Seed Priming with Ulva lactuca L. in Cultivars Grown in Martian and Lunar Regolith Analogues

Space agriculture is critical to the survival of humans outside Earth due to its potential to provide a sustainable source of food and generate income. However, numerous challenges must be faced (Oluwafemi et al., 2018; Cannon Britt, 2019; Verseux et al., 2021). For example, due to the lower luminosity levels, which are crucial for plant development, on Mars and the Moon (Cannon Britt, 2019), lighting for plant growth needs to be supplemented and fully controlled (Ming Brady, 1989; Wamelink et al., 2014; Wheeler, 2017). The high level of cosmic radiation on the Martian surface, which is 17 times higher than on Earth, could significantly impact plant growth and lead to a drastic reduction in plant biomass (Tack et al., 2021; Guo et al., 2017). Another challenge is cultivating plants in a regolith-based system where research is being carried out to optimize the use of In-Situ Resource Utilization (ISRU) (Eichler et al., 2021).

The optimization of ISRU aims to adapt the lunar and Martian environments for plant acclimatization (Eichler et al., 2021). Regolith is unconsolidated material overlying rock and may include dust, broken rocks, and other related materials (Fackrell et al., 2021; Duri et al., 2022). Additionally, regolith contains no associated organic matter or microbiome; therefore, it can only be defined as soil if it has undergone organic processes (Duri et al., 2022). Consequently, it is a stressful substrate for plant growth.

Multidisciplinary studies have explored life-support bioregeneration and food production in extreme space conditions (Miller Ward, 1966; Wheeler et al., 2011; Poulet et al., 2016). For example, the use of marine macroalgae has been studied for their ability to convert carbon dioxide and water into oxygen, as well as research with cyanobacteria fed with Martian analogs (MGS-1) and Mars atmosphere as a source of nutrients to be suitable for photobioreactors of life support systems (Miller Ward, 1966; Verseux et al., 2021). Technologies that aim to measure the gas exchange of the entire plant under the influence of different air velocities allow for understanding the photosynthesis measurements performed by the individual (Poulet et al., 2020).

In space agriculture, the challenges to be overcome to ensure efficient growth of cultivars include the production of successful crop cycles or using crops that accumulate a high nutrient content (Wamelink et al., 2019; Llorente et al., 2018).

For future interplanetary nutrition, it is essential to choose crops that require minimum preparation time, provide essential vitamins (B1, K, C), and do not degrade like most bioactive compounds in stored food systems or require processing (Johnson et al., 2021; Poulet et al., 2022). In addition, producing fresh foods may provide crucial nutritional and psychological information for long-term missions and future human settlers in the ideal choice of vegetables with organoleptic properties (Massa et al., 2015; Zhang et al., 2022).

All essential macro and micronutrients for plant growth are present in sufficient amounts, both in lunar and Martian regolith, except the reactive nitrogen (Duri et al., 2022). Nitrogen in absorbable forms (NO₃ and NH₄) is one of the essential minerals necessary for the growth of almost all plants due to the mineralization of organic matter and nitrogen fixation through mutualistic associations between plants and the terrestrial soil microbiome (Stevens et al., 2011). However, organic matter is absent on Mars and the Moon, although both sites contain graphitic carbon (Parnell, 2005), and no microorganism activity has been recorded. The absence of sufficient absorbable nitrogen can be resolved using nitrogen-fixing species (Llorente et al., 2018).

Martian soil exhibits high levels of perchlorate (NaClO₄) (Hecht et al., 2009) that are toxic to humans; therefore, cultivation in this type of soil will need specific care (Eichler et al., 2021). Studies have been carried out to reduce the levels of this toxic substance. For example, some organisms have been suggested as potential candidates for Martian regolith, such as plants of woody species, non-woody terrestrial plants, aquatic plants, and potential microbes that carry out phytoremediation through phytoaccumulation, phytodegradation, and rhizodegradation (Misra et al., 2021), but these authors recommend further studies in the area so that agriculture based on ISRU (In-Situ Resource Utilization) become viable.

Plant germination and performance can be altered or impaired under atypical conditions in soils with no organic matter, such as those on Mars and the Moon. To ensure a self-sufficient civilization, it will be necessary to improve the cultivation of food plants through biotechnology, robotics, and agriculture (Wheeler et al., 2011; Wheeler, 2017; Cannon Britt, 2019) to associate biological systems with local natural resources (Verseux et al., 2016; Cannon Britt, 2019). Thus, these food mechanisms and biological processes must be fully understood (Verseux et al., 2016). In the processing of seeds, techniques or specific treatments before sowing provide faster and more uniform germination and the generation of seedlings more tolerant to abiotic stresses, higher productivity, and crop quality (Paparella et al., 2015; Sivritepe Sivritepe, 2016).

Seed priming techniques with phytohormones have been reported as an effective complement to minimize the effects of environmental, stressful conditions on vegetables (El-Aziz Kasim et al., 2016; García et al., 2020; Hamouda et al., 2022). This technique consists of pre-soaking seeds with the help of substances that contain bioactives, such as phytohormones, that aid in breaking dormancy and metabolic stimulation of seeds (Rhaman et al., 2020). There is an improvement in shoot length and fresh root mass of wheat seedlings, as well as a higher percentage of chlorophyll a, b, and total, carotenoid and wheat grain carbohydrate (Shahbazi et al., 2015) and, in tomato species, there is an improvement in germination and growth mainly of the stem, and it can act as an antibacterial agent (Sekhouna et al., 2021).

According to du Jardin (2015), a plant biostimulant is any microorganism or substance that is applied to plants to promote nutrient uptake efficiency, enhance resistance to non-living stresses, and improve the qualitative attributes of the cultivated variety without relying on the addition of nutrients. Biostimulants are considered efficient when applied in minimal amounts to promote plant growth, and many of these are derived from marine macroalgae (du Jardin, 2015; Yakhin et al., 2017).

Species of the cosmopolitan marine green macroalgae Ulva spp. are indicated as biostimulants and biorefined products by the presence of bioactive substances (Jaulneau et al., 2011; Hernández-Herrera et al., 2014; Sekhouna et al., 2021; Pappou et al., 2022), as well as macro- and microelements, amino acids, vitamins, phytohormones, betaines and sterols that act in stimulating seed germination (Khan et al., 2009; Hafting et al., 2015; Sekhouna et al., 2021; Pappou et al., 2022). For example, marine macroalgal extracts were used to soak seeds with promising results for organic production in species such as Solanum lycopersicum, Abelmoschus esculentus, Ceratonia siliqua, Triticum aestivum, Vigna radiata, Vigna sinensis, and Zea mays (Ibrahim et al., 2014; Divya et al., 2015; Reis et al., 2020; El Boukhari et al., 2021; Hussein et al., 2021; Zouari et al., 2022).

The use of Ulva spp. extracts as a biostimulant is a promising alternative to improve seedlings’ metabolic capacity and physiological development when they are under stressful conditions (El Boukhari et al., 2021). These macroalgae can be indicated when implemented in seed priming techniques by hydration (Rhaman et al., 2021) because it contains hormones essential for the development and stimulation of germination in its composition and allows the seeds to be prepared to receive this biostimulant before germination and for the improvement of their metabolic processes (El Boukhari et al., 2020; Rhaman et al., 2021).

The use of dry and crushed marine macroalgae in the form of powder may reduce costs due to the facility of production and low production costs when compared to the extraction of the polysaccharide called ulvan from the Ulva species, which has been investigated for use in agriculture (Alves et al., 2013; Reis et al., 2020; Putra et al., 2024). The cost of producing macroalgae extract for agriculture can vary widely depending on the species of macroalgae, the extraction method used, and the production scale since exact numbers for the extraction processes and production of commercially available U. lactuca extract proxies were used to estimate the overall cost of production (Alves et al., 2013; Zhang et al., 2022).

In a study by Shahidi et al. (2017), the cost of producing macroalgae extract from Ascophyllum nodosum using a cold-water extraction method was estimated to be around $14.42 per kg. Another study by Eom et al. (2019) investigated the cost-effectiveness of using macroalgae extract as a biofertilizer in rice cultivation. The authors reported that the cost of producing the macroalgae extract was around $13.16 per kg when using a hot water extraction method.

Multiple studies have estimated the cost of payloads based on various mission designs and launch vehicles (Table 1). For example, Musiolik et al. (2021) estimate the cost per kg to be between $4,000.00 and $40,000.00, depending on the launch vehicle and other factors. Sowers et al. (2020) present a commercial approach to affordable access to lunar resources, including the cost of payloads. They estimate the cost per kg to be between $1,000.00 and $5,000.00 for regular missions and between $5,000.00 and $10,000.00 for long-term missions (used as a Martian proxy). Maruyama et al. (2021) discuss the potential of SpaceX’s Starship to reduce the cost of payloads to Mars and the Moon, estimating the cost per kg to be around $200.00 to $300.00, assuming a fully reusable launch system and cost-saving measures.

Assuming a payload cost of $20,000 per kg (the midpoint of the range provided by Musiolik et al. (2021), the cost of using the tested amounts of 0.2 and 0.4 g · L⁻¹ of macroalgae extract on missions would be approximately $0.40 and $0.80, respectively.

Assessing the germination and emergence of seedlings from cultivars treated with U. lactuca L. is a method to investigate potential crosstalk between the bioactive compounds of marine macroalgae and seed metabolism. (Reis et al., 2020). The germination and emergence of seedlings of cultivars when subjected to the treatment of this macroalgae allows for the assessment of potential effects on seed germination and seedling growth (Hamouda et al., 2022; Hernández-Herrera et al., 2023). By analyzing the presence of bioactive compounds in U. lactuca and its phytochemical extracts, as well as conducting hormonal analysis of seeds, we can understand the mechanisms by which these compounds may influence seed metabolism, providing insights into the potential crosstalk between marine macroalgae, bioactive, and seed physiology (Hamouda et al., 2022). To this end, it is essential to analyze the presence of bioactive compounds of U. lactuca and its phytochemical extracts for compression of its composition and hormonal analysis of seeds.

Therefore, to improve biosynthetic capacity under adverse conditions, as well as biomass production, techniques should be considered that allow for increased vigor and hormonal indices of these plants (Llorente et al., 2018) to play a role in the development of a self-sustaining civilization (Verseux et al., 2016).

Previous studies have demonstrated the success of the cultivation of viable crops in Martian and lunar regolith-based agriculture (RBA) with nutrient supplementation (Wamelink et al., 2014; 2019; Eichler et al., 2021; Kasiviswanathan et al., 2022), but some of these studies did not consider important aspects, such as complete sterilization of the regolith and, in Martian analogs, the addition of perchlorate to the regolith. Although it is considered a viable solution for a continuous and self-sustaining alternative to in-situ cultivation, the use of Martian and lunar regolith as the answer to complete ISRU-dependent missions remains to be determined (Eichler et al., 2021).

In this study, we proposed to conduct experimental verification of the enhancement of seed vigor in sweet pepper (Capsicum annuum L.), lettuce (Lactuca sativa L.), chickpea (Cicer arietinum L.), and pea (Pisum sativum L.) under simulations of Martian and lunar regolith. Furthermore, we chose these crop productions of fresh food, which can provide significant nutritional and psychological benefits for long-term missions and future colonizers, aiding in selecting ideal vegetables with desirable organoleptic properties (Massa Wheeler, 2015).

For interplanetary nutrition in the future, it is essential to select crops that do not require processing, have minimal preparation time, provide essential vitamins, and do not degrade like most bioactive compounds in stored food systems (Johnson et al., 2021; Poulet et al., 2022). Furthermore, fresh food can provide significant nutritional and beneficial organoleptic effects in long-duration missions and for future settlers choosing the ideal vegetables with organoleptic properties (Massa Wheeler, 2015). The simulations will be carried out using different concentrations of Ulva lactuca powder, and the seed germination and development will be conducted in a fully controlled greenhouse incubator. The simulators will serve as stress indicators. Additionally, we will evaluate the phytochemicals of this marine macroalgae to comprehend the potential crosstalk between different phytohormones in generating a stress response for each treatment conducted.

The present work evaluated the effect of different concentrations of U. lactuca powder in the germination and growth of the cultivars C. annuum, L. sativa, C. arietinum, and P. sativum. Terrestrial soil was expected to perform better than both regoliths, Martian and lunar. These results obtained indicate that the technique of seed priming using the marine macroalgae U. lactuca in small amounts (0.2 g · L⁻¹) is considered an excellent biostimulant to increase the percentage of seed germination under stressful conditions, mainly on C. arietinum and P. sativum in both simulated, Martian and lunar. Our results follow the previous study that documented the same patterns of responses using lower U. lactuca concentrations in the treatment of tomato seeds (Solanum lycopersicum L.) under stressful conditions and which related the invigoration of seeds to the bioactive substances present in this macroalga (Hernández-Herrera et al., 2016).

Lactuca sativa seeds developed well in lunar regolith when 0.2 g · L⁻¹ of U. lactuca powder was applied for germination and seedling emergence. In Martian regolith, germination rates were satisfactory for the seeds without any addition of biostimulant (control). However, regarding seedling emergence, it was not satisfactory with the probability of loss of this cultivar.

Macroelements present in marine macroalgae (Hernández-Herrera et al., 2016), as well as in Martian regolith (Caporale et al., 2020; Eichler et al., 2021), may slow or positively affect the osmotic potential of plant seeds (Castellanos-Barriga et al., 2017; Eichler et al., 2021). The matric potential of L. sativa seeds may have been influenced by the presence of these macro elements of the Martian regolith, reducing the metabolic capacity of seeds and the development as seedlings in the culture medium due to high regolith toxicity (MGS-1) and because their pH is significantly more alkaline (pH > 9) (Eichler et al., 2021). Under these conditions, the addition of the macroalga did not increase the percentage of seed germination; including the addition of U. lactuca, to a greater extent, may affect the yield or formation of radicle and plumule (Mzibra et al., 2018; Reis et al., 2020).

In Martian regolith, the seeds of C. annuum performed well concerning seedling emergence when higher amounts of biostimulant (0.4 g · L⁻¹) were applied. The more acidic pH of U. lactuca in higher concentration (0.4 g · L⁻¹, pH= 5.36) probably promoted C. annuum germination in simulated Martian regolith. Most acid extracts of brown seaweed (Ascophyllum nodosum and Fucus vesiculosus) and acid extracts of green macroalgae U. lactuca were also beneficial for Vigna radiata and Vigno mungo seeds (Sharma et al., 2012; Castellanos-Barriga et al., 2017) and influenced the length of the shoots, root length, and fresh weight of seed plants denoting with bioactive of these algae.

The differential responses of Capsicum annuum, Lactuca sativa, Cicer arietinum, and Pisum sativum to the regolith simulants and Ulva lactuca powder may be attributed to their distinct physiological and genetic adaptations. For example, Capsicum annuum’s relatively robust root system may render it more capable of exploiting the biostimulant properties of U. lactuca. In contrast, the faster metabolic rates observed in Pisum sativum may facilitate quicker adaptation to the nutrient stresses imposed by regolith simulants (Kulkarni et al., 2099; Sánchez et al., 2004)

Ulva spp. biostimulants were applied at different concentrations to promote seed germination and root induction in plants that grew under stressful environments or were subjected to stressful conditions (El Boukhari et al., 2021; Zouari et al., 2022). The results were positive for those that were treated at low dosages (e.g., 0.1 and 0.2 %), while high concentrations (e.g., dosages over 1%) inhibited seed performance and seedling development (Hernández-Herrera et al., 2014; Castellanos-Barriga et al., 2017; Reis et al., 2020; Hamouda et al., 2021) according to the methodology used for the preparation of liquid extracts of seaweed. In addition, if applied to seeds before stress occurs, the biostimulant helps the plant to become more tolerant (Borges et al., 2014; Kasim et al., 2022)

Macroalgae of the genus Ulva contain elements such as Na, K, Ca, Mg, Cl, and NO₃ (Khan et al., 2009; Craigie, 2011; Tabarsa et al., 2012; Hussein et al., 2021) and, in simulated Martian and lunar regolith, it contains macro elements such as potassium (K₂O), phosphorus (P₂O₅), magnesium (MgO) and calcium (CaO) (Caporale et al., 2020; Eichler et al., 2021). Notably, lunar regolith (LHS-1) promoted a faster seed germination rate than Martian regolith (MGS-1). The fact of its best performance can be explained by its high concentrations of calcium in its composition (12.8 wt%), as it is known to stimulate the production of gibberellins (Taiz et al., 2015). Furthermore, most seeds grown in lunar regolith with lower amounts of U. lactuca (0.2 g · L⁻¹) had a higher germination potential than the control.

Radish seeds (Raphanus sativus) were pre-soaked with biostimulants based on two macroalgae, Codium taylorii and Pterocladia capillacea, for two hours before sowing. Using these macroalgae in pre-sowing allowed better metabolic development in everyday and stressful situations (Kasim et al., 2022). Similarly, Hamouda et al. (2022) evaluated the effect of seed priming in different concentrations of Ulva linza and Corallina officinalis extracts on wheat germination (Triticum aestivum L.). They provided a good percentage of germination and seedling emergence in relation to control.

Seed preparation increases metabolic activities in pre-germination and antioxidant activities and accelerates metabolic recovery of stress-degraded membranes (El Boukhari et al., 2021). This technique increases the biomass of the adult plant, contributes to better metabolism performance, and adds bioactive compounds necessary for human food (Hamouda et al., 2022), such as phytohormones that are plant growth regulators (PGRs).

García et al. (2020) identified PGRs in extracts of U. lactuca and found higher concentrations of GA₄, highlighting the potential use of this macroalgae as a biostimulant. They were found in species of Ulva auxins (indole-3-acetic acid, IAA, and indole-3-butyric acid, IBA), gibberellins (gibberellic acid, GA₃), ABA, cytokines (ribocytic kinetin, KR), and salicylic acid, which may act in the inhibition or acceleration of seed germination (Crouch van Staden, 1992; Gupta Abu-Ghannam, 2011).

Gupta and Abu-Ghannam (2011) mention that the interactions of the different metabolic pathways of phytohormones regulate various plant physiological processes. Phytohormones play an essential role in the regulatory process of plant growth and development. They are essential chemical messengers, allowing plants to develop when subjected to some stress factor (Rhaman et al., 2020), as in the case of regolith. For example, auxins are essential in plant growth and development, promoting cell division, maintaining meristem, organogenesis, cell standardization, and root and stem development. Cytokines regulate the development of roots and stems, while gibberellins and abscisic acid control germination and stimulate germination and dormancy induction (Crouch van Staden, 1992; Gupta&d Abu-Ghannam, 2011).

For germination to occur in the first stages of development, it is essential to have an antagonistic reaction between ABA and GA to promote dormancy breakdown (Taiz et al., 2015; Tuan et al., 2018). In addition, it is known that both cytokinin and auxin interact synergistically to control specific essential processes, such as the maintenance of the meristem (Su et al., 2011). In the analysis of phytohormones of the seeds (pmol/FW), the expected occurred in relation to this balanced reaction between hormones ABA and GA. There was a decline in ABA and an increased GA sensitivity, which favored germination (Taiz et al., 2015; Tuan et al., 2018). However, when treated at higher concentrations (0.4 g · L⁻¹), phytohormone levels increased significantly, which may have caused stress.

Subjecting seeds to high concentrations can extend or delay plant germination time, and radicle and plumule formation can degenerate (Selvam Sivakumar, 2013; Hernandèz-Herrera et al., 2014, 2016; Mzibra et al., 2018). Hormonal peaks significantly differed in control at ideal concentrations (0.2 g · L⁻¹), which probably favored improved hormonal balance and metabolic development of most seeds in this study.

As in our study, previous phytochemical analysis of U. lactuca also revealed the presence of alkaloids, terpenoids, flavonoids, and fatty acids (Hernández-Herrera et al., 2014; García et al., 2020). These bioactives are present in the secondary metabolism of macroalgae of the genus Ulva (Anjali et al., 2019), and metabolic pathways are essential for forming phytohormones (Taiz et al., 2015). However, their performance in the metabolic pathways of plants remains poorly understood (Gupta et al., 2011). We propose a scheme for the possible action and interaction between the bioactive elements of U. lactuca and their action as biostimulants in seeds (Figure 10). Seed germination metabolism is directly induced by the response to seaweed application (El Boukhari et al., 2021). One can see the crosstalk between the ability of macroalgae secondary metabolite components, such as phenolic compounds, terpenes, and alkaloids, to influence the biosynthesis of phytohormones (Lepiniec et al., 2006; Dumas et al., 2010; Gupta et al., 2011; Jaulneau et al., 2011; Taiz et al., 2015). These compounds are of interest due to their antioxidant properties that act as influential protectors against biotic and abiotic stresses (Lepinec et al., 2006; García et al., 2020) and can act on seed germination. For example, terpenes are responsible for the biosynthesis of gibberellins, cytokinin, and abscisic acid (Taiz et al., 2015).

Flavonoids modulate auxin transport through direct and indirect interactions with cell transport and regulatory mechanisms and, in addition, are of current interest due to their antioxidant properties, which accumulate in seed coatings of various plant species, including peas, and which function as protective agents against biotic and abiotic stresses (Lepiniec et al., 2006). Alkaloids are essential in tryptophan biosynthesis, a precursor in auxin synthesis (Taiz et al., 2015; Jiang et al., 2022).

The nutritional deficiencies of lunar and Martian regolith can be overcome by producing nutrient-rich solutions. However, this cultivation method is not sustainable for space agriculture, as nutrients must be brought from Earth (Cannon Britt, 2019). A solution to mitigate this problem would be biostimulants based on marine macroalgal extracts because they are used in small quantities (Castellanos-Barriga et al., 2017; Reis et al., 2020; Hamouda et al., 2022) and mixed in regolith simulators with organic matter from Ulva spp. Since they contain bioactive substances, they may directly influence the growth of edible plants and the availability of nutrients in regolith-based agriculture (RBA).

Figuring out how to live on the Moon and Mars is the ultimate test of sustainability in agriculture, and learning to grow plants in a hostile environment can maximize the use of valuable and harmful resources, such as water and fertilizers (de Micco et al., 2009; Pickett et al., 2020). In addition, space agriculture will be decisive for the survival of humans outside the Earth, mainly because it is viable as a source of income (Cannon Britt, 2019). Biostimulants offer a potentially new approach to regulating/modifying physiological processes in plants to stimulate growth, reduce stress-induced limitations, and increase productivity (Yakhin et al., 2017; Pardilhó et al., 2022). The raw material of Ulva spp. may be available in natural banks and periodic blooms or from integrated multitrophic aquaculture, which turns our focus to this promising source of biostimulants as a nutritional additive to Martian and lunar regolith simulators.

The metabolism of the studied seeds and the emergence of seedlings were directly stimulated by the response of the application of the macroalgae U. lactuca because they contain chemicals that participate in the biosynthesis of phytohormones and prove the efficiency in stimulating germination. In priming seed techniques under stressful conditions, we recommend the use of U. lactuca in low concentrations (0.2 g · L⁻¹) for chickpea seeds (C. arietinum) and peas (P. sativum) in Martian regolith. Both concentrations were satisfactory for lunar regolith for chickpeas (C. arietinum) and peas (P. sativum). For lettuce (L. sativa), the lowest concentrations were satisfactory. We recommend the lowest concentration (0.2 g · L⁻¹) for the best cost-benefit; we propose further investigations and protocols for applying this biostimulant for its proper use, mainly to investigate the use of different concentrations. The use of Ulva spp. will be essential to connect biological systems with local natural resources (lunar and Martian regolith) to improve the feeding of interplanetary civilization and increase the chances of a self-sustaining establishment.