Agronomic biofortification of waterleaf (Talinum triangulare) with zinc applied via root

Abstract

Zinc (Zn) deficiency affects one-third of the world’s population, and agronomic biofortification is a good way to fight against this problem. Biofortification of leafy vegetables has been driven by their consumption, and, in this scenario, the waterleaf (Talinum triangulare) demonstrates good potential. Thus, this work aimed to verify the efficiency of agronomic biofortification of waterleaf with Zn applied via root. For the experiment, seedlings obtained with vegetative propagation by cuttings were used and cultivated in a mixture of sand and commercial substrate, initially in the laboratory. After acclimatization, the seedlings were transferred to a plant nursery, where NPK and Zn were applied, in six treatments (source Zinc Sulphate Heptahydrate, ZnSO₄.7H₂O), T1: control; T2: 12.5 mg kg^-1; T3: 25 mg kg^-1; T4: 50 mg kg^-1; T5: 100 mg kg^-1; T6: 400 mg kg^-1. The length, number of leaves, shoots, inflorescences, and diameter of the collection were measured. The leaf dry mass (LDM), stem (SDM), root (RDM), root-shoot ratio (R S^-1), leaf weight ratio (LWR), Dickson’s Quality Index (DQI), leaf and soil Zn levels, total proteins and soluble sugars (TSS) were analyzed. The dose of 400 mg kg^-1 provided toxicity to plants, and the dose of 100 mg kg^-1 demonstrated better results in plant growth and development and contents of proteins and zinc, with an increase of 4081% of Zn, indicating which is the most effective dose to be used in the biofortification of this species.

Key words:
growth and development; leafy vegetable; mineral nutrition; UFP; Zn

Resumo

A deficiência de Zinco (Zn) atinge um terço da população mundial, e a biofortificação agronômica é uma forma de combatê-la. A biofortificação de hortaliças folhosas tem sido impulsionada pelo seu consumo e, neste cenário, o cariru (Talinum triangulare) demonstra potencial para esses estudos. Assim, este trabalho teve como objetivo verificar a eficiência da biofortificação agronômica de cariru com Zn aplicado via radicular. Para o experimento, foram utilizadas mudas obtidas com a propagação vegetativa por estaquia, cultivadas em uma mistura de areia e substrato comercial inicialmente em laboratório. Após aclimatação, as mudas foram transferidas para um viveiro, onde foram aplicados NPK e Zn, em seis tratamentos (fonte Sulfato de Zinco Heptahidratado, ZnSO4.7H2O), T1: testemunha; T2: 12.5 mg kg^-1; T3: 25 mg kg^-1; T4: 50 mg kg^-1; T5: 100 mg kg^-1; T6: 400 mg kg^-1. Foram medidos o comprimento, número de folhas, brotações, inflorescências e diâmetro da coleção. A massa seca da folha (LDM), caule (SDM), raiz (RDM), razão raiz-parte aérea (R S-1), razão peso da folha (LWR), Índice de Qualidade de Dickson (DQI), níveis de Zn foliar e do solo, total proteínas e açúcares solúveis (TSS) foram analisados. A dose de 400 mg kg^-1 proporcionou toxicidade às plantas, e a dose de 100 mg kg^-1 demonstrou melhores resultados no crescimento e desenvolvimento das plantas, e teores de proteínas e zinco, com aumento de 4081% de Zn, indicando que é a dose mais efetiva a ser utilizada na biofortificação dessa espécie.

Palavras-chave:
crescimento e desenvolvimento; hortaliça folhosa; nutrição mineral; UFP; Zn

Introduction

Zinc (Zn) is an essential element for human health, with structural and catalytic functions in about 10% of all proteins (Moraes 2020). In plants, Zn is important for the synthesis of proteins linked to the metabolism of carbohydrates, nucleic acids, lipids, and phytohormones, mainly auxin (Hafeez et al. 2013). In humans, Zn can also act in the prophylaxis and treatments against SARS-CoV-2 (Covid-19), due to its antiviral properties (Kumar et al. 2020), in addition to contributing to the proper functioning of the immune system, reducing the incidence of neurological problems, cancer and autoimmune diseases (Skrajnowska & Bobrowska-Korczak 2019).

Therefore, the Zn daily intake of 11 mg day^-1 for an adult male is recommended, while for adult female it is 8 mg day^-1 (11 mg day^-1 for pregnant and 12 mg day^-1 for lactating) and 5 mg day^-1 for children (National Institutes of Health 2016). Despite its importance, it is estimated that one-third of the world population suffers from Zn deficiency (Muthayya et al. 2013), which can affect the growth of children, cause problems in brain development, generate lower performance and productivity in physical activities and increase susceptibility to diseases such as pneumonia and diarrhea (Cakmak & Kutman 2018).

According to White and Broadley (2011), this situation is attributed to the production of food in soils with low levels of Zn, the consumption of crop products with a low concentration of Zn, and the intake of ultra-processed foods. The replacement of micronutrients through nutritional supplements, commonly as capsules, in addition to having a high investment cost, also has greater risks of toxicity from overdose (Morris & Crane 2013).

Thus, an alternative to combat Zn deficiency is agronomic biofortification, a biological method of increasing essential nutrients in crop products (Shahzad et al. 2021), in which the element is supplied to plants through root or foliar fertilization, so that plants can absorb the micronutrient and make it bioavailable to the consumer (Cakmak et al. 2017).

The fertilization via root aims to correct the low amounts of Zn in the soil (Ivanov et al. 2019), whose retention can be interfered with by factors such as pH, texture, organic matter, microbial activity and concentrations of phosphorus and cationic elements (Alloway 2008).

Recent works address the Zn biofortification of cereals, mainly rice, wheat and corn, due to their large global consumption (Nahar et al. 2020; Nadeem et al. 2020; Watts et al. 2020). However, the application of Zn in leafy vegetables such as arugula, lettuce and broccoli has become frequent, due to its great potential for biofortification, purchase and consumption (Moraes et al. 2022; Almeida et al. 2020; Rivera- Martin et al. 2020).

In this scenario, waterleaf (Talinum triangulare) appears as a potential vegetable for studies involving biofortification. Talinum triangulare (Jacq.) Willd (family Talinaceae, formerly Portulacaceae) (Brilhaus et al. 2016), is an unconventional food plant (UFP) and medicinal plant (Agbonon et al. 2010) widely cultivated and consumed in the north and northeast regions of Brazil (Alexandre et al. 2018), mainly by rural populations, as well as in African and Asian countries (Agbonon et al. 2010). The species has considerable amounts of calcium, phosphorus, vitamin C and micronutrients, such as manganese (Araújo et al. 2018), in addition to being rich in proteins, essential oils, flavonoids and polyphenols (Airaodion et al. 2019), which makes it an excellent source of nutrients, but with low levels of Zn (Agbaire 2011).

Thus, agronomic biofortification with Zn is important for this species, to promote a better nutritional quality for the plant and its consumers, aiming to combat Zn deficiency mainly in the Amazon region and enrich its soils with the micronutrient, through application via root. Therefore, this work aims to verify the efficiency of agronomic biofortification of waterleaf with Zn, applied via root, under aspects of growth and development of seedlings, as well as Zn accumulation and production of total soluble proteins and sugars.

Material and Methods

Experiment setup

The experiment was carried out from August to October 2019, at the Laboratory of Plant Physiology and Plant Growing, at the Federal University of Western Pará, in Santarém city, Pará, Brazil. The formation of waterleaf seedlings occurred by vegetative propagation using the cutting technique, and stakes with a length between 10 and 12 cm were removed from the middle portion of the parent plants. The cuttings were placed in 500 mL pots, containing a mixture of sand and commercial substrate (1:1 v/v), considered the most suitable for the growth and development of waterleaf seedlings according to Souza et al. (2021). The physical and chemical attributes of the commercial substrate are shown in Table 1.

Table 1
Physical and chemical attributes of the commercial substrate used for planting Talinum triangulare seedlings. PH = Hydrogenic Potential; K = Potassium; P = Phosphorus; Ca = Calcium; Mg = Magnesium; Al = Aluminum; H+Al = Potential Acidity; SB = Sum of Exchangeable Bases; t = Cation Exchange Capability; O.M. = Organic Matter; V = Base Saturation Percentage.

pH	K	P	Ca	Mg	Al	H+Al	SB	T	O. M.	V	Clay	Silt	Sand
H2O	mg dm-3		cmolc dm-3						dag dm-3		%
5.9	354	49.8	6.6	1.43	0.01	3	8.9	8.9	15.22	75	14.15	10.9	74.95

The seedlings remained for 15 days in a controlled environment, with a 12-hour photoperiod, temperature of 27 ºC and daily irrigation, keeping the field capacity at 75%. After this period, the seedlings were transported to the nursery at the Federal University of Western Pará, Tapajós Unit, where they were transplanted to 5 L capacity pots containing sandy soil, with their physical and chemical characteristics described in Table 2. Previously, this soil underwent a liming process, with lime applied at a dose of 0.7 t ha^-1, 30 days before planting, to increase base saturation to 60% (Luz et al. 2002).

Table 2
Physical and chemical attributes of the soil used for planting Talinum triangulare seedlings. pH = Hydrogenic Potential; K = Potassium; P = Phosphorus; Ca = Calcium; Mg = Magnesium; Al = Aluminum; H+Al = Potential Acidity; SB = Sum of Exchangeable Bases; t = Cation Exchange Capability; O. M. = Organic Matter; V = Base Saturation Percentage.

pH	K	P	Ca	Mg	Al	H+Al	SB	T	O. M.	V	Clay	Silt	Sand
H2O	mg dm-3		cmolc dm-3						dag dm-3		%
4.5	4	3	0.11	0.08	0.32	0.8	0.2	0.5	0.44	20	22	10.3	67.7

NPK and Zn application

The seedlings were acclimated for 10 days, and after this period, they received the first application of NPK 18.18.18 (N-P₂O₅-K₂O), with the second application occurring seven days later, totaling 0.375 g per pot (0.1875 g in each application), whose amounts were adapted from the work by Ndaeyo et al. (2013).

After 15 days of NPK application, fertilization was carried out with Zn via root, having as a source the Zinc Sulfate Heptahydrate (ZnSO₄.7H₂O). The applied concentrations were 12.5 mg kg^-1, 25 mg kg^-1, 50 mg kg^-1, 100 mg kg^-1 and 400 mg kg^-1, in addition to the control (without Zn application). These doses are equivalent to 25, 50, 100, 200 and 800 kg ha^-1 of Zn, respectively, according to Cakmak (2010). Each applied concentration was considered as a treatment, and each treatment had 6 repetitions, totaling 36 samples.

Biometric analysis of seedling growth and development

After 77 days of seedling production, the experiment was finished. Biometric parameters of growth and development were then evaluated, such as the number of leaves, inflorescences, and shoots (new branches in the cuttings), in addition to the stem diameter (mm), with the aid of a digital caliper. The final length (cm) was obtained from the average of the three largest branches of each seedling, and measurements were performed with the aid of a millimeter ruler.

The plants were separated into leaves, stems, and roots, placed in paper bags and taken to a oven with forced air circulation, at a temperature of 60 ºC, until constant weight. After this, each part was weighed separately on an analytical balance, to obtain the leaf dry mass (LDM), stem dry mass (SDM) and root dry mass (RDM), whose sum gives the total dry mass (TDM) (g), with the root-shoot ratio (R S^-1) being subsequently calculated.

Furthermore, with the data, the leaf weight ratio (LWR) (g g^-1) was calculated using the formula: LWR = LDM / TDM, where LDM is the leaf dry mass and TDM is the total dry mass (Benincasa 2003).

Another calculated trait was the Dickson Quality Index (DQI), using morphological parameters of plant height (H), stem diameter (SD), total dry mass (TDM), shoot dry mass (SDM) and root dry mass (RDM) (Dickson et al. 1960). Its formula is expressed by: DQI = TDM / (H/SD) + (SDM/RDM).

Total soluble Zn, protein and sugar contents

Regarding chemical analysis, 0.2 g was separated per sample of leaf dry mass (previously ground in a bench crusher) and 15 g of soil per repetition. The samples were sent to the Laboratory of Soils from the Brazilian Agricultural Research Corporation (EMBRAPA) Eastern Amazon, in Belém-PA, for the analysis of zinc in leaf tissues (through the method of Nitric Digestion by microwave in MP-AES) and the soil [through Mehlich 1 extraction solution (HCl 0.05 mol L^-1 and H₂SO₄ 0.0125 mol L^-1)], with the contents being measured in mg kg^-1. The rest of the leaf dry mass samples were used to perform the analysis of total protein content, according to the method of Bradford (1976), and of total soluble sugars (TSS), with the anthrone protocol (Dische 1962), being measured in mg g^-1 MS.

Statistical analysis

All results were submitted to the Shapiro-Wilk normality test and analysis of variance (one-way ANOVA), with the means submitted to the Tukey test (p < 0.05) and the leaf and soil Zn data submitted to regression analysis, in the statistical program Sigmaplot 12.0 (Systat Software 2010).

Results and Discussion

Toxicity limit

Seedlings submitted to application of 400 mg kg^-1 of Zn showed visible signs of toxicity, such as chlorosis (reduction in chlorophyll contents, causing the leaves to turn yellow) (Silva et al. 2015) and growth inhibition (Tewari et al. 2008) throughout the experiment. As a result, in the end of the experiment, the plants of this treatment were dead, making it impossible to evaluate their growth, development and other analyses. This high dose was applied to assess the zinc toxicity threshold of the species, which is greater than 100 mg kg^-1 and less than 400 mg kg^-1, since most plants have limits between 100 and 700 mg kg^-1 of Zn in their leaf tissues (White & Broadley 2011).

In the work by Jain et al. (2013), it was also shown that the Arabidopsis thaliana had symptoms of Zn toxicity at concentrations above 0.1 mM. In the study by Vijayarengan and Mahalakshmi (2013), soil Zn levels above 150 mg kg^-1 proved to be toxic to tomatoes.

According to Balafrej et al. (2020), the toxic effects of Zn on plants depend on its concentration available to be absorbed, the exposure time, the plant genome, and the development stage in which the seedling is found. Thus, the data found in the present work can be useful for future studies aiming to evaluate the Zn toxicity in T. triangulare and in other similar leafy species.

Foliar and soil Zn contents

Regarding foliar Zn contents, there was a significant difference (p < 0.05) for the 100 mg kg¹ treatment, with an increase of 4081% compared to the control contents (Fig. 1). Leaf Zn contents increased linearly in response to Zn contents applied to the soil, as observed in the work by Almeida et al. (2020). Furthermore, the content of 25 mg kg^-1, despite not differing from the control, also had a large increase (3,346%), demonstrating that there was a high accumulation of the element in the edible parts of the plant, without reducing its productivity.

Increases in Zn content in leaf tissue as a function of the application of increasing doses of the micronutrient have been reported in corn biofortification (Kume et al. 2021), arugula (Rugeles-Reyes et al. 2019), chickpeas (Pal et al. 2019) and beans (Cambraia et al. 2019).

The normal concentrations of Zn in the dry mass of vegetables range from 3 to 150 mg kg^-1 (Dechen & Nachtigall 2006), however, the treatment with 100 mg kg^-1 of Zn provided 560.80 mg kg^-1 of Zn in the waterleaf leaves, values almost 4 times higher than normal conditions.

Currently, biofortification is carried out mainly in cereals, but metals such as Zn must travel a long way from the soil to the edible parts of the plant, which in the case of cereals is the grain (Palmgren et al. 2008). The biofortification of rice with Zn, through its root and foliar application in the maximum stages of tillering and flowering of the cereal, reached levels of 40.8 mg kg^-1 in the work by Saha et al. (2017). However, there were large Zn losses in the crushing, grinding, and cooking processes of the grains, which ranged from 12.6 to 28.7 mg kg^-1, leaving 28.2 to 12.1 mg kg^-1 of Zn in the grains to be absorbed by the consumer. These values are very low compared to those found in this work, carried out with leafy vegetables, further proving the importance of biofortification with these species.

Figure 1
Leaf zinc content in Talinum triangulare seedlings in five zinc application treatments (T1 = control; T2 = 12.5 mg kg^-1; T3 = 25 mg kg^-1; T4 = 50 mg kg^-1; T5 = 100 mg kg^-1). Error bars represent the standard error of the mean (n = 6) and different letters represent a significant difference (Tukey p < 0.05).

Considering the daily demand of 11 mg of Zn for an adult male (National Institutes of Health 2016), it would be necessary to consume 19.61 g of dry waterleaf mass. However, as it is consumed fresh, and its moisture content is 91.83% (Kwenin et al. 2011), it would be necessary to consume 240.02 g of fresh vegetable mass.

The average daily individual consumption of vegetables by brazilians is 49.2 grams per capita^-1 day^-1 (Canella et al. 2018). Thus, if the intake were biofortified waterleaf, 20% of the daily demand for Zn would already be met. Drawing a relationship with the average consumption of rice, which is 131.4 grams per capita^-1 day^-1 Instituto Brasileiro de Geografia e Estatística [IBGE] (2020), if it were the biofortified rice in the work by Saha et al. (2017), only 14.45% of the daily demand for Zn would be met, in a scenario of greater loss of Zn due to its processing.

Thus, regardless of which biofortified species are ingested, the consumption of other sources of zinc in the diet, both animal and vegetable, is suggested to reach the daily recommended amounts, since organic sources of the mineral are more bioavailable to the body (Lima et al. 2019), becoming an advantage over other forms of supplementation.

The application treatments of 50 mg kg^-1 and 100 mg kg^-1 of Zn differed significantly (p < 0.05) from the control about the levels of zinc in the soil, with an increase of 1,037% and 1,904% respectively (Fig. 2), following a linear correlation. In the work by Correia et al. (2008), effects were also observed between the application of Zn and the increase in the nutrient content in the soil. Zinc applied via roots aims to increase the natural content of the element in the soil for a longer period, as opposed to foliar fertilization, which provides the nutrient for just that moment, not producing a considerable residual effect (Alexander & Schroeder 1987). The supply of Zn is important, as both Brazilian and Amazonian soils are poor in this micronutrient (Demattê & Demattê 1996). In addition, Zn is an element that is not very mobile in plants (Marschner 1999), that is, it is not easily redistributed after its assimilation, which further justifies root fertilization due to its constant supply throughout the entire cultivation period.

Figure 2
Soil zinc content in five zinc application treatments (T1 = control; T2 = 12.5 mg kg^-1; T3 = 25 mg kg^-1; T4 = 50 mg kg^-1; T5 = 100 mg kg^-1). Error bars represent the standard error of the mean (n = 6) and different letters represent a significant difference (Tukey p < 0.05).

The increase in Zn in the soil also provided an increase in Zn in waterleaf leaves, demonstrating the responsiveness of plants to the metal, which favorably stimulated their metabolic and physiological processes, resulting in good growth and development of seedlings (Chauhan et al. 2013), a characteristic that is fundamental for agronomic biofortification.

Total soluble proteins and sugars

The total protein contents differed significantly in the 25 mg kg^-1, 50 mg kg^-1 and 100 mg kg^-1 application treatments (Fig. 3a). Due to Zn applications, increases in protein content have also been reported in beans (Phaseolus vulgaris L.) (Kachinski et al. 2022) and coriander (Meena et al. 2016). This may have occurred because zinc is a highly required element as a structural and catalytic component of proteins and enzymes, and with the application of Zn via the soil, there was an increase in the amounts of the micronutrient in the plant, enabling an increase in synthesis to occur cellular protein, and thus, the increase in total protein content in plant dry mass (Pal et al. 2019). Furthermore, it is assumed that Zn is directly responsible for the synthesis of tryptophan, an amino acid precursor of indole-acetic acid and indirectly responsible for the synthesis of proteins (Malta et al. 2002; Castillo-Gonzáles et al. 2018).

There was no significant difference between treatments for total soluble sugars (TSS) (Fig. 3b). However, there was a slight increase in the quantity of TSS contents, reaching its highest values in the treatments of 50 mg kg^-1 and 100 mg kg^-1. Lingyun et al. (2016), evaluating the effects of zinc on the growth and nutritional quality of pea sprouts, also found that the levels of total soluble sugars were gradually increased, until the application of 50 mg L^-1 of Zn, when they began to decline. Also, according to the authors, this increase in the amounts of TSS possibly provided the necessary metabolites for adequate growth and development, which was reflected in a better performance for seedlings submitted to doses of 50 mg kg^-1 and 100 mg kg¹.

Figure 3
Total proteins (a) and total soluble sugars (b) contents in Talinum triangulare seedlings in five zinc application treatments (T1 = control; T2 = 12.5 mg kg^-1; T3 = 25 mg kg^-1; T4 = 50 mg kg^-1; T5 = 100 mg kg^-1). Error bars represent the standard error of the mean (n = 6) and different letters represent a significant difference (Tukey p < 0.05).

Number of leaves, shoots, inflorescences, diameter of collar and length

There were statistical differences in the number of leaves (p < 0.05) in the seedlings from the 100 mg kg^-1 treatment (Fig. 4a). Biofortification with zinc in the species T. triangulare also promoted increases in the number of leaves in the work by Souza et al. (2021). According to Filgueira (2008), this characteristic is very important when buying leafy vegetables, since the leaves are its consumable part, and the consumer’s attention is focused on the appearance, volume, and number of leaves to choosing the product.

However, for the other variables, number of shoots, number of inflorescences, stem diameter and length, there was no significant difference (Fig. 4b-e). Also, no statistical differences were found for the length (height of the plant) in the work by Graciano et al. (2020), for the biofortification of crisp lettuce cultivars, and for the stem diameter, in four of the six forest species exposed to lead and Zn, in the work by Wang et al. (2015).

About the number of shoots (Fig. 4b), the control seedlings showed a tendency to a lower value, whereas the treatment with 25 mg kg^-1 had a propensity to a higher mean. There was also an increase in the number of shoots with the application of zinc in the work by Babajani et al. (2019). This increase in shoots can be an important vegetative characteristic for a plant produced through cuttings, since the greater the number of shoots, the greater the number of branches produced, which will increase the aerial and photosynthetic part of the plant (Carvalho et al. 2015).

The number of inflorescences (Fig. 4c) was slightly influenced by the doses of Zn. Positive results were expected for this variable, as found in the work by Pandey et al. (2013), since zinc is related to reproductive factors, such as pollen production and morphology and stigmatic changes, which could help to induce a greater number of inflorescences in the plant, increasing its chances of reproduction (Pal et al. 2019).

Both stem diameter and seedling length (Fig. 4d-e) were also weakly influenced by Zn doses. Increases in these variables were expected, as Zn participates in the synthesis of tryptophan, one of the precursors of indole acetic acid (IAA), the main phytohormone of the auxin class (Hafeez et al. 2013). IAA has several functions, such as cell multiplication and increases in size, actively participating in the growth of plant species (Majda & Robert 2018).

Dry mass and shoot root ratio

The application of 100 mg kg^-1 of Zn provided a 130% increase in leaf dry mass when compared to the control, with a significant difference (p < 0.05) (Fig. 5a). Haider et al. (2018), in their work on mung bean biofortification, found that the foliar application of a 0.5% Zn solution was the best to increase the quantity of leaf dry mass, while in the study by Oliveira et al. (2020), of zinc content and mass accumulation in green corn fertilized with Zn sulfate, the micronutrient applications did not influence its accumulation.

The present result may have been influenced by the quantity of the number of leaves, which presented in greater quantity in the treatment of the application of 100 mg kg^-1 of Zn. This can show that zinc, at these applied concentrations, provided an increase in both the number of leaves and their mass, as the micronutrient participates in processes such as the synthesis of proteins related to carbohydrates, lipids, and nucleic acids (Palmer & Guerinot 2009), increasing leaf mass.

There was no statistical difference for stem dry mass (Fig. 5b) and root (Fig. 5c), contrary to the work of Teixeira et al. (2019), in which there was a significant interaction between the doses of Zn applied and the types of soil used to produce the root dry mass. However, for the latter, there was a reduction in the values in the treatment with the application of 100 mg kg^-1, which may be related to the balance of auxins in the plant, since at high concentrations of the phytohormone (caused by the application of Zn), there is inhibition of root growth (Davies 2004).

The root shoot ratio (Fig. 5d) had a significant difference in the treatment of 100 mg kg^-1, indicating that the seedlings in this treatment invested more in the shoot (photosynthetically active) than in the root system, corroborating the data from the root and leaf dry mass observed in the present study. In the work by Yang et al. (2011), on grape growth and physiological responses to excess zinc, the shoot root ratio exhibited the highest results in the 21 and 28 mM zinc solution treatments, while in the study by Umar et al. (2020), evaluating the effects of different sources and application methods on the growth and concentration of Zn in corn plants, with the application of foliar Zn at 2% solution, there was a decrease of 3.7% of the variable, about control.

Figure 4
Number of leaves (a), number of buds (b), number of inflorescences (c), root collar diameter (d) and length (e) of Talinum triangulare seedlings in five zinc application treatments (T1 = control; T2 = 12.5 mg kg^-1; T3 = 25 mg kg^-1; T4 = 50 mg kg^-1; T5 = 100 mg kg^-1). Error bars represent the standard error of the mean (n = 6) and different letters represent a significant difference (Tukey p < 0.05).

Foliar weight ratio and dickson quality index

Nonlinear growth analysis in many cases is more reliable, describing growth more objectively. In addition, these analyzes have a capacity for data synthesis, which allows their interpretations in a more practical way (Fernandes et al. 2014). One of the main nonlinear analyzes used is the leaf weight ratio, which represents the portion of dry mass produced through photosynthesis and incorporated into the leaves (Benincasa 2003).

Figure 5
Leaf dry mass (a), stem dry mass (b), root dry mass (c) and root shoot ratio (d) of Talinum triangulare seedlings in five zinc application treatments (T1 = control; T2 = 12.5 mg kg^-1; T3 = 25 mg kg^-1; T4 = 50 mg kg^-1 and T5 = 100 mg kg^-1). Error bars represent the standard error of the mean (n = 6) and different letters represent a significant difference (Tukey p < 0.05).

In this work, the leaf weight ratio (Fig. 6a) had a significant difference for seedlings from the 100 mg kg^-1 treatment, contrary to the study by Jamami et al. (2006), in which there were no statistical differences for the variable. This result indicates that most of the total weight of these seedlings belonged to the leaves, which agrees with the results of the dry mass of leaves, which also differed significantly in this amount of Zn application. According to Falqueto et al. (2009), the increase in the leaf weight ratio indicates an allocation of photoassimilates to the developing leaves, which act as drains, decreasing with maturity. The application of 100 mg kg^-1 of Zn to the seedlings allowed for an increase in the number of leaves, and many were still in the leaf blade expansion phase when they were removed for measurement, which may have contributed to this result.

Regarding the Dickson quality index (Fig. 6b), another nonlinear analysis that serves to assess the robustness of the seedlings (Dickson et al. 1960), it differed significantly for the seedlings from the 100 mg kg^-1 treatment. A positive correlation was also verified between this index and the application of solutions containing Zn in the work of Nordi et al. (2020). According to Azevedo et al. (2010), the DQI is a good indicator of the quality of seedlings, as it assesses their vigor and balance of biomass disposal. Thus, it can be inferred that these plants exposed to Zn application had better growth and development attributes, which can serve as a favorable characteristic for the biofortification of the species.

Figure 6
Leaf weight ratio (LWR) (a) and Dickson Quality Index (DQI) (b) of Talinum triangulare seedlings in five zinc application treatments (T1 = control; T2 = 12.5 mg kg^-1; T3 = 25 mg kg^-1; T4 = 50 mg kg^-1; T5 = 100 mg kg^-1). Error bars represent the standard error of the mean (n = 6) and different letters represent significant difference (Tukey p < 0.05).

Seedlings submitted to a dose of 100 mg kg^-1 of Zn showed better growth and development results, providing an increase of 130% in leaf dry mass, as well as in Zn contents (increase of 4,081% of leaf Zn and 1,904% of Zn in the soil) and proteins, indicating that this is the most effective dose to be applied in biofortification methods for waterleaf, although the dose of 25 mg kg^-1 has also shown positive increment results, mainly of increase in the levels of leafy Zn.

The quantity of 400 mg kg^-1 provided symptoms of toxicity in the plants, indicating that its threshold is between 100 mg kg^-1 and 400 mg kg^-1 of Zn.

New studies are necessary to establish the toxicity threshold for this species, as well as to increase and enable this technique, both for waterleaf and other leafy vegetables in the field, to bring the knowledge produced in the academy to the population.

Acknowledgement

To the Brazilian Agricultural Research Corporation (EMBRAPA), for carrying out the zinc analyses; to the Laboratory of Bioprospecting and Experimental Biology, and the Multidisciplinary Laboratory of Applied Biology, for supporting the use of the equipment and carrying out analyses.

This work was supported by the Federal University of Western Pará (UFOPA) (public notice 10/2018 PROPPIT-UFOPA (Program to Support Course Completion Works - PROTCC) and by the National Council for Scientific and Technological Development (CNPq) (Grant of the Institutional Scholarship Program for Initiation in Technological Development and Innovation (PIBITI) 166522/2019-8).

Data availability statement

In accordance with Open Science communication practices, the authors inform that all data used in this manuscript is publicly available.

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Edited by

Area Editor:

Dra. Georgia Pacheco

Publication Dates

• Publication in this collection
  07 June 2024
• Date of issue
  2024

History

• Received
  19 Aug 2022
• Accepted
  19 Oct 2023