In Vitro Shoot Culture of Sesuvium portulacastrum : An Important Plant for Phytoremediation

: Sesuvium portulacastrum L., a member of the family Aizoaceae , is an important coastal halophyte. Due to its adaptability to salinity and heavy metals, S. portulacastrum has now been widely used for the phytoremediation of saline soils and wastewater and the protection of the coast from erosion. The increasing use of this plant requires a large number of propagules. Stem cutting propagation and seed germination cannot meet this demand, and such propagations can initiate and spread diseases. A recent occurrence of Bipolaris sesuvii J.Z. Zhang and Gibbago trianthemae E.G. Simmons in S. portulacastrum resulted in the substantial loss of the plants during the remediation of aquaculture wastewater. Thus, there is an urgent need for establishing efﬁcient methods of propagating disease-free starting materials. In the present study, we evaluated different growth regulators in the induction of axillary shoots from nodal explants cultured on Murashige and Skoog medium and identiﬁed that zeatin (ZT) and α -naphthaleneacetic acid (NAA) was an appropriate combination for inducing high numbers of axillary shoots. The nodal explants were then cultured on MS medium supplemented with different concentrations of ZT and NAA, and the combination of ZT at 1.0 mg L − 1 and NAA at 0.3 mg L − 1 induced more than 12 axillary shoots per explant. The axillary shoots were excised to produce microcuttings or microshoots, which were rooted on half-strength MS medium supplemented with different concentrations of indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The results showed that IBA at 0.6 mg L − 1 induced 91.7% of the microcuttings to root with root numbers of over 36 per cutting. The rooted plantlets were healthy and true-to-type and grew vigorously in plug trays or plastic containers with a 100% survey rate in a greenhouse. Thus, this established protocol could be used for the rapid propagation of genetically identical and disease-free plants of S. portulacastrum for phytoremediation and the protection of shoreline soils from erosion.


Introduction
Sesuvium portulacastrum L., a member of the family Aizoaceae, is an important halophyte with a chromosome number of 2n = 48.It is a perennial creeping succulent herb grown in coastal sandy or saline soils in the subtropical and tropical regions; thus, it is commonly known as sea purslane [1][2][3].Sea purslane has a thick taproot and many fibrous secondary roots and also produces adventitious roots from the node.Its leaves are evergreen, simple, and succulent, and stems are creeping or prostrate.Sea purslane plants produce small, star-shaped flowers that are white, pinkish purple, or pendent and subsequent capsule fruit containing about 50 seeds.S. portulacastrum is a fast-growing plant and can quickly cover the ground or grow in water with trailing vines or internodes that overlap to create dense evergreen floating plant clusters.
A distinct characteristic of S. portulacastrum is its adaptability to high saline environments [4].Sea purslane plants grow well under a sodium (Na) concentration ranging from 100 to 400 mM [5].In a study conducted regarding the effects of Na and potassium (K) on the growth of S. portulacastrum, stem cuttings grown in a modified Hoagland solution [6] supplemented with 200 mM NaCl produced significantly higher biomass than the control, Na content in the leaves reached up to 100 mg g −1 dry weight, and the authors concluded that Na was more effective than K for cell expansion, shoot development, and leaf succulence [7].
Sea purslane can also take up a large amount of nitrogen (N) and phosphorus (P) and tolerate heavy metals.A study showed that S. portulacastrum plants grown in eutropic marine water on floating beds (60 rooted cuttings per m 2 ) absorbed 377 g N m −2 and 22.9 g P m −2 in eight months [8].Other researchers have reported that S. portulacastrum plants tolerate heavy metals [9,10].S. portulacastrum can be used as a phytostablizer in heavy-metal-contaminated coastal environments [11].Studies have shown that S. portulacastrum is highly tolerant to Cd, Cu, Ni, and Zn [11,12].S. portulacastrum plants have been reported to grow in hydroponic solutions containing 50 µM Ni [13], 90 mg L −1 Zn [14], or 50 µM to 600 µM Cd [15,16] without a significant reduction in dry weight.
Soil salinization is an important problem that seriously affects plant growth and crop productivity [17].Environmental pollution, such as eutrophication and soil and water contamination by heavy metals, poses a great threat to the health of living things [18][19][20][21].Phytoremediation is considered the most effective way to minimize these pollutants [22][23][24][25].Due to its tolerance and accumulation of Na and heavy metals and its absorption of N and P, S. portulacastrum has become a valuable plant for phytoremediation [1].It has also been used for stabilizing coastal erosion and the restoration of shorelines [26].Additionally, organic pollution, such as pesticides and antibiotics, is another ecological and environmental problem in freshwater and coastal water, which represents a challenge to the aquaculture industry.China is a major country in aquaculture and has a total aquaculture area over 7.83 million hectares [27].This large-scale aquaculture has caused severe water pollution problems including enriched N, P, pesticides, and antibiotics in the water.There is an urgent need for the remediation of the wastewater from aquaculture [28].
Today, biological floating-bed technology has been increasingly applied to aquaculture ponds and waterbodies [29] for phytoremediation.S. portulacastrum has been used to construct floating beds for improving water quality in the aquaculture and mariculture industry (Figure 1A).Due to the expansion of aquaculture and the increased use of S. portulacastrum for stabilizing shoreline erosion (Figure 1B), there is a great demand for S. portulacastrum plants.Sea purslane can be propagated by seeds, but seed production is limited [1]; furthermore, there is no commercial supply of seeds.Additionally, the plants propagated from seeds may not be true-to-type, as sea purslane is an open-pollinated plant.As a result, sea purslane is commonly propagated by stem cuttings.Cutting propagation, however, requires a large number of stock plants and can often initiate and spread disease, resulting in large-scale infections and deaths (Figure 1C,D).A new species of Bipolaris known as P. sesuvii has been reported to infect sea purslane plants in Guangdong Province [30], and it has recently been identified that Gibbago trianthemae can heavily infect sea purslane in Fujian Province, China (data not shown).
Micropropagation is the most effective method for achieving a rapid increase in propagules on a year-round basis [31].It includes the culture of preexisting meristems and regeneration of plants through organogenesis or somatic embryogenesis.Indirect organogenesis and indirect somatic embryogenesis through the callus phase may produce plantlets with somaclonal variation [31,32].As a consequence, propagation from preexisting meristems, such as shoots or nodes is the most reliable method, as it produces genetically identical plants without somaclonal variation [33,34].Thus far, there has only been one report on the micropropagation of S. Portulacastrum [35], where nodal explants were cultured on Murashige and Skoog (MS) medium [36] supplemented with different concentrations of BA (6-benzylaminopurine), Kin (kinetin), TDZ or thidiazuron (N-phenyl-N'-1,2,3-thidiazol-5-ylurea), and 2iP (6-(γ, γ-dimethylallylamino)purine).BA at 20 µM and 2iP at 40 µM induced an average of 4.95 and 5.2 axillary shoots, respectively, which were considered the highest induction rates compared to the other growth regulators.However, callus and extensive callus were induced at the aforementioned 2iP and BA concentrations, respectively.As a result, there were two complications with this reported protocol: one being the low induction rates of the axillary shoots and the other being the formation of callus.Callus may lead to the regeneration of adventitious shoots, which may produce somaclonal variants, resulting in genetic infidelity [34].
The objective of the present study was to establish an efficient and rapid protocol for the micropropagation of S. Portulacastrum through existing meristems (node cultures) to provide the aquaculture industry with disease-free, true-to-type, and high-quality plants for phytoremediation.Micropropagation is the most effective method for achieving a rapid increase in propagules on a year-round basis [31].It includes the culture of preexisting meristems and regeneration of plants through organogenesis or somatic embryogenesis.Indirect organogenesis and indirect somatic embryogenesis through the callus phase may produce plantlets with somaclonal variation [31,32].As a consequence, propagation from preexisting meristems, such as shoots or nodes is the most reliable method, as it produces genetically identical plants without somaclonal variation [33,34].Thus far, there has only been one report on the micropropagation of S. Portulacastrum [35], where nodal explants were cultured on Murashige and Skoog (MS) medium [36] supplemented with different concentrations of BA (6-benzylaminopurine), Kin (kinetin), TDZ or thidiazuron (N-phenyl-N'-1,2,3-thidiazol-5-ylurea), and 2iP (6-(γ, γ-dimethylallylamino)purine).BA at 20 μM and 2iP at 40 μM induced an average of 4.95 and 5.2 axillary shoots, respectively, which were considered the highest induction rates compared to the other growth regulators.However, callus and extensive callus were induced at the aforementioned 2iP and BA concentrations, respectively.As a result, there were two complications with this reported protocol: one being the low induction rates of the axillary shoots and the other being the formation of callus.Callus may lead to the regeneration of adventitious shoots, which may produce somaclonal variants, resulting in genetic infidelity [34].
The objective of the present study was to establish an efficient and rapid protocol for the micropropagation of S. Portulacastrum through existing meristems (node cultures) to provide the aquaculture industry with disease-free, true-to-type, and high-quality plants for phytoremediation.

Plant Materials
Fresh mature seeds were collected from coastal-grown S. portulacastrum plants in Fujian Province, China.The seeds were washed in sterile distilled water three times and surface-disinfected with 75% (v/v) alcohol solution for 1 min, followed by immersion in 2% sodium hypochlorite solution for 20 min with occasional agitation, and finally were washed 5 times with sterile distilled water.MS medium [36] (PhytoTechnology, Shawnee Mission, KS, USA) was supplemented with 30 g L −1 sucrose (Morebetter, Hangzhou, China) and 8 g L −1 agar (Solarbio Life Sci., Beijing, China).After the pH was adjusted to 5.8, the medium was autoclaved at 121 • C for 25 min.Autoclaved medium was poured into 250 mL glass culture vessels (Shanghai Zeshine Equipment Co., Shanghai, China), 20 mL each.The disinfected seeds were inoculated on the MS medium in the glass culture vessels, which were placed on the shelves of a growth chamber (PGX, Saifu Experimental Apparatus Tech.Co., Ltd., Ningbo, China) where the temperature was set at 25 • C, relative humidity was adjusted to 70%, and the photoperiod was programmed to 16 h of light, provided by fluorescent lamps at a light level of 25 µmol m −2 s −1, and 8 h of dark.The seeds began to germinate and produced two leaves in two weeks.Germinated seedlings were transferred to culture vessels containing the same MS medium for continuous growth in the same environment for about four months.

Evaluation of Growth Regulator Combinations for Axillary Shoot Induction
To identify appropriate combinations of cytokinin and auxin for inducing axillary shoots, the nodal explants were cultured on the aforementioned MS medium containing nine growth regulator combinations with no growth regulator as a control (Table 1).The MS medium, after supplementation with 30 g L −1 sucrose and 8 g L −1 agar and adjustment of the pH to 5.8, was aliquoted into 10 bottles, 100 mL each, which were autoclaved at 121 • C for 25 min.Three cytokinins, BA, zeatin or ZT (6-(4-hydroxy-3-methylbut-2-enylamino) purine, and TDZ, and four auxins, 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid), IAA (indole-3-acetic acid), and NAA (1-naphthaleneacetic acid), were sterilized by filtration through 0.2 µm membranes (Thermo Fisher Scientific, Waltham, MA, USA), respectively.After the medium was cooled to about 50 • C, the sterilized auxins and cytokinins were added to the respective bottles, resulting in nine growth regulator combinations plus a control listed in Table 1.Nodal explants (about 15 mm in length with three nodes and no leaves) derived from the abovementioned seedlings were cultured on the media, four per culture vessel.The experiment was arranged as a completely randomized design with three replications.Axillary bud number per explant was recorded after 56 days of culture, and plants were photographed.No growth regulators 1.21 ± 0.24 e z Different letters within the column indicate significant differences among treatments based on LSD analysis at p < 0.05 level.

Optimization of Axillary Shoot Induction
The above experiment showed that ZT in combination with NAA induced the highest number of axillary shoots without callus formation.In order to optimize axillary shoot induction, the same MS medium was used to formulate 20 combinations of ZT with NAA at different concentrations, which were used for evaluation of the rate of axillary shoot induction.Additional nodal explants were cultured on MS medium supplemented with 2 mg L −1 ZT and 0.2 mg L −1 NAA for about 60 days to produce a large number of axillary shoots.Nodal explants (three nodes of about 15 mm) were then cultured on the 20 media.There were four nodal explants per culture vessel.The experiment had a completely randomized design with three replications.The culture was maintained in the abovementioned growth chamber.The culture was monitored weekly and photographed, and the number of axillary shoots per explant was scored after 90 days.

In Vitro Rooting and Acclimatization
Axillary shoots were excised from explants cultured on the medium containing 1 mg L −1 ZT and 0.3 mg L −1 NAA, and the resultant microcuttings were cultured on half-strength MS medium supplemented with either IBA or IAA, four concentrations each.There were four microcuttings per culture vessel, and the experiment had a completely randomized design with three replications.After 24 days of culture, the number of microcuttings with roots, root numbers, and root lengths were recorded.The percentages of rooting per culture vessel and mean root length per microcutting were calculated.
Plantlets from the in vitro rooting were washed to remove agar and transferred to plug trays containing a substrate comprised of 40% peat, 20% pine bark, 20% perlite, and 20% sand based on volume (Zhonghe Agriculture, Huaian, China).The plug trays were covered with transparent plastic film to reduce evaporation and placed on the benches of a shaded and evaporative-pad-cooling greenhouse with a temperature ranging from 20 • C to 28 • C, a relative humidity varying from 50% to 80%, and a light intensity of about 400 µmol m −2 s −1 .One month after the plantlets became adapted to the environment, the plastic film was removed.The plants in the plug trays were watered with half-strength Hoagland nutrient solution every 15 days for two months, and then transplanted into 10 cm square plastic pots containing the same substrate for an additional two months.The survey rate of ex vitro grown plants was recorded each month for up to four months.

DNA Flow Cytometry Analysis of Plants Derived from Shoot Culture
Young leaves were collected from three randomly selected plants derived from the shoot culture or three original plants initially used for the shoot culture and analyzed for possible occurrence of somaclonal variants at ploidy levels [37].Tomato leaves were used as an internal reference standard.The analysis was conducted by the Molecular Biology Experiment Center, Germplasm Bank of Wild Species in Southwest China (Kunming, China).The DNA histograms of nuclei from parental plants and shoot-cultured plants were compared.

Data Collection and Statistics Analysis
Data were examined for normality and homogeneity of variance using SPPS Statistics version 20.0 (IBM Corp., Armonk, NY, USA).The data were subjected to analysis of variance (ANOVA) using SPPS.If significance occurred, mean differences among treatments were separated by the least significant difference (LSD) test at p < 0.05 level.All data were presented as means ± standard errors with n = 3.

Effect of Growth Regulator Combinations on Axillary Shoot Induction
Axillary shoots were induced from the nodes of S. portulacastrum explants cultured on MS medium supplemented with different growth regulator combinations (Figure 2).The average number of axillary shoots per explant induced by the growth regulators ranged from 3.0 to 7.4 but was only 1.21 without the growth regulators (Table 1).When the explants were cultured on MS medium containing BA with either IBA, IAA, or NAA, the average number of axillary shoots differed from 3.79 to 5.60, and callus was observed on MS 2-6 and MS 8-9.BA with 2,4-D (MS 4) induced abundant callus at the base of the stem segment, resulting in the lowest number of axillary shoots.The axillary shoots induced on MS 1 exhibited a reddish color (Figure 2).TDZ, as a synthetic phenyl urea derivative possessing strong cytokinin-like activity [38], only induced 4.12 axillary shoots.When the nodal explants were cultured on MS medium supplemented with ZT and IBA, IAA, or NAA, the average number of axillary shoots ranged from 4.31 to 7.41.ZT at 2 mg L −1 with 0.2 mg L −1 NAA induced the highest number of axillary shoots (7.4), which was 42.5% higher than the number of 5.2 induced by 40 µM 2iP, reported by Lokhande et al. [35].Furthermore, no callus appeared at the bases of the explants.
Our results showed that among the growth regulators tested, ZT with NAA was the most effective combination for inducing axillary shoots, with shoot lengths ranging from 10 to 20 mm.Although we did not test 2iP, which was reported by Lokhande et al. [35], we did evaluate BA and TDZ; the number of axillary shoots induced by ZT and NAA was much higher than the 5.2 induced by 2iP.As a result, ZT with NAA was considered to be the optimal cytokinin and auxin combination for the induction of axillary shoots from S. portulacastrum.
IAA, or NAA, the average number of axillary shoots ranged from 4.31 to 7.41.ZT at 2 mg L −1 with 0.2 mg L −1 NAA induced the highest number of axillary shoots (7.4), which was 42.5% higher than the number of 5.2 induced by 40 μM 2iP, reported by Lokhande et al. [35].Furthermore, no callus appeared at the bases of the explants.
Our results showed that among the growth regulators tested, ZT with NAA was the most effective combination for inducing axillary shoots, with shoot lengths ranging from 10 to 20 mm.Although we did not test 2iP, which was reported by Lokhande et al. [35], we did evaluate BA and TDZ; the number of axillary shoots induced by ZT and NAA was much higher than the 5.2 induced by 2iP.As a result, ZT with NAA was considered to be the optimal cytokinin and auxin combination for the induction of axillary shoots from S. portulacastrum.1) on induction of axillary shoots from nodal explants of S. portulacastrum cultured on MS medium.The photos were taken after eight weeks of culture (photos for MS 9 and MS 10 are not shown).

Optimization of Shoot Multiplication
To identify the optimal concentration for inducing axillary shoots, the nodal explants were cultured on 20 combinations of ZT and NAA.Axillary shoots appeared from the node of the explants after two weeks of culture (Figure 3).With the continuation of the culture, more axillary shoots were induced.The number of axillary shoots ranged from 6.44 to 12.11 per explant (Table 2).At the ZT concentration of 0.5 mg L −1 , increased concentrations of NAA did not significantly change the number of axillary shoots.However, at the ZT concentration of 1.0 mg L −1 , increased NAA concentrations from 0.05 mg L −1 to 0.3 mg L −1 resulted in a significant increase in axillary shoots from 8.89 to 12.11, but a further increase of NAA to 0.4 mg L −1 caused a reduction in axillary shoot numbers.At the ZT concentration of 2.0 mg L −1 , the number of axillary shoots induced by 0.2 mg L −1 NAA L −1 was significantly higher than that induced by the NAA concentrations of 0.3 mg L −1 to 0.4 mg L −1 .There was no significant difference in the axillary shoot numbers when the explants were induced by 3.0 mg L −1 ZT with either concentration of NAA (Table 2).  1) on induction of axillary shoots from nodal explants of S. portulacastrum cultured on MS medium.The photos were taken after eight weeks of culture (photos for MS 9 and MS 10 are not shown).

Optimization of Shoot Multiplication
To identify the optimal concentration for inducing axillary shoots, the nodal explants were cultured on 20 combinations of ZT and NAA.Axillary shoots appeared from the node of the explants after two weeks of culture (Figure 3).With the continuation of the culture, more axillary shoots were induced.The number of axillary shoots ranged from 6.44 to 12.11 per explant (Table 2).At the ZT concentration of 0.5 mg L −1 , increased concentrations of NAA did not significantly change the number of axillary shoots.However, at the ZT concentration of 1.0 mg L −1 , increased NAA concentrations from 0.05 mg L −1 to 0.3 mg L −1 resulted in a significant increase in axillary shoots from 8.89 to 12.11, but a further increase of NAA to 0.4 mg L −1 caused a reduction in axillary shoot numbers.At the ZT concentration of 2.0 mg L −1 , the number of axillary shoots induced by 0.2 mg L −1 NAA L −1 was significantly higher than that induced by the NAA concentrations of 0.3 mg L −1 to 0.4 mg L −1 .There was no significant difference in the axillary shoot numbers when the explants were induced by 3.0 mg L −1 ZT with either concentration of NAA (Table 2).
The optimization experiment showed that ZT at 1.0 mg L −1 and 0.3 mg L −1 NAA were the most effective concentrations for inducing axillary shoots from nodal explants of S. portulacastrum, since more than 12 shoots were produced (Table 2) and the shoot lengths varied from 15 to 32 mm.As reported by Fay and Gratton [39], there was little information available on the micropropagation of plants in the family Aizoaceae.Thus far, there has been only one report on the in vitro culture of S. portulacastrum [35].In said study, ZT was not used, and the authors found that 2iP, not BA and TDZ, was more effective in axillary shoot induction.The present study showed that ZT at 1.0 mg L −1 with 0.3 mg L −1 NAA induced 12.11 axillary shoots per nodal explant compared to 5.2 induced by 2iP.It is known that plant species, growth regulators, and even genotypes and explant sources significantly affect in vitro culture [35,40,41].Lokhande et al. [35] showed that clones of S. portulacastrum collected from different geographical regions in India responded differently to axillary shoot induction.Nevertheless, our results are consistent with those reported by Fan et al. [42] on the shoot culture of blueberry and Wei et al. [33] on the shoot culture of Rhododendron fortune, where ZT was more effective in the induction of axillary shoots than BA.Similarly, ZT was reported to induce more shoots from the explants of raspberry [43] and olive [44] than BA.The effectiveness of ZT may imply that ZT, as a native plant growth hormone in the cytokinin family, is more compatible than the synthetic cytokinins with the other native growth hormones in S. portulacastrum for multiplication.Further studies are needed to test this proposition.The optimization experiment showed that ZT at 1.0 mg L −1 and 0.3 mg L −1 NAA were the most effective concentrations for inducing axillary shoots from nodal explants of S. portulacastrum, since more than 12 shoots were produced (Table 2) and the shoot lengths varied from 15 to 32 mm.As reported by Fay and Gratton [39], there was little information available on the micropropagation of plants in the family Aizoaceae.Thus far, there has been only one report on the in vitro culture of S. portulacastrum [35].In said study, ZT was not used, and the authors found that 2iP, not BA and TDZ, was more effective in axillary shoot induction.The present study showed that ZT at 1.0 mg L −1 with 0.3 mg L −1 NAA induced 12.11 axillary shoots per nodal explant compared to 5.2 induced by 2iP.It is known that plant species, growth regulators, and even genotypes and explant sources significantly affect in vitro culture [35,40,41].Lokhande et al. [35] showed that clones of S. portulacastrum collected from different geographical regions in India responded differently to axillary shoot induction.Nevertheless, our results are consistent with those reported by Fan et al. [42] on the shoot culture of blueberry and Wei et al. [33] on the shoot culture of Rhododendron fortune, where ZT was more effective in the induction of axillary shoots than BA.Similarly, ZT was reported to induce more shoots from the explants of raspberry [43] and olive [44] than BA.The effectiveness of ZT may imply that ZT, as a native plant growth hormone in the cytokinin family, is more compatible than the synthetic cytokinins with the other native growth hormones in S. portulacastrum for multiplication.Further studies are needed to test this proposition.
It is noteworthy that callus occurred in the bases of the nodal explants on day 77 (Figure 3, D 77) and more callus appeared on day 90 (Figure 3, D 90) when nodal explants were induced by 1.0 mg L −1 ZT and 0.3 mg L −1 NAA.Since there is a possibility of adventitious shoot regeneration from the callus [45], and shoots derived from indirect organogenesis have the potential to produce somaclonal variants [32,34], it is strongly advised that axillary shoots induced by 1.0 mg L −1 ZT and 0.3 mg L −1 NAA should be excised before day 77 to avoid any adventitious shoots regenerated from the callus.It is noteworthy that callus occurred in the bases of the nodal explants on day 77 (Figure 3, D 77) and more callus appeared on day 90 (Figure 3, D 90) when nodal explants were induced by 1.0 mg L −1 ZT and 0.3 mg L −1 NAA.Since there is a possibility of adventitious shoot regeneration from the callus [45], and shoots derived from indirect organogenesis have the potential to produce somaclonal variants [32,34], it is strongly advised that axillary shoots induced by 1.0 mg L −1 ZT and 0.3 mg L −1 NAA should be excised before day 77 to avoid any adventitious shoots regenerated from the callus.

Rooting of Microcuttings
The induced axillary shoots were excised from the nodal explants, and the resultant microcuttings were rooted on half-strength MS medium supplemented with four concentrations of IBA or IAA.The percentages of adventitious root occurrence varied with the IAA or IBA concentrations (Figure 4).IBA at 0.6 mg L −1 induced 91.7% of the microcuttings to root, and each cutting produced 36 roots with a mean root length of 1.0 cm (Table 3).On the other hand, the highest rooting percentage induced by IAA was 66.7%, and the root numbers were only 15, which were significantly lower than those induced by 0.6 mg L −1 IBA.In the report of Lokhande et al. [35], microcuttings of S. portulacastrum were rooted in four concentrations of NAA or IAA.NAA at 10 µM induced 95% of the microcuttings to form adventitious roots compared to 85% induced by 5.0 µM IAA [35].

Ex Vitro Rooting and Acclimatization
The in vitro rooted plants (Figure 4), after being transplanted into cells of plastic plug tray filled with the aforementioned substrate, were established in one month (Figure 5 upper panel).The plants in the plug trays had a survey rate of 100% after two months of acclimatization (Figure 5, middle panel).The liners (individual young plants derived from tissue culture or seed germination grown in cell plug trays with well-developed root systems) were transplanted into 10-cm square plastic pots filled with the same substrate for further acclimatization in the greenhouse.After two months of growth in the square pots, the plants were healthy, disease-free, and grew vigorously (Figure 5, bottom panel).These  The auxins of IAA, IBA, and NAA are commonly used for inducing adventitious roots.IAA, as the native hormone of plants, was the first to be used to promote the rooting of cuttings, and IBA and NAA are synthesized forms of auxins.IBA was found to be more effective than IAA in the induction of adventitious roots [46].Our data, along with those of Fan et al. [42] and Wei et al. [33], support the claim that IBA is generally more effective than IAA in promoting the rooting of microcuttings.Furthermore, the application of IBA substantially increases the root numbers of microcuttings.

Ex Vitro Rooting and Acclimatization
The in vitro rooted plants (Figure 4), after being transplanted into cells of plastic plug tray filled with the aforementioned substrate, were established in one month (Figure 5 upper panel).The plants in the plug trays had a survey rate of 100% after two months of acclimatization (Figure 5, middle panel).The liners (individual young plants derived from tissue culture or seed germination grown in cell plug trays with well-developed root systems) were transplanted into 10-cm square plastic pots filled with the same substrate for further acclimatization in the greenhouse.After two months of growth in the square pots, the plants were healthy, disease-free, and grew vigorously (Figure 5, bottom panel).These plants were either allowed to grow into stock plants for producing cuttings or transplanted to seashore soils for beach restoration, floating beds for the phytoremediation of wastewater, or saline soils for reducing salinization.

DNA Flow Cytometry Analysis of Plants Derived from Shoot Culture
Flow cytometry analysis showed that the plants derived from the shoot culture (Figure 6A) had the same peaks as the original S. portulacastrum plants initially used for establishing the shoot culture protocol (Figure 6B).The first peak was the internal reference of tomato plants, and the next peak with the count over 120 was derived from S. portulacastrum.There were two additional small peaks, which were probably related to the chromosome characteristics, as type A, C, and D chromosomes were common in S. portulacastrum [47].Regardless, the number of peaks and their positions in the histograms between the original and the shoot-cultured plants were identical, suggesting that there was no ploidy variation in the plants derived from the nodal culture.Additionally, no morphologically different plants were observed among the plants derived from the shoot culture.These results were as expected, as in vitro culture using existing meristems generally produces true-to-type plantlets [34,38].Our results were also consistent with the report of Thiem and Sliwinska [48], where the shoot culture of Rubus chamaemorus produced plantlets without chromosome variation.

DNA Flow Cytometry Analysis of Plants Derived from Shoot Culture
Flow cytometry analysis showed that the plants derived from the shoot culture (Figure 6A) had the same peaks as the original S. portulacastrum plants initially used for establishing the shoot culture protocol (Figure 6B).The first peak was the internal reference of tomato plants, and the next peak with the count over 120 was derived from S. portulacastrum.There were two additional small peaks, which were probably related to the chromosome characteristics, as type A, C, and D chromosomes were common in S. portulacastrum [47].Regardless, the number of peaks and their positions in the histograms between the original and the shoot-cultured plants were identical, suggesting that there was no ploidy variation in the plants derived from the nodal culture.Additionally, no morphologically different plants were observed among the plants derived from the shoot culture.These results were as expected, as in vitro culture using existing meristems generally produces true-to-type plantlets [34,38].Our results were also consistent with the report of Thiem and Sliwinska [48], where the shoot culture of Rubus chamaemorus produced plantlets without chromosome variation.

Conclusions
Sesuvium portulacastrum has been used for the restoration of shorelines [49] and the desalination of saline soils [50].Recently, it has been recognized as an important plant for the remediation of N, P, and antibiotics in aquaculture ponds or waterbodies and of heavy metals in wastewater [1,51].As a result, there has been an increasing demand for propagules of S. portulacastrum used for phytoremediation.This study developed an efficient and simple protocol for propagating S. portulacastrum through nodal culture.The in vitro culture of a nodal explant on MS medium supplemented 1.0 mg L −1 ZT and 0.3 mg L −1 NAA induced an average of 12.11 axillary shoots.The microcuttings derived from the axillary shoots were easily rooted in vitro in 24 days, and the in vitro rooted plants were acclimatized in a greenhouse with a 100% survival rate.Furthermore, the plants were stable, without variation in ploidy levels.As the axillary shoots were induced in vitro under aseptic conditions, they were disease-free.The healthy and uniform plants could be readily transplanted to seashore soils for beach restoration, floating beds for the phytoremediation of wastewater, or saline soils for reducing salinization.

Conclusions
Sesuvium portulacastrum has been used for the restoration of shorelines [49] and the desalination of saline soils [50].Recently, it has been recognized as an important plant for the remediation of N, P, and antibiotics in aquaculture ponds or waterbodies and of heavy metals in wastewater [1,51].As a result, there has been an increasing demand for propagules of S. portulacastrum used for phytoremediation.This study developed an efficient and simple protocol for propagating S. portulacastrum through nodal culture.The in vitro culture of a nodal explant on MS medium supplemented 1.0 mg L −1 ZT and 0.3 mg L −1 NAA induced an average of 12.11 axillary shoots.The microcuttings derived from the axillary shoots were easily rooted in vitro in 24 days, and the in vitro rooted plants were acclimatized in a greenhouse with a 100% survival rate.Furthermore, the plants were stable, without variation in ploidy levels.As the axillary shoots were induced in vitro under aseptic conditions, they were disease-free.The healthy and uniform plants could be readily transplanted to seashore soils for beach restoration, floating beds for the phytoremediation of wastewater, or saline soils for reducing salinization.

Agriculture 2022 , 13 Figure 1 .
Figure 1.Sesuvium portulacastrum plants were used for remediation of pollutants in aquaculture (A) and stabilization of shoreline soil (B) in Fujian, China.The occurrence of disease caused by Gibbago trianthemae in S. portulacastrum grown in coastal soil (C) and during cutting propagation (D).All the photos were taken by Xiangying Wei.

Figure 1 .
Figure 1.Sesuvium portulacastrum plants were used for remediation of pollutants in aquaculture (A) and stabilization of shoreline soil (B) in Fujian, China.The occurrence of disease caused by Gibbago trianthemae in S. portulacastrum grown in coastal soil (C) and during cutting propagation (D).All the photos were taken by Xiangying Wei.

Figure 2 .
Figure 2. Effect of different growth regulator combinations (see Table1) on induction of axillary shoots from nodal explants of S. portulacastrum cultured on MS medium.The photos were taken after eight weeks of culture (photos for MS 9 and MS 10 are not shown).

Figure 3 .
Figure 3. Axillary shoots induced from nodal explants of S. portulacastrum cultured on MS medium supplemented with 1.0 mg L −1 ZT and 0.3 mg L −1 NAA from day one (D 1) to day 90 (D 90).

Figure 4 .
Figure 4. Microcuttings of S. portulacastrum rooted on half-strength MS medium supplemented with 0.6 mg L −1 IBA from day 10 (D 10) to day 24 (D 24) (upper panel).The lower panel shows the increase in root numbers viewed from the bottom of culture vessels.

Figure 4 .
Figure 4. Microcuttings of S. portulacastrum rooted on half-strength MS medium supplemented with 0.6 mg L −1 IBA from day 10 (D 10) to day 24 (D 24) (upper panel).The lower panel shows the increase in root numbers viewed from the bottom of culture vessels.

Agriculture 2022 , 13 Figure 5 .
Figure 5. Acclimatization of plants in a greenhouse where in vitro rooted plants were transplanted into a cell plug tray filled with a substrate and grown in the greenhouse for one month (upper panel, M1 top view and side view, respectively).Middle panel: a top view and side view of plants after two months of growth in a plug tray.Bottom panel: a top view and side view of plants grown in 10 cm square pots at month four in the greenhouse.

Figure 5 .
Figure 5. Acclimatization of plants in a greenhouse where in vitro rooted plants were transplanted into a cell plug tray filled with a substrate and grown in the greenhouse for one month (upper panel, M1 top view and side view, respectively).Middle panel: a top view and side view of plants after two months of growth in a plug tray.Bottom panel: a top view and side view of plants grown in 10 cm square pots at month four in the greenhouse.

Agriculture 2022 , 13 Figure 6 .
Figure 6.Histograms of relative fluorescence intensity of plants derived from the shoot culture (A) and the original S. portulacastrum plants (B).

Figure 6 .
Figure 6.Histograms of relative fluorescence intensity of plants derived from the shoot culture (A) and the original S. portulacastrum plants (B).

Table 1 .
Axillary shoots induced from nodal explants of S. portulacastrum after culturing on MS medium supplemented with different growth regulators for eight weeks.

Table 2 .
Effect of different concentrations of ZT and NAA on induction of axillary shoots from nodal explants of S. portulacastrum cultured on 20 shoot induction media.

Table 3 .
Effect of half-strength MS supplemented with different concentrations of IBA or IAA on in vitro rooting of S. portulacastrum over 24 days.

Table 3 .
Effect of half-strength MS supplemented with different concentrations of IBA or IAA on in vitro rooting of S. portulacastrum over 24 days.