Planta 9 Springer-Verlag1988

Planta (1988) 174:25-29

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae) G. D u c r e u x and B. K l o a r e g 1 Laboratoire de Morphog~n6se V6g&ale Exp6rimentale, UA 115, Universit+ Paris-Sud, Centre d'Orsay, F-91405 Orsay Cedex, and t Centre d'Etudes Oc6anologiques et de Biologic Marine, CNRS LP, 4601 P1. G. Teissier, F-29211 Roscoff, France

Abstract. P r o t o p l a s t were isolated f r o m a filamentous b r o w n alga, Sphacelaria sp. (Sphacelariales, P h a e o p h y t a ) , using alginate-lyases extracted f r o m marine molluscs, and commercial pectinase and cellulase. Yields were a b o u t 4000 protoplasts per gram o f fresh tissue. Different types o f protoplasts, originating f r o m apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells p r o d u c e d a higher percentage o f protoplasts (approx. 2%), c o m p a r e d with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types o f protoplasts divided at least once in culture, but did not develop further. Key words: Alginate-lyase - Apical cell (alga) P h a e o p h y t a - P r o t o p l a s t (plant regeneration) Sphacelaria (protoplast isolation).

Introduction Only a few reports have been published on p r o t o plast isolation f r o m algae. M o s t results were obtained with C h l o r o p h y t a (e.g., Ohiwa 1977; M a r c h a n t and F o w k e 1977; Millner et al. 1979; K u r o d a 1980; Saga 1984). Digestion o f the unique walls o f P h a e o p h y t a and R h o d o p h y t a requires enzymes m o r e specific t h a n pectinases and cellulases. Using extracts f r o m marine molluscs, Polne-Fuller and G i b o r (1984), Saga and Sakai (1984), PolneFuller e t a l . (1986), Cheney e t a l . (1986) and K l o a r e g and Q u a t r a n o (1987) isolated protoplasts f r o m several b r o w n and red algal species. Plant regeneration f r o m seaweed protoplasts was obtained f r o m two genera, Sargassurn and Porphyra (Polne-Fuller et al. 1986). Preliminary experiments with fusion o f algal protoplasts have been r e p o r t e d

(Saga e t a l . 1986). However, m o r e progress is necessary before one can apply to seaweeds the genetic-engineering m e t h o d s which have been developed for higher plants (Cheney 1984). W e r e p o r t here o n regenerating whole plants f r o m protoplasts o f a filamentous b r o w n alga, Sphacelaria sp. Using alginate-lyases extracted f r o m marine molluscs and commercial pectinase and cellulase, viable protoplasts o f Sphacelaria were isolated, mainly f r o m apical cells. M o s t protoplasts f r o m subapical, nodal and internodal cells regenerated a wall and divided once but did not develop further. By contrast, m o s t o f the apical-cell protoplasts grew into whole plants.

Material and methods Plant material. Sphacelaria sp. (courtesy of Dr. L. Provasoli (Haskins Laboratories, New Heaven, Com., USA) and Dr. S. Loiseaux-De Goer (University of Grenoble, France) was grown in 370-ml glass bottles or 5-cm plastic Petri dishes at 12~ with a 12 h/12 h photoperiod (0.8 W.m-2). Culture medium was natural seawater (SW) containing 2% (v/v) Provasoli medium (ESP) modified according to Loiseaux and Rosier (1978). Media were autoclaved at 120~ C for 25 rain. Explants for inoculation were freed from epiphytes using the procedure described by Loiseaux and Rosier (1978). Preparation of alginate-lyases. Alginate-lyases were prepared from hepatopancreas of Haliotis tuberculata (abalone) and Patella vulgata (limpet) and from guts of Aplysia punctata. Molluscs were collected at Roscoff (Bretagne, France) in May ] 985. Enzymes were extracted with a phosphate buffer (pH 6.0) containing 50 mM ethylenediamine tetraacetic acid (EDTA; Muramatsu et al. 1977) and were fractionated with ammonium sulfate. Enzyme activity was monitored according to Nakada and Sweany (1967) using alginate from Laminaria digitata as substrate. One unit of activity (UA) was taken as the amount of enzyme needed to increase the optical density of the substrate by one unit per rain. The most active alginate-lyase fractions were recovered between 45% and 80% of the ammonium-sulfate saturation in H. tuberculata and P. vulgata and between 60-85% in A. punctata. Yields after exhaustive dialysis against

26

G. Ducreux and B. Kloareg: Regeneration of Sphacelaria from protoplast

phosphate buffer ranged from 40-120% of the total activity of ,the crude extracts. Enzymes were stable upon storage at - 2 0 ~ C. As indicated by the comparison of their activities towards either polymannuronate or polyguluronate blocks (Haug et al. 1974), alginate-lyases exhibited a strong specificity toward mannuronic linkages.

Protoplast isolation and culture. Dissociated thalli (1 g fresh weight in 10-mm pieces) were incubated in sterile SW (8 ml) containing 0.8 M mannitol, 2% (w/v) Cellulysin (Calbiochem Hoest, La Jolla, Calif., USA), 0.5% (w/v) pectolyase Y23 (Seishin Pharmaceutical Co., Tokyo, Japan) and 2% (v/v) alginatelyase. Final alginate-lyase activities were 0.2 U A . m l - 1, 0.05 U A . m l 1 and 0.02 UA.m1-1 for H. tubereulata, P. vulgata and A. punctata, respectively. Prior to incubation, the enzymatic solution was adjusted at pH 5.8, centrifuged, sterilized by filtration (0.22 p,m Millipore, Molsheim, France) and stored at - 2 0 ~ C. Incubation was performed at 20 ~ C under constant illumination (0.8 W ' m - Z ) , for 12 h. Preparations were shaken (50 rpm) during the first 6 h of incubation. The non-digested debris were removed by passing through a 100-gm metal sieve and the protoplast suspension was centrifuged at 56.g for 4 rain. Protoplasts were washed three times by centrifugation and resuspension with autoclaved, sterile-filtered SW containing 0.4 M NaC1. Protoplasts were cultured at the approximate density of 2000 protoplasts per ml in sterile SW (pH 7) containing 0.32 M NaC1, 2% (v/v) ESP and glucose and sucrose (250 rag.1 1 each). The cultures were maintained at 20 ~ C with a 12 h:12 h photoperiod (0.8 W. m-1). The state of protoplasts and their development were observed with an inverted microscope (Leitz Diavert, Wetzlar, FRG).

Results and discussion

Protoplast yields ranged from 260-4600 protoplasts per gram fresh weight of thallus. No significant differences (5% level, analysis of variance, five replicates) were detected between enzymes from the three alginate-lyase sources, H. tuberculata, P. vulgata and A. punctata. By contrast, yields were related to the age (i.e., the time after inoculation) of the algal culture. More protoplasts were produced from rapidly growing cultures. For example, protoplast yields from algae sampled 7 d after inoculation into new medium were typically four times higher than samples at 30 d. The efficiency of protoplast release also depended on the algal strain. Very few protoplasts were produced from wild material directly collected from the shore. Protoplasts were released as soon as 2 h after incubation. During this period a hole, from which protoplasts were released, formed in the cell wall, primarily at the distal end of apical cells. Cell-wall digestion was not complete and "cell-wall ghosts" were still visible at the end of the enzymatic treat-

ment (Fig. 1 d, e). Longer incubations (24 h) or different pH (7.0) did not improve yields. Protoplast populations were very heterogenous in size and pigmentation (Fig. I g), corresponding to the various cell types in Sphacelaria (Fig. 1 c). Protoplasts from apical cells were 60-65 gm in diameter and contained numerous physodes. Protoplasts from subapical cells were similar in size (55-60 gin) but did not contain large numbers of physodes. Smaller protoplasts (25-50 gm) were from nodal and internodal cells. The differences in the size and pigmentation allowed direct identification and scoring of each of the protoplast categories (Table 1). Compared with the proportions of apical cells among the different cell-types in the non-digested filaments (3%), protoplast preparations were enriched in apical-cell protoplast, up to 56% of total protoplast population. This proportion did not significantly depend on the nature of the alginate-lyase or the length of incubation. Compared with meristematic tissues of other Phaeophyta, Macrocystis and Sargassum (PolneFuller et al. 1986), or to zygotes of Fucus (Kloareg and Quatrano 1987), protoplast yields were quite low, only 1% of total donor cells. As most protoplasts were produced by digestion of the wall at the tip of apical cells (Fig. 1), protoplast preparations were highly enriched in apical-cell protoplasts (Table 1). In this region, growth is maximal and wall structure is different (Burns et al. 1984), and apparently more digestible by the enzymatic treatment. This is consistent with the observation that protoplast yields were negatively correlated to the age of donor plants. In Sphacelaria, the relative proportions of apical cells depend on the age of the culture. U p o n fragmentation and transfer into a fresh culture medium, the apical cells stop dividing and new, synchronous filaments are initiated from the nodal cells. This increases the proportion of apical cells in the culture (data not shown). Protoplast production shortly after inoculation thus yielded higher numbers of apical-cell protoplasts. Protoplasts were cultured in the same medium as the donor plants, to which osmoticum and sugars were added. Regeneration was better at 20-26 ~ C compared with 12 ~ C, which is the optimal temperature for growth of intact thalli. Within 24-48 h after the removal of the enzymes, most protoplast regenerated a wall and divided once. Only the apical-cell protoplasts, however, devel-

Fig. 1 a-g. Protoplast production from Sphacelaria. a: Piece of Sphaeelar& from an axenic culture (two weeks after inoculation); b: control (non-digested apex); c, d, e: various stages of cell-wall digestion after 1 h incubation; f: isolated apical- and subapical-cell protoplasts; g: protoplast preparation. Notice the different cell-type protoplasts. Apical-cell protoplasts are easily identified by their size and physodes. Ap = apical cell; S.Ap = subapical cell; IN= internodal cell; N = nodal cell

Fig. 2. Regeneration o f a Sphacelaria plant from an apical-cell protoplast. A t t a c h m e n t to the substratum is achieved by a basal disc (arrow). Ap = apical cell; Pr.Ap = initial cell(s) o f the regenerating plant

G. Ducreux and B. Kloareg: Regeneration of Sphacelaria from protoplast Table 1. Relative proportions (%) of the various cell types in the controls (undigested Sphacelaria filaments) and protoplast preparations. In protoplast preparations, cell types were identified by protoplast size and the presence or absence of physodes. Two- to five-hundred cells were scored in each experiment. Results are means of six experiments

Controls Protoplasts

Apical

Subapical

Nodal

Internodal

3 56

3 3

49 24

45 17

oped further (Fig. 2). During the next 1-2 d they underwent one to two additional cell divisions. On the third day a first filament emerged and reached eight to ten cells by the end of the following week. Then a second filament developed and eventually a whole plant was regenerated (Fig. 2). Such plants behaved normally and were propagated as usual by fragmentation. By contrast with apical cells isolated by micromanipulation, which continue normal developm e n t (Ducreux 1984), the development of apicalcell protoplasts (Fig. 2) was very similar to the germination of a spore or a zygote (Van den ttoek and Flinterman 1968, Prud'homme van Reine and Star 1981). Under the culture conditions tested, only apical-cell protoplasts demonstrated totipotency, indicating differences in the morphogenetic competence of the various donor cells. This novel procedure for the isolation of totipotent apical cells provides an opportunity to investigate the possible importance of the cell wall in differentiation, by comparing the development of naked and walled (e.g. Ducreux 1984) apical cells. This work was supported by a grant of the PIRSEM-CNRS (A.I.P. 3233). We thank B. Wolfersberger and M. Joncourt for their technical assistance. We are grateful to our colleagues from A. Gibor's lab (U.C. Santa Barbara) for their critical reading of the manuscript.

References Burns, A.R., Oliveira, L., Bisalputra, T. (1984) A cytochemical study of cell wall differentiation during bud initiation in the brown alga Sphacelariafurcigera. Bot. Mar. 27, 45-54

29

Cheney, D.P. (1984) Genetic modification in seaweeds: application to commercial utilization and cultivation. In: Biotechnology in the marine sciences, pp. 161-175, Colwell, R.R., Pariser, E.R., Sinskey, A.J., eds. Wiley, Chichester Cheney, D.P., Mar, E., Saga, N., Van der Meer, J. (1986) Protoplast isolation and cell division in the agar producing seaweed Gracilaria (Rhodophyta). J. Phycol. 22, 238 243 Ducreux, G. (1984) Experimental modification of the morphogenetic behaviour of the isolated sub-apical cell of the apex of Sphacelaria cirrosa (Phaeophyceae). J. Phycol. 20, 447454 Haug, A., Larsen, B., Smidsrod, O. (1974) Uronic acid sequence in alginate from different sources. Carbohydr. Res. 32, 217-225 Kloareg, B., Quatrano, R.S. (1987) Isolation of protoplasts from zygotes of Fucus distichus (L.) Powell. Plant Science 50, 189-194 Kuroda, K. (1980) Giant protoplasts from Nitella cells. Cell Biol. Int. Rep. 4, 195 199 Loiseaux, S., Rozier, C. (1978) Culture ax6nique de Pylaiella littoralis (L.) Kjellm. (Ph6ophyc+es). Rev. Algol. N.S. 13, 333-340 Marchant, H.J., Fowke, L.C. (1977) Preparation, culture and regeneration of protoplasts from filamentous green algae. Can. J. Bot. 55, 308(~3086 Millner, P.A., Callow, M.E., Evans, L.V. (1979) Preparation of protoplasts from the green alga Enteromorpha intestinalis (L.). L.K. Planta 147, 174~177 Muramatsu, T., Hirose, S., Katayose, M. (1977) Isolation and properties of alginate-lyase from the mid gut gland of Wreath Shell, Turbo cornutus. Agric. Biol. Chem. 41, 1939-1946 Nakada, H.T., Sweany, P.C. (1967) Alginic acid degradation by eliminases from Abalone hepatopancreas. J. Biol. Chem. 242, 845-851 Ohiwa, T. (1977) Preparation and culture of Spirogyra and Zygnema protoplasts. Cell Struct. Funct. 2, 248-255 Polne-Fuller, M., Gibor, A. (1984) Developmental studies in Porphyra. I. Blade differenciation of Porphyra perforata as expressed by morphology, enzymatic digestion and protoplast regeneration. J. Phycol. 20, 609-616 Polne-Fuller, M., Saga, N., Gibor, A. (1986) Algal cell, callus, and tissue cultures and selection of algal strains. Beih. Nova Hedwigia 83, 30-36 Prud'homme van Reine, W.T., Star, W. (1981) Transmission electron microscopy of apical cells of Sphacelaria spp. (Sphacelariales, Phaeophyceae). Blumea 27, 523-546 Saga, N. (1984) Isolation of protoplasts from edible seaweeds. Bot. Mag. Tokyo 97, 423-427 Saga, N., Sakai, Y. (1984) Isolation of protoplasts from Laminaria and Porphyra. Bull. Jap. Soc. Sci. Fish. 50, 1085 Saga, N., Polne-Fuller, M., Gibor, A. (1986) Protoplasts from seaweeds: production and fusion. Beih. Nova Hedwigia 83, 37~43 Van den Hock, C., Flinterman, A. (1968) The life history of Sphacelariafurcigera (Kutz.). Blumea 16, 193-242