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THE UNIVERSITY OF MINNESOTA
GRADUATE SCHOOL
Report
of
Committee on Examination
This is to certify that we the
undersigned, as a committee of the Graduate
School, have given Robert Newton
final oral examination for the degree of
Master of Science We recommend that the
degree of Master of Science be conferred
upon the candidate.
Minneapolis, Minnesota
~--~---·---19"!?/ ~ .. ····--······-·······-·-·-····-·-·-····-·····
Chairman
.11 ... -t. .... ll .. ~-·· ···-Jf.-s£.~#--··-··
THE UNIVERSITY OF MINNESOTA
GRADUATE SCHOOL
Report
of
Committee on Thesis
The undersigned. acting as a Committee
of the Graduate School. have read the accompanying
thesis submitted by Robert Newton
for the degree of l~ster of Science.
They approve it as a thesis meeting the require-
ments of the Graduate School of the University of
Minnesota. and recommer.d that it be accepted in
partial fulfillment of the requirements for the
degree of
10-20 SM
L
A CO~PARATIVE STUDY OF
WINTER lf!IEAT VARIETIES WITH ESPECIAL REFERENCE
TO WINTER KILLING.
By Robert Newton. f
A THESIS
Sub~itted to the Graduate Sc1ool of the University of
:unnesota in partial fulfilment of the requirements
for the degree of
Master of Science •
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St. Paul , :v innesota.
"i!ay 4 , 1 9:... 1 •
' l" • i
ACKNOVTLEOO~NT .
The author tenders sincere thanks to all those who have
helped him in this project. Especial appreciation is due to
Dr . R. A. Gertner under whose direction the work was carried on.
Dr. L. I . Knight and Professor H. K. Hayes contributed many sug-.
gestions to its planning. Professor Hayes and Professor A. C. Arny
placed the necessary plots of winter wheat freely at the disposal
of the writer . Dr. R. B. Harvey made available special equipment
for the thermoelectric freezing point deter~inations and gave
helpful advice . Dr . c. A • • orrow gave special assistance in method~
of analysis, and other members of the Division of Agricultural
Biochemistry have been generous in their help .
Acknowledgment is also made of the substantial help
afforded by election to the Shevlin Fellowship.
10-ZO 5M
..
TABLE OF CONTENTS.
Introductory
Historical
Recent Progress
Experimental
The Problem
Methods
Tables
Collection of Samples
Physical Constants
Preparation of Samples for Analysis
Dry .~atter
Total Nitrogen
Extraction of Sugars and Soluble Nitro en
Amino Nitrogen
Water-Soluble Nitrogen
Clearing Extract for Sugar Analysis
Reducing Sugars
Sucrose
Starch
I Survival under Field Conditions
II Changes duri Storage in Ice Chest
III Physical Constants of Sap, 1st Collection
Page
t
2
3
8
8
9
9
9
, 1
12
12
12
14
14
14
15
16
16
17
18
19
IV Physical Constants of Sap, 2nd and 3rd Collections 20
V The Role of Sugars and Electrolytes in Os~otic Pressure 21
VI Nitrogen 22
VII Sugars 23
10-20 5M
A COMPARATIVE STUDY OF
WINTER WHEAT VARIETIES ITH ESPECIAL REFERENCE
TO INTER KILLING.
By Robert Newton .
INTRODUCTORY.
Winter wheat , where it can be safely grown, usually outyields
spring varieties quite markedly , but unfortunately it is much more
restricted in distribution, due to its liability to Jinter-killing.
Much pro ress has been made with this and other crops in breedino
for cold resistance by empirical methods . It would appear , however,
that greater and more certain progress should be made if the nature
of cold resistance in plants were well understood. This subject
ha s long been of interest to physiologists , and in its practical ap
plications is of widest importance . The northern limit of profitabJ~
growth of our staple crops marks also the limit of profitable ex
ploitation of our agricultural lands . The southern farmer has like
wise to meet the problems of the frost killing of fruit buds and
flowers and the more tender inter cereals .
10·20 SM
HI STORICAL.
The progress of our knowledge of the nature of cold resistance
and frost effects has been reviewed quite fully at different times
by Abbe (1895) , Blackman ( 1909), Chandler ( 1913) and others . Of
the earlier investigations it will be sufficient to note here in
their order the most significant .
The theory of Duhamel ·and Buffon ( 1737) that death from cold was
due to rupturing of the cell walls by expansion on ice formation is
of historical interest . It was almost a century later that Goeppert
(1830) found ice formation to occur in the intercellular spaces .
Sachs (1860) showed this to be the usual occurrence , and developed
the view, now generally considered erroneous , that disorganization
took place on thawing , and might be prevented by warming very slow
ly , allowing time for reabsorption of the ater by the cell .
Later Mliller- Thurgau ( 1886) proved thBt this ice forma.tion in
the tissues was necessary for freezi to death, and concluded that
death was due to the consequent desiccation of the protoplasm. This
hypothesis received support from atruchat and olliard (1901) ho
demonstrated the identity of the modifications in cell structure
produced by frost , plasmolysis and desiccation. The ork of Greely
(1901) supplied si milar evidence . Mez ( 1905) opposed the theory of
death by desiccation, since his investigations indicated that all
solutes crystallize out at a temperature not lo ~r tha~ -6•c. He
concluded that cold desi ccation must therefore be complete at this
temperature , and ca~~ot explain in ury to plants hich resist muc
lower temper tures . He advanced inste d the theory of
minimum temperature for each plant • •
10-20 SM
f t -
Gerke (1906) showed another i mportant effect of the witdr wal
of water , namely, the precipi tation of certain proteins by the in
creasing concentration of the cell sap , aided by its increasing
acidity on cooling. He also showed that the precipitation occurred
at varying temperatures for plants of varying degrees of hardiness.
This was ascribed by Schaffnit (1910) to the splitting in varying
degrees during the hardening process of complex proteins into
simpler, less readily precipitated forms .
Lidforss ( 1907) found most hardy plants to have their starch
reserves converted to sugar during the winter, nd believed this an
adaptation for cold resistance, the sugar having a protective action
in preventing the precipitation of the proteins. Schaffnit ( 1912)
tested the effect of adding sugars and various other substances to
plant saps and to egg albumen solution, and was able to modify very
greatly the precipitation by freezing . Equally striki results
were secured by Maximov ( 1912 ) in increasing the hardiness of sec
tions of red cabbage and Tradescantia by freezing them in solutions
of either organic or inorganic substances, provided these were non
t oxic and had a low eutectic point.
Recent Progress .
Recent investigati ons have dealt mainly with the hypotheses
noted above , extending them in several important respects. Atte~pts
have also been made to determine the correlation of various chemical,
physical , physiological and morphologic 1 characters with apparent
frost hardiness.
Several workers have drawn attention to the possible import nee
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oft' e fact that plant sap is contained in cells of capillary dinen
sions . D'Arsonval (1901) estimated the os~otic pressure in very
s.nall cells at 1000 at~ospheres , and notes that by the ap)lication
of increasing pressure the solidification 'point of water can be
lowered indefinitely . In buds with small cells, dense tissues a.d
meaoer water content, Wiegand (1906c) found no ice at -1 8°C .,
thouoh in ~ost buds it was present in large quantity . He concluded
that the degree of cold necessary to cause the separatio. of ice is
proportional to the force which holds the water in the tissues.
Lewis and Tuttle (1920) reported that livins leaves of Pyrola b .
wrap~d around the bulb of a mercury thermo~eter undercooled to
- 32 . 1°C . before ice for~ation took ,lace . On the other hand, it
has been a universal observation since the ti.:1e of Goep.ert (1 30)
that ice may form in the tissues without injury to hardy pl~~ts,
so that undercoolin3 is not of itself a sufficient expla atio of
hardiness .
The water content of tissues is related to structure, and it
has been shorrn by several investigators ( Schaffnit, 1910; Sinz,
1914; Beach and Allen, 1915; Aker en, 1917; Johnston, 1919; Rosa,
1919) t.at dry. atter content is d rectly correlated ith hardi ess.
Sinz and Beach and Allen noted also the i~portance of struct res
resisting desicc ~tion, while Pantanelli (191 ) found inj ry fro~
frost to be al1ays proporionate to loss oL water from the tissues,
even when freezin..:; was done in a saturated at.:nosphere .
Between the concentration of the cell sap and inter hardi ess,
Ohlweiler (1912) and Chandler (1913) fomd. a direct relationship;
Salmon and Fleming (1918), ~orkin; ith dnter cereals, found none .
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4
Pantenelli (1918) partly reconciled the conflictin· evidence by re
portin...:; a relationship in so:ne crops and none in others, including
wheat . Probably the sap of all plants increases in concentration
durin; the hardening process , but not necessarily in proportio to
the de~ree of hardiness attained. However , the earlier evidence as
to the i ·nportance of the accumulation in the sap of substances of a
protective nature, especially sugars, has received further support .
Gassner and Grimme (1913) and Akerman ( 1917) reported that hardy
varieties of winter wheat and other grains ~ere richer in su ar ,the
differences between varieties correspondin to differences in degree
of hardiness. Pantanelli (1918 , 1919) found that su ar was rapidly
used up durin; exposure to freezL15 temperatures, and that hardi. ess
was rel - ted to the quantity of su5ar retained by the plant . The
association of susar accumulation rl th hardenin- by cold has been
pointe1 out again by Rosa (1919) and Coville (1920) .
On the other hand, Harvey (1918) found that cabbages acquired
hardiness on 5 days exposure to +3 °C., before ru y reat change oc
curred in the carbohydrate equilibrium. He believes the principal
effect of the hardening process is a cha113e in the constituents of
the protoplasm,· as indicated by an increase in the ami o- acid con
tent, and on freezin; the sap by less precipitation of the proteins.
Re :neasured the increase in hydro·en-ion concentration of the sap
01 cooling , and was able to produce the same relativ& preci.itation
of proteins by addin~ equivalent quantities of acid. However , the
ease of precipitation of proteins by freezin; apparently cannot
always be taken as an index of hardiness , since Ch~~dler ( 1913) as
unable to find any difference in this respect bet ee the sap of
10-20 '"'
5
tender and hardy twigs of fruit trees. Again, it must be rememb~red
that only a small fraction of the total proteins is present in the
expressed sap, and at most only 31,2 percent of this is reported •
by Harvey as precipitated by freezing the juice of unhardened cab
bage. To take this as an index of the behaviour of the proteins
Within the cell may be too sweeping a conclusion.
There is some evidence that stability of the dormant condition
may be an import~nt protective adaptation. Lidforss (1907) noted
that a succession of warm days caused regeneration of starch fro~
sugar, with an increase in susceptibility to cold. Chandler (1913)
found some varieties of peaches to have a lon6er rest perioj than
others and to be started into growth more slowly by warn pE~iods in
the winter. Evidently varieties which can maintain continuous
dormancy during the danger period must have a distinct advantage .
Our present concept of the causes of winter -killing may be
briefly SQ~arized. Without doubt, the ultimate cause of death by
freezin3 must be the disorganization of the protoplasm. Irrevers
ible coagulation or precipitation of the colloidal nrotein constitu
ents may be caused by increase in concentration of electrolytes in
the cell sap on withdrawal of water, or by increase in acidity , or
by both f actors acting to ether. The critical minimum te perature
necessary to bring this about must be profoundly modified by rate
of cooling, especially if this be slow enou·h to ive time for the
hardening process, and by the presence of substances which protect
the proteins from precipitation. Splittin3 of the proteins durin
hardening may be a protective adaptation. Si~ce the fundamental
feature of the disturbance produced by freezing is withdra;al of
10-ZO SM
rr===================================================97
water fro.n the cell , intracellular adaptations to resist desiccation
must be of prime importance .
10·20 '"'
EXPERIMENTAL.
The Problem .
The present study seeks to establish a chemical o_ physico
chemical measurement of hardiness for winter wheat varieties . A
number of varieties originated or selected by the Depar.t ent of
Plant Breeding of the University of :unnesota, and known to va.:ry
considerably in hardiness , were co!!l_:Jared 11ith :-eference to the
physical constants of the cell sap , the content of a. ino nitro en ,
water -soluble nitrogen and total n1 trogen , and the content of
SU3ars and starch. All material used as -rown in field plots
under normal conditions . Since it as desired to coapare the var
ieties in the hardened condition rather than to s udy t e harden
in..; process , collections were not ade until ft r the advent of
freezin~ weather .
A preli~inar study of p ysical constants was carried ou•
~ith ei ht varieties . Subsequent study as confi e to fo r of
these , two hardy and two tender . O~e variety, i h ~ i, as col
lected from two plots so~e dist~~ce apart, and these are reported
separately as the effect of loc tlon was quite arked. T ble I
indicates the hardiness of the varieties used as deter i.ed by
survival under field conditions .
10-20 "''
Methods .
Collection of Sa~ules.- All the sa~ples of one series ere
collected fro;n the field the same afternoon, thou.:5h wit 1 the excep
tion of the first series the l eaves ere frozen solid hen collected
so that changes due to vital activities would be very sli~ht . The
pla.nts v1ere growin0 in rows , which were carefully one ov r for t. e
re.noval of dead leaves before taKirl3 the sa.r.1ples . For the col
lection of November 12 ,1 920, it was necessary first to brus: off ~
lisht coverin0 of snow. As the leaves ere cut, they ere thrown
on a wire screen for the re oval of dheril1G bits OJ. irt an ice ,
t en tra sf erred at once to ti 0 ht 0 lass co.tainers . Sa.nples of
ap_roximately 100 ·ra.ns were collected in duplicate , o. e lot _or t. e
st~dy of .hysical consta ts , the other for ru al sis or nitro en
~d carbohydrates . All sa.nples ~ere· ept ~roze. mtil us~.
?!t,rsi cal Consta_ ts.- T e depression o"' the freezL po·. t of
t e first collection was deter i.e b the thermoelectric et~
t.e accuracy of which has been sho b ite :; t ) • •. e conve .-
lent arr"' 0 e:nen of app rntus illustrate b· arvey 9 9 fig . )
was used . The leaves -;ere pac ced into a sect_o. o. t. i - lle'
_, ss tubinr:; hich they hel" b• '""ll - 2 c:n . lo . .;; , in ere 1"' -'"'Ce 2. s _ ..
rubber band, t 1e ther. ocouple t en ein · inserte in + IJ e center.
·ndercoolL _:; sel O.J "'m.ountecl to more t .c 2·c. "'!1 '""S correcte
fo.· i n the usual :/C:: .l· B· t. is etho u_Jlic ... tes o te. v ried ...
much as 0 . 03 °C., and in later collectio s i
of t e sta dard Bec~Cl'"".nn et o , b · hie .. it ·as al a s possible to
obtain c ecks a·reein· wit11n 0 . 0 oc. The worlc of Dixon and At ins ( .., 3), exten e b· Gortn r ,
10·20 5M
La ~renee and ... rri s ( 916) , h s shoYm tht- neccssi ty o=: renderin.:;
the cell membranes per~'lerble by freezin • the tissue previous to sap
extraction in orde~ that ~ representative saQ~le ~ay be obtained.
Eowev...;r , h~vin'-> re.;ard to the observatio.1 o. Enrvey ( 191e) th· t
f r eezin,s permanent! lowered the hydro_;e - ion co centration of
c bba~e juic6 , ~nd 920) th ton the o,her hand cert i ar; ount
of dilutio. did no affect t.~s v lue , the sa ples o. t~e first
collectlo. 7ere not frozen before expressi '-' ~he s"p , as it w"'s
desire , to study particul rly the relationship o. his constant to
hardiness . The follm7in co :1parisons o-: sap expreosed fro:!l up-ic -
te sa. ples 'lith and 1i t .. out previous freezinv i dica"'"ed th~t for
wheat ~t le...,st Harvey ' s observation does not hold true .
V riety ot Prozen Frozen
pH pH
nh"rdi 6 . 580 6 . ~76
Btf.~.um 6 . 465 6 . 2 9
p d.li 6 . 475 6 , ?'-' ...
Therefore in l"ter collectio. s .reli nary freezL ;;.;> of the tissues
was carried out, and the expressed sap used for 11 co .. stan:.s
studie 1 •
The technique of Gertner and .~r~is ( 19 14) w~s follo e i. the
main . The rubber- stoppere bottles cont inin~ the samples ere
packed in slushy mixture of pulverized ice and snlt in an earthen-
.are jar , 1hich fitted s'!'lu ly inside a ell-insul~te "fireless
cooKer ". I n this condition the contents remained frozen solid
until requi red for use , but never i n ny case for less than t·1elve
hours . For the freezi n mi xture , co~~on salt s used t first ,
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10
and later calcium chloride. To thaw the sa~ples, the bottles were
placed under running water , then rinsed 'nth distilled water and
wiped dry before opening . The leaves were folded in pieces of
strong cotton previously boiled in 3 changes of distilled water and
dried free from dust , and the sap expressed either in a hydraulic
press under 400 atmospheres pressure, or in a large hand screw pres
with a small steel cup which enabled heavy pressure to be brou3ht
to bear . The parts of the press with which the juice came in
contact were kept coated with a thin layer of paraffin wax.
The depression of the freezing point was first determined ith
a Beckmann apparatus . Then the conductivity was measured ith a
Wheatstone bridge , using a Freas conductivity c 1, and finally the
hydro en-ion concentration by means of standard Leeds and Northrup
potentiometric equipment . Both of the latter deter~inations were
carried out at 25°C. in a constant temper ture room. Usually the
work on any particular sample as completed within an hour of ex
pressill6 the juice.
Preparetion of Samples for Analysis.- As the aampl~s for
analysis were collected in the field the ·er~ pl~ced direct_· in
tared one-liter erlenmeyers ith rubber stopners . They 1ere frozen
hen collected, and kept in that conditio overni;ht. r~~edi tely
fter th ing in the laboratory next day, s pleo for the deter
minction of dry m~tter were ei-hed out; then in addition, suffic -
ient material as removed to le ve excctly too grams in each flesk.
To this was added 1.5 grams of pure precipitated calcium carbonate
for the neutralization of pl nt acids , and sufficient 95 percent
alcohol to make the final concentration 80 percent after allowine
10·20 5M
1 '
for the dilution due to wcter in the leaves. The sa~ples were
then boiled half an hour under reflux condensers, and put away
ti6 htly stoppered until a convenient time for &nalysis . The pro
cedure fro:n thawing to boilin:; was carried out ith the utmost
expedition.
The advantage of usins calcium carbona te as noted above has
been discussed by Spoehr ( 1919) . Davis , Daish and Swyer (1916)
have pointed out the necessity for rapid destruction of enzymes .
In this connection the present study afforded opportunity for some
observations of interest . An additional quantity of one variety
was collected for experimentation in methods . Part of this ~s
left 5 days in an lee chest , and .as then put through the re·ular
preparative and analytical procedure . In T ble II the results are
co~p red with those for the s~e material disposed of promptly
after collection. en it is considered that the tissues 1ere not
crushed or injured to any appreciable degree , the effect of enzyme
action even in such cold storsve conditions is very striking.
Dry. atter . - Duplicate or triplicate sample of approxim tely
5 rams of reen material were dried to constant ei ht in a vacuuo
oven at 98 •c.
Tota l Nitrogen.- The tot 1 n1tro0 en was determine by the
jeldahl-Gunnin0- Arnold method , usin the residues from the dry
~atter deter. inatlons.
Extraction of Sugars and Soluble itrosen.- L rge extr -ctors
of the Soxhlet type were cede by drilli a s~all hole close to the
bottom of a 750 c . c . ide-mouthed bottle , ~nd fittin in a l a ss
siphon t i ghtl y by maki ng a ground glass joint or by wedging it l t h
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12
a collar of rubber tubing . The bottle was closed with
rubber stopper , in which were fitted two reflux condensers and a
bent lass tube leading to the distilling flask below. The siphon
also passed through the stopper of the disti lling flask . The
sa:nples were transferred to one of these extractors and extracted
v;i th 80 percent alcohol on a steam bath for 30 hours , by which time
the Molisch a - napthol test on the alcohol in the extractor always
became entirely negative . In transferring material from one con
tainer to another , the emptied container was rinsed not less than
3 times with hot alcohol .
The extract was decanted off the small quantity of sedi ent
(chiefly calcium salts ) which collected, the last portion then
bein3 filtered and thoroughly washed lith hot alcohol . The whole
extract and washings ) was concentrated in the apparatus illustrat-
ed by VanSlyke ( 19111ig. 1) under pressure of less than 30 mm.,
with the distillins flask in a water bath ~t a temperature of 40•
to 5o· c . When reduced to a volume of 75 to 100 c . c ., about 200 c . c.
of distilled water was added and the solution reconcentrated to get
rid of the last traces of alcohol. This precaution was found ne-
cessary since the presence of alcohol affected the subsequent
13
deter ination of amino nitrogen. The reconcentrated extract as
then transferred to a 250 c . c . volumetric flask by filtering through
a p ad o f cheesecloth i . a small funnel , aki ng it nearly to volume
by several successive ashings of the di stillin flask ith small
portions of boilin3 ater. A p d of cheesecloth 4 layers thick "tha.
was foundAmost satisfactory fil t er for removing the solid particles
of chlorophyl l hich separated out . All other m terials tried
10-20 Sr.!
clogged a.t once . The extract was then cooled to room temperature
and made to volume .
All volumetric flasks and pipettes used throughout the analyse
were standardized in true cubic centimeters at 2o•c. Amino Nitrogen.- The amino nitrogen was determined by the
usual VanSlyke apparatus , using 10 c . c . portions of the extract .
These were filtered for removal of fine particles which had escaped
the cheesecloth filter . Since preliminary trials had given a some
what higher yield when dea~ination ~as continued for 30 ~inutes
instead of the usual 5 minutes , the former period was adopted. Of
this time , shaking was done durinu the first minute and the last 2
.ninutes .
Water- Soluble Nitrogen.- The total w~ter- soluble nitro en w s
determined by the Kjeldahl -Gunning- Arnold method , using t o c . c .
portions of the extract , filtered as for amino nitrogen.
Clearing Extract for S ~ar Analysis.- After the porti~s re
quired for nitrogen determinati ons had been removed , the reminder
of the extract was cleared ~ith dry po dered lead acet te . The dry
defecation method , first proposed by Horne ( 1904), has the advan
tage not only of largely el iminating the error due to the volume
of the precipi t te , but also of obviati the necessity of making
to volume a second time . The le d acetate as dded in small quan
tities at a time , successive small portions of the extract bei
filtered off and tested for co pl eteness o f precipit tion. The
solut ion was then f i ltered through dry filter paper and deleaded
with a minimum quanti ty of powdered sodium ox late , following the
same techni que as f or clari f ication. A second filtration t hrou0 h a
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t4
15 / r=======================================================,
, ry f i lter paper completed the preparation of the extract for sugar
analysis.
Reduci ng Suge.rs.- The reducing sugars were determi ned by the
very excellent method recently devised by S¢haffer and Hartmann
(1921), in which the cuprous oxide is determined directly by lode
thiosulfate titration in the presence of an excess of potassium
oxala te . The oxal~te inhibits the reaction of cupric ions with
soluble iodides. The reduction of Fehling's solution is carried
out under the standard conditions prescribed for Munson and Walker '
method (Official ~ethods , A. o .A. C.) and the sugar corresponding to
the quantity of cuprous oxide read from the tables . By connecting
the as burners with water manometers , and carefully calibratin6
them in conjunction with the flasks used for the reductions, it was
found possible to keep within t o seconds of the 4 ~inutes prescrib
ed for bringing the solution to boiling.
The thiosulfate was standardized by an adaptation of the simpl
method proposed by Peters {1 9t 2). About t oo mg . of copper foil ,
previously cleaned with dilute nitric acid, as eighed accurately,
placed in a 200 c . c . erlenmeyer , and dissolved in 2 . 5 c . c . of a mix
ture of equal volumes concentr~ted nitric acid and water . After
heatilld until brown fumes ere no longer apparent , 50 c. c . of ~ater
and a knife- point of pure powdered talcum ere added, and the mix
ture boiled vigorously for 10 ~inutes . It was then cooled to room
te.npera ture , 2 . 6 c . c . of coztentrated sulfuric acid as added, the
mixture recooled, 5 . 0 c . c . of a saturated solution of potassium
iodide added, and the whole titrated with thiosulfate . Triplicate
deter~in tions by this method varied less than 0 . 01 mg . copper per
10-20 , ...
c . c . thiosulfate .
The accuracy of this sugar method was tested 71th pure dex
trose obtained from the Bureau of Standards. Duplicate portions
of 100 mu• and of 150 mg. were ~ei0hed out , dissolved in water and
carried through the analyti ca.l procedure , with the follo :ving
results :
Dextrose Present
100
00
150
150
Dextrose Recovered
99. 8
99. 6
149.4
149. 2
The majority of the sugar determinations made fell ithin the
limits of these quantities . Aliquots of t o . c . c . of the cleared
extract ere used for the reductions .
Sucrose.- The sucrose as determined by the increase in re
duction on inversion of the cleared extract . For inversion the
citric acid method of Davis and D ish (1 913) w s employed. Coopar
ative tests with invertase and ith the official hydrochloric acid
method gave results varying by less than 0 . 5 percent. It appears ,
therefore , that sucrose w s the only disaccharide present .
St ca.rch,- A qualitative test for starch, i th the usual iodine ..
potassi~ iodide solution, was made on the dried, ground residue
from the a l cohol extraction.
10-20 SM
16
17 ~=================================,
TABLE I.- PERCENTAGE SURVIVAL UNDER FIELD CONDITIONS
OF VARIETIES STUDIED. (l)
------------------------------------------Variety ~ .1.2.!.2 !2£!. Claaaifi-
cation. U.Farm,Minn. ~occaain, ·on. University Waseca, II Dickinson, N. D. Farm. Gr. Rapids ,tt Fare;o , II
andan, II (Plots used Brookin s ,S.D. in present Highmore, II study.) Archer, Wy. Akron, Col. Ave . 4 Ave. 4 Ashl "'nd, Wis . Variety Breedin Sask .. toon, Can. Plots lots
Ave . % Ave.% %
Turkey 27 eo Tender
Kanred 2) 7 35 7 Tender
• inhardi 71 14 99 97 Very H •. rdy
Buffum 3) 10 84 Very Hardy
Odessa 62 9 3 7 Very Hardy
P"dui 74 7 0 90 Very Hardy
Minturki 5 8 95 91 Hardy
Red Rock (4) 2 3 Tender
1) Data for 191 and 1919, and classification of varieties supplied by Dept. of Plant Breedin0 , University of "innesota.
(2 ) Kanred killed as badly as Turkey in 1917 and 191 t University Farm, when other wheats es Minh rdi lived and produces good crops.
(3 ) Buffum is recognized as a very hardy heat by the U.S. Dept. of Agriculture .
(4) Red Rock killed worse than other hardy whe ta in 1919 variety tests.
10-20 5M
TABLE II. - CHANGES IN AMINO NITROGEN , TOTAL SOLUBLE NITROGEN
AND SUGARS OF WINTER WHEAT LEAVES DURING FIVE DAYS STORAGE
IN ICE CHEST.
Amino Nitrogen
ater- Soluble Nitrogen
Reducing Sugar as Dextrose
Invert Sugar as Sucrose
Total Su ar as Dextrose
Fresh Leaves
% Green Wt.
0 . 050
0 .1 57
3 . 286
4 . 574
7 . 943
Stored Leaves
% Green t .
0. 067
0. 237
4. 174
2.1 96
6. 402
Percentage Change
+34. 0
+51. 0
+27. 0
- 52 . 0
-1 9. 4
---- ·---------------------------------------
10·20 5M
18
r.=======================================================919
TABLE III.- PHYSICAL CONSTANTS OF SAP OF WINTER 'fHEAT LEAVES.
-------------------------------------------------------------
Collected October 29, 1920 .•
Variety Classification ~ p pH
Turkey Tender 2.08 24.99 6.360
Kanred Tender t.97 23.68 6.632
Minhardi ,a Very Hardy 2., 25.35 6.528
nhardi, b Very Hardy 2.15 25.83 6.580
Buffum Very Hardy 2.21 26.55 6.465
Odessa Very Hardy 2.09 25.11 6.457
Padui Very Hardy 2.05 24.63 6.475
inturki Hardy 2.07 24.87 6.536
Red Rock Tender 2.02 24.28 6.632
• ~ determined directly in tissue by thermoelectric method; pH determined on sap expressed after grinding tissue ithout previous freezing.
10·20 5M
TABLE IV.- PHYSICAL CO STA!' TS OF SAP OF INTER WHEAT LEAVES.
--------------------------------------~--------------------
Variety 6. p K X 1o3 pH
Collection of ove::nber 12, 1920 ••
Turkey 2. 26 27.15 11. 6 5. 84o
Kanred 2. 35 28 . 23 14.31 6. 20
nhardi , a 2. 56 30. 74 13.25 6.078
nhardi , b 2.44 29. 30 14. 7 6. 0 6
Buffum 2. 6 32.17 14. 3 5. 980
Collection of Dece. ber 9,, 920 .
Turkey 1.99 23 . 92 14.94 5.4 6
Kanred 1. 4 , 7. 08 14. 04 5. 557
nhardi , a 1. 3 22 . CO 13. 93 5. 54
inhard1 , b 1.69 20.32 15.02 5. 733
Buffum 1. 60 19.24 13.35 5.593
Sap expressed under pressure of 400 t ospheres fter freezing tissues.
• S p expressed by lar e h d sere preos after boilin tissues in pressure flas s.
Jo-ao '""
TABLE V. - THE ROLE OF SUGARS AND ELECTROLYTES IN
OS:OTI C PRESSURE .
-------------------------------------------------
Variety p K X 1cY
Collection of November 12,1 920.
Turkey
Kanred
inhardi , a
inhardi , b
Buffum
27.1 5
28 . 23
30. 74
29 . 30
32.1 7
11. 86
14. 31
13. 25
14. 87
14. 83
11.50
9 . 51
, 1. 54
, o. 51
15. 65
18. 72
, 9. 20
18. 79
19. 85
P - Ps
K X 1o3
1. 32
1. 31
1. 45
1. 26
1.34 --- ------------~-------------~-------------~---------------Avera e 13. 82 1 .oe 18 . 44 1. 34 --------~---~----------------------------------------------
Collection of December 9,1 920.
Turkey
Kanred
inhardi ,
llinhardi , b
Buffum
23 . 92
17. 0
22 . 00
2 • 3
19. 24
14. 94
14. 04
13. 93
15. 02
13. 35
7 . 49
5 . 40
7 · 91
6 . 59
7 . 27
16 .43
1 . 6
14 . 09
3 . 73
1 1. 97
• 1
o. 3
1. 01
0 . 91
0 . 90 ----------------------------------------------------------Avera e 20. 51 14. 26 0 . 95 -----------------------------------------------------------
10- ZO liM
1
2 2
TABLE VI.- NITROGEN OF I TER WHEAT LEAVES .
Variety Dry Amino ater- Total ~atter itro en Soluble lUtro~en Conte!'lt Nitrogen
Green Dry Green Dry Green Dry % "' ,;) I
Collection of ovember 12, 920.
Turkey 37. 54 • 54 .1 4 • 26 (.., ,- I. . 3 4 3. f
Kanred - } 9 45 .14 . , 29 " ~ 1. "'42 3 . 7-.? • • • ~· ........
inhc~.r i , a 37. • 5 • 6 0. 42 1.416 3.73
dinhardi ,b 33 . 51 0.04 o. 14 0.153 0. 46 , • ')2 3. 66
Buffum 5. 83 , " 41 Vo~ 1 • v " ,Jo;.JU , •
Collection of Dece ber 9,192.
Turke r 9 . ns " 9 17 • t 5'1 V• 5' t., 79 3 . 9 .... . ~re 25 .48 0.050 0.20 o. 134 • 53 0. 936 3.67
=-:il ~:r 31.74 0.05"' 0.1 0 9' . 60 1. ~76 4. ") I
'inh~rdi , b ';) . 20 o. 065 0 . 23 0.1 5 ... ., . o ... 1. 049 3. T'
Buffum 29 . 17 0.058 o . ~o o. 149 o. 51 • 93 3.22
IO·ZO
2J
TABLE VII.- SUGAR CONTENT OF INTErt WHEAT LEAVES. ------------------------------------------------
Variety Dry Reducing Invert Total atter Sugar as Sugar as Sugar as
Content Dextrose Sucrose Dextrose
Green Dry Green Dry Green Dry ,(
"' ,
Ia I I
Collection of Nove~ber 12,1 920.
Turkey 37. 54 ? . 992 7o97 5.301 14.13 8.397 2. 37
Kanred 32 . 9 3. 090 9.39 3.900 11. 6 7. 055 21.45
Minhardi , 37· 99 3. 237 • 52 4 . 797 12.63 8.1 27 21 .39
inhardi , b 33 . 51 3. 335 9o95 4.351 12.98 7· 7 0 23. 16
Buffum 35. 3 3.308 9. 23 5.799 , 6 . 19 g. 219 25.73
Collection of December 9,1 920.
Turkey 29. 65 2.347 7. 92 3.598 12. 14 6.004 20. 25
Kanred 25.4 1. 840 7. 22 2 . 660 0. 44 4. 540 17. 2
Minh rdi , a 31.74 2. 579 . 13 3 . 356 1 o. 57 5. 991 1 • 8
ill.nhardi , b 28 . 20 2. 027 7.1 9 3.391 12.03 5. 472 19.40
Buffum 29 .1 7 • , 63 7.42 3.772 ~.93 5. 997 20. 56
----------------------------------------------
10·ZO 5M
DISCUSSION.
The depression of the freezing point , the correspondinb osmot
ic pressure , and the hydrogen- ion concentration of the sap of the
sa~ples collected October 29 are given in Table I II. The os~otic
pressures recorded are based on the freezing point data, use being
made of the published tables of Harris and Gertner ( 19 14). The
hydrogen- ion concentration is expressed in terms of pH value as
read fro~ the tables of Schmidt and oa land (1 919). The cl ssifi
cation of varieties given in Table I is repeated to facilitate com
parison. It will be seen that in this collection at least there
are no si nificant variations in these constants which could be
correlated ith the relative .ardiness of varieties .
The same absence of correlation in physical constants holds
true for the collection of November 12 , reported in Table IV. It
may be oted, ho1ever , that the concentration of the sap had in
creased so~e1hat in the v2rieties used. In the collection of
December 9 , included in the same table , all varieties exhibit
f lling off in the depression of the freezin point and correspond
ing os~otic pressure , probably due in part to si ple dilution of
the sap , as the moisture content of the tissue as reater . 0 c
v iety , Kanred , fell off in this respect ppreci bly ore t a. the
rest . It is perhaps noteworthy th t this variety winter- killed
considerably ~ore than the others duriTh3 the year of this test .
An unexpected difficul ty as encountered i n expressing t e s p
from the sa ples collect~d December 9. The s~~ples were frozen by
10-20 '"'
24
p=====================================================~as
the ethod already described , usin,:, a frcezL · i:.ct· r o_ pulver
ized calci u:1 chloride a: d sno . ixed 1.1 t e proportio.1s hi ch
should thco. etica ly rive a cryoh., rate mixture with a correspo. -
in te perature of - 54. 9•c . Afte1· 7 hours frcezin~ , the s pl s
r;ere thaved under ru.nnin3 ater , and ref.ozcn "'o- a peri.Ol of 11
They \7ere tha Ted again under t:1e tap . Eve. af~er t s trea
ent it ;•1ao foun Lnpossible to express .lore th 2 to 3 c . c . of
juice ... ro.n 1 0 ..;ra:n of natl3rial t.m er 4 at!!lospheres pr ssur •
he data of Table VI s.o th~t t1ese s~ples co ta ne a lo er
percenta0e or ry matter t.1a. did t e earlier collectio , co. se
quently the failure to express the sap as ot ue to lacK O- oia
ture in the .tissues but <>pparently to a failure to :, ca.{ o t. e
co loidal co~plex o ... the protoplasm and to i.crease the er eabilit
of the cell by fre zin· . Since t~e peroeab.lit· o. the tlss es .ad
not been affected by freezi · , it as ecided to tte pt to destroy
t e colloidal complex ad incre se per eabillty by pl ci the
aterial 1 close press re flask .. e"tL - it 1 boilL
ater bath. T e juice as t.en expresse as re ily by the
r ss as by the '1y r lie ress, 30 to 40 c . c . abo t t.- · al
a ount} bel g collecte r~o. each s ple .
se observat o. s ive rise to so ery i port~t co.s_ r -
atio. s . In the f rst 1 ce , t e ._at plats er a p e tl ot
L .. led by exposure to te peratures lo er t . or 11 obtai in
:nany places here the S!.lf .er severe L ter=' .. illi he specific
te perature ust be o~ly o e of a n~ber o! factors i1 o- e • r~
is <>lso ~pare t that t e har e. ng recess conti ued lo ~ fter
the advent of freezi e ther , as this fficult~ as not met it
IO.ZO IM
rr===================~==========================~6
1 the coll ctio of lovember 12. .. t r , an co tr y to th
i d1 a of a ~~ber of ork rs, it a not 1 t is case socl te
with an i cr se in the ry tter content , e1 c as Alr dy ot
t e 'ator content a r,at r 1 the collectio of Dec b r 9.
s it associated ith incre se 1 e · r o t t ; thi a v 1
d cr as
oat
a ill b seen 1 t r .
1 . v fie?. t is the evi e t tenacity it· ch t
or
e t ssue rips its ter co. t . t . ie d 19 a) • ot t t s
t.e te p r tur .alls th q 1tity o at r sep t 1 th for
of ice beco~es co at tl 1 ss d , ess. I. oth r p 1 b),
t e s e t or develops t e t eory th t t SS of t r rom
t c 11 uri fr ezln 1 to of th rc o 1 -
bibitio , act1 · fro t. out c 11 br e t th ce t r of t
syste
l1br1
t s
th t
this follo s as co 8 q c
set up by t fore it ch t
t r rom the surf c of t e c _l.
at is duo to in of t
at r cozt t . Evi 0 0
r 1st
0 t
to
P r .
of
sicc _o.
t r
r1
t 1 at t
s
b
bot
1 so
the 1st c of q 1-
for ic cr t la
it cr1 ic
to of
1.
c 1 c
z tlo o
at b t1 co
it. fore a .ic op alec tion. I t 1 t 0 0 nt
o 1 -e of ~. e s o col1o it 9 st rob b- t t
t e pr1nc1 al .orce is 1 b1b1tion
Spoe ( 919) fo t t 1 c ct1 4 pe. toa s 1. cr _th
ecre si 5 ater s Dply • cDo · 1 (19~0) _so points o t t t
..
F======================================================927
conversion of the diffusible su6 ars to the mucila inous pentosans
is one of the alterations which may result in the cell as a con
sequence of partial desiccation. The latter author pictures 4 lant
protoplas~ as pentosan- protein colloid , and considers that the
character and amount of the pentosans largely determines the hydra
tion reactions of the protoplast . Winter conditions where the soil
freezes solid are really xerophytic conditions , since the plant ' s
usual 1ater supply is cut off . Having this in mind , it may be
reasoned from the evidence of • acDou0 al and Spoehr that under winte
conditions pentosans would accumulate in the cell , contributing
largely to the formation of a protoplasmic gel of hi 0 h imbibitional
powers . Investi ation of this point will be reserved for a later
paper .
The relative importance of su ars and electrolytes in the
development of osmotic pressure is considered in TableV. Unfortun
ately there is no known metho! hereby the relative proportions of
osmotic pressure contributed by electrolytes and non- electrol tea
can be accurately calculated from conductivity nd freezi poi t
data. I n the present instance the sugar percenta es ere kno d
these ~ere assumed to represent the on- electrolyte materials . The
theoretical os~otic pressure exerted by the s ars is calculated
froo t e quantities of reduci s ars and suc_ose determined by
analysis to be present . The difference between this value Ps and
the total osmotic pressur (i . e .,P- Ps ) . ay be attributed ~YobCA. 'j
chiefly , though~not entirely , to electrolytes. In the collection
of Nove~ber 12 , the su3ars present ere sufficient to account for
an average of about 38 percent of the total osmotic pressure ; in
that of De cember 9 , for about 34 percent . The ratio of the value
111-ZO IM
P - Ps to specif~ conductivity (x1o3 ) is given in the last col~~
of the table . This should be a constant in case the cell sap
electrolytes in each variety were identical in composition. For
the earlier collection this r tio varies from 1. 26 to 1· 45 and
avera es 1. 34 , but for the later collection it falls off so~ewhat ,
r anging f~om o.C3 to 1. 10 with an avera e of 0. 95. Possibly the
heatin6 of these later samples to 1oo•c . had released ions which
would otherwise have remained adsorbed by cell colloids . This facto
. ay account in part for the some hat hi ~her value of K, more strik~
ing in view of the dilution which had occurred, since the ater
content of the tissues as greater in the later collection. How
ever , the di inution in the value of P - P8 in this collection is
so decided as to maAe it unlikely that the possible disturbing
factors introduced by the heating could account entirely for the
failure of K to diminish correspondingly . The evidence s eats
that substances other than electrolytes , and variable in nature,
must contribute so~ewhat to the quantity P - P8 , or in other ords
th~su~ars are probably not the only non-electrolytes which con
tribute to the os~otic values .
The amino nitro~en , ater- soluble itro - en and total nitro·en
in percent of ·reen and dry eights are iven in Table VI . The
percent of dry matter content is lso included. It ca~~ot be sai
that any of the figures exhibit a ~arked correspondence to differ
ences in degree of hardiness . The increase in the ater content
of the collection of December 9 is ccounted for by a mild rai.y
period of some days duration which occurred duri the previous
week. I t has been remarked already that Kanred killed orse than the
10·20 SM
28
F===================================================~29
other varieties during the season of this experiment, and this
variety was lowest in dry matter content . inhardi , the hardiest
variety used , had a somewhat lar er content of water- soluble
nitrogen. All varieties show an increase in a~ino nitro en and
water-soluble nitro en in the later collection. This is in har ony
with the evidence of Harvey (1918) that splitti of the proteins
is associated with the hardenin process.
The hish content of sugars , especially sucrose, reported in
Table VII , is quite remarkable . It has been noted already that
sucrose was apparently the only disaccharide present . But here ag
the varieties could not be classified accordi to hardiness on the
basis of the values found. All suffered a loss between the col
lections of lovember 12 and December 9, but Kanred lost decidedly
more than the others. T1e reater degree of killing in this
variety thus lends support to the observ tion of Pantanelli (191 )
that hardiness was proportional to the qua tity of s ar retai ed
duri • exposure to frost.
A qualitative test for st rch on the dried, round residue
fro. the alcohol extr ction -ave e.tirely ne ative results in ever
case . This is s expected from the observ tiona of Miyake (19 2),
Lidforss 1907) and others.
The above discussion indic tea that e are still far fro an
exact analysis of the factors i.fluencing winter hardiness, but
certain of the observations, notably the failure of freezing the
tissues to breaK down the protoplas~ic structure in the hardened
plants, are very aug estive . Further investigation of this
phenomenon will be carried out in the near future .
10-20 , ...
rr=======================~==================~========930
S~:t' ARY .
1 . A nQnber of varieties of inter wheat , kno n to very con
siderably in de•ree of winter hardiness , ere coup red in the
hardened condition with reference to the p~ysic,l const,nts o: the
cell s p , and the content of dry n tter, nitro en, su rs nd st<rc ·
2 . No co'1.stant relation was found bet ·reen depression of the
freezin0 point , specific conductivity,or hydrogen- ion concentration
of the cell sap and rel tive frost hardiness .
3 . Su0 ars rccou te for 34 to 3 perce. of the tot 1 os.o ic
pressure o: the sap .
4 . The ratio of th t part of the os~otic press re not due to
sugo.rs i.e . , P- P 8 ) to t.le specific conductivity x 1 c3 ) is not a
consta t . For the sa. oles collected November 12, this rat o v ried
fro 1. 26 to 1. 45 (~vera•e 1. 34 ) and for those collected Dece. ber 9 1
from o. J3 to 1.1 0 aver e 0 . 95 ).
5. The relation bet een ry ctter content on h rdiness s
not const nt , tho ·h one of the t o tender arie+ies .nd the lo est
percenta e .
6 . All varieties incre se in ino nitroDe and ater - soluble
itrogen durin· the .~rdenit 0 process , but there as little relatio
et ee the a ount of this incre~se and rel~ti re h~rdi eus .
7. The s ·ar co tent did not correspo ~~ifor. ly wit1 the
k own hardiness . The percenta e decreased bet een ove ber 12 d
Dece ber 9 , fall i ng l o est in one of the two tender varieties .
10·ZO 5M
F=====================================================931
8. Sucrose is an important storage material and is apparently
the only disaccharide present.
9. All varieties were entirely free from starch.
10, The colloidal complex of the cell of the fully hardened
tissue could not be broken down by exposure to the temperature of a
ca lcium chloride - snow cryohydric mixture (t~eor.= - 54.9°C.).
11 . The hardened tissue retains its water content with reat
force . From tissue containing about 70 percent of moisture no
appreciable amount of sap could be expressed by 400 atmospheres
pressure , even after severe preliminary freezing.
10-20 SM
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ID-20 ,,..
F====================================================933
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10·20 5M
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10·20 5M
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34
•
•
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10·20 5M
35
36 r.=========================~======================9
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