Isolation and regeneration of protoplasts of Monostroma latissimum Wittrock (Monostromaceae, Chlorophyta)

Yean-Chang Chen and Young-Meng Chiang

Institute of Oceanography National Taiwan University Taipei, Taiwan, Republic of China  

Abstract

Protoplasts of Monostroma latissimum were isolated enzymatically with 4% Cellulase Onozuka R-10, and 2% Macerozyme R-10. The yield of protoplasts isolated increased as the concentration of sorbitol as osmoticum of the enzyme solution was increased from 0.5 M to 1.2 M, at then decreased, with no protoplasts be obtained at 1.9M. The highest number (9X10⁶ protoplasts/g fresh thallus) of normal protoplasts was obtained with 1.2M sorbitol and a 50 rpm shaker speed for 6 hours. The number of isolated protoplasts increased as the speed of rotation increased, however, the relative number of abnormal protoplasts increased when the rotation speed exceeded 50 rpm. The highest percentage (61.3%) of regenerated protoplasts was obtained by incubating freshly isolated protoplasts in 0.85M mannitol-Provasoli's enriched seawater (PES) medium for 5 days then transferring in PES medium. It was found that earlier (less than 5 days) transfer of protoplasts from mannitol-PES medium to PES medium, may cause the death or loss of viability of the protoplasts due to osmotic change. However, longer than 5 days in mannitol-PES medium was also harmful to the regeneration of protoplasts. Protoplasts usually begin to form new cell-walls within 5 hours after isolation and begin to divide from day 7 to day 9 in PES medium, then form cell-clusters from day 14 to day 18. A cell-cluster can form a tubular frond then a leafy thallus through further cultivation in PES medium.

Key words : Calcofluor White - Cell-cluster - Monostroma latissimum -

Protoplast .

Introduction

Monostroma is a genus of widely distributed marine green algae, growing naturally as macroscopic, one cell thick thalli. Among the species of this genus, M. latissimum Wittrock is edible and has been successfully cultivated on a large commercial scale in Japan for many years (Shokita et al., 1991). In Taiwan, M. latissimum is used as food supplement but people are still seeking commercial aquaculture techniques for this plant. The procedures of traditional cultivation of Monostroma are at first, mature thalli are collected from the field during their growing season. After being washed to remove epiphytes, animals and other materials, they are treated to release gametes in a tank under high illumination. Then the gametes are collected for their conjugation to form zygotes. Zygotes are cultured in indoor tanks several months for production of zoospores. Zoospores are liberated and collected to attach on nets. The nets are then hang in the sea for cultivation. As mentioned above, the traditional method is quite tedious and a time consuming task. Sometimes it is also difficult to obtain enough mature thalli and a sufficient number of gametes or zoospores to seed on nets. Therefore, a new method of Monostroma cultivation needs to be developed.

The successful regeneration of plants directly from enzymatically isolated protoplasts of various species of seaweeds (Tang, 1982; Zhang, 1983; Polne-Fuller et al., 1986; Chen, 1987) and cell-suspension cultures of Porphyra (Chen, 1989) have provided a basis for improving aquacultural crops and for genetic manipulations. These techniques might be applied in developing a new method of seaweed cultivation.

Our research has accordingly been directed towards developing new techniques for mass cultivation of M. latissimum in Taiwan.

The present report describes the production of protoplasts and a stable, nonclonal suspension of single cell and cell-cluster cultures of M. latissimum that might provide an alternative propagation method for this alga.

Materials and Methods

Thalli of Monostroma latissimum were collected at Chiang-may, Peng-hu County, Taiwan on April 4, 1990. Freshly collected plants that were wrapped in absorbent paper towels moistened with seawater, were sealed in plastic bags and packed in ice-box for transport to the laboratory of the Institute of Oceanography, National Taiwan University in Taipei.

Preparation of clean and bacteria free plant material

The method of Reddy et al. (1989) was followed with a slight modification for obtaining clean bacteria-free material. Selected pieces of vegetative fronds (2 cm²) were thoroughly cleaned in filtered seawater and placed in an ultrasonic cleaner (Branson 3200) with autoclaved seawater containing 1% KI- I^{2 (}2g KI and 1g I² dissolved in 300ml distilled water) for 5 minutes twice, to remove animals and epiphytes. The pieces were then rinsed several times with autoclaved seawater. Finally they were incubated in 100ml of autoclaved Provasoli's enriched seawater (35°%) (PES) medium (Provasoli, 1968) containing 10ml of antibiotic mixture (Polne-Fuller and Gibor, 1984) for 24 hours at 24°C under a photoperiod of 12:12 L:D regime and a photon flux density of 20 µE m^-2s^-1 in a culture room.

Preparation of an enzyme solution for the cell wall digestion

The enzyme solution was prepared by dissolving 4% Cellulase Onozuka R-10, 2% Macerozyme R-10 ( Yakult Honsha Co. Ltd. Japan) and various concentrations of the osmoticum sorbitol (0.5-1.9M), in 10ml of distilled water. The pH was set at 6.0 using Na₂HPO₄-NaH₂PO₄ buffer.

The enzyme solution was centrifuged at 0°C at 10,000 X g for 10 min (HERMLE Z360K) to remove large particles. The supernatant was sterilized by passing it through a 0.2µm disposable syringe filter unit.

Isolation of protoplasts

To establish the optimum concentration of osmoticum for isolation of protoplasts, healthy fronds were incubated in various concentrations of sorbitol (0.5-2M) for 6 hours, and then immersed in seawater-Evans Blue solution (0.01%) for 1 hour (Millner et al., 1979) under a photon flux density of 20 µE m^-2s^-1 in an incubator at 24°C. The material was examined under the microscope to check whether the cells stained blue (Chen and Chen, 1991). This preliminary test showed that cells of Monostroma latissimum retained their plasmolysis activity after six-hours incubation in 1.3-1.75 M sorbitol solution (=1337.5-1803.7 milliosmo/kg) at 24°C. A concentration higher than 1.75 M caused cell death, and concentrations lower than 1.3M concentration caused cells to swell and lose their viability. Aseptic material of M. latissimum was obtained by washing three times with autoclaved seawater, then cutting into small pieces 0.5-1mm square, with a sterile razor blade under a laminar flow hood. One-hundred milligrams of the material was incubated in 40 sterile 50X80-mm disposable plastic flasks (40ml, Falcon), containing 10ml of enzyme solution of various sorbitol concentrations (0.5, 0.7, 0.9, 1.1, 1.2, 1.35, 1.55 and 1.9 M) that based on the plasmolysis range of the preliminary test, and placed on an orbital shaker (Firstek, Taiwan) rotating at different speeds (40, 50, 60, 70, and 80 rpm), and numbers of normal and abnormal protoplasts were counted. The abnormal protoplasts were distinguished from the normal ones by the shape of their surface. The former had concave or convex surfaces, while the latter had round and smooth ones. All procedures of isolation were carried out in the dark (Marchant and Fowke, 1977; Cheney et al., 1986; Reddy and Fujita, 1991) to promote the sinking of isolated protoplasts (Liu et al., 1992) at 24°C.

Purification of protoplasts

Following the incubation as above for 6 hours, the solution was filtered through a 59µm Nylon mesh to separate protoplasts from undigested cells and cell debris. The filtrate was layered over a 35% (w/v) density buffer (Ficoll-400, Sigma) solution and centrifuged at 100 X g for 30 min (HERMLE Z320), to remove small debris. The protoplasts that collected at the interface were retrieved with a sterile Pasteur pipette.

Yield of freshly isolated protoplasts was counted with a hemacytometer (Bright-Line, improved Neubauer 0.1mm deep) under a light microscope (Zeiss, Axioskop). The experiments in this study were performed three times, and the average and standard error counts were recorded.

Incubation of protoplasts

Purified protoplasts were cultured in Provasoli's enriched seawater (PES) medium with various concentrations (0.5, 0.7, 0.85 and 1M) of the osmoticum mannitol immediately after isolation to determine the most suitable concentration for cultivation of the protoplasts. The number of viable protoplasts incubated in the mannitol-PES media containing 0.01% of Evans-Blue dye was counted with the aid of an inverted microscope (Nikon, Diaphot-TMD) from day 1 to day 10 of incubation. All protoplast cultures were incubated under a photoperiod of 12:12 L:D regime and a photon flux density of 166 µ m^-2s^-1 at 24°C. The regenerated protoplasts were cultured in 0.85 M mannitol-PES medium for 5 days, then they were transferred to pure PES medium for further cultivation under the condition mentioned above.

The cell-clusters regenerated from the protoplasts were sprayed on fine strings made of synthetic fibers and cultured them in an aquarium with PES medium for about 30 days. Then they were transferred to hang in a fish pond for further cultivation.

Protoplasts cell wall formation

A fluorescent brightener reagent, Calcofluor White ST, (Sigma) was used to investigate the course of cell wall resynthesis in cultured protoplasts (Galbraith, 1981; Roberts et al., 1982; Chen and Chen, 1993). We stained the protoplasts with Calcofluor White and found that those with resynthesized walls appeared green, while those without cell walls were red. About 0.01% of Calcofluor White (w/v) was added to each culture of purified protoplasts in 0.85M mannitol-PES medium after 0, 2, 4, 8, 12, and 24 hours in culture. During the first 24 hours, each culture was examined hourly on an inverted microscope with fluorescent equipment (Nikon, Diaphot-TMD, filters, BG-12 with wave length of UV=320-400 nm), and numbers with standard error of green and red protoplasts in five randomly selected microscope fields were counted.

Results and Discussion

As shown in Table 1, protoplast of Monostroma latissimum can be isolated from 0.5 to 1.55M sorbitol-enzyme mixtures containing 4% Cellulase R-10 and 2% Macerozyme R-10 (ca. equal to the osmolarity of 0.6-1.7M pure sorbitol solution) with the speeds of 40-80 rpm on orbital rotary shakers for 6 hours at 24° C. At 50 rpm the number of isolated protoplasts increased to its maximum yield of 9X10⁶ protoplasts /g as the sorbitol concentration of the enzyme solution increased from 0.5 to 1.2M, and then decreased as the sorbitol concentration increased further. At the highest concentration (1.9M) no protoplasts were produced. Fujita and Migita (1985) isolated 105-106 protoplasts per 0.2g thallus from Monostroma nitidum and Ulva pertusa with 10% cellulase (Onozuka R-10) in 0.8M mannitol at 18-20°C for 2 and 4 hours respectively. The differences in the yield of protoplasts from Monostroma and Ulva species could be due to differences in the enzyme components, the osmoticum concentration, pH, or the physiological status and growth stage of the plant materials (Fujita and Migita, 1985; Reddy et al., 1989).

It is necessary to purify the released protoplasts for subsequent study. However, the method of filtration through Nylon mesh (Saga, 1984; Reddy et al., 1989) often resulted in contamination by large amounts of cell debris and broken protoplasts. To avoid this contamination, we tried various methods and found that the utilization of Ficoll-400 (a kind of density buffer for centrifugation) has proven to be most effective in obtaining clean protoplasts due to its low osmotic potential and high density (Chen and Chen, 1991).

The rotating speed of the orbital rotary shaker can affect the yield of protoplasts, irrespective of the concentration of sorbitol-enzyme solutions (Table 1). As shown in Fig.1 the number of normal protoplasts increased from 2.21 to 8.86x10⁶ protoplasts/g as the speed of rotation increased swollen or shrunken increased from 1.16 (17.8 %) to 11.57x10⁶ protoplasts/g (88.2%) when the rotary speed exceeded 60 rpm to 80 rpm. This phenomenon was also found in Ulva fasciata Delile protoplasts (Chen and Chen, 1991).

During this study we found that sorbitol did not form crystals even in the concentration, as high as 1.2 M, but mannitol did. Therefore we used sorbitol for the isolation of protoplasts. Later, we also found that the protoplasts should not be kept in sorbitol-enzyme solution for more than 6 hours, otherwise, they will lose their viability, and the purified protoplast should be transferred to PES medium with an addition of mannitol as soon as possible. This might be due to the toxicity of the sorbitol-enzyme solution at long time digesting. We also found that the addition of high concentration of mannitol in mannitol-PES medium can affect the viability of the protoplasts too ( Fig. 2). Among concentrations of mannitol tested, 0.85 M of mannitol-PES medium was the best for the newly isolated protoplasts, because of the number of viable protoplasts in this concentration were higher than other concentrations of mannitol-PES media in every incubation stage (from day 1 to day 10). Therefore, during this study, as many others did (Saga and Sakai, 1984; Saga, 1984; Cheney et al., 1986; Polne-Fuller and Gibor, 1987; Ducreux and Kloareg, 1988; Reddy et al., 1989, 1992; Fujimura et al., 1989; Fujita and Saito, 1990), we used mannitol for the incubation of algal protoplasts.

For obtaining the highest percentage of regenerated protoplasts, we incubated freshly isolated protoplasts in 0.85M (Ca. 1800 milliosmo/kg) mannitol-PES medium for 1 to 16 days before they were transferred to pure PES medium (Ca. 998 mOs/kg) for 12 days. The results showed (Fig. 3) that the percentage of regenerated cell-cluster increased as the incubation time increased from day 1 to day 5, and decreased as the time increased from day 6 to day 16. This indicates that the protoplasts incubated in 0.85M mannitol-PES for 5 days and then transferred to PES medium produced the highest percentage of regenerated cell-clusters (61.3%). For those incubated in 0.85 M mannitol-PES for 1, 2, 3 and 4 days, the percentage of regenerated cell-clusters was 0.3%, 0.2%, 13.6%, and 45.5%, respectively. The results might be due to the fact that those protoplasts still did not completely form cell walls in 1 to 4 days (Table 2). Thus, sudden transfer to a lower osmotic condition caused the loss of viability or death of the protoplast.

Protoplasts incubated in 0.85M mannitol-PES more than 5 days also showed a lower percentage of regeneration. This may indicate that mannitol is toxic to the protoplasts when they are kept in mannitol solution more than 5 days (Chen and Chen, 1991). The same results were described in Reddy et al. (1989) and Reddy and Fujita (1991) in which, indicated that prolonged cultivation of cells in such, 0.2-0.8M of mannitol, hyperosmotic solutions impaired normal cell division and resulted in abnormal cell structures. The inhibition of cell division in hyperosmotic conditions might be due to the higher concentration of inorganic salts in the cytoplasm which may be inhibiting the various growth processes such as photosynthesis and respiration.

The results of fluorescent staining showed that the protoplasts began to form new cell-walls after 5 hours of isolation, and the number of protoplasts with a cell wall increased relative to the protoplast age (Table 2). Though at 4 hours, about 2% of protoplasts showed the presence of cell wall, this could be a remnant of the old cell wall. The results also show that earlier stain of protoplasts with calcofluor white can result in a lower percentage of protoplasts forming new cell walls, and the negative effects of calcofluor white on cell wall formation in protoplasts seems to be long-lasting. As shown in Table 2, protoplasts which were stained with calcofluor white immediately after isolation accounted for the lowest percentage (56%) of the protoplast population with new cell walls after 5 days of incubation. However, those stained after 24 hours (Table 2, Fig. 4) showed the highest percentage (85.3%). This result (85.3%) is higher than that of Monostroma nitidum (68.3%) in Fujita and Migita's study (1985). The differences in cell wall resynthesis could be due to environmental stresses, such as osmotic pressure and temperature, that affect the physiology of the protoplasts (Galun, 1981).

Protoplasts usually began to divide from day 7 to day 9 in PES medium, and to form cell-clusters (Fig. 5) from day 14 to day 18. Some of them formed tubular fronds (Fig. 6) from day 25 to day 30. Further cultivation in the PES medium for another 30 days induced tubular fronds to become leafy forms (Fig. 7). Saga and Kudo (1989) also obtained cell-clusters from protoplasts of Monostroma angicava. However, Fujita and Migita (1985) reported that the protoplasts of M. nitidum produced gametes after regeneration of cell wall in culture. The differences in the growth pattern of the protoplasts in the species of Monostroma may be affected by the original location of the protoplasts on the frond, as well as by culture conditions. This phenomenon was also reported from Porphyra spp. (Polne-Fuller et al., 1986).

The cell-clusters of Monostroma latissimum can be easily propagated in a large quantity in PES medium. We sprayed them on fine synthetic strings and cultured in fish ponds. In two weeks some thalli grew out attached on the strings. But the yield of thalli varied from ponds to ponds, and from season to season. Apparently, the yield of thalli was affected by natural conditions of the ponds. Therefore, further studies on the environmental factors which affect the cell-clusters to form thalli are needed, before we can develop a new technique for a large scale cultivation of Monostroma with cell-clusters regenerated from protoplasts .

Acknowledements. 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 for useful suggestions. This study was supported by the National Science Council (81-0418-B-002A-503-BG) and the Council of Agriculture (81AC-12.1-F-67(59)), Republic of China.

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