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This is to certify that the

dissertation entitled
Engineering herbicide resistance in creeping

bentgrass and its potential application on the
prevention of fungal diseases

presented by

Chien-An Liu

has been accepted towards fulfillment
of the requirements for

Ph.D degreein Crop and Soil Sciences

 

 

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MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

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ENGINEERING HERBICIDE RESISTANCE IN CREEPING BENTGRASS
(Agroslispalustris Huds.) AND ITS POTENTIAL APPLICATION ON THE
PREVENTION OF FUNGAL DISEASES

By
Chien-An Liu

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1995

ABSTRACT

ENGINEERING HERBICIDE RESISTANCE IN CREEPING BENTGRASS
(Agrostis palustris Huds.) AND ITS POTENTIAL APPLICATION ON THE
PREVENTION OF FUNGAL DISEASES

By

Chien-An Liu

An efficient transformation system to generate transgenic creeping bentgrasses
(Agrostis palustrzk Huds.) by microprojectile bombardment is described. Embryogenic
callus bombarded with the bar gene was cultured on selection medium supplemented with
either five mg/l of bialaphos or fifteen mg/l of phosphinothricin (PPT) for twelve weeks.
38 independent resistant callus lines were generated and found able to regenerate plants.
Except for the plants regenerated from one of the PPT-resistant callus lines, plants from the

rest of the resistant callus lines were shown to be resistant to the application of 1.2%

Ignite®. Resistant callus lines and plants regenerated from them expressed functional
phosphinothricin acetyltransferase, the product of brr. Integration was confirmed by
Southern hybridization analysis and transcripts corresponding to the bar gene were present
in transgenic plants that showed herbicide resistance.

Bialaphos showed a higher level of in vitro antifungal activity against the pathogens
of brown patch (Rhizoctonia solam), dollar spot (Sclerotim'a homoeocarpa) and Pythium
blight (Pythium aphanidennalum), than phosphinothricin (PPT). While PPT suppressed
the mycelial growth of R. solani and S. homoeocatpa, it had no inhibitory effect on P.

aphanidermaturn up to the highest concentration (600 mg/l) in our testing regimes.
Whereas bialaphos was significantly effective in the inhibition of growth of R. solani and
S. homoeocarpa, it was less so in that of P. aphanidennatum. Various concentrations of
bialaphos solutions were applied to transgenic creeping bentgrasses either three hours
before or two days after the fungal inoculation. Bialaphos application was able to
significantly reduce the symptomatic infection by R. solani and S. homoeocarpa. The
inhibitory effect of bialaphos spraying on the suppression of P. aphanidennatwn was not
as effective as that of the other two pathogens; however, disease development was still
reduced to a significant extent. Better control of the disease development of dollar spot and
Pythium blight also occurred when bialaphos was applied before the inoculation. These
results indicated that bialaphos may be used simultaneously and eff icaciously as a herbicide
for weed control and as a fungicide for the protection of turf areas with bialaphos-resistant

creeping bentgrasses against fungal diseases.

To God, my lovely wife and parents

for their love, support and encouragement

iv

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
LIST OF TABLES viii
LIST OF FIGURES x
CHAPTER ONE literature review
Introduction 1
Tradiuonal breeding and plant biotechnology 7
Methods of plant genetic transformation 8
Range of transformed species 34
Markers for plant gene transfer 52
Application of plant transformation to confer insect tolerance 73
Tissure culture and genetic transformation of creeping bentgrass 79
Bibliography 81
CHAPTER TWO: Engineering herbicide resistance in creeping bentgrass (Agrostis
palustrr's Huds.) via microprojectile bombardment and expression of the bar gene in
transgenic plants
Abstract 1 13
Introduction 1 14
Materials and methods 1 18
Plant material 1 18
Evaluation of selective agents 1 18

 

Plasmid vector

 

Microprojectile bombardment

 

Selection of resistant callus lines and plant regeneration

 

Herbicide application

 

DNA isolation and Southern hybridization analysis ---

 

RNA isolation and norhtem hybridization analysis

 

PAT activity assay

 

Detection of ammonia in plant extracts --—

Results

 

 

Evaluation of selective agents

 

Selection of transformed callus lines

 

Plant regeneration

 

Herbicide resistance of transgenic creeping bentgrasses

 

Analysis of PAT activity

 

Southern hybridization analysis

 

Norhtern hybridization analysis

 

Detection of ammonium levels in herbicide-treated plants

 

Discussion

 

Bibliograpgy

CHAPETR THREE: Evaluation of bialaphos on the prevention of fungal diseases in
transgenic bialaphos-resistant creeping bentgrasses (Agrostris palustris Huds.)

Abstract

 

 

Introduction

Materials and methods

 

 

In vitro test

Greenhouse test

 

120
120
121
122
123
124
125
126
129
129
121
136
137
140
140
144
144
149
154

157
159
162
162
163

Results

 

In vitro test

 

Greenhouse test

 

 

Discussion

 

Bibliograpgy

vii

165
165
180
188
192

Table 1.
Table 2.

Table 1.

Table 2.

Table 3.
Table 4.

Table 1.

Table 2.

Table 3.

Table 4.

LIST OF TABLES

 

 

CHAPTER ONE
Examples of transgenic plants and transformation methods used 35
Selectable marker genes for plant transformation 59
CHAPTER TWO

Effects of the duration of selection and the concentration of phosphinothricin

 

 

on the callus growth of creeping bentgrass 131
Effects of the duration of selection and the concentration of bialaphos on the

callus growth of creeping bentgrass 132
Results from transformation experiments of creeping bentgrass 135

 

Ammonium concentration in transgenic and untransformed creeping
bentgrasses after the application of 2.4 g/l glufosinate-ammonium ------------- 147

CHAPETR THREE

Sensitivity of Rhizoctonia solani to bialaphos on potato dextrose agar
medium 167

 

Sensitivity of Rhizoctonia solani to phosphinothricin on potato dextrose agar
medium 168

 

Sensitivity of Sclerotinia homoeocarpa to bialaphos on potato dextrose agar
medium 169

 

Sensitivity of Sclerotinia homoeocarpa to phosphi nothricin on potato dextrose
agar medium 170

 

viii

Table 5. Sensitivity of Pythium aphanidennatwn to bialaphos on potato dextrose agar
medium 17 1

 

Table 6. In vitro inhibition of Rhizoclonia solaui, Sclerotinia homoeocarpa and
Pythium aphanidennatum by bialaphos or phosphinothricin 172

 

Table 7. Effects of bialaphos application on the development of brown patch, dollar
spot and Pythium blight diseases in transgenic creeping bentgrasses ---------- 183

ix

LIST OF FIGURES

 

 

 

 

 

 

CHAPTER TWO

Figure 1. Schematic representation of the plasmid pTW-a 128
Figure 2. Callus responses of creeping bentgrasses to various concentrations of

bialaphos and phosphinothricin and to three durations of selection ----------- 133
Figure 3. Effects of herbicide application on transgenic plants and untransformed

control plants 138
Figure 4. The magnitudes of herbicide resistance of creeping bentgrass regenerated

from three independent resistant callus lines 139
Figure 5. Detection of phosphinothricin acetyltransf erase (PAT) activity by thin layer

chromatography 141
Figure 6. Southern hybridization analysis of transgenic creeping bentgrass plants ----- 142
Figure 7. Northern hybridization analysis of the bar transcript in transgenic

creeping bentgrasses 146
Figure 8. The accumulation of ammonium in transgenic and untransformed

creeping bentgrasses after spraying with 1.2% I gnite® 148

CHAPTER THREE

Figure 1. The inhibition of bialaphos and phosphinothricin on the mycelium of
Rhizoctom'a solani four days after the initial inoculation 173

 

Figure 2. The inhibition of bialaphos and phosphinothricin on the mycelium of
Sclerotim'a homoeocarpa four days after the initial inoculation 175

 

Figure 3. The inhibition of bialaphos and phosphinothricin on the mycelium of
Pythium aphmridemratum four days after the initial inoculation --------------- 177

X

Figure 4. In vitro responses of Rhizoctonia solani, Sclerotinia homoeocarpa and
Pythium aphanidennalum to bialaphos or phosphinothricin-amended potato
dextrose agar medium 179

 

Figure 5. Effects of bialaphos application on fungal infections of transgenic bialaphos
-resistant creeping bentgrasses 184

 

Figure 6. The application of bialaphos on the prevention of pathogen infection by
Rhizoctonia solani in transgenic bialaphos-resistant creeping bentgrasses ---- 185

Figure 7. The application of bialaphos on the prevention of pathogen infection by
Sclerotinia homoeocarpa in transgenic bialaphos-resistant creeping
bentgrasses 186

 

Figure 8. The application of bialaphos on the prevention of pathogen infection by
Pythium aphanidennatum in transgenic bialaphos-resistant creeping
bentgrasses 187

 

CHAPTER ONE

Literature Review

Introduction

Bentgrass (Agrostis spp.) is the cool-season turf grass most commonly used on
golfing greens in cool and transitional climatic regions and in the cooler portions of warm
climatic regions, especially the arid zone (Beard, 1982). The genus Agrostis L. includes a
number of species suitable for many turfs, especially in temperate climates with cool,
moist summers. The common name bentgrass is applied to all turf grass species within
the genus Agrostis, with the exception of redtop. Bentgrass acquired its name from the
characteristic bent of the basal part of each stem when plants are growing in thin stands.
These stems, instead of growing upright, tend to follow the ground for a short distance
and then bend upward. Its growth habit varies from a bunch type with limited
stoloniferous growth to an extensive stolon system (Philipson, 1937). Due to the
prostrate growth habit, bentgrass is the cool season turf grass most tolerant of continuous
and close mowing. Some species are annuals, but most are perennials and have excellent
low temperature hardiness, including all those utilized for turf grass purposes. They form
an extremely fine-textured, dense, uniform, high quality turf when closely mown.
Bentgrass is generally tolerant of acid soils and can thrive at lower levels of soil fertility
relative to that needed for good performance of Kentucky bluegrass or perennial ryegrass.
Future breeding work could significantly expand the areas of adaptation and usefulness of
these grasses (Funk et al. 1994).

There are three widely used turf-type bentgrass species : (a) A. palustris--—
creeping bentgrass, (b) A. tennis-«colonial bentgrass, and (c) A. canina---velvet

bentgrass, which is not used as extensively as the other two. A wide range of divergent
1

2
types and intermediate forms with chromosome numbers ranging from 14 to 42 exist

among creeping, colonial, and velvet bentgrass (Stuckey and Banfield, 1946; Vaatnou,
1967).

The creeping bentgrass (Agrostis palustris Huds.) is native to Eurasia but has
been distributed throughout the world for use on golf putting greens, tees, and closely
mowed fairways. It is the most widely used Agrostis species in North America. The
common name creeping bentgrass is derived from the vigorous creeping stolons that
develop at the surface of the ground and initiate new roots and shoots from the nodes.
Considerable variation exists in the literature concerning the scientific name of the
creeping bentgrass, with some authors referring to it as Agrostis stolonifera var. palustris
(Farwell) (Piper, 1918; Armstrong, 1937; Philipson, 1937; Bradshaw, 1958). It is a
cross—pollinated tetraploid (alloploid) with 28 chromosomes (2n=4x=28; Church 1936).

Creeping bentgrass forms its best quality turf at a cutting height of 1.5 cm or less.
It is a soft, fine-textured grass with a vigorous growth habit and is capable of producing a
very attractive, dense, and compact turf tolerant of very close mowing. The poor wear
resistance of creeping bentgrass is compensated by its excellent recuperative potential.
Its quality surpasses that of any other northern turf grasses. However, such turf s normally
receive very high maintenance inputs, including frequent irrigation and mowing,
aerification and topdressing for thatch reduction, and pesticides for disease and insect
control. The finer textured cultivars tend to be compact and low growing, while the
coarser textured are usually more upright and open (Holt and Payne, 1952). The turf grass
color varies among the cultivars from greenish-yellow to dark green to blue green.

The creeping bentgrass can be subdivided into two types based on propagation
method. Many of the early cultivars of creeping bentgrass for fine putting greens were
selected from old turf areas (Beard, 1973) and propagated by chopping the creeping
stems (stolons) into short sections with one or more nodes. There are a number of

vegetatively propagated cultivars available, and they are preferred where turf grass

3
uniformity is to be maintained for an extended period of time. However, there is an

abundance of commercial seed cultivars available for this species, such as Penncross,
Emerald, Seaside, and Penneagle. Penncross, released in 1954 (Hein, 1958), is a first
generation synthetic cultivar produced by the random crossing of three vegetatively
propagated strains and is the most widely used cultivar throughout North America, except
for the hot and humid southeast (Beard, 1982). The seeded creeping bentgrass cultivars
possess a certain degree of heterogeneity and, over a period of time, tend to segregate
into distinct patches under turf grass conditions (Holt and Payne, 1952). Segregation into
various strains complicates cultivation practices. In spite of this disadvantage, the seeded
cultivars have been widely utilized, primarily because they are more economical to

establish.

Disease Problems of Creeping Bentgrass

Turf grass disease is a greater problem for golf courses than it is for most other
types of turf use, due to close mowing, heavy fertilization, intense irrigation, and constant
bruising from traffic and divoting. Disease problems are most severe on putting greens,
especially those composed of bentgrass in contrast to bermudagrass. Tees are also
subject to high disease activity if the cultural system utilized involves a cutting height and
nutritional level similar to those employed on greens. Fungicide programs are commonly
used on these types of turf during periods when the potential for disease development is
high. The severity of disease problems is typically greater on cool-season grasses than on
warrn-season grasses, especially in the more humid climatic regions.

The creeping bentgrasses are susceptible to a wide range of diseases, including
dollar spot (Sclerotinia homoeocarpa), brown patch and yellow patch (Rhizoctonia
solam’, R. cerealis, and R. oryzae), Helminlhosporium diseases, Fusarium patch
(Gerlachia nivalis), Pythium blight, red thread and pink patch (Laetisaria fucrformis),

stripe smut (Ustilago and Urocystis spp.), takeall patch ( Gaeumannomyces graminis , var.

4
avenue), copper spot (Gloeocercospora sorghi), fairy ring (Marasmius oreades), downy

mildew (Sclerophthora macrospora), and Typhula blight (Typhula incarnata) (Beard,
1973; Sprague, 1982; Smiley, 1983).

Insect Problems of Creeping Bentgrass

There are over two million different kinds of insects, but relatively few pose a
significant problem to turf grasses. The severity of insect attacks on golf course turfs is
not as highly correlated to the intensity of culture as is the disease-causing pathogens.
The severity and frequency of insect attacks tend to be greater on warm~season than on
cool-season turf grasses. The occurrence of insect problems on bentgrasses increased
noticeably during the mid-1970s with the passage of laws that eliminated the use of
persistent insecticides, such as the chlorinated hydrocarbons. Appropriate insecticides
are now applied when potentially serious insect injury symptoms first appear. White
grub (black turf grass ataenius, Ataenius spretulus), chinch bug (Blissus leucopterus),

cutworm (Noctuidae family), and f rit fly ( Oscinella fiits) are of particular concern.

Weed Problems of Creeping Bentgrass

Five major components of turf grass quality are uniformity, density, smoothness,
texture, and color. Plants that disrupt the uniformity of turf s in terms of either texture or
density are considered undesirable in high-quality turf and, thus, are referred to as weeds.
An high number of weeds in the turf results from poor establishment and management
practices. A permanent, properly managed turf grass species makes a tight sod that
prevents most weeds from becoming established. However, perfect turf maintenance is
difficult to achieve, particularly on general turf areas receiving less than intensive care.
Herbicides are usually employed to supplement a good weed-control management

program to make a weed-free turf.

5
The characteristics of turf grass weed species vary tremendously, but all have the

ability to persist even though the grass is cut regularly. Generally, they survive mowing
because of a low growth habit. Weed species are classified as grassy type
(monocotyledon) or broadleaf type (dicotyledon) and are subdivided into annuals,
biennials, and perennials. Selecting the proper herbicide and application time for
controlling a weed species is determined by the life cycle of the weed and whether it is a
broadleaf or grassy-type weed. Generally, weeds fall into three control groups-«annual
grasses, perennial grasses, and broadleaf weeds.

The broadleaf weeds that infest turfs are much more numerous than grass weeds.
Fortunately, they are dicots and can be controlled very effectively with selective and
systematic herbicides, such as 2, 4-D, mecoprop, and dicamba, with little or no injury to
the turf if applied properly. A combination of 2,4-D and dicamba or mecoprop is most
effective since some broadleaf weeds are resistant to one of the herbicides and a mixture
ensures broad-spectrum weed control.

Annual grass weeds can usually be controlled with the application of pre-
emergence herbicides. Most applications occur in the early spring because pre-
emergence herbicides should be sprayed one to two weeks before seed germination.
Post-emergence herbicides are also used to control annual grasses in the early stage after
emergence. However, the post-emergence method of weed control is often unsatisfactory
aesthetically because slowly dying grasses in a turf are unsightly. Another concern is that
post-emergence herbicides can be phytotoxic to desirable turf grasses. Cool season
grasses are especially susceptible to this type of injury. Creeping bentgrass is more prone
to herbicide injury than Kentucky bluegrass (Albrecht, 1947; Skogley and Jagschitz,
1964; Callahan, 1966). Root and leaf injuries frequently result from the applications of
2,4-D and 2,4,5-TP (Callahan and Engel, 1965).

Perennial weed grasses cause serious weed problems because selective herbicides

are not available. Perennial grassy weeds are so similar to desirable turf grasses from a

6
physiological and anatomical standpoint that herbicides used for killing perennial grass in

turf also destroy the perennial turf grass. A nonselective herbicide such as glyphosate or
dalapon must be used with great care. Therefore, the development of a herbicide-
resistant creeping bentgrass variety has become imperative so as to achieve a perfect turf
management program in a convenient and inexpensive way.

Herbicides have become an indispensable tool of modern agriculture in that they
allow economically superior weed control and are more labor- and energy-efficient than
manual or mechanical cultivation methods. In recent years, the demand for
environmental safety has made it necessary to develop less toxic compounds, so that
corporate competition in this field has resulted in the development of several new, better,
and safer herbicides, including a number of selective compounds. However, only a very
few of these fulfill the ideal requirements of a truly selective herbicide in controlling all
plants except the cultivated crop (Schulz et al., 1990).

The information available elucidating the mode of action of several herbicides has
rapidly increased in the past few years (Schulz et al., 1990). This fact, coupled with the
progress achieved in the genetic manipulation of plants and the ability to transfer foreign
DNA from a variety of sources to plants, has opened up an exciting area of research.
These observations are further enhanced in that many possible herbicide resistant gene
determinants are single dominant traits, making them amenable to gene transfer
techniques. Hopefully, the ensuing result will be the expanded use of environmentally
safer herbicides which favor a broader weed control spectrum in response to increasing
concerns over contamination of the environment, toxicity to animals, and persistence of
residues in soil and water. The cultivation of herbicide-resistant turf grasses also brings
economic advantages to the management of golf courses by reducing the quantity needed

and simplifying the use of herbicides.

Traditional Breeding and Plant Biotechnology

Since the beginning of this century, Mendel's law of genetics has provided the
scientific foundation for plant breeding. Traditional plant breeding methods have
resulted in substantial crop improvement and will continue to provide us with useful crop
varieties and enhance our quality of life.

In conventional plant breeding, plant breeders are generally faced with the
following three challenges. First of all, they must attempt to locate and utilize the
maximum possible range of genetic variability that is available. Then, they need to be
able to transfer the trait(s) of interest into the other breeding lines and to select the
recombinants that show the desired characteristics. Finally, they have to generate a large
number of progeny with predictable properties. By crossing parent plants each holding
some of the useful traits and selecting the progeny of those individuals showing the
proper combination of traits, tremendous improvements have been achieved in crop
performance and will continue to be made.

However, the traditional plant breeding method of crossing has its limitations. It
is evident that only traits present within the species or its related species can be
combined, since crosses between distantly related or unrelated species usually yield
sterile plants or, most often, no progeny at all. Besides the problem of compatibility, the
availability of desirable genes and the conservation of genetic diversity are also
limitations to plant breeding research due to the erosion of the natural environment.

During the past twenty years, in addition to the development of plant tissue
culture techniques, much progress has been made in the fields of cell biology and
molecular biology, which, in turn, has had its influence on modern plant breeding
research and has offered alternative routes to resolving some of the limitations of

traditional plant breeding and production. This relatively recent development in plant

8
biotechnology, the technology to manipulate and regenerate plants in vitro and to isolate

and construct specific DNA fragments, more or less at will, has led to advances in the
ability to selectively alter the genetic make-up of certain plant species.

In 1984, stable foreign gene transfer into plant species became possible with the
application of recombinant DNA technology and with the availability of Agrobacterium
tumefaciens transformation methodology (De Block er al., 1984; Horsch et al., 1984).
The ability to transfer genes among organisms without sexual crossing provides plant
breeders with new opportunity to improve the efficiency of production and to increase the
utility of agricultural crops. Plant genetic engineering has become a useful tool to plant
breeding research in that it helps to overcome problems associated with the
incompatibility between different species and to broaden the gene pool plant breeders can
utilize. Plants with new traits, such as herbicide resistance and insect resistance, have
been genetically engineered using genes from unrelated organisms. Scientists are also
trying to improve the quality of our agricultural products by altering the nutritional values
of proteins and oils. However, it should be emphasized that plant biotechnology is not a
substitute or replacement for the conventional plant breeding methods. Rather, it can
supplement the traditional plant breeding approaches to improve crop species. The major
differences between these two methodologies lie neither in goals nor processes, but

rather, in speed, precision, reliability, and scope.

Methods of Plant Genetic Transformation

The revolutionary progress in recombinant DNA technology makes it necessary to
continue to improve the techniques needed for the . genetic engineering of plants.

Improvement of these techniques requires better tissue culture methodologies and gene

9
transfer techniques. Before describing gene transfer and genetic transformation, the

following definitions are adopted to eliminate the ambiguity often found in the literature.

Gene Transfer

Gene transfer (or DNA uptake) refers to a process which moves a specific
fragment of DNA (usually a foreign gene ligated to a bacterial plasmid) into protoplasts
or cells. If the gene transfer is efficient and the foreign gene is introduced into a
sufficient number of cells, transient gene expression can be quantitatively measured
(approximately 12 to 48 hours after the gene transfer). Usually, the level of the protein
encoded by the foreign gene is measured to show the success of gene transfer, because it
is simpler and easier to measure than the level of mRNA. For studying transient gene
expressions, stable integration of the introduced gene is not necessary, but a reproducible

and efficient gene transfer method is needed.

Genetic Transformation

Plant genetic transformation refers to the stable integration of foreign genes
isolated from plants, bacteria, or animals into the genome of a plant regenerated from
DNA-treated protoplasts or intact cells. The plants carrying the stably integrated foreign
genes are defined as transgenic plants. The process of genetic transformation involves
several distinct stages, namely, insertion, integration, expression, and inheritance of the
newly transferred DNA. Integration can be demonstrated by Southern blot analysis. The
analysis should include the use of several restriction enzymes to provide proof of
integration of the foreign gene.

For example, it is useful to include (1) an enzyme that cuts twice within the
plasmid to show the presence of the foreign gene and to show that there is no
reanangement of the foreign gene on the plasmid in the transgenic plants; (2) an enzyme

that cuts once within the plasmid to yield restriction fragments which vary in size

10
depending on the nature of the flanking DNA at the site of insertion of the plasmid in the

transformed plants; and (3) an enzyme that does not cut within the plasmid to distinguish

between the free plasmid (in monomeric or oligomeric form) and the integrated plasmid.

Techniques for Gene Transfer

Although straightforward in principle, in practice plant genetic transformation can
be quite complex. Once a gene of interest is isolated, it is introduced into a plant
transformation vector. The vector DNA facilitates the manipulation of the gene in
Escherichia coli prior to plant transformation, as well as the transfer of the gene into the
host plant. An idealized vector would contain a multiple cloning site, an antibiotic
resistance gene allowing for selection in both E. coli and A.. tumefaciens (e.g., a gene
encoding ampicillin resistance), a broad-host bacterial origin of replication, and an
antibiotic or a herbicide resistant gene for selection of the foreign DNA in transformed
plants. If the Agrobacterium system is used for transformation, the vector may contain
the Ti plasmid virulence gene and T-DNA borders as well. There are many approaches
to introducing foreign DNA into plants, of which the most commonly used are the A.
tumefaciens ”agro-infection" system, PEG- and electroporation-mediated direct gene

transfer, and microprojectile bombardment.

Agrobacten'wn -mediated Genetic Transformation

Agrobacterium is a soil-dwelling bacterium that infects wound sites on a wide
range of plant species and induces the development of crown gall tumors or hairy roots.
These growth responses result from a natural genetic engineering event in which a
specific region of DNA from a Ti (tumor-inducing) plasmid or Ri (root-inducing)
plasmid is transferred from Agrobacterium to a plant cell. This T-DNA (transferred
DNA) is integrated and expressed in the nuclear genome of the plant cells. The

expression of genes located within the T-DNA results in the development of tumors or

1 1
hairy roots. The importance of Agrobacterium for plant genetic engineering is its natural

ability to transfer a segment of DNA into plant cells. Over the past ten years, researchers
have capitalized on molecular biology technology to manipulate the T-DNA of
Agrobacterium for the development of gene vectors to produce transgenic plants.

The molecular mechanisms of plant transformation by A. Iumefaciens have
recently been reviewed (Zambryski, 1988; Kado, 1991; Hooykaas and Schilperoort,
1992). The wild type form of A. tumefaciens induces crown gall tumors on many
dicotyledonous plants when viable bacteria infect wounded plant tissue (Ream and
Gordon, 1982). The crown gall is produced following the transfer of the tumor inducing
T-DNA (transferred DNA) region from the Ti plasmid (140 to 235 kilobases) into the
genome of an infected plant. The T-DNA fragment, which is integrated into plant
nuclear genome and retained in tumor cells, encodes genes for auxin and cytokinin
biosynthesis, and it is these hormones in high concentration that promote growth of
undifferentiated cells in the crown gall. Transfer of the T-DNA to the plant genome
requires the Ti plasmid-encoded virulence genes as well as the T-DNA borders, that is, a
set of direct DNA repeats that delineate the region, to be transferred. The tumor inducing
genes can be removed from Ti plasmid vectors, disarming the pathogenic nature of the
system, without affecting the transfer of DNA fragments between the T-DNA borders.
Therefore, the tumor inducing genes are generally replaced with a gene encoding
resistant to antibiotics such as kanamycin or to herbicides to allow for the selection of
transformants, and with a gene of interest that would confer the desired trait to the
recipient plant.

The Agrobacterium containing the engineered plasmid is co-cultivated with
cultured plant cells or wounded tissue. The de-differentiated cells are then cultured and
propagated on selection medium, and transgenic plants can subsequently be regenerated
from the resistant cells by altering the levels of auxin and cytokinin in the growth

medium. One of the disadvantages of this technique is that most monocots and some

12
dicots are not natural hosts for the bacterium and, for the most part, do not have the

proper wound responses to be agro-infected. They are, therefore, generally excluded

from A. tumefaciens-mediated genetic transformation (Potrykus, 1990, 1991).

Transformation of Protoplasts

Every single cell of a plant is potentially capable of developing into a whole plant.
This phenomenon is known as cell totipotency. Significant advances have been made by
researchers during the past decade in plant regeneration from protoplasts (isolated single
plant cells without cell walls) in both dicots and monocots. This has not only confirmed
the hypothesis of cell totipotency, but has also made it possible to transfer foreign genes
into plants using protoplasts as the starting material. There are two major reasons for
using protoplasts instead of cells or tissues for genetic manipulation. One is that
protoplasts are separated single cells which have been stripped of their cell wall,
facilitating the transfer of foreign genes through the plasma membrane. The other is that
all cells of transgenic plants regenerated from a protoplast will contain the foreign genes
of interest; thus, the transgenic plant will have a uniform genetic makeup, avoiding the
problem of chimerism that sometimes complicates the other transformation methods.

Protoplasts capable of gene integration and plant regeneration may be referred to
as competent. Protoplasts may be induced to become competent by the alteration of
specific experimental conditions. Several methods have been used to stimulate protoplast
competence, such as heat shock (Abdullah et al., 1986), low-dose irradiation (Kohler et
al., 1989), and media ultrafiltration (Davies et al., 1989). These treatments, in addition to
the enzymatic and mechanical treatments used in the protoplast isolation procedure, can
increase gene transformation frequency and protoplast division. When competent
protoplasts with high viability are available, several methods such as polyethylene glycol
(PEG) treatment, electroporation, Agrobacterium infection, and sonication can be used

for gene transfer.

13

PEG- and Electroporation-mediated Direct Gene Transfer

For many years, genetic manipulation in some plants has been accomplished
using direct uptake of foreign DNA by plant protoplasts rather than A. tumefaciens-
mediated gene transfer. The first experiments demonstrating direct gene transfer
included the delivery of plasmid DNA to protoplasts of petunia and tobacco in the
presence of poly-L-ornithine or polyethylene glycol (PEG) (Davey et al. , 1980; Draper et
al., 1982; Krens et al., 1982; Paszkowski et al., 1984). During the following years,
protoplast transformation mediated by PEG (Negrutiu et al., 1987) or electroporation
(Shillito et al., 1985) was substantially simplified, and its efficiency in model systems
was increased by several orders of magnitude (reviewed by Paszkowski etal., 1989).

Various chemical treatments have been used to stimulate DNA uptake by
protoplasts. At present, PEG is the most common chemical treatment used to stimulate
DNA transfer into protoplasts of both dicots and monocots (Negrutiu et al., 1987).

PEG acts to increase the permeability of cell membranes, and has been used as an
efficient protoplast fusion agent in somatic cell hybridization of various plant and animal
species. It has also been effective in delivering DNA into plant protoplast and in
obtaining transformed cells (Wang et al., 1992). Opinion is divided about the mechanism
by which DNA crosses the plasmalemma and then enters the nucleus. However, under
the influence of the elevated osmotic pressures created by the use of high concentrations
of PEG during the transformation of protoplasts, DNA may be incorporated into
membrane-bound vesicles in the cytoplasm in a manner similar to the formation of
cytoplasmic vesicles on the plasmolysis of cells. These may then fuse with the nuclear
membrane systems continuous with the nuclear membrane and effectively transfer DNA
to the nucleus. Evidence for this hypothesis comes from work on cation-mediated

endocytosis and exocytosis in oat prot0plasts. These studies suggest that swelling and

14
shrinking of protoplasts during osmotic adjustment occur by the removal and addition of

membrane from the plasmalemma (Glaser and Donath, 1989).

Various parameters important for gene transfer with PEG have been identified.
First of all, the concentration of PEG can affect the viability of protoplasts and the
efficiency of gene transfer. The optimal concentration of PEG is between 15% and 25%.
If the concentration is too high, cell viability will decrease; if it is too low, the efficiency
of gene transfer will decrease (Hayashimoto et al., 1990). Secondly, the components of
the buffer will also influence gene transfer efficiency (Zhang and Wu, 1988). Finally,
culture media for protoplasts will directly affect protoplast division and plant
regeneration (Jenes and Pauk, 1989). Currently, to transfer the foreign gene, two major
methods, solid culture and nurse culture, are commonly used for efficient protoplast
culture after protoplasts have been treated with PEG (Cao et al., 1991).

The electroporation method involves the application of high-voltage electrical
pulses to a solution containing protoplasts and foreign DNA. DNA transfer by
electroporation is thought to occur as a result of transient changes in the lipid bilayer
structure of the plasmalemma induced by the electric field. The introduced DNA enters
the cells through reversible pores created in the protoplast membranes by the action of the
short electrical pulse treatments. It is known that gene transfer efficiency and protoplast
viability are influenced by the amplitude and duration of the electric pulse and by the
composition of the electroporation buffer. Fromm et al. (1985) first reported gene
transfer into maize protoplasts and Langridge et al. (1985) first reported the stable
transformation of carrot protoplasts with DNA by electroporation. Application of high
voltage either directly (both electrodes immersed in the sample solution) or indirectly
(without anode contact) to a solution containing plasmid DNA and protoplasts of rice,
wheat, or sorghum resulted in different degrees of protoplast viability and gene transfer
efficiency (Ou-Lee et al., 1986). Protoplast size and competent state differ from one

species to another, and, therefore, the optimal electroporation conditions may vary among

15
species. For example, Shimamoto er al. (1989) used a long electrical pulse (10 msec)

generated by a large capacitor and a moderate voltage (300 V/cm) for genetic
transformation of embryogenic protoplasts of rice (10-20 um in diameter). Rhodes et al.
(1988) applied a shorter pulse and higher voltage to maize protoplasts (2540 um in
diameter). As in the case of PEG method, the buffer also greatly influences the gene
transfer efficiency as well as the protoplast survival rate. Tada er al. (1990) demonstrated
that replacing the chloride ions in the buffer with an organic acid, aspartic acid
monopotassium salt, increased the transformation frequency at least 10-fold and
increased the plating efficiency as well.

The production of transgenic plants via direct gene transfer to protoplasts depends
on protoplast-to-plant regeneration and on efficient selection systems for transgenic cell
clones. Early gene transfer experiments focused on protoplasts of Solanaceae species
that are easily regenerable, and on the use of the bacterial gene for neomycin
phosphotransferase (npt II), conferring kanamycin resistance to transformed cell lines.
During the past few years, protoplast-to-plant regeneration was achieved for several
economically important crops such as Japonica and Indica rice varieties (Shimamoto er
al., 1989; Datta et al., 1990; Li et al., 1992a, 1992b), maize (Omirulleh et al., 1993), and
forage grasses (Wang et al., 1992). However, the natural resistance of many
monocotyledonous species to the antibiotic kanamycin (Potrykus et al., 1985; Hauptrnann
et al., 1988; Dekeyser et al., 1989) made the development of other selection systems
necessary. In addition to the npt II gene, the gene for hygromycin-transferase (hpt; Gritz
and Davies, 1983) and phosphinothricin-acetyltransferase (pat; Thompson et al., 1987)
have proven useful for the selection of stably transformed colonies in mono- and
dicotyledonous species (Horn et al., 1988; Masson et al., 1989; Shimamoto et al., 1989;
Datta et al., 1990; Wang et al., 1992; Omirulleh et al., 1993).

PEG- and electroporation-mediated direct gene transfers are simple and efficient:

dozens of protoplast samples can be treated in a single experiment, and thousands of

16
individual transgenic plants can be obtained in model systems with tobacco. Since any

DNA fragment can be delivered into the cell, these techniques have the advantage of
allowing the assimilation of a gene without having to clone the gene of interest into an A.
tumefaciens vector. One disadvantage of these techniques is that during incorporation
into the host nuclear genome, DNA rearrangement sometimes occurs (Paszkowski et al. ,
1989). Furthermore, only plants from which protoplasts can be isolated may be
transformed by these techniques, and not all protoplast systems can be used to regenerate
fertile plants. However, even if a plant cannot be regenerated, these methods still provide

an extremely useful means for studying transient gene expression.

Gene Transfer into Protoplasts Using Agrobacterium

Protoplasts of many dicotyledonous and monocotyledonous (nongraminaceous)
species can be transformed using A. tumefaciens strains harboring foreign gene(s) of
interest. Protoplasts must be prepared using optimal conditions for regeneration, co-
cultivated with the bacteria, and plated on appropriate media for protoplast culture and
regeneration, if desired. Growth of A. tumefaciens in the protoplast culture after co-
cultivation is retarded by the use of antibiotics in the culture media. In general, A.
nanefaciens-mediated transformation of plant tissue requires less effort and expertise in
tissue culture than transformation of protoplasts. However, transformation of protoplasts
using A. tumefaciens is an appropriate method in species where A. tumefaciens-mediated
transformation of tissue explants is difficult or has not been achieved, or in experiments
where expression in regenerated transgenic plants is not the primary goal (T homzik and

Hain, 1990).

Gene Transfer into Protoplasts by Sonication
An alternate transformation method has been reported by Joersbo and Brunstedt

(1990) and by Zhang et al. (1992). Joersbo and Brunstedt introduced DNA into sugar

17
beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) protoplasts by applying a

brief exposure of 20 kHz ultrasound in the presence of a plasmid containing the
chloramphenicol acetyl transferase (CAT) gene fused to the 358 promoter. A maximal
level of CAT activity was achieved in the transformed cells by sonication for 500 to 900
msec at 30 to 70 w electric power. Up to 12% (sugarbeet) and 81% (tobacco) of
maximum transient expression could be achieved with no significant loss of viability.
The concentrations of plasmid DNA (80 to 110 ug/ml) and of sucrose (21 to 28%) in the
sonication medium were also determined for the optimal transient expression. The
protoplasts surviving the exposure to ultrasound were found to have long-term viability
and ability to regenerate to microcalli similar to the untreated protoplasts.

Zhang et al. (1992) succeeded in the regeneration of transgenic tobacco plants
from protoplasts transformed by sonication. They used dimethyl sulfoxide (DMSO) to
raise the transient expression frequency of B-glucuronidase (GUS) gene to 86% and the
shoot differentiation frequency to 60%. The activity of GUS gene in the leaves of
regenerated kanamycin-resistant plantlets was found at a frequency of 22 percent. The
analysis of R1 regenerated seedlings showed that both kanamycin and GUS genes
behaved as typical linked dominant genes. They also found that salmon sperm DNA as
carrier DNA was essential for obtaining stable transformants.

One possible advantage of this method is that the system may be simpler than
electroporation. The efficiency of transformation was comparable to other
transformation methods of protoplasts. It remains to be seen whether this method can be

extended to a greater number of plant species.

Liposome-mediated Transformation of Protoplasts
A variety of approaches have been investigated in order to optimize the delivery
of nucleic acids into eukaryotic cells. In mammalian cells, for example, liposomes were

successfully used to transfer and express DNA and RNA molecules (Fraley et a1 ., 1982).

18
Liposomes spontaneously interacted with the plasma membrane of protoplasts and had

the advantage that they protected the encapsulated nucleic acids from nucleolytic
degradation and, therefore, guaranteed a very efficient delivery of intact nucleic acids
into recipient cells. Stable-transformed, fertile, and transgenic tobacco plants expressing
kanamycin resistance were obtained through the liposome-mediated transformation
method (Caboche, 1990). However, among the various procedures for introducing
foreign DNA into higher plant protoplasts, liposome-mediated direct gene transfer was
one of the most difficult (Caboche, 1990). The frequency of liposome-mediated
transformation was five to ten times less efficient than electroporation in achieving
transformation. The problems lay in the preparation of liposomes and the reliable
encapsulation of plasmids. Caboche (1990) also suggested that due to the rather low
transformation frequency of this technique and to the restriction of the encapsulation
procedure to DNA fragments smaller than ten kilobases (above this size the step of
sonication involved in the procedure was deleterious to the integrity of the encapsulated
material), liposome-mediated transformation of protoplasts was not a recommended
technique.

However, since lipofectin, a stable preparation of cationic liposomes, was
commercially available, the successful application of lipofectin-mediated transformation
has been achieved in animals (Felgner et al., I987; Felgner and Ringold, 1989) and in
plants (Antonelli and Stadler, 1990; Sporlein and Koop, 1991). Complex formation of
liposomes and DNA and their introduction into protoplasts occurred spontaneously.
Since the DNA did not have to be encapsulated into liposomes, this procedure was
significantly simpler than the conventional liposome-mediated transformation method.
The efficiency of this method was also enhanced by its combination with other

techniques such as PEG treatment or electroporation (Spdrlein and Koop, 1991).

19
Microinjection into Plant Protoplasts

Microinjection is one of the most precise and direct techniques for delivering
macromolecules into specific intracellular compartments of living cells (Reich et al.,
1986). Studies with animal cells have shown the utility of intracellular microinjection for
efficient transformation (Capecchi, 1980; Brinster et al., 1985). Early attempts with plant
cells that were hampered by the cell wall or by the fragility of protoplasts. Methods were,
therefore, developed to immobilize protoplasts in agarose or on glass with polylysine or
by holding the protoplasts under suction. Successful transformation of wild-type Ti
plasmid into alfalfa (Reich et al., 1986) and tobacco protoplasts (Crossway et al., 1986)
was achieved. Stable transformation frequencies of 14—66% of injected protoplasts have
been reported, but the absolute number of transformants is always low, due to the low
number of protoplasts which can be microjected.

Basically, microcapillaries and microscopic devices (micromanipulators) are used
to deliver DNA into the protoplasts; if competent, the injected cells can survive and
divide. No other transformation method could compete with microinjection if competent
cells could be visually identified in plant tissue (Potrykus, 1990). However, it is not yet
possible to identify competent cells visually. The disadvantage of this method is that it is
very time consuming and is not amenable when large numbers of transformants are

desired. Moreover, it requires expensive instruments and considerable skill to carry out.

Transformation of Intact Cells by Electroporation

Electroporation has been used for more than ten years for transient and stable
transformation of protoplasts (Fromm et al., 1985; Shillito et al., 1985). Only recently,
however, have electroporation conditions been found that deliver DNA molecules into
intact plant cells which are still surrounded by cell walls (Morikawa et al. , 1986; Lindsey
and Jones, 1987; Dekeyser et al., 1990; D'Halluin et al., 1992b; Kloti et al., 1993;
Songstad et al., 1993; Laursen et al., 1994; Arencibia et al., 1995). Optimization of the

20
system mainly involved eliminating explant-released nucleases, prolonging the DNA-

explant incubation time and expanding the electrical pulse time. In most reported cases,
transformability of intact plant cells or plant tissues depended on pretreatment of the cells
or tissues with hypertonic or enzyme-containing solutions. However, certain cells are
competent for DNA-uptake by electroporation without any pretreatment, for example,
immature embryos of maize and wheat (Klbti et al., 1993; Songstad et al., 1993). The
reasons for cell competence for DNA-uptake by electroporation are still unknown
(Potrykus, 1990). Compared to particle bombardment, the range of tissues that can be
transformed by electroporation, which includes meristem tissue (Dekeyser et al., 1990),
immature zygotic embryos, embryogenic calli (D'Haullin et al., 1992b; Arencibia et al.,
1995), and suspension culture cells (Laursen et al., 1994), seems to be narrower.
However, for tissues that are susceptible to DNA-uptake by electroporation, this is a
simple, fast, and inexpensive method for transient and stable transformation in plant

tissues.

Microprojectile Bombardment

Biolistic gene transfer is a relatively new and promising approach to plant genetic
transformation with the distinct advantages of applicability to any intact plant tissue or
region of the plant and with no host range limitation. The term ”biolistic” (biological
ballistics) was coined to describe the nature of the delivery of foreign DNA into living
cells or tissues through ”bombardment” with a biolistic device (a particle gun). The
concept and process of microprojectile bombardment was first described in detail by
Sanford et al. (1987). These high velocity microprojectiles are capable of penetrating
cell walls. They are coated with a DNA solution, so that when they enter the cells, the
DNA is carried as well. Since thousands of tungsten particles are accelerated

simultaneously, foreign DNA is also delivered into numerous cells. The biolistic process

21
has resulted in successful biolistic transformation of a wide range of tissues in a wide

range of plant species.

Klein et al. (1987) first reported a method in which high velocity tungsten
microprojectiles accelerated by a device known as a particle gun were employed to
deliver nucleic acids into living plant cells. In their experiments, the transient expression
of exogenous RNA or DNA was observed in epidermal cells of onions (Allium cepa).
Evidence that the biolistic process will have broad use has been growing. Following the
original observation by Klein et al. (1987), the biolistic delivery of foreign DNA into
corn cells was also achieved (Klein et al. 1988a; 1988b). The technique of particle
bombardment has been shown to be the most versatile and effective way for the creation
of many transgenic organisms. It has been demonstrated that the process could be used to
deliver biologically active DNA into plant cells that results in the recovery of stable
transformants (Christou et al., 1988; Wang et al., 1988; Cao et al., 1989; Fromm et al.,
1990; Gordon-Karnm et al., 1990). Biolistic transformation of higher animals has also
been demonstrated in vitro (Zelenin et al., 1989), and in vivo (Yang et al., 1990; Williams
et al., 1991).

Microprojectile bombardment has proven to be effective even in very small cell
types, and has, therefore, been useful in transforming diverse microbial species. These
include eukaryotes such as yeast, filamentous fungi (Arrneleo et al., 1990), and algae
(Day et al., 1990); and prokaryotes such as Bacillus megaterium (Shark et al., 1991),
Pseudomonas syringae, Agrobacterium tumefaciens, Erwinia amylovora, Erwinia
stewartii, and Escherichia coli (Smith etal., 1992).

The biolistic process also made the transformation of organelle genomes possible.
Chloroplasts of Chlamydomonas reinhardtii can now be routinely transformed (Boynton
et al., 1988), along with mitochondria of yeast and Chlamydomonas (Johnston et al.,
1988; Fox et al., 1988). Higher plant chloroplasts can be either transiently or stably

22
transformed using the biolistic process (Daniell et al., 1990; Ye et al., 1990; Svab et al.,

1990).

The ability of microprojectile bombardment to deliver foreign DNA into
regenerable plant cells, tissues, or organs appears to provide a reliable and dependable
method for achieving truly genotype-independent transformation in many important
agronomic crops, bypassing the limitations of Agrobacterium host-specificity and the
tissue culture-related regeneration difficulties. Transformed plant tissues include cell
suspension, calli, immature embryos, mature embryo parts, meristems, leaf pieces, and
pollens. Due to the physical nature of this technique, there is no biological limitation and
barrier to the actual DNA delivery process; thus, genotype is not a limiting factor. It
appears that we have a reliable system in place for plant transformation by combining the
relative ease of foreign DNA introduction into plant cells, tissues, or organs with an
efficient regeneration protocol that avoids protoplast or suspension culture.

Important advances and refinements in the process of plant transformation, which
will be described subsequently, have been made using soybean (McCabe et al. 1988;
Christou et al. 1990) and rice (Christou et al. 1991) as model systems for dicotyledonous
and monocotyledonous species, respectively. These systems have demonstrated the
power and versatility of the microprojectile bombardment method in achieving genotype-
independent transformation. Christou (1994) listed four major advantages that make
microprojectile bombardment the method of choice for genetically engineering various
crops: (1) transformation of organized tissue, (2) universal delivery system, (3)

transformation of recalcitrant species, and (4) study of basic plant development

processes.

Instruments
A number of different instruments are currently in use based on various

mechanisms to accelerate microscopic particles to supersonic speeds. Of these various

23
acceleration methods, the only method that has been proven to be of general value thus

far is the acceleration of microprojectiles on the face of a macroscopic carrier, or "
macroprojectile". In all cases, the macroprojectile is driven by a gas shock, which can be
achieved by using a gunpowder (chemical explosion) device (Sanford et al., 1987), an
apparatus based on electric discharge (an electric explosion of a water droplet) (Christou
et al., 1990), a microtargeting apparatus (Sautter et al., 1991), a pneumatic instrument
(Iida et al., 1990), an instrument based on flowing helium (Takeuchi et al., 1992; Finer et
al., 1992), or an improved version of the original gunpowder device utilizing compressed
helium (Sanford et al., 1991). Hand-held devices for both the original Biolistics® device
and the Accell® device are also in use. The most popular and widely-used instrument is
the one currently marketed by Bio-Rad, Inc. (Biolistics®). The macroprojectile may be
any lightweight object that has a front surface that can carry microprojectiles, a back
surface that can receive the energy of the gas shock, and sufficient cohesive integrity to

withstand the gas shock, sudden acceleration, and violent deceleration.

Critical Parameters in Opti_mi_z_igg the Biolistic Process for Different Apmications

In order to optimize the frequency of genetic transformation, a number of
parameters have been identified which need to be considered carefully when using
particle bombardment. The most convenient measure of efficiency for DNA delivery
into plant cells by microprojectile bombardment is the number of cells transiently
expressing a GUS (B-glucuronidase) gene. The GUS reporter system of Jefferson et al.,
(1987) has been the most useful method in determining the number of transiently
expressing cells per bombardment. One to two days after bombardment with the GUS
reporter gene, target cells can be incubated with GUS histochemical substrate (5-bromo-
4-chloro-3-indoyl-B—D-glucuronic acid). Those cells which express GUS stain blue, and
the number and distribution of the cells receiving and expressing the GUS gene are easily

visualized. However, investigators should not over-emphasize the significance of

24
transient expression data. Because optimization or maximization of transient activity

does not necessarily result in optimal stable transformation, transient expression studies
should only be used as a guide to develop systems for the stable transformation of a given
species.
At least five key factors interact to affect the frequency of stable transformation in

the bombarded cells (Birch and Franks, 1991):

1. size and composition of the microprojectiles,

2. DNA attachment to the microprojectiles prior to bombardment,

3. impact velocity of the microprojectile/DNA complex,

4. type of tissue and degree of cell damage suffered on bombardment, and

5. genetic construct.

Microprojectile Size and Composition
Several features need to be considered when choosing materials to be used for

microprojectiles in gene transfer work.

1. Particles should be of high density in order to possess adequate momentum to
penetrate into the appropriate tissue.

2. Microprojectiles should be available in a range of defined sizes.

3. Metals should be relatively inert to reduce the likelihood of explosive oxidation and
to prevent adverse reactions with cell components.

4. There should be no reactivity between particles and DNA and other components of
the precipitating mixes.

5. They should have desirable agglomeration and dispersion properties.

Tungsten and gold microprojectiles meet most of these requirements and have
been widely used in biolistic bombardment experiments. Tungsten particles are

extremely irregular in shape and heterogenqu in size. Although different mean sizes

25
range from 0.4 to 1.7 pm, their distributions overlap extensively. The advantages of

tungsten are that it is very inexpensive, it is available in numerous sizes, each size
represents a broad spectrum of particle diameters, and it is easily coated with DNA. The
disadvantages are that it is potentially toxic to certain cell types; it is subject to surface
oxidation that can alter DNA binding; over time, it catalytically degrades the DNA bound
to it; and it is highly heterogeneous in shape and size, which prevents optimization of size
for a particular cell type.

Gold particles which are available in a very limited range of sizes, are much
rounder and more uniform in size than tungsten. A principal advantage of gold particles
is their uniformity, which allows for optimization of size relative to a given cell type,
assuming one of the few available sizes happens to be optimal. An even more important
advantage of gold is that it is biologically inert. Unlike tungsten, gold does not
catalytically attack the DNA bound to it. A major disadvantage of gold is that it is
relatively expensive. Another concern with gold particles is that it is not stable in sterile
aqueous suspensions and over a period of time agglomerates irreversibly (Sanford, 1993).

The optimal size and impact velocity of the microprojectiles depend on properties
of the target cells such as size, penetrability, and resilience. Those working with plant
cells generally utilize microprojectiles 1.0-2.0 pm in diameter. For example, Klein et al.
(1988b) found higher transient expression levels when maize suspension culture cells
were bombarded with DNA-coated tungsten particles with an average diameter of 1.2 pm
than with average diameters of 0.6 pm and 2.4 pm. However, Casas et al. (1993)
bombarded immature zygotic embryos of sorghum with tungsten microprojectiles 1.7 pm
in diameter or with gold microprojectiles ranging from 1.5-3.0 pm in diameter and

detected little difference based on the types of microprojectiles used.

26
DNA Attachment

Any effective procedure for attaching DNA to microprojectiles is probably
applicable to all cell types regardless of the other optimized bombardment conditions.
However, the microprojectile coating is one of the most important sources of variation
affecting biolistic efficiency. Apparently, each time DNA is precipitated, its pattern of
precipitation and aggregation is unique and nonreproducible. The precipitation occurs so
rapidly that it is nearly impossible to obtain a uniform reaction mixture---especially
because gold or tungsten particles are difficult to keep in suspension.

DNA is usually bound to tungsten particles by variations of the CaClz/spermidine
co-precipitation method developed by Klein et al. (1988a, 1988b). The particles coated
with DNA are washed with 70% ethanol. A second wash with 100% ethanol is
recommended. The DNA-coated particles are gently pelleted, brought up to final volume
with 100% ethanol, and then pipetted onto the surface center of macroprojectiles. It is
usually recommended to wait for the particles to dry thoroughly before using them,
although DNA-coated particles may dry as they are propelled through the vacuum
chamber towards the target. In contrast, DNA-coated gold particles was dried under a
stream of nitrogen before resuspened in 100% ethanol. The gold particle suspension was
then pipetted and dried onto the carrier surface before acceleration (McCabe et al., 1988).
The nature, form, and concentration of the DNA need also be considered. Once particles
have been coated with DNA they should be used as soon as possible. This is particularly
true when tungsten particles are used, because the tungsten can degrade the DNA.
Exposure to humidity during or after drying dramatically reduces transformation
frequency, apparently due to hygroscopic clumping and agglomeration (Smith et al.,

1992).

27
Microprojectile Velocity

Even for a single plant species, it may be necessary to alter microprojectile
velocity for optimal transformation frequencies with different tissue types, depending on
cell wall thickness and the need to penetrate several cell layers. Although there may be
substantial variation between consecutive shots, velocity can generally be controlled to
some degree by altering the accelerating force, the vacuum in the chamber, or the

distance traveled by the microprojectiles.

Tissue Type and Tissue Damage

It is very important to target cells that are competent for both transformation and
regeneration. It is apparent that different tissues have different requirements; extensive
histology needs to be performed in order to ascertain the origin of regenerating tissue in a
particular transformation study. Depth of penetration thus becomes one of the most
important variables, and the ability to tune a system to achieve particle delivery to
specific cell layers may make the difference between success and failure in recovering
transgenic plants from a given tissue. In addition, experiments performed with
synchronized cultured cells indicated that transformation frequencies might also be
influenced by cell cycle stage (Iida et al., 1991).

When a microprojectile bombardment device is employed to transform plant cells,
the velocities of the particles which are sufficient for gene delivery frequently damage
target tissues, especially those near the center of the target. The injury is attributed to
physical trauma to the cells from the gas blast and acoustic shock generated by the
device. The use of baffles or mesh screens reduce cell death and increase transformation
frequency significantly (Gordon-Kamm etal., 1990; Russell et al., 1992).

The addition of an osmoticum (i.e., a supplemental agent increasing osmolarity)
to the bombardment medium can increase the frequencies of transformation. Armaleo et

al. (1990) observed that yeast cells which received bombarding particles usually suffered

28
cytoplasmic extrusions. Higher yeast transformation frequencies were achieved by using

a medium of high osmolarity, possibly due to protection against osmotic disruption
following cell wall damage and improvement of particle penetration. In tobacco cell
suspension cultures, 2- to 10—fold higher rates of both transient and stable transformations
were obtained when at least 300-900 mOsm/kg H20 osmoticum was included in the
bombardment medium (Belefant and Fong, 1989). Osmotic pretreatment of target tissues
have also been shown to be important in other cases (Russell et al., 1992; Vain et al.,

1993).

Genetic Construct

When optimizing a microprojectile bombardment system, it is important to
choose a genetic construct that will be expressed at reasonably high frequencies in the
target tissue. The GUS histochemical assay is sometimes insensitive, and cells
expressing the GUS gene at a relatively low level may not be revealed. It was found that
a change from CaMV 35S to a stronger monocot promoter with sugarcane suspension
cultures resulted in a 100-fold increase in the apparent frequency of transiently
expressing cells based on GUS histochemical staining (Franks and Birch, 1991). Bruce
et al. (1989) also demonstrated the effects of the gene regulatory sequences, the genetic
background of the target tissue, and the environment stimuli on transient expression
levels in bombarded tissues.

As for other direct gene transfer techniques, genes for transfer on high-velocity
microprojectiles need not be arranged between any Agrobacterium T-DNA or viral
sequences, and are generally in a small, high copy number E. coli cloning vector with
appropriate regulatory sequences. Genes can be biolistically delivered as RNA or DNA
(Klein et al., 1987), in circular or linear form (Blower er al., 1989), and as single-
stranded (Blower et al., 1989) or double-stranded DNA. However, there are some

indications that large plasmids (>10 kbp) may be more subject to fragmentation during

29
particle bombardment, resulting in lower expression frequencies and cotransforrnation

frequencies (Mendel et al., 1989; Fitch et al., 1990).

Remaining problems

The powerful biolistic transformation method, however, is still subject to a
number of limiting factors (Hunold et. al.,. 1994). Apart from technical considerations,
including the type of gun and experimental set-up, some tissues may be resistant to
particle penetration due to a strong cuticle, lignified cell walls or a hairy surface. Even
in the best experimental systems, only a small fraction of the total cell population will be
penetrated by microprojectiles and, of these, not all will express the introduced gene.
Transient expression in bombarded suspension culture cells may be observed in 01-03%
of all cells (Iida et. al., 1990; Yamashita et. al., 1991), but the level of stable
transformants is much lower (< 0.05%) (Klein et. al., 1989; Russell et. al., 1993). This
low efficiency requires either a very high number of experiments or an extremely
efficient selection and regeneration protocol. The unfavorable ratio between total cell
population and cells capable of expressing the foreign gene, and the even lower
proportion of stable transformation events are probably the most serious limitations to the

particle bombardment technique.

Microinjection of DNA into Plant Cells

Microinjection of DNA into cells using capillary micropipettes is one of the most
direct methods of delivering foreign DNA into specific cell compartments. The most
successful example is the microinjection of plasmid DNA carrying the nptII gene into
embryoids derived from microspores (12-cell stage) of oilseed rape (Neuhaus, 1987).
Among the 80% of embryoids regenerating to plants, half were stably transformed.
Although many of these plants proved to be chimeric, they segregated in vitro through

embryogenesis to give stable transformants. This clearly demonstrates that cells in

3O
meristematic clumps capable of plant regeneration can be transformed by microinjection

of DNA. Microinjection may also be of some value in transient assays where gene
transfer to particular cell types can give information about cell-specific expression and
cell lineage. A visual marker such as gas can be detected in a single cell. The major
limitations to this method are that it is technically demanding, requires the availability of

small clusters of embryogenic cells and often results in chimeric plants.

Macroinjection

Zhou et al. (1983) injected total DNA from one cotton variety into the embryos of
another cotton variety and obtained a large number of plants. Several of the recipient
plant progeny (R1 through R4) showed morphological characteristics that resembled those
of the donor plant. de la Pena et al. (1987) reported the recovery of transgenic rye plants
containing a foreign gene that conferred resistance to the antibiotic kanamycin. In their
method, plasmid DNA containing a selectable antibiotic resistance gene was suspended
in a buffer medium and injected with a syringe needle directly into the lumen of the
developing inflorescence about 14 days before meiosis. The injected plants were allowed
to produced seeds, which were screened for resistance to kanamycin. Seven seedlings
survived the kanamycin selection and two of these showed neomycin phosphotransferase
activity. Stable integration of the resistance gene was demonstrated in resistant seedlings
by Southern analysis. However, the subsequent work with barley was disappointing and
the frequency of transformation was very low (Mendel et al., 1990). The NPTII activity
corresponding to about one percent of that of stably transformed tobacco plants was

detected, but the genes were not stably inherited.

Pollen Tube Pathway
This method was reported by Lou and Wu (1988) using a plasmid-encoded gene,

neomycin phosphotransferase, as a reporter. Plasmid DNA carrying the selectable

31
antibiotic resistance gene was simply applied to the cut surface of the stigma for some

time after pollination (5-20 minutes to 2-3 hours, depending upon the rate of pollen tube
growth). They presented preliminary molecular evidence of gene transfer by both
genomic blot hybridization and NPTII enzyme assays in transgenic plants. The
transformation frequency was about 2% based on the number of rice florets treated with
plasmid DNA solution. In similar work with barley, transformation frequencies of 103 to
10'4 in F1 seedlings were obtained, but only a low level of expression of the introduced
nptII gene was detected and this was lost in mature plants and in the next generation

(Mendel etal., 1990).

Direct DNA Uptake into lmbibing Dry Zygotic Embryos

The uptake of DNA by dry plant tissue through membranes, whose
physiochemical characteristics change while natural desiccation occurs, was proposed as
an alternative and simple way of gene transfer (Topfer et al., 1989; Senaratna et al.,
1991). Topfer's group isolated embryos of seven cereals and three legumes by blowing
off the bran of the cereals and subsequently separating the endosperrns from the embryos
using cyclohexane and carbon tetrachloride (Topfer et al., 1989). The intact embryos
were selected and imbibed in a solution of plasmid DNA carrying a chimeric NPTII gene.
Molecular evidence was presented for the transient expression of the foreign gene. The
authors suggested that this method was suitable for transient gene expression studies.

Senaratna et al. (1991) also employed this simple strategy for gene transfer which
took advantage of two phenomena occurring in dry tissues : the huge water potential
gradient existing between the cell and a water solution (Hegarty, 1978) and the high
permeability of disorganized cell membranes at low water contents (Simon, 1974;
Hoekstra et al., 1989). Dry somatic embryos of alfalfa (Medicago sativa L) produced in
vitro from cell cultures were induced to become desiccation tolerant, dried to

approximately 10 to 15% moisture, and then used as a target for direct DNA uptake.

 

32
Water uptake into these dry somatic embryos was much more rapid than in a true seed

because they lacked a seed coat and endosperrn (McKersie et al., 1989). To determine
whether it was also permeable to large plasmid DNA molecules, dry somatic embryos
were imbibed in a solution containing a plasmid carrying the B-glucuronidase (GUS)
reporter gene. Transient expression of the GUS gene was observed visually in
germinating embryos and seedlings. Though PCR analysis of plant DNA from epidermal
cells of somatic embryos was presented, further studies are required to test the stability
and hereditability of these transformants. Since this system is suited for transient
expression assays as a simple and quick method of testing the effectiveness of vector
constructs (Lee et al., 1989), it may be necessary to redesign these direct DNA uptake

vectors to allow stable integration of the introduced DNA into the plant genome.

Laser-mediated Gene Transfer

Laser microbeam irradiation has been used successfully for more than twenty
years to manipulate subcellular structure, induce cell fusion, microdissect chromosomes
and perform other subcellular microsurgery (Bems and Rounds, 1970; Bems et al.,
1991). Recently, this application has been expanded to introduce foreign DNA into
mammalian cells (T sukakoshi et al., 1984; Tao et al., 1987). Working with plant cells,
Weber et al. (1989, 1990) reported that they could use a laser to puncture holes in the cell
walls and the membranes of Brassica napus when their chloroplasts and cells were
treated with a hypertonic buffer. Therefore, it was theoretically possible to introduce
foreign DNA into plant cells and cell organelles.

Guo et al. (1995) reported an effective system for introducing exogenous DNA
into cells of embryogenic calli of Japonica cultivar ZYl of Oryza sativa L.. Plant cells
were pretreated in the hypertonic buffer to draw some of the water from the cells, put into
a medium of less negative osmotic potential containing exogenous DNA, and treated

immediately with a laser microbeam (wavelength = 355 nm) to puncture holes in the cell

33
wall and membrane. The pretreatment of the cells generated a gradient of osmotic

pressure between the inside and outside of the cells, which facilitated the uptake of
material into the cells through the laser perforations. Bglucuronidase (GUS) genes were
successfully introduced into rice cells, as indicated by gene expression both in post-
treated cells and in plantlets regenerated from kanamycin-resistant calli that had been

treated by this method.

Silicon Carbide Whisker-mediated Transformation

In contrast to some of the technically demanding methods described above, there
have also been reports on the use of silicon carbide whiskers to develop a simple and
inexpensive system for the regeneration of fertile and transgenic plants. Transformation
of cell lines of maize (Kaeppler et al., 1990, 1992), tobacco (Kaeppler et al., 1990),
Agrostis alba (Asano et al., 1991), and, most recently, Chlamydomonas reinhardtii
(Dunahay, 1993) have been demonstrated using such whiskers. The method involves the
mixing (e. g., by vortex treatment) of cells in a liquid medium with whiskers and plasmid
DNA. The resulting collisions between cells and whiskers appear to lead to cell
penetration and DNA delivery 0(aeppler et al., 1990).

Although stable transformation has been achieved, fertile transgenic plants were
never obtained in any of the cases mentioned above. Frame et al. (1994), however,
reported the production of fertile transgenic maize plants using the silicon carbide
whisker transformation method. Cells from embryogenic maize suspension cultures were
transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial
bar and uidA (GUS) genes, and stable transformants were selected on a medium
containing bialaphos (one m g/l). Integration of the bar gene and activity of the enzyme
phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus

lines analyzed. Fertile transgenic maize plants were regenerated from those cell lines.

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Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a

Mendelian fashion.

Range of Transformed Species

In the late 19808, it became clear that some plant species were amenable to
genetic engineering (Goldberg, 1988; Gasser and Fraley, l989). This is because foreign
genes can be stably introduced into plant chromosomal DNA by a variety of techniques
(Potrykus, 1991). If a given foreign gene contains the appropriate regulatory sequence,
the gene product will be synthesized by the transgenic plant (Benfey and Chua, 1989).
Since most plant cells are totipotent, this allows regeneration of a fertile 'transgenic’ plant
from a single transgenic cell. However, in practice, not all plant species have proven to
be amenable to regeneration from dedifferentiated tissue (Potrykus, 1991).

In general, dicot plants are easier to transform and regenerate from protoplasts or
explant sections than most monocot species (Potrykus, 1991). It has been difficult to
generate transgenic monocot plants, which include the economically important crops
such as rice, corn, and wheat. One reason is that this group of plants is not usually
susceptible to infection by A. tumefaciens. While it is not a problem to deliver exogenous
DNA to protoplasts derived from monocot crop species, it is difficult to regenerate viable
or fertile plants from the transformed cells (Potrykus, 1990; 1991). However, there has
been progress in the area of regeneration, especially in the cases of rye, corn, and rice
(Gasser and Fraley, 1989; Gordon-Kamm et. al., 1990; Lynch et al., 1991). Also, with
the successful development of biolistic transformation methods for plant genetic
engineering, one can obtain transgenic monocot plants if the foreign DNA can be stably
introduced prior to or during embryogenesis and the embryos are still alive after being

bombarded with microprojectiles. For instance, corn and rice embryos bombarded with

 

35

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52
DNA coated microprojectiles have been transformed by this method (Gordon-Kamm et .

al. 1990;Chn'stou et.al., 1991).

By applying plant transformation methodology and recombinant DNA
technology, many transgenic plants have been genetically engineered with different
foreign DNA during the past ten years. Table 1 lists some examples of transgenic plants
that have been produced and the transformation methods, tissue sources, and selective
agents used to transform them. The number of plant species which has been transformed
is increasing rapidly because the technology for stably inserting and expressing foreign
genes in plants has been continuously improved, and more and more genes have been

identified and linked together with the traits they control.

Markers for Plant Gene Transfer

Genetic analysis of plant species would not be possible without the application of
marker genes to distinguish differences between individual plants. Naturally occurring
mutations which can be used as markers range from single base pair differences or
restriction fragment length polymorphisms between homologous stretches of DNA to
altered genes or chromosomes which affect morphological characters or enzyme-
mediated biochemical reactions. In the past, plant genetic research has frequently been
limited by the availability of desirable mutations which could be used as convenient
markers. Now that an increasing number of plant species are amenable to genetic
transformation by foreign DNA, marker genes can be constructed at will by recombining

pieces of DNA from various in vitro sources.

53
Applications of Marker Genes

Confirmation of DNA Delivery and Stable Transformation

Marker genes play a crucial role in the development of transformation procedures
because they can be used to ascertain how effectively the transforming DNA is being
introduced into plant cells using different treatments (Walden, l989). Selectable markers
are generally used for stable transformation. They either allow living cells, tissue, or
whole plants to grow under conditions which would prevent the growth of untransformed
plant cells, or confer a phenotype which could allow transformed and untransformed
tissue to be differentiated. In contrast, assayable markers direct the expression of a
readily detectable gene product (e. g., an enzyme), which is not necessarily
distinguishable in nontransformed living cells.

Assayable markers are frequently employed to optimize DNA delivery in the
early stages of developing a transformation procedure. This is because a high level of
marker gene expression in the first few days following the gene transfer is thought to be
essential for stable transformation. However, a high level of transient gene expression
may not always be sufficient. Some of the variables which affect transient gene
expression (e.g., the number of transformed cells, the number of active templates inside
those transformed cells, and the viability of the transformed cells following DNA
delivery) are important parameters for ensuring stable transformation.

Other parameters, such as the integration of marker genes into the target cell
genome and the cell division following DNA integration, are prerequisites for stable
transformation, but are not necessarily related to the efficiency of transient marker gene
expression. Similarly, the selection of stable transformants can be affected by the
efficiency of marker gene expression at the site of integration, and this may be different
from the level of expression observed prior to integration (Kay et al., 1987 ; Odell et al.,

1988).

54
Quantification of Gene Expression

Assayable markers are frequently used to quantify gene expression for reasons
other than optimizing DNA delivery. Such markers are also commonly referred to as
reporter genes. When fused to plant regulatory sequences in vitro, reporter genes are
indispensable for the analysis of gene expression in transient assays or in stable
transformants. Recombinant gene fusion can be used to determine when, where, and at
what level regulatory sequences direct gene expression in vivo and which intra- or
extracellular signals are involved (Benfey and Chua, 1989, 1990). Reporter genes can
also be fused to peptide coding sequences to determine whether they are involved in
targeting proteins either outside of the cell (Denecke et al., 1990) or into membrane-
bound organelles such as chloroplasts (Schreier and Schell, 1986; Svab et al., 1990),
mitochondria (Boutry et al., 1987), endoplasm reticulum (Iturriaga et al., 1989),

peroxisomes (Gould et al., 1990), or nuclei (Restrepo et al., I990).

Visualization of Gene Expression

Markers which can be directly detected in plant tissues are very important and
powerful tools. In transient expression studies, such markers can indicate the number and
location of transformed cells, both of which are important parameters for optimizing
stable transformation. In stable transformants, it is possible to determine whether an
introduced gene is expressed evenly in all cells of a particular tissue or at higher levels in
certain selected cells or tissue. This information often provides greater insight into the
functional role of a given gene at different stages of plant development and in response to
different environmental stimuli.

The B-D-glucuronidase (GUS) reporter gene system has proven remarkably
versatile in many investigations of this type (Jefferson, 1989). One current limitation of
this system, however, is that the histochemical procedure for detecting GUS expression

usually means sacrificing the tissue. However, markers which can be used to visualize

55
gene expression in living plant tissue have been developed. Some of these have already

proven useful as markers for whole plant transformation. In Arabidopsis, trichome
formation requires the product of the glI gene. This gene has recently been cloned and
reintroduced into a glI mutant which completely lacks trichomes (Herman and Marks,
1989). Trichome formation was restored by gene transfer, indicating that the defect was
complemented by the introduced gene. Plants with trichomes can be easily distinguished
from plants without, so this marker could be very useful for Arabidopsis transformation.
The pigmentation marker gene isolated from maize is another example of the visible

markers which can assist the identification of transgenic plants (Meyer et al., 1987).

Selectable Markers

Selectable markers confer a dominant phenotype on transformed cells because
they result in the addition of a new trait not normally associated with untransformed cells.
Traits which can be used to select transformed cells from those that are not transformed
generally fall into two categories: those which confer either cell viability or lethality in
the presence of a selective agent; and those which have a negligible effect on cell
survival, but which confer transformed cells with some distinguishable physical

characteristic.

Criteria for Choosing Selectable Markers

Plant cell transformation frequencies are generally low, so most stable
transformation systems use markers which ensure the survival of transformed cells in the
presence of a selective agent. The choice of an appropriate selective agent should be
made on the basis of several factors. At the very least, the selective agent should be
readily available and soluble in plant tissue culture medium. However, the most
important factor is whether the frequency at which untransformed cells or plants can

survive at a given level of the selective agent is greater than or similar to the expected or

56
observed transformation frequency. If the frequency of ”escapes" is high, an alternate

selection should be sought. Ideally, the LD50 (i.e., the level at which 50% growth
inhibition is observed) for untransformed cells should be at least an order of magnitude
lower than that for transformed cells (Nutter et al., 1987; Hauptrnann et al., 1988;
Dekeyser et al., 1989). In practice, however, this can only be ascertained when
transformed cells are available.

The nature of the target tissue and the DNA delivery system should also be
considered when developing a selection procedure with a given marker gene. For
example, green tissue may be more sensitive to selective agents which affect plastid-
localized processes than callus cells. Furthermore, exposure of callus cells to high levels
of these agents during the selection procedure can affect the regeneration of green plants
or the subsequent development of plastids. With other selective agents, the survival of
transformed cells under selection conditions can be affected by the release of toxic
compounds from surrounding untransformed cells. With bulky explants, transformed
cells on the surface may also be starved of nutrients if secondary thickening is induced by
the selective agent in adjacent untransformed cells. In these cases, transformants can be
recovered either by subdividing the target tissue or by using suspension cell cultures or
protoplasts where feasible. In addition, large explants or clumps of untransformed cells
often survive for longer periods under selective conditions. This can lead to a higher
frequency of escapes, especially if transformed cells deplete the selective agent in the
surrounding medium. In some cases, such escapes can easily be "weeded" out by
screening procedures which involve rooting or germination in the presence of the

selective agent.

Selectable Marker Genes for Transformation of Monocots
Since monocotyledonous plant species are generally insensitive to

Agrobacterium—mediated transformation, alternate procedures are necessary for genetic

57
modification of these plants. Gene transfer by microprojectile bombardment is currently

one of the successful methods for the genetic transformation of monocots. In this
approach, the efficiency of DNA delivery and especially the efficiency of stable
integration of the introduced genes are rather low compared to those obtained by
Agrobacterium-mediated transformation. For instance, after particle bombardment, only
1.9% of the cells initially expressing the B-glucuronidase gene became transformed by
integration of the gene (Klein et al., 1988b). Although Spencer et al. (1991) obtained
rather high values of stable transformation that ranged from five to ten percent after the
bombardment of maize cells, others obtained very low frequencies of about 0.1%
(Gordon-Kamm et al., 1990). Such a low efficiency of stable integration points out the
importance of an effective selection procedure to identify the small number of the stably
transformed cells from untransformed cells.

The antibiotic kanamycin, which has been most widely used as a selective agent
for the transformation of dicotyledonous species, can not easily be applied to monocots
(Potrykus et al., 1985; Hauptrnann et al., 11988; Dekeyser et al., 1989). The endogenous
resistance of many monocotyledonous species to kanamycin makes the development of
other selection systems necessary. Monocots are often insensitive to relatively high
levels of kanamycin, which allows the regeneration of untransformed plant cells on
kanamycin-containing medium. For example, a concentration at 100 mg/l of kanamycin
allowed the growth of 70% of untransformed rice calli (Dekeyser et al., 1989), and
protoplasts isolated from suspension cells of Lolium perenne were even able to divide in
the presence of 800 mg/l of kanamycin (Potrykus et al., 1985).

Therefore, many attempts have been made to replace the selective agent
kanamycin by other antibiotics or herbicides. This seems to be a reasonable approach,
because the various substances differ in their mode of action and in the way they are
absorbed and transported in plant tissue. The expression level of the resistance gene is

also important in ensuring a sufficient level of resistance in the transformed cells. In this

 

58
way, the transformed cells might be allowed to divide and further develop among the

majority of untransformed, highly susceptible cells. High expression levels can be

obtained by using specific plasmid vectors constructed for monocotyledonous species.

Herbicide and Antibiotic Resistance Markers
The repertoire of selectable markers which result in resistance to antibiotics,

herbicides, or other phytotoxins is now fairly extensive. Table 2 lists those that have
been successfully used for plant gene transfer. Resistance to the selective agents listed in
Table 2 is conferred by one of the following three principle mechanisms.
1. Overexpression of the protein which is the target for the selective agent. This will

cause a plant tolerant to a given level of the selective agent by ensuring a

proportion of the total amount of the target protein synthesized remains

unaffected, thereby allowing the cell to survive.

2. Alteration of the site of action. In this case, mutation of the target protein renders

it less able to bind to the selective agent, but still capable of carrying out its

biochemical function.

3. Introduction of genes from bacteria that can render detoxification either by

enzymatic modification or sequestration of the selective agent .

In general, plant genetic engineers have adapted the first two strategies whereas third
strategy is novel to genetically manipulated plants. In addition to the three mechanisms
mentioned above, some selective agents could also be detoxified by the plant itself, for
example, by glutathione-S—transferase. This enzyme occurs in plants (as well as other
organisms) and renders xenobiotic compounds ineffective by conjugating them to

reduced glutathione.

 

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62
Glutathione—S- transferase has been extensively studied in maize at the molecular

level, but has not yet been exploited in approaches to plant genetic manipulation for
herbicide resistance. Besides these intracellular mechanisms of resistance, tolerance to
inhibitors can be achieved by reducing their uptake into the cell (Harvey and Harper,

1982).

Antibiotics and Resistance Genes

Antibiotic resistance genes of bacterial origin, expressed under the direction of
plant regulatory elements, have been shown to be effective in plant cells. Expression of
such a gene in plant cells resulted in transformed cells showing acquired resistance to the
corresponding antibiotic. Representatives of a major class of antibiotics, the
aminoglycosides, and the corresponding resistance genes of microbial origin have been
widely used for plant transformation research.

The best known member of the aminoglycoside antibiotics is kanamycin, which
has been applied as a selective agent in many dicot transformation experiments. Other
agents are gentamycin, G418, neomycin, paromomycin, and hygromycin. These
antibiotics inhibit the protein synthesis in prokaryotic cells. Kanamycin, gentamycin and
its derivative geneticin (G418), neomycin, and paromomycin bind to the 308 ribosomal
subunit, thus inhibiting the initiation of translation and, consequently, protein synthesis
(Nap et al., 1992). l-Iygromycin occupies the ribosomal binding site of the elongation
factor EF-2, inhibiting peptide chain elongation (Kors, 1991). Ribosomes of
mitochondria and chloroplasts of higher plants are related to bacterial ribosomes, and are
also susceptible to these antibiotics. The most dramatic visible effect on plants is
chlorosis, bleaching of the leaves, caused by the lack of chlorophyll synthesis.

Resistance to kanamycin, G418, neomycin, and paromomycin was obtained by
the aphA2 gene from TnS of Escherichia coli (Bevan et al., 1983). This gene encodes
for the enzyme APl-I(3')II (aminoglycoside 3'-phosphotransferase II), also called NP'TII

63
(neomycin phosphotransferase II), which phosphorylates a specific hydroxyl group of

these antibiotics. Due to this phosphorylation, binding of the antibiotics to the ribosome
is inhibited. Kanamycin can also be detoxified by AAC(3') (gentamycin 3-N-
acetyltransferase), which acetylates the 3'-amino group of ring II. Gentamycin can also
be detoxified by AAC(3'), but, because it does not contain a hydroxyl group at the 3'-
position of ring I, it can not be phosphorylated by APH(3'). However, a gentamycin
derivative, G418, contains an extra hydroxyl group at the 4'-position of ring I, which
enables phosphorylation by NP'I‘II. Genes encoding ACC(3') enzymes have been used
for transformation of Petunia hybrida and Arabidopsis thaliana (Hayford et al., 1988)
and tobacco (Carrer et al., 1991). Hygromycin can be inactivated by phosphorylation of
a hydroxyl group by the enzyme APH(4‘), also called HPT (hygromycin
phosphotransferase). The hpt=aphIV gene which codes for this enzyme was isolated
from E. coli and rendered suitable for expression in plant cells (van den Elzen et al.,
1985; Waldron et al., 1985).

Generally, kanamycin was added to the growth medium in a concentration
previously shown to be inhibitory for the growth and regeneration of untransformed cells.
Little has been known about the transport of kanamycin in plant tissues. It did not seem
to be mobile in the vascular tissue, but rather to diffuse through plant tissue via
intercellular spaces. It was observed that this diffusion only occurred over rather short
distances (Weide et al., 1989), which meant that in large explants, not all parts were
reached by the antibiotic. From prokaryotic studies, it was known that the strongly
positive molecule was bound to the negatively charged cell membrane, then mediated by
carriers, and actively transported into the cell. This last step rendered the process pH
dependent; if the pH dropped below 6, binding was strongly inhibited. However, the
normal tissue culture pH is 5.8 or even lower. Moreover, bivalent ions in the medium
(Ca2+ and Mg2+) competed with the kanamycin for the carrier-binding sites (Nap et al.,
1992).

64
Although the use of kanamycin has proven to be very effective for the

development of transformation protocols for dicotyledonous species, research in monocot
transformation demonstrated that in these species, kanamycin was less effective as a
selective agent. When kanamycin was used for the selection of protoplasts of
Gramineae, it was shown that, for an optimal effect, the antibiotic had to be applied as
soon as possible after protoplast isolation and before restoration of the cell wall
(Hauptmann et al., 1988). This suggests that transport over the cell wall is the limiting
factor. Additional problems have been reported for rice: regeneration of plants from
transformed calli proved to be impossible if kanamycin was used as the selective agent
(Peng et al., 1992). Only when G418 was used for selection could calli expressing the
aphA2 gene be regenerated into plants. It was concluded that kanamycin somehow
impaired the regeneration potential of the rice calli. Similar phenomena were reported by
Toriyama etal. (1988), Zhang et al. (1988) and Battraw and Hall (1992).

G418 (geneticin) has been tested as an alternate selective agent in various
monocots, e.g., rice, Lolium, and other Gramineae, which were highly resistant to
kanamycin (Potrykus et al., 1985; Hauptmann et al., 1988; Dekeyser et al., 1989). In all
cases, G418 was shown to be more effective, perhaps due to the more effective binding to
ribosomes.

Hygromycin was another useful antibiotic for most tissues, including those of
monocots, and showed a higher sensitivity compared to kanamycin (Meijer et al., 1991).
Hygromycin has been applied in establishing transformation procedures for maize
(Walters et al., 1992), rice (Shimamoto et al., 1989; Li et al., 1992a; Datta et al., 1992),
and orchardgrass (Horn et al. , 1988).

65
Herbicides and Resistance Genes

Phosphinothricin (Bialaphos)

In 1971, a novel tripeptide (bialaphos or bilanaphos) containing the previously
unknown amino acid L—2—amino—4-[hydroxyl(methyl)phosphinyl] butyric acid (L-PPT),
together with two L-analine groups, was obtained by two different teams working
independently of one another---Bayer et al. (1972) in Tubingen, from Slreplomyces
viridochromogenes; and microbiologists of the Japanese firm Meiji Seika Kaisha (Kondo
et al., 1973), from Streptomyces hygroscopicus. The Tubingen authors gave the new
amino acid the name phosphinothricin (PPT) (Bayer et al., 1972). Structural proof was
obtained by degradation (Bayer et al., 1972; Ogawa et al., 1973a) and synthesis (Bayer et
al., 1972; Ogawa et al., 1973b).

DL-Phosphinothricin is the active ingredient of a non-selective, broad-spectrum,
postemergent, commercial herbicide formulation and was given the common name
glufosinate ammonium (GLA). It is sold worldwide under the following trade names:
Basta, Buster, Challenge, Finale, Harvest, and Ignite. The active ingredient is highly
stable as a chemical compound, but its degradation is rapid in a microbiologically active
environment, such as soil or surface water. The Disomer is herbicidally inactive. L-
Phosphinothricin was initially identified as the active moiety of the herbicidal tripeptide
bialaphos (L-Phosphinothricinyl-L-alanyl-L-alanine) produced by Streptomyces
hygroscopicus (Kondo et al., 1973). PPT is an analog of glutamate and acts as a
competitive inhibitor of the enzyme glutamine synthetase (GS; glutamate-ammonia
ligase) in both plants and bacteria (Bayer et al., 1972). In plants, GS is critical for the
assimilation of ammonia into L—glutamate, forming L-glutamine, and is central to the
regulation of nitrogen metabolism (Miflin and Lea, 1977). It is the only enzyme in plants
that can detoxify ammonia released by nitrate reduction, amino acid catabolism, and

photo-respiration. Inhibition of this enzyme causes accumulation of ammonia in the

66
cells. The accumulation of ammonia rather than the lack of glutamine caused the death

of plant cells (T achibana etal., 1986).

PPT is synthesized chemically (Basta; Hoechst AG), or by fermentation of
Slreptomyces hygroscopicus (Herbiace; Meiji Seika Kaisha). The latter product, also
called Bialaphos, is a tri-peptide compound, which consists of PPT and two alanine
residues. In contrast to PPT, bialaphos has little inhibitory activity against glutamine
synthetase in vitro, but is converted to PP’I‘ in vivo by intracellular peptidases which
remove the alanine residues (De Block et al., 1987).

Resistance to this herbicide is conferred by phosphinothricin-N-acetyltransferase
(PAT), which inactivates PPT by acetylation, using acetyl coenzyme A as a cofactor.
Two similar genes encode PAT: bar, isolated from S. hygroscopicus (Murakami et al.,
1986; Thompson et al., 1987), and so named because of its resistance to Basta; and pat,
isolated from S. viridochromogenes (Wohlleben et al., 1988).

In transformation studies, Basta, PPT, and bialaphos have each been used to select
for PPT (bialaphos)-resistant plants by spraying full-grown plants or by adding them to
selection medium in earlier stages. In the media, one to three mg/l PP’I‘ is generally
adequate to select for transformed cells (De Block et al., 1987; Gordon-Kamm et al.,
1990). Dekeyser et al. (1989) found a concentration of 10 mg/l optimal in discriminating
between transformed and untransformed rice calli. They also showed that, for an
effective selection, it was important to exclude amino acids from the selection medium,
because several amino acids (glutamic acid, proline, arginine) allowed growth of
untransformed cells in the presence of PPT. This seems to contradict data indicating that
ammonia accumulation leads to cell death (Tachibana et al., 1986). However, it shows
that both the composition of the selection medium and the concentration of PPI‘ are
important factors in the selection for transformed plant cells.

As selective agents, PPT and Bialaphos have successfully been applied with

Brassica napus (De Greef et al., 1989) and Helianthus annus (Escandon and Hahne,

67
1991). Transgenic tobacco, potato, and tomato plants, which expressed the bar gene

from Streptomyces hygroscopicus (Thompson et al., 1987), were tolerant to 410 times
the field rate of application of the herbicide (De Block et al., 1987). They have also
proven to be effective in monocots such as maize (Gordon-Kamm et al., 1990; Spencer el
al., 1991; 1992), oats (Somers et al., 1992), and rice (Dekeyser et al., 1989; Datta et al.,
1992; Cao et al., 1992), which have a high tolerance for antibiotics such as kanamycin

and hygromycin.

Glyphosate
The herbicide glyphosate (N-phosphonomethyl glycine) has a simple molecular

structure, as it is an analogue of the amino acid glycine, and is the active component of
RoundupTM, a herbicide marketed by the Monsanto Company. It is in widespread use as
a nonselective post-emergent herbicide and has the desirability of effectiveness with
short-lived residual soil activity which seems environmentally sound. Glyphosate is
mobile in the phloem and tends to accumulate in the apex of the stem and root, so it
affects meristematic and apical cells especially (Comai et al., 1989). Glyphosate kills
most herbaceous plants, so it must not come in contact with the crop when applied. This
limits its modes of application.

Early investigations into the mode of action of glyphosate indicated that it
inhibited aromatic amino acid biosynthesis (Jaworski, 1972). Under the influence of
glyphosate, bacteria and plant cell cultures accumulated massive quantities of shikimic
acid and shikimic acid 3-phosphate (Amrhein et al., 1980, 1983; Berlin and Witte, 1981),
both of which were intermediates of aromatic amino acid biosynthesis. All these facts
are accounted for by the finding that, in vitro, glyphosate is a potent competitive inhibitor
of the enzyme S-enol-pyruvylshikimic acid 3-phosphate (EPSP) synthase (Steinrticken
and Amrhein, 1980).

68
EPSP synthase has been isolated from a number of plants and microbial

organisms and has probably become the most comprehensively investigated enzyme in
the shikimic acid pathway (Amrhein, 1985). The aroA gene which codes EPSP synthase
has been isolated and sequenced from Escherichia coli (Duncan et al., 1984), Salmonella
typhimurium (Stalker el al.., 1985) and Arabidopsis thaliana (Klee et al., 1987). The
amino acid sequence of EPSP synthase has been derived from the nucleotide sequence of
these genes.

S. typhimurium provided an experimental system based on the availability of
defined mutations in the genetic locus (aroA) coding for EPSP synthase. If EPSP
synthase is the biochemical target for glyphosate, then it should be possible to isolate
glyphosate-resistant mutants by mapping at the aroA locus which encodes a glyphosate-
resistant enzyme. Two types of mutations (an overproduction of EPSP synthase via an
up—promoter mutation and a structurally altered EPSP synthase enzyme which is resistant
to the inhibition of glyphosate) were obtained from ethylmethanesulphate-mutagenized S.
typhimurium cultures (Comai et al., 1983). The enzyme activity of the mutant, which
was shown to synthesize an EPSP synthase with increased resistance to glyphosate, was
50% inactivated at 1.20 mM glyphosate and that of the wild-type at 0.01 mM. A
comparison of the wild-type and mutant gene sequences revealed a cytosine-to-thymine
transition mutation that resulted in a proline-to—serine amino acid substitution at position
101 of the 349 amino acid polypeptide (Stalker et al ., 1985). Another modified EPSP
synthase which was encoded by a mutated EPSP synthase gene was isolated from
Petunia (Shah etal., 1986b).

This information opens up two possible methods of producing glyphosate-
resistant plants by genetic engineering: (1) transfer of a glyphosate-sensitive EPSP
synthase under the control of a powerful promoter, causing overproduction of the enzyme

and/or (2) transfer of a gene which codes a mutated, glyphosate-resistant EPSP synthase.

69
Both methods have been used to generate glyphosate-tolerant plants. Using a

petunia cell line which overproduces EPSP synthase by gene amplification (Shah et al .,
1985; Steinrttcken et al ., 1986), a cDNA of the EPSP synthase gene was isolated and,
under the control of the 358 cauliflower mosaic virus promoter (CaMV), a chimeric gene
was constructed. Leaf discs of petunia incubated with Agrobacterium containing the
chimeric EPSP gene were able to form callus on medium containing 0.5 mM glyphosate,
whereas control leaf discs failed to do so. EPSP synthase activity in transformed callus
was up to 40-fold higher than in the control callus, confirming that the engineered EPSP
synthase gene could confer glyphosate resistance. From these transformed cells,
transgenic plants that show tolerance to glyphosate have been generated (Shah et al .,
1986b). Four independent transgenic plants selected for resistance to kanamycin (the
resistance gene was co—transferred with the chimeric EPSP synthase gene) could tolerate
spraying with Roundup herbicide at a dose equivalent to 0.8 LB per acre, which is two to
four times the level required to kill wild type plants.

The alternate method, i. e., the insertion and expression of a mutated bacterial
aroA gene which codes a glyphosate-resistant EPSP synthase, was also successful
(Comai etal ., 1985; Fillatti et al ., 1987a; Hinchee el al., 1988; Vasil et al., 1991). The
sources of the aroA gene were the strains of Salmonella lyphimurium and E. coli. S.
typhimurium that were able to grow on glyphosate-containing medium and be recovered
as spontaneous or chemically induced mutants. A small percentage of these mutations
mapped to the aroA locus. One of these mutants was shown to synthesize an EPSP
synthase with increased resistance to glyphosate (Comai et al., 1983). Enzyme activity
was 50% inactivated at 0.01 mM glyphosate and 1.20 mM glyphosate for the wild-type
and the resistant EPSP synthases, respectively (Stalker et al., 1985). Comparison of the
derived amino acid sequences of the EPSP synthases from mutant and wild-type S.
typhimurium revealed that the substitution of a proline (Pro 101) for a serine residue

accounted for the decreased affinity of the enzyme for glyphosate (Stalker et al., 1985).

70
A more highly resistant form of EPSP synthase from E. coli has also been described

(Kishore etal., 1986).

Much attention has been paid to improving the selection system based on
glyphosate resistance. This has included isolation of promoters that were effective in
apices and meristems, where glyphosate accumulated (Comai et al., 1989). A higher
transformation efficiency is also expected to be achieved by targeting the enzyme to the
chloroplast, where the shikimate pathway is located. To achieve this, the aroA gene from
S. typhimurium was fused to the region of Rch encoding the transit peptide of the
ribulose biphosphate carboxylase small subunit (Comai et al., 1988), but, so far, this
chimeric gene has not been used in transformation experiments. However, a fusion
between the region of Petunia Epsps encoding the transit peptide and the resistance gene
aroA from S. typhimurium improved the selection of transgenic plants (Della-Cioppa et

al., 1987; Botterman and Leemans, 1988).

mg

Atrazine also inhibits photosynthesis by binding to the Q 3 protein in the thylakoid
membrane, thus blocking electron transport. The resistance gene has been isolated from
chloroplasts of an atrazine-resistant Amaranthus hybridus. The gene is a mutated psbA
gene that encodes a modified QB protein. It has been fused to a transit peptide sequence
for chloroplast targeting. Transgenic tobacco plants appeared to be tolerant to 100 mM
atrazine (Cheung et al., 1988), but no other reports concerning the use of atrazine as a
selective agent in transformation studies are known. There also exists an atrazine-
detoxifying system based on the glutathione-S-transferase gst gene (Shah et al., 1986a),

but this has not been applied in any transformation research.

7 1
Bromoxynil

Bromoxynil is a herbicide that inhibits photosynthesis in plants by binding to
electron-transport components of photosystem II in the thylakoid membrane (Van
Rensen, 1982; Sanders and Pallett, 1986). There may also be a low—affinity binding site
for bromoxynil in the 32-kd polypeptide of the same complex (Szigeti et al., 1982;
Vermaas etal., 1984). The bxn gene (bromoxynil nitrilase gene) from Klebsiella ozaenae
codes for a specific nitrilase that degrades bromoxynil. Transgenic tobacco plants
harboring this gene have been obtained (Stalker et al., 1988). The chimeric bxn gene was
placed in the T-DNA of a binary Ti vector and transferred into tobacco via
Agrobacterium. After the selection of transformants using a co-transferred kanamycin-
resistance marker, the plants were shown to be resistant to bromoxynil up to 104M.
Control tissue is normally bleached between 106M and 105M bromoxynil. Resistant

plants were shown to have inherited one to three copies of the bxn chimeric gene.

Chlorsulfuron

Chlorsulphuron is a selective herbicide that affects amino acid biosynthesis by
inhibiting the enzyme acetolactate synthase (ALS) and can be applied to cereal crops
without detriment to their growth (Sweetser et al., 1982; LaRossa and Falco, 1984). ALS
is involved in the synthesis of branched-chain amino acids. The gene crs l-l, isolated
from Arabidopsis thaliana, confers resistance to Chlorsulfuron by encoding an ALS with
a reduced affinity for the herbicide (Haughn et al., 1988).

Chlorsulfuron has been applied as a selective agent in the transformation of
tobacco (Haughn et al., 1988), maize (Fromm et al., 1990), and sugarbeets (D'Halluin,
1992a). The ALS gene from Chlorsulfuron-resistant Arabidopsis was transformed into
tobacco as a 5.8-kb genomic fragment inserted into the T-DNA of a disarmed Ti plasmid
(Haughn et al., 1988). Unlike all previous examples, this herbicide-resistance gene

required no further manipulation and illustrates an advantage of recovering a resistance

72
gene from a plant such as Arabidopsis than a bacterium. The ALS coding sequence is

flanked by its own transcription regulatory sequences and contains a transit peptide for
targeting the pre-ALS into chloroplasts (Mazur et al., 1987; Haughn et al., 1988).
Transformed plants were selected on kanamycin-containing medium using an aph3'II
chimeric gene which was co-resident with the csr gene in the T-DNA and was present at
two to ten copies per haploid genome. D'Halluin et al. (1992a) compared the use of
Chlorsulfuron, other sulfonylurea compounds, PPT, and kanamycin. They concluded
that, after the selection procedure, 90% of the shoots that survived the application of
Chlorsulfuron were actually transformed, while for PPT and kanamycin, these figures
were 30% and 50%, respectively. These results indicated that Chlorsulfuron was an
efficient selective agent for the transformation of sugarbeets. Transgenic poplars
(Brasileiro et al., 1992) and fertile transgenic rice plants (Li et al., 1992b) have also been

obtained by using the crs 1-1 gene in combination with the CaMV 35S promoter.

23:12

2,4—D (2,4—dichlorophenoxy acetic acid) is an auxin analog that is competitive
with indole acetic acid (IAA) in occupying binding sites of auxin receptors. The
resistance gene tfdA has been cloned from the soil bacterium Alcaligenes eutrophus
(Lyon et al., 1989). This tfdA gene codes for the enzyme 2,4-dichlorophenoxy acetate
monooxygenase that is involved in the degradation of 2,4-D. The gene has been
introduced into tobacco (Streber and Willmitzer, 1989), resulting in transgenic plants
with enhanced tolerance to 2,4—D. However, in this study, kanamycin was used to select
for transformed tissues, and selection by 2,4-D alone did not yield any transformed
shoots, possibly because of the overgrowth of untransformed tissue. Due to the fact that
2,4-D is very toxic to dicotyledons and monocotyledonous plants are known to be
tolerant, the use of 2,4—D as a selective agent has been a question (Streber and Willmitzer,

1989).

73

Methotrexate

Methotrexate causes the deficiency of thymidylate by inhibiting the enzyme
dihydrofolate reductase. Because thymidylate is a precursor of one of the components of
DNA, nucleotide biosynthesis is blocked, and cell death results. Resistance can be
conferred by the mouse dihydrofolate reductase gene (dhfr) (Eichholtz et al., 1987). This
gene encodes an enzyme with a reduced affinity for methotrexate. It has been utilized in
the transformation of Petunia hybrida (Eichholtz et al., 1987), Brassica napus (Pua et al.,
1987), and monocots like rice (Meijer et al., 1991). Also for grass species such as
Panicum maximum , it has shown to be a useful selective agent (Hauptmann et al., 1988).
In all cases, transgenic plants or calli have been obtained. However, Meijer et al. (1991)
questioned the suitability of methotrexate as a selective agent for rice because of an

inhibition of the other enzymes involved in the biosynthesis of nucleotides.

Application of Plant Transformation to Confer Insect Tolerance

If functional insecticides can be genetically engineered into plants, it may be
feasible to develop crops that are intrinsically tolerant of insect pests. Such plants would
not have to be sprayed with costly and potentially hazardous chemical pesticides. In
addition, as in the case with many biologically generated insect pathogens, such
insecticides would be required at much lower concentrations than exogenously applied
synthetic pesticides. Moreover, since biological insecticides are usually specific, they are

generally not hazardous to the intended consumers of the crops.

74
Bt Toxin

Two main strategies have been used to confer resistance against insect pests. The
first involves transforming plants with a gene for an insecticidal protoxin produced by
one of several subspecies of Bacillus thuringiensis (Bt toxin) (Vaeck et al., 1987; Brunke
and Meeusen, 1991; Perlak et al., 1990). The crystalline protein with insecticidal
properties has received much attention because the protoxin does not persist in the
environment nor is it hazardous to mammals. The idea of using such compounds is
nothing new, since Bt toxins in various formulations (usually as whole sporulated
bacteria) have been employed as insecticidal crop sprays for more than twenty years
(Dulmage, 1981), but their application is limited by the high cost of production.
Furthermore, the toxic components, i.e., the intracellular crystals which are produced by
the bacteria during sporulation, are not stable under field spraying and exposure
conditions, and, in particular, under high intensity ultraviolet light.

When ingested by certain insects, the crystals of Bt are partially hydrolyzed under
the alkaline conditions of the midgut, thereby releasing proteins of 65,000-160,000 in
molecular weight; further natural proteolytic processing of these proteins releases smaller
toxic fragments. The mechanism of toxicity appears to depend on the disruption of the
membranes of the cells lining the gut, and this action is only carried out by the cleaved
fragments (Sacchi et al., 1986). These Bt toxins are very species-specific, and, although
commercial toxins effective against more than 50 lepidopteran pest species have been
identified, comparatively few with activity against coleopterans have been isolated.
Despite this possible limitation, the potential for increasing the utility of Bt toxin by the
genetic engineering of plants to express the Bt toxin was recognized, and transgenic
tobacco, tomato, potato, and cotton plants have been obtained (Barton et al., 1987;
Fischhoff et al., 1987 ; Vaeck et al., 1987; Gasser and Fraley, 1989; Perlak et al., 1990;

Brunke and Meeusen, 1991). These transgenic plants have expressed sufficient quantities

75
of insecticidal crystalline protein to provide protection from damage by feeding larvae of

several different insects.

Protease inhibitor

The presence of inhibitors of mammalian digestive protease in plants, particularly
those in legume seeds, has long been known (Read and Haas, 1938). Their
incontrovertible demonstration as proteins came in 1945, when the trypsin inhibitor from
soybean seeds was isolated and crystallized (Kunitz, 1945). This class of compound has
been shown to be very widespread in plants (Richardson, 1977), and those present in the
leaves or seeds of Leguminosae, Graminae, and Solananceae have been extensively
studied and characterized (Richardson, 1991). The possible role protease inhibitors
played in plant defense systems against insect pests was investigated as early as 1947,
when it was observed that larvae of certain pests were unable to develop normally on
soybean products (Mickel and Standish, 1947). Subsequently, the trypsin inhibitors
present in soybean were shown to be toxic to the larvae of the flour beetle, Tribolium
confluum (Lipke et al., 1954). Following these early studies, there have been many
examples of protease inhibitors being active against certain insect species (Birk et al.,
1963; Applebaum, 1964; Steffens et al., 1978; Gatehouse et al., 1979; Broadway and
Duffy, 1986; Gatehouse and Hilder, 1988).

Additional evidence for the protective role of protease inhibitors has been
provided by Ryan and co-workers (Green and Ryan, 1972) when they demonstrated that
damage to the leaves of the tomato and potato, either by insect feeding or mechanical
wounding, induces the synthesis and accumulation of protease inhibitors both in the
damaged leaves (the "local" reaction) and also in undamaged leaves at a distance from
the original wound site (the ”systematic” reaction). This production of inhibitors was
shown to be the result of a wound hormone, protease inhibitor-inducing factor (PIIF),

which is released from the damaged leaves and transported to other leaves where it

76
initiates synthesis and accumulation of protease inhibitors (Shumway et al., 1976;

Walker-Simmons and Ryan, 1977; Brown et al., 1985). This evidence strongly
implicated protease inhibitors in plant protection (Ryan, 1983). Additional evidence
indicating the involvement of protease inhibitor in plant defense systems were provided
by other research groups.

The protection role of protease inhibitor in the "field” was first provided by
Gatehouse et al. ( 1979), who demonstrated that elevated levels of these inhibitors in one
variety of cowpeas were partially responsible for the observed resistance of the seeds to
the major storage pests of this crop, the bruchid Callosobruchus maculatus. This
particular trait was later exploited by conventional plant breeding, whereby bruchid
resistance was transferred to an agronomically improved background (Redden et al.,
1983).

The mechanism of antimetabolic action of protease inhibitors is not yet fully
understood. Direct inhibition of digestive enzymes is unlikely to be the only effect.
Possibly a situation exists that is analogous to the effect of some protease inhibitors on
mammals, where the major deleterious effect is loss of nutrients through pancreatic
hypertrophy and overproduction of digestive enzymes (Liener, 1980). That they affect
the nutritional biochemistry of C. maculatus has been clearly demonstrated, since
methionine supplementation has been shown to overcome the antinutritional effects of
the cowpea trypsin inhibitor (CpTI) (Gatehouse and Boulter, 1983). There is also
evidence to suggest that they are only one part of the complex interaction between plant
nutritional value and the insects' digestive physiology (Broadway et al., 1986). This may
explain why there are some COWpea varieties which bruchids are able to infect even
though they contain high levels of CpTI (Xavier-Filho etal., 1989).

Transformation of plants with the protease inhibitor gene has been considered to
have the potential to enhance resistance against pathogens and insects (Boulter et al.,

1990b; Ryan, 1990) and can also provide information regarding the roles of protease

77
inhibitor in the plant defense mechanism against insects. Direct evidence for the

effectiveness of protease inhibitors in reducing insect damage has been obtained by
transforming tobacco via Agrobacterium tumefaciens with a cowpea protease inhibitor
gene encoding a trypsin/trypsin inhibitor (Hilder et al., 1987). Synthesis of the inhibitor
in transformed tobacco plants was confirmed immunologically by Western blotting.
Transformed tobacco plants which expressed a foreign cowpea trypsin inhibitor gene and
produced about one percent of the leaf proteins as the inhibitor were more resistant to
feeding by larvae of Heliothis virescens than untransformed control plants or transformed
plants that did not express the gene.

The COWpea trypsin inhibitor gene is the first gene of plant origin to be
successfully transferred to another plant species that resulted in enhanced insect
resistance. The protease inhibitor gene was considered to be a particularly ideal
candidate for transfer to other plant species via genetic engineering, since it has been
demonstrated to be insecticidal towards a wide range of economically important field and
storage insects when tested on artificial diets. These include members of the
Lepidopterae such as Heliothis spp. and Anthonomus spp., Coleopterae such as
Diabrotica spp., Bruchidae spp. and Anthonomus spp., and Orthopterae such as Locusta
spp.

Furthermore, and of prime importance, they appear to exhibit low or no
mammalian toxicity (Hilder et al., 1991). These protease inhibitors are small
polypeptides of about 80 amino acids and belong to the Bowman-Birk type of double-
headed serine protease inhibitors, and, while most bind two molecules of trypsin, some of
the isoinhibitors are trypsin/chymotrypsin inhibitors (Gatehouse et al., 1980). They are
encoded by a moderately repetitive gene family in the cowpea genome (Hilder et al.,
1989). One drawback, however, is that relative to the Bt toxin, high concentrations of

cowpea trypsin inhibitor are required for effectiveness (Brunke and Meeusen, 1991).

78
Potato and tomato plants also contain two powerful inhibitors of serine proteases

called inhibitor I and inhibitor 11 (Plunkett et al., 1982). Inhibitor I is an inhibitor of
chymotrypsin that only weakly inhibits trypsin at its single reactive site; while inhibitor 11
contains two reactive sites, one of which inhibits trypsin and the other inhibits
chymotrypsin. Both inhibitors are synthesized as precursors and undergo post-
translational modification (Cleveland et al., 1987) to form the mature proteins which are
sequestered into the vacuole (Shumay et al., 1976). Not only has the gene encoding
protease inhibitor isolated from COWpeas been shown to confer resistance when expressed
in tobacco (Hilder et al., 1987), but the inhibitor II genes of the tomato and potato, and
the inhibitor I gene of the tomato, when expressed in the same model plant, have also
been shown to confer insect resistance (Johnson etal., 1989).

High expression of the protease inhibitor 11 gene severely retarded the growth of
Manduca sexta larvae feeding on the leaves of transformed tobacco plants. Results
showed that the presence of the foreign protease inhibitor 11 gene in tobacco leaves at
levels over 100 pg/g leaf tissue severely retarded the growth of larvae that fed on them,
compared to larvae that fed on untransformed plants or transformed plants that did not
express the inhibitor gene. At lower levels (about 50 pg/g leaf tissue) larvae growth was
retarded to a lesser degree than at higher levels, leading the authors to suggest that there
is a dose-dependent relationship between the levels of protease inhibitor and larvae
growth. In addition to retarding larval growth, the presence of inhibitor 11 appeared to
reduce the amount of leaf tissue consumed. Interestingly, transgenic tobacco plants
expressing the tomato protease inhibitor I gene (a strong inhibitor of chymotrypsin) at
levels greater than 100 yg/g leaf tissue supported similar rates of larval growth as those
found for control plants. Since the protease inhibitor 11 is a trypsin, as opposed to a
chymotrypsin inhibitor, it would appear that it is the trypsin inhibitor which is responsible

for the observed insecticidal activity.

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79

Tissue Culture and Genetic Transformation of Creeping Bentgrass

Plantlet regeneration from embryogenic callus in turf grass was first reported in
species which have a dual use as forage and turf. These include ryegrass hybrids (Lolium
multiflorum x L. perenne and reciprocal crosses) (Ahloowalia, 1975), tall fescue (Festuca
arundinacea, Schreb.) (Lowe and Conger, 1979), and Agrostis slolonifera L. (Wu and
Antonovics, 1978). Krans et al. (1982) first investigated hormonal and environmental
requirements of callus induction from mature caryopses, callus maintenance, and plantlet
regeneration in creeping bentgrass cv. Penncross (Agrostis palustris Huds.). It was found
that prolific callus induction occurred on Murashige and Skoog (MS) medium with 1.0
mg/l 2,4—dichlorophenoxyacetic acid (2,4-D) under light incubation and with 1.0 or 10.0
mg/l 2,4—D under dark incubation.

Kinetin supplements were not required for callus induction. However, caryopses
incubated in the dark with 0.01 mg/l kinetin had greater callus formation than caryopses
incubated without kinetin when combined with 1.0 mg/l 2,4—D. They also indicated that
dark incubation provided better callus production from caryopses, and callus cultured on
1.0 mg/l 2,4-D maintained the best plantlet regeneration ability. It was also found that
callus induced from caryopses originated from the scutellum, root cortex, and along the
coleoptile up to and adjacent to the first node.

Blanche et al. (1986) evaluated the effect of suspension culture on callus growth
and plantlet formation and reported that callus growth was twofold greater in callus
previously cultured in suspension than callus not previously cultured in suspension.
However, plantlet formation was inhibited in callus previously cultured in suspension
(less than ten plantlets per dish) compared to callus not previously cultured in suspension

(250 to 450 plantlets per dish).

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80
Zhong et al. (1991) reported an efficient plant regeneration system via somatic

embryogenesis from mature seeds of creeping bentgrass cv. Penncross. They found that
in combination with 30 uM dicamba (3,6-dichloro-o-anisic acid), the presence of 2.25
pM 6-BA (6—benzyladenine) significantly enhanced the formation of embryogenic callus
and provided the best plant regeneration. Over 80% of these somatic embryos
germinated and formed normal plantlets on half -strength MS basal medium.

Terakawa et al. (1992) produced the first plant regenerates from protoplasts
isolated from embryogenic suspension cultures of creeping bentgrass. Protoplasts
isolated from the cultures were successfully grown on MS basal medium supplemented
with 0.1 mg/l 2,4oD. Although the plating efficiency of protoplasts was rather low
(0.36%), 30% of the protocalli formed normal green plants that were established in soil.

The first transgenic plants of creeping bentgrass were achieved by Zhong et al.
(1993) employing the microprojectile bombardment of embryogenic callus to transfer the
B-glucuronidase (GUS) reporter gene. Transgenic plants were obtained from bombarded
embryogenic calli without the application of any selective agent. The efficiencies for the
expression of GUS activity in the embryogenic calli one month after bombardment
ranged from zero to 28 %. However, of the 500 regenerated plants from their
experiments, only 15 were chosen for assay and four had integrated the reporter gene.

Employing a biolistic procedure, Hartman et al. (1994) transformed creeping
bentgrass with bar gene which confers resistance to the herbicide bialaphos. Suspension
cells were bombarded with gold particles coated with bar gene and transferred to the
selection medium containing two or four mg/ll of bialaphos. A total of 271 plants out of
approximately 900 regenerated plantlets survived the initial spray concentration (1.5
mg/ml) of the commercial herbicide Herbiace“. Of these, 55 survived the higher spray
rate (two mg/ml). The authors speculated that the large number of escapes observed
Could have been due to the interference of amino acids (asparagine and/or glutamine) in

the selection medium.

BIBLIOGRAPHY

Abdullah, R., Cooking, E. C., and Thompson, J. A. 1986. Efficient plant regeneration
from rice protoplasts through somatic embryogenesis. Bio/Technology 4: 1087-1090.

Ahloowalia, B. S. 1975. Regeneration of ryegrass plants in tissue culture. Crop Sci. 15:
449-452.

Akama, K., Puchta, H., and Hohn, B. 1995. Efficient Agrobacterium-mediated
transformation of Arabidopsis thaliana using the bar gene as selectable marker. Plant
Cell Rep. 14: 450-454.

Albrecht, H. R. 1947. Strain differences in tolerance to 2,4-D in creeping bent grasses. J.
Amer. Soc. Agron. 39: 163-165.

Amrhein, N. 1985. Specific inhibitors as probes into the biosynthesis and metabolism of
aromatic amino acids. Recent Adv. Phytochem. 20: 83-117.

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CHAPTER TWO

Engineering Herbicide Resistance in Creeping Bentgrass
(A grostis palustris Huds.) via Microprojectile Bombardment
and Expression of the bar Gene in Transgenic Plants

ABSTRACT

An efficient transformation system to generate large numbers of independent
transgenic creeping bentgrasses (Agrostis palustris Huds.) by microprojectile
bombardment is described. Embryogenic callus was bombarded with a plasmid
containing the bar gene and cultured on selection medium supplemented with either five
mg/l of bialaphos or fifteen mg/l of phosphinothricin (PPT) for twelve weeks. A total of
38 independent bialaphos- or PPT-resistant callus lines were generated and found able to
regenerate plants. Except for the plants regenerated from one of the PPT-resistant callus

lines, plants from the rest of the resistant callus lines were shown to be resistant to the

application of 1.2% Ignite®. All of the resistant callus lines and plants regenerated from
them expressed functional phosphinothricin acetyltransferase, the product of bar.
Integration of bar was confirmed by Southern hybridization analysis in the 37
independent resistant plant lines. Transcripts corresponding to the bar gene were present
in transgenic plants that showed herbicide resistance. Transgenic plants tested were
shown to have the ability to decrease the amount of ammonium accumulated after the
herbicide application. The magnitudes of herbicide resistance were between 20 and 40
times higher than that of untransformed control plants among tested transgenic creeping

bentgrasses.

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Introduction

Allotetraploid creeping bentgrass (Agrostis palustris Huds.; 2n=4x=28) is one of
the species of bentgrasses that have had the widest usage in the united states. It belongs
to the family of Gramineae and is a unique cool-season species exhibiting a vigorous
stoloniferous growth habit and is the primary cool-season grass used for golf course
putting greens, tees, and closely mowed fairways (Beard, 1982).

There exist some major problems, such as multiple applications of herbicides and
pesticides, associated with this important turf grass, both in the golf courses and in
residential areas. All of the presently available cultivars of creeping bentgrass are
susceptible to a wide range of fungal diseases such as brown patch (Rhizoctonia solani),
dollar spot (Sclerotinia homoeocarpa), and Pythium blight. Though most persistent
prostrate-growing broadleaf weeds occurring on established creeping bentgrass greens
and fairways can be controlled with the use of selective herbicides such as 2,4-D. Grassy
weeds, however, are difficult to control since they resemble creeping bentgrass in their
tolerance of herbicidal chemicals. For example, a consistently effective herbicide for
selective annual bluegrass control in bentgrass greens is lacking. Perennial strains of
annual bluegrass are even more difficult to control with selective herbicides than are
annual strains (Beard, 1982; Decker and Decker, 1988).

A number of herbicides exhibits good control of annual bluegrass; however, most
pose a high risk of injury to bentgrass. Though the application of synthetic herbicides
and other pesticides has become an indispensable tool of modern golf course
management in that they allow for economical control of weeds and pathogens, it is
consuming an increasing share of golf course management expenses, and, at the same

time, there are growing concerns about the effects of these chemicals on the environment.

114

1 15
Development of turf grass varieties which could provide consistent effective control of

weeds and pathogens is, therefore, a goal of turf grass breeding programs. The
availability of such varieties could improve weed control by providing flexibility in the
timing of applications and in the selection of efficient herbicides, by reducing the number
of treatments, and provide the protection against the infection of pathogens. However,
the breeding of creeping bentgrass has been limited by the lack of genetic research and its
allotetraploid nature.

Plant genetic engineering has undergone a revolution in the past ten years, and
advanced approaches in plant biotechnology are now beginning to be used to augment
traditional approaches to crop improvement. Development of efficient in vitro
regeneration systems and novel transformation technologies have opened the way for the
engineering of even the most recalcitrant of crops such as maize (Gordon-Kamm et al.,
1990), rice (Christou et al., 1991), wheat (Vasil et al., 1992), oats (Casas et al., 1993),
and barley (Wan and Lemaux, 1994). Genetic engineering has the potential to improve
creeping bentgrass, since it can selectively improve a single trait while still retaining all
of the desirable traits in the parental line, a capability not currently available through
traditional turf grass breeding.

The recent development of the biolistic transformation system has furnished us
with a useful breeding tool to bypass the obstacles impeding the improvement of creeping
bentgrass. The biolistic instrument is capable of accelerating sub-cellular sized
microprojectiles coated with foreign DNA over an array of velocities necessary to
optimally transform many different cell types. Of the several plant transformation
methods available, microprojectile bombardment has the advantage of working with a
wider range of cell and tissue types than any other method. Microprojectile
bombardment as a method to introduce foreign DNA into plant cells circumvents two

major constraints of monocot transformation: the lack of an available natural vector such

116
as Agrobacterium tumefaciens and the difficulty of regenerating fertile plants when

protoplasts are used for transformation.

Bialaphos (L -phosphinothricinyl- L alanyl- L alanine), which is being used as a
non-selective, broad-spectrum and postemergent herbicide, is a tripeptide composed of
two L-alanine residues and an analogue of glutamate known as phosphinothricin (PPT)
(Kondo et al., 1973; Ogawa et al., 1973). PPT was initially identified as the active
moiety of the herbicidal bialaphos produced by Streptomyces hygroscopicus (Kondo et
al., 1973). The active ingredient is highly stable as a chemical compound, but its
degradation is rapid in a microbiologically active environment such as soil or surface
water.

While PPT is a potent inhibitor of the enzyme glutamine synthetase in both plants
and bacteria, the intact tripeptide, bialaphos, has little or no inhibitory activity in vitro
(Bayer et al., 1972; Tachibana et al., 1986a, 1986b). In both plants and bacteria,
intracellular peptidases remove the alanine residues and release active PPT. In plants,
glutamine synthetase is critical for the assimilation of ammonia as well as for general
nitrogen metabolism. It is the major enzyme in plants that can detoxify ammonia
released by nitrate reduction, amino acid catabolism, and photorespiration. Plants treated
with glufosinate-ammonium (ammonium salt of PPT) quickly succumb to the toxic
effects of accumulated ammonia (T achibana et al., 1986a, 1986b).

Transgenic tobacco, potato, and tomato plants, expressing the bar gene from
Streptomyces hygroscopicus (Thompson et al., 1987) were tolerant to 4-10 times the
field rate of application of the herbicide (De Block et al., 1987). If the bar gene which
encodes PPT acetyltransferase (Thompson et al., 1987) could be introduced and
employed as a resistant gene in creeping bentgrass, bialaphos could serve as not only a
selective agent to facilitate the identification of putative transgenic plants in

transformation experiments but also as a selective herbicide on golf courses with

1 17
bialaphos-resistant creeping bentgrasses. The cultivation of environmentally-sound and

bialaphos-resistant turf grass represents significant economic and environmental
advantages for the management of golf courses by reducing the amount and frequency of
herbicide application, and by facilitating the weed control process.

In this paper, we describe the stable transformation and regeneration of creeping
bentgrass by microprojectile bombardment of embryogenic calli and the use of the bar
gene as a selectable marker for the efficient recovery of independent bialaphos-resistant
callus lines. We show that transgenic plants expressing PAT activity are resistant to high
doses of the commercial f ormulation of PPT. The use of the bar gene as reporter gene to

analyze plant gene expression is also demonstrated.

Materials and Methods

Plant Material

Creeping bentgrass (Agrostis palustris Huds.) cultivar "Penncross" which was a
synthetic cultivar produced by the random crossing of three vegetatively propagated
strains (Hein, 1958) was used as the plant material for the transformation experiment.
Friable embryogenic callus was initiated from mature seeds (caryopses) of creeping
bentgrass following the method described by Zhong et al. (1991). Seeds were first
soaked in 70% ethyl alcohol for 15 minutes and then surface-sterilized with 50%
commercial grade Clorox solution containing 0.1% Tween 20 for 15 minutes. Finally,
the seeds were rinsed with sterilized distilled water three times before being transferred
onto embryogenic callus induction medium which contained MS (Murashige and Skoog,
1962) basal medium supplemented with 500 mg/l enzymatic casein hydrolysate, 3 %
sucrose, 30 FM dicamba (3, 6-dicloro-o-anisic acid), 9 ”M BA (6-benzyladenine), and 7
g/l Phytagar.

Cultures were maintained in the dark at 25 °C and subcultured onto fresh-made
callus induction medium once per month. Embryogenic callus was observed after
approximately one month of culture and selected for multiplication. However,
embryogenic calli were used as soon and as fresh as possible and discarded after they had
been initiated from seeds for more than four months. Fresh calli were routinely initiated

every month to maintain the supply of embryogenic callus.

Evaluation of Selective Agents
In order to evaluate the selective agents and determine the appropriate

concentration of the corresponding selective agent needed to distinguish the cells

118

119
transformed with the bar gene from the nontransformed cells, kill curve tests were

performed using various concentrations of bialaphos (kindly provided by Meiji Seika
Kaisha of Japan) and phosphinothricin (D,L-PPT, glufosinate-ammonium; Riedel-

deHaén) (Table 1 and Table 2). Callus initiated from every single seed was maintained

separately, since each seed of the cultivar Penncross potentially represented a different
genotype. Embryogenic calli were first bombarded with tungsten particles without any
plasmid DNA and transferred onto embryogenic callus induction medium.

Three days after the bombardment, bombarded calli were subcultured onto
selection medium (callus induction medium without casein hydrolysate) supplemented
with various concentrations of bialaphos or phosphinothricin. Bialaphos and
phosphinothricin were first dissolved in double distilled water and filter-sterilized
(Nal gene filter) before added into the autoclaved medium. Terr small pieces of callus
(less than three mm in diameter and approximately five mg in weight) were evenly placed
on each Petri dish which contained 35 ml of selection medium. Fifteen dishes which had
a total of 150 pieces of callus were used for every selection concentration. Calli were
transferred onto f resh-made selection medium every four weeks.

Three durations (four, eight, and twelve weeks) of selection were tested to
determine the appropriate length of selection duration. At the end of each selection
duration, calli were carefully broken with forceps and separately transferred onto Petri
dishes containing 35 ml of callus induction medium (pH of the medium was adjusted to
6.0 and containing same amount of corresponding selective agent) with 50 mg/l of
chlorophenol red for four days. The pH indicator chlorophenol red changes from red
through orange to yellow as the pH value decreases from 6.0 to 5.0. When subcultured
onto chlorophenol red medium adjusted to a pH of 6.0, growing maize callus which
survived the PPT selection acidified the surrounding medium, reduced the pH value, and
caused the change in color (Kramer et al., 1993). Calli which caused the color change of

120
the medium from deep red to yellow, orange, or red-orange were transferred to callus

induction medium and cultured for one more month. Calli which continued to multiply
were considered alive, and the number of growing calli was recorded. Culture conditions

were all the same as those of callus induction.

Plasmid Vector

The plasmid used for the biolistic gun transformation method was pTW-a and was
kindly provided by Dr. Ray Wu of Cornell University. The schematic map of this plasmid
is shown in Figure 1. The plasmid pTW-a contains the pin 2 gene downstream of the pin
2 promoter and the rice Act 1 intron, with the pin 2 terminator. In addition, the plasmid
contains the bar gene downstream of the CaMV35S promoter and is flanked by the

nopaline synthase 3' end (nos terminator).

Microprojectile Bombardment

One day prior to the microprojectile bombardment, embryogenic calli were placed

onto a 2 cm2 area in the center of a 15 x 60 mm disposable Petri dish containing 35 ml of
embryogenic callus induction medium. Prior to bombardment, plasmid DNA was
precipitated onto tungsten particles modified from the protocol described by Klein et al.
(1987). Thirty mg of the dry microcarriers (tungsten M17, average diameter
approximately 1.2 pm) were soaked in 1 ml of 100% ethanol in a microcentrifuge tube.
The tungsten particles were then vortexed on high speed for two minutes before they
were centrifuged at 15,000 rpm for one minute. After the supernatant was decanted, one
ml of sterile distilled water was added into the microcentrifuge tube before the tungsten
particles were resuspended and centrifuged.

The deagglomeration process was repeated two times before the microcarriers

were resuspended in one ml of sterile distilled water. 100 pl of the final microcarrier

121
suspension (three mg of the tungsten particles) was transferred into a microcentrifuge

tube and vortexed continuously while pipetting aliquots of the suspension to avoid non-
uniform sampling.

25 yg of plasmid DNA, 100 pl of 2.5 M CaClz and 40 141 of 0.1 M spermidine
(free base, tissue culture grade, Sigma) were added into a microcentrifuge tube in the
above order, while the microcentrifuge tube was continuously being vortexed. The DNA
precipitation mixture was continuously vortexed for ten minutes before the microcaniers
were spinned down and the supernatant was removed from the microcentrifuge tube. The
DNA precipitation mixture was washed with 250 ul of 100% ethanol by brief vortexing
and then centrifuged at 15,000 rpm for ten seconds. Supernatant was removed and the
mixture was resuspended in 100 yl of 100% ethanol.

10 pl aliquots of suspension were pipetted onto the center of the macroprojectile
for bombardment. After the alcohol was evaporated, each Petri dish with embryogenic
calli of creeping bentgrass was bombarded three times with the DNA-tungsten mixture
using the Biolistic PDS-lOOO/He instrument (Bio-Rad Laboratories, Inc.). Following the
same protocol, control plates with embryogenic calli were also bombarded with the same
tungsten mixture except there was no plasmid DNA.

Physical parameters were optimized to increase numbers of gus transient
expressing cells per plate. The following conditions were found to be superior and were
used as a standard bombardment protocol: rupture disc-pressure: 1550 psi; gap-distance
from rupture disc to macrocarrier: 6 mm; macrocarrier travel distance: 16 mm;

microcarrier travel distance: 6 cm.

Selection of Resistant Callus Lines and Plant Regeneration
After the bombardment, calli were separated and transferred onto embryogenic

callus induction medium for three days before being exposed to the selective agent.

122
Three days after the bombardment, calli were subcultured onto selection medium which

contained either five mg/l of bialaphos or 15 mg/l of phosphinothricin (ammonium salt)
and the same components of embryogenic callus induction medium, except without the
enzymatic casein hydrolysate to avoid the possible interference of glutamine or
asparagine with selective agent (Wang etal., 1992; Hartman etal., 1994).

The bombarded calli were transferred to f resh-made selection medium every four

weeks for twelve weeks, and all cultures were maintained in the dark at 25 °C. During
the process of transferring to the second selection plate, individual callus pieces were
broken with forceps into several small pieces and maintained separately. After about
three months of selection, resistant and viable calli showing evidence of more vigorous
growth were transferred onto the same selection medium and maintained under the light
for differentiation for two weeks before being transferred onto MS medium containing
the same concentration of selective agent (bialaphos or PPT) for regeneration.

All resistant callus tissue developed originally from each piece of callus was
defined as a line. Regenerated shoots were maintained and propagated on the MS
medium with the same amount of corresponding selective agent for one to two months
before being transferred to four-inch plastic pots containing peat and perlite growth

medium mixture (Baccto professional planting mix) and placed in the greenhouse.

Herbicide Application

Putative transgenic creeping bentgrass plants regenerated from resistant callus
lines were sprayed with 1.2 % I gnite®, which contained 200 g/l of active ingredient of
glufosinate-ammonium (ammonium salt of PPT), in a spray volume of 150 l/ha.

Symptoms were evaluated 10 days after the herbicide application. Various rates of

Ignite®, ranging from zero to 120 g/l of glufosinate-ammonium, in the same spray

volume were then applied to three of the resistant lines, which had showed no damage

123
after the application of 1.2% I gnite®, to determine the magnitude of their herbicide

resistance. LD50 (lethal concentration that caused 50% of plant damage) was used to

represent the magnitude of herbicide resistance. There were ten replications for each

application rate.

DNA Isolation and Southern hybridization Analysis

The method of isolating genomic DNA from resistant and control turf grass plants
was modified from the CTAB method of Rogers and Bendich (1985). 10 grams of fresh
turf grass tissue was ground with mortar and pestle in liquid nitrogen and transferred into
an oakridge tube before 30 ml of CTAB extraction buffer [0.1 M Tris-HCI, 1.4 M NaCl,
0.02 M EDTA, 2% CTAB (hexadecyltrimethylammonium bromide), 1% 2-

mercaptoethanol] was added. The tube was incubated in a waterbath at 60 °C for one
hour with occasional mixing by gentle swirling.
30 ml of phenol/chloroforrn/isoamyl alcohol solution (25:24: 1, v/v/v) was added

into the oakridge tube after the extraction buffer was cooled down to room temperature.

The tube was then centrifuged at 10,000 rpm for 10 minutes at 10 °C. The top layer of
the extraction buffer was pipetted into another oakridge tube, and an equal volume of
phenol/chloroform/isoamyl alcohol solution was added into the tube.

The extraction procedure was repeated once before the plant DNA was
precipitated with an equal volume of ice cold isopropanol. The precipitated DNA was
spinned down at 10,000 rpm for 10 minutes and washed with 100 % ethanol after the
supernatant was poured off. The DNA pellet was dried and redissolved in 10 ml of TE
buffer (10 mM Tris, 1 mM EDTA). The sample was then extracted three times with
chloroform/isoamyl alcohol solution (24:1). The pellet was washed with 70% ethanol

twice and dried after the DNA was precipitated with ice cold isopropanol. One mg/ml of

124
RNase A was added into the DNA solution after the pellet was dissolved in the

appropriate amount of TE buffer.

To detect the integration of bar gene into the genome of turf grass, 10 fig of the
purified plant DNA was digested with EcoRI, which should release a 0.9 kb fragment
containing the bar coding region if the integration were successful, and with EcoRV,
which has a unique digestion site in the pTW-a. Undigested and digested DNA were
electrophoresed in a 0.8% agarose gel and blotted onto a Nytran membrane, following the
Product Guide & Methods of the Schleicher & Schuell, Inc.

The plasmid pTW-a was used for the isolation of the 0.6 kb bar fragment by

digestion with Smal. The fragments were labeled with 32P using the T7QuickPrime® Kit
(Pharrnacia Biotech) to generate probes for the detection of sequences containing the bar
gene. The Nytran membrane was placed in a polyethylene bag containing
prehybridization buffer (6x SSPE, 10 x Denhardt's solution, 1.0% SDS, 200 yg/ml
fragmented, denatured DNA, 50% formamide pH 7.4, and 10% dextran sulfate 500) for
two hours at 42 0C.

After the prehybridization buffer was poured off, the membrane was incubated
with the hybridization buffer [6x SSPE, 10 x Denhardt's solution, 1.0% SDS, 50%
formamide pH 7.4, and 10% dextran sulfate 500, and 32P-labeled probe (100 )4] per cm2

of membrane)] under continuous agitation overnight at 42 °C. The membrane was then

washed in 150 ml of the following solutions: (1) twice in 7 x SSPE/0.5% SDS for 15
minutes at room temperature. (2) twice in 1 x SSPE/1.0% SDS for 15 minutes at 37 °C.

(3) once in 0.1 x SSPFJ1.0% SDS for 30 minutes at 42 °C.

RNA Isolation and Northern Hybridization Analysis
Total RNA was isolated from the control and the transformed creeping

bentgrasses by the method of Wadsworth et al . (1988). 200 mg of leaf tissue samples

125
were ground to a fine powder in liquid nitrogen with a small mortar and pestle. The

samples were transferred to 1.5 ml microfuge tubes; 500 pl of extraction buffer (25 mM
sodium citrate, pH 7.0, 4 M guanidinium isothiocyanate, 1.5% (w/v) sodium lauryl
sarcosine, 100 mM 2-mercaptoethanol) was added; and the samples were vortexed for 15
seconds. Then 250 pl of phenol and 250 pl of chloroform were added; the samples were
vortexed for 15 seconds and centrifuged at 12,000 rpm for 15 minutes; and 500 pl of
aqueous phase was transferred to a new tube. The samples were extracted twice more
with phenol/chloroform, and, after the final extraction, only 400 pl of the aqueous phase
was transferred to a new microfuge tube. Next, 400 pl of 6 M lithium chloride was

added, and the samples were incubated on ice for one hour.

Following a centrifugation ten minutes at 4 °C, the supernatant was discarded.
The pellets were disrupted with a thin glass rod; 1 ml of 3 M lithium chloride was added;
and the samples were vortexed until the pellet was evenly suspended. The samples were
centrifuged as before, and the supematants were discarded. The pellets were washed

twice in this manner, then resuspended in 400 pl of 2% potassium acetate, and heated to

55 °C for 10 minutes to dissolve the RNA. The samples were then centrifuged for 5
minutes to remove insoluble matter, and the supernatant was transferred to a new
microfuge tube.

The RNA was precipitated by the addition of 1 ml of ethanol and incubated for 15
minutes at -80 °C. The RNA was collected by centrifugation for 15 minutes at 4 oC,

dried under a vacuum, and dissolved in 50 pl of water for 10 minutes at 55 °C. For
northern blot hybridization, total RNA (5 pg) was electrophoretically fractionated through
a 1.2% agarose gel containing formaldehyde (Sambrook et al., 1989) and transferred onto

Nytran membranes which were probed with the bar coding region described above.

PAT Activity Assay

126
Phosphinothricin acetyltransferase (PAT) activity was analyzed using a procedure

modified from Spencer et. al. (1990) and D'Halluin et al. (1992). 200 mg of plant tissue
or callus was ground in Eppendorf tubes, on ice, with 100 pl extraction buffer [50 mM
Tris-HCI, pH 7.5, 2 mM NazEDTA, 0.15 mg/ml phenylmethylsulfonyl fluoride (PMSF),
0.15 mg/ml leupeptin (Acetyl-Leu-Leu-Arg-al), 0.3 mg/ml bovine serum albumin (BSA),
and 0.3 m g/ml ditlriothreitol (DTT)] to which 5 mg of polyvinyl polypyrrolidone (PVPP)
was added. The Eppendorf tube was centrifuged for ten minutes at 4 °C at 14, 000 rpm to
yield a crude extract.

The total protein concentration in the crude extract was measured by employing
the Bio-Rad assay with bovine serum albumin as the standard and was adjusted with the

extraction buffer to reach a final protein concentration of one mg/ml. To 20 mg total

protein, 3 pl 14C-acetyl coenzyme A (48.1 mCi/mmol, NEN), and 1 pl of a 3 mM
phosphinothricin (PPI‘) stock were added.

Reaction mixture was incubated for one hour at 37 °C before 7 pl of reaction
mixture was spotted onto a silica gel thin-layer chromatography (TLC) plate. The plate

was then chromatographed in a 3:2 mixture of l-propanol and NH40H (25% NH3, v/v),

for six hours under room temperature. The 1“C-labeled acetylated PPT was visualized by
autoradiography (XAR-S—Kodakfilm) after a two-day exposure.

Detection of Ammonia in Plant Extracts
Six-month-old nontransgenic control creeping bentgrasses as well as three

transgenic plant lines used to test their magnitudes of herbicide resistance were treated

with 1.2% I gnite®, as described in a previous section on ‘Herbicide Application’. Two
hours after the herbicide spraying, 250 mg young leaf material was extracted in one m1 of

water containing 50 mg polyvinyl polypyrrolidone (PVPP; Sigma). Insoluble material

127
was pelletted by centrifuging for five minutes in an Eppendorf centrifuge. 200 pl of the

supernatant was transferred to a new Eppendorf tube and diluted with 800 pl water.

The ammonia content was determined by the method of De Block et al. (1989)
and D'Halluin et al. (1992). 1.5 ml of reagent A (5 g phenol and 25 mg sodium
nitroprusside in 500 ml water) was added to 20 pl of the diluted plant extract, followed by
1.5 ml of reagent B (2.5 g sodium hydroxide, 1.6 ml of sodium hypochlorite with 13%
available chlorine, to 500 ml of water). The reaction mixture was incubated for 15

minutes at 37 °C. The absorbance was measured at room temperature at 625 nm.

The concentration of ammonia (NH4+-N) was determined on a standard curve (pg
NH4+-N/ g fresh weight = pg determined NH4+-N x 450). The standard curve was made
using Nl-I4Cl in concentrations ranging from 0 to 100 pg NH4+-N (3.82 g NH4Cl = l g

NI-I4+-N), with 20 pl leaf extract from an untreated control plant added to the reaction

mixtures. The measurement of ammonium concentration was taken at several times after
the herbicide spraying (as indicated in Table 4). There were five replications in each

measurement.

128

 

 

 

 

 

 

SmaI
Barnl-II ECORI
HindIII BamlIl I-lirrdlll EcoRV Smal EcoRI
—J-[ pin2-5' >1 acu>lfi2in2 (31> pin2-3' >JF 35S-5'>lrbar>Jjnos-J3>L— pUC19
1.5 kb 1.5 kb 0.8 kb 0.6 kb 0.3 kb

Figure 1 . Schematic representation of the plasmid pTW-a.

Results

Evaluation of Selective Agents

Embryogenic calli of creeping bentgrass cultivar Penncross were bombarded with
tungsten particles which had no plasmid DNA and exposed to various concentrations of
selective agents for three selection durations to evaluate the efficiency of bialaphos and
phosphinothricin in the killing of nontransgenic calli. Before being transferred onto the
selection medium, embryogenic calli were first transferred onto callus induction medium
and given three days after they were bombarded to recover from the damage caused by
the biolistic bombardment.

At the end of each selection duration, calli were separately transferred onto Petri
dishes containing callus induction medium (with the same amount of corresponding
selective agent) with 50 mg/l of chlorophenol red for four days. Calli which caused the
color change of the medium were transferred to callus induction medium. Calli which
continued to multiply and grow were considered alive, and the number of surviving calli
was recorded one month after they had been on callus induction medium.

Calli of creeping bentgrass were not very sensitive to either bialaphos or
phosphinothricin (PPT) after they had been exposed to the selection medium for four
weeks (Table 1, Table 2, and Figure 2). The concentration of bialaphos (17.5 mg/l) and
of PPT (25 mg/l) to achieve 95% of selection efficiency were relatively high after the
first selection duration. Both bialaphos and PPT were not very effective in the killing of
embryogenic callus, especially at lower concentrations. However, the number of growing
calli which survived the selection decreased as the duration of selection increased for
both selective agents. For instance, after four weeks of selection on 15 mg/l of PPT,
there were still 32 growing calli on the selection medium. The selection efficiency was

less than 80% at this concentration. However, as the selection time increased to eight

129

130
weeks, the number of growing calli dropped to eight. After twelve weeks of selection on

15 mg/l of PPI‘, there were only two calli surviving at this concentration of PPT. More
than 95% of the tested calli were killed. This phenomenon was also observed with the
other concentrations for both bialaphos and PPT.

Bialaphos was found to be a more effective selective agent in killing control calli
of creeping bentgrass. Not only was the necessary concentration of bialaphos to kill the
same percentage of calli lower than that of PPT, but also the duration needed for
bialaphos to achieve the same efficiency was less than that of PPT. After four weeks of
selection, bialaphos was able to kill more than 95% of the tested calli at the concentration
of 17.5 mg/l (54.0 pM). However, the concentration needed for PPT to obtain the same
efficiency after the same duration of selection was 25 mg/l (126.3 pM). The fact that
bialaphos was a better selective agent than PPT was even more significant when the
duration of selection increased from four weeks to twelve weeks. There were only seven
growing calli on bialaphos selection medium at the concentration of 5 mg/l (15.4 pM);
however, there were still nine calli surviving the PPT selection at the concentration of
12.5 mg/l (63.1 pM).

Both bialaphos and PPT were employed as selective agents to distinguish calli
transformed with the bar gene from those nontransformed calli. The concentration of
bialaphos and of PPT used in the selection medium was 5 and 15 mg/l, respectively,
which could achieve 95% of selection efficiency after three transfers on the selection

medium and twelve weeks of selection.

Selection of Transformed Callus Lines
To test whether the expression of the phosphinothricin acetyltransferase (PAT)
encoded by the bialaphos resistant gene (bar) from Streptomyces hygroscopicus allowed

for the selection of transformed plant cells, embryogenic calli of creeping bentgrass cv.

131

Table 1. Effects of the duration of selection and the concentration of phosphinothricin on
the callus growth of creeping bentgrass

 

Number of callus growing on callus induction medium after each duration of selection on
callus induction medium containing various concentrations of phosphinothricin*

Concentration of phosphinothricin (mg/l)

 

Weeks of selection” 0 2.5 5 7.5 1012.5 1517.5 20 25 30 35 40

 

4 150 88 64 62 51 41 32 19 14 6 4 1 0
8 150 67 43 30 23 14 8 3 2 O O O 0
12 150 48 37 22 14 9 2 O O O 0 O O

 

‘There were fifteen Petri dishes in every concentration, and each Petri dish contained ten
small pieces of callus. At the end of each selection duration, calli were carefully broken
into small pieces and separately transferred onto Petri dishes containing 35 ml of callus
induction medium (with the same amount of PPT) with 50 mg/l of chlorophenol red for
four days. Calli which caused the color change of the medium from deep red to yellow,
orange, or red-orange were transferred to callus induction medium and cultured for one
month. Calli which continued to multiply on callus induction medium were considered
alive, and the number of growing calli was recorded.

#Calli were transferred to fresh-made selection medium every four weeks.

132

Table 2. Effects of the duration of selection and the concentration of bialaphos on the
callus growth of creeping bentgrass

 

Number of callus growing on callus induction medium after each duration of selection on
callus induction medium containing various concentrations of bialaphos’“

Concentration of bialaphos (mg/l)

 

Weeksofselection# 0 2.5 5 7.5 1012.5 1517.5 2025 30 35 4o

 

4 150 48 36 3O 12 11 8 6 4 3 O 0 O
8 150 29 17 14 ll 7 4 0 0 O O 0 O
12 150 21 7 3 1 0 O O 0 0 0 0 0

 

x"There were fifteen Petri dishes in every concentration, and each Petri dish contained ten
small pieces of callus. At the end of each selection duration, calli were carefully broken
into small pieces and separately transferred onto Petri dishes containing 35 ml of callus
induction medium (with the same amount of bialaphos) with 50 mg/l of chlorophenol red
(CR) for four days. Calli which caused the color change of the medium from deep red to
yellow, orange, or red-orange were transferred to callus induction medium and cultured
for one month. Calli which continued to multiply on callus induction medium were
considered alive, and the number of growing calli was recorded.

#Calli were transferred to f resh-made selection medium every four weeks.

133

 
   
   

+Four weeks on Bialaphos
—-B--Four weeks on WP
+Eight weeks on Bialaphos
‘94—Eight weeks on PPT
+«Twelve weeks on Bialaphos
—e—Twelve weeks on PPI‘

  

number of calli alive

 

concentration (mg/l)

Figure 2. Callus responses of creeping bentgrasses to various concentrations of
bialaphos and phosphinothricin and to three durations of selection.

134
Penncross were bombarded with tungsten particles coated with the bar-containing

plasmid pTW-a. Calli were transferred onto callus induction medium and given three
days after the bombardment to allow them to multiply and recover from the damage
caused by the bombardment of tungsten particles before they were exposed to selection
medium containing either 5 mg/l of bialaphos or 15 mg/l of PPT.

Calli that did not turn brown or black and survived the selection were transferred
onto fresh-made selection medium every four weeks. After twelve weeks of selection
and three transfers onto fresh-made selection medium, no growth was observed on calli
from the control plates. Thus, the low dose of 5 mg/l of bialaphos and 15 mg/l of PPT
were both sufficient to inhibit the growth of sensitive callus.

Resistant callus lines appeared four to six weeks after the bombardment, as they
could be easily distinguished from those non-growing or dead calli. In order to avoid the
possible problems of chimerism and escape, those resistant calli were still maintained on
selection medium and transferred every four weeks for a total of twelve weeks. During
the process of transfer, resistant calli were carefully broken into small pieces with forceps
and evenly distributed on selection medium.

Results from transformation experiments are shown in Table 3. The number of
callus lines surviving the selection decreased as the duration of selection increased for
both selective agents. A large percentage of the bombarded calli were killed during the
first selection period on 5 mg/l of bialaphos (four weeks of selection). However, it took a
little longer for PPT (15 mg/l) to distinguish resistant callus lines from sensitive ones.
After four weeks on the selection medium containing bialaphos or PPT, there were 170
and 163 resistant callus lines, respectively.

It was not until the end of the second selection duration that the resistant callus
lines could be easily discriminated from the sensitive lines for both selective agents.
There were still 84 callus lines showing resistance to bialaphos and 92 to PPT at the end

of the second selection duration.

 

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136
However, the number of resistant callus lines dropped dramatically during the last

selection duration. Twenty-two and sixteen bialaphos and PPT-resistant callus lines,
respectively, were recovered from 250 plates (125 plates for each selective agent) of
bombarded calli after about three months of selection on either bialaphos or PPI‘.
Resistant callus lines from both selection schemes were able to multiply and grow
vigorously on selection medium during the last two weeks of selection.

Though it was difficult to keep track of each bombarded callus lines, there were
two reasons to suspect that most of these resistant callus lines probably arose from
independent integration events. Firstly, bombarded calli were separated before being
transferred onto selection medium, and resistant calli were collected and maintained as
individual cell lines. Secondly, very few callus pieces from the same plate showed

resistance to either selective agent after three months of selection.

Plant Regeneration

After three months of selection in the dark, embryogenic resistant callus lines
were placed under the light for two weeks before they were transferred onto regeneration
medium (MS medium) containing the same level of bialaphos or PPT and grown under
the light. A high proportion of calli from each resistant line remained viable, and shoot
regeneration was observed one to two weeks after they were transferred to the
regeneration medium. All of the resistant callus lines from both selection schemes were
able to form shoots and roots without the addition of any growth regulator (T able 3).

Most of the creeping bentgrass plants regenerated from resistant callus lines were
morphologically similar to seed-derived plants, and their growth was indistinguishable
from nontransformed control plants (either seed-derived or regenerated from calli
bombarded without plasmid DNA of pTW-a). The total period of time from initiation of

the cultures to acclimation of the plants in the greenhouse was about six months.

137
Herbicide Resistance of Transgenic Creeping Bentgrasses

To evaluate whether the level of PAT activity in putative transgenic creeping

bentgrasses was sufficient to confer resistance to the herbicide Ignite®, which contained
200 g/l of glufosinate-ammonium, transgenic and nontransformed control plants were
first sprayed with 1.2 % Ignite (2.4 g/l glufosinate-ammonium) in a spray volume of 150
l/ha. Ten days after the herbicide application, the results showed that this application rate
was able to kill all of the control plants (Figure 3).

There was almost no plant damage observed on most of the transgenic plants at
that screening rate. A few of them did show some damage; however, they were all able
to recover from the damage by two weeks after spraying. Plants regenerated from one of
the PPT-resistant callus lines were all killed by the herbicide at the application rate of 2.4
g/l glufosinate-ammonium.

Plants of three resistant lines showing no damage after the screening application

of herbicide were multiplied and tested for their tolerance to various concentrations of

Ignite® to evaluate the magnitude of herbicide resistance. The level of PAT activity for
transgenic plants regenerated from three independent resistant callus lines appeared to be
different, as reflected by the extent of plant damage caused by various rates of herbicide

application and their values of LD50 (Figure 4).
The LD50 values of tested transgenic plants were about 20 to 40 times higher than
that of nontransgenic plants (Figure 4). Among tested plants, transgenic plants from

transformation event A (resistant callus line A) showed the highest level of herbicide

resistance and transgenic B plants the lowest. These three clones were also tested for

their concentrations of ammonia after they had been sprayed with 1.2% I gnite® to
determine whether there was a correlation between their magnitudes of herbicide

resistance and the abilities to detoxify the herbicide.

138

 

Figure 3. Effects of herbicide application on transgenic plants (on the right) and
untransformed control plants (on the left) at an application rate of 1.2 ’70 lgnitc® (2.4 g/l of
the active ingredient glul'osimite-ammonium). Plants were photographed two weeks after
the herbicide spraying.

139

  
      
 
   

 

 

a) 100

.3 90 +Control

Lé ‘ +Transgenic A3

o 80 .

.c: 70 +Transgenrc B

H .

g m 60 I +TransgenicC
c:

g? 50 > LD 50

.2 Q. 40 __

t I'D

a 30 -~

5. f3 "

Q, -.

5° 0 \:-___ __l

 

 

O

20 4O 6O 80 100 120

concentration of glufosinate-ammonium (g/l)

Figure 4. The magnitudes of herbicide resistance of transgenic creeping bentgrasses
regenerated from three independent resistant callus lines.

a: A, B, and C indicate three independent transgenic lines.

140
Analysis of PAT Activity

Evidence for the expression of the bar gene was obtained by the analysis for the
phosphinothricin acetyltransferase (PAT) activity by thin layer chromatography (TLC).
Resistant callus lines, as well as transgenic plants regenerated from them which had
shown resistance to the herbicide application were tested for their PAT activity. The
assays were performed using a mixture of D- and L-phosphinothricin, in which L-PP'I‘ is
the active moiety of bialaphos and is generated in plant cells through cleavage of
bialaphos by intracellular peptidases. L-PPT, a potent inhibitor of glutamine synthetase
in plants and bacteria (De Block et al., 1987), is acetylated by the bar gene product in the
presence of acetyl-CoA to yield an inactive product, N-acetyl-PPT.

Protein extracts from independent resistant callus lines and young leaves of

regenerated transgenic plants from each resistant callus line all showed PAT activity.

The expression of the bar gene was observed in the enzymatic assay in which l4C-
labeled acetylated PPT was detected after separation by TLC (Figure 5). Minor
differences were found in the levels of PAT activity between independent resistant lines
of callus and plant, and there was a very low background activity detected in the controls.
The resistant callus lines were maintained on the corresponding selection medium and

repeatedly tested positive for PAT activity during a six-month period (data not shown).

Southern Hybridization Analysis
Southern hybridization analyses were performed on control plants as well as

transgenic creeping bentgrasses obtained by bialaphos or phosphinothricin selection for

the presence of the introduced bar gene. A 32P-labeled purified DNA fragment
containing the coding sequence of the bar gene from pTW-a was used as the probe.

Undigested genomic DNA from transgenic plants was hybridized with the bar probe, and

141

 

Figure 5. Detection of phosphinothricin acetyltransferase (PAT) activity by thin layer
chromatography. Lane A: untransformed control plant; Lane B through G: transgenic
creeping bentgrasses regenerated from independent resistant callus lines. Position of the
N -acetyl-PPT is indicated by the arrow.

 

 

 

 

Figure 6. Top: Southern hybridization analysis of transgenic creeping bentgrass plants
from independent resistantcallus lines. Lane A, B, C, and D indicate different transgenic
lines and lane N indicates nontransgenic control plant. Lane 1: undigested genomic DNA;
Lane 2: EcoRI digested genomic DNA; Lane 3: EcoRV digested genomic DNA.

Bottom: Southern hybridization analysis of transgenic creeping bentgrasses from same
transgenic resistant callus line A. P: positive plasmid control; Lane Al through A4 indicate
transgenic plants from same transgenic resistant callus line A. Lane 1: undigested genomic
DNA; Lane 2: EcoRl digested genomic DNA; Lane 3: EcoRV digested genomic DNA. The
fragment of bw coding region is indicated by the arrow.

143
hybridization was observed only in the region of high molecular weight DNA, providing

evidence that the bar gene was integrated into genomic DNA, as exemplified in Figure 6.

Genomic DNA was digested with EcoRI, which releases a 0.9-kb fragment from
pTW-a that contains the coding sequence of the bar gene (0.6 kb) and the nos 3’ end (0.3
kb), and with EcoRV, which has a unique digestion site in the pTW-a. Genomic DNA
from resistant plants was shown to contain bar-hybridizing sequences. The variation in
the banding pattern and copy numbers was exemplified in Figure 6. Hybridization
patterns varied from line to line. All transgenic lines contained an expected 0.9-kb
fragment, although most also contained extra hybridization fragments of different sizes
that could represent rearranged copies of the bar expression unit or be the result of partial
digestion of the genomic DNA.

Furhtermore, the hybridization banding pattern of genomic DNA digested with
EcoRV suggested that there were multiple integrations in some of the transgenic lines.
The hybridization patterns of transgenic lines were unique, consistent with the
expectation that all these lines were from independent transformation events. However,
the hybridization patterns could not be used to determine whether a resistant callus line
arose from more than one independently transformed cell. There was no apparent
correlation between the copy number of the intact or rearranged bar genes and the level
of expression (PAT activity and herbicide resistance) in transgenic plants.

Southern analysis with the bar gene as the probe was also performed on genomic
DNA isolated from plants regenerated from the same resistant callus line, since the
polymorphisms of the multiple restriction fragments hybridizing to the bar gene can
provide us with more accurate information to determine whether they regenerated from
the same transformation event. The banding patterns were identical and suggested that

analyzed transgenic plants were regenerated from the same unique transformation event.

144
Northern Hybridization Analysis

Northern analysis was performed to determine if bar transcripts accumulated in
transgenic plants. Total RNA was isolated from young leaves of untransformed creeping
bentgrasses and of transgenic plants showing resistance to herbicide application, then
separated on a formaldehyde agarose gel, and probed with the Smal fragment
corresponding to the bar coding sequence (Figure l). PAT-specific mRNA was detected
in the analysis of total RNA, as shown in Figure 7.

No signal was detected in the untransformed control plants, while resistant
transgenic plants contained expected size transcripts of around 600 bp. Although all of
the analyzed transgenic plants which regenerated from independent transformation events
contained the same bar gene, they showed some variation in the accumulation of PAT-

specific mRNA, as shown by their different densities on the autoradiogram (Figure 7).

Determination of Ammonium Levels in Herbicide-treated Plants
For a more sensitive indicator of glutamine synthetase inhibition, we measured

the accumulation of ammonium in transgenic and untransformed plants treated with 1.2%

of Ignite®. in order to determine whether there was a correlation between their
magnitudes of herbicide resistance and the ability to detoxify glufosinate-ammonium,
transgenic plants tested for their concentrations of ammonia after the herbicide spraying
were from the same transgenic lines which had been tested for their magnitudes of
herbicide resistance.

Ammonia accumulated rapidly in treated control plants and increased 30-fold
only two hours after the herbicide spraying (Table 4 and Figure 8). The concentration of
ammonium in treated control plants continued to increased sharply during the two-day

testing period. Two days after the herbicide application, the ammonium level reached

145

4,380 pg NH4+-N per gram of fresh leaf sampl,e and there was substantial necrosis

observed on control plants.

The levels of ammonium in the tested transgenic plants did increase after the
herbicide treatment. The levels of ammonium increased 10, 15, and 11 times for
transgenic plants from A, B, and C transformation events, respectively, two hours after
the spraying of the herbicide. However, transgenic plants were able to detoxify the
glufosinate-ammonium and decrease the amount of ammonium, as shown in Table 4.

Transgenic plants from independent transformation lines showed differential
responses to the herbicide application and different ability to detoxify the herbicide. Four
hours after the herbicide application, transgenic A plants were able to start decreasing the
amount of ammonium accumulated after the spraying (Table 4). Though it was a little
slower, transgenic C plants showed the same ability to decrease the amount of
ammonium as transgenic A plants. However, the ability of transgenic B plants to
detoxify the herbicide was not as good as those of transgenic A or C plants. The level of
ammonium in transgenic B plants continued to rise for six hours after the spraying and
began to decrease 12 hours after the herbicide application.

The abilities for independent transgenic plants to detoxify glufosinate-ammonium
corresponded to their magnitudes of herbicide resistance (compare Figure 4 and Table 4).
Transgenic A plants had the highest level of herbicide resistance and were able to
decrease the accumulation of ammonium in a more efficient way. Transgenic B plants,
which showed the lowest level of herbicide resistance, were found to be the fastest in the
accumulation and the slowest in the decreasing of ammonium among the three tested

independent transgenic lines.

 

Figure 7. Northern hybridization analysis of the bar transcript in transgenic creeping
bentgrasses. Top: Lane P: positive control; Lane N: untransformed control plants; Lanes
A through F: independent transgenic plants. Bottom: mRNA isolated from additional
independent transgenic plants. Position of the 600 bp bar transcript is indicated by the
arrow.

147

Table 4. Ammonium concentration in transgenic and untransformed creeping bentgrasses
after the application of 2.4 g/l glufosinate-ammonium

 

Concentration of ammonium (pg NH4+-N / g f resh weight).

 

 

 

 

Control plants Treated transgenic plantsA
Hours after
application Untreated* Treated A B C
O# 13 16 18 16 20
2 14 450 180 250 225
4 13 720 155 280 180
6 12 855 80 295 90
12 15 1350 55 230 68
18 14 21 15 43 168 45
24 12 2700 22 l 10 23
30 17 3250 14 80 17
36 12 3750 14 50 14
42 15 4160 15 35 12
48 18 4380 l 1 26 15

 

‘: There were five replications in each measurement of ammonium concentration.
A: A, B, and C indicate transgenic plants from three independent transgenic events.
*: Untreated means there was no herbicide application on untransformed control plants.

#: Ammonium concentrations of transgenic and untransformed plants measured before the
herbicide application.

148

4500 '

l

4000 -

3500 -

T

   
  
  

I

3000 ‘ +Con trol-Un*

+Con trol-T

+Transgenic—A
+Transgenic—B
+Transgenic—C

2500 '

1

2000 ‘

1500 ‘
1000 '

Concentration of ammonium z“:

500 r
0;

 

0 10 20 30 40 50

Hours after herbicide application

Figure 8. The accumulation of ammonium in transgenic and untransformed creeping

bentgrasses after spraying with 1.2% Ignite®. Samples were taken before the herbicide
application and at the times indicated in Table 4.

#2 The concentration of ammonium is yg NH4+-N per gram of fresh leaf samples.

*: Un : Untreated untransformed control creeping bentgrasses; T: treated untransformed
control plants. Transgenic A, B, and C indicate transgenic plants from three independent
resistant callus lines.

Discussion

These results demonstrated that both bialaphos and phosphinothricin (PPT) can be
used as an efficient selective agent in the selection of callus which had been transformed
with the bar gene by microprojectile bombardment. After three transfers and twelve
weeks of culture on selection medium containing 5 mg/l of bialaphos, it was possible to
select out calli which had been transformed with the bar gene. There was no escape
detected in this selection scheme, and all of the resistant callus lines were able to
regenerate transgenic plants expressing the enzyme phosphinothricin acetyltransferase
(PAT) which detoxifies PPT by acetylation. However, 15 mg/l of PPT was necessary to
distinguish transformed calli from nontransgenic ones under the same selection scheme.

The reason that a higher concentration of PPT was necessary to achieve the same
selection efficiency than of bialaphos might be partially due to the application of D, L-
PP'I‘ in the selection medium. Bialaphos was a tripeptide antibiotic, which consisted of
L-PP'I‘ and two L-alanine residues. Upon removal of these residues by peptidases, L-PPT
was released and was the potent inhibitor of glutamine synthetase. The D-isomer of D,
L-PPT was not active in the inhibition of glutamine synthetase (Kondo et al., 1973;
Hoerlein, 1994). The exact percentage of D-isomer in the mixture of D, L-PPT was
unknown, so it was unsure that the reason for this phenomenon was solely the presence of
D-PPT, which reduced the percentage of active components in the mixture. With the
availability of both pure D-PPI‘ and L-PPT, it should be possible to access the role of the
D-isomer related to the L-isomer in the inhibition of glutamine synthetase.

Several factors must be refined to develop a routine transformation system for
creeping bentgrasses. The length of selection duration and the concentration of selective

agent appear to be important in the selection of transgenic callus lines. Concentration of

149

150
selective agents need to be carefully chosen to avoid being either too low and thereby

allowing an undesirable number of escapes to develop, or too high so that transformants
expressing low or moderate levels of resistance are lost. The length of selection, another
factor in the development of a transformation system, has to be judiciously determined.
It should not be too long, since the chance of somaclonal variation will increase with
culture age, and cultures may loose their regeneration capacity due to prolonged culture.
However, if the length of selection is not long enough, and the selection pressure is not
strong, there may still be a lot of escapes.

Hartman et al. (1994) reported the transformation of creeping bentgrasses with the
bar gene after suspension cells were bombarded with vectors containing the gene and
placed under selection for eight weeks without transfer. Initially, a concentration of 2
mg/l of bialaphos was used in the selection medium, but it was subsequently raised to 4
mg/l to reduce the number of escapes in their experiment. However, the percentages of

recovered plants after selection that survived the herbicide application (2 mg/ml of

Herbiace® which contains bialaphos) ranged from 0-13.7%. In our selection scheme,
though embryogenic callus was employed as the bombarded material instead of
suspension cells, 5 and 15 mg/l of bialaphos and PPT, respectively, were used in the
selection medium, and the bombarded embryogenic calli were under selection for twelve
weeks. All of the recovered plants which regenerated from bialaphos-resistant callus
lines were able to survive the application of glufosinate-ammonium at the rate of 2.4 g/l.
There was only one clone from the PPT selection scheme unable to survive the herbicide
spraying. The other advantage of our transformation strategy is that callus tissues are
easier to initiate and are more likely to regenerate plants compared to suspension cultures.

It was apparent that the length of selection duration was also a crucial factor in the
selection of transgenic callus lines in our experiments. The number of resistant callus

lines decreased as the length of the selection duration increased for both bialaphos and

151
PPT selection schemes (Table 3). The number of resistant callus lines decreased

dramatically during the last selection duration (between nine and twelve weeks). Some
callus lines of creeping bentgrass cultivar Penncross sensitive to bialaphos or PPT did not
turn brown until after five to six weeks of exposure to the selective agent, so it was
difficult to discriminate transformed callus lines from nontransformed ones. Twelve
weeks of selection appeared to be the appropriate length of selection duration, since it
maintained the regeneration capacity of resistant callus lines, and all them were able to
regenerate plants.

These results also suggested that the frequency of transfer after callus lines had
been bombarded might play an important role in the success of discriminating transgenic
callus lines from untransformed ones. Bombarded suspension cells were maintained on
the same selection plate for eight weeks without transfer in the experiment of Hartman et
al. (1994). Embryogenic callus tissues were transferred every four weeks in our selection
scheme, and only one escape was detected after regenerated plants were sprayed with
herbicide. The frequent transfer onto fresh-prepared selection medium provided some
advantages to the selection of resistant callus lines with the desired transgene sequence
over the selection scheme with no transfer. On the one hand, it may avoid problems
associated with cross-feeding. Transgenic callus tissue might provide neighboring
nontransgenic tissue with glutamine and detoxify the selective agent for them. On the
other hand, toxic substances such as excess amounts of ammonium accumulated in
sensitive tissue might diffuse to transgenic tissue and cause difficulty in the identification
of transformed callus lines. Another possible advantage is that the frequent transfer and
separation of bombarded callus into small pieces may reduce the possibility of
chimerism, one of the common problems found in transformation research, and provide a
more even selection pressure on all of the callus lines. The frequent transfer can also
reduce the possibility of the degradation of PPT and bialaphos and increase the selection

efficiency.

152
The phenomenon of heterogeneous responses of callus lines initiated from mature

seeds of cultivar Penncross was possibly due to the breeding history of this cultivar.
Penncross is a first generation synthetic cultivar produced by the random crossing of
three vegetatively propagated strains selected by H. B. Musser (Hein, 1958). The
synthetic cultivar is genetically heterogeneous, and each seed of Penncross possibly
represents a different genotype. The results from the evaluation of selective agents
reflected the heterogeneity of callus lines initiated from Penncross (Table 1 and Table 2).
The decision concerning the concentration of the selective agent and the length of the
selection duration again played an important role in the selection of transgenic resistant
callus lines of Penncross, since the selection strategy should provide a dependable and
repeatable scheme to select transgenic callus lines from a heterogeneous population.

Kramer et al. (1993) used a successful selection scheme with PPT as the selective
agent and with the pH indicator chlorophenol red for the identification of transformed
protoplast-derived colonies of maize. Chlorophenol red also proved to be a useful tool in
determinating the appropriate concentrations of selective agents needed to kill
embryogenic callus of creeping bentgrasses and in shortening the time necessary to
discriminate surviving colonies from dead ones in the evaluation of selective agents. We
also employed chlorophenol red to distinguish transgenic callus lines from nontransgenic
ones; however, the identification frequencies were not satisfactory, especially during the
early stages of selection (data not shown).

PPT- or bialaphos-resistance conferred by the bar gene was a useful selectable
marker for the isolation of transformed creeping bentgrasses. By using the Southern
hybridization analysis to detect stably integrated bar gene sequences, we showed that all
of the plants regenerated from resistant callus lines, except plants from one PPT-resistant
callus line, were transformed. The bar gene also proved to be a good reporter gene in
creeping bentgrasses. PAT activity and PPT-specific mRNA were detected in all of the

transgenic plants, and three of independent resistant plant lines tested were able to

153
decrease the accumulation of ammonium within a short period of time. The bar gene can

not only successfully confer resistance to transformed plants but can also be used as a

selectable marker in the transformation of creeping bentgrasses with other useful genes.
Penncross, the cultivar used in this experiment, is widely used on golf greens and

has been the most popular creeping bentgrass cultivar. The transgenic plants showed a

high level of resistance to glufosinate-ammonium, which is the active ingredient of

commercial herbicide I gnite®, Rely®, LibertyTM and FinaleTM, and, therefore, have
great commercial potential. The utilization of a PPT-resistant cultivar on golf courses
can not only reduce the amount of the toxic herbicide 2,4D applied on golf courses, but
can also be more environment-friendly, since PPT was degraded rapidly by microbes in
soil or surface water to 3-methyl phosphinicopropionic acid and ultimately to C02
(Humburg et al., 1994). With transgenic creeping bentgrasses expressing the bar gene,
the spraying of PPT or bialaphos on golf courses cultivated with resistant creeping
bentgrasses can kill both monocot and dicot weeds and provide a more efficient control

of weed problems.

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callus. Plant Cell Rep. 13: 1-6.

CHAPTER THREE

Evaluation of Bialaphos on the Prevention of Fungal Diseases
in Transgenic Bialaphos-resistant Creeping Bentgrasses

(Agrostis palustris Huds.)

ABSTRACT

Bialaphos showed a higher level of in vitro antifungal activity against the brown
patch disease pathogen, Rhizoctonia solani; the dollar spot disease pathogen, Sclerotim'a
homoeocarpa; and the Pythium blight pathogen, Pythium aphanidermalum, than
phosphinothricin (PPT). While PPT, an inhibitor of glutamine synthetase, suppressed the
mycelial growth of R. solani and S. homoeocarpa, it had no inhibitory effect on Pythium
aphanidermalum up to the highest concentration (600 mg/l) in our testing regimes.
Whereas bialaphos, the precursor of PPT and also an inhibitor of glutamine synthetase,
was significantly effective in the inhibition of growth of R. solani and S. homoeocarpa, it

was less so in that of Pythium aphanidermatum. Mean ED50 values for inhibition of

mycelia growth of R. solani and S. homoeocarpa were 5.54 and 33.04 mg/l (17.10 and
101.98 yM), respectively, for bialaphos, compared with 292.18 and 270.06 mg/l (1475.66
and 1363.94 yM), respectively, for PPT.

Various concentrations of bialaphos solutions were applied to transgenic
bialaphos-resistant creeping bentgrasses either three hours before or two days after plants
were inoculated with mycelia of R. solani, S. homoeocarpa and Pythium
apham'dermatum. Bialaphos application was able to significantly reduce the

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158
symptomatic infection by R. solani and S. homoeocarpa. Though the inhibitory effect of

bialaphos spraying on the suppression of Pythium aphanidermatum was not as effective
as that of R. solani and S. homoeocarpa, disease development was still reduced to a
significant extent. Better control of the disease development of dollar spot and Pythium
blight also occured when bialaphos was applied three hours before the pathogen
inoculation. These results also indicated that bialaphos may be used simultaneously and
efficaciously as a herbicide for weed control and as a fungicide for the control of diseases
caused by fungal pathogens in turf areas with transgenic bialaphos-resistant creeping

bentgrasses.

Introduction

Turf grasses have been recognized for their importance to our quality of life for a
long time and are cultured in nearly all inhabited regions of the world. Bentgrass
(Agrostis spp.) is the cool-season turf grass used commonly on greens in the cool and
transitional climatic regions and in the cooler portions of the warm climatic region.
Creeping bentgrass (Agrostis palustris Huds.) is the most commonly used Agrostis
species on golf putting greens and similar closely cut turf areas in North America (Beard,
1982). Among the existing cultivars of creeping bentgrass, Penncross is the major
cultivar employed today and is being used increasingly on greens of many golf courses.

Turfgrass plants are affected by numerous diseases, insects, and physiologic
disorders resulting from environmental stresses. Although diseases and the costs of
controlling them are difficult to estimate, the science of turfgrass pathology,
management, and breeding have frequently focused on disease control as a primary
research objective (Smiley, 1983). Turf grass diseases are a greater problem on golf
courses than on most other types of turf use. This is due to close mowing, heavy
fertilization, intense irrigation, and constant bruising from traffic and divoting. Disease
problems are most severe on putting greens, especially those composed of bentgrass or
annual bluegrass in contrast to bermudagrass turfs. The creeping bentgrasses are
susceptible to a variety of diseases such as brown patch (Rhizoctonia spp.), dollar spot
(Sclerotinia homoeocarpa), Pythium blight (Pythium spp.), Fusarium blight (Fusarium
roseum and F. tricinctum), and takeall patch (Gaeumannomyces graminis) (Smiley,
1983).

Bialaphos, the active ingredient of the commercial formulation herbicide

Herbiace® (Meiji Seika Kaisha) which is now being used in agriculture as a non-

159

160
selective and broad—spech contact herbicide, is a tripeptide composed of two L-alanine

residues and an analogue of glutamate known as phosphinothricin (PPT) (Ogawa et al.,
1973; Kondo et al., 1973). PPT inhibits the amino acid biosynthetic enzyme glutamine
synthetase (GS) of bacteria and plants (Bayer et al., 1972; Leason et al., 1982).
Bialaphos is a precursor of PPT produced by some strains of Streptomyces, and the intact
tripeptide has little or no inhibitory activity in vitro (Bayer et al., 1972; Tachibana et al.,
1986a). In both plants and bacteria, the active PPT moiety is released by intracellular
peptidases which remove the alanine residues from bialaphos.

Glutamine synthetase plays an important role in the assimilation of ammonia both
in plants and bacteria (Miflin and Lea, 1977; Joy, 1988). Inhibition of GS by these
compounds causes a rapid buildup of intracellular ammonia levels and an associated
disruption of chloroplast structure, resulting in the inhibition of photosynthesis and plant
cell death (T achibana et al., 1986b). Bayer et al. (1972) reported that the tripeptide
bialaphos has a certain antibiotic effect in Escherichia coli and Bacillus subtilis, which is
neutralized by the addition of glutamine. Experiments with GS isolated from E. coli
confirmed that PPT inhibits the GS enzyme in competition with glutamate as varying
substrate. Leason et al. (1982) determined GS inhibition by PPT in peas and
demonstrated that the mode of action is the same in bacteria and plants.

PPT resistance has been achieved by introducing a gene that codes for a
detoxifying enzyme (PAT: phosphinothricin acetyltransferase) into several plant species
(De Block et al., 1987, 1989; Spencer et al., 1990; Christou et al., 1991; Somers et al.,
1992; Casas et al., 1993; Wan and Lemaux, 1994; Akama et al., 1995). The gene,
designated bar, was found in strains of Streptomyces that produce bialaphos and isolated
from S. hygroscopicus by Murakami et al. (1986). The bar gene product protects these
strains from the action of their own antibiotic by metabolizing PPT to an inactive,

acetylated derivative (Thompson et al., 1987).

161
We have previously generated transgenic creeping bentgrasses showing high

levels of bar gene expression, which resulted in herbicide (either PPT or bialaphos)
resistance. It has been reported that bialaphos treatment of transgenic rice plants
expressing the bar gene could prevent infection by the sheath blight fungal pathogen
(Rhizoctonia solani) (Uchimiya et al., 1993). Since R. solani is the etiologic agent of
brown patch, one of the severe fungal diseases of creeping bentgrass, we were prompted
to assess its application to bialaphos-resistant transgenic creeping bentgrasses as a means
of protecting against fungal diseases. If the application of bialaphos could be shown to
reduce the plant damage due to fungal infection, a novel and economical usage for the
herbicide would be to provide weed control in turf areas with bialaphos-resistant creeping
bentgrasses while simultaneously and cumulatively reducing levels of fungal pathogens.
In this communication, we present the evidence that the application of bialaphos
on transgenic bialaphos-resistant creeping bentgrasses can prevent or alleviate the
infection by several fungal pathogens, including Rhizoctonia solani (brown patch),
Sclerotinia homoeocarpa (dollar spot), and Pythium aphanidermatum (Pythium blight).
This experiment indicates that it may, therefore, be possible to provide an opportunity for
the simultaneous control of weeds and fungal diseases in golf courses of transgenic

creeping bentgrasses expressing the bar gene.

Materials and Methods

In Vitro Test
The sensitivity of different pathogens to phosphinothricin (PPT; glufosinate-

ammonium from Riedel-deHaén of German) and bialaphos (kindly provided by Meiji

Seika Kaisha of Japan) was first tested in vitro by culturing mycelium of fungi on PPT-
or bialaphos-supplemented medium. Fungus isolates of Rhizoctonia solani , Sclerotinia
homoeocarpa, and Pythium aphanidermatum were kindly provided by Dr. J. M. Vargas
of Michigan State University and cultured on potato dextrose agar medium (PDA, 39 g/l;
Difco Laboratories, Detroit, MI) as stock plates.

PPT and bialaphos were dissolved in double-distilled water and filter-sterilized
(Nalgene filter) before adding into molten PDA (60 OC) medium after sterilizing at 121

0C for 15 minutes. A dilution series was used to obtain the needed concentrations. The

amended medium was mixed thoroughly and poured into 10—cm-diameter Petri dishes.
Inoculum for the study, which consisted of seven-mm-diameter agar plugs with

mycelium of each fungus isolates, was taken from stock plates and transferred onto

freshly prepared PDA medium. After the plates were incubated for at least one week

under the light at 25 oC and the growths of mycelium had completely covered the plates,
plugs were taken aseptically from plates with actively growing mycelium of Rhizoctonia
solani, Sclerotinia homoeocarpa, and Pythium aphanidermatum on PDA medium.
Inverted plugs were then transferred to the center of test plates (one plug per plate)
containing 20 ml PDA medium supplemented with various concentrations of PPT or

bialaphos ranging from 0 to 600 mg/l. Test plates with inoculum were wrapped with

162

163
parafrlm and placed under the light at 25 °C. There were 15 replications in each

treatment.

The growth of different fungi determined by measuring the radial length (mm) of
the colonies on corresponding plates after incubation for four days was used as an
indicator of the sensitivity of the fungus to the addition of PPT or bialaphos into the PDA
medium. Mycelial growth on bialaphos- and PPT-supplemented medium was compared
with that on non-amended medium. Percent inhibition was plotted as a function of the
bialaphos or PPT concentration. Linear regression was used to fit a line to the points and

determine the concentration causing a 50 % reduction in growth (ED50). Tukey’s

Honestly Significant Difference Test at P = 0.05 was employed to perform the statistical

analysis to detect differences in the mean values of radial length.

Greenhouse Test

Twelve grams of dry wheat seeds were autoclaved twice for 30 minutes in a 125
ml glass flask containing 25 ml of distilled water. Five 7-mm-diameter PDA plugs with
actively growing mycelium of Rhizoctonia solani, Sclerotinia homoeocarpa, or Pythium

aphanidermatum were put into the flask and mixed with the autoclaved wheat seeds. The

fungal culture was incubated under the light at 25 0C for one week after the inoculation.
In order to have uniform plants for fungal inoculation, three-month old healthy
transgenic (all from the same transgenic event) and nontransgenic control creeping
bentgrasses (Agrostis palustris Huds.) cultivar Penncross which were grown in four-inch
plastic pots were trimmed (about one inch in height) then fertilized with Peter’s 20-20—20
one week before the pathogen inoculation. To inoculate creeping bentgrasses with
different pathogens, about 500 mg of wheat seeds with mycelium of the corresponding

fungus were preweighed and evenly distributed on top of each plant

164
Various concentrations of bialaphos solution ranging from 200 to 2,400 mg/l were

prepared before the application and sprayed on the creeping bentgrasses with a hand
sprayer either three hours before or two days after the fungus inoculation. After the
bialaphos application, plants were wrapped with plastic bags with holes to raise the
humidity. Transgenic and nontransgenic control plants which were not treated with
bialaphos were subject to the same pathogen infection procedures as well. There were
ten replications in each treatment.

Disease rating on a zero to ten scale with the smallest increment of 0.5 was
recorded on the basis of the percentage of plant damage due to fungal infection one week
after the initial pathogen inoculation, for example, 0: no damage; 5: 50 % plant damage;
10: death. Differences in mean disease rating values between two spraying times were
detected using F test at P = 0.05 and Tukey’s Honestly Significant Difference test at P =
0.05 were employed to perform the statistical analysis to detect differences in mean
disease rating values between different concentrations of bialaphos spraying with the

same spraying time for each fungal pathogen.

Results

In Vitro Test

Fungal pathogens Rhizoctonia solani, Sclerotinia homoeocarpa, and Pythium
aphanidermalum, the etiologic agents of brown patch, dollar spot, and Pythium blight
diseases, respectively, of creeping bentgrasses were inoculated and cultured on PDA
medium supplemented with various concentrations of bialaphos or PPT to assess their
responses to these two inhibitors of glutamine synthetase.

Rhizoctonia solani was very sensitive to the addition of bialaphos into the PDA
medium (Table 1 and Figure 1). Even at the lowest concentration of one mg/l, the
mycelial growth of Rhizoctonia solani was significantly suppressed as compared to that

on PDA medium with no bialaphos supplement. Only about five mg/l (EDSO = 5.54

mg/l) of bialaphos amendment was needed to reduce the fungal growth by 50 % (Table 1
and Table 6). There was almost no fungal growth observed four days after the
inoculation when the bialaphos concentration of PDA medium was 60 mg/l. There was
still no significant growth of Rhizoctonia solani even two weeks after the initial
inoculation when the concentration of bialaphos was 60 m g/l or higher (data not shown).
PPT was also very effective in the suppression of the mycelial growth of
Rhizoctonia solani (Table 2 and Figure 1). However, the presence of PPT was not as
effective as that of bialaphos in suppressing the growth of Rhizoctonia solani on PDA
medium . Bialaphos inhibited mycelial growth of R. solani more than PPT did, as
reflected by their values of ED 50 (Table 6). The growth of mycelium was significantly
reduced at the concentration of 25 mg/l PPT. More PPT amendment (292.18 mg/l or
1475.66 17.10 pM), as compared to the amount of bialaphos supplement (5.54 mg/l or
17.10 yM), was necessary to reduced the growth of Rhizoctonia solani by 50 %. There

was still some mycelial growth of R. solani observed even when 600 mg/l of PPI‘ was

165

166
amended into the PDA medium. The same trend was also evident for Sclerotinia

homoeocarpa and Pythium aphanidermatum , where the ED50 values for S. homoeocarpa

and Pythium aphanidermatum were higher for PPT than for bialaphos (Table 6).

The mycelial growth of S. homoeocarpa was also sensitive to the presence of
bialaphos and PPT (Table 3, Table 4 and Figure 2), though its responses were apparently
different from those of R. solani (Table 6 and Figure 4). Higher concentrations of
bialaphos and PPT were necessary to significantly reduce the mycelial growth of S.

homoeocarpa than that of R. solani. The ED 50 value of S. homoeocarpa for bialaphos

was higher than that of R. solani (33.04 and 5.54 mg/l, respectively) (Table 6). More than
150 mg/l of bialaphos amendment was necessary to completely suppressed the mycelial
growth of S. homoeocarpa on PDA medium. However, S. homoeocarpa responded a
little more sensitively to higher concentrations of PPT (between 400 and 600 mg/l) than

R. solani (Table 2, Table 4 and Figure 4). The ED50 value for PPT of S. homoeocarpa

was lower than that of R. solani (270.06 and 292.18 mg/l, respectively) (Table 6). In
general, the effect of bialaphos or PPT amendment into PDA medium on the inhibition of
mycelial growth of R. solani and S. homoeocarpa was effective with the highest
concentration resulted in the least growth of mycelium.

Compared with R. solani and S. homoeocarpa, Pythium aphanidermatum was the
least sensitive fungus to both bialaphos and PPT (Table 5, Table 6, Figure 3 and Figure
4). At least 500 mg/l of bialaphos supplement was needed to significantly reduce the
mycelial growth on the PDA medium (Table 5) and the whole plate was covered with the
mycelium of Pythium aphanidermatum one week after the initial inoculation. The
presence of PPT had no effect on the inhibition of Pythium aphanidermatum up to the

highest concentration (600 mg/l) amended in PDA medium.

167

Table 1. Sensitivity of Rhizoctonia solani to bialaphos on potato dextrose agar medium

 

concentration of bialaphos (mg/l)

 

0 l 5 10 20 40 60 80

 

40.0 :r: 0.0# 31.6 x 0.7 12.5 i 0.7 7.3 :9: 0.5 4.4 2 0.2 1.5 1- 0.2 0.4 i O.la* O : 0.0a

 

#T he radial length (mm) of the colony growing on various concentrations of bialaphos-
supplemented potato dextrose agar medium four days after inoculation was used as an
indicator to measure the sensitivity of Rhizoctonia solani to bialaphos.

*Mean 3: SE. (standard error) for 15 replications. Means with the same letter were not
significantly different according the Tukey’s test at P = 0.05.

168

Table 2. Sensitivity of Rhizoctonia solani to phosphinothricin on potato dextrose agar
medium

 

concentration of phosphinothricin (mg/l)

 

0 25 50 100 200 300 400 500 600

 

8
40.0t0.0# 35011.23 30811.03 25.4:l.8b 20.211.0bc 16.8tl.0cd l3.0:t:0.8de 12.011.1e 6.81:0.5

 

#T he radial length (mm) of the colony growing on various concentrations of
phosphinothricin-supplemented potato dextrose agar medium four days after inoculation
was used as an indicator to measure the sensitivity of Rhizoctonia solani to
phosphinothricin.

*Mean 1 SE. (standard error) for 15 replications. Means with the same letter were not
significantly different according the Tukey’s test at P = 0.05.

169

Table 3. Sensitivity of Sclerotinia homoeocarpa to bialaphos on potato dextrose agar
medium

 

concentration of bialaphos (mg/l)

 

O 20 40 60 80 100 150 200 250 300

 

40.0:0.0# 26.8113 20.0tl.4 15010.5; l3.2t0.4a 10.8:0.5ab 7.010.7b 1.8:t0.4c 0.610.2c 0:0.0c

 

#T he radius length (mm) of the colony growing on various concentrations of bialaphos-
supplemented potato dextrose agar medium four days after inoculation was used as an
indicator to measure the sensitivity of Sclerotinia homoeocarpa to bialaphos.

1"Mean 2 SE. (standard error) for 15 replications. Means with the same letter were not
significantly different according the Tukey’s test at P = 0.05.

170

Table 4. Sensitivity of Sclerotinia homoeocarpa to phosphinothricin on potato dextrose

 

 

 

agar medium
concentration of phosphinothricin (mg/l)
0 50 100 150 200 250 300 350 400 500 600
40.00.0011" 37.8:06a 33011.0 29020.7 23311.1 17.620.6b 15.2.0031» 120110.9cd 10.21031 3.33.071: 2.4.0.7.:

 

#T he radial length (mm) of the colony growing on various concentrations of
phosphinothricin-supplemented potato dextrose agar medium four days after inoculation
was used as an indicator to measure the sensitivity of Sclerotinia homoeocarpa to
phosphinothricin.

*Mean : S.E. (standard error) for 15 replications. Means with the same letter were not
significantly different according the Tukey’s test at P = 0.05.

171

Table 5. Sensitivity of Pythium aphanidermatum to bialaphos on potato dextrose agar
medium

 

concentration of bialaphos (mg/l)

 

0 100 200 300 400 500

 

40.0 :1: 0.0 a# * 40.0 1 0.0 a 40.0 :t: 0.0 a 39.0 :1: 0.4a 38.0 x 0.5 a 31.0 i 1.0

 

#T he radial length (mm) of the colony growing on various concentrations of bialaphos-
supplemented potato dextrose agar medium four days after inoculation was used as an
indicator to measure the sensitivity of Pythium aphanidermatum to bialaphos.

*Mean :1: SE. (stande error) for 15 replications. Means with the same letter were not
significantly different according the Tukey’s test at P = 0.05.

172

Table 6. In vitro inhibition of Rhizoctonia solani, Sclerotinia homoeocarpa, and
Pythium aphanidermatum by bialaphos or phosphinothricin

 

 

Amending Correlation ED 50

Fungus ingredient Linear regre ssion# coefficient (mg/l)
Rhizoctonia bialaphos Y = 0.91X + 44.96 0.72 5.54
solani PPT Y = 0.11X + 17.86 0.97 292.18
Sclerotinia bialaphos Y = 0.28X + 40.75 0.88 33.04
homoeocarpa PPT Y = 0.17X + 4.09 0.98 270.06
Pylhium bialaphos Y = 0.037X - 4.28 0.79 1467.18

*

 

aphanidermatum PPT -..-- .......

 

#Percent inhibition [Y] was plotted as a function of bialaphos or phosphinothricin (PPT)
concentration [X].

*There was no inhibition of Pythium aphanidermatum by phosphinothricin up to the
highest concentration (600 mg/l) amended in PDA medium.

173

Figure 1 . The inhibition of bialaphos and phosphinothricin on the mycelium of
Rhizoctonia solani four days after the initial inoculation.

Top: the growth of mycelium on PDA medium.

Middle: the growth of mycelium on PDA medium contained 400 mg/l of bialaphos.

Bottom: the growth of mycelium on PDA medium contained 400 mg/l of phosphinothricin.

 

R. 400 m5]? PPT

175

Figure 2 . The inhibition of bialaphos and phosphinothricin on the mycelium of
Sclerotinia homoeocarpa four days after the initial inoculation.

Top: the growth of mycelium on PDA medium.

Middle: the growth of mycelium on PDA medium contained 400 mg/l of bialaphos.

Bottom: the growth of mycelium on PDA medium contained 400 mg/l of phosphinothricin.

r‘“

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177

Figure 3 . The inhibition of bialaphos and phosphinothricin on the mycelium of Pythium
aphanidemratum four days after the initial inoculation.

Top: the growth of mycelium on PDA medium.

Middle: the growth of mycelium on PDA medium contained 350 mg/l of bialaphos.

Bottom: the growth of mycelium on PDA medium contained 600 mg/l of phosphinothricin.

178

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However, the amendment of bialaphos and PPT did show some inhibitory effect

on the growth of Pythium aphanidermatum when the amount of mycelium, instead of the
measurement of radial length of mycelium, was employed as the indicator to represent

the growth of Pythium aphanidermatum. (Figure 3).

Greenhouse Test

Various concentrations of bialaphos solution were applied on transgenic creeping
bentgrasses expressing the bar gene to assess the effects of bialaphos spraying on the
development of the three different pathogens (Table 7). The application of bialaphos had
a very significant effect on the suppression of brown patch disease development when the
disease rating was taken one week after the fungus inoculation. When bialaphos
application was executed three hours before the inoculation of Rhizoctonia solani on
transgenic plants, disease symptoms were rarely observed, and there was only minimal
plant damage due to the infection of Rhizoctonia solani. At the concentration of 200 m g/l
of bialaphos solution, about one-tenth of the recommended herbicide spraying rate to kill
untransformed creeping bentgrasses, the application significantly reduced the plant
damage due to pathogen infection.

Transgenic plants that were not treated with bialaphos showed typical symptoms
of brown patch disease and a significant amount of plant damage. Even two days after
the pathogen inoculation, when the disease began to develop, the application of bialaphos
could still significantly restrain the growth of mycelium and the development of brown
patch disease. The untreated control plants, either transgenic or nontransgenic, were
severely damaged by the infection of R. solani. The grass blades became water soaked
and dark at first but soon became dry, wither, and turned light brown. The disease was
able to continuously develop even after plastic bags were taken off and a lot of the

untreated plants were dead three weeks after the pathogen inoculation (data not shown).

181
There was no significant difference observed between two different timings of bialaphos

application (F = 0.29 < F .05 (1’ 63) = 4.00).

The bialaphos application, either three hours before or two days after the
pathogen inoculation, was also very effective in preventing the disease development of S.
homoeocarpa, the etiologic agent of dollar spot, on transgenic creeping bentgrasses. The
plant damages on transgenic bialaphos-resistant creeping bentgrasses after the bialaphos

application were significantly less than those on transgenic plants not treated with

bialaphos. The difference between the two application times was significant (F = 8.23 >

F _05 (1, 81) = 3.98), and there was more plant damage caused by the infection of S.

homoeocarpa when the bialaphos spraying on transgenic plants was done two days after
the pathogen inoculation.

The development of disease symptoms of dollar spot on transgenic and
nontransgenic control plants which were not applied with any bialaphos solution was not
as‘rapid and severe as that of brown patch, and plant damage of untreated plants caused
by the fungal infection was also less severe than that of R. solani in our testing system.
Most untreated control plants showed small, circular, sunken white patches when covered
with plastic bags and a few of them were able to recover from the damage caused by the
infection of S. homoeocarpa when plastic bags were taken off two week after the data of
disease rating had been collected.

The bialaphos application was more effective in protecting against the infection of
R. solani than against the infection of S. homoeocarpa. There were more plant damages
observed on transgenic bialaphos-resistant creeping bentgrasses due to the disease
development of the dollar spot pathogen than those of the brown patch pathogen after the
application of bialaphos. However, the disease development was significantly restrained

and most plants were able to completely recover from the infection and grew normally.

182
Though the spraying of bialaphos, applied either three hours before or two days

after the pathogen inoculation, was effective in the prevention of disease development as
reflected by the results that the increases in the concentration of bialaphos did reduce the
plant damage caused by either brown patch or dollar spot, the treatment means were not
significantly different. The lowest applied rate of 200 mg/l of bialaphos was high enough
to suppress the disease development of both fungal pathogens.

The results also showed that a single application of bialaphos could suppress the
disease development of Pythiwn blight, though not as effectively as with brown patch and
dollar spot (Figure 5). When 200 mg/l of bialaphos was applied three hours before the
pathogen inoculation, it significantly restrained the infection of Pythium aphanidermatum
and reduced the amount of plant damage one week after the initial inoculation. Better
disease control was achieved when higher rate of bialaphos spraying was applied on
transgenic plants. Higher concentration of bialaphos (at least 800 mg/l) was needed to
significantly reduce the plant damage due to the infection of Pythium blight when
bialaphos was applied two days after the inoculation. Bialaphos application on

transgenic plants before the pathogen inoculation provided a little better protection

against the infection by Pythium aphanidermalum (F = 25.57 > F .05 (1, 153) = 3.96).

However, no matter which application time and concentration of bialaphos were
employed in this study, the infection of Pythium blight was severe and caused more plant
damage than the other tested pathogens when disease symptoms were examined two

weeks after the initial inoculation (data not shown).

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Figure 6. 'l‘hc uppllczttmn ol‘ bmluphns un the plmcnunn nl' puthugcn IHICCUHH h}
Rhinu'hmiu ,w/(mi In transgenic bmluphus—resistant creeping bcntgt‘usscs.

Tup: transgenic plant Much “as not sprancd \ch bulldphns \‘hO\\Cd <C\'c1'cl_\ dummgvd h}
the inl‘ccuon at R. mlam.

Bottnm: transgenic plant \\ hlch “as «pun Cd \\ 1th 200 mg“! nt’ hmluphm‘ lhl't‘C hntn'x‘ hCIHIC
the pathogen tmwulutmn <htmcd littlcdumagc by the inl‘cctmn ml' R. ,w/(mi.

186

 

Figure 7. The application of hialaphox on the prevention of pathogen rnl‘cction h}
Srleruliniu lmmmlururpu in transgenic bialaphos-resistant creeping bentgrasses.

Top: transgenic plant \\ hich was not spra§ ed \xith bialaphos shoued \‘e\erel}' damaged by
the infection ol' S. lmmncnmr/xr.

Bottom: transgenic plant \\ htch “as spra} ed \\ ith 3th mg/l ol' bialaphox' three hourx helorc
the pathogen tnoculatton showed almost no damage b} the tnlectron ol' .V. /I()Ill/)cu('(lr/’(l.

187

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' . ; tea 'or o' ta; (s or to revenion o' m togen in ‘ec ior
Flure8 The 1)l tti 1 lblth) 1tl t I ll g l t 1h}
l’yl/rirmr (Ip/Iunidz'rrmuum in transgenic bialaphosa‘esistant creeptng bentgrasses

. t t- c ; ic ';s < s r; 'e '1 la a os stowe severe \' LllTLL’C ,,
To "tr'lnsgent‘ |1ntwh h w 1 nit t_\ d w th b l h l d l‘ d 11g d h}
the inleetron ol l’vl/Iium (ll)/l(llli(/(’N”(l/II"!t
Bottom: transgenic plant w hich was sprayed with 400 mg/l ol' bialaphos one day alter the
pathogen inoculation showed some damage by the inl’eetton ol~ I’wllium (I[,)/l{lIIi(/(’rll11IIIIIII.

Discussion

Bialaphos exhibited inhibitory activity in vitro against the growth of R. solani , S.
homoeocarpa, and Pythium aphanidermatum that was superior to PPT, as reflected by

their ED 50 values. Increasing concentrations of bialaphos and PPT were of greater

effectiveness, with the highest rates resulting in the smallest colonies of mycelium.

When working with mean values of ED 50 for R. solani and S. homoeocarpa, the 51350

values for PPT were higher than those for bialaphos. It is surprising that the mycelial
growth of R. solani was significantly inhibited at the concentration of one mg/l bialaphos
(21 % reduction).

The same trend was also evident in the case of Pythium apham'dermatum, though
the inhibition of the mycelial growth due to the inclusion of bialaphos in PDA medium
was not as significant as with R. solani and S. homoeocarpa. There was no significant
inhibition of the mycelium of Pythium aphanidermatum detected up to the highest
concentration of supplement (600 mg/l) in our treatment regimes of PPT when radial
length was used to measure the growth of mycelium. However, both bialaphos and PPT
did show certain degree of inhibitory effect on the amount of mycelial growth of Pythium
apham'dermatum. (Figure 3). The inhibition by bialaphos of growth of Pythium
aphanidermatum was corroborated by the protection that the application of bialaphos on
transgenic plants provided against the infection by Pythium aphanidermatum and by the
reduction on the plant damage due to Pythium blight

It is intriguing to observe that different fungal pathogens showed the same trend
in differential in vitro responses toward bialaphos and PPT. Bialaphos is a tripeptide
precursor of PPT, an analogue of glutamate, in which two alanine residues are linked to

the PPT. While PPI‘ is an inhibitor of glutamine synthetase in both plant and bacteria,

188

189
the intact tripeptide has little or no inhibitory activity in vitro (Bayer et al., 1972;

Tachibana et al., 1986a). In both bacteria and plants, intracellular peptidases remove the
alanine residues and release active PPT (L-phosphinothricin).

Though the PPT used in this study was a mixture of D- and L-phosphinothricin
(ammonium-DL-homoalanin-4.ylmethylphosphinat), in which the D-isomer is the
inactive inhibitor of glutamine synthetase and the L-isomer is the active moiety of
tripeptide bialaphos, it is still difficult to explain the significant differences shown in the

magnitude of ED 50 values where the sensitivities of R. solani and S. homoeocarpa for

bialaphos were higher than those for PPT. Though it has not been reported, we
speculated, however, that the D-isomer, the inactive inhibitor of glutamine synthetase,
might have interfered with the L-isomer, the active moiety of tripeptide bialaphos, in the
binding of glutamine synthetase and reduced the inhibition efficiency of L-PP'I‘.

It is even more surprising to know that the bialaphos provided a better selection
efficiency in killing nontransgenic callus of creeping bentgrass than the PPT did (Chapter
Two of the dissertation). Though it has been remarked that the inefficiency of PPI‘
selection was due to the interference of glutamine or asparagine in the culture medium
with herbicide activity (Wang et al., 1992), it seems to us that it is more or less due to the
nature of PPT. The composition of selection media used to establish the kill curves for
bialaphos and PPT were identical except for the selective agent in our experiment.
Compared to PPT, bialaphos was able to kill nontransgenic callus more effectively, at a
lower concentration, and within a shorter period of selection. It would be interesting to
see whether the same trend could be applicable to other plant materials and fungus
species.

Certainly, the relative sensitivity to bialaphos and PPT was significant, but the

basic difference in the values of ED50 for the three different fungi was significant as well.

In our in vitro test, the three pathogens also showed different responses to bialaphos and

190
PPT. R. solani was most sensitive to the presence of either bialaphos or PPT and

Pythium apham'demzatum was the least. It has been noticed for some time that the
application of one of several fungicides, such as Benomyl, Chlorothalonil,
Cycloheximide+Thiram, PCNB, and Triadimefon, could provide an efficient control of
brown patch and dollar spot diseases at the same time. However, they could not
effectively control Pythium blight in most cases. Most the fungicides designated to
control Pythium blight, such as Chloroneb, Ethazole, Metalaxyl, and Propamocarb, were
not able to prevent infection by either R. solani or S. homoeocarpa (Beard, 1982).

The in vitro sensitivity data help explain some of the efficacy trends observed in
the greenhouse study. Application of bialaphos on transgenic bialaphos-resistant
creeping bentgrasses, even at the lowest rates, showed universal effects in suppressing
disease development and reducing plant damage due to fungal infection. Bialaphos
spraying was most significant in restraining the disease symptoms of R. solani. Both
spraying times showed significant effectiveness in the suppression of the disease
development of brown patch.

S. homoeocarpa was also significantly sensitive to the in viva application of
bialaphos; however, the application before the pathogen inoculation provided a better
control of dollar spot But even if bialaphos was applied two days after the pathogen
inoculation when the pathogen had started to develop and spread, it still provided good
plant protection and was able to significantly restrain the disease symptoms of S.
homoeocarpa.

Though it was not as effective as in the cases of R. solani and S. homoeocarpa,
bialaphos spraying still limited plant damage due to infection by Pythium
aphanidermatum. The timing of bialaphos application was also important in suppressing
the disease symptoms of Pythium blight. Better results in reducing plant damage were

obtained when bialaphos was applied three hours before the pathogen inoculation.

191
Interactions between herbicides and plant pathogens have been well documented

(Altman, 1985; Ben-Yephet et al., 1991). The main cause of this phenomenon is that the
biological activity of pesticides may extend beyond its effects on the target organisms.
Upon treatment with herbicides, plant diseases caused by fungal pathogens have been
reported to increase (El-Khadem et al., 1979 ; Altman, 1981) or decrease (Grinstein et
al., 1979, 1984; Cohen et al., 1986). More research needs to be done not only to assess
the applicability of the antifungal activity of bialaphos toward other fungi, but also to
investigate the mechanism of its inhibitory effect so that we might better understand the
interactions between bialaphos and fungal pathogens and explain the differing reactions
of the various fungi to the application of bialaphos.

Bialaphos has mainly been used as a broad—spectrum contact herbicide and as a
selective agent in plant transformation experiments. However, it has been reported that it
could be used as an effective selective agent to improve the transformation frequencies of
Cercospora kikuchii , a fungal pathogen of soybean (Upchurch et al., 1994). Their report
and the results of our in vitro study, where the bialaphos showed significant inhibitory
effects toward R. solani and S. homoeocarpa, suggest that bialaphos could be used as an
efficient fungicide for a variety of fungal pathogens.

The application of 200 mg/1 of bialaphos, which is about one tenth of the
recommended concentration to kill untransformed turfgrass plants, was enough to
significantly reduce plant damage due to the infection of both R. solani and S.
homoeocarpa. The low rates at which bialaphos was effective present a novel and
economical means for the control of some fungal pathogens. These facts coupled with
the results presented in this paper which show that the application of bialaphos could
prevent or suppress the infection by several fungal diseases indicate that it may,
therefore, be possible to combat fungal infections and weed infestation simultaneously in
fields of bialaphos-resistant creeping bentgrasses by a judicious choice of concentration,

frequency, and time of application.

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