TER VERKRIJGING VAN DEN GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE AAN
DE RIJKS-UNIVERSITEIT TE UTRECHT, OP GE-
ZAG VAN DEN RECTOR-MAGNIFICUS Dr. TH.
M. VAN LEEUWEN, HOOGLEERAAR IN DE
FACULTEIT DER GENEESKUNDE, VOLGENS
BESLUIT VAN DEN SENAAT DER UNIVERSITEIT
TEGEN DE BEDENKINGEN VAN DE FACULTEIT
DER WIS- EN NATUURKUNDE TE VERDEDIGEN
OP MAANDAG 20 MAART 1939. DES NAMIDDAGS
TE 4 UUR

Gaarne maak ik van de mij hier geboden gelegenheid gebruik,
om mijn welgemeende dank te betuigen aan allen, die medegewerkt
hebben aan mijn wetenschappelijke vorming.

In de eerste plaats denk ik hierbij aan U, Hooggeleerde
Koningsberger, Hooggeachte Promotor. De jaren, bij U
als assistent doorgebracht, zullen bij mij steeds in dankbare her-
innering blijven. Naast Uw wetenschappelijke leiding, heb ik Uw
medeleven ten zeerste gewaardeerd. Ook dank ik Mevrouw Ko-
ningsberger en U voor de gastvrijheid, bij U ondervonden.

Hooggeleerde Jordan, eveneens reken ik het mij tot een
voorrecht, gedurende eenige jaren bij U als assistent werkzaam te
zijn geweest. Deze tijd heeft mijn inzicht in algemeen biologische
vraagstukken zeer verdiept. Mevrouw Jordan en U zal ik steeds
erkentehjk bUjven voor Uw gastvrijheid.

Wijlen mijn leermeesters Went en Nierstrasz zullen
in mijn dankbare herinnering bUjven voortleven.

Hooggeleerde W e s t e r d ij k. Hooggeleerde P u 11 e, U zeg
ik dank voor Uw leerzame practica en colleges.

Zeergeleerde M i 1 a t z. Zeergeleerde Ter H o r s t, ik ben
U ten zeerste verphcht voor Uw hulp op physisch gebied.

Verder richt ik mijn dank tot allen, die op de een of andere wijze
aan het tot stand komen van dit proefschrift hebben bijgedragen.

Mijn mede assistenten ben ik ten zeerste dankbaar voor de pret-
tige samenwerking.

Het personeel van het Botanisch Laboratorium dank ik voor de
groote hulpvaardigheid, die ik steeds van hen mocht ondervinden.

Ten slotte rest mij nog een woord van dank te brengen aan U,
waarde A. de Boute r, voor de keurige verzorging van het
teekenwerk.

The aim of this research was to investigate, to which extent the
problem of polarity can be elucidated by studying the variations
of bio-electric potentials during regeneration processes.

To that purpose we had in the first place to try to get clear on
the source or sources of bio-electric P.D.'s in plants. Opinions
concerning their origin are rather divergent. In literature these
potentials are indicated as redox-potentials as well as concentration-
potentials while other, less defined possibilities have been discussed
too. This divergency of views may be caused partly by contradictory
experimental results, partly by the use of different electrode
systems.

The first part of this paper only will deal with the nature of the
potentials. Not until their principles are known it will be possible
to valuate the importance of the bio-electricity with regard to plant
physiology.

The next mootpoint is, whether the P.D. is cause or effect of
the vital processes concerned. With this we touch the question of
electrophoresis. Are the P.D.'s large enough to cause a migration
of electrolytes? In that case the possibiUty of their formative influence
might not be ruled out. If, however, the potentials only are by-
products of the vital processes, they merely can be considered as
indicators of functions and conditions of the cells.

We know two kinds of potentials: action currents (belter action
potentials) and rest potentials. The potentials studied here are rest
potentials.

The problem of polarity has been studied in several manners.
So has been controlled: the influence of gravity, centrifugal force,
light, humidity, assimilation, transport of assimilates, auxin and
so on. It seems interesting to look after a possible relation between
the regeneration processes and variations of the P.D. If this con-
nection exists it might be possible to reveal something more about
the internal alterations of the cells during the period of regeneration.

The problem of bio-electric phenomena was studied already in
the end of the past century. In confining ourselves to^ the most
important papers of this period, we cite first K u n k e 1 's research
(1878). This author was of opinion that the water transport must
be considered as the cause of plant electricity. Haake (1892),
however, showed that alterations in transpiration are not correlated
with variations of the electric currents. By changing the intensity
of transpiration, fluctuations in the current occurred indeed, but
no conformity could be obtained as to the sense or as to the magnitude
of the variations of both phenomena. Rejecting K u n k e 1 's view
on plant-electricity as capillar-electricity Haake considered
respiration and assimilation the source of the currents. Increase of
the Oa-pressure caused increase of the currents independently of
its direction. In 100 % Ng the amperage did not decrease down to
zero. This was explained as a result of intramolecular respiration.
Influence of COg-pressure could not be detected satisfactorily, but by
darkening the objects, the amperage was diminished. So Haake
concluded that assimilation might play a part as well as respiration.
It should be mentioned here that Haake was measuring currents.
His instruments did not enable him to measure potentials.

More than 30 years passed until a more detailed research was
set up. Waller (1925, 1929) studied the effect of light and COg.
From the fact that illumination of a chlorophyll containing part
(leaf) causes a photoelectric reaction Waller considered this
reaction quantitatively related to the COg-percentage of the sur-
rounding air, and concluded that the currents were a product of
the oxidation and reduction of a hypothetical acid. It is not clear
why the effect was only short-lasting. As a rule exposure to light
caused a positive wave, darkening a negative one but the reverse
occurred also. So it seems to be a more complicated phenomenon.

The relation between metabolism and bio-electric currents has
been studied profoundly by Lund and his co-workers. In a
series of five papers (Lund and K e n y o n 1927, Lund
1928a and b. Marsh 1928, Lund 1928c) it was stated that
the electric polarity shows a quantitative relation to the metabolism

of the cells. The parts in the roots of Allium Cepa which show the
highest P.D.'s also are the parts of the most intensive methylen
blue reduction and discoloration of phenol red. KCN and ether
reduce the respiration and with it the potential differences. ^^

According to Lund, the bio-electric potentials are due to the
flux equilibrium of the oxidation-reduction systemsquot;. With flux
equihbrium is meant the dynamic balance between oxidation and
reduction and so the P.D. is fluctuating continuously.

The P.D. of a tissue is considered as the algebraic sum of the
P D's of the individual cells, which are connected to each other
partly in series, partly parallel. This is stated once more by Lund
and Bush (1930) and by Miss R o s e n e (i935)- . ^ . ,
Rosene and Lund (1935) suggest that the electric, functional
and morphological polarities are coupled. In the roots ot Allium
Cepa the maxima of P.D., O^-consumption, methylen blue reduction,
concentration of sulphydrilcomponents, COa-production and growth
(undifferentiated tissue) are found to coincide. The authors empha-
size that the metabohsm is rtot the only factor to which potentials
are due in cells, quot;but it (the flux equihbrium) is the only type thus
far considered in the literature of electro-physiology which can
mantain in a direct manner a continuous output of electric energy .

The theory of Lund is supported by many experiments by
Marsh. According to Marsh (1930a) the recovery curves after
an applied current are due to a redox system. Ether and
reduce the potential of Valonia. In a discussion Marsh (1930b)
agrees with the idea of Lund (1927) that the recovery cannot
be explained clearly by assuming that the permeability of the
membranes, increased by a stimulus, should be restored to its normal
value by metabohsm. The hypothesis of alterations of permeability
is based on changes found in the resistance of the membranes. It
is possible, however, that the decrease of the resistance might be
due to a counter-E.M.F. If this were true the resistance cannot
be considered to be a measure for the permeability, bo Marsh
concludes once more to the flux equihbrium theory of L u n d.
The formula of Lund (1931) expressing the electric polarity

Pc being a function of the Og-pressure, is treated mathematically
by Marsh (1935, 1937) and is shown to be in accordance with

did not use noble metal electrodes. So a real redox potential cannot
be measured. Lund, however, accepts certam quot;structures (or
interfaces)quot; in the protoplasm bearing the properties of noble metal.

Marsh also studied the effect of CO^ (1935— 36) and of light
(i936-'37) on the potentials of Valonia. These results match those

° P o n d e r and M a c 1 e o d (1937)5 however, could inhibit the
respiration of the frog skin by certain carbamates and lysms without
changing its P.D. According to the authors, this needs not necessarily

Another argument for the bio-chemical character of the source
of the P.D. is furnished by experiments on the influence ot Ae
temperature on it. Marsh (i934-'35. 1936) showed that the Qio
at low temperatures amounts to about 4 and at high temperatures
to about 2. It seems difficult to me, however, to draw conclusions
from the experiments, the results being rather divergent. As can
be seen from his curves, in many cases no change at all occurred
(the quot;undershootingquot; and quot;overshootingquot; effect excepted).

Umrath (1934) studied the effect of temperature on inherent
(= rest) potentials and on action currents. The increasing phase
of the latter lasted 2—3 times longer at a sinking of the temperature
of 10° C. The Qio of the inherent potential also is an indication
that this potential too depends on a chemical process. U m r at h
(1933) interprétés the P.D. as follows: quot;Im Plasmalemma sind ober-
flächenaktive Molekeln orientiert eingelagert, welche durch eine
elektrische Polarität das Plasmalemmapotential bedingen, bezieh-
ungsweise einen Beitrag zu demselben hefernquot;. With reference to
the effect of temperature Umrath (1934) specified: Es hegt
nahe, anzunehmen, dass die Menge der orientiert in das Plasmalem-
ma eingelagerten Molekeln von Stoffwechselvorgängen abharigt,
wodurch die beobachtete Temperaturabhängigkeit des Potentials

The rate of conduction of action currents in Mimosa is increased
also two to three times by rising the temperature 10° C. (U m r a t h

^^Quite a different hypothesis is formulated by O s t e r h o u t
(1928a). The protoplasm would consist of three layers: an outer
one (X), an inner one (Y) and between them the rest of the proto-
plasm (W). X and Y are semi-permeable membranes. X is in con-
tact with the cell-wall, while Y is surrounding the vacuole. So this
system may give rise to diffusion potentials, boundary potentials
and D 0 n n a n-potentials. From a calculation Osterhout
(1930a) concluded the diffusion potentials to predominate.

As in general a more concentrated solution is negative to a more
diluted one, the conclusion seems justified that the membranes are
impermeable for anions (Osterhout and Harris 1930).
U m r a t h (1934a) described the formation of such a membrane
on the surface of his electrodes after insertion in the protoplasm of
Spirogyra and Vaucheria.

There are exceptions, however. Damon and Osterhout
(i93o)showed the more diluted solution to be negative in Valonia.
Amberson and Klein (1928) found the sign of the concen-
tration potentials across the sldn of the frog depending on the pn.
At the alcahne side of the iso-electric point of the membrane the
dilute solution is positive, at the acid side it is negative.

As to the nature of the protoplasmic surfaces Osterhout
concluded (for a survey see O s t e r h o u t 1936):

They behave as a liquid. In plasmolysis and after removal of the
cell-wall the protoplasm rounds up quot;like an oily hquidquot;.

They are non-aqueous. This is concluded from the fact that electro-
lytes enter the protoplasm as molecules chiefly. (Jacques and
Osterhout 1930, Osterhout 1933, Jacques 1936).

They are layers more than one or two molecules thick. Bases seem
to combine chemically with certain constituents of the layers.
(Osterhout 1933, 1937, Kraus 1934, Keller 1936).
Furthermore it seems doubtful whether a thickness of one or two
molecules only suffices to cause the very slow penetration of many
substances (Jacques 1936-'3 7).

They are not homogeneous. The reversible loss of the potassium
effect in distilled water is used as an argument. With potassium
effect is meant the decrease of P.D. when NaCl is substituted by
KCl. If, however, cells are placed in distilled water for some days
this effect vanishes. This fact is explained by assuming that a
certain organic compound (or compounds), R, which sensibilizes
the layers for K , diffuses away. It is possible to restore the potas-
sium effect by placing the cells in a concentrated solution of these
diffusable substances (Osterhout and Hill i933-'34gt; Hill
and Osterhout 1938 and 1938a). So in normal circumstances
the membranes would consist of at least two compounds.

The sign and the magnitude of the P.D. (when considered as
a diffusion potential) would be determined by selective pernieability.
On its turn the selective permeability would be determined by
diffusion constants and concentration gradients which depend upon
paritition coefficients. As diffusion constants and partition coefficients
vary with alterations of temperature, concentration, the presence of
electrolytes, othersubstancesandadditionalfactors,theP.D. varies too.

The temperature coefficient for penetration of substances proves
to be high in many cases. For dyes the Qio amounts to 4 or more
(Irwin 1925-26, i926-'27) while for bromide has been found
— 3(Hoagland, Hibbard and Davis 1926-27).

Organic substances can affect the P.D. to a great extent. This
is shown, for example, for guaiacol (O s t e r h o u t 1936a, i936-'375
1938a). So it is possible that metabolites in the protoplasmic surface
influence the P.D. The relation between the Og % and P.D. is not
a direct one. As to this O s t e r h o u t (1936b) remarks: quot;Redox
potentials do not enter into the picture since no metallic electrodes
come in contact with the hving cell (but oxidation and reduction
may affect the P.D. by changing the organic composition of the
protoplasmic surface or the concentration of ions in contact with it).
Redox potentials would not change with change of KCl concen-
trations as does the living cellquot;.

Blinks, D e r s i e Jr., and S k o w (i938-'39) suppose the
sensibilizing substance Rof Osterhouttobea product of
respiration. So Og influences the P.D. via the amount of this
metabolite.

The membranes X and Y are unlike to each other. This appears
when a cell is placed in vacuolar sap one electrode being apphed
outside while the other one is pierced into the vacuole. So we have
a chain: sap | X | W | Y | sap. The fact that a P.D. results indicates
that the chain is not symmetric (Osterhout and Harris
1928, Osterhout, Damon and Jacques 1928). Damon
(1930) too concluded on a dissimilarity of the membranes from the
shape of the action current in Valonia.

Osterhout and co-workers used single cells m their experi-
ments. The objects were Valonia, Halicystis and Nitella. Of course
it is of great interest to study the bio-electric phenomena in large
single cells and not only in more comphcated tissues. Since this
paper, however, deals with higher plants, only the results which
seem to be of general significance are quoted here.

Blinks studied the nature of the membranes by measuring
the resistance of single cells. The hving protoplasm offers a very
high resistance (in Nitella up to 3i megohm). An electric stimulus
reduces it temporarily (Blinks 1930). This is supposed to be
due to alteration of the permeability of the membranes. G 1 c k 1-
horn and Dedjar (1931)5 however, became negative results
on Spirogyra. S u o 1 a t h i (1937) also could not find any influence
of an electric current on the permeability of Char a.

Blinks (i935-'36a, b, c) showed that the resistance largely
is due to the development of a counter-E.M.F. caused by the flow
of the apphed current. The ohmic resistance only plays a subor-
dinate role. This quot;polarizationquot; is found at Valonia, Halicystis and
Nitella. So Blinks concluded that the protoplasmic surfaces
may exist of ions (probably of fatty acids) which can be transported
electrophoretically.

The capacity of the protoplasm might be based on two principles.
It may be caused by a (lipoid) film with none or little (in the latter
case an equal) permeability to anions and cations. Secondly it may
be caused by one or more boundaries through which one kind of
ions can pass faster than the other. Probably both exist at the same
time; the second case, however, is dominant (Blinks 1936)-

B r a u n e r (1927, 1928) described the geo-electric effect. The
lower side of a membrane always is positive to the upper side.
It is the result of the migration of the cations under influence of
gravity, while the anions are adsorbed to the membrane to a high
degree. This effect, however, has nothing to do with vital processes.

Recapitulating one can state that the bio-electric potentials are
influenced by oxygen pressure, KCN, ether and chloroform and
furthermore by the differences in concentration of electrolytes and
certain organic substances at the inner and outer surface of the
protoplasm.

According to L u n d the P.D.'s are redox potentials, according
to Osterhout diffusion potentials.

The arguments of Lund are the relation between P.D. and
respiration, the decrease of the potentials by KCN, ether and
chloroform. The Qio indicates the chemical nature of the cause
of the P.D.

The arguments of Osterhout are based on the influence
of salts and some other substances on the potentials. A calculation
points out that the potentials can be considered as diffusion poten-
tials. Moreover it is possible to explain the shape of action currents
as the result of diffusion through the membranes X and Y separately.
The temperature coefficient is high, but this does not necessarily
indicate a chemical reaction, though this seems to occur in some
cases. The influence of the oxygen tension does not indicate a
redox potential because no noble metal electrodes were used and
because of the salt effects. The fluctuations are due to the quantita-
tive or (and) qualitative variations of metabolites in the protoplasm,
by which the diffusion constants and the partition coefficients of
the electrolytes can be affected.

The potentials are measured with the aid of a potentiometer
with a valve amplifier (Philips 4060). The diagram is given in
fig. I. The apparatus was built according to E 1 e m a (1932) as far
as the plate circuit is concerned. The grid circuit was arranged as
described by Kordatzky (1934 P- 126). In this way the advan-
tages of an easy and exact control of the plate current on the one
side and a minimal risk of leakage currents on the other side
were obtained.

As one can see in the diagram the measurements were done
according to the compensation method. The unknown potential of

the object (X) was taken up into the grid circuit. This caused a
deflection of the galvanometer (H.). Then a counter-E.M.F. to the
potential of X was supplied until the galvanometer pointed to zero
again. The value of the counter-E.M.F. could be read from the
scale of a Cambridge A potentiometer (P, indicated by the part
surrounded by the dotted hne).nbsp;.

The instrument was adjusted as follows. After connection ot the
batteries the grid was connected to I. The plate current, regulated
by the quot;free gridquot; potential caused a deflection of the galvanometer
(G). Then this deflection was compensated by regulating the
• resistance R^. Now the grid was connected to II. So it loosed the
charge when R4 is short-circuited. The deflection of G caused by this

manipulation was compensated by adjusting R4. In this way the
apphed grid potential was exactly the same as the quot;free gridquot; poten-
tial. So the danger of leakage currents was minimized. Finally the
grid was connected to III. The galvanometer deflected again,
affected by the E.M.F. of the object. The resistance of the potentio-
meter (P), which provides the counter-E.M.F. to the potential of
the object, was regulated till G pointed to zero again. (The zero-
point of G was controlled before each reading). One division on
the scale of the potentiometer P indicated 0,2 mv.

The Z e r n i k e galvanometer (G) was made aperiodic by
shunting it with a resistance of 150 ohm (not shown in the figure).
The deflection caused by i M amounted to 12 cm at a distance
from scale to mirror of 40 cm.

The electrodes used have been described already in a previous
paper (D e G r o o t Jr. and Thomas 1938). So a brief des-
cription will suffice here. The U-shaped part of a glass tube (fig. 2)
was filled with a solution of 30 % pure gelatin in tap water. An
amalgamated zinc rod was immersed in saturated ZnS04 solution
in redistilled water in one of the horizontal hmbs, which was closed
by means of paraffin-soaked cork. The other limb was left open
and filled with tap water. Its end was paraffined to prevent the
water from flowing out. By placing the open end of the electrode
over the contact-tube, the contact with the plant is made. The
contact-tube existed of a narrow glass lube which was filled with
tap water and closed at one end by means of gelatin.This end was
fixed to the plant by a drop of gelatin (15 %). By adjusting the'
contact-tubes to all spots required before the beginning of the

experiment, it was possible to measure tile P.D.'s of different points
by placing the open end of the electrode over the successive tubes
without stimulating the plant.

The objects were placed in moist chambers. As, however, dif-
ferent types of chambers are used they will be described with the
experiments on which they refer. If, in some cases, the plants were
not placed in the dark, they were illuminated by the light of a loo-
watt bulb at a distance of i m.

It is important to know whether the potential measured with
these electrodes is due to the diffusion of all kinds of ions or that the
influence of particular ions is predominating. In this respect H -ions
are meant especially.

To investigate this, a model was made in which two solutions
were kept separated by a semi-permeable membrane (cellophane).
Two contact-tubes were dipped into the liquids and the measure-
ments were done just as in experiments with plants. Table i gives
the results. As appears from the table the potential measured is due
to all kinds of ions present in the solution. Dilutions as compared
to the original solutions are positive and the value of the potential
is as to be expected from the equation of N e r n s t for diffusion
potentials. If, however, solutions are used of nearly the same con-
centration but of different pn a litrie predominance of the H -ions
is shown, but it is not permitted to draw any conclusion as to the pn.
Thus measuring the P.D. on plants in this way, it is not justified
to conclude on the diffusion of H -ions particularly although in
some cases this seems to be probable, (see table i, page 384).

No mean errors were calculated. In this respect I refer to a pre-
ceding paper (De Groot Jr. and Thomas 1938). It has
been pointed out there, why it is difficult to determine them: the
P.D. is fluctuating continously. However, the fluctuations of the
potentials of the objects used here were much less than in Phaseolus.
We confine ourselves to show this in the next way. Seven contact-
tubes (numbered i up to 7 including) were fixed along a stem of
Bryophyllum calycinum. At first was measured the P.D. of i (the
apical contact-tube) to 7 (the basal one). Then the electrode in
contact with i was placed over 2, the other electrode resting over 7.
Going on in this way the P.D.'s are measured as indicated in the
upper row of table II. Furthermore the P.D. at two contact-tubes
in succession to each other was determined by moving both electrodes
(table II, middle row). Finally the electrode placed over i was kept

TABLE I. Diffusion potentials caused by various
ions passing though cellophane, measured with
Zn-ZnSOi electrodes.

at its place, the other one was moved and as a final control 6 to 7
was measured again (lower row).

One reading was made pro minute while the measurements of
the different combinations followed each other immediately. So the
whole experiment lasted 84 minutes.

It appears from table II that the P.D. of i to 7 increased 0,48 mv
during 70 minutes, while that of 6 to 7 decreased 0,13 mv during
50 minutes. The data from this table enable to calculate the P.D.
between every pair of contact tubes desired and to compare this
value with the measured one. Table III shows the accordance between
the P.D.'s measured and calculated.

As one sees, the maximal divergence between the computed
values and the experimental ones amounts to 1,12 mv (with the
contact-tubes 3 and 4). Taking into account, however, the slow
natural variations of the potentials the results are most satisfactory.

TABLE III. P.D.'s measured and calculated compared to each other.
The measured values are data from table II.

Vicia Faba. The beans were soaked during 24 hours in tap water.
Then they were planted in moist saw dust and cultivated at 23° C
in a dark room. The roots were used when they had reached a
length of 4 to 7 cm.

Coleus hyhrida var. Bertha Grosze was cultivated in the green-
house. In most cases plants were used with a stem of 7 to 10 nodes.

Of Bryophyllum calycinum grown
likewise in the greenhouse specimens
were used with a stem of 10 to 14
nodes.

The isolation, defoliation and ad-
justment of the objects occurred at
least 3 hours before the beginning
of the experiment. In long lasting
regeneration experiments the gelatin
bridges between the contact-tubes
and the plant were renewed each
3 days. This was necessary because
of the moulding of the gelatin.

As was cited in the review of lite-
rature Lund and co-workers con-
sider the bio-electric potentials as
redox potentials, while Oster-
hout and his school are regarding
them as concentration phenomena.
So, in the first place, it seemed of
interest to repeal the experiments
on the influence of narcotica on the
P.D.

To this purpose pieces of the stem
of Bryophyllum calycinum and Coleus
hyhrida were defoliated and placed
in a moist chamber as shown in fig. 3. The contact-tubes to which
the plant is adjusted pierced through the paraffin wall. The other
walls were covered with wet filtering paper. A current of air.

saturated with moisture, was led in through the bottom. If required,
the air was also saturated with ether.

In total lo experiments were done; 7 of them on Bryophyllum
and 3 on Coleus. Moreover 4 controls were made. To this purpose

the stems (2 of each species) were killed by dipping them into boiling
water for 15 minutes.

Fig. 4 shows the results of one experiment on Bryophyllum. In
each case the basal electrode was placed on an internode, while
the apical electrode was connected to a node or an internode alter-
natively. In the diagrams the position of the contact tubes is indi-
cated by arrows. So the potentials of both apical spots refer to that
of the basal one. The nodes are represented by horizontal lines.

Considering fig. 4 one can see the effect of air saturated with
ether. Immediately a negativation at the apical contact tubes re-
sulted. After 5 minutes the ether supply was stopped and fresh air
was allowed to enter. It then lasted 3 minutes until the ether was

removed completely. The P.D. began to restore itself 15 minutes
afterwards. The same procedure was repeated after 90 minutes.

In fig. 5 an experiment is shown in which the plant was staying
in ether vapour for more than one hour.

From the experiment of fig. 4 one may conclude that the ether
acted as a quot;stimulusquot; only, time being too short to allow a thorough
penetration. In that case the respiration would not have decreased

considerably but the membranes of the peripherical cell layers would
have been injured to such an extent that an action current resulted.
If this is true, still attention must be paid to the fact that after
removal of the ether the recovery begins not until 20 minutes
passed on.

In long lasting treatments with ether, however, the metabohsm
must have been affected. Also in these cases the P.D. changed; in

the fully drawn curve of fig. 5 the P.D. decreased but did not reach
a zero value. In only one more experiment the P.D. decreased as
much as it did in that case of fig. 5. As even in killed objects (fig. 6)
the P.D. does not disappear completely, these two cases may be
considered as an argument in favour of the redox-theory. But this
cannot be said of the other experiments and even not of the dotted
curve of fig. 5, obtained from the same object.

short time intervals only the first exposure causes an effect. In the
meantime the P.D. increases so that the apical part of the plant

The other experiments of
this kind showed similar
results. It cannot generally
be predicted whether the
P.D. at the apical electrode
will become more negative
or more positive.

From all this we may con-
clude that the P.D. measured
in this way is not resulting
from a redox system. The
results of experiments of
others which seem to affirm
the flux equilibrium theory
perhaps can be explained in
another way. This will be
discussed once more in the
end of part I.

The next question is
whether the source of the
bio-electric potentials con-
cerned is of a chemical nature
at all.

This problem was studied
by controling the influence
of temperature on the P.D.
As cited above (p. 377) the
Qio has been described as
rather high, chiefly at rela-
tively low temperatures.
Above 18° C the Qjo ranges
between 2-3. This would
indicate that the P.D. is
caused by chemical proces-
ses. However, as stated by

U m r a t h, in some cases more than one hour of exposure to a
different temperature is required until an effect occurs. Moreover
the experiments of Marsh seem to diverge to some extent.

So it seemed useful to study the influence of temperature once
more. To this purpose a defoliated stem piece of Coleus was placed
in a moist chamber as indicated in fig. 8. This chamber consists
of two copper cylinders telescoped into each other. The ends of
the inner one are closed by rubber- and those of the outer one by
cork stoppers. A flow of water of the temperature required is led
between the two cylinders. Both cylinders are perforated by brass
tubes coated with paraffin to enable the adjustment of the contact-
tubes. Also a thermometer is fit into the inner cyHnder. The tempera-
ture of the water is regulated by means of an apparatus after
Hille Ris Lambers (1926).

Fig. 9. Influence of temperature on the P.D. Sign of the potential: b related
to a. Exp. 4).

This series consists of 10 experiments. As the results are uniform
in all cases, we confine ourselves to the discussion of one of them
(fig. 9). The situation of the contact-tubes is shown in the diagram.
A rising of the temperature from 17,3 to 22,3° C is followed imme-
diately by a positivation of the P.D. (b related to a). This effect,
however, is short-lasting and, the temperature being kept constant
at its new level, the P.D. returns to its original value. Lowering
the temperature to 17,3° C again, the P.D. curve follows at once,
but also here for a short time only. It then returns to its former level.

The initial alteration of the P.D. after a change in the temperature
cannot be considered as an action current. This is demonstrated
by fig. 10, showing an action current superimposed on the pheno-
menon described above. In this experiment the temperature was

be clear that this effect is not
identical with the slow vari-
ations and therefore cannot be
due to the same process.

It might be possible, how-
ever, that the slow variations
are due to unequal heating or
coohng of the electrodes and
thus have nothing to do with
the object.

Fig. II showing a control on
a killed stem piece, proves that
this is not the case.

It seems very doubtful to me
whether it is permitted to con-
clude from this on the chemical
nature of the origin of the P.D.
The temporary effect could be
explained as the result of an
alteration in the rate of pro-
cesses which alteration would
not proceed simultaneously at

Rapid sinking of the temperature
causes an action current. Sign of the
potential: a related to b. Exp. 4c.

mv
control

Coleus

40 SO
time in mm

Fig. II.

Control: killed stem piece. Sign of the
potential: b related to a. Exp. 4k.

both contact-tubes. But if so, it would be impossible that the value
of the P.D. always returns
to its original level after
20 to 30 minutes.

According to U m r a t h
the influence of the tem-
perature on the P.D. of
Nitella is only noticeable
a much longer time after
a change in temperature.
A small object hke Nitella
undoubtedly accepts the 1 mv
temperature of its medium 5
in less than 5 minutes.
Also in this case it seems
questionable to me
whether one may con-
clude on the chemical

The discussion on the short lasting effect is deferred to the end
of part I. We only state here that it is not possible to conclude from
these experiments on a P.D. caused by chemical processes.

So we must try to find another explanation of the problem.
There are three obvious possibilities left. Firstly the bio-electric
potentials may be diffusion potentials. Further they may be Don-
nan potentials. Finally there is a possibility that the P.D. is due
to membrane potentials. Of course, it is not quite excluded that
other phenomena, such as
streaming-potentials (caused
by the water-transport in the
vascular system), play a part
but it seems not very pro-
bable that they can be in-
fluenced in such a way as
has been and will be shown
in the next experiments.

Osterhout (1930a) de-
monstrated by mathematical
reasoning that the influence
of diffusion potentials is pre-
dominating. He admits that
D o n n a n potentials as
well as membrane potentials
too may play a part, but, if
so, they are of minor impor-
tance.

So the influence of varying concentrations of electrolytes was
studied next. The root of Vicia Faba proved to be a suited object
for this purpose (for its cultivation see p. 386 quot;Materialquot;). The
bean was adjusted on a paraffin table by means of gelatin (fig. 12),
the root piercing downward through a hole. Over the bean a
tumbler, coated with wet filtering paper, was placed. The root
was covered previously with vaselin to prevent drying out. One
and a half centimeter next to its tip was left uncovered. This end
dipped into the test solution. Moreover the advantage of this proce-
dure was that when changing the solution exactly the same part
of the tissue was in contact with the liquid.

One contact-tube was adjusted to the basal part of the root, while
the other one was put into the solution.

The method used required a most careful treatment. The P.D.
immediately reacted on the slightest vibration of the table. So a
little disturbance could not be avoided at the replacement of the

solutions as can be seen in
the graphs. Some practise
could reduce this incon-
venience to a minimum.

When studying concen-
tration-effects, the concen-
tration can be modified in
two ways. Firstly the con-
centration of ions can be
changed in the surrounding
solution and secondly the
vacuolar sap can be diluted
or concentrated. As it had to
be discriminated whether the
P.D. is caused by the diffu-
sion of ions, present in the
plant, the second procedure
was chosen and happened as
follows.

The root tip was put in
tap water first. Two to three
hours later the P.D. had
reached a constant level. In
this state an equilibrium
must be reached between
the entrance of water into
the vacuole and the turgor.
In this phase the concen-
tration of the electrolyte
solution in the root is the
lowest possible. By applying
a solution of a non-electro-
lyte as medium, water will
be withdrawn from the cells
the root will become more
naturalquot;

and the solution of electrolytes in
concentrated. In this way only concentration effects of the
ions are studied.

As was cited above, the more concentrated solution is negative
to the more diluted one as the anion-permeability of the protoplasmic
membranes is relatively small. Consequently, it can be expected

15-
10-

Fig 14 Difference between the effects of glucose and of sodium phosphate. During the periods, indicated by the
dotted lines, no observations were made. Sign of the potential: root tip related to root base. txp. 5e.

that the root tip will become more negative (or less positive) in
relation to the base of the root in a more concentrated solution.

At first a lO % glucose solution in tap water was used; fig. 13
shows a result of one experiment. A strong decrease of the positivity
of the root tip immediately following the apphcation of glucose
matches the expectation. After replacement of the glucose solution
by tap water the root tip is positivated again. The fact that the
recovery proceeds slowly may indicate a partial disturbance of the
membranes by the rapid water movement. This indication is sup-
ported also by the continuous fluctuations during the exposure to
the glucose solution. Another experiment showed the same results.
Therefore the use of a less concentrated solution seemed advisable.

Moreover the next point was taken into account. If the inter-
pretation of the phenomena described above is right, an electrolyte
solution of the same osmotic value as the sugar solution sho^d
cause a greater effect. For, besides the osmotic effect, a migration
of ions from the medium into the cell will occur. This will cause
a negativation of the external solution to the plant, as relatively the
membranes are impermeable to anions. Relatively, since anion-
impermeabihty means only a greater motility of the cations in the
protoplasmic surfaces.

The solutions of the same osmotic values used are glucose 2,39 %
and Na2HP04 0,83 %, both dissolved in tap water. At each change
of the medium the root is washed by dipping it three times carefully
in the liquid to be applied.

Six experiments gave perfectly uniform results. So we confine
ourselves to the discussion on two of them.

As can be seen in fig. 14 the P.D. was reduced (negativation of
the root tip) 20 mv after the substitution of water by glucose. If,
however, the root was placed in the phosphate solution, the nega-
tivation increased considerably. No doubt can exist about this
phenomenon since repeated exposure to glucose caused positivation.
The P.D. does not return to its former quot;glucose levelquot; (A). This
could be predicted since during the exposure to phosphate ions
these ions penetrated into the vacuoles thus causing at B a higher
concentration of salts in the cell sap as compared to A. At a final
substitution of the glucose by water the P.D. continued to rise
(positivation of the root tip) until, in course of time, it would return
to its original value.

The objection could be made that the action of the phosphate
is superimposed on the glucose-effect. This does, however, not seem
very probable in view of the restoring effect of repeated exposures
to glucose and water. A conclusive proof is delivered by experiments

in which the water was directly replaced by the phosphate, as shown
in fig. 15. The root tip was negativated considerably. After apphca-
tion of the glucose solution the negativation decreased. The sub-
stitution of the glucose by water gradually restored the P.D. to its
original value.

So we may conclude from these experiments that the P.D. also
in the roots of Vicia Faba is determined by the concentration of the
ions in the vacuoles.

It must therefore generally be possible to influence the P.D. by
concentrating or diluting the ions in the cell sap; in other words
by moving water to or from the vacuole. It was still tried to cause
this water movement by replacing the root pressure by an artificial
hydrauhc pressure. By varying the latter, it will be possible to alter
the water content of the cells mechanically. The objects used were
stems (the leaves were left attached to them) of Bryophyllum caly-
cinum, in which the pericycle is well developed to a solid layer,
which is necessary in these experiments. At the base the cortex
is removed and the stem is placed on the hydraulic press as shown

in fig. i6. By turning A tighter, the rubber piece B is tightened up
against the pericycle. The pressure can be regulated by C and is
controlled by the manometer D.The hydrauUc press has been devised
and carried out by P. A. d e B o u t e r, mechanist of the Botanical

Adjusting the plant on the press in this way, the water is sqeezed
into the xylem only. So the natural pressure is imitated as well as
possible. The results, however, are published with some restriction.
Firstly they depend on the changes in the cell sap in the tissue next

	-H
Y////////////}	Y///////////^A

to both contact-tubes. These changes are determined by the hy-
draulic pressure, the osmotic pressure and the turgor. If both the
latter are equal in all parts of the tissue between the contact-tubes,
one cannot expect a great effect. If, however, they are not, the
question whether, by variation of the pressure, the tissue at one
of the contact-tubes is positivated or negativated will depend on
the values of the osmotic pressure and of the turgor as compared
to those of the other part of the tissue. Further, in most cases,
action currents could not be avoided as the pressure had to be

regulated constantly during the experiment. Finally, the vascular
system sometimes being pinched, no effect occurred at all.

One of the positive results is shown in fig. 17. In this case the
more apical tissue was positivated by applying an effective pressure
of i atmosphere.

As is pointed out above, it is difficult to valuate these experiments.
So we only state that it probably is possible to influence the P.D.
by alteration of the pressure in the vascular system.

Finally an object was looked after, in which consistent alterations
of concentration naturally occur. As the results of these experiments,
made in collaboration with D e G r o o t, have been published in
a previous paper (D e G r o o t and Thomas 1938), a short
summary will do here.

Phaseolus multiflorus is capable of quot;variation movementsquot; by
means of pulvini. These movements are achieved by an alternating
increase and decrease of the cell volumes of the motile tissues in
the upper side or in the lower one of the pulvini. It has beeri made
probable that the changes of volume are the result of alterations in
osmotic pressure in the cells caused by a more or less intensive
conversion of starch into glucose. The glucose constantly is removed
by metabolism as well as by diffusion (D e G r o o t 1938). So it

will depend on the rate of each of the both processes whether the
osmotic value of the cells will increase (water intake, enlargement
of the volume) or whether it will decrease (loss of water, decrease
of the volume). So the concentration of the electrolytes in the
vacuole will vary too. Assuming an ideal semi-permeabihty of the
membranes, the electrolyte concentrations at different times are to
be considered proportional to the reciprocal of the quotient of the
volumes of the cells at the same moments. By measuring the volumes
of the cells of the upper and lower sides of the pulvinus at a high
and at a low position of the leaf, it thus is possible to calculate,
according to the formula of N e r n s t, the difference m P.D.
between upper and lower side of the pulvinus at both stages. The
values obtained in this way fairly well agree with the experimentel
ones. The difference of the means of ii experiments on the P.D.
between the upper and lower side of the pulvinus at a sinking and
a rising state of the leaf amounts to 24,85 mv, the theoretical
value being 20,90 mv. The theoretical value being based on the
assumption that the semi-permeability is ideal, the agreement seems

2 the action of ether results either in positivation or in negativa-
tion but not in a quot;lossquot; of potential (by using no lethal

4.nbsp;the potential is showing a short-lasting increase when the
temperature rises, while a short-lasting decrease is caused by
sinking of the temperature.

5.nbsp;by moving water to or from the vacuole it is possible to in-
fluence the P.D.

When assuming that the P.D. is the result of a flux equihbrium
according to Lund we cannot explain why the potentials are not
reduced to zero by ether as was the case in the experiments by his
co-workers. Apparently a real temperature coefficient does not
exist. In our objects a Q^o = 2—4 was not stated. Finally m the

experiments on the influence of the concentration of the cell sap
on the P.D., the dipping of the root tip of Vicia F aba in a glucose
solution caused a negativation. If glucose would act as an oxidizable
substance and be a limiting factor in the respiration, the latter had
to be increased and the P.D., if it were a redox potential (the tip
being positive), had to be increased too instead of being decreased.
If the glucose would not hmit the oxidation process, no effect on
the P.D. had to occur at all. The negativation found, however,
cannot be explained in the terms of the redox theory of Lund.

Considering the P.D. as a diffusion potential according to O s-
t e r h o u t one can readily interprete the stated effects. As is
argued above, the apphcation of a glucose solution instead of water
negativates the tissue by the withdrawal of water thus increasing
the electrolyte concentration in the vacuole. An electrolyte solution
(phosphate) of the same osmotic value causes a much more
pronounced effect, the removal of the water being attended with a
diffusion of cations into the cells. Correctly Osterhout (1936b)
remarks that, if the bio-electric potentials were redox potentials,
they would not change with changes of electrolyte concentrations.
Osterhout mentions this especially for the concentration effect
of KCl, but this statement probably has a more general bearing.

In experiments with Phaseolus multiflorus the formula of N e r n st
for diffusion potentials proved to be valid.

The question arises whether the temperature effect and the
influence of ether fit in this explanation. In our experiments no
permanent effect of changes of temperature has been found;
only a temporary variation of the P.D. was stated. If a process of
continuous diffusion in the plant were involved, one might expect,
as permanent effect, a Q^ of about 1,2—1,3 as normally occurs in
diffusion phenomena. Nothing of this kind could be stated; there
is no question of a Q^ For the present it is not possible to explam
with certainty the temporary effect. The rate of the transport of
cations and anions being different, I may suggest — as a preliminary
attempt to explain this effect — that the rate of their migration
in and through the membranes too is differently affected by changes
in temperature. The latter difference, however, only could be
ascribed to a reversible alteration of the membranes themselves
caused by the change in temperature. Such an akeration might be
due to changes in the adsorption-equilibrium and hydratation and
consequently in the relative permeability to ions; factors, that could
be responsible for temporary changes in the P.D., until a new sta-
tionary state is reached. The hypothesis given by Marsh can-
not explain our case. His quot;overshootingquot; and quot;undershooting

effect (with sinking and rising of the temperature respectively)
consists of a short lasting counter effect, which was not found
by us.

The experiments with ether too do not conflict with our opinion.
The permeabiUty of the membranes is influenced by ether. It
therefore will depend on the concentration of electrolytes in the
vacuoles of a tissue whether it will be negativated or positivated
in relation to another one.

The objection can be made that it is also possible to explain the
effect of ether by assuming an unequal rate of penetration of the
ether into the tissues at both contact-tubes. In this case the part
in which the respiration is inhibited less would become positive.
This, however, does not hold true since even at prolonged exposure
the P.D. is not reduced in most cases.

So we conclude that, using our methods, no evidence is shown
of protoplasmic interfaces acting Uke electrodes of noble metal
according to Lund. The theory on redox potentials fails to
explain various results which can be understood by considering
the bio-electric P.D. as a diffusion potential.

Of course it is not possible to draw more detailed conclusions
from our experiments as to the nature of the membranes. It seems
doubtful whether one, working with complicated tissues, can go
further into details at all. To this purpose one would have to use
single cells in which one of the electrodes could be pierced into
the vacuole. In that case the results would not be troubled by the
shunting effect of the cell walls.

Also in single cells, however, one has to reaUse that no absolute
certainty as to the interpretation of the results can be obtained.
In chemistry the data on membranes are derived from models in
a dynamical balance. Selective permeability in vivo and in vitro
are not absolutely the same, since in living cells the balance con-
tinuously is shifted by the metabohsm.

The membranes are interfaces of the protoplasm. Since surfaces
are, chemically spoken, the most reactive parts it seems probable
that the metabolism chiefly occurs in or near the membranes. As
further their structure immediately and profoundly changes at
death, a close relation seems to exist between metabolism and
membranes. We believe that the structure of the protoplasmic inter-
faces is determined and regulated by the redox processes.

We will compare our point of view with that of Osterhout
and that of Umrath. We can resume the hypothesis of the
authors (see review of literature p. 377) shortly as follows:

the ions {e.g. organic metabo-nbsp;arrangement in the membranes,
htes) in contact with it.

It seems questionable to me whether the dependency on the
Og-pressure can be explained satisfactorily by the hypothesis of
Osterhout, whithout assuming that the structure of the
membranes largely depends on the state of the metabohsm.

On the other hand U m r a t h's explication is not quite satis-
fying for the understanding of concentration phenomena. U m-
r a t h does not deny absolutely the influence of selective cation-
permeability on bio-electric potentials. His admittance, however,
is not sufficient to explain quantitative relations between P.D. and
electrolyte concentration. Moreover, as shown above, the absence
of a Qio in our experiments excludes a direct dependency of the P.D.
on a chemical process.

The results of the experiments described in this paper were
explained by the movement of water causing a change in the electro-
lyte concentrations in the vacuolar sap and in the protoplasm.

Osterhout, however, considers the influence of non-electro-
lytes from quite a different point of view. According to this author
(1938) the alterations in P.D., caused by organic molecules, may be
due to a number of possibilities, such as: alterations of partition

coefficients, mechanical rupture of the membranes, production of
organic ions in the protoplasm, changes in phase boundary potentials
or in membrane potentials.

The effect of the movement of water has been studied by him
by placing Valonia cells in diluted sea water. A negativation resulted
as compared to the P.D. in normal sea water. The exposure to the
solution in question lasted very short (about 5 minutes). However,
these results cannot be compared with the initial alterations of the
P.D. in Vicia roots after changing the solutions. The rapid changes
of the P.D. occurring during the first minutes are not taken into
account by me, as at this object they undoubtedly are the result
of an inevitable irritation, caused by the replacement of the solutions.

Bio-electric potentials and polarity may be correlated in two
different ways. In the first place the P.D. may be caused by various
processes. In this case it only would be a by-product, useful to
control its sources. In the second place the potentials possibly are
responsible for the transport of electrolytes in the organism. If this
were true, electrophoresis would be one of the most important
phenomena in life.

In discussing this latter point of view we first will quote the
work of the „Biologisch-Physikalische Arbeits-
gemeinschaft Pragquot;, which is summarized by Keller
(1932, 1938). In this work, carried out on vegetable and animal
tissues, two methods have been used to control the bio-electric
behaviour. In the first place the sign of the enlarge of tissues or of
cellular parts has been determined by examining whether they
were coloured by positive dyes or by negative ones. Secondly these
results are controlled by means of bio-electric measurements. It
appeared that in various cases, in which organic and inorganic ions
are moving against concentration gradients, the direction of this
transport could be explained by electrophoresis. A number of
substances, however, moved just in a way opposite as expected if
caused by electric phenomena. Now it appeared that the direction
in which those substances move depends on the presence of colloids.
So e.g. some metals such as potassium, copper and magnesium
are moving towards the anode in the presence of electro-negative

colloids at which they are adsorbed. Xeller (1936) classifies
the organic and inorganic substances into two groups. Biologically
negative is the potassium-group, while the sodium-group is bio-
logically positive. This classification includes undissociated mole-
cules too. It can be considered as a supplement to the classification
of antagonistic ions. The reported facts are very suggestive. When
a negative compound enters the cell, a positive one leaves it. They
never are transported in the same direction. The potential of the
cells of diseased animal tissues changes of sign as related to the
surrounding liquids. In such cases the direction of transport of both
groups is reversed (Keller 1938).

As to the question of the source of E. M. F.'s Keller (1932)
remarked: quot;. . . , dass im lebenden Gewebe in der Regel auf beiden
Seiten einer Membran eine für das betreffende Gewebe und für
seinen Zustand charakteristische Ladung fortwährend aufrecht-
erhalten wird durch einen chemischen oder elektrischen Mechanis-
mus, dessen Wesen bis jetzt unaufgeklärt bleibt und der, wie L u n d
vermutet, mit der Atmung der lebenden Zellen im Zusammenhang
ist und mit ihr steigt und fälltquot;.

We have pointed out in the preceding part, that in our opiiiion
the bio-electric potentials are not directly related to respiration.
We beheve them to be concentration potentials. If only mere diffu-
sion of charged complexes (or ion§) would occur, it would be difficult
to understand in which manner electrophoresis can play a part.
But we will not absolutely deny electrophoresis and its significance
since redox potentials must exist in living matter, they are, however,
not measured in our experiments. Moreover we are dealing here
only with plants.

Furthermore it should not be forgotten, that one is working under
controlled conditions. In nature plants are irritated constantly by
the action of wind, rain etc. and action currents will disturb the
inherent P.D. considerably. So under natural conditions the transport
of all substances would be very irregular if not temporarily reversed,
if electrophoresis were responsible for it.

On the other hand the influence of electrolytes on the P.D. and
chiefly the quantitative relation between their concentration and
the variation of the potentials are facts indicating diffusion effects.
As to this, Keller (1932) points to the fact, that molecules can
penetrate the protoplasm more easily than ions. Osterhout
(1936) assumed the membranes to be an oily hquid in which dis-
sociation is minimal. Kraus (1934)5 however, showed that for-
mation of charged complexes occurred in media of low dielectric
constants.

In our opinion, the results obtained up to the present affirm the
hypothesis on diffusion potentials while others are not in contra-
diction to it.

The idea, that polarity in plants might be due to electric pheno-
mena, has been suggested by F. W. Went (1932)- This author
supposed the polar transport of the negatively charged auxin-ion
to be caused by the potential differences between top (—) and
base ( ). Experiments with acid and basic dyes seemed to affirm
this hypothesis. Things became more complicated as Rams-
horn (1934) discovered that plants are electrically tripolar. Shoot
tip and root tip both are positive while their base is negative. Growing
tissues appear to be positive in relation to resting ones. Rams-
horn therefore concluded that auxin cannot be transported
electrically.

The theory of Went has been rejected by various authors.
Their objections are based partly on the different sign found for
the charge in the tip and the base of the plant. An enumeration of
these contradictory data is given by Hellinga (i937)- This
author also discarded W e n t ' s hypothesis. Hellinga did
experiments with stem pieces of Coleus, placed in water partly in a
normal position, partly in an inversed one. Always the end pointing
upward was found to be negative. Unfortunately his method shows
an error as the electrodes are not of the same nature. An Ag-AgCl
electrode was inserted into the upper end, while the contact with
the other pole is performed by dipping a platinum wire into the
water in which the stem piece had been placed. So the negativity of
the upper part is not due to geo-electric phenomena but to the
method used.

We will not cite all literature concerning the question whether
the auxin transport is directed by bio-electric potentials. We will
confine ourselves to cite Clark, a student of F. W. Wen t.
This author (1937a) measured the P.D. on Avena and other objects.
The shoot tip was found to be negative. This agrees with W e n t ' s
theory on auxin transport. Clark (1937b), however, came to the
conclusion that it is impossible for the present to show a direct
relation between the electric polarity and the auxin transport.
Artificially the potentials of Avena were changed by shunting, by
counter-E.M.F.'s and by varying the influence of the geo-electric
effect. From these experiments negative results were obtained.
Moreover, the auxin transport could be stopped by applying sodiuni-
glycocholate (i/ioo.ooo) which had not any influence on the electric
polarity, respiration and semipermeabiUty (Clark 1938)- So,
according to Clark, a discrimination on W e n t ' s theory is

As was cited Clark showed the tip of Avena to be negative.
According to Ramshorn (1937) on the contrary it is positive.
As was found by the latter, this discrepancy is due to the fact,
that C 1 a r k's objects were kept in the dark. So the mesocotyl
will grow out more rapidly as compared to the tip. This will cause
a positivity of the base of the coleoptile. But, if the seedlings are
cultivated in light, the development of the mesocotyl is suppressed
and the tip is found to be positive. The positivity of growing tissues
was not found by Ramshorn (1934) only, but in this respect
the data as a rule are not contradictory. Lund and K e n y 0 n
(1927), Marsh (1928), Miss Rosene and Lund (1935),
D r a w e r t (1937) and others stated this too.

It was tried to show the correlation between electric and morpho-
logic polarity by applying currents. Lund (1921) succeeded to
reverse the polarity of Obelia commissuralis in this way. It has been
demonstrated to be possible with the eggs of Fucus too (Lund
1923). The plain of the first division could be affected by the
direction of the current flow. Furthermore a quantitative relation
appeared to exist between current density and orientation of the
axis of symmetry of Obelia (Lund 1924). For this object Lund
(1925) demonstrated that the threshold-potential for inhibiting
growth is of the same value as that of the inherent P.D. So, for this
object, Lund (1931) drew up the hypothesis that quot;the oriented
process of cell oxidation which is associated with this electric
polarity should be subject to control or modification by an appro-
priate application of an E.M.F. of external originquot;. In this way
correlation is explained without intervention of hormonal processes
or of the nervous system.

Also, according to Schechter (1934)5 the polarity of the red
alga Griffithsia bornetiana can be reversed. The rhizoids always
develop at the anodal side.

Neilson Jones (1925), however, could not obtain positive
results on seakale roots {Salicornia maritima).

Also the P.D. in plant cells can be regulated by an E.M.F. of
external origin. In this respect I mention the way in which an action
current is propagated. Osterhout and Hill (1930) showed
this clearly by connecting two cells of Nitella by means of salt
bridges. An action current of one of the cells passing through the
salt bridges caused stimulation of the other cell.

In higher plants the P.D. can also be influenced by an E.M.F.
applied externally, as was shown by Miss Rosene (1937). In
the Douglas Fir the inherent P.D. was increased by applying a

potential in the same sense while it was decreased by an opposite
one. It further appeared that the longitudinal electric polarity of
the xylem and the cortex are opposite to each other.

The influence of electric potentials on the auxin transport has
been studied by Brauner and Bünning (1930) by placing
Avena coleoptiles between two plates in an electric field. The tip
of the coleoptile curved towards the negatively charged plate, while
the root tip showed a curvature to the positive plate.

Katunskij (1936) succeeded in affecting the growth of the
same objects by placing them in an electric field in such a way,
that the condensor-plates were adjusted perpendicularly to the
longitudinal axis of the coleoptile.

Experiments were reported by Kögl (i933) m which an h.M.t.
was appHed to Avena coleoptiles. It was concluded, that the growth
could be influenced by accelerating or retarding the auxin transport
by means of electricity. According to Cholodny and Sanke-
witsch (1937) this effect would be temporary only. At prolonged
current flow in both senses retardation of growth resulted. A current,
flowing from base to tip of the Avena coleoptile, initially caused
an acceleration of the growth but a short time afterwards growth
was retarded. When the current went from tip to base in most
cases a retardation of the growth occurred. At breaking of the current,
however, the growth was retarded once more. So the authors believe
that, by applying a current, the translocation of auxin is not a direct
consequence (electrophoresis) but an indirect one, via the complex
system of the living protoplasm.

Du Buy and Olson (1938a) showed the influence of apphed
currents on the protoplasmic streaming in the coleoptiles of Triticum
and Avena. According to these authors the auxin transport will be
influenced too as it largely depends on the protoplasmic strejming.
So a relation may exist between growth and electric polarity via
this streaming. The same authors (Du Buy and Olson 1938b)
stated the auxin transport to be regulated by three causes. In the
first place the protoplasmic streaming, further electrophoresis and
finally diffusion through the membranes between neighbouring
cells. The first process is retarded by an applied current. It is
indifferent in which direction the current flows. The next one, of
course, is dependent of this direction, while the last one (Clark
1938), the charge of the membranes being responsible for the rate
of the auxin transport through the protoplasmic surfaces, is depen-
dent on the direction too. So, according to D u B u y and O 1 s o n.

the auxin transport depends on a comphcated system. This agrees
with the results of Cholodny and S a n k e w i t s c h (i937)-

Clark (1938), however, succeeded in inhibiting the protoplasmic
streaming in Avena coleoptiles without influencing the auxin
transport by applying saponine in a concentration of 0,05 saturated.
On the contrary sodium-glycocholate (i : 5000) entirely stopped
the auxin transport without affecting the protoplasmic streaming.

In the literature cited above, auxin has only been considered
in its function of ^roro^/? regulator. Olson and Du Buy (1937)
have shown, however, that growth substance can play a role in the
polarity of Fucus. If a solution of hetero-auxin (500 mgr[L) is
applied to one side of the eggs, the rhizoids always develop on this
side. The egg-cells and the spermatozoids appear to contain
auxin in a high concentration (D u Buy and Olson 1937)-
According to D u B u y and O 1 s o n, the group effect (Miss
H u r d 1920) and the influence of pH (W h i t a k e r 1937,1938gt;
W h i t a k e r and Lowrance 1937) on the rhizoid formation
of the same object is due to growth substance.

Rehm (1938) studied the relation between electric polarities
and bud regeneration in Phaseolus multiflorus. At decapitation the
node below the cut surface became strongly negative during the
first hour. Then it positivated and remained positive during the next
days. The temporary negativity of the base of the regenerating buds
seems to be an effect of wounding. The positivity following after-
wards and lasting for some days, however, probably is an indication
of starting growth after decapitation. As has been cited already,
growing tissue, as a rule, is positive in relation to resting cells. The
base of a regenerating bud is positive to that of a resting sister
bud; the tip is negative to that of the resting bud. Applied currents
do not inhibit their growth. Only a hyponasty towards the negative
electrode and an epinasty towards the positive one is the result.

As to regeneration phenomena in general an extensive review of
literature is given by Van der Lek (1925), to which I refer.

Resuming, we state that the data on correlation in plants caused
by bio-electric potentials are rather contradictory. Polarity cannot
be considered to be based on the direct electrophoretic transport
of growth substance. As to the question whether the P.D. acts in-
directly in polarity phenomena, the opinions do not match each

As was mentioned above, we beheve that the P.D. is due to a
great extent to concentration differences. In that case the correlation

between P.D. and regeneration, if existing, must consist in either
alterations in concentration of the vacuolar sap either alteration of
the membranes, or in both.

Before studying the problem of regeneration by means of bio-
electric measurements it is
necessary to show their mutual
correlation. As cited above, a
correlation between growth and
P.D. exists. This could be
stated by me too. Roots of
Vicia Faha were adjusted in
the moist chamber as shown
in fig. 3. The contact-tubes
were connected to the roots at
a distance of 0,6 cm apart. The
experiments were started one
hour after the adjustment of
the objects. The values ob-
tained are the means of 5
readings (one reading pro mi-
nute). An interval of one mi-
nute was taken between each
set of measurements at succes-
sive points. One electrode was
connected to the contact-tube
at the base of the root, while
the other one was placed over
the successive tubes. Five roots
were tested in this way. The
objects were aged 3 to 4 days.
One of the results is represented
in fig. 18. In all cases the ex-
treme end of the root tip was
strongly negative as compared
to the other tissue. The growing
zone always proved to be the
most positive part of the root.

in the P.D. As in regenerating tissue growth is started, one might
expect a positivation of the regenerating part. For our experiments
we looked for an object which is showing unipolar regeneration
and found it in the leaf of Coleus.

id Leaves ofColeus were adjusted in the moist chamber (see fig. 3).
As isolated leaves of these plants are forming roots on the petioles
while shoots never develop, it is certain that only unipolar regener-
ation occurs.

One contact-tube was adjusted to the middle of the lower side of
the lamina, while a second one was connected near the base of
the petiole. The leaves were allowed to recover during half an
hour before the beginning of the first reading. The results are shown
in fig. 19. As one sees, 20 hours after isolation of the leaves, all
petioles are negative in relation to the laminae. Then in all but one
cases the negativity decreases. After 4 days 9 petioles are positive.
One object (K) only shows a slight variation during the first days.
After 8 days this positivity decreases slightly. At the moment of
the positivation of the petiole, regeneration cannot yet be observed.
This will be clear since this P.D.-variation occurred 2 to 4 days
after the isolation of the leaves. We are inclined to consider this
positivation to be coupled with the initial processes of growth.

The initial negativity of the petioles, however, possibly may be
due to processes preceding the formation of the new meristems. If
this were true, this negativation could be caused by preparatory
processes of regeneration.

Experiments on shoot-regeneration only did not succeed. Since
the plants were placed in air, saturated with water, root formation
occurred too, even if the root system was left intact.

To study bipolar regeneration stem pieces of Bryophyllum
calycinum and Coleus hybrida were adjusted in the moist chamber
as indicated above. At least 3 contact-tubes were connected to the
objects. In this way the P.D. between tip and base could be estimated.
Moreover it was possible to control the P.D. between tip or base
and the middle of the plants. So it could be discriminated whether
for instance a positivation of the tip to the base was due to a positiva-
tion of the apex or to a negativation of the base.
The results on the regeneration of Bryophyllum-^ttm pieces are
shown in table IVa.

TABLE IVa. P.D. between apical and basal node in mv in Bryophyllum
calycinum during regeneration. The signs refer to the apical node.

8,51
14,50
55,18
—30,22

5,00

— 1,50

4,16
0,51
44,68
—18,04

4,84
— 1,78

—nbsp;1,04
14,98
5,05

—nbsp;1,69
1,18

7,27

—nbsp;3,83

-15,28

-nbsp;2,77

-nbsp;5,08

-nbsp;0,87
-11,18
-10,30

In this table the potential differences between the apical node
and the basal item are given. In the first place it can be observed,
that the apical node can be positive or negative as related to the
basal one. Apical parts also do not prove always to be negative as
they were supposed to be by some authors. As to the variation
of the P.D. during 9 days after the isolation no uniformity seems
to exist. It is of interest to know whether at least the basal or the
apical nodes behave uniformly in an electric sence. To this purpose
the variations of the P.D. between the apical node to the middle
of the stem piece are given in table IV^;, while table IVc shows the
same in relation to the basal node.

TABLE IVZgt;. P.D. between apical and middle node, in mw in Bryophyllum
calycinum during regeneration. The signs refer to the apical node.

6 a
3

3
5
3

3
3
3

TABLE IVc. P.D. between basal and middle node in mv in Bryophyllum
calycium during regeneration. The signs refer to the basal node.

6,12
-10,62
-48,83
19,83

-nbsp;0,22
-18,27

-nbsp;3,34

0,11

—nbsp;5,32
—13,93
8,57

1,62
—16,68

—nbsp;2,08

—nbsp;7,03

—nbsp;4,37
—58,80
24,21

-nbsp;1,22

-nbsp;5,53

-nbsp;3,60
-27,38

-nbsp;0,88
-11,22

— 1,21

—nbsp;3,79
—24,84

27,51

0,69

14,71

—nbsp;5,86

—10,34
4,71

1,73
— 4,03
1,30
8,40

It will be clear that no general conclusion can be derived
from these data. The apical nodes seem to behave as irregularly
as the basal ones do. It is of interest only, in connection with
the next experiments, to state the negativity of the basal nodes on
the day after the isolation of the cuttings. In this respect only exp.
8d is an exception.

Fig. 20 shows the P.D.-variation of different points in one object
{Bryophyllum). The situation of the contact-tubes is illustrated in
the diagram. The nodes are indicated by the horizontal lines. The
potentials refer to tube o. Any other example, however, would be
different. We only want to demonstrate, that the potential of the
nodes is not principally different from that of the internodes.
Clark stated (1937a) this only for young tissues. Only the basal

As Bryophyllum proved not to be a suitable object, the followmg
experiments were carried out with Coleus.

Firstly 5 stem-pieces were treated in the same way as mdicated
above. The results are presented in fig. 21. The apical node m all
cases proved to be positive as compared to the basal one durmg the
first day after isolation. After 5 days the curves diverge. In studymg

the P.D.-variations of the same nodes as compared to the middle of
the stem piece one finds (fig. 22) that the basal nodes during the first
3 days show a uniform electric behaviour by all growing negative
to about the same extent. Then a slow positivation follows and
afterwards the curves begin to diverge. These phenomena suggest
that there is some correlation between regeneration and P.D. It
is clear that we have not to deal with a reaction of the tissues in the
neighbourhood of the cut surface since the apical nodes do not
show a temporary negativation (a excepted) while they are situated
near to a wound surface as well as the basal ones.

In considering the behaviour of the apical nodes no regularity
can be observed, b, c and d show a shifting of the P.D. opposite to
that of the corresponding basal nodes. This is very interesting
indeed. However, exceptions are too frequent to base a conclusion
on this phenomenon. So from this experiment we only will conclude
that the initial positivation of the apical nodes to the basal ones (fig.
2i) in general is due to a negativation of the basal nodes, while the
reaction of the apical nodes can be different.

It is of interest to know to which extent the P.D. is affected by
influencing the regeneration. Regeneration of roots is accelerated
by the contact with water. However, a disturbing effect can be
expected here, since the water will enter the cells. Consequently,
the electrolyte concentration of the soaked tissue will decrease.
According to our theory this will cause a positivation, independently
of the regeneration.

The experiments were carried out in the same way as the pre-
ceding ones, but the bases of the stem pieces were placed in water
at a length of 0,5 cm. We confine ourselves by reproducing the
readings concerning the P.D.-variation between base and middle
during the first 2 days only. The data represented in table V fit
our expectation.

TABLE V. Influence of water on the P.D. of regenerating
Coleus stem pieces. P.D. between basal node (sign) and
middle node in mv.

9a only remained negative, but in comparison to its P.D., when
intact, a positivation occurred. The negativation which could be
expected from the regeneration is superseded by a positivation
due to the entrance of water into the basal tissue.

According to Voechting (1878) and others gravity influences
the polarity to a certain extent. When cuttings of several plants
are placed in an invarsed position in moist air, polarity is not reversed,
but the zone of the root formation is elongated much more in the
apical direction. Therefore it will be interesting to control the P.D.

alterations in regenerating, inversely placed stem pieces. 5 Experi-
ments were taken in this way. The results are shown in fig. 23. As can
be seen, the positivity of the apical nodes to the basal ones during
the first period did not predominate any more. This does not mean
that the apical nodes were negativated to about the same degree
as the basal ones (fig. 24). The effect seems to be a disturbing one.
The nodes of both, apex and base, did not behave accordirg to
any rule. From this we may conclude that the P.D.-reaction in

normally placed cuttings is influenced by gravity, but it is not
caused by it. In the latter case the P.D. had to change its sign and
that apparently does not happen. So,quot;the phenomena described
here are not due to a geo-electric effect. This effect, moreover,
causes much weaker potentials.

Fig. 24. Individual potential variations of the bases and tips of the objects
of exp. II. See fig. 23. Sign of the potential: see text fig. 22.

hat the movement of water in stem pieces is determined by gravity
since the root-pressure and the transpiration have ceased. This
seems not very probable since the initial positivation of the apex is
due to a negativation of the base. If the water is moving downward
a basal positivation could be expected. The experiment indeed
proved that this transport does not occur.

To examine the water distribution, 10 Coleus-stem pieces were
suspended in a moist chamber immediately after isolation and left

in it during 5 days. Then from apex, base and middle of each object
a piece of i cm length was cut out. Of each 10 pieces together the
fresh-weight and the dry-weight have been determined. The same
has been done with controls of equal stem pieces. The results are
shown in table VI.

TABLE VL Water distribution in Coleus stem-pieces suspended in moist
air during 5 days, and of controls ( ).

The apex proves to contain the highest content of water, while
at the base this content is smallest. So we conclude that gravity is
not influencing the P.D. during regeneration by redistribution of
the water.

Another method of affecting regeneration was used by applying
hetero-auxin in a concentration high enough to cause root formation.
To this purpose hetero-auxin paste (0,5 % according to F i s c h -
n i c h 1935) was applied to the bases of 5 Coleus-sl^xa. pieces and
renewed daily. The results are shown in fig. 25. Now the P.D.
between apical and basal nodes proved to alter much more regularly
in comparison to those of the untreated stem pieces (fig. 21). The
mean resulting phenomenon, seems to be that the maximal positivity
is reached the day after the isolation instead of reaching it 2 to ^
days later on.

Moreover, the curves are almost uniform even at prolonged
regeneration. In all cases a minimum occurs in which the apex is
negativated to the base. This minimum is then followed by some
positivation. Only the synchronism is lost after the fifth day.

Considering fig. 26 showing quot;individualquot; P.D.-alterations of the
apical and basal nodes, it is obvious that the basal reaction during
the first day is the most striking phenomenon. Moreover, it appears

that the minimum after 6—9 days in fig. 25 meanly is caused by a
negativation of the apex. However, it seems expedient to be cautious
in drawing detailed conclusions.

Fig. 27 and fig. 28 show the controls to which water-paste had
been applied and renewed daily. They show the same as is shown
in the figures 21 and 22. It is obvious, however, that the paste acts
in a somewhat disturbing way.

From these experiments, the conclusion can be drawn, that the
basal reaction is the most determined one. The negativation of the
base following the isolation of the cutting, reaches its maximum
after 2 to 3 days without hetero-auxin. With hetero-auxin this
maximum is reached in one day. The hetero-auxin seems to accelerate
the process and to master it during the first time. The latter fact
is derived from the initial synchronism. So a parallehsm occurs
between the influence of hetero-auxin on the P.D. and the regener-
ation of roots as far as its accelerating action is concerned.

To investigate this, 5 stem pieces of Coleus were placed in the
moist chamber (see fig. 3). On their apical ends 0,5 % hetero-
auxin paste was applied. The paste was renewed daily. The results
are shown in fig. 29, giving the P.D.-variations between the apical
and basal nodes.

moreover, shows that also the quot;individualquot; reactions of the base
and the apex are disturbed. The initial negativation of the basal
nodes, occurring in normal cases, cannot be observed here.

It will be of interest to try to explain this disturbing effect caused
by the apical application of hetero-auxin and to compare this distur-
bance with the regulating influence of hetero-auxin paste of the
same concentration applied to the base.

In the first place we want to mention that growth substances
are transported polarly. According to Van der Wey (1932)
they move in the basal direction only. As to the polar transport of
hetero-auxin, we further refer to H e 11 i n g a (1937). Theoretically
hetero-auxin cannot move in the tissue when it is applied from the

base as happened in our experiments. Perhaps an uptake is possible
but a significant transport cannot occur, even not in elements of
the vascular system, since the water movement will be reduced consi-
derably in isolated stems.

An intake of hetero-auxin by the basal tissue certainly did occur.
Besides of all regeneration phenomena this intake was shown by a
swelling of the basal tissue. Consequently we may expect that also
the electric behaviour of only the basal part has been affected.

When the paste is apphed to the apical cut surface things are
quite different. The hetero-auxin will enter the tissue and will be
transported basally. Therefore we cannot expect a locahsed influence
here. This means that also the quot;potential-distributionquot; along the
object will be affected, if hetero-auxin influences the bio-electric
P.D. in a direct way. We will return to this subject later on.

Secondly the concentration of the hetero-auxin in the objects is
important. If pastes of the same concentration of hetero-auxin are
applied to the basal or to the apical cut surface, the concentrations
in the plant will not be comparable. Probably the hetero-auxin will
diffuse much more easily into the plant from the apical end than
from the base. At basal apphcation it will remain near to the base,
while when apphed apically it will be constantly removed from
the tip. Considering these facts, it is not surprising that an initial
negativation of the apical nodes fails to appear after application of
hetero-auxin on the apex.

The irregular behaviour of the P.D.-variations when hetero-
auxin is apphed apically, can be understood by assuming that the
hetero-auxin influences the P.D. between all cells, which are
reached by this substance. As probably it flows along all cells, we can
imagine that the P.D.-fluctuations, occurring in the controls, are

According to our theory the disturbance of the P.D.-variations
by hetero-auxin may be due to two reasons. Firstly migration of
the ions of the hetero-auxin through the membranes could cause
a diffusion potential. Secondly the hetero-auxin could change
the properties of the membranes. It seems difficult to me to under-
stand the disturbing influence by assuming a direct diffusion effect.
For, in this case we might expect a correlation between hetero-
auxin concentration in the plant and variation of the P.D.: the
concentration in the apical part will be higher — at least in the
beginning — than that in the middle part of the stem piece. So we
might expect, during the first days, some uniformity of the curves
representing the variations of the P.D. between apical node and
middle one. This, however, does not hold true.

Considering as the next possibihty the hetero-auxin as a modifier
of the properties of the membranes, the P.D. can be supposed to
vary with the varying degree of permeability of the protoplasmic
surfaces. In this case the rate and the sense of the alteration of the
P.D. depends partly on the concentration of the hetero-auxin too.

but chiefly on the concentration and the nature of the ions already
present in the protoplasm and in the vacuolar sap.

Assuming such an effect on the membranes, the disturbance
of the P.D.-variations by apical application of hetero-auxin can be
understood by admitting that the diffusion potential of all cells
is altered successively.

auxin can influence the P.D. by affecting the membranes. We studied
this by applying hetero-auxin to the objects in such a low concen-
tration that no diffusion potential of any importance could be ex-
pected. To this purpose the effect of hetero-auxin was compared
to that of another electrolyte both in a concentration i in 10«.

Roots of Vicia Faba proved to be suitable objects. They were
adjusted as shown in fig. 12. Seven experiments were done. In all
cases the results were equal. We therefore will confme ourselves
to discuss the experiments shown by fig. 31 and 32. Before the
beginning of the experiment the roots were placed m tap water
and were allowed to recover for 2 to 3 hours. Then the water was
replaced by a NaH^POi-solution in a concentration of i in 10 .
This phosphate was used since it is eagerly absorbed by roots.

The concentration used, however, is too low to cause any noticeable
effect The temporary variation is only due to stimulation. Ihe
P.D. having restored itself, the former level is attained. This proves
that the effect of the transport of ions is too weak to cause a registrable
P.D.-variation. If, however, hetero-auxin in the same concentration
of i in 10« was appUed a pronounced decrease of the positivity ot
the root tissue followed. Although the ion-concentration was less
than in the applied phosphate solution, an effect was clearly recorded.
We only can interprété this phenomenon by assuming that the
membranes are altered. The permeability of the protoplasmic
surfaces being changed, the diffusion potential caused by the elec-
trolytes in the cells, will change too. There are two more facts
affirming our hypothesis. Firstly the degree of the decrease of the
potential depends on the value of the original P.D. (fig. 32)^ S^o
it is not of an absolute magnitude. Secondly the P.D. is affected for

a long time. As can be seen in fig. 13 and 14, immediately after
replacing an quot;effectivequot; phosphate solution by an isosmotic glucose
solution a restoration of the P.D. sets in. Hetero-auxin being
applied, the recovery sets in only one hour and a half after substi-
tuting it by water. This only can be interpreted by assuming that
the hetero-auxin is taken up in the membranes and causes a long-
lasting alteration of the surface structure.

Fig. 33. Difference between the influence of hetero-auxin dissolv d in the
solution (s) according to Kögl and Haagen Smit and that
of the blank solution, on the potential of the root tip of Vicia Faha.
Exp. 15b.

It seems worth while mentioning that the after-effect of hetero-
auxin does not occur when it is solved in a solution as indicated
by Kögl and Haagen Smit (1931) and consisting of
0,2 cm^ acetic acid and 150 mgr KCl in 1000 cm® water. This
solution is used in experiments on growth since the hetero-
auxin is much more active when dissolved in it. By this solution
the measured P.D. is negativated. Fig. 33 demonstrates the immediate
recovery of the potential after replacing hetero-auxin, dissolved in
the mentioned solution, by the blank solution. The fact that in

this solution the P.D. is decreased by hetero-auxin (and not negati-
vated as in fig. 31 and 32) also is a strong argument that the
membranes are changed; i.e. their permeability is increased. We
w^ill not discuss this phenomenon further.

From these results we conclude that hetero-auxin acts in the
membranes. This fact seems also important in relation to the effect
of auxins on the plasticity of the cell walls.

It will be clear that many problems on the influence of hetero-
auxin still remain unsolved. We mention in the first place the
effect of various concentrations of it in the regeneration experiment
as well as in those on Vicia roots. Further experiments on this
subject must be left to the future. In our research it only was ot
interest to study whether hetero-auxin, which affects regeneration,
affects the P.D.-variations too. Further we only wanted to control
whether an influence on the properties of the membranes could
be demonstrated. The results obtained seem to open an interesting
field of research in relation to the growth substance problem itself.

1.nbsp;the zone of maximal growth in the roots of Vicia Faba is positive
as compared to the other tissue. So a correlation between growth
and P.D. exists.

2.nbsp;the petioles of Coleus leaves are negative during 5 days alter
their isolation as compared to the middle of the under side of
the lamina. Then they become positive. If we consider the positi-
vity caused by the outgrowth of the root initials, the negative
period seems to indicate the initial reactions of the cells preceding
root formation. We are inclined to assume that this negativity
is correlated with regeneration.

3.nbsp;in isolated stem pieces of Bryophyllum calycinum the basal
nodes are negative as compared to the middle ones during the
first days after isolation. There is one exception in seven experi-
ments. The apical nodes react very irregularly.

4.nbsp;the potential-quot;distributionquot; along stem pieces of Bryophyllum
and Coleus (the latter has not been reported here) does not show
an essential difference in the behaviour of nodes and internodes
during the first stage of regeneration.nbsp;.

5.nbsp;during regeneration of Coleus stem pieces the basal node is
negativated as compared to the apical one. This has been studied

by comparing the P.D. between apical and basal nodes as well
as that between apical, resp. basal node to the middle one. The
apical node yields either positivation or negativation.

7.nbsp;the distribution of water in the stem pieces is not responsible
for the value and the sign of the P.D.

8.nbsp;hetero-auxin in a concentration causing root formation seems
to accelerate the process, when apphed to the base. The negativity
of the base reaches its maximum after the first day after isolation.
No influence could be observed on the apical node. When
hetero-auxin is apphed apically, the regularity in the electric
behaviour disappears entirely.

9.nbsp;in roots of Vicia Faba the P.D. is decreased by hetero-auxin in
I in 10®; it is made probable that the hetero-auxin acts in the
membranes and increases their permeability to ions.

In discussing these data we have to start from our conception
on the origin of the bio-electric potentials. Considering them as
diffusion potentials according to O s t e r h o u t, we beheve them
to depend on two factors. Firstly the degree of the permeability
of the membranes must be important. Secondly the presence of
diffusible ions is necessary. Though other potentials may occur,
we here will consider diffusion potentials only, since they appeared
to prevail.

The positivity of growing tissue as compared to a resting one
probably will be caused by both factors. Young membranes may be
of a different composition as older ones, since the cell wall of the
former consists of pectin, while at older stages cellulose or other
substances are quot;excretedquot;. Moreover, growing tissue is extremely
rich in water. In this way the electrolyte solution may be different
too.

The preceding period of temporary basal negativity seems not
to be caused by alterations of the water content. For, if this were
so, the water movement would be very comphcated: we had to
suppose that the water is transported apically during the first time
after isolation of the stem piece and basally later on. The same, though
in a reverse sense, could be said of the transport of the electrolytes.
Such a transport, moreover, cannot be significant since the reasons
of the movement of water and of salts in intact plants are absent
in isolated stem pieces. Gravity proved not to affect the distribution
of water. So we conclude that the P.D.-fluctuations during regener-
ation are not due to concentration effects.

variations during regeneration must he caused by alterations of the
properties of the membranes.

Since it was shown that growth is correlated with maximal
positivity of the tissue, the initial negativity of the bases of regener-
ating stem pieces cannot be caused by this process. This phenomenon
was found clearly too in experiments on root regeneration in the
petioles of isolated Coleus leaves. The initial negativity of the
petiole was followed by a pronounced positivity. The latter
phenomenon was shown much more clearly in these objects pro-
bably since unipolar regeneration occurred here.

We consider the positivity correlated with the productivity of
the formed meristem, while the negativity is attended with the
initial processes of regeneration. We have shown, that the negativ-
ation in the regeneration experiments on stem pieces is not caused
by the distribution of water in the plant. Moreover, it seems
improbable that it is caused by transport of electrolytes since the
root pressure and the transpiration are failing. We therefore con-
cluded that the negativation is due to alteration of the membranes.
This conclusion seems to be supported by the results of the expe-
riments on the influence of hetero-auxin paste (0,5 %) when applied
to the base. In this concentration hetero-auxin promotes root
formation and at the same time it accelerates and regulates the
basal negativation. This seems to indicate that the process occurring
spontaneously is accelerated and, since we have shown that hetero-
auxin influences the P.D. in Vicia roots by affecting the mem-
branes, it is probable that this initial negativation is due to an
alteration of the membranes.

We therefore conclude that the polarity of regenerating stem
pieces is correlated with typical changes of the membranes at the base,
preceding the outgrowth of the formed meristems.

With the method used such a specific reaction could not be
detected in the new apex.

1.nbsp;The P.D. along stem pieces of Bryophyllum calycinum and of
Coleus hybrida var. Bertha Grosze is influenced by ether.

2.nbsp;If no lethal quantities of ether are applied, no quot;lossquot; of P.D.
is stated. Either a positive or a negative variation of the inherent
P.D. occurs. From this it is concluded that the bio-electric
potentials, measured in our experiments, are not redox poten-
tials.

3- No Qio is found. Only a short-lasting influence of alterations
of the temperature is observed. We therefore conclude that
the potentials measured are not due to chemical processes.

4.nbsp;It is shown that the P.D. can be influenced considerably by the
motion of water into — or out from the cells.

5.nbsp;From these results it is concluded that the potentials in our
objects are diffusion potentials according to Osterhout.

6.nbsp;In the roots of Vicia Faba the zone of maximal growth coincides
with the most positive potential.

7.nbsp;The regenerating petioles of Coleus leaves negativate during
the first days after isolation. After 5 days they positivate.

8.nbsp;As a rule the basal nodes oiBryophyllum stem pieces are negative
during the first time after isolation. In Coleus this phenomenon
is much clearer.

id. No redistribution of water in stem pieces could be stated; this
factor is not responsible for the alterations of the P.D. in
regenerating stems.

11.nbsp;Hitero-auxin, applied basally in lanolin paste 0,5 %, acceleratp
and regulates the spontaneous variations of the P.D.'s in
regenerating stem pieces of Coleus. When applied apically,
however, the normal P.D.-alterations are disturbed.

12.nbsp;It is concluded that in initial regeneration typical changes of
the membranes at the base occur. In the new apex such a
specific reaction could not be detected.

A m b e r s o n, W. R. and H. Klein. 1928. The influence of PH upon
the concentration potentials across the skin of the frog. J. gen. Physiol.

II.nbsp;Halicystis. J. gen. Physiol. 19, 867. , , . , • . 1
_ i935^'36c. The effects of current flow on the bioelectric potential.

Jahrb. wiss. Bot. 66, 381.nbsp;, , • ^ ™ ••nbsp;tt
_ 1028 Untersuchungen über das geoelektrische Phänomen. II. Mem-
branstruktur und geoelektrisches Effekt. Jahrb. wiss. Bot. 68, 711.

Buy, G. H. du and R. A. O 1 s o n. 1937- The presen^ of grovrth
regulators during the early development of Fucus. Am. J. Bot. 24, amp;09.

growth in coleoptiles of Triticum and Avena. Science 87, 490.
Cholodny, N. G. and E. C h. Sankewitsch. 1937- Influence
of weak electric currents upon the growth of the coleoptile. Plant 1 hysioi.

C 1 a^i^W.quot; G. 1937. Electrical polarity and auxin transport. Plant Physiol.

Elema, B. 1932. De bepaling van de oxydatie-reductie potentiden m
bacteriencultures en hare beteekenis voor de stofwisseling. Itiesis.

G i c k 1 h o r n, J. und E. D e d j a r. 1931. Beobachtungen an elektrisch
gereizten Pflanzenzellen und die Frage des Nachweises reversibler
Permeabilitätserhöhung. Protoplasma 13, 592.
De Groot Jr., G. J. 1938. On the mechanism of periodic movements

of variation. Ree. Trav. bot. neerl. 35, 758.
-and J. B. Thomas. 1938. On bioelectric potentials and the move-
ments of the motile leaf pulvini in Phaseolus multiflorus L. Proc. Ken.
Ned. Akad. Wet. 41, nr. 4.
Haake, O. 1892. Über die Ursachen electrischer Ströme in Pflanzen.
Flora 75, 455.

elektrische bei Coleus-Stecklingen. Thesis, Wageningen.
Hill, S. E. and W. J. V. Osterhout. 1938. Calculations of bio-
electric potentials. II. The concentration potential of KCl in Nitella.
J. gen. Physiol. 21, 411.

Hoagland, D. R., P. L. Hibbard and A. R. Davis. 1927.
The influence of light, temperature, and other conditions on the abiUty
of Nitella cells to concentrate halogens in the cell sap. J. gen. Physiol.

H u r d, A. M. 1920. Effect of unilateral monochromatic light and group
orientation in the polarity of germinating Fucus spores. Bot. Gaz.

I r w'l'n, M. 1925. On the accumulation of dye in Nitella. J. gen. Physiol.
8, 147.

J. gen. Physiol. 10, 75.
Jacques, A. G. 1936. The kinetics of penetration. XII. Hydrogen sul-
fide. J. gen. Physiol. 19, 397.nbsp;. ^.

into Valonia. J. gen. Physiol. 20, 737.
- and W. J. V. Osterhout. 1930. The kinetics of penetration.

11.nbsp;The penetration of CO2 into Valonia. J. gen. Physiol. 13, 695.
Jones, W. Neilson. 1925. Polarity phenomena in seakale roots. Ann.

Kögl, F. 1933. Über Auxine. Angew. Chem. 46, 469.
-and A. J. Haagen Smit. 1931. Proc. Kon. Akad. Wet. Amster-
dam. 34, 1411.nbsp;, ^
Kraus, C. A. 1934. Tr. Electrochem. Soc. 66, 179 (»ef. Osterhout).
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lebenden Pflanzentheilen. Arb. bot. Inst. Würzburg. 2, i.
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strooming. Thesis, Utrecht.
L e k, H. H. A. van der. 1925. Over de wortelvorming van heutige
stekken. Thesis, Wageningen.

Lund, E. J. 1921. Experimental control of organic polarity by the electric
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Protoplasma 13, 236.

Bryophyllum. Plant Physiol. 5, 491.
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cell respiration. IV. J. exp. Zool. 51, 309.

Olson, R. A. and H. G. du Bu y. 1937. The rôle of growth substance
in the polarity and morphogenesis of Fucus. Am. J. Bot. 24, 611.

Osterhout, W. J. V. 1925. Is living protoplasm permeable to ions?
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J. gen. Physiol. 13, 47-nbsp;. .
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P o n d e rf^E. and J. M a c 1 e o d. 1937- The potential and respiration of
frog skin. I. The effect of the homologous carbamates. II. The effect
of certain lysins. J. gen. Physiol. 20, 433.nbsp;• , • ,

Wachstumstheorie. Planta 22, 737.nbsp;.nbsp;u • a
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by polar tissue and cell oxidation. Plant Physiol. 10, 27.
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S u o 1 a t h i, O. 1937. Uber den Einfluss des elektrischen Stromes auf die

Plasmapermeabilität pflanzlicher Zellen. Protoplasma. 27, 496.
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über Potentialmessungen an Pflanzenzellen. Protoplasma 22, 193.
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Voesch Ving, H. 1878. Über Organbildung im Pflanzenreich. II. Bonn.
Waller, J. C. 1925. Plant electricity. I. Photoelectric currents associated
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Waller, J. C. 1929. Plant electricity. II. Towards an interpretation of
the photoelectric currents of leaves. New Phytologist 28, 291.

Wey, H. G. van d e r. 1932. Der Mechanismus des Wuchsstofftrans-
portes. Ree. Trav. bot. neerl. 29, 379.

Whitaker, D. M. 1937. The effect of hydrogen ion concentration upon
the induction of polarity in Fucus eggs. I. Increased hydrogen ion con-
centration and the intensity of mutual inductions by neighboring eggs
of Fucus furcatus. J. gen. Physiol. 20, 491.

of polarity in Fucus eggs. HI. Gradients of hydrogen ion concentration.
J. gen. Physiol. 21, 833.

tration upon the induction of polarity in Fucus eggs. II. The effect of
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Physiol. 21, 57.

• vr.esW'Wt-nbsp;fï'/àuBfcciMOJcaarnbsp;'-iquot;• uli in't!-«•.'«-quot;in;-'«--.

Het is waarschijnlijk, dat de polariteit bij regenereerende planten-
deelen berust op een specifieke basale reactie.

Hetero auxine beïnvloedt de ionenpermeabiliteit van de grens-
lagen van het protoplasma.

Een gelijktijdig apicaal transport van stikstof en basaal transport
van koolhydraten in het phloeem is niet aangetoond.

De opvatting van F. W. Went, dat de semipermeabele mem-
branen zich binnen het protoplasma bevinden, is in strijd met
verschillende verschijnselen.

Het buitengewoon speculatieve karakter van de evolutiegedachte
maakt het ongewenscht een plantensysteem op deze leer te baseeren.

Ten onrechte meenen Krueger en Northrop bewezen
te hebben, dat de phaag productie een continu proces is.

De hypothese van Garrett, dat het parasitair vermogen van
Ophiobolus in negatieven zin correleert met de activiteit en het
aantal van de bodem organismen is niet afdoende bewezen.

De opvatting, dat de haemolyseerende werking van lymphe te
wijten is aan glycerine, is onwaarschijnlijk.

De bij rekkingsproeven aan de voet van Helix pomatia L. ver-
kregen rustkromme en volwaardige herhaUngskrommen zijn vrij
van verschijnselen, welke berusten op de dynamische component
van de viscosoide tonus.

Solutions measured to each other.	mv.
tap water to tap water	I gt;43
HCl 0,1 n to HCl 0,01 n	— 55^20
Phosphate buffer pH = 6,00 to same solution di- luted 5X.	— 27,93
Phosphate buffer pH = 6,24 to same solution pH = 7,38	— 8,32
Glycocoll HCl pH = 1,2 to Glycocoll NaOH pH = 12,9	— 24,88

Readings in mv.	—nbsp;5.30 —nbsp;5=29 —nbsp;5gt;03 —nbsp;5,17 —nbsp;5.30	—nbsp;8,18 —nbsp;1,11 —nbsp;7.98 —nbsp;8,06 —nbsp;7gt;92	—nbsp;1.95 —nbsp;1,88 —nbsp;1,96 —nbsp;1.95 —nbsp;1,88	—nbsp;4.53 —nbsp;4.59 —nbsp;4.56 —nbsp;4,60 —nbsp;4.53	—nbsp;2,51 —nbsp;2,32 —nbsp;2,42 —nbsp;2,41 —nbsp;2,47	—20,98 —21,14 —21,02 —21,06 —20,98
Mean	— 5,22 — 7,98 1 — 1,92 — 4,56 1 — 2,43 —21,04
Contact-tubes	5 to 6 1 4 to 5		3 to 4 2 to 3 1 I to 2 1
Readings in mv.	18,07 18,03 17.93 18,08 18,00	—nbsp;2,88 —nbsp;1,11 —nbsp;2,90 —nbsp;2,88 —nbsp;2,91	1.52 1.34 1.46 1.66 1.64	—nbsp;7.46 —nbsp;7.34 —nbsp;7.48 —nbsp;7.32 —nbsp;7.40	3.26 3.20 3.12 3.04 3.20
Mean	18,02	— 2,86	1.52	— 7.40	3.16
Contact-tubes \| i to 3 \| i to 4 i to 5 1 to 6					I to 7 1 6 to 7
Readings in mv.	—nbsp;4,04 —nbsp;4.03 —nbsp;4,00 —nbsp;4,12 —nbsp;4,13	—1,62 —1,62 —1,61 —1,60 — 1.57	—nbsp;4.14 —nbsp;4,09 —nbsp;4.14 —nbsp;4,18 —nbsp;4,10	14,42 14.43 14.43 14.44 14.43	—nbsp;5.72 —nbsp;5.69 —nbsp;5.69 —nbsp;5.69 —nbsp;5.73	—20,91 —20,92 —20,89 —20,87 —20,94
Mean	— 4,06	—1,60 1 — 4,13 1 14,43 — 5gt;70				—20,91

Contact-tubes	P. D. figured out in mv.		P.D. measured in mv.
I to 2	(I to 7) — (2 to 7) (I to 3) — (2 to 3)	2,76 3.34	3.16
2 to 3	(2 to 7) — (3 to 7) (I to 3) — (i to 2)	—nbsp;6,06 —nbsp;7.22	— 7.40
3 to 4	(3 to 7) — (4 to 7) (i to 4) — (i to 3)	2,64 2,46	1.52
4 to 5	(4 to 7) — (5 to 7) (I to 5) — (I to 4)	—nbsp;2,13 —nbsp;2,53	— 2,86
5 to 6	(5 to 7) — (6 to 7) (i to 6) — (i to 5)	18,61 18,56	18,02
6 to 7	(5 to 7) — (5 to 6) (i to 7) — (i to 6)	—nbsp;20,45 —nbsp;20,13	—nbsp;21,04 and —nbsp;20,91

m\/\				_
W\
							quot;C
\ \			temp.		I b		ÎU
5							22
		;					10
							ta
0			eO gg		m	m
5		/ \	/
		V	, y m y K f
to	-		\ /
	1-	Coleus
15	r

	li-i 0
d	1_\| to Sg p a	s
u	1_\| to Sg p a	fl
8a	3	18,77
8b	5	—30,97
8c	3	48,51
8d	2	4,11
8e	3	24,90
8f	3	— 9,78
8g	3	—24,49

17,56	7,42	— 0,66	—0,15	10,28	1,48
—21,26	9,35	13,06	9,66	11,16	9,77
23,57	—	—	—8,88	— 4,15	—3,62
31,62	0,25	—	6,88	1,79	—6,01
25,59	—2,64	—	2,80	—	—8,40
4,93	—9,49	—	—9,41	—13,43	—8,40
—30,25	—4,23	—	—5,89	— 5,12	—4,22

		number of days after isolation
exp.	intact
exp.	intact	I	2
9a 9b 9c 9d 9? 9f	—nbsp;13,46 5,59 —nbsp;6,01 —nbsp;8,78	— 1,77 8,30 1,69 10,43 29,46 3,67	— 3,80 4,51 12,59 5,63

	10 X I cm cut from
	base	middle	apex
fresh-weight in mgr.	2243 (2495)	1754 (2235)	1135 (1809)
dry-weight in mgr.	224 (227)	149 (168)	71 (128)
water in mgr.	2019 (2268)	1605 (2067)	1064 (1680)
water in %	90,01 (90,51)	91,51 (92,49)	93.74 (92,90)



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