HORTSCIENCE 55(4):436–443. 2020. https://doi.org/10.21273/HORTSCI14584-19 sion and biodiversity maintenance, and for reduction of energy and water consumption, whereas their potential salinity tolerance In Vitro Propagation and NaCl could be an important commercial feature for reducing production costs (Cassaniti and Tolerance of the Multipurpose Romano, 2011). It is noteworthy that there are 1 billion hectares of salt-affected land Medicinal Halophyte Limoniastrum worldwide that may be resource opportuni- ties for halotechnologies, such as halophyte monopetalum crops and landscape plants, which grow bet- ter under high salinities (Yensen, 2008). Aikaterini N. Martini and Maria Papafotiou Halophytes adapt to salinity through complex Laboratory of Floriculture and Landscape Architecture, Department of Crop mechanisms of avoidance, evasion, or adap- tation processes and tolerance (Breckle, Science, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, 2002). L. monopetalum adsorbs salts and then Greece secretes them through salt glands found on its leaves, a strategy that makes it a typical Additional index words. ex vitro acclimatization, in vitro rooting, Mediterranean native plant, halophyte (Akoumianaki-Ioannidou et al., salinity tolerance, shoot multiplication 2015). Several halophytes deal with fre- Abstract. Limoniastrum monopetalum is an evergreen perennial shrub native to Medi- quent changes in salinity level and synthe- terranean coastal sands and salt marshes. It has adapted to a variety of environmental size several bioactive molecules (primary stresses and is used in traditional medicine and as an ornamental plant. In the present and secondary metabolites) that display study, an efficient micropropagation protocol for this species was developed to facilitate potent antioxidant, antimicrobial, anti- the production of selected genotypes and promote its wider use. Research has focused on inflammatory, and antitumoral activities the effects of various cytokinin types [benzyladenine (BA), zeatin, 6-furfurylaminopurine (Ksouri et al., 2012). (kinetin) or 6-g-g-dimethylallilopurine (2iP)] and concentrations (0.0–4.0 mg·LL1) and L. monopetalum is adapted to water def- various NaCl concentrations (0.0–20 g·LL1) during all stages of in vitro culture. For in icits, high light intensity, and high tempera- vitro establishment, Murashige and Skoog (MS) medium supplemented with 0.5 mg·LL1 tures (Neves et al., 2008), and it is capable of BA and 0.0 or 5.0 g·LL1 NaCl was most appropriate (100% explant response, 3–4 shoots growing on soil poor in organic matter per explant, 2 cm shoot length). The best results for shoot multiplication (100% response, (Salama, 2007), which could be attributed 9 shoots per explant, 0.8–1.0 cm shoot length) were obtained with low (0.5 mg·LL1)BAor to various structural and chemical character- relatively high (2.0 mg·LL1) kinetin concentrations in the medium; however, 0.5 mg·LL1 istics that provide defense and protection kinetin should be preferred in the case of production of multiple rooted microshoots (Akoumianaki-Ioannidou et al., 2015). Its during one stage. The addition of NaCl at relatively low concentrations (2.5 or 5.0 g·LL1) ecological value as a sand accumulator, in a medium supplemented with 0.5 mg·LL1 BA doubled shoot multiplication but did not salt-tolerant, and wind-breaker (Salama, improve shoot elongation (100% explant response, 16 shoots per explant, 0.8 cm shoot 2007) should not be ignored. It can grow in length). For in vitro rooting, half-strength MS medium supplemented with 1.0 mg·LL1 oil-contaminated soils (El-Bakatoushi, 2011; IBA was most appropriate (97% rooting, 9.4 roots per microshoot, 1.2 cm root length). Hussein and Terry, 2002) and has the poten- Regarding the effects of NaCl on in vitro rooting, microshoots were relatively tolerant to tial of phytoremediation of heavy metals NaCl concentrations up to 10.0 g·LL1. The effects of NaCl depend on the micro- from polluted sites (Cambrolle et al., 2013a, propagation stage; they are synergistic during shoot multiplication and tolerant during 2013b; Manousaki et al., 2014). All these rooting. However, explants responded satisfactorily in its absence, indicating that NaCl adaptations make L. monopetalum an ideal was not necessary as a medium component. Ex vitro acclimatization and establishment of ornamental plant for xeriscaping and land- plantlets was 100% successful in a mixture of peat:perlite 1:1 or 2:1 (v/v). scape architecture in arid and semiarid Med- iterranean areas with adverse conditions, such as restoration of quarries or roadsides, Limoniastrum monopetalum (L.) Boiss branched spikes during June to August green roofs, archaeological sites, and other (Statice monopetala L., Plumbaginaceae) is (Blamey and Grey-Wilson, 1993). It is a disturbed areas. a small, silvery, blue-green evergreen peren- valuable multipurpose halophyte used in tra- We have successfully propagated L. nial shrub with much-branched, leafy stems ditional medicine as an antidysenteric agent monopetalum by stem-tip cuttings, more that is native to coastal sands and salt marshes against infectious diseases or parasites that effectively during winter and spring in southern Greece and other Mediterranean cause painful and bloody diarrhea (Chaieb (Akoumianaki-Ioannidou et al., 2016). Our countries. It has fleshy leaves, is covered with and Boukhris, 1998), for hair tinting and skin preliminary results regarding its micropropa- white scales and bright pink flowers that tanning (Ksouri et al., 2012), and as an gation (Martini and Papafotiou, 2016) indi- · –1 become violet after drying and are borne in ornamental plant (Lieth and Mochtchenko, cated that BA at 0.5 mg L induced the most and longest shoots of rather small length 2002). It is rich in phenolics and antioxidants (they did not exceed 0.8 cm). (Aboul-Enein et al., 2012; Trabelsi et al., Biotechnological tools are important to Received for publication 25 Sept. 2019. Accepted 2010, 2012, 2013); therefore, it has been for publication 16 Dec. 2019. select, multiply, conserve, and genetically proposed as a novel source of natural antiox- enhance the critical genotypes of medicinal Published online 20 February 2020. idants for human consumption and for agro- This work was supported by the NSRF 2007–2013, plants in particular (Debnath et al., 2006; Operational Program ‘‘Education and Lifelong food, cosmetic, and pharmaceutical industries Tripathi and Tripathi, 2003). Plant tissue Learning’’–funded research project: Thales— (Trabelsi et al., 2010, 2012, 2013). Further- culture techniques offer an integrated ap- ‘‘Integrated management of vegetation at archaeo- more, it has high potential to be used as a proach for the production of standardized logical sites to protect monuments and enhance the fodder-producing plant because it is rich in quality pharmaceuticals through the mass historical landscape’’—‘‘ARCHAEOSCAPE,’’ MIS nutritive values (carbohydrates and protein) production of consistent plant material with code 380 237. and has low water requirements (Neves et al., M.P. is the corresponding author. E-mail: mpapaf@ qualitatively and quantitatively uniform aua.gr. 2007; Zahran and El-Amier, 2013). chemical constituents. Furthermore, an This is an open access article distributed under the Halophytes, due to their adaptation to ever-increasing demand for uniform medici- CC BY-NC-ND license (https://creativecommons. salinity, are mainly suitable for restoration nal plant-based medicines warrants their org/licenses/by-nc-nd/4.0/). of disturbed landscapes, control of soil ero- mass cloning through plant tissue strategies 436 HORTSCIENCE VOL. 55(4) APRIL 2020 (Chaturvedi et al., 2007; Debnath et al., shige and Skoog (MS) (Murashige and lected after 40 d of culture. Two more subcul- 2006). Skoog, 1962) medium with 30 g·L–1 sucrose tures on MS medium with 0.5 mg·L–1 BA During ex situ conservation of wild plants and 1.0 mg·L–1 benzyladenine (BA) (Martini without the addition of NaCl were performed from salt-affected coastal habitats using tis- and Papafotiou, 2016) after two subcultures to produce microshoots for rooting experiments. sue culture, it is important to search for a on the same medium were used as explants. In vitro rooting. For rooting, microshoots possible specific requirement of NaCl in the They were cultured on MS medium with 30 0.8 to 1.0 cm long excised from fourth to cultivation medium (Freipica and Ievinsh, g·L–1 sucrose, supplemented with various sixth subcultures of the culture established by 2010). Therefore, NaCl was a key factor for concentrations (0.5, 1.0, 2.0, and 4.0 mg·L–1) Martini and Papafotiou (2016) on MS me- in vitro propagation of Salicornia europaea of BA, zeatin, 6-furfurylaminopurine (kine- dium with 0.5 mg·L–1 BA were cultured on (Shi et al., 2006) and Salicornia brachiata tin) or 6-g-g-dimethylallilopurine (2iP) as half-strength MS medium with 20 g·L–1 su- (Joshi et al., 2012), whereas in Crithmum well as on plant growth regulator-free crose and various (0.0, 0.5, 1.0, 2.0, and 4.0 maritimum shoot proliferation was gradually (PGR-free) medium (control) to find the op- mg·L–1) concentrations of indole-3-butyric reduced at higher concentrations of NaCl, timum cytokinin type and concentration for acid (IBA). Three replications of 15 micro- although shoot height was enhanced shoot multiplication and elongation. A cul- shoots were used for each treatment. Data (Grigoriadou and Maloupa, 2008). Relatively ture was also maintained with successive were collected after 35 d of culture. high NaCl tolerance in conditions of tissue subcultures on MS medium with 30 g·L–1 Effects of NaCl on in vitro rooting. To culture was also found for three rare and sucrose and 0.5 mg·L–1 BA for the production investigate the effects of NaCl on in vitro endangered coastal plant species of the Baltic of microshoots for rooting experiments. rooting, the concentration of NaCl both during Sea: Glaux maritima, Dianthus arenarius During the shoot multiplication experi- the stage of microshoot production and during spp.