Cadmium (Cd) is a trace element found in the earth’s crust and is considered a natural pollutant in agricultural soils. In the aqueous phase of the soil, Cd is easily absorbed by plants and bioaccumulated in different tissues, which makes it easier for it to enter the food chain (Zhao et al., 2020). The soil-to-plant transfer rate of Cd in cocoa cultivation is greater than 1 (Oliva et al., 2020), which leads to bean Cd concentration above 10 mg kg⁻¹, which is a serious concern for chocolate production (Argüello et al., 2019). Consumed Cd is harmful is known to cause serious health issues, such as cardiovascular disease, bone disorders, renal dysfunction, and DNA damage and, consequently, cause cancer (Davidova et al., 2024).

The European Union (EU) has set a maximum permissible limit of 0.80 mg kg⁻¹ of Cd in cocoa by-products to reduce risks to its citizens (Florida Rofner, 2021). However, countries like the Russian Federation, New Zealand, and Australia have established more stringent restrictions of 0.50 mg kg⁻¹ of Cd in chocolate products (Meter et al., 2019). Currently, in Ecuador, Peru, and Colombia there is no set limit for Cd in cocoa beans, although high levels have been found in cocoa derivatives (Argüello et al., 2019; Bravo et al., 2014; Chávez et al., 2015; Florida et al., 2018; Mite et al., 2010). Ecuador’s El Oro, Manabí, and Esmeraldas provinces are the most affected by high levels of Cd contamination in cocoa-growing soils, reaching critical values ≥ 2 mg kg⁻¹ (Argüello et al., 2019; Mite et al., 2010). Ecuador’s coastal regions rely heavily on cocoa for high-value chocolate production, but increased Cd contents could hinder global trade, especially under EU rules (Morales-Rodriguez et al., 2025).

Cocoa plants are affected by heavy metals like Cd, which disrupt various biological functions such as metabolism, leaf gas exchange, chlorophyll synthesis, cell division, electron transfer rate, and photochemical efficiency in the light reactions of photosynthesis (Haider et al., 2021; Borjas-Ventura et al., 2022; Vera Pinargote et al., 2025). Plants lacking Cd tolerance experience higher toxicity, leading to decreased leaf water content, reduced stomatal conductance (g_s ), and increased reactive oxygen species production, necrosis, and cell damage. CCN-51 seedlings exposed to different Cd concentration showed altered mineral nutrient absorption, chlorophyll content, photosynthetic apparatus damage and reduced quantum yield, and the expression of the psbA gene was also disrupted, biomembrane rupture occurring in root and leaf cells (Pereira de Araújo et al., 2017).

It has been recently reported that the cultivation of the BN-34 genotype grafted onto the CCN-51 rootstock shows a high tolerance to Cd, which was associated with a high activity of ascorbate peroxidase, a protein that removes free radicals from plant cells, suggesting that some combinations of cocoa scions and rootstocks may be used to mitigate Cd toxicity (Almeida et al., 2023). Also, the EET-62 genotype showed strong tolerance to Cd content at 6 and 12 mg kg⁻¹; while PA-46 and IMC-67 genotypes had lower Cd accumulation, suggesting their potential use to limit Cd buildup in cocoa tissues (Galvis et al., 2023). Juvenile cocoa plants grown in soils with high Cd levels or combined Mn + Cd levels exhibit impaired photosynthesis, oxidative stress, metabolic alterations, and increased Cd absorption, transport, and accumulation in roots and leaves (Barroso et al., 2023).

Genetic differences in Cd tolerance were also noted among cocoa rootstocks, with the EET-103 genotype exhibiting lower Cd extraction and accumulation capacity compared to CCN-51 (Reyes-Perez et al., 2023a). Genetic variations in cocoa hybrids during seedling growth exhibit differences in Cd partitioning, biomass accumulation, and photosynthetic efficiency, suggesting that cocoa responses to Cd stress are primarily determined by genetic variations (Borjas-Ventura et al., 2022; Vera Pinargote et al., 2025). Previous research findings highlight the role played by genetic enhancement programs in obtaining genotypes that accumulate less Cd and achieve better physiological performance, growth, and development of cocoa plants. Agronomic strategies aim to reduce Cd transfer rates by applying organic and inorganic amendments to precipitate or adsorb Cd, decreasing its bioavailability in soil solution, and leveraging interspecific variations in Cd phytoextraction and accumulation among cocoa genotypes (Meter et al., 2019; Engbersen et al., 2019).

However, limited documentation exists on the Cd absorption capacity of commercial cocoa genotypes from Ecuador. We hypothesize that: 1. The genotypic variation among the cocoa genotypes studied will result in significant differences in physiological and agronomic characteristics, and 2. there will be significant differences among the genotypes of cocoa in Cd absorption, transfer, and bioaccumulation. Therefore, this study aimed to evaluate the physiological characteristics (SPAD values, gas exchange, macronutrient concentrations in the shoot, and Cd bioaccumulation) and biomass distribution in seedlings of seven cocoa genotypes (CCN-51, EET-103, IMC-67, POUND-12, EET-399, EET-95, and EET-400) cultivated in greenhouse conditions.

There were no significant differences in soil pH and EC among the genotypes. However, all genotypes exhibited strongly acidic soil pH values (≤5.5) and high EC levels (6 dS m⁻¹) ( Figure 1 ).

Chlorophyll concentrations, measured at 40, 55, and 80 DAS, showed no statistically significant differences between genotypes ( Figure 2 ). Nevertheless, a significant reduction in SPAD units, amounting to 31.5%, was observed at 80 DAS.ç

In terms of leaf gas exchange, no significant differences were found between genotypes at 40 and 80 DAS, except for transpiration, which was statistically significant at 80 DAS. The EET-103 genotype showed the highest E at 0.39 mmol m⁻² s⁻¹ ( Figure 3 ). At 80 DAS, a reduction of 94% in A, 37% in E, 45% in g_s , and 71% in WUE was observed, while C_i increased by 61%.

There were no significant differences between cocoa genotypes in the production of dry biomass in shoots and roots ( Figure 4 ). However, for aerial dry matter, IMC-67 produced 5% more biomass than CCN-51, while the clones POUND-12, EET-103, EET-399, EET-95, and EET-400 produced 36%, 27%, 22%, 25%, and 19% less biomass than CCN-51, respectively.

Similarly, in root dry weight, IMC-67 exhibited 5% higher biomass compared to CCN-51. In contrast, the POUND-12 and EET-400 genotypes produced 29% less biomass than CCN-51, followed by EET-103 (14% less), EET-399 (10% less), and EET-95 (5% less).

Macronutrient concentrations in the shoot ( Figure 5 ) showed statistically significant differences only for N, P, and S. Among the genotypes, the highest concentrations were observed in CCN-51 for N (6.1 dag kg⁻¹), EET-399 for P (0.54 dag kg⁻¹), and IMC-67 for S (0.61 dag kg⁻¹).

Cocoa genotypes did not exhibit significant differences in Cd absorption and translocation efficiencies or Cd content in shoots, except for root Cd accumulation, which was statistically significant ( Figure 6 ). Among the genotypes, EET-95, POUND-12, and EET-399 absorbed 26%, 24%, and 22% less Cd, respectively, compared to CCN-51, the control and the genotype with the highest Cd absorption ( Figure 6A ). Cadmium uptake efficiency ranged from 68.7 to 79.7 µg Cd g^-1 of dry root matter, with the lowest value found in EET-95 and the highest average in CCN-51, respectively.

In Cd translocation, values ranged from 5.8 to 9.5, with the lowest average obtained in EET-399 and the highest in CCN-51, respectively. In terms of Cd translocation efficiency from roots to shoots, EET-399, EET-103, and EET-95 exhibited 39%, 23%, and 17% lower translocation rates, respectively, than CCN-51 ( Figure 6B ). Additionally, the POUND-12 genotype bioaccumulated 49% less Cd in the shoot compared to CCN-51, followed by EET-399 (39%) and EET-400 (32%).

The study revealed no significant differences among the evaluated cocoa genotypes in physiological variables or biomass distribution, thereby rejecting Hypothesis 1. In contrast, significant genetic differences were evident in Cd absorption efficiency, translocation, and content, supporting Hypothesis 2. Notably, POUND-12 genotype producing the least biomass and exhibited reduced Cd accumulation in shoots. This genetic effect in Cd bioaccumulation aligns with findings by Galvis et al. (2023) and Reyes-Pérez et al. (2023a), who identified similar trends in genotypes with lower biomass production, such as PA-46 and EET-103, respectively.

At the end of the experiment, chlorophyll reduction correlated with decreased leaf gas exchange. This was likely due to salt stress induced by high soil salinity (6 dS m⁻¹). A similar response was reported by Reyes-Pérez et al. (2023a) under high EC conditions (5 dS m⁻¹), which led to reduced chlorophyll (90 DAS), and gas exchange (80 DAS), along with reddish-brown leaf margins. This leaf margin color was also observed in our study. Comparable reductions in A, g_s , E have also been observed in juvenile cocoa plants under water stress and Cd stress (Ortiz-Álvarez et al., 2023; Pereira de Araújo et al., 2017).

Among physiological parameters, only E at 80 DAS showed significant genetic variation, with EET-103 exhibiting the highest transpiration rate (0.39 mmol m⁻² s⁻¹). In fact, most cocoa genotypes showed low E due to small g_s , probably in response to the salt stress experienced by the cocoa seedlings. Higher transpiration values were reported in cocoa genotypes by Reyes-Pérez et al. (2023a). The physiological performance of the genotypes that were evaluated in this study is not documented in the literature under the same conditions. However, the greater transpiration in IMC-67 observed by Ortiz-Álvarez et al. (2023) under water stress, linked to higher Cd translocation, was not corroborated under the present study. The physiological traits of 18 cocoa genotypes under irrigation effects, found higher gas exchange performance (A, g_s and E) in genotypes such as CP-41, CP-43, and CCN-51 (Araújo et al., 2024).

Dry biomass is a key indicator of plant growth, directly linked to water content and the synthesis of primary metabolites (Huang et al., 2020). In this study, the IMC-67 and CCN-51 genotypes demonstrated the highest accumulation of dry biomass in both shoot and root compared to the other genotypes evaluated (EET-95, EET-399, EET-103, EET-400, and POUND-12). These findings are consistent with those reported by Reyes-Pérez et al. (2023a, 2023b), who observed that CCN-51 produces greater biomass than domestic cocoa genotypes like EET-103 and EET-801.

However, contrasting results were noted in the work of Borjas-Ventura et al. (2022), where POUND-7 outperformed CCN-51 in shoot biomass production. This variation highlights the influence of environmental adaptability and genetic plasticity on biomass outcomes. Studies by Héctor Ardisana et al (2018) and Palacio-López and Rodríguez-López (2007) suggest that biomass variation among genotypes is often driven by their ability to adapt to specific climatic conditions, which, in turn, affects the expression of phenotypic traits such as leaf plasticity.

Although this study was conducted with juvenile plants, the Cd content in the shoot may be related to the findings of Lewis et al. (2018), who observed a 2:1 relationship between Cd concentrations in the leaves (2.15 mg kg⁻¹) and beans with controls (1 mg kg⁻¹) in adult plants of 100 cocoa genotypes from eight genetic groups. This relationship was supported by a significant correlation coefficient (r = 0.61; P < 0.05). Furcal-Beriguete and Torres-Morales (2020) reported a similar pattern, with Cd concentrations of 3.44 mg kg⁻¹ in leaves and 2.31 mg kg⁻¹ in beans, showing a correlation coefficient of r = 0.90. Therefore, regardless of the plant’s age, genetic load, or edaphoclimatic conditions, it is considered that the beans accumulate less than 50% of the Cd found in the leaves.

Ortiz-Álvarez et al. (2023) found that increases in dry matter, biomass output, leaf area or proliferation, and Cd bioaccumulation in terms of content are all linked to Cd accumulation under greenhouse conditions. These authors also showed that when transpiration increases, there is greater Cd accumulation because sap flow carries more Cd to the leaves and shoot (Sterckeman, 2025), but it is diluted in terms of concentration (Ortiz-Álvarez et al., 2023). These explanations are reinforced by the results reported in POUND-12, IMC-67, and CCN-51. The first genotype had lower dry matter and Cd content, and the other two clones were superior in terms of shoot dry matter production and Cd bioaccumulation. In cacao seedlings, was found in CCN-51 had a greater capacity to produce dry matter and absorb nutrients and Cd (Arias-Contreras et al., 2024).

In the present study, there were differences in root Cd bioaccumulation, with the highest accumulation found in the roots of EET-399, IMC-67, and CCN-51 and the lowest allocation in POUND-12. Similarly, significant variations in Cd bioaccumulation were observed in the roots of young plants cultivated in greenhouses; the PA-121 x IMC-67 progeny exhibited the highest accumulation (Fernandez-Paz et al., 2021). Growing in a greenhouse with soil Cd concentrations of 12 mg kg⁻¹, PA-121 was shown to have the maximum Cd allocation in root tissues at 30 and 60 DDS, in comparison to the genotypes ETT-61, EET-62, IMC-60, IMC-67, PA-150, PA-46, SCC-85, and SCC-86 (Galvis et al., 2023).

All cocoa genotypes had Cd translocation > 1, but EET-399, compared to the other clones (EET-103, IMC-67, EET-95, CCN-51, EET-400, and POUND-12) had the lowest translocation. This result is consistent with the higher Cd accumulation in the roots of this genotype, showing that it restricts Cd translocation to the aerial parts by capturing it in the root system. Reyes-Perez et al. (2023a) also detected a similar phenomenon: the EET-103 clone had higher Cd accumulation in the roots and lower Cd translocation to the shoots, compared to CCN-51, which obtained the opposite response. Also, were found a genotypic variations in Cd accumulation in the roots of five fine aroma cocoa genotypes, detecting the highest amounts of Cd (156.75 mg kg⁻¹) and lower translocation to the leaves in the INDES-38 genotype (Meléndez-Mori et al., 2023).

It was also observed that the Cd content in the shoot was nine times higher than that in the root. Similarly, Castro et al. (2015) found the highest accumulations of Cd in the offspring of two cocoa progenies (CCN-10 x SCA-6 and Catongo x Catongo) at the juvenile stage. In contrast, Oliva et al. (2020) reported that in mature trees, the leaves accumulate 123 times more Cd than the roots. However, other studies involving juvenile plants (Calva Jiménez et al., 2022) and adult trees (Llatance et al., 2018) found that the roots accumulated 4.84 and 3.36 times more Cd than the leaves, respectively. These findings demonstrate that Cd accumulation in cocoa plants varies between organs, influenced by age, genotype, and environmental conditions that affect Cd migration from one organ to another.

Of the seven genotypes studied, CCN-51 exhibited the highest absorption and accumulation of Cd in the offspring. In Ecuador, this genotype is widely used as a rootstock, covering 75% of the planted area (Latin American Center for Rural Development (RISMIP), 2023). These findings support the results of Reyes-Pérez et al. (2023a), who reported that the EET-103 genotype accumulates less Cd than CCN-51. Additionally, due to its resistance to machete sickness, EET-103 was recommended as a standard.

The POUND-12 genotype exhibited the lowest Cd content in both shoot and root. Due to its resistance to C. cacaofunesta, it is proposed as a key genetic rootstock strategy to mitigate Cd in soils with high metal concentrations. Additionally, EET-399 and EET-400 are viable alternatives, as they demonstrate low Cd accumulation, resistance to C. cacaofunesta (Suárez Capello et al., 1993), and, according to Jaimez et al. (2021), tolerance to water stress.

In contrast, Arévalo-Hernández et al. (2021) in Peru reported results that differ from the present study, finding that CCN-51, with a Cd concentration of 1.56 mg kg⁻¹, accumulated less Cd in the shoots compared to EET-400 (18.56 mg kg⁻¹). Despite its susceptibility to root diseases, CCN-51 was included in a list of 11 genotypes recommended as rootstocks for Cd mitigation, alongside AYP-22, PAS-105, UGU-126, ICT-1026, ICT-1087, ICT-1189, ICT-1292, PH-17, ICS-39, and TSH-565. Furthermore, Borjas-Ventura et al. (2022) found in the Central Jungle of Peru that CCN-51 bioaccumulates more Cd in the shoots compared to genotypes like ICS-95, ICS-60, VRAE-99, and POUND-7. These latter genotypes were recommended as standard due to their low Cd accumulation and partial resistance to Verticillium (Braga and Silva, 1989; End et al., 2021).

The highest N concentration in the shoot was detected in CCN-51, consistent with the findings of Reyes-Pérez et al. (2023a), who also reported that CCN-51 exhibited the highest N concentration in the shoot compared to the EET-103 genotype. Similarly, under field conditions, Carrillo et al. (2010) found that CCN-51 accumulated more leaf N compared to national genotypes such as EET-95 and EET-103.

Genetic variation was observed in the extraction and accumulation capacity of Cd in cocoa rootstocks. These results suggest that genotypes with limited Cd absorption could serve as an agronomic strategy for producing cocoa beans in soils contaminated with this metal. In leaf gas exchange, significant differences were found only in transpiration at 80 DAS, with the EET-103 genotype showing the highest average at 0.39 mmol m⁻² s⁻¹. Overall, at 80 DAS, a reduction in A, E, g_s , and WUE was observed, while C_i increased, which were associated to a decrease in chlorophyll content. In this study, the CCN-51 genotype exhibited greater Cd absorption and bioaccumulation in the shoot, while also showing the highest leaf N concentration. On the other hand, EET-95 showed lower Cd absorption. The POUND-12, EET-400, and EET-399 genotypes, with their low Cd extraction and accumulation capacity and resistance to C. cacaofunesta, are proposed as suitable candidates for use as cocoa rootstocks and for introduction into genetic improvement programs aimed at developing Cd-excluding genotypes. The primary research constraint, however, was the use of cocoa seedlings with limited root volume and a brief exposure to Cd. This could have an impact on the results when extending these findings to mature, fruitful plants in the field. Thus, we think that more research in this area in field settings on plantations is very important.