same trends in tolerance and susceptibility observed in Experiment 1 were expected to be replicated. Experiment 3. Evaluation of freezing tolerance in S. sitiens introgression lines (SILs) The freezing tolerance of 24 SIL accessions were evaluated, S. sitiens and LA4354 were included as tolerant and susceptible controls. Due to poor germination and stunted growth observed in some genotypes, an incomplete randomized block design (IBD) was used. The experiment consisted of eight blocks in total, with four blocks assigned to each treatment (Figure 6). 24 Figure 6 Block arrangement used in experiment 3 to evaluate freezing tolerance in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), conducted at Cornell University in 2025 in chamber #62 Each of the 26 genotypes was randomly assigned to a plot, each plot representing a specific position within a plot holder (Figure 7). Figure 7 Plot arrangement in potholders used to evaluate freezing tolerance in 26 genotypes of tomato: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), with one replication per block, conducted in chamber #62 at Cornell University, 2025 Chamber, (-2°C) Chamber, (-4°C) Block 1 Block 2 Block 5 Block 6 Block 3 Block 4 Block 7 Block 8 25 Each potholder had 15 available positions. To accommodate all 26 genotypes, two potholders were used per block. As a result, each block included 26 plots, allowing one replication of each genotype per block. The experiment was conducted in chamber #62, with temperature monitored using a thermo- hygrometer Govee device. Temperature readings were recorded every 15 minutes throughout the exposure period to establish the average temperature to which the plants were subjected. This was done because small fluctuations were observed within the chamber, likely caused by the cooling and heating mechanisms used to maintain the set temperature. The experiment was carried out over three days. Initially, it was planned to be conducted in two days, with four blocks per day. However, it was observed that when treated at -2 °C for 6 hours in the positions corresponding to blocks 3 and 4, a phenomenon of dry pots occurred, which, combined with the stress from low temperatures, caused the death of the plants, including the tolerant control. Consequently, for Treatment -4°C for 6 hours, a redistribution was made, and the blocks for this treatment were relocated, using only the positions corresponding to blocks 1 and 2. As a result, Treatment 2 was conducted over two days (Figure 8). 26 Figure 8 Block distribution used in treatment 2 to evaluate freezing tolerance in 26 genotypes of tomato: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), with one replication per block, conducted in chamber #62 at Cornell University, 2025 Data Analysis A linear model was fitted to evaluate the effects of Day, Block, line, on the dependent variable freezing damage using formula: 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷~𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 + 𝐷𝐷𝐷𝐷𝐷𝐷 + 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 + 𝐷𝐷𝐷𝐷𝐷𝐷: 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 [ 1 ] All factors were included as fixed effects; additionally, the interaction between Day and Block was evaluated to account for potential combined effects. The model was fitted using linear regression in R with the command lm (). Model fit and significance were assessed using the summary () function, which provided coefficient estimates, standard errors, t-values, and p-values, as well as overall model statistics, R-squared (R²), adjusted R-squared, F-statistic, and its p-value. An analysis of variance (ANOVA) was performed to assess the significance of the predictors in the model and to identify which factors contributed significantly to the variability in freezing damage among the genotypes. Model assumptions were checked by examining residuals using diagnostic plots and tests (QQ-plots) to ensure normality. Chamber(-4°C), Day 1 Chamber(-4°C), Day 2 Block 5 Block 6 Block 7 Block 8 27 Estimated Marginal Means (EMMs) were calculated using the emmeans package. Pairwise comparisons between lines were conducted with p-values adjusted by the Benjamini-Hochberg (BH) method to control the false discovery rate. 28 Results and Discussion Experiment 1. Freezing tolerance in S. sitiens. In all treatments, an initial increase in temperature was noted. This can be attributed to the time required to position the experimental units inside the chamber, which involved opening the chamber and consequently causing a temporary rise in temperature. This effect was particularly observable during the first two hours of exposure. After approximately this interval, the temperature within the chamber stabilized. The 0 °C treatment for 4 hours had an average temperature of -0.23 °C, with a minimum of -1.1 °C and a maximum of 2.1 °C. The 0°C treatment for 6 hours resulted in an average temperature of -0.37°C, a minimum of -1.3°C, and a maximum of 2.1 °C. The 6-hour treatment includes the data from the 4-hour treatment plus an additional 2 hours. -2 °C treatment for 4 hours had an average temperature of -2.13 °C, a minimum of -3 °C, and a maximum of -0.1 °C. The 6-hour -2 °C treatment showed an average of -2.35 °C, a minimum of -3.1 °C, and a maximum of -0.1 °C. -4 °C treatment for 4 hours resulted in an average temperature of -3.88 °C, with a minimum of -5.2 °C and a maximum of -1.4 °C. The -4 °C treatment for 6 hours showed an average of -4.00 °C, a minimum of -5.2 °C, and a maximum of -1.4 °C (Figure 9). Figure 9 Temperature tracking in the 0°C, -2°C, and -4°C treatments during experiment 1 on freezing tolerance in Solanum sitiens at Cornell University, 2025 -0.6 1 2.1 1.9 1.2 0.4 -0.3-0.2-0.5-0.9-0.7-0.8-0.8-0.9-1.1-0.8-1.1 -0.5-0.8 -1.3 -0.8 -1.3 -0.8 -1.3 -2.8 -1.3 -0.1-0.3 -0.9 -1.6 -2.3 -2.8-2.5 -3 -2.5 -3 -2.5-2.9-2.8-2.8-3.1 -2.5 -3 -2.5-2.8-2.8-2.8 -3 -2.4 -1.4 -2 -2.1 -2.8 -3.3 -3.8-4.2-4.5-4.8 -5 -5.2 -3.9-4.3-4.6-4.9-4.8-5.1 -4.3-4.2-4.5-4.8-5.1 -4.1 -6 -5 -4 -3 -2 -1 0 1 2 3 1 2 3 4 5 6 Te m pe ra tu re (° C) Exposure(Hours) 0°C for 6 hrs 0°C for 4 hrs -2°C for 6 hrs -2°C for 4 hrs -4°C for 6 hrs -4°C for 4 hrs 29 It was found that S. sitiens showed greater tolerance to freezing temperatures unlike the accession LA4354. It was observed that as the temperature decreased and exposure times increased, the damage affected the commercial line more and eventually caused mortality. Treatments with temperature factor of 0 °C caused initial damage in the genotype L4354. This damage occurred in small regions of the leaflets, and the plants eventually recovered. Treatments at a temperature of around -2 °C show more pronounced signs of freezing stress, with 20% mortality at 4 hours and 60% at 6 hours. In contrast, S. sitiens maintained a 100% survival rate at both exposure times, suggesting that the wild species had greater tolerance to freeze. At lower temperatures, approximately -4°C, the results were even more evident. After 4 hours of exposure, the commercial accession LA4354 exhibited an 80% mortality rate, whereas S. sitiens maintained a 100% survival rate. After 6 hours, LA4354 suffered complete mortality (100%), while S. sitiens continued to show its tolerance. (Table 3). Table 3 Treatments used to evaluate freezing tolerance of 2 genotypes: the commercial accession of tomato (Solanum lycopersicum L.) LA4354 and the wild species Solanum sitiens, conducted at Cornell University, 2025 Treatment Accession EU Survived % Survived Died % Died 0 °C / 4 h S. sitiens 1 1 100 % 0 0 % 0 °C / 4 h LA4354 5 5 100 % 0 0 % 0 °C / 6 h S. sitiens 1 1 100 % 0 0 % 0 °C / 6 h LA4354 5 5 100 % 0 0 %  –2 °C / 4 h S. sitiens 1 1 100 % 0 0 %  –2 °C / 4 h LA4354 5 4 80 % 1 20 %  –2 °C / 6 h S. sitiens 1 1 100 % 0 0 %  –2 °C / 6 h LA4354 5 2 40 % 3 60 %  –4 °C / 4 h S. sitiens 1 1 100 % 0 0 %  –4 °C / 4 h LA4354 5 1 20 % 4 80 %  –4 °C / 6 h S. sitiens 1 1 100 % 0 0 %  –4 °C / 6 h LA4354 5 0 0 % 5 100 % Note. EU = experimental units used in each treatment. 30 Plants manage freeze-induced damage through different strategies: cold tolerance and cold avoidance, tolerance mechanisms involve acclimatization to foster gradual accumulation of cold resistance. In contrast, avoidance mechanisms rely on cryoprotectant molecules like potassium ions (K+), proline, glycerol, and antifreeze proteins (AFPs) (Jahed et al., 2023). Some freezing tolerant plants mitigate this risk and effects by expressing ice-binding proteins (IBPs), that adsorb ice crystals and modify their growth (Bredow & Walker, 2017). In the absence of these ice-binding proteins (IBPs), large ice crystals can form in the apoplast, potentially causing mechanical damage to plasma membranes. Lipid composition and lipid metabolism during cold stress influence the degree of cold damage in plants as well as other species. It is estimated that increased unsaturated fatty acids content in the plasma membrane of the acclimated plants possesses more fluidity for stabilization of the membranes (Adhikari et al., 2022). S. sitiens likely possesses one or more of the mechanisms mentioned above. On the other hand, LA4354 seems to have a few ways to deal with the harmful effects of freezing stress. Experiment 2 Validation of Freezing Tolerance in S. sitiens at Early Developmental Stages It was observed that both genotypes survived exposure. S. sitiens showed no apparent visible damage, while LA4354 suffered from small lesions observed on the leaflets. The damage was not severe, likely because the treatment did not reach the target temperature of -2 °C; instead, an average of -1.6 °C, with a higher recorded temperature of -1,8°C (Figure 10). Figure 10 Temperature tracking -2°C for 6 hours treatment: Validation of freezing tolerance in Solanum sitiens at early developmental stages (Experiment 2), Cornell University, 2025 -1.85 -1.8-1.8 -1.75-1.75 -1.6 -1.65-1.65-1.65 -1.6 -1.55 -1.65 -1.55 -1.65 -1.6 -1.7 -1.65 -1.6 -1.65 -1.55 -1.7 -1.55 -1.65 -1.55 -1.9 -1.8 -1.7 -1.6 -1.5 1 2 3 4 5 6 Te m pe ra tu re ( ° C) Exposure (Hours) 31 This explains why the effects were less pronounced compared to the first experiment. Nevertheless, the absence of visible damage in S. sitiens indicates that it still exhibited a degree of freezing tolerance. Plants use avoidance and tolerance strategies to mitigate cold stress, by preventing the formation of ice crystals inside the cell and are primarily associated with structural aspects (Satyakam et al., 2022). Morphological adaptations of S. sitiens to drought have been described: thick, leathery leaves that are small, as well as the ability to regenerate from the base of the stem (Molitor et al., 2021). Morphological adaptations could explain S. sitiens’ ability to tolerate low-temperature stress. Plants respond to stress by reducing their height, limiting both the size and number of leaves, and increasing epidermal thickness (Satyakam et al., 2022). Experiment 3 Evaluation of freezing tolerance in S. sitiens introgression lines Different temperatures were recorded across the blocks during the treatments. It was observed that the blocks included in the -2 °C for 6 hours treatment showed more fluctuation in their calculated average temperatures. Blocks 1 and 4 registered -1.72 °C and -2.4 °C; these blocks were the closest to the target temperature of -2 °C. In contrast, blocks 2 and 3 did not reach that close to the desired temperature, with average temperatures of -1.1°C and -0.39°C (Table 4). Table 4 Average temperature by block during –2 °C for 6-hour freezing treatment in experiment 2: Validation of freezing tolerance in Solanum sitiens at early stages, Cornell University, 2025 Treatment Blocks Temperature (°C) SD -2 °C / 6 hours 1 -1.72 0.759 2 -1.1 0.749 3 -0.39 1.196 4 -2.4 0.731 Note. SD= Standard Deviation. 32 The developed freezing-damage scale proved effectiveness for gathering qualitative data on genotype responses to freezing stress, simplifying both data collection and subsequent analysis. Visual rating scales have been employed to assess frozen injury in vegetative organs, particularly in shoots, which were evaluated based on observable damage symptoms (Centinari et al., 2016). Data analysis showed that Treatment -2°C for 6 hours had irregularities and several outliers. It is suspected that it was likely caused by an issue with the positions of blocks 3 and 4 in the chamber, where some of the plots were affected by a phenomenon of pot dehydration and freezing. It was considered that the presence of air currents or other factors contributed to this phenomenon, since some plots in those positions did not show problems. Due to this, treatment 1 was excluded from the analysis. For treatment -4°C for 6 hours the chamber was unable to reach the target temperature. The distribution of blocks in this treatment showed average temperature values that were closer to the target (set) temperature. Fluctuating temperatures around -2 °C were observed on both days. On Day 1 of the new block distribution, temperatures of -1.48 °C and -1.45 °C were recorded in blocks 5 and 6, respectively. On Day 2, an average temperature of -2.3 °C was recorded in block 7 and -1.9 °C in block 8 (Table 5). Table 5 Average recorded temperatures during –4 °C for 6-hour freezing treatment over two days in experiment 2: validation of freezing tolerance in Solanum sitiens at early stages, Cornell University, 2025 Treatment Day Block Temperature (°C) -4 °C / 6 hours 1 5 -1.48 6 -1.45 2 7 -2.3 8 -1.9 Data analysis revealed greater consistency in the response of genotypes when evaluating only the -4°C for 6 hours treatment. The overall model explained approximately 42.9% of the variability (R² = 0.4294), with an adjusted R² of 0.1836. The overall model was statistically significant (p = 0.033) (Table 6). 33 Table 6 Summary statistics of the linear model assessing freezing damage in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), using the predictors line, day, block, and the day × block interaction, fitted in R Model Summary Multiple R-square 0.4294 Adjusted R-squared 0.1836 F-statistic 1.747 p-value 0.03339 The gap between the R-squared and the adjusted R-squared suggests that not all predictors meaningfully contribute to explaining the variation in the data. The S. sitiens introgression library consists of 56 overlapping introgressions that together represent approximately 93% of the S. sitiens genome: 65% in homozygous ILs and 28% in heterozygous (segregating) (Chetelat et al., 2019). Indicating a source of variation that may not be fully captured by the predictors. The ANOVA performed to assess the impact of predictor effects on the dependent variable showed a statistically significant effect of the factor "Line" (p = 0.02947), indicating that 'Line' influences the plants’ response to freezing damage and that there are significant differences between the lines. The other factors were not significant, with p-values of 0.31804 for Day, 0.50672 for Block, and 0.14027 for the Day: Block interaction (Table 7). 34 Table 7 Analysis of variance (ANOVA) to assess significance of predictors (line, day, block, and day × block interaction) in the linear model assessing freezing damage in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) Fixed effect Df Sum Sq Mean Sq F value Pr(>|t|) Line 25 109.243 4.3697 1.809 0.02947* Day 1 2.446 2.4457 1.0125 0.31804 Block 1 1.077 1.0768 0.4458 0.50672 Day: Block 1 5.384 5.3845 2.2291 0.14027 Residuals 65 157.01 2.4155 Note. Fixed effect = source of variation; Df = degrees of freedom; Sum Sq = sum of squares; Mean Sq = mean squares; F value = ratio of mean square of the effect to the residual mean square; Pr(>|t|) = p-value indicating statistical significance, with values < 0.05 denoted by “*”. Out of the 26 genotypes tested, only two were significantly different from the reference line LA4354. S. sitiens had a p-value of 0.03149, and LA5287 had a p-value of 0.04237 (Table 8) (Figure 11). Significant differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs harbor highly divergent allelic variation (Chetelat et al., 2019). Which explains why some of the lines did not express cold tolerance as expected. Lines such as LA5287 likely possess adaptations or specific mechanisms derived from S. sitiens that help mitigate the adverse effects of freezing stress. A third locus associated with elevated anthocyanin accumulation, atv-3, was mapped to the short arm of chromosome 10 in line LA5287 (Chetelat et al., 2019). It confers high anthocyanin accumulation in stems and leaves. Anthocyanins are typical chilling-induced metabolites with vigorous antioxidant activity and photoprotective capacity (Ye et al., 2025). Under extreme stress conditions, ROS are overproduced, causing oxidative damage to plants. In such conditions, Plants produce anthocyanins after ROS signaling via the transcription of anthocyanin biosynthesis genes, these anthocyanins are then utilized in antioxidant activities by scavenging excess ROS for sustainability (Naing & Kim, 2021). 35 Table 8 Estimated differences in freezing damage response of the 26 genotypes included in experiment 3: evaluation of freezing tolerance in 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), Cornell University, 2025 Estimate Std. Error t value Pr(>|t|) (Intercept) 3.07425 0.93188 3.299 0.00158 ** Estimate Std. Error t value Pr(>|t|) LineLA5243 -1.21759 1.19174 -1.022 0.31071 LineLA5246 0.33333 1.269 0.263 0.79363 LineLA5247 0.03241 1.19174 0.027 0.97839 LineLA5252 0.53241 1.19174 0.447 0.65654 LineLA5254 -2 1.269 -1.576 0.11987 LineLA5256 -0.13194 1.42468 -0.093 0.9265 LineLA5257 0.53241 1.19174 0.447 0.65654 LineLA5258 0.28241 1.19174 0.237 0.81342 LineLA5259 -1.21759 1.19174 -1.022 0.31071 LineLA5263 1.03241 1.19174 0.866 0.38951 LineLA5264 -1.96759 1.19174 -1.651 0.10356 LineLA5265 0.33333 1.269 0.263 0.79363 LineLA5270 -0.71759 1.19174 -0.602 0.54918 LineLA5272 0.03241 1.19174 0.027 0.97839 LineLA5275 0.53241 1.19174 0.447 0.65654 LineLA5276 -0.96759 1.19174 -0.812 0.4198 LineLA5277 -0.71759 1.19174 -0.602 0.54918 LineLA5282 1.28241 1.19174 1.076 0.28587 LineLA5287 -2.46759 1.19174 -2.071 0.04237 * LineLA5289 -0.71759 1.19174 -0.602 0.54918 LineLA5290 -1.33333 1.269 -1.051 0.29729 LineLA5295 -1.33333 1.269 -1.051 0.29729 LineLA5297 -0.96759 1.19174 -0.812 0.4198 LineLA5298 1.03241 1.19174 0.866 0.38951 LineSitiens -3.13194 1.42468 -2.198 0.03149 * DayDay2 0.11538 0.43106 0.268 0.78979 LocationB 0.66186 0.44432 1.49 0.14117 DayDay2: LocationB -0.98113 0.65714 -1.493 0.14027 Note. Intercept = baseline value of Damage when all predictors are at their reference levels (control line, Day = 0, Location = A); Estimate = predicted mean change in Damage for each predictor relative to the baseline; Std. Error = standard deviation of the estimate (precision); t value = test statistic for difference from zero; Pr(>|t|) = two-tailed p-value; * and ** indicate significance levels at p < 0.05 and p < 0.01, respectively. 36 Variation in tolerance levels was observed among the different genotypes. A mean tolerance score of 3 was estimated for the commercial tomato accession or susceptible control, LA4354, indicating moderately severe negative effects on its tissues after exposure to low temperatures. On the other hand, genotypes falling below the line or point marked by LA4354 can be considered more freezing tolerant, as they exhibit lower mean damage than the reference variety. Genotypes such as LA5287 display lower median damage values. Indicating that this line has a degree of tolerance to freezing stress. It is believed that introgression of wild alleles into cultivated backgrounds may result in complex gene interactions. Introgression lines (ILs) carry beneficial QTLs; however, linkage between beneficial genes and deleterious ones can reduce the potential of ILs due to introgression depression (Tripodi et al., 2021). Line LA4354 performed better under freezing stress compared to some SILs, possibly due to the absence of linked deleterious alleles that may be present in certain introgression lines. Figure 11 Boxplots comparing freezing tolerance among 26 genotypes in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354), under the -4 °C for 6 hours treatment (actual average temperature: -2 °C) 37 After applying multiple test corrections, no line pairs were found to differ significantly (Figure 12). The current study is likely underpowered given the limited number of line observations and the relatively large number of pairwise comparisons. Figure 12 Heat map of adjusted p-values for pairwise comparisons in freezing tolerance in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) The observed trends in freezing tolerance among SILs, particularly the superior performance of lines such as LA5287 and S. sitiens, highlight the potential of wild genetic resources in improving abiotic stress tolerance in cultivated tomatoes. However, the lack of statistically significant differences in pairwise comparisons, despite visible phenotypic variation, is likely due to limited statistical power resulting from 38 small sample sizes and the high number of comparisons. In addition, in some of the evaluated genotypes, it was not possible to obtain all four plants per treatment, which is likely related to the introgression from the wild species. Introgression lines (ILs) between Solanum pennellii and the cultivated tomato variety shows that some ILs enhance germination while others reduce it, depending on the specific genomic segment introgressed (Rosental et al., 2016). Freezing tolerance was assessed using an average of 8.3 plants, emphasized that an insufficient number of samples per block or genotype significantly compromises the statistical validity of results (Sanderson et al., 2020). Moreover, while the Benjamini- Hochberg (BH) procedure is less conservative than methods like Bonferroni and effectively controls the false discovery rate (FDR), it can still reduce statistical power in studies with small sample sizes or subtle phenotypic effects (Stephens, 2017). The QQ-plots indicated that the residuals were approximately normally distributed, supporting the validity of the model assumptions (Figure 13). Figure 13 Q–Q plot of residuals from the linear model assessing freezing damage in the study population: 24 introgression lines of Solanum sitiens, one accession of Solanum sitiens, and one commercial tomato line (Solanum lycopersicum L., LA4354) Linear models are usually applied in agronomic studies to analyze both quantitative and qualitative traits due to their simplicity, interpretability, and statistical efficiency. These include normality of residuals, linearity between predictors and response, and homoscedasticity. 39 Conclusions A preliminary timeline of S. sitiens physiological development was established, and ordinal damage scale based on phenotypic characterization was developed and proven effective in evaluating freezing tolerance. These contributions will support the Cornell team in future studies of the wild species S. sitiens and their introgression lines. It was demonstrated in various experiments that the wild species S. sitiens exhibits a degree of tolerance to freezing, being capable of withstanding short exposures of 4–6 hours to temperatures around 0°C and –2°C without showing signs of stress or apparent damage. This suggests that S. sitiens could be a valuable source of genes for improving important freezing tolerance traits in cultivated plants. Some of the introgression lines also showed signs of this freezing tolerance, although to a lower degree, lines such as LA5287 stood out for their performance at temperatures around –2°C during exposures of 6 hours, suggesting that it is a promising line for future studies. On the other hand, some lines, such as LA5282, performed even worse than the susceptible control LA4354, possibly due to depression associated with the introgression of S. sitiens in their genetic background. 40 Recommendations It is advisable to sow S. sitiens two weeks prior to the sowing of other tomato accessions to compensate for its slow start and to ensure the production of experimental material under similar conditions. Increase the number of replicates and repetitions of the experiment to obtain more conclusive data. Conduct experiments focusing on the lines that showed greater tolerance than the reference accession LA4354. Evaluate freezing tolerance at more than one time (multiple or consecutive freezing events). It is recommended to complement this approach with quantitative measurements in future experiments. 41 References Adhikari, L., Baral, R., Paudel, D., Min, D., Makaju, S. O., Poudel, H. P., Acharya, J. P., & Missaoui, A. M. (2022). 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Acta Horticulturae(1372), 1–8. https://doi.org/10.17660/ActaHortic.2023.1372.1 44 Appendixes Appendix A Chamber #62 45 Appendix B Chlorine treatment for S. sitiens 46 Appendix C Experimental units for experiment 3 47 Appendix D S. Sitiens Acknowledgement List of Tables List of Figures Appendix Index Abstract Resumen Introduction Materials and Methods Experimental Site Experimental Units Material Preparation Treatments Variables Experimental Design Experiment 1. Freezing tolerance in S. sitiens. Experiment 2. Validation of freezing tolerance in S. sitiens at early developmental stages Experiment 3. Evaluation of freezing tolerance in S. sitiens introgression lines (SILs) Data Analysis Results and Discussion Experiment 1. Freezing tolerance in S. sitiens. Experiment 2 Validation of Freezing Tolerance in S. sitiens at Early Developmental Stages Experiment 3 Evaluation of freezing tolerance in S. sitiens introgression lines Conclusions Recommendations References Appendixes