Development of protoplasts from

Grateloupia sparsa and G. filicina

(Halymeniaceae, Rhodophyta)

Yean-Chang Chen and Young-Meng Chiang

Institute of Oceanography

National Taiwan University

Taipei, Taiwan, Republic of China

ABSTRACT

For isolation of protoplasts from Grateloupia sparsa and G. filicina, commercial enzymes of cellulase, macerozyme, agarase, abalone acetone powder and papain were used in seven combinations. A maximum yield of protoplasts of 7x10⁸ g^-1 (fr. wt.) was obtained with a mixture of 4% cellulase, 2% macerozyme, 50U/mL or 100U/mL agarase and 4% papain. Very few protoplasts (10³ g^-1 fr. wt.) were isolated with mixtures of cellulase and macerozyme. After isolation, protoplasts were incubated in 0.7 M mannitol-PES medium for 10 days then transferred to PES medium to obtain the highest number of regenerated protoplasts. Protoplasts excreted amorphous matrix on their surface as soon as they were isolated, and deposited cell wall after 4 days of culture. A protoplast began to divide after 7-9 days in culture to form a radially expanded disc with a meristematic marginal portion of one-celled layer thick. The disc issued erect thalli or filaments after 60 days of culture. This is the first report of the regeneration of protoplasts from species of the Florideophycidae.

INTRODUCTION

Protoplasts have been produced from a number of higher plants and subsequently used as tools in physiological and cytological studies, as well as in crop improvement research involving genetic engineering techniques (Galun 1981; Ahuia 1982). However, protoplast research in seaweed is a relatively new field and lags far behind that of land plants and unicellular algae (Berliner 1981, 1983; Cheney 1986). The basic methodology of protoplast isolation and culture must be successfully developed before such techniques can be practically applied to marine plants (Cheney et al. 1986). At the present, there are about 33 species of multicellular marine algae from which protoplasts have been isolated (Polne-Fuller and Gibor 1987). Most of these species belong to Chlorophyta and Phaeophyta. Among the Rhodophyta, protoplasts have only been successfully regenerated from Porphyra species (Polne-Fuller and Gibor 1984, 1987; Fujita and Migita 1985; Chen 1987, 1989). Protoplasts from more complex multicellular red algae, such as Gracilaria tikvahiae McLachlan (Cheney 1986) and Chondrus crispus (L.) Stackhouse (Le Gall et al. 1990) have also been isolated, and their division has been reported. However, little information about protoplast regeneration to full plantlets has been reported.

Grateloupia sparsa (Okamura) Chiang (Chiang 1970) and G. filicina (Lamouroux) C. Agardh which grow on the rocks in the intertidal zone, are two common red algae along the coasts of northern Taiwan during the spring season. They are annual and have macroscopic, isomorphic gametophytes and tetrasporophytes.

Both species of Grateloupia have been studied for commercial scale cultivation for food and extraction of their carrageenan. This paper reports on the isolation and regeneration of protoplasts of G. sparsa and G. filicina.

MATERIALS AND METHODS

Thalli of Grateloupia sparsa and G. filicina were collected at Keelung, Taiwan, on May 12, 1991. Freshly collected plants that were wrapped in absorbent paper towels moistened with seawater in a plastic bag, were brought back to the laboratory of the Institute of Oceanography, National Taiwan University in Taipei.

Preparation of Axenic Plant Material.

Selected pieces of vegetative fronds (2 cm²) were thoroughly cleaned in filtered seawater. They were then put in an ultrasonic cleaner (Branson 3200) with two changes of autoclaved seawater containing 1% KI-I² (2g KI and 1g I² dissolved in 300mL distilled water as 100% KI-I) for 5 minutes each, to remove animals and epiphytes (Reddy et al. 1989). The pieces were then rinsed several times with autoclaved seawater. Finally they were incubated in 100mL of autoclaved enriched seawater (Provasoli 1968) containing 10mL of antibiotic mixture (Polne-Fuller and Gibor 1984) for 24 hours at 18ºC under a photon flux density of 20?mol m^-2s^-1 and 12:12 L:D photoperiod in a culture room.

Preparation of Enzyme Solutions for the Cell Wall Digestion

Commercial enzymes (cellulase Onozuka R-10 Yakult Honsha Co. Ltd., macerozyme R-10 Yakult Honsha Co. Ltd., agarase Sigma A-6300, abalone acetone powder Sigma A-7514 and papain Sigma P-3375) for cell wall digestion were used in this study.

Seven combinations of enzyme mixtures as shown in Table I were applied for cell wall digestion. The enzyme mixtures were dissolved in 0.6 M mannitol-seawater (35‰) solution and were then centrifuged at 0ºC at 10,000 xg for 20 min (HERMLE Z360K). The supernatants were filter-sterilized by passing through a 0.2µm disposable syringe filter unit. During preparation, the enzyme solutions were kept in an ice-bath.

Isolation of Protoplasts

Cleaned, vegetative material of Grateloupia sparsa and G. filicina were washed three times with autoclaved seawater. They were then cut into small pieces of 0.3-0.5 mm² using a sterilized razor blade on a clean bench. Five hundred milligrams (f.w.) of each species was incubated in each of fourteen sterile 50X80mm² disposable plastic flasks (40 mL, Falcon) with 10mL of enzyme solution as described above and rotated with an orbital shaker at 50 rpm. In order to prevent the toxicity of papain, 0.5% (w/v) dextran sulfate and 50 m M of phenyl methyl sulfonyl fluoride (P.M.S.F.) were added to the enzyme solution. All procedures for isolation were carried out in a laminar air flow cabinet in darkness for 18 hours at 18ºC.

Purification of Protoplasts

After incubating for 18 hours, the solution was filtered through a 59 µM nylon mesh to separate protoplasts from undigested tissue and cell detritus, and then centrifuged at 150 xg for 20 min (HERMLE Z320). The protoplast pellet was washed gently with autoclaved 0.7M mannitol-seawater (35‰) three times to eliminate the remnant of enzyme solution.

Incubation of Viable Protoplasts

Purified pellets of protoplasts (ca. 7x10⁸ protoplasts or equal to 2500 protoplasts per 0.2mm2 area) were cultured in 10 mL of Provasoli's enriched seawater (35‰) (PES) medium with 0.6M, 0.7M and 0.85M of mannitol respectively in 40 mL-flasks immediately after isolation. After incubation in this mannitol-PES medium for 10 days, 10 mL of pure PES medium was added to the 40 mL-flask at days 12, 14 and 16, to dilute the mannitol concentration of mannitol-PES medium gradually. The total volume of 40 mL diluted mannitol-PES medium was discarded and another 40 mL of pure PES medium was added into the flask at day 18. The medium was then refreshed with 40 mL of pure PES medium every 4 days. Protoplasts were incubated at 18ºC in the dark for the first 4 days to promote sinking (Liu et al. 1992), and were then cultured under a photon flux density of 166 m mol m^-2s^-1 and a photoperiod of 12:12 L:D at the same temperature in a culture room.

Yields of viable protoplasts were counted with a hemacytometer (Bright-Line, improved Neubauer 0.1mm deep) under a light microscope (Zeiss, Axioskop) with 0.25% Evans-blue staining. The experiments in this study were performed three times, and the average counts were recorded.

The regeneration of viable protoplasts was observed and photographed under an inverted microscope (Nikon, Diaphot-TMD) with Nikon camera equipment .

Numbers of discs which were formed from the protoplasts were counted in five randomly selected microscope (Nikon, Diaphot-TMD) fields.

Cell Wall Formation in Protoplasts

A fluorescent brightener reagent (Calcofluor White M2R; Tinopal LPW, Sigma F-6259) was used to follow the course of cell wall resynthesis in the protoplast population (Galbraith 1981; Chen and Chen 1993).

During this study, we stained the protoplasts with calcofluor white and found that those protoplasts with resynthesized walls appeared bright-blue when viewed with a fluorescent microscope, while those without walls appeared red. About 0.01% of calcofluor white (w/v) was added to cultures of purified protoplasts in 0.7M mannitol-PES medium immediately after isolation. During the first 24 hours of incubation, protoplasts were examined under an inverted microscope with fluorescent equipment (Nikon, Diaphot-TMD) at hourly intervals, and the numbers of bright-blue and red protoplasts in five randomly selected microscope fields were counted.

RESULTS AND DISCUSSION

As shown in Table I, protoplasts of both Grateloupia sparsa and G. filicina can be isolated with the seven combinations of enzyme solutions investigated. In those solutions, the combination of 4% cellulase, 2% macerozyme, 4% papain and 50 or 100 U/mL of agarase produced the highest number of protoplasts (7x10⁸ g^-1 fw.), while the combination of 4% cellulase, 2% macerozyme produced the lowest number of protoplasts (0-103 g-1 fw.). This result is similar to that for Chondrus cripus gametophyte protoplasts studied by Le Gall et al. (1990). In their study, the enzyme solution contained agarase and several kinds of carrageenase. But in this study, no carrageenase was used and the mixture of agarase and papain was sufficient enough to stimulate the production of Grateloupia protoplasts. It may indicate that the constituent of cell wall of Grateloupia differ from that of Chondrus or at least C. cripus.

There were two sizes of protoplasts isolated from G. sparsa and G. filicina. One of them was about 10µm, and the other was 15-20 µm in diameter (Figs 1 and 2). They are presumably produced from the inner and outer cortical cells respectively, since both of them showed the same pattern in their growth and regeneration. Cheney et al. (1986) also found two varieties of protoplasts from the Gracilaria tikvahiae and G. lemaneiformis (Bory) Weber-van Bosse, but no further information was reported.

As shown in Figure 3, when protoplasts of G. sparsa and G. filicina were incubated in 0.6 M, 0.7 M and 0.85 M mannitol-PES medium for 10 days, those in the 0.7 M medium produced the largest number of viable protoplasts, while those in the 0.85 M medium produced the least. The result indicates that a 0.7 M mannitol-PES medium is the most favorable condition for the newly isolated protoplasts of the two species of Grateloupia. Protoplasts that were suspended in those mannitol-PES media had a tendency to sink and adhere on the bottom of the flask to form discs. Those that did not sink ultimately disintegrated.

It was also found that the concentration of mannitol in mannitol-PES medium can influence the sinking time of the protoplasts. In 0.85 M mannitol-PES medium, it took 10 days for most of the protoplasts to reach the bottom of the flask, and some of them shrunk. Protoplasts in both 0.6 M and 0.7 M media took only 4 days to reach the bottom, but those in 0.6 M medium, swelled slightly.

Those shrunken and swollen protoplasts could be stained easily with Evan's blue (0.25%), and retained their blue color when they were returned to the same concentrations of Evan's blue-free mannitol-PES media. The result indicated that those protoplasts had died (Saga et al. 1989).

Fluorescent brightener reagent (Calcofluor White) was applied to examine the development of new cell walls in protoplasts (Galbraith 1981; Chen and Chen 1993). Protoplasts were incubated in 0.7 M mannitol-PES with 0.01% calcofluor white medium immediately after isolation. During the first hour of treatment, the color of a large number of protoplasts changed from bright red to orange-red, then two hours later about 50% of them turned bright-blue, and this number increased over time. On the 6th hour, up to 98% of G. sparsa and 93% of G. filicina protoplasts showed bright-blue emission (Fig. 4). The bright-blue emission changed gradually to yellow-green, then to pale green after day 4. The change of color in protoplasts was probably due to the presence of mucilage and wall materials on the surface of the protoplast. From our electron microscopic study (Fig. 11), we found that a small amount of mucilaginous-like material was excreted and deposited on the surface of the protoplast as soon as it was isolated. The amount of mucilaginous material increased rapidly and covered the whole surface of the protoplast prior to production of microfibrils which form the cellulose cell wall. The same pattern of color changes with calcofluor white in the regenerating walls was also reported for those of Porphyra protoplasts (Polne-Fuller and Gibor 1990).

It was found that the protoplasts of G. sparsa and G. filicina usually began to divide between day 7 and day 12 in PES medium (Figs 5 and 6), and radially expanded discs with meristematic marginal portion of one-celled layer thick (Fig. 7) were formed from day 12 to day 28. The shape and size of the discs were almost the same in the two species and the yield of discs was about 0.01% of the initial number of protoplasts. Around the 60th day, the discs regenerated either leafy thalli (Figs 8 and 9) or filaments (Fig. 10). The filaments were sparsely branched, composed of a line of cells about 15 m m in diameter. Frequently, the terminal portion of a branch was swollen to form a ball-like body.

However, development of the protoplasts are different from those of spores which released from the mother thalli. According to the description of Inoh (1947), the freshly released carporspores and tetraspores of Grateloupia spp. sunk and produced mucilage material to adhere the substrate within 12-24 hours. Those spores protruded germtube at one side of the spore and the protoplasm flew into this newly formed germtube. Then a septum was formed between the empty spore and the germtube. The germtube finally formed a disc by continually dividing with the empty spore. The protoplast does not form the germtube, and disc is formed by cell dividing directly, thus, no empty spore remained on the side of the disc. Therefore, it is easy to distinguish the development pattern between spores and protoplasts by the appearance of the germtube and empty spore.

This is the first report on protoplasts isolated from species of Florideophycidae, Rhodophyta regenerating to produce leafy thalli. The same results were also obtained from the protoplasts isolated from seedlings developed from carpospores or tetraspores and young thalli that were regenerated from the protoplasts isolated in this study.

ACKNOWLEDGMENTS

We wish to express our sincere thanks to Dr L. C.-M. Chen, Institute for Marine Biosciences, National Research Council of Canada for reading the manuscript and offering useful suggestions. This study was supported by the National Science Council Grant No. NSC (81-0209-B-002A-04), Republic of China.

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