PROEFSCHRIFT TER VERKRIJGING VAN
DEN GRAAD VAN DOCTOR IN DE WIS- EN
NATUURKUNDE AAN DE RIJKS-UNIVER-
SITEIT TE UTRECHT, OP GEZAG VAN DEN
RECTOR MAGNIFICUS DR. H. BOLKESTEIN,
HOOGLEERAAR IN DE FACULTEIT DER
LETTEREN EN WIJSBEGEERTE, VOLGENS
BESLUIT VAN DEN SENAAT DER UNIVER-
SITEIT TEGEN DE BEDENKINGEN VAN DE
FACULTEIT DER WIS- EN NATUURKUNDE
TE VERDEDIGEN OP MAANDAG 3 DECEM-
BER 1934, DES NAMIDDAGS TE VIER UUR

Nadat ik vele jaren werkzaam ben geweest in Zuid Afnka.
miin aangenomen vaderland, ben ik zeer dankbaar dat ik. door
het van wege het Departement van Landbouw, Pretoria, mij
verleende studieverlof, de gelegenheid kreeg om aan deze Uni-
versiteit, waar mijn wetenschappelijke opleiding begon, mijn

Dit doel heb ik steeds voor oogen gehad en het is een belofte
aan U, Hooggeleerde Went, welke ik hiermee heb vervuld. Vele
botanici, die Uw leerlingen waren, hadden langer de gelegenheid
onder Uw leiding te werken; toch reken ik mij met hen gelukkig
U als leermeester gekend te hebben en Uw invloed te erkennen

De wijze waarop Gij deze laatste periode van studie voor mij
hebt wiUen voorbereiden en mogelijk maken stemt mij tot groote

heb ik te danken voor de mogelijkheid mijn studieonderwerp in
betrekkelijk korten tijd te beëindigen. Uw voortdurende belang-
stelling en aanmoediging hebben mij gesteund bij mijn werk.
Nadat U vele jaren geleden als assistent mij hebt ingeleid in
de botanische studie mag ik nu het voorrecht hebben deze stu-
diën te voltooien door bij U te promoveeren.

Hooggeleerde Pulle, de wijze waarop Gij mij zijt tegemoet
gekomen bij het hervatten van mijn studie is mij een oorzaak
van groote dankbaarheid, zooals ik U ook steeds dankbaar ge-
weest ben voor Uw colleges die ik vroeger heb bijgewoond, daar
zij mij veel geholpen hebben om spoedig vertrouwd te raken
met de problemen der Zuid Afrikaansche Flora.

Hooggeleerde Westerdijk, ook voor Uw onderwijs dank ik
U, daar het mij de wetenschappelijke basis gaf voor de practi-
sche ervaringen in Zuid Afrika opgedaan.

Hooggeleerde Nierstrasz en Jordan, vergun mij hier ook
U te danken voor wat ik in vroegere jaren van U mocht ont-

Hooggeleerde Kruyt, dat U over het eerste deel van mijn
proefschrift Uw critisch oog liet gaan stel ik zeer op prijs.

Van die geleentheid, wat die publikasie van hierdie proef-
skrif my gee, maak ek gebruik om ook alger te bedank in Bloem-
fontein, aan die Grey Universiteits Kollege, die aan mij weten-
skaplike vorming bygedra het.

In particular I want to thank you, Prof. Dr. Geo. Potts,
for the botanical training which I received in Bloemfontein.
Many pleasant memories are associated with that period. I ap-
preciate your continued interest in my subsequent career.

Gaarne wil ik mijn dank uitbrengen aan de assistenten van
het Botanisch Laboratorium voor hun gewaardeerde hulp en
prettige samenwerking.

Het personeel van het Laboratorium wensch ik te bedanken
voor hun bereidwillige hulp bij verschillende gelegenheden.

The object of these investigations was to determine the in-
fluence of some seed desinfectants upon the germination of the

The possibiUty that desinfectants might have a stimulating
effect on the germination process made it essential that atten-
tion should be paid to the absorption of water and the subsequent

Since the temperature is an important factor in all physio-
logical processes, the temperature influence on both the swelling
and the germination process was investigated.

The apparatus consisted of an incubator, fitted with four
zinc trays, 45 X 30 x 5 cm, lackered inside. Over each tray
three slabs of plate glass, 8 cm wide, could be placed. Each
slab could carry five germination beds side by side. The ger-
mination beds were circular pieces of filter paper, 8 cm in
diameter. The paper was of the same brand as used for the
germination tests at the Rijksproefstation voor Zaadcontrôle
(Government Seed Testing Station) at Wageningen, and is

Each tray was filled to a certain level with distilled water.
Over each glass slab narrow strips of filterpaper were placed,
which dipped into the water on either side, one strip to every

The quantity of water in the tray and the width of the strips
were adjusted, so as to keep the germination beds soaked with

The air in the thermostat was saturated with water-vapour
before the experiments were started, the seeds consequently
did not need to be enclosed altogether in filter paper. This has
many advantages, as was pointed out by Pringsheim (1928),
whose method resembles mine very closely.

As part of the object was to investigate the influence of
desinfectant solutions on the rates of water-absorption and of
germination it was important to choose a certam number of
seeds as standard quantity, which would ensure both compara-
tive reliable mean values and easy manipulation. The standard
methods for seed testing, e.g. Rules for Seed Testmg (1928),
prescribe the use of 4 X 100 seeds for a single sample or m
Le of large seeds 4 X 50 each. Such quantities would have
involved a cumbersome apparatus. Quanjer and Oortwyn
Botjes (1915) and Pringsheim (1928) used smaller quantities

in their experiments and the latter author pointed out that for
comparative analyses small quantities may be used. He did
not use larger samples than of 50 seeds each, irrespective of the
size of the seeds. A fair degree of accuracy can be obtained with
such samples, as is demonstrated in Table I.

The weight increase of the seeds, as a result of steeping, was
determined with analytical accuracy by weighing the seeds,
before and immediately after steeping, in tared weighing bottles.

The seeds were steeped in water and in three desmfectmg
solutions, viz. 0.1 per cent CuSO^, 2 per cent CuSO^, 0.25 per
cent Uspulun.

The solution of 2 per cent coppersulphate was apphed for one
hour only, thereafter the seeds were thoroughly washed with
distilled water. According to Gassner and Rabien (1926) and
Kotowsky (1926) practically all traces of a salt solution can
thus be removed. After washing the seeds were steeped in
distilled water.

The other solutions were applied till the seeds were removed
to be weighed. Before weighing they were washed, rapidly dried
between filter paper and transferred into the stoppered weighing
bottles. After reweighing they were arranged on the germination
beds. The seeds were always placed with the scutellum on the
filter paper, so that uniformity of arrangement excluded varia-
tions due to the positions of the seeds.

The vessels used for steeping were small glass dishes with a
capacity of about 15 cm^. A measured quantity of liquid was
used in every case which just covered the seeds. This quantity
was 8 cm3. The influence of this arrangement on the process of
germination will be discussed later.

The seeds could absorb water during various lengths of time.
By these means seeds with varying quantities of water were
put to germinate and the influence of presoaking on the ger-
mination process could be observed.

The seeds were steeped for 1, 2, 3, 4. 5, 6. 9, 12, 18, 24, JO
and 36 hours. In some cases no determination of the quantity
of water which was absorbed was made after 18 hours and a
duplicate determination after 24 hours was made. Only where
a 2 per cent CuSO^ solution was used, no determination could

In table I are recorded the percentages of weight increase
after 24 hours steeping in water at 22.5°, for ten samples The
average germination time, in days, is given in the second column.

quot;?i!;\trtgVrbtervation appears to be quite accurate, but
in a few cases, in particular those marked with asterisks, the
variation is much larger than the mean error.

This however, is a phenomenon frequently met with in in-
vestigations on seeds and larger samples nor anbsp;^
samples would eliminate this. Due allowance wiU have to be

made for a striking deviation from the mean. In general, it
does not influence the accuracy of a number of readings, as the
general tendency of a series of observations determines the
value to be attached to occasional deviations.

In the following table a number of duphcate determinations
of weight increase show that great accuracy can be obtained.

The figures in the tables endorse the assumption that samples
of 50 seeds each may produce results of a fair degree of accuracy.

The influence of the temperature on the water absorption
and on the subsequent germination was studied at 15°, 20°, 25°,
30°, 35°, 40° and 45°. The seeds were soaked and laid out to
germinate at the particular temperature.

A germination experiment was taken to be started from the
moment of steeping. The number of germinated seeds was
determined every 24 hours, those seedhngs only being considered
which had a developed root system as well as a plumule standing
away from the scutellum. This is in agreement with the general
rules for seed testing. In some cases the points of the radicles
could be observed outside the seeds, but no further development
took place afterwards. This was very striking at 45°, where
several seeds came to this stage only and no further growth
followed; the seeds died, owing to the length of exposure to this
high temperature. It would have been erroneous to consider
them as germinated. The same phenomenon was observed with
seeds which were badly infected with parasitic fungi. The
embryos developed to the first stages of germination, where
root development can be observed, but before complete develop-
ment could take place they were killed by the infection.

For aU samples the average germination time, as defined by
Gassner, was calculated. This proved to be of feat value m
determining the effect of the treatment the seeds had under-

^°Sterile conditions were maintained as far as practicable
Before every experiment the incubator was desinfected with
dilute alcohol and with formaline. The trays and glass plates
were sterilised in the same manner. The filter paper and other
apparatus was sterilised in a drying oven at 110° for six hours.
On the whole, very little infection from outside could be obser-
ved during the course of the investigations. The only fungi
which did occur were those inherent to the material itself.

Three varieties of A. sorghum were used during the experi-
ments, viz. Klerksdorp Kort Rooi, Dwarf Hegari and Dwarf
Yellow Milo. The first was obtained from the Faculty of Agri-
culture of the University of Pretoria, cropped March 1934 at
Pretoria. Samples of the other two varieties were kindly fur-
nished by the Division of Plant Exploration and Introduction
of the Bureau of Plant Industry, U.S. Department of Agri-
culture, Washington D.C. They were grown at the Woodward
Oklahoma Field Station of the Division of Cereal Crops and

the investigation of the temperature influence on the rate of
absorption, germination etc. Together with these experiments
duplicate tests with the American varieties were made for
comparative purposes, so that some interesting data could be

In some additional experiments the American varieties have
been used because of the fact that the South African sample
became badlv infected with grain weevils. The required selecting
of undamaged, sound seed required more time than was justified
since the other varieties remained free from this source of
trouble.

I want to express my thanks to Prof. A. R. Pullen, of the
University of Pretoria and to Mr. B. Y. Morrison, of the
Bureau of Plant Industry, Washington, for their courtesy and
assistance by supplying me with the required material.

The absorption of water by seeds is a process which is con-
trolled by a number of factors. Because of the complex nature
of the material involved it is possible that other factors than
the following influence the swelhng process:

a.nbsp;permeabihty of the layers enveloping the embryo and en-
dosperm, as weU as the permeabihty of the cellwaUs and
protoplasm of the seed contents,

It is stated by Lehmann und Aichele (1931), that Heinrich
(1913) should have indicated that live seeds absorb water at a
faster rate than dead ones. Whereas this might be taken to
indicate that vital forces are of influence on the absorption
process, it is necessary to point out that this question will be
referred to in due course.

The permeability of the composite layer of pericarp and testa,
— for convenience sake frequently called testa, while the grain
for identical reasons will be referred to as seed, — is of principal
importance for the absorption process.

This permeabihty is associated with the anatomical structure
and detailed information on the structure of the testa of A.
sorghum seeds has been given by Swanson (1926, 1928), to whose
articles one is referred.

It was Brown, who indicated in 1907, that the testa functions
as a semipermeable membrane. After having demonstrated its

nature in barley, he was able to do the same for rye, wheat and
oats Soon afterwards semipermeable membranes were observed
in a large number of seeds, e.g. rice by Valeton (1907) and
Nagai (1916), maize by Nagai (1916), Xanthium by Shull
(1913), Cucumis by Van der Marel (1919), while a list of other
seeds vnth semipermeable membranes was published by Rippel
(1918). In all these cases the absorption of totally submerged
seeds gained attention. It was Schroeder (1911), who deter-
mined the exact nature of the semipermeable membrane and
described the phenomena of selective permeability and locaHzed

In his initial study on barley. Brown describes how he
observed, that the grains of Hordeum vulgare, var. caerulescens
contain k blue pigment in the aleuron layer. This pigment
changes colour and becomes red in acid medium. When such
barley grains were steeped in dilute sulphuric acid, they retamed
their blue colour and cracked or damaged seeds only showed a

He noticed that the seeds absorbed water from the dilute
acid, thereby increasing its concentration. This simple obser-
vation has been repeated many times with several solutions
of electrolytes and organic substances and an increase in the
concentration of the solution was always noticeable.

Brown concluded, that the acid did not enter the seeds but
water only. This proved even to be the case when a solution of
sulphuric quot;acid of 36 per cent was used.

Brown was able to indicate the position of the semipermeable
membrane by steeping the seeds first in a 3 per cent solution of
silver nitrate and thereafter in a 5 per cent solution of sodium
chloride. A depository layer of silver chloride was formed inside
the testa. On exposure to hght. after sectioning, it was found
that the precipitated silver oxide occurred in the testa itself
only and the layer on the inner side of the precipitate was taken
to be the semipermeable membrane. Brown gave it the name of
quot;spermodermquot; and did not investigate its morphological nature.

The spermoderm was found to be ahnost impermeable, but
at or near the micropyle some substances could enter the seed.
Here permeabihty was greater.

Brown observed that a solution of iodine entered the seed
at the germinal end and other substances were soon observed

Of the mineral acids, sulphuric acid was never noticed to
enter the seeds. Normal hydrochloric acid entered at high tem-
peratures only (Reichard, 1909, not seen). Dilute nitric acid,
1 per cent solution, did not enter during the first 24 hours of
steeping, but, according to Brown, appeared to enter with
length of time. Collins (1918) found no traces of nitric acid
inside the seeds, even when a 10 per cent solution of the acid
was applied.

Several organic acids were found to enter easily: acetic acid,
formic acid, lactic acid, butyric acid, trichloracetic acid, picric
acid and (by Schroeder) osmic acid.

The only salts that have been observed to pass through the
membrane are mercuric chloride and mercuric cyanide (Brown),
traces of mercury from desinfectants like germisan and uspulun
(Heubner, 1928), traces of copper from copper salts (Lunde-
GâRDH, 1924, 1925), and potassium iodide (Brown).

Brown was able to distinguish between the behaviour of an
iodide solution which entered readily, as was noticeable by the
staining of the seed contents, and the behaviour of a solution
of thiosulphate, which decolourised the testa, but not the seed
contents.

Several organic .substances pass through the membrane, e.g.
formaldehyde in solution, alcohols, aether, chloroform, phenohc
substances and glycocol (Brown and Tinker, 1915). It was
established soon, that in all cases permeation depends upon the
presence of water, a dry membrane being altogether imper-
meable.nbsp;.

A few tests were made by me to verify the behaviour of mi-
neral acids and picric acid. Van der Marel stated, that while
picric acid was found to enter the grains of barley, wheat, maize
and Penicillaria sficata, it did not enter the grains of sorghum.
I have tested the grains of the three varieties of A. sorghum at
my disposal. They were steeped in a saturated aqueous solution.
This did not enter the seeds during the f^rst 6 hours, but after
24 hours it could be observed in all seeds. (Temp. 18°).

The mineral acids nearly all enter the seeds, though the rate
of entrance appears to be very slow and unequal for sulphuric,
nitric and hydrochloric acid. After 24 hours, at a temperature
of 18° traces of sulphuric and nitric acid only were observed.
While'it appeared that hydrochloric acid had entered further
into the seeds, this observation is obscured by the fact, that the
seeds contain a certain amount of soluble chlorides. When due
allowance was made, it was found that traces only could have

beans He found that the absorption of water by living and dead
seeds was identical, until the beginning of germination of the
living seeds, at which time the osmotic phenomena became mani-
fested. According to this author, the forces which are concerned
in the initial stages of water intake are those of capiUanty and
imbibition. On germination osmotic forces begin to influence
the amount of water taken up by the living seeds. Atkins,
however, did not take into account the open micropyle of the
material with which he worked, as was pointed out by Schroe-
der (1911).

This investigator contributed some very important data, ne
identified the semipermeable membrane v/ith the inner integu-
mentary layer. He observed that seeds, that were steeped m an
iodine solution, did not absorb the iodine over their whole sur-
face but the blue colour could be seen to spread progressively
from the germinal end of the grain. This indicated that the solu-
tion was absorbed at the germinal end at a rate much higher
than that with which it is absorbed over the general surface. To
describe this he introduced the term quot;selective permeabihtyquot;.
This was illustrated by an experiment with halved seeds. These
were first soaked in a solution of cobaltous chloride, then dried
and thereafter made to absorb water. In both parts the cut sur-
faces changed colour, but in the germinal parts of the grains an
additional change was observed round the embryo.

Absorption of water, therefore, takes place principaUy at the
germinal end of the seed and the water spreads inside the gram

The other part of the seed coat is not so much impermeable to
water as very httle permeable. Further evidence was given by

This proved the existence of semipermeable cellulose mem-
branes. Rippel (1918) drew attention to this fact, since semiper-
meability in plants had been associated always with Hving
matter only.

Collins pointed out that the distribution of water mside the
grains always takes place from the germinal towards the apical
end. This distribution is precisely the path of enzyme desinte-
gration during the germination of the embryo. Water thus pre-
pares the way for the distribution of the enzymes or even may
carry the enzymes which are active in dissolving the food reserves.

He noticed, that the impermeability to any solute is not per-
fect lt;=ince with time, small quantities enter the grain at the
germinal end. This is the place where selective permeability is
located. The cutinised inner integument, on the other hand,
remains impermeable to salts for a considerable time. It is be-
cause of this property that osmotic cells could be constructed
to good effect with these parts of the testas of several seeds:
the general testa, e.g. Collins (1918), Brauner (1928), Gure-
wiTSCH (1929). According to Rippel (1918), however, the inner
integument of grains should prove to be httle suited for such
purposes, because it is cutinised and does not readily imbibe
water. Pfeffer stated, in 1877 already, that quot; kern Kork und
keine Cuticula absolut impermeabel für Wasser istquot;. Rippel s
opinion did not keep the other investigators back.

Gurewitsch (1929) determined the permeability of the ge-
neral wheat testa, by measuring electrically its degree of per-
meabihty to various ions. An electric current which passes
through a solution of an electrolyte causes the ions to move in
the direction of the poles. When the vessel contaimng the solu-
tion is divided in two by a diaphragm constructed with a piece
of testa, the ions have to move through this diaphragm and the
resistance offered is quantitavily a measure for the lonpermea-
bility of the membrane.

Ions which increased the rate of swelling of colloids did per-
meate at a faster rate than those which retarded the swelling.

The membrane itself does not swell easily. The effect of cer-
tain ions, therefore, could be studied with accuracy.

The membrane should consist of a fine network of micels with
intermicellary pores. The solvent is taken up between the mi-
cels, the ions influence the volume of the intermicellary system.
Alcohol causes an irreversible increase in size of the intermicel-
laries. Substances, like iodine, mercuric chloride, osmic acid and
organic dyes, which always pass through the membrane, are
marked by their adsorption by the membrane. These substances,
therefore, should pass through the micels themselves.

This explanation of Gurewitsch shows a complete parallelity
with the one of Schonfelder (1930) for the permeabiUty of
protoplasm. In both cases the explanation requires a joint appli-
cation of the ultrafilter and adsorption theories.

Little information is available about the osmotic values of
the seed contents. Because of the dry condition of the seeds,
they cannot easily be determined. Bouyoucos and McCool
measured osmotic values of ground seed after mixing the meal
with water. They found osmotic pressures for A. sorghum of
about 7 atmospheres. In how far this value corresponds with the
one prevailing under natural conditions escapes analysis.

Shull (1913) determined the capillary and imbibition forces
of Xanthium seeds and found these to be very high. Very little
water only needed to be imbibed, however, to cause a conside-
rable drop in these forces.

The sole reason for mentioning these factors is to emphasize
that the Çio of the osmotic value is only Httle influenced by a
change in temperature. The rate of uptake of water varies
chiefly because of the presence of non-osmotic factors regulating
the rate of entrance of water.

The water that enters the grain causes swelling. This swelling
will be caused principally by the swelling of starch. Both the swel-

ling force and the osmotic force influence the absortion of
water. The maximum capacity of the grain to absorb water
depends largely upon the elasticity of the testa. The behaviour
of the germ, however, is also of great influence.

Own experiments. Seeds, that are steeped in water which just
covers them, hardly suffer from oxygen deficiency. The varieties
of A. sorghum, used in the experiments, germinated at a very
fast rate. At 25°, 30°, 35° and 40° the absorption process had
practically come to an end after 30 hours steeping. About that
time the radicles appeared and absorption was no longer studied
on seed, but on germinating embryos joined to their reserves.
It was only in case the exchange of oxygen between the seeds
and the air was prevented, by using deep steeping vessels filled
to utmost capacity, that no germination took place.

It is probably due to the use of deep vessels for steeping, that
Brown (1909, 1912) was able to plot absorption curves over

The absorption of water by seeds may be represented by a
graph. Brown (1909), Brown and Worley (1912) and Shull
(1920) have tried to give an explanation of the curves they found.
As the influence of the temperature will be discussed separately,
the general information to be obtained from any one curve will
be given first.

Shull (1920) produces a curve for the moisture intake in
Xanthium, to which he applies the formula:

wherein y =■- total percentage of water already taken up at any
given moment, % -- time elapsed since the beginning of soaking,
a, b, c are constants.

Shull observed that the constants a, b, c are not the same
for every part of the curve, since he could divide his curve into
three component parts, each having its own values for the con-
stants. This formula, with different constants, could be applied
to all absorption curves which Shull plotted for Xanthium
seeds, split peas and maize.

The formula does not tell us anything about the processes
which take place inside the seed during absorption, nor does it

give an idea of the exact place where the three components
meet at different temperatures. It would be interesting to know
if a relation exists between these places and if they express some

I am not able to contribute anything to this side of the pro-
blem since the formula of Shull does not appear to be appli-
cable to my curves. This, however, does not prevent an ana-

The absorption of water shows an initial rate which is very
high Without doubt this is largely due to imbibition by the
testa The absorption rate thereafter decreases gradually, as
saturation increases. The absorption rate is not a logarithmic
function of the time.

The process becomes still more complicated, when the influence
of the temperature on the swelling process is considered.

From a physico-chemical point of view the temperature influ-
ence on sweliing has not been investigated. Hennemann (1929)
has stated that swelling is influenced by the antagonistic influ-
ences of cohesion and hydration.

Because the sweUing of seeds is such a comphcated process
it was hoped to simplify the case by observing the swelhng of
ground-up seeds. The experiments performed to this purpose
cannot claim a high degree of accuracy, they merely give quah-
tative information.

A small quantity of meal was shaken with water in a graduated
measuring cylinder of 10 cm«, divided in tenths of a cm«. As soon
as the material had settled, the volume was read. Ihe cylmders
were then placed in incubators, kept at different temperatures
and the volumes were read again after certain intervals. Ihe
results are tabulated in table III.

The sweUing of meal is influenced by the temperature. This
sweUing takes place at a very fast rate. F.qailibrium is reached
in about 30 minutes time. This equüibrium proves to depend

Meal was shaken with water and allowed to settle. The cylm-
ders were placed immediately afterwards in incubators at dif-
ferent temperatures. After one hour they were removed, a ter
the volume had been read, and transferred to a room at 15
An increase in volume was observed in those cylinders which
came from cooler places, while in the others a decrease m volume

It had still to be determined in how far the sweUing of the
meal was due to water only, for dissolved material from the seed
might influence the process. About equal weight quantities of
meal were shaken with water and with N: 1000 solutions of
NaCl and CaCl^. The results are presented in Table IV.

The swelling of meal in water differs from that in dilute solu-
tions of electrolytes. In the latter instance there is a continuous
decrease in the volume, while with water only the original
volume of the swollen material remains practically constant.
The shght decrease in volume, noticeable towards the end of
the observations in some cases of Table III, has to be ascribed
to the passing into solution of the electrolytes which are present
in the meal.

The seed contents absorb different quantities of water at
different temperatures. This deduction is of importance for the
explanation of the absorption curves of the seeds.

Towards the end of the absorption period the curves run
parallel The general tendency shows, that a continued, very small
absorption may be expected. For the curves at 25° and higher,
the absorption rate, at least during the last six hours, showed
an increase over the expected one. This must be ascribed to
the fact that the seeds were actually germinating at the tmie,
as could be observed by the appearance of a number of roottips.
The curves have been plotted as they would probably have
continued their course, in case this compHcation had not arisen.
The parallelity of the last parts of the curves indicates that the
swelUng maximum for the various temperatures has been
approached. This maximum, however, proves not to be mdepen-
dent from the temperature. This contradicts the statement of
CouPiN (1896) that seeds always swell to the same maximum

This intake represents the amount of water which is absorbed
by the testa. This precedes the swelling of the seed contents
and the two processes can hardly be separated from one another,
in particular when a number of seeds are used.

The testa itself wil hardly affect the swelling process, as the
localised absorption of water by the seed contents is of predo-
minating influence.

The influence of the temperature on the swelling process is
very striking. This can be demonstrated best by comparing the
velocities of water intake at various tem.peratures, after certain

15%

3.8

2.3
1.7

1.4

17%	20%	23%	25%
4.8	2.6	2.5	2.4
3.0	2.2	1.8	2.0
2.3	2.1	1.9	2.0
1.6	1.5	1.8	1.9
1.4	1.4	1.6	1.8

0,0 ratios at different stages of the swelling process.

equal weight percentages of water have been absorbed. The Q^^
ratios have been determined for the times that 15, 17, 20, 23
and 25 per cent of water had been absorbed.

The remarkable feature of the swelUng process is. that the
temperature ratios increase with a rise in temperature, which
indicates that the intake of water is inhibited at low tempera-
tures, but that the responsible factor decreases in influence as
the temperature rises. These ratios, however, show a steady
decrease as the amount of absorbed water increases and this
must express that during the first stages of the absorption pro-
cess something takes place inside the seeds at a diminishing rate
which accounts for this phenomenon.

When considering what material is used, it seems possible
to find an explanation by advancing the following hypothesis.
The temperature ratios give expression to the differences in the
rates with which water is absorbed by the colloids, men equal
weight percentages of water have been absorbed, more water
can be bound at higher temperatures than at lower ones, wth
constant temperature the quantity of water that can stiU be
bound decreases with time. The sweUing of dehydrated colloids
takes place at a very high rate when the temperature is high,
— the membrane being more permeable, — at a low tempe-
rature the permeabihty limits the absorption. Thus permeabi-
hty is a hmiting factor. It is due to the nature of our material
that some of the temperature ratios appear remarkable. The
drier the material, the higher these ratios are at high tempera-
tures. The more the seeds approach germination, the more the
temperature ratios approach, which are usually considered to

In aU investigations, where temperature ratios Q^^ have been
determined, the material consisted of organs or tissues which
were functioning normally, i.e. they were saturated with water.
In the present instance, however, little water only is present
and that in such a small quantity that the material cannot be
taken to be quot;normaUyquot; active. The only physiological process
that can be traced in seeds is respiration, and even that takes

place at an extremely low rate. The moment that water enters
the seeds, aU fimctions of the protoplasm can start, and the
intensity largely depends upon the amount of water present.
This saturation again depends upon the temperature, as is
shown in the next table.

The fact, that the water absorption depends upon the tem-
perature, should show a parallel in observations made on plas-
molysis. A large degree of parallelity does exist between the pro-
cesses of sweUing and plasmolysis, though the direction of the
passage of water is opposite in the two. If a principal difference
does exist, it must be looked for in the membranes, for in swel-
Ung a semipermeable cellulose membrane regulates the process,
while in plasmolysis the protoplasm is the layer involved. The
influence of the protoplasmic membranes inside the seed may
be neglected for the moment. Before comparing my observa-
tions with those of other authors, who studied sweUing on seed,
I shaU refer to some observations on plasmolysis.

Delf (1916) published an almost classical study on the sub-
ject. She observed that the shrinkage of a tissue followed an
almost logarithmic course, but as she probably expected such
a relationship between the percentage contraction and the time
of contraction, she selected from several duplicate determina-
tions those data which actually did show this. Apart from this,
she observed that the rate of plasmolysis was high at high tem-
peratures and low at low temperatures, in particular during
the first stages of plasmolysis. Her calculated coefficients of
increase are reproduced here:

The ratios increase with a rise in temperature. In either expe-
riment the protoplast showed a tendency to contract at high
temperatures. This tendency became noticeable already at 35 .
and the values for the higher temperatures have been compu-
ted from short time observations, during which the protoplasm
could be taken not to be injured by the temperature.

De Haan (1933) showed that the increase in permeabiUty
with temperature is due to the influence of the temperature
on the swelling of the protoplasm. He summarised his obser-
vations as follows: quot;Die Quellung erfolgt bei niederer Tempe-
ratur langsamer als bei höherer, folglich wird auch die Permea-
bilitätszunahme bei niedriger Temperatur langsamer statt inden.
Das (3,0, das dabei erhalten wird, ist auch für einen QueUungs-

While this observation in itself can be corroborated, it is
probably the explanation offered by Irwin (1928), which will
prove to be most helpful. In her study: On the accumulation
of dye in Nitella, she describes that the ceUs of Nitella show a
remarkable faculty of absorption. The initial absorption rates
show temperature ratios which agree with those found m my
experiments, viz. 5.9 to 4.6. She considers that the absorption
is the result of a chemical combination of the dye and the cell
contents In the theoretical discussion of the experimental
data, she comes to the conclusion that this explanation fits the

There is no reason for supposing that water, which is absor-
bed by seeds does not combine with the coUoids; as a matter
of fact' this would be contradictory to the experimental evidence.

The remarkable temperature ratios may be explained by the
sweUing of the coUoids inside the seed; the rate of sweUing of these

A short survey of previous observations made on sweUing
seeds may be given at present.

Dimitrievicz (1875) observed already that the swelling rate
was increased by a rise in temperature, and he pointed out that
this had to be of influence on the velocity of the germination
process, since a certain quantity of water has to be present before

Eberhart (1906) noticed that the germinal parts of grains
absorbed more water within a certain period than the apical
parts. This phenomenon itself was independent from the tem-
perature. He stated that seeds of wheat and barley sweU to the
same maximum volume, though the time required to reach
this state is dependent on the temperature. His observations
were made over 240 hours! We are not informed how it was
possible to suppress germination.

Brown and Worley (1912) measured the weight increase of
samples of seeds which were steeped in water at 3.8°, 21.1° and
34.6°. The weights were determined after fairly long intervals,
altogether five over a period of 90 hours. They were presented
as graphs. The temperature ratios are fairly high, as they remar-
ked, viz.:

They explained the fact, that the velocity with which water is
absorbed nearly is an exponential function of the time, with
the Hydrone Theory of Armstrong.

Denny (1917) found a much lower temperature ratio, 1.6
to 1.3, while the ^^o decreased with rising temperature.

Shull (1920, 1924) found a close agreement between his ob-
servations and those of Brown and Worley. He considers that
the lt;2io values of 1.55 to 1.83 should indicate that absorption

KonÏdo (1923) again noticed that the total volume of rice
seeds depends upon the temperature and the time, the swelling
rate again being dependent upon the temperature.

Other important observations on swelling are those of Stiles
and j0rgensen (1917), while Stiles (1924) gave a survey of
Permeability.

Stiles and J0rgensen immersed smaU discs of storage tissue
of potatoes and carrots in water at different temperatures.
Changes in weight were recorded and the amount of water
absorbed plotted against the time. They noticed an increase in
the absorption rate with rising temperatures up to a critical
temperature. Prolonged exposure led to a rapid loss of weight,
probably due to protoplasm becoming injured and no longex
functioning as a semipermeable membrane. This agrees mth
the observations of Delf. Stiles and J0RGENsen find the follo-
wing temperature coefficients:

The effect of high temperatures made it difficult to obtain
rehable information about the absorption under those circum-
stances In the graphs which they produce, it is evident that
some pronounced change takes place in the material. Conside-
ring that the material was saturated to utmost capacity with
water is is not remarkable that it should prove to be very sen-
sitive to heat. The loss in weight was the result of a contraction
of the tended cellwalls, which pressed out water.

In seeds I have not obtained a clear confirmation of this
point, though some obvervations show that the same may hap-
pen Seeds that absorbed water at 45° did so very rapidly. Only
a few embryos developed, and not further than the initial stages
of root growth. Not a single one produced a seedhng. A remar-
kable large number of seeds showed drops of water oozing out
some time after being put out to germinate. The appearance of
moisture at first was taken to be a sign of infection by some
micro-organism. As none could be observed shortly after their
appearance, another explanation had to be looked for. and he
most hkely one is that of Stiles and J0rgensen. viz. that the

living seed had died after exposure to heat and that a contraction
of the walls caused excessive water to be pressed out.

As seeds which did show such drops never did germinate,
the presence of drops was taken to indicate death after sweUing.

Another quantity of the seeds became cracked. The latter
condition is the result of absorption being carried to such a
point that the elastic seed coat could extend no further and had
to give way. This also was fatal. I consider it likely, that seeds
with lowest viability wiU succumb due to exposure to high
temperatures much sooner than perfectly sound seeds. The first
wiU be kiUed before the seed contents have absorbed so much
water that the testa cracks, the others wiU die only afterwards.

It was of interest to know what manner of sweUing cracked
seeds would show. A hundred seeds — Dwarf YeUow Milo —
were steeped in water of 55°, and the absorption of water was
determined after certain intervals. After about 5 hours, many
seeds showed cracked testas. As the weight remained more of
less constant from six hours steeping onwards, the seeds were
dried back to approximately the original weight in vacuo over
concentrated sulphuric acid. Thereafter they were steeped once
more in water, now at 30°, and the weight increase was compared
with that of another sample of sound seed. These last determina-
tions are reproduced in table VII.

While the rate of absorption at first is much faster for the
cracked seeds than for the normal ones, the total quantity of
water absorbed by the first is less. The osmotic forces which are
active in the normal seeds have been eliminated in the cracked
material. This then may explain the difference in the rate of
absorption as noticed by Heinrich (1913).

We may summarize the factors which are of influence on the
swelHng of seeds as follows:

1 The permeability of the selective semipermeable membrane.
This permeabUity, as well as that of the protoplasm is
increased by a rise in temperature, cf. Hennemann and
de haan.

2.nbsp;The sweUing force of the coUoids. The final amount of water
' absorbed by the coUoids increases with a rise in temperature.

3.nbsp;The osmotic forces. These are only Uttle increased by higher
temperatures.

The influence of salt solutions hardly needs a lengthy discussion
after the absorption of water has been analysed. The seed is
enclosed by a semipermeable membrane which shows a locaUsed
area of greater and selective permeability. By various experi-
ments it has been shown that this part of the testa is fuUy
semipermeable for a certain Umited time. This time has been de-
termined to be approximately 24 hours. This is in agreement with
observations by other authors on gramineous seeds.

When a grain is steeped in a salt solution, the water from the
solution is absorbed and the rate depends on the osmotic con-
centration of the steeping solution.

In the experiments very dilute concentrations have been used,
viz. 0.1 per cent CuSO^, 0.25 per cent Uspulun and 2 per cent
CuSO^. The latter solution was appUed for one hour only and
cannot have had any noticeable influence upon the absorption
rate after the seeds had been thoroughly washed. The molar
concentration of Uspulun cannot be given, since the exact formula
is unknown. Thus the 0.1 % solution of coppersulphate only
remains. The molar concentration is iV/125, and this cannot
have influenced the absorption rate to any large extent.

In no case can one expect an acceleration of the water ab-
sorption to result from the appUcation of desinfectant solutions.

The term germination does not lend itself to a clear definition.
Kisser (1932) has given a contribution in which he tries to clear
this point. He gives a survey of the definitions of previous
authors and this is shortly reproduced here.

Nobbe (1876) considered the swelling of seeds as a mechanical
process, preceding the development of the embryo. As a result
of swelling the ceUwaUs were stretched but no change m cell
numbers nor growth of the ceU walls was caused by it. The
chemical changes in the food reserves of the f.eed facihtated sub-

sequent growth of the embryo. Germination commenced at the
moment, that the radicle, which had increased in size by cell
division, pierced the testa.

Detmer (1880) identified the absorption of water by the seeds
with the beginning of germination.

Klebs (1885) regarded germination as a number of consecutive
processes, as water absorption, piercing of the testa etc.

ScHMiD (1902) defined germination as the moment at which
the testa was broken by the embryo.

Lehmann and Aichele (1931) describe germination as the
beginning of a new phase of life, under influence of external
conditions. Inside the germinating seed a number of processes
take place, which separately cannot be considered as germination,
but only the sum total of all individual processes constitutes
germination.

Kisser himself considers that germination commences at the
moment at which the embryo passes from its phase of relative
rest to a phase of growth. He suggests that this should be de-
termined by measuring the length of the radicle.

However correct this definition may be, it is but hkely that
it will not find ready application, owing to practical difficulties.

It is still more difficult, according to Lehmann and Aichele,
to determine the end of the germination process. Gradually the
embryo develops into a seedhng and the seedling into a plant.
Clear limits do not exist. They cite the quot;Technische Vorschriften
für die Prüfung von Saatgut, 1928quot;, which like the quot;Rules for
Seed Testing, 1928quot;, consider those seeds as germinated which
show a normal sprout and a root with roothairs, but principally
those which may reasonably be expected to continue their
development under favourable conditions. General experience is

The official ruhngs, to a certain extent, have simphfied the
general position and while the definition remains rather vague it
is more readily apphcable than the one of Kisser for the be-
ginning of the germination process.

A seed was considered as germinated when either the radicle
had developed normally or adventitious roots had developed,
so that the embryo possessed organs for absorption of water and

other substances which could function if the seedlings were
developing in soil. The plumule had to develop so far that it did
stand away from the scutellum; this includes that it did react
to its position and showed geotropic response. The length of the
plumule was not considered of importance, neither the fact that
the first blade could still be enclosed by the coleoptile.

When counting the number of seeds that had germinated, I
foUowed, as far as possible, the Rules for Seed Testing. Because
the average germination time had to be determined, the germi-
nated seeds were counted and removed every 24 hours.

In the tables presenting the results of the germination ex-
periments No. XII—XVIII, the first double column deals with
untreated seeds, steeped in water only. Each table gives the
results obtained at one temperature. As the influence of des-
infectants was studied at the same time, the various columns

For every sample the average germination time has been cal-
culated. These figures have been used to calculate the means for
the samples soaking from 1 to 6 hours, from 1 to 12 hours and
from 12 to 36 hours. They are presented in the following table.

While there is hardly any difference between the values in the
first two columns, a striking difference is noticeable between
them and the values in the last column. It is only at 15°, where
germination proceeds very slowly, that the difference between
the values is smaU. On the whole, the longer period of soaking

has a retarding influence on the rate of germination. When
comparing this with the total number of seeds germinated in
each case, it is clear that the duration of soaking does not in-
fluence the flnal count, except at 40°, where a decrease in the
germination percentage demonstrated the injurious effect of
long soaking.

This was put to a test. 100 seeds were soaked in the usual way,
while a second sample of 100 seeds was placed in a flask with
little water on a shaking machine. 24 hours later the seeds were
put out to germinate and the average germination time was
calculated. The test sample germinated in 3.05 days, the control
in 3.25 days. The temperature varied, since the shaking machine
could not be placed in an incubator at constant temperature.
The difference was not due to a greater absorption of water,
though the shaken seeds absorbed water at a slightly faster rate
than the controls.

The amount of oxygen available to the seeds is increased by
shaking, the shaking itself is of no influence. This could be proved
by another experiment. One set of samples were soaked in water,
about 25 cm3 to 100 seeds, with an oxygen atmosphere above the
water, a second group in stoppered vessels, filled to utmost

capacity with water and a third one in stoppered vessels filled
with boiled water. The samples in boiled water were placed on
the shaking machine. After 24 hours the percentages of water
absorbed were 30.08, 28.85 and 29.37 respectively, while the
average germination times in the same order were 2.80, 2.97

the rate of germination. Whenever the layer of water over the
soaking seeds prevents or obstructs the gas exchange of the seeds,
the germination rate is retarded and the average germmation
time is lengthened. The method as employed in my experiments
gave nearly optimum conditions for development.

Observations of this kind were made by Just. Haberlandt
(1877) had stated that presoaking had no influence on the rate
of germination. Just (1877) remarked that Haberlandt had
not stated the height of the water column in his experiments. J ust
found this to be an important item, since the presence of water
might influence the gas exchange. He observed that seeds which
had been covered by a water layer of i/o-l cm in depth germi-
nated more readily than seeds which had been covered by 4—6

Eberhart (1906) observed that presoaking barley at 10
during 100 hours had about the same effect as 48 hours presoaking
at 20°. If the seeds were soaked for longer times, germination was

Geiger (1928) noted that soaking seeds show inhibited gas
exchange, as graduaUy aU oxygen is used; anaerobic conditions

The influence of the temperature on the rate of germmation
is very pronounced. The influence on the absorption of water
has been discussed already and the effect of the absorption on

the living organism is left for discussion. From the experimen-
tal data we may deduce that a relatively small quantity of
water suffices to start the germination process. How small that
quantity actually is, could not be determined by my experi-
ments, for the seeds could absorb water from the filter paper
after having been soaked.

The average germination time clearly shows the effect of the
temperature. This has been presented in a graph (Klerksdorp
var.), fig. 3. The average of the germination times for the samples
soaking from 1—6 hours has been plotted together with the
averages for the samples soaking from 12—36 hours. The dif-
ferences become more and more pronounced as the optimum
temperature is approached. This must be ascribed to the
inhibiting effect of presoaking. When the optimum has been
passed the injurious effect of the temperature causes a lengthe-
ning of the average germination time.

The averages for 24 hours soaking are about the means of
the values shown in the graph. The varieties Dwarf Yellow MUo
and Dwarf Hegari have been soaked for 24 hours at all tempera-
tures. The values for the absorption of water, average germina-
tion time and final count have been hsted with those of Klerks-
dorp, which underwent the same treatment.

Absorption, Av. Germination time and Final count of the
varieties after 24 hours soaking at different temperatures.

Final Count out

of 50
1	2	3
42	48	29
43	46	39
48	49	47
47	43	47
45	37	33
41	12	31
—	—	—

3 Dwarf Yellow Milo.

three

For Klerksdorp Kort Rooi, the optimum temperature lies
between 30° and 35°. The final counts do not show any difference
in the germination percentages. From the graph, fig. 3, however,
it is evident that the optimum temperature is influenced by the

presoaking period. WTien this period is short, there is no dis-
tinction to be made between the influences of the two tempera-
tures.

For Dwarf Hegari the shortest germination time equals two
days. The final counts at the temperatures 25°, 30° and 35° show
such pronounced differences, that the optimum temperature
appears to be nearer 25° than 30°. Strictly this applies only to

the experimental conditions. Dwarf Hegari proved to be the
fastest in germination of the three varieties at medium tempe-
ratures. The radicle-tips became visible: at 15° after 48 hours.
20°, 24 hrs.. 25°, 12 hrs.. 30°, 9 hrs., 35°, 5 hrs., 40°, 12 hrs. The
variety appears to be very sensitive to heat.

Of the three varieties the South African one seems to be the
most resistant to heat. Another important character of this
material is that they absorb relatively less water than the

Swanson (1926) ascribed the rate of water absorption by
A sorghum to the structure of the seed coat. The absorption
rate of the three varieties showed great variation. SmaU diffe-
rences in the structure of the testas could be observed, but these
cannot be held to be responsible for the observed variation.
All evidence, as has been stated, points to the absorption taking
place at the micropylar end of the seed, and here no remarkable
difference could be observed.

My observations agree weU with those of Tjebbes (1912).
who observed that the germination rate of sugar beet seeds
became accelerated by temperatures higher than 30°. but the
seedhngs were sometimes found to be abnormal. He saw that
at 40° a noticeable decrease in germination could be observed,
while at 50° no germination at all took place.

Abnormal seedlings were noticed in my experiments frequently
at temperatures above the optimum. The Klerksdorp variety,
with a high optimum, showed relatively few abnormal seedhngs
while the two American varieties germinated badly at high
temperatures. The abnormal feature was that the radicle did
not grow more or less straight, but developed spiral-wise. for-
ming either a longitudinal or a ring-spiral. Because this was
observed more frequently in Klerksdorp var. m desmfected
seeds, it was at first taken to be a result of desinfection. Expe-
riments showed that this phenomenon could result as weU
after short time exposure to high temperatures and seeds which
had been soaked at 55°, for one hour, showed large numbers of
abnormal roots. This has to be regarded as a general symptom

of injury to the root or radicle, irrespective of the cause of the
injury GeneraUy the radicles recovered after a few days at
lower temperatures, as was shown by their further growing
straight out.

Somewhat similar observations were made by Friesen, and
Pringsheim (1928). Friesen observed that the radicles of seeds
which had been exposed to heat or had been treated with che-
micals showed less geotropic response than normal radicles. He
considers this to be the result of a reduction of number of starch
grains in the calyptra. He does not want to draw the parallel
between heat influence and that of chemicals too close, because
the alteration in the behaviour of the root is caused by different

My own observations have shown that the reaction of the
root is independent of the cause of the reaction and is merely
symptomatic.

Reference has to be made to the experiments of Wassink
(1934), which induced my own experiments with a shaking-
machine. Wassink was able to grow Phycomyces in a liquid
medium by shaking an inoculated culture solution. He suggested
that the development of the spores normally is retarded because
they sink in the solution and suffer from oxygen deficiency. The
rate of gas exchange by diffusion is very slow and the oxygen
supply in the immediate neighbourhood of the spores is exhausted
after some time. By shaking the solution, the spores are brought
in continuous contact with a medium with normal oxygen
content, so that diffusion of oxygen from the air needs to take
place through a thin layer of water only and this hardly influ-
ences the diffusion rate.nbsp;.

Seeds also depend upon the oxygen supply for germination.
The paraUel with the material of Wassink is obvious. In this
case however, the conditions which suppress germmation could
not all be eliminated. This may be due to the fact that the seeds

as they are in immediate contact with the air, at most separated
from it by a thin film of water.

As stated in the introduction, the influence which desinfec-
tants may have on the seed and on the germination process has
drawn much attention during the last 20 years.

Some investigators tried to demonstrate that seeds could be
treated in such a manner that, as a result, the growth of the
embryo became accelerated and germination took place, in a
shorter time than normally, while because of stimulation of the
embryo the seedlings might show more vigorous growth than
untreated ones.

Others again considered the stimulation of seeds as not pro-
ved, or otherwise not sufficiently controllable to warrant advo-
cating its general apphcation in practical agriculture.

Many opinions have been aired and data have been collected to
support the one or the other theory.

The principle of stimulation itself has ruled botanical physio-
logy for quite a long time. The controversy was started by the
publication of Raulin (1869), who indicated that some elements,
up to then considered of no value to plant nutrition, were
essential for plant growth. The list of these elements, started
by Raulin with zinc and silicon, has become much extended
since and a large number of elements are now accepted as
essential which formerly were considered as perfectly useless.
Raulin, already, pointed out that the concentration of the
elements in the culture solution was of importance and that
some substances in every concentration had to be considered
as poisons, e.g. silver and mercury. It was Richards (1897),
who interpreted the observations of Raulin by suggesting that
elements like zinc, cobalt, nickel, manganese and others, were
not so much of essential value for nutrition itself as they might
be stimulants for metaboHsm. This remark was made in the
days that Pfeffer and Czapek had built up a physiological
terminology in which the term stimulus had a principal value
and was applied and abused in several instances. Phototropism
and geotropism were processes, wherein a stimulus was per-
ceived by the plant. A minimum stimulus was required to start

off any reaction. The chemical stimulation of plants was reduced
to a phenomenon comparable with others in human and animal
physiology. A substance could be poisonous in large quantities
and would, sometimes, show an enhancing influence on some
process in lesser concentration. Later on this was defined by
Pringsheim (1914), who distinguished between chemical sti-
mulants and nutritive substances by applying the following test:
WTien a substance causes an increase of one third in the dry
weight of a plant when apphed in double the normal quantity
the substance is of nutritive value; when the increase is no more
than one seventh the substance is a stimulant. That this distinc-
tion is merely arbitrary needs no comment.

This hne of thought may have been fruitful to a certam
degree, it has never explained what did happen. Seeds of diffe-
rent plants and, in case of cereals, of many varieties have been
subjected to numerous treatments, so that an extensive htera-
ture has been produced on the subject. The conclusions and
deductions of the investigators sometimes escape analysis,
because of the scantity of information suppHed. Many have been
pubhshed before a standard method for analysis and obser-
vations on seeds was demanded and described by Gassner
(cf. page 40).

The term stimulation is justly vague. Pfeffer (1897—1904)
uses it extensively, likewise Czapek (1922). Benecke-Jost
(1923) state that the term stimulation will have to be
dropped most probably in seedwork, as the observed pheno-
mena could be reduced to catalytic functions. According to
these authors, chemical stimulants have no nutritive value
From this one might deduct that they consider it essential
that no stimulation experiments should be performed
nutritive salts. In Kostytschew-Went (1931) a similar
opinion is stated. The principal experiments which would
support this statement are those of Stephan and Lantz.
Stephan (1929) observed a more rapid germination after stimu-
lation and a greater catalase activity. By what method the ger-
mination rates are determined and compared is not commented.
The catalase activity needs not to be a result of the previous
treatment, but may be a parallel phenomenon with a more

rapid germination in some samples. Lantz (1927) not^ced tha
0 25 per cent Uspulun has very httle influence on the amount
of water absorbed within a given period. No direct relation
between desinfection and catalase production can be found m

^'seTsWation with chemicals began to receive a great
deal of attention since the publications of Popoff and his

'quot;quot;^he basic principle for his investigations was the assumption
that in the course of its life, every organism looses water and
the loss of water causes ageing. Thus ageing is linked to a pro-
cess of dehydration. The rate of vital processes depends upon
he degree of hydration of the protoplasm. When the latter is
increased a greater activity results and such an increase can be
induced by applying certain, weU-chosen stimulants

This point of view, in itself a rather fascinating theory has
started Search workers looking for the adequate chemical sti-
mulants. At first they were found to be rather
increasing accuracy in experimental methods ^n^ thorough
interpretation of the results have gradually reduced the number
to a dwindling few. Some nitrates are stiU considered as stimu-
lants though the objection of Benecke-Jost should apply to
them', while others again look for stimulating substances prm-
cipally among the poisonous seed-desinfectants.

As Senf (1925) stated the case of the adherents of the
stimulation-idea, a treatment of the seed with chemicals may

4.nbsp;increasing the viability of the embryo.
These points may be analysed as follows:

1 The practice of using nutritious substances should be abo-
lished for this purpose, since the object is to study stquot;aulaUon
of the embryo and not the best method of supplying the young
seedhng with food.

2 The observations on the existence of semipermeable mem-
' branes provide the answer. The testa is not easily made more
permeable and if so, at great risk to the embryo only. Greater
permeability may be caused by the application of alcohol and
other substances which cause an irreversible change in the
membrane. These substances are all very poisonous to the
embryo. The use of known seed-desinfectants does not in-
crease the permeabihty and never causes stimulation, much
sooner retardation of the germination process.

3.nbsp;The destruction of spores of fungi can only ameliorate the
conditions for the seedling after development and thus may
influence the germination percentage, as well as the rate of
germination. This will be demonstrated by the results from

Bredemann (1924, 1926), in his plot- and field-expenments.
failed to find a stimulating effect for salts of magnesmm, manga-
nese and potassium, when appUed in concentrations of 3 to 4
per cent during from 4 to 24 hours.

LuNDEGäRDH (1924) Considered that Cu was absorbed from
solutions of copper sulphate. The absorption should take place
periodically and in a serial experiment the periodical stimu-
lations should correspond with the absorption of minute quan-
tities of copper. His method, however, leaves doubt about the
value of the observations, for the small variations in the percen-
tage of coppersulphate in the steeping solution may come within

Kotowsky (1927) drew attention to the fact that the theory
of popoff opposes the theory of semipermeability. He observed
that the testa of cereals prevented the absorption of potassium
nitrate and other salts from solution and washing the seeds for
1 minute resulted already in very heavy losses in the amount
of salts absorbed in the testa. The behaviour of the testa m the
presence of stimulants should be known, before any further
attempt should be made to apply stimulation on a larger scale,

opinion. While for some time associated with Popoff's periodical,
she has apparently become convinced by Gassner and considers
that since the desinfectant, in this case Uspulun, has a killing
effect on the spores of several micro-organisms, it is very likely
that its stimulating effect should be regarded as a result of its
desinfectant properties.

Kisser (1933), in a largely theoretical contribution, considers
stimulation not proved and the possibility of stimulation not
likely.

The present author is in no way advocating the use of any
particular desinfectant and it is partly because of the historical
value of coppersulphate that its effect has been chosen as an
object for closer investigation and comparison with that of one
of the products of modern industry, Uspulun.

The influence of coppersulphate has attracted the attention
of numerous investigators.

VoGT (1926) records the use of this salt in the year 1761, and
consequently it is impossible to give a complete survey of the
development of its application. Many statements have been
made which discourage the continuation of the application of
this fungicide in seed practice. One of the most recent is that
of Heald (1932):

quot;The injury from coppersulphate treatment has generally been
measured in terms of the reduction in the percentage of viable
seeds, which may frequently show a drop from 90 to 100 per cent
germination of untreated wheat to 36 to 60 per cent germination
when given the standard bluestone treatment (1 pound to 5
gallons for 5 to 10 minutes)quot;. This is practically a 2 per cent
solution.

quot;It has been shown that the toxic action of the copper also
causes a pronounced retardation of growth when the treated seed
is planted in the field and that many seedlings which do grow
make an abnormal development, with curved, deformed plumule
and poor root growthquot;.

The question arises, whether the influence of coppersulphate
really is as injurious as stated here. For it has been in use so
long, that one wonders why it has remained so at all, if its action
is as dangerous as stated by Heald; it would be no compliment

In the middle of the last century, Kühn advocated the use of
coppersulphate. Some years afterwards, in 1872, Nobbe wanted
to determine the influence of a coppersulphate treatment on the
germination of seeds which had been threshed by hand and by
machinery. Machine-threshed seed were found to contain some-
tmies up to 20 per cent of seeds which were damaged, with
cracked testas. These seeds, under normal conditions and when
not vitally injured, germinated faster as they could absorb water
more rapidly than sound seeds. They were, however, much more
sensitive to the desinfectant. Nobbe used a solution of 0.1 per
cent and steeped for 24 hours. Hand-threshed seed germinated
faster, after steeping, than machine-threshed. The differences
between the two groups became less pronounced in about three
weeks time. Wheat was relatively more sensitive than rye, barley
and timothy.

Kühn (1873) repHed by stating that Nobbe should have
applied a 0.5 per cent solution for 12 hours only, since then no
injury to wheat was noticeable. In this way the effect of copper-
sulphate became the object for exact investigations and all kinds
of concentrations have been apphed as well as different treat-
ments after the apphcation thereof.

Nobbe noticed that the treatment had impaired the quahty
of the seedlings. Some were rootless, others had injured roots.
This, however, was observed in both the machine- and hand-
threshed material and had to be ascribed to the influence of the
coppersalt on the germ. As Nobbe stated: quot;Die vom gebeiztem
Korn erzeugten Pflänzchen sind nämlich durchweg entweder
gänzlich wurzellos, oder doch mit einem sehr geschwächten Wur-
zelsystem versehenquot;.

Tjebbes (1912) observed that a 2 per cent solution did not
cause any injury to the seeds of sugar beets, provided the seeds
were spread out to dry before sowing, immediately after steeping.
When dry, they could be sown without any danger. Sowing in
moist condition led to heavy losses.

Quanjer and Oortwyn Botjes (1915) used coppersulphate
in a very concentrated solution, which was apphed as a spray.
The argument in favour of this treatment was that the seeds do

not swell in such a solution and no salt can enter the micropyle.
The germs on the testa are killed effectively. In all their experi-
ments. the treated seeds show a shghtly retarded germmation as
compared with the controls. The method itself, from their
accounts, leaves little to be desired. They noticed that a more
dilute solution of coppersulphate, a 2 per cent solution, caused
injury. They present here and there slightly flattered results by
including rootless seedhngs in their germination counts.

This point will always remain a subject for discussion, for
seeds which have produced normal and vigorous plumules may
reasonably be expected to continue growth. The present inter-
national rules have been laid down fairly recently (1928). My
own experiments show that rootless seedlings were not retarded
in their complete development by more than about 24 hours.

During the following years a great effort was made to increase
the size of crops to the utmost. The prevailing conditions
favoured the publications of Popoff and his school. Accordmg
to his theories, one should be able not only to increase the quan-
tity but also the quality of crops, by applying small amounts of
inorganic substances. The outlook was decidly promising, tiU
exact investigations began to shake the foundations of the

It was Gassner (1923, and following years), who exploded
the theory. In a series of pubhcations he gave a reUable basis to
seed work. Germination tests had to be made on 4 X 100 seeds.
The average germination time had to be calculated for every
sample, to enable comparison, together with the germination
percentage. The quotient of the percentage and the germmation
time gave a figure which he caUed the quot;Wertungszahlquot; —
germination value. By comparing these values the results of
different treatments could be determined.

All field tests should be preceded by laboratory tests under
controlled conditions. The effect of any treatment could thus be
studied accurately, interfering environmental factors had to be
eliminated.

In a laboratory experiment it was necessary to wash the seeds
after desinfection to prevent injury and this washing was found
to be quite effective.

In this way he prepared the way for the determination of the
dosis toxica and dosis curativa, the concentrations of the des-
infectant which caused injury to the seed and which prevented

temperature and studied the action of formaldehyde and of
organic mercury compounds. He noticed, that while washing
Xved practically all desinfectant from the testa small
oultities,'traces, could stay behind. As a result root injury
could occur, the other parts of the germ were not injured. Ihis is
indirect evidence in favour of localized absorption. The explana-
tion offered was that some of the desinfectant became adsorbed
bv the testa. A similar observation had been made ah^dy by
Kurd (1921) who observed this for coppersulphate. It is but
likely that the desinfectant wiU enter more or less deeply into
the Lropvle during steeping and that subsequent washing wall
remove only the most readily accessible deposit.
TasLer observed that the influence of the desinfectant
depends upon the temperature. While this quot;^t refers mo^^^
in particular to the mercury compounds and formaldehyde, the
same could be observed for coppersulphate. At high tempera-
tures swelling and development of the embryo take place at a
higher rate and the radicle will pierce the testa much sooner than
at low temperatures, consequently root-injury may become more
frequent. LssNER considered that low temperatures shoT^d
favour desinfection, but other observations made it
to lay down a hard and fast rule. A simple relation between
lengtl of time, temperature and concentration m a desmfectmg

of uspulun, 'W—
_ a mixture of mercuric chloride and coppersulphate -, and
coppersulphate was not influenced by a change in temperature.
TAur(1925) and Nagel (1925) both noted that the tox,c
effect of desinfectants increased with a rise in temperature and
r dosis curativa had to be reduced for both Uspulun and copper-
slhate. No stimulation was observed by Plaut m sugar beets,
:^her in seedling growth or in sugar content of the beets after
applying various desinfectants and magnesramchlonde.

Bfxker (1926) used coppersulphate on wheat and applied
concentrations of 0.05,0.1, 0.25 and 0.5 per cent. After 36 hours(!)
he counted the number of germinated seeds and found a larger
percentage germinated after the 0.1 per cent treatment than in
all others, while 0.5 per cent retarded germination. After 3 days
the difference had disappeared, the injurious effect of the con-
centrated solution remained visible. The time of steeping was
4 hours, at 18°, germination taking place at 10°.

With Uspulun concentrations of 0.05 and 0.1 per cent stimula-
ted slightly, while those of 0.25 and 0.5 per cent retarded germi-
nation. After 48 hours the effect of the highest concentration was
still noticeable, that of the others was about the same.

Rice was found to be stimulated by 0.02 to 0.03 per cent
coppersulphate after 24 hours steeping, but the seedlings were
injured. During later stages of development this effect wore off
altogether. Similar observations were made with Uspulun.

Niethammer advanced the opinion that the stimulating effect
of Uspulun might be due to sterilisation rather than to
stimulation.

Some attention must be paid to the statements of Senf (1925).
His observations on the effect of coppersulphate require careful
analysis. He treats his material, amongst others, with the
following concentrations of coppersulphate:

0.25 % for 30 min. germination percentage 85.3
0.25% „ 60 „nbsp;„nbsp;„ 90.8

Material: wheat, Rimpaus Dickkopfweizen. From these figures
he concludes that the seeds have been injured by the treatment.
The average germination time was found to vary from 3.1 to
3.4 days.

The results with Uspulun have been treated in a similar off-
handed manner. The material for attention is wheat, Kirsche
Dickkopf. With the normal treatment, 0.25 per cent Uspulun

in the average germination time should make one rather cautious
not to rely too much on the deductions made. The material shows
marked variations and the mean error, apparently, has not

treatment are based on the means of the results obtained with a
number of different varieties, which each show a different be-
haviour towards the desinfectant, expose his deductions to
serious objections. It is of httle importance that he finds that
coppersulphate should be unsuitable for desinfection.

To determine the influence of time, temperature and concen-
tration of a desinfectant solution, two concentrations of copper-
sulphate were used, viz.: 0.1 and 1.2 per cent. The seeds were
steeped for 1, 6 and 12 hours at 0°, 30° and 45°, and laid out to
germinate at 30°. A control test was made wherein water took the
place of the coppersulphate solution.

If any simple relation did exist, 12 hours steeping m 0.1
per cent should have an equal effect as 1 hour steepmg m 1.2
per cent The influence of the temperature should become
evident from the results. The results have been tabulated m
Table XI.

Sample No. 24, steeped in water for 12 hours, at 30°, was taken as the
one with which the others might be compared.

Effect of time, temperature and concentration of CuSOt, upon further
development at 30°.

The highest germination values are obtained where the seeds
are steeped in water for one hour only, as long as the tempera-
ture is high. Little difference exists between the values of 30°
and 45°. The low temperature retards the rate of development
and this effect has not yet worn off after about a day and a
half. Longer exposures to 45° cause a rapid drop in the germi-
nation percentage and everything points to the material being
vitaUy injured. At 30° the longer steeping causes lengthemng
of the germination time, which phenomenon is less pronounced
at 0°, though quite prominent.

The temperature influence is here of the same nature as m
the controls. In the dilute coppersulphate series the different
top values agree closely with those of the controls. This agrees
well with the observations made on the rate of water absorp-
tion and germination with the Klerksdorp variety. A slight
retarding influence of dilute coppersulphate is evident. The
more concentrated solution has a pronounced inhibiting influence
on the average germination times and consequently on the ger-
mination values.nbsp;,

seeds exposed to high temperatures. When comparing the fib-
res of samples 8 and 9 with those of 26 and 27, it is clear that
the first values show a great advance over the second ones.
Considering all sets of samples, it appears as if ^he^oppersu^hate
in this case has assisted in the development of the seedlings.
The only explanation which I could find is, that the seedcoat
of the seeds, during the swelling process, cracked and tha
some small quantity of the salt itself has corne -to -nt-^
with the embryo and has counterbalanced the effect of the
temperature. To a certain extent this might be considered as
stimulation, though it is preferred not to use that term since
the term has generally been applied for annbsp;^

mination percentage at normal temperatures. At and below the
optimum temperature there is no stimulation.

The relative toxic effect of cofpersulphate is not increased by
a rise in temperature.

The general development of the experiment was indicated
already after 12 hours. At that time the following samples
were germinating, showing the tips of the radicles: 1, 4, 5, 6,
7. 10, 13, 16, 19, 22, 23, 24 and 25.

This is an ideal illustration of the effect of the opthnum tem-
perature and of the inhibiting action of the concentrated salt
solution, when applied for 6 and 12 hours (14, 15).

The effect of the treatment on the development of parasitic
fungi was carefully noted. Dwarf Yellow Milo, at least the
sample at my disposal, was infected with Helminthosporium.

The controls showed no infections on seed which had ger-
minated within 24 hours. It is possible that some of these seeds
were infected, but the development of the embryo left no
time to observe this. After 48 hours the foUowing observations
could be made:

sample 19, on germinated seeds 23 infections, on rest of 18, 8 mf.
20 „
21 „
22 „

The exposure to 45° for one hour or longer reduces the num-
ber of infected seed to practically nil. It is in agreement with
the general observations on desinfection that the percentage
infection decreases when the temperature is raised above the
optimum of the parasite. The hot water treatment of cereals
requires a 10 minutes steeping in water of 51° to 53°. and a
longer period at a lower temperature appears to be equally
effective. This could be observed on Klerksdorp as well, where
the seeds that had been soaked longer than 12 hours at tempera-
tures close to and above the optimum showed no infection
at all.

The following observations were made on the seeds treated
with 0.1 per cent coppersulphate:

38	„ „ 16,	13
48	„ 16.	8
14	„ „ 9.	5 „
10	„ „ 10,	4 „
13	„ 6,	2 „

sample 2. and 3, roottips, in particular the tip of the coleorhiza,
slightly brown, roots developing normally, though
slower than in controls.

„ 4. 5, 6, 7, roottips slighly brown, roots developing slo-
wer than controls, root-hair development delayed.

8 and 9, germinating slowly, no signs of injury.
After 72 hours more infection appeared on 1, 2 and 4, the
others remained perfectly free from infection. The radicles were
generally thinner than in the controls and in some the develop-
ment of root-hairs took place rather late. It was impossible to
determine in how far this is an important feature, it did occur
in some of the controls as well.

The treatment with 1.2 per cent coppersulphate resulted in
complete desinfection. After 48 hours many seeds had deve-
loped normal plumules, but as no radicles were present or any
other roots, the seeds were not considered to be germinated. It
appeared as if the coleorhiza could not be pierced by the radicles
and in some cases the radicles themselves were injured. The
colour of the affected parts was deep brown to black.
After 48 hours the following notes were made:
sample 10, plumules normal in 63 seeds, but roots undeveloped.

Table XI shows that within 24 hours these seeds had all
produced roots. In some the radicle had finally pierced the
coleorhiza and was visible as a white tip, in others adventitious
roots had developed.

A much better comparison between the effects of coppersul-
phate and Uspulun could be made on the Klerksdorp seeds, as
reported in the tables XII to XVII. They are shown in the
germination values.

38
41

41
43

39
43

37
39

34
37
41
41
41
44

44
36
36

41
39

Before considering these data, however, we have to know the
mean error of the material. The figures of Table I give a mean
germination value of 100 ± 1.1, the mean error does scarcely
exceed one per cent. The germination values are calculated and
tabulated for the main experiments, in the same manner as
the average germination times of table VIII have been calcu-
lated, and are presented in Table XIX.

The values have been calculated to the basis of 1 to 6 hours
steeping in water at 35°.

The values obtained at 15° have not been included in the
table, since the desinfected seeds develop at a much slower
rate than the controls and do not require any further discus-
sion.

Considering the mean error of the material nothing points
to a favourable stimulating effect. Furthermore these figures
show that the effect of coppersulphate about equals that of
Uspulun, so that there is httle preference to be made for the
one or other desinfectant. Both have a decidedly depressing
influence on the germination process.

Very little distinction can be made between the values of
the controls at 30° and 35°, except that prolonged steeping at

the higher temperature depresses the germination value. This
is a regular occurrence, but most striking at high tempera-
tures. The values for 1 to 6-, and for 1 to 12 hours soaking are
practically identical. While Table XI showed that a one
hour's period of soaking may be considered more advantageous,
the desinfection of the seed is frequently not completed within
such a short period. The notes made during the course of the
experiments support this.

At 15° the controls showed, after 3 days, 16 per cent of the
total number of seeds to be infected with Helminthosporium,
and one seed infected with Fusarium. Those treated with 0.1
per cent coppersulphate showed only 1 per cent infected. A
shght increase in the number of infected seeds was observed
during the foUowing days, in the first case by 1 per cent, in
the second 1/2 per cent. One infected seed only was found in
the series treated with 2 per cent coppersulphate, in the Uspulun
series nearly 8 per cent were infected, and these seeds had all
been treated less than 6 hours.

At 20° the infections were counted after 48 hours. At that
time only 6 per cent were infected in the controls, but this

number increased to about 8 per cent during the next 24 hours.
Of the seeds treated with 0.1 per cent coppersulphate only 1
per cent became infected, all when treated less than 6 hours;
2 per cent coppersulphate caused complete sterilisation; Uspulun
again required about 6 hours before desinfection was completed.

At 25° the conditions for the development of the seed became
much better, while those for the infection had gone backwards.
The required times to bring about desinfection are reduced and
no fungi were noticeable on seeds which had been desinfected
longer than 4 hours.

At 30° infection of the controls was visible here and there
after 24 hours. In the one sample, steeped in water for 1 hour
only, infection was rather severe, in all others the seedhngs
outgrew the fungus.

On some treated seeds Helminthosponum developed, but not
until another 24 hours later. The total percentage of infected
seed, in all series together, did not exceed % per cent, and
again the 2 per cent coppersulphate was free from infection.

At 35° hardly any fungi did develop. The desinfected seeds
showed none at all. Some of the seeds of the american varieties
were killed by heat as was concluded from the appearance of
droplets on the surface, in particular the seeds of the variety
Dwarf Hegari appeared to be injured. Some of the seedlings of
this variety showed rootlets with injured tips. This injury re-
sembled that caused by 0.1 per cent coppersulphate and by
Uspulun, the tips of the radicles were black. Adventitious roots
appeared very soon and a complete seedling emerged well within
24 hours later. In the desinfected seeds several showed pronoun-
ced signs of root-injury, though here as well the injury was of
passing nature.

At 40° the two american varieties were perfectly free from
infection, but nearly killed by heat. All the seeds that did not
develop showed droplets. Klerksdorp had practically no infec-
tions at all, the influence of the temperature was noticed by
the curling of the roots, in particular of those seeds which had
been treated with Uspulun. 2 per cent coppersulphate and the
more dilute concentration also caused root-injury and root-cur-
ling but not to the same extent.

From these data no deduction can be arrived at which would
support the opinion that seed desinfection causes stimulation.
In all cases the desinfected seeds show a retarded development,
the degree of retardation being about the same for the three

The toxic action of coppersulphate does not increase with a
rise in temperature in my experiments, Uspulun, however, has
a pronounced toxic effect when the seeds are steeped for periods
longer than 12 hours. This effect might perhaps be ascribed to
decomposition of the mercury-compound at high temperatures,
as a result of which inorganic mercury salts pass into solution
and injure the germs. Coppersulphate is not influenced by a
change in temperature and an increase of the toxic effect of
coppersulphate could be associated only with a direct influence
of this salt on the radicles of the germinating embryos.

Observations have shown that generally those seeds which
are either dead or show a low viability are infected with Hel-
minthosporium and succumb to the infection. In desinfected
samples the percentage of seeds which might have been conside-
red vitally injured by the treatment corresponds with that of
the seriously infected seeds of the controls. The amount of
damage, as a result of desinfection, closely corresponds with the
percentage of seeds with low \iability.

Injury can hardly be prevented when a desinfectant solution
is apphed. If such a solution has any killing effect on the spores
of micro-organisms, it must be a potential danger to the em-
bryos. If desinfection leads to selection of the material, this
should not be considered a disadvantage. In agricultural practice
this will be preferable to an infected crop.

While the apphcation of mercury compounds wiU have to
be preceded in tropical and sub-tropical countries by a deter-
mination of the dosis toxica and of the dosis curativa of these
substances, the simpler inorganic salts do not require these tests

The results of the experiments described here show, that the
practicability of the apphcation of coppersulphate should obtain
further attention.

1.nbsp;Three varieties of Andropogon sorghum Brot. have been used
for the investigations: Klerksdorp Kort Rooi, from South
Africa, and Dwarf Hegari and Dwarf Yellow Milo, from the
United States. Of these the first one was employed in the
major number of experiments.

2.nbsp;The swelling of seeds at different temperatures has been
observed. The swelling maximum was found to be dependent
on the temperature. Ground-up seeds, meal, showed a
similar phenomenon.

3.nbsp;The principal factors controlling the swelling process have
been analysed: the rate of permeability of the testa is a
limiting factor in this process, while the degree of permea-
bility is influenced by the temperature,

4.nbsp;The temperature ratios of the sweUing process have been
indicated to be determined by the degree of saturation of
the material.

5.nbsp;The germination rate has been observed to be inhibited by
a period of presoaking of more than 12 hours, shorter periods
of presoaking causing no principal differences.

6.nbsp;The optimum temperatures for germination have been
found to differ in the three varieties.

7.nbsp;Abnormal root development in the seedlings has been obser-
ved and interpreted as a symptom of a sUght degree of root
injury; the causal factors in these experiments were the
influence of heat and of desinfecting solutions.

8.nbsp;The appUcation of desinfecting solutions, coppersulphate:
0.1 per cent and 2 per cent and Uspulun: 0.25 per cent, did
not increase the germination value of the samples which
were tested; no stimulation could be noticed.

9.nbsp;The temperature has no influence on the degree of toxicity
of a 0.1 per cent coppersulphate solution; the dosis toxica
of Uspulun decreases at higher temperatures.

10 A remarkable effect of 0.1 per cent coppersulphate at 45°
quot; has been observed (page ), for which a tentative expla-
nation is offered.

The study of the behaviour of ground-up seeds in the presence
of solutions of electrolytes offers scope for further investiga-

*'°amp;^eUing and germination are linked processes. To determine
the moment at which germination commences, it is suggested
that this may be effected by exposing the seeds, after different
periods of presoaking, for a short time, to a high teiin^)erature
of about 55°; seeds which are germinating will then be killed,
those which have not yet started growth wiU survive.

I want to express my sincere appreciation of the hospitaUty
extended to me at the Botanical Institute of the University
to Prof. Dr. F. A. F. C. Went and Prof. Dr. V. J. Konings-
berger, as successive Directors of the Institute.

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Het temperatuurquotient, Q^t^, is, bij processen van water-
opname en afgifte, afhankelijk van de graad van verzadiging
met water. Bij alle proeven over Q^q's dient men zich hiervan
rekenschap te geven.

Het is onmogelijk in de voedingsphysiologie der planten te
onderscheiden tusschen voedingsstof en prikkelstof.

Er is geen reden om aan te nemen dat kopersulfaat in het
bijzonder beschadiging van zaden tijdens het beitsen veroorzaakt.

De proeven van Waldron laten niet toe te besluiten dat een
toename van het 1000 korrel gewicht aUeen aan Helminthospo-
rium-infectie wordt toegeschreven.

De proefopstelling van Sumner, om het bestaan van be-
schuttende kleuren te bewijzen, is foutief.

De proeven van Sierp maken waarschijnlijk dat de beweging
van huidmondjes aan een photochemisch proces is toe te
schrijven.

De onderzoekingen van James wijzen er op dat het geenszins
zeker is dat de sapstroom bij worteldruk door de houtvaten
plaats vindt.

De samenstelling van de Flora capensis en die van de Zuid
Afrikaansche flora in het algemeen wijzen op een migratie in
zuidelijke richting onder invloed van klimaatsverandering.

De omgrenzing van de famüies Liliaceae en Amaryllidaceae
van Hutchinson verdient aanbeveling.

1	\v. Germ. Time	Seeds
% Water	in days	germinated
28.61	3.96*	45
29.18	3.68	46
28.33	3.70	43
28.42	3.70	44
29.56	3.77	44
26.85*	3.80	44
28.92	3.67	42
27.45*	3.72	46
28.76	3.70	42
28.61	3.78	46
Av.	Av.
28.67 ± 0.26	3.75 ± 0.025	44

Time of steeping in hrs.....	I	2	4	5 6		12
per cent weight increase. . .	11.01 11.17	14.58 14.72	20.74 20.53	23.21 23.11	23.85 24.35	28.86 28.88
	11.09	14.65	20.64	23.16	24.10	28.87

Temp.	Volume in cm®						% Incr.
0	6.5	6.5	6.5	6.5	6.4	6.4	0
16	6.4	6.6	6.6	6.6	6.6	6.5	3
20	6.5	6.85	6.85	6.85	6.8	6.8	5 fi
30	6.2	6.8	6.8	6.8	6.8	6.8	9 11
35	6.3	7.0	7.0	7.0	7.0	7.0	9 11
Time	0	30	60	90	120	300	min.

Temp.	0	30	60	150	210	minutes
0	6.55	6.55	6.55	6.55	6.5	Hfi
0	7.5	6.85	6.8	6.75	6.7	NaCl
	7.5	6.8	6.8	6.7	6.7	CaCl^
20	6.75	6.8	6.8	6.8	6.8	H^O
20	7.1	6.85	6.8	6.75	6.75	NaCl
	7.6	6.8	6.65	6.6	6.55	CaCli
30	6.4	6.6	6.6	6.6	6.6	HJD
30	6.9	6.8	6.75	6.75	6.7	NaCl
	7.35	7.0	6.9	6.9	6.85	CaCl-i
43	6.4 6.8	6.8	6.8	6.8	6.85	up
43	6.4 6.8	6.7	6.7	6.7	6.7	NaCl
	7.3	7.1	7.05	7.0	7.0	CaClz

Hours of steeping	per cent weight increase
Hours of steeping	seeds killed at 55°	normal seeds
1.5	27.68	20.56
3	31.74	26.44
4	32.12	31.61
5	34.21	34.04
6	35.38	36.42
9	35.71	38.10

17%	20%	23%	25%
4.8	2.6	2.5	2.4
3.0	2.2	1.8	2.0
2.3	2.1	1.9	2.0
1.6	1.5	1.8	1.9
1.4	1.4	1.6	1.8

Percentage	45°	40°	35°	30°	25°	20°	15°
of water	45°	40°
25	2.2	4.9	7.2	10.8	14.7	18.7	24.0
20	1.5	2.5	3.7	5.8	8.1	10.2	12.9
15	—	1.3	1.8	3.0	4.2	5.8	7.4

Temp.	Aver. 1st. 6 hrs.	Aver. 1st. 12 hrs.	Aver, last 24 hrs.
15	5.55	5.57	5.62
20	3.03	3.07	3.48
25	2.33	2.32	2.65
30	1.70	1.77	2.15
35	1.70	1.74	2.40
40	1.98	2.03	2.83

Time in hours	per cent water absorbed
Time in hours	shaken	control
2	11.72	10.19
4	15.92	14.41
6	18.73	16.68
12	25.31	23.33
24	29.18	29.10
36	32.64 1)	30.61

2	3	1	2	3
34.92	30.39	5.6	5.8	6.1
38.85	34.04	3.4	3.0	3.2
43.23	38.26	2.3	2.0	2.3
43.31	40.56	2.1	2.0	2.0
44.04	41.55	2.1	2.0	2.3
43.06	44.57	2.4	2.7	2.3
43.26	46.14	—

1 % for	15 min.	99 %, time	3.5 days
0.5 % „	30 „	98 %, „	3.3 „
0.25 % „	120 „	97 %, „	3.3 „
0.25 % „	30 „	96 %, „	3.5 „
0.1*)% „	120 „	98%, „	3.1 „
0.1*)% „	60 „	98 %, „	3.2 „
water „	120 „	95 %, ,,	3.5 „
water ,,	60 „	96 %, ,	3.2 „
water „	30 „	95 %, „	3.7 „
untreated		95 %, „	3.5 „

Sam-	Temp.	Time	Treat-	Germinated after hrs.					Germ.	A.G.T.	Germ.
pie No.	Temp.	hrs.	ment	24	48	72	96	120	%	A.G.T.	value
			CuSO^
1	0	1	0.1% gt;gt;	49	42	1	—	2	92	1.59	116
2		6	0.1% gt;gt;	5	75	4	3	—	84	2.13	79
3		12	fi	—	81	10	2	—	91	2.20	83
4	30	1	gt;t	56	35	8	—	—	99	1.52	130
5		6	7i	40	36	8	—	—	84	1.62	104
6		12	fi	—	89	3	—	—	92	2.03	91
7	45	1	if	56	31	4	—	—	91	1.43	127
8		6	gt; f	4	36	6	—	—	46	2.04	45
9		12	11	—	19	12	2	1	31	2.81	22
			CuSO^
10	0	1	1.2% 1gt;	21	14	56	—	—	91	2.38	76
11		6	1.2% 1gt;	—	4	84	—	—	88	2.95	60
12		12	fgt;	—	3	76	—	—	79	2.97	53
13	30	1	tgt;	12	9	69	—	—	90	2.63	68
14		6	tf	—	7	83	2	1	90	3.07	59
15		12	igt;	—	8	59	5	—	67	3.18	42
16	45	1	igt;	15	10	67	—	—	92	2.57	72
17		6	gt;gt;	—	1	21	—	4	26	3.27	16
18		12	gt;gt;	—	—	4	3	2	9	3.78	5
19	0	1	water	53	34	2	_	_	89	1.43	124
20		6		—	84	4	—	—	88	2.05	86
21		12		—	84	2	—	—	86	2.02	85
22	30	1		59	32	—	—	—	91	1.35	135
23		6	a	38	52	1	—	—	91	1.59	114
24		12	fgt;	11	83	1	—	—	96	1.92	100
25	45	1	ff	63	25	—	—	—	88	1.28	138
26		6		1	21	5	—	—	27	2.15	25
27		12	gt;3	—	3	2	—	—	5	2.40	4

1	7.80	5.5	8.28	6.0	—	—	7.59	6.2
2	9.14	5.5	8.84	6.3	7.34	6.6	9.10	6.4
3	10.58	5.6	11,09	6.3	9.93	6.7	10.86	6.4
4	11.64	5.5	11.78	6.6	10.42	6.7	11.19	6.8
5	11.68	5.6	11.39	6.7	11.58	6.7	12.49	6.3
6	12.99	5.6	12.18	6.6	12.92	6.7	13.52	6.3
7	14.48	5.6	14.56	6.5	14.16	6.9	14.21	6.3
8	15.56	5.5	14.66	6.5	14.04	6.9	15.32	6.6
9	16.51	5.8	15.55	6.6	14.18	6.8	15.44	6.6
12	18.17	5.5	17.24	6.6	16.57	6.7	21.09	6.5
24	22.14	5.6	22.31	6.6	23.30	6.7	24.14	6.5
24	22.45	5.6	22.16	6.6	23.30	6.6	24.05	6.4
30	25.67	5.6	25.06	6.6	23.76	7.0	25.30	6.3
36	27.10	5.7	26.42	6.8	26.17	6.7	25.82	7.0
Av. 1st. 6
hrs.		5.55		6.42		6.68		6.40
Av. 1st. 12
hrs.		5.57		6.47		6.74		6.44
Av. last
24 hrs.		5.62		6.65		6.75		6.55

Time of soaking in hrs.	HJ3		0.1% CuSO^		2% CuSOi		0.25% Uspulun		I	II	III	IV
Time of soaking in hrs.	O	H Ü	O s?	H d lt;	O	H d	O öT ^	H d	I	II	III	IV
1	8.46	3.0	7.97	3.9	_	_	7.33	3.3	40	39	—	45
2	11.42	2.9	10.77	3.4	10.34	3.8	10.01	3.3	44	44	42	45
3	13.54	3.1	11.53	3.7	12.52	3.8	12.54	3.6	45	45	44	40
4	14.03	2.9	13.67	3.7	13.83	3.6	14.00	3.5	43	45	41	47
5	16.16	3.0	14.25	3.7	15.29	3.8	14.68	3.4	43	45	42	45
6	17.25	3.3	16.65	3.8	15.25	3.5	16.63	3.5	42	41	46	46
7	17.94	3.1	18.16	3.7	17.27	3.5	17.20	3.5	45	41	42	47
8	19.01	3.1	18.03	3.7	17.28	3.6	17.91	3.5	44	40	47	42
9	20.51	3.1	20.08	3.7	19.51	3.7	18.86	3.8	41	43	41	47
12	21.57	3.2	21.35	3.9	20.38	3.7	22.19	3.8	44	40	45	41
18	24.35	3.3	27.46	4.2	24.79	3.8	24.95	3.9	44	40	40	43
24	27.26	3.4	28.06	4.2	26.01	3.7	28.13	3.9	43	40	43	43
30	28.88	3.4	29.53	4.0	28.70	4.0	28.11	4.0	45	42	42	46
36	30.53	3.8	29.88	4.2	28.89	4.1	31.02	4.6	41	43	42	36

Av. 1st 6 hrs.	3.03	3.70	3.74	3.43
Av. 1st 12 hrs.	3.07	3.72	3.69	3.52
Av. last 24 hrs.	3.48	4.15	3.90	4.10

Hrs.	Hgp	0.1% CuSO^	2% CuSOt	0.25% Uspulun
2	21.90	20.05	18.14	14.13
4	27.75	25.73	26.01	23.60
6	29.61	28.91	27.89	28.14
8	31.26	30.89	30.53	30.11
10	32.73	32.73	31.09	32.08
24	36.29	37.06	36.14	37.32
30	36.23	36.79	37.08	37.66
36	37.05	36.42	36.62	38.16



	f ^ 1 -SI

17%	20%	23%	25%
4.8	2.6	2.5	2.4
3.0	2.2	1.8	2.0
2.3	2.1	1.9	2.0
1.6	1.5	1.8	1.9
1.4	1.4	1.6	1.8

Time of soaking in hrs.	H^O		0.1% CuSOi		2% CuSOi		0.25% Uspulun		I	II	III	IV
Time of soaking in hrs.	O ^	H Ü	O tlT vO o^	H Ü	o	H Ö lt;i	O ^ 1	H 6	I	II	III	IV
1	8.69	2.4	6.83	3.2	_	_	7.43	2.9	45	45	_	44
2	10.18	2.3	8.74	2.7	8.60	3.2	10.23	2.7	47	49	48	46
3	13.64	2.3	11.14	2.7	11.26	2.8	13.04	2.9	46	45	47	45
4	14.45	2.3	13.71	2.7	13.72	2.8	16.43	2.2	48	48	47	41
6	17.70	2.4	14.97	2.8	14.95	2.6	17.67	2.4	47	46	45	46
6	17.66	2.3	16.00	2.8	16.00	2.7	18.04	2.1	48	48	42	49
7	18.35	2.3	18.37	2.8	18.58	2.6	20.82	2.3	46	47	44	45
8	18.96	2.3	18.60	2.8	18.32	2.7	20.62	2.4	49	46	44	44
9	20.90	2.3	19.62	2.7	20.16	2.7	22.31	2.3	48	48	46	47
12	21.82	2.3	22.15	2.6	22.51	2.5	25.41	2.2	49	48	44	42
18	27.28	2.3	27.27	2.6	27.78	2.6	28.00	2.3	46	44	48	46
24	28.76	2.3	28.88	2.9	30.02	2.6	29.87	2.6	48	47	45	44
30	28.56	3.0	30.97	2.9	31.60	2.7	30.23	3.0	47	46	46	47
36	31.27	3.0	31.52	3.0	—	3.1	32.43	3.1	46	46	46	46

Av. 1st 6 hrs.	2.33	2.82	2.82	2.53
Av. 1st 12 hrs.	2.32	2.78	2.73	2.44
Av. last 24 hrs.	2.65	2.85	2.75	2.75

Time of soaking in hrs.	H^O		0.1% CuSOt		2%, CuSOt		0.25% Uspulun		I	II	III	IV
Time of soaking in hrs.	O ■vP	H d lt;lt;	O ^	H Ü	O	H 6	^	H 6 lt;i	I	II	III	IV
1	10.38	1.7	8.97	2.1	_	_	7.55	2.1	43	45	_	47
2	12.72	1.7	10.82	2.0	10.57	2.0	10.09	2.1	48	48	47	47
3	14.47	1.7	12.80	1.8	14.08	1.9	11.93	2.0	48	45	48	42
4	17.34	1.8	16.36	1.8	15.68	1.9	17.48	2.0	49	47	46	49
5	19.24	1.6	18.04	1.8	16.34	1.9	19.52	2.0	46	46	48	47
6	19.00	1.7	19.46	1.8	20.93	1.9	21.87	2.0	46	46	48	45
7	21.52	1.8	20.07	2.0	21.27	1.8	23.20	2.0	47	48	44	47
8	23.27	1.9	21.84	2.0	22.29	2.1	25.03	2.0	40	48	46	4a
9	23.53	1.9	22.98	2.0	23.14	2.0	24.12	1.9	47	48	44	48.
12	26.90	1.9	27.07	2.0	25.31	2.1	26.38	2.0	48	46	48	47
18	27.66	2.0	29.68	2.1	28.54	2.1	30.10	2.0	46	48	47	42
24	29.68	2.1	30.50	2.1	30.06	2.1	31.64	2.0	47	48	49	45
30	29.91	2.1	30.51	2.0	32.26	2.0	31.74	2.2	45	46	46	46
36	32.75	2.4	32.16	2.2	31.86	2.2	32.09	2.4	48	48	45	45

Time of	H^O		0.1% CmSOj		2% CuSO^		0.25% Uspulun		1
soaking in hrs.	amp;	H d lt;i	O tqquot;	H d	O lt;m tu	H d	O im	H d lt;5	I	II	III	IV
1	11.82	1.7	9.51	2.3	_	_	7.85	2.0	46	46	_	46
2	15.33	1.8	13.17	1.8	11.75	2.5	16.22	1.8	45	46	45	45
3	17.82	1.7	15.42	2.4	14.58	2.4	16.48	2.1	50	46	47	46
4	21.67	1.8	18.55	2.2	18.74	2.3	20.76	1.8	43	48	47	44
5	22.75	1.6	20.36	2.0	18.77	2.2	20.23	2.0	50	47	50	47
6	25.29	1.6	23.86	2.1	21.60	2.2	22.94	1.8	43	48	47	50
7	25.08	1.8	23.05	2.0	22.39	1.9	24.54	2.0	45	46	44	46
8	25.74	1.7	24.75	1.9	25.60	2.0	26.22	2.0	46	46	40	40
9	25.73	1.9	26.60	1.9	26.11	1.9	25.29	2.0	48	46	44	42
12	29.37	1.8	32.39	1.9	28.35	2.0	28.81	2.0	46	47	39	39
18	30.66	2.0	30.64	2.2	30.20	2.1	30.37	2.2	42	41	43	38
24	31.52	2.1	31.50	2.1	32.11	2.2	31.71	2.4	45	45	43	41
30	32.18	2.5	31.82	2.3	32.56	2.5	30.01	2.9	43	41	44	28
36	33.48	3.0	33.26	2.9	34.35	2.8	31.97	3.0	41	46	39	22
Av. 1st 6
hrs.		1.70		2.13		2.32		1.92
Av. 1st 12 hrs.		1.74		2.05		2.16		1.95
Av. last 24 hrs.		2.40		2.38		2.40		2.63

Av. 1st 6 hrs.	1.98	1.93	2.14	2.10
Av. 1st 12 hrs.	2.03	2.02	2.12	2.16
Av. last 24 hrs.	2.83	2.90	2.92	3.20

Temp.	water	0.1% CuSOi	2% CuSO^	0.25% Uspulun
20	52	43	42	48	1— 6 hrs. soaking
	52	42	43	47	1—12 hrs.
	46	37	39	46	12—36 hrs.
25	74	61	60	66	1— 6 hrs.
	75	62	61	68	1—12 hrs.
	65	59	62	62	12—36 hrs.
30	101	90	91	84	1— 6 hrs.
	96	89	87	85	1—12 hrs.
	80	83	83	76	12—36 hrs.
35	100	81	76	88	1— 6 hrs.
	98	84	76	84	1—12 hrs.
	66	67	65	45	12—36 hrs.
40	86	84	77	80	1— 6 hrs.
	83	80	76	74	1—12 hrs.
	52	53	44	16	12—36 hrs.

17%	20%	23%	25%
4.8	2.6	2.5	2.4
3.0	2.2	1.8	2.0
2.3	2.1	1.9	2.0
1.6	1.5	1.8	1.9
1.4	1.4	1.6	1.8