Understanding the Tetrazolium Test in Seed Analysis

Written by: Gabriele Vieira; Reviewed by: Olga Kildisheva

The tetrazolium test (TZT) has been developed and refined since the early 20th century, originally designed to assess seed viability. Over time, methodological advances have expanded its application to include the evaluation of seed vigor across various species, including forest species.

Seed viability and seed vigor

Seed viability refers to the ability of a seed to remain alive and capable of germination under ideal conditions. It indicates the presence of metabolically active and structurally intact embryonic tissues capable of developing into a normal seedling. Seed vigor, on the other hand, is a broader concept that encompasses the seed’s potential to germinate rapidly, uniformly, and produce strong seedlings even under suboptimal or stressful environmental conditions. While all vigorous seeds are viable, not all viable seeds are necessarily vigorous. Both attributes are essential for successful seedling establishment and can be evaluated through physiological and biochemical tests, such as the tetrazolium test.

Principles of the test

The TZT indirectly assesses the respiratory activity of the cells that make up seed tissues. It is based on the activity of dehydrogenase enzymes, which catalyze key reactions in mitochondrial respiration—particularly during glycolysis and the Krebs cycle. These enzymes reduce the colorless tetrazolium salt in metabolically active (living) tissues.

When immersed in the colorless tetrazolium solution, the dye penetrates seed tissues and interferes with the reduction processes of the living cells by accepting a hydrogen ion. In its reduced form, the tetrazolium solution becomes a red-colored, stable, non-diffusible substance called triphenyl formazan, or simply formazan.

Tetrazolium reduction reaction, Source: França-Neto, J. B.

Procedures of the test

Seeds are initially preconditioned using moist germination paper (slow moistening) to reach moisture levels that activate mitochondrial respiration. This method is preferred for larger seeds, as it allows tissues to imbibe water without damage and prevents death from anaerobic conditions like full water immersion. For smaller seeds, water immersion is a faster alternative but requires careful timing to avoid tissue damage.

Preconditioning may also include dormancy-breaking treatments. Seeds often need cutting, perforation, or seed coat removal to facilitate water and tetrazolium solution uptake. Sharp tools must be used to avoid damaging seeds and compromising test accuracy.

Tetrazolium solutions at 1% or 0.5% concentrations are common, with pH maintained between 6.5 and 7.5 to ensure proper staining. A 1% solution is prepared by dissolving 10 g of tetrazolium salt in 1 liter of distilled water.

During staining, seeds must be fully submerged and protected from light to prevent unwanted salt reduction. After staining in a light-proof container, seeds are incubated at species-specific temperatures and durations, following established protocols when available. Concentrations can vary if results remain reliable. Finally, seeds are rinsed to remove excess solution.

Tetrazolium test interpretation

The primary objective of the tetrazolium test (TZT) is to distinguish viable seeds from non-viable ones. This distinction is based on staining patterns and tissue integrity, allowing for a clear separation between the two groups. Moreover, it is critical that the technician has a thorough understanding of seed structures, which may vary depending on the species.

A. Embryo structure of tomato/bell pepper seed; B. embryo stained by the tetrazolium solution. (tg) tegument/seed coat; (ct) cotyledons; (en) endosperm; (rh) radicle-hypocotyl axis. Source: França-Neto, J. B.

In color interpretation, faint normal red typically indicates vigorous tissue, intense red signifies deteriorating tissue, and absence of staining denotes dead tissue. Viable seeds are those capable of producing normal seedlings in a germination test under favorable conditions, once dormancy has been overcome. These embryos typically exhibit uniform staining; however, partially stained embryos may also be considered viable if their staining patterns indicate metabolic activity. Necrotic tissues may be present in different regions of these embryos, and the viability classification depends on the size and position of such areas—not necessarily on the intensity of the staining. Tissue firmness is another crucial criterion that must accompany staining patterns to ensure proper identification and classification of viable seeds.

Non-viable seeds, by contrast, do not meet the above criteria. They often display irregular or poorly defined staining and contain flaccid or unstained essential structures. Seeds exhibiting abnormal embryo development—or other defective vital structures—are considered non-viable, regardless of their staining. Empty seeds must also be classified as non-viable. In the case of conifers, seeds with rudimentary embryos are likewise regarded as non-viable.

Another application of the TZT is to assist in categorizing the vigor levels of different seed lots and in estimating their performance under both optimal and stressful field conditions. Vigorous seeds are capable of developing into normal seedlings, whereas non-vigorous seeds are not. The TZT can also be used in conjunction with germination tests to account for potential dormancy-related discrepancies.

Tomato seeds stained by tetrazolium solution. A, viable and vigorous seed; B, viable non vigorous; C, non-viable seed; D, non-viable (dead) seed. Source: França-Neto, J. B.

In some forest seed laboratories in Brazil, such as the Laboratório de Sementes e Mudas (LASEM) at the Federal University of São Carlos, Sorocaba campus, standardized evaluation forms are used to support the assessment of seed viability and vigor. However, interpretation can still be subjective, as the accuracy of the test relies heavily on the technician’s skill and experience. Expanding the use of the test to a broader range of native species from the Brazilian flora would be extremely beneficial—especially in ecological restoration projects, where high seed diversity and time-sensitive decision-making are common. This highlights the importance of developing and publishing more species-specific TZT protocols, since morphological characteristics, dormancy-breaking requirements, and other factors play a critical role in achieving accurate evaluations.

In forest restoration projects, the tetrazolium test has proven to be a promising tool for evaluating the physiological quality of forest seeds due to its low operational cost and faster results when compared to germination tests. It enables a quick assessment of seed viability, especially for species that require long periods to complete germination, thus supporting timely decision-making by restoration teams—particularly in projects that employ the muvuca technique (see 'Muvuca' Direct Seeding Restoration Method for Biodiversity and People).

Given the test’s practical importance and interpretative challenges, technological solutions are increasingly relevant. With advances in computerized image analysis techniques, efforts should be directed toward developing software tools capable of digitally estimating seed viability and vigor, enhancing the accuracy and objectivity of TZT evaluations.

References

Association of Official Seed Analysts (AOSA). 2010. Seed vigor testing handbook. East Lansing, MI: AOSA.

Brasil. Ministério da Agricultura, Pecuária e Abastecimento. 2025. Teste de Tetrazólio. In Regras para análise de sementes (Chapter 5). Brasília, DF: Secretaria de Defesa Agropecuária. https://wikisda.agricultura.gov.br/pt-br/Laborat%C3%B3rios/Metodologia/Sementes/RAS_TZ 

França-Neto, J. B., Krzyzanowski, F. C., & Costa, N. P. 1998. O teste de tetrazólio em sementes de soja. Londrina: EMBRAPA-CNPSo. https://www.infoteca.cnptia.embrapa.br/infoteca/handle/doc/461306 

França-Neto, J. B. 1999. Testes de tetrazólio para determinação do vigor de sementes. In F. C. Krzyzanowski, R. D. Vieira, & J. B. França Neto, Vigor de sementes: conceitos e testes. Londrina ABRATES. https://loja.abrates.org.br/vigor-de-sementes 

França-Neto, J. B., & Krzyzanowski, F. C. 2018. Metodologia do teste de tetrazólio em sementes de soja. Londrina, PR: Embrapa Soja, Ministério da Agricultura, Pecuária e Abastecimento. (Documentos/Embrapa Soja, ISSN 2176-2937; n.º406). 
 https://www.infoteca.cnptia.embrapa.br/infoteca/bitstream/doc/1098452/1/Doc406OL.pdf 

International Seed Testing Association (ISTA). 2025. International rules for seed testing (Ed. 2025, Chapter 6: The tetrazolium test). Wallisellen, Switzerland: ISTA.

Fish as Seed Dispersers in the Amazon

Written by: Gabriele Vieira; Reviewed by: Olga Kildisheva

Seed dispersal plays a vital role in maintaining biodiversity and ensuring the ecological balance of forest ecosystems. While birds and monkeys are commonly associated with this process, in the seasonally flooded forests of the Amazon, an unexpected group also helps spread plant life: fish.

Many tropical tree species depend heavily on animals to transport their seeds. In fact, around 75% of them would struggle to survive in their natural state without these plant-animal interactions. In flooded tropical forests, numerous plants have adapted to the rhythms of rising and falling waters, timing their flowering and fruit production to coincide with the flood season.

Fish may have been the first vertebrate seed dispersers. During these seasonal floods, frugivorous fish venture into the forest in search of food, inadvertently aiding in seed dispersal. This process, known as ichthyochory, is especially important in such dynamic and complex landscapes. There, dispersal occurs not only through water currents (hydrochory) but also thanks to the crucial ecological role played by fish, which help maintain plant populations and promote the colonization of new areas.

Ichthyochory

Ichthyochory refers to the process of seed and fruit dispersal carried out by fish. This specific form of zoochory—in which animals act as dispersal agents—plays a crucial role in the regeneration and maintenance of aquatic ecosystems and seasonally flooded environments. One of the most iconic examples of such habitats is the igapó, a type of Amazonian floodplain forest that becomes submerged for several months each year due to the rise of blackwater rivers like the Rio Negro. In these nutrient-poor and waterlogged areas, many plant species have evolved to release their seeds during the flood season, taking advantage of water currents and aquatic fauna to spread their offspring.

Ecologically, ichthyochory supports the survival and diversity of many plant species that depend on water-based dispersal routes. Some fish swallow whole seeds or parts of fruits, and the seeds that pass through the digestive tract intact may be deposited far from their parent plant, where they can successfully germinate. This interaction is especially common among frugivorous fish, which are found in both freshwater and marine environments. Their feeding behavior not only nourishes them but also strengthens the connectivity between aquatic fauna and plant reproduction in flood-prone landscapes.

Key Species Involved

Researchers have identified at least 276 fish species as potential seed dispersers in tropical flooded forests. However, this estimate is based primarily on studies conducted in Neotropical floodplain areas, so the actual number may be even higher. In a study aimed at evaluating how different fish species contribute to seed dispersal in oligotrophic blackwater floodplain forests of the Amazon, a total of 41 fish species were found to contain seeds. Among them were Brycon amazonicus, Myloplus asterias, and Serrasalmus rhombeus (Weiss et al., 2023.).

Species of seed-dispersing fish found in the Uatumã River basin. Source: Weiss et al., 2023. 

Another study showed that seed size negatively influences the number of fish species that act as dispersers, with only a few capable of dispersing larger seeds. Among Brycon species, the probability of dispersal increases with individual body size. Large-bodied frugivorous fish also tend to masticate seeds less frequently than smaller species, which favors seed survival. Moreover, they can disperse a wider range of seed sizes—including non-floating and disperser-limited large seeds—playing a key role in the regeneration of flooded forests (Correa et al., 2015).

Brycon amazonicus measuring 35 cm. Source: Correa et al., 2015.

This feeding behavior, central to ichthyochory, varies according to fish and seed traits, and can lead to both dispersal and predation. During the seasonal floods in the Amazon, frugivorous fish enter the flooded forests to feed on fruits and seeds that are either floating on the water surface, submerged, or attached to vegetation. By consuming whole fruits or seeds, these fish inadvertently disperse seeds when they excrete them intact in new locations, often far from the parent plant. However, depending on the fish species and seed characteristics, some seeds may be crushed or digested, resulting in seed predation rather than dispersal. This dynamic feeding behavior highlights the dual role of fish as both seed dispersers and seed predators, making them key players in the regeneration and maintenance of flooded forest ecosystems.

Conservation Challenges for Frugivorous Fish

Despite the ecological importance of ichthyochory, the frugivorous fish responsible for this ecosystem service remain largely overlooked in conservation strategies. Several factors pose direct threats to these aquatic seed dispersers, particularly large-bodied species such as Colossoma macropomum, which play a key role in dispersing large, non-buoyant seeds. Overfishing has significantly reduced their populations, diminishing the effectiveness of seed dispersal across vast areas of flooded forests.

In addition to fishing pressure, the degradation of aquatic habitats represents an increasing threat. Deforestation along riverbanks and floodplain forests compromises the availability of fruits during the flood season, directly affecting the diet and feeding behavior of these fish. The construction of dams and other infrastructure also disrupts natural flooding regimes, fragmenting ecological corridors and limiting access to areas traditionally used for foraging and seed dispersal.

Although these impacts are still insufficiently quantified, they highlight the vulnerability of a mutualistic interaction that sustains much of the biodiversity in tropical flooded forests. Therefore, including frugivorous fish in conservation policies and sustainable fisheries management is essential to ensure the continuity of the natural regeneration cycle that connects aquatic and terrestrial ecosystems in the Amazon.

References

Correa, S. B., Araujo, J. K., Penha, J. M. F., Nunes da Cunha, C., Stevenson, P. R., & Anderson, J. T. (2015). Overfishing disrupts an ancient mutualism between frugivorous fishes and plants in Neotropical wetlands. Biological Conservation, 191, 159–167. https://doi.org/10.1016/j.biocon.2015.06.019 

Correa, S. B., Costa‐Pereira, R., Fleming, T., Goulding, M., & Anderson, J. T. (2015). Neotropical fish–fruit interactions: eco‐evolutionary dynamics and conservation. Biological Reviews, 90(4), 1263-1278. https://doi.org/10.1111/brv.12153 

Weiss, B., D. Santana, F., Petene Calvi, G., Costa, G., Zuanon, J., & Piedade, M. T. F. (2024). Effectiveness of fish assemblage as seed dispersers in Amazon oligotrophic flooded forests. Austral Ecology, 49(1), e13330. https://doi.org/10.1111/aec.13330 

Call for papers on "Impacts of Climate Change on Long Term Viability of Trees Species with Recalcitrant Seeds"

Narkis Morales San Martin, Adjunct Professor in the Departamento de Ecosistemas y Medio Ambiente at the Pontificia Universidad Católica de Chile, is co-editing a new article collection, “Impacts of Climate Change on Long Term Viability of Trees Species with Recalcitrant Seeds” on the journal Frontiers in Forests and Global Change. You can learn more about the scope of the topic and its accepted article types here: Impacts of Climate Change on Long Term Viability of Trees Species with Recalcitrant Seeds.

If you are interested in contributing to the special issue, please feel free to register via the link above. Frontiers will shortly provide further details.

Whilst not mandatory, you are encouraged to submit an abstract (or short outline of the manuscript) by 12 August 2022. The deadline for submissions is 12 October 2022, but Frontiers will aim to accommodate extensions on a case-by-case basis.

For further queries you can reach the dedicated specialist, Lily Campbell at orestsandglobalchange.submissions@frontiersin.org should you need any help.