A STUDY OF COLD RESISTANCE
' AND
STARCH RESERVES 1N ALFALFA

Thesis for the: Degree of M. S.
MICHlGAN STATE COLLEGE

Richard W. Bell
1940

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A STUDY OF COLD mSISTANCE AND STARCH RESERVES
IN ALFALFA

RI CHART) W. BELL

A THESIS
Submitted to the Graduate School ot'michigan
State College of Agriculture and Applied

Science in partial fulfilment of the
requirements for the degree 0!

MASTER OF SCIENCE

Department of Farm Crops
1940

THESIS

ACKNOWLEDGEHENT

The writer is grateful to Dr. E. E. Down, Kr. H. M.
Brown, Dr. C. M. Harrison and Dr. S. T. Dexter for their
assistance during this experiment and in the preparation

of the manuscript.

CONTENTS

INTRODUCTION
LITERATURE
MATERIALS AND METHODS
A. Technique of Cross-pollination
B. Iechanical Injury in Root Tissues
C. Determination of Cold Resistance
D. Determination of Starch Reserves
EXPERIMENTAL RESULTS AND DISCUSSION

A. Mechanical Injury to Roots as a Result of
Freezing

B. Trend of Starch Reserves and Cold Resistance
During the Fall of 1939

C. Changes in Intensity of Starch Reserves and
Cold Resistance

D. Relation of Intensity of Starch Reserves to
Cold Resistance

SUMMARY
LITERATURE CITED

Page

'0 03 (fl OI cs u> #4 14

15

18

24
29
51

INTRODUCTION

Under Michigan conditions some varieties of alfalfa,
such as Hardigan and Grimm, exhibit a high degree of winter
hardiness, while Hairy Peruvian and others are decidedly
non-hardy and consequently have no place in the agriculture
of the state.

iany workers have attempted to establish the relation-
ship between physiological and morphological characteristics
of alfalfa and its capacity to resist the injurious effects
of freezing temperatures. The present experiment was de-
signed to further such studies by obtaining additional infor-
mation regarding the relation of starch reserves to cold re-
sistance in alfalfa roots. In view of the plant material on
hand and the findings of other workers to date, it appeared
that the problem might be attacked by studying this relation-
ship as it existed within individual plants. For this purpose,
the plants of‘Fz progenies resulting from original crosses
between Hardigan and Grimm (female parents) and Hairy Peruvian
(male parent) were used, rather than individual plants of a

variety relatively pure, genetically, for winter hardiness.
LITERAIURE

Literature dealing with the subjects of organic reserves
in alfalfa and the cold resistance of plants is voluminous.
No attempt is made here to review it in detail. For comprehen-

sive reviews of the subject of organic reserves in alfalfa the

2.

papers of grandfield (5), Graber et al. (4), mark (9), and
Willard et al. (80) should be consulted. Papers dealing with
the low temperature relations of plants have been thoroughly
reviewed by Harvey (6), Rose (15), Newton (12), maximov (10),
and others.

Other pertinent literature will be referred to as the

several phases of the experimental results are discussed.

MATERIALS AND METHODS

In September, 1937, several plants of Hardigan and Grimm
alfalfa were dug from field plots, brought into the greenhouse,
and transplanted into clay pots. Of these, only two plants
of each variety were later used as female parents in the crosses
made. Two Hairy Peruvian plants which had been growing in the
greenhouse were used as the male parents. Throughout the course
of the experiment all plants grown in the greenhouse were in
clay pots or flats containing coarse sand, and were given nu-
trient solution at weekly, or more frequent, intervals.

The crosses between the parent plants were made during
the first three months of 1938. For the purpose of maintain-
ing identity, they were given numbers as followsi Grimm 4
X.Hairy Peruvian 1, Cross 4; Grimm.l.X.Hairy Peruvian 2,

Cross 5; Hardigan 4 X Hairy Peruvian 2, Cross 6; and.Hardigan
l X Hairy Peruvian 1, Cross 7. Individual P1 plants of the
first-named cross were then designated as 4-1, 4-2, 4-3, etc.

The same system of identifying Fl plants was used throughout.

5.

The 31 seedlings were grown in flats in the greenhouse-
and were later transplanted into clay pots. During the last
two months of 1938 they were self-pollinated by manually
"tripping" the flowers with a needle. F3 seed was harvested
from.them.during January and February, 1939.

Cn May 9, 1939, all F1 plants, about 830 in number, were
transplanted into the field. These plants were spaced at 30-
inch intervals in rows 30 inches apart. At the same time,

22 plants resulting from self-fertilizing Hairy Peruvian l
were transplanted. Seed from these inbreds and from all Fl
plants was harvested by cutting the plants down to the crown
in early September, 1939. After harvesting the plants, growth
was resumed and considerable root storage took place previous
to killing frosts.

F2 seedlings were grown in the greenhouse until July 3,
1939, when, at the age of six weeks, they were transplanted
into the field. They were spaced at 8- to 10-inch intervals
in.rows 30 inches apart, and were not harvested for either hay
or seed after the above date.

The field coil varied from.sandy loam to loam, and had

been uniformly cropped during the previous five years.

A. Technique of Cross-pollination
The technique employed in controlled cross-pollination
was similar to that used by many workers, and involved the
use of suction in emasculation of the flowers. The suction
equipment consisted of a glass tube having an inside bore of

approximately 1 millimeter, and gum.rubber tubing which con-

4.

nested the capillary tube with an.ordinary laboratory filter
pump. The glass tube had been drawn out at one and until the
inside bore was only slightly greater than the sins of an al-
falfa anther.

Only flowers in which the anthers had dehisced were
chosen for crossing operations. Essence of the female parent
were thinned to from three to six flowers. Using small scis-
sors, the standard of each flower was then clipped off as
short as possible. By exerting a slight, steady pressure
against the base of the keel, the staminal column was made to
emerge slowly so that little pollen was thrown from.the anthers.
Emasculation.was accomplished by holding the smaller and of
the glass tube in contact with the exposed anthers. With
short upward strokes of the tube the ten anthers were quickly
removed by the suction and drawn within the tubing. Follow-
ing emasculation, a fine stream of water from a syringe was
directed upon the face of the stigma to remove any pollen which
mdght have adhered. Final examination of the stigma was made
with a small hand lens to determine completeness of’pollen re-
moval. A single flower from.the male parent was then picked
from its raceme and held at its base with forceps while the
standard was gently removed.with the fingers. Using a needle,
the flower was "tripped" as gently as possible. The anthers
were then brushed directly against the stigma of the emascu-
latcd flower until it was apparent that the entire stigmatic
surface was well covered with pollen. The cross-pollinated
flowers of the female parent plant were tagged for identifi-

cation.and allowed to remain unbagged. Emasculated flowers

5.

were left occasionally as a check upon the thoroughness of

the technique.

B. mechanical Injury in Root Tissues

To compare the nature of'mechanical injury resulting
from freezing under field conditions, as reported by several
investigators, with that resulting from artificial freezing,
the following procedure was adopted. Sections of unfrozen
roots from.six cold resistant Hardigan plants and from four
non-resistant Hairy Peruvian plants, all of which had been
dug from.the field on December 8, were imbedded in paraffin
in the usual manner. other sections of the same roots were
artificially frozen at a temperature of -7°C. to -8°c. for
3% hours and then were likewise imbedded. All roots were
sectioned transversely at a thickness of 15 microns, mounted
on slides, and stained with safranine 0, crystal violet and

orange G.

C. Determination of Cold Resistance
Dexter, Tottingham, and Graber (2) have described a

method of determining cold resistance of alfalfa root's. They
point out that tissue injury as a result of freezing involves
disorganization of cells and that, upon disorganization, the
cells lose their ability to control diffusion of soluble con-
tents. The outward diffusion of electrolytes can be deter-
mined by electrical conductivity measurements and from.these
the degree of injury estimated.

The method of determining cold resistance in this experi-

ment was based upon the work of the above writers and differed

6.

only in details from.the procedure they describe. Each samp
ple consisted of l gram.of root tissue from.a single plant,
and comprised three adjacent sections of root from either
side of a middle section located 1% inches beneath the crown.
The middle section was placed in a formalin alcohol fixative,
and preserved for starch determination. The test tubes, each
containing the six sections of root tissue, were placed in a
water bath at-+2° C. for ll to 15 hours so that all sections
might attain a uniform temperature before freezing. The roots
were frozen at -7°C. to ~800. in an alcohol slush, for a per-
iod of 3% hours. Upon removal they were allowed to stand at
rocm.temperature for a period of 1% hours. Five cc. of dis-
tilled water were then added to each test tube, and exosmosis
was allowed to proceed at-+8°c. for a period ranging tram 25
to 27% hours before resistance measurements were made with a
conductivity cell and a Wheatstone bridge.

0n five of the dates of sampling, test tubes containing
sections of Hardigan roots were handled in the manner describ-
ed above, except that they were not subjected to freezing.

This was done to provide a check upon the effects of freezing.

D. Determination of starch Reserves
Chemical analysis is the usual method of determing quan-
titatively the presence of starch in plant tissue. In this
experiment, however, the method of determination was based
upon the fact that starch grains assume a dark blue color when
treated with an iodine solution.
The mdddle section from.each root, previously fixed in

formalin alcohol, was imbedded in paraffin in the usual manner

7.

and cut transversely at a thickness of 15 microns. The sec-
tions were then fixed to glass slides, stainedin Gram's io-
dine soluticn for a period of ten minutes, mounted in Canada
balsam.and covered with a Number 2 cover glass.

Plate 1 shows the equipment, and its arrangement, used
in the determination of intensity of starch reserves. By
means of a projecting microscope the image of an individual
root was focused directly upon a photronic cell. This in
turn was connected with a sensitive galvanometer, the read-
ings from.which indicated, relatively, the amount and inten-
sity of light falling upon the cell. It was found necessary
to use an automatic voltage stabilizer because of sharp fluc-
tuations of voltage in the current delivered to the micros-
cope lamp.

Those roots containing large amounts of stored starch
intercepted a greater portion of the light beam than did the
roots containing lesser amounts, and.this in turn resulted
in lower galvanometer readings. Preliminary trials indicated
that the "depression" values obtained in this manner corre-
lated closely with the comparative amounts of starch as ob~
served with the naked eye. Because the iodine stain faded,
particularly in the presence of light, starch determinations
were made within six hours after staining.

It was found after numerous trials that reliable read-
ings could be obtained only when the intensity of the uninter-
rupted light beam.was constant and when interruption by a glass
filter resulted in a constant "depression" as indicated on the

galvanometer. In order to eliminate variations in readings

k

Plate 1.

8.

 

Arrangement of equipment used in determination
of intensity of starch reserves in alfalfa roots.
A, voltage stabilizer; B, projecting microscope;

C, microscope stage; D, photronic cell; E, galvan-
ometero

9.

between slides, cover glasses and balsam layers of different-
thicknesses, the following precaution was taken. Several
readings were obtained from each slide when the light beam
was made to pass between adjacent root sections and was inter-
rupted only by the slide, cover glass and the balsam layer.
The average of the "depression" values so obtained was then
deducted from the "depression" value for each root section
upon that slide. These differences, then, represented the
comparative amounts of the light beam intercepted by the

root sections.r Only one reading was obtained from each root,
the light beam passing through as near the center of the root
as possible. This procedure was decided upon only after it
had been noted that intensity of starch reserves in the cen-
tral area correlated closely with the intensity throughout
the entire root. There were exceptions, however, particu-
larly noticeable in one progeny in which starch was lacking
from the central portion of many of the root sections. An
attempt was made to correct for this by allowing the beam of
light to pass through an area slightly off-center.

EXPERIMENTAL RESULTS AND DISCUSSION

A» mechanical Injury to Roots as a Result of Freezing
Numerous workers have observed that root tissue of al-
falfa is subject to injury by freezing, particularly when or-
ganic reserves have been depleted (14) (4) (20). In Michigan,
this latter condition may be expected most commonly during
late winter and early spring. Death of plants following root
injury appears to be largely dependent upon the extent and

10.

severity of cell disorganization.

Jones (8) describes the nature of injury in roots of
alfalfa plants that had been severely frozen under field con-
ditions. He also describes repair of parenchymatous tissue
by meristematic activity of adjacent parenchmma cells. Injury
was always found where cells had been separated by rifts along
the middle lamella. However, from.his observations he inferred
that injury was primarily a direct effect of freezing, and not
primarily, or necessarily, a consequence of mechanical injury.
Weimer (19) found that splitting of tissue was so commonly
associated with death of the cells that the two must be con-
sidered together. He further observed in histological studies
of 16 varieties that splitting of rays was more or less severe
in all alfalfa plants during the winter, but that, if the in-
jury was not too severe, practically all of the cracks were
closed during the following growing season.

Plates II, III, IV and V show the condition of roots of
Hardigan and Hairy Peruvian varieties before being frozen and
after having been artificially frozen at -7°C. to -8°C. for a
period of 3% hours.

It will be noted that there was no apparent injury in
the unfrozen roots of either variety. This was exPected as
the soil had been frozen to a depth of only one-half inch
prior to the date of sampling.

In the case of the frozen Hardigan roots, considerable
injury was apparent, chiefly in the form.of rifts which extend-
ed radially along the parenchyma cells of the rays. Paren-

chymatous tissue in the center of the root (not shown) was also

11.

l

s. a

it. .‘w

try

 

alfalfa root as dug from the field on December

Photomicrograph of typical unfrozen Hardigan
8, 1939.

Plate II.

12.

_. 9 fee

a
C .

 

aph of typical unfrozen Hairy Per-

uvian alfalfa root as dug from the field on

Photomicrogr

Plate III.

1959.

December 8,

Plate IV.

Photomicrograph of typical Hardigan alfalfa
root dug on December 8, 1939, after being
artificially frozen at -7°c. to -8°C. for
3% hours.

13.

 

Plate V.

14.

 

Photomicrograph of typical Hairy Peruvian alfal-
fa root dug on December 8, 19696 after being art—
C

ificially frozen at -7°C. to -8 . for 3% hours.

15.

disorganized. Although there was almost a complete absence
of starch in the roots, the cambium had suffered no injury.

Injury in the roots of the Hairy Peruvian variety was
somewhat different in nature, most of it having taken place
in the vicinity of the cammium layer. The abundance of starch
present may account in part for the less extensive injury in
the ray tissue. Whereas the cambium layer of the Hardigan
roots showed no effects of freezing, the cambium of the Hairy
Peruvian roots was badly Split and many of the meristematic
cells were collapsed.

Because of the tendency of Hairy Peruvian alfalfa to
grow until late in the fall, and because the fall months of
1939 were abnormally mild, the meristematic tissue of this
varietyxmay have been still active on the date of sampling
(December 8). In this condition, the cambium would be es-
pecially subject to injury by freezing. In the literature
reviewed, no description of repair of injured cambium tissue
was found. Further study of alfalfa grown in southern Mich-
’igan probably would reveal considerable injury of this type
during winter and early spring in non-hardy varieties. This
injury in turn would appear to be associated with failure of
the plant to recover.

B. Trend of Starch Reserves and Cold Resistance

During the Fall of 1939

It has been established by many workers that alfalfa
increases in its resistance to cold during the fall months
under the influence of shortening day lengths and lowering

temperatures. Peltier and Tysdal (l3) and others observed

16.

that under normal conditions hardy varieties became dormant
earlier and.hardcned more rapidly than non-hardy varieties.
This suggests a correlation between dormancy and phsiolog-
ical processes associated with hardening.

In a study of root reserves and resistance to cold in-
jury mark (9) found that in each of the varieties of alfalfa
analyzed, starch began disappearing in early fall and consti-
tuted a very small portion of root reserves after October.
However, the varieties showed differences with reapect to
rate of disappearance of starch during the fall.

Figure 1 shows the intensity of starch reserves and the
degree of cold resistance (as indicated by electrical conduc-
tivity) in Hardigan and Hairy Peruvian alfalfa roots during
the fall of 1939. Although there were fluctuations in both
starch reserves and cold resistance from.week to week in the
two varieties, it will be noted that the trends were as fol-
lows: ,

l. Intensity of starch reserves in Hardigan at the

close of the experiment was decidedly less than at

the beginning, and less than on any date during the

course of the experiment.

2. Intensity of starch reserves in the Hairy Peruvian

roots on December 8 was greater than at the beginning

of the experiment, and represents the average intensity
for the entire period.

3. contrasting the intensity of starch reserves in the

two varieties, Hardigan plants showed a more rapid de-

crease than did Eairy Peruvian plants.

 

18.

4. Cold resistance in the case of Hardigan showed only
a slight increase from.November 14 to December 19, the
date upon which the experiment was terminated. The

sharp decrease in cold resistance (increase in electri-
cal conductivity) during the period December 5 to De-
cember 8 can be explained by the fact that one of the
ten roots used in this determination was entirely devoid
of starch and was decidedly lacking in hardiness. The
other nine roots contained visible amounts of starch.

The dotted line represents the trend when this plant was
omitted from the calculations.

5. Cold resistance in the Hairy Peruvian plants increas-
ed markedly from.November 14 to December 8.

6. Hairy Peruvian plants hardened more rapidly than
Hardigan plants, although at no time did they attain the
degree of cold resistance possessed by the latter. It

is probable that during late summer little difference
existed between the two varieties in cold resistance (2).
From late summer until the beginning of the experiment,
November 14, hardening must then have progressed much

faster in the Hardigan variety.

C. Changes in Intensity of Starch Reserves and Cold Resistance

Referring to Figure 1, it is interesting to note that

increases in starch content in both varieties during the course

of the experiment were accompanied by decreased cold resist-

ance, while decreases in intensity of starch reserves were

accompanied by increased cold resistance. Under the treatment

1-

19.
received by these plants, they made considerable growth be-
fore killing frosts in October rendered them.incapable of pho-
tosynthetic activity. Ensuing warmer weather failed to stimp
ulate vegetative growth to a visible extent. It is believed,
therefore, that these fluctuations in starch content represent
conversion of starch to sugar and reconversion to starch rather
than marked.changes in total carbohydrates. Since the harden-
ing process in many plants has been associated with increased
concentration of sugar, it is probable that the sharp decreases
of starch during the fall months represent physiological pro-
cesses directly associated with increased cold resistance.

Figures 2, 3, 4 and 5 present similar data for the four
F2 progenies studied. The mean air temperature, as reported
by the East Lansing station of the U.S. Weather Bureau. for
each of the 7 days preceding each date of sampling were aver-
aged together, and these computations are also presented since
it is believed they provide an eXplanation, in part, for the
behavior of the plants.

In three of the four progenies an apparent correlation
existed between average mean temperature and intensity of starch
reserves (er = +.e4, +89 and-)z78 for progenies 7-19, 6-29 and
5-12 respectively). This relationship is backing in progeny
4-18 (rI ; +303), and it has been shown statistically that this
is the only instance in which changes in intensity of starch
reserves might easily have been the result of inadequate sampling.

Likewise, the correlation between the average mean temper-
ature and electrical conductivity of the water extracts is ap-

parent (rxy a -h?0, -h65, -h52 and 4360 for progenies 7-19, 6-29,

 

21o

 

 

 

 

24.

5-12 and 4-18 reapectively).

Abrupt decreases in the intensity of starch reserves
can best be explained by assuming that the starch was convert-
ed to sugar, as it is not probable that the decreasing temper-
atures would result in an increased reapiratory rate within
the plantS. The F2 plants made no visible amount of growth
(which would involve utilization of carbohydrates) during
the course of the experiment.

Increases in starch.may have been the result of conver-
sion of sugar already present in the roots to starch, or the
result of photosynthetic activity. Either, or both, are pos-
sibilities as most of the F2 plants on the dates of sampling
possessed living above-ground growth. Had the increases been
the result of photosynthesis activity they should have been
accompanied by increased cold resistance, since, as Dexter (1)
states, hardening progresses under conditions favorable for
synthesizing of carbohydrates but unfavorable for utilization
of synthesized materials. In most of the cases, however, in-
creases in starch were accompanied by loss of cold resistance.
Therefore, it is reasonable to assume that the increasing temp
peratures resulted in conversion of sugar to starch and decreas-
ed cold resistance.

D. Relation of Intensity of Starch Reserves to Cold Resistance

Numerous experiments have shown that winter injury in el-
falfa is closely associated with management practices (4)(5)(14)
(20). The explanation for this observed fact is that, to es-
cape winter injury, alfalfa plants must be well supplied with

starch at the time they become dormant in the fall. However,

25.

as Hegee (ll), Steinmetz (16), and others have pointed out,

the presence of abundant organic reserves does not insure
against winter injury, since the plant must also have an he-
reditary ability to successfully withstand cold. Mark (9)
states this postulate somewhat differently when he says that
within the Grimm variety a correlation was found to exist be-
tween hardiness and root reserves, but that when varietal (gen-
etic) differences were introduced no correlation was found.

It has not been established that, in the hardened state,
hardy varieties of alfalfa, as such, are significantly higher
in any reserve fraction than non-hardy varieties. 0n the con-
trary, several workers have shown that the non-hardy were con-
sistently higher in some of the reserve fractions than the har-
dy alfalfas. mark (9) reports that Ladak alfalfa was higher in
dextrins, starch, and insoluble nitrogen than arizona common
during the fall, winter, and early spring of 1934 and 1935. He
also found that hardy varieties digested starch reserves more
rapidly in the fall than did non-hardy varieties. Janssen (7)
found that the starch content of Hairy Peruvian alfalfa was
higher than that of Grimm during the winter of 1937-38.

In connection with utilization of reserves, Tysdal (18)
concluded from.his work that hardy varieties have a greater pro-
tected diastatic power when in a hardened condition than do
non-hardy varieties. This finding, however, does not serve to
explain differences in rate of starch digestion between alfal-
fas differing in inherited hardiness.

I It appears from the above and from other work that the

level of starch reserves attained by alfalfa before becoming

26.

dormant in the fall is governed largely by environment (includ-
ing cultural practices), whereas differences between varieties
in the rate of digestion of starch during the fall and winter

is primarily a response to hereditary make-up. During the fall
months of most years complete and continued dormancy is not at-
tained. Following relatively short periods of dormancy, weather
conditions may be such that the plant resumes photosynthetic
activity and even vegetative growth. The effects of alternat-
ing dormancy and non-dormancy complicate the relation of starch
reserves and winter hardiness to heredity.

As a test for the correctness of the belief that alfalfa
plants having the hereditary capacity of comparatively rapid
digestion of starch during the fall months also possess the
inherited capacity to resist cold injury, simple correlation
coefficients were determined between specific conductivity of
the water extracts and intensity of starch reserves for each F2
progeny and for row-grown Hardigan. The results are given in
Table 1.

In the row-grown Hardigan, three of the five correlation
coefficients were negative. As the season advanced the degree
of negative correlation increased, changing from.+ad3 on Novem-
ber 14 to -.72 on December 8.

In no instance did progeny 4-18 exhibit correlation co-
efficients which were statistically significant, although all
were positive.

Correlations for all four dates of sampling were positive
in the case of progeny 5-12. Of these, the plants dug on No-

vember 22 and on December 8 exhibited coefficients significant
at the 1% point.

Table 1 e

Progeny

Hardigan
Hardigan
Hardigan
Hardigan
Hardigan

4-18
4-18
4-18
4-18

5-12
5-12
5-12
5-12

6-22

6-29
6-29
6-29
6-29
6-29

7-5

7-19
7-19

7-19 -

7-19

Note: 1,2,3, and d asterisks indicate significance to 10%,
5%, 3W. and 1% points, reSpectively (3))

27.

Coefficients of variability for intensity of starch
reserves and electrical conductivity, and correlation coeffi-
cients between the two on each date of sampling.

Date of
Sampling

NOV.
PEOVO
NOV.
D63.
Deco

Weighted

NOV.
Nov.
NOV.
D300

Weighted

Nov.
Nov.
NOV.
Dec.

Weighted

Dec.

Nov.
NOV 0
NOV.
D33 0
D30.

Weighted

Dec.

Nov.
NOV 0
NOV 0
D60.

14
22
50
5
8

14
22
50
8

14
22
50
8

19

14
22
50
8

19

12

14
22
50
8

Coefficient of
variability -

Mean
22

9
10
13
10

Weighted.mean

conductivity of
water extracts

55.6

()3
H

e

b‘
ԤWfi

19.8%
19.8%

14.6%
11.6%
18.4%
40.0%
20.5%

24.2%

28.1%
26.0%
22.3%
26.2%

22.5%
24.5%

23.3%

13.4%
12.8%
13.5%
31.9%
17.7%

Coefficient of
variability -
intensity of

starch reserv-

Coefficient of
Correlation

+.28
+.70****
+.50*
+.sz****

+.55****

+. 53**
'1'. 52***
+.54***
h80****
+. 30*

+602

+.47
-914
-021
-051

38.

In.progeny 6-29 positive correlations between intensity
of starch storage and electrical conductivity were significant
at the 5%;point or more on four of the five sampling dates.

The only F2 progeny that exhibited negative correlations
was 7-19 although none of these was statistically significant.
As in the row-grown Hardigan, the magnitude of the negative
correlation coefficients increased as the season advanced. The
fact that the weighted mean of the several coefficients of var-
iability in progeny 7-19 was less than that of any other pro-
geny may indicate a more nearly homozygous condition with re-
spect to starch storage and winter hardiness. On the basis of
the work of.Mark (9), and Stewart (17) a negative correlation
mdght be eXpected.

The extremely high coefficients of variability found in
the Hardigan plants are believed to be the result of environ-
mental differences. Roots and top growth varied greatly in
size as a result of the thick row-planting and some of the
plants appeared to have thrived at the expense of others. Even
on.November 7, the first date of sampling the Hardigan, the lev-
el of starch reserves was highly variable from.plant to plant.

Although the evidence presented here is not conclusive,
it appears that those plants which have the hereditary charac-
teristic of comparatively rapid digestion of starch during the
fall also have the hereditary ability to resist injury from
freezing. It also appears that within the Hardigan variety
higher levels of starch reserves are associated with increased
hardiness. Whether or not this relationship exists within such
non-hardy strains as Hairy Peruvian and Arizona Common is prob-

lemat 5. Cal 0

29.

That extreme hardy alfalfas, even though low in starch
reserves, possess greater cold resistance, when in the hard-
ened condition, than do extreme non-hardy alfalfas having an
abundance of starch is evidenced by the fact that, of all the
roots containing visible starch, only two Hardigan plaits fail-
ed to show greater cold resistance than the most resistant non-
hardy alfalfa plant. A.few Hardigan roots containing no vis-
ible starch were decidedly lacking in cold resistance and were

comparable to the least resistant Hairy Peruvian plants.
SUHMARY

A study of cold resistance and intensity of starch re-
serves was made during the fall of 1939 with Hardigan and Hairy
Peruvian alfalfas and with F3 progenies resulting from origi-
nal crosses involving hardy and non-hardy varieties.

Hardigan alfalfa roots, having a low intensity of starch
reserves, when artificially frozen exhibited mechanical injury
of ray tissue, but no injury in the cambiwm. Hairy Peruvian
roots, having a high intensity of starch reserves, when fro-
zen exhibited not only injury of ray tissue in the vicinity of
the cambium, but of cambium.tissue as well.

The decrease in starch was more rapid in Hardigan alfal-
fa than in Hairy Peruvian from.November 7 to December 8.

Hairy Peruvian plants hardened.more rapidly than Hardi-
gan plants after November 14. However, the Hardigan variety
had already attained a much higher degree of cold resistance
on November 14, and it maintained this superiority over Hairy

Peruvian until December 8, at which.time conductivity deter-

minations on the latter were terminated.

30.

In.most cases, changes in intensity of starch reserves
were accompanied by correSponding changes in electrical con-
ductivity of the water extracts. The several F3 progenies
did not react alike in this respect.

In three of the Pg progenies intensity of starch stor-
age was positively correlated with field temperatures, while
in all four Fa progenies cold resistance was negatively cor-
related with field temperatures.

It is believed that fluctuations in intensity of starch_
reserves were due to conversion of starch to sugar and recon-
version to starch rather than to photosynthetic or metabolic
activity.

Evidence is presented which indicates that rapid loss of
starch reserves during the fall months is associated with in-
herited cold resistance, but that within the Hardigan variety
intensity of starch reserves is correlated positively with cold
resistance. This is believed to be an indication that within
individual plants, level of starch reserves and level of cold
resistance are positively correlated.

Hardigan roots having no visible starch were comparable
in cold resistance to the least resistant Hairy Peruvian roots.

No Hairy Peruvian roots were found to be devoid of starch.

l.

2.

3.

4.

5.

6.

8.

9.

10.

11.

12.

15.

14.

LITERATURE CITED

Dexter, S. T., Effect of Several Environmental Factors
on the Hardening of Plants. Plant Phys. 8:125-159. 1955.

Dexter, S. T.. Tottingham, W. E., and araber, L.F., Pre-
limdnary Results of Measuring the Hardiness of Plants.
Plant Phys. 2:215-225. 1950.

Fisher 8. A., Statistical Methods for Research Workers,
5rd edition. Oliver and Boyd, London, 1950.

Grabcr, L. F. Nelson, N. T., Luekel, W. A., and Albert,
W. B., Organic Food Reserves in Relation to the Growth
of Alfalfa and Other Perennial Herbaceous Plants. Wis.
Agro EXP. Sta. Res. Eula 80. 128 PP. 1927.

Grandfield, C.0., The Trend of Organic Food Reserves in
Alfalfa Roots as Affected by Cutting Practices. Jour.
Agr. Res. 50:697-709. 1955.

Harvey, R. B., An Annotated Bibligraphy of the Low Tem-
perature Relations of Plants. Burgess Publishing 00.,
Minneapolis, Hinn. 1955.

Janssen, G., The Relationship of Organic Root Reserves
and Other Factors to the Permanency of Alfalfa Stands.
Jour. Am. Soc. Agron. 21:895-911. 1929.

Jones, F. R., Winter Injury in Alfalfa. Jour. Agr. Res.
57:189-211. 1928.

Mark, J. J., The Relation of Reserves to Cold Resistance
in Alfalfa. Iowa Agr. Exp. Sta. Res. Bul. 208. pp. 501-
555. 1956.

maximov, N. A.. Internal Factors of Frost and Drought
Resistance in Plants. Protoplasma 7:259-291. 1929.

Megee, C. R.. A Search for Factors Determining Winter Hard-
iness in Alfalfa. Jour. Am. Soc. Agron. 27:685-698. 1955.

Newton, R., Colloidal Preperties of Winter Wheat Plants
in Relation to Frost Resistance. Jour. Agr. Sci. 14:178-
191. 1924.

Peltier, G. L. and Tysdal, H. M3, Hardiness Studies with
3-Year-01d Alfalfa Plants. Jour. Agr. Res. 45:951-955.
951.

Rather H. C. and Harrison, C. Mt, Alfalfa Management with
Special Reference to Fall Treatment. Mich. Agr. Exp. Sta.
Spec. Bul. 292. pp. 18. 1958.

15.
16.
17.

18.

19.

20.

52.

Rosa T. 3.. InVestigations on the Hardening Process in
Vegetable Plants. Mo. Agr. Exp. Sta. Res. Bul. 48. 1921.

Steinmetz, F. H. ‘Winter Hardiness in Alfalfa‘Variettas.
Minn. Agr. Exp. Sta. Tech. Bul. 58. 35 pp. 1926.

Stewart, 0., Effects of Inbreeding on Variability in
Alfalfa. Jour. Agr. Res. 49:669-694. 1954.

Tysdal H. M.. Determination of Hardiness in Alfalfa
Variet as by Their Enzymatic Reaponses. Jour. Agr. Res.
48:219-240. 1954.

Weimer, J. L.. Alfalfa Root Injuries Resulting From Freez-
ing. Jour. A81. Res. 40:121-145. 1950.

Willard, C. J., Thatcher, L. E.. and Cutler J. 8.. Alfal-
fa in OhiOQ Ohio Agra EXP. Sta. Bul. 540. £46 PP. 1954.

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