Characterization and scarification of Neltuma spp. Seeds from soil and trees

Caracterización y escarificación de semillas de Neltuma spp, procedentes del suelo y de árboles

S. A. Atarama-Castillo

Universidad Nacional de Piura, Peru

R. G. Chanduví-García

Universidad Nacional de Piura, Peru

H. Morocho-Romero

Universidad Nacional de Piura, Peru

Instituto Nacional de Innovación Agraria, Perú

R. A. Peña-Castillo

Universidad Nacional de Piura, Peru

Ana Montero Salazar

Universidad Nacional de Piura, Peru

Madai Ruesta-López

Universidad Nacional de Piura, Peru

L. Maza-Córdova

Universidad Nacional de Piura, Peru

M. García-Zapata

Universidad Nacional de Piura, Peru

D. A. Morales-Pizarro dmoralesp@unp.edu.pe

Universidad Nacional de Piura, Peru

The carob tree is a tree species native to the Piura region, initially known as Prosopis pallida and renamed as Neltuma piurensis (L. Vasquez, Escurra and Huaman) CE Hughes and GP Lewis (Hughes et al. 2022). Currently, this forest tree belongs to Fabaceae family, subfamily Caesalpinioideae (Hughes et al. 2022). The carob tree's distribution in the Americas extends from arid to tropical zones. In Peru, the carob tree is a representative species of the dry forests of the north, extending from the Tumbes region to the north of La Libertad (Rivera et al. 2020). The carob tree is of great socioeconomic and ecological interest in the Piura region (Caycho et al. 2023). It is used as forage for goats, sheep, and cattle. It is also used as a construction material, in artisanal medicine, and in local cuisine (Depenthal and Meitzner Yoder 2018). The carob fruit is composed of pod (90 %) and seed (10 %) and is used in human and animal feeding (Silva et al. 2000 and Zarzosa et al. 2021). Given its high nutritional value (carbohydrates, fiber, proteins and sugars) (Ludeña Gutiérrez et al. 2021), this fruit has been shown to be a good forage food and to increase the weight of goats (Silva et al. 2000) and rabbits (Macías-Rodríguez and Usca-Méndez 2017), in addition to reducing production costs. Several studies have reported that carob trees improve the physicochemical and biological characteristics of arid and semi-arid soils (Santos-Jallath et al. 2012 and Depenthal and Meitzner Yoder 2018).

In recent decades, this species has been threatened by unsustainable anthropogenic activity such as agricultural expansion, urban sprawl, climate change, and stress from biotic and abiotic factors. Because of this, it has been categorized as a vulnerable species, according to Supreme Decree N^o. 043-2006-AG, which categorizes threatened species of wild flora (Organismo de Supervisión de los Recursos Forestales y de Fauna Silvestre 2013). Currently, there is limited research focused on sexual propagation through carob seeds. Therefore, it is necessary to explore scarification strategies that increase seed germination (Asif et al. 2020) and technical information on the propagation of this species.

Seed germination is the initial phase of plantlets development, which begins with water absorption, giving rise to a sequence of metabolic processes until the emergence of the radicle (Huang et al. 2020, Xu et al. 2023 and Ruesta-López et al. 2024). A Good initial seed development favors the adaptability and initial vigor of plantlets and directly influences on crop yield (Vieira et al. 2019 and Morales Pizarro et al. 2023). The main form of propagation of carob tree is through seeds (sexual form), which have a very rigid testa (Ewens et al. 2022), which is why different scarification (stratification) strategies have been used: mechanical, thermal and chemical, which favor seed germination (Bhansali 2010). Based on the above, the objective of this study was to characterize the carob fruit and seed and evaluate chemical and biological scarification in the germination of carob seeds collected from the soil and the tree

The study was carried out in the laboratory of the Academic Department of Plant Morphophysiology, belonging to the National University of Piura, Peru, with coordinates 5°11'40.2'' S, 80°37´58.2'' W at 30 m o.s.l. (figure 1). The research was conducted from November 2023 to January 2024.

Plant material: In the collection of seeds, healthy, vigorous trees were selected from the Pariñas district, Talara province, Piura Region, located at 4°31'26.6" S, 81°12'26.2" W, at 15 m o.s.l. (figure 1). The fruits were collected based on two criteria: criterion 1 (directly from the tree) and criterion 2 (fallen on the soil).

Figure 1
Carob harvesting and processing area.

Characterization of fruits and seeds: Fruit characterization was performed on 50 pods (fruits). The following were evaluated: length (L), width (W), and weight (W). For seed characterization, a total of 100 seeds were selected. Their L, W, W, and shape were determined.

The shape of the seed was determined by the relation between the width (W) and length (L): W/L>1 (flattened or ovoid), L/W=1 (spherical), W/L<1 (elongated or oblong), using the scale proposed by Peña-Castillo et al. (2023).

Products used: It was worked with commercial Thinner products (toluene, methyl acetate, methyl alcohol, 2-butoxyethanol and 4-methylpentan-2-one; Peruvian Chemical Products Corporation S.A. Av. César Vallejo 1851 - El Agustino Lima - Peru) and biol or biofertilizer was provided by the Department of Plant Morphophysiology, prepared using the technique of Chanduví-García et al. (2023).

Seed scarification treatments: The seeds were disinfected with 70 % alcohol for five seconds. Then, they were subjected to various scarification treatments: soaking in distilled water for 48 h (T0), 5 % tinner for 1 h (T1), 15 % tinner for 1 h (T2), 25 % tinner for 1 h (T3), 35 % tinner for 1 h (T4), 25 % biol for 20 min (T5), 50 % biol for 20 min (T6) and 75 % biol for 20 min (T7). Later, the seeds were sown in Petri dishes containing sterile paper towels moistened with distilled water. Each Petri dish contained 20 carob seeds and formed a repetition. Seed germination was evaluated for 15 d at 25±2 °C using the following variables:

Germination percentage (GP): The GP was determined using the formula proposed by Brown and Mayer (1988), where GP = (total germinated seeds/total sown seeds) * 100. Seeds were considered germinated when they had 2 mm of radicle length.

Germination speed (GS): It was calculated using the formula proposed by Maguire (1962), where GS = number of germinated seeds / germination time.

Germination index (GI): It was calculated with the formula proposed by Scott et al. (1984), where:

GI = ∑ (the number of germinated seeds on the nth day * number of days after sowing) / total number of seeds sown.

First day of germination: The germination start time was evaluated using the method proposed by Gutiérrez-Gutiérrez et al. (2022).

Statistical analysis: For the characterization of fruits and seeds, the arithmetic mean (mean), standard deviation (SD) and coefficient of variation (CV) were used. The study was carried out in a completely random design (CRD) with eleven treatments and five repetitions per treatment. The ​​obtained values were analyzed using ANOVA (analysis of variance) and Tukey's multiple comparison of means test (P ≤ 0.05).

Characterization of fruit and seed: The carob fruit (pod) had a length of 19.12 cm, a width of 12.10 mm, and a fruit weight of 7.58 g. The seed showed a length of 6.62 mm, width of 2.39 mm, weight of 0.05 g and the L/W ratio was 0.36 (table 1).

Table 1
Biometric description of fruits and seeds of carob tree Neltuma piurensis (L. Vasquez, Escurra & Huaman) CE Hughes & GP Lewis.

Germination variables: In the GP (tree), T4 with 32.5 % was significantly higher than the other treatments, followed by T3 with 23.75 % (table 2). In the GP (soil), T3 exceeded T0 by 20 %, being significantly higher than the other treatments (table 2). The highest GP was from seeds, whose fruits were collected from the soil compared to those harvested directly from the tree.

Table 2
Analysis of germination variables of carob seeds Neltuma spp.

In the GS (tree), T4 with 0.43 germinated seeds per day significantly exceeded the other treatments (table 2), and in the GS (soil), the treatments did not has significant differences, with values between 0.5 to 0.87 germinated seeds per day (table 2). The highest GS values were obtained from fruits collected from the soil.

In the GI (tree), T4 with 3.41 obtained the highest GI, being superior to the other treatments. The GI (soil) treatments did not has significant differences with values between 3.34 to 5.36 (table 2), which showed that the highest GI is obtained from the samples extracted from the soil.

In the FGD (tree), the T0 and T5 treatments germinated in a shorter time by 1.5 days (table 2). However, the FGD (soil), T0 germinated in 1 day, being its germination significantly faster, showing that the FGD was in seeds from the soil zone.

In the characterization of fruits and seeds are agree with the research carried out by Prokopiuk et al. (2000) in the identification of Prosopis pallida, currently renamed Neltuma piurensis (L. Vasquez, Escurra & Huaman) CE Hughes & GP Lewis (Hughes et al. 2022). However, the seed width was smaller at 2.39 mm (table 1), showing that the environment influences on the carob genotype. The seed's L/W ratio indicates an oblong shape (Peña-Castillo et al. 2023).

Germination variation is influenced by the collection area, as well as the fruit (pod) maturity, sugar content, and storage time (Ludeña Gutiérrez et al. 2021). Likewise, Henciya et al. (2017) mention the presence of phytochemicals: alkaloids, flavonoids, terpenes, tannins, phenolic compounds (quinones) in fruits and seeds of Neltuma/Prosopi spp., which are in higher concentrations in freshly harvested fresh fruits, which influences on seed germination. However, dried pods used in animal feeding should not exceed 40 % of phytochemicals substances of those freshly harvested (Ruiz-Nieto et al. 2020). De Sousa Leite et al. (2018) show that the physiological maturity of the seed directly influences on the germination and vigor of the seed.

Regarding the results of this research, seeds from the soil showed the highest GP with chemical scarification with Thinner at 25 % (T3) and biological with Biol at 75 % (T7). These results are consistent with Abdala et al. (2020) in that chemical scarification based on sulfuric acid for three minutes had the highest GP compared to physical and thermal scarification. The effect of chemicals alters, degrades and softens the seed coat composed of hydrophobic and lignified substances (macrosclereids) present in the seeds of Neltuma/Prosopis genus. This lignified layer is responsible for preserving, protecting and preventing the imbibition of the seed. In addition, De Sousa Leite et al. (2018) increased the GP of Bromelia laciniosa seeds to 83 % with acetone for 90 min in mature seeds and to 60 % in immature seeds compared to the control with 40 % and 35 %, respectively. Similarly, these authors reduced the germination days to 15 (mature seed) and 25 (immature seed), days after sowing (DAS), compared to a control that began germination at 45 and 55 DAS, respectively. Rivera Curi et al. (2020) obtained the highest GP with 33.3 % in P. pallida seeds, treated with 100 % acetone for 10 min, being higher than the control with a GP of 30 %.

Carob seeds treated with biol significantly improved GP. These results are consistent with those described by Ruesta-López et al. (2024) in passion fruit (Passiflora edulis S.) seeds treated with 5 % biol, increasing GP to 80 % during 12 h of imbibition. However, at 24 h the GP was lower with 8.33 %. According to Chanduví-García et al. (2023), Galecio-Julca et al. (2023) and Morocho-Romero et al. (2024), the biol has carboxylic acids, humic acids and microorganisms, which weaken the seed coat and increase the GP. Utello et al. (2023) show that heat treatments reduce the growth of embryonic roots compared to chemical or mechanical treatments performed on seeds.

The highest germination speed is in seeds extracted from the soil with treatments based on Thinner at 25 % (T3) and Biol at 75 % (T7). These products weaken the seed layer of carob seed, which facilitates seed imbibition or hydration; activating various metabolic processes that initiate seed germination and gas exchange (Batista Sánchez et al. 2017 and Utello et al. 2023). Ruesta-López et al. (2024) mention that passion fruit seeds exposed to 5 % biol for 12 h, increase the GS to 1.5 germinated seeds per day, surpassing the control. The positive effect of the biostimulant (biol) significantly influences on cell multiplication, promoting the development and emergence of the radicle (Mendivil-Lugo et al. 2019). The highest GI was obtained with seeds collected from the soil with chemical treatments T3 (tinner 25 %) and organic treatments T6 (biol 50 %) and T7 (biol 75 %). These results coincide with those reported by Abdala et al. (2020), indicating that chemical scarification (sulfuric acid) improves the germination speed index (GSI). On the other hand, Utello et al. (2023) report that mechanical scarification (sanding) was superior to chemical (sulfuric acid) and thermal scarification, improving the uniformity and spontaneity of the germination process, being a key factor in the propagation stage in the nursery. Coa et al. (2014) mention that the combined effect of mechanical scarification and imbibition at 12 and 24 h of the seeds, improved the GI compared to chemical treatments (sulfuric acid and muriatic acid) and without scarification with imbibition. On the other hand, Ruesta-López et al. (2024) improved the GI in passion fruit seeds treated with 5 % biol.

The FDG was lower at T0 (48 h imbibition) in seeds from the soil (1 d) and from the tree (1.5 d). The results of the study differ from those obtained by Abdala et al. (2020), who show that chemical scarification (sulfuric acid) accelerates the beginning of germination. Morales Pizarro et al. (2023) show that the imbibition time and the concentration of stimulants influences on the FDG. Similarly, Ruesta-López et al. (2024) accelerated the FDG by imbibition of seeds for 12 h and 24 h. However, when passion fruit seeds were treated with higher concentrations of biol, the FDG was delayed.

The morphometric characteristics of the fruit and seeds coincide with those from Neltuma piurensis genus. However, the seed width shows to be larger than those described for this species. Seed maturity is a key factor in seed germination of Neltuma spp. genus. The chemical pre-germination treatment with Tinner (T3), followed by the organic treatment with biol (T7), improved the germination variables GP (62.5 % and 56.25 %), GS (0.87 and 0.75 germinated seeds per day) and GI (5.36 and 4.79) and the FDG was given a DAS with T0, the best values ​​were showed by seeds from the soil.

Thanks to the technical staff of the Department of Plant Morphophysiology, belonging to the Faculty of Agronomy of the National University of Piura, Peru, for their support to this research.