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This is to certify that the
thesis entitled

REGENERATION 0F CAPSICUM ANNUUM AND SCREENING
OF THE R1 GENERAI IUN FUR—RESISTANCE T0
CUCUMBER MOSAIC VIRUS AND A SURVEY
OF PEPPER VIRUSES IN MICHIGAN

presented by

Janette Lynn Jacobs

has been accepted towards fulfillment
of the requirements for

 

 

M.S. degree in Plant Pathology

M 5421‘};va

Major professor

 

Date October 28, 1991

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ix

MSU Is An Affirmative Action/Equal Opportunity Institution
cMuna-q

 

 

 

 

 

 

REGENERATION OF CAPSICUM ANNUUM AND SCREENING
OF THE R1 GENERATION FOR RESISTANCE TO
CUCUMBER MOSAIC VIRUS AND A SURVEY
OF PEPPER VIRUSES IN MICHIGAN

BY

Janette Lynn Jacobs

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1991

682' 67/4

ABSTRACT
REGENERATION OF CAPSICUM ANNUUM AND SCREENING
OF THE R1 GENERATION FOR RESISTANCE TO

CUCUMBER MOSAIC VIRUS AND A SURVEY
OF PEPPER VIRUSES IN MICHIGAN

BY

Janette Lynn Jacobs

Differences in morphogenetic response of 58 pepper
(Capsicum annuum) genotypes was examined on Murashige and
Skoog (Physiol. Plant. 53:319-326) medium (MS) supplemented
with indole-3-acetic acid (3 mg/L), 6-benzylaminopurine (4
mg/L), and silver nitrate (10 mg/L). Explants were
incubated at 28 C for one month, transferred to MS medium
supplemented with napthaleneacetic acid (0.1 mg/L), with
whole plants regenerated from 19 of 58 genotypes. A total
of 95 R1 generation lines from the sexual offspring of
plants regenerated from g. annuum PI178849 were screened for
resistance to cucumber mosaic virus (CMV). Extensive
variability in infection of plants was observed between
inoculation and screening methods. Resistance to CMV was
not observed among the 95 R1 lines. A survey of randomly-
selected pepper from commercial fields in Michigan in 1989
and 1990 demonstrated that CMV is an important virus endemic
in Michigan and tomato spotted wilt virus is not presently

endemic, but imported to the state in pepper transplants.

ACKNOWLEDGEMENTS

I would like to sincerely thank Dr. Christine T.
Stephens, my major professor for her support and guidance
throughout my studies at Michigan State University. I would
like to thank Dr. Donald Ramsdell and Dr. Joseph Saunders
for their time and guidance while serving on my graduate
committee.

My sincere gratitude is extended to Dr. Harry Murakishi
and Dr. Richard Allison for their friendship and for kindly
providing the use of their laboratory.

I would like to express sincere thanks to my husband,
family, and friends for their constant encouragement and
support.

In addition, I am grateful for the technical assistance
from Sheila Linderman, Gongzhi Li, and John Kusnier with my
research project.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . .
LIST OF FIGURES. . . . . . . . . . . . . . . . . .
LITERATURE REVIEW. . . . . . . . . . . . . . . . . .
LITERATURE REFERENCES . . . . . . . . . . . . . .
CHAPTER 1: SURVEY OF CUCUMBER MOSAIC, POTATO VIRUS Y,
TOBACCO ETCH, AND TOMATO SPOTTED WILT VIRUS IN
PEPPER IN MICHIGAN USING ELISA . . . . . . . . .
ABSTRACT O O O O O O O O O O O O O O O O O 0 O O
I NTRODUCT I ON C O O O O O O O O O O O O O O O O 0
MATERIALS AND METHODS . . . . . . . . . . . . . .
Field Survey Procedures . . . . . . . . . . .
Sample Collection . . . . . . .
Virus Identification Using ELISA . . . . . . .
RE SULT S O O O O O O O O O O O O O O O O O O O 0
DISCUSSION . . . . . . . . . . . . . . . . . . .
LITERATURE REFERENCES . . . . . . . . . . . . . .

CHAPTER 2: REGENERATION OF PEPPER (CAPSICUM ANNUUM)
PROM PRIMARY LEAP EXPLANTS . . . . . . . . . . .

ABSTRACT . . . . . . . . . . . . . . . . . . . .
INTRODUCTION . O O O O O O O O O O O O O O O O 0

MATERIALS AND METHODS . . . . . . . . . . . . . .

w Genotypes
In vitro Seedlings
Source of Explants
Culture Medium . .

Page
vii

ix

14

20
20
20
22
22
22
23
24
29

34

36
36
37
38
38
38

39
39

Rating System . . . . . .
Growth Hormone Experiment
Silver Nitrate Experiment
Temperature Experiment . .
Shoot Elongation Experiment
Genotype Experiment . . .
Acclimation of Somaclones

RESULTS . . . . . . . . . . .
Growth Hormone Experiment .
Silver Nitrate Experiment .
Temperature Experiment . .

Shoot Elongation Experiment
Genotype Experiment . . . .

DISCUSSION. . . . . . . . . .
LITERATURE REFERENCES . . . .

CHAPTER 3:
CUCUMBER MOSAIC VIRUS

ABSTRACT . . . . . . . . . .
INTRODUCTION . . . . . . . .
MATERIALS AND METHODS . . . .
Virus 0 O O O O O O O O O 0
R1 Seedlings . . . . . . .
Inoculation of R1 Seedlings

Mechanical Inoculation . .
Aphid Inoculation . . . . .

Virus Screening of R1 Pepper Progeny Using

ELI SA 0 O O O O O O O 0

RESULTS . . . . . . . . . . .

DISCUSSION . . . . . . . . .

LITERATURE REFERENCES . . . .

CHAPTER 4: PROTOPLAST INFECTION WITH VIRUS

ABSTRACT . . . . . . . . . .
INTRODUCTION . . . . . . . .
MATERIALS AND METHODS . . . .

Protoplast Isolation . . .

SCREENING THE R1 GENERATION WITH

Page
39
42
42
44
44
45
45
46
46
47
49
53
53
62

65

66
66
66
67
67
68
68
68
69
69
70
83
86
87
87
87
91

91

Protoplast Viability and Yield . . . . . . .

Purification of Virus . . .
Inoculation of Protoplasts .
Protoplast Viability . . . .

Preparation of the FITC-Conjugate . . .

Cross Absorption of FITC- Conjugate with Acetone

Powder of Healthy Tobacco

Leaves . . . .

Preparation and Staining of Infected

Protoplasts . . . . . . .
Detection of Virus Infection
Microscopy . . . . . . .
Photography . . . . . . . .
RESULTS . . . . . . . . . . . .
Inoculation of Protoplasts .
DISCUSSION. . . . . . . . . . .

LITERATURE REFERENCES . . . . .

vi

by Fluorescence

Page
. 94
. 94
. 96

. 97
. 97

O 100

. 101
O 101

. 101
. 101
. 102

. 108

LIST OF TABLES

Page
CHAPTER 1

Table 1.1 Identification of viruses in pepper
survey samples using enzyme-linked immunosorbent
assay (ELISA) . . . . . . . . . . . . . . . . . . . . 25

CHAPTER 2

Table 2.1 Analysis of variance of a 3 X 6 X 6
factorial experiment concerning the effects of
indole-3-acetic acid, 6-benzylaminopurine, and
pepper genotype on regeneration . . . . . . . . . . . 48

Table 2.2 Analysis of variance of a 4 X 4

factorial experiment concerning the effect of

silver nitrate and pepper genotype on

regeneration . . . . . . . . . . . . . . . . . . . . 50

Table 2.3 Differences in the effect of silver

nitrate concentration amended to Murashige and

Skoog medium on shoot regeneration of four pepper
genotypes . . . . . . . . . . . . . . . . . . . . . . 51

Table 2.4 Analysis of variance of a 4 X 4

factorial experiment concerning the effect of
temperature on shoot regeneration of four pepper
genotypes cultured on Murashige and Skoog medium
supplemented with 3 mg/L indole-B-acetic acid,

4 mg/L 6-benzylaminopurine, and 10 mg/L silver

nitrate . . . . . . . . . . . . . . . . . . . . . . . 54

Table 2.5 Differences in the effect of temperature

on shoot regeneration of four pepper genotypes

cultured on Murashige and Skoog medium supplemented

with 3 mg/L indole-3-acetic acid, 4 mg/L 6-
benzylaminopurine, and 10 mg/L silver nitrate . . . . 55

Table 2.6 Differences in the percentage of explants
forming shoots and the number of shoots per explant

of pepper genotype cultured on Murashige and Skoog
medium supplemented with 0.1 mg/L naphthaleneacetic

acid or 4 mg/L 6-benzylaminopurine . . . . . . . . . 57

vii

Page
Table 2.7 Shoot initiation and elongation
responses of Capsigum genotypes when cultured
on Murashige and Skoog medium supplemented
with 3 mg/L indole-3-acetic acid, 4 mg/L 6-
benzylaminopurine, 0.1 mg/L napthaleneacetic acid,
and 10 mg/L silver nitrate . . . . . . . . . . . . . 59

Table 2.8 Regeneration capacity of fifty-eight

Capsicum genotypes when cultured on Murashige

and Skoog medium supplemented with 3 mg/L

indole-3-acetic acid, 4 mg/L 6-benzylaminopurine,

0.1 mg/L napthaleneacetic acid and 10 mg/L silver

nitrate . . . . . . . . . . . . . . . . . . . . . . . 60

CHAPTER 4
Table 4.1 Treatment conditions of successful
experiments infecting pepper (Capsipum appppm)

protoplasts with pepper mild mottle virus using
electroporation . . . . . . . . . . . . . . . . . . . 103

viii

LIST OF FIGURES

Page
CHAPTER 1

Figure 1.1 Distribution of cucumber mosaic (CMV),

potato virus Y (PVY), tobacco etch (TEV), and

tomato spotted wilt virus (TSWV) identified in

survey pepper samples in 1989-1990 . . . . . . . . . 26

Figure 1.2 Seasonal occurrence of cucumber mosaic

(CMV), potato virus Y (PVY), tobacco etch (TEV), and
tomato spotted wilt virus (TSWV) in survey pepper
samples in 1989 . . . . . . . . . . . . . . . . . . . 27

Figure 1.3 Seasonal occurrence of cucumber mosaic

(CMV), potato virus Y (PVY), tobacco etch (TEV),

and tomato spotted wilt virus (TSWV) in survey

pepper samples in 1990 . . . . . . . . . . . . . . . 28

CHAPTER 2

Figure 2.1 Four to six week-old seedlings of pepper
(Capsicum annuum) grown ip yitro . . . . . . . . . . 40

Figure 2.2 A rating system for the regeneration
response of pepper (Capsicum annuum) from leaf
explants after 30 days in culture . . . . . . . . . . 41

Figure 2.3 Primary leaf explants (5 x 10 mm) of
pepper (Capsicum annuum) cultured on Murashige and
Skoog regeneration medium . . . . . . . . . . . . . . 43

Figure 2.4 Regenerated somaclones of pepper
(Capsigpm annuum PI178849) from primary leaf
explants . . . . . . . . . . . . . . . . . . . . . . 47

Figure 2.5 Qualitative differences in the response

of four pepper genotypes cultured on Murashige and

Skoog medium supplemented with 0, 10, 20, and 30

mg/L silver nitrate . . . . . . . . . . . . . . . . . 52

ix

Page
Figure 2.6 Qualitative differences in the response
of four pepper genotypes cultured on Murashige and
Skoog medium supplemented with 3 mg/L
indole-3-acetic acid, 4 mg/L 6-benzy1aminopurine,
and 10 mg/L silver nitrate at 24, 26, 28, and 30 C . . 56

Figure 2.7 Shoot elongation of pepper (Capsicum
appuum P1178849) on Murashige and Skoog medium
amended with 0.1 mg/L naphthaleneacetic acid . . . . . 58

CHAPTER 3

Figure 3.1 Experiment 1. A comparison of the
percentage of infected plants of R1 generation

lines of pepper (Capsicum pppppm P1178849)

obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis

as detection methods for virus-infection . . . . . . 71

Figure 3.2 Experiment 2. A comparison of the
percentage of infected plants of R1 generation

lines of pepper (Capsippm annuum P1178849)

obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis

as detection methods for virus-infection . . . . . . 72

Figure 3.3 Experiment 3. A comparison of the
percentage of infected plants of R1 generation

lines of pepper (Capsicum annuum P1178849)

obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis

as detection methods for virus-infection . . . . . . 73

Figure 3.4 Experiment 4. A comparison of the
percentage of infected plants of R1 generation

lines of pepper (Capsipum annuum P1178849)

obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis

as detection methods for virus-infection . . . . . . 74

Figure 3.5 Experiment 5. A comparison of the
percentage of infected plants of R1 generation

lines of pepper (Capsicpp pppppm P1178849)

obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis

as detection methods for virus-infection . . . . . . 75

Page
Figure 3.6 Experiment 6. A comparison of the
percentage of infected plants of R1 generation
lines of pepper (Capsicum apnuum P1178849)
obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis
as detection methods for virus-infection . . . . . . 77

Figure 3.7 Experiment 7. A comparison of the
percentage of infected plants of R1 generation

lines of pepper (Capsicum annuum P1178849)

obtained using mechanical or aphid-inoculation
techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis

as detection methods for virus-infection . . . . . . 82

CHAPTER 4
Figure 4.1 Fluorescence microscopy detection of
pepper mild mottle virus infected protoplasts of
pepper (Capsicum annuum) by electroporation . . . . . 104
Figure 4.2 Viability of protoplasts of pepper

(Capsicum annuum) after exposure to various
voltages during electroporation experiments . . . . . 105

xi

LITERATURE REVI EW

In terms of economic importance, pepper (Capaippp
annuum) is a significant crop placing fifth in the world-
wide production of vegetables. The pepper crop in the
United States has a value of 169 million dollars annually.
There are 110,000 acres in pepper production and 686,750
tons of pepper fruit are harvested each year (Marshall,
1977). Michigan has a relatively small acreage (3850) of
pepper with a value of 3.2 million dollars annually
(Michigan Agricultural Statistics, 1989). The majority of
peppers grown in Michigan are of the bell fruit type and are
produced for fresh market. Peppers are mostly grown in the
southwest, central and southeast regions of the Lower
Peninsula.

Internationally, severe losses in the pepper industry
occur due to the presence of virus diseases. Virus diseases
damaging to pepper have been reported in California
(Abdalla, 1985), Florida (Anderson and Corbett, 1957;
Zitter, 1973), Georgia (Benner et a1, 1985), Texas
(Villalon, 1975, 1981) and in several countries including
the Netherlands (Rast, 1982), Italy (Conti, 1977), Canada
(Lana and Peterson, 1980), and India (Deol and Rataul,
1978). Due to virus infection, pepper crops are
occasionally abandoned without harvesting in some of the
major production areas in the southwestern United States
(Villalon, 1981). Pepper production has been eliminated
from some growing regions in Massachusetts (Moorman and

Woodbridge, 1983) and Texas (Villalon, 1975) as a direct

1

2

result of virus disease pressure.

Thirty six viruses are known to infect pepper
(Villalon, 1975), but only alfalfa mosaic (AMV), cucumber
mosaic (CMV), pepper mottle (PeMV), potato virus X (PVX),
potato virus Y (PVY), tobacco etch (TEV), and tobacco mosaic
(TMV) viruses have been economically important (Marco and
Cohen, 1979; Horvath and Nienhaus, 1982; Agrios et al.,
1985; and Villalon, 1981).

Although not serologically confirmed, a preliminary
field study demonstrated that CMV was involved in natural
infection of pepper in Michigan (C.T. Stephens and T.
Stebbins, personal communication). CMV is a highly
destructive plant pathogen known to have a number of
strains. CMV attacks a wide range of crops (191 species in
40 families), and is world-wide in distribution. CMV is
transmitted in a nonpersistent manner by more than 60
species of aphids (Francki et al., 1979). The virus belongs
to the cucumovirus group and has a tripartite genome
encapsidated in three types of isometric particles of about
28 nm in diameter. All three components are necessary for
infection (Francki et al., 1979).

Pepper is one of the major economic crops affected by
CMV. In virus surveys, CMV has been detected in pepper
fields in Texas (Villalon, 1975), Florida (Simons, 1957),
Louisiana (Barrios et al., 1971), New Jersey (Doolittle
andnd Zaumeyer, 1953), Georgia (Benner et al., 1985), and

Quebec (Lana and Peterson, 1980). Symptoms of CMV on pepper

3

include mottling, chlorosis, oak-leaf or circular necrotic
markings on the leaves, and tan rings or spots on the fruit.
These rings become brown and necrotic and result in fruit
spotting. Leaf symptoms develop when peppers are infected
at a young stage of growth (Simons, 1957). Fruit symptoms
are not always present, particularly when plants become
infected at an early or late stage of development (Pasko et
al., 1984).

The major strategy for virus disease control is the use
of vigorous aphid control programs. Various cultural
practices have been developed for reducing the incidence of
aphid transmitted viruses in pepper: oil sprays which
decrease the ability of aphids to acquire and transmit
stylet-borne viruses (Vanderveken, 1977); soil mulching with
reflective surfaces which repel the aphids (Smith and Webb,
1969); and attraction of the aphids to yellow sticky
polyethylene traps, thus keeping the aphids from the plants
(Cohen and Marco, 1973). Each of the above mentioned
practices, however, has certain limitations. Additionally,
aphid control programs for virus disease management are
usually unsuccessful because virus transmission only
requires one short feeding.

Genetic resistance remains the principal solution for
controlling virus diseases in pepper. Disease-resistant
crop species have long been produced by identifying
resistant genotypes and then crossing individuals exhibiting

resistance with cultivars that possess horticulturally

4

acceptable traits. Traditional breeding has been successful
and in many cases, resistance to PeMV, PVY, TEV, and TMV has
been found and introduced into different pepper cultivars
(Villalon, 1986; and Holmes, 1937). Capaippm fpppaapapa
cultivar LP-l showed a high degree of tolerance to CMV, and
the tolerance was determined to be a recessive trait
(Barrios et al., 1971).

Limitations do exist, however, to conventional plant
breeding methods. For example, the desired resistance is
not always available, it may only exist in a species that is
sexually incompatible, the genes for resistance may be
tightly linked to undesirable traits, or may be multigenic
and difficult to transfer (Knott and Dvorak, 1976; Wenzel,
1985). Also, virus-resistance is subject to breakdown due to
the appearance of other viral strains. Significantly, in
spite of active pepper breeding programs in the United
States, few lines are commercially available that contain
multiple virus resistance.

Genetic variability is essential to plant breeding;
continual input of new genes is vital for the improvement of
commercial crop species. A relatively recent method of
developing new genetic material is through the use of plant
cell culture-generated genetic variability, which is termed
somaclonal variation (Larkin and Scowcroft, 1981). Plant
cell culture generates genetic variation as well as
epigenetic variation in regenerated plants. Genetic

variation is the result of pre-existing genetic differences

5

in somatic cells in the explant tissue, or evolves during
cell proliferation on the explant. Genetic variant
characters that are expressed in plants regenerated from
plant cell culture are transmissable to progeny in sexually
propagated crops (Evans and Sharp, 1983). Examples of
somaclonal variation include cytoplasmic gene changes,
chromosome rearrangements, mitotic crossing over, and
changes in gene expression (Evans, 1989). These genetic
changes are either unique to somaclonal variation or occur
at a much higher frequency than in spontaneous or induced
mutagenesis. Epigenetic changes are induced by specific
components of the cell culture medium, and although almost
unique to somaclonal variation, they are not useful for crop
improvement, as they are not expressed in the R1 progeny
(Evans, 1989). Examples of epigenetic variation include,
dwarfing, alteration in leaf shape, or other changes in
growth habit.

As a result of genetic changes, somaclones with altered
genotypes may express levels of disease resistance which are
greater than the parent genotype. The first report
recognizing the potential of tissue culture-derived variants
as a source of variability for crop improvement occurred
with sugarcane (Heinz and Mee, 1969) and later with potatoes
(Shepard et al., 1980). In these studies researchers
foundthat plants regenerated from callus and protoplast
cultures varied in a number of traits, including

morphological characteristics, maturity date, yield and

6

response to pathogens. Although this early work was done
with asexually propagated crops, other investigators have
since shown that somaclonal variation occurs in many plant
species, including tobacco, rice, carrot (Larkin and
Scowcroft, 1981), alfalfa (Latunde-Dada and Lucas, 1983),
celery (Wright and Lacy, 1985), and tomato (Evans and Sharp,
1983).

The causes of somaclonal variation are still being
investigated, but a number of facts are known. Somaclonal
variation is usually genetic and heritable, and variants
occur at a high frequency. In addition, changes occur both
in monogenic and polygenic traits, and in both nuclear and
organelle genomes (Daub, 1986). In somaclonal studies, the
frequency of variation has been estimated to be as high as
30-40% for the number of plants showing some type of
variation, and from 0.2 to almost 3% for variation in a
particular trait (Daub, 1986). Somaclonal variation
frequencies are high enough that desirable variants can be
identified and selected if an efficient screening system
exists at the whole-plant level.

Somaclonal variation may be used to introduce the best
available varieties into plant cell culture and to select
among regenerated plants (R0) or their self-fertilized
progeny (R1) for incremental improvements in resistance
overexisting commercial varieties. This procedure has been
utilized in selecting tomato somaclones resistant to TMV

(Barden, 1986), and sugarcane subclones with a greater

.7

degree of resistance to Fiji virus (Krishnamurthi, 1974).
The phenomenon of somaclonal variation offers a means
to select variability without exposure to a mutation agent.
The use of protoplasts offers a means of directly exposing
single plant cells to a selection agent. Utilization of
plant protoplasts in an in yippp selection system may offer
a means of creating or isolating plant genotypes with
increased resistance to disease. Screening for resistance
to plant pathogens at the single cell level offers unique
advantages. Millions of cells can be screened in a short
time, the technique holds promise for selection of
resistance from horticulturally susceptible cultivars
(Shepard, 1981), and may allow for the recovery of increased
levels of disease resistance (Murakishi and Carlson, 1982).
To date, viral pathogens offer the most promise as

direct selection agents in the use of ip vitro screening

 

systems because protoplasts can be synchronously infected
with viruses (Hibi et al., 1986; Maule et al., 1980;
Motoyoshi and Oshima, 1975; Nishiguchi et al., 1987).
Infection of protoplasts has been demonstrated in a variety
of plant species such as barley, Brassica, cowpea, cucumber,
tobacco, and tomato (Okuno and Furusawa, 1978; Maule, 1983;
Koike et al., 1977; Maule et al., 1980; Hibi et al., 1986;
Nishiguchi et al., 1987; Motoyoshi and Oshima, 1975). Also,
studies using protoplasts isolated from resistant host
plants of tomato and cucumber infected with TMV and CMV

respeCtively, revealed that resistance to the pathogen

8
functions at the single cell level (Maule et al., 1980;

Motoyoshi and Oshima, 1975). In addition, Murakishi and
Carlson (1982), working with protoplasts isolated from
systemically infected tobacco plants, demonstrated that
viral pathogens can be used as selection agents at the
single cell level to isolate variant cells, if conditions
are manipulated to allow for uniform infection and for the
preferential growth and selection of virus-resistant cells.

Although studies examining the manipulation of pepper
ip yippp have been moderately successful, plant regeneration
has often restricted to only a few genotypes. To date, the
regeneration of whole plants from somatic cells is not
sufficiently routine for efficient application of technology
such as somaclonal variation and genetic transformation.

Manipulative variables in pepper regeneration systems
include factors such as growth medium components, light
duration and intensity, and temperature. Experiments in the
literature involving pepper plant regeneration utilize the
same basal medium but disagree on the type and
concentrations of hormones in the regeneration process.
Shoot bud formation of hypocotyl and cotyledon sections was
reported on Murashige and Skoog (MS) medium supplemented
with 1 mg/L indole-3-acetic acid (IAA) and 2 mg/L 6-
benzylaminopurine (BAP) (Gunay and Rao, 1976), but,
quantitative response data were not included in this report,
and 58 - 62% of cultures produced a mass of callus

overgrowth which suppressed shoot primorida elongation in

9

another study using the same medium (Fari and Czako, 1981).
A continuous light and constant temperature treatment at
28.5 C was reported as stimulatory for extended shoot and
root organogenesis in pepper tissues incubated in MS medium
supplemented with 0.5 mg/L IAA and BAP (Phillips and
Hubstenberger, 1985). Glucose as a carbon source has also
been reported as stimulatory for shoot regeneration
(Phillips and Hubstenberger, 1985). A concentration of 3 -
7 mg/L of BAP in MS medium induced the largest number of
shoots per explant in another study, with auxin (1AA or
naphthaleneacetic acid, NAA) inhibiting shoot formation when
used in combination (Sripichitt et al., 1987). Finally, 5
mg/L BAP in MS medium resulted in the best shoot
differentiation, however, the number of shoots increased but
did not elongate (Agrawal, et al., 1989). Also, in this
study, 0.1 mg per liter of IAA or NAA was critical to obtain
complete plants (Agrawal et al., 1989).

One factor common to all of these reports is the low
frequencies of the number of explants producing plantlets as
well as the low number of plantlets obtained per explant.
Also, regarding all of the above reports, regeneration of
pepper genotypes was not reproducible using these methods.

A thorough understanding of how explant sources, medium
components, and environmental conditions affect shoot
initiation and elongation is necessary to provide a rapid
regeneration technique which will be applicable to a wide

range of pepper genotypes.

10
A factor in these studies which had not been explored,

was the role of ethylene in pepper regeneration. Ethylene
is a gaseous plant hormone produced from essentially all
parts of higher plants, including leaves, stems, roots,
fruits, tubers, and seedlings (Abeles, 1973). Ethylene
production plays an important role in regulating many
developmental processes, ranging from germination to
senescence.

Few studies have addressed the role of endogenously
produced ethylene on organogenesis in higher plants (Horner
et al., 1977). However, with respect to the role of
exogenously applied ethylene, it was shown that shoot
forming callus tissues contained significantly lower
concentrations of the ethylene precursor 1-‘
aminocyclopropane-l-carboxylic acid (Grady and Bassham,
1982) and produce less ethylene (Huxter, et al., 1981)
compared to non-shoot-forming callus tissues. Exogenously
applied ethylene precursor in high concentrations inhibited
shoot primordium formation in tobacco callus cultures
(Huxter et al., 1981).

Also, auxins can promote the endogenous production of
ethylene (Yang and Hoffman, 1984). The promotion of
ethylene production by auxin was initially discovered by
Zimmerman and Wilcoxon (1935). Since auxin and ethylene
cause a number of similar responses, and auxin is capable of
promoting ethylene production, many responses previously

attributed to auxin can be traced to ethylene produced in

11
response to auxin treatment. In vegetative tissues, the

rate of ethylene production is thought to be regulated by
the internal level of free auxin (Abeles, 1973). It is
known that ethylene production rate in IAA-treated tissue
declines when IAA is withdrawn from the incubation medium
(Yoshii and Imaseki, 1982). Auxins, which are essential for
callus induction, can play a negative role in plant
regeneration. The results of previous studies suggest that
in non-regenerating plant tissue cultures auxin induced
ethylene production may be responsible for the suppression
of shoot regeneration.

Silver ions are known to inhibit ethylene action in
plants (Beyer, 1976). Silver ions applied foliarly as
silver nitrate, effectively blocked the action of
exogenously applied ethylene in such classical ethylene
responses as abscission, senescence, and growth retardation
in pea, orchid, and cotton (Beyer, 1979). The basis for
this protection by silver ions is unknown. It has been
suggested that silver ions interfere with the incorporation
of ethylene at its receptor site, but not its oxidation to
(KL, in etiolated pea seedlings (Beyer, 1979). An effect of
silver nitrate on liverwort organogenesis was interpreted as
a desuppression of potential development of local
populations of cells suppressed by ethylene (Basile and
Basile, 1983). Silver nitrate effectively promoted shoot
regeneration in callus cultures of wheat and tobacco

genotypes with poor regeneration ability (Purnhauser, et

12
al., 1937).

Another factor of importance in plant regeneration is
the variation observed among genotypes of a particular
species. This genotypic variation in the organogenic
response is widespread among plant species. Callus cultures
of monocots have poor regeneration ability, and great
differences in organogenic response exist between genotypes
(Sears and Deckard, 1982; Duncan, et al., 1985). Genotypic
specificity for regeneration capacity in legumes is
well-documented in alfalfa (Chen and Marowitch, 1987), white
clover (Bhojwani et al., 1984), and soybeans (Parrott et
al., 1989). Genotypic variation is even observed in
Nicotiana, which has been used as a model in tissue culture
systems (Purnhauser et al., 1987).

Researchers have attempted to find indicators of a
genotype's ability of regenerative capacity. A study using
soybean genotypes showed no relationship between maturity
group, seed coat color, flower color, and disease
susceptibility or resistance to a genotype's ability to
produce high numbers of somatic embryos (Parrott et al.,
1989). Genotype-specific capacity for regeneration has been
exploited to breed alfalfa with a high regeneration capacity
(Bingham et al., 1975).

The objectives of this study were to: 1) determine
which pathogenic viruses are present in pepper fields in
Michigan, 2) to develop a reproducible whole plant

regeneration system for pepper, 3) to assess the use of

13

somaclonal variation as a means of selecting virus-resistant
pepper germplasm at the whole plant level, and 4) to
investigate those variables which influence the uptake of
virus particles into protoplasts of pepper by

electroporation.

14

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15

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Doolittle, S.P., and Zaumeyer, W.J. 1953. A pepper ring
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Fari, M., and Czako, M. 1981. Relationship between
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16

Francki, R.I.B., Mossop, D.W., and Hatta, T. 1979.
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Grady K.L., and Bassham, J.A. 1982. 1-Aminocyclopropane-1-
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Gunay, A.L., and Rao, P.S. 1978. In vitro Plant
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Holmes, F.0. 1937. Inheritance of resistance to tobacco
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Horner, M., McComb, J.A., and Street, H.E. 1977. Ethylene
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Horvath, J., and Nienhaus, F. 1982. Separation of viruses
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Huxter T.J., Thorpe, T.A., and Reid, D.M. 1981. Shoot
initiation in light-and dark-grown tobacco callus: the
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Jourdan, P.S., and Earle, E.D. 1989. Genotypic variability
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protoplasts of four Braasipa spp. and of Raphapus
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Koike, M., Hibi, T., and Yora, K. 1977. Infection of conea
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Virology 83:413-416.

17

Krishnamurthi, M., and Tlaskal, J. 1974. Fiji disease
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Lana, A.F., and Peterson, J.F. 1980. Identification and
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Phyto-protection. 61:13-18.

Latunde-Dada, A.0., and Lucas, J.A. 1983. Somaclonal
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Larkin, P.J., and Scowcroft, W.R. 1981. Somaclonal
variation -- a novel source of variability from cell
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Lieberman, M. 1979. Biosynthesis and action of ethylene.
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Marco, S., and Cohen, S. 1979. Rapid detection and titer
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Maule, A.J., Boulton, M.I., Edmunds, C. and Wood, K.R. 1980.
Polyethylene glycol-mediated infection of cucumber
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Gen. Virol. 47:199- 203.

Maule, A.J. 1983. Infection of protoplasts from several
Baassipa species with cauliflower mosaic virus
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Gen. Virol. 64:2655-2660.

Mendoza, A.B., and Futsuhara, Y. 1990. Varietal
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Michigan Agricultural Statistics. 1989. Vegetables. pp.37-
41. Michigan Dept. Agric., Lansing, MI.

Moorman, G.W., and Woodbridge, W.C. 1983. Morphogenesis of
cucumber mosaic virus-induced crystalline inclusions in
peppers. Phytopathology 73:1106-1108

Motoyoshi, F., and Oshima, N. 1975. Infection with tobacco
mosaic virus of leaf mesophyll protoplasts from
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18

Murakishi, H. and Carlson, P. 1982. la vipro selection of

Niggtiapa,§ylxa§tri§ variants with limited resistance
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Pasko, P.J., Nicklow, C.W., and Moorman, G.W. 1984.
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19

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57:991-994.

CHAPTER 1

SURVEY OF CUCUMBER MOSAIC, POTATO VIRUS Y, TOBACCO ETCH, AND
TOMATO SPOTTED WILT VIRUS IN PEPPER IN MICHIGAN USING ELISA
ABSTRACT

Cucumber mosaic (CMV), potato virus Y (PVY), tobacco
etch (TEV), and tomato spotted wilt virus (TSWV) were
detected in Michigan pepper fields in 4/6, 2/6, 1/6, and 3/6
respectively in 1989. CMV and TSWV only were detected in
1/7 and 2/7 fields in 1990. CMV was detected throughout the
four major pepper growing regions in the state while the
other viruses were only detected in one or two geographic
regions. TSWV was detected for the first time in Michigan,
early in the growing season on pepper transplants from the
southeastern United States. CMV, PVY, and TEV were not
detected on Michigan-grown transplants until later in the
growing season. The data suggest that CMV is a virus
endemic to Michigan and TSWV is not presently endemic, but
has been imported to the state in pepper transplants.

INTRODUCTION

The importance of virus diseases in pepper production
has been recognized for many years. A number of surveys in
the United States have been conducted on virus diseases of

pepper; these studies reported that cucumber mosaic virus

20

21
(CMV), pepper mottle virus (PMV), potato virus Y (PVY),

tobacco etch virus (TEV), and tobacco mosaic virus (TMV)
were most frequently responsible for economic losses in
pepper production (Abdalla et al., 1985; Agrios et al.,
1983; Anderson and Corbett, 1957; Benner et al., 1985;
Makkouk and Gumpf, 1974; Steepy and Averre, 1971; Steepy et
al., 1967; Villalon, 1975; and Zitter, 1973). Other
naturally occurring pepper viruses identified to be present
in the United States at lower frequencies included alfalfa
mosaic virus (AMV), aster ringspot virus (ARSV), potato
virus X (PVX), tobacco ringspot virus (TRSV), and tomato
spotted wilt virus (TSWV) (Abdalla et al., 1985; Anderson
and Corbett, 1957; Makkouk and Gumpf, 1974; and Villalon,
1975).

In Michigan, reports of an increase in the incidence
and severity of mosaic or virus-like symptoms have been
received from commercial producers of pepper in recent
years. In general, efforts to control aphid introduction
and movement in the field have not been successful.
Accurate virus identification and information regarding
which viruses are most likely to be prevalent helps
producers make informed decisions on disease management. In
this study, a survey was conducted to determine the presence
and prevalence of the four viruses most likely to be found
in Michigan pepper fields, those being CMV, PVY, TEV, and
TSWV. AMV, ARSV, PMV, PVX, and TRSV have not been found in

the midwestern United States, and most common pepper

22
cultivars are now resistant to TMV (Sherf and Macnab, 1986);

therefore, these viruses were not included in the survey.
The natural distribution of the viruses, and their seasonal
occurrence was also investigated.
MATERIALS AND METHODS

Field Survey Procedurea

Surveys were conducted during the growing season (June-
September) of 1989 and 1990 in six and five fields
respectively, encompassing the four pepper-producing
geographic localities in Michigan. These fields represented
a total of 480 acres or 12.5% of the state pepper production
area. The fields were selected prior to or at the time of
planting. All fields had been planted with local or
southern-grown transplants of common commercial fresh market
and processing pepper cultivars (Bell Tower, Cheese, Harris,
Jalapea, Jupiter, Keystone Resistant Giant, Marengo and
Mayata). None of these cultivars are resistant to any of
the viruses in the study.
Sappie Coiiection

Prior to planting, transplants were tested for all
viruses. In 1989, the fields were sampled four times during
the growing season at approximately three week intervals.
One field was sampled two additional times late in the
season due to high incidence of CMV infection. In 1990,
fields were sampled eight times during the growing season at
two week intervals. The number of samples increased in 1990

because fields were sampled both earlier and later in the

23
growing season. Samples were collected along intersecting

field diagonals and consisted of 50 randomly chosen leaves
taken from shoot apices of 50 individual plants. Also, some
leaves were collected from plants showing virus-disease
symptoms as a separate sample. Additional pepper fields not
included in the survey in which virus infection was
suspected were sampled late in the growing season during
1989. Leaf samples were combined, placed in plastic bags,
and transported to the laboratory on ice for testing.
WM

Leaf samples were weighed and ground in a 10:1
volume:weight ratio of extraction buffer which contained 2%
Tween 20, 2% egg albumen, 2% polyvinyl pyrrolidone, and
0.13% sodium sulfite in 0.015M phosphate buffered saline, pH
7.2. Samples were then tested for the presence of viruses
using the enzyme-linked immunosorbent assay technique
(ELISA) described by Clark and Adams (1977). ELISA
PathoScreenm‘virus test kits were purchased from Agdia,
Inc. (Elkhart, IN) and used following the instructions of
the manufacturer. Strains of virus used for detection were:
1) crocus and vinca strain of CMV, 2) unknown strain of PVY,
3) unknown strain of TEV, and 4) lettuce strain of TSWV.
Assay controls were sap from healthy pepper plants and sap
from plants with known viruses. Samples having an
absorbance reading (490 nm) of at least 3X the absorbance
reading for sap from healthy plants were considered to be

positive.

24
RESULTS

Over time, all four viruses assayed were detected in
random samples from at least one or more of the geographic
localities of pepper production in Michigan. TSWV was the
only virus detected in transplants prior to planting. CMV,
PVY, TEV, and TSWV were detected in 1989 and only CMV and
TSWV were detected in 1990 in the fields sampled randomly.
CMV was present in 4/6 and 1]? fields in 1989 and 1990
respectively; PVY was present in 1/6 fields in 1989; TEV was
present in 1/6 fields in 1989; TSWV was present in 3/6 and
2/7 fields in 1989 and 1990 respectively (Table 1.1).
During the survey period, CMV was detected in all geographic
locations sampled, TSWV was detected in locations 3 and 4,
PVY was detected in locations 1 and 2, and TEV was detected
only in location 1 (Fig. 1.1). In 1989, TSWV was first
detected on 23 June, and PVY, CMV, and TEV were first
detected 5 July, 10 July, and 31 July respectively. In
1990, TSWV was first detected on 6 July; CMV was not
detected until 31 August (Fig. 1.2, 1.3).

From the four additional fields with suspected virus
infection sampled, CMV was present in 2/4 and TSWV was
present in 3/4 fields in 1989. PVY and TEV were not
detected in these fields. The fields in which CMV and TSWV
were detected were located in geographic locations 3 and 4
(Fig. 1.1).

DISCUSSION

The 1989 and 1990 survey results using ELISA support

25

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26

 

Figure 1.1 Distribution of cucumber mosaic (CMV), potato
virus Y (PVY), tobacco etch (TEV), and tomato spotted wilt
virus (TSWV) identified in survey and symptomatic pepper
samples in 1989-1990.

27

 

 

        

  

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Figure 1.2

and tomato spotted

wilt virus (TSWV) in survey and symptomatic pepper samples

in 1989.

Seasonal occurrence of cucumber mosaic (CMV),
tobacco etch (TEV),

potato virus Y (PVY),

28

 

N]

C)
I

01
I

.p
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00
I

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a
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Number of Virus Diseased Pepper Fields

 

 

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0

Figure 1.3 Seasonal occurrence of cucumber mosaic (CMV),
potato virus Y (PVY), tobacco etch (TEV), and tomato spotted
wilt virus (TSWV) in survey and symptomatic pepper samples
in 1990.

29
the conclusion that the most widespread and principal virus

disease of pepper in Michigan was caused by CMV. CMV and
PVY were identified as the most predmoninant viruses of
pepper in Massachussetts (Agrios et al., 1983), while PVY
and TEV were identified as the most predominant viruses in
pepper in California (Makkouk and Gumpf, 1974), Florida
(Zitter, 1973), Georgia (Benner et al., 1985), New Jersey
(Steepy et al., 1967), North Carolina (Steepy and Averre,
1971), and Texas (Villalon, 1975).

Plants with obvious disease symptoms exhibited large
necrotic rings or oak-leaf markings on mature leaves and
severe mosaic on the new growth. Symptoms on the fruit
consisted of tan to necrotic spots, or yellowish concentric
rings. Plants expressing these symptoms tested positive to
either or both the crocus and vinca strain(s) of CMV.

Studies by Anderson (1959) identified virus-infected
plant species bordering pepper fields as important sources
of primary inoculum in initial infection of aphid-borne
viruses by their vectors. They identified 16 plant species
as reservoirs for CMV in Florida (Anderson, 1959). Many of
the CMV-host reservoirs identified are common weed or crop
plants in Michigan. CMV was predominantly detected in the
latter part of the growing season with the earliest
detection on 10 July in isolated plants within pepper
plantings. The date of detection and increased incidence in
the latter part of the growing season suggests that pepper

transplants are not the source of CMV. The most likely

30

source of primary inoculum of CMV in Michigan is weed
reservoir hosts bordering pepper fields. The increased CMV
titer in reservoir plants and primary infected pepper
plants, along with the movement of the aphid vectors into
the fields probably accounts for the incidence of secondary
spread of CMV in the latter part of the growing season. The
distribution of CMV is facilitated by the number of aphid
species (over 60) which are capable of vectoring the virus,
and also by its naturally wide host range, (40 families
which include 191 species) (Francki et al., 1979). Such
factors as virus host reservoirs and the presence of
efficient aphid vectors contribute to the survival and
dissemination of CMV which is endemic in Michigan.

TSWV was detected in Michigan (1989) for the first time
in pepper. TSWV was detected early in the growing season
only in samples from plants purchased as transplants from
suppliers in Georgia. In 1989, unusually high populations
of the thrips Epapkiiniaila occidengaiis were known to have
infested large areas of transplant production in Georgia.
Pepper transplants were then shipped north with an
undetermined percentage of plants infected with TSWV as well
as thrips. Transplants from Georgia generally were
symptomless prior to transplanting in Michigan. Transplant
shock results in defoliation of transplants making it
difficult to detect virus diseases until new growth is
present to assay. In the 1990 survey, we were not able to

detect TSWV until 6 July due to transplant shock defoliation

31

and unseasonally cold temperatures, which suppressed new
growth on plants. In the majority of Michigan fields, the
occurrence of TSWV was minor (<5% of the plant stand). In a
few fields, however, TSWV infected up to 40% of the
transplants. Field symptoms as observed in Michigan were
severe stunting, in some cases almost complete cessation of
growth, many plants did not produce any fruit; distortion
and mottling of young leaves, and ringed patterns on mature
leaves; necrotic markings were present on the fruit. Visual
observation revealed that secondary spread of TSWV within
infected fields was neglible. The lack of secondary spread
may have been a result of low populations of the western
flower thrips (E. occideppaiis) transported with the
transplants or migration of the thrips due to a lack of
foliage or flowers to feed on.

TSWV is distributed worldwide largely because of its
wide host range, the establishment of its main vector, the
western flower thrips in new geographic areas, and movement
of virus-infected plant material, especially in the
floricultural and ornamental industries. Additionally, the
ability of TSWV to form hybrid strains by the process of
recombination has allowed its diverse strains to flourish in
many environments.

Michigan has natural populations of the onion and
celery thrips, which also vector TSWV, however, not as
effectively as the western flower thrips. If the western

flower thrips becomes established outside of greenhouses in

32

Michigan, TSWV could pose a serious threat to the vegetable
industry. TSWV has been reported to cause severe economic
losses in vegetable production in Hawaii (Milbrath and Cook,
1971).

In fields where PVY and TEV were detected, infection
sites remained isolated within pepper plantings. PVY and
TEV currently do not appear to seriously threaten Michigan
pepper production, however, these viruses have caused major
economic losses in California (Abdalla et. at, 1985, and
Makkouk and Gumpf, 1974), Florida (Zitter, 1973), Georgia
(Benner et. al, 1985) New Jersey (Steepy et. al, 1967),
North Carolina (Steepy and Averre, 1971), Texas (Villalon,
1975), and in other countries (Conti and Masenga, 1977, and
Stover, 1951). Therefore, the possibility exists that PVY
and TEV may become important pathogens on pepper in Michigan
in the future.

Viruses in field-infected pepper were difficult to
identify based on symptom development. Plants showing
classic symptoms of CMV infection, necrotic rings or oak-
leaf markings on mature foliage could be diagnosed.
Southern transplants infected early with TSWV could be
detected based on the severe stunting and formation of
concentric rings on the foliage, however, if infection took
place later in the season, positive identification would be
difficult. The identification of pepper viruses based on
field symptom expression alone is not accurate due to

symptom variability. The ELISA technique was found to be

33

efficient and reliable for virus disease detection in pepper

field surveys.

34

LIST OF REFERENCES

Abdalla, O.A., Desjardins, P.R., and Dodds, J.A. 1985.
Survey of pepper viruses in California by the ELISA
technique. Plant Disease. 75:1311.

Agrios, G.N., Walker, M.E., Ferro, D.N., and Corredor, D.
1983. Virus incidence and spread in pepper field plots
treated with reflective mulch and oil. Phytopathology
73:361.

Anderson, C.W. 1959. A study of field sources and spread of
five viruses of peppers in central Florida.
Phytopathology 49:97-101.

Anderson, C.W. and Corbett, M.R. 1957. Virus diseases of
peppers in central Florida survey results 1955. Plant
Dis. Reptr. 41:143-147.

Benner, C.P., Kuhn, C.W., Demski, J.W., Dobson, J.W.,
Colditz, P., and Nutter, F.W. Jr. 1985. Identification
and incidence of pepper viruses in northeastern
Georgia. Plant Dis. 69:999-1001.

Clark, M.F. and Adams, A.N. 1977. Characteristics of the
microplate method of enzyme-linked immunosorbent assay
for the detection of plant viruses. J. Gen. Virol.
34:475-483.

Conti, M. and Masenga, V. 1977. Identification and
prevalence of pepper viruses in northwest Italy.
Phytopathol. Z. 90:212-222.

Francki, R.I.B., Mossop, D.W., and Hatta, T. 1979. Cucumber
mosaic virus. Descriptions of Plant Viruses, NO. 213.
Commonwealth Mycological Institute, Association of
Applied Biologists, Kew, Surrey, England.

Makkouk, K.M. and Gumpf, D.J. 1974. Further identification
of naturally occurring virus diseases of pepper in
California. Plant Dis. Reptr. 58:1002-1006.

Milbrath, G.M. and Cook, A.A. 1971. Virus diseases of pepper
(Capsicum sp.) in Hawaii. Plant Dis. Reptr. 55:783-785.

35

Sherf, A.F. and Macnab, A.A. 1986. Vegetable diseases and
their control. pp 520-536. John Wiley & Sons, New York,
N.Y.

Steepy, T.L. and Averre, C.W. 1971. Prevalence of pepper
viruses in North Carolina. Plant Dis. Reptr. 55:751.

Steepy, T.L., Lewis, G.D., and Varney, E.H. 1967.
Identification of the viruses affecting commercial
varieties of pepper (Capaicum apnuum) in New Jersey.
Plant Dis. Reptr. 51:709-711.

Villalon, B. 1975. Virus diseases of bell peppers in south
Texas. Plant Dis. Reptr. 59:858-862.

Zitter, T.A. 1973. Further pepper virus identification and
distribution studies in Florida. Plant Dis. Reptr.
57:991-994.

CHAPTER 2

REGENERATION OF PEPPER (CAPSICQM amappm) FROM PRIMARY LEAF
EXPLANTS
ABSTRACT

Growth regulator combinations and silver nitrate
concentrations were examined for their effect on shoot
regeneration of 58 pepper genotypes. Primary leaf explants
from ip yippp seedlings were cultured on a revised Murashige
and Skoog medium amended with auxin, cytokinin and 1.6%
glucose. Combinations of of indole-3-acetic acid (IAA), 0-5
mg/L, and 6-benzylaminopurine (BAP), 0-5 mg/L, were tested
to determine the most effective medium for shoot initiation.
When cultured on MS amended with specific growth regulator
combinations, the Capsicum genotypes examined showed varied
shoot initiation and elongation responses. No specific
growth regulator combination resulted in shoot regeneration
of all genotypes tested. Ten mg/L silver nitrate resulted
in the best shoot and leaf differentiation and also reduced
hard callus formation. Differences in morphogenetic
response of individual genotypes were evaluated on a single
shoot initiation medium. Overall, whole plants were
regenerated from 19 of 58 genotypes examined. Based on

these experiments, a reproducible regeneration system for

36

37
pepper was developed with a total of over 500 plants

regenerated.
INTRODUCTION

Although studies examining the manipulation of pepper in
yippp have been moderately successful, shoot regeneration
has been restricted to only a few genotypes. In some of the
previous studies data presented concerned the formation of
shoot buds on explants, but not shoot regeneration
frequencies. To date, the regeneration of whole pepper
plants from somatic cells is not sufficiently routine for
the application of technologies such as somaclonal variation
and genetic transformation.

Manipulative variables that have been studied in pepper
regeneration systems include culture medium constituents,
length of photoperiod, temperature, and explant age and
source (Agrawal et al., 1989; Fari and Czako, 1981; Gunay
and Rao, 1978; Phillips and Hubstenberger, 1985; Sripichitt
et al., 1987). Previous studies involving pepper
regeneration have utilized Murashige and Skoog (MS) medium
(Murashige and Skoog, 1962), but differed on the source of
explant and in regard to the function and importance of
auxin and cytokinin in the regeneration process.

The goal of this study was to develop a protocol for
efficient regeneration of whole plants from primary leaf
explants of Capsicum spp. The objectives of this research
were: 1) to examine the role of growth hormones in pepper

shoot initiation and elongation; 2) determine the effect of

38

silver nitrate on morphogenetic responses of pepper; 3)
examine the effect of environmental conditions on ip yippp
pepper culture; and 4) screen Capsicum genotypes to test for
regeneration capacity.

MATERIALS AND METHODS

Ca c e ot es
The Capaippm genotypes surveyed encompass a wide range

-of vegetative morphologies and horticultural qualities
including traditional open-pollinated cultivars as well as
hybrids. Most of the C. appppm cultivars examined are
commonly grown by commercial producers in the United States.
The plant introductions (PI) used in the study were obtained
from the Southern Regional Plant Introduction Station
(Experiment, GA).
Ia vippp saadiinga

Seeds of C. appppm were surface sterilized by immersion
in 95% ethanol for 2 min followed by 30 min in 50% aqueous
(v/v) commercial bleach (2.62% sodium hypochlorite)
containing 1-2 drops/250 ml Tween 20 (Sigma Chemical Co.,
St. Louis, MO). The seeds were rinsed three times and
imbibed for 30 min in sterile distilled water. Nine seeds
per container were sown in GA-7 vessels (Magenta Corp.,
Chicago, IL) containing 50 ml of revised MS medium
(Murashige and Skoog, 1962) modified by omitting the growth
hormones, reducing the amount of NHJKE to 165 mg and Bacto
agar (Difco Laboratories, Detroit, M1) to 8 g, and

increasing the thiaminedflzl to 0.2 mg per liter. The pH of

39
the medium was adjusted to 5.8 using 1N NaOH or 1N HCl prior

to autoclaving. Vessels were placed in a growth room at 25
C under cool-white fluorescent lights supplying 50 umoluw
2~s‘l on a 16 hr photoperiod to allow seed germination and
seedling growth.
£22122_21_2§21sB£§

For all experiments explants (5 x 10 mm) were excised
from fully expanded primary leaves of 4-6 wk-old seedlings

(Fig. 2.1), resulting in 2-4 explants per leaf, depending on

the genotype used.

t e um

For all experiments MS medium was modified by changing
the carbon source to glucose (16 9), increasing the amount
of nicotinic acid, pyridoxin-HCl and thiamine-HCl to 1.0 mg,
and decreasing the amount of Bacto agar to 8 g per liter.
The pH of the medium was adjusted to 5.8 using 1N NaOH or 1N
HCl before the agar was added, and 25 m1 of medium was
dispensed into 100 x 15 mm petri dishes. This modified MS
medium will be designated MSc medium.

etc

After 30 days in culture, the explants were rated for
shoot regeneration response. A rating system of 1-5 was
used: 1 = no callus formation, 2 = callus formation, 3 =
callus and shoot-bud formation, 4 = callus, shoot bud and
leaf formation, and 5 = callus and elongated shoot formation

(Fig. 2.2). This rating system was employed in all

40

 

Figure 2.1 Four to six week-old seedlings of pepper
(M film) grown in Vitrp.

41

 

 

 

 

Figure 2.2 A rating system for the regeneration response of
pepper (Capsicum annuum) from leaf explants after 30 days in
culture. Regeneration rated on 1-5 scale; 1 = no callus
formation, 2 = callus formation, 3 = callus/shoot bud
formation, 4 = shoot bud/leaf formation, and 5 = elongated
shoot formation.

42
experiments. Explants with a rating of 4 or 5 were

subcultured for shoot elongation to GA-7 vessels in 50 m1 of
MSc medium supplemented with 2 mg/L or 4 mg/L of BAP or 0.1
mg/L NAA depending on the experiment. Elongated shoots were
excised and transferred for rooting to GA-7 vessels in 50 ml
of MSc medium without growth hormones. Each experiment was
repeated at least once. As the results obtained were very
similar, the results of only one experiment are presented in
this study.
W

Auxin, in the form of indole-3-acetic acid (IAA), and
cytokinin as 6-benzylaminopurine (BAP) were added alone or
in various combinations at concentrations of 0, 1, 2, 3, 4,
and 5 mg/L each to MSc medium. The variables IAA, BAP and
genotype were arranged in a factorial combination in a
completely random design. The three C. annuum genotypes
used in the experiment were P1178849 and the cultivars
Mayata and Paprika. Three explants were placed per petri
dish on MSc medium and incubated at 26 C under cool-white
fluorescent lights supplying 20 umol-m‘z-s’l on a 16 hr
photoperiod (Figure 2.3). Eighteen explants were employed
in each growth hormone treatment.
Silver nitrate emperiment

MSc medium was amended with IAA (3 mg), BAP (4 mg) and
silver nitrate at concentrations of 1, 10, 20, and 30 mg per
liter. The variables silver nitrate concentration and

genotype were arranged in a factorial combination in a

43

 

Figure 2.3 Primary leaf explants (5 x 10 mm) of pepper
(Capsicum annuum) cultured on Murashige and Skoog
regeneration medium.

44

completely random design. The four C. appppm genotypes
examined were P1178849 and the cultivars Bell Star, Mayata,
and Memphis. Three explants were placed per petri dish on
MSc medium and cultured as above. Forty eight explants were
used in each silver nitrate treatment.
Temperature experiment

Temperatures of 24, 26, 28 and 30 C were used to
culture pepper explants placed on MSc medium supplemented
with 1AA (3 mg), BAP (4 mg), and silver nitrate (10 mg) per
liter. The variables temperature and genotype were arranged
in a factorial combination in a completely random design.
The four C. annuum genotypes used were P1178849 and the
cultivars Espana, Mission Belle, and Paprika. Three
explants were placed per petri dish on MSc medium and
incubated at the treatment temperature under cool-white
fluorescent lights supplying 20 umol-Iii'2-5'l on a 16 hr
photoperiod. Forty eight explants were employed in each
temperature treatment.
Shoot elongation emperimenp

Primary leaf explants from P1178849 were cultured on
MSc medium supplemented with IAA (3mg), BAP (4 mg) and
silver nitrate (10 mg) per liter. After approximately 30
days in culture explants with a rating of 4 or 5 were
transferred to MSc medium supplemented with BAP (4 mg) or
NAA (0.1 mg) per liter. The number of explants forming
elongated shoots and the number of elongated shoots per

explant were counted. Elongated shoots were excised and

45
rooted in MSc medium without growth hormones.

W

Primary leaf explants of Capaippm genotypes Bell Star,
California Wonder, Crispy Hybrid, Jalapeno, Jupiter,
Marengo, Mayata, Memphis, P1178849, and PIP were cultured on
MSc medium supplemented with IAA (3 mg), BAP (4 mg), and
silver nitrate (10 mg) per liter. Three explants were
placed per petri dish on MSc medium and incubated at 28 C
under cool-white fluorescent lights supplying 20 11mol-m”-s‘1
on a 24 hr photoperiod. Twenty four explants per genotype'
were employed. Cultures were transferred onto shoot
elongation medium containing 0.1 mg/L NAA.

During this study, 58 Capsicum genotypes were screened
on MSc medium for their shoot regeneration capacity.

cc at o a lone

Regenerated pepper somaclones were planted in moistened
soilless peat mix (Baccto Professional Planting Mix,
Michigan Peat Co., Houston, TX) in 2 or 3" plastic pots and
placed in plastic bags. Plants were grown at room
temperature (24 C) under cool white fluorescent lights
supplying 60 ;Imol-rri'2-S'l 'with a 16 hr photoperiod. Plants
were gradually acclimated by opening the plastic bag for
increased periods of time. After plants were acclimated
they were placed in the greenhouse and allowed to self

pollinate and form seeds (Fig. 2.4).

46
RESULTS

MW

An analysis of variance (Table 2.1) shows that the
factors genotype, IAA, and BAP were significant at P = 0.01
level. There was also a significant interaction (P = 0.05)
between factors, indicating the factors were not
independent.

The following visual observations were made while
rating cultures. Without the presence of auxin (IAA) and
cytokinin (BAP) in the MSc medium explants of all Capsicum
genotypes examined showed no morphogenetic response in
culture. MSc medium containing 1-5 mg/L IAA resulted in
callus and in some cases root formation on primary leaf
explants. With only the amendment of cytokinin (BAP) in the
MSc medium primary leaf explants of all Capsicpm genotypes
examined formed callus and shoot buds when BAP was included
at 3-5 mg/L. When auxin (IAA) and cytokinin (BAP) were used
in combination, morphogenetic responsiveness increased,
however, response varied among genotypes with different IAA
and BAP combinations. IAA in a range of 0-5 mg/L and BAP in
a range of 3-5 mg/L resulted in better regeneration means
for the genotypes tested.

8 ve ni ate a er ment

An analysis of variance (Table 2.2) shows that the
factors genotype and silver nitrate were significant at the
level P = 0.05 and 0.01, respectively. There was no

significant interaction between the two factors. A Duncan's

47

 

Figure 2.4 Regenerated somaclones of pepper (Capsicum
anpuum P1178849) from primary leaf explants.

48

Table 2.1. Analysis of variance of a 3 X 6 X 6 factorial
experiment concerning the effects of indole-3-acetic acid,
6-benzylaminopurine, and pepper genotype on shoot
regeneration.

 

 

Source df mean square F value
Genotype 2 7.35‘ 37.43*
1AA 5 0.97 4.96*
Genotype x IAA 10 0.30 1.54
BAP 5 12.34 62.82**
Genotype x BAP 10 0.38 1.91*
1AA x BAP 25 0.36 1.81*
Genotype x IAA x BAP 50 0.33 1.66*
Error 108 0.20

 

‘Regeneration rated on 1-5 scale; 1 = no callus formation,
2 = callus formation, 3 = callus/shoot bud formation,
4 = shoot bud/leaf formation, and 5 = elongated shoot
formation.

*Significant difference at P = 0.05
**Significant difference at P = 0.01

49
multiple range test (Table 2.3) revealed that all genotypes

responded the best when 10 mg/L of silver nitrate was
amended to the MSc medium. Although genotype and silver
nitrate treatment means were not significantly different,
there were qualitative differences seen in the cultures
(Fig. 2.5). Visual observations of the cultures showed a
decrease in callus formation and/or the callus formed was
more friable. Also, shoot buds and leaves were more
differentiated with the presence of 10 or 20 mg/L silver
nitrate in the medium. At 30 mg/L silver nitrate, some of
the leaves looked thicker, abnormal in shape, and water-
soaked.
Temperature experiment

An analysis of variance revealed that the factors
genotype and temperature were not significant at the level P
= 0.05 (Table 2.4) A Duncan's multiple range test showed
similar regeneration means in all genotypes examined at 28 C
(Table 2.5). Although treatment means were not
significantly different, visual observations revealed shoot
and leaf structures were more differentiated after
approximately 30 days in culture at 28 C than at lower
temperatures (Fig. 2.6). At 30 C some of the leaves
produced on cultures began to turn yellow, showing signs of
stress before being subcultured.
Shoot elongation-emperiment

Both BAP (4 mg/L) and NAA (0.1 mg/L) were effective in

promoting shoot elongation of shoot buds initiated on

50

Table 2.2. Analysis of variance of a 4 X 4 factorial
experiment concerning the effect of silver nitrate and
pepper genotype on shoot regeneration.

 

 

Source df mean square F value

Genotype 3 4.72‘ 15.44**

Silver nitrate concentration 3 0.86 2.81*

Genotype x silver nitrate 9 0.19 0.61
concentration

Error 48 0.31

 

‘Regeneration rated on 1-5 scale; 1 = no callus formation,
2 = callus formation, 3 = callus/shoot bud formation,
4 = shoot bud/leaf formation, and 5 = elongated shoot
formation.

*Significant difference at P = 0.05
**Significant difference at P = 0.01

51

Table 2.3. Differences in the effect of silver nitrate
concentration amended to Murashige and Skoog medium on shoot
regeneration of four pepper genotypesfi

 

 

Ipeatmemt Treatment
Genotype Silver Nitrate (mg/L) mean
P1178849 0 2.4 c‘
P1178849 10 3.2 abc
P1178849 20 2.7 c
PI178849 30 2.8 bc
Bell Star 0 3.6 ab
Bell Star 10 3.9 a
Bell Star 20 3.9 a
Bell Star 30 3.8 a
Memphis 0 2.9 bc
Memphis 10 4.0 a
Memphis 20 3.7 ab
Memphis 30 3.8 a
Mayata 0 4.0 a
Mayata 10 4.0 a
Mayata 20 4.0 a
Mayata 30 4.0 a

 

fMeans within the column followed by the same letter do not
differ by Duncan’s multiple range test (P = 0.05).

‘Regeneration rated on 1-5 scale; 1 = no callus formation,
2 = callus formation, 3 = callus/shoot bud formation,
4 = shoot bud/leaf formation, and 5 = elongated shoot
formation.

52

Mimi?"

' 20 30

 

 

 

 

 

 

 

 

 

 

Figure 2.5 Qualitative differences in the response of four
pepper genotypes cultured on Murashige and Skoog medium
supplemented with O, 10, 20, and 30 mg/L silver nitrate.

53
primary leaf explants of Capgipum genotypes. A larger

percentage of P1178849 explants formed elongated shoots
after approximately 2 wk in culture on the NAA treatment
(63%) than the BAP treatment (12%) (Table 2.6). There was
no significant difference in the number of shoots formed per
explant between the treatments. Explants continued to form
elongated shoots up to 56 days after subculture to MSc
medium amended with 0.1 mg/L NAA (Fig. 2.7).
MPG—M

The Capsicum genotypes examined showed varied shoot
initiation and elongation responses when cultured ip yip_p.
All genotypes tested formed shoot buds, however only four of
the genotypes formed elongated shoots (Table 2.7). The
genotype P1178849 showed the lowest shoot initiation
response, but formed the highest number of elongated shoots.

Of the 50 C. annuum genotypes cultured on MSc medium,
13 formed elongated shoots (Table 2.8). Of the 8 genotypes
of other Capaicum species, 6 formed elongated shoots (Table
2.8) when cultured on MSc medium.

DISCUSSION

Successful and efficient regeneration in plant tissue
culture is dependent on the thorough understanding of how
the plant species, explant source, composition of the
culture medium and environmental conditions affect shoot
initiation and elongation. Among these factors, the
composition of the culture medium and especially growth

regulators play a key role in shoot initiation, elongation

54

Table 2.4. Analysis of variance of a 4 X 4 factorial
experiment concerning the effect of temperature on shoot
regeneration of four pepper genotypes cultured on Murashige
and Skoog medium supplemented with 3 mg/L indole-3-acetic
acid, 4 mg/L 6-benzylaminopurine, and 10 mg/L silver
nitrate.

 

 

Source df mean square F value
Genotype 3 0.655‘ 2.20 ns
Temperature 3 0.670 2.25 ns
Genotype x temperature 9 0.218 0.73 ns
Error 48 0.297

 

‘Regeneration rated on 1-5 scale; 1 = no callus formation,
2 = callus formation, 3 = callus/shoot bud formation,
4 = shoot bud/leaf formation, and 5 = elongated shoot
formation.

ns = nonsignificant at P = 0.05 or 0.01

55

Table 2.5. Differences in the effect of temperature on
shoot regeneration of four pepper genotypes cultured on
Murashige and Skoog medium supplemented with 3 mg/L indole-
3-acetic acid, 4 mg/L 6-benzylaminopurine, and 10 mg/L
silver nitratefi

 

 

Ipeatmepp Treatment
Genotype Temperature (C) mean
PI178849 24 2.9 bc'
P1178849 26 3.4 ab
P1178849 28 4.0 a
PI178849 30 3.3 ab
Mission Belle 24 3.5 ab
Mission Belle 26 3.7 a
Mission Belle 28 3.9 a
Mission Belle 30 3.8 a
Paprika 24 3.8 a
Paprika 26 4.0 a
Paprika 28 3.8 a
Paprika 30 3.8 a
Espana 24 3.6 ab
Espana 26 3.5 ab
Espana 28 3.9 a
Espana 30 4.0 a

 

fMeans within the column followed by the same letter do not
differ by Duncan's multiple range test (P = 0.05).

‘Regeneration rated on 1-5 scale; 1 = no callus formation,
2 = callus formation, 3 = callus/shoot bud formation,
4 = shoot bud/leaf formation, and 5 = elongated shoot
'formation.

56

 

Figure 2.6 Qualitative differences in the response of four
pepper genotypes cultured on Murashige and Skoog medium
supplemented with 3 mg/L indole-3-acetioc acid, 4 mg/L 6-
benzylaminopurine, and 10 mg/L silver nitrate at 24, 26, 28,
and 30 C.

57

Table 2.6. Differences in the percentage of explants
forming shoots and the number of shoots per explant of
pepper genotype cultured on Murashige and Skoog medium
supplemented with 0.1 mg/L naphthaleneacetic acid or

4 mg/L 6-benzylaminopurine.

 

 

Explants Shoots
Medium‘ forming shoots per explant
4 mg/L BAP 4/30(12%) 3.5
0.1 mg/L NAA 19/30(63%) 3.7

 

‘A basal medium of Murashige and Skoog supplemented as
stated.

58

 

Figure 2.7 Shoot elongation of pepper (Capsicum annuum
PI178849) on Murashige and Skoog medium amended with 0.1
mg/L naphthaleneacetic acid.

59

Table 2.7. Shoot initiation and elongation responses of
Capsipum genotypes when cultured on Murashige and Skoog
medium supplemented with 3 mg/L indole-3-acetic acid, 4 mg/L
6-benzylaminopurine, and 10 mg/L silver nitrate.

 

 

No. explants No. explants
forming forming
Genotype shoot buds elongated shoots
Bell Star 21/24 1/24
California Wonder 7/24 0/24
Crispy Hybrid 11/24 1/24
Jalapeno 14/24 2/24
Jupiter 5/24 0/24
Marengo 9/24 0/24
Mayata 12/24 0/24
Memphis 10/24 0/24
P1178849 5/24 2/24

PIP 12/24 0/24

 

60

Table 2.8. Shoot regeneration capacity of Capsicum
genotypes when cultured on Murashige and Skoog medium
supplemented with 3 mg/L indole-3-acetic acid,

4 mg/L 6-benzylaminopurine, and 10 mg/L silver nitrate.

 

Genotype Regeneration‘

 

Q . annuum

Annabelle -
Bell Star +
Bell Tower -
Big Belle -
California Wonder
Cadice

Calumet

Capri

Crispy Hybrid

Cuban

Early California Wonder
Espana

Giant Ace -
Gloria —
Green Boy -
Gloria Pepper -
Hidalgo

Honeybelle

Hybrid Hot

Jalapeno

Jupiter

Keystone Resistant Giants
Lady Bell

Marengo

Mayata

Memphis

Midal Pepper

Mission Belle

Mr. Bell

Orobelle

PI173769

P1174114

PI178849

P1201571

PI206949

PIP

Paprika

Pepper Mexibell

Pimiento Perfection
Purple Bell

I++II

I-++-+ I+-+-+I I

+

r +-+I + l

 

61

 

Genotype Regeneration‘

 

Ranger -
RioGrande Gold -
Scarlet Pendant —
Sironofi -
Skipper -
Summer Sweet Brand -
Sweet Belle —
Tambel-Z -
Yolo Wonder -
Yolo Wonder L -

C. baccatum
PI238061

P1281407
P1439383

9.93m

P1260435 +
C. cpipepse

P1360724 +
9W

P1358968 _ +
C. pragtazmissum

P1260595 -

+-+-+

C. pubascens
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regeneration of whole pepper plants

= regeneration of whole pepper plants did not occur on
the medium used

' +

62
and rooting. In the present study, no one specific

combination of IAA and BAP resulted in optimal shoot
initiation in primary leaf explants of all Capaippm
genotypes tested. The genotypes initiated shoots when IAA
was used at 0-5 mg/L and BAP was used at 3-5 mg/L.
Concentrations of 3 mg/L IAA and 4 mg/L BAP were chosen for
the shoot initiation medium since all genotypes examined in
the experiment formed shoot buds to some extent at these
concentrations. The results of this study support previous
reports concerning the importance of auxin and cytokinin in
regeneration of pepper ip zippp (Fari and Czako, 1981; Gunay
and Rao, 1978; Phillips and Hubstenberger, 1985). These
results disagree with other reports stating that 1AA is
inhibitory to shoot initiation in pepper (Agrawal et al.,
1989; Sripichitt et al., 1987).

NAA at 0.1 mg/l induced shoot elongation with some
Capaippm genotypes. Agrawal et al. (1989) reported the
importance of NAA in pepper shoot elongation, however, NAA
at 0.1 mg/L was not sufficient for elongation of all
genotypes examined in this study. BAP at 2 or 4 mg/L also
induced shoot elongation in some genotypes (data not shown).

The production of ethylene in plants is induced by
wounding and external factors such as auxin. Wounding and
auxin are factors which are present in most ip yippp tissue
culture systems. Induction of ethylene production in plants
can in turn bring about important physiological consequences

(Yang and Hoffman, 1984). Exogenously applied ethylene or

63

ethylene precursors inhibited shoot initiation in tobacco
callus cultures (Huxter et al., 1981). Silver ions are
potent inhibitors of ethylene action in plants (Purnhauser
et al., 1987). Silver nitrate effectively promoted shoot
regeneration in wheat and tobacco cultures, which previously
had poor regeneration ability (Purnhauser et al., 1987).
Silver nitrate did not affect shoot initiation in Capsipum
leaf explants; however, shoots and leaves were more
developed. Also, callus formation was reduced or the callus
formed was more friable. Callus overgrowth suppressed shoot
primordia in 58-62% of Capaippm cultures in a study by Fari
and Czako (1981). Hard callus overgrowth was observed in
Capsipum cultures on medium lacking silver nitrate. Because
of positive effects on in yippp culture of Capaippm spp., 10
mg/L silver nitrate became a routime addition to the shoot
initiation medium.

Leaf tissue of pepper has been used as an explant
source in previous regeneration studies. However, explants
were obtained from older plants grown in a greenhouse
(Phillips and Hubstenberger, 1985), or from field grown 3-
to-4 month old plants (Agrawal et al., 1989). Also, in
these earlier works the data reported involves shoot bud
formation on leaf explants and not data on shoot elongation
or whole plant regeneration (Fari and Czako, 1981; Gunay and
Rao, 1978; Phillips and Hubstenberger, 1985). The present
study reports whole plant regeneration from primary leaf

explants, and includes data on the number of explants

64
forming elongated shoots and the number of elongated shoots

formed per explant.

The shoots regenerated from primary leaf explants of
pepper are adventitious shoots. Microscopic observation
revealed that the shoot primordia were differentiated
directly on the explants around the cut edges of the leaf.
This observation is in agreement with the histological

evidence provided by Agrawal et a1. (1989).

65

LIST OF REFERENCES

Agrawal, S., Chandra, N., and Kothari, S.L. 1989. Plant
regeneration in tissue cultures of pepper (Capsicum
annuum L. cv. maphania). Plant Cell Tiss. Org. Cult.
16:47-55.

Fari, M., and Czako, M. 1981. Relationship between
position and morphogenetic response of pepper hypocotyl
explants cultured in vitro. Sci. Hortic. 15:207-213.

Gunay, A.L., and Rao, P.S. 1978. In vitro plant
regeneration from hypocotyl and cotyledon explants of
red pepper (Capsicum). Plant Sci. Lett. 11:365-372.

Huxter, T.J., Thorpe, T.A., and Reid, D.M. 1981. Shoot
initiation in light- and dark-grown tobacco callus: the
role of ethylene. Physiol. Plant. 53:319-326.

Murashige, T., and Skoog, F. 1962. A revised medium for
rapid growth and bio assays with tobacco tissue
cultures. Physiol. Plant. 15:473-496.

Phillips, G.C., and Hubstenberger, J.F. 1985.
Organogenesis in pepper tissue cultures. Plant Cell
Tiss. Org. Cult. 4:261-269.

Purnhauser, L., Medgyesy, P., Czako, M., Dix, P.J., and
Marton, L. 1987. Stimulation of shoot regeneration in
Triticum aestivum and Nicotiana plumbaginifplia Viv.
tissue cultures using the ethylene inhibitor AgNOr
Plant Cell Rep. 6:1-4.

Sripichitt, P., Nawata, E., and Shigenaga, S. 1987. In
vitro shoot-forming capacity of cotyledon explants in
red pepper (Capsicpm annuum L. cv. Yatsufusa). Japan.
J. Breed. 37:133-142.

Yang, S.F., and Hoffman, N.E. 1984. Ethylene biosynthesis
and its regulation in higher plants. Ann. Rev. Plant
Physiol. 35:155-189.

CHAPTER 3

SCREENING THE R1 GENERATION WITH CUCUMBER MOSAIC VIRUS

ABSTRACT

Four hundred plants were regenerated from primary leaf
explants of pepper (Capaippm ampppm P1178849). R1
generation lines of the somaclones were assessed under
growth chamber and greenhouse conditions for variation in
response to Cucumber mosaic virus (CMV) using mechanical and
natural aphid vector inoculation techniques. The screening
of R1 generation lines for resistance to CMV revealed
extensive variation in response among the lines and also
within the parental control line. There was a poor
correlation between visual ratings for symptom development
and assessment using ELISA for CMV infection. R1 lines and
parental controls contained a high proportion of plants
which did not develop symptoms (escapes). Plants remaining
uninfected were visually observed in the parent and R1
generation lines during the experiment. In view of the
small sample size and the fact that parental controls also
contained escapes, no significance was attached to this. A
total of 2/95 lines in the study were not infected, however,

repeated inoculation of these lines resulted in infection.

66

67
INTRODUCTION

Traditional methods of plant breeding in pepper
(Capsicum spp.) have yielded few cultivars presently
resistant to cucumber mosaic virus (CMV); none that are
widely used in commercial production (Barrios et al., 1971).
Traditional plant breeders utilize many sources of
variability such as land races and wild species for input of
new genes. Somaclonal variation holds promise as an
alternative source for genetic variation in plant breeding
(Larkin and Scowcroft, 1981). Somaclonal variants of plant
species generally range from highly-susceptible to highly-
resistant when challenged with disease organisms to which
the plant was originally susceptible (Brown et al., 1986;
Larkin, 1981). The phenomenon of somaclonal variation has
been utilized in selecting tomato somaclones resistant to
tobacco mosaic virus (Barden, 1986), lettuce somaclones with
reduced susceptibility to lettuce mosaic virus (Brown et
al., 1986), and sugarcane subclones with a greater degree of
resistance to Fiji virus (Krishnamurthi, 1974).

The objectives of this study were 1) to determine
whether variation in disease response to CMV occurred in
pepper somaclones regenerated from leaf explants and 2) to
compare the efficiency of aphid versus mechanical
transmission of CMV to R1 pepper lines in a screening

system.

68
MATERIALS AND METHODS

line

A vinca strain of CMV was provided by Dr. Robert Davis
(Agdia, Inc., Elkhart, IN). This strain of CMV was used
because it was virulent on pepper and was used by Agdia,
Inc. to develop the CMV pathoscreen kit, which was employed
in this study. The virus was increased in P1178849 pepper
plants for use in inoculations. The vinca strain of CMV
will be designated as CMVvi hereafter.
Rim—411111.99

Seeds were collected from all mature self-pollinated
fruits of each somaclonal plant and pooled. Fifty seeds
from the parent and each somaclone were sown in a soilless
peat mixture in 3" plastic pots covered with clear plastic
wrap. The seeds were allowed to germinate at room
temperature (24 C) under cool-white fluorescent lights
supplying 30 umoluNLSJ. Thirty-six parent seedlings and
twenty-four R1 seedlings of each somaclone were transplanted
in soilless peat mixture in 8 oz styrofoam cups (Dart
Container Corp, Mason, M1) at 12-18 days after sowing.
inoculatiop o; R; seedlings

Twelve seedlings from the P1178849 parent and each R1
line were used in each treatment in studies screening for
resistance to CMVvi. The two uppermost leaves of seedlings
were inoculated between 24-31 days after sowing. Controls
consisted of both inoculated and healthy parent plants.

Seedlings of the parent and R1 lines were reinoculated if

69
they tested negative to CMV upon ELISA assay. These

seedlings were subsequently observed visually for symptom
development 14 and 28 days after mechanical and aphid
inoculations respectively.
Machanical inoculation

Leaves of seedlings were dusted with carborundum and
rub-inoculated with a cotton swab dipped in a solution
containing 1 g fresh weight of CMVvi infected pepper tissue
which was ground in 10 ml of 0.2 M Na-phosphate buffer pH
7.0 using a mortar and pestle. The leaves were allowed to
air dry and were rinsed with distilled water to remove
excess carborundum and virus inoculum. After inoculation,
the plants were grown in a growth chamber at 27 C under
cool-white fluorescent lights at 60 umolurLSJ.
Aphid inoculapion

Green peach aphids (Myapa persicae) were obtained from
Dr. Donald Ramsdell. Aphids were reared in cages on CMVvi-
infected pepper plants. Five aphids that had been reared
continously on infected pepper plants were transferred to
each seedling and allowed a 48 hr inoculation access period
after which aphids were removed or killed. After
inoculation, the plants were grown in a greenhouse under
spring weather conditions.
virus screening of R1 pepper lines using ELISA

Assays were done at approximately 14 and 28 days after
mechanical and aphid inoculations, respectively. This time

frame was determined necessary based on symptom development

70
and ELISA studies using mechanical versus aphid inoculation

techniques. The uppermost leaf tissue (0.2 - 0.5 g) was
placed in a plastic bag and ground at a 1:10 dilution in
extract buffer. Sample aliquots of 100 pl were loaded into
microtiter plates, 2 wells per sample and subjected to ELISA
according to the protocol supplied by the manufacturer
(Agdia, Inc., Elkhart, IN).

RESULTS

Virus infection was assessed visually for symptom
expression and assessed using ELISA. Symptoms were rated on
a scale of 0 - 2 where 0 = plants without symptoms, 1 =
plants with indistinct symptoms, and 2 = plants with
distinct, characteristic symptoms. Plants with a rating of
1 or 2 were considered CMV-positive. Plants were considered
CMV-positive using ELISA if their absorbance value (490 nm)
was 3X the mean of the healthy control.

Each of 95 R1 generation lines were screened with CMV
using mechanical and aphid-inoculation techniques. The
visual rating and ELISA absorbance values were converted
into percentage of CMV-positive plants (Fig. 3.1 - 3.7).
Extensive variation was observed among and within the parent
and R1 lines. Also, variation was observed between the
inoculation techniques and between the assessment
techniques. Among the seven experiments, infection in the
parental line monitored with ELISA varied from 0 to 67% and
0 to 92% using the mechanical inoculation and aphid

inoculation techniques respectively. A total of 93/95 lines

Infection (96)
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annuum P1178849) obtained using mechanical or aphid-
inoculation techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis as
detection methods for virus-infection.

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enzyme-linked immunosorbent assay (ELISA) analysis as
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infected plants of R1 generation lines of pepper (Capsicpm
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detection methods for virus-infection.

77

Figure 3.6 Experiment 6. A comparison of the percentage of
infected plants of R1 generation lines of pepper (Capsicum
appuum P1178849) obtained using mechanical or aphid-
inoculation techniques and a comparison of visual rating or
enzyme-linked immunosorbent assay (ELISA) analysis as
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83
were considered susceptible to CMV because at least 10% of

the plants of these lines were CMV-positive using one of the
inoculation techniques. Only 2/95 lines were rated CMV—
negative during the initial experiment. However,
reinoculation of these two lines resulted in >40% plants
visually rated as CMV-positive.

DISCUSSION

The screening of R1 generation lines for resistance to
CMV revealed extensive variation in response among the lines
and also within the parental line. Variation was also
observed in the same line using rub and aphid inoculation
techniques when infection was assessed using a visual rating
system for symptom development or ELISA. In one experiment,
none of the plants in the parental line were determined to
be infected using a visual rating system and ELISA.
Variability among parents such as this was also observed by
Brown et al. (1986) when screening lettuce R1 lines for
resistance to lettuce mosaic virus.

Most of these uninfected plants are believed to be
escapes, i.e. inoculated plants in which infection was
unsuccessful. Because escapes occurred in both parental and
R1 generation lines within the same experiment, these were
not viewed as being significant. Unsuccessful infection of
pepper leading to escapes can be caused by a number of
factors. Pepper leaves contain a protein and a phenolic
substance which act as natural inhibitors against subsequent

mechanical inoculation of virus. In a study by Horvath and

84
Nienhaus (1982), pepper plant extracts reduced the activity

of CMV up to 100% in cowpea. In our experiments, attempts
to mechanically transmit CMV from infected pepper to local
lesion hosts (Chenopodium and cowpea) were repeatedly
unsuccessful probably due to these inhibitors. This may
also explain the variation in infection of the parent and R1
generation lines which were inoculated mechanically.

Infected plants used as inoculum in experiments may
have contained a low virus titer or the titer of the virus
may have differed among the experiments, leading to
variation in infection.

Variability among aphid-inoculated plants may have been
caused by differences in virus titer among experiments or
uncontrollable variation among aphid feeding.

In a study of resistance to TMV in tomato somaclones,
Barden et a1. observed a delay in symptom development of 46
days in 18/376 somaclones. Among the 93 lines studied in
this experiment, only two remained uninfected after 30 days.
Plants from these two lines were re-inoculated and visual
assessment revealed >40% infection. Plants from those two
lines were most likely uninfected escapes.

If somaclonal lines were to be rated as resistant to
CMV, a delay in symptom development and virus detection must
be observed. Therefore, lines in which as little as 10% of
the plants were infected would likely be discarded as
susceptible and the unsuccessful infection attributed to

escapes.

85
Somaclonal variation may hold promise as a means for

generating virus resistance. The screening regimen,
however, is labor-intensive, and tremendous variability may

be observed.

86

LIST OF REFERENCES

Barden, K.A., Smith, S.S., and Murakishi, H.H. 1986.
Regeneration and screening of tomato somaclones for
resistance to tobacco mosaic virus. Plant Sci. 45:209-

213.

Barrios, E.P., Mosakar, H.I., and Black, L.L. 1971. The
inheritance of resistance to tobacco etch and cucumber
mosaic viruses in Capsicum frutescens. Phytopathology
61:1318.

Brown, C., Lucas, J.A., Crute, I.R., Walkey, D.G.A., and
Power, J.B. 1986. An assessmant of genetic
variability in somacloned lettuce plants (Lactuca
sativa) and their offspring. Ann. Appl. Biol. 109:391-
407.

Krishnamurthi, M., and Tlaskal, J. 1974. Fiji disease
resistant Saccharum officianarum var. Pindar subclones
from tissue cultures. Proc. Int. Soc. Sugarcane
Technol. 15:130-137.

Larkin, P.J., and Scowcroft, W.R. 1981. Somaclonal
variation -- a new source of variability from cell
cultures for plant improvement. Theor. Appl. Genet.

60:197-214.

CHAPTER 4

PROTOPLAST INFECTION WITH VIRUS PARTICLES

ABSTRACT

Protoplasts were consistently isolated from pepper
(Capsicum annuum) with an average viability of 85%.
Variables studied among electroporation experiments included
virus concentration, protoplast density, and power levels.
Infection of protoplasts was achieved in two experiments,
one using a protoplast density of 5 x 105/ml, virus
concentration of 50 ug/ml, and power levels of 250, 300,
350, or 400 volts/cm, and the other using a protoplast
density of 6.5 x 162 virus concentration of 125 ug/ml, and
power level of 250 volts/cm. Subsequently, voltage was
shown to sharply reduce protoplast viability above 100
volts/cm.

INTRODUCTION

Disease-resistant plants have traditionally been
produced by identifying resistant genotypes and then
crossing these individuals with cultivars that possess
traits which are agriculturally valuable. Although
traditional breeding has been highly successful in many

cases, there are limitations to classical plant breeding

87

88
methods. Some of the limitations include: 1) the desired

disease resistance is not always available, 2) the
resistance may only exist in a species that is not
interfertile with the crop, 3) the resistance genes may be
tightly linked to other undesirable traits or 4) the
resistance may be multigenic and difficult to transfer
(Knott and Dvorak, 1976; Wenzel, 1985).

Tissue culture techniques may be used to overcome some
of the factors limiting traditional plant breeding.
Protoplasts are currently being used as a source of plant
material for a range of in yitgg genetic manipulations aimed
at studying and developing disease-resistant crop plants.
One technique, protoplast fusion, involves the transfer of
resistance genes from a resistant to a susceptible host,
resulting in the production of interspecific plant hybrids
(Glimelius, 1988). Virus resistant transgenic plants
containing cloned viral genes have been produced using plant
transformation and molecular genetic technologies (Powell-
Abel et al., 1986). The most significant contribution of
tissue culture technologies has been as a tool to study
basic mechanisms of pathogen virulence and host defense.
The most extensive studies used protoplasts to study the
mechanism of infection and replication of plant viruses
(Zaitlin and Hull, 1987).

Utilization of plant protoplasts in an in giggg
selection system may offer a means of selecting plant

genotypes with increased resistance to disease. Screening

89
for resistance to plant pathogens at the single cell level

offers unique advantages. Millions of cells can be screened
in a short time, the technique holds promise for selection
of resistance from susceptible cultivars (Shepard, 1981),
and may allow for the recovery of increased levels of
disease resistance (Murakishi and Carlson, 1982).

To date, viral pathogens offer the most promise as
direct selection agents in the use of in yitrg screening
systems because protoplasts can be synchronously infected
with viruses (Hibi et al., 1986; Maule et al., 1980;
Motoyoshi and Oshima, 1975; Nishiguchi et al., 1987).
Infection of protoplasts has been demonstrated in a variety
of plant species such as barley, Brassica, cowpea, cucumber,
tobacco, and tomato (Okuno and Furusawa, 1978; Maule, 1983;
Koike et al., 1977; Maule et al., 1980; Hibi et al., 1986;
Nishiguchi et al., 1987; Motoyoshi and Oshima, 1975). Also,
studies using protoplasts isolated from resistant host
plants of tomato and cucumber infected with TMV and CMV
respectively, revealed that resistance to the pathogen
functions at the single cell level (Maule et al., 1980;
Motoyoshi and Oshima, 1975). In addition, Murakishi and
Carlson (1982), working with protoplasts isolated from
systemically infected tobacco plants, demonstrated that
viral pathogens can be used as selection agents at the
single cell level to isolate variant cells, if conditions
are manipulated to allow for uniform infection and for the

preferential growth and selection of virus-resistant cells.

90
For selections to be successful at the single cell

level, each cell must be exposed to the selection agent,
otherwise there will be too many escapes. Therefore, the
ability to achieve a high efficiency of infection by the
virus is a key concern. Electroporation has been adapted as
a genetic technique for simultaneous and uniform infection
of plant protoplasts with viruses. Viral infectivity
greater than 90-95% has been demonstrated with the use of
electroporation (Hibi et al., 1986; Nishiguchi et al., 1987;
Watts et al., 1987).

In order to obtain resistant plants from this
selection, shoot regeneration of the crop plant after
genetic manipulation is necessary. Shoot regeneration from
protoplants has been successful in solanaceous crops with
tobacco, potato and tomato included in the first crop
species regenerated from protoplasts. Pepper (Capsicum
annuum) a member of the solanaceous family, was used as the
host plant in this study. Protoplasts of pepper have been
isolated and regenerated into whole plants (Diaz et al.,
1988; Saxena et al., 1981.).

A number of plant viruses have been used in host-
pathogen-interaction studies. Pepper mild mottle virus
(PMMV), a more virulent strain of tobacco mosaic virus
(TMV), that has been isolated from TMV- and tomato mosaic
virus-resistant pepper in Italy and Spain was used in this

study (Wetter et al., 1984). The interaction of TMV and

 

91
protoplasts of various hosts has been studied extensively

(Murakishi et al., 1984).

The research performed in this study was aimed at
identifying a method for the development of virus-resistant
germplasm in Q. appppm. The development of a system for
uniform, simultaneous infection of cells with virus also
offers a means of studying basic methods of pathogen
virulence and host defense, in the area of viral infection
and replication in plant cells.

The goal of this study was to assess the use of virus-
infected protoplasts as a means of selecting for virus-
resistant plants at the single cell level. To achieve this
goal, those variables (power level, virus concentration, and
protoplast density) which influence the uptake of particles
of PMMV into protoplasts of pepper by electroporation were
investigated. Also the cultural conditions (light
intensity, temperature, and culture medium) which would
preferentially permit the growth of uninfected cells or of
variant cells less affected by virus-infection were
investigated.

MATERIALS AND METHODS
E£QSQEl£§£_l§2l££i22

Seeds of Q. appuum were surface sterilized by immersion
in 95% ethanol for 2 min followed by 30 min in 50% aqueous
(v/v) commercial bleach (2.62% sodium hypochlorite)
containing 1-2 drops/250 m1 Tween 20 (Sigma Chemical Co.,

St. Louis, MO). The seeds were thoroughly rinsed three

92
times with sterile distilled water, and soaked in sterile

distilled water for 30 min. Nine seeds per container were
sown in GA-7 vessels (Magenta Corp., Chicago, IL) containing
50 ml of Murashige and Skoog (MS) medium (Murashige and
Skoog, 1962) which was amended with 165 mg NHJKR, 100 mg
myo-inositol, 2 mg glycine, 0.5 mg nicotinic acid, 0.5 mg
pyroxidineHCl, 0.2 mg thiamine-RC1, 30 g sucrose and 8 g
Bacto agar (Difco Laboratories, Detroit, MI) per liter. The
pH of the medium was adjusted to 5.8 using 1N NaOH or 1N
HCl. Pepper seedlings were grown in a growth room at 25 C
under cool-white fluorescent lights supplying 50 umol-m’z-s'1
on a 16-hr photoperiod.

Fully expanded cotyledons from 3 wk-old seedlings were
used for isolation of protoplasts. The following steps were
performed using sterile technique. The cotyledons were
removed from the seedlings with a scalpel, allowed to become
flaccid, and the lower epidermis of the cotyledon was
removed using fine forceps. The cotyledons were placed
peeled-side down in a petri dish containing 20 ml of a
filter-sterilized enzyme solution containing 1.0% (w/v)
Cellulysin, 0.5% (w/v) Driselase, 0.1% (w/v) Macerase
(Calbiochem, Behring Diagnostics, San Diego, CA; Kyowa Hakko
Kogyo Co., Japan; Calbiochem), CPW salts ( consisted of: ),
and 0.4 M sorbitol at pH 6.0. Cotyledons were incubated at
25 C in the dark on a gyrotory shaker at 45 rev/min for 4-5

hours. The petri dish containing the enzyme solution was

 

93

weighed before and after the addition of cotyledons to
determine the amount of plant tissue.

The enzyme solution containing protoplasts and
cotyledon debris was filtered through a 60 um nylon sieve
into a petri dish, and the remaining cotyledon debris was
gently rinsed with W5 salts (consisted of: 0.11 M CaClv
0.13 M NaCl, 5 mM KCl, 5 mM glucose, 5 mM MES) at pH 5.8.
The protoplasts were transferred to 15 ml round-bottomed
centrifuge tubes which were centrifuged at 35 x g or 400 rpm
in an HNS-II IEC No. 2355 tabletop centrifuge for 10 min.
The supernatant was gently removed by suction using a
disposable pipette, and the pellet was resuspended by
rolling in a small amount of W5 salts solution. The
protoplasts were rinsed in 10 ml of W5 salts solution,
centrifuged again at 35 x g and the supernatant removed by
suction using a disposable pipette. The protoplasts were
rinsed a total of three times in this manner. The final
step in isolation of the protoplasts was to float the intact
protoplasts on a density buffer made of Lymphoprep (9.6% w/v
sodium metrizoate and 5.6% w/v ficoll; Accurate Chemical and
Sci Corp, Westbury, NY) :0.5 M sucrose-10 mM CaCl2 in W5
salts at a 1:2 ratio. The intact protoplasts formed a band
at the top of the solution which was removed by suction and

placed in a clean centrifuge tube.

 

94
Proto at b d d

The viability of protoplasts after isolation was
determined using an inverted microscope and rating the
protoplasts for their general appearance. Viable
protoplasts remained spherical with evenly distributed
chloroplasts. Non-viable protoplasts were not spherical and
were misshapen. The percent viability was determined by
dividing the number of viable protoplasts by the total
number of protoplasts.

The yield of protoplasts was determined per gram of
plant tissue using a hemocytometer to count the viable-
looking protoplasts. The concentration of protoplasts was
then adjusted to the proper amount for each experiment.

Pu on virus

A virus solution of 20 ug/ml in 0.2 M phosphate buffer
(pH 7.2) was mechanically inoculated onto leaves of 6-8
week-old nipppiapg plgyglapdii plants and allowed to
multiply for 3 wks. The tobacco leaves were removed and
weighed. The leaf tissue was frozen and stored at -20 C
until further use. For purification, the method of Gooding
and Herbert (1967) was followed with slight modifications.
The frozen tissue was thawed and homogenized (1 g plant
tissue/2 ml 0.1 M phosphate buffer, pH 7.0) in a blender for
3 min. The homogenate was filtered through four layers of
cheesecloth and the filtrate distributed into 250 ml
centrifuge bottles. The filtrate was centrifuged at 10,000

rpm for 15 min at 4 C in a Sorvall GSA rotor (16,400 x g) to

95
remove cellular debris. The supernatant was collected,

adjusted to 8% w/v polyethylene glycol (PEG) MW 8000 and to
0.2 M NaCl, and stirred at room temperature for 30 min to
precipitate the virus. The entire mixture was centrifuged
at 10,000 rpm in a Sorvall SS-34 rotor (9400 x g) for 10 min
at 4 C, and the virus precipitate resuspended in 0.01 M
phosphate buffer, pH 7.0 at 1/5 the volume used in the PEG
precipitation.

Three cycles of high (108,000 x g) and low speed
(12,000 x g) centrifugations were performed to further
purify the virus. Ultracentrifugation of the virus solution
was performed at 25,000 rpm for 2.5 h at 4 C in a Beckman
Ti70 rotor to pellet the virus. The pellets were
resuspended in 0.01 M phosphate buffer, pH 7.0 after
overnight incubation at 4 C and centrifuged for 10 min at
10,000 rpm (SS-34 rotor) to remove more cell debris.

Concentration of the virus was estimated based on
absorbance at 260 nm. At this wavelength, the extinction
coefficient (1 cm pathlength) for purified PMMV absorbance
(1 mg/ml) is 3.18 (Wetter, 1975). The purified virus was
filtered through a Morton ultra-fine fritted disc filter
(0.9-1.4 u pore diameter) to remove microorganisms, and
stored in 0.01 M phosphate buffer. After filtering, the
absorbance at 260 nm was measured and the virus was diluted

to a concentration of 1 mg/ml and stored in 1 ml aliquots at

4C.

96
ocu at n o asts

Protoplasts of pepper susceptible to PMMV were
inoculated by electroporation using the method of Nishiguchi
et al., (1987) with slight modifications. Immediately
before inoculation, the protoplast pellet was resuspended in
a solution of 0.7 M mannitol, followed by centrifugation at
35 x g for 10 min. The supernatant was removed and an ice
cold solution of 0.7 M mannitol containing PMMV particles
was added to the protoplast pellet to give a concentration
between 1-6.5 x 105 protoplasts/ml. The mixture was then
transferred into a microcuvette tube and incubated on ice
for 10 min.

An exponential pulse was applied to the solution of
protoplasts in the microcuvette using an electroporation
system (Prototype Design Services, Biotechnology Equipment,
Madison, WI 53705). The distance between the electrodes
was approximately 0.3 cm. The voltage varied in
experiments. After electroporation, the protoplasts were
transferred to a 15 ml culture tube and kept on ice for at
least 10 min. Protoplasts were resuspended in several m1 of
0.7 M mannitol and centrifuged at 35 x g for 10 min to
remove any excess virus particles. The protoplasts were
resuspended in 1.0 ml of KMT medium and were cultured in a
petri dish at 28 C under continuous cool-white fluorescent

illumination at 50 umoluwzsq.

 

97
0t 88 V lb

Protoplasts were harvested 24 hr after electroporation
and used to determine viability and infection. Protoplast
viability was determined using Evan’s blue, a non-permeating
pigment which penetrates dead or damaged protoplasts and is
excluded by live intact protoplasts (Gaff and Okong'O-Ogola,
1971). Protoplasts were incubated in Evan's blue dye (0.25%
w/v Evan's blue in W5 salts, pH 6.0) solution for at least 5
min to allow penetration of the pigment before being viewed
using an inverted microscope. The percentage of viability
was determined using the above described method.

r t o he FITc-con u ate

Conjugation of immunoglobulin g (IgG) with fluorescein
isothiocyanate (FITC) was carried out by using modifications
of a published technique (Spendlove, 1967). Serum was
collected from the rabbit following three virus injections
at 2-wk intervals. The IgG fraction was precipitated by the
dropwise addition of 25 ml (NI-1,02504 (saturated solution) to
50 ml of serum with stirring. This was mixed for 3 h at 0
C. The precipitate was collected by centrifugation at 5000
rpm in a SS—34 rotor for 15 min. The pellet was then
dissolved in 40 ml of phosphate buffered saline (PBS, which
was 0.01 M K- phosphate buffer, pH 7.0, containing 0.85%
NaCl), and precipitated a second time by the dropwise
addition of 20 m1 saturated (NI!,)2SO4 while stirring for 1 h
at 0 C. After centrifugation at 6000 rpm for 15 min, the

pellet was dissolved in 6 m1 PBS. The IgG was then dialyzed

 

98

against five changes of PBS over a period of 24 h. The
solution was then clarified by centrifugation at 14,000 x g
for 30 min and the supernatant containing the IgG was
collected. The protein concentration was determined by
measuring the absorption at 280 nm, using the extinction
coefficient of 1.8 for rabbit IgG (McGuigan and Eisen,
1968).

To label the IgG with FITC (ICN Immunobiologicals,
Lisle, IL) 2 ml of the purified IgG was stirred while adding
1.2 ml of FITC solution dropwise. For the FITC solution, 15
mg FITC was mixed with 1 g protein, made in freshly prepared
0.1 M NaZHPOP An additional 0.4 ml of freshly prepared
NazPO, was added, followed by the addition of 0.4 ml 0.04 M
NaOH (pH 9.4-9.6). The solution was loaded onto a Sephadex
G-25 column (2.5 x 15 cm) which was previously equilibrated
with PBS to free the conjugated IgG from uncoupled dye. The
conjugate was then eluted with PBS and the first visible
(greenish-yellow) band was collected in fractions. The
eluate was centrifuged at 15,000 rpm for 30 min to clarify.
The supernatant containing the conjugate was adjusted to

0.1% NaN3 and stored at 4 C.

onsg gpgorptiop of FITC-copjugggo with acetope powde; of
healthy pobacco legvgs.

To minimize non-specific staining, the conjugated IgG
was adsorbed with acetone-extracted powder of healthy
tobacco leaves. Ten g of healthy leaves were picked, placed

in 100 ml cold acetone (0 C) and homogenized at maximum

99
speed in a Waring blender for 5 min. More acetone was added

to make a volume of 300 ml. The homogenate was washed on a
Buchner funnel lined with Whatman No. 1 filter paper with
cold acetone until the filtrate was nearly colorless. The
homogenate was further pulverized by grinding in a dry
mortar, resulting in an almost powder-like substance. This
was dried under vacuum overnight and stored in a desiccator
at room temperature.

The acetone powder was prepared for cross-absorption by
several washes with PBS, pH 7.0. To 40 ml PBS, 0.2 g of
powder was added and stirred for 10 min. The suspension of
powder was centrifuged at 3000 rpm in an SS-34 rotor for 7
min, and the pellet was resuspended in 40 m1 PBS, and
centrifuged again as before. The pellet containing the
acetone powder was saved, resuspended in 35 ml PBS and
divided into two tubes for cross-absorption of the different
FITC-conjugate bands collected from the Sephadex column. To
determine the optimum amount of cross-absorption necessary,
one tube contained approximately twice the amount of the
pellet as the second tube. Each fraction was centrifuged
again and 2 ml of FITC-conjugate solution was added to each
pellet. This was stirred with the aid of a glass rod and
the mixture was incubated for 30 min at 37 C. After
unwanted antibodies were adsorbed to the tobacco acetone
powder, a centrifugation at 3000 rpm for 7 min removed the
solids, and the supernatant fraction containing the FITC-

conjugate was saved. Tests for the optimum amount of

 

100

acetone powder needed for cross-absorption were made by
trial and error method using identical sets of isolated
infected protoplasts. The various fractions were labelled
and stored at 4 C.
W

The percentage of virus-infected protoplasts was
quantified by sampling the protoplasts after specified post-
inoculation periods and staining with viral coat-protein
specific antibodies conjugated to FITC using the procedure
outlined by Otsuki and Takebe (1969). Infected protoplasts
from approximately 0.5 ml of the cultured suspension were
pelleted by centrifugation at 100 x g for 2 min and the
pellet was resuspended in a small volume of KMT medium.
Duplicated drops of this concentrated preparation were
placed on a microscope slide previously coated with a thin
film of Mayer's albumin (egg white + phenol) and allowed to
dry. Cells were fixed by immersion in 95% ethanol for 5
min, followed by a rinse for 5 min in PBS. Slides were
carefully blotted with filter paper and protoplast spots
were stained with one drop of 1:6 dilution of FITC-conjugate
in PBS. These slides were incubated at 100% relative
humidity at 37 C for 1 hr in a water bath, following which
the unbound FITC-antibody conjugate was removed by rinsing
the slides in a large volume of PBS for 10 min. The
specimen was mounted for viewing by carefully blotting away
excess PBS, adding a drop of PBS:glycerol (9:1) and placing

a coverslip over the cells.

 

101
a o f v u t o b uoresce c c os o

FITC-labelled cells were viewed with a Zeiss—GFl
epiluminescent microscope equipped with exciter filters KP—
490 and LP-445, dichroic reflector 510 and barrier filter
520. Infected cells contained specks which were a bright
fluorescent yellow-green in color. Uninfected cells
exhibited slight light green fluorescence, and dead cells
appeared dull orange-brown in color. When chlorophyll was
present, cells fluoresced red. The percentage of infected
cells was determined by rating at least 300 viable
protoplasts in the sample as fluorescent or non-fluorescent.
The ratio of fluorescent to total viable protoplasts counted
gave the percent fluorescent protoplasts.

EDQEQQISEAX

Photographs of the fluorescing cells were taken with
ASA 400 daylight film. Exposures were between 10 and 30
min.

RESULTS
Inoc t roto sts

Protoplasts were consistently isolated using the
protocol with average yields of 5.1 x 106/9 of tissuNhe
average viability of the isolated protoplasts was 85%.

A total of 26 electroporation experiments were
performed using the following varying parameters: virus
concentration 50,- 300 ug/treatment; protoplast density 1 x
105 - 6.5 x 105/ml; and power levels of 100 - 400 volts/cm.

Infection was achieved in two experiments with the treatment

 

102
conditions outlined in Table 4.1. Infected protoplasts were

detected using antibodies conjugated to FITC (Fig. 4.1).
Because positive results were obtained in only two
experiments and only a low percentage (1-2%) of protoplasts
were infected in these experiments, the effect of voltage on
protoplast viability was studied. As the voltage increased

above 100 volts, viability of the protoplasts was reduced
sharply (Fig. 4.2). In fact, in the successful experiments,
total protoplast viability after electroporation was
probably <10% which would explain why the total percentage
of infection was low.

Also, the infectivity of the virus inoculum was
retested in local lesion assays on tobacco and Qpepppodium
and the virus was infective at a level of 0.01 ug/ml.

DISCUSSION

A requirement in the development of an ip 115:9 tissue
culture selection system is efficient regeneration of the
source material of the crop plant in study. There have been
two reports of regeneration of pepper from protoplasts,
however, whole plant regeneration was limited to Q. annuum
cv. California Wonder and cv. Dulce (Diaz, 1988; Saxena,
1981). Using the protocols presented, our laboratory was
unable to replicate whole plant regeneration from
protoplasts; extensive efforts resulted in only the
formation of callus tissue (S. Linderman, personal
communication). Although there have been many virus-host-

interaction investigations, only a few crop plants have been

103

Table 4.1. Treatment conditions of successful experiments
infecting pepper protoplasts with pepper mild mottle virus
using electroporation.

 

 

Protoplast density Virus concentration‘ volts/cm
5.0 x 105 so 250
300
350
400
5.5 x 105 125 250

 

‘ Concentration of virus in ug/ml.

 

104

 

Figure 4.1 Fluorescence microscopy detection of
protoplasts of pepper (Capsicum annuum) infected with
pepper mild mottle virus by electroporation.

105

 

60

50

8

Viability (%)
8

20

 

10

 

 

 

O 1 l 1 l l
O 50 100 150 200

Volts/cm

Figure 4.2 Viability of protoplasts of pepper (Capsicum
annuum) after exposure to various voltages during
electroporation experiments.

106
used. In these protoplast/virus studies, crop plants such

as potato, tobacco and tomato, in which the culture and
regeneration requirements have been worked out and a number
of successful genetic manipulations have been employed, were
chosen.

Obviously, for selections to be successful at the
single cell level, each cell must be exposed to the
selection agent. Of the plant pathogens, viruses appear to
be the selection agent of choice, since simultaneous
infection as great as 95% has been achieved (Watts et al.,
1987). However, based on our current understanding of virus
infection and replication processes, it has proven
unobtainable to achieve 100% infection of protoplasts in
culture.

Selection for disease resistance would be most
efficient at the single cell level, but this requires that
the specific resistance genes be expressed during the
culture process. The selection agent would have to kill or
inhibit growth of the cell to the point that the resistant
cells could divide, form callus and regenerate faster than
the susceptible cells and thus be identified. Infection of
protoplasts with viral pathogens rarely leads to death of
the cell. Also, the percentage of possible resistant
variants is so much lower than the 5-10% virus-free
protoplasts that escape infection, the method is unsuitable

for selecting variants during culture. This type of

107
selection system would require a large number of protoclones

to be indexed to identify the variant.

lg yippp single cell selection systems could be
successfully used to isolate variants if the discussed
conditions could be manipulated to allow uniform infection,
preferential growth of the variant cells, and regeneration

of the variant crop plant.

 

108

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