The population growth rate on the planet is 1.08% per year. The current population count is around 7.6 billion [1
]. It is estimated to exceed nine billion by 2050 [2
]. Considering that the primary sources of nutrients come directly or indirectly from foods produced by agriculture, this population increase strongly pressures the environment to meet such a demand [3
]. Increasing crop yields has been important for reducing global undernourishment from about 19% to 13% between 1990 and 2010 [5
]. However, micronutrient deficiency currently affects almost half of the world’s population, and Zn deficiency is one of the major public health concerns in developing countries [6
In humans, Zn deficiency is associated with poor dietary diversification [7
] and is exacerbated by its poor availability in soils [8
]. Tropical soils present naturally low fertility and low Zn concentration (0.6 to 2.0 mg kg−1
), affecting approximately 90% to 95% of the native Brazilian Cerrado soils [9
]. Moreover, the availability of this micronutrient is dependent on several soil properties, such as pH or capacity for phosphorus adsorption [10
]. Thus, fertilization with Zn is a good strategy to enrich agricultural products and increase the daily Zn intake of the population [11
], especially for those who consume it in small amounts daily. According to the National Research Council, the recommended dietary allowance (RDA) of Zn that an adult needs is about 11 mg; however, one-fifth of the world’s population consumes deficient quantities of this micronutrient [6
In this sense, agronomic biofortification seeks to increase the nutrient concentration and bioavailability in the edible parts of plants. It is considered the most economical solution for micronutrient deficiency in humans and animals [14
]. Several studies have shown that Zn fertilization is efficient at increasing the Zn concentration in foods [13
]. Yet, leafy vegetables are more suitable for achieving higher Zn concentrations than fruits, tubers, or seeds, since Zn is mainly transported through the xylem of plants [8
]. White et al. [16
] found a linear increase in shoot Zn concentrations in different cabbage and broccoli genotypes grown under Zn applications with the Zn nitrate as the substrate. Those authors found a wide range of critical shoot Zn concentrations for cabbage (74–1201 mg kg−1
) and broccoli (117–1666 mg kg−1
), which exceeded the estimated potential for Zn biofortification of leafy vegetables (up to 700 mg kg−1
]. For lettuce, the range of optimal leaf Zn concentrations is 20 to 60 mg kg−1
]. However, Padash et al. [19
] found increases up to 185 mg kg−1
without toxicity effects on lettuce growing with 10 mg L−1
of Zn. This could be a significant contribution as a biofortified crop, since lettuce is the main leafy horticultural produce worldwide, with a production increase of about 62% in the least two decades [20
Although studies have been carried out to prove the agronomic efficiency of Zn biofortification in agricultural products, it is interesting to address genotype–soil interactions and their effects on agronomic biofortification efficiency [8
Thus, a better understanding of how Zn biofortification of lettuce is influenced by production factors may provide greater efficiency for this technology. This study evaluated the response of lettuce genotypes to Zn application for biofortification in two soils.
2. Materials and Methods
2.1. Location and Experimental Design
The experiment was carried out in greenhouse conditions at the Federal University of Lavras, Brazil (21°13′35″ S, 44°58′43″ W, altitude of 918 m). According to Koppen’s classification (1936), the climate is humid subtropical (Cwa), mesothermal, with dry winters and the temperature of the coldest month is between −3 and 18 °C, with a rainy subtropical summer. The temperature of the hottest month is higher than 22 °C.
The experiment was completely randomized and distributed in a 5 × 3 × 2 factorial design, as follows: Five doses of Zn (0, 5, 10, 20, and 30 mg kg−1) as Zn sulphate (ZnSO4), three lettuce genotypes (‘Grand Rapids’, ‘Regina de Verão’, and ‘Delícia’), and two soil types (Red-Yellow Latosol, RYL; and Dystroferric Red Latosol, dRL) in three replications.
2.2. Installation and Conduction of the Experiment
Samples from two soils were collected at a depth of 0–20 cm. The dRL and RYL samples were collected from a native forest and grazing soil, respectively, of the Lavras municipality. They were air-dried, sieved to <2 mm, and subjected to physicochemical soil analysis according to Corguinha et al. [10
] and mineralogical analysis according to Souza et al. [21
] (Table 1
Liming was performed with lime (CaO: 38.8%, MgO: 13.4%, PRNT: 94.4, Ca: 27.72%, and Mg 8.0%). The soil remained incubated for 30 days, with 70% of the total pore volume occupied by deionized water. After the incubation period, soil pH was 6.0. Then, soil samples received the following basic fertilization: 100 mg kg−1 of N, 200 mg kg−1 of P (MAP—60% of P2O5 and 11% of N), 100 mg kg−1 of K (KCl), 37.5 mg kg−1 of Mg, 50 mg kg−1 of S (MgSO4 7H2O), 0.5 mg kg−1 of B (H3BO3), 1.5 mg kg−1 of Cu (CuSO4 5H2O), 5 mg kg−1 of Mn (MnSO4 H2O), and 0.1 mg kg−1 of Mo (Na2MoO4 2H2O). Along with the basic fertilization, the treatments (Zn doses) were applied.
Samples of each soil were again incubated for 10 days with 70% of the total pore volume occupied by deionized water. After this second incubation, the soil samples were individually harrowed. Then, the lettuce was sown, and after seven days, the most vigorous plant was left in each pot containing 5 dm−3 of soil. The water was replaced daily. At 25 and 40 days after sowing, 100 mg kg−1 of N (NH4NO3) and 100 mg kg−1 of K (KCl) were applied as a cover fertilization. Harvesting was performed 55 days after sowing by cutting lettuce plants close to the soil.
2.3. Growth and Biofortification with Zn
After harvesting the plants, the shoots and roots were washed with running deionized water, and the samples were placed to dry in a forced air oven at 65 to 70 °C. The shoots (SDM) and roots (RDM) dry matter were determined. Only the shoots were ground and subjected to Zn analysis. Subsequently, the dried tissues (approximately 200 mg) were weighed and acid-digested in 2.0 mL of HNO3 with 2.0 mL of HClO4 at 120 °C for 60 min, and then at 220 °C until HClO4 fumes were observed. The Zn concentration in SDM was determined by an atomic absorption spectrophotometer (PerkinElmer Inc., AAnalyst 800®, San Jose, CA, USA). A standard reference material from the National Institute of Standards and Technology (NIST 1573a containing 30.94 mg kg−1 of Zn), as well as a blank sample were used in each batch of digestion for quality assurance and quality control in Zn determinations, which were considered satisfactory (>90% recovery).
2.4. Statistical Analysis
Data were subjected to analysis of variance and, when significant at 5% by the F test, a polynomial regression analysis was performed for Zn doses. The significant equations with the highest coefficient of determination were chosen (R2
). Linear correlation analyses were also performed between the Zn contents of the lettuce genotypes and the Zn doses applied to the soil. Analyses were run on the AgroEstat software [22
According to the results, all three genotypes of lettuce studied have a different ability to be biofortified in the same soil. By applying the dose for highest SDM yield, the ‘Grand Rapids’ (15.6 mg kg−1
), ‘Regina de Verão’ (4.4 mg kg−1
), and ‘Delícia’ (0 mg kg−1
) genotypes accumulated 781.3, 265.4, and 61.9 µg of Zn in the shoots of plants grown in RYL, respectively. Therefore, the ‘Grand Rapids’ genotype was enriched more with Zn without a loss of yield and presented a greater capacity for Zn biofortification. Micronutrient fertilization has been more successful in increasing the Zn concentration in the edible portion of leafy vegetables [13
] than plants whose edible parts are reserve organs [17
A similar linear increase of the leaf Zn concentration as in the three lettuce genotypes in both soils (Figure 2
) was also found by White et al. [16
] on genotypes of cabbage and broccoli grown in substrate. This is because the plants are natural extractors of minerals from the soil, without the distinction of how much is absorbed. This leads to phytotoxicity and consequently low crop yields, as found in our results (Figure 1
). The increased availability of this micronutrient in soil coupled with its predominantly xylem transport causes a greater concentration in the organs (leaves) with s higher transpiratory rate [24
]. However, the SDM yield was significantly reduced in all three genotypes at the dose of 30 mg kg−1
, because, although lettuce leaves were strongly Zn enriched, their biofortification capacity was limited by phytotoxicity.
Moreover, the Zn availability from fertilizer depends on the soil conditions or types. The soils used in our study (Table 1
) differed regarding their ability to make Zn available to plants and consequently biofortify lettuce genotypes. The clay-textured dRL has a lower concentration of P and and Mn2+
, higher buffer capacity, higher Fe and Al oxide contents, and higher organic matter and gibbsite (35.9%) than RYL. These features increase Zn adsorption and decrease its availability to plants [23
]. In addition, increasing pH by liming in both soils may also reduce the availability of this micronutrient, especially in dRL [26
]. Pongrac et al. [23
] found that soil types affected red cabbage growth more than phosphorus and Zn treatments. Therefore, these authors concluded that more studies in different types of soils are necessary to investigate their impact on the biofortification of the edible parts of crops. This suggests that the increases in the concentrations of essential mineral elements in the edible portions of crops are subject to the plant absorption capacity and to the restrictions imposed by the environment [16
The biofortification methods should be used considering the genotype × soil interaction, since genotypes distinctly respond to different soils [10
]. ‘Regina de Verão’ and ‘Delicia’ grown in RYL had the lowest tolerance to an increase in micronutrient concentrations, as demonstrated by the negative correlation found in these genotypes between biomass production and leaf Zn concentration (Figure 3
c,e). Thus, we recommend biofortification with 7.8 and 3.7 mg kg−1
of Zn to the RYL and dRL, respectively, since higher doses resulted in a 10% loss of SDM yield of these genotypes. At these doses, we reached 31.2 and 17.1 mg kg−1
of Zn in the SDM of the ‘Regina de Verão’ and ‘Delicia’ genotypes, respectively. By contrast, the Zn dose of 15.6 mg kg−1
in the RYL maximized the SDM yield of ‘Grand Rapids’ and increased the leaf Zn concentration up to 50.3 mg kg−1
, almost a 250% higher micronutrient concentration than in plants not fertilized with Zn. If we consider the possibility of a 10% yield loss in SDM as in the other genotypes, ‘Grand Rapids’ would have its biofortification increased to 83.2 mg kg−1
Zn. On the other hand, ‘Delícia’ was the genotype with the highest Zn concentration (27.1 mg kg−1
) in SDM, followed by ‘Grand Rapids’ with 25.7 mg kg−1
, both grown in dRL soil.
The average concentration of Zn (43.8 mg kg−1) from the highest leaf Zn concentrations were found for ‘Regina de Verão’ (31.2 mg kg−1), ‘Delicia’ (17.1 mg kg−1), and ‘Grand Rapids’ (83.2 mg kg−1) without a loss of SDM yield. Therefore, if we assume that a daily serving size of the Zn-enriched lettuce is approximately 30 g fresh weight (or 3 g dry weight due to its 90% water content), consumption of the average Zn-containing lettuce (43.8 mg kg−1) would result in the ingestion of 0.13 mg Zn, equivalent to 1.2% of the recommended dietary allowance for Zn (RDA; 11 mg day−1 Zn). Although these increments pose a small contribution to people’s daily Zn intake, due to being the most widely produced and consumed leafy vegetable in the world, its biofortification will contribute to Zn intake.
Our results confirm the hypothesis of interaction between lettuce genotypes and soil conditions on the efficiency of agronomic biofortification supported by other studies [8
]. Therefore, genetic and agronomic biofortification programs should be improved together, as their success depends on each other. Regardless of whether a new cultivar developed can absorb Zn, its success will depend on the availability of the Zn pool in the soil. In addition, plant breeding seems to be the most viable and economical way to reach rural populations with limited access to agronomically biofortified crops [14