Introduction
Mesembryanthemum crystallinum, commonly known as ice plant or glacier lettuce, is regarded as a stress-tolerant plant. During the dry season and periods of water stress, the photosynthesis mode switches from C3 to Crassulacean Acid Metabolism (CAM) 9. This halophytic plant possesses specialised trichomes called epidermal bladder cells, which have various functions including: water storage, salt accumulation, protection from UV rays and a role in plant defence 7. When mature plants die, the stored salt is leached into the soil thereby dramatically increasing salinity, making it difficult for other native species to grow 10,20,32. However, due to its ability to accumulate salt, the plant has been employed for soil desalination and proposed for bioremediation 2. The ice plant can also rapidly absorb soil moisture and help to build up high nitrate levels 13.
The ice plant’s numerous biological properties include antioxidant activities, performed by betacyanin and other flavonoids, as well as antimicrobial activities 18,25. Leaves and stems are used raw or cooked. With its succulent, mellow, slightly salty-tasting leaves, the species is considered a fashionable plant sold in delicatessen shops 16,30. The product, however, is highly perishable. The wild nature of this species suggests it could be culti vated throughout the year, with little watering and care, based on a different maintenance approach 1. Therefore, the objective was to conduct a greenhouse production experiment and assess optimum ice plant growth conditions in soilless media.
Materials and methods
Plant material and growth conditions
Mesembryanthemum crystallinum seeds were collected from wild plants in Alicante (in South East Spain). They were surface disinfected with 0.5% NaClO for 2 h and pre-hydrated with aerated, distilled water for 22 h. Germination subsequently took place in vermic ulite, hydrated with distilled water and maintained in a growth chamber at 24°C day/night (D/N) air temperature (T) and 70% D/N relative humidity (RH) 29. Chamber light condi tions were 16 h light-8 h dark cycle with photosynthetically active radiation of 400 μmol m-2s-1, provided by a combination of fluorescent tubes (Philips TLD 36W/83, Germany and Silvana F36W/GRO, USA). After 20 days, seedlings were transferred to a greenhouse under semi-controlled conditions of T D/N: 25/18°C; RH D/N: 60/80% and received natural daylight (mean photosynthetic photon flux rate of 400 μmol m-2 s-1) 3 (Photo 1, page 49).
During the transplant, plants were divided into three homogeneous groups of 10 plants. Each group underwent different growth conditions: 1) seedlings transplanted to plastic peat pots (V=1 L) watered weekly with 300 mL demineralised water; 2) seedlings inserted into 1 L plastic vermiculite pots watered weekly with 300 mL Hoagland nutrient solution 17; 3) seedlings grown in hydroponic containers (V=1 L) with Hoagland aerated nutrient solution, which was replaced weekly. Hoagland solution has been widely used for glacier lettuce growth 3,5,24,29. Peat was mainly composed of pine bark, with 86% of organic matter based on dry weight, 49 mS m-1 conductivity, pH 7 and grain size <10 mm. After 45 days, the plants were harvested and the different determinations were performed. At the end of the experiment, the electrical conductivity (EC) of the hydroponic, vermiculite and peat culture was 1.38, 0.57 and 0.63 mS cm-1, respectively.
Growth parameters
Plant dry weight (DW) was determined after oven drying at 80°C until constant weight. Shoot and root lengths were also measured. Leaf area was measured using the app “Easy Leaf Area Free” 12, while Specific leaf area (SLA) was calculated as the ratio of the leaf area to leaf dry weight.
Water status
Leaf relative water content (RWC) was calculated according to Weatherley’s method (1950), using the following equation:
where:
FW = fresh weight,
TW = turgid weight,
DW = dry weight of the tissue, respectively.
This determination was performed on expanded young leaves collected at noon. Foliar succulence was measured according to Atzori et al. (2017) as the ratio of leaf FW to leaf area.
Photosynthetic pigments
Foliar photosynthetic pigment concentration was determined in young, recently expanded leaves collected at noon, as described in Sesták et al. (1971). Samples (20 mg FW) were placed in 5 ml of 96% ethanol at 80° C for 10 minutes to extract the pigments. The absorbance of the extracts was spectrophotometrically measured and the equations reported by Lichtenthaler (1987) were used to calculate chlorophyll and carotenoid concentrations.
Mineral analysis
Leaf samples (0.5 g DW) were dry-ashed and dissolved in HCl in accordance with Duque (1971). Phosphorus, potassium, calcium, magnesium, manganese, zinc, iron and sodium concentrations were determined using a Perkin Elmer Optima 4300 inductively coupled plasma optical emission spectroscopy (ICP-OES) (Perkin Elmer, USA). The ICP-OES oper ating parameters were: radio frequency power 1300 W, nebulizer flow 0.85 L min-1, nebu lizer pressure 206.84 kPa, auxiliary gas flow 0.2 L min-1, sample introduction 1 mL min-1 and three replicates per sample. Total nitrogen and carbon were quantified after leaf DW combustion (950°C) with pure oxygen using an elemental analyser with a thermal conduc tivity detector (TruSpec CN, Leco, USA).
Statistics
To compare all three treatments, a one-way analysis of variance (ANOVA) (SPSS v.26, IBM Corp., USA) was conducted. The means ± standard deviation (SD) were calculated. When the F-ratio was significant (p<0.05), Duncan’s multiple range test was applied. When only two treatments were compared, Student’s t-test was performed. Significance levels were always set at 5%.
Results
Growth parameters of Mesembryanthemum crystallinum showed significant differences according to the culture medium (Table 1).
Plants in vermiculite presented greater shoot growth and leaf DW than those under hydroponic conditions, but the difference was espe cially notable compared to peat-grown plants. Vermiculite treatment also enhanced ice plant root length and biomass, while peat growth presented significantly low values. In fact, peat pot culture did not seem to be an adequate substrate to cultivate M. crystallinum. Despite the same environmental conditions and age, plants did not grow properly. Therefore, it was not possible to obtain sufficient plant matter from the peat treatment to perform further deter minations such as: RWC, succulence, photosynthetic pigments or mineral analysis. On the other hand, vermiculite-grown plants exhibited a significant increase in leaf area compared to the hydroponic culture, despite their similar SLA values.
Regarding leaf water status, ice plant foliar RWC did not differ according to the culti vation mode (Table 2, page 51).
Vermiculite-grown plants, however, presented the highest edible leaf FW production values together with greater succulence. As for photosynthetic pigments (Table 3, page 51), both chlorophyll and carotenoid concentrations increased in vermiculite-grown plants.
Foliar mineral concentration is summarised in Tables 4 and 5 (page 51).
While C concen tration did not differ among the plants grown in different media, foliar N concentration was higher in the ice plant cultivated in vermiculite (Table 4, page 51). Moreover, vermic ulite-grown plants had significantly higher Mg (Table 4, page 51), Mn, Fe and Na (Table 5, page 51) concentrations compared to the hydroponic treatment. Nevertheless, K and Zn concentrations were greater in plants grown in hydroponics, while P and Ca contents were similar across all treatments.
Discussion
Peat is a widely used substrate for plant cultivation. In fact, Mesembryanthemum crys tallinum has been successfully grown in peat-based substrate or mixed with vermiculite 3,4,8. However, our results showed that peat was not a suitable medium for ice plant pot-growing. Plant growth was very poor, making it impossible to determine many param eters and leading to significantly reduced shoot length, root length and leaf area. These results could be given by the fact that peat-grown plants were irrigated with deminer alised water, while those grown in vermiculite and hydroponics received Hoagland nutrient solution. This may have generated an essential nutrient deficit in peat-grown plants, since the limited 1 L potting space increases nutrient needs 28. Moreover, according to Morales and Casanova (2015), peat is poorly drained. This probably leads to insufficient root oxygen ation, causing growth limitations.
On the other hand, hydroponic and vermiculite cultures led to optimum M. crystallinum growth. In addition, vermiculite-grown plants irrigated with Hoagland nutrient solution presented a significantly greater yield. Vermiculite has a high cation exchange capacity 19, while in hydroponics, the nutrients are very accessible 22. It is worth noting that the hydro ponic culture’s EC levels at the end of the experiment were more than two times greater than that of the other two substrates; despite this, EC levels were within an optimum range regarding root water and nutrient absorption 15. In addition, the nutrient solution was replaced every week in order to ensure good nutrient balance in the hydroponic culture.
Vermiculite-grown plants presented higher values across all growth parameters, except for SLA. The reason may be that the nutrient intake of vermiculite-grown ice plants is greater due to longer roots and higher root DM, which would improve water and nutrient acquisition and/or use efficiency 26. In fact, plants grown in vermiculite presented higher leaf N, Mg, Mn and Fe concentrations.
Regarding the water status, cultivation mode was not found to influence foliar RWC in any way. On the contrary, leaf succulence improved in vermiculite-grown plants, probably due to greater nutrient intake than in hydroponics. Increases in succulence, that is, water content per unit area, allows osmotic adjustment 14. According to our results, vermicu lite-grown Mesembryanthemum leaves also exhibited higher Na concentrations. The latter increases the salty taste of leaves and is highly appreciated by consumers. In fact, leaf succulence and glistening bladder cells, in particular, provide the edible leaves with a taste, consistency and appearance that make ice plants particularly valued by consumers 6. In addition, Agarie et al. (2007) worked on an ice plant mutant lacking epidermal bladder cells (EBCs). The authors concluded that EBCs contribute to succulence by serving as a water storage reservoir. They also contribute to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.
Significant differences were found regarding pigment concentrations, with enhanced levels in total chlorophylls and carotenoids of vermiculite-grown leaves. This fact could imply that ice plants grown in vermiculite have a greater photosynthetic capacity, together with a greater allocation of energy in thermal dissipation over photochemistry 6.
Conclusions
Clear evidence was found that vermiculite pot culture irrigated with nutrient solution allows obtaining M. crystallinum yield for edible purposes. Under greenhouse conditions, this cultivation mode leads to greater biomass - including the production of edible fresh leaves - than hydroponics or peat culture. In addition, increased leaf succulence and Na concentration of vermiculite-grown plants offer consumers a more interesting taste, consis tency and appearance.