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SOME BIOCHEMIC’XL STUDIES ON
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SOME BIOCHEMICAL STUDIES ON SEED VIAVILITY

THESIS

Submitted to the Faculty of the Michigan
State College in partial tulxillment of'
the requirements for the degree of
Doctor of PhilOBOphy
By

Ereton Vinton 19.1163:

May 25, 1926.

TH ESIS

TABLE OF CONTENTS

 

”age No.
.ACkhOWledgements .................. -L ................ 52
Apparatus Set - Up --~~-~~«-——~- ~~~~~~~~~~~~~~ -. ~~~~~ 8
Bibliogrdphy. —————————————————————— . ~~~~~ ~~ ------------ 58
Colloidal Silver ———————————————————————— - ~~~~~~~~ ~-- 49
Discussion of Electrolytic Results - --------------- 53
Discussion of Reduction of KMnO4 —————————————————— 46
Experimental Procedure --------------------- ~~~«~- 9
Introduction ---———~~-———~-——-;~~~- ———————— ~ ------ 1
Reduction of Potassium Permanganate ——————————————— 57
Results of Electrolytic Work --------------- ~ -------- 10
Summary of Electrolytic Work ---------------------- 34
Summary of KMnO4 Method -------------------------- 47
Summary of Colloidal Silver'EXperiments ———————————— 56

SOME BIOCHEMICAL STUDIES ON SEED VIABILITY

Introduction

 

The object of this investigation has been to obtain
a shorter method for determining viability of seeds. An
abbreviated.method would certainly mean a great saving of
time and.money. This may not be so true of seeds like
pea, bean, corn, and wheat, which germinate in three or
four days, but there are other seeds of economic impor-
tance which require a longer period for germination. Seeds
like those of grass and celery usually require three weeks.
Hybrid rose seeds may require six months for germination
and the Hawthorne may require a period of from one to three

years. Many other examples might be given.

There are occasions when a shorter method for deter-
mining viability may be useful even for seeds whose nermal
germination period is comparatively brief. Last fall in.
this state there was considerable rain,and cold weather
began very early. The corn crOp was exposed to freezing
conditions and every grower was interested to know whether
his seed crOp had survived or not. It is quite evident
then, under abnormal conditions like these, how advantageous
it would'be to the seed analysts to be able to shorten

their seed-testing method and thus conserve both time and

space.

Earlier work on this problem was terminated by

Fick and Hibbard in 1924 and is reported in the former's

-2...

Master's thesis presented to the Michigan State College, as

well as in a paper prepared for the Michigan Academy of

Science, Arts and Letters (1;)-

The present work began in January 1925, when a
large number of seed lots was secured for material. The
problem has been develOped along several lines, first
taking up the method of electrical conductivity, modifying

it, and finally evolving an entirely new method.
Historical

The use of electrical conductance methods for
measurements in physiological research dates back to the
work of Eduard weber (1,) in 1836 and du Bois Reymond (2 )
in 1849. Ranks (25) in 1865 studied the decrease in re-
sistance of plant and animal tissues upon death.

Brooks (44) in 1922 worked with Laminaria, yeast,
bacteria and'blood cells. He showed that during the pro-
gress of heating of Laminaria the "net' conductance“
approached a constant value which he considered indicative
of death. With Bacillus coli his results are rather var-
iable.

Johnson and Green (38) have shown that upon
death the conductivity of yeast cells increases, this being
due both to exosmosis of salts and to decrease in 813° of
cells-

The outstanding investigator in the field of

electrical conductivity measurements of permeability in

plant cells is Osterhout ( 5) who recommends this method

-S;

and shows that the results do not vary more than one percent
from the mean. In a later work ((3) he pointed out that the
time curve eXpressing the increase in the permeability of
Nitella during the progress of death is practically the same
whether derived from measurements of exosmosis or of elec-
trical resistance.

' Many valuable contributions concerning the appara-
tus to be used in conductivity measurements have been made by
Washburn (7 ), Taylor and Acres (8‘) , Hibbard and Chapman (9)
and Green ( 1°) .

Other Methods

 

The problem of viability of seeds has been attacked
from several different points of vantage.7 Pierce (13,15,14)
and his coworkers in 1914 noted that the heat of respiration
was greater for live seeds than for dead ones. Heat measure-
ments were made under adiabatic conditions, the seeds being
placed in silvered Dewar flasks under suitable conditions fiar
germination. He claimed that there was a "normal temperature"
for each Species of'plant and that departures from this tem-
perature indicated. departures from the best conditions of
the organism. Excess of normal indicates infection, while
subnormal is indicative of' lessened vigor,usually due to
increased age. The author did not make any great claims as
to the accuracy of this method for determining viability,
but it is evident that seeds Qf high, low and medium viability

only could be thus selected.

-4-
Lesage (15) evolved a method in which seeds were soaked
in solutions of XOR of strengths varying from normal to N/682.
Non-germinating seeds imparted a color to all solutions,
while the viable seeds colored the strong solutions and those

down to N/52, but had no noticeable effect upon weaker

solutions.

Many workers have attacked the problem of seed
viability from the standpoint of enzyme activity. Kastle (57)
in his classical work on ”Oxidases" states that peroxidases
and catalases are even.more widely distributed in living

tissues than are oxidases.

Peroxidases attack Hydrogen Peroxide and liberate
atomic Oxygen. Their presence may be demonstrated by the
fact that they cause the bluing of guaicum. Samples are
ground, a drop or two of guaicum added, and then two to three
cubic centimeters of neutral Hydrogen Peroxide added. If the
peroxidases are present the sample becomes blue, the inten-
sity of color being prOportionate to the intensity of the
enzyme action.

McHargue (16) has applied this test on seeds of
corn, hemp, tomato, tobacco, oats, cow peas, soy beans,
eastor beans and lettuce. This investigator claims that
these seeds, when exhibiting zero germination, displayed no
peroxidase reaction. He goes still farther and claims that
the peroxidase reaction might be used for seed-testing and

that seeds of high, low, and medium viability might be thus

classified.

b‘

~5—

On the other hand Brocq-Rosseu and Gain (17) studied
the peroxidase activity of seeds ranging from two years to
five thousand years old. They found.peroxidase exhibited in
a wheat sample 2000 years old and c1ain.that wheat will retain
peroxidase .activity 106 years after it loses its ability to

germinate.

The enzyme catalase has also been employed for deter-
mining viability in seeds. This enzyme attacks hydrogen perox-
ide and liberates molecular oxygen. A known weight of the
powdered sample is mixed with a known volume of neutral hydrogen
peroxide and the volume of oxygen evolved is measured.

Crocker and Harrington (18) determined the catalase activity of
Johnson and Sudan grass seeds and then made additional deter—
minations on other seeds of the same sample after they had
germinated. It was found that the c talase activity increased
with germination, thus paralleling respiratory intensity. Eith
Amaranthus seeds no correlation was found to exist between
catalase and respiratory intensity, vitality and age. They
conclude that generally there is a close correlation between
catalase activity and respiratory intensity, but not a very
close relation between either and the vitality of the seeds or
the vigor of the seedlings.

Names: and Duchon (19) demonstrated a close correla’do n
between catalase activity and viability of seeds, being able to
obtain a difference between seeds varying ‘but two or three
per cent in germination. ‘These workers employed cereal grains,

legumes and other seeds.

In 1924 Marotta and Kaminka (20) found that ‘Nemec's

-6-

catalase method could not be applied to wheat seeds. Shull
and Davis (21) have found a relationship between catalase
activity and delayed germination in XanthiUm seeds. There is
a decrease in catalase activity in delayed germination. In
a later work with Xanthium seeds Shull (381 showed that oxygen
accelerates germination and that a greater amount of oxygen is
absorbed with-the seed coats removed. Crocker (39) in 1906
showed that oxygen increases respiration and in this way ini—
tiates germination.

Since catalase is an oxidizing enzyme and believed to
participate in respiration, it may be this connection with
vital activities that suggested the large amount of work attempt-

ing to correlate catalase with viability.

All ef the evidence herein quoted seems to furnish
direct confirmation of the conception of enzymatic activity
and its relation to vital phenomena as held by Palladin (22)
who says: "Life processes are not to be interpreted simply as
enzymatic activity“. This writer continues to show how orga-.
nisms might be killed without destroying the enzymes and that
enzyme activity is always exhibited by freshly killed tissues
where precaution was not previously taken to destroy the en—
zymes, and that the only difference between enzyme activity in
living tissues and dead ones is that in the former they are

organized in their work.

We have thus attempted to review briefly the work
that has been done toward shortening the method for detenmin-

ing viability of seeds. The majority of the workers quoted

here have sought to correlate enzymatic activity with viability.

-7-

Though enzymes are originally associated with living organisms,

we have attempted to show in this historical review that they

are not necessarily to be taken as indices of life in organisms.
Catalase and peroxidase might be classified as respiratory

enzymes and for this reason may be thought to be concerned with

vital phenomena. 0n the other hand.both dead and live seeds

respire and respiratory measurements would be of little value

for determining viability. Again any carbon compound might

be oxidized to carbon dioxide and the‘ process thus be mistaken

for one of reSpiration.

The deeper we penetrate into this problem the more com—
plex it grows. It practically resolves itself into the question
of “When is a seed dead?" or a matter of evolving a physical
or chemical means for determining the difference between life
and death. Has this been determinined even for the higher

orgenisms? We are told that death is gradual even in man.

Whether there be a chemicoaphysical difference between
life and death, it Seems that there should be a correlation
between viability and.permeability of plant cells. it seems
reasonable to suppose that non-living cells should be more per-
meable than living enes and that the salts should leach out
more rapidly from the dead cells than frcm the live ones. The
amount of salts diffusing out of the cells 'of the seeds should
then modify the resistance of distilled water in which the seeds

were soaking and this change of resistance could thus be measured

electrically. An attempt was made to measure this change in

resistance and to correlate it with viability of seeds. Suitable

-8-

apparatus was assembled for making the determinations.

Apparatus

 

The apparatus used was similar to that recommended by
Hibbard and Chapman ( 9), there being a few minor changes.
The rotary converter was discarded and the 110 alternating
current from the College Power Plant used as a source of"power.
A variable transformer, manufactured by the Holtzer Cabot
Electric Company of Boston, “stepped down" the voltage to 4.4
volts. This led directly to the bridge and galvanometer.
The bridge, of the Kohlrausch roller type, was manufactured
by the Leeds and Northrup Company. The wire of the bridge
is 470 centimeters long with the scale divided into a thousand
divisions, each division being divided into halves. The known
resistance was ”plug decade” type, manufactured by the Leeds
and Northrup Company. This had a range of from 0.1 ohm to
20000 ohms.

The alternating current galvanometer was of the Rowland
electrodynamometer.type, also a Leeds and Northrup product.
The determination of the balance in the resistance on the Wheat-
stone bridge was made by the galvanometer instead of employing
the usual telephone receiver method. The stationary coil of
the galvanometer is connected on the main circuit, while the
swinging coil is placed across the bridge.
A telegraph key was inserted in the line between the

bridge and the swinging coil. Thus the circuit could be broken

as soon as the deflection on the galvanometer was noted. Since

galvanometers of this type are extremely sensitive to external

-9-

fields, great care was exercised in removing the chance for
error. The transfonaer was placed under the table and the
wiring system was confined to as small a space as practicable
and kept as orderly as possible.

The electrolytic cells were of the immersion type
made by Eberbach Brothers of Ann Arbor from specifications
furnished them. The electrodes were not platinized as was
done in the work by Hibbard since it was found that small seeds
would come in contact with the platinum black and possibly
remove it.

Another improvement over the method as used by Hibbard
and Pick was added in the form of a stirring apparatus. The
seeds were poured into Pyrex beakers containing one hundred
cubic centimeters of conductivity water and the beakers placed
in a constant temperature water bath at 25 degrees Centigrade.
A Cenco friction-drive universal motor was connected to four
stirrers so that the four samples could be run simultaneously.
The electrodes which were immersed in the beakers could be

connected to a four-way switch.
Emperimental Prgcedure

Beakers containing one hundred cubic centimeters 6f
doubly distilled water were placed in the water bath, the elec-
trodes inserted and connected, and the stirrers started. One
gram of clover seeds was added to each beaker, permitting one
lminute to elapse between addition of subsequent samples, in
order that the resistance readings might be made in the same
order. For the sake of brevity this resistance whichwill-rep-

resent that of the conductivity water plus that of the salts

leached out of the seeds, will hereafter be designated as

-10-

”solution resistance". Readings were recorded at the end

of 60 and 80 minutes. The results appear in table 1.

Table 1
Comparison of the germination (per cent) of clover seeds

With the solution resistance for definite time periods.

Sample No. 1 Eermination Resistance in Ohms :

60 minutes I 80 minutes;

 

 

 

 

3‘ i 92 : T7760 ; 16671 -
III:IIeI IIIIIIII ——————— 8 I5 fifififififififi I “““““ 1 IeIeIeIo fififififi I fififififi 1 77I1I7Is -----
a " """"" 7 “g “““““ : """ I; 19701
IIIIIII """ IIIIIIIIEII """" I """ 2 331% """" I """" s 631?"?
§_:__2_§_:_I:II: III}; """"""" I IIIII 1 Tie} IIIII I IIIIII 9 EEIsIIIII
24 I II; IIIIII 5 I IIIIIII I IIIIII a Isto IIIII IIIIII'éEI-BII":
31 I 53 "III IIIIII e IsIsIe IIIII IIIIIIEIIaIsIIIIE
:_::3§i“_-:::_- ::—3i;-I ..... I ----- lzgggIIIIIIIIIEEEQEIII-I
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1--.?3. _____ §_:_:I_I_I:5 IIIIIIII I IIIII iI 8.5.9.7 IIIII I IIIII 1 IeIéIsIeIIII I
E 50 I 1 IIIIIII I ...... 43:} ..... 3----IE££E:I—I;

 

From the results in this table there appears to
be no correlation between viability and solution resistance,
either at the end of 60 or 80 minutes. (These particular time
periods were selected because Fick showed that the rate of
leaching out was very uniform and that reliable readings could

be taken at the end of 80 minutes.) The seeds with a germination

- ll...

of from 61 to 100 per cent showed resistances of from 17000
to 23000 Ohms and the 1% germination showed 4347 ohms.
However the 5 and 7 per cent germination came in with resis-
tances as high as 12000 to 18000 ohms. The resistance of the

conductivity water was around 81000 ohms.

éince there appeared to be no correlation between
solution resistance and viability of seeds an investigation
was made on the rate of leaching out of salts from seeds.
Glover and alfalfa seeds were permitted to soak in conductivity
water and resistance measurements taken at regular intervals.
The results, recorded in tables 2 and 3 , show that the drop
in resistance is regular and continues over an extensive penod
of time. In fact it was later found that the salts leached

out for over 24 hours.

Table 2
Clover (Samp. 24; Germ 55-78)

Fall in Solution Resistance (Ohms) Produced by Seeds

 

Time Resistance (Ohms) Time Resistance (Ohms)
Emlmigpsl: ....... .3___6.2_ ..... _; ....... 7 5.9.: ...... __II
Isa ..... 4.-..-.1..5§.49. ......... .§.-.aa ..... .I. ..... .easa ....... -3
Illa ..... glasses. ......... guru. ..... .1. ..... 55m ......... I
1.4a ...... = ..... 1. 0.3.8.5. ......... 4......8.§...----.3 ...... ham ..........

Table 3
Alfalfa

Fallfiin Resistance of Solution Producedpby Seeds

 

Egme Resistance (Ohms? Time Resistance (0hms¥
I20 Min I 12153 i 220 Min I- 5726 I
IZBIIIIIIIIIIIIIeeoi IIIIIIIII E'""e;6 IIIII IIIIISEBE IIIIIIIII :
:56“”""?’“”"“86§6 IIIIIII IIIIIIIEEB IIIII IIIIISESE IIIIIIIII I
:36 IIIIII :IIIIIIEIZE IIIIIIIII g”“"é§6 IIIII IIIIIEZEE IIIIIIIII I
166 IIIIII ‘IIIIIIEEEEIIIIIIIIIEIIIIBoo IIIII IIIIISEEZ IIIIIIIIII
1E6 IIIIII fIIIIIIZe;s IIIIIIIII IIIII525 IIIII IIIIIEEEB IIIIIIIII I
125 IIIIII IIIIIIIZZEs IIIIIIIII IIIIIBBB IIIII IIIIISESE IIIIIIIII I
ieBIIIIIIIIIIIIIEaEB IIIIIIIII IIIIIBEB IIIII IIIIIEBEB IIIIIII III

The question arose as to whether it was absolutely
essential to employ conductivity water. Accordingly an orper-
iment was conducted in which the rate of diffusion of salts
was determined for two samples of seed, one in ordinary dis-

tilled water and the other in conductivity water. The results

appear in Table 4.

Table 4
Comparison of Distilled Water with Conductivity Water For

Measuring Solution Resistance (Alfalfa Seeds )

 

 

 

DiStilled Water Time Conductivity Water
2. 6038 Ohms I I 81000 Ohms I
"3:335; """"""""" 2mm ''''' £5355 ““““““ i
"f“szgg """""""""" 4 “““““ 1 3%?) """"""" .

~13-

Table 4 (Continued)

 

Distilled Water Time Conductivity water
; 4798 Ohms ; 8 Min . 12575 Ohms I
; 4990 I 10 ° 11850 3

The resistance of the conductivity water is much
higher than that of the distilled water yet both are lowered
by the introduction of the sedds and both continue to drop
regularly. Also the figures given by the distilled water

offered a better working level.

In order to verify original results a new supply
of seed samples was secured, sending to several different states
for supplies. The original methods were employed: one hundred
cubic centimeters of conductivity water, one gram of seeds stirred
in solution and readings in resistance taken regularly. The

results appear in Table 5.

Table 5

Comparison of Germination (Clover) to Solution Resistance(0hms)

 

Sample Germination Resistance Original Fall in

Resistance Resistance
40 0% 28610 Ohms 65190 56580
42 1 17520 73640 55320
30 1. 11050 64070 53020
55 2 24010 72640 48650
54 2 81560 74750 55200
56 5 .17620 81740 65120
29 4 23500 75470 51970

414-

Table 5 (Continued)

 

Sample Germination Resistance Original Fall in
Resistance Resistance

54 4 II 59500 70650 51150
57 4 50490 75470 45100
51 4 54520 80910 26590
52 6 14450 75470 61020
48 6 55250 71970 56720
4 7 25550 76960 55650
45 11 39030 91010 51990
47 12 21950 75470 55520
28 17 25460 7 5470 50010
52 19 20400 73640 52240
45 33 25550 89010 65680
46 22 15200 76210 65010
49 50 6026 76210 65010
26 41 25090 70000 65974
6 52 42650 91010 67680
10 55 18550 80910 38380
51 55 19150 77720 59590
24 56 24250 82590 65440
55 58 24250 85240 60990
44 59 15640 76960 65640
9 61 55670 76210 42540
11 61 54840 77720 42880
25 61 54050 86150 52100
5 9 62 5514 28 500 55500
58 70 21450 7 47 50 55500
8 75 68740 81740 15000
S 8f 51150_ 92040 60890
5 2: 2:23:53 3:722 72922

-15-

It will be noticed from table 5 that still no correla-
tion'between viability and resistance is found. In this case
the test has been applied to 36 samples.
It was noticed that there existed a slight differenCe

in the "specific conductivity” of the different electrode cells.
Though the difference was not great, it was feared that this
might be the source of some greater or inexplicable error. Hence
the same electrode was employed on nine different samples of

clover. The results will be found in table 6.

Table 6
Solution Resistance of Clover Seeds Compared

to Genmination. Same Electrode on all Readings

 

Sample :Germination; Solution : Resistance 3 Fall in f

: ; Resistance ;.of H30 1 Resistance;

, ; (15 Min.) _;' A _I _i
I 50 I 1 % f 11550 Ohms; 15640 I 4090 i
; 55 I 3 5 22790 f (58780 I 15990 f
I 56 I 5 § 11880 § .41550 § 29670 I
I 29 I 4 f 25900 3 42650 I 18750 i
I 4 I 7 I 25840 E 42650 § 16790 g
I-45 I 11 f 24970 3 40250 3 15280 f
I 47 I 12 I 10660 E 49880 i 59220 I
I 28 I 17 I 21450 § 50240 § 28790 I
E 53 : 19 3 16880 § 51550 § 54470 :

Fram the results in table 6 it is again evident that
there

 

is no oorrelation'between resistance and viability,

there being high and low resistances for samples of all via-

bilitiel.

115;

Thue far red clover (Trifolium pretense) has been.most
frequently employed in the work. It will be recalled that this
particular type of seed. possesses a hard seed coat and for
this reason salts may not diffuse out very readily. To sur-
mount this difficulty the seeds were scarified in H3804 (sp. g.
1.83) for fifteen minutes, They were next washed thoroughly
with tsp water and finally rinsed three times in distilled
water. Three minutes after removal from the H2804 the seeds
were plunged into distilled water preparatory to determining

solution resistance. The results are presented in table 7.

Table 7

Solution Resistance of Clover Seeds Scarified With R3804

 

*SOIution fleeistance :

 

 

 

 

 

 

 

I ISample 3 (Gene. 92%) I Sample 4 (Germ. 7%)I
I 25 Min. I 12570 Ohms I 15560 Ohms I
III}? IIIIII IIIIIIIiEIéIéB IIIIIIIIIII IIIIIIIiIéIsIéB IIIIIIIIIIII
50— I IIIIIIIIEBBEB IIIIIIIIIII IIIIIIIBSES IIIIIIIIIIII I
I--EE: I 10570— --------- I-----16656 ------------ I
IIII5I6IIIIIII- II 9455 IIIIIIIIIII IIIIIII8I975IIIIIIIIIIIII
IIIIZO IIIIII I 80I5I1 IIIIIIIIIII IIIIIIII7I51I5 IIIIIIIIIII i

 

The resistance of the scarified samples (92% and 7%
germination) were almost identical despite the great difference
in germination. It is pessible that fifteen.minutes was not
long enough to thoroughly scarify clover, yet the seeds ger-

minated.much more readily after the treatment with H3304

.417-

In all of the readings, whether the same electrode was
used or not, there was a slight variation in the duplicates
of the same sample. Hence an experiment was conducted in which
10 replicates of the same sample were run, this time employing
timothy seed. Otherwise the usual.prooedure was followed.
The figures for resistance in table 8 represent the average of
ten readings. In these results the average solution resistance
for all samples is surprisingly similar. The lowest is 3875

ohms and the highest is 7118 ohms and show no correlation.

Table 8

Comparison of Germination (fi) of Timothy
Seed With Solution Resistance (Ohms)

 

 

 

 

 

 

: Sample : Germination : Solution Resistance :
g : g (Average of ten) 9
:“5 """"" {mm‘gn “““““ :m""5177155‘55‘533
5""; IIIIIIII IIIIIIIIII'si' ”I“ 7113"“"“"; """" j
IIIIIEO IIIII II I 52IIIIIIIIII I 6007 IIIIII: IIIIIII
II 65 IIIIIII IIIIIIIIII'is IIIIIIII IIIIIIIISEEEIIIIII'I IIIIIII
EMIQB """""""" II'I'I'"""6 """""" I""""E,Z;EB"""'I """" "'
IIIII'é'é IIIIIIII IIII 6 IIIIIIII IIIIIII'Ei'éi""'""I ’’’’’ “’ ‘

In all of the eXperiments thus far described the per-
meability or change in resistance has been.measured by the egress
of salts, the seeds having been immersed in distilled water.

Most other investigators in this field have employed salt
solutions in place of the water for the measurement of permea-

bility of living and dead cells.

-185.

Osterhout (83) , in some of his work, found that salts
could be divided into two classes according to their effect
upon conductivity of cells: (1) salts (bivalent and trivalent
cationsfflgibduce a rise in resistance, followed by a fall;
(2) those (monovalent cations) that produce only a fall in
resistance. When the tissue is placed in artificial sea
water in which NaCl has been replaced.by LiCl, the LiCl
molecule penetrates the cell and the resistance is lowered.

An attempt was made to determine the variation in solu—
tion resistance When the seeds were placed in solutions of
electrolytes of known strength. Two samples of timothy seed

were placed in N/lO solutions of LiCl and the resistance meas-

ured at regular intervals. The results may be found in table 9.

Table 9

Comparison of Germination of Timothy

 

With Solution Resistance in N/lO LiCl

 

!

 

Resistance in Ohms

 

 

e a. 00 00.. no 00 ~

 

 

I (LiCl) I 48.81 I 47.28

3 ISampie 57 Isamgi1e65

; Time ;(Germ. 5442(611111. 0%);
10 1115,: 47.71 I 49.01 E
;IIIII1I5IIIIIIII§IIIZ7II2I8IIII IIIIEIIBSIIII
fIIIIIEBIIIIIIfIIZEIEEIIIIIIIIIEEIIII
:IIIIIIB IIIIII :IIZIIZIIIIIIIZIIZEII'I

The resistance was not only similar for both samples,

-19-

but remained practically the same throughout the erperiment
for both the 0 t and the 54 % germination. It is probable
that the solution was too strong. Experiments were performed
with greater dilutions (N/lOO) but with similar results.

The LiCl solution was next replaced by solutions of
KOH. It will be recalled that Lesage (15) employed this
alkali for testing seeds, claiming that the viable seeds
differed from the non-viable ones in their inability to color
solutions of XOR whose strength was less than N/52. It was
thought by the present writer that this behavior of the seeds
toward KOH might be correlated with permeability of the cells
and it was this which occasioned the following experiment.

One gram of timothy seeds was added to 100 cubic

centimeters of N/800 KOH solution, as in the preceeding ex-
periment, and the beakers placed in a water bath at 25 degrees

Centigrade. The results appear in table 10.

Table 10
‘ggmparison of Germination of Timothy

With Solution Resistance of N/800 KOH

 

 

 

_:_Time I Sample 3 Germination I Solution Resistance

I__________; KOH E I 731.5 I
10_Min. I 58 IIIEIIIIIIIIEIIIIIIIIIIIIIIIIIIIIIIII, IIIIIII
25 ''''' .0 ““““ ‘7“.
.......... .1--_----_--__s-_--_----____-__s---_-_----_---___-----_-‘
I__________;____§E§ _____ ; I 687.5 I
I___1_o_ 57 fIIIIIIIIEZ IIIIII IIIIIIIIIII IBEISIIIIIIIII
a; """ """" """""" 55:; """"
= 1m}. """ ; """""""""" t‘ “““““““““ 5753"”

1|- one
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J.

-20...

Table 10 (Continued)

 

Time ; Sample : Germination - Solution Resistance:
. KOH 3 673 . 5 Ohms
5511:?“31 """ """"" at """" T """"" 5551'; """""
:5}; """" T"? """""" """"""""""""" 3"-"m52'7'3 ------ _3
f """"" TEES}; """"" """"""""""" 3 """"" 52KB """""""
gag """"" :meg """ o ''''' :""""'gga‘:5“"""‘=
3.; """" """" ““““““““““““ ;"“""a;3':; """" '-

The samples represented germinations of O, 6, 54, and
81 % yet the resistances varied only from 675.5 to 847.5
ohms for 25 minute readings. There was no correlation between
viability and solution resistance.

Since our samples varied among themselves, an attempt
was made to compare the living and dead cells of the same
sample. Osterhout (6) showed that the time curve expressing
the increase in permeability of cells of Nitella during the
progress of death is practically the same whether derived from
measurements of exosmosis or from electrical resistances.

He derived the constant from the formula K : -% log a
a - x

 

Where a : total amount of chlorides and 5:: the amount d1f_
fusing out in'the time 2 .

Samples of the same seed were divided into two lots.
Lot 1 was run through the usual routine of conductivity
measurements. In lot 2 the samples were first killed by dry
heat ( 90 to 100 degrees for 5 days ) and then run.

421:

It has also been suggested that foreign substances"
might adhere to the seeds and thus alter the conductivity of
the solutions. To obviate this difficulty the seeds were first
washed by spreading on a filter and pouring distilled water
through them. Table ll includes the results of both these pro-
cedures.

Table 11
Comparison of Viability and Solution Resistance of

timothy. Seeds Living and Dead; lashed and Untreated

-——-.—‘--—u—-—---o~.—-fi—~o—-O ., ——-"“——-~’--~-—-~'---’& -Q-~,-.-O-.-——.

;Samp.. Germ. 3 Normal Seeds 3 Killed Seeds

- _____ 4---—---a--_-___~--------»~---; ........... 7 ...........

2 f f Untreated . Washed f Untreated ; Washed

I 57 : 54 7 =“”555“2’"“§§i§"?"“§7§i"" f” {6850 ”’"

;-___-£ ....... i ........... 1 ......... L ........... a ...........

. 57 f 54 f 5512 f 7857 3 4881 ; 9380

~-_—-;sn——-~~.f——-~.ee-———:-———_----f——--——_~~~»}——---—-~--.
58 f 8 ; 8215 ; 15580 3 7075 ; 13420

O
o ’ 0 °
~--n..~-.-.-POUOAF O-- O . .— o 0-.-. .—--—-.--—~.—--hr--~~--’~fio-'p- _~.b--.—-.v'-
O
O
O

58 f 81 3 7007 f 12800 f 5937 f 12850 .
f”5i""§"”é"‘”§““é§;5““"E‘ - “£5656"§'"’”"“ "5-30770” .....
f’e‘i"§’"’e’"'§ “”5755" “"?"i§o’§o'”§”" ° . --_..-...:.. ”17050 ' . "
T'é'i"§"""6“”§'““;6;5’ PM? "$5080“? """""""""" : "5658'" .
f’§;“’f'““6' "f" "£055” ' ”f'"i‘1'o'io’“3" """""""""" f ”-7.731"..—
T—€5~”?-+0"—?""§§Z5” "E- “i? 5 50 "E— ”-§50 f ”-15, "$07 50 ' ”1
Tag”§‘“"'6""§'”"gagi""?”igaza"§“”;;;5“”§“‘5;55“‘ ;
3"57"‘”3‘"é';'“"?"£66s'6""§" ———————— _ “”5837”? “ii-£5 ““““
7-57-_?"8; ”Eh—"5158M"? hhhhhhhhhh f-——§08§-'-”§Wfo'f§0"""
"‘ """""""" '3 """""" e """"""""" :
:76"?“”o"”2 ---------- 5' """""" 3" "WEEK”? """"""" '3

 

Reference to table 11 indicates that washing increases

the resistance but there is stills no correlation.between

426— -

resistance and viability either before or after washing. Killing
the seeds did not seem to alter the resistance of the solutions.
At this point the method was modified in such a way that_
resistance was measured by actual contact with the seeds. The
immersion electrode cell was replaced by a glass cylinder con—
taining cepper pistons for electrodes, one of these pistons being
adjustable. The cylinder was placed in an upright position,
the adjustable piston removed, and one gram of seeds poured in.
One cubic centimeter of distilled water was added, the piston
replaced, and the electrodes connected to the Wheatstone bridge.
The cylinder was 1 centimeter in diameter and about 10
centimeters long. The piston was permitted to rest upon the seeds
in the cylinder by its own weight. Samples of O and 84 %
germination were used and the readings recorded at intervals of
19, 35, and 50 minutes. There was no significance in the dif—

ference in the resistances of the two samples. See table 12.

Table 12

Comparison of Viability and Resistance of Timothy by Contact,

”----.-hO-~--------.npo----~—---

3 Time = Sample 3 Germination 3‘ Resistance

....... a 33.1.1-3 ..... 81..-: _______ 9 _____ 3 4091 Ohms
I ....... £2 ______ E _____ §Z___-E 84 3 2645 3
3----...i§ ...... E _____ €§----E-- 0 3 2987 3
. _______ éé ______ ; _____ §z_-__; ...... ss,-_i__--; ---§E3? ...... ;
j__,____§9___~_’3___”'§§m~_'f 0 E 2704 3
= 50 =' 67 = 84 3 2407 f

 

 

r" ' H
~25-

A number of references has been made to the work on en-
zymatic activity as related to viability. Kastle ( 37), in
his excellent mon0graph on Oxidases, mentions the fact that
catalases, which occur in plant extracts and which liberate
molecular oxygen from hydrogen.peroxide, will reduce other
oxidizing agents, among them being KMnO4 {/ While making
some preliminary tests for catalase it was noticed that seeds
reduced very dilute solutions of potassium permanganate
at different rates. The thought occurred that the substances
leaching out of seeds might be organic in nature and hence
not measurable by conductivity methods. If this were true
the distilled water'might be replaced by KMnO4 solution
and the change in resistance noted as the permanganate is
reduced.

Accordingly experiments were set up in which the
reduction of Khno4 by seeds could be measured electrolytically.
The strength of the KMn04 was M/ZOOOO. Timothy, as well as
larger seeds, were employed. The quantities used were one
gram of timothy, 50 peas, or 100 wheat seeds. Resistance
was determined as in the original solution.methods, readings
being taken.at the end of 25 minutes. The results are recorded

in tables 15, 14 and 15.

-25—

Table 14 (Continued)

I
--—--~-------~----”b‘“--'&—--t—o- '-- ’Dr- ‘ .- ”-*~~--h~---u~O—b~.p Q.--

 

 

: Sample : Germination : Solution - :
: : : Resistance 3 Average :
1 g 85 g :9048 Ohms é E
;---3:93 ——————— : —---—-—--f ----- : ID":- ~~~~~~~~~~~ : 972g .
: I z I. : 10410 : ~ .
E 105 : 16 E 10660 E - :
: ”””””””””” : ““““““““““““““ : “““““““““““““““ : 10$ 5 2
g . 3 ' : 8051 2 g
E 105 ; 75 § 11850 E I
: ------------- : --------------- :- ------------- : 10072
: ' : ' : 8515 :- 2
Q 104 E 65 E 5699 E :
: ------------- : ——————————————— e ——————————————— 0 5518 I
: ' : ' : 5557 ~ .
Table 15

Comparison of Viability of Timothy Seed

With Solution Resistance in.KMnO4 (M/ZOOOO)
; Sample 3 Germination 3 Solution = 2
: 1 ; Resistance 3 Average :
Q 58 i 81 % I 6540 Ohms I 6540 3
2 68 § 70 f 4970 I ‘
7 """"""""" r """"""""""""" r """""""""""" ; 4795 F
5 ' z " 1 4530 ' 3
r ------------ E “““““““““““““““ 2 “““““““““““““ a “““““““““ 5
E 57 Z 54 : 5974 f g
g ------ —=* v—? ””‘“”"”? ************ ‘: 5715 3
: ' L " , 5456 . ‘
§ 65 '§ 0 3 4205 f '
: u . '“'; “““““““““““““““““““““ 4266

E

--—-_.-------— ------—~-h-.’--~-

----—--u-~~o-r--— ---—-—-—--.

134;

able 13
Comparison of Viability of Wheat With
Solution Resistance in KHnO4 (M/BOOOO)

l
--—-O-——-o--~v-r-o -——ocu~r-o-.-won--.o-—oo“uncut-onO-o-o—oow-c—o-bho—‘n"put—ooq-o-o- --

: : Solution : .
: Sample : Germination g Resistance : Average .
:“"155“""“”55"7 """"""""" 7553612."; """"" 758?" I
1.18.7464 ““““““““ ; 7564 :
3"""166"mm”EEMMM'M’EEEZ """"""" 3 """""" ‘
---------------------------------------------- g 7292 4
- 100 81 7561 . .
"'"165"""”"E's-"mmwmlééé' """""" 3 """"""""
.............................................. . 5119
. 102 22 5576 .
Table 14
Comparison of Viability of Peas With
Solution Resistance in KMnO4 (M/ZOOOO)
: Sample : Germination . Solution : 3
: _ : . Resistance : Average;
1-3.9-..- 97% ..... 1 P_ii0_.9.h.a.a-: 3
E e 3 a . 15090 § 12985 3
2"‘Ia;'”':""'";3 ““““““““ t """" 1 as """"" ’’’’’ “'3
E.2:25:23:III::ZEIIEEGEO::3-__i319._4i_-
f 107 : 93 I 16520 ° .
""" 1 Z575 """"" 2 16145 ‘
11192 """""""" """" 1 6.5—2.0 """"" 3 """"" "T;
E~-—--:---:----—-:----fi---~-? ----- 16590 ....... 3 13555 i
E _ iii..- 5’9..- .. -3 1,1600 '
..... Z-_-_é_-,-_-f---__-__--§,---355551----_-£-_3ffff1_3
108867361 ....... £1,156.13

-----uv--—-—~--—~~no~- --.-v.—.—-—

Comparison of Viability of Wheat With

- rs .

Table 16

Solution Resistance in KMnO4 (M/BOOOO)

--—-------.--0-.—._..”"’-—.--

e. e. e. 00 e. 00 or e. o. o. e. o. 00 9Q 90 e. e. e. o. o. to e. on o. oo o. a, o. e. e. e. e. e. e. e. e. e. 0

Solution
Resistance

-—-—.—--...--—-0-~uu--—~——-~~-*-'-——"— ”—“~--—'

~.—~’~--.—-~--~~I--"

u~~----_.—~ ”flour-OI-

e. e. e. no 0. e. e. o. 09 e. 00 00 e. no co oo 00 e. o. 00 ea 00 e. 0‘ .0 '0

00 N e. e. e. "‘ 05 e. 00‘ 0‘ O. O. 00 o.

---~-.fi —~- 0..-

\ 00 Q. \

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—m7-

Greater encouragement was received from the resistance
measurements in Kln04 than from any thus far. In table 13
the wheat shows true correlation. The viability varies directly
with the resistance. In the case of the peas (table 14 ) the
samples of from 90 to 97 % germination show high resistance.
With but one or two exceptions the rest of the figures for
resistance fall in their preper places in the table. (Ehe
second experiment with wheat (table 16 ) and the one with
timothy (table 15 ) are not entirely in accord with the first
results.)

The same procedure was employed with corn seeds
except that three different temperatures ( 15, 25 and 35 )
were maintained in the water bath while the conductivity
readings were being made. The results appear in table 17
and indicate as much correlation between solution resis—

~94
tance and viability as did those with the peas. ’pll‘a y I

IX X””
Table 17

Comparison of Viability of Corn With
Solution Resistance in KMn04 (M/BOOOO)

. Samp 1 e Ge rm inat ion Solution. limitatama 3
. . 15 degrees , 25 degrees, 35 deg. ;
::-]fi§§"'-:-’"7fii§?t ’’’’’ f ''''' :fflifiiYfimfi533 ---------- f7fiifl§ '"'—3
....................... 4------..--..........-J----_---..-.......:....-..-..__ .-
, 156 ; 99 ‘3 18410 3 14940 f 12940
g 157 i 98 § 12120 : ° 8085 2
I 158 E 96 3 3 12220 E ........ §
--------- e—--~—----—----+---—--—--~--~~—:---—--—--~---:———---—~~-:
: 152 : 96 : 12570 : : 5031 2:
--------- +-~~---——--—---e-—---—--—--—~--+-----------—-:-——~~——-~~:
: 160 : 93 : : 8085 .

-----~—--’--'---—------------- ~-----------------“-------

~--~~ ’---~—-—
--——.--..
----—-.—.—.-
“IO-’O-O"-.—-—-----—.—-—-------
—~-.—.¢-—-—-

Table 1? (Continued)

. —------ .-_—-“—--"'-’---~-“-nun“--.—h-’-—--—--~---------~0—---.—-- 0-.- .

Germination Solution Resistance

 

25 degrees - 35 deg. °

3 15 degrees

; 159 : 79 g I 3 4490 ohms; -
THEE-776 """""" T ""3336 """" 3’"“’§€E'é”"?3m§'éi'é”
i"'322"§""86 """"""" 3"”"831'5' """"" """""""""" """""""" 3
3"3'52'33umi '''''''' 2"”"2852 """"" i‘mi'é’é'i """ 3""5621'”:
"""""""""""" :fisa ''''' """""" s

 

Next corn seeds were divided into two lots. Lot 1 was
placed on a window ledge over night when the temperature was 0
degrees Fahrenheit. ( subsequent tests showed that these seeds
did not freeze. Hence they will be used as cheeks against the
killed seeds ).

Lot 2 was kept in an air oven at 60 to 130 degrees
Centigrade over night. These seeds were killed by the process.
The results appear in table 18.

These results reveal the fact that heating lowered
the resisoance slightly. In the samples 150 and 144 the grains
were‘Sbrowned‘by the heat treatment and the resistance was
naturally much lowered. There is about as much correlation
between viability and resistance evinced in this case as was
true of the pea experiments. All of the samples in the 90
to 100 % germination class as awhole rank high in resistance,
extending from 15,980? ohms to 31,150 ohms, though not always
in the prOper order among themselves. Samples 148 and 144,

which respectively represent 70 and 65 % genninations, are

129-

reversed in order though both are lower than those of the 90%
germination class. Sample 154, germination 1 %, was the lowest

in resistance, being 6449 ohms.

Table 18

Solution Resistance of Live and Dead Corn Seeds in KMnO4

g-------~-~~—-Nu_-¢-—~~~--—-‘--------_—--------~u‘—-‘—_n- .

: Sample : Germination : Solution Resistance (50 Min.);
: 3 E Lot 1 (Alive) 2 Lot 2 (Dead) :-
§""1537""3"-"166-% ''''' 2"“‘;;;;a“5;;;":"';;3;; ''''' 3°
:"’1;;““:""“‘;; """""" 2"“";;;;5"“ ''''' :"‘;;;;5 ‘3"‘3
:"’1;;"‘:""";; """"""" 33""17226 """"""" :"';;;;s 33333 i
:"’1;;“"":"“';; ------ 3”""18516 3333333 3'3-355663 """" 3
:"‘1;ef“:“‘“‘;;. 3333333 ;""';;;;5 """"""" 3""32582F3"“'§
:"'1;;"";““‘;3 """"""" 2""1£1§6 """"""" 3""12555 """""" 3
3'-'12§T"3-_'3'ee ””””””” 3""312356”"'"‘—3""1683511333"3
I‘"'Ze;7""3""'"1 ''''''' :""';2;5 """""" 3'-"E§1£ """" 3
* Duplicate discarded. **Browned by heat.

At this point a deviation from the usual method
. I
of procedure was made. Instead of'measuring the solution

resistance of the seeds, a few experiments were performed

to determine the rate of moisture intake and rate of dessi—

cation of seeds.

Atkins (24) has shown that the absorption of
water by living and dead seeds is the same until germination
commences. Shull (25) claims that selective semipermeability

is a phenomenon of the seed coat and.hot related to vital processes.

L50;

Crocker and Harrington (18) remind us of the suggestion
that the gradual loss of viability of air-stored seeds with age
is due to time denaturing or time coagulation of embryo proteins.
Hence an attempt was made to induce the seeds to absorb water
from a moist atmosphere and to measure the degree of absorption.
Also the tine rate of dessication was determined.

Air - dried.seeds (clover) were placed over H3304 (c.p.)
in a dessicator and weights computed at regular intervals, until
the seeds stepped losing weight. The figures in table 19 indi-
cate the percent of’moisture a loss ‘ based on the original

weight.

Table 19

Comparison of Viability of Clover Seeds to Water Loss

: Sample - Genmination ; Loss of Water (Based on t
g g : Original Air-Dry Weight) 2
4 : 7 % 2 7,24 % E I
.---------7 ------------------------------- : 7.09 (Av) -
fl . ” ° 6095 t .

' 10 : 55 - 9.59 E' -
.-——-——-—-7 --------------------------------- g 8.40 .
n : ° 7 o 21 : .

: 5 9 93 . 7.28 ; Z
:""-—--""'": --------------- ' -------------- g 8 o 23 ‘
: ' : " - 9.18 g .

 

Though the duplicates are not close in the results

in table 19, the percent of water loss was nearly the same
for all samples, irrespective of their viability.

The rate of’moisture absorption was determined
by placing air - dry timothy seeds in a chamber in which the

air was saturated with moisture. Weighings were made at

-3 1..

regular intervals. At the end of twenty-four hours the seeds
were placed in a dessicator over sulfuric acid and kept there
for an additional twenty-four hours to note the rate of water

absorption again. The results appear in table 20.

Table 20

Comparison of Viability of Timothy Seeds to Moisture Absorption

-—-----—------—---—----—----——-———_~—-—--—---——-—-——-—-—_—--—~—.w~~

28amp. : Germ. : Percent of Water Absorption : Percent of Loss .
g g : Figured on Air—Dry Weight : (Per air—Dry Wt.):
""" If """" f"‘i’i§???£§§”l§£2§52.71;?"i""EIEET‘Efigéaf-‘i
§"52""?"6%":"3T63’E'Z52'3'éT'éi’fI'éTB?"§ """" 3T58“"""“2
TEES":""6"?'"T55"§'£T8§"T§‘I2§'§EET§1""I'm"£735 """""" 2
72'7""? "52""? "TBZEE'ZEB'TZEIEiffmg"""3'115 ““““““
3'85"”? "7'6"": "SEBTETEETEBB’,1311""?"m"3255""""-i
2’35“‘:“;15“'i“‘:a;‘:‘2:;;f:';:sa‘:i1:“3"': """"" 3T3§"’""“"’
2'23""TE?"I""IBEETZQETZSETEETEB’"I””"E'I'Zé """""""""

 

The degree of water absorption was uniform for samples
of various viabilities, as was true of the figures for dessica-
tion in the previous experiment. There was no difference where
the seeds were re-dessicated; that is, they lost water in the

usual manner.

-52-

Discussion

 

Evidence has been presented in the first part of this
paper indicating that there is no correlation between viability
of seeds and the electrical resistance of solutions of these
seeds or ”solution resistance". Why is it not possible to
develOp a conductivity method for determining viability of
seeds 7 A number of reasons might be advanced.

(l) The method, as applied, presupposes that the mineral
content is the same for different samples of seeds of the
same species and that the change in solution resistance would
be dependent solely upon the change in.permeability in the
seeds.

(2) Brooks (4) elhhinated the individual difference
factor by employing the same plant for measurements of both
live and dead cells. He developed the terms "not conductivity”
and "dead conductivity”.

' (3) Another possible reason for the failure of a con-
ductivity method might be found in the chance for minerals to
adhere tenaciously to the exterior of the seed costs, their
presence being due to previous conditions of handling.

(4) In practically all of the literature citations

on electrical conductivity measurements of'permeability, the

actual tissues were measured rather than solutions.

Actual contact methods were attempted in this
work with both whole seeds and with the powder. However, an
adequate type of electrode has not as yet been developed to

suit the problem. It might be possible to use disks of the

-So-

larger seeds, following the routine of Osterhout (23),
but we are then deviating from the path of practicability.
@JAL/The work with potassium permanganate and conductiv—
ity seemed the most promising as far as correlation was
concerned. Though there were a few discrepancies, resis-
tance for the most part rose with viability. Curves plotted
from any of the individual tables indicate as great a degree
of consistency as most workers seem to obtain from other
methods. Seeds of high viability repeatedly exhibit a pro—

if}; " v ‘t

- . . . _. TR. , 1;; ’
portionately high resistance in solution. it -»n~.434«L wg ,

r {‘1‘

T

Why should potassium permanganate solutions furnishéstfh?;.39
better results than distilled water 7 It is possible that ‘
the substances leaching out of the seeds and which are in—
dicative of viability might be non-electrolytes. Say, for
instance, that they are readily oxidizing substances of
organic nature, such as amines, and that the oxidation would
be brought about by the potassium permanganate. The resist-
ance would then be lowered in.pr0portion to the amount of
KMnO4 reduced.

The reduction of the permanganate takes place as a
result of soaking the seeds in the solution. It has been
shown that soaking seeds hastens germination only because
the imbibition of water is the first step in germination.
Now the initial chemical change in the process of germina-
tion involves enzymatic activity. Could it not be possible
that in soaking the seeds, the first step in germination is
begun and the byproducts of the enzymatic processes are

leaChed out.

;34-

Again, with the entrance of water in the seeds, we
might have new products formed by hydrolysis, other than those
hydroleed by the enzymes.

On the other hand we must not attempt to seek one
method that is applicable to all seeds. We cannot apply one
method of sterilization, germination or cultivation to all
seeds, for they are embryos of vastly different kinds of
plants. Also we have carbohydrate, fatty, and.proteinaceous

seeds and this alone should be responsible for different

chemical behavior.

Summary

(1). The literature dealing with the application
of electrical conductivity measurements in living and dead
cells is briefly reviewed.

((2). Recent methods for determining viability of
seeds are enumerated.

(3). The electrical conductivity method for deter-
mining viability of seeds is attempted.

(4). A description of the apparatus employed is pre—
sented.

(5). No correlation was found between viability of
clover seeds and solution resistance (resistance of solution
in which the seeds were soaked).

(6). Experiment showed that the regiétancetdffiwater
in which clover seeds were soaking continually drOpped,

even after twenty-four hours.

-55-

(7). Distilled water was substituted for conductivity
water (doubly distilled water), similar results being ob—
tained.

(8). Experiments were repeated, using a larger number
of samples, noting the original resistance of the water
and the drOp in solution resistance after addition of seeds.
No correlation between viability and solution resistance
could be found.

(9). No better results were obtained by first scarifying
the seeds or by using the same electrode for all determi—
nations.

‘ (lO). Timothy soaked in salt solutions (LiCl) lowered
the resistance as when soaked in pure water. In acids and
bases the resistance increased.

(11). Live seeds were compared with seeds of the same
sample which had been artificially killed. Killing seemed
to lower the resistance.

(12). Timothy seeds were rinsed with distilled water
before adding to solution. Washing the killed seeds raised
the resistance but exhibited no marked effect on the live ones.

(13) An.improvised.method.making actual contact with
timothy seeds revealed no uniform results.

(14). A temporary departure from the original method
was'made. A study was made of the tendency of seeds to
give up moisture in a dry atmosphere and to absorb moisture
from a moist atmosphere. No correlation with viability was

noted.

(15). The conductivity method was resumed. Determi-

nations were made of the solution resistance of timothy

Wheat, peas, and corn in dilute potassium permanganate solu-
tions. Greater indications of correlation between viability
and solution resistance were obtained than in any of the
previous experiments. .

(16). Corn, soaked in potassium.permanganate at 15, 25,
and 35 degrees Centigrade, produced no new data.

(1?). Killing corn seeds seemed to have no marked effect

upon the resistance measurements in KMnO4 .

-37..

Reduction of Potassium Permanganate by Viable and NonLViable Seeds

In determining the solution resistance of seeds in
potassium permanganate it was noticed that the permanganate
was reduced at different rates by different samples, the end-
point being an easily-recognized amber color. Resistance
measurements taken at regular intervals did not take into ac~
count the color changes, 'being concerned only with the change
in solution resistance.

‘ The method was accordingly altered in such a way
that the time rate of reduction of KMn04 by the seeds could
be measured. One hundred corn seeds were ground to the fine—
ness of meal, one-gram samples weighed out and.permitted to
stand in twenty cdbic centimeters of distilled water for an
hour. At the end of this time the mixture was filtered, one
cubic centimeter of the filtrate drawn off and added to 1/2
cubic centimeter of’M/BOO KMn04 . The time when adding to the

permanganate was recorded as well as the time When it was

completely reduced.

In

L...‘

The seeds wereiground because the substances reduc-
ing the permanganate were disselved in the water more readily
and the process thus hastened.< The cells would hardly be
destroyed ‘by this coarse grinding since it was performed in
a meat chOpper. Usually material must be ground with quartz
sand in order to rupture the individual cells Grinding didlrmm
influence the relative rates cf reduction‘by the different

samples.- The results will be found in table Bl.

Table lg;

 

Relation of Viability of Corn Seeds to Time Rate

0f Reduction of Kun04‘by Aqueous Extracts (Powder)

 

: Sample : Germination : Time to Reduce é
§"'iéé"""i"""""'§§TB'%T'""§"""E§T5'fi23:::'-§,La‘
:""EEET"'i5"'”"'EE""""'E'""'EB:§77"'Th"i3;.~
i”'i§§""§""'"’“'§é ””””””” §"“""25i§ """" 3"”;
i”'”i§é’"'”;"”""'§3f§ """" i """ 58?? """""" i
i”"1§3""§"""""5§ """""" §""'EB:EFF """" 3 a/ ,
E"’iéé""i"""""é6i2. """"" Z""'Eé?é """""""" i
Z""12s7'"'§7"""""32 """""" i’"""13:3"7"”” “““ § ,3
°"'3257'"'§-"""""33 """""" i'""'13:6 """"""" i
125559 “““““ _;

_----—-~~—u--’--~-_---‘..~--——_~n-_‘-—--—-‘O.---——

It was found that, with the exception of but a few
of the high-germination samples, there was direct corre-
lation between viability and time of reduction of perman-

ganate. The higher the germination the longer the time
required for complete reduction of the permanganate.
A larger number of samples was secured and the corn
meal was passed through a twentyémesh sieve before soaking.

As was true in other eXperiments, where a greater number of

.9»

samples was employed, the results are not nearly so uniform.

These results are presented in table 22.

.39;

.1

Table 22

Relation of Viability of Corn Seeds to Time Rate of Reduction % [m’

of Potassium Permanganate by Aqueous Extracts of Meal(20 Mesh) .“*

: samp. E Germ. 3 Eggicgo E Samp. g Germ. : Téggucgo :
2"REEMTTBSTi'””51?5”§5§53”156'"'3'32""I 15.5 ‘i
7115‘"?"ESQ-“7725?; "T155 55 Q 15.5 ;
5"155"?"’55"'§"15T'5""§"556’":'“53"""§""'1'2T'1':"§
1575515535575 25.5""; ,7,
145551414575155
:--157-- (“-9.63 "1:, 12.5m- : 141 : 71 : "jog/,4“
155555515555 1575”? ,
§"1'55”?"-55”"T"'1§T5""i“122’"§"'55""3" "' 16'3”";
55?:51t'5mz'1‘54": 1 “"3 fl

 

Grinding the seeds and extracting the powder is a
rather intricate process for a simplified method. An attempt
was made to use the Whole seeds. Ten seeds of corn were
placed in twenty cubic centimeters of N/lOOO potassium per-
manganate and the time recorded. The time was again recorded
when the permanganate was completely reduced. It was found
that if a few dr0ps of N/lO oxalic acid were added to the
mixture the end point would be clear and colorless instead

of the amber color. The results may be found in table 83.

-40;
Table 25

Relation of Viability of Corn to Time Rate

0f Reducing Potassium Permanganate(Whole Seeds)

 

: Sample : Germination . Time To Reduce :
3"152 """" §""”'§5’% """"" I"""'é§?5’i§£ """" i
§"I'51"""§"""§5 """""" im'm'éiié """""
3"125 """ ;"""5’5 """""" Z“"'"'16'.'§ """""
T7123 """ I'm-"5'75 """""""" I"“""i?'."é """""""
57121 """" 2"""71'1"""""I""""16T6 """"""
3"125'm'°""""6""""""1 """""" 577's """"""

 

In the six samples included in table 25 there is
only one (146) which does not occur in the regular order.
There is here almost complete correlation between viability

and the reduction of potassium permanganate.

To hasten the time of reaction the seeds were first
soaked over night in water and one cubic centimeter aliquots
withdrawn for the test instead of using the seeds. One cubic
centimeter of this extract was treated with one drop of N/Z
KMn04 . The time required for complete reduction was recordeds
(The results will be'an d in table 24?) . '

\__/

/‘ , , --5-'
{ here, too, low viability seemed to be consistent
I

with the rapidity of reduction of potassium permanganate,
altho h there was a great amount of non-uniformity in the
-_ 0.11: 11;. 55c” 1’
table. he results fin table 25 fvereobtained in the same
"i

manner as those in table 34. Here again the low-germinating

seeds were first to reduce the potassium permanganate.

Relation ofKViability of Corn Seeds to Time Rate of Reduction
0f Potassium Pe‘rmanganate by Aqueous Extracts of Whole Seeds_

Samp. 5 Gem. {Mme To - Samp. 5 Gem. ,5 Time To 5

g : 86211100 :. 2 I 3 Reduce

--------v-~*~—~--:--*-~5**~~-----~"'ctr-r‘----:**~~'--~~~w
155 5 100 5 5 23 Min 5 167 5 ’81 5 20.5 ,

----~_-.~'—u-OO.-o-O- o urn-urn». .- 0 h—v—-—.—-~o—-.—'--o—u—o~ 0-- -t—vo—w- -—-———-a

161 ° ' 25 5 168 5 65 5 19 5

' 5 21.5 5 171 E 25 E 13 5

tn—Oo--.-.—o .--——--—-’—. ~-.-~--—-'----—o—wb--'I-—»--. , '-~—--‘
0 O O

+4 +4
o: c»
01 01
I. O. O. O. O.
a) (O
a) (O

5 24.5 5 175- 5 s 15 7 5

—-—...-‘--_. '-pw---~.--~HQ—- hfifisfl‘-’v~-_wb .hu’O—U O Q G F.~-~‘”&-§-.-' p-—J

172 5 97 5 11.5 5 169 5 p o ; 1e: 1

---—&—'.-h-..r- “cu—-1 r - .— ..-—---O o~'—-.-.- . -—~--“—--.---~.—“'- . ---—I.--’.¢'.-‘ .

164 5 86 5 24.5 -5 170 5 o 5 12

—---~.~.~.&

I
\
"~.’.’~."'.~.~—v-."fi.'- .--~fi-‘—p.--”"-E~" ’
O O

2100 t : :

0.--”..o-uo- %.

O. O.
p

O)

O}

O. O O
CD

01

Table 25

Relation of Viability of Corn to Reduction
0f Kun04'by Aqueous Extracts of Whole Seeds

5 Sample 5. Germination 5 Time To Reduce 1 5

“----—u——-.~u—ho---——------——-—.-—O -—.-o—o-o- -—~—-o—'-o- o o r h..

163 : 9s : 54 5

—---~—--ho—~.~-——-~——--—---—-—--—-. ---’-"~» -0..- o-u-M 0-- .— .
O

164 ' 86 . 3 8

O.
p
0}
q
m
p

I
I
I
I
I
I
I
I
I
I
I
I
E
I
I
I
I
I
I
I
I
I
I
I
I
I
I
O oOeo‘e. O. o
I
I
I
I
I
I
1
'
I
I
I
I
I
I
I
I
I
I.

:- ---------- . ------------------------ 1 ~~~~~~~~~~ - ~- -~-—:
, 168 ' 6 5 3 31 g
.......-........._...:.. ................... L .................. :
170 . O 3 22 .

:

.

; 154 : o = 19 5

O -----—~-----~hh.-.~Cv .- v 5"..- ~~--.---o- u-o-ro-w--—.—~o. .--—_o.
O

A ".4‘ ; : . /
- ‘5- I" I. -' '4:

At the conclusion of this experiment on reduction

of potassium permanganate some additional experiments were
conducted with beans. The results (table 26 ) eXhibit a
moderate amount of correlation. The figures for germination
were obtained by selecting fifty normal - appearing seeds,
sterilizing and germinating. This was done because it was
later learned that these were not true samples but had been
more or less adulterated by the growers. This is quite
evident from the results of several different germinations.
In one set a hundred seeds were selected at random; in
another the twenty seeds that had been used in the reduction
of the KMn04 were subsequently germinated. The variation

may also be due to the difference in methods. See table 27.
Table 86

Relation of Viability of Bean Seeds

To Time Rate of Reduction of KMnn4

 

 

: Sample ; Germination.: Time to Reduce:
EI-125,_-§ _______ 199-5--.i ...... €1;1221_-_§
i--1zs_--i ______ 199-----2 ...... E1 ......... 3
i__iz_---i__--_-_9a---,-§ ______ §§1§ ...... 2
:-_lZZ__-: ....... §§-_-_-: ...... §§i§ ...... :
§-_1za_-_i ...... 199 ______ ;-,-_--}? ________ 5
§_-1zs_--;--__-_-s§ ....... i ______ as ........ i
i 180 : 2 l4 3

~“--“F””*W-ah ._ ”on- ~._ .. ..-..---’.,'_' -~._. ._ . . .

I

-43-

Table 87

Experiment on Germinat ion

: Sample - Percent Germinating :
; . 100 Seeds : so Seeds 3; so Seeds :
: = g - (Selected and 3
: : e : Sterilized ) :
g 174 - 90 : 100 - 100 .
g 175 : 89 : 95 : lOO :
0""”'”—””!"""”"""?””””“”“’7""“”“'"""“”-g
: 175‘ : 85 : 85 : 98 ;
. ......... 'rt"”'*""‘*'"r""t"'"""""'"”**““"“”‘
: 177 3 7e : so : 98 ,
o ---""""‘-""”' ': ""‘"" '°“"'""""""‘"‘. """"""’"""‘?“""" ""’"”“"’ ’ "'”_ S
: 178 - 27 2 65 : 100 3
. ---------- r ~~~~~~ '-~ ~~~~~ r~~~ ~~~~~~ 2~~~ ------------- e
- 179 - 15 2 15 : 46 :
. ---------- v-~*~**-'~——~v—h-—~-~'-7-~~*--'--""~'~~f
: 180 : O 0 0 : O :
(I f

x \/ ‘ I [L"( 4 L ’. f/ -’; f .3.

“I 1 1“ 1 VWW ‘ ‘ .r

As will be shown in the latter part of this
paper the reducing substance has been found to be present
in large quantities in the seed cost of the bean. Accord-
ingly these same sngles were soaked over night in distilled
water, the seed coats removed and.placed in the permanganate
solutions. Ten seed coats were placed in 10 cubic centimeters
of N/lOOO KMn04 and the time for reduction recorded. These
results will be found in table 27. The ten seeds, after the
coats had been removed, were germinated. Not only was direct
correlation between viability and rate of reduction found, but
sample 179, which formerly did not fit into the proper order,

\.
appeared in its prcper place in the table.'-

- A 1 .
- of;—

Table 29
Relation of Viability of Beans To Time Rats

Of Reduction of KMnO4‘by Their Seed Coats

0—9-va1 ~".~p.—~p.—~~~b~.

Ed-
p
O
(D
£3
5
d-
p.
O
:5
P3
5'
(D
d"
0
CU
(D
p.
G
O
0

r-n—’--"~- .» . fiuw—u— ...... -~---—-.—.-—.-.-—-_o—'---’p--'-.-.

O O

'7 ‘ O 24

O O

l. 5 : 4.0 o
.al'---'~.~.-.—..——.-.~-’.‘"'W—.—.—--~.**--' '-- -‘O .--.-"-h.--’.
C O
7“ ioo - 25 .

1:0 . V o

----~~-~~- —-~——-~,~.—---~--—.~ . ----~-~--.'.L ." .‘O - .u-“--

, 177 . 100 I s

-u—-.~"---—-k‘n'- --'---—-.".--".I' .‘..~"..-.——.‘ —-.—~- - 0-- -
O O
0
fin 9"
. O
o 1:7EB : uMJ . ‘5\/
0
.'——-~ vvvvv 'h-O-r—O-C-O----—~-~“'—--b-~-~fiO-fi-"Ivfi-O-Cv -D—uo.
.
179 50 7 ‘ 7
: . 4.0

. ----..o..- On. "hh'b~‘-—~.~---~-—-.—-~- -—-—-—.—--.—*v~.— ’ .v 0—. c- _.
. 0

g 180 ; O : 1

-’--—~'.-- . pr ~-~--——hr’-'~--fi-~‘- . . ‘-’--~--."‘.—"---u

Table 50
Relation of Viability of Been. to Time Rate
Of Reduction of KMn04_ by Their Seed Coats

0 -—--——-~....‘.—...—, .n-fiyvhr .- 0 or.- O—. ub-‘fi—finp’Q—bw—o—-~~n~—O~Q—M
0 O Q

- Sample Germination Time To Reduce 3

----—~~%- -’-—-—‘-

I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
O.

1403 Min :

175 100

-“------¢w fl------W---.--D-—.-.- .- h.----_-‘-—~_h'r ‘0. ’ ”-5- .
O

Q 175 : 100 : 16.3 ;

177 ;

-----~--'-

100 3 16.0

--——-.--.—.u—o—.-_~-.-’— ---—--.—~---D.-- --~——

- ---—..'u-.—.-.—fi—.--' —.—.— Q --—-——-—-.——- -—_.—-- ‘

I
I
I
I
I
I
I
I
I
I
o. co co 00 oo e. 0Q 00 o

The results in table 30 were obtained in the
sane manner as those in the preceeding table. The same
seeds were germinated after the seed coats had.been re-

moved for the reduction experiment.

440-

An experiment was next performed in which the three
parts of the seed (seed coat, cotyledons, plumule and.hypo-
cotyl) were treated with the potassium permanganate. In
this case there were employed 5 seed coats, lO cotyledons,
and 10 embryos. The figures for germination are those ob-
tained in selecting and sterilizing 50 seeds. The results
in table 31 show almost complete correlation in all cases.
The seeds germinating above 90 percent do not always occur
in the proper order yet they are usually higher in the table

than those of low germination.

Table 31
Relation of Viability of Beans To Thne Rate of

Reducing KMnO4 by Different Parts of Seed

I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

:Sample ; Germination : Time Required For Reducing :
332222-; “EBEESEBQS’EEQESET
7'132-"3-"765’72 ''''' 3"32’21’n"mii """ T"???
7 “““““ o ““““ 11
:“1;g“‘:“”";g """""" teats":"""“‘é':g"“:“"";”‘";
:"19'7'"“:"”“”;; ------ TEE?§"'""ETEM§""§'IB”3
:“1I;~'e““:""‘155 """"" §"ES”"§"""§ """" TEE“
:"1;;‘“:"“"“;; """""" 3"EETE'E"""E """ i""§""§
i'"1§6""3""m6 """""" 5127mm}. """"" 3min“?

 

 

 

-46_

Discussion on Reduction of Potassium Permanganate

v»- .- — ---..._ -- .—

 

 

—_

Frdm the results presented in this paper it is ob-

‘ vious that seeds of low viability exhibit a tendency to
reduce potassium permanganate in less time than is required
by seeds of higher viability.- It does not matter whether
the seeds of zero germination have been.killed by heat,
frost, disease, or have died of old age.* They are nearly
always the first to reduce the permanganate. It is possible
that this ,me-thod,migh?‘be employed for determining seeds of
high, low and medium viability, but, like other*methods,
-mnn$aened above, it needs considerable refinement before it

will show differences in seeds varying 5 or 10 percent in
germination.

It is of interest to know the nature of this sub-
stance which reduces the permanganate. Reed (26) believes
that the peroxidases in plant juices, having the power to
absorb oxygen from oxygenases, will attack Klino4 in the
sump manner. Bunzel and Hasselbring (27) note that
Khn04, may be reduced to a straw color by peroxides of'man-

, ganese and, further to a clear solution by organic sub-
stances, but they hold that the oxidations are brought
about by peroxides of’manganese rather than by activated

plant peroxidasee.

That the reduction mentioned in this paper is not
enzymatic may be demonstrated by first boiling the aqueous
extract of seeds for fifteen.minutes. The reduction will

take place just as readily after'hoiling.

-47-

There is evidence supporting the view that these reduc-
ing substances might belong in the group of peptides, acid
amides, and amino acids. If the proteins are precipitated
by lead acetate and the excess lead removed from the filtrate
by means of sodium carbonate, the filtrate will still reduce
the potassium permanganate. Furthermore, an aqueous extract
of the precipitate brought down by the lead acetate will not
produce the reduction unless boiled with 10 %~HCl or incu-

bated with pepsin solution.

Proteoses would remain in the filtrate after the pro-
teins had been removed with lead acetate. However, phospho—
tungstic acid brought down no precipitate and it was assumed
that in this case there were no proteoses present.

For a long time it was believed that ungerminated seeds
contained no protein cleavage products. This view has now
been changed by Jodidi and coworkers (28), who have found amino
acids and peptides to be present in ungerminated kernels of
maize, rye, wheat and oats. Bushey (29) showed that frosted
and "hailed" corn, besides being high in certain proteins, was

also much higher in amide content than normal grains.
Summary

(1) Another method for determining viability of
seeds is suggested. The method is based upon the relative
time required by seeds of different viability for reducing
dilute solutions of’potassium permanganate.

(2) The method has been used on corn and beans

and as yet is not recommended for other seeds.

_48..

(5) This method is suggested for use in selecting vdar
ble from non—viable seeds. Probably greater refinement of the
method will permit its use for selecting samples of smaller
differences in viability.

(4) Proof is given that the substance which reduces
the permanganate is not an enzyme and that it might belong in

the group of substances known as aminbwacids, peptides, and

amino acids.

AQUEOUS EXTPACTS or serve As Acrrrs IN 133

PREPARATION OF gILVER SOLS

Another phase of this problem develOped in this work
was the prOperty of reducing molecular silver to the col-
loidal phase as exhibited by aqueous extracts of seeds.
This peculiar property of the extracts was discovered While
testing for chlorides. Silver nitrate had been added to
the solfitions in Which the seeds had'been soaked and the
solutions accidentally permitted to stand over night. In
the morning a dark brown color, characteristic of silver_

in the colloidal phase, had appeared in the solution.

Many methods have been suggested for the preparation
of colloidal metals by condensation with the use of organic
compounds as stabilizers (50). For the latter might be
mentioned gum arabic, gelatin, sugar, glycerol, sodium
citrate, saponin, barium arabinate, sodium protalbinate,
and sodium lysalbinate.

Probably colloidal silver has received as much atten—
tion as has colldidal gold. Wool-fat has been employed as
a stabilizing agent in preparing colloidal silver from
Organic solutions. Kohlschutter (51) reduced AgOH by means
of hydrogen. Luppo-Cramer (52) obtained a series of'beauti-

fully colored silver sols by the reduction of silver nitrate

with hydroquinone in the presence of gelatin. Carey Lea (55)
reduced AgNOg with ferrous citrate, dissolving the the
deposit in water and reprecipitating with ammonium nitrate.
Svedberg (54) observed that a silver plate sub—
merged in water or alcohol produced a silver colloid when
illuminated by ultra-violet light or x-rays. According
.to Traube-Mengarini (35) a certain amount of silver col-
loid.may be produced by boiling in water .
Nordensen (36) showed that silver is oxidized
by both water and alcohol and is dissolved as AgOH or
some other compound. This silver solution, Svedberg has
shown, may be reduced by illumination, while Traube-Mengarini
produced srnilar results with traces of reducing agents.
The dissolution (or oxidation) is accelerated by light,

especially ultra-violet light.

The method described in this paper depends upon
the ability of aqueous extracts of seeds to reduce silver
nitrate. A number of methods for preparing colloidal silver
have already been.mentioned. This is an addition to the
list and is highly recommended because of its simplicity of
manipulation. It should therefore be of’more than.passing

interest to Plant Physiologists and Botanists as well as to

Chemists.

The Method

 

One gran.of timothy seeds is stirred into 100
cubic centimeters of distilled water and allowed to stand

about an hour. fhetsolution id filterdd'and two drops of

~51-

N/io AgN65 added. If the solution is then permitted to
stand in diffused light for an hour a dark brown color will
appear, this being due to colloidal silver. The Speed of
the reaction.may be increased.by exposure to sunlight.
However, too great exposure will precipitate the silver.

If the aqueous extract is placed over a Bunsen'burner and
heated as soon as the AgNO3 is added, the colloidal silver
will form within a few minutes.

If the colloidal silver is not placed in the strong

sun light the sol remains indefinitely stable. A solution
in the laboratory at present has held up for over nine
months.

The following seeds were later employed for reduc-
ing the silver, positive results being obtained in all cases
except clover: pea, bean, tomato, clover, corn, wheat,
'buckwheat, grass, sunflower, lettuce and beet. Later in-
vestigation revealed that though there was a precipitate
formed in the clover solution, the supernatant liquid was
colored a faint brown like that of other sols.

In a subsequent experiment colloidal silver solutions
were prepared from the following: corn, oats, Wheat, rice,
peas, beans, soy beans, cotton, best, and gladialueffcbrh),
In this experiment 50 and 100 seeds were soaked in 100
cubic centimeters of water. These were all placed under
the ultraemicroscope and were found to exhibit Brownian
movement. In the sol prepared.frcm: the cats the particles
were so numerous that the field.presented the appearance of

a confused mass of seething particles.

RXlO AQNOJ added. If tile solution is then permitted to
stlni in iiifuael light for in hour a dark brown 0010 will

afyoar, this hair 3 due to callniial silv

(3

r. The speed of
the reaction may be increased by exposure to sunl ig.t
Hovover, too great oxpoiurc will prec‘pitute tho silv
he agueous extract is plac;i over a Bunson'burnor and
heated as soon is the A ‘Jfld is aided, the colloidal silver
will form :ithin a few Linutos.

If the colloiill Silver is at placed in the strong
sun light the sol romaine indefinitely stable. A sclution

in th; laboratorv at ~reoent has held up for over nine
J I

Lent..30
The following seeds were later employed for reduc-
ing the silver, pgsitive results being obtained in all cases

except clover: pea, bean, toarto, clover, corn, aheat,
buckahOit, grins, su r.flov.r, lott co and best. atar in-
vestigation revealed thit though there was a precipitate
fended in th: clover solution, the supernatant liquid

colore' a faint rosn like that of other sole.

0
i

In a subsequent x arisent colloidal silver solutions
were prepared from the following: corn, oats, waezt, ricn,
peas, beans, soy beans, cotton, boot, and gladiclus (comm),

'—

In this expo fluent he and 100 oil; were seized in 106

A)

cubic eonthneters or r.to:. Thais were all placed under
he ult r.~m icros mp0 ani were found to exhibit Brovnian
0

LJVUS ant. In tho sol.prepared iron the cats the particles

were so numerous that the field.prodentod the appearance of

-52,-

-A_ platinum wire. was: placed on each side of the
ultrarmioroscopic field and connected to storage batteries
by means of a key. If the circuit were closed while the
field was being observed the charge on the particles could
be determined by their behavior in the electric field. All

of the above samples possessed negative charges.

An outstanding feature of these colloidal solutions
is the elaborate coloring. Colors varying from a dark amber
to a rich rose or orchid were obtained. It is possible
that different classes might be characterized'by the color
of their colloidal solutions. Corn, Wheat, oats and rice
all produced colloidal silver in a rose-colored suspension.
Peas, beans, and soy beans were all characterized.by a‘brown
color. Other seeds were examinod‘but could not be compared

because no other closely related groups were included.

In an effort to locate the exact source of this
reducing substance different parts of bean seeds were soaked
in water and the solutions treated with AgNOs. Beans were
first soaked in distilled water over night. The next morn-
ing they were taken from the water, the seed coats remaved,
and the plumule and.hypocotyl out out of the cotyledons.
These three parts ( seed coat, cotyledons, and hypocotyl
and.plumulo ) were again soaked over night. On the follow-
ing morning AgNOoB was added to the extracts.

The intensity of the eclloidal silver color was
greatest with the seed coats, next with the cotyledons

and least with the hypocotyl andfplumuls. In the last, the

-r55-

Color did not form until after several hours.

Shull (59) and Crocker (40) both hold +hat delayed
germination is due to the inability of oxygen to reach the
embryo. Is it possible that we have here a reducing sub-
stance in the seed coat that tends to decrease the amount

of oxygen reaching the embryo during the period of dormancy?
The Nature of the Reducing Agent

About ten cubic centimeters of the aqueous extract
of timothy seeds was enclosed within a collodion sack and
lowered into a beaker of distilled water. Two or three drOps
or AgN05 were added to this distilled water and in a few
days the extract within the collodion sack had assumed a
brown color characteristic of the colloidal silver. This
would indicate non-diffusibility of the part of the reduc-
ing agent .

A.more concentrated solution of the timothy ex—
tract presents a dark brown color, indicative of tannin.

In the presence of ferric chloride a faint blue-black pre-
cipitate is formed, the intensity of color resembling that
produced by a .001%'tannin solution When treated in the
same way.

Following this a timothy extract was treated with
lead acetate to remove the proteins. The excess lead was
removed with H38 . A few drape of AgNO5 were added and
a brown colloid, similar to the original, was formed. How—
ever, the colloid soon settled out.

The experiment was repeated, first adding enough

+54;

itnnin to make it a..OOl% solution. The same brown colloid
was formed, appearing permanent at first, but settling out
after a longer interval. This filtrate did not reduce
Fehling's solution.

The instability of this last colloid was no doubt
largely due to the presence of acid formed.by the method
employed for removing the excess lead. Later sodium carbo-
nate was used instead of’hydrogen sulfide and the filtrate
treated with AgNog. This time a colloid formed which was
not quite the same color as the original. This colloid
lasted longer than the others ( about a week ) but finally
settled out like the rest.

Difficulty in identifying the reducing agent arises
from the fact that many substances are extracted from the
seeds by both water and alcohol. In some instances sugar
has been found and in others it has not..

An alcoholic (90%) extract of corn.meal has been found
to reduce silver nitrate, producing a colloid identical in
appearance with the original aqueous solution colloid. This
silver sol thus far rivals the aqueous extract sol in stabilé
ity.

In another expernnent an alcoholic extract of corn
meal was evaporated and the alcohol replaced with water as
the alcohol evaporated. The zein was precipitated in this
process. This impure zein was dissolved in alcohol (ethyl)

and AgNOs added to the solution. A silver sol, very similar

to the original was fonned. After a period of a week this

sol became cloudy and appeared to be precipitating.

From the above results it seems possible that both
sugar and zein would be extracted by the alcohol. When the
alcohol is replaced by water the sugar would go into solu-
tion, leaving the zein to be precipitated. Hence it might
be suspeeted that the silver can be reducdd'by either the
alcohol- or water-soluble proteins and the sol stabilized
-by sugar or vice versa.

An aqueous extract of corn.meal was made after the
meal had first been extracted with 90 % alcohol. This aqueous
extract reduced Fehling's solution when tested for sugars.
There were also proteins present, as was evidenced.by the
precipitate formed in clarifying the sugar solution. A col—
loidal silver solution was produced by this aqueous extract
but the sol was unstable.

The zein thus far'mentioned in this paper has been
designated as impure zein. It was prepared by extracting
corn.meal with alcohol, evaporating off the alcohol, replac-—
ing the alcohol with water as the alcohol evaparated, and
collecting the zein as it precipitated.

A little later some pure zein was prepared. An a1-
cdholic extract of corn meal was made, the zein salted out
with dilute NaCl, and the NaCl removed.by dialysis. It is
possible that some of the carotinoid pigments were brought
down with the zein but we shall distinguish between the two
by calling the last one ”pure zein”.

‘4

-336...

A stable colloid has been.prepared by dissolving
the "pure zein" in alcohol and adding AgNOg. With the
impure zein the colloid was not stable unless a small amount
of arabinose was added. These last two colloids have been
called stable because they have held up longer than any of
the other artificially prepared sols. As a matter of fact,
their appearance at the end of two weeks would indicate that
though they are stable, they probably will not last as long

as the aqueous and alcoholic extract sols.

8mm

(1) The literature dealing with the preparation
of colloidal metals by condensation has been.briefly reviewed.
(2) A simple method has been described for pre-
paring colloidal silver from aqueous extracts of seeds.
(5) Colloidal silver may also be prepared from
. alcoholic extracts of seeds, alcoholic solution of’pure zein,
and an alcohelic solution of impure zein plus arabinose. The
stability of the last two is not guaranteed.
(4) The reducing agents are not as yet known but
it seems probable that they might be alcohol- and water—

soluble proteins with sugar as the stabilizer.

-57-

Acknowledgements

 

Appreciation is herewith expressed to Dr. E.A.
Bessey and Dr. R.P. Hibbard of the Department of Botany of
the Hichigan State College for many helpful suggestions

offered while the work on this problem was in progress.

Appreciation is also expressed to the following
who kindly supplied seed samples for use in these exper-
‘iments: Messers J.R. Duncan and.A.R. Marston, Dept. of
Farm CrOps., Mich. State College; Prof. F.S.Holmes,
University of Md.; Dr. L.H. Pammel, Dept. of Botany, Ames,
Iowa; Miss Edith M. Patt, Seed Analyst, Lafayette, Indiana;
Dr. H.C. Young, Wooster, Ohio; D.M. Ferry Seed 00., Detroit.

'Work on this problem has been.made possible by
the generosity of the D.M. Ferry Seed Co. of Detroit Who
have kindly contributed funds for a fellowship in.this

department.

.. 58-

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