The Caesalpinia genus includes 150 species, of which 40 are present in South America (Neri Chávez et al. 2018). C. spinosa, also known as tara or guarango, is a tree from the Caesalpiniaceae family, native to Peru (Castro et al. 2017) and found in Venezuela, Colombia, Ecuador, Peru, Bolivia and Chile, growing in areas with average annual precipitation between 230 and 500 mm and temperatures ranging from 12 to 18°C (Guerrero et al. 2016).

C. spinosa is a plant species known for its environmental benefits, as it contributes to soil conservation in the sloped areas of the Andean Basin (Murga et al. 2023). It can grow in degraded soils unsuitable for conventional agriculture (De La Torre 2018), suggesting its potential as an interesting species for ecological soil restoration (Sangay and Robin 2018). Additionally, it contributes to fog capture in forests, which improves soil water availability (Cordero et al. 2017). Furthermore, various vegetative parts of C. spinosa contain biomolecules used in different industries: polyphenolic compounds from dry pods are used in the cosmetic and pharmaceutical industries (Salirrosas et al. 2021), as biopesticides for controlling phytopathogens in the agricultural industry (León and Mancheno 2020) and tannins are employed in the leather industry (Ibieta and Peñarrieta 2021).

C. spinosa exhibits a low natural regeneration rate in both remnant forests and agroforestry systems (Murga et al. 2023). This population decline is driven by land-use changes caused by deforestation, desertification, invasive species and the expansion of agricultural, livestock and urban boundaries (Isbell et al. 2017). In light of this situation, in vitro plant propagation technology has emerged, where micropropagation refers to the multiplication of plants from germinated seeds or vegetative tissues cultured with the correct combination of nutrients and growth regulators (Kulak et al. 2022). To obtain high-quality seedlings, it is important to consider parameters such as seed viability and germination, as well as seedling growth, with the latter two assessed at the in vitro level. Several authors have conducted research on these parameters in various species of the Caesalpinia genus.

Viability studies have been conducted by Guerrero et al. (2016) with mature and immature seeds of C. spinosa, Neto and Barbedo (2015) with mature seeds of C. echinata and Ferro et al. (2019) with mature seeds of C. pulcherrima. These studies evaluated seed physiological quality using the tetrazolium test, which is based on the activity of specific dehydrogenase enzymes and indirectly measures the respiratory capacity of living seed tissues (França and Krzyzanowski 2022).

Germination studies were carried out by Romero et al. (2016) with C. glabrata seeds, achieving the highest germination percentage by applying seed coat scarification and by Vishnu (2020) with C. bonduc seeds, where mechanical scarification yielded the highest germination percentage. Da Silva et al. (2018a) worked with C. ferrea seeds scarified with emery stone and achieved a higher germination rate compared to explants of zygotic embryos.

Several authors have also worked on tissue culture of specific species within the Caesalpinia genus. Núñez et al. (2017) established an in vitro propagation protocol for C. spinosa from axillary buds obtained from selected trees with superior morpho-agronomic characteristics. In C. bonduc, Cheruvathur et al. (2010) achieved shoot organogenesis from callus derived from the epicotyl, while Santosh et al. (2012) established a high-frequency shoot induction micropropagation protocol using cotyledons as explants. Da Silva et al. (2018b) developed a protocol for the rapid in vitro multiplication of C. ferrea.

Location and Plant Material

Experimental processes were carried out at the Biotechnology Laboratory of the Life Sciences Laboratories at Politécnica Salesiana University, Cuenca campus. Pods were collected from 10 randomly selected trees in the Monay parish of Cuenca (17M 725585 UTM 9679559) and from the only two trees in the Jima parish of Sigsig (17M 0729048 UTM 9645248 - 17M 0727560 UTM 9646970). Both locations are situated in the Azuay Province, Ecuador. The pods were classified into three groups, based on their colour (Fig. 1):

Figure 1.

Colouration of Caesalpinia spinosa pods. Immature pod (reddish with greenish-yellow), mature pod (light red with yellow) and overripe pod (dark red).

1. Immature pod (red with greenish-yellow coloration);
2. Mature pod (light red with yellow);
3. Overripe pod (dark red).

They were collected between November and December 2022, January and February and May and June 2023, respectively.

Seed Viability

Seeds were manually extracted from C. spinosa pods and classified into three groups based on their colour (Fig. 2):

Figure 2.

Colouration of C. spinosa seeds. SI (greenish-yellow), SM (light brown) and SSM (dark brown). [SI: immature seed, SM: mature seed and SSM: overripe seed].

1. Immature seed (yellow-green colouration);
2. Mature seed (light brown colouration);
3. Overripe seed (dark brown colouration).

The seed viability test was conducted using the tetrazolium test (ISTA 2016). A total of 1,000 mature and overripe seeds were submerged in water for 48 hours for hydration, with 30% of the seed coat softening. Following the method established by Salazar et al. (2020), the embryo and cotyledons from the various seeds were extracted using a scalpel and placed in a 1% aqueous tetrazolium salt solution for 5 hours, stored in the dark at room temperature. The test was conducted in triplicate, with 100 immature seeds, 100 hydrated mature seeds and 100 hydrated overripe seeds. After the incubation period, each embryo and the condition of the cotyledons were evaluated individually based on colour.

Seed Water Content

Pre-germinative Treatments and In Vitro Growth

Statistical Analysis

Seed Viability

The origin of the plant material did not significantly affect the results according to the experimental design; seed quantity and availability were considered, so seeds from C. spinosa trees located in the City of Cuenca were used.

According to the evaluation of C. spinosa seed staining, variation was found in the three groups, indicating that the condition of the tissues is influenced by exposure to tetrazolium salt. Based on this, the seeds were classified into different categories according to the characteristics of the embryo and cotyledons as viable or non-viable.

Immature (SI) and mature (SM) seeds showed uniform embryos of light pink colour, with the tissues exhibiting a firm texture and were considered viable and vigorous seeds. In contrast, overripe seeds (OS) showed deteriorated embryos with a deep red colouration; the cotyledons displayed the presence of deteriorated tissue, characterised by a deep red colour and the tissues exhibited a soft texture (Fig. 3).

Figure 3.

Seed viability of C. spinosa using the tetrazolium test. SI and SM were viable, while SSM was not viable.

The average viability rates for SI, SM and SSM were 97.72%, 73.45% and 26.76%, respectively. The treatments showed a significant effect (P = 4.32e^-08). The viability percentage of SI was 1.33 and 3.65 times higher compared to SM and SSM, respectively (Fig. 4).

Figure 4.

Effect of 1% tetrazolium salt evaluated after 5 hours on C. spinosa seeds. The results are expressed as the mean percentage of seed viability ± ED. Treatments sharing a common letter do not differ at a 5% level according to Tukey's HSD test [SI: immature seed, SM: mature seed and SSM: overmature seed].

Water Content of Seeds

Germination and In Vitro Growth

The results of this research showed that the phenological state of the seeds, type of scarification and days of evaluation are important factors in the in vitro germination of C. spinosa seeds. The highest percentage of germination was observed in immature seeds (SI) with testa removal (ST) at 48.55%, followed by mature seeds (SM) with ST at 33.01%, evaluated at 4 days compared to other treatments. The minimum percentage of germination was recorded in overripe seeds (SSM) with cotyledon cut (CT) and SSM with ST, both at 0%, evaluated on the same day. In the various treatments (SI-CT, SM-CT and SSM-CT), germination was observed between 20.82% and 7.41% over 12 days (Figs. 2B and 4A, Fig. 5 and Fig. 6).

Figure 5.

In vitro germination of C. spinosa seeds: SI-CT, SI-ST, SM-CT, SM-ST, SSM-CT and SSM-ST, evaluated at 6, 4, 5, 3, 3 and 3 days, respectively.

Figure 6.

Germination rate of C. spinosa seeds according to phenological state and pre-germination treatment.

The germination of C. spinosa seeds is conditioned by the phenological state of the seeds and the type of scarification. It was observed that the highest percentage of germination was achieved with the SI-ST treatment at 93.16%, followed by the SI-CT treatment at 48.84% and the control treatment at 0%. A similar trend was observed in mature seeds, with the highest percentage of germination obtained from the SM-ST treatment at 84.52%, while the SM-CT treatment yielded 44.75% and the control treatment at 0%. For overripe seeds, the percentage of germination did not differ significantly between treatments, with SSM-ST and SSM-CT achieving 32.40% and 27.03%, respectively and the control treatment at 0% (Fig. 7 and Fig. 8).

Figure 7.

Germination of C. spinosa seeds in relation to phenological state and pre-germination treatment factors.

Figure 8.

Germination of C. spinosa seeds in relation to phenological state and pre-germination treatment factors.

The average growth for SI-CT, SI-ST, SM-CT and SM-ST was 52.26 mm, 54.06 mm, 64.79 mm and 61.26 mm, respectively. The treatments did not show a significant effect (P = 0.223) (Fig. 9 and Fig. 10 - Table 2).

Table 2.

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Germination percentage of C. spinosa seeds in relation to phenological stage and type of scarification.

Phenological stage	Type of scarification
	ST	CT	Control
SI	93.16% Aa	48.84% Ab	0% Ac
SM	84.52% Aa	44.75% Ab	0% Ac
SSM	32.40% Ba	27.03% Aa	0% Aa

Figure 9.

In vitro growth of C. spinosa seedlings at 21 days post-sowing [SI: immature seed, SM: mature seed and SSM: overmature seed. CT: cut in the distal region of the cotyledon and ST: seed coat removal].

Figure 10.

Germination of C. spinosa seeds in relation to phenological state and pre-germination treatment factors.

Results obtained in this study indicated that the viability and in vitro germination of C. spinosa seeds depend on the phenological stage of the seeds. Our results showed that the water content in the seeds is an important parameter. In this context, immature seeds (SI) achieved a higher percentage of viability, germination speed and germination percentage compared to mature (SM) and overripe seeds (SSM). We observed that, as the phenological maturation process of C. spinosa pods progresses, the water content in the seeds decreases, which could affect seed vigour. It is important to understand that water content is specific to each plant species (Li et al. 2020).

The tetrazolium test is an efficient alternative for determining seed viability and vigour (Ferro et al. 2019). As shown in Fig. 2A, visual inspection made it possible to assess the state of the embryo and cotyledons of the different types of C. spinosa seeds for correct discrimination. Our observations were corroborated by Guerrero et al. (2016) in mature C. spinosa seeds and by Neto and Barbedo (2015) in C. echinata seeds with 10% water content. Ferro et al. (2019) evaluated and classified the embryos and cotyledons of C. pulcherrima seeds, where a viable seed exhibited a uniform pink colour and firm-textured tissue, while a non-viable seed showed an intense red colour and flaccid tissues with deformation. A dead seed appeared milky white. Similar characteristics have also been observed in other Fabaceae species, as reported by Nunes et al. (2022) in Tamaridus indica L. According to Fernandes et al. (2019), red or light pink colours indicate viable tissue and the terminology used to determine the colours of seeds observed in the tetrazolium test is generally established by the authors.

Based on the results in Fig. 3 and Table 1, a relationship could be established between the percentage of viability and water content. SI and SM with average water contents of 52.23% and 7.60% obtained average viabilities of 97.72% and 73.45%, respectively. Contrasting these results with the observations of Guerrero et al. (2016), we found differences; in the present research, using SI resulted in a higher percentage of viability compared to SM seeds, while the aforementioned authors reported average viabilities of 78% and 86% in SI and SM, respectively. Therefore, the differences found in seeds at the SM phenological stage could be attributed to seasonal variations in environmental conditions (De Oliveira et al. 2010). In contrast, the differences found in seeds at the SI phenological stage could be due to the ideal humidity level in our study, resulting in high metabolic activity (Ferreira and Barbedo 2017). Research does not report viability and germination in seeds at the SSM phenological stage, possibly because their dehydration level causes damage to the cytoskeleton (Ranganathan and Groot 2023).

Various studies report that seeds of the genus Caesalpinia are recalcitrant, meaning they lose viability and germination capacity if their moisture content is below 20% (Romero and Pérez 2016). These seeds, upon detaching from the tree with high moisture content, are metabolically active and need to remain hydrated (Berjak and Pammenter 2017; Ranganathan and Groot 2023). Water is crucial for their metabolic and enzymatic reactions (Kibinza et al. 2006). Recalcitrant seeds germinate and establish rapidly under humid conditions due to their large energy reserves (Colville 2017; Pritchard et al. 2022). During drying, they experience negative changes such as reduced respiration, toxin accumulation and DNA degradation (Kibinza et al. 2006, Yuniarti 2015), which can lead to dysfunction of cellular organelles and extensive vacuolisation, predisposing the seeds to deterioration (Ranganathan and Groot 2023). In overly dry seeds, extreme dehydration alters the water film interface covering the cellular structure and exposes macromolecules in the membranes, such as lipids and proteins, making them vulnerable to reactive oxygen species (ROS) (Ramtekey et al. 2022). In this context, Li et al. (2016) and Castro et al. (2017) extracted oil from the seeds and fruits of C. spinosa. Damage caused by free radicals leads to a breakdown of genomic integrity within the nucleus (Shvachko and Khlestkina 2020). This occurs because antioxidant enzymes and non-enzymatic antioxidants, such as ascorbic acid, decrease during maturation and glutathione acts as the main redox buffer, with its redox state (E GSSG/2GSH) being a marker of viability. As seeds age, this marker becomes more positive; viability is lost when (E GSSG/2GSH) exceeds -160 mV (Nadarajan et al. 2022), allowing us to predict whether a seed will survive and germinate or die. Therefore, E (GSSG/2GSH) is a universal marker of plant cell viability and allows us to predict whether a seed will survive, germinate and produce a new plant or if it will die (Kranner et al. 2006).

In the interaction between the phenological state of the seeds and the type of scarification, germination in this study began 2 days after sowing, achieving its highest germination rate at 4 days. These results are similar to those obtained by Alvarez et al. (2014), who observed germination starting at 3 days in C. cacalaco seeds with a scarification that involved a small cut in the testa. Meanwhile, Sánchez et al. (2016) reported germination beginning at 1 day and the peak at 4 days in C. platyloba seeds with a scarification involving rubbing the seeds on sandpaper. In contrast, Da Silva et al. (2018b) achieved germination beginning between 7 and 10 days with mechanical scarification by rubbing C. ferrea seeds on an abrasive surface. These results suggest that mechanical scarification with CT and ST is effective in removing the dormancy of C. spinosa seeds, allowing for a higher germination rate.

Regarding the relationship between the phenological state of the seeds and the type of scarification, although there was no significant difference between treatments, SM-CT achieved a seedling growth of 64.79 mm in 21 days. These results are superior to those reported by Assis et al. (2012) and Marín and Iglesias (2022) with the woody species Anacardium othonianum and Pinus pseudostrobus, which achieved seedling lengths of 20.00 mm and 30.26 mm evaluated at 30 days, respectively.

This research demonstrated that the phenological state and the scarification applied to C. spinosa seeds are important for the parameters of viability, speed and percentage of germination. In immature seeds (SI) and mature seeds (SM), the viability results were 97.72% and 73.45% with water contents of 52.23% and 7.60%, respectively. On one hand, regarding the relationship between phenological state and type of scarification, the germination speed for SI-ST and SM-ST achieved the best averages of 48.55% and 33.01% at 4 days after sowing. On the other hand, in the relationship between phenological state and type, the best results for SI-ST and SI-CT were 93.16% and 48.84%, while for SM-ST and SM-CT, they were 84.52% and 44.75%, respectively; the control treatments showed no germination. Finally, the results of in vitro growth for SI and SM with the different types of scarification evaluated at 21 days after sowing ranged between 52.26 mm and 64.79 mm. This research provides a foundation for selecting SI and SM of C. spinosa for efficient in vitro seedling production, which could later be allocated to reforestation programmes, conservation and sustainable economic development.

The authors of this research express their gratitude to the Vice-Rectorate of Research at Politécnica Salesiana University, the Research and Biodiversity Assessment Group (GIVABI), the Rufford Foundation, the Jima Ltda Community Development Cooperative and the Tambillo Forestry Initiative for their support and collaboration, which were instrumental in the successful development of this study.

Politécnica Salesiana University, Jima Ltda Community Development Cooperative