SOME BEOCHEMICAL STUCIES
0N SEED WABILITY
Thesis for the Degree of M. 5.
Oman E. Street
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SOME BIOCHEMICAL STUDIES ON SEED VIABILITY
. 11. Reduction of Potassium Permanganate

As a Measure of Seed Viability

Thesis presented for the Degree of
luster of Science.
Michigan State College.
by
Oman E. fireet

1927

THEsxs \

 

sc-‘i: PICTCY’::"ICIil STUD I38 97' 52m . ‘~.'I1LBII_.ITY

 

II. Reduction of Potassium Permanganate

As a ”easure of Seed Viability

Introduction

 

 

The possibility of develOping a simrle method to serve as a
measure of seed viability was the basis of this study. In spite of
the wide divergence in the chenical and physical composition of dif-
ferent seeds within a group or class, it was h0red that there might be
some simple relation, that would Operate as a function of the germ-
inating power of the seed, and thus serve to measure that rower.

The previous work conducted at this Institution on this problem
has been along two lines: the first, the measurement of tie elictrioal
conductivity of seed extracts; the second, the detersination of the
reduction of chemical reagents, notably potassium permanganate.
DeveIOpment of the method of electrical conductivity measurement is
described in a paper presented by Fick and Hibbard before the Vichigan
Acadeny of Science, Arts, and Letters(12). This work was continued
by Eiller and is reported in his thesis, presented in 1926. The latter
paper contains in addition the study of the chemical methods.

The present study was planned as a continuation of the per-
manganate reduction method, with a View to refining it sufficiently
to establish differences of germination of two or three per cent.
Considered from that viewpoint, the results are rather conclusively
negative, but with further studies it has been possible to demonstrate

some of the factors responsible for the results obtained.

-2-

.Tisturlfiul

Of the long list of rea"ents used in oxidrtion-reduction
reactions, potassium permanganate is undoubtedly tie most favored, both
for inorsanic and orsanic reductions. Its ability to react in acid,
alkaline, or neutral solutions, corhired with its intense coloration,
which serves as its own indicator, leads to its adention in a wide
ranse of situations.

Within the sore restricted range of plant materials, it is

4.1.
e 8

lften used. Reichert(?§) lists a nurber of methods for its use in
preyrration of soluble starch. Lasser-Cohn(2u) in his "Fanual of
Cree ic Chemistry", gives the reactions of potassium permanganate with
a number of aromatic compounds, including a quantitative estimation
of glycerol. Reed(34) reported a reduction of conCentratad potassium
permangaiate by horseradish extract, in which the peroxidases were-held
to be the reducing scents. Bunzel and Passelhring(4) refuted the
eviience of Reed very soon after his publication, insofar as the per-
oxidase is concerned as they list ten orsanic compounds that reduce
permanganate. In all the reactions the formation of hydrated peroxides
of manganese was noted.

Its direct application to seed extracts, however, is new, hence

the feasibility of attempting to establish a quantitative relationship

between it and some component of the seed extract.

vethods Other Than Permanganate Reduction

Waller(43) made use of the after-currents aroused by single
induction shocks to determine whether seeds of beans were dead or alive
Dead seeds always dBVBIODed an after-current, or "blaze current", in the
Opposite direction to the induced current. If the after-current aroused
by induced currents of both directions were in the same direction, or if
there was no change in current direction between induced and after-currents,
the seed was alive..

The electrical conductivity of plant materials has been studied
by a number of workers, but not all this work is applicable to this
problem. Osterhout(32) studied the resistance of disks of Lsminaria,
using a modification of the Wheatstone bridge. Brooks(3) made a definite
contribution to the field in his studies of conductivity as a measure of
vitality and death. He defines ”net conductance” as the conductance of
the tissue, independent of the conductivity of the bathing fluid. In
most cases, conductance of dead tissues was only 35% to 60% of that of
live tissues.

Fick and Hibbard(12) applied the method of electrical conductivity
to determinations of seed viability. The relative exosmosis of salts was
greater in seeds of low germination, and hence the resistance of the ex-
tracts to the passaqs of an electric current was less. A positive cor-
relation was found for timothy and red clover. With a larger number of
samples, and improvements in the technique, Miller reports in his thesis

that he has not been able to correlate viability and solution resistance,

-4-

and that no definite equilibrium is reached, even after twenty four hours.

The decrease in the heat of respiration has been used by Darsie,
Elliott, and Pierce(10) as an indication of the laws of germinating power.
The temperature which was develOped by germinating seeds in silvered Dewar
flasks, under conditions suitable for germination, was taken to indicate
the viability. A "normal temperature” for each species of plants studied,
was used as a basis of comparision. Another application of thermal relations
is suggested by Munerati(26) who finds that as seeds of wheat age, they
germinate better at temperatures above their normal. This could be taken
as an approximation of the age of the seeds, within a relatively narrow
range.

Lesage(22) presented a method which had as its basis the ability
of seeds to color solutions of KOH. He used solutions ranging from N/l
to N/683 and found that dead seeds colored all, while live seeds colored
only the solutions stronger than N/32. He suggested the use of this re-
agent in a concentration range between N/32 and N/683, as a means of
determining viability.

Brocq-Rosseu and Gain(2) were among the earliest workers on the
relation of enzymes to viability. They reported peroxidase in wheat
estimated as being from two to five thousand years old. All samples of
lesser antiquity showed the presence, in an active state, of the enzyme.

McHargue(24) did not verify these findings, as he states that in
every case where the seeds showed a weak or zero germination, they showed

also a weak or zero peroxidase test. Using tests described by Kastle(18),

he found peroxidases in twenty species, but oxidase and peroxidase in only
three species, namely: lettuce, alfalfa, and soy beans. His tests enabled
him to classify seeds as high, medium, and low germination.

The relation of enzymes to germination is mentioned by Crocker(7)
in his study of the delayed germination of Xanthium. He was unable to
detect any differences in digestive activity of extracts from the upper
or lower seeds, altho the latter germinated a year sooner. A more extensive
study of catalase and oxidase activity appeared in a later work, Cracker
and Herrington(8), in which they study Johnson and Sudan Grass seeds.

They do not find a very close correlation between either of the enzymes
and the vitality of the seed, but stated that the decrease of catalase
activity with age was a fair measure of age in continuously dry-stored seeds

Shull and Davis(40), in contrast to Crockerfs results, find that
the lower seeds show greater catalase activity than the upper seeds,
both under laboratory and field conditions. The enzymatic differences
were held to be in harmony with other physiological differences, which
soaperate to delay germination.

Names and Duchon(27) reported investigations of catalase activity
which gave much promise. Working on oats and peas, they obtained a re-
markable correlation of catalase activity and germinating power. They
used 2 grs. of meal, to which was added 15 ccm of neutralized 0.3%
hydrogen peroxide. The net volume of oxygen liberated in 5 minutes
was about equal in cubic centimeters to the percent germination.

The attempts of other investigators to duplicate these results

have not been uniformly successful. In the same year, de Vilmorin and

-6-

Cazaubon(42) applied the test to different varieties of peas. They
concluded that there was no consistency between the relation of catalase
activity and germinability.

Wilmer Davis(ll) found that the meal from dead and live seeds
of lettuce often showed a catalase activity nearly parallel. If the
meal was soaked in water over night, that from the live seeds showed
slight change in catalase activity, while that from dead seeds showed
a decrease. This he interpreted as a reduction or chemical decomposition
of the catalase. A more reasonable eXplanation would be to ascribe

the difference to greater permeability in the dead cells.

E§perimental @935.

At the point where work on this problem was terminated by Miller,
the procedure in the potassium permanganate method was as follows: The
seeds were soaked over night in water and one cubic centimeter aliquots
withdrawn for the test. To this was added one drOp of N/2 Khn04 and
the time required for reduction noted. In order to obtain a clear and
point, a few drape of h/lO oxalic acid was added when the reaction was
nearly complete.

It was deemed advisable to germinate the seeds after soaking,

as the irregularities within a sample were often greater than the
differences between samples. In view of this, a further effort was
made to evaluate the vitality of the seedling produced. A system of
scoring was arbitrarily sat up, based on the vigor of the seedling.
As 20 Seeds were used, a perfect score for a strong seedlirg would Le
Efl. Those of medium vigor were given 4%, the weak sprouting ones, 3%,
and those which failed to germinate, Ufi. While it is recognized that
this is subject to a personal error, it was nevertheless more reliable
than a rare plus or minus rating. Further, it is more in harmony with
the order of accuracy of a chemical reaction.

After a few preliminary tests, with varying amounts of aqueous
extract, and varying amounts and concentrations of potassium permanganate
as the subject, the following test was tried. Twenty seeds were soaked
in 40 c.cm. of distilled water for a period of 24 hours at room terperature.
The extract was decanted, the seeds sterilized with Chlorozene, and
placed in sterile dishes to germinate. To 5 c.cm. of the extract, é c.cn.
of V/bl.2 KVnC4 was added, and the tire of reduction noted. Oxalic acid

\: ~-.e~¢ ~ - a (.r‘ '. , 1 ,~-‘ 4- ~«‘« .V’r“ ‘, ,\ ‘ ‘ M~ ' ...
lsfyinp agent He: also added b0 e-e a part of ,c.cr. lhls in

Q
I'.

{.3 a C .L‘.;

1

l.\,.. . .. ~., ‘ '
u-e permanganate in 1 hr. ?5 W1n., sense any

1 4‘04

solutions requirinf near.y ova. time for reduction would have zero

reducing

type.

power. Table

1 shows the rajer asvects of a reac

Time Rate of Reduction

As a Yeasura of Viability

 

Sample

Percentage

 

Germinatinn_

eduction Sample Percentage Reduction

 

lime in Min- G .Iima_in_Min‘_.

 

9 100 44.5 14 80 30-2
10 95 45.3 7 72 34.5

 

11

42.2 6 65 26.3

 

12

37.7 4 5 21.2

 

o
k )1

 

 

31-7 16 45 11.4

 

27.6 2 42 210.6

 

17

32.2 1 35 27.8

 

 

 

 

 

 

 

 

not at all. cox-xsistent.

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28.0 15 0 17.7

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1y 8 d f.‘Jo' .;:.u :n t a IoSultS, unO t'au is

An and reint was found difficult to obtain as
I

inner fer the for ation of a colloidal brown
J )

sustersion of PnOZ, which Was relatively stable. Yo reason was apparert

to eXplain why a time rate neaSurement should have any advantace over

a conventional titration method. Fevertheless, several more series were

run with very mediocre results.

Oxidation-reduction reactions of permanganate are usually
conducted at a temperature of 70°C. Since at this temperature, oxalic
acid reacts quantitatively with permanganate, it was omitted.

In this and subsequent experiments, several concentrati.ms of
KKnO4 were used as work on the method prcgressed. However, these will
all be reduced to a basis of N/lO KNn04, computed on the pentavalent
reactivity of this reagent in an acid medium.

Table 2 shows the results of a run made under the following
conditions: 10 c.cm. of extract were heated to 70°C. on a water bath

and permanganate added until an end point of a brown suspension was

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

obtained.
Table 2
Titration of Aqueous Extracts of Corn
By Neutral KMnO4
at 70°C.
Sample Percentage KNn04 Sample Percentage KMn04
Germination in p-pm1 Germination in e. c
m 95 -130 4 4; .155
11 94 113g 8 45 -130
41 89 .163 1 412 2.371
3 85, .1791, 6 31 .146
12 82 .179 5 21 .139
13 75 .130 16 12 .285 ,
2 54 .146 14 10 .122
7 51 .114 15 0 .465

A discrepancy in the method was apparent.

to reach any definite end point in its absorption, as there was apparently

-10-

Sample No. 1 failed

Ian accelerativs formation of the colloidal hydrated oxides of manganese.

The total absorption was low, as may be seen by comparison with Table

3, using the same samples, but with 2% by weight of sulphuric acid

added to the extract.

Table 3

Titration of Aqueous Extracts of Corn

By Acid KNnO4 at 70°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample ercentaas KVnO4 Sample Percentave KMnO4

Germination in c. cm. Earninatinn in 3. cm-

10 95 1-13 4 45 2121
11 94 1.21 8 45 2.2111

17 89 61.74 1 42 2-08

1, 85 2.22 6 31 2-19

12 B2 1.68 5 21 2.16
13 75 1.75 16 12 2.42__

2 54 2.26 .14 10 1.36

7 51 1.67 15 O 2.h0

 

The greater amount of reduction was a favorable feature of this

series, but the fact that an end point of a clear solution was not

attainable would indicate that the reaction was not reaching a point of

equilibrium.

Inasmuch as the conditions established are exactly com-

parable with those prescribed for a permanganate oxalate reaction, it

was next decided to titrate the excess permanganate with sodium oxalate.

 

-11-

Table 4 shows the results of this procedure, as well as the modification

used:

(acidified to 2% H280

4

Permanganate in excess (lc.cm. F/2 KYnO4) was added to the solution,

and kept at 70°C.) and at the end of exactly

ten minutes the unreduced permanganate was titrated back with sodium

 

 

 

 

 

 

 

 

 

 

oxalate.
Table 4
Application of Standard Oxidation-Reduction
Reaction to Aqueous Extracts of Corn

Sample Percentage Net c. cm. Sample ercentage Net c. cm.
Germination KMnOx, .gzminagign__gyng‘_____4

12 96 1.25 -_4; 169 2-00

11 95 3-00 6 57 3-9n

10 95 1.15 7 60 2-65

8 91 3.15 5 51 1 -15

17 88 2-30 1 50 4-30

1 81 2.75 14 42 1-75

11 74 4.6g 16 21 4 .51

2 71 2.95 15 0 4-25

 

 

 

 

 

 

 

 

VIt is hardly politic to claim that there is any correlation

between the reduction as carried on in this experiment, and the viability

of the seed.

If there is a relation between reducing power and viability,

it does not appear to be a direct function.

 

If” I

-12-

Three samples from the same lot of corn were extracted under
different conditions. No. 1 was allowwd to soak for 24 hours at
room temperature, No. 2, placed in a constant temperature oven at 50°C.
and No. 3, left in the ice box. The hot-water treatment killed the
seeds, but increased slightly their reducing power, as will be shown
by table 5.

Table 5
Effect of Temperature Upon

Reducing Power of Extracts

 

Sample Percentage Net c. cm.
Germination KMnO.

 

 

 

 

 

 

 

3 98 1-20
1 90 1.4.5.—
2 0 4-05

 

An attempt was made to apply the test to beans, but no con-
sistent results were obtained altho wide differences in reducing power
of the extracts were noted. Steps were also taken to adapt the alkaline
permanganate method to these extracts. It took several days to obtain
differences in reduction, which fact in itself would diSqualify the
method.

As permanganate absorption, within definite time limits,
closely parallels methods used for iodine absorption, it was thought

feasible to try the latter method. In this case, the reagents used

were of the normality recommended in the A.O.A.C. Handbook(l), but the net
iodine absorption is computed on the basis of N/lO solution. The solution
was left in contact for 15 minutes, and the excess iodine titrated with

sodium thiosulphate, using starch as an indicator. The results are shown

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

in Table 6.
Table 6
Absorption of Iodine
by Aqueous Extracts of Corn
Sample - Percentage Net Sample Percentage Net
Germination Igdine "srminaiion___lndina___.
12 96 ‘16 4 69, .20
ll 95 -24 6 57 -10
10 95 -11 7 60 -11
8 93 .17 5 51 .17
17 88 .13 1 50 -20
3 61 .17 14 42 -19
-13 74 -16 16 21 -20
2 73 -14 15 0 -23

 

 

 

There seems to be even less possibility of correlation by this
reaction than by the permanganate tests. The low reactivity of the
extracts with this reagent, would indicate that there was not a definite

reaction, which observation is fortified by the inconsistencies in

-14-

amount of net iodine absorbed.

At this point it was decided to substitute an electrometric
titration method for the colorimetric titration method, which had failed
to give consistent results. The potentiometer set-up which was used is
described by Sherrill(39). A mechanical stirrer was connected with a
Cenco motor, as a means of insuring a uniform solution. The reference
electrode was not the conventional calomsl electrode, as it was desired

to swoid the presence of the Cl ion. Instead the half-cell, Hg (metal),

H82504, H2504 (1N), technically known as the mercurous sulphate cell,

was used.° A platinum foil, carefully cleaned and not platinizsd, was
used as the other electrode. This was later changed for a platinum wire,
because the latter did not collect bubbles, which usually cause incorrect
voltage readings.

Altho diffusion proceeds from the greater to the lesser concen-
tration, and the liquid electrode was 1 N, which was greater at all
times than the titrating solution, the electrode yet suffered contamination
from the permanganate in the solution. Other salts might also proceed
up the arm of the electrode, and being colorless could not be detected.
To overcome this error, it was necessary to allow a small amount of the
liquid in .the electrode to drain into the beaker at intervals during
the titration.

A solution temperature of 70°C. was maintaired by the use of

 

‘_ --..‘-..— ”u“

0 Credit for the suggestion to use this electrode is due Mr. A. M. Malloy,

of the Chemistry Department, M.S.C.

lllL'll i .l

-15-

a "micro-burner", regulated to give a minimum flame. This feature, to-
gether with the use of the special electrode and the mechanical stirrer,
were the only deviations from the usual assembly employed in this work.

The method next passed through a period of trial variations.
Direct titration of the solution was not possible, because it possessed
an apparently endless ability to partly reduce permanganate. The first
few runs were made by adding a set amount of Kuno4 and after ten minutes
at Optimum temperature, titrating the excess with sodium oxalate. ‘The
formation of colloidal brown Mnoz was often a deterring factor, as it
was characterized by a sharp drop in voltage at a time when none was
justified. It was necessary to add as much as l c.cm. of oxalate in
excess of the end point, when the colloid would be broken down, and the
excess could be run back with permanganate.

The acid concentration was raised to 5% by weight, in order to
keep the cell wall supplied with active ions, and not have a sluggish
change in voltage readings.

The standard solutions used in this phase of the work were more
carefully prepared than those formerly used. The potassium permanganate
was standardized repeatedly, and the sodium oxalate was electrometrically
titrated against the permanganate. The permanganate was .1141 normal,
and the oxalate was .0985 normal, but again the results are computed to
the basis of .lOOO normal permanganate.

These trials led to the development of a technique where-in the

permanganate was added at the rate of 1-1.5 c.cm. per minute, so that a

-16-

purple tinge was noted in the solution at all times. At the end of ten
minutes, the beaker was placed in the apparatus, and oxalate solution
added drOp by drOp until the fall in voltage indicated the end point.
By these discrete additions of permanganate, the colloid was eliminated.
While it would be possible to show titration curves for all the

reactions studied by this method, there would be no particular justification
for the inclusion of such a mass of data. Instead, a few charts, ( Fig.1-4)
are to be found on the following pages of this paper. It is to be noted
that the general form of these curves is nearly identical. Often the
identical reading in millivolts wns noted for the end-points of different
reactions.

Table 7 shove the first series run by this method, with only the
net KKnO4 column of the ori~inal data sheet included.

Table 7
Electrometric Titration

As a Measure of Viability

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Se"ple Ierceqtcge Yet c. or Samrle Perenntave Net c. cm.
Germination ?”n“, Berrination KHnOA

17 100 16.53 L 64 10:33
11 9” (-16 / 51. 7-66
12 9§ 7-21 ;, 7gb 4-50
7 91 13470 14 33 49.20
10 91 16.90 1 26 1,64
13 90 .5.99 6 24 411.90
1 75, 2-08 415 M 46135
16 75 13-48

 

4 . .444. a

MlLLIVOLT:

-17-

Figure l

 

900*-

wd

700

500

4-00

300

200

'100’-

 

000
O

 

 

 

I l l i d l .70

 

.IO .20 .30 . 50 .60
CUBIC CENTIMETE35 5ODIUM OXALATE

 

Representative Electrometric Titration Curve

of Excess Permanganate Against Sodium Oxalate.

 

 

MILL a van:

a

-13-

Figure 2

 

 

e

 

§
l

 

4g:

 

 

 

 

oath .1. Heaven.

CUBIC CENTIMETERG SODIUM OKALATE

Representative Electrometric Titration Curve

of Excess Permanganate Against Sodium.Oza1ate.

 

 

 

-19-

Figure 3

 

900—

700—

‘00--

MILL! VOLTS

i

200--

«oo—

 

Ea3

 

T

COLD

 

 

 

.oogo

.I‘o. fir )a at dr—t

CUBIC Csurlmzrs'fls sooum OXALATE

 

Representative Electrometric Titration Gurve

of Excess Permanganate Against Sodium Oxalate.

 

 

MILLIvoLrS

e 9L.

' 3

Figure 4

-20..

 

 

70

2i

i

 

COOL I
.10

l
' 10 v
CUBIC CENTIMETERB SODIUM

l
.30

   

.1

 

 

l
50

l
T

.OXALATI:

 

A

Representative Electrometric Titration Curve

of Excess Permanganate Against Sodium Oxalate.

The extremely high reactions shown Ly most of the serples in this
series was hardly eXplicable. In searching about for a possible flaw in the

technique employed, blanks were run on all he reagents employed. As 3,

.—
w. . .

phenol had been used to preserve tle solutions of this series, this was
included in the test. The results are Shown below:
Table 8

Reactivity of phenol with Fermanganate

 

Tast_Mixtuce Nst_KMnfl4___

Water, acid, 5 drops

 

 

 

 

 

 

 

phenol 6.27
Janet 943-—
Extract, 0.2 c. cm. ohgno 16.51

 

he the phenol v43 added to this series with no particular pains
to regulate the amount, it is evident that the results are thereby
vitiated.

Replicate determinations were run an samples without phe-ol,
and the reductiin of per anrnnate checked exactly in several runs, while
in none did it Very more than a few hundredths of a c.cm.

Table 9 shows the results of a series of fresh extracts, run
immediately after the completion of the period of soaking. The
titrations made here are of a decree of atcuracy wlich would be worthy
of better ends. There is every reason to believe that the technique is
such that the maximum reducti n of permanganate occurs, and the ease
with which check are secured on duplicate determinaticns leads the

‘

writer to relieve tint the method is cle'ically sound.

-22-

Table 9
Electrometric Titration of Aqueous Extracts

of Corn as a Yeasure 3f Viability

 

 

 

 

 

 

 

 

 

Sample Percentage Net c.cm Sample [Percentage Net c.cm.
Germination KhnOA, bermination Krtno4
11 100 1-95 1% 74 1.10
7 100 1-61 9 an 4-54
10 93 1.90 5 16 2.48
l? 93 1.89 1 36 8-78
12 91 1-80 3 35. 3-7a
15, 81 1:19 16 25 7.95
8 76 9.96 15 0 12.38
14 75 1.88

 

 

 

 

 

 

 

 

 

u
.

Tte results of table 9 indicate that u are is no sir 1e relation
between viaaility and reduction of permansanuze. From the Graph, (Fig. 5),
there is possible evidence that the total reduction is due to a number
of components in the extract, and tiese may be present in Such a multi-
plicity of proportTO s that it would be impossible to establish a correlatiui

The determinations reported thus far are not conclusively negative.
Yet they are even less conclusively positive. During the time that the
work on improvement of the method was in Progress, some secondary deveIOp-
ments appeared, the pursuit of which justified the continuance of the work.

First among these was the fact.that the ability of the solutions

to reduce permanoanate was diminished quite rapidly by excosure to room

conditions. This is apparent in Table 10, the history of which could be

 

 

 

 

-23-

Figure 5

 

 

~I

«h
I

cuelc CENrmerRs KMn04
b I

N
T

 

 

l . 1 J I
0 IO 20 30 40. O ‘ O 0 M

PERCENTAGE GERMINATION

Correlation Curve Between Viability and Roduction 01’

Pomangauato an [awed by Electromotric Titration.

multiplied by that of every other extract used.
Table 10
Effect of Prolorged Standing

0n Reducing Power of Extracts

 

 

 

 

 

 

 

Egg 9: Sample ? vv
] day 0-64
__2__dm 0 -39
3 days 0.39
3 weeks 0.00

 

It is evident that tiese reactions, involving probably the
oxidation of unstable organic compounds, may go on with atmospheric
0 oxygen at room temperatures. Where fungi appeared on the extracts, the
loss was hastened. Coons and Klotz(6) report the lowering of the content
of certain classes of nitrOgenous compounds in the diseased leaves of
celery. The loss of reducing power in seed extracts may be due to a
pragressive break down of protein compounds into a-amino acids.

Inasmuch as the results thus far presented have shown that there
are large discrepancies in the correlations attempted. it follows lOgically
that some attempt to discover the bases of these discrepancies should be
made. The physical state of the extracted materials might give a clue.
It is a primary concept of colloid chemistry that the ability of a
material to pass through the pores of a semi-permeable membrane is

governed by the state of division in which the material is found. Kon-

dialyzable compounds are usually of a high molecular weight, so the

possession of the reducing power by that fraction might first point to

proteins as the reducing agents. The results of the first experiment in

this direction are shown in Table 11. Collodion sacks formed on the in-

side of a larre test tube were used in this experiment. .
Table 11

Dialysis of Aqueous Extracts

in Colloidon Sacks

 

 

 

 

 

 

 

 

 

 

PeriOd of Nature of ___EaL_K‘L1nC.¢—Leducti n

Dialysis l’em‘urane Dip-14: 4- Colloid r'ginal
16 hrs. medium O-l? 0-61 0-78
16 ' " 0-17 0-30 n_4g
16 " " -4g'17 0-94 974%
40 .. thick 0.08 0-4.3; n-aa

 

 

 

 

‘

This would indicate that both fractions have the ability to
reduce perrangannte, altho the larsnr part of that ability lies with the
material found in s colloidal state. In the above series, there was no
attempt to remove the products of dialysis. The sack, with extract, was
placed in a heaher containing 75 c.cm. of distilled water and left at
room conditions.

The question then arose as to whether tie reducins power of the

colloidal fractiun risht not SHFVa as a measure of viabili€V. It would

seem from the above that tle dialgsate did not vary greatly in its re-
ducinr power, while the collgical "aterial showed considerable variation.

‘ 0 l

A complete series was attempted, in wnicn the extracts were Llaced in

uniform collodion Sacks, which were t‘rea; fitted xvi t‘r tubes and 12131350

LJo

n
an apparatus f:r continuous dia ysis. Distilled water was si finned
throunh the bankers from an overhead surply at t“e rate of l litvr per
hour. At the end 3f a week, the contents of t‘e brss were tested fer

‘

reducinr Lower, tie results of tuese tests bein: slrwn in Table 12.

Reduciar Power

of Dialyzed Se:d Extracts

 

Percentage Net Percentage net
Sample Germination KMnQ4yf sample, Germination KMn04

 

 

 

7 100 0:i9a 3 ?9 0,32

0 11 A an 0-95

v—v— v

11

no
no

 

tJ

1Q 98 e-le, 61 (t1;

 

17 an, a C1 14 57 0.00

«7

 

47 0.27

0‘)
U.)
I)
O
;4
0‘
H

 

LS 0-‘0 16 24 0.53

O

 

kn

0.15 15- 5 0-27

LJ
i»)
L.‘

,»

 

 

 

 

 

 

 

 

 

1.4
‘1

93 0.39

 

A check series, consistinq of aliquots of the same solutions

(

kept in steppered flasks, Was tested sinilinrlg 2t tie and of an equal
time period, and the reSults appear in Table 13.

While theoretically, dialysis misht hate stabilized the extracts
by removing oxidizing scents, he only evidence from these tables is that
tie loss of redacins power is more rapid wlen the products of oxidation

1

are removed Ly dialysis and hydrolysis. The renewa- of ti» products

enables t‘: e reaction to proceed in (we directim until nothim I‘{~-;l"!€;ihS

to be oxidized or broken dosh. There is no applicability to measurements
of viability.
Table 1}
Reducing Power

of Undielyzed Seed Extracts

 

 

 

 

 

 

 

 

 

 

Percentage Net Percentagi Net
l__5§mpli_1_G§:Ql§§&i22n. KMn04, Seaple Germinatio Runs.
7 110 9‘53 3 ?9 1 17
11 99 o.17 6 as c-44
lo {)8 {1.21.1 2 61 (1-36.
11 90 0-35 114 57 ‘%;;____
E so 1-951 1 47 12-23
1; 05 0.54 15 z; 2.24
13 L3 c-61. 15 ; 1L15L
12, 0% olgo

 

 

 

 

 

 

 

 

 

Reducing Power of Extracts of Corn Feel

It is an obvious conclusion that tke extract of corn meal Would
possess greater reducing power than the extract of whole grains. Because
it was desired to germinate all the seeds which were tested for reducing
power, it had been impossible to work with meal. Comparative results
on this were desirable, however, so 500 grains of sarple No. 17 were
coarsely ground. This sawple had an average of 90% germiLetion during
the period of eXperimentation. From.Teble 9, it will is seen that

whole seeds of this serple had s net reduction of 1.89 c.cm. of per-

-28-

manganate. When meal was extracted at room temperature for 24 hours,
care being taken to use a prOportionate amount of water, the net reduction
was 22.84 c.cm.; and when the extraction of meal was made at 30°C. for
12 hours, the net reduction was 37.29 c.cm.
Comparision of Electrometric and Colorimetric End-Points

As valuable as the electrometric titrations proved to be in the
develOpment of the technique of the method, it is evident from the
charts, (Fir. 1-4), that the color and point coincides very closely with
the point of maximum change in voltage. The very abrupt drOp in voltage
was not any more striking than the fading of the purple color, when the
apprOpriate amount of oxalate had been added. In the interest of simpli-
fication of methods, there was no reason why a return to colorimetric
titrations should not be effected. Thus it is that all succeeding
determinations of permanganate reduction are on that basis.

Isolation of Reducing Compounds in Seed Extracts

On the basis of the preliminary experiments so far reported,
there seemed ample justification for an attempt to isolate the compound
or compounds, which were responsible for the reduction of permanganate.
The loss of reducing power on prolonged standing would indicate organic
compounds. But such an assumption is not the basis for any conclusions,
because of the number and complicity of organic compounds that might
diffuse out of the seed. It becomes necessary to examine farther as to
the nature of the compounds in question. Empgrical considerations would

indicate that proteins have no monOpoly on the property of reduction,

yet they would seem to occupy a commanding position in the study.

-29-

Hawk(15) lists lead acetate and ammonium sulphate among the
common precipitants for proteins. Saturation with these reagents is
supposed to bring dorn all the proteins. In the case of the former salt,
the excess lead in solution is removed by anhydrous sodium carbonate.
Table 14 gives the results of tests with these reagents.

Table 14
Reducing Power of Fractions

of Extract of Corn Meal

 

 

 

 

 

 

 

 

 

 

 

 

Description Net Description net
of Fractign KMn04 of Fraction KMnOA
Original Original
mtract 37 . 29 mm 140—,
Filtrate of ' Filtrate of
Lead acetate 4.q1 (NH4)2°§Q¢ 2-52
" Redissolv
Precipitate of Preci itate
Lead acetate 0.00 {334)E soI 2446

 

 

In the ammonium sulphate precipitation, the failure of the two
portions to equal the original in reducing power may be attributed to
the fact that some of the proteins were denaturalized and failed to re-
dissolve in the dilute solution of ammonium sulphate which resulted.
The lead precipitate was entirely insoluble in the concentration of acid
employed in these tests.

Osborne and his associates(28, 29, 30, 31) devoted a lifetime
to the study of the vegetable proteins. Their classifications, nomen-
clature, and methods of isolations are standard, hence any procedure
dealing with proteins well be borrowed 'en tout' from their works. In
relation to the proteins of corn, their amounts and prOperties, the

following classification is valuable.

-30-

Classification of the Proteins of Corn, ”Zea Mays"
1. Protein soluble in pure water--- -------------- - Proteose ------- 0.06%

2. Protein soluble in aqueous extract
(Very dilute salt and acid solution)

.’

A. Re-precipitated by dialysis --------- ------ Maysin --------- 0.25%

B. Coagulable by heat in presence of haCl---- Maize globulin-0.04%

3. Protein soluble in 10% NaCl --------- --- -------- Maize Edestin--O.10%
4. Protein soluble in 60-90% alcohol -------------- Zein----- ------ 5.00%
5. Protein soluble in dilute alkalines and acids-- Glutelin -------- 3.15%

In attempting to isolate these compounds, one must follow a
laborious scheme, the essential features of which are shown in the
description of the actual technique. Forty grams of the ground meal
of sample No. 17 were weiched out ard extracted in 200 c.cm. of distilled
water for a period of twelve hours. The extract was decanted, an equal
amount of water added to the meal, and the extraction repeated. The
fractions were then combined.

All the proteins and proteoses were precipitated by complete
saturation of the solution with (NH4)ZSO4. The precipitate was redis-
solved by dilution with water. It was then dialyzed in a collodion sack
for a period of ten days, using running distilled water. Faysin separated
out within a few days, and filtrate showed only a faint trace of maize
globulin by testing with 10% haCl in H01 solution and heatina to 80°C.

Filtrate tested for proteoses and their presence indicated in
slight amount upon dialysis of the filtrate into concentrated alcohol,

and further concentration of the alcohol to high percentages by addition

 

of 95% alcohol.

The meal was then extracted for a similar period of time with
10% NaCl, the extraction repeated and the solutions combined and dialyzed.
A small amount of maise edestin was obtained, when sufficient salt had
been removed to cause precipitation.

The meal was next extracted with 80% alcohol. Zsin was obtained
in abundance, combined with some alcohol soluble pigments. Petrol ether
removed part of the yellow carotinoid pigments, but not enough to justify
fractionation. By evaporation of the alcohol and replacement with water,
the zein was precipitated in large masses. Redissolution in 85% alcohol
and evaporation left the zein as a flaky, horny, hyaline layer on the
cover glass.

After these treatments, the meal was devoid of color, and was a
powdery, granulated raterial. No extraction with dilute alkali or acid
was attempted. Such treatment would not furnish a protein whose reactions
would be characteristic of the compound in its natural state.

It is not claimed that the proteins were obtained in even
approximately a pure state, but they were nevertheless recognized as
entities, and it was possible to measure their reducing power. Because
of the fact that precipitation of the globulins and albumins is apt to
cause the formation of irreversible colloids, it was found more valuable
in sons cases to conduct the tests upon the solutions. In the case of
maysin, which is coagulated oy removal of the prote tive ions, it was

imrossible to entirely redissolve it, only a s all fraction being

—v--

 

-32-

ameanable to boilins with 5% H2804. Maize globulin was entirely refractory
when once precipitated, but it was possible to keep it in solution. The
results of the tests are shown in Table 15.
Table 15
Reducing Power

of Proteins of Corn

 

 

 

 

 

 

Amount Net

Material in gms. Kung4
Protease ,ou1 0:09

Maize Globulin '

plus Protease .004 0.80
Maysin .020 L96
Edestin -006 0.65
Zein -020 4 . 24

 

 

 

 

 

Thus, instead of finding a single protein capable of reducing
permanganate, all were found to possess the ability. Osborne(29) reports
the reduction by zein of ferric chloride in an alcoholic solution, but
the failure of that protein to reduce potassium ferricyanide. The writer
was not able to secure reduction of potassium dichromate, using aqueous
extracts which reduced permanganate strongly.

If the proteins reduce permanganate, might it not be that they
do so by beinq themselves broken down by the rigorous conditions of the
test, If such an assumption is sound, then amino-acids should be able
to reduce permanganate as well. Osborne and Clapp(30) give the products

of hydrolysis of the proteins of maize. It was not possible to obtain

 

 

1

all tee amino-acids listed, but several were available. In this connection,
it might be noted that non-protein compounds of nitrogeneous nature are
found in corn, Schulze and Castoro(38) reporting 0.90 per cent of non-
proteins. Schulze(37) found that maize contains 0.25 per cent of
lecithin, while Czaoek(9,8d.l, p. 157), reports the same amount in
yellow maize and 0.28 per cent in white maize. Jodidi(l7), studying the
non-proteins of the ungerminated seeds of maize, found polypeptides,
free amino acids and acid amides present. An indication of the re-
ducing power of some of tiese compounds is found in Table 16.
Table 16
Reducino Power of Primary

hitroseneous Compounds

.4

 

 

 

 

 

 

 

 

 

 

Material Net 0. cm. Material Net c. cm.

h m 20 . of each M mo; ,
._Leu.c_ine 9431 :uclaiLAciLLIaast) L49
Agrartic Arid 0.05 Sodium @mocholete 0-57
.ASparagina 0-00 ucine 13-1?
,lynasima 213;" x thine 0419
ypt Lip-name ll . :4 Cr eat inc 0 .00

cithin l .474

 

 

 

 

 

 

The first half of Table 16 deals with amino-acids found as
products of tte hydrolysis of the proteins of corn. Included in this
orOup is the phOSpho-protein, lecithin. The latter half of the table

is not directly ap licaole to tnis study, out has seneral interest in

demonstrating the wide ranoe of reactivity of permansanate.

 

 

-34-

It is interesting to note tLat all of the high reactinq compounds
contained a rino structure. According to the classification of Haas
and Hill(l4, p. 324) these are as follows: tyrosine, an aromatic compound,
B-parahydroxyphenyl, a-amino proprionic acid; tryptOphans, a hetero-
cyclic compound, B-indole a-amino prOprionic acid; brucine, a complex
alkaloid of the quinoline yroup, characterized by two six-membered
rings condensed tooether. Lesser-Cohn(20) mentions also that permangan-
ate reactivity is a means of distinquishino between unsaturated acids
and saturated acids containing Open or closed chains, and carboxylic
acids of benzene or similar bodies.

The role of this group of compounds was thus sufficiently
.established, but there had also been indications that the simple sugars
were not lacking in reducing ability. Qualitative tests on glucose
confirmed the suspicion, so the tests tabulated below were performed.

Table 17
Permansanate Reduction

by Common Sugars

 

 

 

 

 

 

 

 

 

 

 

 

 

Sugar Net <3. cm. Sugar 1m c. cm.
WEE—Mk 20 ch u _;1g_m_g‘_,
tratiePQp 9'13 Galactosa, 9,33
leose 3.b0 Sucrose 9-96
Dextrose 0.10 Paltose 5-13
Hannose b401, Lactose 3- 4
Isvulose £3-35 admins; 4.43
Sorbose 4§.6E

 

While the data on reducing power of sugars in no way constitutes
a scientific novelty, the direct application of permansnnate is not
mentioned in the literature. The nearest approach is the indirect
method wherein the reduced c0pper is measured by permansanate titration.
In this connection, mention mioht adain be made of t‘e action of per-
mannanate on starch, noted by Reichert(35). The sivnificance of sugar

n the aqueous extract is differently interpreted by Filler and Hibnard(2§).

Ho

1 0 0

They considered it as a sterilizinc e~ent in the foxwetion of silver sols

by reduction of silver nitrate, while Droteiys Ware given t”e cover of

reduction.

\
5—1.
,4
1
'u—J
‘

v o)
s,
U)
U)

eke fiu-dfi7 cresence 0F SJ” F in ii; AQun-JS n.o;‘Cn

LII

J. _V,_- - ~ ., ,.. ..,\ , _;3,,., A1,, 0 - '.- ,. 4.1. ,- .,J. ,y . ,. ‘ 1‘ y ..
d‘suerfl;1hfxi ‘Q' a Eabflluhurd run.. o3 . u,LfiIT.fl”LlLlOV1 oP u e fibf’xct vus My .18d

of Horne's anhydrous lead Sib-fiCBtaCB’ w‘ick would remove the pro

1‘.. .. n4- ‘ A .3 4- ~, , . , , ,, , g . ' J 5.-. ,. ‘ .
U'Jt 1$UU It .5} ‘ .l or3"-3'; T'3\48 PJ"'.‘0:"1\!9 O; 7.;‘n'pr3 .4511)! C .arv'LC eel ' or TAG

- .. " r "tn 4‘ ,. ... ..2.L‘ v T._',’- "Wu +;nt 4
suNaxs. Fae excess lend Jns c L .Jve.rd : u .ngnlun- LnJ ~Uav f0!

. .. .. x." a ‘ 4.2,. I' . My 1" 1. ML. , , r _‘ ' ° ‘. ...° .l . (1‘
SUN'C'.:‘ “(1:13 1/- 7:73 k)?1 u.."z' I'll-118;)Il EL'Lkl aid]. tar. rt!‘.'1l;v_'\’)¥1 ‘f‘ 0‘ I‘\.c:1 "1 LE1 UJL‘11(‘ 1"; ol)‘\_)) ,

and tie Schaffer-Hsifiwnnna iodometric titratitnx:of no per(32). The ally

 

 

Inodific.AJ.n1‘vas ix: 3 9.3 N Hzfifigl to tVe {cunnit of l? (3“, . '"‘ NT 3,
insteo3 of 5V to file Cj‘. 3.9ant.0 Fadias cf tTe ere-voint vs“ cw recr’
lg t" is c‘ cmr .

’Nith proteins and Svszrs alisned in rashect to their pre;erty
01' red;n3i2f“ gsxxuc r :J.L3, ‘fiie (filly 12¢ mfr.in” "roup. of "n‘t:z-sollfi,le
cowvonfids of any ir“ortence, a) *‘u zen-rrvtein Litter, sens 3-riv Lives.
A r527 r “rritgs "ethod of rroof was s"rlr; d ‘r t‘eir ?*?a. The

__ ..,..,_.,_....,_,.*..w.."..__

0 or "5 ’: 3 "r. H. F. Clev~ is, sf t‘u “u;a y cht. ’.Q.J., for tPis

1L

‘- «4“
. .3 P? ‘ -' £- ..l , ,.(~ .3 4 M L- .. s .. . ' 4- #1
cl 1; _+e uYCr‘fi. 'r>':.,i as 3e:er. rd e:ove 'Hs Sun atted to “Jul the
L . J ‘ L “L - . - 'L ‘4‘ ' 0 V .' - ‘ ' r 1 x ‘ ,- J' -. " " '1‘ -‘ " ' I‘I ‘ ‘ - 1'
seendrro si"fir test and ».e -erw-w~3raoe TcCu'b.Uh cunt. By means of tre

’5' J. ’c _’_ ‘f i“. ,, ‘. ‘ r»; , V ‘3’. I “_ , ‘ .—L‘,L, .~
dl_z~,"‘§ ()f J‘r":)-] ., J7, ,‘ ‘.) ":;171.L{}!~,‘}A:1‘,{‘2 L“ \iucbl\)rl f-‘Ir U Vl‘u LW’L’LAI‘J; ’4', DE

"1J1FHJSE‘ «U:5 <33‘_rn',9d . Irltfi d if‘feannc': iJn } 9.53: TC‘.H h}. r l3s115"33 :“i'“‘t
t‘"'*’ ' 0 wt tr‘5“. t (i ‘tf t' ino?1-‘ C’“Z *i‘-s . .J;:LI‘-\,t o F “ er. ”‘-" T ‘vs-s 3"c5
for ~his fi:~t, :2 o'ie“ t‘at iirsrr d’ffsr‘nccs “f"ht :H C‘C?:a.0 ‘ 3
t‘is "five a f‘;“>r luais ">r c.rn.rlsigr. T7: 9C‘r"P 1~ S‘Jvu j. L‘s
fol?» 7‘“ t.;7~.
Tduid 1;
1'0 1"}1‘0'335113 £33 1’1"..‘.'.i.3 i‘d
tle Rednetian of Pernansn ace
in

 

DWW

 

 

 

Permanepnntp on giggifiifi;_mafiluextract 34-46 n-o

 

Standard sneer on semel£2c-4 re- glncnag)

 

 

(301114311th I’Vnofi fOL?!. ‘4 m7 qlnggqg 11.66 C;

 

 

 

 

 

' . .- .2 -- , "z” ' ‘,- 4 1:. +4 . .-.
It 13 not (ossiels, -L x U .1 .f 4,.3. firm lsc, d ~,\, sch
"” . ""5 L ' "L ‘ "- ‘ ‘ fi 4’ n I‘ r“ ‘\ I: .' , L . . t‘, ' I . ' z in N -. J - ‘ ‘ J ‘ , vs
V' I? 1 " 4 ’J .h J 1 (-3 a. S U l, U D \_I‘ F t V . 1. \‘f ,1. ’ f 1( ‘: ‘ ’ . \J j .YA C ‘4 I. ‘3 r... 1 .L 1 ' .L Q ‘ ‘ L1 1 \2 C‘- :. L .

1':~.LL .1 . 1' _ “1-, av : _0 VAL .: 1, ,_ t; ‘,i J.‘, . ,"
'.u 1 Uf: O'-_L:"" r1 ' .4' {$313, _| t- is a'l\. LI ' C’Srf' s3 :1. U‘J‘ (1‘ u Line vs 'J;~O'-

-37-

influential in the reduction, yet not in equal measure, or even intensity
of reaction. The fact t'at the TOdJClK" go er is quite rapidly dininishe
upon standins may he in vart a matter of actual decrease of the materials,
or it may be an oxidation without any other quantitative differences.

A test of the correlation of the content of protein, non-prntein,
and susar, with the reduction of permansanate, was next attenpted. It
was heped that some clue to the unusual reactivity of several samples of
good germination, misht Fe 0 tained.

The methods were conventional. The sugar test was as given in
the precedins paves of this paper. Total nitrocen was run by the
Kjeldahl method, usinq CuSO4 as the catalyst. The acid used to absorb
the NH3 wiven off was found to be exactly N/lO by aravimetric determination.

On the first few samples, it was attempted to run the non-protein

nitrosen from the clarified sonar-test extract. This proved impossible
because the lead contained 0 nsidera31e nitrogen as a contamination.
Precipitation ny phospho-tunsstic acid proved more consistent. The test
is as follows: 5% phOSpho-tungstic acid in 5% H2804 is added to a seed
extract made acid to 5% with H2504 and heated to boiling. Fortunately,
a sufficient amount of the original extract from the samples remained to
repeat the test with the latter reagent. The protein nitrogen was
obtained by difference. Direct determination was unaccountahly incon-
sistent, and it was not considered germane to this study to spend time
on that problem.

Permanganate reduction was run on the original extract and the

sugar-test extract. The reacting power of the latter to permanganate

-38-

exactly equaled that prOperty in the extract from the phOSpho-tungstate

precipitation, so that the results skown in table 19 are as applicable

as if run on the identical solutions.

Table 19

Test of Correlation of Content of Proteins,

Non-Proteins, and Sugars, with Reducing Power

in Terms of Permanqanate

 

,-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Percentage Total Total Non-Pro. Glucose Second
MMinetio mm); kin mg. 11- in mg. 1% MOL
10 94 0-53 -195 -130 0129 g-gz_
1 85 1.16 .146 .122 1.60 0.91
a 83 6.32 .12 .204 8.81 2.73
7 13, 0.71 -130 -082 2-28 ng1_
2 91 1-08 .163 -130 ,0-80 33_0-48
6 64 0.52 -114 -082 9 21 Q;L2_
5 60 0-97 .212 -203 1 59 0-42_
1, 29 5-05 -489 .155. 2-92 2.951
16 21 5349 .619 .521 6.53 3.82
1: 0 9-15 1-695- _1.305 12.77 1-82

 

In this series, 100 seeds of each sample were placed in a flask

with 200 c.cm. of distilled water and allowed to soak for 24 hours at

room temperature. The extract was decanted,

filter paper, and made up to 200 c.cm.

filtered throuch a coarse

5O c.cm. of this was precipitated

'ith lead for sugar and permanranate tests, 50 c.cm. with phOSpho-tuncstic
acid for non-protein nitrogen, 50 c.cm. used for du;licate determinations
of total nitrooen, and the remainder devoted to total permanganate re-
duction tests.

As the data in previous tables is all on the basis of 10 c.cv.
of extract, the amount obtained from five seeds, the results of these
determinations will be similarily reduced. In that pronortion the amounts
of some of the constituents would he too small for detection, but as the
results were obtaised on sanples averasino five or ten times the minimum
amount, clecks were very consistent.

From table 19 it may be computed tlat the amount of sucar in the
extract varied from .02i to .EOfi of the averace weight of the corn, while
the amounts of nitrooen were of a lower order. But within the ranoe
presented, wide differences are evident.

The small amounts of soluble rate.fal in the unoerminated seed
was the subject of early investirations. In 1385, Portele(33) made a
study of the chemical nature of yellow corn at various snares in its
growth. Starting from the time of flowering and runninr to the time
when tie kernels were lard, there Was 2 st! dv decrease in surars Lhd
soluple nitrocehe us comCOunds, :nd a steed; rise in 8
But it is recorrized til: the act of soakihc seeds would encourace
enzyratic activity and initiate c eaicnl ctriocs tendin" towrrd tle
formation of simpler compOunds. The diffusion of these procucts into

the SOldtlun Visit Dd at differeht rites, loveiez. as wall, as 1L9 ,

‘E

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0 f £0 I .457 ‘10 J]? ‘10 7‘5 a; [a]? mo

PERC IN 1"“ C‘RMINATION

Correlation Curves Between Viability and Various Chemical
Components as follows: A. Total Nitrogen, B. Total may
0. Non-Protein Nitrogen, D. Glucose, E. 2nd KMnO4(Reduction

of Protein- Free Extract).

Comrarstive

Table

-42-

20

uci‘c Power

of Su~nrs and litreseneous Compounds

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Total 1'0““ Glucose
Sam] a 1mm H-ln Qg In as y.
5 0.52 ..;;4 _ 2.24
10 0°53 .195 0.29
7 0.71 ..lgo 2.2g
5 ‘o.77 .212 1‘52,
2 1.08 .163 0.80
3 1 . 16 9 146 3. 80
1 5.05 .489 2.12
15 5.49 .619 6.53
8 6.32 .326 8.83
¥;§ 9.15 A1£§95 12.77
Even grantinc tke fact that tle nitro~ene0us comnounds hrs

present as proteins or Similar corpounds,
active, rram for gram, than the sucnrs.
nitrogen or .50 mg. of protein, between samples No. 6 and E0. 10 is
compensated by 1.95 ms. of sugar to give an equal permansenate reduction.
But between samples lo. 7 and No. 5, an equally great difference of

nitrogen is compensated by .76 mg. of sucar, so it is hardly wise to

draw any hard and fast conclusions.

tley are nevertheless more

A difference of .08 mg. of

-43-

With only general relations under consideration, it is convenient
to consider the samples in :airs of nearly equal reducing power. In all
cases of this sort, an excess of suvars in one is compensated cy an excess
of nitrogen-bearing compounds in the other. In the latter part of the
table, amples of maxtudly greater reducing power are found to surpass
those of lesser activity in content of both sugars and nitrogeneous
compounds. That the reducing power of aqueous extracts rests on the
sugars and nitr0geneous compounds is clearly demonstrated.

The variation in germinability of a sample of seed upon the
conventional method of growinp the seeds ray amount to as much as five
or ten per cent. If there was any correlation between viability and
reducing power, it might be sXpeuted to stay wittin the same limits.

But when the variation in reducing power is very much greater, as was
found in table 19, it is sound to conclude that a positive correlation

is lacking.

-44-

D scussion on Experimental Data

-._.—_ A

On the basis of results presented in this paper, it cannot be
assumed that there is any correlation between the viability of seeds and
the reducing power of their aqueous extracts. As far as the relative
position of samples in a series was concerned, there was a fair con-
sistency in the reaction when tested from time to time. But it was
only infrequently possible to find a group of Samples which would give
gradations in reactivity at all comparable to the viability.

Why are the results inconsistent? A number of reasons miwht be
advanced, and no one alone suffice to interpret the situation. For the
first line of encroach let us consider the chericsl phases of the

question. A great amount of work on the chemical corpositicn of corn

‘

has been published. With Special reference to the variations encountered,

mention nickt be wade of the analyses reported by Pushey(5), Ladd(l9),
Leach(21), Lindstrom and flerhardt(23), and Purtele(33). This group
night be greatly ausnented, but the type conclusions found are much the
same.

The content of all the important constituents may be varied
by a host of circumstances. The state of maturity enters very strongly
in influencino the covpcsition. Rushey(5) found that corn killed by
frost hat a hish oercent of non-proteins in the form of polypeptides and
amino-acids. Immature corn is also known to have wore susars and less
starch than riser samples. flenetic differences are the bases of pr at

differences in composition, as has been shoun,amono others, by Lindstrom

-45- '

and Gsrhardt(23). Lack of chemical uniformity is so conclusive that
no further mention need be made of it.

Added to these differences, the fact that in permanganats
reduction, ssvsral groups of compounds were active, makes the task of
establishing a correlation on chemical grounds sell nigh.impossibls.
Tbs various possible combinations in.smount of thsss components, com-
bined with the differential reactivity of tbs groups, makss tbs rs-
lstion still more canplex.

Tbs physical stats of plsnt membranes is not tbs least im-
portant factor in establishing differences. Shull(40),in his study
of the semipermeability of seed costs, found that even dead plant
membranes might be semipermeabls. Tho sntirely impermeabls naturs
of tbs coats of many seeds, and tho deterrent sffsct of this on garb
mination, hss bssn ths subject of investigation.

The effect of the colloidal stats on.ths permeability is none
too clearly defined. Whether changes in permeability ars due to tho
coagulation of the proteins, a view advanced by Crocker(7), is hardly
definitely provsn.

The rscsnt findings of Hottss and Huslson(16) on swsst corn
constituts an interesting study of physics-chemical stats. Although
their studios wsrs on tho rslation of ths sssdling vigor to tbs
colloidal properties of the aqueous extract, tbs relation of the
latter to viability was also indicated. Between samples of zero
germination and those of 95% to 100% germination, the colloidal indsx,

as measured with a Lsits nephelometsr, varied considerably. The

results show that denser suspensions were found in extracts from seeds
of low viability, but as far as the applicability of these findings

to measurements of lesser differences in germination power is con-
cerned, the method shows little promise.

In consideration of the data presented in table 19, it would
seem that the most important change accompanying loss of germinating
power is an increase in permeability. ‘Yet even this rule is violated
in at least two cases out of ten, and it is necessary to assume some
uncommon occurrences in the history of samples like No.8 and No.3 in
order to explain their high rate of exosmosis. Subject to these
deviations, the difference in permeability seems to bear a fundamental

relation to the phenomonon of death.

M

l. The literature on methods for determining viability was
reviewed.

2. The permanganate reduction method as a measure of viability
was made the subject of a process of refinement.

3. Electrometric titrations were substituted for colorimetric
titrations with a view toward deveIOpment of a more discriminating
technique.

4. The method was perfected sufficiently to insure consistent
results.

5. No correlation between differences of viability of as much
as ten per cent, and pemanganate reducing power of extract, was
established in any case.

6. Colorimetric titrations under the conditions established
were provem as accurate as electrometric.

7. In a supplementary experiment, iodine absorption of aqueous
extracts was measured, but even less promise was shown by this method.

8. The reducing power of dialysed extracts was found to have
no correlation with viability.

9. Isolation of compounds causing reduction of permanganate
was attempted.

10. The following classes of compounds were found to have
reducing power: 1, proteins found in corn; 2, some amino-acids found
in corn, and other primary nitrogeneous compounds; 3, common sugars;

4, nitrogeneous compounds of non-protein character found in corn.

-43-

11. A correlation between content of proteins, nonpproteins,
and sugars of extract, and reducing power of extract, was attempted
on basis of standard analyses.

12. Positive correlation between permanganate reducing power
of solution and total nitrogen plus sugar content was indicated.

13. No correlation between viability and amounts of any of

the canstituents was found.

-49..

Acknowledgement s.

To Dr. E. A. Bessey, the writer is
indebted for kindly insight and inspiration
at all times, and to Dr. R. P. Hibbard, the
writer is indebted for patient guidance and
cooperation both during the course of the
experiments and the period of preparation
of the manuscript.

To Dr. D. T. Ewing, the writer is
indebted for the generous disposal of equip-
ment and materials for electrometric titrations.

To the D. M. Ferry Seed Co. of Detroit,
for the kind contribution of funds for a fellow-
ship in this department, the writer is further

1114013131 0

9—1‘ In

 

 

l.

2.

3.

4.

5.

6.

7.

8.

9.

2113110th

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F—_-_._--._

O I
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O
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IA
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-51..

viability by electrical conductivity measurements. Mich. Adad.
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Lindstrom, B. W. and Gerhardt, F. Inheritance of carbohydrates

-52-

and fat in crosses of dent and sweet corn. Iowa State Res.
Bull. No.g§,l926.

24.2nc Hargue. J. S. The significance of the peroxidase reaction
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25. Miller. E. V. and Hibbard, R. P. Aqueous extracts of seeds as
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_: 409-413, 1926.

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-53—

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