43 1. Introduction The world’s cultivated lands are increasingly affected by drought and salinity (Barrett-Lennard, 2000). Halo- phytes growing in highly saline soils could thus serve as a resource for the identification and development of new crop systems for marginal saline soils (Debez et al., 2011; Ben Hamed et al., 2013). The actual yield of halo- phytes remains largely unknown since their domestica- tion is still limited, yet the economic potential of some of these plants has been positively assessed by various groups (Aronson, 1989; Ashour and Thalooth, 1993; Ab- delly et al., 2006). Numerous Brassicaceae species are current and emerging biodiesel crops: in addition to the oil-rich seed, the ability of Brassicaceae species to grow on marginal land with minimal inputs make them par- ticularly attractive and potentially viable for this applica- tion. Among halophyte species studied for their potential as oleaginous plants, the Brassicaceae Cakile edentula (O’Leary et al., 1985), Crambe abysinnica (Mandal et al., 2002) or Cakile maritima (Ghars et al., 2005) have been reported to contain high amounts of oil. Cakile ma- ritima, the sea rocket, is an annual succulent halophyte frequently found from the Black Sea coasts to the Medi- terranean region, and from the Atlantic coasts of North Africa to the north of Europe (Clausing et al., 2000). Tunisian accessions of C. maritima contain up to 40% seed-oil (Ghars et al., 2005). Plant growth, harvest index, silique number and seeds produced per fruit segment is maximal at 100 mM NaCl (Debez et al., 2008) but C. maritima can survive at up to 800 mM NaCl (Ellouzi et al., 2013) and successfully reproduces till 500 mM NaCl salinity (Debez et al., 2004). Seed-oil content did not seem to be affected by salinity, although erucic acid level could increase (Debez et al., 2006). These facts highlight the need to better understand the basis of adaptation to saline environments, as well as traits associated with oil production itself. In previous works we described some aspects of the response of C. maritima to salt stress (De- bez et al., 2004, 2006, 2008; Ellouzi et al., 2011) and our data point out that C. maritima adopts a complex survival strategy at high salinity, however numerous data concerning the cellular mechanisms allowing this plant to resist salinity are still lacking. Suspension culture cells offer an in vitro system that is widely used in plant biology as a convenient tool to inves- tigate a wide range of phenomena. It consists in a model system as suspension culture provide a ready source of a homogenous cell type and avoids the complications of multicellular tissue types in planta (Moscatiello et al., 2013). Suspension culture cells were recently used in vari- ous studies and models concerning plant responses to sa- linity, such as proteomic studies (Chen et al., 2012; Liu et al., 2013), metabolomic studies (Liu et al., 2013) or Establishment of a cell suspension culture of the halophyte Cakile maritima I. Ben Hamed*, **, B. Biligui*, D. Arbelet-Bonnin*, C. Abdelly**, K. Ben Hamed**, F. Bouteau*(1) * Université Paris Diderot, Sorbonne Paris Cité, Institut des Energies de Demain, UMR 8632, Paris, France. ** Laboratoire des Plantes Extrêmophiles, Centre de Biotechnologie de Borj Cedria, University of Carthage-Tunis, BP 901, 2050 Hammam Lif, Tunisia. Keys words: biofuel, Cakile maritima, cell suspension, halophytes, oil. Abstract: Cakile maritima is a member of the Brassicaceae family also known as sea rocket. It is an annual succulent halophyte frequent in coastal dune vegetation in Mediterranean regions and Atlantic coasts from North Africa to the north of Europe. This halophyte presents a complex survival strategy at high salinity and its seeds contain up to 40% of an oil which could be suitable for biofuel production and other industrial applications. However, data concerning the cellular mechanisms allowing this plant to resist salinity are still lacking. Cell suspension cultures offer an in vitro system convenient for cell biology studies and biotechnological methods are still not developed for this putative crop. The present paper reports initiation of C. maritima cell suspension cultures from callus obtained from aerial parts of seedlings. The establishment of a suspension culture which preserves its salt resistance provides an opportunity to gain insights into C. maritima biology. Adv. Hort. Sci., 2014 28(1): 43-48 (1) Corresponding author: francois.bouteau@univ-paris-dodierot.fr Received for publication 17 March 2014 Accepted for publication 4 June 2014 44 transcriptomic studies (Matsuura et al., 2010; Bae et al., 2012). It is also a convenient means to study transport sys- tem regulations and oxidative responses to various biotic and abiotic constraints (Kadono et al., 2010; Baz et al., 2012; Yukihiro et al., 2012; Tran et al., 2013), comprising salinity (Cessna et al., 2007; Wang et al., 2010; Pons et al., 2011; Queirós et al., 2011). Here, we report the development of a cell suspension culture of C. maritime; growth performance was evaluated on control and NaCl cultured cells to validate the biologi- cal system. These suspension culture cells could be a valu- able tool to gain further insights into halophyte studies and their potential applications. 2. Materials and Methods Establishment of callus of Cakile maritima In this study we used Cakile maritima seeds harvest- ed in the Raoued region in the north of Tunisia. Callus cultures were initiated from the aerial part of 14-day-old, light-grown seedlings. Seeds were submerged in 70% ethanol for 1 min, then rinsed with sterile distilled water, submerged in chlorine bleach for 10 min and then rinsed three times (5 min each) with sterile distilled water. The seeds were placed in petri-dishes containing Murashige and Skoog medium including vitamins (MS) (Murashige and Skoog, 1962), supplemented with 30 g.L-1 sucrose, 8 g.L-1 agar. The pH was adjusted to 5.8 with KOH. Stem segments were finely cut and then placed on a solid callus-inducing medium (CIM) containing 6.2 g.L-1 Gamborg B5 (Gamborg et al., 1968) supplemented with 20 g.L-1 glucose, 8 g.L-1 agar and with growth regulators 9.06 µM of 2.4 D and 0.46 µM of kinetin. The pH was adjusted to 5.7 with KOH. After two to three weeks, cal- lus appeared on the sides of the segments. When the size of callus became larger than 1 cm, they were divided and transferred to a new medium. Establishment cell suspension cultures of Cakile maritima Approximately 5 g of callus were transferred to 125 mL flasks containing fresh Gamborg B5 medium supple- mented with 30 g.L-1 glucose, 0.2 µM 2,4-D and 0.45 µM kinetin. The pH was adjusted to 5.7 with KOH. Flasks were incubated on a rotary shaker at 120 rpm and main- tained at 22°C in the dark. Feeding of the cultures with fresh medium was done at 10-14 day intervals during which time the suspensions were allowed to settle under agitation (rotary shaker at 120 rpm). This procedure was repeated for about eight weeks. Then, the suspension was subcultured every seven days by transferring 20 mL of the culture into 50 mL of fresh medium in 250 mL Erlen- meyer flasks. Arabidopsis thaliana cell suspension culture conditions Arabidopsis thaliana L. cell suspensions were prepared from calluses of the cell line T87 generated from the eco- type Columbia plant as previously described (Tran et al., 2013). The suspension cells were obtained after about two months and five to six subcultures in 1 L round-bottom flasks containing 350 ml liquid Gamborg B5 culture me- dium (pH 5.8). Cell suspensions were sub-cultured weekly using a 1:10 dilution. Growth evaluation Growth of the culture was evaluated by measuring the fresh weight of cells and the density of cells using a Na- geotte cell. Cell viability Cell viability was assayed using the vital dye neutral red. Cells (100 µL) were incubated for 5 min in 400 µL phosphate buffer pH 7 with neutral red to a final concen- tration of 0.001% (w/v). Cells that did not accumulate neutral red were considered dead. At least 500 cells were counted for each replicate and the procedure was repeated at least three times for each treatment. External pH measurements Measurements of extracellular pH were performed with pH-sensitive electrodes every 24 h for six days from 5 mL of cultured medium cells. The procedure was repeated on three independent subcultures. Cell size Cell images were recorded with a camera (Kappa CF11DSP) on a light microscope (Labophot-2 Nikon) and sizes were measured using image analysis software KappaImageBase-2.2SP2-Metreo (Kappa Optoelectronics GmbH, Gleichen, Germany). Protoplast Isolation Protoplasts were isolated from suspension cultures six days after subculture. Fifteen mL of suspension cells were used. After cell sedimentation, the supernatant was removed and replaced by 5 mL of Gamborg B5 fresh me- dium containing 0.1 g cellulysin, 0.05 g macerase and 0.3 M sorbitol. The digestion was carried out under shaking at 120 rpm at 22°C for 30 min. After incubation, protoplasts were collected by centrifugation at 300 rpm for 3 min and re-suspended in 5 mL of Gamborg B5 fresh medium sup- plemented with 0.6 M sorbitol or 0.3 M sorbitol. 3. Results and Discussion The different steps in the development of Cakile ma- ritima callus from stem segments are reported in figure 1. Callus appeared on the sides of the discs after two to three weeks. Although produced in light, callus became non-chlo- rophyllian and pale yellow in color (Fig. 1C, D). Thus Cakile maritima loses its power of chlorophyll synthesis during its passage from plant stage to callus stage. To generate cell suspension, approximately 5 g of calluses were transferred in Gamborg medium. These calluses progressively disinte- grated to the smallest cell aggregates in the liquid medium. 45 Large quantities of cells could be obtained after two months feeding (Fig. 2A, B). At this stage suspension cells were subcultured every week in fresh Gamborg medium and after an additional 1.5 months the culture became more homo- geneous as the cell aggregates became smaller and the cell size increased (Fig. 2C, D). The level of dead cells in the culture also progressively decreased reaching about 10% after four months of culture, in the same range as observed for tobacco or A. thaliana suspension cells (Baz et al., 2012; Yukihiro et al., 2012; Tran et al., 2013). Using four-month-old C. maritima culture, growth curves were established by measuring the fresh weight of cells (Fig. 3A) and the cell density (Fig. 3B). Both curves showed a typical sigmoidal shape with a latency period of 24 h, then an exponential phase lasting about four days prior to a plateau phase (Fig. 3A, B) supposed to be due to nutrient depletion. We monitored the medium pH during the culture procedure (Fig. 3C). After a slight acidification at the beginning of the exponential phase, the pH became more alkaline, reaching 6.5 at the end of the culture (Fig. 3C). This alkalization could be involved in the decrease in biomass production as Ling et al. (2008) pointed out that pH of 6.7 lowered the growth of suspension culture of Ficus deltoidea. However, the pH variations recorded during the culture should remain suitable for nitrate uptake as reported for Ipomoea suspension cells (Martin and Rose, 1976). Histological analysis on six-day-old suspension cells re- vealed distinct morphological features of the cultured cells (Fig. 4). Although the cell aggregate size diminished dur- ing culture establishment, a few large aggregates (>20 cells) remained. However the largest part of the culture consisted of compact small groups of less than 20 cells, 60% corre- sponding to groups of three to 10 cells, single cells repre- senting about 10% of the population (Fig. 4A). Most of the cells were rounded in shape (Fig. 4B, left and center); less than 2% were elongated (Fig. 4B, right). Interestingly, as observed during the establishment of the culture (Fig. 2B, D), the size of the cells seemed to be dependent on the size of the groups, the isolated cells being the largest (Fig. 4C). It is known that protoplasts can be used for transient expression, trafficking assay or ion homeostasis analysis, notably in studies on plant resistance to salinity (Laohavis- it et al., 2012; Haro et al., 2013; Morgan et al., 2013; Mot- taleb et al., 2013; Son et al., 2013). Thus, we evaluated the usefulness of the cell cultures for the isolation of proto- plasts. Protoplasts were isolated from six-day-old suspen- sion cultures. Based on the different cell sizes measured (Fig. 4B, C) we observed protoplasts of different sizes. Most of these protoplasts maintained in 0.6 M sorbitol ap- Fig. 1 - Establishment of Cakile maritima calluses. (A) Three-week- old C. maritima seedling grown from sterilized seed. (B) Slices of leaf and rod were finely cut for callus generation. (C) One- month-old calluses derived from rod and leaf slices. (D) Mag- nification of a callus. Fig. 2 - Establishment of Cakile maritima cell suspensions. (A) Dense suspension cells grown in flask after two months feeding and (B) corresponding cell aggregates magnified 385X. (C) Dense sus- pension cells grown in flask after 1.5 months of subculture after feeding and (D) corresponding cell aggregates magnified 385X. Fig. 3 - Growth pattern of Cakile maritima cell suspension determined by fresh weight (A) or cell density measurements (B). Evolu- tion of the medium pH during cell suspension growth (C). Bars indicate mean ± SD of at least three experiments. 46 peared plasmolyzed and numerous protoplasts seemed to be shrunken (Fig. 5A). We then used 0.3 M sorbitol and obtained rounded protoplasts with large vacuoled evi- denced by neutral red staining (Fig. 5B). Finally, Cakile maritima being a halophyte, we checked the salinity resistance of the suspension cells. Suspension cells were subcultured in Gamborg medium complement- ed with 100, 400 or 800 mM NaCl. Sigmoidal growth curves were obtained and no significant differences were observed between the control and the suspension cells growing in presence of 100 mM NaCl when 400 mM and 800 mM strongly decreased the growth of the suspension cells (Fig. 6A). These data are in accordance with what was described for seedlings, C. maritima even requiring the presence of a moderate salt concentration (50-100 mM NaCl) to maintain a significant growth activity and plant development (Debez et al., 2004, 2008). Although strong- ly reduced at 400 mM or 800 mM NaCl, the growth of the suspension cells was also in accordance with previous data indicating that C. maritima can survive up to 800 mM Fig. 4 - Morphology of six-day-old Cakile maritima suspension cells. (A) Frequency of cell groups in the culture. (B) Different mor- phologies of cells. (C) Sizes of the cells according to the size of the cell groups. At least 250 cells were analyzed; bars indicate mean ± SD. Fig. 5 - Protoplasts derived from six-day-old Cakile maritima cell sus- pension maintained in 0.6 M sorbitol (A) or 0.3 M sorbitol (B). Fig. 6 - Evaluation of Cakile maritima cell suspension resistance to salinity. (A) Growth pattern of cell suspensions grown in pres- ence of 100, 400 or 800 mM NaCl determined by fresh weight measurement (B). Comparison of cell death extents of C. ma- ritima and Arabidopsis thaliana cell suspensions treated 6 h with NaCl concentrations ranging from 100 to 800 mM. Bars indicate mean ± SD of three experiments. 47 NaCl (Ellouzi et al., 2013) and successfully reproduces till 500 mM NaCl salinity even if the biomass was reduced (Debez et al., 2004). We further compared the extent of cell death induced 6 h after the addition of various NaCl concentrations on C. maritima and A. thaliana suspension cells. Cell death began to increase from 100 mM NaCl to almost 100% at 600 mM for A. thaliana (Fig. 6B). For C. maritima, the increase in cell death was significant only with 400 mM NaCl and reached only 60% at 800 mM, remaining largely inferior when compared to A. thaliana (Fig. 6B). It is worth noting that 40% of surviving cells probably go on dividing, which explains the growth of the culture, although reduced, at 800 mM NaCl (Fig. 6A). As a whole, these data demonstrate that C. maritima suspension cells preserve their ability to resist salinity. 4. Conclusions The present study reports the development of C. mariti- ma cell suspension cultures which maintained their salt re- sistance, offering greater understanding about adaptation to saline environments, as well as traits associated with biofuel production. Acknowledgements IBH was supported by fellowships from Tunisian Higher Education and Scientific Research Ministry and Université Paris-Sud. 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