Phyc%gia (1977) Volume 16 (4), 451- 455

Growth regulating effects of phenylacetic acid and p-hydroxy­

phenylacetic acid on Fucus spiralis L. (Phaeophyceae, Fucales)
in axenic culture

LISBETH FRIES

Institute of Physiological Botany, Uppsala University, Uppsala, Sweden

FuclIS spiralis L. has been brought into axenic culture by treatment of young frond tips in saturated calcium hypo­
chlorite solution. In axenic culture the FuclIS plants grow mostly with cylindrical thalli, which branch once or twice
and remain about I cm high for several months.
Additions of p-hydroxyphenylacetic acid (p-OHPAA) in a concentration range of 10-7-10-5 M as well as of
phenylacetic acid ( PAA) in concentrations around 10-7 M could induce normal morphology. Broad and slightly
wavy fronds were formed and a growth was induced quite comparable to that in bacteria-infected cultures. As
these two phenylacetic acids have been identified previously in the brown alga Undaria, the current experiments
show thatp-OHPAA and PAA may be natural growth regulators in brown algae.

Introduction Fucus vesiculosus as well as of fertilized eggs had been

Members of the family Fucaceae are dominant
among the algae in the littoral zone on the Swedish
west coast. The seawater collected from this zone has
been found to be especially rich in substances pro­
moting growth in other multicellular algae (Kylin,
1941; Fries, 1970; Pedersen, 1973; Fries, 1973).
These substances have been presumed to emanate
from members of the two dominating genera, Fucus
and Ascophyllum. It was thus considered to be of
interest to cultivate some species of these genera in
axenic culture for further investigation of their
exudation and possible demand for growth sub­
stances. The role of their epiphytic bacteria could
also be elucidated in this connection.
Material and methods
On the Swedish west coast, where the difference
between ebb and tide is only 10-20 cm, Fucus spiralis
L. grows on cliffs just below the lower water level.
Segments of fronds from different Fucus species show
a remarkable ability in wound healing even in culture
and new adventitious branches are formed (Moss,
1963, 1974; Fulcher & McCully, 1969). Fronds
should thus be suitable as initial material to obtain
axenic cultures. As several experiments with anti­
biotical treatments of frond tips of Fucus spiralis and
performed previously proved ineffective, some more
drastic sterilization method seemed necessary.
In September small green fronds of Fucus spiralis
appear in a fringe along the cliffs. Such fronds were
collected and 3-5 mm long tips were cut and treated
with saturated calcium hypochlorite solution for
2-5 min. Thereafter they were rapidly washed in
sterile seawater and placed in Petri dishes containing
20 ml ASP6 F2 medium with 5 g agar per litre. After
about 1 week the pieces that appeared to be bacteria
free were transferred to new agar plates. After a
second transfer about a. week later, the tips were
moved to 100 ml Pyrex flasks containing 25 ml
ASP6 F2 medium and then left in an incubator at
15°C with 15 hr light per day.
The segments had now lost their colour with the
exception of a few cells in the apical pit region, at
some spots along the mid rib or around the crypto­
stomata. Within a month such living cells could start
dividing and form a callus-like tissue which differen­
tiated into one or, usually, several shoots. Some cells
could also rest for months before new shoots
·
appeared. The new plants gr�w very slowly and
started as cylindrical thalli from the original growing
point.
The axenity of these plants was then carefully
tested (Fries, 1970). Several plants proved bacteria
free in the first test but showed infections later.
Obviously, single bacteria survive in the mucilage of

29 451
452 Phycologia, Vol. 16 (4), 1977

the algal cell walls for months and develop when the B12 are sterilized separately and mixed after auto­
alga is cut into pieces for an experiment. At the end claving.
only four axenic plants remained from many hundred
treated tips. Each of these plants had produced
several fronds. Results and discussion
About 2 mm long apices from plants cultivated
under short day conditions (16°C, 10 hr L) were used Under short day conditions (16°C and 10 hr light)
as inocula in cultivation experiments. The same the axenic Fucus plants grow very slowly and form
number of flasks in each series was inoculated from only cylindrical or very slightly flattened fronds,
the same plant to diminish the differences between which branch only once or twice (Figs 1 and 2). Such
inocula. This means that the plants in the horizontal an axenic plant grew about 10 mm in 4 months, while
rows in Figs 3 and 4 originate from the same mother a plant isolated at the same moment but found to be
plant. The axenity was tested on the inocula as well bacterized developed 30 mm long and flattened
as on the developed algae at the end of an experiment. fronds in the same time.
Fronds were cultivated in 100 ml Pyrex flasks The plant in Fig. 1 has a well-developed rhizoid
containing 25 m] nutrient medium ASP6 F2 at 15°C system which attaches the plant to the glass wall of
with 15 hr light from a light bank with Philips light the flask. Many tips, cut for inocula, developed
tubes 33 giving 20-25 W m-2. Incubation time was rhizoids but no regularity in this formation could be
70 days. In the last experiment the fresh weight of the observed. Infected plants could develop whole
algae at the end of the experiment is given. bunches of rhizoids, which was mentioned for other
The phenylacetic acids were dissolved in water, Fucus species by McLachlan et al. ( 1 97 1 ).
autoclaved separately and added to the experimental The bacteria-free plants apparently need external
flasks. Their purity was checked by GLC. additions of one or several growth substances for
normal growth. In contrast to what was expected,
Fucus spiralis can not be a major donor of growth

Medium ASP 6 F2
substances in the coast water. From the brown alga
Undaria pinnatifida (Harvey) Suringar (Laminaria­
NaCl 24g ceae) Abe etal. (1972, 1974) isolated not only indole-
MgSO•. 7H20 8g 3-acetic acid (IAA) but also phenylacetic acid (PAA)
KCI 0·7g
and p-hydroxyphenylacetic acid (P-OHPAA). These
CaCI..H20 0'55 g
NaN03 0·1g two substances were known previously to give auxin
Na2-glycerophosphate 50mg effects in higher plants (Hansen, 1954; Lee & Skoog,
K2HPO. 2mg 1965). The axenic culture of Fucus spiralis now gave
KI 0·065 mg
an opportunity to investigate whether these sub­
KBr 96·9mg
stances play some role in the metabolism of brown
TRIS (hydroxy methyl) aminomethane 1g
Micronutrient solution 1 ml macroalgae.
Vitamin solution 1ml In Fig. 3 we can see how additions of p-OHPAA in
B12 ll-'g concentrations of 1O-LlO-5 M had a remarkable
Redistilled water 1000ml
influence on growth as well as on morphology. In the
control flasks only a minimal increase in the length
of the plants appeared during the experimental time,
Micronutrient solution: NTA (nitrilotriacetic while p-OHPAA induced branching and a slight
acid) 50 mg, Fe (as chloride) 1 mg, Zn (as chloride) broadening of the fronds. This experiment was run
0·25 mg, Mn (as chloride) 0'5 mg, Co 5 /Lg, Cu 10 /Lg, during December-January. The algae still keep a
B (as HaBOa) 1 mg, Mo (Na-salt) 0·25 mg per m] seasonal variation in growth for a long time even if
H20. cultivated under controlled conditions. The next
Vitamin solution: Thiamine . HCI 0·2 mg, nicotinic experiment was run during April-June and here we
acid 0·1 mg, Ca pantothenate 0'1 mg, pyridoxin can see a much better growth even in the control
HCl 0·04 mg, p-aminobenzoic acid 0·01 mg, biotin series (Fig. 4). The results obtained in the previous
0'5/Lg, thymine O·g mg, inositol 1 mg, orotic acid experiment with p-OHPAA were confirmed. Inocula
0·26 mg per m] H20. from plants with tendencies for branching were
The micronutrient solution, vitamin solution and further branched or broadened and slightly wavy
 Fries: Effects of PAA and p-OHPAA on Fliclis 453

p-OHPAA

FIG. I. An axenic plant of Fl/Cl/S spiralis, one year old, attached to the bottom of a 100ml flaskby rhizoids. The
fronds are slightly flattened.
FIG. 2. The same plant 3 months later at 16°C, 10 hr light per day. x 1·65.
FIG. 3. Gro wth regulating effects of p-hydroxyphenylacetic acid (p-OHP A A) on axenic plants of FUCl/S spiralis.
The plants are photographed from belo w when gro wing in their experimental flasks at 15°C and 15 hr light per
day; incubation time 70 days. Scale 15 mm.
=

fronds, characteristic of Fliclis spiralis, were formed. PAA and p-OHPAA have functions as growth
Additions of P AA gave more striking effects. This regulators in Fliclis spiralis.
substance was tested only at concentrations of 10-7 The values of the fresh weights of the algae in dif­
and 10-6 M. As the strongest effects were observed at ferent series support the visual observations about
the concentration 10-7 M we might expect PAA to the morphology as seen in Fig. 4. At a concentration
also show activity at lower concentrations and thus in of 10-7 M, PAA increased weight by nearly 200%,
completely hormonal amounts. At the most active while p-OHPAA at the same concentration gave a
concentrations of p-OHPAA and PAA the Fliclis 100% increase. The concentration of 10-6 M of
plants had a quite natural appearance and formed p-OHPAA gave a lower fresh weight increase than
25-30 mm long fronds of normal breadth in 70 days, the higher concentration of 10-5 M. A similar
a growth quite comparable to that of bacteria­ observation was also made on Porphyra tenera
infected plants. Comparable effects have been shown (Fries & Iwasaki, 1976) and it might indicate that
in the 'conchocelis' stage of the red alga Porphyra p-OHPAA in higher concentrations plays another
tenera (Fries & Iwasaki, 1976). It is thus clear that role than that of a growth regulator.

29*
454 Phyc% gia,Vol. 16 (4),1977

�
15mm PAA p-OHPAA

Control 10-7 10-6 10-7 10-6 10-5M

.
,,
.......
� tr -> �
..,r

Vt- � '-' � (....

�
'-r

--( r
* + f\
"--
� "-
�. � �
.......
� � � .. ..-�

1 17 330 220 219 167 218

Fresh weight (mg)

FIG. 4. Gro wth regulating effects of phenylacetic acid (P A A) and p-hydroxyphenylacetic acid (p-OHP A A) at
15°C and 15 hr light per day; incubation time 70 days. Five parallels per series with one plant per flask; the
horizontal ro ws represent plants originating from the same mother plant.

In higher plants PAA and p-OHPAA have been IAA as well as the phenylacetic acids in Undaria.
identified as naturally occurring substances (Feld­ These algae were naturally growing plants and thus
man & Hanks, 1965; Okamoto et aI., 1967) and bacteria-containing. The question remains whether
Wightman & Rauthan (1975) showed the bio­ PAA and p-OHPAA are produced by bacteria epi­
chemical pathway for l4C-phenylalanine to 14C_ phytically growing on the wild Fucus plants or
phenylacetic acid in tomato shoots. In tobacco callus whether the algae are induced to produce them by the
the activity of PAA and p-OHPAA was found addi­ activity of these bacteria. An answer to this question
tive to IAA but Milborrow et al. (1975) suggest the is very important for the classification of P AA and
possibility that IAA and PAA affect different sites p-OHPAA as hormones in algae.
although in some materials they give similar effects. Further investigations are being made into the
Davidson's (1950) results with Fucus sporelings activities of PAA and p-OHPAA in combination
might indicate that IAA has an important influence with IAA and other growth regulators. Hitherto the
on the morphology too. In the sea the bacteria play a shortage of axenic inoculum material has limited all
dominating role in IAA synthesis (Schiewer, 1967) experiments.
and the production of IAA by macroalgae themselve�
has been questioned by Buggeln & Craigie (1971�.
After his experiments with Alaria (Laminariacea�) Acknowledgments
Buggeln (1976) even doubts the role of auxin as an
endogenous growth regulator in algae. However by This investigation was supported by a grant from the
more refined methods Abe et al. (1974) identified Swedish Natural Science Research Council.

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