EFFECT OF THE HARDENING PROCESS
ON THE WATER CONTENT AND
ANATOMY OF SOME
HERBACEOUS PLANTS

Thesis for the Degree of M8.
DONALD ARTHUR KIMBALL
June 1927

 

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Thesis.

 

Submitted to the Faculty of the Tichigun State
5011039 of lgriculture afid .‘-.‘mlie'q Sci-
ence in partial fulfillment of the
requirements for the degree
of Easter of Science.

by
Donald Arthur Vimball
JTlne 19270

THFSTS

\

 

 

(1.)

_;ntroduction.

The annual loss that results from the injury of cultivated
plants by low temperatures is great, the effect being spread
over a wide range of territory. in localities where the mini-
mum temperature is seldom below zero the injury is frequently
as severe as in the more northern areas. This nukes the problem
of ascertaining the cause of the killing of plant tissue by cold
one of general interest and immortance. In these investigations
a study was made of the relation of anatomy and bound water con-

tent to the condition termed hardiness.

101888

(2.)

Historical.

Theories regarding cause of Death from freezing.

There have been many theories advanced to account for the
death of plants resulting from low temperature. The first of
these was the opinion of Duhamel and Buff n (15), that death
was due to the crushing of the cell wall by ice formation.

This was disproved by Goeppert (18}, Sachs (48), Vageli (38),
Wiegand (56), and others, as will be shown later.

Muller-Thurgau (36) held that the cause of death was
water withdrawal from the cell and ice formation usually in
the intercellular Spaces. Wiegand's work (56) supported this
theory. In the light of more recent investigations this must
be broadly interpreted to include such secondary effects as
would in some way change the character of the cell contents.
Molisch (35) agreed with this broader internretation ofihuller-
Thurgau's hypothesis.

Gorke (l9) advanced the theory that death from freezing is
due to "salting out" of proteins. The removal of water from the
cell causes increased concentration of the cell sap, and pro-
teins are precipitated much as from strong salt solutions in
vitro. Schaffnit's results (50) supported this explanation.

Gorke (19) noted increased acidity in the sap upon freezing

and thought this might be a factor in protein precipitation.

Harvey (25) noted Horke's observation; accumulated consid-
erable data, and concluded that increased acidity is the cause of
protein precipitation.

Rosa (4?) considered nentosans (hot water soluble) to be an
important factor in protecting plant cells ani tissues from water
loss. Hooker (27) and fewton (59) give evidence in support if
this theory.

Chandler (ll) writes: "The theory that the disorganization
of the protoplasm is a mechanical injury caused by the pressure
of the ice mass seems to explain more of the experimental results
with the freezing of plants than does any of the others". He

admits, however, that the proof of this is not conclusive.

Evidence on Water Loss and Ice Formation.

many investigators have observed water withdrawal from the
cell and subsequent ice fonnation.

huller-Thurgau (36) pointed out that the loss of water from
the cell when ice formation commences is very rapid at first,
becoming gradually sluwer. It ceases when there is equilibrium
between the force of crystallization and imbibitional forces plus
osmotic pressure within the cell. "or apple tissue he give the

following water losses at stated terperatires:-

 

 

 

Temperatire. % Togalflwater frozen out.
0p '7
"4050uo 60.8
‘800 C. 7204

-15020Co 79.3

Iflller—Thurgau also found that ice formatiin usxally occurs
in the interceliular Spaces. Ice did not begin to form at all
points simultaneously, but water moves from adjoining or nearby
scaces to points at which ice fornation commences. even when ice
mass becomes so large that the cells are torn away from each
other, they may not be killed. Sachs (48) and Molisch (35) noted
ice fornation as chiefly confined to the intercellular spaces,
although it occurs within the cell when the temperature fall is
very rapid.

Uiegand (56) agreed with Lullerhuhurgau. he observed that
ice forms in broad prismatic crystals perpendicular to the ex-
creting surface. it -1800. to -25°Q. the cells near the ice mass
are, in certain cases, in a state of collapse but upon thawing
re-absoro the water and resume their normal condition. he re-
ported ice firmation in twigs and buds of wooiy species in win~
ter as taking place chiefly within the intercellular spaces. Ice
is at times found within the cell. )he spaces between the cud
scales were packed with ice crystals. )orsey (14) also reported
this condition in plum fruit buds, noting that there is no ice
in the space aaove and around the floral parts, between them and
the bud scales.

idims (1) workinp with moist seed observed conditions of
water withdrawml and re-absorption similwr to those previously
mentioned. He stated, as did fliegand, that if the tissue is

killed no re-absorption of water takes place.

(0.)

Greeley (23) found that water is extruded from Spirogyra
cells at l to 20 above freezing and a condition similar to
plasmolysis is brought about. .orphological changes similar to
plasmolysis are produced at will by lowering the temperatur

Wiegand (5?) advanced two theories to account for cellular
water loss on freezing. These were: (1) Extrusion (2) Attraction.

The first theory is based on the supposition that the cell
gives up water at low temperatures by contraction and scueezlng.
This condition may be due to change of permeability as Osterhout
(42) has shown that freezing, among other ayencies, greatly changes
cellular permeability. Yiegani favors his "attraction theory" as
the more probable explanation. Fe considers a film of pure water
to be adhering to the outer cell wall, around the intercellular
spaces. Che cell wall ani cytoglasm contain water of imoibition
while the vacuole contains a water solition of various substances.
The outer film of water freezes first. The whole system was in a
state of equilibrium and to restore this water is drawn from the
cell wall. She latter draws on the cytoplasm and the cytoplasm
takes from the vacuole. Water exchange goes on until the force
of crystallization and the forces within the cell are again in
equilibrium. *0 more water freezes out until the temperature is
lowered. The forces holding water in the cell become stronger as
the amount of water is reduced and less water is frozen out at
each degree of lowering of temperature. This agrees closely with
M311er—Thur*au's findines with apple tissue. Uiegand also states
that the forces withholding water from freezing are dependent on the

water content, and inversely proportional to it. in plants with

high water content the surface film is thicker and therefore, more
weakly held against freezing. he thought that, in succalent tissues,
a large part of this water is withdrawn at the initial freezing.

Huller-Thurgau (36) and Voigtlander (55) cooled tissue well
below the temperature at which it killed. When the tissue was
warmed backzwithout ice fonnation, no dmnage occurred. Wright and
Taylor (59) proved this to be true with potatoes, and reported that
where ice fonnation occurs before warming, typical frost injury
appears. Jith woody plants ice fonnation would occur well above
the death point.

Chandler (10) stated that leaves of tender plants whose sur-
faces are wet, kill much more severely than those which are dry.
Harvey (25) demonstrated that cabbage leaves cooled below the
freezing point may be caused to freeze internally in Spots by
droplets of water on the surface freezing and inoculating the
tissqe directly ueneath them. injury is grextest at the point on
the leaf where crystallization commences. This agrees close y with
certain findings of Nuller-thrgau and Jiegand given previously.
Harvey (26) also deronstrated with Hinereria, schiveria ani other
plants the imjortance of waxy or hairy coverings in preventing the
inoculation mentioned above. Wndercooling to -5 to -6°C. can be

attained without injury, when these coverings are not removed.

Ice Pressure Effects.
It will be recalled that under "ice formation" mention was
made of the work of Wiegand, MHller—Thurgau and others, reporting

the collapse and pressing out of shape of cells or groups of

(7-)

cells by ice pressure. Cells might be torn from each other and
still return to normal on thawing, if death had not ensued.

Chambers (9) stated that the cytoplasm of the egg of some
marine animals ceagulates with ease on mechanical injury. Com-
pression often 081883 the whole egg to cougilate in a solid mass,
although continuous gradual pressure is much less harmful than
short, rapid apolicltion. injury is accompanied by swelling and
an increase in acidity of the part involved.

Bancroft (3) quotes Ehrenberg's Opinion that the coaguhation
of colloidal solutions on freezing is due to the pressure of the
expanding ice.

Veith (29) carried an work with Bacillus coli held at low
tempe~atures in water. At -ZOoC. most of them are killed in five
days and all are dead in a few weeks. If during freezing the ice
is kept worked into a loose mass, a large percentage of the bacter-
ia remain alive for many months at ~2000. Lany survive over a long
period when 40 and 4bfi glycerin is added to the water.

Dorsey and Strausoaugh (14) studying flower buds of the plum
that had been killed by freezing found that the plasma membrane
of the cell shows as a dense spreading smudge of stain. In the
Case of sections similiarly treated, but killed by laboratory
reagents, a distinct limiting membrane is observed. in many of
the cells killed by freezing details of the nuclear organization
are completely obliterated. The authors state, "It is apparent,
that the killing of the embryonic buds by low temperature brings

about fundamental changes in the cell organization, particularly

in the nucleus".

(8.}

Differences between hardy 31d Tendeggfiissues.

Considerable data are available dealing with differences
between tissues of varying degrees of resistance to low temper-
ature, either measured or stated from observation. Dhese differ—
ences may be of water content, rate of water loss, sup density,

carbohydrate changes, etc.

Water Content and Dry Matter of Tissues.
Bauke (2) gives the following percentage of water (average

of 17 varieties) found in apple twigs at various periods.

 

?eriod; % Hater
Dormant 45.765
Bud dwelling 52.56
Blossoming 64.19
Summer Growth 58.92
Hood wipening 52.55

Chandler (10) could find but slight differences in the water
content of the cortex of Alberta peach and Jonathan qiple stems
from november to may, nor were the differences consistent.

Johnson (28) reports a marked seasonal increase in.water
content of peach buds in haryland, the increase being parallel
with the increase in tenderness of the buds toward spring. Greens-
boro, a hardy variety, has a lower percentage moisture than £1-
berta, a more tender variety.

Chandler 610), Potter (46) and Garrick (8) report that apple
roots which are allowed to absorb moisture suffer somewhat more
injury by cold than unsoaked roots and much more than roots which

are dried.

(9.)

Yiesselbach and Ratcliff (30) found freezing to death of
seed corn is directly prooortional to the moisture content and
duration of exposure. they also noted that the temperature at
which ice formation commences depends largely on moisture content.
With 60 to 80% wate", ice forms just below 32‘p,, while with 18%
moisture in air dry seed, no ice is formed at -lOOF.

Rosa (47) found a greater percent; e of dry matter in hardy
than in tender plants. the differences do not parallel the kill-
ing temperatures, h wever, and are small in many C1593. Jhe follow-

ing table compiLed from his data for cabbage plants shows this.

TABLE I.

 

 

 

 

 

Treatment __% dry mapper. Relative hardiness.
Greenhouse plants 9.58 ‘illed at ~40“. in 2 hrs.
not hardened 10.52
Xinimxm moisture. 9.22 fininjured at ~401. in 2fihrs.
greenhouse 10.9 " " -5 C. in 2 hrs.
12.8 " " -5°C. in 1 hr.
In cold frame 3 wks. 13.25 Slightly injured at ~60”.
in 2 hrs.
—, / —.. , . . O
Compost Mylo 33.03 9.51 Slightly inJured at -6 C.
in 50 min.
Sand M/lO YaYO 6.52 (Killed at -6OT. in 50 min.
5 (Not injured it -300. in
50 min.

..

 

It would seem that in the last two cases shown in Table l.

the percentage of dry matter is not the important factor.

(10.)

Schaffnit (50) reports the amounts of dry matter in wheat
varieties in direct proportion to their known hardiness. Corks
(19) states that more hardy plants have a greater percentage of
dry matter.

Lany other quotations might be given showing increased dry
matter content for nardy varieties. it would appear that high
percentage of dry matter may be correlated with hardiness but
other factors may have as great or greater importance in enabling
a plant to withstand low temperature. host of the data deal with
different varieties and not the same viriety in a hardened and
tender condition. Lay it not be that water content and not dry
matter is the important factor? Thus the amount of water may in-
crease in woody plants as they become more tender but it does not
necessarily follow that the actual .mount of dry matter changes,
altho it would decrease when measircd as percentage of green
weight or ratio of water to total solids.

Chandler (10) conc uded after experimenting with a number of
tender plants and fruits of the peach, when in both a turgid and
a wilted condition, that wilting did not increase cold resistance,
except in occasional instances. Slow loss of water in dormant
peach buds increased resistance to low temperature. It is pointed
out, however, that this is entirely different from rapid wilting,
in that the slow drying permits of changes other than a decrease in
the amount of water. However, it is problematical if any changes
would occur at the low temperature prevailing.

Beach and Lllen (4) found that drying apple twigs before freez-

ing lessened the injury. Salmon and Fleming (49) working with

(11.)

greenhouse grown cereals found slightly less injury in vzlted
than turgid plants.

Irmscher (58) showed thxt drying any of the species of moss
he used increases their resistance to cold.

fiiegand (57) considered that the greater the water content
of plant tissue, the thicker would be the :ilm of water on the
surfaces of the imbibing substance. Therefore, the force by
which the outer layers of this film are held would be weaker,

hence more easily withdrawn to form ice.

Rate of Hater Loss.

 

Beach and Ellen (4) found that in general the wood of the
hardiest varieties of apples was more resistant to water loss than
more tendér varieties.

Straasbaugh (51) reported that semi—hardy varieties of plums
have a fluctuating moisture content in twigs and buds, while the
w_ter retaining power of hardy varieties is constant. During
warm periods, (practically never above COG.) the buds of semi-
hardy varieties increase in water content while at a lowering
of temperature the process is reversed with equal rapidity. duds
of hardy varieties have a nearly constant water content. This
was further demonstrated by drykg twigs with buds attached, over
sulphuric acid of various concentrations. The tests covered a
period from Yovember to Karch.

Boswell's (5) work shows a faster rate of dehydration for
tender than for hardy plants. Hardy tomatoes lose water at a

slightly more rapid rate than tender cabbage.

  
 

V u Lvaul

1 "I'TVEMIH _

Freezing Point Depression of Sap.

Lewis and Turtle (31) ex mined leaves of Picea canadensis,
Linnaea, Pyrola and bark of Populus. There is but slight diff-
erences in sap concentration from October to April, the maximum
occurring in Xarch after cold weather is past. Harris and Popenoe
(24) found that the hardiest species of avocado have the lowest
freezing point depression.

Ohweiler (4a) investigating the results of a late Spring
frost at the Missouri Botanic Gardens, concluded that in the ma-
jority of cases plants with the greatest sap concentration suffer-
ed least, altho there were many exceptions.

Yewton (41) could find no relation between the freezing point
depression and resistince to cold of wheat varieties. Salmon and
Fleming (49) came to similar conclusions using oats and wheat.

Rosa (47) gives data on this subject similar to that here-
after quoted from Chandler. Fis differences are quite large but
the lowest freezing point of any of hlS samples of Cabbage or
tomatoes is that for medium and hardy lots of tomatoes. These
killed at -ZOC., a higher temperature than any of the cabbage and
only slightly different from tender tomatoes. Differences within
a lot of similarly treated plants were frequently as great or
greater than differences between different lots.

'Chandler (10) and Harvey (25) found a lower freezing point
for expressed sap of hardy than for tender plants. Table II.
taken from Chandler shows some of the variations of freezing point

of sap in relation to temperature injury. We subjected plants for

(15.)

24 hours with roots in solutions of strengths measured by the

freezing point noted.

(Chandler (10) pp. 157-)

L e a v e st_

 

 

 

 

 

 

3 killed 8
% partially £5

TPEItPERt- Temp;_ 0 killed killed

Tomato.
Glucose (.460) -3 65.0 67.6 .785
Cane Sugar (.455) -3 86.3 88.3 .925
Glycerine (.450) -3 72.8 88.5 1.070
Wgter -3 100.0 100.0 .700

Cabbage
Glucose (.44) -4 100.0 100.0 1.190
Cane Sugar (.77) -4 100.0 100.0 1.250
Glycerine (.66) -4 66.6 66.6 1.270
Water -4 66.6 66.6 1.080

Lettuce.
Cane Sugar (.677) -3 0. 25.0 .652
Glycerine (2.820) -3 00. 35.0 .728
Water -5 20.0 65.0 .597

 

 

 

 

 

 

 

 

(14.)

There is considerable difference in injury to plants from
the same solution but subjected to different temperatures. Pow-
ever the extract given in Table 11. is fairly representative.

From Table 11. one would conclude that altho the freezing point
may vary irectly as the temperature required to kill, yet this
relation is incidental. Other factors than those which make for
hardiness, have a greater influence in some instances on the
depression of the freezing point.

Jiegand (57) considered sap concentration of slight importance
and then only at the inception of freezing.

Rosa (47) states "It may be said that the apparent increase
in osmotic concentration of the sap in plants on hardening is
merely co-incidental".

The findings of Garrick (7) are rather indicative, especially
from the standpoint of studies with expressed sap. He reports
that the freezing point of expressed apple-fruit juice, measured
with the aeckmann apparatus, was significantly higher than the
freezing point of the cell sap as determined within the normal

unfrozen apple by thermo-junctions.

Acititg, Carbohydrate and Protein change§_on hardening.

 

Gorke (19) suggested that sap concentration in freezing acted
on the protOplasm and precipitated the proteins. He reported pro-
. O O 0 o O
teins prec1pitated from Begonia sap at -3 while -40 was required
to precipitate them from pine needles. He also noted increased
acidity on freezing expressed sap. Schaffnit (50) in his experi-

ments with rye could not precipitate protein from hardy plants.

at the higher temperature which will precipitate proteins from
the sap of tender plants. Furthermore he could prevent proteins
being precipitated from the expressed juice by the addition of
snail quantities of sugar. The conclusions were that starch in
hardy plants changes to sugar on the approach of winter, and
protects the protoplasm from changes similar to protein precip-
itation.

Pojarkova (45) dealt with 14 Ribes, lO Lonicera, 11 icer
also Aeroeris, imelanchier, and Corylus. Those species in which
a large preportion of starch is transformed into sugar at the
awproach of winter manifest profound donnancy. The relation of
the instensity of winter dormancy to cold resistance, as reported
elsewhere for plums, is shown only in vibes. Relatively cold re-
sistant species have a profound dormancy.

Lidforss (52) found with evergreen plants in South Sweden that
most of the starch is changed to sugar in cold weather. Starch
again appears on the return of warm weather.

KHller—Thurgau (36) Hichael Jurand (34) and other investiga-
tors have noted the accumulation of sugars in plants on the approach
of winter.

Dorsey and Strausbaugh (14) found chiefly protein substances,
fat and dextrin in plum fruit buds in winter.

Rosa (47) showed that total and reducing sugars increase in
hardened plants, using greater in cold frwne hardened than those
hardened by other means. The increase in sugars is greater in

, .- .: (v ,1‘ ' . '—
cabbage and lettuce than in the tomato. ”e sxcs, "Cnere 18 no 41

rect evidence that the absolute quantity of sugars present in a
plant is directly related to its cald resistance". in lettuce,
cauliflower and cabbare the amount of total polysaccharides is
slightly less in hardened than tender plants, the increase being

d e to the reduction in starch. Iith tomatoes the? show a large in~
crease in the hardened plants due ta starch accumulation. 1051
points out this larger acc2mnlitian of starch as an indication of
differences between plants which can be hariened and those which
Cannot. He stmted, however, "It appears probable that an increased
sugar content in hardened plants is more likely one of the mani-
festations of the condition of being hardy than a direct cause of
cold resistance". Aosa also found ‘ncreased pentcsnn centent (hot
water soluble), in plants which hid been hardened but his analytical

thnayh considered

(1'
pJ.
m
re
' x
’L
C)
<+
C
'1
62/!
O

methods, as he stated later, were unsa
the best at that ime.

hooker (27) and newton (39), using iosa's method of analysis,
found pentosan content to incre se in the hardier parts of apple
wood and in more cold resistant varieties of whett.

Harvey (25) in his work with cabbaae, stated that the aciditv
increases greatly on freezing. "e precipitated a much greater per-
centage of protein frum the excressei sap of tender than of hardy

o
cabbage, both by freezing at —4 C. for two hours and treatinfl with
sulphuric acid safficient to increase the acidity equal to the in—

O O
crease caused by freezing at -3 3. He conclzded that the increased
acidity causes protein precipitation and death. Varvey also found
an increase in the amino nitrogen in hardened plants and considered

this a significant result of the hardening process, indicating a reason

for nonwprecipitatian.

(17.)

Maximow (33) froze thin sections from the upper side of red

caboage leaves and Tradescantia in pure water and solutions of

various substances. The sections were placed in the solutions

and immediately frozen, as it was found this gave as much pro-

tection as when they were soaked in the materials previous to

freezing. Some of his results are given in the following table

taken from Chandler (ll).

'Jiats

t4
L4

 

freezing Temperatures of sections of Cabbage Leaves.

“-———- .—

 

Kuterial in “hich frozen

J

 

Pure Water

Glycerine 2E conc
Glucose 23 "

Kethyl ilcohol 23 conc.
YaCl 27 conc.

CaClg EH con .

TTaT-TO lTT "

I/‘TO:5 l?! "

Sodium Oxalate 1? conc.
Potassium " l? "

Exoressed Aed Cabbage Sap

" and Boiled Red Cabbage Sap

 

Einimum temo.
withstquL_

 

 

n... ———

-11.1 to-17.3OC.
-32°c.
-22.0 to —32°C.
0
”17.3 C.
-7.a°c.
-508 to “708°C.

—7.8 to -11.1°c.

-11.1 to-17.3OC.

o
-11.1 to-l7.3 C.

 

 

(18.)

The figires are interesting overuse of the fact that some
substaaces which would Kill the tissue at tevneratzres above
freezing gave protection equal to that afforded by glucose uni
other sugars not Shaun. 'oth Schatfnit (50) and Kidforss ($2
consider d that su: rs nroteet against salting out -f proteins.
The sap of the ctobage exhibited a distinct nrotective action
which dies not alt rath=r agree with findings of Harvey (25),
for the acidity must have been high. Doxic action is apparent-
ly gre;tly reduced by low temperature.

Vass (54) found a protective action exhioited by dextrose
ani glycerine bacteria. He concluded, as did Eaximow, that the
protective action was due to the keeping of a film of unfrozen
water in contact with the outer layer of the protonlasm, not

therefore to any direct effect on the protonlasm or cell con-

tents.

(19.)

Obiects of fingeriments.

 

The aim of this work Was to ascertain the effect of the har-
dening process on the hydrophyllic colloid content of the sap and
gross anatomy of certain plants. ?he freezing noint of expressed
sap, rate of tissue dehydration, percentave of solids and water
in sap and tissue were studied in their relation to Killing tem-
perature. The use throughout of plants of like variety and con-
dition with controlled methols of finding cold resistance render
the investigttions more valuable. Jhe object throughout was to
add something to the available information on 331d resistance in

plants.

(20.)

Uaterials.

Cabbage was used to represent plants which could be hardened
to withstand a fairly low temperature. The tomato, the other
plant chosen, can be grown so that it is to all appearances very
hardy, yet it will survive temperatures but little below 0°C.

The work here reported Wis carried on during the summers of
1925 and 1926. The plants used were uniform in vigor and size be-
fore undergoing treatment and were of the same age. All were
grown in outdoor cold frames, covered only when protection from

rain was necessary.

12.25 timer-13m 8 ._

 

egies A.

U)

 

A good loamy soil, except lot 4, reaction rendered distinct-

ly alkaline with Ca(OH)2.

Lot 1. Plants watered freely. Tender
Lot 2. Watered only enough to prevent wilting. Well hardened.
Lot 4. The soil was pure sand to Which had been added a very

little of that used for other lots in this series.
hlants watered freely.
hot 5. Ten days before being used plants were taken from Lot 1.

and given an application of Yafio one gram in 2Q 0.0.

39
water to each 4" pct. This was repeated in five days..
The result of the first application was evilent in two

days and plants at the time tested were succulent in

appearance.

Series B.
Soil similar to A. except that it gave a slightly acid re-

action. Soil test pH 6.2 to 6.7. (soiltex).

Lot 1. Plants watered freely. fender.

Lot 2. Watered only enough to prevent wilting.

 

 

All plants were grown in soil very similar to that used in
1925, being nearly neutral in reaction. Five inch pots were used,

and both cabbage and tomatoes treated as follows.

Lot 1. Plants watered freely.

Lot 2. Hardened by withholding water.

Lot 3. Bot bound. Water supplied freely.

Estinntion of Villing_Temnerature.

 

Tests of killing temperature were carried on in a four-
chambered "Frigidaire" freezer. It Wis very easy with this map
chine to lower the temperature gradually and steadily.

At the start the temperature was at 0°C. or just below. Four
pots were put in each compartment. The temperature rose at once
to around 5°C. due to the heat from the plants, soil and pots.

In from 1} to 2 hours it was down to the starting point. In 1926
the temperature was lowered 1% to 2°C. every hour. It took there-

fore approximately four hours to reach ~3boc. at which point all

point all the tomatoes had succumbed. The cabbage plants were
frozen slightly faster, as their minimum temperature of ~7QC.

wxs reached in five hours. the temperature in i925 was lowered
more slowly. It does not seem, however, that even the former lowh
erings would be considered excessively rapid freezi.g. Possibly
the differences in killing temperatures would have been greater
had the fall been less rapid. Chandler (10) points out the great
injury even to dormant buds and twigs by a very rapid lowering in
temperature as compared with slower fall. Fe lowered the temper-
ature to -20°J. in from 1 to 12 hours with rapid freezing, while
in slow freezing 7 to 10 hours‘ ere required to reach that temper-
ature.

Readings were taken every 50 minutes, plants being removed
from the freezer at each reading, starting when the leaves first
became stiff. They were exposed for ooservation of injury at room
temperatu‘e, 21 to 27°C., out of direct sznlight. Half an hour
was usually sufficient to bring out any injury.

A part of this work consisted in a study of the anatomy of
the plants used in 1926. The object was to determine such gener—
al differences as might exist between different treatments and
correlate them with cold resistance. The stem between the second
and third nodes, leaf petiole, and middle part of leaf blade were

selected for sectioning. The parrafin method was used throughout.

 

the

.—
lb
’ C
H
D.)
n
F,
U]
L—J‘
a
m
p
O
3.)
H
o
('1’
s
G)
a
o
O
c
(-+
o
.,
vw
2.)
("f
1+
z:
.‘D
(D
d
,fi
m
0
(D
O
*5

ground, weighed, cut into fine nieces with a knife and thoroughly
mixed. A fifty gram samqle was taken for rate of dehydration de-
terminations. dnother portion was put in a tightly corked uottle,
and suspended in an ice and salt mixture, at -180C. She container
with the freezing mixture was insulated from room temperature with
hair felt between it and an outer vessel. :11 samples were taken
in the late afternoon and left in freezing mixture until the follow-
ing morning. The bottles were then removed, washed clean, dried
an: the contents thawed by placina the bottle in warm water. :0
moisture was lost from the tissue before pressing, altho it was
wet and soggy. ifter thawing the material was packed in a clean
steel cylinder and immediately pressed in an hydraulic press.
Pressure was anlled only fast eiough to keep the sap dripping,
this operation taking from 15 to 25 minutes. The 1926 sanples
were W‘tpped before placing in the cylinier in unbleached muslin,
which had been boiled several hours in changes of distilled water
and thoroughly driei. in 1925 a pressure of four tons, and in
1926 one of 16 tons to the square inch was used. dhe residue in
each case was dry to the touch.

The eXpressed sap was at once centrifiged for ten minutes at
1500 r.p.m. to remove debris. She liguid remaining was kept at a

temperature close to 0°C. until used.

The remainder of the procedure and apdaratus used for es-

timating hydrichilic colloids was as used by “rist (1° pp. 15,

16, following the methods of Cortner and his co—worke

*1

59 (20)!
(21), (22), (40).

The samples of tissue for drying were put in 600 0.0.
beaks s in an electric oven held at 900?. Weixhts were taken

at stated intervals.

(25.)

, ' Q Y V“ 1
-b 7' 03 IJA‘J I Or.
.— ——-————-——.——

Killing Temperature, Freezing 9oint
Percentage Eater in the Sap and Tissme of Cabbage and fiomatoes.

Treatment

—‘

Killing Temp.

 

CABBAGE. 1925

Watered freely

 

tlkaline soil
Slightly acid soil

Hatered spjringly

 

Alkaline soil
Slightly acid soil

Poor soil
Treated with Va203

3926

 

Watered freely
Hatered sparingly

 

 

Pot bound
TO‘iTODS 1925
Watered freely

 

Alkaline soil
Slightly acid soil

watered sparingly

Alkaline soil
Slightly acid soil

Poor soil
Treated with Naflos
1°26

=2:—

Watered freely
Watered sparingly
Pot bound

 

A

 

00.

1 hr. at -5to-5§
1 n n -5; 3

-; hr. at —6to-7
1 " 'u_4?to-51

1.1.th at ‘6
1 to n '43:.13‘0-5}:

_1 n n -7

n n _5

1 hr. at -3%

l n n -3;
3 hr. at -3
1 n n -21
L

4

» hr. at -3%

J: n n -336:
g. n N -3

 

.747
.815

.834
.883

.806
.980

.766
.896
.843

.610
.653

.838
.900

.624
.780

.620
.880
.621

 

r77

)epression of sap and

"'53 water in -

 

Express- Oven dried
ed sap_ tissue
93.6 88.2
93.2 86.8
94.1 90.0
94.1 90.0
93.4 89.2
93.5 88.0
g
93.7 88.2
93.3 g 83.0
92.5 : 81.2
94.9 88.1
94.2 85.7
93.4 90.0
92.1 86.3
95.0 90.0
93.1 86.2
94.6 86.0
93.0 84.2
94.1 85.4

 

 

 

Killing Tenneratire ag;“freezins 2:igt_§ip:essign;

Data in Table IV. show that the freezing point may vary di-
rectly as the killing temperature but that it does not consistently
do so. The differences in the freezing point of expressed sap are
larger between lots of tomatoes than lots of cabbage yet the former
had nearly equal killing temperatures. The lots treated with sodium
nitrate proved the most tender, but the cabbage so treated had th
lowest freezing point of any lot. The freezing point was always

L

higher for plants grown on acid than on alkaline soil.

nelation between Villing Tempergtlre_and Percentage of “otal Tater.

 

 

Instances can be found in Table IV. in wtich the percentage
of total water drOps as the killing temperatire lowers. This is,
however, the exception with cabbage. Tomatoes show this lowering
of rater content censistently for lots which would be hard; in
plants that can oe indi‘ed to resist temperature lowering to any
degree. Vowever, the tomatoes showed very little zifferenees in
killing temperature. There was also as great a Variati n in water
content among lots of hardy plants in different years or on differ-
ent soils as between lots receiving different treatments.

In the 1925 experiments the "freely watered" cabbage had a
lower water content than the "sparingly watered" lots. In 1926 this
WLS reversed. Taking all lots into consideration there is no con-
sistent relation between Killing temperature and percentaye of
total water. Any correlation between the two, then, would have
to be considered merely incidental, at least as far as these exper-

iments are concerned.

Relation Between Killing Temperature, Bound Water, and
Percentlge Solids in Juice and Tissue.

«7

o solids in
Bound Express— Oven dried

 

 

 

 

 

 

 

 

 

 

 

 

Treatment. Tilling‘tempr__ Tater ed juice tissue
fltotal
0163293 1925 02. water
'Saterei freely
Alkaline soil 1 hr. at -5to-5L 6.46 6.4 11.7
Slightly acid soil 1 n n - g 6.09 6.8 13.2
'.'.'at ered sod ri 11511
Alkaline soil 1 hr. at -6to-7 4.50 5.9 10.0
Slightly acid soil 1 n " -4gto-6; 3.20 5.9 10.0
Poor soil 1; hr. at -6 2.50 6.6 10.8
Treated with :1'a::03 1 n " -4Ito-5; 5.10 6.5 12.0
1926
Watered freely -% hr. at -5 10.99 6.3 11.8
Watered sparingly §-" " -7 10.99 6.7 17.0
?Ot bomd :2 H ” -5 3045 P’05 1808
TOXATOES 1935
'Hatered free1y_
Alklline soil -§ hr. at -3to-3; 7.59 5.1 11.9
Slightly acid soil 1} n " -2 5.40 5.8 14.5
Jatered sparingly
Alkaline soil 1 hr. at -3; 10.80 6.6 10.0
Slightly acid soil 1 " " -3§ 7.20 6.9 13.7
Poor soil -g hr. at -3 5.0 10.0
Treated with nanoy 1 N " -2; 6.57 6.9 13.8
1926 °
Watered freely -} hr. at -3§ 7.45 5.4 14.0
Watered Sparingly fi-" " -3; 8.44 7.0 15.8
Pot bound g»" " - 6.43 5.9 16.6
Cauliflower 1. 4/5 dead 1hr.-8 6.5 5.9 12.0
Cauliflouer 11‘ dead 1 hr. -8 9.1 6.1 15.0

 

 

 

 

 

# These plants were originally, I. tender;

had become badly pot bound before use.

11 hardy, but

(28.)

Bound filter, tillinngemperature and Percentage Solids.

The cabbage and cauliflower have a percentage of "bound water"
varying inversely as the killing temperature, with few exceptions.
Tomatoes show a greater percentage of "bound" or colloidally held
water in "hardened" than tender lots, altho there is little or no
difference in killing temperature.

"Pot bound" and "poor soil" lots of cabbage show a very low
percenta e of bound water, altho the killing temperature is equal
to or greater than tender lots or those showing much greater percen-
tages of bound water.

Plants treated with sodium nitrate were tender yet they showed
a relatively higher amount of "bound water".

These dlta show chiefly a negative correlation between "bound
water" and killing temperature, altho exceptions occur.

There is little relation between "bound water" and percentage
solids in tissue anl juice.

'Tne percentage of bound water is low throughout on acid soil,

as compared with that of plants on alkaline soil.

(29.)

71:111‘8 10

)ehydration of fabbage Tissue.
5C) 533. 81.313103 at 95°C
100 *

90

80

7O // ,/‘

60

;/:/
50 ¢/ ,, .
4o ./ /
, / Cabbage.

 

 

 

30 ' / ./ Hardy
’/'i/ Tender

 

. z”, I i not Bornd
2 O / A (
//

 

 

fl“
% 1° 5 /
Total ,5?“
Water o A .
lOSto
é» 1 1%;- 233- 3;;- 4; V; 5.3;— 173 22-;-

Time in hours.

4

)0
Total
Water
IOSto

100

90

80

70

60

50

40

50

20

10

O

F: b~u1‘[.e 2 O

Dehydration of Tomato Tissue.
50 gm. samples at 95°”.

 

(30.)

 

 

 

 

.. .1
2/]!
/;
/ x
/ /
fl”
/ /
/ /
// /f'
/ 2
f’
,I/ /
/
/ /
,l/
/
/ Hardy
// Tender aid
///7 Pot bound __ __
l/J/
,///
/
92‘ 1 1% 3}; '3}; 4% 5;; 6,32: 17;; '22;

Time in hours.

100
90
80
70

60

50

30

<20

% 10
Total
Water 0
lost.

(31.)

Eigure 3.

Dehydration of Cabbage and Tomato Tissue.
50 gm. samples at 95°C.

"Q .
nabgege.

 

 

 

 

//‘/// “am-V
/ K¢/ Tender ________
)7C’ Tomato.
/ 2/ '
/’/// Hardy
/ .-
,/ . Tender
/ ,-'/ ——-—-———_———-
/'////'
f ,»
2/,/<f/
,
t 1 1% 2%; 3;- 49: 5% 6}; 1.7% 22-};

Time in hours.

Relation Between Rate of Dehydration, sound Jater,

and ”illing Temperature.

The graph in Fig. 1, shows the rate of dehydration of cabbage
tissue. Although the variations in all dehydrations, including

this, were not large, the order from the greatest to the least

was tender, pot bound, hardy. There is no relation to bound water

nor any significant relation to killing temperature.

Data similar to the above is given for tomato tissue, in Fig.

2. The results are the same as for cabbage except that the rate of
dehydration of pot bound and hardened plants is almost identical.

In Fig. 5. are presented the rates of dehydration for hardy and
tender cabbage and tomatoes. The order from the greatest to the least
rapid in loss of water is tender cabbage, tender tomato, hardy tom-
ato, hardy cabbage. The latter at first lost water faster than
the hardy tomato. There is no definite correlation between rate of

dehydration and bound water, or between rate of dehydration and

killing temperature.

(33.)

Anatomy.

In plates 4 to 9, inclusive, are shown bne series of cabbage
and one of tomato tissue. Other sections are not included because
of lack of space. however, these show differences similar to the
sections included.

The chief points of difference between the tender and hardy
tissues are in size of cell, thickness of cell wall and compact-
ness of cells. These are much the same as differences noted by
Sruner and Weaver Lg), Pool (44), and others as existing between
tissues of xerophytic and meSOphytic plants.

The leaf petiole of the hardy cabbage show smaller, much
thicker walled cells in the cortex. These are also more closely
packed tOgether than is the case in the tender cabbage. In the
latter the cortex cells are loosely arranged. The pgt bound plants
occupy an intermediate position. The cells of the -epidermis of
the leaf petiole exhibit like conditions‘but more marked even than
in the cortex. The collenchyma directly below the epidermal layer
is so small celled in the hardy tissue as to give the appearance of
a double or triple layered epidermis. The pot bound plants have
even smaller, more closely packed cells in the epidermis and sub-
epidermis than the hardy tissue. .

The cabbage stem cross-sections (not included) show similar and
evident differences. These are not quite as marked as in the leaf
petiole. Jhe pot bound plants have, in this case, smaller, thicker
walled, more closely packed cells than even the hardy lot.

Tomato sections do not differ markedly from the cabbage, in com-

(54.)

parison with each other. the fibre vascultr system as shown in
cross section, appears to be larger in the tender than either of
the other two tissues.

In comparing hardy and tender tissues, there are certainly
differences Which appear to be correlated with the ”hardened"
condition. These are similar to those noted by other workers as
being associated with xerOphytic or meSOphvtic conditions. Dhis
would be expected from the treatments the plants received. It is
evident from a study of the gross anatomy of these plants, that
there are distinct differences which may be associated with cold
resistance. These differences are not causes of hardiness nor
can thex be taken as measures of cold resistance. Pot bound plantS,
relatively tender, approached the hardy lots very closely in

structure, and tomatoes did not differ greatly from caobcce in

comparisons between lots.

 

From the material submitted in the review of literature it
is clear that the weight of evidence supports changes caused by
water loss from the cell as the reason for death by low temperature.
There is considerable evidence pointing to important changes in the
protoplasm of the cell resulting fn;m death by freezing.

The salient points of the cold resist11ce problem, from the
water relations standpoint are firstly: Is increased water re-
taining power, of hardy plants, the imiortant factor or is it the
amount of freezaole water? For example, in a hardy tissue, which
has a higher water retaining power than a more tender one, this
hardy tlSSle can be made more tender by allowing it to imbibe water.
13 the original water retaining power changed? If not, this hardy
tissue would show a higher percenta e of freezzble water, “at it
would be due entirely to the higher water contvnt. Secondly: Is
water content always the important factor? :hirdly: Joes death
reellt from changes within the cell caused by its lowered water
content or are these changes caused by ice pressure from without
the cell?

Chandler, Garrick, Potter, Rosa, Salmon and Fleming, Beach and
Allen, lrmscher, and others found that with various plants hardiness
could be increased by decreasing the water content and the first
three, that increasing the rater content decreased hardiness. Uhis
was in most cases accomplished quickly so that in all prooability
little change other than that of water content occurred. fihandler

did not find this to hold true for many of the tender tissaes he

'—'\
L;
L“:
o
5

used. Other workers have found increased water content corre-
lated with a condition of decreased hardiness, while some in-
vestigators have found increased dry matter in hardy plants. is
has been pointed out (pp. 8.) this latter might be considered
another way of stating decreased water.

The work reported in this paper indicates very little differ-
ence in the total WAtQ? content of hardened and non-hardened
tlSSJe and no regular relation between it and the killing tender-
ature. it is clear then that under some conditions the amount of
total water is not the important factor in cold resistance. Some
condition other than water content made the difference in cold
resistance between both similarly treated lots of different kinds
of plants and different lots of the same plant.

It has been demonstrated by Strausbaugh, also Seach and Allen,
that woody tissues, of certain tender varieties are less retentive
of water than corresponding tissues of hardy varieties. doswell
found this to be true with cabbage and tomato tissue, in which
case the same variety was hardened to resist cold. The findings of
this work do not bear out that point of view exceot with cabbage.
It is significant that rate of dehvdration had no relation to the
percentare of bound water. This agrees with the work of “rist (12)
in which he foznd that bound water did not parallel the water con—
tend or rate of iehydration.

Kewton (41) fOLNd that sup was strongly held against pressure
in green tissues of wheat plants in late Fall, when such was not

the case in early Fall. Other reports from dilwtometer experiments.

indicate that o; thit methad more water is held in hardy tissue than
tender. In another paper Yewton reports hydrOphyllic colloid con—
tent to as directly proportional to hardiness. The writer's work
shows no relation oetreen cold resistance and bound water (hydro~
phyllic colloid content) as measured by the methods used. It is
clear that conditions other than those affecting cold resistance
can change the percentage of bound water. Llso the hardened con-
dition may give an increase in bound water over succelent tissue
without increasing the cold resistance, as the tomato plants show.
The conclusion seem justified in view of the data swbmitted, that
where the percentage of bound water varies directly as the hardin ss,
this relation may be only incidental. It is possible, however,
that thcre may he need for firther standardization and perfecting
of technique in expressing Sip from tissues. ”ewton, Brown and
Mirtin (60) in a recent paper have shown the uifterences which
exist in sap extracted from identical tissues at various pressures.
knong other variations the greater the pressure, the lower was
the percentage of solids, although electrolytes in the sap in-
creased. They idvise a pressure that causes the pressure gauge on
a hydriulic press to barely register. it is stated in their paper
that there is no proof that the extract is a true representative
of the cell sap, even when expressed and carried at 0°C. Carrick's
work would indicate that this difference may be considerable.

The imbibition theory, in which cold resistance is credited
to cell colloids, is not entirely satisfactory in accounting for
greater injury through rapid temperature fall; nrotection from
death by cold due to rapid drying of the tissue; or increased

tenderness on greater water content. The data submitted in this

(.2; . )

work Show that increased water 0 ntent is not always accomptnied
by increased tenderness nor did the imbibitianal theory hold good
as an explanation of cold resistance. This does not exclude
certiin factors from standing as measures of cold resistance,

yet it does not necessarily follow that such factors are more than
indications of the hardened condition. Koreover, as previously
mentioned, it remains to be proved that sap extracted is repre—
sentative of the cell sap or that such samples are suitable {or
intercomparison. Juice from tissues Virying in cold resistance
might be reasonably expected to be affected in different degrees
and ways by laboratory methods.

Refinement of method, Sich as will reduce disorganization of
and changes in the material used, is iesirable before consistent
results can be hOped for.

The theory of ice pressure as a cause of death thrOigh low
temp retire apparently explains more of the finiings of hardiness
and correlated studies than any other. However, it does not

‘0

necessarily follow that freezing to death always results iron the
same cause, or that the same factor or group of factors are alway
responsible. Different methods of inducing: resistan‘e to low tem-
perature apparently bring into play different factors which are

responsible for this result.

l.

3.

c't‘fi'f'iv.

(I

 

 

There was little correlation and no censistent relation be-

tween oercentlge of water in the tissue, freezing point
depression of exeressed sup, rate of tissue dehydration

and the killing tem erature with caboage and tomato nladts.

The percentage of bound water (by method used) showed no corre—

On

It

Q

I
A-

lation with the rate of tiSSie dehydration and if anything,
negative correlation with the killing temperature.

acid soils the freezing point depression of exnressed sap

is higher, the "b-und water" lower and with one exception,

the percentage of total water lower and solids higher than

on alkaline soils.

is evident from the results obtained by different investigators
that there is need for standardizltion and perfection of
methods of extracting juice from plant tissues, with the aim,

if possible, of maintaining more nearly normal conditions.

study of the gross anatomy of t e plants used in 1925

showed distinct differences between hardy and tender plants
altho these m‘e not necessarily causes of tenderness or

hardiness.

 

 

(41.)

 

 

“ ""79 "-
Luca.“ u.

0
ht “'6 do

, ,

Ho
3‘

o
Caboage after exposure at -5 C. Plant at r

c.)

(4

 

 

to exposed at -3°C.

cm

"I
A.

(43.)

 

 

PLATE 4.

Cross-section of Leaf Petiole of Hardy Cabbage.

 

 

 

BLATE 5.

Cross—sect ion of Leaf Petiole of Tender Cabbage.

(45.)

\ I .‘\'\‘ ~

PD

-.’ ,1 ~1

‘\

‘ . “ - (O Q k,\ ._
< -‘ ‘ ‘ K Qib‘a‘. N f. i \
w . were .3. F

‘-
Q.

 

 

 

PLATE. g.

Cross-section of Leaf Petiole of Pot Bound Cabbage

 

 

 

 

PLATE 7.

Cross-section of Leaf Petiole of Hardy Tomato.

(47.)

 

 

 

 

 

. u. .05.... ,. J...

\ 4“ rl‘.‘ .vukv .

. .701.»er 7.1 .
I...

v-:;

1‘.

 

. ‘ J, JU.A.. . ..
D ‘11 ~H.Ii'll€} ..-s..ru.Q_..w} lQJAm.
Ilv. ..lrl..u.\| . . .. \\ _

 

omato.

m

&

Cross-section of Leaf Potiole of Tender

 

(48.)

 

 

 

 

 

PLATE 9.

Cross-section of Leaf Petiole of Pot Bownd 'L‘omato.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10,

Bib

ioeranhv. .

 

Adams, J. The ’ffect of Ver, Low Temperatures on Koist
Seed. Sci. Proc. toy. )uolin Soc. ”.3. Vol. lI, p.1,
1905

Saake A. L. et al. A New Factor in the )etermination of
Hardiness in the Apple. Proc. L. S. H. S. 17, 1920,
pp. 279.

Bancroft, W. D. Applied Colloidal Chemistry. kcflraw Hill
00., pp. 222. 1922.

Beach, S. A. and Allen, F. N., Hardiness of the Apple as
Correlated With Structure and Composition. Iowa Agr.

Exp. Sta. Mes. fiullo 21. 19150

eoswell, V. R., Jehydration of Certain Plant Tissues. bot.

gazette, V01. 75, pp. 86, 1923.

Bruner, W. E. and Weaver, J. 3., Size and Structure of Cereal
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Studies, Vol. XXIII. No. 3 and 4.

Garrick, D. 3., Some Affects of Freezing on mature Fruits of
the Apple. Cornell Agr. Sxp. Sta. Lemoir 81, 1924.

Garrick, D. 3., Iesistance of the ioots of Some Fruit Species
to Low Temperature. Cornell figr. gxp. Sta. hemoir 36, 1920.

Chambers, 1. 0., uicrodissection Studies 1. Visible Structure
of Cell Hrotoplasm and Jeath Changes. Amer. dour. Physiol-
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Chandler H. H., The Killing of Plant Tissue by low Temperature.

X0. Agr. SXPO Sta. Mes. 3u110 8’ 19130

 

11.

13.

14.

15.

1530

17.

19.

20.

”handler, J. 2., fruit nrowing. Houghton Li

(:1
(— )
o

0 ' I ‘7
Lil-1T1 "009.10 0

“rist, a. J., affect of “utrient ‘onditions on folloidal

’rooerties of Hertain vegetable (rcps. nich. lgr. Exp.
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Jixon and gall. extraction f Sap from Living leaves 03 hemns
of domoressed Air. zpt. or. 133. ldv. Sci. 1933, pp. 486.

Jorsey, 1. J. and Stransbatgh, H. )., ”lum investigations.
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)ihamel, Ju [ouceau, H. 1. ani 8 ffon, G. L. I. Cbservation
dos Uifferents Effects que ?roduisent snr les Vegetaux les
Grundes Gelees D'hiver et les Petite Gelees du “rintemns.
Hem. Kath. and Vhys. Load. Toy. Soc. (Paris) 1737. pp.
237. “noted by Chandler (11) and Rosa (4?).

Ewart, i. J. On the Power to Jithstand Jessicrtion in Plants.
Liverpool 3iol. Soc. 701. 11., pp. 51. 1897.

Gardner, 7. R., Braiford, 7. C. and Hooker, E. 3. Fundamentals
of Fruit Production. Kcfiraw Tv'ill 00., 1922.

Seopnert, N. R. fiber oer Uarme Entwickerlung in dene ?flanzen
deren Cefrieren and die Schutzmittel flegen Dusselbe. Book,
pp. 274. fireslau, 1839. ‘uoted by 2053 (47).

Corke, H. 7ber Chemisohe Vorgange beim Exfrieran des Pflanzen.
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Grotner, 1. A. and Harris, J. A. Totes on the Technique of
the Determination of the Jepression of the Freezing Point

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{O
{0

25.

IO

26.

27.

30.

Gortner, 2. A., and iawrence, f. V. The Extraction of San

J

from Plant Tissue by Pressure. Biochem. Full. 5, pp. 139,

O

1916.

V

fiortner, A. 1., and ’offmin, j. T. Determination of the Fois~
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701. 74, pp. 308, 1922.

Greeley, 1. U. Cn the Analogy ietween the Sffects of the Loss
of Uiter and towering of Temperature. Amer. Jour. Phys.
Vol. 6, pp. 22. 1901.

Harris, J. A., and 2openoe, U. Freezing >oint Lowering of the
Leaf Sap of Horticultural Types of Persea Americana.

Jour. Agr. Res. Vol 7, pp. 261. 1916.

Harvey, R. B. Hardening Processes in Plants and Developments
from frost Inju y. Jour. Apr. Res. Vol. 15. pp. 83, 1918.

Harvey, M. 3. Importance of Epidermal Coverings. Bot.
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Hooker, n. D. ’entosan Cont nt in ielation to Uinter Verdi—
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Johnson, 3. S. in Index to Tlardiness in Peach fiuds. lmer.
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Seed Corn. febraska Agr. Exp. Sta. Res. Bull. 16, 1920.

 

Q.
}—4
0

CC.
to

36.

37.

(.3
O)
O

39.

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a‘ f‘y‘rv'l’V-‘T |T“r1 2w IEIW‘IS .
4- _ . a 4.4-. -- L -

.
‘ .l-J-—J./ t

The writer wishes to make acknowledgement to Dr. John
N. Crist for criticism and advice in planning and conduct-
ing this work and for reviewing the manuscriwt; to Professor
V. R. Gardner for many helpful sugge tions and also for re-
viewing the manuscript; and to Professor E. 0. Bradford for

assistance in some phases of the work.

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