Microgreen is a collective term used for vegetable or herb seedlings consumed at a young growth stage, with expanding cotyledons or the first pair of true leaves, harvested 7 to 21 days after germination [1
]. As a new specialty crop, the market demand for microgreens has been rapidly increasing in recent years [5
]. Microgreens are popular items at local markets and are used by chefs and consumers to enhance flavor, color, and texture in various foods [2
]. The market value of microgreens is 30 to 50 USD per pound [7
], drawing interest among vegetable growers for the high value and short production cycles [8
]. A number of plant species in the families of Amaranthacea, Apiaceae, Asteraceae, Brassicaceae, Fabaceae, and Lamiaceae have been produced as microgreens [5
Microgreens are considered to be a functional food and have high concentrations of mineral nutrients and phytochemicals that contribute to health benefits [11
]. For example, microgreens in the Brassicaceae family are rich sources of mineral nutrients including potassium (K), Ca, iron (Fe), and zinc (Zn) [5
]. Basil (Ocimum basilicum
) and Swiss chard (Beta vulgaris
) microgreens have been reported to be excellent sources of K and Mg [14
microgreens also have high contents of healthy phytochemicals including ascorbic acid, phylloquinone, carotenoids, tocopherols, glucosinolates, and polyphenols [15
]. Species vary in their nutrient profiles. Cultural practices including pre-sowing seed treatments, seeding rate, and fertilization, along with the microenvironment including temperature, light, and growth medium may all affect yield and nutrient compositions of microgreens [3
Fast and uniform germination is a key aspect in successful production of microgreens. Pre-sowing seed treatments including seed priming and soaking were examined for their effects in advancing seedling emergence [16
]. Lee et al. [19
] found priming seeds in grade five vermiculite was the most effective pre-sowing treatment in advancing germination of microgreen beet (Beta vulgaris
) and chard compared to seed soaking in a variety of solutions including water, hydrogen peroxide, or hydrochloric acids. Seed soaking in cold water was recommended for large seed crops including pea (Pisum sativum
) and radish (Raphanus sativus
), but not for small seed species including a number of Brassica
crops, or mucilaginous seeds such as basil that develop a jelly-like coating when wet [18
]. Imbibing seeds can leak several compounds including amino acids, inorganic ions, sugars, phenolics, and proteins due to the inability of cellular membranes to function normally until seeds are fully hydrated [22
]. There is lack of research-based information regarding the effects of seed soaking on crop yield and nutritional content of microgreens.
Fertilization management is another important aspect in promoting fast growth and high yield of microgreens [23
]. Fertilizer premixed in growing substrate or applied post-emergence through soluble fertilizer were both investigated for their effect in the production of microgreens [3
]. Daily post-emergence fertigation with 150 mg·L−1
N was found to be of the most economical fertilization treatment for improving fresh shoot weight of arugula (Eruca vesicaria
) microgreens [16
]. Kou et al. [24
] found 10 mM of calcium chloride solution applied daily for ten days increased biomass of broccoli (Brassica oleracea
) microgreens as compared with water and improved visual quality during storage. Plant species, slow versus fast growing, may differ in their requirements for fertilization [7
]. There is concern that some crops such as arugula tend to accumulate excessive nitrate [23
]. Crop-specific fertilizer requirements with respect to shoot yield and mineral nutrients in microgreen production remain unclear.
The objective of this study was to investigate the effects of pre-sowing seed soaking and post-emergence fertilization on shoot growth and mineral nutrients of ten microgreen species.
2. Materials and Methods
2.1. Plant Materials and Microgreen Culture
Ten species were grown as microgreens (Table 1
) and evaluated for shoot growth and mineral nutrient concentrations, in a greenhouse on the campus of Mississippi State University in Starkville, MS, USA (33.4552° N, 88.7944° W). Microgreen seeds of all selected species were purchased from True Leaf Market (Salt Lake City, UT, USA). Seed sowing rate for each species was as determined by the supplier’s recommendation and summarized in Table 1
. Hundred-seed weight of each species was measured in triplication. This study included two experiments with the first experiment conducted on 15 November 2018, and then repeated on 7 January 2019.
Seeds of appropriate weight were measured and manually sown into black plastic trays with drainage holes (width 25.72 cm, length 25.72 cm, depth 6.03 cm, T.O. Plastics, Clearwater, MN, USA) and filled with a peat-based soilless substrate (PRO-MIX BX general purpose, Premier Tech Horticulture, QC, Canada). After sowing, seeds were gently pressed into the medium with the bottom of another tray for maximum substrate contact and covered with an additional thin layer of substrate. The temperature in the greenhouse was set at 25 °C with natural light.
Prior to seed sowing, half of the seeds for each species were soaked with tap water (250 mL of water for pea seeds, and 100 mL for all other species) at room temperature (approximately 20 °C) for 6 h. Soaked seeds were drained and mixed with a handful of growing substrate and evenly distributed into the growing tray. All microgreens were hand watered as needed, approximately, once per day until harvest. After seed germination, half of the trays from each species were fertigated once with 120 mL of water-soluble fertilizer 20N-8.7P-16.6K (Peters® Professional 20-20-20 General Purpose, also containing (wt/wt) 0.05% Mg, 0.05% Fe, 0.025% Mn, 0.013% boron (B), 0.013% copper (Cu), 0.005% molybdenum (Mo), and 0.025% Zn, ICL Specialty Fertilizers, Tel-Aviv, Israel) at a rate of 100 mg·L−1 N four days after planting. The fertilizer solution had a pH of 6.56 and electrical conductivity of 0.41 mS·cm−1. As a control to the fertilization treatment, the other half of the trays were irrigated with the same volume of water (pH 7.54, EC 0.15 mS·cm−1).
2.2. Shoot Harvest and Data Collection
Microgreens grown in each tray were carefully harvested above the substrate surface, with the expanding cotyledons (microgreen stage 1) or with the first pair of true leaves (microgreen stage 2), as described by Waterland et al. [4
]. Fresh shoot weight of microgreens harvested from each tray was measured. Then, fresh microgreen shoots from each tray were oven dried at 60 °C until constant weight and measured for dry shoot weight (DW). Dry weight percentage (%) was also determined. Plant height was measured in each tray before being harvested from the substrate surface to the highest point of shoot growth. Each tray of microgreen species was given a visual quality rating from 1–5, where 1 = seedlings cover 20% of the growing surface area or less, 2 = seedlings cover 20% to 40% of the growing surface area, 3 = seedlings cover 40% to 60% of the growing surface area, 4 = seedlings cover 60% to 80% of the growing surface area, and 5 = seedlings cover over 80% of the tray surface area with healthy plant growth.
2.3. Mineral Nutrient Analyses
Dry microgreen samples were ground to pass a 1 mm sieve with a grinder (Wiley mini mill, Thomas Scientific, Swedesboro, NJ, USA) for mineral nutrient analyses. Combustion analysis was used for the determination of total N concentration with 0.25 g of dry tissue using an elemental analyzer (vario MAX cube, Elementar Americas Inc., Long Island, NY, USA). A dry tissue sample of 0.5 g was digested with 1 mL of 6 M hydrochloric acid (HCl) and 50 mL of 0.05 M HCl for the concentrations of phosphorus (P), K, Ca, Mg, Cu, Fe, Mn, Zn, and B using inductively coupled plasma optical emission spectrometry (SPECTROBLUE, SPECTRO Analytical Instruments, Kleve, Germany). Microgreen samples were tested at the Mississippi State University Extension Service Soil Testing Laboratory. Concentrations of macronutrients (mg·g−1 DW) and micronutrients (µg·g−1 DW) in microgreens were presented on a dry weight basis.
2.4. Experimental Design and Statistical Analyses
This experiment was conducted in a randomized complete block design with a factorial arrangement of treatments. Microgreen species (10 species), pre-sowing seed treatment (seed soaking or not), and fertilization (fertilizer or not) served as the three main factors. Each treatment had four replications with an individual growing tray as the experimental unit. Significance of any main effect or the interaction among main factors were determined by analysis of variance (ANOVA) using GLMMIX procedure of SAS (version 9.4, SAS Institute, Cary, NC, USA). Where indicated by ANOVA, means were separated by Tukey’s honest significant difference at α ≤ 0.05. Data from the two experiments were compared as repeated measures, where experimental date was used as a factor to analyze its effect. All statistical analyses were performed using SAS.
The ten tested microgreen species varied in their yields in terms of fresh and dry shoot weights, consistent with reported ranges [3
]. Several factors affect yield of microgreens, including seeding rate, fertilization, growing medium, pre-sowing seed treatment, harvest stage, and microenvironment including temperature and lighting conditions [3
]. Low yield has been considered to be one of the limiting factors in microgreen production [24
]. Microgreen yield of a given species varied between the two experiments (November 2018 and January 2019) in this study, likely due to changing microenvironment in the greenhouse. Air temperature in the greenhouse mostly fluctuated within 5 °C as compared with the setting of 25 °C, and relative humidity ranged from 30% to 70% within the experiment duration. Light conditions in the greenhouse were not recorded in this study and fluctuated drastically within a year. According to past records, daily light integral between November and February was generally the lowest of the year in the local area [28
], which limits microgreen growth without supplemental lighting. Growers could experience fluctuations in microgreen yield between production cycles throughout a year.
A meaningful evaluation of microgreen yield should take seed cost into consideration, since seeds are used in large quantity and represent a major part of production costs [9
]. Seeding rate in this study ranged from 52.9 g·m−2
in basil to 1285 g·m−2
in pea (Table 1
), equivalent to 8378 seeds per m2
for pea to 69,807 seeds per m2
for amaranth, mostly consistent with ranges of 10,000 to 40,000 seeds per m2
, as reported by Di Gioia and Santamaria [29
]. Increasing seedling rate increases microgreen fresh yield but decreases mean shoot weight [3
]. Crop-specific information investigating the interaction between seeding rate and yield, and quality of microgreens merits further investigation. This is especially challenging with the expanding microgreen industry and the constantly increasing number of species and varieties being produced as microgreens [5
Microgreens varied in their nutrient profiles including mineral nutrient and phytochemical concentrations [13
]. The mineral nutrient concentrations of microgreens tested, in this study, were generally within the ranges as reported by Waterland et al. [4
] and Xiao et al. [13
]. There are a number of reports regarding how light quality and quantity affect nutritional content of microgreens [5
], with fewer reports investigating the effects of cultural practices on such variables.
One-time fertilization increased fresh shoot weight and concentrations of N, P, K, Fe, Zn, and B in microgreens in one or both experiments in this study. On the one hand, the fertilization treatment decreased Ca concentration in November 2018, decreased Mg in both experiments, and decreased Mn concentration in January 2019. The water-soluble fertilizer, used in this study, provided macronutrients (20N:8.7P:16.6K), 0.05% Mg, and micronutrients including 0.013% Cu, 0.05% Fe, 0.025% Mn, 0.013% B, and 0.025% Zn, and therefore increased concentrations of macro- and micro-nutrients in the microgreens. The decreased Ca concentration could be due to the dilution effect in microgreens without a source of Ca from the fertilizer. Bulgari et al. [23
] reported that low Ca concentration was commonly found in some microgreens. On the other hand, calcium chloride application was reported to increase Ca concentration, increase biomass, and improve postharvest quality in broccoli microgreens, making it a better Ca source to human nutrition [24
Further investigation is needed regarding crop-specific requirements for fertilization rate and frequency with respect to microgreen yield and quality. There are concerns that some microgreens such as arugula accumulate high levels of nitrate, which is considered to be an unhealthy factor [23
]. However, this was believed to be controlled by lowering fertilization rate or selectively controlling N fertilizer form [28
]. A positive aspect is that microgreen Swiss chard and arugula were reported to have lower nitrate concentrations as compared with their baby leaf or adult stage counterparts [23
]. In other cases, seeds were intentionally biofortified to increase certain beneficial micronutrients, for example, selenium (Se), to increase Se concentration in basil microgreens [35
The seed soaking treatment consistently decreased fresh and dry shoot weights, shoot height, and concentrations of macro- and micro-nutrients in tested microgreen species when there was an effect, with the exception that soaking increased Cu concentration in pea in November 2018 and January 2019. This possibly resulted from the fact that dehydrated cellular membranes in imbibing seeds are dysfunctional, resulting in a leak of nutrients such as inorganic ions, amino acids, carbohydrate, and phenolics [22
]. When evaluating pre-sowing seed treatments including seed soaking or priming, their effects on shoot production and nutritional content of microgreens should be examined in addition to the efficacy in advancing seedling emergence [19
Most microgreen species, investigated in our study, were considered to be fast growing microgreens. Nine species germinated within 36–48 h regardless of seed soaking, with basil having the slowest germination. We observed advanced germination of approximately 12 to 24 h, but similar harvest dates within a given species. Seed soaking might be valued more in slow growing species like basil than fast-growing species for accelerating germination and shortening the production cycle. The use of seed soaking treatment should be weighed against the fact that it may decrease microgreen yields, since, in our study, fresh shoot weight and shoot height of microgreens were decreased by soaking in January 2019. Human pathogen contamination has become one of the major food safety concerns in microgreen and sprout production [36
]. It is important to purchase high quality seeds from reliable suppliers that have a high germination percentage, 85% to 98% in this study according to the label, and the seeds should be certified for microgreen and sprout production for reduced pathogen risks [5