Space Flight Cultivation for Radish (Raphanus sativus) in the Advanced Plant Habitat

To select the most reliable variety of Raphanus sativus, we tested germination rates of seeds from various cultivars (Table 1). Fast and reliable germination (24 h after imbibition) and uniform seed size were the main selection criteria. Based on the projected maturation time and reliable germination, we chose the variety “Cherry Belle” for our experiments.

To minimize the effect of surface-borne microorganisms, we sanitized seeds based on several protocols, including bleach and ethanol (Table 2), and noticed high mortality after as little as 1-minute exposure to 70% ethanol. Therefore, sanitation was based on immersion in 20% bleach for 10 min, and three times rinsing (5 min) in autoclaved (121°C, 20 min) deionized water and draining. After complete removal of the last rinse, seeds were blotted dry with Kimwipes, and air dried for >5 h. Subsequently, the sanitized seeds were stored in autoclaved polypropylene tubes.

Prior KSC-based experiments used arcillite (calcined Montmorillonite commercially available as Turface Pro League). Its porous structure, neutral pH, low density (~0.63 g · cm⁻³), and hydrophilicity suggested its use as growth substrate (Adams et al., 2014). The commercial material was sifted to obtain grains between 1 mm and 2 mm. Preliminary studies showed that arcillite does not contain necessary nutrients to support plant growth. Therefore, arcillite was supplemented with half-strength, modified (chloride-free, because of corrosion concerns) Murashige and Skoog medium (Table 3). Equal weights of dried and autoclaved arcillite and modified Murashige-Skoog (MS) medium at final concentration were combined, soaked for 24 h and dried (70°C for 72 h). The fertilized, dried arcillite can be stored indefinitely.

Each of the four quadrants of the SC was filled with ~1600 mL of fertilized, dry arcillite. The substrate was filled and tamped down to fill all the available spaces in and around the sensors and porous tubes. Medical gauze was placed above the arcillite to keep the substrate in place. A hole (~5 cm diameter) was cut in the gauze to accommodate OASIS foam (Smithers-Oasis, Kent, OH) pucks that accommodated the seeds. Another layer of medical gauze was placed on top to secure the oasis foam (Figure 1). The top-layer gauze was split by two perpendicular cuts of ~2 cm length each. In addition to retaining the foam, the gauze also provided visual feedback of the wetness and thus the water level of the entire setup. A layer of orthopedic foam with cutouts for the floral foam secured everything under the SC covers. Dry sanitized seeds were glued with water soluble glue (polyvinyl acetate, Elmer's clear glue) ~5 mm deep into the foam such that the micropyle was positioned toward the arcillite. The foam provided water for seed imbibition and germination, and its flexibility allowed for the expansion of the developing bulb (used here instead of the anatomically correct description of “swollen hypocotyl,” Figure 2).

Figure 1

Figure 2

The ECS of the APH unit was set to the following parameters: Photoperiod: 16 h light/8 h dark; Temperature: 24°C day/20°C night; Relative humidity 65%; CO₂ Concentration: 3500±3% ppm; and Air speed: 0.9 m/s based on fan rotational velocity.

One of the parameters that strongly affects the development and growth of radishes is light quality and fluence (Samuolienė et al., 2011; Yorio et al., 2001). Since the GLA is APH-specific, we used an alternate, programmable, high intensity LED based light fixtures (Heliospectra RX30), which provides up to 1000 μmol m⁻² · s⁻¹ photosynthetically active radiation (PAR). These light fixtures provide a more versatile light spectrum and higher light output than the APH system but are of smaller size. The relatively lower fluence rates of the GLA were adjusted by reducing the output of the LEDs and increasing the distance between the light fixture and the growth surface. Laboratory experiments produced comparable results to the experimental units [APH and the lower-fidelity Engineering Development Unit (EDU)] at the KSC.

Dried and ground leaf and bulb tissues (~100 mg) were digested in 2 mL aliquots of 70% trace metal grade nitric acid (Fisher Chemical A509-P212) for 72 h. The digested samples were diluted with nanopure water (>10 MΩ) to 10 mL, filtered, and analyzed via Inductively Coupled Optical Emission Spectrometry (Perkin Elmer, Optima 5300 DV). A multielement standard (Inorganic Ventures, Christiansburg, VA) was diluted to the same matrix concentration and used for calibration.

The total nitrogen (N) content from dried and ground leaf and bulb (~100 mg) tissue material was analyzed via LECO TruMac Nitrogen analyzer using EDTA as calibration material.

Arcillite is one of the favored rooting systems for space because it reduces the chances of root zone hypoxia (Porterfield et al., 2000). However, it does not release sufficient minerals, and therefore cannot support long-term plant growth (Figure 3). Our approach of infusing arcillite with MS medium not only produced sizeable radishes but also allowed quantitation of the amount of nutrients utilized by the plants (Figure 4). Such information from space grown plant materials will be extremely valuable in understanding the mineral uptake of plants past the seedling stage under space environment.

The susceptibility of radish growth to plant density and spacing clearly showed improved plant performance when the number of plants per quadrant was reduced to five plants (Table 4). Although these data support the view that improved plant performance can be obtained as a result of substrate conditions and reduced shading, they do not address any below-surface competition. The APH design prevents variation and thus optimization of the root space, a notion especially important for plants that develop root or hypocotyl-based storage tissue. In radish, high planting densities are likely to affect photomorphogenic mechanisms as total light fluence and red to far-red ratios are known to reduce vegetative growth (i.e., bulb formation) and accelerate flowering (Weston, 1982). Thus, by decreasing the number of plants, at least two factors were affected; (1) relatively large-sized bulbs were obtained, and (2) the tendency of bolting was reduced. Equally important was the provision of larger growth areas rather than elongated slots as the modification reduced restrictions and injuries (Figure 5). Enhanced mechanical stress as well as irregular expansion due to inconsistent watering resulted in defects on the bulb surface. Abrasion and reduced integrity of the bulb epidermis increases the chances of microbial growth. The overall roughness of arcillite may also contribute to an indirect, but testable, increase in potential microbial contamination. Such effects were reported in growth experiments performed with the VEGGIE hardware (Khodadad et al., 2020) and also reported in Daikon radishes (Shiina et al., 2013). High humidity but absence of precipitation in space may contribute to higher microbial loads than on earth. Thus, microbial contamination needs to be examined, especially for below-ground tissue.

The water loss data (Figure 6) demonstrates two stages of evapotranspiration. Stage 1 (day 1–12) water loss during germination and seedling establishment is minimal and essentially consists of evaporation of water from the SC surface. During stage 2 (day 13–28), the water consumption increases exponentially. Both stages are approximations, as the evapotranspiration rates are known to be affected by humidity and temperature (Nonnecke et al., 1971), plant size (Kim et al., 2011), and light quality (Lim and Kim, 2021). Under constant temperature and humidity levels in our tests, the major driving factor for water usage was leaf size and plant density (Table 4). The watering schedule in the APH was based on the age of the plants such that initial flood filling of the SC provided sufficient water for the first week. The flooding also resulted in reduced activity of the watering system and prevented drought stress during the early stages of plant growth. Additional adjustments involved the air velocity. During the flood-filled stage, the air speed was adjusted to 0.6 m · s⁻¹ but increased to 0.9 m · s⁻¹ during the remainder of the cultivation time.

The sensitivity of radishes to the spectral composition of light shows a prominent role of red and blue light. Increased blue light fluence increased the biomass of radishes (Table 6) and reduced the leaf area, which also resulted in a smaller overall canopy. A similar effect of blue light was described in several studies. Tezuka et al. (1994) reported that radish growth was promoted by UV-A light (λmax = 360 nm) and resulted in higher chlorophyll and vitamin content and larger radish formation. A study on the effect of red and blue light illumination examined combinations of red LEDs (638 nm) supplemented by 455 nm (blue) light on radish physiological indices and it showed that radishes grown under red light alone were elongated, with poor hypocotyl formation (Samuolienė et al., 2011). Interestingly, a small supplement of blue light (5%) enhanced leaf area formation. Higher proportions of blue light (10%) enhanced non-structural carbohydrate distribution between storage organs and leaves and resulted in enhanced radish formation. Thus, supplementation of the blue light not only promotes growth and development in radishes but also plays an important role in nutrient allocation. Light is a dominant stimulus for plants in weightlessness and affects phototropic and gravitropic responses (Sindelar et al., 2014; Vandenbrink et al., 2016). However, to date, the effects of light on biomass production under weightlessness conditions have not been investigated. The light effect also underscores the need to study growth beyond the seedling stage, which has been the focus of most space experiments. During early seedling development, effects of light on biomass accumulation, growth habit, and photomorphogenesis in general cannot be assessed, but these factors are important for adequate biomass production and nutritional value. Future space experiments should therefore include experiments that test the effect of light fluence, spectral composition, and (extended) photoperiod to optimize plant cultivation under space conditions. Similarly, effects of elevated CO₂ concentrations under weightlessness on mineral, vitamin, and lipid content are needed to optimize nutrition of spacefarers.