Development of protoplasts from holdfasts and vegetative thalli of Monostroma latissimum (Chlorophyta, Monostromataceae) for algal seed stock

Yean-Chang Chen

Department of Aquaculture,

National Taiwan Ocean University,

Keelung, Taiwan, R. O. C.

Received 9 April 1998. Accepted 31 August 1998

Email:ycchen@mail.ntou.edu.tw

Abstract

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock, and subsequently induce them to form algal filaments to act as an algal 'seed' stock. Protoplasts of the alga were isolated enzymatically with 4 % cellulase Onozuka R-10 and 2 % Macerozyme R-10. The highest number of protoplasts was obtained on a 50 RPM shaker with 1.2 M sorbitol after 6 h of incubation, with a yield of 9x10⁶ protoplasts per gram of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May about 1 % of protoplasts isolated from vegetative thalli developed directly to form tubular thalli and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, while protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than three years at 24ºC under the low irradiance of 66 μmol photons.m^-2.s^-1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons.m^-2.s^-1 at 18 to 30º C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons.m^-2.s^-1 and at 18 to 22 ºC. Therefore, the filaments could serve as 'seed' stock for algal mass culture.

Key index words: algal seed stock, holdfast, Monostromataceae, Monostroma latissimum, protoplast.

Introduction

Most economically important macroalgae have been harvested from natural habitats. This has resulted in a mass decline of those algal populations with a low regeneration rate which could not replace the harvested material. It is thus important to develop means of cultivating these algae for future needs. However, marine algae do not have structures that are the physiological equivalent of the resistant, dormant seeds of higher plants. Besides, algae frequently have rather complex life histories, often with alternation of phases (Polne-Fuller and Gibor 1987). Therefore, it is necessary and important to develop algal cultivation methods involving the seed stock, growing on artificial substrates (Polne-Fuller and Gibor 1987). In view of the above, I used an economically important macroalgae, Monostroma latissimum Wittrock, for this investigation on the development of algal 'seed' stock.

Monostroma spp. are widely distributed marine green algae with macroscopic thalli which are only one cell thick. Among the species of the genus, M. latissimum 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 a food supplement. However, commercial aquacultural techniques are still being developed for this alga. The traditional procedures of cultivation of Monostroma mentioned in our previous paper (Chen and Chiang 1994) are quite tedious and time-consuming, and it is sometimes also difficult to obtain enough mature thalli and a sufficient number of gametes or zoospores to 'seed' onto nets for the cultivation of M. latissimum. Therefore, developing new cultivation procedures of this alga is of great importance.

The algal protoplasts can be cultured to form seedlings quickly (Chen and Chen 1991, 1993, Chen and Chiang 1994). This new method may accelerate the success of algal cultivation. However, according to our previous reports (Chen and Chen 1991, 1993, Chen and Chiang 1994), the germlings regenerated directly from protoplasts have to be transferred immediately to the seashore for further mass culture, otherwise they will die because the laboratory cannot offer the vast area with essential high irradiance and renewed seawater (nutrients) that occur in their natural habitat. If the germlings regenerated from protoplasts can be treated like higher plant seeds, it will be much more convenient than the tradition way of algal cultivation. This means that we can keep the algal 'seeds' in the laboratory for longer periods, seeding areas when necessary.

The main purpose of this study was to obtain a stock of seedlings that can survive many years in an incubator, whereas maintaining their ability to develop into leafy thalli for algal mass culture.

Materials and Methods

Thalli of Monostroma latissimum were collected at Chiang-May, Peng-Hu County, Taiwan in March and May 1994. Freshly collected algae were wrapped in absorbent paper towels moistened with seawater, sealed in plastic bags, and packed in an ice-box for transport to the laboratory of the Department of Aquaculture, National Taiwan Ocean University in Keelung, Taiwan.

Preparation of clean and bacteria-free algal material. The method of Reddy et al. (1989) was followed with a slight modification (by using autoclaved seawater and antibiotic mixture) for obtaining clean bacteria-free material. Selected complete algal thalli were thoroughly cleaned in filtered seawater and placed in an ultrasonic cleaner (Branson 3200) with autoclaved seawater containing 1 % KI-I2 (2 g KI and 1 g I2 dissolved in 300 mL distilled water) for 3 min to remove animals and epiphytes. The thalli were then rinsed several times with autoclaved seawater. Finally, they were incubated in 100-mL of autoclaved Provasoli's enriched seawater (35 ‰) (PES) medium (Provasoli 1968) containing 10-mL of antibiotic mixture (Polne-Fuller and Gibor 1984) for 6 h at 24°C under a photon flux density of 66 μmol photons.m^-2.s^-1 in a plant incubator (Firstek, Taiwan).

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) in 10-mL of 1.2 M sorbitol. The pH was set at 6.0 using Na₂HPO₄-NaH₂PO₄ as 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 0.2 μm disposable syringe filter unit.

Isolation of protoplasts. Aseptic material was obtained by washing three times with autoclaved seawater. Holdfasts were separated from the rest of the algae; holdfasts and vegetative thalli (thalli without holdfasts) were then cut separately into pieces of 0.5-1 mm², with a sterile razor blade under a laminar flow hood. One hundred milligrams of the materials were incubated in sterile 50x80-mm disposable plastic flasks (40-mL, Falcon), containing 10-mL of enzyme solution and placed on an orbital shaker (Firstek, Taiwan) rotating at 50 rpm for 6 h (Chen and Chiang 1994). All isolation procedures were carried out at 24 C in the dark (Marchant and Fowke 1977, Cheney et al. 1986, Reddy and Fujita 1991) to promote sinking of isolated protoplasts (Liu et al. 1992).

Purification of protoplasts. Following incubation as described above for 6 h, the solutions were filtered through a 59-μm 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) (Chen and Chen 1991), to remove small pieces of debris. The protoplasts collected at the interface were retrieved with a sterile Pasteur pipette.

Yields of freshly isolated protoplasts were counted with a hemacytometer (Bright-Line, improved Neubauer 0.1 mm deep) under a light microscope (Zeiss, Axioskop).

Protoplasts cell wall formation. A fluorescent brightener reagent, Calcofluor White ST, (Sigma) was used to investigate the course of cell wall resynthesis in the cultured protoplasts (Galbraith 1981, Roberts et al. 1982, Chen and Chen 1993). The protoplasts were stained with Calcofluor White and those with resynthesized walls appeared green, while those without cell walls were red. Approximately 0.01 % of Calcofluor White (w/v) was added to a culture of freshly isolated protoplasts in a 0.85-M mannitol-PES medium. The culture was examined hourly on an inverted microscope with fluorescent equipment (Zeiss, Axiovert 135, Excite filters: Lp510-Kp560; Chromatic beam splitters: Ft580; Barrier filter Lp590) to determine whether the protoplasts had regenerated new walls or not.

Incubation of protoplasts. Purified protoplasts were cultured in PES medium with 0.85 M of the osmoticum mannitol immediately after isolation (Chen and Chen 1991, Chen and Chiang 1994). Eighteen groups of protoplast cultures were incubated under a photoperiod of 12:12 L:D and three photon flux densities of 66, 166 and 300 µmol photons.m^-2.s^-1 at six temperatures of 18, 20, 22, 24, 26 and 30°C (=3x6 groups). The regenerated protoplasts were cultured in the 0.85-M mannitol-PES medium for 5 days and then transferred to a pure PES medium for further cultivation under the conditions mentioned above (Chen and Chiang 1994). An inverted microscope (Zeiss, Axiovert 135) with camera equipment (MC 80DX) was used at intervals to observe and photograph the regenerated and differentiated state of protoplasts.

Culture of filaments. The regenerated filaments were transferred to 500-mL flasks with cotton plugs and kept in a plant incubator (Firstek, Taiwan) with a photoperiod of 12:12 L:D and a photon flux density of 66 μmol photons.m^-2.s^-1 at 24 °C. Development of the filaments to form leafy thalli was promoted by transferring the filaments to two photon flux densities of 166 and 300 μmol photons.m^-2.s^-1 combined with six temperatures of 18, 20, 22, 24, 26 and 30°C.

Culture of leafy thalli. When most of the leafy thalli had reached about 0.5-1 mm in length they were moved from the flasks to glass aquarium jars (40x30x20 cm) with fine nylon ropes (1 mm in diameter) on the bottom and with 12 L of PES medium, with a photoperiod of 12:12 L:D and a photon flux density of 300 µmol photons.m^-2.s^-1 at 22°C. A stereo microscope (ZEISS, SV11) with camera equipment (MC80DX) and a camera (Contax 167MT) with Zeiss Makro-Planar lens (60mm) were used to observe and record growth.

Results

Protoplasts of Monostroma latissimum could be isolated enzymatically with 4 % cellulase Onozuka R-10 and 2 % Macerozyme R-10 (Fig. 1). The highest yield of protoplasts was obtained on a 50-RPM shaker with 1.2 M sorbitol after 6 h of incubation, which gave a yield of 9x10⁶ protoplasts per gram of fresh holdfasts and vegetative thallus.

Some of the freshly isolated protoplasts began to regenerate new walls within 5 h of incubation. The regeneration of cell walls among the protoplast populations was variable, though the regenerated percentage of cell walls of protoplasts generally increased with increasing time of incubation. Almost all of the protoplasts (85%) had regenerated complete cell walls after 4-5 days of incubation.

Those protoplasts with a new cell wall began to divide from day 6 to 9 in PES medium. Protoplasts from vegetative thalli formed cell clusters and/or small thalli from day 14 to 18, while protoplasts from holdfasts regenerated to form filaments.

About 60 % of the protoplasts isolated from the vegetative thalli of M. latissimum collected in March and May retained the ability of regeneration under further culture. However, different differentiation patterns were observed as a function of time of collection of algae from the shore. On day 18 of cultivation the material collected in March regenerated directly to form tubular thalli which grew erect from their holdfasts, and were composed of a cylinder with several rows of cells, tapered at the apex (Fig. 2). Approximately 1 % of the protoplasts from material collected in May developed directly to form tubular thalli, while further another 59 % of protoplasts regenerated to form cell clusters (Fig. 3) without the differentiation.

In contrast to protoplasts from the vegetative thalli, on day 18 of culture about 45 % of the protoplasts obtained from holdfasts collected in March developed to form filaments (Figs. 4 and 5). Initially, the filaments were short and sparsely branched, and composed of a row of cells about 15-20 μm in diameter. Protoplasts isolated from holdfasts collected in May failed to develop and ultimately disappeared.

As shown in Table 1, variation in irradiance affected the development of filaments from protoplasts derived from holdfasts. Filaments kept in the low irradiance of 66 µmol photons.m^-2.s^-1at 18 to 30°C grew to form thick and thin filaments (Fig. 5), but developed no other kinds of structure. Filaments that were initially thick (Fig. 4) became thinner (Figs. 5 and 6) after being kept in the incubator for more than one year without the medium being renewed. The cells of the thin filaments were 4-8 μm in wide and 20-25 μm in long, and contained less cytoplasm than did the cells of thick filaments. Some cells of the thin filaments (Fig. 6) are empty since they had released monospores. However, apart from the empty cells, thin filaments recovered to form thick ones after the PES medium was renewed. In addition, the monospores (Fig. 6) that were released by the thin filaments germinated to form filaments under further cultivation at 66 µmol photons.m^-2.s^-1 and 18 to 30°C.

In contrast to those kept under low irradiance, erect tubular thalli developed from entangled tufts of filaments (Fig. 7; Table 1) incubated in an irradiance of 166 µmol photons.m^-2.s^-1at 18 to 30°C, from day 20 to day 45 of culture.

Temperature also affected the number of filaments that developed to form tubular thalli at an irradiance of 166 µmol photons.m^-2.s^-1. As shown in Fig. 8, with an increase in temperature from 18 to 30°C, the mean number of filaments forming tubular thallus increases from 31.2 to 115.4 per mm² area of the flask bottom, but their mean length decreases from 73 to 6 mm (Fig. 9).

The tubular thalli never developed to form leafy ones at temperatures above 24 C. At high temperatures (24-30°C) and irradiance of 166 and 300 µmol photons.m^-2.s^-1, these thalli increased their length continuously (Fig. 10) and became long tubular ones.

As shown in Table 1 and Fig. 10, increasing the irradiance from 166 to 300 µmol photons.m^-2.s^-1at 18 to 22°C induced long tubular thalli to form initials of leafy thalli on day 45 of cultivation. With further cultivation under the same conditions they finally formed complete leafy thalli (Fig. 11) with a frond composing of several lobes on day 90 of cultivation.

In this study, the stock filaments were kept for more than three years and still retained the ability to develop leafy thalli.

Discussion

The yield of protoplasts of Monostroma latissimum is different from that found by Fujita and Migita (1985). The difference in the yield of protoplasts 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, Chen and Chiang 1994). However, the yield of protoplasts in this study is consistent with the results of our previous paper (Chen and Chen 1991, 1993, Chen and Chiang 1994).

Purification of the isolated protoplasts is an essential step in the subsequent procedures. However, the method of filtration through nylon mesh (Saga 1984, Reddy et al. 1989) often resulted in contamination. Instead, density buffer, Ficoll-400 (Sigma), was used and proved to be most effective in obtaining clean protoplasts owing to its high osmotic potential and high density (Chen and Chen 1991, Chen and Chiang 1994).

The percentage (85 %) of cell wall regeneration by protoplasts in this study is higher than that of Monostroma nitidum (68.3 %) in Fujita and Migita's study (1985). This variability in cell wall resynthesis could be due to environmental stresses, such as osmotic pressure and temperature, which affect the physiology of protoplasts (Galun 1981).

Protoplasts cultured in a 0.85-M mannitol-PES medium for less than 5 days and then transferred to normal sea water lost their viability or died under these lower osmolarity condition since they still had not completely formed their walls. On the other hand, protoplasts cultured in a 0.85-M mannitol-PES medium for more than 5 days also showed a lower percentage of regeneration. This may indicate that a high concentration of mannitol is toxic to protoplasts stored in it for more than 5 days (Chen and Chen 1991, Chen and Chiang 1994). Reddy et al. (1989) and Reddy and Fujita (1991) similarly reported that prolonged cultivation of cells in hyperosmotic mannitol-seawater solutions at concentrations of 0.2-0.8 M impaired normal cell division and resulted in abnormal cell structures.

In view of these considerations, this study adopted the method used in our previous investigation (Chen and Chiang 1994), that is, the protoplasts were first incubated in a 0.85-M mannitol-PES medium for 5 days, and then transferred to normal enriched sea water (PES) medium for further cultivation. This method resulted in a greater quantity of isolated protoplasts of Monostroma latissimum which could retain the ability to regenerate (see also Chen and Chiang 1994).

Most of the protoplasts isolated from vegetative thalli and holdfasts collected in May regenerated to form cell clusters without the ability to differentiate. However, protoplasts from vegetative thalli and holdfasts collected in March were capable of differentiation into tubular thalli (Fig. 2) and filaments (Figs. 4 and 5) respectively. Both of these forms could develop into leafy thalli (Figs. 10 and 11) at high irradiance (300 µmol photons.m^-2.s^-1) and low temperatures (18-22 °C). In contrast to this finding, Saga and Kudo (1989) isolated protoplasts from M. angicava which did not have the ability to differentiate. Fugita and Migita (1985) reported that protoplasts of M. nitidum developed to form gametes after regeneration of new cell walls in culture. These diverse growth patterns of protoplasts among the genus Monostroma may be due to the original location of the protoplasts on the algal body, as well as different culture conditions, such as irradiance and temperature. This phenomenon has also been reported for Porphyra spp. (Polne-Fuller et al. 1986).

There were differences in differentiation between the isolated protoplasts of Monostroma latissimum thalli that were collected in March and those collected in May. This may be due to the aging of the algae (Galun 1981, Polne-Fuller et al. 1986). Generally speaking, in Peng-Hu Country, Taiwan, M. latissimum grows luxuriantly in March, and the population of this alga decreases gradually in May. In other words, the alga is young in March while it is senescent in May. Therefore, although the protoplasts were isolated from the same species, their physiological status varied, giving rise to various forms of algal bodies during their subsequent culture.

To date, Monostroma latissimum has not been reported to form individual filaments under natural conditions. However, multiseriate filamentous cells were confined to form the holdfasts of the thalli that were observed here. Protoplasts of holdfasts should regenerate to form free filaments as inferred from the filamentous cells of the holdfasts. Irradiance and temperature could be used to control the dedifferentiation and differentiation of those filaments. Therefore, filaments could potentially be a valuable storage form (seed stock) of this alga for further mass culture.

This study is also the first to report on the production of filaments from protoplasts of green macroalgae. At the time of writing, the filaments had remained viable for more than three years.

In brief, factors which those affect the regeneration and the differentiation abilities of the isolated protoplasts are the age of the parent algae (those collected in March and May) and irradiance combined with temperature. Filaments in the current study were observed to form tubular thalli at various irradiance irrespective of temperature. However, only high irradiance combined with low temperature induced tubular thalli to form leafy ones.

Conclusion

This report demonstrates the successful culture of the algal protoplasts to form typical thalli. Only protoplasts of holdfasts cultured under low irradiance (66 µmol photons.m^-2.s^-1) differentiated to form filaments. These could act as stock for future use. The developed filaments can re-adhere to the substrate and grow well during further cultivation. When these filaments adhere to ropes, they develop firm holdfasts from which small tubular thalli are continually produced. The ropes were fully covered with thalli with the average density of 25 thalli/cm on day 30 of cultivation (Fig. 12), and can be subsequently transferred to the seashore for further cultivation. The new technique described in this study could facilitate the mass cultivation of this delicious and economically important green alga.

Acknowledgements

Financial support from the National Science Council (NSC-87-2313-B-019-031; NSC-88-2313-B-019-046) of the Republic of China is highly appreciated.

References

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Figure legends

Figs. 1-6. Fig. 1. Freshly isolated protoplasts of Monostroma latissimum. Fig. 2. Tubular thalli (T) developed directly from the protoplasts after 18 days of culture. The protoplasts isolated from the thalli of Monostroma latissimum were collected in March. Culture conditions were 166 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium (H: holdfasts area). Fig. 3. The cell clusters (cc) developed from the protoplasts of Monostroma latissimum were collected in May, after 18 days of culture. Culture conditions were 166 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium. Fig. 4. The 18-day-old initial thick filaments (IK) and thin filaments (IN) developed from the protoplasts of holdfasts were collected in March. Culture conditions were 66 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium. (P: protoplast from holdfast). Fig. 5. Thick (TK) and (TN) thin filaments which had been kept in the plant incubator for more than one year without the medium being renewed. Culture conditions were 66 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium. Fig. 6. Thin filaments (TN) with empty cells (E) and their released monospores (M) that had been cultivated for more than one year without medium being renewed. Culture conditions were 66 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium.

Fig. 7. The erect tubular thalli (TT) developed from the entangled mass of tufted filaments (TF), after 20 to 45 days of culture. Culture conditions were 166 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium.

Figs. 8-9. Fig. 8. The relationship between the average number of tubular thalli that developed from the filaments and the temperatures, after 28 days of culture. Culture conditions were 166 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D in PES medium, at 18-30 °C. Fig. 9. The relationship between the length of tubular thalli and temperatures after 28 days of culture. Culture conditions were 166 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D in PES medium.

Figs. 10-12. Fig. 10. The initial leafy thallus (L) developed from the long tubular thallus. Culture conditions were 300 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 22°C in PES medium, on day 45 of culture. Fig. 11. A leafy thallus. Culture conditions were 300 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 22°C in PES medium, on day 90 of culture. Fig.12. Many tubular thalli attached on the nylon ropes. Culture conditions were 166 µmol photons.m^-2.s^-1 photon flux density and a photoperiod of 12:12 L:D at 24°C in PES medium, on day 30 of culture.