Glassworts : From Wild Salt Marsh Species to Sustainable Edible Crops

Halophytes are naturally adapted in saline environments, where they benefit from the substantial amounts of salt in the growth media. The need for salt-tolerant crops increases as substantial percentages of cultivated land worldwide are affected by salinity. There are few protocols, guidelines, or trials for glasswort (Salicornia (L.) and Sarcocornia (Scott), belong to the Amaranthaceae) field cultivation. The high salt tolerance and content in bioactive compounds make glassworts one of the most important candidates for future use both for fresh and processed food, due to their functional and health properties. This review describes the glassworts respect to their biodiversity and the most important factors affecting propagation, salt tolerance traits, agro-techniques and yields, food uses and nutraceutical properties.


Introduction
The depletion of natural resources due to human activity, global change, and the increasing population have led to a great reduction in arable lands along with salinization of the soil and unavailability of freshwater.Only cultivating conventional crops that have a low tolerance to salinity and drought is therefore not recommended in the future [1]; new sustainable crops, with low inputs and strong abiotic tolerance, are thus needed.
Halophytes need a high-salt soil composition to develop properly [2,3]; their salt tolerance is related mainly to the ability to control ion uptake and the vacuolar compartmentalization of Na + , K + , and Cl − , in order to maintain the osmotic balance between vacuoles and cytoplasm by the synthesis of osmotic active molecules [4,5].
Today edible halophytes are one of the most interesting groups of plants as they can be cultivated in marginal areas with seawater, or in saline areas near the sea, in beginning a potential cash crop [6].
This review describes the glassworts in respect to their biodiversity and the most important factors affecting propagation, salt resistance traits, agro-techniques and yields, food uses, and nutraceutical properties.
They are a difficult taxonomy because of the richness of the microspecies, subspecies, hybrids, and varieties.Kadereit et al. [12] found that Salicornia and Sarcocornia were separated from each other near the Middle Miocene (14.2-9.4 mya); however, actual lineages were diversified during the Early Pleistocene (1.4-1.8 mya).

Habitat and Apulian Biodiversity
Salicornia and Sarcocornia develop mainly in sandy or muddy saltmarshes that are flooded by the tide, in mudflats, sandflats, and sometimes in open saline areas.Salicornia is found around much of the coastline of Europe from the Arctic to the Mediterranean, as well as on the shores of both the Black Sea and Caspian Sea; it is also present sporadically where inland salines occur across Europe [13].
Annual Salicornia species have a much wider distribution than Sarcocornia in the Northern hemisphere and in South Africa.They are absent from South America and Australia [12].Table 1 shows the origin of some species belonging to Salicornia and Sarcocornia genus [12,[14][15][16].In south of Italy (Apulia Region), S. europaea L., known as common sea asparagus, is a characteristic pioneer species of the association Thero-Salicornietea [17]; the brackish lakes of Lesina (Figure 1) and Varano (Foggia Province) are rich in this wild plant species, that colonize damp and brackish soils, flooded by the tide for most of the year and dry during the warmer seasons [18].There are S. glauca Delile and Sarcocornia fruticosa (L.) A.J. Scott too.[13].
Annual Salicornia species have a much wider distribution than Sarcocornia in the Northern hemisphere and in South Africa.They are absent from South America and Australia [12].Table 1 shows the origin of some species belonging to Salicornia and Sarcocornia genus [12,[14][15][16].
In south of Italy (Apulia Region), S. europaea L., known as common sea asparagus, is a characteristic pioneer species of the association Thero-Salicornietea [17]; the brackish lakes of Lesina (Figure 1) and Varano (Foggia Province) are rich in this wild plant species, that colonize damp and brackish soils, flooded by the tide for most of the year and dry during the warmer seasons [18].There are S. glauca Delile and Sarcocornia fruticosa (L.) A.J. Scott too.In the south of Apulia, the "Torre Guaceto" coastal lagoon (Province of Brindisi-Figure 2) is a National Natural Reserve characterized by a wide number of halophyte plants; among these, Sarcocornia fruticose is one particular example.In the province of Taranto, "Salina dei monaci", Sarcocornia sp.grows in globe-like shrubs (Figure 3).It is richly branched, with long sterile shoots (in the current year) and fertile shoots (from the previous year) with leaves small and green.In the Province of Lecce, "Le Cesine" another glasswort was found by [19] on cliffs, where the salinity is the same as the sea: Sarcocornia fruticosa L.

Morphological Characteristics
As glassworts are characterized by an extremely reduced morphology, they can grow prostrate, erect, or branched [13].Figure 4a shows that the leaves are very small and the assimilatory surface is composed of succulent branches with cylindrical internodes [20].Axillary buds on the main stem develop into primary branches which produce secondary ones [21].Annual species are usually green, but in autumn, near the end of the cycle, the stems turn red.In Salicornia spp.flowers are aggregated in a dense terminal, spike-like thyrses composed of three flowers: one central and two lateral (Figure 4b).In Sarcocornia, these are placed in horizontal rows [12].Glassworts differ from all other Amaranthaceae since the seeds that lack perisperm [22].The Salicornia seed is small, ellipsoid, dark (Figure 4c) and is characterized by heteromorphism: i.e., seeds from the same plant have a difference in size, color, or shape [23].In S. europaea L. there is a wide variation in size: 0.4-0.9mm

Morphological Characteristics
As glassworts are characterized by an extremely reduced morphology, they can grow prostrate, erect, or branched [13].Figure 4a shows that the leaves are very small and the assimilatory surface is composed of succulent branches with cylindrical internodes [20].Axillary buds on the main stem develop into primary branches which produce secondary ones [21].Annual species are usually green, but in autumn, near the end of the cycle, the stems turn red.In Salicornia spp.flowers are aggregated in a dense terminal, spike-like thyrses composed of three flowers: one central and two lateral (Figure 4b).In Sarcocornia, these are placed in horizontal rows [12].Glassworts differ from all other Amaranthaceae since the seeds that lack perisperm [22].The Salicornia seed is small, ellipsoid, dark (Figure 4c) and is characterized by heteromorphism: i.e., seeds from the same plant have a difference in size, color, or shape [23].In S. europaea L. there is a wide variation in size: 0.4-0.9mm [24,25].Heteromorphism is also widespread in Asteraceae and Amaranthaceae [26,27] and is the result of an adaptation to desert environments, because each seed morph germinates under different conditions [28][29][30][31].

Propagation
Propagation can be carried out by both gamic and agamic techniques.

Propagation
Propagation can be carried out by both gamic and agamic techniques.

Gamic Propagation
Halophytes usually inhabit extreme soil parameters.This implies that seeds need strategies in order to overcome these situations: dormancy under hypersaline conditions and germination at high salinity levels [32].
Dormancy starts during the late autumn and ends with seed germination of glassworts in late April-early May [33].The main advantage of germination in early spring is the abundance of water which involves low salt concentrations, ideal for germination.S. europaea (L.) and Spergularia marina (L.) have two or more different seed morphs and each seed has a different dormancy and salt tolerance [25,34,35].In fact, the lateral seeds of S. europaea (L.) are more dormant than the central seeds (which are more salt tolerant), in order to maintain a long-term soil seed bank [25].Orlovsky et al. [36] found that the germination of large seeds was three to four times higher than small seeds in the control and under 0.5-2% salt (both NaCl and 2NaCl + KCl + CaCl 2 ).This high dormancy typical of smaller seeds can be partly broken with chloride or sulfate salts (concentrations of 0.5 to 2%).
In order to overcome the dormancy period in specialized nurseries, seeds need to undergo a cold stratification: T = 5 • C for 30 days in the dark.Aghaleh et al. [37] and Lv et al. [38] reported that Salicornia seeds were exposed to sunlight and salinity using 30:70 or 50:50 seawater:freshwater.This phase ended in 14 days, leading to appropriate seedlings (Figure 4d).
The percentage and rate of germination decreased as salinity increased which is true for both seed morphs [36].Although there is a greater success of germination under freshwater, halophytes can also germinate at high saline concentrations: for example, S. europaea can germinate at 600 mM NaCl [39,40].
Ungar [34] found that treatments with GA 3 , play an important role in overcoming salt stress-induced dormancy in Salicornia, Suaeda, and Spergularia.

In Vitro Propagation
In vitro propagation of S. bigelovii and S. europaea was studied [41,42] but "in vitro" culture system of succulent halophytes was not very successful so far. Lee et al. [41] developed in vitro propagation of S. bigelovii using the shoot tip culture method but the regeneration rate was very low; however, Shi et al. [42] established a method of S. europaea regeneration from mature embryos.Tissue culture of succulent halophytes using a mature embryo is not promising because of the microscopic size and non-availability of explants throughout the year.

Growth
Glassworts, in North Apulia, are typically cultivated from February-March to August-September in the acidic marshes near the brackish lake of Lesina, Cagnano Varano, and San Nicandro Garganico in open fields [44].In Israel, Salicornia is grown using nests or greenhouses in soil, floating systems or plastic sheets [45] with perlite, dune sand, coconut fiber, or a commercial substrate mixed with sand (1:1 v:v) [46].Singh et al. [3] showed that production (fresh and dry biomass) of S. dolichostachya moss, in hydroponic cultivation, was higher than plants grown in sand media; on the other hand, hydroponic cultivation can lead to chlorosis events.
A notable amount of biomass for S. ramosissima woods was achieved using artificial seawater containing 257 mM NaCl [3]; on the other hand, Rozemaa et al. [47] found that growth was depressed at low salt concentrations.Regarding irrigation, Singh et al. [48] showed that cultivation of S. brachiata in a greenhouse using reverse osmosis reject water with and without fluorine ion could be a sustainable solution for reject water management.
The main problem related to wild species introduced to cultivation, is the prevalence of non-commercial characteristics due to genetic diversity, for example, production homogeneity.Besides breeding programmes, with plant growth promoting bacteria (PGPB) could be an alternative in order to increase production.Bashan et al. [49], found that S. bigelovii increase production (height and weight) when inoculated with some PGPB, i.e., Azospirillum halopraefrens; A. brasilense (two different strains); Vibrio aestuarianus + V. proteolyticus; Bacillus licheniformis + Phyllobacterium spp.It is worth underlining that the cultivation of Salicornia could be beneficial in order to increase soil fertility, due to the presence of rhizobacteria in its roots [50].
In Apulia, harvesting, in open field, takes place in July-August; in Israel in August-September, and it is carried out manually in order to maintain the high quality of the final product; only fresh and tender parts can be sold.Salicornia can be collected several times during the year in greenhouses: The harvest can be repeated every two or three weeks (depending on the level of development).Plants are cut above 5 cm from the ground at a height of 10-15 cm [51].This repeated harvesting enables the same plant to be cut from three to four times depending on the level of growth.The yield can reach 10-15 tons per hectare.

Flowering
Under natural conditions, when the day length decreases, the reproductive phase of glassworts begins with a differentiation in terminal fruiting spikes [52].The optimal day length is different for high latitude and short latitude plants: in fact, northern glassworts (S. europaea) need 18 or more hours to prevent flowering; for S. bigelovii, 13 h are enough [51].Flowering induction has many negative effects on both the production (intense decrease in growth) and production of non-saleable shoots and nutraceutical properties [51].For perennial Sarcocornia, flowering can be controlled with a repeating harvesting regime [51].Based on the results of Ventura and Sagi [46], nutrition management can also be used for flowering control.The regulation of Mo and N-NO 3 can prevent the reproductive stage, in fact, ammonium fertilization promotes flowering in S. bigelovii.In addition, the sowing date can be useful in preventing flowering: Seeds planted in November or December repress the reproductive stage; whereas, planting in January results in inhibition, because during the growing cycle, the day length increases [51].

Salinity Tolerance, Water Relations, and Gas Exchange
Glassworts are salt-tolerant species: Redondo-Gòmez et al. [21] showed that S. fruticosa has a considerable salt tolerance, compared with other halophytes, resulting in the greatest growth with 510 mM NaCl treatment.Similar results, regarding the optimal salt concentrations, have been achieved for different genotypes: for Salicornia persica Akhani, e.g., 200 mM NaCl [53]; 200 mM NaCl for S. brachiata [54]; 170-340 mM NaCl for S. europaea, S. bigelovii, S. herbacea, and S. brachystachya [55].The photosynthetic apparatus of S. fruticosa also appears capable of tolerance prolonged high external salt exposure, suggesting plasticity.
The main salt tolerance responses of plants relate to an increased leaf thickness.Katschnig et al. [56] found that leaf succulence and diameter in S. dolichostachya were higher when plants were grown in 300 mM NaCl of nutrient solution compared to other treatments grown in from 0 to 500 mM NaCl.This is due to an increased accumulation of Na + and Cl − ions in the vacuoles [57], which leads to a reduced leaf surface area and consequently an increased water use efficiency.In saline environments, in order to ensure the water uptake, a low water potential in the cells is needed [56].Beside Na + and Cl − ions, organic compounds such as glycine betaine are stored in the cytoplasm in order to guarantee a water potential equilibrium in the cells [58].Organic solute as malate dehydrogenase can also act as an enzyme that stabilizes at high salt concentrations [59].Jolivet et al. [60] also found that glycine betaine inhibits the betacyanins efflux when present in the external medium along with the oxalates.Glycine betaine is perhaps the main organic compound with osmoregulation properties for Salicornia spp.[61].
Another mechanism used to maintain a better water uptake is gas exchange regulation: Stomata conductance generally decreases for halophytes as a reaction to increasing salt concentrations.At high salt concentrations, as reported by Ayala and Oleary [62] for S. bigelovii, stomata conductance decreases, consequently the carbon dioxide fixation and transpiration rate also decrease, in order to obtain the best efficiency in terms of water use.

Bioactive Compounds
Halophytes can be used for many commercial purposes: Not only as fresh and processed foods, but also in terms of their high salt content, as biofuel, high nutritional oilseed, medical raw materials, as well as nutraceutical and functional foods [63,64].
Chenopodiaceae members are known to contain high amounts of crude proteins, sulfur, and minerals [65], which favor glasswort as an edible plant.Salicornia, developed in extremely saline environments, produces antioxidative metabolites, which are desirable in the human diet.Simple and complex sugars, alcohols, quaternary amino acid derivatives, tertiary amines, and sulfonium compounds are also useful for human nutrition, and are found on these plants [45].
Guil et al. [66] showed that sea asparagus has high levels of ascorbic and dehydroascorbic acids (more than 100 mg 100 g −1 ), as well as carotenoids (5 mg 100 g −1 ). S. bigelovii also show high content of β-carotene (15.9 mg 100 g −1 of fresh weight), and a high amount of vitamin A [67].Both Salicornia and Sarcocornia showed a high content of polyphenols (1.2 and 2.0 mg GAE g −1 of fresh weight, respectively) [51].Kang et al. [68] found that the freshwater cultivation of Salicornia had a higher phenolic and flavonoid content, than seawater cultivation.
Due to the specific biochemical composition of Salicornia seeds, they are very good for human health.The main molecules include linoleic (75.6%) and oleic acids (13.0%); in addition to palmitic, linoleic and stearic acids [63].Proteomic analysis has also revealed a high protein content of the seeds [69].Considering the main components, palmitic acid, tetracosanol, and octacosanol showed significant levels [70].Octacosanol is a high-molecular-weight aliphatic alcohol and belongs to cholesterol-lowering drugs such as polycosanol [71].
In addition, the leaves of Salicornia are also a good source of fatty acids such as linoleic and oleic acids [72].Finally, the presence of selenium has also been detected; which is an important micronutrient both for growth and has good antioxidant effects [73].
Abiotic stress, including salt stress, leads to the production of reactive oxygen species in the plants, which damage cellular membranes and enzymatic activity [75]; to combat salt-oxidative stress, antioxidative enzymes and small antioxidant molecules are produced.These molecules give medicinal properties to halophyte plants, which are used for several treatments for chronic disease, including diabetes, cancer [74].Polysaccharides from S. herbacea at 0.5-4 mg mL −1 have been found to be antiproliferation of human colon cancer HT-29 [76].Another study found that pentadecylferulate from S. herbacea has an antioxidant effect and, an anti-cancer response to human hepatocellular liver carcinoma HepG2 and human lung adenocarcinoma epithelial A549 cells [77].The methanol extract of S. herbacea has shown antibacterial activities, mediated by interference with cytochrome and several enzymes [78].Osteoporosis is a bone disorder, caused by a higher bone adipogenesis (differentiation of stem cells into mature adipocytes): S. herbacea extract has been found to inhibit adipogenesis [69].
Besides the health properties of Salicornia, there are also some negative aspects: Chenopodiaceae members are known to contain a high oxalate content, which could be harmful to consumers [65].

Food Uses
The edible parts of glassworts have tender leaves and shoots can be used in a fresh salad, or boiled like spinach without salt, and then coated with extra virgin olive oil (Figure 5).The color, after cooking, resembles seaweed, and the flavor and texture are similar to young spinach or asparagus.Sea asparagus is often used as an accompaniment to fish or seafood [45].

Conclusions
In the last few years, interest in halophytes cultivation has increased; however, scientific papers and multiple large scale experiments are very limited.There are few protocols for glassworts field cultivation and few guidelines for small cultivations or trials.
The high salt tolerance and content in bioactive compounds makes glassworts one of the most important candidates for future use both for fresh and processed food, due to their functional and health properties.S. europaea L. is currently included in the list of traditional Apulian vegetables.In Hawaii, it is often blanched and used as a topping for salads or as an accompaniment for fish [79].The seeds of S. bigelovii can be used to make edible oil; however, it is not always edible because it contains saponins, which can be toxic.S. brachiata is used as fodder for cattle, sheep, and goats [80].In Sri Lanka, it is used to feed donkeys [81].
In India, shoots can also be transformed into beverages such as nuruk (fermentation starter), makgeolli (Korean rice wine), or vinegar [82,83].One study found that Salicornia enhances fermenting microbe propagation and improves vinegar quality [84].Other authors have found that these plants can be used as a source of salt for several dishes.Indeed, S. herbacea powder can be transformed into spherical granules, which may be used as NaCl [85].

Conclusions
In the last few years, interest in halophytes cultivation has increased; however, scientific papers and multiple large scale experiments are very limited.There are few protocols for glassworts field cultivation and few guidelines for small cultivations or trials.

Figure 5 .
Figure 5. Apulian traditional dish: boiled Salicornia with extra-virgin olive oil and garlic.
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