Stable Protoplast Isolation and Its Regeneration into ...

Nippon Suisan Gakkaishi 58(1), 153-157 (1992)

Stable Protoplast Isolation and Its Regeneration into Thallus

of the Marine Green Alga Ulva pertusa

Aritsune Uchida,*1 Takeshi Yoshikawa,*1 Yuzaburo Ishida,*1and Naotsune Saga*2

(Received July 24, 1991)

Protoplasts were isolated enzymatically from female gametophytes of the marine green alga Ulva pertusa. The best enzyme compositions to yield protoplasts were 5% Abalone Acetone Powder, 2% Cellulase Onozuka R-10, and 0.1% Macerozyme R-200 in 1.2M sorbitol at pH 5.5.

The isolated protoplasts regenerated in PES medium and two types of developmental process were observed; thallus forming type and rhizoid and thallus forming type.

Seaweeds are important marine resources and

have many uses. They have been regarded as

promising biomasses for fine chemicals and

alternative energy production. Therefore, the

demand for high quality and fast-growing sea-

weeds has been increasing. Recently biotechno

logical methods have been applied to seaweeds

and isolations of protoplasts from them have

been achieved in 9 genera and 16 species.1)

Though marine green algae have been popularly

used in these studies,2-6) it is still very difficult

to obtain stable number of protoplasts and

regenerate them into complete thalli.

We report here the efficient isolation and re

generation of protoplasts of Ulva pertusa Kjell

man with several enzymes.

Materials and Methods

Alga

The unialgal culture of Ulva pertusa female

gametophytes was used in this study. The

cultures were subcultured every month for several

years in PES medium7) in 300ml plastic cups at

15•Ž under illumination of cool white fluorescent

lamps (14L: 10D, 3,000lx). Natural thalli of

Ulva pertusa were collected at beach of Katsurakoi,

Kushiro, Hokkaido Prefecture and Takahama,

Fukui Prefecture.

Preparation of Enzyme Mixture for Protoplast

Isolation

The enzyme mixtures used in this study were

prepared according to a method described pre

viously.8) Sorbitol-MES (2-(N-morpholino) eth

anesulfonic acid) buffer solution was used as a

basal solution of enzyme mixture. The com-

position of enzyme solution is shown in Table 1.

Abalone Acetone Powder (AAP) was purchased

from Sigma Chemical Co. Cellulase Onozuka

R-10 and Macerozyme R-200 were purchased

from Yakult Honsha Co. Ltd.

Protoplast Isolation

Vegetative parts of leafy thalli were cut into

small pieces and homogenized with a Waring

blender. After this, the cells were maintained

in sorbitol-MES buffer solution for 1h in order

to introduce plasmolysis. The cells (250mg)

were added to 2.5ml of enzyme solution and

incubated at 20•Ž for 5h with reciprocal shaking

(30 strokes/min). After being separated from un-

digested cellular debris by fitration through 40ƒÊm

Nylon mesh, protoplasts were centrifuged at 50•~g

for 10 min and washed several times with sorbitol-

MES buffer solution and diluted with PES medium

at 2ml/h for 5h in order to make osmotic pressure

lower.

Count of Protoplasts

Isolated protoplasts were counted with a

hemocytometer. The viability of protoplasts

*1 Laboratory of Microbiology, Department of Fisheries, Faculty of Agriculture, Kyoto Universiky, Kita

-shirakawa-oiwake, Sakyo, Kyoto 606, Japan(内田有恆,吉川 毅,石 田 祐 三 郎:京 都 大 学 農 学 部 水 産

微 生 物 学 研 究 室).

*2 Department of Marine Science, School of Marine Science and Technology, Tokai University, Orido

, Shimizu, Shizuoka 424, Japan(嵯 峨 直恆:東 海 大 学 海 洋 学 部 海 洋 科 学 科).

was evaluated by Evans Blue (Sigma) staining.9) Cell walls were stained with Calcofluor White M2R (Fluorescent Brightener 28; Sigma).10)

The stained cell walls were observed with fluorescent microscope.

Table 1. Basal composition of enzyme solution

for protoplasts isolation

Fig. 1. Effect of Abalone Acetone Powder (AAP) on the yield of protoplasts from cultured Ulva

pertusa. Each AAP enzyme mixture contained 2% Cellulase Onozuka R-10 and 0.1% Macero

zyme R-200. •œ•\•œ, Yield of protoplasts; •›------•›, protoplast viability.

Fig. 2. Effect of AAP on the yield of protoplasts from natural Ulva pertusa . Each AAP enzyme

mixture contained 2% Cellulase Onozuka R-10 and 0.1% Macerozyme R-200. •œ•\•œ,

Yield of protoplasts; •›------•›, protoplast viability.

Protoplast CultivationProtoplasts were suspended with 2ml of PES

medium in a 24 wells microtiterplate and incubated

at 20•Ž under illumination of cool white flu

orescent lamps (14L: 10D, 3,000 lx). After 2

weeks incubation, regenerated protoplasts were

transferred to 15ml culture tubes containing

8ml PES medium. After one month, plants were

transferred to 300ml disposable sterilized cups

containing 200ml of PES medium. The medium

was exchanged for a new one once a month.

Fig. 3. Freshly isolated protoplasts of Ulva pertusa and their regenerated cells.

(A) Freshly isolated protoplasts. (B, C) Freshly isolated protoplasts which were stained

with Calcofluor White M2R. (B) Bright field illumination, (C) fluorescent illumination.

(D, E) Regenerated cells after 2 days incubation which were stained with the same dye

as B, C. (D) Bright field illumination, (E) fluorescent ilumination.

Scale bar shows 100ƒÊm.

Results and Discussion

It is generally said that the cell wall of Ulva

contains glucose polymer with smaller quantities

of xylose and that Ulva has mucilage which

consists of D-galactose, L-alabinose, D-xylose,

and L-rhamnose complex on the cell surface.11)

Therefore, we tested several enzyme compositions

to digest the cell wall of Ulva pertusa and chose

the best enzyme composition. Recently it has

become popular to use AAP which can degrade

cellulose, ƒÀ-mannan, alginate, ƒÈ-carageenan, and

ƒÀ-1, 4- xylan12) as an indispensable enzyme. There-

fore, the effect of the enzyme concentration on

the isolation of protoplasts was tested. The

higher the concentration of AAP, the more the

yield of protoplasts. At more than 5.0% of AAP,

the yield of protoplasts reached plateau and

about 4.0•~105 protoplasts were obtained from

1g fresh weight of the culture (Fig. 1). The

Fig. 4. The thallus forming type protoplast regeneration of Ulva pertusa . (A) Cells after 2 days

incubation of protoplast. (B) Cells after 3 days incubation. (C) Cells after 4 days incubation.

(D) Cells after 6 days incubation. (E) Cells after 15 days incubation.

Scale bar shows 100ƒÊm.

Fig. 5. The rhizoid and thallus forming type protoplast regeneration of Ulva pertusa. (A) Cells

after 2 days incubation of protoplast. (B) Cells after 3 days incubation. (C) Cells after 4

days incubation. (D) Cells after 6 days incubation. (E) Cells after 10 days incubation . (F) C

ells after 15 days incubation.

Scale bar shows 100ƒÊm.

reason for this phenomenon is considered that

AAP is a crude enzyme and contains some low

molecular toxic substances against the proto

plast.13) Therefore, if AAP is purified the

yield of protoplasts and viability are improved. From the stain test , the percentage of viable

protoplasts was generally 40-50. The same result was obtained in the case of natural thalli

and about 2.0•~106 protoplasts per 1g fresh

weight of thalli were obtained under the best

condition (Fig. 2). From the above result, the

enzyme mixture containing 5% AAP was adopted

for further studies.

Freshly isolated protoplasts were green, spher

ical (20-50ƒÊm in diameter), and mostly had

large vaculoes (Fig. 3A). Cell walls were com

pletely digested with the enzymes since we could

not observe the stained cell walls with Calco-

fluor White M2R (Fig. 3B, C). After 2 days

incubation of protoplasts, new cell walls were

synthesized (Fig. 3D, E). One day after cell

wall formation, each cell divided into 2 cells.

Two types of the developmental process following

cell division were distinguished: 1) thallus forming

type; after cell wall regeneration, the walled cells

divided, and produced leafy thalli (Fig. 4), and

2) rhizoid and thallus forming type; the cells

repeated divisions in the same direction to form

filamentous tissues with branches, and thalli grew

from the center of the rhizoidal tissues (Fig. 5).

Regenerated cells formed Enteromorpha-like

monolayer tubular thalli. Saga & Kudo14) re

ported that several types of the development were

observed in protoplast regeneration. As the

protoplasts were not incubated axenically, the

different morphogenesis may be due to the dif

ferent coexisting bacteria. Provasoli & Pintner15)

also reported that cocultivation of U. lactuca

with bacteria which attached to the algal surface

caused different shaped thalli, and suggested the

interaction of morphogenesis and bacterial flora.

In both types, after 4 months incubation, re

generated thalli grew 15-20cm long.

Acknowledgment

This work was supported by a grants-in-aid

from the Ministry of Education, Science, and

Culture of Japan, No. 63440015.

References

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Nippon Suisan Gakkalshi: Formerly Bull. Japan. Soc. Sci. Fish.