Introduction

Seaweeds are a rich source of valuable compounds including food additives and biomedicinals. In Europe and North America, economically important species of algae are still collected from wild populations leading to the depletion of the natural resources by overharvesting (Sahoo and Yarish 2005; Neori et al. 1998). Moreover, the growing demand for reliable crop quantity and quality and the possible destruction of natural stocks by potential pollution events require the development of in vitro culture systems which could be a relevant alternative for the production of valuable compounds. Furthermore, while tissue culture and protoplast technology represent a source of novel and potentially useful genetic variations, either by somaclonal variation or by somatic hybridization, efforts to develop programs of genetic improvement by conventional techniques are being hindered by the complex life histories of algae and by the difficulty of cross-breeding (Polne-Fuller and Gibor 1986). Calluses and tissue cultures could also be used as a means for the maintenance and propagation of systems of production of value-added secondary metabolites (Rorrer and Cheney 2004) and finally used as highly efficient techniques for germplasm storage in selection programs (Garcia-Reina et al. 1991). Some economically interesting marine macroalgae belong to the order of Laminariales and are used either for the industrial extraction of phycocolloids and iodine (Laminaria, Macrocystis) or for food (Laminaria, Undaria). In France, thalli of Laminaria digitata (Linnaeus) Lamouroux are collected extensively for industrial production of alginate, a gel-forming polysaccharide used in a large variety of pharmaceutical, food and industrial applications (Onsoyen 1996). In addition, some molecules such as laminarine and its oligomers exhibit different biological properties with potential applications in medicine and in agronomy (Miyanishi et al. 2003; Torosantucci et al. 2005; Aziz et al. 2003).

In Laminariales species, many reports have described tissue culture, particularly in the genera Undaria (Kimura and Notoya 1997; Kawashima and Tokuda 1993; Notoya and Aruga 1992a; Yan 1984; Fang et al. 1983; Zongxi et al. 1983), Ecklonia (Kimura and Notoya 1996; Notoya et al. 1994; Kawashima et al. 1992; Notoya and Aruga 1989, 1992b; Kawashima and Tokuda 1990; Lawlor et al. 1987, 1989; Notoya 1988), Macrocystis (Ar Gall et al. 1996; Polne-Fuller and Gibor 1987), Eckloniopsis (Notoya 1997; Notoya et al. 1994), Egregia (Polne-Fuller and Gibor 1987), Eisenia (Notoya and Aruga 1990) and Agarum (Notoya et al. 1994). Tissue cultures of the genus Laminaria have been reported in seven species: Laminaria angustata (Saga and Sakai 1983; Saga et al. 1978), L. digitata (Asensi et al. 2001; Ar Gall et al. 1996; Folefack and Cosson 1995; Liu and Kloareg 1992; Butler 1989; Fries 1980), L. hyperborea (Fries 1980), L. japonica (Wang et al. 1998; Yan 1984; Fang et al. 1983; Zongxi et al. 1983; Saga and Sakai 1977), L. saccharina (Ar Gall et al. 1996; Butler 1989; Lee 1985), L. setchellii (Qi et al. 1995) and L. sinclairii (Polne-Fuller and Gibor 1987). Callus formation in these highly differentiated seaweeds is relatively rare. It occurred on 0.5–27% of the sectioned tissues depending on the plant and on the type of tissue studied (Polne-Fuller and Gibor 1987). The common developmental pattern from in vitro tissue culture of sporophytes of Laminariales is the outgrowth of aposporous gametophyte-like filaments with generally differentiated fertile branches from which sporophytes were often regenerated (Ar Gall et al. 1996).

A few studies have reported the production of calluses from protoplast culture of brown macroalgae exhibiting a complex morphological organization. In Laminariales, the developmental processes from protoplast culture are generally either direct regeneration into plantlets (Undaria pinnatifida, Matsumura et al. 2001; L. japonica, Matsumura et al. 2000; Sawabe and Ezura 1996; Sawabe et al. 1997) or indirect regeneration of plantlets after dedifferentiation through a filamentous stage (U. pinnatifida, Matsumura et al. 2001; L. saccharina, Benet et al. 1997) or from a callus-like mass (U. pinnatifida, Matsumura et al. 2001).

In this paper, we report on tissue and protoplast culture from meristematic areas of L. digitata. The different morphogenetic responses are described. In addition, in order to isolate protoplasts, we have also developed a one-step enzymatic protocol.

Materials and methods

Axenic treatment

Sporophytes of Laminaria digitata, ranging in length from 30 to 50 cm and corresponding to about 1-year-old thalli, were collected from the French Channel coast at Cap Levy in Normandy (Manche), France. Freshly collected algae were used immediately for protoplast production and tissue culture or were stored submerged in bubbled seawater aquariums for 2 weeks under controlled conditions (12 h photoperiod at 50 μmol photon m−2s−1, 10°C). Different parts of the thallus (frond, stipe and intercalary meristem) were cut off and selected for experiments. Excised pieces were then first cleaned several times with filter-sterilized seawater and wiped vigorously with absorbent paper to remove all epiphytes. Axenic material was obtained by steeping them for 5 min in Betadine (1%) prepared in filter-sterilized seawater and then rinsing in sterile seawater for 1–2 min in a 1% solution of commercial bleach (Domestos®) and finally rinsing three times in sterile seawater. Explants were then incubated overnight in sterile seawater containing 1 μM GeO2 to eliminate diatoms (Lewin 1966) and 2% Provasoli’s antibiotic concentrated solution (Sigma, with 240,000 units penicillin G, 1,000 μg chloramphenicol, 6,000 units polymyxin B and 1,200 μg neomycin per L) to control growth of bacteria. Explants were then rinsed in sterile seawater and cut into small sections thickness ranging between 2 and 4 mm. Explants treated were used either for tissue culture on solid medium or for protoplast production.

Tissue culture

Only the intercalary meristem was used for tissue culture. This zone of thallus of Laminaria is located at the junction between the blade and the stipe. We have distinguished meristematic explants excised at the side of the blade and those excised at the side of the stipe. The terms of stipe and blade are then used to name these explants. After axenic treatment, the explants were transferred onto a solidified culture medium containing, in natural seawater, 30 mM sucrose, 2% (v/v) Provasoli medium modified according to Loiseaux and Rozier (1978), 2 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 0,6% (w/v) agar and supplemented with auxins and cytokinins, alone or in combination. The pH of the medium was adjusted to 7.8. The growth regulators were added individually to autoclaved culture medium as filter-sterilized stock solutions. The following auxin/cytokinin combinations were tested over the concentration range (0.04 μM–53 μM): 2,4-D (0.45–45 μM); NAA (0.53–53 μM); Pi (0.04–4 μM)/BA(0.44 μM); NAA (0.53 μM)/CPPU (0.04–4 μM); Pi (4 μM)/KIN (0.46–2.3 μM); Pi (4 μM)/CPPU (0.4–2 μM); Pi (4 μM)/ ZEA (0.45–0.9 μM); Pi (4 μM)/ TDZ (0.45–0.9 μM). The cultures were incubated at 15°C under a 12-h photoperiod at 30 μmol photon m−2 s−1. Explants were subcultured at regular intervals (4–6 weeks) onto fresh media.

Protoplast isolation and purification

The enzyme solution consisted of filtered natural seawater containing mannitol 0.5 M, trisodium citrate 50 mM, bovine serum albumin 0.4% (w/v), cellulase R-10 Onozuka (Yakult Pharmaceutical, Tokyo, Japan) 2% (w/v), 1% (v/v) of centrifuged crude extract of the gut gland of Aplysia vaccaria Winckler rich in alginate-lyases and 0.08% (w/v) 2-(N-morpholino) ethanesulfonic acid (MES) buffer at pH 5.8. The enzyme solution was centrifuged (1,500 g, 45 min) and sterilized by Millipore filtration (0.22 μm). Different parts of the thallus were slightly scarified, and then transferred into the filter-sterilized enzyme solution. Digestion was performed for 3 h on a gently swirled rotary shaker (50 rpm) at 25°C in darkness. At the end of the digestion, protoplasts were then separated from undigested tissues through a metal sieve (38 μm) with a washing solution consisting of filter-sterilized seawater containing 0.4 M NaCl and 5 mM CaCl2 at pH 7. The resulting suspension was centrifuged at 167 g for 8 min, the supernatant was removed and the protoplasts resuspended in the washing solution. This procedure was repeated twice.

Protoplast culture

The last pellet with protoplasts was resuspended in washing solution containing 2 mM N-(2-hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid) (HEPES), 1% (v/v) Provasoli medium modified according to Loiseaux and Rozier (1978), 0.25 M glucose, 0.15 M sucrose, 0.025% (w/v) casein hydrolysate, 20 μM putrescine, 20 μM ornithine and supplemented with different combinations of growth regulators. The following auxin/cytokinin combinations were tested over the concentration range (0.4–45 μM): Pi (0.4–40 μM)/CPPU (0.4–4 μM); Pi (0.4 μM)/ZEA (0.45 μM); 2.4-D (0.45–45 μM)/CPPU (0.4–2 μM); Pi (4 μM)/KIN (0.46 μM); Pi (0.4 μM)/2,4-D (0.45 μM)/KIN (0.46 μM). The cell density was adjusted to 1 × 104 protoplasts mL−1. The cultures were kept in darkness at 15°C for 2 days, afterwards they were exposed to a 12 h photoperiod at 50 μmol photon m−2s−1 and 15°C. Calcofluor White (10 μg mL−1) was used for checking the cell wall removal after isolation and for investigating the course of the cell wall synthesis in the cultured protoplasts. When cell wall regeneration was complete, the medium was changed once a week for a month.

Results

The results described here show two interesting responses to culture from meristematic tissues of Laminaria digitata on media supplemented with plant growth regulators. Different morphogenetic responses were obtained from tissue culture and, interestingly, thalloid-like structures were produced in a medium containing a combination of picloram and zeatin. Friable calluses developing from protoplasts were observed only in a medium containing a combination of CPPU and 2,4-D. The sequential treatment of algal material with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO2 for 16–18 h successively enabled viable axenic explants as high as 55%.

Tissue culture

Explants excised from the intercalary meristem were cultured on solid media containing different combinations of plant growth regulators for 7 months. Results obtained after 7 months of culture are summarized in Table 1. A few weeks after inoculation, according to the media and the standard, 50–100% of the explants lost their pigmentation and bleached. Consequently, they did not develop morphogenetic responses. Otherwise, explants showed three kinds of morphogenetic responses: filamentous calluses, compact calluses and thalloid-like structures comparable to adventitious bud structures. The main morphogenetic response was the production of filamentous calluses. The explants showed a filamentous type of outgrowth from cortical and medullary cells mostly from stipes (Fig. 1a). The filaments were uniseriate, unpigmented, mostly unbranched and apical in growth. These structures appeared very quickly after the beginning of the culture, after approximately 3 weeks, and occurred on all media tested except for media containing 4.14 μM Pi combined with either 2.3 μM KIN or with 0.44 μM BA. Percentages of filaments regeneration ranged from 2.5 to 50% according to the media, the highest percentages being obtained on media containing a high level of auxins: 2,4-D alone (0.45, 4.5 and 45 μM) or Pi (4 μM) combined with high concentration of CPPU (2 μM). Dark green compact calluses were formed only from stipe explants incubated in media supplemented with 4 μM Pi + 2 μM CPPU and 0.04 μM Pi + 0.44 μM BA, the percentages being 12.5% and 2.5%, respectively. These calluses were compact and differentiated on a white, primary callus (Fig. 1b). This type of callus appeared approximately after 6 months of incubation. Thalloid-like structures occurred after 6 months of culture with a low frequency (5.5%) from only blade explant incubated in medium supplemented with Pi (4.14 μM) and ZEA (0.46 μM). These structures appeared as adventitious buds which developed from medullary tissue on the section of the blade. On this medium, the cells of the meristoderm did not lose their pigmentation even after more than 6 months of culture (Fig. 1c,d). The different regenerated structures, particularly adventitious bud structures, were transferred onto liquid medium consisting of natural seawater supplemented with 1% Provasoli medium modified according to Loiseaux and Rozier (1978). No further development was observed and the structures quickly bleached.

Fig. 1
figure 1

a Production of filamentous outgrowths from stipe explants (scale bar = 1.5 mm). b Dark green compact callus developing on stipe (scale bar = 600 μm). c General view of explant bearing 3 thalloid-like structures (→) (scale bar = 1.5 mm). d Detail (scale bar  = 90 μm)

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Table 1 Percentage of explants exhibited morphogenetic responses (uniseriate filaments, thalloid-like structures, dark green compact callus, no response) after 7 months of culture
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Callus regeneration from protoplasts

Protoplasts from different parts of the thallus of L. digitata were isolated enzymatically using a one-step procedure. As shown in Table 2, the enzyme solution enabled the production of protoplasts from stipes, blades and intercalary zones, but highest yields were obtained from meristematic zones which gave a mean yield of 22 × 106 protoplasts g−1 fresh weight. Protoplasts were obtained from directly field-collected seaweeds as well as from plants stored in an aquarium which yielded protoplasts, up to 44 × 106 protoplasts g−1 fresh weight after 2 weeks storage. Afterwards, in order to induce regeneration, only protoplasts isolated from meristematic zones were cultured. Release of protoplasts from the meristematic part of the blade began as early as 1 h after the transfer of the explants into the enzyme mixture and continued for up 24 h. Freshly isolated protoplasts from the meristematic zone were varied widely in size and in structure (Fig. 2a,b). Two types of protoplasts were purified using a 38 μm sieve. Small (8–10 μm in diameter), yellowish, highly pigmented protoplasts with densely packed plastids released from the meristoderm and approached 80% of the total number of isolated cells. A second type was larger (14–22 μm in diameter), highly vacuolated and with scattered plastids. These protoplasts were originated from the cortex. Hyphae exhibiting a typical trumpet shape were also released from medulla. No fresh protoplast was stained by Calcofluor White indicating that the protoplasts obtained were free of cell wall. After 36–72 h in medium culture, the deposition of new cell wall material appeared by Calcofluor staining. One month after protoplast isolation, friable calluses were developed from cells only cultured in medium supplemented with 0.40 μM CPPU + 0.45 μM 2,4-D (Fig. 2c). No development was observed when protoplasts were cultured on media supplemented with the others combinations of plant growth regulators as well as for the standard medium. Friable callus masses developed directly by cell divisions of meristoderm-isolated cells. Such calluses were first recognizable 6 weeks after protoplast isolation. The cells of calluses were brown-pigmented and nearly loosely packed. Calluses were fast-growing and, 2 months after protoplast isolation, quickly reached up to 2–3 mm in diameter. Fragmentation of the microcolonies was frequently observed in culture. It occurred spontaneously in the liquid culture medium leading to the formation of numerous microcalluses which also increased quickly in size (Fig. 2d). This fragmentation then allowed the micropropagation of calluses. Protoplasts-derived colonies were transferred at regular intervals onto fresh liquid medium. No further development was observed, and after 1 year of culture calluses turned white. Protoplasts cultured on other media only regenerated cell walls and remained spherical without starting a morphogenetic pathway.

Fig. 2
figure 2

a Populations of protoplasts of Laminaria digitata from meristematic zone after isolation (scale bar = 10 μm). b Small protoplasts from meristoderm (×) and larger protoplasts from cortex (scale bar = 10 μm). c Friable callus developed from protoplasts (scale bar = 400 μm). d Fragmentation of calluses (→) (scale bar = 200 μm)

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Table 2 Effect of the different parts of Laminaria digitata thalli employed on protoplast yields
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Discussion

The present study reports on protoplast regeneration into calluses and on morphogenetic responses from tissue culture of sporophytes of Laminaria digitata on media supplemented with combinations of plant growth regulators. In these experiments, sporophytes were 1-year-old and the tissue source for protoplasts and tissue culture was the meristematic region of the frond. It is now well known that, in land plants, embryogenic and meristematic tissues have a higher regeneration potential for tissue and protoplast cultures compared to mature explants because of their totipotency (Davey et al. 2005). Likewise, in seaweeds with highly differentiated thalli, meristematic areas and juvenile thalli were reported to be effective for micropropagation from protoplast and tissue culture (Rhodophyta, Titlyanov et al. 2006a, b; Phaeophyta, Mussio and Rusig 2006; Benet et al. 1997; Kawashima and Tokuda 1993; Kloareg and Quatrano 1987).

Tissue culture

A variety of combinations of growth regulators were used for tissue culture of L. digitata. In this study, we observed three forms of regeneration from culture of the meristematic zone: uniseriate filaments, thalloid-like structures and dark green compact callus. Except for the majority of explants bleaching and despite the different nutritional and growth factors conditions, the main morphogenetic process observed was the production of filamentous calluses from cortical and medullary stipes occurring on almost the totality of media tested. The development of filamentous calluses has been frequently described from Laminariales (see reviews in: Ar Gall et al. 1996; Reddy et al. 2007), from Fucales (Hizikia, Hwang et al. 1994; Sargassum, Rajakrishna Kumar et al. 2007; Kirihara et al. 1997; Turbinaria, Rajakrishna Kumar et al. 2007) and from red seaweeds (Gracilaria, Hypnea, Rajakrishna Kumar et al. 2004, 2007; Kappaphycus, Reddy et al. 2003; Solieria, Yokoya and Handro 2002; Agardhiella, Bradley and Cheney 1990). In this study, no further development of the filamentous calluses was observed, contrary to other studies. In L. digitata, Asensi et al. (2001) established a cell-filament suspension from the in vitro culture of stipe medullary explants leading to the regeneration of juvenile sporophytes. Fries (1980) and Folefack and Cosson (1995) mentioned a sporadic regeneration of plantlets from chlorophyllous filaments developed from thallus culture. In Kappaphycus alvarezii (Rhodophyta), regeneration of somatic embryos was demonstrated from pigmented uniseriate filamentous callus (Reddy et al. 2003). Here, the production of filamentous outgrowths was enhanced by plant growth regulators. According to the media, 2.5–50% of the explants produced filamentous callus with the highest percentages occurring on media supplemented with 2,4-D (38–50%). In L. setchellii, 30% of stipe medullary explants produced filamentous callus-like tissue on solid medium without plant growth regulators (Qi et al. 1995). More interestingly, thalloid-like structures comparable to adventitious bud structures were developed on medium supplemented with picloram (4 μM) and zeatin (0.45 μM) directly at the cut surface of the thallus medullary explant. This morphogenetic response occurred at low frequency (5.5%) and only from a thallus medullary explant. Production of morphologically-normal sporophytes from tissue culture of L. digitata has been already described, but only from gametophyte culture (Ar Gall et al. 1996) and from the outgrowth of aposporous gametophyte-like filaments (Asensi et al. 2001) and never directly from the medullary zone at the cut surface. In seaweeds, two kinds of adventitious shoots have been described: those arising from medullary cells at cut surfaces and those formed by renewed growth of the lateral filaments that constitute the thallus cortex (Collantes et al. 2004). The third morphogenetic response was compact green dark calluses which were produced on two media (4 μM Pi + 2 μM CPPU and 0.04 μM Pi + 0.44 μM BA) at low frequency (2.5% and 12.5%, respectively) which is in agreement with Polne-Fuller and Gibor (1987) who reported that the percentage of callus formation in Laminariales ranged from 2 to 10%. Generally, the occurrence of calluses from the in vitro culture of seaweeds is sporadic and the percentages are often very low (see review in Reddy et al. 2007).

Callus regeneration from protoplasts

A one-step protocol was developed for protoplast isolation whereas the procedures previously described by Butler et al. (1989) and Benet et al. (1994, 1997) were comprised of two steps including first an incubation in a calcium chelation medium followed by transfer onto digestion medium. In comparison with these previous studies, yields obtained were very similar, about 10–100 × 106 protoplasts per gram of fresh weight. The enzyme composition was similar to those used for Ectocarpales (Ducreux and Kloareg 1988; Mejjad et al. 1992), Fucales (Mussio and Rusig 2006) and Chlorophyceae (Rusig and Cosson 2001). The main difference was the absence of macerozyme for production of protoplasts from L. digitata. Only protoplasts obtained from the meristoderm produced calluses while protoplasts from cortical cells died without dividing, which was also observed from other Laminariales (L. japonica, Matsumura et al. 2000; L. longissima, Matsumura 1998; U. pinnatifida, Matsumura et al. 2001). In our study, callus regeneration was only induced by a single combination of growth regulators (CPPU and 2,4-D). Forchlorfenuron (CPPU), as thidiazuron (TDZ), is a non-purine, synthetic phenylurea compound with a strong cytokinin-like activity. CPPU and other diphenylurea derivatives have been recently utilized for regenerating several recalcitrant and difficult-to-regenerate higher plant species such as trees and monocotyledons from protoplasts (Wakita et al. 2005; Sasamoto et al. 2002) and from tissue culture (Carra et al. 2006; Zhang et al. 2006). Up to now, in previous studies concerning algae culture, only classical plant growth regulators were used, particularly adenine-cytokinin (BAP and KIN) (Collantes et al. 2004; Reddy et al. 2003; Yokoya and Handro 1996). Callus formation from protoplast culture of seaweeds, particularly of brown seaweeds, is relatively rare. As pointed out by Matsumura et al. (2000, 2001), the development of callus from protoplast culture is induced by the absence of polarity of the cells during their developmental pathway. Generally, in Laminariales, the protoplasts regenerated into normal sporophytes via different development processes: direct regeneration into plantlets (L. digitata, Benet et al. 1997; U. pinnatifida, Matsumura et al. 2001; L. japonica, Matsumura et al. 2000; Sawabe et al. 1997, Sawabe and Ezura 1996), indirect plantlet regeneration either after dedifferentiation through a filamentous stage (U. pinnatifida, Matsumura et al. 2001; L. saccharina, Benet et al. 1997) or after development of callus-like masses (U. pinnatifida, Matsumura et al. 2001; L. japonica, Matsumura et al. 2000). The direct and indirect regeneration from protoplasts of L. japonica and U. pinnatifida were closely related to the water temperature in culture (Matsumura et al. 2000, 2001). In L. digitata, production of calluses was reported but the type of callus varied widely in structure. Benet et al. (1997) described a pattern of regeneration from protoplasts of juvenile L. digitata thalli involving budding and mitoses leading to various regenerated forms ranging from rhizoid-bearing callus-like structures to abnormal bladelets. In L. japonica, Matsumura et al. (2000) observed the production of callus-like amorphous masses which never grew over 5 mm in length nor differentiated rhizoids and which finally died. Kloareg et al. (1989) observed the development of three types of callus from protoplasts of Macrocystis pyrifera (Laminariales): small callus growths without pigment, large callus-like masses with brown pigment and masses with differentiated rhizoids. Callus regeneration was also observed from protoplasts of Fucales, notably protoplasts of Sargassum (Polne-Fuller and Gibor 1986). Only three to five divisions were observed leading to the formation of a small mound of cells. In any case, callus development from protoplasts did not require medium supplemented with plant growth regulators. Benet et al. (1997) showed that the culture of protoplasts in supplemented media resulted in a marked decrease in survival rates.

In order to micropropagate seaweeds, direct regeneration from protoplasts is the efficient and relevant developmental pathway while callus regeneration represents a morphogenetic stalemate. But indirect regeneration of somaclones from callus can be a possible alternative to conventional breeding, callus culture inducing genetic variability (Larkin and Scowcroft 1981).

Our results show that synthetic growth regulators, active in land plants, are also capable of stimulating growth in cell cultures of L. digitata, namely of inducing callus regeneration from protoplasts. But the low frequencies of morphogenetic responses, particularly of the adventitious bud structures, suggested a genotype-dependent response as stated by Polne-Fuller and Gibor (1987) and Aguirre-Lipperheide et al. (1995). In higher plants, the recalcitrance, i.e. the inability of plant cell, tissue and organ to respond to tissue culture, is well known to be greatly genotype and environment dependent (Cassells et al. 2001). The genetic variability of the starting plant material collected from natural populations could probably explain for the most part the low occurrence and the absence of the control of organogenic potential of the algal species in in vitro culture.