finh‘éwfiim
. . m. . _ Mum.

 

 

  

 

.
v?
i . x .. . . L\tefi).¢.k.r. a
.. . , . . flanksuilwwq
Li? H kg
. a... rwfifwwrrwfi.
1...... . 1.}. , .
I. r, “mm“ #3137“ S .
. . .. L," .
. - . L 5",”

_. . w... 1.1 :1
Rafi

#33,?
«w... rfiwmwumq
l h‘ - ‘ma'um.-R ”bi-”Q

.4 . {If}... .
.. Mummy? L .fihwfi
flaw-A “ . |% 31:43.:
2 , .
I .
12m. in.
r ‘IGI... .A . .n l. .

.1... .. :35!

if»: “NH—WA;
.51...

.. u: I.

navy.

4. i ,5 , .

l. 5:2,; ' n .

.332.‘l . .. . .
5L . . Ea: . . . . .
._ {a 2 a . .. _ . . .
253......" a! . V . .. . I» L14 1
diasm! k... . . , . , Lawn}.
.» ,. . . .

.ufi . _ .

.. 4.4%.... . .pne

. .15

 

an; Lh.

.1 vatfian

{viva-v5”. 3.35.7.5

.2 Riki}!!! 3...}.
WW. .. ..:ic£i..u 0d.

9. 1“. .'¥-1vh . t
«6... $3.... $5... ....
. g Emu}.
\‘E‘ ta

. 2Q. »

1 .

1

 

 

. ’11... s!
. (“ab .
. . tail" 1. ‘15.
II. :11. irwqaflhqfi
.Lfrfit?v?l‘
II\1”.IJUQW

EVA? 1

 

 

0 1.9.4. fin I‘V‘
, 13.... n3.» .1:
5.9.3501... .,t.«4a...‘t1u3n. ., F
1......) .i.. €53 .h
. , .119:

. Junk

 

. .kz’ilf-Vul l
. . ...Z . .L..:..uv.x.v.mm.
. . x ..5ivtg.m.l.u¢. {2:31 .

n (3 $1...
. .1: Ila-HY;

 

 

 

 

. .71.}! 11.91 I. 5‘: . ‘ . .5 . .
- . a..- ...- Ii‘kflhfipfl .. .2... 11111 .
. 1.....- . . n.......i..!...v<. r. ,

.. .. 5.3-. a

 

 

.rli... ..

     

   
     

 

 

 

LL: .
JP. .2433?

a .. 1: . . :v .

r I. . ......o. 131! an); .1 . é
. . .I.|7 lysnfirunimuu .mwr.."....:...1 l

. ,.I x . . u. u . a . A. . 1...! 1. 1:1 .
II. M L - ...... . J .
, ll . . - . . . - J21...“ .331: .,.v..a0v-..n A... . ..
| I I .l i i
I 0 r 3‘ lb

r ‘

 

 

Wit-753:8

Date

0-7 639

llllllllllllll|||H||llHlllllllllllllllllllllllllllllllllllll

3 1293 02080

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the

thesis entitled

Inheritance and Intercellular Fluid Protein of
a Foliar Disease Lesion Mimic Trait in
Sugarbeet (Beta vulgaris L.)

9/9/99

presented by

Goran Srnic

has been accepted towards fulfillment
of the requirements for

8.3. degree in W011 Sciences

by;

 

‘ I
0 major professor

MS U is an Affirmative Action/Equal Opportunity Institution

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11/00 mummy.“

INHERITANCE AND INTERCELLULAR FLUID PROTEIN OF A FOLIAR
DISEASE LESION MIMIC TRAIT IN SUGARBEET (BETA VULGARIS L.)

BY

Goran Srnic

A THESIS
Submitted to
Michigan State University
in partial fulfilment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Crop and Soil Sciences

1999

ABSTRACT

INHERITANCE AND INTERCELLULAR FLUID PROTEIN OF A FOLIAR
DISEASE LESION MIMIC TRAIT IN SUGARBEET (BETA VULGARIS L.)

BY

Goran Srnic

Disease lesion mimic (DLM) phenotypes in crop plants
are characterized by water-soaked spots and lesions on
foliage, but close association with forms of disease
resistance has been discovered in most such cases. A single
DLM sugarbeet from a breeding population was used as a
parent in determining the inheritance of the DLM phenotype,
using segregation patterns of F1, F2, F3, and BCl progenies
from a single DLM X wild type cross. This DLM trait is
proposed to be conditioned digenically, by homozygosity of a
recessive allele at one locus, and by the simultaneous
presence of at least one dominant allele at a second
independently segregating locus (i.e., dlmL/dlmerlm2/—z).
DLM occured on older leaves, but never on shoots and
plantlets grown on various media in vitro. When
intercellular fluid (ICF) proteins were Visualized, defense
proteins, including those with chitinase activity, appeared
several fold more abundant in leaves from DLM than from WT

plants.

Dedicated to my parents, Mira and Slavko, and to my
sisters, Katarina and Jelena, for all of their love and

support.

ACKNOWLEDGMENTS

I would like to thank to all people and schools who
influenced, encouraged, and supported me in my studies:

I had a great honor working last two and a half years
under my major adviser Dr. Joseph W. Saunders. I want to
thank him for all the time, energy as well as patience in
teaching me about plant research, science, and life in
general. It was from him teaching me plant breeding in such
a way that I developed a great attraction to the discipline.
I would like to thank deeply my committee members: Dr. John
M. Halloin for his valuable advice and discussions, and for
always being available for me, and Dr. Raymond Hammerschmidt
for his input, and guidance during my studies.

My thanks go to: Dr. Mitchell J. McGrath for his advice
with inheritance and protein studies and for his generous
technical support; Peter S. Hudy for laboratory support and
help with experiments; Gretchen Yurk and Lisa Snyder for
help with in vitro cloning; doctoral candidate Luis
Velasquez for teaching me spectrophotometric and
electrophoretic techniques; and doctoral candidate in
physics and my close friend Emil Bozin for his patience in
helping me with statistical problems.

I would like to thank Michigan State University where I

spent four years and was awarded BS and MS in Crop and Soil

Sciences, and "Agronomski Fakultet-éacak", Yugoslavia, where
I completed the biggest part of my undergraduate studies. I
also would like to thank: "Gimnazija Filip Filipovic"—Cacak,
Yugoslavia, where I finished the final two years of high
school; Srednjoskolski Centar "Niko Andjus"—Budva where I
completed the first two years of high school; and elementary
school "Stjepan Mitrov Ljubiéa"—Budva, Yugoslavia, where I
completed eight years of elementary education.

Thanks to all of my friends who supported and
encouraged me during my studies: families Bakic, Bozin-
Tomic, Drndarevic, Popovic, Rankovic, as well as Susan
Redwine, Dr. Djordje Tomasevic, Lazar Nesié, Slobodan Eric,
Aleksandar Krstic, Milan Pajovic, and Zoran Terzic. Thank
you Wendy A. Pline for your love and unselfish support
through all of these years.

I would like to thank all of my cousins in families
Radmilac and Srnic for all their love, care and help during
all of my life. Thank you my dear uncle Branko Radmilac for
being like a father to me and for developing love for
village and agronomy in my childhood. Thanks to my family
Mira, Slavko, Jelena and Katarina, to whom this thesis is
dedicated, for being always there for me.

At the end I beg for forgiveness to many of those whom

I did not mention and who deserved it.

TABLE OF CONTENTS

Page
LIST OF TABLES .......................................... viii
LIST OF FIGURES ............................................ x
CHAPTER I
LITERATURE REVIEW
Description of the Species ................................. 2
Disease Lesion Mimics ...................................... 6

Inheritance of Foliar Disease Lesion Mimic (DLM) Traits....8
Pleiotropy Between DLM and Disease Resistance in

Plants ............................................... 10
Chemical and Molecular Markers of DLM ..................... 12
Objectives, Approaches and Methods........ ................ 13

CHAPTER II

INHERITANCE OF A FOLIAR DISEASE LESION MIMIC (DLM) TRAIT IN
SUGARBEET (BETA VULGARIS L.)

INTRODUCTION .............................................. 19
MATERIALS AND METHODS ..................................... 24
Plant Material ....................................... 24
Propagation of Plant Material in vitro ............... 30
RESULTS ................................................... 32
DLM Occurrence in Puqm: E3, E5, BC1, and F3
Families ........................................... 32
DISCUSSION ................................................ 36

w

CHAPTER III

ICF PROTEIN OF DLM IN SUGARBEETS

INTRODUCTION .............................................. 77
MATERIALS AND METHODS ..................................... 81
Plant Material ....................................... 8l
Intercellular Fluid (ICF) Isolation .................. 82
Measurements of Total Protein in ICF Samples ......... 82
SDS Polyacrylamide Gel Electrophoresis ............... 83
Protein and Chitinase Activity Staining .............. 85
RESULTS ................................................... 87
SDS PAGE of ICF Proteins ............................. 87
Spectrophotometric Determination of ICF Protein
Concentrations .................................. 91
DISCUSSION ................................................ 94
BIBLIOGRAPHY ............................................. 108

Wi

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

Page
Some characteristic single—gene traits in
sugarbeet (Beta vulgaris L.): characters and
gene symbols (modified from Smith, 1980) ....... 15
Foliar DLM mutants ............................. 17
F1 individuals and respective F2, F3, and BCl
progenies involved in segregation analysis ..... 48
Genotypes used as controls for phenotypic
categories for segregation analysis of
progenies from 106-C1 X REL-2 .................. 49
Chi-square tests for 3:1 (12:4) (WT:DLM)
segregation ratio in F2 families ............ 50

Chi-square tests for 9:7 (WT:DLM) segregation
ratio in F2 families ........................ 51

Chi-square tests for 10:6 (WT:DLM)
segregation ratio in F2 families ............ 52

.d Chi-square tests for 13:3 (WT:DLM)

segregation ratio in F2 families ............ 53

Chi-square tests for 15:1 (WT:DLM)
segregation ratio in F2 families ............ 54

Chi-square tests for 1:1 (WT:DLM) segregation
ratio in BC1 families ....................... 55

.g Chi-square tests for 3:1 (WT:DLM) segregation

ratio in BC1 families ....................... 56

Chi-square test for 1:3 (WT:DLM) segregation
ratio in BCl families ....................... 57

Chi-square tests for 5:3 (WT:DLM) segregation
ratio in BC1 families ....................... 58

Chi~square tests for 1:0 (WT:DLM) segregation
ratio in F2 families ........................ 59

WH

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

.k Chi-square tests for 3:1 (WT:DLM) segregation

ratio in F3 families ........................ 60

.1 Chi—square tests for 0:1 (WT:DLM) segregation

ratio in F3 families ........................ 61

.m Chi-square tests for 13:3 (WT:DLM)

segregation ratio in F3 families ............ 62

.n Chi-square tests for 1:3 (WT:DLM) segregation

ratio in F3 families ........................ 63

.a Summary of chi—square analyses for fit of F2

families to possible digenic segregation
ratios ....................................... 64

.b Summary of chi-square analyses for fit of BC1

families to possible digenic segregation
ratios ....................................... 65

.c Summary of chi-square analyses for fit of F3

families to possible digenic segregation
ratios ....................................... 66

.a Proposed lineage of'EH, E2, and BC1

individuals based on segregation ratios in
F2 and BC1 families (Tables 2.3.a-i) ......... 67

.b Proposed lineage of E3 families, based on

their segregation ratios (Tables 2.3.j-n)....67

Spectrophotometric determination of ICF protein
concentrations, experiment #1: Protein
concentrations in foliar ICF of WT and DLM
individuals .................................... 101

Spectrophotometric determination of ICF protein
concentrations, experiment #2: Protein
concentrations in foliar ICF of WT and DLM
individuals for each of two flushes from the

same leaves .................................... 102

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

(A)

LIST OF FIGURES

Page
DLM phenotype of an F2 plant in the form
of water-soaked spots on foliage ............ 68
DLM phenotype of an F2 plant in the form
of necrotic lesions on foliage .............. 68

Monogenic recessive segregation ratios in F1,
F2, F2, BC1, and in 81 of P2 if no phenotypic
segregation for DLM is observed in F1.

Underlined are proposed DLM genotypes ......... 69
.a All possible digenic segregation patterns
in F2 generation, if 106-C1 is aa -— genotype,
and REL-2 is A—--. Underlined are proposed
DLM genotypes ............................... 70

Expected digenic segregation ratios in F2
generation, if 106-C1 is aaBb genotype,

REL-2 is AABb, and DLM does not segregate

in F2 generation. Underlined are proposed

DLM genotypes ............................... 71

Expected digenic segregation ratios in BCl
generation, if 106—C1 is aaB- genotype,

REL-2 is AABb, and DLM does not segregate in
the F2 generation. Underlined are proposed

DLM genotypes ............................... 72

Expected digenic segregation patterns in F3
generation, if 106-C1 is aaBb genotype,

REL-2 is AABb, and DLM does not segregate in
F2 generation. Underlined are proposed DLM
genotypes ................................. 73

DLM segregation percentage in F2 vs. BCL ...... 74

Frequency distribution of DLM segregation

in F2 families. Three groupings (around 25%,

18%, and 0%) evident here are characteristic

of digenic segregation from.F2 genotypes
(Dlml/dlm1:Dlm2/Dlm2),(Dlml/dlm1:Dlm2/dlm2), and
(DlmI/dlm1:dlm2/dlm2), respectively ........... 75

Figure

Figure

Figure

Figure

Figure

3.

.1

2

.4

.5

Foliar ICF protein SDS PAGE separation

profile following Coomassie Blue staining.

ICF taken from WT vs. DLM individuals, with
equal ICF volumes applied to gel ............. 103

Foliar ICF protein, SDS PAGE separation

profiles following Coomassie Blue staining.

ICF taken from WT vs. DLM individuals, with
equal protein amounts applied to gel ......... 104

Foliar ICF protein, SDS PAGE separation

profiles following Coomassie Blue staining.
Consecutive ICF isolates taken from WT vs.

DLM individuals, with equal ICF volumes

applied to gel ............................... 105

Foliar ICF protein, SDS PAGE separation

profiles following a) Coomassie Blue staining,
and b) chitinase activity staining.

Consecutive ICF isolates taken from WT vs.

DLM individuals, with equal ICF volumes

applied to gels .............................. 106

Foliar ICF protein, SDS PAGE separation

profiles following a) Coomassie Blue staining,
and b) chitinase activity staining.

ICF taken from WT vs. DLM individuals, with
equal protein amounts applied to gels ........ 107

fl

CHAPTER ONE

LITERATURE REVIEW

Description of the Species

The species Beta vulgaris belongs to the family
Chenopodiaceae. The species is highly variable and consists
of four groups of agricultural significance: leaf beet,
garden beet, fodder beet and sugarbeet (Winner, 1993). The
species was named for the first time in 1753 by Linnaeus,
who classified it into three "varieties": var. perennis
(wild type), var. rubra (garden beet), and var. cicla
(foliage beet) (Winner, 1993). Wild beet, the ancestor of
sugarbeet, is considered to have originated from
Mediterranean coasts and Western Asia, but extended to India
and Scandinavia.

Sugarbeet is a unique widely grown product of breeding
within the species Beta vulgaris. Production of this crop
has spread extensively to all populated continents except
Australia. Sugarbeet and sugar cane are the only two crops
that are economically important sources of sucrose. The
world production of sugar has at least doubled during the
last thirty years (Winner, 1993). Despite production of
alternative sweeteners, whether artificial or
monosaccharide, demand for sucrose continually increases.

Sugar beet is a dicotyledonous plant, considered to
have a biennial growth habit. The mature beet consists of

crown, root neck and tap root. Being a crop of temperate

regions, it is mainly grown between 30° and 60°N. If left in
the ground, it reaches flowering in the second year. A
process of vernalization, artificially achieved by 10-16
weeks at 4-5 °C, is needed for flowering with the majority
of sugarbeet germplasm. Some of the wild Mediterranean
forms, such as B. maritima, B. macrocarpa, and B.
atriplicifolia, are annuals, and do not require the
vernalization process (Bosemark, 1993). Hayward (1938)
described variability of biennial habit in B. maritima under
different environments, so that any individual plant could
behave as annual, biennial or perennial, depending on
environmental conditions. Saunders (1982) reported on
flowering in several sugarbeet genotypes being induced by
continuous incandescent light and moderate temperatures,
without vernalization.

The sugarbeet tap root produced in the first year is
succulent, and serves as a fleshy food reserve. The crown
stem is short with a rosette of leaves. In the second year
or following vernalization, the plant first forms a second
rosette of leaves, then a bushy shoot, and finally flowering
branches. Food reserves from the tap root are used during
this process (Hayward, 1938).

All groups of B. vulgaris are diploid with chromosome
number 2n=2X=18. Haploid beets can be created by ovule

culture (Hosemans et al., 1983). Polyploid beets can be

created from: unreduced gametes, colchicine treatment, or
from crosses between polyploid and diploid beets (Bosemark,
1993). Identification and use of the first (auto)tetraploid
beets (2n=4X=36) was done in the 1930's (Bosemark, 1993).
The first triploid beets (3X=36) were obtained by crossing
diploid with tetraploid plants. Diploid and triploid hybrid
varieties are currently marketed. Some parental combinations
are more productive as diploids, while others are more
productive as triploids. Principles of Mendelian inheritance
apply in tetraploid and diploid beets. In tetraploids, each
locus is present four times in somatic nuclei, and
inheritance is more complicated than in diploids, which
makes breeding and genetic manipulation in general difficult
at the tetraploid level.

Sugarbeet is considered to be mainly self-sterile and
sets few to no seeds under strict isolation. Sugarbeet is
very sensitive to inbreeding depression. Germplasm that has
undergone only several generations of exclusive self
pollination often yields less than 50% of comparable non-
inbred families. This is consistent with cross-pollination
being the primary mode of sexual reproduction within the
species. In contrast to this character, sugarbeet plants
that carry the dominant Sf allele for self-fertility are
almost obligately self-fertile. The trait controlled by the

Sf allele is used to develop inbred lines. Pollination

control for hybrid seed production in sugarbeets depends on
cytoplasmic-nuclear male sterility, and requires development
of monogerm parental lines, usually inbreds, that are O—type
maintainer (N)xxzz genotypes and their male sterile
"equivalents" (S)xxzz genotypes, which are created by up to
8 repeated backcrosses of the developing cytoplasmic-nuclear
male sterile lines to their respective maintainer lines.
This way each male sterile line has its near—isogenic O—type
counterpart or "equivalent," which is used to increase seed
of the sterile line when needed. The most common system in
creating diploid commercial hybrids is to use the F2 hybrid
between an inbred male sterile (MS) line and an unrelated,
but good specific combining inbred O—type, as female parent,
and an open pollinated multigerm line or population as the
male parent (Bosemark, 1993).

A recent genetic map of sugarbeet contains 177
identified markers, including 2 morphological traits, 7
isozymes, and 168 RFLP markers (Pillen et al., 1993). This
did not include the locus identified through the process of
somaclonal cell selection for sulfonylurea herbicide
resistance (Sur) (Saunders et al., 1992). Two further
alleles (Sir-13 and 93R30B) at the locus were identified by
Wright et al. (1998), following isolation of two forms of
imidazolinone herbicide resistance by somatic cell

selection. All three mutants are allelic. The use of

molecular linkage maps which identify agronomically
important traits would provide important information of
practical significance. Known markers closely linked to
important loci with desirable variation can help to reduce
the number of generations needed to introduce a new gene,
when using traditional breeding procedure such as
backcrossing. Sugarbeet markers have been organized into 9
linkage groups, with an average marker spacing of 6 cM
(Pillen et al., 1993). Each of those linkage groups
contained between 13 and 24 markers, with the length of the
groups ranging from 80.7 cM (Group VIII) to 167.4 cM (Group

VII) (Pillen et al., 1993).

Disease Lesion Mimics

Foliar disease lesion mimicry (DLM) is a heritable
spontaneously occurring phenotype in many species which is
expressed as the production of water-soaked and/or necrotic
areas on leaves. Plants expressing the DLM trait often are
less vigorous and have reduced leaf area, with less
photosynthetically active foliage. Many require special care
in order to survive, which adds to research efforts.

Localized foliar cell death and lesion formation, as a
general phenomenon in plants, can be caused by physiological
stress, wounding, and/or disease. In addition to these

factors, DLM phenotypes involve formation of lesions even in

the absence of stress, wounding or disease (Walbot et al.,
1983). The report of Emerson (1923) on "blotch leaf" in corn
was one of the first reports on DLM. There are a number of
reports from plant species (Table 1.2), of genetic variants
that form areas of water—soaked and lesion tissues on
foliage in the absence of pathogens. In sugarbeet, Ulrich
(1961) reported a "red-spot disease of genetic origin," but
the study did not focus on the phenomenon nor demonstrate a
genetic origin, and the "red—spot" plants were discontinued
from the study.

In some cases, the water-soaked and/or lesion areas
resemble those resulting from the hypersensitive response
(HR). Others resemble specific disease symptoms caused by
pathogens (Dangl et al., 1996). In Arabidopsis, plants with
the DLM phenotype were named either as "accelerated cell
death” (acd) mutants (Greenberg et al., 1993) or "lesion
simulating disease" (lsd) mutants (Dietrich et al., 1994).
Both types of mutants involved similar spontaneous foliar
phenotypes, but were non—allelic to each other. In
transgenic tobacco plants expressing either vacuolar or
apoplastic yeast invertase, DLM was expressed as
spontaneously formed HR-like lesions (Herbers et al., 1996).
Kosslak et al. (1996, 1997) reported 3 allelic root disease
mimic mutants in soybean, that expressed root lesions

whereas the foliage appeared normal.

Inheritance of Foliar Disease Lesion Mimic (DEM) Traits

In corn, barley, soybean, Arabidopsis, tobacco,
tomato and rice a large number of loci have been identified
to be involved in DLM phenotypes (Walbot et al., 1983;
Wolter et al., 1993.; Dietrich et al., 1994). Hoisington et
al. (1982) projected 60 to 70 independently segregating loci
responsible for DLM in corn alone, “based on the frequency
of mutant recovery thus far.” Overall such a large number of
loci could involve a wide range of DLM inheritance patterns
both between and within species.

In corn, DLM inheritance has been viewed as either
dominant or recessive, depending on the locus. In at least
one case (Ullstrup et al., 1967), the trait in corn showed a
monogenic recessive mode of inheritance, with segregation in
3:1 (wild type:mutant) ratios in F2 and F2 single-ear
progenies. DLM there was not expressed in F2, but was
expressed in ~25% of F2progenies. Neuffer et al. (1975)
created a corn population by pollinating a vigorous genotype
with pollen treated with ethyl methanesulfonate (EMS). They
identified two disease lesion leaf types (Lesion—1 and
Lesion-2) in the NH population. Both mutant types were
inherited as dominant, demonstrated by reciprocal crosses
between DLM and wild type plants. Lesion—1 (Les—l) and

Lesion-2 (Les—2) mutants clearly differed in lesion

characteristics, such as size and frequency. Crosses between
double heterozygote (Lesion-1 X Lesion-2) plants as well as
backcrosses of the double mutant to the wild type parents
were made to test for allelism. In the resulting generation,
all phenotypes, including Lesion-1, Lesion-2, Lesion-
1Lesion-2 and wild type were obtained, indicating that the
two lesion mutants were non-allelic.

Dietrich et al. (1994) described six Arabidopsis
mutants (lsdl, lsd2, lsd3, lsd4, lsd5, and T5121), defining
four loci, that spontaneously formed necrotic lesions on
leaves. Mutants lsdl, lsd3 and lsd5 were each inherited as
monogenic recessive, and lsd2 and lsd4 as monogenic
dominant. In all cases, segregation patterns were consistent
with traits conditioned by single loci, in the homogenous
Arabidopsis genetic background of ecotype Columbia.

Wolter et al. (1993) identified a locus with multiple
recessive alleles (mlol, mlo3 and mloS) responsible for a
DLM trait in barley after mutagen treatment of barley
cultivars that previously had carried a heterozygous or
homozygous dominant wild type M10 allele. Near-isogenic
lines that carried dominant M10 as well as recessive mlo3,
mlo7, mlo9, mlolO, mlol3, mlol7, and m1026 alleles did not
express the DLM phenotype. All 10 mlo recessive alleles were

involved in gene to gene resistance to Erysiphe graminis,

and thus three of them were expressing pleiotropy for DLM in
barley.

Another rare example of multiple alleles being
responsible for DLM expression in plants was reported in
corn by Hu et al. (1996). Four mutant or recombinant alleles
(Rpl—D21, Rpl—MD19, Rpl-KrlN, and rpl-NC3) at the Rpl locus
in corn control a DLM phenotype. The first three alleles
segregated codominantly, so that the DLM phenotype was
strongly expressed in heterozygous combinations, and the
homozygous condition was lethal. Allele rpl-NC3 segregated
as recessive (Hu et al., 1996). This appears to be the only
report of codominant inheritance of alleles at a DLM locus
in plants.

Langford (1948) reported more complex inheritance of a
DLM trait in tomato, one that included at least 2 major
independently segregating loci, Cfbland Ne, as well as an
undetermined number of loci that prevented and/or suppressed
the development of that DLM phenotype. The DLM trait was
inherited digenically, with heterozygous or homozygous
dominant alleles at one locus, and homozygous recessive

allele or alleles at other locus (i.e., Cflfl—;nene).

Pleiotropy Between DLM and Disease Resistance in Plants

Some DLM traits, despite foliar damage, may not be

entirely undesirable. Association between DLM and greater

lO

systemic acquired resistance (SAR) in Arabidopsis has been
reported (Dietrich et al., 1994; Greenberg et al., 1994). In
the Dietrich et al. (1994) study, four out of 6 lsd mutants
expressed greater systemically acquired—like disease
resistance to various fungal and bacterial pathogen isolates
than the WT plants. Two acdl mutants were inherited as
monogenic recessive, and both produced HR—like lesions
(Greenberg et al., 1993). These mutants were more
susceptible to opportunistic infections than were wild type
plants. Some of the avirulent races of Pseudomonas syringae
pv. maculicola promoted rapid spread of lesions on foliage
of acdl mutants.

Langford (1948) reported that a foliar DLM as a form of
"a spontaneous and destructive necrosis” in tomato could be
seen only in lines that carried the dominant Cflfl allele for
resistance to tomato leaf mold, caused by races 1-4 of
Cladosporium fulvum Cooke. Only resistant plants segregated
for the spontaneous necrosis phenotype. Presence of the Cflfl
allele appeared to be necessary for both disease resistance
and spontaneous necrosis formation.

In barley, the Nflo locus with recessive mlo resistance
alleles controls race non-specific resistance to powdery
mildew caused by Erysiphe graminis. Near-isogenic barley
lines carrying the mlol, mlo3 or mloS alleles had a lesion

mimic phenotype and underwent spontaneous foliar cell wall

11

appositions (Wolter et al., 1993). All other recessive mlo
alleles studied were involved in resistance to E. graminis,
but without expressing DLM.

Some degree of pleiotropy between spontaneous lesion
phenotypes and altered levels of resistance has been
reported in Arabidopsis (Dietrich et al., 1994), and, in
other species, different levels of association between
spontaneously formed lesions and greater or, rarely, lesser
levels of resistance have been established. Yet, it is very
important to thoroughly dissect these phenomena, and to be
able to separate genetic factors for water-soaked and lesion
appearances on the foliage from any individual genetic
factors for disease resistance that could be used in

breeding more resistant crops.

Chemical and.Mb1ecu1ar Markers of DLM

In five out of six Arabidopsis lsd mutants, lesion
formation and expression of four markers of disease
resistance response (Dietrich et al., 1994) such as
accumulation of autofluorescent materials, presence of
callose at the sites of necrosis, irreversible membrane
damage, and expression of SAR genes, happened at the same

time as lesion formation. Autofluorescence and callose

12

deposition were examined after serial incubations and
staining of leaves, by observing them under ultraviolet
light and differential interference contrast microscopy.
Irreversible membrane damage as a sign of cell death was
measured by trypan blue uptake. Expression of SAR genes was
visualized by extracting RNA from leaf material,
electrophoresis of the RNA through agarose—formaldehyde
gels, and transfer of the RNA to nylon membranes where it
was probed for SAR genes PR—l, PR—2, and PR-5 using cDNAs

(Dietrich et al., 1994).

Objectives, Approaches and.Methods

Objective 1 of this study with sugarbeet was to

 

determine the mode of inheritance of a DLM from one source.
The approach for this objective was to investigate the
transmission genetics characteristics of the DLM trait. The
method used was segregation analysis of F2, F2, F3, and BCl
progenies from a DLM X wild type (WT) cross.

Objective 2 was to investigate some physiological

 

processes that might be characteristic of DLM expression.
The approach to this objective was to study some defense
protein characteristics of the DLM phenomenon. Methods used
to achieve this objective relied on the comparison of
protein patterns and protein concentrations using sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS

13

PAGE) to describe differences between wild type and DLM

phenotypes.

14

Table 1.1 Some characteristic single-gene traits in
sugarbeet (Beta vulgaris L.): characters and gene
symbols (modified from Smith, 1980).

 

 

Genetic Character
_3gmbols
Y,Y‘Ly Yellow pigment

R,Rt,Rp,Rh, r Hypocotyl color

Cl,cl Colored leaf

Tr,tr Trout or spotted leaf

Cv,cv Colored vein

B,b Annual growth habit

V2,V3 Variegated foliage

C,c Partially dominant curly top resistance

cr,Cr Crinkled foliage, reduced plant size

p,P Non-production of color

m,M,MBr,M1,M2 Monogerm seed

lb Late bolting

C2 Partial curly top resistance

a1,A1 Mendelian male sterility

luLLu2 Lutescens, green cotyledons followed by death

ru,Ru Russet root

ch2,Ch2 Chlorina cotyledons and all leaves are yellow
green

cfln,Ch1 Chlorina, more reduction in root yield than ch2

viLVd4 Virescens, pronounced delay in chlorophyll

production in first true leaves, white to
light green leaves

X,x Restores male fertility in sterile cytoplasm

Z,z Produces partial fertility in sterile
cytoplasm

Sh,sh Enhances pollen production

y1,Yl Yellow leaf

bl,Bl Black root

d,D Dwarf mature plants. Seedlings have thick
hypocotyls

pl,Pl Plantain leaf; semi-parallel venation

33,31' Self fertility

Sa,Sb Self sterility

Sapfiwék,sd Self-incompatibility, 4 gametophytic loci

w,W Albino lethal

V2,V2 Variegated cotyledons

V3,V3 Variegated foliage and root fleshfl

 

0

table continues on the next page

15

Continued from the previous page:

 

 

Sur, Sirl3,

93RBB, sur

Table 1.1 Some characteristic single-gene traits in
sugarbeet (Beta vulgaris L.): characters and gene
symbols (modified from Smith, 1980).
Genetic Character
_ggmbcls
f,F Flaccid leaf
n,N Nana plants, leaves thick and leathery
and Miniature, lethal, conflicts with monogerm
re,Re New leaves of mature plants reduced in size
and lanceolate shaped
W2,W1 Albino seedlings
Au,au Golden-yellow cotyledons, semilethal
heterozygote, lethal homozygote
lu,Lu Lutescens, leaves produced after second pair

become progressively yellow, some survival
Virescens, golden-yellow seedlings, varying
degrees of delay of chlorophyll production
Sulfonylurea, imidazolinone, and combined
sulfonylurea and imidazolinone herbicide
resistance.

16

Table 2.1 Foliar DLM mutants.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Disease
Species IDLMzmutants Inheritance Resistance
b1 blotched leaf in CORN Monogenic Not available
(Emerson 1923.) recessive
b1 blotched leaf in CORN Monogenic Not available
(Ullstrup et al., 1967.) recessive
ns necrotic leaf spot in Monogenic Not available
CORN recessive
(Hornbrook et al., 1970.)
lesion-1,2 in CORN Monogenic Not available
(Neuffer et al., 1975.) dominant,
non-allelic
rpl-related lesion mimic Monogenic Increase in gene
mutants in CORN dominant and to gene to
(Hu et al., 1996.) recessive, .Puccinia sorghi
multiple
alleles
lls lethal leaf spot in Monogenic Increase in SAR—
CORN recessive like
(Simmons et al., 1998.)
no, Cf spontaneous necrosis Digenic: Increase in gene
in TOMATO recessive to gene to
(Langford 1948.) and dominant Cladosporium
alleles fulvum
s1 Segiguchi lesion in RICE Monogenic Decreased
(Marchetti et al., 1983.) recessive
.mlo in BARLEY Monogenic Increase in gene
(Wolter et al., 1993.) recessive, to gene to
multiple Erysiphe
alleles graminis
lsd lesion simulating Monogenic Increase in SAR-
disease in ARABIDOPSIS dominant and like
(Dietrich et al., 1994.) recessive
acd accelerated cell death Monogenic Increase, and
in ARABIDOPSIS dominant and decrease, in
(Greenberg et al., 1993.) recessive SAR-like
cwlnv, vaclmv in transgenic Not Increase in SAR-
TOBACCO available like

(Herbers et al., 1996.)

 

 

 

 

17

1px;: ~—-:: +1

CHAPTER TWO

INEERITANCE OF A FOLIAR DISEASE LESION MIMIC (DLM) TRAIT IN

SUGARBEET (BETA VULGARIS L.)

18

INTRODUCTION

The discoveries in plants of heritable spontaneous
lesion formation, generally referred to as: "disease lesion
mutants" (Walbot et al., 1983; Kosslak et al., 1996),
"lesion mimic mutants" (Herbers et al., 1996), and "cell
death mutants" (Dangl, et al., 1996), have broadened an
earlier definition that: "the formation of local lesions on
plant leaves can result from a variety of factors, including
physiological stress, wounding, and pathogen infection" (Hu
et al., 1996). Programmed and localized cell death can be a
form of the hypersensitive response (HR), usually at a
pathogen infection site. Easily seen are water-soaked leaf
areas of various sizes, depending on the cause and plant
genotype, that progressively become necrotic (Agrios, 1997).
HR is an important feature that is closely associated with
systemic acquired resistance (SAR) to disease. Both HR and
SAR usually are initiated by either avirulent or virulent
pathogens during plant-microbe interactions (Greenberg et
al., 1993).

Variants and mutants with characteristic phenotypes of
multiple foliar lesions resembling damaged areas produced in
HR, and possessing constitutively altered levels of disease

resistance have been reported in quite a few plant species

19

(reviewed by Dangl et al., 1996). Some of these phenotypes
were discovered as spontaneous variants, as in corn
(Emerson, 1923), tomato (Langford, 1948), sugarbeet (Ulrich,
1961), rice (Marchetti et al., 1983), and barley (Wolter et
al., 1993). Others were induced by a) mutagenic irradiation
or chemicals as in corn (Hornbrook et al., 1969; Neuffer et
al., 1975), Arabidopsis (Greenberg et al., 1993; and
Dietrich et al., 1994), or b) genetic transformation as in
tobacco with transgenes for yeast-derived invertase (Herbers
et al., 1996). The Arabidopsis mutants have been used for
study of plant responses to pathogen attack, because of the
constitutive expression of some of the disease resistance
markers such as a) cell death and oxidative burst, b)
deposition of callose and lignin, c) accumulation of
phytoalexins, d) induction and accumulation of SAR gene
transcripts, and e) accumulation of PR proteins (Dangl et
al., 1996; Ryals et al., 1996).

As a general rule, expression of HR is induced during
the onset of SAR. A number of exceptions have been reported.
Yu et al. (1998) reported a dndl mutant in Arabidopsis that
underwent SAR after induction with Pseudomonas syringae, but
without expressing HR. A series of acd and lsd mutants of
Arabidopsis expressed both HR and SAR constitutively,
without being challenged by biotic or abiotic inducers

(Dietrich et al., 1994; Weymann et al., 1995; Greenberg et

20

al., 1994). Moreover, the two acdl mutants in Arabidopsis
that spontaneously formed foliar lesions, were more
susceptible to opportunistic infections, meaning that SAR in
both acdl mutants was less expressed than in wild type
plants (Greenberg et al., 1994). Numerous allelic and non-
allelic lesion mimic mutants have been identified in several
plant species. (see Introduction). In at least 7 out of 10
different DLM sources in 7 plant species, such as tomato
(Langford, 1948), corn (Neuffer et al., 1975; Hu et al.,
1996; Simmons et al., 1998), rice (Marchetti et al., 1983),
barley (Wolter et al., 1993), Arabidopsis (Greenberg et al.,
1993; Dietrich et al., 1994), and soybean (Kosslak et al.,
1996) in which the association with disease resistance was
investigated, were reported to possess greater expression of
SAR, and/or greater gene-to-gene specific resistance,
compared to wild types.

Similarities and differences of these mutants with
genetically controlled expression of the lesion phenotype
have not been compared elswhere in the literature. In
Arabidopsis some of the acd mutants have shown greater
similarity in disease response for example, to lsd than to
other ACD mutants. In corn, lesion mimic mutants have been
classified based on the method and date of isolation, rather
than based on physiology and genetics. Altogether, these

mutants can be considered as Disease Lesion Mimic mutants,

21

because all reports have agreed that the lesions and water—
soaked spots on foliage resembled those of pathogen-attacked
plants. The exceptions are 3 allelic necrotic root mutants
in soybean that show disease—like symptoms only in root
tissue (Kosslak et al., 1996, 1997). For sugarbeet, the term
"Disease Lesion Mimic (DLM)" is considered the proper one,
as well. The foliage of DLM sugarbeet plants clearly
expressed disease symptoms, similar to pathogen-induced
ones, and the plants show less vigor compared with WT
relatives. The similarity in appearance of the water-soaked
spots and lesions of sugarbeet DLM plants to the
descriptions of other lesion—mimic mutants is large.
Additional characteristics of DLM mutants such as a)
Mendelian segregation of one to few loci conditioning the
DLM phenotype, and b) enhanced levels of accumulation of at
least some of the defense chemicals, and perhaps altered
levels of disease resistance, could be used as
distinguishing criteria for classification of this sugarbeet
foliar phenotype in the DLM category.

Such spontaneous lesion formation is genetically
controlled, and does not require direct plant pathogen
interactions (reviewed by Dangl et al., 1996).
Identification, isolation, and manipulation of genes
involved in spontaneous lesion formation has prospects for

improving not only intensity but also speed of disease

22

resistance responses. In most cases, spontaneous lesion
formation was controlled by as few as one or two loci. Study
of inheritance patterns is a way of identifying the minimum
number of loci involved, and of indicating efficient methods
for introduction of critical alleles into other genetic

backgrounds of interest.

23

MATERIALS AND METHODS

Plant Meterial

This research was based primarily on a cross between
the original DLM clone (106-C1) and the wild—type (WT) clone
REL—2, with segregation analysis of progeny generations. All
F2, F2, F3, and BCl progenies referred to were generated from
the foundational cross (106-C1 X REL-2), unless otherwise
indicated. Vegetatively propagated ramets (copies) of 106-C1
and REL-2, and 81 progeny of REL—2 were used as controls in
scoring progeny for the DLM phenotype. An advantage of using
a single DLM source in initial determination of the
inheritance was the avoidance of complications that may
arise from involvement of multiple loci or DLM systems that
independently of each other could condition the DLM
phenotype.

The DLM parent (P1) 106—C1, considered from past
experience to be pseudo self-fertile, trait not associated
with DLM, and thus capable of setting a small number of
seeds if self-pollinated under strict isolation, was the
seed parent in the foundational cross. This parent was
selected for root smoothness from progeny of the full sib
cross of two plants in the B3—7 family in a smooth root

breeding program in 1991. Clone 106-C1 is biennial and

24

diploid. Genotype 106-Cl, when pollinated in a pair—cross by
REL-2, a plentiful pollen producer, very likely sets no
self-seed. All Flplants from this cross were classified as
WT and there was no segregation for DLM as would be likely
in cases of self-fertilization, presuming some form of
recessive inheritance of DLM. Lack of any progeny derived
from self-fertilization in such cases could be expected due
to broad sense gametophytic selection in which abundant REL-
2 pollen completely outcompetes the pollen of 106-C1 for
access to eggs of 106-C1.

REL-2, originally known as LTR—41, is a WT clone, used
as the male (pollinator) (P2) in the foundational cross with
DLM parent 106-C1. The clone was developed and released to
the public by Saunders (1998). This clone is unusual because
of its intense shoot regeneration and somatic embryogenesis
from callus tissue. REL—2 was created as a Flindividual
from a cross between an EL 45/2 individual, which was an
excellent shoot regeneration monogerm genotype, and REL—1,
which is an internationally used sugarbeet genetic
background (Saunders 1998). REL-2 is annual, diploid, self—
fertile with N cytoplasm. REL-2 is heterozygous for the
annual/biennial, multigerm/monogerm, and red/green hypocotyl
traits, thus being genotypically Bb,Mm,Rr. REL—2 is itself a

candidate for recognition as an international standard, and

25

was chosen because the annualism it carried would accelerate
genetic studies with DLM.

All progenies were grown in the greenhouse under
plentiful artificial lighting: high intensity sodium,
incandescent and/or fluorescent lights. The seeds were
planted and grown on “Bacto” professional planting mix
(Michigan Peat Co., Houston TX). Watering was daily and
nutrients were provided weekly with Peters fertilizer
formulated as 20:20:20 N:P:K with micronutrients. Under
greenhouse conditions, seeds germinated 5 to 10 days after
planting into two inch square peat pots, depending mainly on
seed quality, temperature, and depth of planting. Newly
emerged seedlings were in numbers 1 to 3 per a single
planted "seed" (dry fruit would be a more accurate term, but
is not used because of clarity and consistency with some of
the cited publications). At the time when recently emerged
plants had reached a height of about 5 cm and 3 to 4 leaves,
approximately one month after planting, multiple plants
occupying the same peat pot were separated or "split" to
individual 3 inch pots. The separated plants were kept
covered for 2-4 days to slow water loss until the plants
became established. Very few plants were lost during the
procedure. All plants were grown for at least 4 months

before phenotypic classification for DLM and WT was done.

26

Isolation for controlled self—pollination of F2 and F2
individuals, and for cross-pollination of F2 individuals
backrossed onto P1, was achieved by placing a white paper
bag over staked flower stalks, immediately prior to first
flower opening. The bags were shaken every few days in order
to increase pollen movement inside. After from one to one
and a half months the bags were removed, and the tips of
floral branches were clipped immediately above the youngest
swollen seeds on each branch. Seeds were ripened on plants
for an additional month and then hand harvested.

F2 seed from 106-C1 X REL-2 was generated in 1995. More
than 120 F1 seedlings were started in the greenhouse the
same year by Dr. Saunders. Those F2 plants were grown for
about a year, and 22 of the plants were grown for more than
two and a half years, being observed for presence of the DLM
and the annual/biennial trait. Annual plants bolted under
the greenhouse environment. Biennial plants, that is, those
not spontaneously flowering in the greenhouse, were placed
in a cold room at 4 °C under incandescent lights for at
least 10 weeks in order to be vernalized, then were returned
to the greenhouse for flowering under incandescent lighting
or natural long days.

In 1997 and 1998, 22 F2 plants were used for further
progeny analysis. Both self-fertility/self—sterility and

annualism/biennialism segregated in the F2 generation. Only

27

self-fertile F2 plants were used in further generations for
inheritance studies. With respect to the annual/biennial
trait, 13 plants were biennial (bb), 4 were annual (B—), and
5 were unrecorded for the trait. Seventeen self-pollinated
F2 plants produced seed of 17 respective F2 families, which
were planted to obtain segregation ratios in the F2(Sl)
families. The number of plants from each of the 17 F2
families ranged from 18 to 198, with an average of about 92,
standard deviation (OX) of 56.3 and total of 1568 plants
(Table 2.1).

Furthermore, 13 BC1 families were generated by
backrossing each of 13 different F2 individuals to ramets of
the original DLM parent, 106—C1. Ten F1 individuals, of the
22 used to produce seed, were thus each in production of
both types of progenies: BCl and F2. The number of plants
per BCl family“was from 33 to 91, with an average of about
64, standard deviation of 16.2 and total of 833 plants.

Ten F3 families were generated by self—pollination
(i.e., as S2 generation) from 2 F1 individuals (Table 2.1).
Five F3 families, derived from self-pollination of F2 WT
individuals, had 30—79 plants per family, with an average of
44 plants, standard deviation of 18.9 plants, and total of
220 plants. Five F3 families, derived from self-pollination
of F2 DLM individuals, had 22 to 50 plants per family, with

average of about 41 plants, standard deviation of 10.3

28

plants and total of 203 plants. WT controls were over 20
ramets and 67 81 individuals from REL-2 (Table 2.2). DLM
controls were over 40 ramets of 106-C1 (Table 2.2). All WT
and DLM control progenies were always grown under the same
environment, either greenhouse or growth chamber.
Chi-square analysis was used for determining the
probability of fit between the observed and the tested
segregation ratios for various models of inheritance in F2,

F3, and BC1 families (Srb et al., 1952). The chi-square for

all classes was calculated by the formula x%%ZH(observed—
expected number)—HQ]2/expected number} (Strickberger, 1968).

Each chi-square value has a probability of occurrence, which
depends on the number of degrees of freedom (df) (Srb et
al., 1952, Strickberger, 1968). If the probability of a chi-
square calculated from segregation data is smaller than the
corresponding probability that the deviation from the tested
ratio is 1/2o(0.05), it is generally accepted that the
deviation is non-significant (NS) and that the observed
ratio "fits" the expected (tested) ratio. On the contrary,
if the calculated chi-square is greater than corresponding
probability that the deviation from expected ratio is 590
(0.05), it is generally accepted that the deviation is
significant [** if (<0.01), or * if (0.01<p<0.05)
probability that the deviation is not due to chance alone].

In such a case, it is considered that the observed ratio

29

"does not fit" the expected ratio (Srb, et al., 1952). Thus,
a significant deviation from the expected and tested

segregation ratio warrants rejection of the model tested.

Propagation of Plant Material in vitro

Both parents in the foundational cross, 106-C1 and REL—
2, and at least 7 Fland 20 F2 individuals were cloned in
vitro as shoot cultures, in order to maintain and produce
adequate numbers of their ramets (copies) for crossing, for
controls, and for evaluation of intercellular fluid (ICF)
protein parameters. The availability of these ramets
facilitated speed and number of backcrosses, and generated
sufficient numbers of genetically identical plants of each
clone for leaf protein studies over the course of study.
Some of the genotypes involved were maintained for several
years, with tens of ramets produced in vitro, and used in
growth chambers and greenhouse.

The system used for maintenance, multiplication and
rooting of the shoots was that adapted for sugarbeet shoot
culture by Saunders (1982). The shoot cultures were
established from elongated flowering stalks' lateral buds,
about 1 cm long. The buds were sterilized twice for 15
minutes each in a solution of 15% chlorox and 0.01% sodium
lauryl sulfate, followed by four washes in sterile distilled

water and placement on modified MS agar medium (Murashige

30

and Skoog, 1962), containing 0.25 mg Iflrf-benzyladenine.
This medium formulation is used for maintenance and
multiplication of axillary shoots. For production of rooted
shoots for use as whole plants, well developed shoots about
3-4 cm long were transferred onto MS medium containing 3 mg

I."1 arnaphthalenacetic acid (35 ml in 125 ml Erlenmeyer

flasks). These two media differed only in the hormone
constituents. Organic compounds of the basal medium were
sucrose 30 g L4, myo-inositol 100 mg L4, Bacto Difco agar 9
g L4, pyridoxineMKfl.l mg L“, thiaminefiKfl.2 mg L4, and
nicotinic acid 1 mg L4. Mineral macroelements were NHth
1.65 g L‘l, KNO3 1.9 g L'l, CaCl202H2O 440 mg L'l, Mg80407H2O
370 mg Ld, and KHflXh 170 mg Ld. Mineral microelements were
H3BO3 6.2 mg L'l, MnSO40H2O 16.8 mg L'l, ZnSO4u7H2O 10.6 mg L‘l,
KI 0.88 mg 1:1, Na2MoO402H2O 0.25 mg L'l, Cu80405H2O 0.025 mg
III, and COClflOHflD 0.025 mg L4; Shoot and rooted shoot
cultures were kept in controlled environments, under
indirect fluorescent lighting and at about 18 C. After 2 to
4 weeks, rooted shoots in the form of plantlets were

transferred to the greenhouse.

31

RESULTS

DLM Occurrence in Pug) , F1, F2, 3C1, and F3 Families

When DLM plants, either ramets of 106-C1 and F2
individuals, or in segregating populations were developing
at a normal rate, some expressed the first signs of the
water-soaked spots (Figure 2.1.a) and/or necrotic lesions
(Figure 2.1.b) on well developed and older leaves within 5
weeks. Although it was not uncommon for scattered lesions or
colorations to occur on the oldest leaves of some wild type
plants, the DLM phenotype was unique by its high intensity
and extensive presence of the water-soaked spots and
necrotic lesions. The DLM phenotype was not observed on
incompletely unexpanded leaves. Ramets of DLM genotypes such
as: 106—C1, and at least 4 DLM F2 individuals from the
foundational cross always expressed the DLM phenotype, under
the greenhouse and growth chamber environments.

Over 120 F2 individuals from the cross of 106-C1 X REL-
2 were grown for at least a year, without displaying any DLM
appearance. Sixty-seven 81 plants from self pollination of
the WT parent were all WT. Two models for DLM inheritance
received focus, based on the absence of DLM progeny in the
F2 generation, as well as in the 81 from P2: a) monogenic
recessive (Figure 2.2), and b) digenic with DLM plants being

homozygous recessive at at least one locus, and having at

32

least one dominant allele at another independently
segregating locus (Figures 2.3.a-e).

In all 17 F2 families evaluated, segregation for DLM

was in the range 0-27%, Y=13.88%, ox=7.95 (Table 2.1). Chi-
square analysis (Tables 2.3.a-e) for either mono- or digenic
recessive (for at least one locus) segregation in each of
the 17 F2families showed that: 9 families fit a 3:1 (12:4)
ratio, one fit a 9:7 ratio, 4 fit a 10:6 ratio, 13 fit a
13:3 ratio, and five families fit a 15:1 ratio, all with
0.05<P (Table 2.4.a). Overall, 6 F2 families fit only a
single segregation ratio, 5 fit two, 4 fit three, and two
families each fit 4 of the 5 tested segregation ratios
(Table 2.4.a). Two F2 families produced 0% DLM, but it was
not possible to use the chi-square test in their cases,
because it requires a number higher than zero for each of
observed segregation category.

Five out of 10 F2 families had been generated by
selfing F2 DLM plants, and the other 5 had been generated by
selfing WT F2 plants (Table 2.1). Three out of five F3
families generated from F2DLM individuals produced all DLM
progeny. The other two F2 families segregated 89% and 85%
for DLM. The five F2 families generated by selfing E2WT
individuals segregated 20%, 22%, 30%, 34%, and 37% DLM. Chi-
square analysis (Tables 2.3.j-n) indicated that segregation

in all 5 F2 families generated by selfing WT F2 individuals

33

fit a ratio of 3:1 (WT:DLM) and segregation in 3 of those 5
families also fit the 13:3 ratio. Out of 5 F3 families
generated by selfing DLM F2 individuals, one fit the 1:3
ratio, one (—1U8 -14DLM80, with 89% DLM, did not fit any of
1:0, 3:1, 0:1, 13:3, or 1:3 ratio, and all progeny in the
other 3 families were all DLM, i.e. true breeding. Ratio 1:0
(WT:DLM) was not observed in any F2 family (Table 2.4.b)

In 13 BC1 families, segregation for DLM ranged from 21%

to 54%, x=40.23%, ox=10.05. Two Bleamilies segregated with
42% DLM plants, while the remaining 11 Bleamilies
segregated 21%, 25%, 28%, 34%, 39%, 43%, 45%, 48%, 50%, 52%,
and 54% for DLM plants. Chi—square analysis indicated that 9
Bleamilies fit a 1:1 (WT:DLM) segregation ratio, 4

families a 3:1 ratio, no families fit a 1:3 (WT:DLM) ratio,
and 10 families fit a 5:3 ratio (Table 2.4.b). Overall, 3
BCl families fit only a single segregation ratio, and 10
families each fit different combinations of 2 of the 4
tested segregation ratios (Table 2.4.b).

Both types of families, F2 and BC1, were successfully
generated from each of 10 Flindividuals. The ten F2 families
stemmed from 5 WT F2 and 5 DLM F2 individuals, each from one
of two F1 individuals that also produced BC1 progeny. Ten F2
families that corresponded to 10 BC1 families satisfied five
different segregation ratios (Table 2.4.a) and three

different models (Table 2.5.a). Six F2 families satisfied

34

only one segregation ratio, two satisfied two, and two F2
families satisfied three different segregation ratios (Table
2.4.a). Further on, five F2 families satisfied two models
each, and five F2 families satisfied only one model per
single family (Table 2.5.a). Ten BC1 families, that
corresponded to 10 F2 families, satisfied three different
segregation ratios (Table 2.4.a) and three different models
of inheritance as well (Table 2.5.a). In seven BCl families,
each family satisfied two segregation ratios, and three BC1
families satisfied only one segregation ratio (Table 2.4.a).
Also, in seven BCl families, each family satisfied only two
models, and three BCl families satisfied only one model
(Table 2.5.a). Five cases satisfied fit to both 3:1 (WT:DLM)
in F2 and the corresponding 1:1 (WT:DLM) in the BC1, five
cases fit to both 13:3 in the F2 and corresponding 5:3 in
the BCl generations, and two cases fit to both 1:0 in the F2
and corresponding 3:1 in the BCl generations (Tables 2.3.a,-
b). Distributions of DLM in F2 and corresponding BCl
families, and frequency distribution of DLM segregation
frequencies in F2 families are presented in Figures 2.4 and

2.5 respectively.

35

DISCUSSION

A large number of lethal and sublethal mutations have
been reported in many organisms. Many of the alleles for
such mutations are eliminated from gene pools because the
phenotypes that they encode lack competitive abilities in
comparison to the wild type. Some alleles are eliminated as
early as the gametophyte, others are lost by embryo
lethality, while most can be carried by plants through to
later stages of development. Recessive mutations of these
types are more frequent in populations than are dominant
ones. Such recessive traits can be carried from one
generation to another in heterozygous condition without
being expressed. Examples are albino mutations that occur in
both plants and animals.

Potential associations of uncompetitive or sublethal
phenotypes with certain beneficial biological
characteristics are worthy of investigation, so that close
linkage and/or pleiotropy of lethal and sublethal genes to
"good" genes and desirable phenotypes may be utilized. In
many plant species, association of the hypersensitive
response (HR), which is a form of controlled cell death,
with elevated resistance to pathogens, reflects a balanced

association between locally harmful and systemically healthy

36

responses. Hypersensitive response is reported to be closely
associated with gene-for-gene resistance and systemic
acquired resistance (Yu et al., 1998; Dangl et al., 1996).
In many cases, plants recovering from a disease can exhibit
very high resistance to further possible infections caused
by a broad spectrum of pathogenic viruses, fungi, and
bacteria (Delaney, 1997).

In normal, resistant plants, HR is initiated by the
attempted infection of host tissue by a non-pathogen or race
non-specific microorganism. Jabs et al. (1996) pointed out
that reactive oxygen intermediates (ROI) are a regulating
factor not only for HR in plants, but also in apoptosis
(programmed cell death) during normal development and
disease in animals. More precisely, superoxide is considered
to be a critical signal for initiation of programmed cell
death (Jabs et al., 1996). The role of HR in increased
levels of resistance in plants is still speculative. Two
possibilities and their interactions have been studied by
Dangl et al. (1996): one that HR deprives pathogens of
nutrients, the second that cell-initiated release of toxic
compounds kills pathogens. Once the plant recognizes an
attack, it responds with HR, and subsequently with intense
production of phytoalexins, oxidants, lytic enzymes,
antimicrobial proteins, and strengthening of the cell wall

as means of halting the further pathogen spread (Tenhaken et

37

al., 1995). The defensive substances or mechanisms mentioned
above are not detectable, or are present in very low
amounts, in healthy plants.

In the majority of DLM cases, the lesions are formed
without apparent need for a pathogen-type triggering.
Ectopic expression of a transgenic yeast invertase gene gave
rise to a "lesion-mimic" mutant phenotype in tobacco
(Herbers et al., 1996). By one view, plant diseases can be
classified as "low and high sugar diseases," so that
concentrations of specific sugars in plants are correlated
with magnitude of resistance (in Herbers et al., 1996).
According to that concept, the sugar status in the plant or
organ is an important factor for the regulation of
resistance, and potentially for DLM expression. Many DLM
phenotypes were expressed during growth in sterile culture,
as a test that expression of some of DLM phenotypes did not
need a biotic trigger. Such DLM cases were mlo barley
(Wolter et al., 1993), and acd and lsd in Arabidopsis
(Greenberg et al., 1994, Dietrich et al., 1994). The rp—l
mutant in corn did not show any signs of lesions when grown
in an aseptic environment (Hu et al., 1996). In the
sugarbeet DLM case, no DLM phenotype was observed in various
kinds of sterile shoot and plantlet culture including low
sucrose, even after 24 weeks. After transfer to greenhouse

conditions, no DLM ramet failed to express the

38

characteristic phenotype. This indicates that some sort of
biotic, physical, or dual trigger may be needed for lesion
initiation in the DLM system studied here. These DLM plants
may thus be much more responsive to the presence of at least
some microorganisms than WT plants, possibly due to failure
to shut down the activated defense mechanisms after early
and successful defense. In addition, the responses can be
very quick, because it took 1-3 days from the first visible
spots on the foliage until the formation of extensive water-
soaked and lesion areas. Lesion formation in this case can
be considered as a dynamic process, which does not appear to
correlate with the rate of leaf expansion, which is a many
fold slower process. Rate of responsiveness to
microorganisms is an important defense characteristic,
considering that many successful infections take place
because plants are unable to recognize and rapidly respond
to pathogen (Delaney, 1997). Gene sources of greater
responsiveness to pathogen attack in crop plants have
prospects for use to genetically increase resistance to one
or more diseases.

Inheritance of the disease lesion mimic phenotypes in
diverse species has been reported to be in simple Mendelian
fashion. In a majority of reported cases, the mutant
phenotypes segregated as at a single locus, with either

dominant or recessive alleles (Emerson, 1923; Wolter et al.,

39

1993; Hornbrook et al., 1969; Neuffer et al., 1975;
Marchetti et al., 1983; Greenberg et al., 1993; Dietrich et
al., 1994; Herbers et al., 1996). Thus the first working
hypothesis on inheritance of DLM in sugarbeets to be tested
was that of segregation at a single locus, with both parents
homozygous for different alleles, because REL-2 selfed were
all WT, i.e., P2 was not heterozygous in a monogenic model.
Segregation data was somewhat supportive, that the DLM
character was inherited as monogenic recessive (dlmL/dlml)
with no segregation in the F2 family, up to 27% in some of
the F2 families, and up to 54% in some of the BC1 families
(Figure 2.2). The model was indicated as insufficient, by
chi-square analysis, because it could not account for low
segregation ratios in some F2 families. Segregation ratios
in 8 out of 17 F2 families did not satisfy fit to 3:1
(WT:DLM) (Tables 2.3.a., 2.4.a.), and 4 out of 13 BC1
fandlies did not satisfy fit to 1:1 (Tables 2.3.f., 2.4.b.).
The next step was to test existence of at least one
nmnre segregating locus involved in DLM inheritance. The
second model accounted as if there were 2 unlinked loci, one
homozygous recessive (dlmphihm) and the other
simultaneously with at least one dominant allele (Dlm2/afl
(Figures 2.3.a—d). Such DLM parental genotypes could produce
1i3EXDssible combinations of F1 genotypes, depending on the

POSSilole WT parent genotypes. Six hypothetical F2 genotypes

40

[(Dlml/d1m1:D1m2/Dlm2), (Dlml/dlmllem2/dlm2),
(Dlml/d1m1:dlm2/d1m2), (dlml/dlm1:DIM2/Dlm2),
(dlmL/dlm1:Dlm2/dlm2), and (dlmL/dlm1:dlm2/d1m2)] were
possible according to the scheme in Figure 2.3.a. The first
step to narrowing the list of acceptable genotypes was to
exclude those that segregated for DLM in F2, because all F1
plants were WT. Values of percentage of DLM were plotted on
the X-axis, with frequencies of distribution [f(X)] plotted
on the Y—axis, and three groupings (~0%, ~18%, and ~25%)
were evident (Figure 2.5). That suggested the possibility
that each F2 family was derived from anyone of 3 F1
genotypes.

Segregation in matched F2and.BC1 families was very
supportive of digenic model. Chi-square tests supported the
existence of three F2 genotypes with fit in 9 out of 10
matched pairs of F2 and BCl families (Tables 2.10.a-c). One
family that did not show consistency with the model differed
in correspondence between F2and.BC1, but if observed
separately, both this F2 and the corresponding BC1 were
consistent with the DLM genotypic model that involves 2
unlinked loci, one being homozygote recessive (dlmi/dlml)
and the other simultaneously having at least one dominant
allele (Dlmmfiu). The allelic scenario created in such a way
resolved the genotypic nature of the DLM parent, as well as

of the WT parent. Heterozygosity at the second locus in both

41

parents was responsible for the multiplicity of F2 and BC1
ratios, due to the heterogeneous F2 population produced. The
conclusion is that 106-C1 is homozygous recessive for the
first (hyperstatic) DLM locus and heterozygous for the
second (hypostatic) DLM locus (dlml/d1m1:Dlm2/dlm2). The wild
type parent, REL-2, is homozygous dominant for first and
heterozygous for the second locus (LUnu/Dlm1:Dlm2/dlm2).
According to this model, all F2, F3, and BCl progenies from
individuals derived from the foundational cross and having a
dlml/dlm1:D1m2/-2 genotype express the phenotype. Also, F2
genotypes Dlm1/—1:D1m2—2 express the WT phenotype, but their
F2 progenies segregate for the DLM phenotype, thus DLM can
skip two generations and reappear in the third.

It can be hypothesized that in addition to the Dlm2 and
Dlm2 loci, one or more other loci could be involved in
controlling this DLM phenotype in sugarbeets. Any additional
loci that are homozygous recessive or dominant, but
identical for both parents, will not segregate in subsequent
generations, and the DLM phenotype would appear to be
inherited as digenic. For example, if both parents are
homozygous recessive for the hypothetical Dlm3 locus, their
genotype for DLM would be: a) 106—Cl
(dlml/dlm1:Dlm2/dlm2:d1m3/dlm3), and b) REL—2

(Dlml/Dlm1:Dlm2/dlm2:dlm3/dlm3).

42

The present investigation of inheritance of the
spontaneous lesions of a DLM phenotype in sugarbeet points
to control at a minimum of two independently segregating
loci. The allelic constitution needs to be homozygous
recessive at one, as well as heterozygous or homozygous
dominant at the other locus, so the genotypes conditioning
the trait are dlmphihszlmmfi7. This is one of only two
reports on involvement of more than one locus in inheritance
of a naturally occurring DLM trait.

The only two-locus model of DLM inheritance in the
literature is that proposed in tomato by Langford (1948),
where spontaneous lesion formation was observed on some
plants carrying a dominant allele at the Cf locus
conditioning gene for gene resistance to Clavidosporum
fulvum. The dominant allele had been introgressed from wild
germplasm. Langford analyzed segregation for lesion presence
among plants with the Cf allele, and concluded that a second
locus in homozygous recessive condition (nene) was also
necessary for spontaneous lesion formation. Similar to the
presently proposed DLM genotypic model in sugarbeet (i.e.,
dlml/dlm1:D1m2/-2), the DLM genotype proposed in tomato
called for a combination of homozygous recessive alleles at
one locus and at least one dominant allele at the second
locus (i.e., nene:Cfifl—) (Langford, 1948). All plants with

the necrotic foliage expressed resistance to C. fulvum. All

43

tomato plants with the Ne—;Cfifl- genotype were resistant and
non-necrotic. Thus, variability at both loci permitted
genetic separation of disease resistance and necrosis in
tomato, and it may be similarly possible in sugarbeet to
deal with any resistance encoded by the Dlm2 allele separate
from the lesion presence. Interestingly, two independently
segregating loci controlling individual DLM phenotypes
similar to this one in sugarbeet and the Ne—;Cfifl— tomato
system, in other species would exhibit a single—locus
segregation pattern if both parents were identically
homozygous for either of the two loci. Whether the trait
appeared to be inherited monogenically as either recessive
or dominant would depend on which locus happened to be
homozygous for the identical allele in both parents. This
possibility could be tested by using various WT genotypes as
WT parent in crosses with a single DLM parent, attempting to
detect allelic variation at the second locus.

In sugarbeets, two important and well studied traits
are cytoplasmic (CMS)-nuclear male sterility (Owen, 1945)
and betalain pigmentation (Keller, 1936). Each of these
traits is controlled by at least two distinct loci. Both
traits are utilized as tools in sugarbeet breeding (Smith,
1980). Cytoplasmic-nuclear male sterility is used for
creating pure hybrid populations, which are the most

productive marketed type of cultivar. For this type of

sterility to be fully expressed, cytoplasm should carry the
S factor for sterility and both nuclear loci, X and Z, have
to be homozygous recessive (i.e., S xxzz) (Owen, 1945). If
the cytoplasm is normal (i.e., N), the plants will be
normally male fertile regardless of the nuclear loci's
allelic constitutions. If the cytoplasm is S, then presence
of the dominant X and Z alleles is crucial for the
expression of male fertility. Genotypes S X-Z- will always
be fertile, but combinations such as S XXzz and S xez will
be semi-male-sterile, usually without any viable pollen
(Owen, 1945). Under different environmental regimes, some S
XXzz and S xez plants may behave as male-fertile. The two
segregating nuclear loci conditioning male—sterility with S
cytoplasm can be considered complementary to each other, and
epistatic (specifically, hypostatic) with the cytoplasmic
factor. This complementarity might be explained by
similarities of function common to each locus.

Betalain pigmentation in beet is known to be controlled
by three major loci: P, R, and Y. Multiple alleles R, Rt,
193 RE, and r have been reported for the R locus. The Y
locus conditions intense color in beets. Two recognized
alleles, Y and y, occupy this locus. Expression of Y locus
alleles is modified by R locus alleles and vice versa
(Smith, 1980), which indicates interaction between Y and R

loci. A third locus, P, is tightly linked to Y and R and is

45

epistatic with (hyperstatic to) them (Linde-Laursen, 1972).
The white pp phenotype was only decisively identified from a
small range of beet germplasm.

In comparison to these two fairly well recognized
digenic and trigenic systems, respectively, in sugarbeets,
this sugarbeet DLM phenotype study showed that the Dlml
locus is hyperstatic (i.e., dominant to) to Dlm2 with
regards to the DLM phenotype. Two or more distinct
phenotypes for various DLM traits in some other species have
been used to study not only conditions needed for HR
initiation, but also physiology and intensity of HR
reactions (reviewed by Dangl, et al., 1996). The possible
role of DLM genes in disease resistance has been studied,
and in some cases pleiotropy with pathogen-specific
resistance has been discovered: tomato Cf (Langford, 1948),
barley mlo (Wolter et al., 1993), corn rpl (Hu et al.,
1996), and soybean RN (Kosslak et al., 1996). Disease lesion
mimic mutants have expressed elevated types of resistance in
comparison to WT plants: corn lesion-1 and lesion—2 (Neuffer
et al., 1975), rice sl (Marchetti et al., 1983), some of
Arabidopsis acd (Greenberg et al., 1993), Arabidopsis lsd
(Dietrich et al. 1994), tobacco transgenic cwlnv and vaclnv
(Herbers et al., 1996), and corn lls (Simmons et al., 1998).

On the other hand, a single acd mutant of Arabidopsis had

46

depressed levels of SAR, and was more susceptible to
opportunistic infections (Greenberg et al., 1993).

Study of the expression and function of loci involved
in DLM traits may lead to use of these genes in breeding or
genetic engineering for disease resistance. It has been
reported that certain defense genes in plants have promotive
effects on initiation of defense responses, while other loci
function in suppression of the response by limiting the
intensity, location, and rate of the responses, not letting
them to go too far (Bent, 1996). If defense reactions would
not be restricted to certain intensities and be shut off
after a time, the plants undergoing them would likely kill
themselves by their own defense responses. It would seem
important to be able to modify the expression of defense
genes so that the defense reaction rates can be programmed
and altered in a such manner that pathogens are disarmed at

the lowest cost to the plant.

47

Table 2.1

F1 individuals and respective F2,

F3!

and BC1 progenies involved in segregation

 

 

 

 

 

 

 

 

analysis.
P1 9 106-Cl X REL-2 6 P2
scl F1 F2 1137
(106-C1 X Fl)‘ (106-C1 X (F1 ®)‘
REL-2)

[# of DLM % [plant no.] DLM % [# of
plants] plants]
53 28 -1 - -*
60 42 -3 12 113
91 54 -6 18 97
69 52 -8 13 144
77 45 -11 14 114‘ 1 WT 2 DLM

- - -15 6 78
67 25 -18 1 135
64 34 -19 - -
80 50 -2o 20 1985 4 WT 3 DLM
33 21 -21 0 197
- - -33 11 99
— - -34 18 121
- - -38 0 18
33 42 -71 26 47
63 48 -81 16 32
- - -88 27 30
76 43 -89 - -
- - -90 18 22
67 39 -99 17 102
- - -100 19 21
Total 2:40 . 23 2:13. 9 Total
833 1568
i=64; ox=10.0 ox=7.9 2:92;
ox=16.2 ox=56.3

 

’ No F2 progeny created from the genotype.
* From ®s of single F2 plants.
g F2 families were produced from these two F2 families.
‘ Family number corresponds to F2 plant number.

48

 

Table 2.2 Genotypes used as controls for phenotypic
categories for segregation analysis of progenies

 

 

from 106-C1 X REL-2.
Genotype Phenotype Propagation method # of
plants
REL-2 WT self-pollination and 67
shoot culture cloning >20
106-C1 DLM shoot culture cloning >40

 

 

 

 

49

 

 

 

 

Table 2.3.a Chi-square tests for 3:1 (12:4) (WT:DLM)
segregation ratio in F2families.
Family F Jr a” a“ 7“ P5
-3 113 13“ 28.30“ -15.30 10.32 <0.01 **
100‘p 84.80” 15-30
-6 97 17 24.30 -7.30 2.54 >0.05 NS“
80 72.80 7.30
-8 144 19 36.00 -17.00 10.08 (<0.01 **
125 108.00 17.00
-11 114 16 28.50 -12.50 6374 <0.01 **
98 85.50 12.50
-15 78 5 19.50 -l4.50 13.40 <0.01 **
73 58.50 14.50
—18 135 2 33.80 -31.80 .38.66 <0.01 **
133 101.30 31.80
-20 198 40 49.50 -9.50 2.18 I>0.05 NS
158 148.50 9.50
-21 197 0 49.30 -49.30 64.42 <0.01 **
197 147.80 49.30
-33 99 11 24.80 -l3.80 9.51 <0.01 **
88 74.30 13.80
-34 121 22 30.30 —8.30 2.68 >0.05 NS
99 90.80 8.30
-38 18 0 4.50 —4.50 4.74 <0.05 *
18 13.50 4.50
-71 47 12 11.80 0.30 0.00 I>0.99 NS
35 35.30 -0.30
-81 32 5 8.00 -3.00 1404 :>0.20 NS
27 24.00 3.00
-88 30 8 7.50 0.50 0.00 2>0.99 NS
22 22.50 -0.50
-90 22 4 5.50 -1.50 0.24 2>0.50 NS
18 16.50 1.50
-99 102 17 25.50 -8.50 3.35 >0.05 NS
85 76.50 8.50
-100 21 4 5.30 -1.30 0.16 >0.50 NS
17 15.80 1.30
I Number of plants.
h Observed number.
n Expected number.
x o-e.
* x2=2(d-1/2)2/e.
5 df=1.
& DLM.
9 WT.
1

Non-significant deviation of observed to expected ratio.

50

Table 2.3.b Chi-square tests for 9:7

(WT:DLM)

segregation ratio in F2families.

 

 

 

 

Family Nf ch .21 dx 2 * PS
-3 113 13¢ 49.40& -36.40 46.35 <0.01 **
1004 63.60‘p 36 40
-6 97 17 42.40 —25.40 25.98 <0.01 **
80 54.60 25.40
-8 144 19 63.00 —44.00 53.40 (<0.01 **
125 81.00 44.00
-11 114 16 49.90 -33.90 39.76 <0.01 **
98 64.10 33.90
-15 78 5 34.10 -29.10 42.62 (<0.01 **
73 43.10 29.10
-18 135 2 59.10 -57 10 96.41. <0.01 **
133 75.90 57.10
-20 198 40 86.60 —46.60 43.62 {<0.01 **
158 11.40 46.60
-21 197 0 86.20 -86.20 151.49 <0.01 **
197 110.80 86.20
-33 99 11 43.30 —32.30 41.51 <0.01 **
88 55.70 32.30
-34 121 22 52.90 -3o.9o 31.04 <0.01 **
99 68.10 30.90
-38 18 0 7.90 —7.90 12.35 (<0.01 **
18 10.10 7.90
-71 47 12 20.60 —8.60 5.67 <0.05 *
35 26.40 8.60
-81 32 5 14.00 -9.00 9.17 (<0.01 **
27 18.00 9.00
-88 30 8 13.10 -5.10 2.87 :>0 05 Ns‘
22 16.90 5.10
-90 22 4 9.60 -5.60 4.81 <0.05 *
18 12.40 5.60
-99 102 17 44.60 -27.60 29 26 <0.01 **
85 57.40 27.60
-100 21 4 9.20 -5.20 4.27 <0.05 *
17 11.80 5.20
I, Number of plants.
h Observed number.
n Expected number.
N o-e.
* x2=2(d—1/2) 2/e.
9 df=1.
* DLM.
9 WT.
‘l

Non-significant deviation of observed to expected ratio.

51

Table 2.3.c

Chi-square tests for 10:6 (WT:DLM)

segregation ratio in F2families.

 

 

 

 

Family NI oh 621 d“ 5* PS
-3 113 13“ 42.405 -29.40 31.53 <0.01 **
100: 70.60¢ 29-40
-6 97 17 36.40 —19.40 15.71 <0.01 **
80 60.60 19.40
-8 144 19 54.00 -35.00 35.27 '<0.01 **
125 90.00 35.00
-11 114 16 42.80 —26.80 25.79 <0.01 **
98 71.30 26.70
-15 78 5 29.30 -24.30 30.84 <0.01 **
73 48.80 24.20
-18 135 2 50.60 —48.60 73.14 <0.01 **
133 84.40 48.60
-20 198 40 74.30 -34.30 24.55 ‘<0.01 **
158 123.80 34.20
-21 197 0 73.90 -73.90 116.67 <0.01 **
197 123.10 73.90
-33 99 11 37.10 -26.10 28.25 ‘<0.01 **
88 61.90 26.10
-34 121 22 45.40 —23.40 18.49 <0.01 **
99 75.60 23.40
-38 18 0 6.80 —6.80 9.24 (<0.01 **
18 11.30 6.70
-71 47 12 17.60 -5.60 2.36 >0.05 NS‘
35 29.40 5.60
-81 32 5 12.00 -7.00 5.63 (<0.05 *
27 20.00 7.00
-88 30 8 11.30 -3.30 1.08 >0.20 NS
22 18.80 3.20
-90 22 4 8.30 -4.30 2.73 >0.05 NS
18 13.80 4.20
-99 102 17 38.30 -21.30 18.01. <0.01 **
85 63.80 21.20
-100 21 4 7.90 -3.90 2.35 >0.05 NS
17 13.10 3.90
I Number of plants.
h Observed number.
n Expected number.
x o-e.
* X2=2(d"1/2)2/e.
5 df=1.
0‘ DLM.
° WT.
‘1

Non-significant deviation of observed to expected ratio.

52

Table 2.3.d

Chi-square tests for 13:3 (WT:DLM)

segregation ratio in F2families.

 

 

 

 

Family Nf oh .2‘ d“ x5 * P5
-3 113 13‘ 21.20& —7 00 3.44 >0.05 ms'I
100” 91.80° 7-00
-6 97 17 18.20 -0.70 0.03 I>0.80 NS
80 78.80 0.70
-8 144 19 27.00 -7.50 2.56 >0.05 NS
125 117.00 7.50
-11 114 16 21.40 —4.90 1.38 >0.20 NS
98 92.60 4.90
-15 78 5 14.60 -9.10 6.98 ‘<0.01 **
73 63.40 9.10
-18 135 2 25.30 22.80 25u29 <0.01 **
133 109.70 22.80
-20 198 40 37.10 2.40 0.19 >0.50 NS
158 160.90 -2.40
-21 197 0 36.90 36.40 44.18 (<0.01 **
197 160.10 36.40
-33 99 11 18.60 -7.10 3.34 >0.05 NS
88 80.40 7.10
-34 121 22 22.70 —0.20 0.00 :>0.99 NS
99 98.30 0.20
-38 18 0 3.40 —2.90 3.05 :>0.05 NS
18 14.60 2.90
-71 47 12 8.80 2.70 1.02 .>0.20 NS
35 38.20 —2.70
-81 32 5 6.00 -0.50 0.05 I>0.80 NS
27 26.00 0.50
-88 30 8 5.60 1.90 0.79 >0.20 NS
22 24.40 —1.90
-90 22 4 4.10 0.40 0.05 >0.50 NS
18 17.90 -0.40
-99 102 17 19.10 -1.60 0.16 >0.50 NS
85 82.90 1.60
-100 21 4 3.90 -0.40 0.05 >0.80 NS
17 17.10 0.40
If Number of plants.
h Observed number.
3 Expected number.
x o-e.
* x2=2(d-1/2)2/e.
5 df=1.
& DLM.
‘9 WT.
a

Non-significant deviation of observed to expected ratio.

53

 

 

 

 

Table 2.3.e Chi—square tests for 15:1 (WT:DLM)
segregation ratio in F2families.
Family NI oh 621 d” 2* PS
-3 113 13& 7.1057 5.90 4.38 <0.05 *
1004 105.909 -5 90
-6 97 17 6.10 10.90 18.92 3<0.01 **
80 90.90 10.90
-8 144 19 9.00 10.00 10.70 1<0.01 **
125 135.00 10.00
-11 114 16 7.10 8.90 10.60 3<0.01 **
98 106.90 -8.90
-15 78 5 4.90 0.10 0.03 >0.80 NS)1
73 73.10 -0.10
-18 135 2 8.40 -6.40 4.42 <0.05 *
133 126.60 6.40
-20 198 40 12.40 27.60 63.18 ‘<0.01 **
158 185.60 27.60
-21 197 0 12.30 12.30 12.07 <0.01 **
197 184.70 12.30
-33 99 11 6.20 4.80 3.18 >0.05 NS
88 92.80 —4.80
-34 121 22 7.60 14.40 27.13 <0.01 **
99 113.40 14.40
-38 18 0 1.10 -1.10 0.35 >0.50 NS
18 16.90 1.10
-71 47 12 2.90 9.10 27.18 <0.01 **
35 44.10 —9.10
-81 32 5 2.00 3.00 3.33 >0.05 NS
27 30.00 -3.00
-88 30 8 1.90 6.10 17.62 (<0.01 **
22 28.10 -6.10
-90 22 4 1.40 2.60 3.36 >0.05 NS
18 20.60 -2.60
-99 102 17 6.40 10.60 17.01. <0.01 **
85 95.60 10.60
-100 21 4 1.30 2.70 3.97 <0.05 *
17 19.70 —2.70
I Number of plants.
h Observed number.
n Expected number.
x o-e.
* x2=2(d-1/2)2/e.
5 df=1.
& DLM.
‘P WT.
a

Non-significant deviation of observed to expected ratio.

Table 2.3.f Chi-square tests for 1:1 (WT:DLM)
segregation ratio in Bleamilies.

 

 

 

 

Family IfF oh 321 dx {1* PT
-1 53 15‘ 26.50& —11 50 8.96 <0.01 **
38° 26.50” 11 50
-3 60 25 30.00 -5.00 1.35 >0.20 NS‘1
35 30.00 5.00
-6 91 49 45.50 3.00 0.39 >0 50 NS
42 45.50 —3.00
-8 69 36 34.50 1.00 0.06 >0 80 NS
33 34.50 -1.00
-11 77 35 38.50 —3.00 0.46 >0.20 NS
42 38.50 3.00
-18 67 17 33.50 -16.00 15.06 <0.01 **
50 33.50 16.00
-19 64 22 32.00 -9.50 5.64 (<0.05 *
42 32.00 9,50
-20 80 40 40.00 0.50 0.01 >0.80 NS
40 40.00 —0.50
-21 33 7 16.50 -9.00 9.53 <0.01 **
26 16.50 9.00
-71 77 32 38.50 -6.00 1.85 >0.05 NS
45 38.50 6.00
-81 63 30 31.50 -1.00 0.06 >0.80 NS
33 31.50 1.00
-89 76 33 38.50 -4.50 1.07 >0 20 NS
43 38.50 4.50
-99 67 26 33.50 -7.00 2.88 .>0.05 NS
41 33.50 7.00
* Number of plants.
h Observed number.
3 Expected number.
x o-e.
* x2=z(d-1/2) 2/e.
5 df=1.
& DLM.
° WT.
‘l

Non-significant deviation of observed to expected ratio.

55

Table 2.3.g Chi—square tests for 3:1 (WT:DLM)
segregation ratio in BC1families.

 

 

 

 

Family Nf oh an d.“ {5* PS
-1 53 15° 13.30‘ 1.80 0.17 >0.50 NsTI
38° 39.80° -1 80
-3 60 25 15.00 10.00 8.02 '<0.01 **
35 45.00 -10.00
-6 91 49 22.80 26.30 38.94 <0.01 **
42 68.30 -26.30
-8 69 36 17.30 18.80 25.82 <0.01 **
33 51.80 -18.80
-11 77 35 19.30 15.80 16.18 <0.01 **
42 57.80 -15.80
-18 67 17 16.80 0.30 0.00 >0.99 NS
50 50.30 -0.30
-19 64 22 16.00 6.00 2.52 .>0.05 NS
42 48.00 -6.00
-20 80 40 20.00 20.00 25.35 <0.01 **
40 60.00 -20.00
-21 33 7 8.30 ~1.30 0.10 >0.50 NS
26 24.80 1.30
-71 77 32 19.30 12.80 10.46 <0.01 **
45 57.80 —12.80
-81 63 30 15.80 14.30 16.08 <0.01 **
33 47.30 -14.30
-89 76 33 19.00 14.00 12.79 <0.01 **
43 57.00 -14.00
-99 67 26 16.80 9.30 6.15 <0.05 *
41 50.30 -9.30
I Number of plants.
h Observed number.
n Expected number.
x o-e.
* x2=2(d-1/2)2/e.
5 df=1.
& DLM.
8 WT.
a

Non—significant deviation of observed to expected ratio.

56

Table 2.3.h Chi-square tests for 1:3

(WT:DLM)

segregation ratio in BC1families.

 

 

 

 

57

Family NT oh 3” dx i“ P5
-1 53 15& 39.80° —24 80 59.23 <0.01 **
38° 13.30° 24 80
-3 60 25 45.00 —20.00 33.80 (<0.01 **
35 15 00 20 00
-6 91 49 68.30 3—19.30 20.68 <0.01 **
42 22.80 19.30
-8 69 36 51 80 —15.80 18.05 (<0.01 **
33 17.30 15.80
-11 77 35 57.80 —22.80 34.47 (<0.01 **
42 19.30 22.80
-18 67 17 50.30 —33.30 85.43 (<0.01 **
50 16.80 33.30
-19 64 22 48.00 -26.00 54.19 <0.01 **
42 16.00 26.00
-20 80 40 60.00 -20.00 25.35 <0.01 **
40 20.00 20.00
-21 33 '7 24.80 -17.80 48.13 <0.01 **
26 8.30 17.80
-71 77 32 57.80 -25.80 44.24 <0.01 **
45 19 30 25.80
-81 63 30 47.30 -17.30 23.83 <0.01 **
33 15.80 17.30
-89 76 33 57.00 —24.00 38.75 (<0.01 **
43 19.00 24.00
-99 67 26 50.30 -24.30 44.98 ‘<0.01 **
41 16.80 24 30
I Number of plants.
h Observed number.
m Expected number.
N o-e .
* x2=>:(d-1/2) 2/e.
5 df=1.
° DLM.
° WT.

Table 2.3.1 Chi-square tests for 5:3 (WT:DLM)
segregation ratio in BC1families.

 

 

 

Family NI ch .21 d” x51: P5
-1 53 15& 19.9o° —4 90 1.56 >0.20 Ms‘r
38° 33.10° 4-90
-3 60 25 22 50 2.50 0.28 >0.50 NS
35 37.50 -2 50
-6 91 49 34.10 14.90 9.73 -<0.01 **
42 56.90 -14.90
-a 69 36 25.90 10.10 5.70 <0.05 *
33 43.10 —1o.10
-11 77 35 28.90 6.10 1.74 >0.05 NS
42 48.10 -6.10
-18 67 17 25.10 —8.10 3.68 >0.05 NS
50 41.90 8.10
-19 64 22 24.00 -2 00 0.15 :>0.50 NS
42 40.00 2.00
-20 80 40 30.00 10.00 4.81. <0.05*
40 50.00 -10.00
-21 33 7 12.40 -5.40 .3.10 >0.05 NS
26 20.60 5.40
-71 77 32 28.90 3.10 0.37 :>o.50 NS
45 48.10 -3 10
-31 63 30 23.60 6.40 2.36 :>0.05 NS
33 39.40 -6.40
-89 76 33 28.50 4.50 0.90 >0.20 NS
43 47.50 -4 50
-99 67 26 25.10 0.90 CLCH. >0.80 NS
41 41.90 -0.90

 

Number of plants.

Observed number.

Expected number.

o-e.

x2=2<d-1/2)2/e.

df=1.

DLM.

WT.

Non—significant deviation of observed to expected ratio.

norm-*XISJ"

58

Table 2.3.j Chi-square tests for 1:0 (WT:DLM)

segregation ratio in F2 families.

 

 

 

 

Family N" 0*! % DLM .3 d“
-11’®-10‘A‘ WT' 79 27° 34 0.00“ 27.00
52° 79.00° -27 00

-20®-7A WT 30 9 30 0.00 9.00
21 30.00 —9.00

-20® 34m WT 32 12 37 0.00 12.00
20 32.00 -12 00

-20®-18A WT 49 11 22 0.00 11.00
38 49.00 -11.00

-20®-7 WT 30 6 20 0.00 6.00

24 30 00 -6.00

-11®-14 DLM' 47 42 89 0.00 42.00
5 47.00 —42.00

-11®—181\ um 47 40 85 0.00 40.00
7 47 00 -40 00

-208-6 um 37 37 100 0.00 37.00

0 37.00 -37.00

-20®-8 DLM 50 50 100 0.00 50.00

0 50 00 -50.00

-20®-25A DLM 22 22 100 0.00 22.00
0 22.00 -22.00

 

«shaggy-ones

.II'

F1 individual from which F2 family is derived.
F2 individual plant from which F2 family is derived.
Planting batch.

Number of plants.

Observed number.

Expected number.

o-e.

df=1.

DLM.

WT.

Phenotype of the F2 parent of the F2 family.

Chi-square can not be calculated for a class when the
frequency of any category is 0.

59

Table 2.3.k Chi—square tests for 3:1 (WT:DLM)
segregation ratio in F3 families.

 

 

Family N? oh 321 d“ xf* PS

-11*®-10°A° WT"' 79 27° 19.75° 7.25 3.08 >0.05 Ns‘
52° 59.25° -7-25

-20®-‘7A WT 30 9 7.50 1.50 0.18 >0.50 NS
21 22.50 -1.50

-20®-34CA WT 32 12 8.00 4.00 2.04 >0.05 NS
20 24.00 -4.00

-20®-18A WT 49 11 12.25 —1.25 0.06 >0.80 NS
38 36.75 1.25

-2o<8>-7 WT 30 6 7.50 —1.50 0.18 >0.50 NS

24 22.50 1.50

-11®-14 DLMIr 47 42 11.75 30.25 100.43 <0.01 **
5 35.25 -30.25
-11®-18A DLM 47 40 11.75 28.25 87.38 <0.01 **
7 35.25 -28.25
-20®-6 DLM 37 37 9.25 27.75 107.04 <0.01 **
O 27.75 -27.75
-20®-8 DLM 50 50 12.50 37.50 146.03 <0.01 **
O 37.50 -37.50
-20®-25A DIM 22 22 5.50 16.50 62.06 <0.01 **
O 16.50 -16.50

 

 

F2 individual from which F2 family is derived.
F2 individual plant from which F2 family is derived.
Planting batch.

Number of plants.

Observed number.

Expected number.

o-e.

x2=2(d—1/2) 2/e.

df=1.

DLM.

WT.

Phenotype of the F2 parent of the F3 family.
Non-significant deviation of observed to expected ratio.

m<epm+x53+oe~g

60

Table 2.3.1 Chi-square tests for 0:1
segregation ratio in F2 families.

(WT:DLM)

 

 

 

 

Family N" 0*! a” d“ P5
-11’®-10‘A‘ WT' 79 27° 79.00‘ -52.00 —
52° 0.00° 52 00
-20®-7A WT 30 9 30.00 -21.00 -
21 0.00 21.00
-20®-34CA WT 32 12 32.00 —2o.00 -
20 0.00 20 00
-20®-1BA WT 49 11 49.00 -38.00 -
38 0.00 38.00
-20®-7 WT 30 6 30.00 -24.oo -
24 0.00 24.00
-11®-14 nm' 47 42 47.00 -5.00 >0.99 NSF
5 0.00 5.00
-11®-18A um 47 40 47.00 -7.00 -
7 0.00 7.00
-20®-6 DLM 37 37 37.00 0.00 >0.99 NS
0 0.00 0.00
-20®-s um 50 50 50.00 0.00 >0.99 NS
0 0.00 0.00
-20®-25A DLM 22 22 22.00 ' 0.00 >0.99 NS
0 0.00 0.00

 

«ehm*xg:+¢‘0<

11'

hi

F2 individual from which F2 family is derived.

F2 individual plant from which F2 family is derived.

Planting batch.
Number of plants.
Observed number.
Expected number.
o—e.

x2=2(d-1/2) 2/e.
df=1.

DLM.

WT.

Phenotype of the F2 parent of the F2 family.
Chi-square can not be calculated for a class when the

frequency of any category is 0.

Non-significant deviation of observed to expected ratio.

61

Table 2.3.m Chi-square tests for 13:3 (WT:DLM)
segregation ratio in F2 families.

 

 

-11°®-10°A°WT7 79 27° 14.81“ 12.19 11.35 <0.01 **
52° 64.19° -12-19

-20®-7AWT 30 9 5.63 3.38 1.81 >0.05 NS!
21 24.38 -3.38

-20®-34CAW‘1' 32 12 6.00 6.00 6.21 <0.05 *
20 26.00 -6.00

-20®-18AWT 49 11 9.19 1.81 0.23 >0.50 NS
38 39.81 —1.81

-20®-7W'1' 30 6 5.63 0.38 0.00 >0.99 NS
24 24.38 -0.38

 

-11®-14 DLM' 47 42 8.81 33.19 149.22 <0.01 **
5 38.19 -33.19
-1l®-18A DLM 47 40 8.81 31.19 131.52 <0.01 **
7 38.19 -31.19
-20®-6 DLM 37 37 6.94 30.06 155.04 <0.01 **
0 30.06 -30.06
-20®-8 DLM 50 50 9.38 40.63 211.37 <0.01 **
O 40.63 -40.63
-20®-25A DLM 22 22 4.13 17.88 90.07 <0.01 **
O 17.88 -17.88

 

 

F2 individual from which F2 family is derived.
F2 individual plant from which F2 family is derived.
Planting batch.

Number of plants.

Observed number.

Expected number.

o-e.

x2=2(d-1/2) Z/e.

df=1.

DLM.

WT.

Phenotype of the F2 parent of the F3 family.
Non-significant deviation of observed to expected ratio.

nflfifllmfixggqnea

62

Table 2.3.n

Chi—square tests for 1:3

(WT:DLM)

segregation ratio in F2 families.

 

 

 

 

 

Family NI oh 321 dIt 1* PS
-11'8>-10T°WTi 79 27° 59.25° -32.25 68.05 <0.01 **
52° 19.75° 32 25
-20®-7AW'1‘ 30 9 22.50 -13.50 30.04 <0.01 **
21 7.50 13.50
-20®-34CAWT 32 12 24.00 -12.00 22.04 <0.01 **
20 8.00 12.00
-20®-18AW'1' 49 11 36.75 -25.75 69.39 <0.01 **
38 12.25 25.75
-20®-7 WT 30 6 22.50 —16.50 45.51 <0.01 **
24 7.50 16.50
-11®-14 13m" 47 42 35.25 6.75 4.43 <0.05 *
5 11.75 -6.75
-11®-18A DLM 47 40 35.25 4.75 2.05 >0.05 NSI
7 11.75 -4.75
-20®-6 DLM 37 37 27.75 9.25 11.04 <0.01 **
0 9.25 -9.25
-20®-8 DLM 50 50 37.50 12.50 15.36 <0.01 **
0 12.50 —12.50
-20®-25A DLM 22 22 16.50 5.50 6.06 <0.05 *
0 5.50 -5.50
7 F2 individual from which F2 family is derived.
I F3 individual plant from which F2 family is derived.
‘ Planting batch.
I Number of plants.
h Observed number.
a Expected number.
x o-e.
I x2=2(d-1/2)2/e.
5 df=1.
& DLM.
I WT.
¥
1

Phenotype of the F2 parent of the F3 family.
Non—significant deviation of observed to expected ratio.

63

.Ho.ovo um ucmoflmecoflm

>Hamoflumflumum mum mosses coflummmummm omuommxm ocm om>ummno cmozpmn mmocouTMMHo
.ucmoHMHcmHm
>Hamoflumflumum Doc mum moaumu coflummmuomm oouomaxo ocm oo>uomno cmozumn mTUCTHTMMAQ

 

 

 

 

*4HO.oV *mo.OIHo.o mme.OIom.o mZON.OImo.o *mo.OIHo.o mZom.OIom.o OOH!
*«Ho.ov 4*H0.0V mZom.OIom.o .*«Ho.ov *THO.OV mZON.OImO.o mml
*4HO.OV mZON.OImo.o mZom.OIom.O mZON.OImo.o *mo.OIHo.o mZom.OIom.o om!
*5HO.OV Tsao.ov mZom.OION.O mZom.OION.O mZON.OImo.o mme.oA mm!
TTHO.OV mZON.OImo.O mme.OIom.o Tmo.OIHo.O «*Ho.ov mZom.OION.o Hm!
3*HO.OV 4*Ho.ov mZom.OION.o mZON.OImo.o *mo.OIHo.o mme.OA th
mme.oA mZom.OIom.o mZON.OImo.o *3HO.OV *THO.OV *5HO.OV QM!
*TH0.0V **H0.0V mme.OA 3*Ho.ov *3HO.OV mZON.OImo.o fiMl
TTHO.OV mZON.OImo.o mZON.OImo.O TTHO.OV stfio.ov *3HO.OV MMI
mme.OA *THO.0V TTHO.0V «*Ho.ov *THO.OV *4HO.OV HNI
*4HO.OV *4HO.ov mZom.OIom.o *iHo.OV *4HO.OV mZON.OImo.o ONI
emme.OA «mo.OIHo.o *WHO.0V *«HO.OV TTHO.OV *3HO.OV mHl
*THO.OV mme.OIom.o tsao.ov *3HO.OV TTHO.OV *«Ho.ov mHI
«TH0.0V *THO.oV mZom.OION.O ¥¥H0.0V «*Ho.ov *3H0.0V HHI
*THO.OV *«HO.ov mZON.OImo.o *3HO.OV *3HO.OV *THO.OV ml

TTHO.OV *THO.OV mzmm.OIom.o *4HO.OV TTHO.OV mZON.OImo.o ml

»**H0.0V *mo.OIHo.o mZON.OImo.O 4*Ho.ov TTHO.OV *9HO.OV MI

AVHNHV
one m Hume m mums m 93 m t... m in m 33am
.mOHumu coaummoumom
oscomfio manflmmoa ow mmHHflEmwmm mo new mom mmmxamcm mumsomlflco Mo xumEEom m.v.m manme

64

.Ho.ovo um

>Hamofiumflumum mum mowpmu coaummonmom Uouomaxm cam om>ummno cmmzumn

.mo.ovovao.o um

maamoflpmflumum mum moflumu coaummwnoom oouomoxo ocm om>uomno consume

>Hamofiumflumum Doc mum moaumu coaumomnmom Umuomaxw cam om>ummno cmozuon moocmHTMMHo

pcmoHMHcmHm
mmocmuowwflo
ucmoflwflcmfim
mooconomwflo
.ucmoHMHcmHm

 

 

mme.OIOm.o *TH0.0V *mo.OIHo.o mZON.OImo.o mm!
mZom.OION.o *TH0.0V *«Ho.ov mZom.OION.O mm!
mZON.0!mo.O T¥H0.0V *«H0.0V mme.OIOm.O Hm!
mZom.O!om.o TTH0.0V *«Ho.ov mZON.O!m0.0 HP!
mZON.OImO.O *«H0.0V mZOm.0!om.o TTHO.OV HNI
*mo.OIHO.o TTHO.OV *«Ho.ov mme.OIom.o ON!
mZom.0!om.o *THO.OV mZON.OImO.O *mo.OIHo.o OH!
mZON.O!mo.O *¥H0.0V mme.oA *5H0.0V OH!
mZON.OImO.O 4*H0.0V *3H0.0V mZom.O!ON.O HH!
meo.OIHO.O *«H0.0V *«Ho.ov mme.OIom.O m!
*TTH0.0V *4H0.0V TTH0.0V mZom.O!om.o m!
mZom.O!om.O «*H0.0V *5HO.OV mZom.OION.o M!
emZom.OION.O *4HO.OV mZow.0!om.O *3HO.OV H!
mum m muH m Hum m HuH m nOHHflfiflh

 

 

owcmoflo mabflmmom ou mmflaflfimw Hom mo

.mOADmu cofiumomummm

use now momxamcm mnmoomufino mo >um§Esm b.v.m Tammy

65

.Ho.ova um ncmoflweamem

>Hamoflumflumum one moaumn coflumomnomm oouommxm pom oo>ummno comzumn mooconTMMHo
.mo.ovavHo.o um ncmoemncmem

>Hamoflumflumum mum woeumn coflummmnmmm oouoomxo pom om>uomno cmozpmn moocouomwflo m
.DCMUAMflcmflm

>Hamoflumflumum uoc mum moaumn coeummoumom omuoooxm cam oo>uomno CTTZDTQ mTUCTHTMMHQ

 

 

 

 

*mo.O!HO.o .IH0.0V mme.OA **H0.0V tTC‘HO.OV an flmNI®ONI
h._T¢._HO.OV «.«Ho.ov mme.OA «.«H0.0V h.Ce._HO.oV an OI®ONI
«.3HO.OV b.C.:HO.OV mme.0A «*H0.0V «.3HO.OV an UI®ONI

emZON.OImO.o «.«H0.0V *¥Ho.ov 3*H0.0V #TH0.0V an del®HHl
meO.OIHO.O *TH0.0V L:LHO.OV *THO.OV .14H0.0V an— GHI®HHI
*3HO.OV mme.OA h:LHOAVV mZOm.OIom.O «:«HO.oV B3 PI®ONI
«.TH0.0V szm.0!om.o «*Ho.ov Wme.0!om.o *TH0.0V B3 gHI®ONI
«.THO.OV *mo.OIHO.O 4*Ho.ov mZON.OImo.o TTHO.0V H3 6‘MI®ONI
*5H0.0V mZON.OImO.o «*H0.0V mZom.0!om.o 3*Ho.ov B3 flhl®ONl
ntsH0.0V «*Ho.ov *4H0.0V WZON.OImO.o «TH0.0V B3 wHI®HHI

m5 mums To Hum o5 35.4. mm

 

.mOHumH coflummonomm
oecmmflo manflmmom ou mmflHflEmw mm mo Dam How mmmxamcm mumoomuflno wo xumEEDm o.v.m magma

66

Table 2.5.a

Proposed lineage of F2 individuals based on

segregation ratios in F2 and BCl families
(Tables 2.3.a-i).

 

 

I . D . & Flt F2I 3C1?
-3 AaBb AaBb AaBB AaBb
-6 AaBB AaBb AaBB AaBb AaBB AaBb
-8 AaBB? AaBb? AaBb AaBB
-11 AaBb AaBb AaBB AaBb
-18 Aabb Aabb Aabb
-20 AaBB AaBB AaBb AaBB
-21 Aabb Aabb AaBb Aabb
-71 AaBB AaBb AaBB AaBb AaBB AaBb
-81 AaBB AaBb AaBB AaBb AaBB AaBb
-99 AaBB AaBb AaBB AaBb AaBB AaBb

 

 

 

 

I A, a; B, b
alleles at

AaBb (based on segregation in F2 family),
aaBB (based on segregation in BC1 family)
& F1 individuals from 106-C1 X REL-2 cross.

respectively.
I F2 genotype of (106-C1 X REL-2)-8 is:

or

generic symbols for dominant and recessive
Dlml and Dlm2 loci,

 

 

 

 

 

 

Table 2.5.b Proposed lineage of F2 families, based on their
segregation ratios (Tables 2.3.j-n).
106 x REL-2*
F1® -11® AaBb® -20® AaBB®
l l i l 1
-10A WT® AaBB -'7 WT® AaBB
-7A WT® AaBB
-34CA WT® AaBB
-18A WT® AaBB
F2 -14A DLM® aaBb -6 DLM® aaBB
-18A DLM® aaBb -8 DLM® aaBB
-25A DLMO aaff

 

 

 

 

I A, a; B, b generic symbols for dominant and recessive
alleles at Dlm2 and Dlm2 locus,
Underlined are DLM genotypes.

67

 

respectively.

Figure 2.1.a DLM phenotype of an F2 plant in the form of
water-soaked spots on foliage.

 

Figure 2.1.b DLM phenotype of an F2 plant in the form of
necrotic lesions on foliage.

 

68

Figure 2.2 Monogenic recessive segregation ratios in F1,

F2; 5'3: 3C1:

and in 81 of P2 if no phenotypic

segregation for DLM is observed in F1.
Underlined are proposed DLM genotypes.

 

Scheme of generations

Genotypic frequenciess

 

 

Parents 106-C1 (DLM): REL-2 (WT):
_a_g_ AA
106-C1 X REL-2 22 X AA
4 l
F1 A3 1/1
DLM=O%
F1 Aa
l8 to
F2 AA 1/4
Aa 2/4
§§_1/4
DLM=25%
F2 .AA.1/4 .Aa 2/4 aa 1/4
18 lo $8 “to
:5 AA 1/1 (1m AA 1/4 (2/16) 23 1/1 W.)
Aa 2/4(4/nfl
.22 1/4 (2/16)
DLM=O% DLM=25% DLM=100%
106-Cl X F1 32 X Aa
l
13c1 Aa 1/2
fig 1/2
DLM=SO%
REL-2 AA
is 18
S1 ILA 1/1
DLM=O%

 

 

5 A, a; B, b generic symbols for dominant and recessive
alleles at Dhm and Dlm2lrmj, respectively.

69

Figure 2.3.a All possible digenic segregation patterns in

F2 generation,

if 106-C1 is aa-- genotype, and

REL-2 is A---§.'Underlined are DLM genotypes.

 

 

 

 

 

 

 

 

 

REL-2 AABB AABb AaBB AaBb AAbb Aabb

106~C1

aabb AaBb 1/1 AaBb Aabb 1/1 bb /2
Aabb 1 2

11812 AaBB 1/2 AaBB AaBb 1/2

AaBb 1/2 AaBb Aabb 1/2

Aabb

aaBB AaBB 1/1 AaBB AaBb 1/1 2

 

 

AaBb

 

 

 

 

 

 

 

Crossed out are aa-—
because there was no segregation for DLM in F2 families.
§ A, a; B, b generic symbols for completely dominant and

recessive alleles at Dlm1 and Dlm2 loci,

7O

(DLM) genotypes in.F2 families,

respectively.

Figure 2.3.b Expected digenic segregation ratios in F2
generation, if 106-C1 is aaBb genotype, REL-2
is AMflfifi, and DLM does not segregate in F1
generation. Underlined are DLM genotypes.

 

 

(F1) AaBB§ (F1) AaBb (F1) Aabb
$8 $8) $8
AABB 1/, AABB 1/16 AAbb 1/,
AaBB 2/, AABb 2/16 Aabb 2/,
m5 AaBB 2/16 aabb 1/4

AaBb 4/16
AAbb 1/16
Aabb HQ,
1 1.6
2
1.6
aabb HQ,
DLM=25% DLM=18.75% DLM=O%

 

I A, a; B, b generic symbols for completely dominant and
recessive alleles at Dlm1 and Dlm2 loci, respectively.

71

 

Figure 2.3.c

generation,

Expected digenic segregation ratios in BC1
if 106-Cl is aaB— genotype,

is AABba and DLM does not segregate in the F1
generation. Underlined are DLM genotypes.

 

 

 

 

 

 

 

. 106-C1
\ aaBb eeBB
(106-c1) (FJ
(106-c1) (FJ aaBB x AaBB
ang X AaBB i
J, AaBB 1/2
AaBB AaBB 1/, 223341:
AaBb 1/,
new,
aaBb—11.3
DLM=50% DLM=50%
(106-C1) (Ffl (106-c1) (FJ
aaBb X AaBb aaBB X AaBb
l l
AaBB 1/8 AaBB V,
AaBb 2/8 AaBb V,
AaBb Aabb 1/8 WA
1 3 483317.11,
2 fl
aabb U2
DLM=37.5% DLM=50%
(106-C1) (FJ (106-C1) (FJ
gng X Aabb 3353 X Aabb
l l
AaBb 1/, AaBb 1/2
Aabb Aabb 1/, flaw;
383L113
aabb HQ
DLM=25% DLM=50%

 

§ A, a; B, b generic symbols for completely dominant and

recessive alleles at Dlm1 and Dlm2 loci,

72

respectively.

REL—2

 

Figure 2.3.d

Expected digenic segregation patterns in F3
generation,

if 106-C1 is aaBb genotype, REL—2
is AABUI and DLM does not segregate in F1

 

 

 

 

generation. Underlined are DLM genotypes.
F2 AABB F2 AABb F, AaBB
18 l8 l8
AABB V1 AABB V, AABB V,
AABb V, AaBB V,
AAbb 1/. mu,
DLM=0% DLM=0% DLM=25%
F2 AaBb F2 AAbb F2 Aabb
l8 l8 l8
AABB V16 AAbb V1 AAbb V,
AABb V,, Aabb V,
AaBB 2/16 aabb 1/,
AaBb V,6
AAbb V16
AAbb V1,
1 1.6
2 1.6
aabb 1/16
DLM=18.75% DLM=0% DLM=0%
F2 aaBB Fzgng F2 aabb
$8 18 ~L®
9.58m, 52.215111, aabb V.
812m,
aabb 1/,
DLM=100% DLM=75% DLM=O%

 

 

 

 

5 A, a; B, b generic symbols for completely dominant and
recessive alleles at Dlnh and Dlng loci, respectively.

73

.Hovofi o>fimnooou Dadomonofi Haw modafifidm Hum one uh nookuon sudden uoomnomg 4 w

seesaw «m as snow

 

 

 

 

 

 

on mm on ma as m o
P — — —

o

I as
H~.e %
I U
m~.H e on m
o T.

u n
- om m
I
mm.sH m
s 0 e T.
we own «w «H - o... M.
m¢.¢a
O
om.on ww.ma
‘ .I.
em.ma o om
w 0 Nm~MH
om

.Hum .m> «h Ga omuunouuom coaummwumou tan ¢.N ohfimfih

 

& SAG
bnwuvaNMNNNHNONmHmHhHmeHwHMHNHHHOH m w b m m ¢ m N H o
_ _ _ _ _ _

 

 

 

 

no

991nm; 3:! :0 'ON

 

 

$33333» .336156353458 e5 .ANEEAESEAEEHE
. AufiHn\ufiHnnflfiHu\Hfiae moguo‘nom Hm fiouu nofiummoumom 9.2396 mo
owumwuouomumno who one: vampire 30 on.» 33..” Jim Ugoumv panama—Ohm ooh—An.

.umfifiwfium «h a.“ womanoflvonu nowummoumou San mo coauanfluumwu hodosbonh

m . a 053m

75

CHAPTER THREE

INTERCELLULAR FLUID PROTEIN OF A DISEASE LESION MIMIC IN

SUGARBEET (BETA VULGARIS L.)

76

INTRODUCTION

In many cases, systemic acquired resistance (SAR) to
disease, and the hypersensitive response (HR), are induced
when the products of a pathogen's avr genes are recognized
by the products of the R genes of a resistant plant, in what
can be viewed as a form of allelic interaction (Dietrich et
al., 1997). In that interaction, an avr gene conditions the
avirulent phenotype of the pathogen, and the R allele is
responsible for the resistance of the plant, as currently
articulated in the theory of "gene to gene resistance"
(Staskawicz et al., 1995). Physiological and molecular
responses thought to be coordinated with HR include: a) a
rapid oxidative burst (Tenhaken et al., 1995), b) ion
exchanges and fluxes (Staskawicz et al., 1995), c)
strengthening of the cell wall by lignification, by
accumulation of appositions, and by early accumulation of
phenolic compounds (Nicholson et al., 1992; Elliger et al.,
1994), d) production of antimicrobial compounds known as
phytoalexins (Agrios, 1997), and e) induction of a variety
of pathogenesis-related (PR) proteins, such as chitinases
(Staskawicz et al., 1995).

Intensive production and/or accumulation of PR proteins

can be induced by a) biotic agents such as necrosis-inducing

77

pathogenic viruses, bacteria, and fungi (Van Loon, 1983;
B01, 1988), or b) abiotic agents such as specific chemicals:
acetylsalicylic acid, amino acid derivatives such as 1-
aminocyclopropane—l—carboxylic acid, salts of barium and
manganese, benzoic acid, mannitol, polyacrylic acid, and
salicylic acid (Van Loon, 1983; B01, 1988), and c)
physiological conditions such as flowering and callus
culture (Van Loon, 1983). PR proteins can be distinguished
from other pathogen-induced plant enzymes because of these
characteristics (B01, 1988): a) secretion by plant cells
into intercellular spaces, b) protease resistance, and c)
solubility at low pH. PR proteins of some categories have
been found in a range of plant genera, following infection
by various pathogens. Ideally PR proteins can be recognized
as extra bands present compared with protein patterns of
non—infected plants on polyacrylamide gels following
electrophoresis, when stained with general protein stains
such as Coomassie Blue (Van Loon, 1983). The majority of PR
proteins are monomers, with the exception of a single
dimeric type which was classified as a PR-5 (thaumatin—like)
protein (Linthorst, 1991). Most PR proteins have enzymatic
activity following isolation and/or separation. Several
methods have been developed for visualization of specific PR
proteins on polyacrylamide gels, based on their catalytic

interaction with added substrates: a) chitinases (Trudel et

78

al., 1989; Pan et al., 1991; Kalix et al., 1995), and b)
glucanases (Pan et al., 1991; Mathew et al., 1992).

One of the most important roles of chitinases, as a
category of plant PR proteins, is considered to be their
involvement as a defense mechanism against pathogen attack,
especially fungal pathogens (Punja et al., 1993). Chitinases
are known to catalyze the hydrolysis of chitin (B-l,4-linked
homopolymer of N—acetyl-n-glucosamine), a major constituent
of fungal cell walls (Coolinge et al., 1993). Other
substrates for plant chitinases are bacterial
peptidoglycans, glyco-chitin (a soluble chitin derivative),
and chitosan (Coolinge et al., 1993). Based on primary
structure, three classes of chitinases have been proposed:
1, II, and III (Coolinge et al., 1993), although Nielsen et
al. (1994) described a chitinase class IV, found in
sugarbeet. Many chitinases are localized apoplastically. A
few chitinases, such as one basic belonging to class I, are
localized in vacuoles (Coolinge et al., 1993).

Knowing that defense proteins accumulate in
intercellular (ICF) fluid, and that their concentration
increases as a plant's defenses reach a higher level (Parent
et al., 1984), ICF protein investigation in DLM and wild
type plants was a relevant and feasible approach for partial
characterization of the disease resistance physiology of

this sugarbeet DLM phenotype. Protein mobility patterns on

79

SDS PAGE gels, and protein concentration, from isolated
foliar ICF of WT and DLM plants, mostly of F2 individuals
from the foundational cross 106—C1 X REL-2, were determined.
These tests were employed to observe consistency of relative
expression of PR proteins as markers of disease resistance-
related response in DLM plants, and also to ascertain if
important defense proteins are constitutively more expressed

in the DLM phenotype than in normal (WT) plants.

80

MATERIALS AND METHODS

Plant Material

Twelve cloned F2 individuals (nine WT and 3 DLM) and
the DLM parent 106-C1 from the foundational cross (i.e.,
106-C1 X REL-2) plus two additional DLM clones were used in
the SDS PAGE separation of ICF proteins. The two clones were
a) bmsr-F2—dlm-1, an independently identified DLM plant not
closely related to 106-C1, but having common ancestors in
the smooth root breeding germplasm, and b)'a single F2 plant
from the cross (106-C1 X bmsr-F2-dlm-1) that was an intense
DLM phenotype. All of the ramets (copies) were clonally
propagated in vitro, which allowed repetition of the
experiments, and availability of plants at the desirable
growth stage at the time of sampling. Leaves were taken from
plants grown in 3 inch peat pots, with 6 fully developed
leaves. The growing conditions included 2 months in a growth
chamber with a weekly nutrient provided, constant

temperature of 68 F and continuous fluorescent lighting.

81

Intercellular Fluid (ICF) Isolation

Fully expanded non-senescent leaves with petioles were
excised from individual plants immediately before use. In
each case, 5-7 leaves, weighing a total of 10-14 g, were
washed in distilled water and placed in a 500 ml beaker, so
that the leaf blades were completely submerged in water, and

the petioles stuck out of the water. The beakers were placed

”.1 “ um"_‘ll_-\1
;

in a vacuum jar which was evacuated for 25 minutes, so that
water would enter the intercellular spaces. Next, the leaves
were taken out and separately blotted on paper towels, then
rolled and placed inside 50 ml centrifuge tubes, one for
each set of leaves. The tubes were centrifuged for 15
minutes at 1000g, and then an additional 5 minutes at 200g.
After centrifugation, the liquid on the bottom of the tube
was considered the ICF isolate. Up to 1,500 pl of ICF was
obtained with a single such isolation, and this was somewhat
variable. Whenever multiple isolates were obtained from the
same sample of leaves (up to 4 times), the procedure was

repeated starting with the immersion in the beaker of water.

Measurement of Total Protein in ICF Samples

The method for measuring total protein in samples was
based on the binding of proteins to a specific dye
(Coomassie Brilliant Blue G-250) (Bradford, 1976) whereby

the protein-dye complex causes a shift of the absorption

82

maximum, proportional to the concentration of protein
present. The corresponding absorbance readings were measured
by a Varian Cary 50 Bio spectrophotometer at A=595 nm.

To measure concentration of protein per sample, a
standard curve was created, for every experiment separately,
so that known (standards) and unknown (samples) protein

concentrations were processed simultaneously. This was also

1.. oer-LL: A 1.: '

a way of testing that the spectrophotometer was operating

‘ ..
m.
I

1

normally, and that the protein assay solution (from Bio—Rad)
was not non-specifically binding to other classes of
molecules (e.g., carbohydrates). To create the standard
curves, bovine serum albumen (BSA, Sigma Chemical Co.) was
used in concentrations 0.0, 0.2, 0.6, 0.9, 1.2, and 1.5 mg
ml*, each amount mixed with 1.5 ml of the Bradford reagent
solution. The concentrations were plotted against the
absorbance readings. Each 20 ul of ICF was mixed with the
1.5 ml of Bradford reagent solution and absorbance readings

were taken immediately. Absorbance readings were converted

to concentration by using the standard curve.

SDS Polyacrylamide Gel Electrophoresis

A reducing buffer was used to reduce protein disulfide

 

bonds of all ICF proteins, so that the proteins were

unfolded (Payne, 1976). This buffer contained 47.5% v/V dd

83

EEO, 6.25 mM TrisHCl pH 6.8, 10% vol. glycerol, 4% m/v

sodium dodecyl sulfate, 5% v/v 2-mercaptoethanol, and 5% m/v

bromophenol blue (Payne, 1976). Redissolving buffer was used

 

to redissolve proteins after the freeze drying of ICF in
order to adjust their concentrations. This buffer consisted

of 0.01 M Na acetate in water, pH 5.0. Separation gels

 

(12.5% acrylamide-bis) were prepared with 40% m/v
acrylamide-bisacrylamide (30:0.8), 0.375 M TrisdmCl pH 8.8,
2.80% v/v dd Hg), 0.125% v/v N, N, N',N'-tetramethyl-
ethylenediamine, and 0.00088% ammonium persulfate (Payne,
1976). Chitinase gels were prepared to contain 0.01% m/v
glyco-chitin, as a substrate used for visualization of

chitinase activity (Trudel et al., 1989). Stacking gels were

 

prepared with 15% m/v acrylamide-bisacrylamide (30:0.8),
0.062 M Tris-HCl pH 6.8, 31.2% v/v dd H2O, 0.001% m/v sodium
dodecyl sulfate, 0.25% v/V N, N, N',N'-tetramethyl-
ethylenediamine, and 0.0009 m/v ammonium persulfate (Payne,

1976). Running buffer was prepared with 3% m/v Tris-base,

 

14.4% m/v D-glycine, 0.6% m/v sodium dodecyl sulfate, and
77% v/v dd Hg) (Payne, 1976).

After mixing, gels were left to polymerize for 30-45
minutes at room temperature, prior to sample loading. For
each sample, 30 ul of ICF was mixed with 10 ul of reducing
buffer in 1 ml microtubes. All samples were placed in

microtubes and placed in boiling water for 4 minutes to

84

“IT.

1"
.._

If . T. 75

permit 2-mercaptoethanol-assisted denaturation of protein
quaternary structure, thus converting proteins to linear
forms. The role of SDS was to make a film on the linear
protein molecules, keeping them linear during the
electrophoresis process. Electrophoretic mobility of the
protein molecules was size-dependent, thus smaller molecules

migrated farther during the electrophoresis process. About

”I.-.“
. _ _ a . 1
‘
r . v - .m

35 ul for each sample (ICF+reducing buffer) was loaded into

V—L
I

each of the 10 wells on each 9X6 cm gel, respectively. In
both cases involving electrophoresis, the samples contained:
a) same volumes of ICF per sample, and/or b) equalized
amounts of proteins in ug uld'of a sample. The
electrophoretic protein apparatus was model EC142 (Savec
Company). A continuous type (2 buffer chambers) of
electrophoresis was run at 45 V for 12.5% polyacrylamide
gels (~2 hours), and at 70 V for 15% (~2 hours) and 20%

polyacrylamide gels (~3.5 hours).

Protein and Chitinase Activity Staining

Separation gels for visualization of general ICF

 

protein patterns were stained in a standard solution of

 

Coomassie Blue for two hours. Coomassie Blue is a dye that
covalently binds to proteins (Schfigger et al., 1988). The

solution contained 0.25% m/v Coomassie Brilliant Blue R 250,

85

45.4% v/v methanol, 9.2% v/v glacial acetic acid, and
45.15% v/v dd H2O (modified from Payne, 1976). Following
that, the gels were de-stained for approximately 10 hours at
room temperature, in a solution that contained 5% v/v
methanol, 7.5% v/v glacial acetic acid, and 87.5% v/v dd Hg)
(Payne, 1976). Gels were photographed under light from a

fluorescent lamp, using the Eagle Eye II system (Stratagene

 

Ltd.), and stored in 7% aqueous acetic acid solution.

Separation gels for visualization of chitinase activity

 

were incubated immediately after electrophoresis in 200 ml
of a buffer solution (100 mM sodium acetate, pH 5.0, with 1%
v/v Triton X—100) for 2 hours at 37°C. Gels were rinsed once
for about a minute with dd H30 and incubated in 200 ml of a
second buffer solution (500 mM TrisHCl, pH 8.9, 0.01% m/v
calcofluor white), for 5 minutes in the dark at room
temperature (modified from Trudel et al., 1989). Gels were
stored in dd H33 and photographed under UV light, using the

Eagle Eye II system (Stratagene Ltd.).

86

RESULTS

Investigation of intercellular fluid (ICF) proteins was
done in order to determine if the DLM phenotype differed
from the WT phenotype in the expression of those proteins. A
benefit of using ICF rather than total leaf protein extract
is that PR proteins are specifically and easily extractable
from intercellular spaces, absent of both structural
proteins, including membrane and cell wall constitutive
proteins, and intracellular soluble proteins (Parent et al.,
1984). Three primary methods of protein comparison between
WT and DLM plants were used: 1) electrophoretic separation
patterns of ICF proteins on SDS PAGE gels visualized with
Coomassie Blue R 250, and 2) electrophoretic localization of
chitinase activity from ICF, visualized on SDS PAGE gels and
3) spectrophotometric determination of total ICF protein

concentration.

SDS PAGE of ICF Proteins

Denaturing SDS PAGE of ICF proteins with subsequent
Coomassie staining was used as a protein separation and
visualization technique. Results of 5 separate SDS PAGE-
based experiments were analyzed in order to compare a)

general protein patterns, intensity, and their associations

87

with spectrophotometrically determined protein
concentrations and b) differences in visualized chitinase
activity.

SDS PAGE Experiment #1 involved 12.5% polyacrylamide

 

SDS gel, equal volumes of unadjusted ICF for each sample,
stained with Coomassie Blue for visualization of total ICF

protein (Figure 3.1). Lanes 1-6 represent ICF of 6 F2 WT

1*"?- Ai DA 0 (‘1'
C

individuals. Lanes 7 and 8 were from two DLM F2 individuals,
and lanes 9 and 10 were from cloned DLM individuals not
derived from the foundational cross. Protein patterns for
each of the 4 DLM individuals were of higher overall
staining intensity than for any the 6 WT individuals.
Isolates from DLM individuals showed more intensity in at
least two specific lanes: F2DLM2 (lane #8) and DLMa (lane
#9) than any of isolates from the WT individuals (Figure
3.1). The lowermost stained areas on the gel are accumulated
peptides and amino-acid chains, both of which are probably
short and/or broken. Total staining intensity on SDS gel
generally corresponded to the concentration of ICF protein
measured spectrophotometrically (Table 3.1).

SDS PAGE Experiment #2 included ICF from 8 plants out

 

of the 10 used in experiment #1. Lanes were equalized for
protein concentration (2 mg mlq) and run in 20%
polyacrylamide SDS gel, to facilitate the differential

separation of proteins with low molecular weight (i.e., the

88

ones of greatest mobility). The clearest differences between
WT and DLM protein patterns were observed in a single
protein band (Figure 3.2) that was visualized as an
intensive one in DLM plants whereas in WT plants it was
completely absent or present only as a weak protein signal.
The lowermost stained areas on the gel are probably broken
peptides and amino-acid chains.

SDS PAGE Experiment #3 compared ICF general protein

 

patterns between different consecutive isolates of WT and
DLM plants in 12.5% polyacrylamide SDS gel. The experiment
included 3 genotypes: 1) 106-C1, 2) a single DLM F2
individual, and 3) a single WT F2 individual. Four
consecutive ICF isolations from the same leaves were done,
and isovolumes of ICF obtained from the first, second, and
fourth isolations were used in the gels (Figure 3.3). Both
DLM individuals showed higher protein staining intensity
than the F2 WT individual and expressed at least one
specific protein band that was not present in WT samples.
This band was estimated to be a protein of about 29 kDa,
based on molecular weight standards run on the same gel.
Consecutive ICF isolates of all genotypes had greater
staining intensity and reflected greater proportionate
intensity of all bands rather than individual band presence

or absence, or differential intensities of specific bands.

89

SDS PAGE Experiment #4 was done to visualize protein

 

patterns between WT and DLM individuals, in 12.5%
polyacrylamide SDS gel. Four consecutive ICF isolations from
comparable leaves of a single WT and a single DLM individual
that was not derived from or part of the foundational cross
were used to separate and visualize proteins, as well as
chitinase activity (Figure 3.4). With all 4 ICF isolates
from the DLM plant, the proteins stained with Coomassie Blue
were much more intensive for all visible bands, than with
corresponding isolates of the WT plant. Chitinase activity
appeared localized in one band, which was intense for all 4
DLM samples, whereas it was barely detectable in the WT
samples. Staining intensity of total protein and chitinase
decreased with each consecutive flush, but there were no
clear differences in the proportions of the specific bands
between flushes of the same leaves.

SDS PAGE Experiment #5 was a comparison of general

 

protein patterns from ICF, and visualization of chitinase
activity therein, for 4 WT F2 individuals, 2 F2 DLM
individuals, and 2 DLM individuals not derived from the
foundational cross. Protein concentrations of all samples
were adjusted to 2 mg per one ml of sample by redissolving
the ICF isolates in adequate amounts of redissolving buffer.
ICF protein concentrations prior to their equalization were

determined by the protein concentration assay #1,

90

corresponding with Table 3.1. Adjustment of protein
concentrations allowed for visualizing differences in
intensity or presence of specific protein bands, at 12.5%
polyacrylamide SDS gels. Protein staining showed a clearly
higher intensity of at least 2 individual bands in all DLM
samples compared to WT samples (Figure 3.5). Chitinase
activity visualization from ICF samples from at least three
out of four DLM individuals showed conspicuous chitinase
activity bands, while samples derived from all four WT

individuals showed no conspicuous chitinase activity.

Spectrophotometric Determination of ICF Protein
Concentration

Two experiments for comparing the protein
concentrations in ICF of WT vs. DLM plants were done: ICF

Protein Concentration Experiment #1 was done to compare

 

single ICF isolates between WT and DLM individuals. Eight F2
individuals (six WT and two DLM), plus two individuals
(bmsr-F2-dlm-1, and F2 individual #50 from the cross 106 X
bmsr—F2—dlm-1) were used in the experiment. The
concentration range of proteins in the ICF of F2 WT

individuals was in the range 0.011 to 1.330 mg ml“,

x=0.570 Ox=0.397, CV=69.6% (Table 3.1). ICF protein in the

samples of the four DLM plants ranged from 0.835 to 2.190 mg

91

mld, R=l.6l, Ox=0.488, CV=30.4% (Table 3.1). The average
protein concentration in ICF of DLM individuals was 2.82
times higher than the average protein concentration of the
WT individuals. Yet, the protein concentration for the DLM
individual with the lowest concentration was 1.59 fold lower
than the protein concentration (1.33 mg mld) for the WT
individual with the highest concentration. Analysis of
variance (ANOVA) using the F test could not be applied, due

to limited size of the experiment and lack of replication.

ICF Protein Concentration Experiment #2 was a

 

comparison of two consecutive ICF isolations from the same
leaf material of the WT and DLM phenotypic categories. WT
individuals were represented by three F2 and one F2 clone
from the foundational cross (106-C1 X REL-2). DLM
individuals were two F2 clones from the foundational cross,
the DLM parent (106-C1), and one DLM clone (106 X bmsr-F2-
dlm—l) that was neither derived from the foundational cross,
nor closely related to 106-C1. Protein concentrations in the

ICF of the four WT individuals ranged from 0.00 to 0.568 mg

m1‘1, with 2:0.311 and Ox=0.203, CV=65.3% (Table 3.2). ICF

protein in the samples of the four DLM plants ranged from

0.192 to 1.690 mg mld, x=0.842, Ox=0.60, CV=71.21% (Table
3.2). Average protein concentration in ICF of the DLM clones

was 3.3 times greater than in that of the WT ones. The

92

protein concentration for the DLM individual with the lowest
concentration (0.192 mg mld) was 3.0 fold lower than the
protein concentration (0.568 mg mld) for the WT individual
with the highest concentration. Analysis of variance (ANOVA)
could not been applied because of limited size of the
experiment and lack of replication.

Both ICF protein concentration experiments were
initiated as a technique for determining and adjusting
protein concentrations per volume of sample, for use in SDS
PAGE experiments #2, and #5, respectively. The results of
both experiments showed large standard deviations and
coefficients of variation, due to lack of replication of the
treatments and small range of the genotypes used. A larger
range of genotypes was not achieved due to limited
availability of different clonal genotypes. In each
experiment, mean ICF protein concentration of DLM plants was
greater (2.7 and 3.3 fold) than that of WT plants. Even if
not statistically testable, the total mean ICF protein
concentration of DLM plants was more than twice greater than
the mean ICF protein concentration of WT plants in each
experiment.

Overall, ICF protein including chitinase was generally
several fold more abundant in leaves from DLM sugarbeet with
water-soaked and/or necrotic areas than in leaves from WT

plants.

93

DISCUSSION

Early reports of four, and later six, novel foliar
proteins visualized by polyacrylamide gel electrophoresis
(PAGE) concerned tobacco plants expressing the
hypersensitive response (HR) following infection with
tobacco mosaic virus (TMV) (Van Loon et al., 1970; Bol,
1988). The proteins were termed "pathogenesis-related (PR)"
proteins by Antoniw et al. (1980), to help classification of
similar proteins, known to be a) induced upon pathogen
infection, b) spread widely through the phloem to the
infection sites, and c) characterized by enzymatic activity
(Parent et al., 1984; Bol, 1988). Since then, similar
inducible PR proteins have been reported and described in
many monocotyledonous and dicotyledonous species. Inducing
factors have included: necrosis-inducing pathogens, such as
viruses, bacteria, and fungi, as well as a number of
specific chemicals (Van Loon, 1983; Bol, 1988). Van Loon
(1983) also reported induction of PR proteins in several
plant species under physiological conditions such as
flowering and in vitro growth on cytokinin- and auxin-rich

media.

94

In the case of the sugarbeet DLM phenotype
investigated in this thesis, foliar ICF protein
concentration was measured, and the proteins were
electrophoretically separated and visualized in the case of
WT and DLM phenotypes in the same genetic background.
Concentrations of total protein in ICF measured in two
separate experiments were less than half from WT compared
with DLM plants (Tables 3.1, 3.2). Visualization of ICF
protein patterns on SDS PAGE gels indicated that DLM plants
accumulated higher levels of total protein (Tables 3.1, 3.3,
3.4), but also of at least one specific protein (Figures
3.1-5). A single band or zone was visualized to be with
chitinase activity (Figures 3.4.b, 3.5.b). In one case
(Figures 3.4.a,b), chitinase activity was visualized as a
thick intensive band and appeared to correspond to a major
ICF protein band (fraction) visualized by the general
protein stain (i.e. Coomassie Blue) that accounted for a
large portion of the protein in the ICF.

There are few reports connecting induced resistance
responses and PR proteins with the DLM phenotype. Some DLM
cases have constitutively elevated levels of PR gene
transcripts: PR-l and PR-5 in accelerated death (ACD)
mutants of Arabidopsis (Greenberg et al., 1994), PR-l in
lesion simulating disease (LSD) mutants of Arabidopsis

(Dietrich et al., 1994), PR-l, PR-2, PR—5 in lesion

95

simulating disease mutants of Arabidopsis (Weymann et al.,
1995), and both PR-l and PR—Q (chitinase) in lesion-mimic
transgenics in tobacco (Herbers et al., 1996). Thus, this
present report appears to be the first, dealing with a case
of DLM in a crop plant, to involve ICF total protein
patterns and chitinase activity. Although ICF protein
including chitinase was generally more abundant in leaves
from DLM sugarbeet with water—soaked and/or necrotic areas
than in leaves from WT plants, chitinase could not be used
as a unique molecular marker of this sugarbeet DLM, because
many leaves of WT plants contained chitinase. Because of
their antimicrobial role in active defense mechanisms, PR
proteins in plants have been extensivelystudied (Linthorst,
1991). A knowledge of the genetic control of either the
constitutive, or of the more easily inducible PR proteins as
appear to be expressed in DLM sugarbeet has prospects for
use in enhancing some aspects of disease resistance in
commercial sugarbeet cultivars. The possibility exists that
either or both of the two Dlm loci identified here encode
defense functions that have not been described yet. To
realize such potential, an important step might be to
genetically separate the extensive tissue damage of water-
soaked and lesion areas on DLM foliage from the presence of
elevated levels of PR proteins, and of other defense

metabolites as well.

96

The most widely used classification of PR proteins is
still that of Van Loon et al. (1987). Each of the five PR
protein groups (PR-1 to PR—5) contains proteins that are
serologically related, with very similar molecular weights,
and with similar amino acid sequences at a "domain" at C-
terminus: a) PR-la, -1b, and -1c (molecular weight 17 kDa)
have been identified in at least 8 mono- and dicotyledonous
plants (Linthorst, 1991), as basic and acidic monomeric
proteins. Their specific function has not yet been
determined. However, because of their wide-spread occurrence
and high inducibility by necrotizing and non-necrotizing
pathogen attack, it is thought that they have an important
role in defense mechanisms, and also in general stress
responses; b) PR—2a, -2b, and -2c are related acidic
proteins, and two additional acidic and one basic protein
have been found with different degrees of serological
relationship to PR-2a, -2b, and -2c (Linthorst, 1991); it is
not clear if they should be classified as PR-2 proteins, but
they as well as all PR-2 proteins have expressed B-1,3-
glucanase activity (Linthorst, 1991); c) PR-3a and -3b are
extracellular (as are all acidic PR proteins) and protease
resistant, with similar molecular weights; PR-3 proteins
have chitinase activity, that is they hydrolyze chitin, a
polymer of B-l,4-N—acetylglucosamine; PR-3 proteins are also

reported to accumulate in some floral organs (Linthorst,

97

1991); d) PR-4 proteins are acidic with molecular weights of
13 to 14.5 kDa; proteins known as r1, r2, $1, and $2 belong
to this group; all three were induced in tobacco leaves by
inoculation with TMV (Linthorst, 1991); e) PR-5 proteins are
referred to as thaumatin-like proteins; three distinct
proteins are known in this group; two are monomers with
molecular weight of 24 to 25 kDa (Van Loon et al., 1987),
and the other is a dimer with subunits of about 14 kDa each;
all three were found in TMV infected tobacco plants
(Linthorst, 1991).

PR-3 chitin-(hydrolyzing) degrading enzymes, abundant
proteins found in many species, are important and well
studied PR proteins. Most are 25-35 kDa monomeric proteins
(Coolinge et al., 1993; Graham et al., 1994), classified
into 4 different classes (I-IV). The majority of isolated
chitinases function as endo-hydrolases, meaning that they
catalyze hydrolysis of chitin from within that polymer
(Graham et al., 1994). The presence of chitinases is
developmentally controlled. In addition to their defense
function(s), chitinases have a role in the inactivation of
lipo—oligosaccharide signal molecules produced by some
Rhizobium species (Coolinge et al, 1993), in early embryo
development (Coolinge et al., 1993; Graham et al., 1994),
and in pollination and sexual reproduction (Graham et al.,

1994).

98

The differences in protein patterns of healthy and
pathogen-infected sugarbeet tissues have been reported in
several cases (Fleming et al., 1991; Rousseau—Limouzin et
al., 1991; Coutts et al., 1994). Fleming et al. (1991)
described tobacco-necrosis Virus (TNV)-infected sugarbeets'
major intercellular leaf proteins in SDS PAGE gels. Rosseau—
Limouzin et al. (1991) described total protein extracted
from leaves by low pH buffers, after infection with
Cercospora beticola. Once selectively isolated, the acidic
and basic proteins were separated by cation and anion
exchange chromatography, and visualized on polyacrylamide
gels without use of SDS. Coutts et al. (1994) visualized
differences in general protein patterns between a) Polymyxa
betae-infected and non-infected sugarbeet seedling roots,
and b) suspension cultures treated and untreated with
Botrytis cinerea cell walls using two dimensional iso—
electrofocusing SDS PAGE. All of the described experiments
cases (Fleming et al., 1991; Rousseau-Limouzin et al., 1991;
Coutts et al., 1994) confirmed major differences present in
general protein patterns between healthy vs. plants infected
by various pathogens. In all three experiments, infected
organs and cells gave higher general protein staining
intensities and also higher intensity of some individual
bands, or spots. When Rousseau-Limouzin et al. (1991)

compared general leaf protein patterns from a) sugarbeet

99

infected with leaf spot pathogen Cercospora beticola, with
b) purified tobacco PR proteins from TMV-infected tobacco,
the similarities between proteins from the infected
sugarbeets and the infected tobacco plants were much greater
than those between healthy and infected sugarbeet plants.

The Dlm2 locus, with the dominant Dlm2 allele, more
likely than the Dlm2 locus, which is homozygous recessive in
DLM plants could be a PR protein locus. It's function may be
in encoding for PR protein production and/or accumulation in
intercellular spaces. It is more likely that Dlm2 is a early
acting regulatory locus and that it's function is in
regulation of expression of PR protein loci, and other
defensive loci. The role of Dlml locus in the DLM phenotype
is unknown, however the involvement suggests some kind of
loss of function, possibly of a feedback regulatory

operation.

100

Table 3.1 Spectrophotometric determination of ICF protein
concentrations, experiment #1: Protein
concentrations in foliar ICF of WT and DLM

 

 

 

 

individuals.

Genotype Protein [mg ml'l]

r2 WT—J. 0. 011"

F2 “2 0 . 54 0

F2 WT3 0 . 34 0

F2 WT4 1 . 330

F2 WTS 0 . 580

F2'WT6 0.620

WT Phenotypic mean 0.570
Ox=0.397 CV=69.6%

F2 DLMl 0 . 835

F2 DLMZ 1 . 690

bmsr-F3-dlm-1 2 . 190

(106-C1 Xbmsr-F3-dlm-l) -50 1 . 715

DEM Phenotypic mean 1.605

Ox=0.488 CV=30.4%

 

101

Table 3.2 Spectrophotometric determination of ICF protein
concentrations, experiment #2: Protein
concentrations in foliar ICF of WT and DLM
individuals for each of two flushes from the same

 

 

 

 

 

leaves.
Genotype ICF Protein [mg ml'l]
isolate 2
F2-1 1 0.274 0.568
2 0.862
F2 WTl 1 0.000 0.180
2 0.359
F2 WT2 1 - 0.282
2 0.282
F2 WT3 1 0.000 0.000
2 0.000
'WT Phenotypic 0.2575 Ox=0.206
mean CV=80%
106-C1 1 1.497 1.119
2 0.741
F2 DDMl 1 1.825 1.690
2 1.554
F2 DLMZ 1 0.185 0.369
2 0.553
(106-Cl x.hmsr- 1 0.227 0.192
F3-dlm-l) -50 2 0 . 157
DLM Phenotypic 0 8425 Ox=0.600
mean CV=71.21%

 

102

.mmemH zen .m> as me “mouse
umnu mpnmn Osweowmm OD ucflom OD mom: who m30HHm HounONfiHomo

.mmOHU HMGOHDMUGSOM Eonm Doc SAG .m> mm SAG .m> am 83
mo mmUH nufi3 mmeH oumummom 0» com: mum mzonhm HMOfluHo>o

H

mBS mm .
#83 mm .
MHZ «m .
NBS um .
HHS mm .H

 

.Hom Cu poflflmmm moESHo> mUH Hodge Suez
.mHosvfl>Hpcfl SAD .m> Hz Eoum coxmu mUH .mcflafimum osHm oflmmmEooo
mnflonHOM THHHOHQ Gofiumummom mwdm mom aflououm moH HMHHom a.m ousmwm

103

.mmouo HmcofiumpGSOM Eouw uoq Eda .m> um SAD .m> am as
no mmUH Sues monH euphmmom 0» tom: one msonnm Hmofluuo>o

I-i

semen .

MEAD
«SAD um
HEAD um

\DPQO‘O

104

me: am
semen
«as am .
Has an .H
"mama

NMV‘

 

.Hom

Cu Umfiammm maoflumuucoucou aflououm Hodge sues .mHmsofl>HUGH

Eda .m> Hz Eoum coxmu moH .mcficflmum TSHm mammosoou
mnflsoHHOM moHHMOHQ GOADMHmmom m0<m mam .cflouowm mUH HmHHom N.m ohsmflm

.mman SAD .m> 93 CH
Houqu omen meson UHMHome uGHom Ou mom: who msoHHm HouGoNHHomo

.HBS mm .m> HEAD mm .m> HUImOH
Mo mmUH onHuHSE nuHB mmeH mumummwm ou com: one mzohhm HMUHuHo>o

mUHMUcmum
uanmz smHsomHos .OH HHH HH H HHH HH H HHH HH H
>H Hes um .m 0H m m e m m e m m H
HH Hes ”m .m
H Hes «m .s
>H HEEnm.m
HH quo Nm .m
H quo «m .4
>H Ho-moH .m
HH Ho-GOH .m
H Ho-moH .H
GUMHOmH .mUH "UCMHH

 

HHS um HEAD mm HUImOH

.Hmm OD ooHHmmm moEsHo> mUH Hodge nuHs .mHmspH>Hch 2A0 .m>
Hz Eon“ coxmu moumHomH mUH 0>Husoomcou .meHchum dem onmmEooo
mcHsoHHOM moHHuoum GoHumummmm Home mam .CHTDOHQ mUH HmHHom m.m mudmfim

105

.mocmH mo mHHmm mcHDWMHucou 3onm Cu pom: mum monHm HmoHuHm>o

xGmHQ . m

>H HEAD . m

>H HEB . b

HHH HEAD . m

HHH HHS . m

HH HEAD . v

HH HEB . m

H HEAD . N

H HEB . H
.mUH OCMA

 
 

,3

.mHom Cu pwHHQO mmESHo> mUH Hodge
ADA; .mHmDUH>HUGH SAD .m> BB Eouu cmxmu moumHOWH mUH 0>Hudoomsou
.mchHMDm huH>Huom ommcHuHeo AA one .mchHmum ode onmmEooo

Am mGHBOHHOM mmHHMOHQ GOHDMHmmmm mofim mam .GHTDOHQ mUH HmHHom v.m mmhzmHm

106

mpHMUGMHm
uAmHTS HdeooHoe .OH

xann .m DEAD .m
.mmeH EAD .m> BB Ga HOMMHU DEAD .m mEAD .m
omen meson oHuHoomm mu uchm mZAQ .b NSAQ mm >
CD pom: ohm mEowum HMDCONHwomo NEAD mm .m HSA mm .m
. quo ”H .m 3.3 mm m
.mmouo HMGOHDMUGSOM EOHM 393 mm .w mHS «h .v
Doe EAD .m> mm .SAQ .m> «m .53 2.3 mm .m «93 «m M
AC mmUH suds mocmH oumummom «a: «m .N H93 «m N
OD poms ohm msouhm Hmoauhm>o HHS mm .H xGmHA .H

. "TcMA TGMA

OH m w b m m w m N H 0H m m b m m ¢ m N H

 

Q.mEAD EAD «m BS mm A D.MEAD EAD um BS mm Am

A
.mHom ow poHHmmm

mcoHumnucoocoo GHouonm Hmsoo AuHs .mHm5oH>Hch 2AA .m> Hz Eonm norm»
mUH .mcHGHmum >DH>Huom ommcHuHAU An one .manHMDm ode onmmEooo
Am mCHBOHHou moHHwonm GOHDMHmmom mw<m mam .CHTHOHQ mUH HmHHom m.m mousmHm

107

BIBLIOGRAPHY

108

BIBLIOGRAPHY

Agrios, G.N. 1997. Plant pathology. 4th. Edition. Academic
Press, San Diego.

Antoniw, J.F., C.E. Ritter, W.S. Pierpoint, and L.C. Van
Loon. 1980. Comparison of three pathogenesis—related
proteins from plants of two cultivars of tobacco
infected with TMV. Journal of General Virology 47:79-
87.

Bent, A.F. 1996. Plant resistance genes: function meets
structure. The Plant Cell 8:1757-1771.

Bol, J.F. 1988. Structure and expression of plant genes
encoding pathogenesis-related proteins. In Verma
D.P.S., and R.B. Goldberg (ed). Temporal and spatial
regulation of plant genes. Springer, New York. pp. 201—
221.

Bosemark, N.O. 1993. Genetics and breeding. In Cooke, D.A.
and R.K. Scott (ed). The sugar beet crop. Chapman and
Hall, London. pp. 67-119.

Bradford, M.M. 1975. A rapid and sensitive method for the
quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding.
Analytical Biochemistry 72:248—254.

Coolinge, D.B., K.M. Kragh, J.D. Mikkelsen, K.K. Nielsen, U.
Rasmussen, and K. Vad. 1993. Plant chitinases. The
Plant Journal 3:31-40.

Coutts, R.H.A., D.J. Linton, and G.P. Bolwell. 1994.
Patterns of protein synthesis in infected and stressed

sugar beet (Beta vulgaris L.). Journal of
Phytopathology 142:274-282.

Dangl, J.L., R.A. Dietrich, and M.H. Richberg. 1996. Death
don’t have no mercy: cell death programs in plant-
microbe interactions. The Plant Cell 8:1793-1807.

Delaney, T.P. 1997. Genetic dissection of acquired
resistance to disease. Plant Physiology 113:5-12.

Dietrich, R.A., T.P. Delaney, S.J. Uknes, E.R. Ward, J.A.
Ryals, and J.L. Dangl. 1994. Arabidopsis mutants

109

simulating disease resistance response. Cell 77:
565-577.

Elliger, C.A., and J.M. Halloin. 1994. Phenolics induced in
Beta vulgaris by Rhizoctonia solani infection.
Phytochemistry 37(3):691-693.

Emerson, R.A. 1923. The inheritance of blotch leaf in maize.
-Cornell University Memoir 70:1-16.

Fleming, T.M., D.A. McCarthy, R.F. White, J.F. Antoniw, and
J.D. Mikkelsen. 1991. Induction and characterization of
some of the pathogenesis-related proteins in sugar
beet. Physiological and Molecular Plant Pathology
39:147-160.

Graham, L.S., and M.B. Sticklen. 1994. Plant chitinases.
Canadian Journal of Botany 72:1057-1083.

Greenberg, J.T., and F.M. Ausubel. 1993. Arabidopsis mutants
compromised for the control of cellular damage during
pathogenesis and aging. The Plant Journal 4:327-341.

Greenberg, J.T., A. Guo, D.F. Klessig, and F. Ausubel. 1994.
Programmed cell death in plants: a pathogen-triggered
response activated coordinately with multiple defense
functions. Cell 77:551-563.

Hayward, H.E. 1938. The structure of economic plants.
Macmillan, New York. pp. 246-250.

Herbers, K., P. Meuwly, W.B. Frommer, J-P. Métraux, and U.
Sonnewald. 1996. Systemic acquired resistance mediated
by the ectopic expression of invertase: possible hexose
sensing in the secretory pathway. The Plant Cell 8:793-
803.

Hoisington, D.A., M.G. Neuffer, and V. Walbot, 1982. Disease
lesion mimics in maize. Developmental Biology 93:
381—388.

Hornbrook, A.R., and C.O. Gardner. 1970. Genetic study of
'necrotic leaf spot' mutation induced in an inbred line
of maize (Zea mays L.) by thermal neutron irradiation
of seed. Radiation Botany 10:113-117.

Hosemans, D., and D. Bossoutrot. 1983. Induction of haploid
plants from in vitro culture of unpollinated beet
ovules (Beta vulgaris L.). Zeitschrift f0r
Pflanzenzfichtung 91:74-77.

110

Hu, G., T.E. Richter, S.H. Hulbert, and T. Pryor. 1996.
Disease lesion mimicry caused by mutations in the rust
resistance gene rpl. The Plant Cell 8:1367-1376.

Jabs, T., R.A. Dietrich, and J. Dangl. 1996. Initiation of
runaway cell death in an Arabidopsis mutant by
extracellular superoxide. Science 273:1853-1856.

Kalix, S., and H. Buchenauer. 1995. Direct detection of B-
1,3-glucanase in plant extracts by polyacrylamide gel
electrophoresis. Electrophoresis 16:1016-1018.

Keller, W. 1936. Inheritance of some major color types in
beets. Journal of Agricultural Research 52(1):27-38.

Kosslak, R.M., J.R. Dieter, R.L. Ruff, M.A. Chamberlin, B.A.
Bowen, and R.G. Palmer. 1996. Partial resistance to
root-borne infection by Phytophthora soyae in three
allelic necrotic root mutants in soybean. The Journal
of Heredity 87:415-422.

Kosslak, R.M., M.A. Chamberlin, R.G. Palmer, and B.A. Bowen.
1997. Programmed cell death in the cortex of soybean
root necrosis mutants. The Plant Journal 11:729-745.

Langford, A.N. 1948. Autogenous necrosis in tomatoes immune
from Cladosporium fulvum Cooke. Canadian Journal of
Research 26:35-64.

Linde-Laursen, I., 1972. A new locus for colour formation in
beet, Beta vulgaris L. Hereditas 70:105-112.

Linthorst, H.J.M. 1991. Pathogenesis-related proteins of
plants. Critical Reviews in Plant Sciences 10(2):123-
150.

Marchetti, M.A., G.N. Bollich, and F.A. Uecker. 1983.
Spontaneous occurence of the Segiguchi lesion in two
American rice lines: its induction, inheritance, and
utilization. Phytopathology 73(4):603-606.

Mathew, R., and K.K. Rao. 1992. Activity staining of
endonucleases in polyacrylamide gels. Analytical
Biochemistry 206:50-52.

Murashige, T., and F. Skoog. 1962. A revised medium for

rapid growth and bio assays with tobacco tissue
cultures. Physiologia Plantarum 15:473-497.

lll

Nicholson, R.L., and R. Hammerschmidt. 1992. Phenolic
compounds and their role in disease resistance. Annual
Review of Phytopathology 30:369-389.

Nielsen, K.K., K. Bojsen, P. Roepstorff, and J.D. Mikkelsen.
1994. A hydroxyproline-containing class IV chitinase of
sugar beet is glycosylated with xylose. Plant Molecular
Biology 25:241-257.

Neuffer, M.G., and O.C. Calvert. 1975. Dominant disease
lesion mimics in maize. The Journal of Heredity 66:
265-270.

Owen, F.V. 1945. Cytoplasmically inherited male—sterility in
sugar beets. Journal of Agricultural Research
71(10):423-440.

Pan, S.Q., X.S. Ye, and J. Kuc. 1991. A technique for
detection of chitinase, B-1,3-glucanase, and protein
patterns after a single separation using polyacrylamide

gel electrophoresis or isoelectrofocusing.
Phytopathology 81:970-974.

Parent, J.G., and A. Asselin. 1984. Detection of
pathogenesis-related proteins (PR or b) and of other
proteins in the intercellular fluid of hypersensitive
plants infected with tobacco mosaic virus. Canadian
Journal of Botany 62:564—569.

Payne, J.W. 1976. Electrophoresis of proteins on sodium
dodecyl sulphate polyacrylamide gels. In Smith, I.(ed).
Chromatographic and electrophoretic techniques.
Interscience, New York. pp. 321-346.

Pillen, K., G. Steinrficken, R.G. Herrmann, and C. Jung.
1993. An extended linkage map of sugar beet (Beta
vulgaris L.) including nine putative lethal genes and
the restorer gene X. Plant Breeding 111:265-272.

Punja, Z.K., and Y.Y. Zhang. 1993. Plant chitinases and
their roles in resistance to fungal diseases. Journal
of Nematology 25:526-540.

Ryals, J.A., U.H. Neuenschwander, M.G. Willits, A. Molina,
H-Y. Steiner, and M.D. Hunt. 1996. Systemic acquired
resistance. The Plant Cell 8:1809-1819.

Rousseau-Limouzin, M., and B. Fritig. 1991. Induction of
chitinases, 1,3-8-glucanases and other pathogenesis-

112

related proteins in sugar beet leaves upon infection
with Cercospora beticola. Plant Physiology and
Biochemistry 29:105-117.

Saunders, J.W. 1982. A flexible in vitro shoot culture
propagation system for sugarbeet that includes rapid
floral induction of ramets. Crop Science 22:1102-1105.

Saunders J.W., G. Acquaah, K.A. Renner, and W.P. Doley.
1992. Monogenic dominant sulfonylurea resistance in
sugarbeet from somatic cell selection. Crop Science
32:1357-1360.

Saunders, J.W. 1998. Registration of REL-l and REL-2
sugarbeet germplasms for tissue culture genetic
manipulations. Crop Science 38:901-902.

Schégger, H., H. Aquila, and G. Von Jagow. 1988. Coomassie
blue-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis for direct visualization of
polypeptides during electrophoresis. Analytical
Biochemistry 173:201-205.

Simmons, C., S. Hantke, S. Grant, G.S. Johal, and S.P.
Briggs. 1998. The maize lethal leaf spot 1 mutant has
elevated resistance to fungal infection at the leaf

epidermis. Molecular Plant-Microbe Interactions
11:1110-1118.

Smith, G.A., 1980. Sugarbeet. In Fehr, W.R., and H.H. Hadley
(ed). Hybridization of crop plants. American Society of

Agronomy and Crop Science Society of America, Madison.
pp. 601-616.

Srb, A.M., and R.D. Owen. 1952. General genetics. W.H.
Freeman, San Francisco and London.

Staskawicz, B.J., F.M. Ausubel, B.J. Baker, J.G. Ellis, and
J.D.G. Jones. 1995. Molecular genetics of plant disease
resistance. Science 268:661-667.

Strickberger, M.W., 1969. Genetics. Macmillan, New York.
pp.127-149.

Tenhaken, R., A. Levine, L.F. Brisson, R.A. Dixon, and C.
Lamb. 1995. Function of the oxidative burst in
hypersensitive disease resistance. Proceedings of
National Academy of Science USA 92:4158-4163.

113

Trudel, J., and A. Asselin. 1989. Detection of chitinase
activity after polyacrylamide gel electrophoresis.
Analytical Biochemistry 178:362-366.

Ullstrup, A.J., and A.F. Troyer. 1967. A lethal leaf spot of
maize. Phytopathology 57:1282-1283.

Ulrich, A. 1961. Variability of sugar beet plants grown in
pots without competition for light, water and
nutrients. Journal of The American Society of Sugar
Beet Technologists 7:595-604.

Van Loon, L.C., and A. Van Kammen. 1970. Polyacrylamide disc
electrophoresis of the soluble leaf proteins from
Nicotiana tabacum var. 'Samsun' and 'Samsun NN.‘ II.
Changes in protein constitution after infection with
tobacco mosaic virus. Virology 40:199-211.

Van Loon, L.C., S. Gianinazzi, R.F. White, Y. Abu-Jawdah,
P. Ahl, J.F. Antoniw, T. Boller, A. Camacho-Henriquez,
V. Conejero, J.C. Coussirat, R.N. Goodman, E. Maiss, P.
Redolfi, and T.M.A. Wilson. 1983. Electrophoretic and
serological comparison of pathogenesis-related (b)
proteins from different plant species. Netherlands
Journal of Plant Pathology 89:293-303.

Van Loon, L.C., Y.A.M. Gerritsen, and C.E. Ritter. 1987.
Identification, purification, and characterization of
pathogenesis-related proteins from virus-infected
Samsun NN tobacco leaves. Plant Molecular Biology
9:593-609.

Walbot, V., D.A. Hoisington, and M.G. Neuffer. 1983. Disease
lesion mutations. In Kosuge, T., C.P. Meredith, and A.
Hollaender (ed). Genetic engineering of plants. Plenum,
New York. pp. 431-442.

Weymann, K., M. Hunt, S. Uknes, U. Neuenschwander, K.
Lawton, H.Y. Steiner, and J. Ryals. 1995. Supression
and restoration of lesion formation in Arabidopsis lsd
mutants. The Plant Cell 7:2013-2022.

Winner, C. 1993. History of the crop. In Cooke, D.A., and
R.K. Scott (ed). The sugar beet crop. Chapman and Hall,
London pp. 1-35.

Wolter, M., K. Hollricher, F. Salamini, and P. Schulze-

Lefert. 1993. The mlo resistance alleles to powdery
mildew infection in barley trigger a developmentally

114

controlled defense mimic phenotype. Molecular General
Genetics 239:122-128.

Wright, T.R., and D. Penner. 1998. Cell selection and
inheritance of imidazolinone resistance in sugarbeet

(Beta vulgaris). Theoretical and Applied Genetics
96:612-620.

Yu, I-C., J. Parker, and A.F. Bent. 1988. Gene-for-gene
resistance without the hypersensitive response in
Arabidopsis ndnl mutant. Proceedings National Academy
of Science USA 95:7819—7824.

115