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ABSTRACT

AN EVALUATION OF SELECTED HARDINESS TESTS
IN RELATION TO PEACH BREEDING AND
MINIMIZATION OF NON-GENETIC WOOD
HARDINESS VARIATION
THROUGH SAMPLING

BY

David Wayne Cain

This research deals with winter injury to woody
tissues of peach trees. When developing a program to
improve wood hardiness, it is necessary to develop satis-
factory sampling techniques and viability evaluation

methods. A single peach genotype (Prunus persica Batch.

 

'Redhaven') was used to study non-genetic hardiness varia-
tion and evaluate tissue browning, regrowth, and
electrolytic conductance. The three evaluation methods
were studied to determine their suitability as evaluation
methods for handling large volumes of plant materials
encountered in a breeding program for improving wood
hardiness.

Electrolytic conductance was unsatisfactory for
evaluating large amounts of material because experimental
techniques could not be satisfactorily controlled when

handling such large quantities. Tissue browning was more

David Wayne Cain

closely correlated with regrowth than with electrolytic
conductance. Both tissue browning and regrowth tests
appear well suited to rapid evaluation of large volumes of
plant material.

Each of several randomly chosen trees were divided
into two parts; the upper southwest (sector 1) and the
lower northeast (sector 2). Twigs were removed from each
sector. They were frozen and divided into three sections;
the basal, middle, and tip sections. Tissue browning was
used to determine hardiness.

An appropriate statistical model was devised which
would separate the variance components of interest. Statis-
tical analysis revealed significant hardiness differences
among twig sections, tree sectors, and between trees.
Examination of variance components indicated that in most
experiments trees and tree x sector interaction constitutes
only a small portion of the total random variation. Twigs
and residual error accounted for 57 percent to 95 percent
of the total random variation. The browning rating system
had a repeatability of .79. An appendix illustrating the
browning rating is included.

-Variance estimates were used to estimate sample
sizes needed to detect hardiness differences of a desired
magnitude. It is suggested that sampling uniformly from
one location within all trees and within one part of all
twigs would eliminate a considerable amount of non-genetic

variation. The upper southwest sector contains more twigs

David Wayne Cain

and is less variable than the lower northeast sector. The
base twig sections were more differentiated and were easier

to rate visually than were the middle or tip sections.

AN EVALUATION OF SELECTED HARDINESS TESTS
IN RELATION TO PEACH BREEDING AND
MINIMIZATION OF NON-GENETIC WOOD
HARDINESS VARIATION

THROUGH SAMPLING

BY

David Wayne Cain

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1975

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude
to Drs. G. S. Howell, C. E. Cress, and C. R. Olein for
serving on his committee and for their guidance and many
helpful suggestions given during the course of this study.

Special thanks are extended to Dr. Robert Andersen
for serving as my major professor and for being a good
friend who always found time to help me with both my

professional and personal goals.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . .

LIST OF FIGURES. . . . . . . . . . .

INTRODUCTION. . . . . . . . . . . .
LITERATURE REVIEW 0 O O O O O I I O I

Hardiness variability in Relation to Plant
Breeding. . . . . . . . . . . .

SECTION I: AN EVALUATION OF THE SUITABILITY OF
TISSUE BROWNING, REGROWTH, AND ELECTROLYTIC
CONDUCTANCE WOOD HARDINESS TESTS FOR PEACH
BREEDING . . . . . . . . . . . .

Introduction . . . . A
Materials and Method . . . . . . . .

Freezing Techniques . . . . . . . .
Evaluation Methods . . . . . . . . .

Electrolytic Conductance . . . . . .
Tissue Browning . . . . . . . . .
Regrowth. . . . . . . . . . . .

Individual Experiments. . . . . . . .
November 24, 1973 (Expt. 1) and
December 15, 1973 (Expt. 2). . . . .
February 7, 1974 (Expt. 3). . . . . .
March 20, 1974 (Expts. 4 and 5) . . . .

Results and Discussion. . . . . . . .

iii

Page

vii

10

13

14
15

16
l7
17
17
18
19
19
20

20

Page

SECTION II: MINIMIZING NON-GENETIC WOOD HARDINESS
VARIATION IN REDHAVEN PEACH (PRUNUS PERSICA

 

BATCH.) THROUGH SAMPLING . . . . . . . . . 32
Introduction . . . . . . . . . . . . . 33
Materials and Methods . . . . . . . . . . 35
Results. . . . . . . . . . . . . . . 37
Discussion. . . . . . . . . . . . . . 40
SUMMARY AND CONCLUSIONS . . . . . . . . . . 48
APPENDIX . . . . . . . . . . . . . . . 52
BIBLImRAPHY. O O O O O O O O O O O O O 62

iv

Table

LIST OF TABLES‘

SECTION I

Overall experiment means for each evaluation
method. . . . . . . . . . . . .

Simple correlations between evaluation
methods . . . . . . . . . . . .

Twig section means within each sector of
the tree for experiments 1 and 2 . . . .

Twig section means within each sector of
the tree for experiment 3 . . . . . .

Regrowth and conductance means at each
browning level in experiment 3. . . . .

Random variation in experiment 3 expressed
as a percentage of the grand mean. . . .

SECTION II

Analysis of variance table with the expected
mean squares used to calculate F values and
variance components . . . . . . . .

Analysis of variance table showing degrees of
freedom, mean squares, and significance
values of the F statistic for effects of
interest in all experiments. . . . . .

Estimates of variance components in all
experiments . . . . . . . . . . .

Page

28

28

29

30

31

31

44

45

46

Table Page

4. An estimate of the number of twigs (n)
needed to detect a desired difference (6)
where m = .05, p = .8, and t has infinite
degrees of freedom. . . . . . . . . . 47

vi

LIST OF FIGURES

Figure Page

1. Regression of browning on wood weight for
sector 1 twig section 1 and sector 2
twig section 3. . . . . . . . . . . S3

2. Photographs illustrating the 5 unit
browning scale. A through E represent
a rating of 1 through 5 respectively . . . 55

3. Illustration of a tree divided into
sectors 0 O O O O O O O O O O O O 59

4. Illustration of a twig showing its
division into twig sections . . . . . . 61

vii

INTRODUCTION

Peaches are among the most cold susceptible peren-
nial fruit crops able to be commercially grown in the
northern U.S. Their range is much more restricted than
apples, pears or other fruit trees, primarily because they
are more subject to winter injury.

In the northcentral and northeastern United States
much winter injury occurs in late fall or early winter,
resulting from freeze injury to immature tissue before it
has fully acclimated. Conversely, the southern peach
growing regions receive much cold damage during late winter
due to early deacclimation (Andersen, 1974).

In Michigan, boundaries of the peach producing
regions can be delineated by winter low temperature iso-
therms (Kessler, 1971). Winter injury can include
sunscald, black heart, root killing, death of flower buds
and even death of the entire tree. Winter killing of
flower buds causes losses to growers both by reducing crops
and by over production during years when no injury occurs.

These factors cause disruption of orderly marketing.

Winter injury to woody tissues is sometimes dra-
matic, killing thousands of trees in a single freeze
(Bradford and Cardinell, 1952; Kessler, 1971). Often,
however, winter injury to woody tissues is more subtle,
causing injury to cambium and xylem which reduces tree
vigor and makes the tree more susceptible to attacks by
insects and diseases. This reduces the economic life of
the orchard and increases production costs.

Bradford and Cardinell (1952) have surveyed winter
injury occurring in Michigan from 1846 to 1926. They
report numerous winters in which severe winter injury has
occurred. Spectacular winter injury resulting in the death
of thousands of peach trees throughout Michigan occurred
after the winters of 1855-56, October 1906, 1917-18, and
Thanksgiving Day 1950. Kessler (1971) reports that there
were 12,500,000 peach trees in Michigan in 1889. In
October 1906 a severe freeze killed 73 percent of the peach
trees in Michigan after which the peach never regained its
former prominence. In 1949 there were 3,603,800 peach
trees in Michigan but again a severe freeze in November 1950
killed thousands and in l972_there were 1,630,000 trees.

Thus, winter injury to the wood has limited peach
production in Michigan to favorable sites located in a
narrow strip of land protected by Lake Michigan. Even
within this area winter injury problems often occur as the
winters of 1971-72 and 1972-73 point out. Furthermore, as

human p0pu1ation pressure eliminates many of these

favorable lakeside sites, peach production will be forced
to move to less favorable land.

Improved wood hardiness is essential for the suc-
cessful long-term commercial production of peaches in
Michigan and should be of prime concern in breeding new
peach varieties for Michigan.

In setting up a breeding project to identify hardy
genotypes one of the first logical steps is to decide on a
suitable method of injury evaluation. Any evaluation tech-
nique used in breeding must be adaptable to handling large
sample sizes, have reasonable accuracy and precision, and
must be able to evaluate a single plant without completely
destroying that genotype.

When trying to identify genetic differences in
hardiness it is important to know the amount of non-genetic
variation which may be encountered. Knowing the magnitude
and sources of non-genetic variation, sampling techniques
and sample size estimates which allow detection of genetic
differences of a given magnitude can be calculated.

This work was a preliminary step in development of
a program for breeding peaches which would be winter hardy
in climates similar to Michigan. There were three specific
objectives to this thesis research. The first was to evalu-
ate the suitability of tissue browning, tissue regrowth,
and electrolytic conductance as possible techniques of
injury evaluation when handling large volumes of plant

materials. The second objective was to identify some

sources and obtain some estimates of the magnitude of non-
genetic hardiness variation occurring within a represen-
tative peach genotype. The third was the develOpment of a
satisfactory sampling scheme and estimation of a reasonable
sample size needed for detection of hardiness differences

of a desired magnitude.

LITERATURE REVIEW

Fruit growers and agricultural scientists have
learned the importance of good cultural practices in
limiting the extent of winter injury problems (Chandler,
1913; Bradford and Cardinell, 1913). Varietal hardiness
differences have also been recognized (Chandler, 1913;
Hedrick, 1916). These varietal differences showed up in
field survival results after test winters. Such test win-
ters have been widely used to study plant hardiness and
many researchers have issued reports dealing with injury
to fruit crOps after such winters (Chandler, 1913; Dorsey
and Strausbaugh, 1923; Dorsey and Bushnell, 1925; Potter,
1938; Lantz and Pickett, 1942; Cooper, 1953; Fogle and
Overley, 1954). At most locations a good test winter may
occur on an average of once in ten years (Levitt, 1972).
Researchers realized that test winters alone occurred too
infrequently to provide a continuous reliable means of
determining hardiness of new genotypes. Therefore, they
sought some method of artificially cold stressing plants.

Chandler (1913) described a salt ice bath in

which a temperature of ~12 to -15°C could be reached. This

system could not be programmed to control freezing rate.
According to Levitt (1972) an artificial freezing chamber
in which material could be quickly and quantitatively frozen
was introduced by Harvey in 1918. Edgerton (1960)
described an antifreeze bath freezer which is programmable.
Meader (1945) described a modified ice cream freezer using
an ethanol mixture for coolant. Weaver et a1. (1968) have
used a cryostat chamber. Weaver et a1. (1969) have also
used a liquid nitrogen system. Scott and Spangelo (1964)
described a portable freezing chamber for use in the field.
Weiser (1970) had used insulated thermos bottles placed in
a Revco freezer. To give the best research information the
system must allow controlled freezing at a predetermined
rate.

The evaluation of injury to buds is relatively
easy. Live buds remain green and healthy while dead buds
quickly become water soaked, turn brown, and eventually
dry up. Number dead versus number alive can be expressed
as a percent live or dead buds (Chandler, 1913; Edgerton,
1960). The percentage of live or dead buds over a range
of temperatures can be expressed graphically and the LT50
can be estimated (Proebsting and Fogle, 1956). Bitten-
bender and Howell (1974) have recently adapted the
Spearman-Kfirber method to estimate the LT50 using an
equation rather than a graph. This equation also provides
information on the slope of the curve through the LT50

point. Evaluation of woody parts is difficult because the

injury ratings are a continuous gradient rather than dis-
crete in nature.

Some methods try to measure a plant character which
appears to be correlated with differences in plant hardi-
ness. Some characters which have been so measured include:
osmotic pressure (Chandler, 1913), cell size (Wiegand,
1906), moisture content (Hildreth, 1926), various carbo-
hydrates (Cooper, 1953), fatty acids (Ketchie, 1966), and
protein content (Siminovitch and Briggs, 1949; Craker et a1.,
1966).

One of the oldest and most widely used methods is
to rate the survival of whole plants or plant parts on an
arbitrary scale. These subjective ratings may be expressed
verbally (Hildreth, 1926; Fogle and Overly, 1954; Brierly
and Landon, 1954), or as a percentage of live versus dead
tissue (Watkins and Spangelo, 1970; Ketchie et a1., 1972),
or the ratings may be given a numerical rating on an
arbitrary scale (Lantz and Pickett, 1942; Lapins, 1962a,
1962b). Numerical data can be analyzed using analysis of
variance procedures providing the assumptions of homogene-
ous variance and normal distribution of residual deviations
are not violated. Often such data are skewed toward one
end of the rating scale. In such cases some type of
scaling procedure can be used to give approximate solutions
(Snell, 1964) or some type of nonparametric statistic such
as Friedman's two-way analysis of variance can be used

(Steel and Torrie, 1960). It should be noted that

regrowth does not directly measure injury. It describes
the ability of the plant to survive injury and is closely
related to the amount of injdry induced but injury and
recovery are two separate phenomena.

Another subjective rating system involves visual
estimation of tissue damage via tissue browning. Usually
the wood or cambium tissue is given a numerical rating
describing the severity of browning (Lapins, 1962a, 1962b;
Blazich, 1974). The data are then handled in the same
manner as for recovery ratings.

Regrowth and browning ratings have been considered
reliable but they have been criticized for being slow
(Wilner, 1955; Stushnoff, 1972; Blazich, 1974). Individual
bias can influence tissue browning results (Steponkus,
1967). Workers searched for evaluation techniques which
would be simple, fast, quantitative, and free from indi-
vidual bias. Many methods have been developed, each having
its own advantages and disadvantages.

Cell plasmalysis and neutral red staining methods
have been used to determine cell viability (Siminovitch and
Briggs, 1953; Lumis et a1., 1972). Stepokus (1967) has
described a refinement of the triphenyl tetrazolium chloride
(TTC) method of determining cold injury involving measure-
ment of the reduced TTC using spectrOphotometric techniques.
Staining techniques, however, are not well adapted to

handling large volumes of plant materials.

Heat is given off when supercooled liquid water
suddenly freezes. This heat can be measured as an exo-
therm. Quamme et al. (1972a) have associated exotherm
analysis with seasonal changes in hardiness of apple xylem.
In blueberry stems exotherms were associated only with xylem
injury which was not as critical for survival as the bark
tissues (Quamme et a1., 1972). Stergios and Howell (1973)
found that exotherm analysis worked well for some species
but not for others and the method was not adapted to quan-
titative analysis.

Electrical resistance or impedance has been used to
measure plant hardiness in_§itg_(Wilner, 1960a). This
method involves placing the tissue to be tested between two
electrodes and passing a small electrical current through
the tissue and measuring the resistance. Results of this
method are closely correlated to results from.e1ectrolytic
conductance methods (Wilner, 1961). Blazich et a1. (1974)
found that electrolytic conductance was more closely
associated with tissue browning than was electrical
impedance. Evert and Weiser (1971) using electrical
measurements at two frequencies found that stem sections
exposed to lethal temperatures could not consistently be
separated from sections exposed to non-lethal temperatures
when tested immediately after thawing.

Dexter et a1. (1930) described a method of studying
injury by measuring electrolytic conductance of water

leachates of frozen tissues. This method has been used by

10

a large number of researchers (Emmert and Howlett, 1953;
Wilner, 1955; Edgerton, 1960; Lapins, 1962a, 1962b; Ketchie
et a1, 1972). Results are usually expressed as absolute
conductance or as a percentage of the initial leachates in
relation to the total amount of leachates in a boiled
sample (Wilner, 1960b). Flint et a1. (1966) have expressed
electrolytic conductance as an index of injury where an
unfrozen sample has a value of zero and a heat-killed sam-
ple has a value of one hundred. The injury is expressed as
a relation between the frozen sample minus the unfrozen
sample, and the heat-killed sample minus the unfrozen
sample.

Wilner (1955) states that electrolytic conductance
is more desirable than the more tedious and time-consuming
examination of sectioned tissues. Lapins (1962b), however,
found that recovery tests were much more sensitive in
differentiating plant hardiness. He also found a higher
correlation between recovery and browning than between
either recovery or browning and electrolytic conductance.

Hardiness Variability in Relation
to Plant Breeding

 

Dorsey and Bushnell (1925) found that winter hardi-
ness in plums was controlled by a multiple allelic series.
watkins and Spangelo (1970), using apple, investigated the
polygenetic trait called plant survival. Thus, winter
hardiness can be considered a quantitative trait. Such

traits are often involved in development of new varieties.

ll

Breeding new superior varieties of asexual propa-
gated plants involves two distinct aspects. One involves
selection and asexual propagation of commercially acceptable
seedlings which are superior to and are expected to replace
a standard variety. The other involves selection of
seedlings with some characteristics superior to the old
variety but are themselves of little commercial value.
These plants constitute parents of the next breeding cycle.
They are used in an effort to recombine their different
favorable genes into a single genotype.

The progress which can be made for both aspects of
variety development is a function of the heritability of
the characters concerned. Broad sense heritability is the
genetic variance as a fraction of the phenotypic variance.
If genotypes are randomly placed into the environment, the

2 2 2

total phenotypic variance can be expressed as: op = 0y + Ge“
2 2

where Up is the phenotypic variance, 0y is the total genetic
variance and a; is the environmental variance (Comstock and
Robinson, 1948). The environmental variation is a
nuisance and masks the genetic differences of interest.
Therefore, any reduction in environmental variation will
result in the genetic variation contributing proportion-
ately more to the total phenotypic variation. This
increases the heritability of the trait in question.
Differences in wood hardiness within twigs,

between twigs within a tree, and among trees of the same

genotype have been found (Dorsey and Strausbaugh, 1923;

12

Dorsey and Bushnell, 1925; Fogle and Overley, 1954; Wilner,
1960; Wilner, 1961; Lapins, 1962). ‘Using an apprOpriate
experimental design, such differences can be measured on a
quantitative scale and the data analyzed via standard
analysis of variance techniques (Steel and Torrie, 1960;
Sokal and Rohlf, 1969). The identified sources of variance
and the estimates of their magnitude can then be used to
develop optimum sampling procedures to reduce this nuisance
variation (Marcuse, 1949; Schultz, 1955). Sample sizes
necessary to detect differences of'a desired magnitude can
be calculated using the variance estimates (Sokal and

Rohlf, 1969; Schultz, 1955).

SECTION I

AN EVALUATION OF THE SUITABILITY OF TISSUE
BROWNING, REGROWTH, AND ELECTROLYTIC
CONDUCTANCE WOOD HARDINESS

TESTS FOR PEACH BREEDING

13

Introduction .

 

In hardiness research some method of injury evalu—
ation must be devised. Methods suitable for physiological
research on wood hardiness may not be well adapted as mass
screening techniques for identifying wood hardy plants
(Lapins, 1962a, 1962b; Stergios and Howell, 1973; Stushnoff,
1972). While an evaluation method used as a screening
technique need not yield immediate results, it must be able
to handle large amounts of plant materials with ease.

Tissue browning, regrowth, and electrolytic conductance have
been widely used and are well suited as evaluation tech-
niques for mass screening (Lapins, 1962a, 1962b; Wilner,
1960b). Tissue browning and regrowth have been used to
standardize other tests but have been criticized as being
qualitative and subject to individual bias (Ketchie et a1.,
1972; Lapins, 1962a; Stergios and Howell, 1973).

Electrolytic conductance of leachates provides a
qualitative test which has correlated well with regrowth
and visual methods (Blazich et a1., 1974; Ketchie et a1.,
1972; Wilner, 1960b). Some workers have claimed that

while browning and regrowth were qualitative, they provided

14

15

a better estimate of injury than conductance (Lapins,
1962a, 1962b; Stergios and Howell, 1973).

Most studies which have compared different methods
utilized relatively small sample sizes (Blazich et a1.,}
1974; Stergios and Howell, 1973; Wilner, 1960b; Wilner,
1961). This study was included as part of a larger experi-
ment to study wood hardiness variation occurring within a
single peach genotype. Tissue browning, regrowth, and
electrolytic conductance methods were studied to determine
their suitability as evaluation methods for handling large
volumes of plant materials similar to the amounts which
would be encountered in a breeding project for improving

wood hardiness.

Materials and Methods

 

Estimates of wood hardiness were determined on
November 24, 1973; December 15, 1973; February 7, 1974; and
March 20, 1974. Plant material was collected near Hartford,
Michigan, from a typical commercial orchard of four-year
old Redhaven peach trees grafted to Siberian C rootstocks.
Trees selected at random represented a wide range Of
orchard elevations differing by over 9m. Variation in
elevation, fertility, moisture, soil, and tree vigor as
well as the effects of the seedling rootstocks contributed
to microenvironmental variation which would be expected to
lead to random differences in wood hardiness among trees.

Different trees were used at each sampling date.

16

Each tree was divided into eight sectors. Hardi-
ness of the current years twig growth in the upper
southwest and the lower northeast sectors was evaluated
(see Figure 3). They were chosen because they exhibit
maximum differences in the interception of solar radiation.
A number of twigs were taken at random from within each
sector. Each twig was divided into three equal parts and
the test sections were removed from within each of these

parts (see Figure 4).

Freezing Techniques

 

A11 twigs from within each sector were taped
together and properly labeled. The strips of whole twigs
were wrapped in several layers of aluminum foil and foam
rubber to allow uniform removal of heat during the freezing
process. Three such packages were frozen to different
test temperatures at each date to be sure at least one
received the desired stress.

Temperature was monitored by inserting 26 gauge
copper-constantan thermocouples into the pith of several
twigs in the bundle. The thermocouples were connected to
a 24 point recorder. All bundles were placed in a Revco
freezer and temperature reduction was maintained at approxi-
mately 3°C/hr. Each bundle was removed immediately upon
reaching the desired test temperature and was allowed to
thaw completely at room temperature. Upon thawing, stressed

twigs were unwrapped and stored under humid conditions

17

until they were evaluated for injury. Conductivity tests
were performed within twenty-four hours after thawing and

browning was usually done within seventy-two hours.

Evaluation Methods

 

Electrolytic Conductance

An electrolytic conductance test of leachates,
similar to the technique described by Wilner was used
(Wilner, 1960a, 1960b; Wilner, 1961). Each twig section
was weighed then cut into approximately 1 cm sections.

The sections were placed into individual test tubes and a
volume of water equal to seven times the wood weight was
added to each tube. All tubes were stored at room tempera-
ture for approximately twenty-four hours.

After twenty-four hours initial conductance was
taken using a solu-bridge soil tester (Model RD-lSXI
Industrial Instruments, Inc.). The solu-bridge had 1 cm2
platinum electrodes enclosed in a glass bulb. After
recording initial readings, all tubes were autoclaved for
twenty minutes. The final conductance was taken after
another twenty-four hours. Injury was eXpressed as:

Initial Conductance x 100 = % specific conductivity
Final Conductance

 

Tissue Browning

 

A visual estimation of injury was made by examining

a cross section of each twig section under a binocular

18

microscope (see Figure 2). The extent of tissue injury was
expressed on the following 1 to 5 tissue browning scale:

1--No injury. All tissues appear bright green and
alive.

2--Primary phloem fibers brown but surrounding tissue
still green, giving a circle of brown spots sur-
rounding the cambium area. These were judged fully
capable of recovery.

3--Primary phloem fibers brown and some browning in
the secondary phloem forming a continuous ring
between the cortex and cambium areas. It is con-
sidered alive but with moderate injury.

4--All phloem cells brown and the cambium area showing
some discoloration. Viability is questionable.

5--Completely brown and discolored with only the cortex
possibly being green. These were judged to be dead.

On the basis of regrowth tests, browning ratings of
l to 3 were considered to be alive and able to recover from
injuries. A 4 rating was considered very severe injury and

recovery was uncertain. A 5 was considered dead.

Regrowth

Materials for the regrowth tests were properly
labeled and taped together, then placed in flats of moist
peat moss. The flats were incubated in a 26°C greenhouse
for fourteen to twenty-one days to allow development of
callus formation. The following rating system was used:

l--No injury, very prolific callusing covering the
entire cut surface.

2--Callus protruding from the cut surface around the
entire cambium area.

 

 

 

 

19

3--Moderate callus formation not protruding above the
cut surface and not necessarily forming a continuous
ring around the entire twig section.

4--No callus formation but the twig bark appeared green
and intact with no or only slight degeneration.

5--No callus formation with the Outer bark brown and
showing extensive tissue degeneration and often
being invaded by saprophytic fungi.
A regrowth rating of 1 to 3 was considered alive
and fully capable of recovery. Twigs with a 4 rating were

considered severely injured and chance of recovery was

considered very low. A rating of 5 indicated death.

Individual Experiments

 

November 24, 1973 (Expt. 1) and
December 15, 1974 (Exp_. 2)

 

 

Twelve twigs were removed from each of the two
sectors in each of six trees. Each twig was divided into
three equal parts and the tip section discarded. A 5 cm
portion was removed from the middle of both remaining sec—
tions. The distal 2 cm of each section was used in the
browning test. Regrowth data were incomplete and were
not used.‘ The artificially induced test temperature in

both experiments was -28°C.

February 7, 1974 (Expt. 3)

Fifteen twigs from each sector within eight trees
were used. Twigs were divided into three equal parts. A
9 cm twig section was removed from the middle of each

part. The distal 2 cm portion was utilized for the

20

browning and regrowth tests. A thin cross-section slice
was removed from this piece and evaluated for browning,
the remaining portion was placed in moist peat moss. The
remaining 7 cm portion was used in the conductance test.

Twigs were frozen to -34°C.

March 20, 1974 (Expt. 4 and 5)

 

The sampling procedure was the same as in experi-
ment 3 except no conductance tests were performed and the
whole 9 cm twig section was used in the browning and
regrowth tests. Twigs in experiment 4 were frozen to -25°C

while those in experiment 5 were frozen to -27°C.

Results and Discussion

 

For statistical and discrimination purposes it was
desirable to induce an intermediate level of injury in
the plant material. This level would be represented by a
3 on the browning and regrowth scales. This was desired
because it would produce a normal distribution about the
mean. Either very little or very severe injury would pro-
duce a skewed distribution because of the finite scale.
This could seriously distort assumptions underlying the
analysis of variance, biasing, results, making application
of nonparametric statistics necessary.

Table 1 shows the overall experiment means for each
evaluation method. Experiments 2, 3, and 4 have mean
browning ratings very close to the desired 3.0, while

experiments 1 and 5 show more injury than desired. The

21

regrowth means indicate more severe injury than the
browning means. However, as the browning ratings increase,—
regrowth ratings increase in a similar magnitude. '
Browning tests indicate twigs in experiment 1 were more
severely injured than those in experiments 2 or 3 but the
percent conductance did not indicate any substantial
change in injury.

Simple correlations between the three methods»
(Table 2) indicate a much closer relationship between
browning and regrowth than between either browning or
regrowth and electrolytic conductance. Experiment 1 indi-
cates no relationship between browning and electrolytic
conductance while experiments 2 and 3 indicate a low but
highly significant (testing the null hypothesis of zero
correlation) negative correlation of about -.25. The
correlation between regrowth and conductanCe (-.29) is
similar to that of browning and conductance. These nega-
tive correlations indicate that as browning and regrowth
rating increase, indicating more severe injury, the percent
conductance decreases indicating decreased injury. This
conflicts with results obtained by other researchers using
electrolytic conductance (Blazich et a1., 1974; Ketchie
et a1., 1972; Lapins, 1962a, 1962b; Wilner, 1960a, 1960b;
Wilner, 1961). The highest correlation between browning
and regrowth was .67 (Expt. 4). This is not an extremely
high correlation but it is good considering the subjec-

tivity of both rating systems.

22

In experiments 1 and 2, tissue browning reveals
significant differences among twig sections within a given
sector as well as between sectors (Table 3). Twig sections
in sector 1 are hardier than comparable sections in sector
2. The base sections (section 1) are hardier than the
middle sections of the twigs. The electrolytic conductance
test in experiment 1 indicates that only the bases of the
twigs in sector 2 were less injured than the tip sections
in sector 2. In experiment 2 conductance fails to reveal
any differences in injury. This conflicts with results
of the browning tests.

In experiment 3 browning and regrowth means
(Table 4) indicate a significant hardiness gradient exists
within the twigs from both sectors, the basal sections
being hardiest and the tip sections being least hardy.
Browning and regrowth also indicate that twig sections in
sector 1 are hardier than comparable twig sections in
sector 2.

Electrolytic conductance tests in experiment 3
(Table 4) indicate that within a sector basal twig sections
have higher conductance values than tip sections. The
basal sections in sector 1 have a higher conductance than
those in sector 2 while other twig sections show no dif-
ferences from one sector to the other. The conductance
means in Table 4 show an inverse relationship to both the
browning and regrowth means so that those twig sections

having the severest injury according to browning and

23

regrowth show the lowest conductance values. This disagrees
with conclusions reached by other researchers (Blazich

et a1., 1974; Ketchie et a1., 1972; Lapins, 1962a, 1962b;
Wilner, 1960a, 1960b; Wilner, 1961).

Table 5 shows regrowth and conductance means for
all the cases of a given browning rating in experiment 3.
While regrowth ratings indicate more severe injury to the
twig sections than browning ratings, both methods increase
in a similar manner. The conductance means are again
shown to decrease as browning and regrowth ratings indicate
more severe injury.

The results show that browning and regrowth yield
similar results even though regrowth often indicates more
severe injury than browning. One explanation of this dis-
crepancy is that control of regrowth conditions in the
greenhouse was not ideal. Fungi and bacteria infected
many of the cuttings and spread to adjacent twig sections
even though a fungicide was used. This caused some bias
in the estimate of injury in some trees and sectors
because twig pieces were taped together according to tree
and sector. Even healthy twigs were often invaded and
killed by microorganisms. There was also some evidence
that callus formation occurred more readily after the rest
period had been satisfied.

Electrolytic conductance proved to be a very unsat-
isfactory evaluation method. It is not understood why the

results from conductance tests show an inverse relationship

24

to browning and regrowth or why they disagree with other
researchers' conclusions (Blazich et a1., Ketchie et a1.,
1972; Lapins, 1962a, 1962b; Wilner, 1960a, 1960b; Wilner,
1961). Some factors which may have contributed to unusual
conductance results are listed here. The xylem was injured
much more severely than the phloem and cambium areas. The
relatively large amount of leachates released by the xylem
could have largely masked any relatively small differences
caused by release of leachates from a small number of
injured cells in the phloem and cambium. Lapins (1962b)
indicated that this is a problem with the conductance
method. The solu—bridge used to make the readings was not
as accurate as desired and led to a reduction in the
ability to detect small differences. The small amount of
material used (3 or 7 cm sections) and the small amount of
water in which they were immersed led to relatively large
measurement errors which further reduced the ability to
detect small differences.

Control of experimental conditions when such a
large number of samples was involved (n=720 samples) was
very difficult. The time delay between reading the first
sample and the 720th was several hours and led to
increased error.

The large random variation about the grand mean
for sectors (51.34%) shown in Table 6 is the result of
having all twig sections from one sector of one tree

placed together in one test tube rack and thus being

25

analyzed as a group. This resulted in large random dif-
ferences between racks which increased experimental error.
The large random variation for sectors in the regrowth
test (Table 6) is also a result of not having complete
randomization of all twig sections.

The time involved in preparing samples and calcu-
lating conductance values combined with the difficulty in
controlling experimental error effectively eliminates
electrolytic conductance as a method of mass screening
large numbers of seedlings in a breeding program.

Tissue browning proved to be the most satisfactory
evaluation method. About thirty-six hours after thawing
tissue browning was well developed and changed very little
over the next several days as long as the twigs were not
allowed to dessicate. This provides some flexibility
concerning time of evaluating injury. Browning is much
faster and less technically involved than conductance.

The time involved in determining browning value is no
greater than the time involved in rating the amount of
regrowth, but browning eliminates the disease control
problems associated with the regrowth test. Another
advantage of browning is that it permits multiple observa-
tions on one twig section.

A major criticism of tissue browning is the sub-
jectivity and individual bias which may be associated with
the test (Blazich et a1., 1974; Lapins, 1962a; Stergios

and Howell, 1973). In experiments 4 and 5, two browning

26

observations were made on each twig section. To eliminate
any memory bias each of the 720 twig sections were rated
once before the second rating on any section was performed.
A determination of the repeatability of the browning test
was then made by using estimates of variance components in

the following equation:

 

 

Repeatability of _ 32 32 32
determinations trees + trees x sectors + twigs +
32 32 32
trees + trees x sectors + twigs +
32
error
A2 A2

0 I 0
error + determinations

As the 32 determinations increases the repeatability
decreases from unity. This gave a repeatability of .76 for
experiment 4 and .82 for experiment 5, giving an average
repeatability of .79 for 2880 observations. This suggests
that the determination error was as large as any of the
other components but it is still small enough to be con-
trolled reasonably well through pr0per experimental design.
It should be noted that this expresses nothing about the
accuracy of the method.

By observing browning of specific tissues rather
than overall intensity of browning, subjectivity of the
test is reduced. The possibility exists that killing
temperature of specific cell types which cause only partial

injury are not a true reflex of the ultimate killing

27

temperature of the whole twig involved. The close rela—
tionship between browning and regrowth minimizes this
danger.

Tissue browning and regrowth tests appear well
suited to rapid evaluation of large volumes of materials
involved in preliminary screening of large numbers of seed-

lings in a hardiness breeding program.

28

Table l.--Overall experiment means for each evaluation

 

 

method.
Test .
Experiment Temperature Percent
Number °C BrOwning Regrowth Conductance

2 -28 2.86:.06 23.29:.25

3 -34 2.84:.03 3.62:.03 22.851.17

4 -25 3.10:.03 3.77:.05

5 ~27 3.59:.03 4.20:.04

 

Table 2.—-Simp1e correlations between evaluation methods.

 

 

Experi- Browning ’ Regrowth
ment Conduc- Browning Conduc-
Number tance P Regrowth P tance P
1 .05 N.S.
2 -.25 <.001
3 -.26 <.001 .54 <.001 -.29 <.001
<
4 .67 <.001
5 .64 <.001

 

n = 720 observations in each experiment.

P = Significance level of the null hypothesis of
zero correlation.

29

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30

Table 4.--Twig section means within each sector of the

tree for experiment 3.

 

 

Twig
Sector Section Browning Regrowth Conductance
l l 2.26 2.83 26.34
1 2 2.60 3.32 23.87
1 3 2.94 3.77 21.05
2 1 2.58 3.63 22.72
2 2 3.02 3.91 22.91
2 3 3.63 4.29 20.16
LSD z
.05 .ll .14 .81
Y
LSD.os .15 .28 2.19

 

sector.

ferent sectors.

zLSD value used to compare twig sections within a

yLSD value used to compare twig sections in dif-

31

Table 5.--Regrowth and conductance means at each browning
level in experiment 3.

 

 

Browning Percent Number
Rating Regrowth Conductance of Cases
1 2.5 i .50 25.94 t 1.45 4
2 3.06 i .06 25.34 1 .33 226
3 3.77 t .03 22.41 i .22 400
4 4.37 i .07 20.96 i .55 60
5 4.83 i .06 20.90 i .84 30

 

Table 6.--Random variation in experiment 3 expressed as a
percentage of the grand mean.

 

 

Browning Regrowth Conductance
Trees 29.06 26.64 20.65
Sectors 24.87 39.55 51.34

Twig Sections 15.86 15.70 14.08

 

SECTION II

MINIMIZING NON-GENETIC WOOD HARDINESS
VARIATION IN REDHAVEN PEACH

(PRUNUS PERSICA BATCH . )

 

THROUGH SAMPLING

32

Introduction.

 

Lack of winter hardiness is a major limiting factor
in fruit production in northern latitudes (Bradford and
Cardinell, 1926; Chandler, 1912). Stushnoff (1972) has
recently reviewed cold hardiness breeding and previous
efforts have sought to select hardy genotypes either after
test winters (Dorsey and Strausbaugh, 1923; Dorsey and
Bushnell, 1925; Fogle and Overley, 1954; Lantz and Pickett,
1942) or by use of artificial freezing techniques (Lapins,
1962a, 1962b; Watkins and Spangelo, 1970; Wilner, 1960b,
1961).

Researchers have often used excised twigs of the
previous season's growth as their experimental material to
evaluate wood hardiness of the entire plant (Lapins, 1962a,
1962b; Wilner, 1960b, 1961). Twigs provide large amount’
of material for sampling and a means of testing wood hardi-
ness of seedlings without destroying entire plants.

Differences in wood hardiness over the length of a
twig, between twigs within a tree, and among trees of the
same genotype have been found (Dorsey and Strausbaugh,
1923; Dorsey and Bushnell, 1925; Fogle and Overley, 1954;

Lantz and Pickett, 1942; Lapins, 1962a, 1962b; watkins and

33

34

Spangelo, 1970; Wilner, 1960b, 1961), but, few attempts
have been made to systematically measure the sources and
magnitude of this non-genetic variation. Lapins (1962a,
1962b) has stressed the use of uniform material for evalu-
ating wood hardiness. He gave estimates of sample sizes
needed to select specific percentages of hardy seedlings
from a population of apple seedlings (Lapins, 1962b)
Detection of genetic wood hardiness differences
of a given magntitude among seedlings in a breeding program
has been the ultimate goal. A knowledge of sources of
non-genetic wood hardiness variation would be important
if an efficient sampling method was to be developed. An
estimate of the amount of nonégenetic variation would
allow determination of sample sizes needed for detection of
genetic wood hardiness differences of a desired magnitude.
This study was undertaken to identify sources of
wood hardiness variability and to ascertain the extent of
variation within and among trees of a single peach genotype.
This information would permit development of sampling
methods to reduce non-genetic wood hardiness differences.
It would also provide estimates of the magnitude of random
variation. These would be used to develOp sample size
estimates needed to detect differences of a desired magni-

tude in future experiments.

35

Materials and Methods

 

Twigs of the previous season's growth were removed
from the upper southwest (sector 1) and lower northeast
(sector 2) sectors (see Figure 3) of four-year old peach

(Prunus persica Batch. 'Redhaven') trees being grown on a

 

good commercial peach site at Hartford, Michigan.

All twigs were wrapped in several layers of alumi-
num foil and foam rubber to allow slow uniform heat
removal. They were frozen to a predetermined test tempera-
ture designed to induce the proper stress and were
immediately removed upon reaching the desired temperature.
Each twig was cut into three equal length sections. From
within the middle of each of these twig pieces, a uniform
length twig section was removed and used as the basic
experimental unit (see Figure 4). A thin cross section of
‘each twig section was microscopically examined for tissue
discoloration. Extent of injury was based on an arbitrary
1 to 5 browning scale (see Figure 2).

In experiments 1 and 2 twelve twigs were removed
from each sector of six randomly selected trees. Data from
only the base and middle sections (twig sections 1 and 2)
were analyzed. Fifteen twigs from the southwest and north-
west sectors of eight trees were evaluated in experiments 3,
4, and 5. All three twig sections were examined. In
experiments 4 and 5 two browning observations were taken
on each twig section. More details of materials and

methods are given in section I.

36

An appropriate statistical model was devised which
would separate the variance components of interest. In the
model the value of an individual observation (Yijkl) can be

described as:

Yijkl = u + Ti + Sj + (TS)ij + A(ij)k + B1 + (SB)jl

+ E(ijk1)

Where:
T--symbolizes Trees
S--symbolizes Sectors of the tree
A—-symbolizes twig sections
E--sumbolizes residual error
And:
u = the true pOpulation mean which is a constant

1, 2...t where i refers to the random effect of
the ith tree '

H.
II

j = l, 2...5 where j refers to the fixed effect of
the jth sector of the tree

k = l, 2...a where k refers to the random effect of
the kth twig within the jth sector of the ith
tree

1 = l, 2...b where 1 refers to the fixed effect of
the 12h twig section

The model is mixed. It also contains nested and
crossed factors. The statistical implications of a mixed
model and nested elements have been discussed in detail
(Henderson, 1953; Marcuse, 1949; Schultz, 1955; Sokal and

Rohlf, 1969).

37

Table 1 shows the analysis of variance table with
the expected mean squares. Observed mean squares were
equated to their expectation and the proper F test for each
effect of interest was determined from Table l. The
residual error term is composed of higher order interactions
as well as several two-way and three-way interactions which
were not of interest.

In experiments 4 and 5 two browning observations
were taken on each twig section. This effect was denoted
as: determinations. It was symbolized in the model as
F(ijkl)m where m = 1,2. It had an expected mean square of
0;. It was not shown in Table 1 but its effect would be
added to all higher effects in the table. This effect gave
an estimate of the variance due to inaccuracies in the

rating system.

Results

Table 2 shows the experimentally obtained estimates
Of mean squares with their associated degrees of freedom
plus the Significance level of the F statistic. Signifi-
cant differences among trees were found in all experiments
except experiment 2. The two sectors of the tree also
differed significantly. Because of the significant tree by
sector interaction, sector differences should be examined
separately within each tree. Closer examination revealed

that although the magnitude of differences between sectors

38

changes from tree to tree, only three trees out of thirty-
six examined in all five experiments showed sector 2 to be
hardier than sector 1.

In every experiment twig sections showed a highly
significant gradient exists within them. The highly sig-
nificant sector by twig section interaction in experiments
3, 4, and 5 indicated that differences among twig sections
were not consistent from one sector to the other. Such an
interaction is of interest only if the order of hardiness
ranking of twig sections is changed from one sector to
another. Relative magnitudes of differences between twig
sections were not of interest. Closer examination showed
that the gradient was less pronounced in twigs from sector 1
than in those from sector 2. However, in all cases the
basal section of the twig was the least injured while the
tip section always exhibited the most injury.

The variance components have been listed in Table 3.
Values are expressed as the actual estimate of random vari-
ation due to the component and as a percent of the total
random variation. It can be seen that differences among
twigs and residual error accounted for a major portion of
the total random variation while trees and the tree by
sector interaction make up a relatively small proportion
of the total random variation. Both trees and the tree
by sector interaction showed increased magnitude in the

March 20 experiments (expts. 4, 5). This may indicate an

39

increase in variability within and among trees as they
begin to deacclimate in the spring.

Multiple browning determinations on the same twig
section contributed about 20 percent of the total random
variation in experiments 4 and S. This gives an average
repeatability of .79 (see Section I). This variability
arises because different browning ratings are assigned to
a single twig section when multiple observations are made.
These inconsistencies appear at random throughout the
entire experiment so blocking cannot be used to eliminate
this source of error. In many instances where the second
Observation was different, it was obvious that it was a
border-line case which could possibly fall into either of
two categories. If such difficult to rate twig sections
were discarded, a large part of the 20 percent of total
random variation due to poor repeatability would be elimi-
nated. This would decrease the determination variance
component and increase the repeatability of the rating
system.

The weight of a 7 cm portion of each twig section
in experiment 3 was used as an estimate of the twig section
size. Using this as the independent variable and tissue
browning as the dependent variable a regression analysis
was performed separately for each twig section within a
sector. Figure 1 shows the regression slopes and their
95 percent confidence belts for sector 1 twig section 1

and sector 2 twig section 3. It can be seen that for a

40

given change in wood weight the browning values in the tip
sections of sector 2 show a greater change than the base
sections of twigs in sector 1. Twig size is much less
critical if samples are remOved from sector 1 twig section
1. Other sector-twig section combinations are not shown

but have intermediate regression lepes.

Discussion

 

Previous research has indicated that wood hardiness
is a complex genetic trait (Dorsey and Bushnell, 1925;
Watkins and Spangelo, 1970). Its expression is controlled
by many physiological and environmental processes (Brierley
and Landon, 1954; Cooper, 1953; Craker et a1., 1969;
Ketchie, 1966; Levitt, 1972; Siminovitch and Briggs, 1949).
When breeding for a quantitatively inherited trait, it is
important to obtain an estimate of the trait's heritability.
Broad sense heritability specifies the portion of total
variation caused by genetic influences and that portion due
mainly to environmental influences. Environmental and
experimental variation may arise from a whole array of
causes which are non-genetic in nature. Some, such as
sampling technique, may be controlled by the experimenter
while others such as microclimate effects cannot. It was
desirable to reduce this non-genetic variation as much as
possible because environmentally caused mistakes in iden-
tification of hardy plants reduce heritability and the

amount of genetic progress which can be made.

41

A single peach genotype was used to eliminate any
genetic variation due to scion variety. All variation
obtained, therefore, should be non-genetic except that
caused by genetic variation amongst seedling rootstocks.
This variation was further broken down into variation which
can be minimized by the experimenter through proper experi—
mental design and sampling procedures and that which is
beyond control of the experimenter.

Since there are significant wood hardiness dif-
ferences between sectors of the tree and between twig
sections it is important to remove samples from equivalent
areas of all trees. If samples were removed at random
from any sector and section, the sector and twig section
hardiness differences are more likely to mask genetic
differences. The other favorable effect of sampling one
twig section in one sector of all trees is that the
residual error component, which accounts for about 50 per-
cent of the total random variation in the first three
experiments (Table 3), is largely eliminated. This is due
to the residual error being composed of two-way, three-way
and higher order interactions not partitioned elsewhere in
the analysis. Uniform sampling of a standardized sector
and twig section would eliminate these interactions.

The base sections of uniform sized twigs removed
from the upper southwest sector appear to be best suited
to use. Figure 1 showed that differences in twig size

were much less critical in sector l-section 1 than for

42

those twigs in sector 2-section 3. The base of the twigs
showed more uniformity in maturity and differentiation
of tissues making it easier to rate the amount of injury in
this part of the twigs. Finally, the standard deviation
of the base twig sections in the upper southwest sector
were generally smaller than the standard deviation of
other sector-twig section combinations.

Variance estimates obtained by summation of twig,
error, and determination components of Table 3 are shown
in Table 4. These variance estimates can be used to calcu-
late the sample size necessary to detect a given size
difference between means in a future experiment. The sample
size can be calculated using the equation (Sokal and

Rohlf, 1969):
J. z.

(a. I,

where: O is an estimate of the variability

o.|o >

, “'2‘

>

6 is the difference to be detected

t is students t value

a is the desired probability level

p is power of the test

n is the estimated sample size, and

v is the degree of freedom of the sample

In any program where seedlings are being screened

for wood hardiness there will probably be at least 120

43

observations so t has infinite degrees of freedom and no
iteration is required.

Table 4 shows some estimates of the sample size
needed to detect a given size difference (6) between
browning means. Using a reasonable sampling size of five
to twenty twigs per seedling, differences between .5 and
1.0 browning units should be detectable. It is not known
exactly what difference in stress tolerance one browning
unit equals but preliminary estimates indicate that one
browning unit may denote a 2 to 4°C difference in hardiness.
The scale is not linear throughout its range. These sample
size estimates are slightly larger than estimates developed
by Lapins (1962b) using apple seedlings but the two esti-
mates are not directly comparable.

The stated sampling method and sample size estimates
do not take into account the variation among trees. Since
a wood hardiness breeding program would involve many seed-
lings it would be economically unsound to replicate each
genotype. Since trees cannot be replicated over time or
space, microenvironmental differences unique to a given
spatiotemporal arrangement can never be exactly duplicated.
Such differences give rise to variation which will be
confounded with genotypic differences. Good experimental
design including blocking and treating all seedlings in as
similar a manner as possible will minimize this nuisance

variation.

44

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47

Table 4.--An estimate of the number of twigs (n) needed to
detect a desired difference (6) where a = .05,
p = .8, and t has infinite degrees of freedom.

 

A

 

Experiment O 6 n
.1 499
.6536 .5 +6
1 .14-404 4 .75 4
1.0 4'
.1 21461195
.8735 .5 ~8-7
2 .1415 .75 39 5.1
1.0 22
.1 245568
.134 .5 +1 .23
3 .3515 .75 5
1.0 —3
.1 546- 865
0390 .5 ea
4 4.09.: .75 to
1.0 -s 9
.1 6‘5 300
.7/37 .5 as
5 .5461 .75 11

1.0 ~4?

 

SUMMARY AND CONCLUSIONS

A single representative peach genotype was used to
study non-genetic wood hardiness variability and to evalu-
ate tissue browning, regrowth and electrolytic conductance.
The three methods were studied to evaluate their suitability
as viability tests for use in a breeding program in which
large amounts of plant material would be handled.

The electrolytic conductance tests involved many
measurements of water extracts from the frozen twigs. The
procedure was too involved to be well adapted to large
volumes of plant materials. Careful experimental technique
needed to obtain accurate and reliable conductance valves
could not be achieved because of the difficulty of handling
large numbers of samples. The use of blocking procedures
might have eliminated some of the variability associated
with experimental techniques but it probably would not
wholly eliminate the problem.

Tissue browning ratings on a 5 unit scale were made
by microsc0pic examination of thin cross sections of the
twigs. Regrowth ratings were based on a 5 unit scale depen-

dent on the amount of callus develOped at the cut ends.

48

49

Browning and regrowth were more highly correlated
(R2=.6l) than either browning and conductance (R2=-.15) or
regrowth and conductance (R2=.29). Problems of controlling
fungi and bacteria were associated with the regrowth test.
Also, callus formation appeared to occur more readily after
the cold requirement had been satisfied. Careful control
of standardized regrowth conditions is needed to produce
satisfactory results.

Tissue browning had a repeatability of .79. This
value is high enough to give reasonable precision if this
source of error is considered when establishing a sampling
procedure.

One alternative approach to determine the feasi-
bility of using these evaluation methods with large sample
sizes would be extrapolation from a small, more carefully
controlled experiment. A small timed experiment would
permit more careful laboratory technique. Knowing the time
needed to analyze a small number of samples, the time
needed for a large number of samples could be estimated.
Feasibility of the technique based on time and results
obtained could then be determined.

The approach used in this study may not have
allowed as careful control of experimental techniques as
alternative methods. It did, however, provide a practical
test of the techniques under conditions expected to be
encountered in a breeding project with limited available

manpower. This approach also provided an estimate of

50

problems and sources of error which might not arise and
could not be measured in a smaller more controllable experi-
ment.

Other sources of non-genetic hardiness variation
were also identified using an apprOpriate statistical
model. Using tissue browning to assess injury, significant
differences were found among trees in all five experiments.
This indicates the need for uniform field conditions.
Significant differences between sectors of the tree and
between sections of the twig were also found. In thirty-
three of the thirty-six trees examined the upper southwest
sector (sector 1) was hardier than the lower northeast
sector (sector 2). In all cases a pronounced injury
gradient was found to exist over the length of the twig.
The bases of the twigs were injured least while the tips
exhibited the most injury. This gradient was less pro-
nounced in twigs located in sector 1 as opposed to those in
sector 2.

Variance components were used to estimate the
amount of variability contributed by each of the sources.
Trees and the tree by sector interaction made up a rela-
tively small portion of the total variation. Twigs and
the residual error accounted for the largest portion.
Uniform sampling from a given sector and twig section
would eliminate much of the residual error by eliminating
many of the interactions composing it. The upper southwest

sector should be used because twig size was much less

51

critical in this section and more twigs were available for
sampling. The base twig sections were more mature and
differentiation of tissue made these sections easier to
observe and rate. Finally, the standard deviation of
sector 1-twig section 1 was generally smaller than other
sector-twig section combinations. Thus, it is recommended
that samples be removed from this area of the tree.
Variance estimates obtained by summation of twig,
error and determination components were used to calculate
sample sizes needed to detect differences of a desired
size. Using a reasonable sample size of between five and
twenty twigs, differences of .5 to 1.0 browning units could

be expected to be detected.

APPENDIX

52

.m unmEHummxm sH m :oHuomm mHsu.N Houomm pom
H coHuomm 0H3» H Houomm MOM uanOS @003 so mGHG3OHn mo :onmemmm

.H,musmHm

53

8:8. £8.35 tam; 803

000. 000. 000

 

 

_ 8:8» 23. I. .226...
n 355 33... I N 385 -u

 

 

SNINMOBB BOSSII

54

Figure 2. Photographs illustrating the 5 unit browning
scale. A through E represent a rating of 1
through 5 respectively.

 

55

 

 

56

 

57

 

58

Figure 3. Illustration of a tree divided into sectors.

59

 

UPPER SOUTHWEST SECTOR

.
"|"|

(Sector l )

 

 

 

( Sector 2 )

60

Figure 4. Illustration of a twig showing its division into
twig sections.

 

 

 

 

 

 

 

 

 

 

61

- 1? -
TWIG SECTION 3
kiTip twig section)

 

- I‘L-

 

 

 

 

TWIG SECTION 2
(Middle twig section)

 

TWIG SECTION I

(Bose twig section)

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