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

Discussion

Summary

Bibliography

10-20 5M

..

age

24

30

32

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

10-20 5 M

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 .

10-20 5M

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 ,

10-20 '"'

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

10-20 5M

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

10-20 5M

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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Pla.11Ze • !..and ) a.'ld Chandle

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10·20 5M

1914. Notes on the calculation of the os~otic pressure of ex-• n

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l'action du ~roid . Co pt . Rend.Acad. Sci .{Paris) 13 : 7 - 791 . 1901 . Sur l 1 iden~e des nodific tions de str cture prod itea

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10·20 5M

1905. eue Untersuchun3en uber das Erfrieren eisbest di er Plan zen. Flora94: 89 . Cited by Blaclcnan( 1 909) and Chandler( 191

34

•

•

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1919 . Alterations of cellular exchange and permeability at te~peratures near freezin~ point . Ibid.~ Ser. 5 , 28 : 205-209 .

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10·20 5M

35

36 r.=========================~======================9

Schmidt , C. L. A., and Hoagland, D. R. 1919. Table of pH, H+ and OH- values corresponding to electro~o

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Sinz E. ;914. Connection between dry matter content and resistance to

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Tuttle , G y.ethe , • 1913 . Induced ch~~ges in reserve materials in ever-reen herb­

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Uphof , J . C.T., and Thornber,J. J . 1916. Cold resistance in spineless cacti . Ariz . Exp. St . Bul. 79:

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. est , F. L., and Edlefsen, . • E. 1917. The freezin~ of fruit buds. Utah Agr . ~xp.Sta, Bul. 1 51:1-~4 1921. Freezi ·· of fruit buds. Jour . r . Rese rch 20 : 655-662.

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10-ZO 5M

25-39 · 1906b T: e ass ··e of ·7ater fro the pl t cell duri .; freezing.

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