KR20220170686A - Composition for preparing of protoplasts from Undaria pinnatifida sporophyte - Google Patents

Abstract

The present invention relates to a composition for preparing of protoplasts from Undaria pinnatifida sporophytes. More specifically, the composition of the present invention contains cellulase and alginate lyase as active ingredients. Accordingly, a large amount of protoplasts can be produced in a short period of time by establishing optimal conditions for the enzyme combination through the present invention Therefore, the composition of the present invention can be used for mass-cultivation of Undaria pinnatifida.

Description

Composition for preparing protoplasts of wakame sporophyte {Composition for preparing of protoplasts from Undaria pinnatifida sporophyte}

The present invention relates to a composition for producing protoplasts of seaweed sporophytes.

Brown algae are a diverse group of multicellular photosynthetic organisms comprising about 2,000 species worldwide. Seaweed ( Undaria pinnatifida ) is a brown algae belonging to the seaweed family and mainly inhabits Far East Asia. Seaweed is used in food, medicine, cosmetics, and pharmaceutical fields in countries such as Japan, China, and Korea. In Korea, in 2004, about 700 tons (wet weight) were harvested as wild products, and about 260,000 tons (wet weight) were harvested as farmed products. In domestic farms, gametes are generally cultivated during the summer and used as seedlings in order to cultivate seaweed sporophytes in the winter.

A protoplast is a plant cell wall from which the cell wall has been removed, and is particularly useful in economic plant breeding because it offers the possibility of crop improvement and in vitro manipulation that circumvents sexual reproduction. There has been a resurgence of interest in protoplasts in recent years due to their utility in single-cell RNA sequencing and genome editing and gene-silencing techniques. Protoplasts are mainly produced using terrestrial plants, but can also be obtained from bacteria, fungi and algae.

The main components of the cell wall of brown algae are alginates and fucoidans, which are not only different from those of terrestrial plant cell walls, but also have special characteristics that distinguish them from green and red algae. Conventionally, protoplast production in brown algae has been achieved by a combination of non-commercial enzymes and commercial enzymes or by non-commercial enzymes. Non-commercial enzymes refer to enzymes obtained by direct extraction from marine herbivores (eg, abalones and chimneys), and commercially available cellulase and macerozyme were used as commercial enzymes.

Although production of seaweed protoplasts has been attempted in the past, only commercial enzymes have not yet been used, and all studies have involved non-commercial enzymes directly extracted from organisms. As a result, the production results of seaweed protoplasts in previous studies showed problems in that the yield (yield) of protoplasts was low and reproducibility was poor.

Therefore, it is essential to develop optimal conditions for enzymes that can be usefully used in seaweed farming without being affected by the season by simplifying the protoplast production process and increasing the yield and reproducibility of protoplasts.

Accordingly, the inventors of the present invention completed the present invention by establishing optimal conditions for enzyme combinations as a result of efforts to increase the yield and reproducibility of protoplasts.

Korean Patent Registration No. 10-1594950

An object of the present invention is to provide a composition for preparing protoplasts of seaweed sporophytes that can be usefully used for breeding seaweed by establishing optimal conditions for enzyme combinations for production of protoplasts of seaweed sporophytes.

In addition, another object of the present invention is to provide a simplified process for producing protoplasts of seaweed sporozoites.

1. A composition for preparing protoplasts of wakame sporophytes containing seawater of pH 5.0 to pH 7.0 containing 0.1 to 5 w/v% of cellulase and 0.01 to 1 w/v% of alginate lyase.

2. The composition according to 1 above, wherein the composition comprises seawater of pH 5.5 to pH 6.0 containing 0.5 to 2 w / v% of cellulase and 0.04 to 0.08 w / v% of alginate lyase.

3. The composition for producing protoplasts of seaweed sporophytes according to 1 above, wherein the sporophytes are sporozoites cultured for 1 to 5 months.

4. A method for producing protoplasts of seaweed sporophytes comprising the step of culturing the cut seaweed sporophytes under the composition of 1 or 2 above.

5. The method for producing protoplasts of seaweed sporozoites according to 4 above, wherein the culturing is performed for 2 to 6 hours.

6. The method for producing protoplasts of seaweed sporophytes according to 4 above, further comprising the step of obtaining the chopped sporophytes by cutting the sporophytes cultured for 1 to 5 months.

7. The method for producing a protoplast of the seaweed sporophyte according to 4 above, wherein the sporophyte is not chelated.

The present invention can produce a large amount of protoplasts in a short time by establishing the optimal conditions of the enzyme combination for producing protoplasts of the seaweed sporophyte. The protoplasts prepared according to the present invention can be used for mass farming of seaweed, thereby contributing to the increase in income of fishermen.

Figure 1a shows the protoplast yield of seaweed spores according to the seawater pH in the composition of the present invention.
Figure 1b shows the protoplast yield of seaweed sporozoites according to the cellulase concentration change in the composition of the present invention.
Figure 1c shows the protoplast yield of seaweed sporozoites according to the concentration of alginate lyase in the composition of the present invention.
Figure 1d shows the protoplast yield of wakame spores according to the presence or absence of the addition of driselase to the composition of the present invention according to the osmolarity.
Figure 1e shows the protoplast yield of the seaweed sporophyte according to whether or not the chelate pretreatment was performed on the seaweed sporophyte of the present invention.
Figure 1f shows the protoplast yield of the seaweed sporophyte according to the culturing time of the seaweed sporophyte of the present invention.
Figure 1g shows the yield of protoplasts of seaweed sporophytes according to tissue sites of seaweed collected outdoors.
Figure 1h shows the protoplast yield of seaweed sporophytes according to the present invention and existing protocols.
Figure 2a shows the cell wall melted thallus after 2 hours after treatment with the composition of the present invention to the seaweed sporophyte.
Figure 2b shows the initial spherical protoplasts formed from the composition of the present invention.
Figure 2c shows protoplasts (epidermis (E), glandular cells (G), cortex (C)) formed from sporophytes.
Figure 2d shows protoplasts recognized as red by fluorescence microscopy.
Figure 2e shows live protoplasts stained with FDA showing green fluorescence.

Hereinafter, the present invention will be described in detail.

The present invention may provide a composition for preparing protoplasts of wakame spores containing seawater of pH 5.0 to pH 7.0 containing 0.1 to 5 w/v% of cellulase and 0.01 to 1 w/v% of alginate lyase.

"Cellulose" refers to a lattice-like polysaccharide in which linear chains of D-glucose units linked by β(1→4) glycosidic linkages are overlapping, and is used in green plants, many types of algae and oomycetes. Structural component of the primary cell wall of oomycetes.

"Cellulase" is an exoglucanase, exocellobiohydrolase, endoglucanase or β-glucosidase that catalyzes the hydrolysis reaction of β(1→4) glycosidic linkages in cellulose. Enzymes that do

The origin of the cellulase is not limited as long as it is an enzyme capable of decomposing the β(1→4) glycoside bond of cellulose, which is a cell wall component, and, for example, Trichoderma sp. cellulase (eg, Onozuka RS) derived from, but is not limited thereto.

The concentration of cellulase can be adjusted to increase the protoplast yield, for example, the concentration of cellulase is 0.1 to 5 w / v%, 0.2 to 5 w / v%, 0.3 to 5 w / v%, 0.4 to 5 w / v% v%, 0.5 to 5 w/v%, 0.1 to 4 w/v%, 0.2 to 4 w/v%, 0.3 to 4 w/v%, 0.4 to 4 w/v%, 0.5 to 4 w/v% , 0.1 to 3 w/v%, 0.2 to 3 w/v%, 0.3 to 3 w/v%, 0.4 to 3 w/v%, 0.5 to 3 w/v%, 0.1 to 2 w/v%, 0.2 to 2 w/v%, 0.3 to 2 w/v%, 0.4 to 2 w/v%, or 0.5 to 2 w/v%, but is not limited thereto.

"Alginate lyase" means an enzyme that decomposes alginate, which is a cell wall component of brown algae, and decomposes alginate by cleaving the glycosidic bond between mannuronic acid and guluronic acid constituting alginate.

The alginate lyase is used to decompose alginate, which is a cell wall component of brown algae, and may be a commercially available alginate lyase (eg, #A1603, Sigma Aldrich), but is not limited thereto.

In order to increase the protoplast yield, the concentration of alginate lyase can be adjusted. For example, the concentration of the alginate lyase is 0.01 to 1 w/v%, 0.02 to 1 w/v%, 0.03 to 1 w/v%, 0.04 to 1 w/v%, 0.01 to 0.09 w/v%, 0.02 to 0.09 w/v%, 0.03 to 0.09 w/v%, 0.04 to 0.09 w/v%, 0.01 to 0.08 w/v%, 0.02 to 0.08 w/v%, 0.03 to 0.08 w/v%, 0.04 to 0.08 w/v%, 0.01 to 0.07 w/v%, 0.02 to 0.07 w/v%, 0.03 to 0.07 w/v%, 0.04 to 0.07 w/v% v%, 0.01 to 0.06 w/v%, 0.02 to 0.06 w/v%, 0.03 to 0.06 w/v%, 0.04 to 0.06 w/v%, 0.01 to 0.05 w/v%, 0.02 to 0.05 w/v% It may be 0.03 to 0.05 w/v%, or 0.04 to 0.05 w/v%, but is not limited thereto.

"Seawater" means a solution containing salts used to grow marine organisms or to maintain marine organism tissues in a state close to normal. The seawater includes artificial seawater or natural seawater prepared to have an ionic composition, osmotic pressure, and pH close to seawater. The seawater may be sterilized.

The seawater contains cellulase and alginate lyase. The content of cellulase and alginate lyase may be within the aforementioned range.

Seawater is pH 5.0 to pH 7.0, pH 5.0 to pH 6.7, pH 5.0 to pH 6.5, pH 5.0 to pH 6.0, pH 5.3 to pH 7.0, pH 5.3 to pH 6.7, pH 5.3 to pH 6.5, pH 5.3 to pH 6.0, pH 5.5 to pH 7.0, pH 5.5 to pH 6.7, pH 5.5 to pH 6.5, pH 5.5 to pH 6.0, pH 5.7 to pH 7.0, pH 5.7 to pH 6.7, pH 5.7 to pH 6.5, pH 5.7 to pH 6.0 , but is not limited thereto.

Seawater may further include inorganic salts normally included in seawater, such as NaCl, MgCl ₂ 6H ₂ O, MgSO ₄ , KCl, and CaCl ₂ 2H ₂ O.

Seawater may further include a buffer. For example, MES (2-(N-morpholino)ethanesulfonic acid) or the like can be used.

The composition of the present invention is a composition for producing protoplasts of seaweed spores, and can be used to prepare protoplasts by culturing seaweed spores in the composition.

"Spophyte" refers to a diploid (2n) individual that forms spores in the heterogeneous life cycle of seaweed, and gametophyte refers to a single (n) individual that forms a gamete. Between winter and early summer, zoospores are released from the thallus, and zoospores form gametophytes, and male and female gametophytes are fertilized, germinate, form spores, and grow into sporophytes.

"Protoplast" means a plant cell from which the cell wall has been completely or partially removed and its lipid bilayer membrane is exposed. The protoplast may include a spheroplast having a partially removed cell wall. Typically, protoplasts refer to isolated plant cells without cell walls that have the ability to regenerate into cell cultures or whole plants.

Sporophytes to be cultured may be obtained by crossing gametophyte plants and then cultured in a medium for a certain period of time and for a certain length. Here, the medium may be PES (Provaoli Enriched Seawater).

The period of time is, for example, 1 to 5 months, 1 to 4 months, 1 to 3 months, 1 to 2 months, 2 to 5 months, 2 to 4 months, 2 to 3 months, 3 to 5 months, 3 to 5 months It may be 4 months, 4 to 5 months, but is not limited thereto.

The predetermined length is about 0.3 to 1.5 cm, 0.3 to 1.3 cm, 0.3 to 1.0 cm, 0.3 to 0.7 cm, 0.5 to 1.5 cm, 0.5 to 1.3 cm, 0.5 to 1.0 cm, 0.5 to 0.7 cm, 0.7 to 1.5 cm, It may be 0.7 to 1.3 cm, 0.7 to 1.0 cm, but is not limited thereto.

The incubation time of the sporophyte in the composition of the present invention is, for example, 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 4 hours. It may be 6 hours, 4 to 5 hours, but is not limited thereto.

In addition, the present invention can provide a method for producing protoplasts of seaweed sporophytes comprising culturing the cut seaweed sporophytes in the composition for producing protoplasts of seaweed sporophytes.

"Slicing" may include various grinding or cutting processes to increase the surface area of the sporophyte so that the cell wall degradation of the sporophyte occurs rapidly in seawater, and the size of the sporophyte is 2 to 8 mm ² , 4 to 8 mm ² , 6 to 8 mm ² , 2 to 6 mm ² , 4 to 6 mm ² , but is not limited thereto.

The chopped seaweed sporophyte is cultured under the composition for preparing the protoplast of the seaweed sporophyte.

The "incubation time" for culturing the cut seaweed sporophytes is 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours time, but may be 4 to 5 hours, but is not limited thereto.

The "temperature" of seawater for culturing the cut seaweed sporophytes is, for example, 18 to 22 ° C, 18 to 21 ° C, 18 to 20 ° C, 18 to 19 ° C, 19 to 22 ° C, 19 to 21 ° C, 19 to 20 ° C , 20 to 22 ℃, may be 21 to 22 ℃, but is not limited thereto.

Culturing may be, for example, culturing in a shaking incubator, but is not limited thereto.

In the method of the present invention, spores of wakame that are not treated with a chelate may be used. In general, in the method for producing protoplasts of seaweed sporophytes, protoplasts are prepared with such sporophytes after pre-treating the sporophytes with a chelate such as calcium chelate to secure the yield. The method of the present invention can produce protoplasts in high yield without such chelate pretreatment can

The method of the present invention may further include the step of crossing gametophyte plants to obtain sporophytes and culturing them in a medium for a certain period of time to obtain said sporophytes.

The medium may include Provaoli Enriched Seawater (PES).

The "cultivation period" of culturing the spores of a certain length in the medium is, for example, 1 to 5 months, 1 to 4 months, 1 to 3 months, 1 to 2 months, 2 to 5 months, 2 to 4 months, 2 to 4 months. It may be 3 months, 3 to 5 months, 3 to 4 months, 4 to 5 months, but is not limited thereto.

The "length" of the sporophytes growing during the incubation period is about 0.3 to 1.5 cm, 0.3 to 1.3 cm, 0.3 to 1.0 cm, 0.3 to 0.7 cm, 0.5 to 1.5 cm, 0.5 to 1.3 cm, 0.5 to 1.0 cm, 0.5 to 0.7 cm. cm, 0.7 to 1.5 cm, 0.7 to 1.3 cm, and 0.7 to 1.0 cm, but is not limited thereto.

The present invention may further include the step of cutting the sporophyte grown to the predetermined length. In addition, the present invention may include a step of removing the rhizoidal portion before or after mincing the sporophyte.

The present invention includes the step of incubating the cut sporophytes in a shaking incubator under the composition for producing protoplasts of seaweed sporophytes according to the present invention. It can be set differently as appropriate.

In the specification of the present invention, when it is said to "include" a certain component, it means that it may further include other components unless otherwise stated.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and the present invention is not limited thereby.

Example 1. Securing the spores of seaweed

Seaweed ( Undaria pinnatifida ) used in the present invention was distributed and cultured at the Marine Brown Algae Plant Resources Deposit Registration and Preservation Institute of Chosun University (Resource ID Numbers: MBRB0049TC17136 and MBRB0049TC17223).

The seaweed sporophyte used in the present invention is a sporophyte obtained by crossing gametophyte plants of Heuksando and Jindo. Sporophytes obtained by crossing were cultured (primary culture) in Provasoli Enriched Seawater (PES) at 16° C. for 2 to 3 months to obtain sporophytes having a length of 0.5 to 1 cm.

The sporophyte with a length of 0.5 to 1 cm obtained by primary culture was rinsed three times with sterile seawater, and then the sporophyte was cut into 4-6 mm ² using a clean razor blade. At this time, the rhizoidal part was discarded because the protoplast yield was low.

Example 2. Preparation of sterile artificial seawater

The composition of sterilized artificial seawater used in the present invention is shown in Table 1 below.

[Table 1]

Example 3. Preparation of protoplasts from seaweed sporophytes

Commercially available enzymes, 1 w/v% cellulase (Onozuka RS) and 0.04 w/v% alginate lyase (Sigma Aldrich) were added to sterile artificial seawater. 300 mg of shredded seaweed sporophyte pieces were added to 5 mL of sterilized artificial seawater containing the commercial enzyme, and cultured (secondary culture) in a shaker at 70 rpm for 2 to 4 hours at pH 6.0 and 20 ° C under dark conditions. After 2 hours, the seaweed sporophytes were completely decomposed to form protoplasts.

Example 4. Purification of protoplasts and calculation of protoplast yield

The protoplasts were filtered using a 25 μm nylon mesh, and the filtrate was received in a 15 mL sterile conical tube and centrifuged at 100 g for 10 minutes. After centrifugation a total of three times, the supernatant was removed, and the pellet (protoplast) was washed and purified with sterile artificial seawater containing 130 mM magnesium chloride and 160 mM potassium chloride, and then the protoplast was finally maintained in 1 mL culture medium. In the present invention, the final protoplast yield was calculated using a hemocytometer as follows:

[Equation 1]

Protoplast yield (protoplasts/g fresh weight) = {protoplast concentration (protoplasts/mL) * volume of protoplast solution (mL)} / fresh weight of samples (g)

The amount of protoplasts harvested according to Example 3 was 2-4 x 10 ⁷ protoplasts/g (Table 2).

[Table 2]

Example 5. Evaluation of various conditions affecting protoplast yield

5-1. Comparison of protoplast yield according to seawater pH

The effect of seawater pH on the protoplast yield of wakame sporophytes was studied. The pH of seawater was changed to pH 5.5, pH 6.0, pH 6.5, pH 7.0 or pH 7.5, respectively. Experiments were performed under the same conditions as in Examples 1 to 4 except for changing the pH of seawater. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

In seawater pH 6.0, the yield of protoplasts from wakame sporophytes was the highest (Fig. 1a).

5-2. Comparison of protoplast yield according to cellulase concentration

The effect of the concentration of cellulase (Onozuka RS) on the protoplast yield of wakame sporophytes was studied. The concentration of cellulase (Onozuka RS) contained in 5 ml of artificial seawater was 0 w/v%, 1 w/v%, 2 w/v%, and 3 w/v%, respectively. Experiments were performed under the same conditions as in Examples 1 to 4 except for changing the concentration of cellulase. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

The highest yield of protoplasts was obtained from seaweed sporozoites at a cellulase concentration of 1 w/v% (FIG. 1b).

5-3. Comparison of protoplast yield according to alginate lyase concentration

The effect of the concentration of alginate lyase (Sigma Aldrich) on the protoplast yield of seaweed sporophytes was studied. The concentration of alginate lyase contained in 5 ml of artificial seawater was 0 w/v%, 0.02 w/v%, 0.04 w/v%, and 0.06 w/v%, respectively. Experiments were performed under the same conditions as in Examples 1 to 4 except for changing the concentration of alginate lyase. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

At 0.06 w/v% of alginate lyase, the yield of protoplasts from seaweed sporophytes was high (Fig. 1c).

5-4. Comparison of protoplast yield according to Dricellase addition and osmotic pressure

The effect of dricellase addition and osmotic pressure on the protoplast yield of wakame sporophytes was studied. Artificial seawater having an increased osmotic pressure was prepared by increasing the concentration of the components of the artificial seawater while maintaining the same ratio of each component of the artificial seawater (Table 3). At this time, the artificial seawater contained 1 w/v% cellulase (Onozuka RS) and 0.04 w/v% alginate lyase. As in Example 1, the primary cultured 0.5 to 1 cm long sporophyte was cut into 4 mm ² . The chopped sporophytes were cultured (secondary culture) in artificial seawater with normal osmotic pressure (1Х = 1570 mOsm/LH ₂ O) and increased osmotic pressure (1.6Х = 2512 mOsm/LH ₂ O), respectively. In addition, the same experiment was conducted by adding driselase to each artificial seawater. The secondary culture was performed for 6 hours under dark conditions, pH 6.0, and 20°C. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

[Table 3]

a normal osmotic pressure (1x) = 1570 mOsm/LH ₂ O; b Increased osmotic pressure (1.6x) = 2512 mOsm/LH ₂ O

It was shown that no driselase other than cellulase and alginate lyase are required for the production of protoplasts of wakame sporophytes, and more cell wall degradation occurred in normal osmotic (1x) seawater than in increased osmotic (1.6x) seawater.

5-5. Comparison of chelation pretreatment

The effect of chelation pretreatment on the protoplast yield of wakame sporophytes was studied. As in Example 1, the primary cultured 0.5 to 1 cm long sporophyte was cut into 4 mm ² . The cut sporophytes were cultured in a calcium chelate solution having the composition shown in Table 4 below for 20 minutes. Subsequently, the culture was subcultured in artificial seawater containing 1 w/v% cellulase (Onozuka RS) and 0.04 w/v% alginate lyase. The secondary culture was performed for 6 hours under dark conditions, pH 6.0, and 20°C. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

[Table 4]

The present invention showed that pretreatment with calcium chelate was not required in preparing protoplasts from seaweed sporozoites (Fig. 1e).

5-6. Comparison of protoplast yield according to incubation time

The effect of incubation time on the yield of protoplasts of seaweed sporophytes was studied. In the secondary culture, the incubation time was set to 1 hour, 2 hours, 4 hours or 6 hours, respectively, at 70 rpm in a shaking incubator. Experiments were performed under the same conditions as in Examples 1 to 4 except for changing the incubation time. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

The present invention showed the highest protoplast yield from seaweed sporozoites when cultured for 2 to 4 hours at 70 rpm in a shaking incubator (FIG. 1f).

5-7. Comparison of protoplast yield according to tissue site

In order to compare the protoplast yield of seaweed sporophytes according to the parts of seaweed thallus, 20 cm long seaweed sporophytes were collected in Jindo on April 14, 2018. Areas free from epiphytes were selected and rinsed 10 times in sterile seawater. Henroot, stem, basal meristem and distal blade were cut into 4-6 mm ² using a clean razor blade, and then treated with 1 w/v% cellulase (Onozuka RS) and 0.04 w/v% Incubated in artificial seawater containing alginate lyase under dark conditions, pH 6.0, at 20 ° C for 6 hours. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

The basal meristem of the seaweed sporophyte showed the highest protoplast yield (Fig. 1g).

5-8. Comparison of protoplast yields of protocols

1) As in Example 3 of the present invention, when cultured using commercial enzymes, 1 w / v% cellulase (Onozuka RS) and 0.04 w / v% alginate lyase (Sigma Aldrich), 2) Implementation of the present invention In Example 3, only the enzymes were different, that is, when cultured using 4 w/v% cellulase (Onozuka RS) and 2 w/v% macerozyme (R-10), 3) chelate pretreatment, the chopped sporophyte , Protoplast yields were compared when cultured in sterile artificial seawater containing no enzymes. Protoplast production was performed at the same time, and experiments were performed under the same conditions as in Examples 1 to 4 except for the type and use of enzymes. The protoplast yield was calculated using a hemacytometer and expressed in units (protoplasts/g fresh weight).

The present invention showed a higher protoplast yield than previous studies (Fig. 1h).

Example 6. Evaluation of protoplast usability and cell wall removal

Protoplasts were analyzed by red chlorophyll autofluorescence using an inverted microscope (Leica DMI8, Germany) equipped with an external light source (Leica EL6000) for fluorescence excitation and equipped with emission filters (470/40 nm) and suppression filters (515 nm). Viability was assessed.

The protoplasts were stained with 0.01% calcofluor white M2R (USA Sigma Aldrich) fluorescent staining solution and examined using a Leica DMI8 equipped with an inverted microscope.

Protoplast viability was further confirmed with a 2.4 μM diacetate (FDA; Sigma, USA) fluorescent staining solution, and an inverted microscope (Leica DMi8, Germany) equipped with an emission filter (540/46 nm) and a suppression filter (590 nm). Observed. Protoplast viability (%) and cell wall removal rate (%) were calculated with a minimum of 100 protoplast samples.

Figures 2d and 2e show viable protoplasts with red chlorophyll auto-fluorescence and green fluorescence due to FDA staining, respectively. The viable protoplasts were not stained with blue fluorescent Calcofluor staining solution. The viability of freshly isolated protoplasts ranged from 98 to 100% for red chlorophyll autofluorescence and about 65% for FDA staining. Cell wall removal rates ranged from 99 to 100%.

Claims (7)

0.1 to 5 w / v% cellulase, 0.01 to 1 w / v% alginate lyase, pH 5.0 to pH 7.0 seawater containing a composition for producing protoplasts of seaweed sporozoites.
The method according to claim 1, wherein the composition is cellulase 0.5 to 2 w / v%, alginate lyase 0.04 to 0.08 w / v% pH 5.5 to pH 6.0 seawater containing the protoplast preparation composition of seaweed sporozoites.
The composition of claim 1, wherein the sporophyte is a sporozoite cultured for 1 to 5 months.
A method for producing protoplasts of seaweed sporophytes comprising the step of culturing the shredded seaweed sporozoites under the composition of claim 1 or 2.
The method of claim 4, wherein the culturing is performed for 2 to 6 hours.
The method for producing protoplasts of seaweed sporophytes according to claim 4, further comprising the step of obtaining the chopped sporophytes by cutting sporophytes cultured for 1 to 5 months.
The method of claim 4, wherein the sporophyte is not chelated.

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