Apolar Embryos of Fucus Resulting from Osmotic and Chemical Treatment
Author(s): John G. Torrey and Esra Galun
Source: American Journal of Botany, Vol. 57, No. 1 (Jan., 1970), pp. 111-119
Published by: Botanical Society of America
Stable URL: http://www.jstor.org/stable/2440384 .
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Amer.J. Bot. 57(1): 111-119.1970.
APOLAR EMBRYOS OF FUCUS RESULTING FROM OSMOTIC
AND CHEMICAL TREATMENT'
JOHN G. TORREY AND ESRA GALUN2
The BiologicalLaboratories,HarvardUniversity,
Cambridge,Massachusetts
A B S T R A C T
L. weregrownas populationsin glasspetridishes
alga Fucusvesiculosus
Embryosofthebrowni
forperiodsup
illumination
unilateralfluorescent
low-intensity
in seawaterat 15 C in continuous
to 2 weeks.A quantitativeestimateofincreasein nuclearnumberwas madefromacetocarmine
Overtheperiodof2-6 daysembryos
ofsamplestakenat 12-or-24hrintervals.
squashpreparations
eachembryo
showeda doublingtimeofabout12-18hr.Undernormalseawatercultureconditions
above
withsugarconcentrations
formeda singlerhizoid.Whengrownin seawatersupplemented
sphericalembryoslackingrhizoids.In 0.6 M
0.4 M, Fucus embryosdevelopedas multicellular
97% of the embryoswereapolar at 2 days; only37% wereapolar at 4 days,
sucrose-seawater,
Some embryosremainedapolaraftergrowth
manyhavingrecoveredfromthesucroseinhibition.
inhibitedthe
markedly
in 0.6 M sucrosefor2 weeks.Nuclearcountsshowedthatsucrose-seawater
D-galactoseand the sugar
rate of cell division.OthersugarsincludingD-glucose,D-fructose,
were
When apolar embryosgrownin sucrose-seawater
were also effective.
alcoholD-mannitol
to seawater,embryogrowthresumedat thenormalseawaterrate,judgedfromnuclear
returned
counts.Such embryosformedmultiplerhizoids,varyingfromtwo to eightrhizoidsper embryo,
whichdevelopedon the embryoquadrantor halfaway fromthe unilaterallight.Each of the
spherica
ofthemulticellular
froma singlesmallcellin theperiphery
multiplerhizoidsoriginated
stimulusapparentlyhad been subdividedamonga number
embryo.Thus the rhizoid-forming
of thisfindingare discussed.Attemptsto
of the cells of the apolar embryos.The implications
of embryoswithindoleaceticacid or 2,4-dichlorophenproducemultiplerhizoidsby treatment
oxyaceticacid failed.However,embryostreatedwith10-4 M O- 5 X 10-5 M 2,3,5-triiodobenzoic
thatsomechemical,perhaps
suggesting
acid formed40 and 30% multiplerhizoids,respectively,
in Fucusembryogenesis.
is involvedin polarizationand rhizoidinitiation
mechanism
hormonal,
DEVELOPMENT of embryos of protuberancein one-halfof the initiallyspherical
the brownalga Fucus has servedas a classicalsub- cell. By about 18 hrthenucleusundergoesmitosis,
ject forthe studyof the influenceof the physical formingtwo nuclei with the spindle orientedin
and chemicalenvironmenton the determination the plane ofthe protuberance.By 20-24 hra cross
of polarity. Factors influencingorientationof wall formsat right angles to the plane of the
polarity were studied in extenso by Whitaker protuberanceand two quite dissimilarcells result,
(e.g., 1931, 1936, 1938) and more recentlyby the pyramidalrhizoidcell and the hemispherical
Nakazawa (1959, 1960, 1962) and by Jaffe thallus cell. Further divisions of the rhizoid
rhizoidwhichultimately
(1968) who reviewedthis work and the related cell lead to a filamentous
of the thallus cell
Divisions
holdfast.
the
diam)
forms
(70-80,uO
large
the
nature
In
literature.
eggs are released into the seawater where they give rise to a pear-shaped multicellularyoung
are fertilizedby motile sperm. Each zygote plant. By the 6th to the 10th day, depending
settlesand adheres to the substratumby means upon illuminationand temperature,apical hairs
of a rapidly formedsticky polysaccharidewall. are formed(see Galun and Torrey,1969); these
Within about 12 hr at 15 C, depending upon hairs mark the beginningof the formationof
the physical and chemicalgradientsto which it the apical meristemof the vegetativethallus.
is exposed,the zygote develops an asymmetrical The firstdivision is asymmetricaland establishes the fate of the cell progeny.AlthoughreI Received
18July1969.
forpublication
in part by the Maria centlymuchinteresthas centeredupon thenature
was supported
This research
cell divisionin ultrastructural
Harvard of this differential
Research,
forBotanical
MoorsCabotFoundation
and in partby the PublicHealthService terms(Neushul and Liddle, 1968; see also Jaffe,
University,
ResearchgrantGM-08145to JGT.
to Mrs.JoanMillerandMr. 1968) and in chemicalterms(Petersonand Torrey,
areindebted
The authors
assistance. 1968; Quatrano, 1968), most attentionhas been
technical
Jr.,forcompetent
HarryL. Phillips,
at the Mount paid to the factorscontrollingthe initial polariwas conducted
Most of the research
SalisburyCove, zation of the fertilizedegg.
Desert Island BiologicalLaboratory,
Maine.
of Science, The initiationof the protuberance,and hence
Institute
2Permanentaddress:Weizmann
the determinationof the site of the rhizoid,is
Israel.
Rehovoth,
ASYMMETRICAL
ill
112
AMERICAN
JOURNAL OF BOTANY
IVol.57
debrisandgroupsofreceptacles
factors. removeall surface
subject to a varietyof environmental
seawaterin 10-cm
on thesideawayfromunilateral werefloatedin fresh,filtered
forms
Therhizoid
illuninated
illumination(Whitaker,1936; Whitakerand petridishesplacedina 15 C incubator
fluorescent
Lowrance,1936),blue lightbeingmosteffectivefromone side by an 8-w cool-white
(580-700nm)havingno lamp. Oogonialreleasefollowedby egg release
and longerwavelengths
effect(Hurd, 1920). Rhizoidsformtowardthe usually occurredwithin several hours; eggs
positivepole in an imposedelectricalgradient releasedmorethan12 hrwerenotusedforexperi(Lund, 1923), towardthe higherconcentrationments.Male receptacleswere placed moistin
.ofa potassiumion gradient(Bentrup,Sandan, 10-cmpetri dishes.Usually antheridialrelease
in therefrigerator
at 4 C in thedarkas
and Jaffe,1967), on the acidic side of the pH occurred
sidein a manifestby a brightorangeexudate on the
(Whitaker,
gradient
1938),onthewarmer
temperaturegradient(Lowrance,1937), and receptaclesurface.Sperm releasewas effected
receptaclescoveredwith
or a pieceofthallusrather readilyby transferring
towardotherembryos
-thanaway(theso-called"groupeffect")(Whita- such exudateinto a small volumeof filtered
was kept
these con- seawaterat 4 C. The spermsuspension
ker, 1931). Jaffe(1966) interpreted
trollingfactorsin termsof electricalgradients,in a flaskon ice untilused,usuallywithina few
ofchemical minutes
ofsuspension.
differences
relatedperhapsto internal
organization, In orderto achievea cleanpreparation
and/orultrastructural
ofeggs
,constituents
in or near the plasmamembraneor freeof oogoniaor debris,the eggswerefiltered
-especially
througha Nitexnylonfilterwitha 102,u pore
outercortexofthecell.
apolar embryos size (Tobler,Ernst,and Traber,Inc., 71 Murray
Developmentof multicellular
was observedearlierin Fucus. Farmer and Street,New York,N. Y. 10007).Spermsuspento removeantheridia.
filtered
of rhizoid sionsweresimilarly
Williams(1898) describedinhibition
in embryosculturedin seawaterat Spermand egg suspensionswere combinedin
-formation
flaskon ice,werevigorously
-densitieshigherthan normal. Kniep (1907) a 125-mlErlenrmeyer
spher- swirled,and thenwereallowedto standon ice
of multi-cellular
thedevelopment
reported
of the forabout1 hr.Although
wasachieved
fertilization
destruction
aftermechanical
ical embryos
allowed
(1931) observedoccasional veryrapidly,a delaybeforefinalfiltering
rhizoidcell.Whitaker
eggsto developa rigidwall.Filtraapolar embryosdevelopingin the centerof a thefertilized
eggpopulation
dense populationof normal embryos.Sussex tionand washingofthefertilized
(1967) producedapolar embryosin the brown at the end of 1 hr on a 35 .ANitex nylon filter
eggsto allowedthe spermand unfertilized
thefertilized
eggsto pass
by subjecting
alga Homosira
agitation.The predictablelack of throughthe 35 A pores but caughtthe rigid
-continuous
in fresh,
polarizationof developingembryosof Fucus zygoteswhichwerethenresuspended
ofa selectedvolumeandaliquots
a filtered
seawater
offers
conditions
experimental
underspecified
Most
usefultoolin the analysisof the stimuliacting were placed in dishes for experiments.
were carriedout in 5-cm Pyrex
on rhizoidinitiation.The workreportedbelow experiments
susofthenatureoftherhizoid- petridishes.Small dropsof concentrated
thequestion
,concerned
stimulusas deducedfromexperimentspensionsofzygoteswerepipettedwitha Pasteur
initiating
pipetteinto 10-mIvolumesof the experimental
withapolarembryos.
incubated
solutionsand the dishesimmediately
releaseand at 15 C in7thelightedincubator.Each dishwas
MATERIALSAND METHODS-Gamete
to the
of plants,therelease markedwith respectto its orientation
collection
fertiltzation-The
were essentially fluorescent
illuminalamp.One-sidedcontinuous
of gametes,and fertilization
of all embryos
thosedescribedby Petersonand Torrey(1968). tion caused preciseorientation
Plants of Fucus vesiculosusL. were collected withthe rhizoidgrowingaway fromthe light.
verymuch
rocksat Sea Wall, Mount Desert Island, Such orientedembryodevelopment
-from
stagesand assured
scoringembryonic
Maine, duringebbingand low tidesduringthe facilitated
of rhizoidformation
would
months.Whole plantswereplaced in thatthe asymmetry
:summer
storage be quite'imanifest
Embryoswere
to theobserver.
plasticbags,packedon ice in polystyrene
eontainers,and carriedto the laboratoryat observedat regular,usually at 24 or 48 hr,
binocular
SalisburyCove, Maine. Plantsof thisdioecious intervalsand scoredusinga dissecting
withtransmitted
lightat about 50
-specieswere separated accordingto sex by microscope
In experiments
carriedmore
samplingand examiningfree-handsectionsof X magnification.
wererenewed
solutions
receptaclesof everyplant specimen.Wholere- than3 days,experimental
ceptacleswere cut fromthe plants and male at least at 3-day intervals.Several series of
in 10-cm experiments
storedseparately
using seawaterwith or without
andfemalereceptacles
glass petriplatesat 4 C in the darkfora mini- sucrosewereconductedin the presenceof an
mum of 24 hr but not longerthan about 7 added antibioticmixture(25,ug streptomycin
per ml). The presence
plus 50 unitsof penicillin
days.
did not changethe observed
werewashed of theseantibiotics
To obtaineggs,femalereceptacles
behavior.
seawaterin a funnelto developmental
in Millipore-filtered
January,
1970]
TORREY AND GALUN-APOLAR
EMBRYOS OF FUCUS
113
Cytologicalpreparations-Fertilized eggs ad- over an alcohol lamp flame for a few seconds,
hered to glass withina few hours afterfertiliza- avoiding boiling, and allowed to stand. The
tion and remained attached, especially at the preparation was then gently resquashed with
rhizoid end of the embryo,as developmentcon- filterpaper taking up the excess stain. In some
tinued. This feature served a useful purpose preparationsthis proceduregave good nuclear
since it allowed petri plates to be emptied and stainingwith relativelylight cytoplasmicstainreplenishedsimplyby pouringoffsolutionswith- ing. The nuclear stain could be intensifiedwith
out loss of the plants. Also unfertilizedeggs, a dropofverydiluteirontartratesolutionwhich,
withtime to produce
spermand debriswerereadilywashed away. This however,acts progressively
same featurewas used forcytologicalpreparations. overstaining. Most cell walls stained poorly
Fertilized eggs were pipetted onto albumen- but could be discernedby theirexclusionof stain.
coated 22-mm-squarecover slips attached to The rhizoidcell and its adherentpolysaccharide
the bottomof 10-cmpetriplates (six per dish). On stained dark red. Also in apical-hairstage emeach cover slip was placed a 20-mm-diamglass bryos an internal skeleton-likewall component
van Tieghemring to restrictthe embryosto the took up the carminestain. Nuclear counts were
cover slip. When the dish was filledwith sea- usually made on temporarymounts. However,
water or the experimentalsolution,all embryos in some cases, air-driedslidesweremade permawerewashed with a commonsolution. The glass nentsimplyby poppingoffthe coverslip,drying,
ringscould be removedat about 12 hr when the and mountingin Permount.
embryos were well affixedto the cover slips.
of Fucus emREsuLTs-Normal development
The albumen coat facilitated later squashing
proceduresand was not essentialforadherenceof bryosin seawaterat 15 C-In Fig. 1 is presentedin
terms of the number of nuclei per embryothe
the embryos.
For cytologicalpreparationsthe coverslip was time course for normal developmentof Fucus
simply picked up from the culture dish with embryosgrownin seawaterat 15 C in continuous
unilateralwhite light. In Fig. 2-7
stainless-steelforcepsand transferred
to a small low-intensity
screw-capbottlecontaininga smallvolumeof10 % are illustratedsquash preparationsof embryos
formalinin seawater at room temperature.The
embryos could be stored indefinitelyin this
fixingsolutionbut wereheld at least 24 hr before
100
furtherprocessing.
Detailed quantitative informationabout the
course of cellular events during embryogenesis
necessitated our developmentof a method of
80 _
accurate nuclear or cell counting,based on a
large sample size. We developed the following 0
squash techniquemodifiedfromproceduresdes- co
SEA WATER
cribed by Roberts (1966). Embryoswere carried W
CONTROL
throughall the proceduresadheringto the cover slip. Checks were made after each step to see a- 60
that treatmenthad not removedthe embryos.
After24-hrfixationin 10% formalin-seawater,
the embryoswere given at least three changes
of tap water duringone hour. The embryoswere z 40
transferredto 6 % Na2CO3 solution in distilled 0LL
waterand warmedgentlyon a hot plate; boiling
was avoided. Higher heat treatmentcould be
tolerated by the young embryos than by the
Z20
older embryos which macerated very readily
(Fig. 12). After15 min in Na2CO3 solution,the
TRANSFERRED
embryos were given at least three tap water
.TO SEA WATER
/
f
rinsesduringat least 15 min.
o.6 M SUCROSE
The cover slip was then placed embryo-side
I
I
I .__
I -J
down on a clean glass slide and the cover slip
4
5
6
7
3
2
1
gentlypressed to squash the embryosin a drop
of tap water. The amount of squashingrequired
TIME IN DAYS
depended upon the age of the embryosand the
Fig. 1. Developmentof Fucus embryosculturedin
of the carbonate maceration.After continuouslow-intensity
effectiveness
lightat 15 C in seawateror in
squashing, drops of acetocarminesolution (2 g 0.6 M sucroseplus seawaterfor4 days thentransferred
to
carminein 100 ml 45 % acetic acid) were allowed seawater.Developmentis expressedas numberof nuclei
to seep under the cover slip reaching all the perembryoand eachpointis based on the mean of 25-30,
embryos. Then the slide was heated gently embryos.Fertilizationoccurredat day 0.
1,14
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January,1970]
TORREY AND GALUN-APOLAR
EMBRYOS OF FUCUS
115
.showingsome of these stages. Note that orien- TABLE 1. Effectof seawatersupplementation
withsugars
or sugaralcoholsof different
concentrations
on polaritation of the embryos was disturbed in these
zationof Fucus embryos
preparationsby the squash procedure. Polarization of the embryodeveloped between12 and
Numberof
%
-20hr afterfertilization(Fig. 2, 3) and by 24 hr
embryos
counted polarized
mitosisand cell divisionhad occurred.By 48 hr
concentration
a mean value of 6.5 nuclei per embryo was Sugar
in seawatera
48 hrb 96 hr 48 hr 96 hr
observed. In some series accelerated rateswere
,observed;for example,in one set a mean value o sucrose
130
124
100
100
fornuclearnumberof 3.9 at 36 hr and 8.5 at 48 0.45 M
100
125
51
86
hr was recorded(Fig. 4, 5). With a cell doubling 0.5 M
136
132
32
80
time of approximately12-18 hr between day 2 0.55Mm
135
157
24
84
and day 6, mean nuclear number increased to 0.6Mm
123
116
3
63
133
153
7
35
10.6 at 60 hr, 20.7 at 72 hr, and 42.1 at 96 hr 0.65M "
(Fig. 6, 7). The rhizoid cell which initially
153
154
44
77
represented almost one-half of the dividing 0.4 M D-mannitol
"
0.5M
144
5
118
23
zygote,divided again parallel to the initialplane 0.6 M
102
1
134
11
of division(Fig. 4); thesecellselongatedmark-edly
.and divided again to forma single long rhizoid 0.6 M D-galactose
216
75
made up of six to eightlargeelongatecells almost 0.6 M D-glucose
162
68
devoid of chloroplastsand quite poor in cellular 0.6 M D-fructose
239
88
organelles(Fig. 7). These rhizoid cells produced
a thick outer layer of sticky polysaccharide a Embryoswereculturedin 10 ml of mediumin 5-cm
glass petridishesat 15 C in continuousunilateralwhite
which cementsthe embryoto its substratum.
light.
The thallus cell of the two-celled embryo fluorescent
b Embryoswere transferred
to freshsolutionsof the
proceededto cleave into many small cells as the
at 48 hr.
thallusbodyincreasedslowlyin size (cf.Fig. 2, 6). same constitution
The pattern of cell divisions was not precise.
No multinucleatecells wereobservedduringthis seawater,with increasedNaCl content,and with
study, and the nuclear counts representcell sugaradditionsto seawaterweredone in collaboranumbers.By the 6th to the 7th day, the firstsign tion with MargaretMcCully, whose help on the
withsucroseis acknowledged.
was the developmentof a earlyexperiments
of apical hairformation
small papilla on the thallus body approximately In Table 1 are presentedin summaryforn the
180? fromthe rhizoid end of the embryo.This results of experimentsshowing the effectsof
hair developed by intercalarygrowthfrom in- sugar concentration in seawater on embryo
ternal cells and usually more than one hair polarization. Embryos were transferredto the
developed (Galun and Torrey,1969). Apical hair experimentalconditions1 hr after fertilization
was precededby internaldifferentiationand were culturedin 10 ml of mediumin 5-cm
formation
of cells (Nienburg,1931). Such internaldifferen- glass petri plates at 15 C in continuouslowintensityunilateralwhite fluorescent
light.Ran-tiationwas not apparent at 4 days (Fig. 7).
dom counts of two dishes from each treatment
The formationof apolar embryosin sucrose- were made at 48 and 96 hr of continuousexpo-seawater-Earlyin our studies it was observed sure to the treatmentsolutionsafterfertilization.
that Fucus embryos cultured in small-volume In the seawater controlsall embryosshowed
dishes (e.g., Bureau of Plant Industry dishes polarized developmentat 48 hr and continued
whichhold 1 ml) unpredictablyproducedmulti- to develop thereafterin a polarized fashion as
cellular spherical embryos completely lacking already described. In all the sugar solutions
rhizoids.A searchof the literaturesuggestedthat testedat 0.4 M or highersome embryosdeveloped
osmotic conditionsinfluenceembryogenesisand which were apolar. In 0.6 M sucrosein seawater
led us to test the effectof increasingthe osmotic 97 % of the embryosshowed apolar development
pressureof the seawateron embryodevelopment. at 48 hr (Fig. 13). In 0.6 M mannitolonly 1 % of
withconcentrated the embryoswere polarized. Thus, these high
experiments
These preliminary
"?
=
fixedin
lightat 15 C for variousperiodsoftimeafterfertilization,
Fig. 2-12. EmbryosofFucusculturedin continuous
treated withsodiumcarbonatesolution,squashed and stainedwithacetocarmine.All X 180
10% formalin-seawater,
exceptFig. 12, X 245.-Fig. 2. Embryosin seawater12 hr.-Fig. 3. Embryosin seawater24 hr.-Fig. 4. Embryosin
seawater36 hr.-Fig. 5. Embryosin seawater48 hr.-Fig. 6. Embryosin seawater72 hr.-Fig. 7. Embryosin seawater
36 hr.-Fig. 9. Embryosin 0.6 M sucrose-sea,96hr. Note singlerbizoids.-Fig. 8. Embryosin 0.6 M sucrose-seawater
to seawater2 days.Note multiple
4 days; thentransferred
water48 hr.-Fig. 10. Embryosin 0.6 M sucrose-seawater
to seawater4 days.-Fig. 12. Fucus em4 days; thentransferred
-rhizoids.-Fig.11. Embryosin 0.6 M sucrose-seawater
possiblewiththesodiumcarbonatetreatment.
bryosin seawater4 days,thenfixedand squashed,showingthemaceration
116
AMERICAN
[Vol.57
JOURNAL OF BOTANY
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
7~~~~~~~~~~.7
-........
... ........
K
_
:
:
z
0...
...
- .:..
*-
A
surs-sewtr
tiei0.M
.....
i:/ .t
Al X 20:
0.6 M sucroseseawaterfor4 das
2 d
.
18.
.?:e
,
. ....................
.. ..
,,:
,'1..7X,;'~-8
B'ss_
.......... _.si.
.;.
;--'_.:.........
..
i
i
5Eby
_r *
nq,
19a
3
mroi
n0
: ],J.
0.6 M surs
:1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
....
:.;
]i.
.s,
.....
....
,:
sewae
surs-saae
o
mont
s in
of Fucus fixed
0.6M
4ater
sucrosei-seawater
embryo
fo
......
...
..
..
.
....
-s
.
2v'P.,-14.,Embry in.
........
for2.days Fi.
ay 16
.
mroi
4 das.-Fig.
folt.3r
in 0.6 m sucrose-seawaer for 4 days then transferredto seawater for 6 days.
Note beginningof apical hair formation.
concentrationsof sugars added to the seawater
dramaticallychangedthe developmentalpattern,
almost completelysuppressingrhizoidformation.
With time, an increasing proportionof the
populationrecoveredfromthe osmoticinhibition
of rhizoid formation.Thus, in 0.6 M sucrose in
seawater only 37 % of the embryos were still
apolar after 4 days. These embryos were enlarged, multicellularspheres (Fig. 14). If maintained in high-sucroseseawater a proportionof
the embryos continued to grow as spherical
embryos, showing progressiveincrease in cell
number and in total volume but showing no
polarization and no rhizoid initiation (Fig. 15,
16). Similar responses were observed in treatmentsof embryoswith othersugars at the same
concentration. In the high concentration of
mannitol, what were apparently toxic effects
interferedwith embryodevelopment;hence this
reagent was not useful for furtherexperiments.
,,.,,.
.
19. mro.o
ig. 20. Embryo treated as in Fig. 19.
Since mannitolis metabolizedby Fucus (Bidwell
and Ghosh, 1962), its uptake at these high
concentrationsmay have "swamped" normal
metabolicprocesses,causing inhibitionand toxicity.
For subsequent experiments,sucrose at 0.6 M
in seawater was selected. At this concentration
sucrosedelays cell division.In Fig. 1 is shownthe
time course of an experimentin which embryos
were culturedin 0.6 M sucrose in seawater for 4
days and thenwere transferred
to freshseawater
alone. During the first36 hr in sucrose-seawater
no cell divisionwas seen (Fig. 8). M\litosis
occurred
at about 48 hr (Fig. 9). By 4 days the mean
nuclear numberwas 11.2 as comparedto 42 for
seawatercontrols.
Release of the sucrose-inducedinhibitionby
transferof the embryos to seawater led to a
rapid restoration of the nornal rate of cell
division as is apparent in the curve in Fig. 1..
i
0
January,1970]
TORREY
AND GALUN-APOLAR
EMBRYOS
117
OF FUCUS
Thus the sucrosetreatmenthad no residual In thehighestTIBA concentrations
(10-4and
effects.The most strikingeffectof 5 X 10-5M in seawater)embryoswithmultiple
deleterious
sucrosetreatmentwas that when normalcell rhizoidswere formedin good numbers,i.e.,
the apolarembryos about 40 and 30%, respectively,
divisionrateswereresumed,
after6 days
,showedpolarizationand multiplerhizoidinitia- (Table 2). Thesenumbers
werelowerthanthose
tionon the side of the embryoawayfromlight. producedby thesucrosetreatment,
but in other
theembryos
After2 days in seawatertwo or morerhizoids respects
werecomparable.
At 2 days
wereobserved;by 6 days up to
wereevident,each developingfroma separate apolarembryos
sphere(Fig. 10, fiverhizoidsper embryowereobservedin some
cellofthemulticellular
peripheral
arosefromadjacentor casesof TIBA treatment,
17). Usually,theserhizoids
althoughthemajority
cells, withinone quadrantof the of multirhizoid
neighboring
embryosformedtwo or three
.embryo.As many as seven or eightseparate rhizoids.
rhizoidswereobservedin someembryosarising It is evidentthat TIBA at a concentration
cellsof of 10-4M had an inhibitory
fromsevenor eightseparateperipheral
effect
on celldivision
in the averagenumberof nucleiper
thesphericalembryo(Fig. 11, 19, 20). Thus the as reflected
whichin the uni- embryo.Here again the effectwas less marked
stimulusto rhizoidinitiation,
to one of than that causedby 0.6 M sucrosein seawater.
distributed
zygoteis normally
-cellular
the two cells formedat the firstdivision,was The strikingfact is that with relativelylow
duringthe periodof sucrosetreat- concentrations
of TIBA (10-4M) we were able
,distributed
amongseveralor manycellsoftheembryo. to reproducea responseelicitedby veryhigh
-ment
Mul- concentrations
(0.6M) ofanother
expressed.
Onlylaterwas rhizoidinitiation
substance
which
acts by osmoticmeansratherthan
tiple rhizoidsoriginatedand developedsimul- presumably
a complexembryo(Fig. 11, by influencing
eventsat metabolicor hormonal
forming
-taneously,
its levels.
approximately
followed
17-20).Hairformation
-normaltime courseand typicallyoccurredat
the normalsite 1800 removedfromthe position DIscussIoN-Normal developmentof Fucus
embryosproceedswith remarkableprecision.
of the rhizoids(Fig. 20).
Undercontrolled
environmental
conditions,
even
Attemptsto initiate multipolar embryosby 6-day-oldembryos
showconsiderable
synchrony
hormonetreatments-Davidson (1950) reported in theirchanging
morphology
and anatomy.Up
-thatembryosof Fucus culturedin seawater
of auxins TABLE 2. The effect
concentrations
containingdifferent
of2,3,5-triiodobenzoic
acid (TIBA) on
acid or
-suchas indoleaceticacid, indolebutyric
increasein averagenuclearnumberper embryoand
theinitiationof multiplerhizoidsin Fucus embryos
naphthaleneacetic acid rangingfrom106 to
-10 M, werestimulated
to formmanyrhizoids
Hr afterfertilization
on each embryo.No mentionwas made of an
apolar stage in this development;however,only
Treatmenta
24
48
96b
144
one observation was made which was at the
end of the experiment40 days afterfertilization.
Average
ofnucleiperembryoo
As auxinswere invokedby Davidson as probably Seawatercontrol number
1.7
6.6
33.8
92.5
rhizoidinitiation,we wereinterestedto 0.6 M sucrosein
controlling
test these observations.
seawater
1.0
1.9
11.2
58.1
Fertilized Fucus eggs were transferredafter TIBA at 10-4 Min
seawater
1.0
3.6
18.8
filtrationand washing into solutions of auxins
70.6
preparedin seawaterin 5-cmpetriplates. Several
Percentoftotalnumber
ofembryos
with
hundredembryosper dish were set up in triplimultiple
rhizoidsd
of
ml
medium.
10
cate, each dish containing
Seawater control
4%
4%
Tests were made of 2,4-dichlorophenoxyacetic0.6 Msucrosein
.acid (2,4-D) at 10-5 and 10-6 M; of indole-3- seawater
60%
62%
aceticacid (IAA) at 10-5, 10-6, 10-7, 10-8 and
(apolar)
acid (TIBA) at TIBA at 10-4M in
10-9 M, and 2,3,5-triiodobenzoic
seawater
0.1, 0.5, 1, 5, 10, 50 mg/liter(2 X 10-7M to
38%
42%
104 M). Embryos were grown in continuous TIBA at 5lX 10* M
with
unilaterallightat 15 C. In the experiment
IAA, a duplicateset was run in total darknessat
15C.
with2,4-D and IAA the
In the experiments
singlerhizoidsand weredeembryosall formed
from the seaindistinguishable
velopmentally
Therewas no indicationovera
watercontrols.
had inperiodof 12 days that auxintreatment
thenumberofrhizoidsformed.
fluenced
in seawater
-
18%
29%
a Embryos
wereculturedat 15 C in continuous
unilateral
whitefluorescent
light.
b All embryoswere transferred
after96 hr to fresh
filtered
seawater.
"Nuclear counts are based on 25 embryosfor each
time.
treatment
d Percents formingmultiple rhizoids are based
on
randomcountsof > 200 embryos
for"
eachtreatment
time.
118
AMERICAN
JOURNAL OF BOTANY
[Vol. 57'
until4 days, nuclearcountsin any given popula- sumablysucrosewas absorbedand as the intemal
tionshowonlyslightvariability.Thus, embryonic and external osmotic pressure became more
development in Fucus is an excellent system nearly equalized, the localized softeningbecame
for quantitativestudies. The embryoalso has a apparentthroughnormalrhizoidformation.The
in susceptibility
of embryosto sucrose
plasticity in its developmental pattern which difference
inhibitionof embryodevelopmentis not underallows experimentalmanipulation.
Inhibitionof protuberanceformationand sub- stood. At presentwe have no information
about
sequent rhizoid formationrepresenta dramatic the fate of the sucroseprovidedin the external
upset in the normal well-ordereddevelopmental solution.
The moststrikingand potentiallymostinformaprogram.The sphericalembryoswhich formare
reminiscentof globular-stageembryos in some tive observationin these experimentsis the fact
dicotyledonous plants. Osmotic inhibition of that sucrose-inhibitedapolar embryos formed
since effective multiple rhizoids when returned to seawater.
rhizoid formationis non-specific,
inhibitioncan be achieved with a number of Rhizoid initiationoccurredamong a numberof
osmoticagents. Even seawater concen- peripheralcells of these multicellularspherical
different
trated above its usual osmoticpressureprevents embryosso that several rhizoidsdeveloped.This
on how to
rhizoidformation.Sucrose appears to be particu- responsemeans that the information
larlyusefulforexperimentalworksince it has no formrhizoidcellshad been distributedto a whole
Tulecke (1957) reported series of cells in a portionof the multicellular
deleteriousaftereffects.
that high sucrose concentrationscaused Ginkgo embryo.This responseis no longerthe definitive
cell divisiondetermining
male gametophytes to develop as spherical unequal or asymmetrical
structures.A similareffectof sucrosetreatment cell fateswhichone observesin thefirstdivisionin
Rather, several to
was reportedby Valanne (1966) in apolar embryo normalFucus embryogenesis.
productionin mosses.These effectsof sucroseare many cells on one side of the multicellularemin some respects reminiscentof the effectsof bryoare informedby the environmental
stimulus
high sucrose concentrationon isolated embryo of unilateralilluminationto dividein such a way
culturesof Capsella describedby Raghavan and that each will forma rhizoid.Thus the rhizoidTorrey (1963) in which osmotic conditionsde- stimulushas become dividedamonga numberof
terminedwhethernormal or abnormal embry- cells.
In accountingfor the polarizationmechanism
onic developmentoccurred.
Nakazawa (1959) has postulatedthe following which results in rhizoid initiation, one must
sequence of events leading to rhizoidinitiation: now take into accountthis phenomenonthat the
stimuluscan be subdividedand
a localized area of increased surface energy rhizoid-initiation
develops in the corticallayer of the cell as the expressedin a numberof cells of the multicellustimulussuch lar embryo. Careful analyses of the timing of
resultof an externalenvironmental
as unilaterallight;an accumulationoccurslocally multiple rhizoid orientation and initiation in
embryosmay make it possible
of a surface-activeagent and potential mem- sucrose-inhibited
branecomponentsuch as lecithin;thisresultsin a to clarifythe nature of these rhizoid-forming
ofmembranepermeability, differences.
localizedenhancementa lowered surfacetension,and then a localized
That auxin of the IAA-type is the chemical
bulging leading to rhizoid formation.
basis for rhizoid initiationseems unlikelyfrom
Protuberanceformationprobablyresultsfrom the experimentsdescribedhere. No multirhizoid
a localized softeningof the cell wall of the zygote embryoswere formedin any of the experiments
and a distentioncaused by internal pressures. with exogenouslysupplied IAA or 2,4-D. One
Presumably concentratedsolutions outside the can surmise that Davidson (1950) probably
cell prevent protuberanceformationeither by observed multirhizoidformationin Fucus emprocess,i.e., inhibitionof bryosarisingfromosmoticeffectsof evaporating
preventingthesoftening
a chemicalprocess,or by preventingthe expres- seawatersolutionsin his small-volume,long-term
sion of the internalpressure,i.e., by a physical experiments.
process. In the experimentsreportedhere, both
Yet one can elicit multirhizoidformationby
osmotic inhibition by concentratedsugar-sea- treatmentwith TIBA at near-hormonallevels;
water solutionsand chemicalinhibitionby TIBA these concentrationsare equivalent to those
treatment resulted in apolar embryos. In the effectivein many higher plant experiments.
formercase cell divisionwas markedlyinhibited; How TIBA acts in Fucus is not known. It is
in the latter case only a-slightdelay in the rate not reasonableto interpretthese resultsin terms
of cell division was noted. It would seem that of blockingauxin transportbetweencells,as has
inhibitionof cell divisiondid not play a critical been shown in higherplants (Keitt and Baker,
role in producingapolar embryos.
1966). However, TIBA has been reported to
In all these experimentsonly a portionof the act directlyon individualcells in some cases as,
population responded to the treatment. With forexample,on the growthof root hairs (Gorter,
sucrosetreatmentsa good proportionof the em- 1949), where the mechanism-remainsunknQwn.
bryos recovered from initial inhibition. Pre- Perhaps the TIBA is acting in Fucus as a sulf-
January,
1970]
TORREY
AND GALUN-APOLAR
EMBRYOS
OF FUCUS
119
S. 1959. General mechanism of the
hydryl-inhibitingagent (Leopold and Price, NAKAZAWA,
in some fucoideggs. Naturpolaritydetermination
1956). That one can effect the formationof
46: 333-334.
wissenschaften
multiple rhizoids by such chemical treatment
-.
1960. Nature of the protoplasmicpolarity.
encouragesone to believe that a directattack on
Protoplasma52: 274-294.
-the nature of the chemical stimulus initiating
. 1962. Polarity,p. 653-661. In R. A. Lewin
rhizoidsin Fucus embryosshould be possible.
ofalgae.Academic
and biochemistry
[ed.],Physiology
Press, New York.
1968. A light- and
M., AND L. LIDDLE.
studyof primaryheterogeneity
electron-microscopic
BENTRUP, F., T. SANDAN, AND L. JAFFE. 1967. Inin the eggs of two brownalgae. Amer.J. Bot. 55:
ductionof polarityin Fucus eggs by potassiumion
1068-1073.
gradients.Protoplasma64: 254-266.
der Keimlinge
BIDWELL, R. G. S., AND N. R. GHOSH. 1962. Photo- NIENBURG, W. 1931. Die Entwicklung
von Fucus vesiculosusund ihre Bedeutungfuirdie
synthesisand metabolismin marinealgae. IV. The
Phylogenieder Phaeophyceen.Wiss. Meersunters.,
fate of C14-mannitol
in Fucusvesiculosus.
Can. J.Bot.
Kiel, N. F. 21: 49-63.
40: 803-807.
1968. Amino
DAVIDSON, F. F. 1950. The effectsof auxins on the PETERSON, D. M., AND J. G. TORREY.
acid incorporationin developingFucus embryos.
growthofmarinealgae. Amer.J. Bot. 37: 502-510.
Plant Physiol.43: 941-947.
FARMER, J. B., AND J. L. WILLIAMS. 1898. Contributions to our knowledgeof the Fucaceae: theirlife- QUATRANO, R. S. 1968. Rhizoid formationin Fucus
zygotes: dependence on protein and ribonucleic
historyand cytology.
Phil.Trans.Roy.Soc. (London)
Science162: 468-470.
acid syntheses.
190B: 623-645.
GALUN, E., AND J. G. TORREY. 1969. Initiationl and RAGHAVAN, V., AND J. G. TORREY. 1963. Growthand
of globular and older embryosof
morphogenesis
suppression
ofapicalhairsofFucusembryos.
Develop.
Capsellain culture.Amer.J. Bot. 50: 540-551.
Biol. 19: 447-459.
GORTER, C. J. 1949. Action of 2,3,5-triiodobenzoicROBERTS, M. 1966. The Phaeophyceae,p. 149-167.
acid on growthof roothairs.Nature164: 800.
of the
In M. B. E. Godward[ed.],The chromosomes
HURD, A. M. 1920. Effectofunilateralmonochromatic
algae. Edward Arnold,Ltd., London.
lightand grouporientation
on thepolarityof germi- SUSSEX, I. M. 1967. Polar growthof Homosirabanksii
natingFucus spores.Bot. Gaz. 70: 25-50.
zygotesin shakeculture.Amer.J. Bot. 54: 505-510.
JAFFE, L. F. 1966. Electrical currentsthroughthe TULECKE, W. 1957. The pollen of Ginkgobiloba: In
developingFucus eggs.Proc. Nat. Acad. Sci. U.S.A.
vitro cultureand tissue formation.Amer.J. Bot.
56: 1102-1109.
44: 602-608.
. 1968. Localization in the developingFucus VALANNE, N. 1966. The germination
phases of moss
egg and the generalrole of localizingcurrents,p.
sporesand theircontrolby light.Ann.Bot. Fennici
295-326. In M. Abercrombie[ed.], Advances in
3: 1-60.
morphogenesis.
AcademicPress, New York.
WHITAKER, D. M. 1931. Some observationson the
KEITT, G. W., JR., AND R. A. BAKER. 1966. Auxin
in the
eggs of Fucus and upontheirmutualinfluence
benzoicacids and theireffect
activityof substituted
of the developmental axis. Biol. Bull.
determination
on polar auxin transport.Plant Physiol.41: 156161: 294-308.
1569.
1936. The effectof whitelightupon the rate
KNIEP, H. 1907. Beitrage zur Keimungs-physiologie
and the
of the rhizoidprotuberance
of
development
undBiologievonFucus.Jahrb.Wiss.Bot.44:635-724.
firstcell divisionin Fucus furcatus.Biol. Bull. 70:
LEOPOLD, A. C., AND C. A. PRICE. 1956. The influence
100-107.
of growthsubstancesupon sulfhydryl
compounds,
ion concentration
ofhydrogen
1938. The effect
p. 271-283.In R. L. Wain and F. Wightman[ed.],
upon the inductionof polarityin Fucus eggs. III.
The chemistry
and mode of actionof plant growth
J. Gen.
Gradientsof hydrogenion concentration.
Butterworth
and Co., London.
substances.
Physiol.21: 833-845.
E. W. 1937. Effectof temperature
LOWRANCE,
gradi1936. On the periodof
, AND E. W. LOWRANCE.
entsuponpolarityin eggs of Fucusfurcatus.J. Cell -in the eggs of Fucus furcatuswhen
susceptibility
Comp. Physiol.10: 321-337.
polarityis inducedby briefexposureto directedwhite
LUND, E. J. 1923. Electricalcontroloforganicpolarity
light.J. Cell Comp. Physiol.7: 414-424.
in theeggofFucus. Bot. Gaz. 76: 288-301.
LITERATURE
CITED
NEUSHUL,