TH .

THESfi

IES

“‘°““?‘"t‘ll\‘ mm W" \\\l
‘l‘lllllllllil‘lll r

l

.1

This is to certify that the

thesis entitled
Localization of T-DNA Inserts of Petgnia
hybrida Chromosomes by g situ Hybridization

 

presented by

JIMEI WANG

has been accepted towards fulfillment
of the requirements for

Master's Plant Breeding and

degree in

 

 

Genetics-Horticulture

Mai/44%

 

 

Major professor

i/Mfia

MSU is an Affirmative Action/Equal Opportunity Institution

 

 

LiBfififiY
Michigan State
i University

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative ActiorVEquei Opportunity Institution
cmmd

 

LOCALIZATION OF T-DNA INSERTS ON Eetunig hxbgidg

CHROMOSOMES BY 13 SITU HYBRIDIZATION

BY

Jimei Wang

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1992

ABSTRACT

LOCALIZATION OF T-DNA INSERTS ON Petunia hybrid;

CHROMOSOMES BY IN SITU HYBRIDIZATION

BY
Jimei Wang

The locations and distribution of the T-DNA region in
plant chromosomes were detected by in situ hybridization.
The T-DNA sequence was introduced into VR hybrid of Eetunig
nybzigg genome by inoculation of leaf explants with
Agrgbacterium tumefaciens plasmid pMONZOO. The genes for
kanamycin resistance and nopaline synthase are carried in
T-DNA region. Putative transformed shoots were selected by
their ability to grow in the presence of kanamycin.
Transformed plants were confirmed by leaf-disc assay,
nopaline synthase activity and slot blot assay.

Ten transgenic plants were used to determine the sites
of T-DNA fragment in the host chromosomes. Nine plants
showed one location of T-DNA sequence on chromosomes. One
plant contained two T-DNA inserts in different chromosomes.
The results indicated that T-DNA was integrated into
different locations and distributed randomly among
chromosomes in independent transformants. Each chromosome

could be as a target for T-DNA insertion.

DEDICATION

To my parents
my husband
and

my daughter

iii

ACKNOWLEDGEMENTS

I would like to thank my major professor, Dr. Kenneth
Sink for his guidance and support throughout this research
program. I would also like to thank Dr. Joanne Whallon for

her guidance in the i_ situ hybridization and microscopic

 

studies.
Appreciation also goes to the members of my guidance
committee: Drs. Lowell Ewart and James Hancock for their

direction and assistance during this research.

iv

TABLE OF CONTENTS
Page
LISTOFTABLESOOOOOOOOOOOO....0...OOOOOOOOOOOOOCOOOOOOOVi

LISTOF FIGURES. ..... O...O...0.0.0.0...OOOOOOOOOOOOOOOOVii

LITERATURE REVIEW

Introduction...........................................1
Biology of Agrobacterium-mediated transformation.......1
T-DNA as selectable markers............................4
T-DNA as a selectable marker in protoplast fusion......7
Site of integration of T-DNA...........................8
T-DNA used as an insertion mutagen.....................13
In situ hybridization............... ...... .............14
T—DNA in use of plant breeding.... .......... ...........17
Literature Cited............. ................. .........19

LOCALIZATION OF T-DNA INSERTS ON Petunia hybrid;
CHROMOSOMES BY 1N SITU HYBRIDIZATION

Introduction........... ....... ......................... 28
Materials and Methods...................................30
Eetunia Karyotype..................................30
Transformation and Confirmation....................31
In Sign Hybridization..............................32
Results and Discussion.... ........ ......................34
Literature Cited........... ..... ........................58

APPENDIX ................................................ 62

LIST OF TABLES

Table Page

1. Verification of kanamycin resistance,
nopaline production, slot blot and T-DNA
locations on chromosomes in transgenic VR
hybrid 2; hybrida................................45

APPENDIX
I. Assay results of 2; hybrid; transformants

for nopaline production, response to
kanamycin and slot blot test... ..... ....... ..... .62

vi

LIST OF FIGURES

Figure Page

1. The karyotype of 2; h brida.......................46

2. Leaf explant transformation of Petunia
VR hybrid co-cultivated with pMONZOO .............. 47

3. Leaf explants from untransformed and
transgenic Petunia plants on MS medium
containing 300 ug/ml kanamycin...... ........... ...48

4. Transgenic Petunia plant 38 rooted in

MS containing 300 ug/ml kanamycin ...... . .......... 49
5. Nopaline synthase assay on non-transformed

and transformed leaf tissue ...... .. ...... . ........ 50
6. Slot-blot analysis of Petunia transformants ....... 51

7. in situ hybridization of T-DNA probe to
metaphase chromosomes prepared from each
transgenic Petunia plant..........................52

8. In situ hybridization with a T-DNA probe in
transformed 2; hybrida.. ..... ........... .......... 55

9. Localizations of T-DNA inserts by in situ
hybridization indicating on the karyogram
of Petunia in 10 independent transgenic
plants. The T-DNA locations labelled
with arrows. Arabic numbers indicated
plants 1-10..... ........... .......................56

10. in situ hybridization with a ribosomal DNA
probe in non-transformed g; hybrida...............57

vii

LITERATURE REVIEW

Plant cell and molecular genetic technologies provide
opportunities to supplement traditional plant breeding.
Foremost among these biotechnologies is gene isolation and
subsequent transformation into crop species for
improvement purposes (Perani et al. 1986; Gasswe et al.
1989). The transfer of foreign DNA into plants can be
achieved in a variety of ways (Potrykus 1991). One
prominent way is to engineer the T—DNA, transferred DNA, of
Agrgbacterium tumefaciens (Paul et al. 1988). The natural
transformation capability of A. tumefaciens enables this
organism to be used extensively as a vector of foreign

genes into plants (Gheysen et al. 1985).

Biology of Agrobacterium-mediated transformation

A. tumefaciens infection causes tumorous plant growths,
commonly called crown galls, by introducing T-DNA into the
plant cells at a wound site. The ability of A; tumefaciens
to transform plants is determined by the presence of two
regions of the Ti, tumor-inducing, plasmid. These regions
are the T-DNA and the virulence (gig) ones. The T-DNA has
flanking sequences, each 25 bp long, termed the left border
(LB) and right border (RB) that are essential for the

transfer to occur. These borders are involved in excision

2

of the T-DNA fragment (Chilton et al. 1977; Koukolikova-
Nicola et al. 1987). When Agggbggtgrium infects a plant, an
interaction between the bacterium and the host plant cell
stimulates excision of the T-DNA sequence. Some, but not
all, steps in this process are known. Transfer begins with
the nicks in the two borders producing the T-DNA molecule.
Following activation of the 21; genes, the T-DNA molecule
is excised from the plasmid DNA and transferred into the
nucleus of a plant cell where it eventually becomes
integrated into one or more host plant chromosome(s) (Kerr
et al. 1977; Matsumoto et al. 1990; Watson et al. 1992).

The gig genes, which consist of six loci (21; A, B, G,
C, D and E), are required for transferring the T-DNA into
plant cells (Klee et al. 1982; Stachel et al. 1986). They
produce trans-acting proteins that are essential for plant
cell transformation. These proteins must function within
the agrobacterial cells for the T-DNA to be excised from
the plasmid DNA (Zambryski et al. 1988; Watson et a1.
1992). 1;; Dl/Dz endonucleases recognize the T-DNA border
sequences (Albright et a1. 1987; Wang et a1. 1987) that
generate the processed T-DNA molecules (Yanofsky et a1.
1986; Yamamoto et al. 1987). Some proteins such as those
encoded by 2;; D2 and El; E2 may also function later on in
the plant for the conditioning of the plant cells during
infection and for subsequent transfer of the T-DNA (Perani

et a1. 1986; Zambryski et a1. 1988; Hohn et al. 1989). In

3
addition, another set of genes are indirectly involved in
transformation (Watson et a1. 1992). These genes are
carried on the Agrobacterium chromosome and are responsible
for proteins that bind the bacteria cells to the plant
cells.

Native T-DNA also contains genes referred to as
oncogenes which are responsible for tumor induction (Caplan
et al. 1983; Perani et al. 1986). The oncogenes code for
enzymes that lead to the production of phytohormones. Three
of these genes are directly responsible for tumor formation
(Caplan et al. 1983; Watson et al. 1992). Genes 1 and 2
code for enzymes that cause the production of an auxin.
Gene 4 codes for an enzyme that leads to the production of
cytokinin. Overproduction of these endogenous hormones
results in tumorous growth of plant cells (Parani et al.
1986). Expression of the T-DNA genes in plant cells has
been shown to be responsible for the observed hormone-
independent cell growth (Lichtenstein et a1. 1987).

The T-DNA also carries genes that code for the synthesis
of unusual amino acids called opines. Depending on the
opines that are synthesized, A; tumefaciens strains are
classified as nopaline or octopine. The strains contain
either the nopaline synthase gene or octopine synthase
gene, respectively, in different types of Ti plasmids
(Greve et al. 1984; Nester et al. 1984; Rogers et al.

1988). It has been demonstrated that the tumorous state is

4
characterized by the presence of an Agrobacterium plasmid
fragment, T-DNA, which is transcribed into RNA. This makes
it likely that plant cells are transformed by plasmid DNA,
and are transferred during tumor induction. The enzymes
mentioned above are encoded by one of the plasmid genes.
Therefore, the presence of these enzymes can be considered
as a marker for cell transformation. The opines offer a
second marker in transformation studies (Otten et al.
1978).

A major breakthrough in the development of Ti plasmid
vectors was to "disarm them" by deleting the oncogenes in
the T-DNA sequence that elicit the typical crown gall
tumors (Rogers et al. 1986; Misra 1990). This change in T-
DNA sequences allowed shoot regeneration from transformed
cells. Furthermore, the insertion of selectable marker
genes, such as antibiotic resistance, aids in the selection
of transformed tissues and in the confirmation of
regenerated plants (Barton et al. 1983; Fraley et al. 1986;
Zambryski et al. 1983). The selectable marker and the
engineered genes of a disarmed T-DNA region of the Ti
plasmid can insert and become stably integrated into the
host nuclear DNA (Hooykaas et a1. 1984; Horsch et al. 1988;
Watson et al. 1988). Thus, various plant species have been
transformed by disarmed vector strains of A; tumefaciens

(De Block 1988; Ledger et al. 1991; Sitbon et al. 1991).

T-DNA as selectable markers

A very important factor in the development of T-DNA-
based vectors is the availability of selectable markers
which impose antibiotic resistance (Hauptmann et al. 1988;
Misra 1990). Such genes give high levels of resistance in
transformed cells to enable their selection from among
wild-type cells. A variety of chimeric genes that express
bacterial coding sequences under control of plant promoters
have been used as selectable markers (Reynaerts et al.
1988). For example, the neomycin phosphotransferase gene
(NPTII) confers resistance to kanamycin (Horsch et al.
1985). Other markers include: hygromycin phosphotransferase
(HPT) giving resistance to hygromycin (Van Den Elzen et al.
1985), the bleomycin resistance gene confers resistance to
bleomycin (Hille et a1. 1986), the dihydrofolate reductase
gene (DHFR) confers meththotrexate resistance (Eichholtz et
al. 1987), and the chloramphenicol acetyltransferase gene
(CAT) confers resistance to chloramphenicol (Komari et a1.
1990). These markers are included within the 25-bp repeats
of the T-DNA, so that they too are transferred and
integrated into the plant cells (Watson et al. 1992). The
marker probably used most extensively is the NPTII gene
(Bevan 1984; Fraley et a1. 1983; Rogers et a1. 1988). Thus,
transformed plant cells carrying the NPTII gene are
kanamycin resistant such that they grow in the presence of

kanamycin (Klee et al. 1987; Misra 1990). Many engineered

6
co-integrated vectors also carry the nopaline synthase gene
(NOS), which provides a second marker for confirming
transformed cells and plants (Bevan et al. 1983; Horsch et
a1. 1985).

The chimeric NPTII gene has been very useful as a
selectable marker in transformation of different plant
species, including several Solanaceae such as Nicotiana
spy. (Klee et al 1987), Petunia (Horsch et al. 1985; Shah
et al. 1986), tomato (McCormick et al. 1986), potato
(Shahin et al. 1986; Stiekema et a1. 1988), and eggplant
(Guri et al. 1988). Numerous other dicot plants including
soybean, Brassica napus cv., and Arabidopsis have been
transformed to date (Misra 1990; Potrykus et al. 1991).

Kanamycin resistance has been used to optimize different
transformation systems: co-cultivation of cultured plant
cells with A; tumefaciens (Marton et al. 1979; De Black et
al. 1984), infection of leaf discs (Horsch et al. 1985),
callus transformation (An 1985), direct gene transfer to
protoplasts (Paszkowski et al. 1984; Saul et al. 1987), and
symmetric and asymmetric protoplast fusions (Toki et al.
1990; Bates et al. 1987). Kanamycin resistance has provided
a stringent selection means both at the cell and plant
levels (Herrera-Estrella et al. 1983a). The production of
transgenic plants can be divided into the following main
steps: introduction of genes into modified A; tumefaciens

strains; transformation of T-DNA into plant cells or

7
tissues; selection and regeneration of transformants; and
verification and analyses of gene expression in transformed
plants and the offspring (Rogers et a1. 1986; Power et al.
1986). Due to the efficiency of this selection procedure,
stable transformants can be routinely obtained in many

dicot species (Hauptmann et a1 1988).

T-DNA as a selectable marker in protoplast fusion

The T-DNA sequences can also be used as a marker for
selecting somatic hybrids. For example, the general utility
of T-DNA has already been demonstrated in both symmetric
and asymmetric nuclear genome transfers via protoplast
fusion (Bates et al. 1987; Ichikawa et al. 1990). The
transfer of the T-DNA of A; tumefaciens, previously
introduced into plant cells, via protoplast fusion from T-
DNA donor Nicotiana paniculata, to recipient N; tabacum has
been reported (Muller-Gensert et al. 1987). The hybrid cell
lines survived the kanamycin selection treatment by
exhibiting sustained growth. Dot-blot analysis showed that
besides the T-DNA other nuclear genomic DNA of the donor
species had also been transferred in various amounts.
Moreover, asymmetric somatic hybridization in Nicotiana by
fusion of irradiated and T-DNA inserted donor protoplasts
of N; plumbaginifolia with N; tabacum was reported by Bates
et al. (1987). Selection on kanamycin medium allowed the

recovery of asymmetric somatic hybrids retaining the

8
chromosome, or chromosome fragment(s), with the gene
encoding kanamycin resistance. A single chromosome can also
be transferred by donor—recipient fusion as long as the
donor contains a nuclear encoded marker gene (Dudits et a1.
1980; Gupta et al. 1984; Bates et al. 1987). T-DNA was
detected in asymmetric somatic hybrids between Brassigg
313;; and g; a us, and cytological analysis showed the
addition of donor chromosomes (Sacristan et al. 1989). The
T-DNA marker may also be used in the transfer of clustered
nuclear genes in the form of isolated chromosomes (Dudits
et a1. 1985).

Since the T-DNA becomes a marker for the tagged genes or
chromosomes, a system of selecting those genes and
chromosomes is available for somatic hybridization. Such a
system is possible by the use of T-DNA in a dual role as a
marker for plant chromosome or gene(s) of interest and as
cell level selectable trait (Herrera-Estrella et al. 1983b;
Misra 1990). Therefore, it might be possible to use the T-
DNA as a chromosome and/or gene marker in transformation

experiments with crop plants.

Site of integration of T-DNA

It is of practical and theoretical value to know whether
the T-DNA integrates only at a limited number of specific
sites or random throughout the host genome. It has been

reported that the T-DNA insertions integrate randomly into

9
the plant genome (Wallroth et al. 1986). Wallroth et al.
confirmed the presence of the expected T-DNA fragments in
six transformed Petunia plants. Southern hybridization and
genetic analyses showed that the size of the plant/T-DNA
junction fragments are different, thus, suggesting the
integrations occurred in different sites (Wallroth et a1.
1986). Agrobacterium-mediated T-DNA insertions in the
Lygopersicon, tomato, genome were at random locations based
on the genetic analysis (Chyi et al. 1986). Based on
genetic analysis, 10 independent T-DNA insertions were
located on 10 different chromosome loci. Herman et al.
(1990) also found that the T-DNA sequences integrated into
plant chromosomes at random positions. They measured the
frequency with which a promoterless reporter gene (NPTII)
is activated after T-DNA insertion into the Nicotiana
tabacum SR1 genome. When adjacent to the left or right T-
DNA border sequences, about 35% of the transformants
expressed the marker gene. Therefore, T-DNA integration is
random in respect to sequence specificity. Another evidence
for random insertion is that the integration of foreign
genes in the transgenic plant genome commonly occurs at
random sites. For example, Ambros et al. (1986b) detected
the locations of the T-DNA of the Ri plasmid in the host
chromosomes of Qrepis by in situ hybridization. The results
indicated that the T-DNA region of A; rhizogenes Ri plasmid

also integrated randomly in the four chromosomes of Crepis.

10

On the other hand, Paszkowski et al. (1988) presented
evidence for directed T-DNA integration into a predicted
location in the host plant genome. They constructed five
strains of transgenic tobacco plants containing chromosomal
copies of a non-functional, partially deleted APH(3')II
gene. Protoplasts of these plants were used for DNA-
mediated direct transformation with the missing part of the
gene in order to form an active gene. Molecular and genetic
data confirmed the integration of foreign DNA directed to
specific loci through homologous recombination.

The integration of previously missing sequences resulted
in the formation of an active gene in the host chromosome.
This suggested the possibility of integration of the T-DNA
into an homologous location in the plant genome. The
integration of Acrobacterium T-DNA into a plant chromosome
was also investigated in tobacco tumor cell lines
(Matsumoto et a1. 1990). Nucleotide sequence analysis
showed significant homology between the region adjacent to
the integration target site and both external regions of
the T-DNA breakpoints. In addition, a short stretch of
plant DNA near the integration site was deleted. This
deletion seems to have been promoted by homologous
recombination. Analyses of T-DNA/plant DNA junctions have
shown that in some cases plant sequences on both sides of
the integration site are homologous to the plasmid

sequences outside of the T-DNA breakpoints. These results

11
indicated that the T-DNA integrated by a mechanism similar
to that for homologous recombination. Therefore, sequence
homology between the incoming T-DNA and the plant
chromosomal DNA may have an important function in T-DNA
integration. The homology may promote close association of
both termini of T-DNA molecule on a target sequence; then
T-DNA may in some case be integrated by a mechanism at
least in part analogous to homologous recombination
(Matsumoto et al. 1990).

Genetic analysis has shown the deletion of the right
border abolishes tumorigenicity. This result indicates the
polarity of T-DNA transfer (Wang et al. 1984; Zambryski
1988). IN addition, Mouras and Negroid (1989) located the
T-DNA insertion site in a tobacco crown-gall line by in
situ hybridization: the hybridization signal was found on a
small metacentric chromosome. Chromosome and molecular
analyses showed there were specific chromosome
rearrangements in the transformed cells. The chromosome may
translocate onto other chromosomes to make marker
chromosomes through specific rearrangements. The insertion
of T-DNA is random within the plant genome and the activity
of the introduced genes may be affected by adjacent plant
DNA (Hobbs et al. 1990). The expression of the T-DNA
introduced genes was studied in ten tobacco transformants.
They found the T-DNA was apparently integrated at different

sites in the plant genome. The insertion site of the

12
foreign DNA (Km? in two independent transformed tobacco
plants was localized on different homologous chromosomal
pairs by in sign hybridization (Mouras et al. 1987).

When the genes of interest are introduced into the plant
genome in relatively high copy number, in order to have a
significant effect, this presents no technical problem.
Usually, multiple copies of T-DNA integrate at a single
random site in the plant chromosome (Watson et al. 1992).
The T-DNA can integrate in the form of tandem repeats
(Watson et a1. 1988). Transformed tobacco plants containing
3-5 multiple copies of the T-DNA sequence were analyzed at
the molecular level (Paszkowski et al. 1984) The genetic
(Potrykus et al. 1985) and in sign hybridization data
(Mouras et a1 1987) of the same plants confirmed that the
T-DNA was integrated at one chromosomal location in
multiple copies. Combined molecular and genetic analysis of
transformed SRl tobacco plants have shown that the
integration of the foreign DNA usually takes place at one
physical location within the genome as a multiple of both
functional and unfunctional copies (Negrutiu et al. 1987).
One transgenic Petunia plant was reported to have two
possible tandemly repeated T-DNAs (Fraley et a1. 1985).

On the other hand, Wallroth et al. (1986) reported that
another transformant Petunia possibly had two T-DNA
inserts. The integrations were in different sites of the

Petunia genome. In transformed Crepis plants, Southern

13
hybridization showed that each root line was the result of
one or more independent Ri T-DNA insertion events (Ambros

et al. 1986b). The results of in situ hybridization

 

indicated that the transformed root line contained one or
two A; zhigogenes Ri T-DNA insert(s) on the host

chromosomes (Ambros et al. 1986b).

T-DNA used as an insertion mutagen

The T-DNA is used as a gene/chromosome marker since the
T-DNA inserts can integrate into the host genome. T-DNA-
mediated gene tagging in plants was investigated on T-DNA-
linked mutation (Koncz et al. 1989). T-DNA is proving to be
a very efficient insertion mutagen that causes inactivation
of plant genes. The potential of T—DNA as an insertional
mutagen was studied in Arabidonsis (Lijsebettens et a1.
1991). Arabidopsis lines transformed with T-DNA vector were
generated by selection on kanamycin medium. One mutation,
named p£;, was found to affect mainly morphology of the
first seeding leaves and was mapped to chromosome 1. The
detection of recombination between 9;; and T-DNA, and
analysis of genetic distance suggested that the pf;
mutation was induced by insertion of the T-DNA.

Insertional mutagenesis may be used to tag genes (Marks
et al. 1989). Marks et al. found the mutation causing via
T-DNA insertion is tightly linked to a T-DNA insert.

Complementation analysis with genetically characterized

l4
trichome mutant revealed that the new mutation in an allele
of the GL1 locus. Thus, the T-DNA tagged with the GL1 gene
in Agabidopsis thaliana. Those T-DNA-linked mutations have
been selected and isolated. Furthermore, the use of T-DNA
as gene/chromosome marker was demonstrated in another
experiment. Transgenic tobacco plants carried copies of a
partial, non-functional kanamycin-resistance gene in the
nuclear DNA (Mouras et al. 1987). Such plants were used as
recipients for DNA molecules containing the missing part of
the gene. Molecular and genetic data confirmed that the
kanamycin resistant gene integrated into the protein coding
region, and resulted in the formation of an active gene.
The selection for kanamycin resistance conferred by the
marker gene made the detection of target gene possible

(Paszkowski et a1. 1988).

In sign hybridization

When T-DNA inserts are used for tagging genes of
interest, it is necessary to establish the location of the
T-DNA inserts among the chromosomes of a particular genome.
1n sign hybridization provides the means to detect and
localize the position of specific gene sequences and makes
it possible to visualize DNA fragments on metaphase
chromosomes (Huang et al. 1989). Since the first in sign
hybridization of nucleic acid probes was used by Gall et

al. (1969) to detect an RNA-DNA hybrid molecule on

15
chromosomes, this technique has undergone continuous
refinement. Some refinements have enhanced the efficiency
and sensitivity of hybrid detection (Shen et al. 1987).
Thus, it is possible to localize single gene sequences or
low copy numbers. The use of biotin-labeled probes produced
higher resolution and lower background interference than
did conventional in sign hybridization using isotope
labeling and autoradiography (Manuelidis et al. 1982;
Singer et al. 1982). The technique can indicate exactly
which chromosome(s) and the particular site(s) of the
chromosome(s) where specific DNA sequences are located
(Simpson et al. 1990).

The repetitive DNA sequences such as tandem repeats of
the pea SS rDNA in pea (Simpson et al. 1990), 188.268 rDNA
gene repeat unit in wheat (Mulai et al. 1991), and a TGRI
satellite repeat in tomato (Lapitan et a1. 1989) have been
detected. Low copy number and single-copy endogenous plant

genes have also been detected by i_ situ hybridization. A

 

6.6 kb of the chalcone synthase gene was observed on
chromosomes in parsley by in sign hybridization (Huang et
al. 1988). A legumin gene DNA fragment of 13.5 kb was
localized in pea (Simpson et al. 1988). To test the
sensitivity of the method, a cloned 10.8 kb genomic DNA
fragment encoding the waxy locus mRNA and the same DNA
fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and

0.8 kb were used for 1 situ hybridization on maize

 

16
pachytene chromosomes. The data showed that single copy
sequences longer than 1.9 kb could be detected at the
correct position (Shen et al. 1987). A 900 bp sequence from
a cDNA clone of the rye endosperm-storage-protain gene Sgg;
1 was visualized on the chromosome in rye (Gustafson et al.
1990).

The T-DNA sequences have also been localized in the host
genome by in SLLQ hybridization. A 17 kb T-DNA unique
sequence was detected on chromosomes of transformed Cgenis
anillaris by Ambrose et al. (1986a). The results indicated
that T-DNA was present in a different location in each root
line, and that each chromosome had been a target for T-DNA
insertion at least once (Ambrose et al. 1986b). A 5.4 kb
kanamycin resistance gene (Kmfl, with 3-5 copies, was
localized on different homologous chromosome pairs in
transgenic tobacco (Mouras et al. 1987). And, a 6 kb T-DNA
was detected in a tobacco crown-gall line by Mouras and
Negroid (1989). The hybridization signal was found on a
small metacentric chromosome, which could translocate onto
other chromosomes. The smallest target T-DNA sequence
detected to date was a 1-2 kb fragment (Mouras et al.

1989).

T-DNA in use of plant breeding
In spite of rapid developments in genetic engineering of

plants which have allowed many genes to be cloned and

17

successfully introduced into transgenic plants, most genes
of agronomic importance have not yet been isolated and
characterized. The genes that have been successfully
engineered to date are single-copy and well-defined,
whereas, agronomically important traits such as yield,
disease resistance and flower color may be controlled by
polygenes. Since the biochemical products of these genes
are not known, it will be some time before their isolation
for gene transfer is possible (Le et al. 1991). An
alternative method is to tag the gene(s) of interest. Thus,
the T-DNA inserts may be used as a selective marker on the
chromosome(s) mapping the gene of interest. Subsequently,
the tagged gene, chromosome or chromosome fragment may be
transferred by protoplast fusion. This approach provides an
efficient means for chromosome engineering of desired
traits in crops (Dudits et al. 1985). The system is
potentially applicable to crop species where Aggobacterium
based transformation and plant regeneration are available.

Many transgenic plants including Petunia have been
produced by the leaf-disc transformation method (Horsch et
al. 1985; Rogers et al. 1988). The fate of the foreign
genes in such transformants has been studied. Since
regenerates are often fertile and transmit the newly
inserted gene in a Mendelian fashion to the progeny (Horsch
et al. 1984; Rogers et a1. 1986; Uchimiya et a1. 1986),

transgenic plants can be readily used in genetic studies.

18

The objective of the current study was to use the in
sign hybridization technique to directly visualize the
location of T-DNA on the host chromosomes of transformed P;
nynnign plants. The distribution of T-DNAs among the
chromosomes was an necessary step for their further use in
chromosome tagging in cell fusion. 2; nybrida was chosen
as the material since it is one of the most-studied
dicotyledons in terms of both genetics and cytology and as
an experimental subject to investigate molecular aspects of
biological phenomena (Cornu et al. 1983). Hence, it has
become an ideal model system for molecular biological
studies. It has a diploid chromosome number of 2n=2x=14.
Flower color genetics has been well established and some of
these genes are mapped (Cornu 1984). Moreover, plant
transformation and protoplast regeneration have been
successful (Horsch et al. 1985; Frearson et al. 1973). But

the distribution of T-DNA in chromosomes by ;_ situ

 

hybridization has not been reported. Analysis of several
different transformed plants should allow a valid
comparison of independent T-DNA insertion events. In
addition, the growing root tips are an abundant source of
metaphase chromosomes, a feature which is essential for in

sign hybridization.

l9

LITERATURE CITED

Albright L, Yanofsky M, Leroux B, Ma D and Nester E. 1987.
Processing of the T-DNA of Agrobacterium gumefagiens
generates borders and linear, single-stranded T-DNA. J
Bacteriol 169:1046-1055.

Ambros P, Matzke M and Matzke A. 1986a. Detection of a
17 kb unique sequence (T-DNA) in plant chromosomes by in
situ hybridization. Chromosoma 94:11-18.

Ambros P, Matzke A and Matzke M. 1986b. Localization of AP

Inigogenes T-DNA in plant chromosome by in situ
hybridization. EMBO 5:2073-2077.

An G. 1985. High efficiency transformation of cultured
tobacco cells. Plant Physiol. 79:568-570.

Barton K, Binns A, Matzke A and Chilton M. 1983.
Regeneration of intact tobacco plants containing full
length copies of genetically engineered T-DNA and
transmission of T-DNA to R1 progeny. Cell 32:1033-1043

Bates G, Hasenkampf C, Contolini C and Piastuch W. 1987.
Asymmetric hybridization in Nicotiana by fusion of
irradiated protoplasts. Theor. Appl. Genet. 74:718-726.

Beven M, Barnes W and Chilton M. 1983. Structure and
transcription of the nopaline synthase gene region of T-
DNA. Nuc. Acids Res. 11:369-389.

Caplan A, Herrera-Estrella L, Inze D, Van Haute E, Van
Montagu M, Schell J and Zambryski P. 1983. Introduction of
genetic material into plant cells. Science 222:815-821.

Chilton M, Drummond M, Merlo D, Sciaky D, Montoya A,

Gordon M and Nester E. 1977. Stable incorporation of
plasmid DNA into higher plant cells: The molecular basis of
crown gall tumorigenesis. Cell 11:263-271.

Chyi Y-S, Jorgensen R, Goldstein D, Tanksley S and
Loaiza-Figueroa F. 1986. Locations and stability of
Agggnngggninn mediated T-DNA insertions in the
Lygonegsion genome. Molec. Gen. Genet. 204:46-49.

Cornu A and Maizonnier D. 1983. The genetics of Petunia.
Plant Breeding Reviews 2:11-57.

Cornu A. 1984. Genetics. In: Sink K. (Ed) Petunia 9:34-48.
Springer-verlag, Berlin.

20

De Black M, Herrera-Estrella L, Van Montagu M, Schell J and
Zambryski P. 1984. Expression of foreign genes in
regenerated plants and in their progeny. EMBO J 3:1681-1689.

De Block M. 1988. Genotype-independent leaf disc
transformation of potato using Agrobacterium
tnmefaciens. Theor. Appl. Genet. 76:767-774.

Dudits D, Fejer O, Hadlaczky G, Koncz C, Lazar G and
Horvath G. 1980. Intergeneric gene transfer mediated by
plant protoplast fusion. Molec. Gen. Genet. 179:283-288.

Dudits D and Praznoysky T. 1985. Intergeneric gene transfer
by protoplast fusion and uptake of isolated chromosomes. In:
Zaitlin M, Day P and Hollaender A. (Eds). Biotechnology in
Plant Science: relevance to agriculture in the eighties.
Academic Press. Orlando. p115-127.

Eichholtz D, Rogers 8, Horsch R, Klee H, Hayford M, Hoffmann
N, Braford S, Fink C, Flick J, O'Connell K and Fraley R.
1987. Expression of mouse dihydrefolate reductase gene
confers methotrexate resistance in transgenic Petunia
plants. Somatic Cell Molec. Gen. 13:67-76.

Fraley R, Rogers S, Horsch R, Sanders P, Flick J Adams
8, Bittner M, Brand L, Fink C, Fry J, Galluppi G,
Goldberg S Hoffmann N and Woo S. 1983. Expression of
bacterial genes in plant cells. Proc. Natl. Acad. Sci.
USA 80:4803-4807.

Fraley R, Rogers 5, Horsch R, Eichholtz D, Flick J, Fink C,
Hoffmann N and Saders P. 1985. The SEV system: a new
disarmed Ti plasmid vector for plant transformation.
Bio/Technology 3:629-635.

Fraley R, Rogers S and Horsch R. 1986. Genetic
transformation in high plants. CRC Critical Rev. Plant
Sci.4:1-46.

Frearson E, Power J and Cooking E. 1973. The isolation,
culture and regeneration of Petunia leaf protoplasts. Dev.
Biol. 33:130-137.

Gall J and Pardue M. 1969. Formation and detection of RNA-
DNA hybrid molecules in cytological preparations. Proc.
Natl. Acad. Sci. USA 63:378-383.

Gasswe C and Fraley R. 1989. Genetically engineering
plants for crop improvement. Science 244:1293-1299.

Gheysen G, Dhaese P, Van Montagu M and Schell J. 1985.
B.Hohn and E.S. Dennis, Genetic flux in plants, Advances in

21
plant gene research. 2:11-17 Springer-Verlay, Wien.

Greve H, Dhaese P, Seuinck J, Lemmers M, Van Montagu M
and Schell J. 1983. Nucleotide sequence and transcript
map of the Agrobacterium tumefaciens Ti-plasmid encoded
octopine synthase gene. Molec. Appl. Genet. J. 1:499-511.

Gupta P, Schieder O and Gupta M. 1984. Intergeneric
nuclear gene transfer between somatically and sexually
incompatible plants through asymmetric protoplast
fusion. Molec. Gen. Genet. 197:30-35.

Guri A and Sink C. 1988. Agrobacterium transformation
of eggplant. J. Plant Physiol. 133:52-55.

Gustafson J, Butler E and McIntyre C. 1989. Physical mapping
of a low-copy DNA sequence in rye (Secale cereale L.). Proc.
Natl. Acad. Sci. USA. 87:1899-1902.

Hauptmann R, Vasil V, Ozias-Akins P, Tabaeizdeh Z, Rogers 5,
Fraley R, Horsch R and Vasil I. 1988. Evaluation of
selection markers for obtaining stable transformants in the
gramineae. Plant Physiol. 86:602-606.

Herman L, Jacobs A, Van Montagu M and Depicker A. 1990.
Plant chromosome/marker gene fusion assay for study of
normal and truncated T-DNA integration events. Molec. Gen.
Genet. 224:248-256.

Herrera-Eatrella L, De Block M, Messens E, Hernalsteens J,
Van Monyagu M and Schell J. 1983a. Chimeric genes as
dominant selectable markers in plant cells. EMBO J. 2:987-
995.

Herrera-Estrella L, Depider A, Van Montagu M and Schell J.
1983b. Expression of chimeric genes transferred into plant
cells using a Ti-plasmid-derived vector. Nature. 303:209-
213.

Hille J, Verheggen F, Roelvink P, Franssen H, Kammen A and
Zabel P. 1986. Bleomycin resistance: a new dominant
selectable marker for plant cell transformation. Plant
Molec. Biol. 7:171-176.

Hobbs S, Kpodar P and DeLong C. 1990. The effect of T-DNA
copy number, position and methylation on reporter

gene expression in tobacco transformants. Plant Molec.
Biol. 15:851-864.

Hohn B, Koukolikova-Nicola Z, Bakkeren G and Grimnsley N.
1989. Agrobacterium-mediated gene transfer to monocots and
dicots. Genome 31:987-993.

22

Hooykaas P and Schilperoort R. 1984. The molecular genetics
of crown gall tumorigenesis. Adv. Genet. 22:209-230.

Horsch R, Fraley R, Rogers S, Sanders P, Lloyd A and
Hoffmann N. 1984. Inheritance of functional foreign genes in
plants. Science 223:496-498.

Horsch R, Fry J, Hoffmann N, Eichholtz D, Rogers S and
Fraley R. 1985. A simple and general method for
transferring gene into plants. Science 227:1229-1231.

Horsch R, Fry J, Hoffmann N, Neidermeyer J, Rogers 8 and
Fraley R. 1988. Leaf disc transformation. Plant molec.
biology. Manual. 5:1-9.

Huang P-L, Hahlbrock K and Somssich E. 1988. Detection
of a single-copy gene on plant chromosomes by in sitn
hybridization. Molec. Gen. Genet. 211:143-147.

Huang P-L, Hahlbrock K and Somssich E. 1989.
Chromosomal localization of parsley 4-coumarate: CoA
ligase genes by in situ hybridization with a
complementary DNA. Plant Cell Reports 8:59-62.

 

Ichikawa H and Imamura J. 1990. A highly efficient selection
method for somatic hybrids which used an introduced dominant
selectable marker combined with iodoacetamide treatment.
Plant Sci. 67:227-235.

Kerr A, Manigault P and Tampe J. 1977. Transfer of
virulence in vivo and in vitro in Agrobacterium. Nature
265:560-561.

Klee H, Gordon M and Nester E. 1982. Complementation
analysis of Agrobacterium tumefaciens Ti plasmid mutations
affecting oncogenicity. J. Bacteriol. 150:327-331.

Klee H, Horsch R and Rogers S. 1987. Agrobacterium-mediated
plant transformation and its further application to plant
biology. Ann. Rev. Plant Physiol. 38:467-486.

Komari T, Saito Y, Nikaido F and Kumashiro T. 1990.
Efficient selection of somatic hybrids in Nicotiana tabacum
L. using a combination of drug-resistance markers introduced
by transformation. Theor. Appl. Genet. 77:547-552.

Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z,

Korber H, Redei G and Schell J. 1989. High-frequency T-DNA-
mediated gene tagging in plants. Proc. Natl. Sci. USA
86:8467-8471.

Koukolikova-Nicola Z, Albright L and Hohn B. 1987. The

23

mechanism of T-DNA transfer from Agrobacteriun
tumefaciens to plant cell. In Hohn T and Schell J. Plant
gene research. 4:109-148. Springer, Wien.

Lapitan N, Ganal M and Tanksley S. 1989. Somatic chromosome
karyotype of tomato based on in sitn hybridization of the
TGRI satellite repeat. Genome 32:992-998.

Le H and Armstrong K. 1991. in situ hybridization as a
rapid means to assess meiotic pairing and detection of
alien DNA transfer in interphase cells of wide crosses
involving wheat and rye. Molec. Gen. Genet. 225:33-37.

Ledger S, Deroles S and Given N. 1991. Regeneration and
Agrobgcgerium-mediated transformation of Chrysanthemum.
Plant Cell Reports. 10:195-199.

Lichtenstein C and Fuller 8. 1987. Vectors for the
genetic engineering of plants. Genetic engineering
6:104-171.

Lijsebettens M van, Vanderhaeghen R and van Montagu M.
1991. Insertional mutagenesis in Arabidonsis thaliana:
isolation of a T-DNA-linked mutation that alters leaf
morphology. Theor. Appl. Genet. 81:277-284.

Manuelidis L, Langer-Safer P and Ward D. 1982. High-
resolution mapping of satellite DNA using biotin-labeled DNA
probes. J. Cell. Biol. 95:619-625.

Marks M and Feldmann K. 1989. Trichome development in
Anabidopsis thaliana. 1. T-DNA tagging of the GLABROUSI
gene. Plant Cell. 1:1043-1050.

Marton L, Wullems G, Molendijk L and Schiperoort R. 1979. In
vitro transformation of cultured cells from Nicotiana
tabacum by Agrobacterium tumefaciens. Nature 227:129-130.

Matsumoto S, Ito Y, Hosoi T, Takahashi Y and Machida Y.
1990. Integration of Agrobacterium T-DNA into a tobacco
chromosome: Possible involvement of DNA homology between T-
DNA and plant DNA. Molec. Gen. Genet. 224:309-316.

McCormick M, Niedermeyer J, Fry J, Baranson A, Horsch R and
Fraley R. 1986. Leaf disc transformation of cultivated
tomato using A; tumefaciens. Plant cell Rep. 5:81-84.

Misra, S. 1990. Transformation of Brassica napus L. with a
disarmed octopine plasmid of Agrobacterium tumefaciens:
Molecular analysis and inheritance of the transformation
phenotype. J. Exp. Bot. 41:269-275.

24

Mouras A, Saul M, Essad S and Potrykus I. 1987.
Localization by in situ hybridization of a low copy
chimeric resistance gene introduced into plants by
direct gene transfer. Molec. Gen. Genet. 207:204-209.

Mouras A, Negroid I, Orth M and Jacobs M. 1989. From
repetitive DNA sequences to single-copy gene mapping in
plant chromosomes by in sitn hybridization. Plant Physiol.
Biochem. 27:161-168.

Mouras A and Negroid I. 1989. Localization of the T-DNA
on marker chromosomes in transformed tobacco cells by in
sign hybridization. Theor. Appl. Genet. 78:715-720.

Mukai Y, Endo T and Gill B. 1991. Physical mapping of the
188.288 rRNA multigene family in common wheat:Identification
of a new locus. Chromosoma 100:71-78.

Muller-Gensert E and Schieder O. 1987. Interspecific T-DNA
transfer through plant protoplast fusion. Molec. Gen. Genet.
208:235-241.

Negroid I, Shillito R, Potrykus I, Biasini G and Sala
F. 1987. Hybrid genes in the analysis of transformation
condition. Plant Molec. Biol. 8:363-373.

Nester E, Gordon M, Amasino R and Yamada Y. 1984. Crown
gall: a molecular and physiological analysis. Ann. Rev.
Plant Physiol. 35:387-413.

Otten L and Schiperoort R. 1978. A rapid microscale
method for the detection of lysopine and nopaline
dehydrogenase activities. Biochem. Biophys. Acta.
527:497-500.

Paszkowski J, Shillito R, Saul M, Mandak V, Hohn T, Hohn B
and Potrykus I. 1984. Direct gene transfer to plants. EMBO J
3:2717-2722.

Paszkowski J, Baur M, Bogucki A and Potrykus I. 1988. Gene
targeting in plants. EMBO J. 7:4021-4026.

Paul J and Hooykaas J. 1988. Agrobacteriun molecular
genetics. Plant Molec. Biol. Manual. 4:1-13.

Perani L, Radke S, Wilke-Douglas M and Bossert M. 1986. Gene
transfer methods for crop improvement: Introduction of
foreign DNA into plants. Physiol. Plant. 68:566-570.

Potrykus I, Paszkowski J, Saul M, Petruska J and Shillito R.
1985. Molecular and general genetics of a hybrid foreign
gene introduced into tobacco by direct gene transfer. Molec.

25
Gen. Genet. 199:169-177.

Potrykus I. 1991. Gene transfer to plants: Assessment of
published approaches and results. Ann. Rev. Plant Physiol.
42:205-225.

Power J, Davey M, Freeman J, Mulligan B and Cocking E.
1986. Fusion and transformation of plant protoplasts.
Methods in Enzymol. 118:578-594.

Reynaerts A, De Block A, Hernalsteens J and Montagu M.
1988. Selectable and screenable markers. Plant Molec. Bio.
Manual. 9:1-16.

Rogers 8, Horsch R and Fraley R. 1986. Gene transfer in
plants. Methods in Enzymol. 118:627-640.

Rogers 8, Klee H, Horsch R and Fraley R. 1988. Use of
cointegrating Ti plasmid vectors. Plant Molec. Biol. Manual
2:1-12.

Sacristan M, Gerdemann M and Schieder O. 1989. Incorporation
of hygromycin resistance in Bgassica nigra and its transfer
to a. nanus through asymmetric protoplast fusion. Theor.
Appl. Genet. 78:194-200.

Saul M, Paszkowski J, Shillito R and Potrykus I. 1987.
Methods for direct gene transfer to plant. Plant Physiol.
Biochem. 25:361-364.

Shah D, Horsch R, Klee H, Kishore G, Winter J, Turner N,
Hironaka C, Sanders P, Gasser S, Aybent N, Siegel 8, Rogers
8 and Fraley R. 1986. Engineering herbicide tolerance in
transgenic plants. Science 233:478-481.

Shahin E and Simpson R. 1986. Gene transfer system.
HortScience 21:1199-1201.

Shen D-L, Wang Z-F and Wu M. 1987. Gene mapping on maize
pachytene chromosomes by in situ hybridization. Chromosoma
95:311-314.

Simpson P, Newman M-A and Davies R. 1988. Detection of
legumin gene DNA sequences in pea by in situ
hybridization. Chromosoma 96:454-458.

Simpson P, Newman M-A, Davies R, Ellis N and Matthews P.
1990. Identification of translocations in pea by in sitn
hybridization with chromosome-specific DNA probes. Genome
33:745-749.

Singer R and Ward D. 1982. Actin gene expression visualized

26

in chicken muscle tissue culture by using in sitn
hybridization with a biotinated analog. Proc. Natl. Acad.
Sci. 79:7331-7335.

Sitbon F, Sundberg B, Olsson O and Sandberg G. 1991. Free
and conjugated indoleacetic acid (IAA) contents in
transgenic tobacco plants expressing the innM and innH IAA
biosynthesis genes from Agrobacterium tumefaciens. Plant
Physiol. 95:480-485.

Stachel S and Zambryski A. 1986. Aggobacterium, gumefggigns
and the susceptible plant cell: a novel adaption of
extracellular recognition and DNA conjugation. Cell. 47:155-
157.

Stiekema W, Heidekamp F, Louwerse J, Verhoeven H and
Dijkhuis P. 1988. Introduction of foreign genes into potato
cultivars Bintje and Desiree using an Agrobacterium
tumefaciens binary vector. Plant Cell Rep. 7:47-50.

Toki 8, Kameya T and Abe T. 1990. Production of a triple
mutant, chlorophyll-deficient, streptomycin-, and kanamycin-
resistant Nicotiana tabacum, and its use in integenetic
somatic hybrid formation with Solanum melongena. Theor.
Appl. Genet. 80:588-592.

Uchimiya H, Hirochika H, Hashimoto H, Hara A, Masuda T,
Kasumimoto T, Harada H, Ikeda J.-E and Yoshioka M. 1986. Co-
expression and inheritance of foreign genes in transformants
obtained by direct DNA transformation of tobacco
protoplasts. Molec. Gen. Genet. 205:1-8.

Van Den Elzen P, Towsend J, Lee K and Bedbrook J. 1985. A
chimeric hygromycin resistance gene as a selectable marker
in plant cells. Plant Molec. Biol. 5:299-302.

Wallroth M, Gerats A, Rogers 8, Fraley R and Horsch R.
1986. Chromosomal localization of foreign genes in
Petunia hybrida. Molec. Gen. Genet. 202:6-15.

Wang K, Herrera-Estrella L, Van Montagu M and Zambryski P.
1984. Right 25 bp terminus sequence of the nopaline T-DNA is
essential for and determines direction of DNA transfer from
Agrobacterium to the plant genome. Cell 38:455-462.

Wang K, Stachel 8, Timmerman B, Van Montagu M, Zambryski P.
1987. Site-specific nick in the T-DNA border sequence as a
result of Agrobacterium yin gene expression. Science
235:587-591.

Watson J, Hopkins N, Roberts J, Steitz J and Weiner A. 1988.
Molecular biology of the gene. p817-831. The

27
Benjamin/Cummings Publishing Company, Inc.

Watson J, Gilman M, Witkowski J and Zoller M. 1992.
Genetic engineering of plants. Recombinant DNA. W.H.
Freeman and Company.

Yamamoto A, Iwahashi M, Yanofsky M, Nester E, Takebe I and
Machida Y. 1987. The promoter proximal region in the yinD
locus of Agrobacterium. tumefaciens in necessary for the
plant-inducible circularization of T-DNA. Molec. Gen. Genet.
206:174-177.

Yanofsky M, Porter 8, Young C, Albright L, Gordon M and
Nester E. 1986. The yiPD operon of Aggobagteriun,
Iumefagiens encodes a site-specific endonuclease. Cell
47:471-477.

Zambryski P,Joos H, Genetello C, Leemmans J, Van Montagu M
and Schell J. 1983. Ti plasmid vector for the introduction
of DNA into plant cells without alteration of their normal
regeneration capacity. EMBO J 2:2143-2150.

Zambryski P. 1988. Basic processes underlying Agrobacterinn-
mediated DNA transfer to plant cells. Ann. Rev. Genet
22:130.

Localization of T-DNA Inserts on Petunia nybzida

Chromosomes by in situ Hybridization

INTRODUCTION

The production of transgenic plants using Agrobactegium
:nngfngigng as a vector is well established for many
species (Klee et al. 1987; Potrykus 1991). The introduced
T-DNA sequences are known to integrate stably into plant
chromosomes (Perani et al. 1986; Matsumoto et al. 1990). As
well, the T-DNA loci have been genetically mapped and found
to be transmitted in a Mendelian manner in two solanaceous
genera (Chyi et al. 1986; Wallroth et al. 1986). These
genetic analyses indicated that the T-DNA inserts were
distributed randomly in chromosomes of the Lycongrsicon and
Pegunia genomes, respectively. Although the T-DNA inserts
have been used principally as a vector of genes to date,
they also show promise as a tag of genes (Andre et al.
1986), or of a chromosome or chromosome fragment(s)
(Wallroth et al. 1986) and as a cell selectable trait via
an antibiotic resistance. Such a role may be particularly
useful for the introduction or identification of
economically important genes where the molecular biology is
not established. Thus, an efficient means to localize the

T-DNA insert sites and determine their distribution among

 

chromosomes of transgenic plants is needed. I situ

28

29
hybridization is such a technique that has potential for
this purpose.

In gign hybridization allows for visualization of
labelled DNA fragments on the chromosomes, and has already
been successfully used on plants (Rayburn et al. 1985;
Huang et al. 1989). In SLED hybridization provides an
efficient means for mapping and physical identification of
chromosomes, chromosomal segments, and transformed genes
(Bergey et al. 1989). To date, In sign hybridization has
already allowed both T-DNA and genomic DNA sequences as
small as 1-2 kb to be detected (Mouras et al. 1989; Shen et
al. 1987). For example, a legumin gene DNA sequence and a
multiple copy rDNA gene were identified in pea (Simpson et
al. 1988; 1990). The chalcone synthase gene was localized
on parsley chromosomes (Huang et al. 1988). Ambros et al.
(1986a) identified the site of a 17 kb A; rhizogengs Ri T-
DNA on the chromosomes of a transformed Crepis root line.
The detection of T-DNA inserts containing the K_n' gene on
the chromosomes of transformed tobacco cells was reported
by Mouras et al. (1987), the Km'insertion sites were found
to be localized on different homologous chromosome pairs in
two independent transformed tobacco plants. Recently, a 0.9
kb DNA fragment was detected on the chromosomes of rye
(Gustafson et al. 1990). In addition, In sign hybridization
has been used for detecting species-specific repetitive DNA

in order to identify recombined donor chromosome fragments

30

in asymmetric somatic hybrid plants (Piastuch et al. 1990).

While most studying In sIIn hybridization indicates
that the detection of foreign DNAs in the plant genomes,
the distribution of physical location for the insertion
sites has only been reported for A; rhizogenes Ri T-DNA in
transformed Qrenis canillaris plants (Ambros et al. 1986b).
The Ri T-DNA inserts in four separate transformed g;
gnnIIInnIs (2n=2x=6) were distributed randomly among all
three chromosomes.

In this study, i

situ hybridization was used to

 

visualize the location and distribution of T-DNA inserts on

chromosomes of ten independent transgenic plants of P;

112321219;-

MATERIALS AND METHODS

Petunia karyotype

The VR (R51 X V23) hybrid of Petunia h brida, a gift
from T. Gerads, Vrije University, was examined
cytologically to establish the karyotype. Root tips were
collected in dHZO and pretreated at 0°C for 24 h. They were
fixed in 3:1 ethanol:acetic acid (v/v) solution at 4°C for
24 h. After rinsing with distilled water, the root tips
were incubated in 1.0 M HCl at 6UT!for 10 min, rinsed again
in distilled water and placed on a microscope slide. A drop

of 1% acetocarmine in 45% acetic acid was added and the

3l
meristematic region which was cut off from the root tip was
gently squashed. Chromosomes were observed on a Carl Zeiss
microscope (CARL ZEISS, Oberkochen/West Germany) using 40X
objective and phase contrast. Photographs were taken on

Kodak Technical Pan film (ASA 125)(Eastman Kodak Company).

Transformation and Confirmation

The VR hybrid was transformed with plasmid pMON200 (9.5
kb) which contains chimeric NOS:NTPII:NOS genes. The
Agrobacterium tumefaciens containing pMON200 was cultured
as described by Guri et al. (1988). The transformation was
carried out by the leaf-disc method of Horsch et a1.
(1985), with some modifications. The leaves were sliced
into 1.0 x 0.6 cm sections and soaked in the bacterial
culture about 5 min. to ensure that all leaf edges were
infected by the bacteria. The inoculated explants were
placed on solidified MS salts and vitamins medium
(Murashige et al. 1962) plus benzyladenine 1.0 ug/ml,
naphthaleneacetic acid 0.1 ug/ml, and 100 ug/ml kanamycin
without a nurse culture. Uninoculated VR hybrid leaf
explants were cultured identically as the control.

To confirm transgenic status, 50 regenerated plants, the
original shoots taken from separate sites of origin, were
tested for the presence of kanamycin resistance by leaf-

disc and rooting assays as described by Rogers et al.

32
(1986). Analysis of nopaline synthase activity in leaf
tissue was done according to Otten et al. (1978) except
that the samples were run at 300 V for 80 min. For slot
blot hybridization, total genomic DNA was extracted from
young leaf tissue of putative transformed and non-
transformed plants using the procedure of Dellaporta et al.
(1985). An isolated 900 bp Pstl fragment of plasmid pBlOS,
encoding the NPTII sequence, was used as the probe (gift
from Dr. W. Kopachik, Michigan State Univ.). The probe was
32P-dCTP labelled (approx. 1 X 108 cpm) by a random primed
DNA labeling system according to the supplier's instruction
(Boehringer Mannheim Inc.). Slot blot hybridization was

performed as described by Rivin (1986).

In Situ Hybgidization

Ten independent transgenic plants that were confirmed by
the previous four assays were used for the detection of T-
DNA inserts by In sIIn hybridization. The root tips of
transformed and non-transformed plants grown in the
greenhouse were collected 10 to 14 days after the plants
had been repotted. The plants were watered 18-24 h before
the root tips were taken, and the root tips were harvested
at 10:30 am. Samples for In §I§n hybridization were
prepared according to the method described by Mukai et al.
(1990) with several minor modifications. The root tips were

pretreated in water at JTBfor 18-20 h and fixed in 3:1

33
ethanol:glacial acetic acid (v/v) at mmrfor 24 h. After
washing with distilled water, the root tips were incubated
in 1.0 M HCl for 15 min at 3Tktand rinsed in distilled
water. They were soaked in 0.7% acetocarmine in 45% acetic
acid solution for 30 to 60 min and placed in 45% acetic
acid on acid-cleaned slides. The meristematic region cut
off from a single root tip was gently squashed under a
acid-cleaned coverslip, placed on dry ice for 10 to 15 min
followed by removal of the coverslip. The slides were
dehydrated in an ethanol series (70-95-100%, 5 min each)
and then allowed to air dry.

The 900 bp NPTII gene fragment described previously was
used as the probe to visualize the T-DNA inserts. The probe
was labelled by nick translation with biotin-16-dUTP
according to the instructions of the supplier (Borhringer
Mannheim GmbH, West Germany).

The protocol of hybridization and post-hybridization
washes followed that of Rayburn et al. (1985) with a few
changes. The chromosomal DNA on the slides was denatured by
incubation in 70% formamide, 2x SSC at 70TrfOr 2 min. The
slides were immediately dehydrated through prechilled
(-2W%n 70, 95 and 100% ethanol for 5 min each and then air
dried. The hybridization solution consisted of 50%
deionized formamide, 10% dextran sulfate, 2x 88C, 500 ug/ml
salmon sperm DNA, 500 ug/ml yeast tRNA, and 2.5 ng/ul

biotinylated probe DNA. This mixture was denatured in

34
boiling water for 10 min and quenched on ice. The
hybridization solution, 15 ul, was applied per slide and
covered with an acid-cleaned coverslip. Slides were
incubated at 37°C in a humidified chamber for 12-16 h.
Following hybridization, the samples were washed twice in
2X SSC at 25°C, each for 15 min, 2x SSC at 37°C for 10 min,
2x SSC at 23%:for 10 min, 0.1% Triton in phosphate-buffer
saline (PBS) at Zymrfor 2 min and finally in phosphate-
buffer saline (PBS) at zyttfor 5 min; twice.

After PBS post-hybridization washing, the slides were
drained, but not allowed to dry during the detection
process. The DETEK I-hrp signal generating system (ENZO
Diagnostics, Inc.) was used for locating hybridization
sites. The supplier's instructions were followed except
that the DETEK I-hrp complex was diluted 1:150 with 1X
complex dilution buffer. To reduce background
hybridization, slides were additionally washed at Zyrtin 4x
SSC, 0.1% Triton in 4X SSC, 4X SSC, 10 min for each
solution. The chromosomes were stained with 2% Giemsa for 5
min and a coverslip positioned using double distilled water
as the mounting medium.

To test the procedure of In sIIn hybridization working
well, a ribosomal DNA of wheat (gift of Dr. H.J. Price and
Mr. Charles Crane, Texas A&M University) was used as a
probe on Petunia.

The chromosomes and hybridization regions were observed

35
on a Zeiss 10 Laser Scanning Confocal Microscope (Carl
Zeiss, West Germany) by using 100x objective and
brightfield. Black-and-white photographs were taken with

Kodak T-max (ASA 100)(Eastman Kodak Company).

RESULTS AND DISCUSSION

Both transgenic and non-transformed VR hybrid plants had
2n=2x=14 chromosomes which is consistent with previous
reports for P; hybrida (Maizonnier 1984). In order to
assign the T-DNA inserts to specific chromosomes, it was
necessary to distinguish all 7 pairs. Maizonnier et a1.
(1979) and Smith et al. (1973) previously described the
Petunia karyotype and the VR hybrid is consistent with
their description (Fig. 1). Briefly, chromosome I is the
longest; with a median centromere. Chromosome II is nearly
the same length as chromosome I, but has a sub-telocentric
centromere and satellites of varying size. Chromosome III
is similar to chromosome II with respect to length and
centromere position but without satellites. Chromosome IV
is slightly shorter than the three preceding ones and has a
sub-median centromere. The centromere of chromosome V is
intermediate between sub-telocentric and sub-median. Since
chromosome VI is very similar to chromosome V, they can not
be distinguished by morphology. Maizonnier (1984) also
reported that chromosomes V and VI of Petunia were

indistinguishable although they were discerned by Smith et

36
al. (1972; 1973) through fluorescence. Chromosome VII is
the shortest and has a median centromere.

Calluses appeared on pMON200 inoculated leaf explants in
2 weeks; between 1 to 2 weeks later about 5 to 10 shoots
arose per explant (Fig. 2). When the shoots were
subcultured on root-inducing medium, the shoots grew roots
in the presence of 100 ug/ml kanamycin (Fig. 3). Non-
transformed leaf explants did not produce callus or shoots
on MS with kanamycin at 100 ug/ml and eventually turned
yellow and died.

Leaf explants taken from 49/50 independent putative
transformed plants formed callus on MS medium containing
300 ug/ml kanamycin (Fig. 43, Table I). Conversely, leaves
from non-transformed plants did not produce callus (Fig.
4A). Nopaline was detected in leaf extracts of 48/50
transformed plants, but not in a non-transformed plant
(Fig. 5, Table I). Horsch et al. (1985) reported that 20/25
transformed VR hybrid Petunia plants were resistant to
kanamycin, and nopaline was detected in 17/25 of those
plants. The loss of expression of kanamycin resistance and
nopaline synthase genes in certain transformants may be due
to influences from surrounding DNA or chromatin structure
at the site of insertion of the foreign genes into a plant
chromosome (Horsch et al. 1985). Of 50 regenerated plants,
37 (74%) had a positive slot blot assay (Fig. 6, Table I).

Some putative transformants that were negative in the slot

37
blot assay may be due to incomplete transformation or the
T-DNA may be lost (Klee et al. 1987). The slot blot assay
also revealed various T-DNA intensities. Such plants may
contain variable numbers of T-DNA inserts. The 37 verified
transgenic plants were eventually grown in the greenhouse.
They were pollen fertile and had leaf and flower morphology
identical to control plants. The results of plant
transformation and confirmation were shown in the Appendix.

Ten of 37 plants which were positive in all three
transformation assays were used for In gILn hybridization.
The signals obtained with the T-DNA probe by In sIIn
hybridization were visible as twin spots, one on each
chromatid of a single chromosome (Fig. 7). The T-DNA
locations on the chromosomes were detected in three
independent cells of every plant.

For each plant, a signal was clearly present at a
particular chromosomal location. In plants 1 and 6, the
signals were present at the satellite of chromosome II
(Fig. 7A & 7F). The T-DNA insert of plant 2 integrated into
the middle of an arm of chromosome I (Fig. 78). For plant
4, the signal was found near the telomere of chromosome I
(Fig. 7D). The signal observed in plant 5 was at a
centromere position on chromosome V or VI (Fig. 7E). In
plants 3, 7 and 8, the T-DNA inserts were localized at the
telomere of the short arm of chromosomes V or VI (Fig. 7C),

chromosome VII (Fig. 7G) and chromosome IV (Fig. 7H),

38

respectively. The T-DNA insert in plant 10 was integrated
into a proximal position of the short arm of chromosome III
(Fig. 7J). In plant 9, two T-DNA inserts were observed on
two different chromosomes: One insert was near the end of
chromosome IV, and the other integrated in the long arm of
chromosome III (Fig. 71). As expected, no signal of T-DNA
was observed in the non-transformed plant (Fig. 8). The

summary of verifying assays for transformation and

 

detection of the T-DNA inserts by I_ situ hybridization
were shown in Figure 9 and Table 1.

The signal of ribosomal DNA was observed on chromosome
II proximal to the satellite in a non-transformed plant
(Fig. 10). As a rule, the ribosomal DNA is located in the
nucleolus organizing region (NOR) (Mukai et al. 1991). This
was the case herein where the NOR region in Petunia is
cytologically discerned as a secondary constriction
associated with a nucleolus and a distal satellite on
chromosome II. Therefore, the signals of rDNA were obtained
at expected positions.

Sometimes, the results were interfered by the background
hybridization. Based on the followings, we may distinguish
the signals of T-DNA from the background. The signal was
twin spots (Fig. 7C, black arrow indicated), but the
interference was not (Fig. 7C, white arrow indicated). The
color of the signal visualized on microscope was black-

brown twin spots, and the dirt was seen to be black single

39
spot. Also, the signal was observed at the same position in
the repeated experiment, however, the site of background
hybridization was not repeatable.

The important factors contributing to the success of In
gIgn hybridization in this study mainly involved in the
following aspects. First one was the preparation of clean
metaphase chromosome spreads from root tip cells. The
signals observed by In sIIn hybridization only occurred on
those chromosomes and nuclei which were free of cellular
contaminants such as cell wall and cytoplasmic debris.
Similarly, Ambros et al. (1986a), using plant protoplasts
to prepare chromosome spreads for In §I§n hybridization,
found that those preparations without the cell wall were a
prerequisite for visualizing the Ri T-DNA signal. In
addition, vigorous cell division in root-tip cells was
found at 10:30 am and good cell division phases were also
found at 12:30 am and 4:00 pm. Watering the plants 18-24
hours before collection at 10:30 am helped to obtain more
dividing cells.

Secondly, the degree of background hybridization
influenced the efficacy of detecting the T-DNA probe
signals. Thus, an additional post-hybridization wash of
slides in 4X SSC, 0.1% Triton in 4x SSC, 4X SSC before
Giemsa staining helped to eliminate such interference.

Since the procedure of i situ hybridization were

 

enzymatic reaction, the third important factor was giving

40
longer reacting time and supplying enough substrate for
each reaction. Thus, more products were formed and the size
of the signal were enhanced.

In situ hybridization allowed the clear identification

 

and localization of T-DNA sequences in the chromosomes of
transgenic P; hybrida plants. Since this technique allowed
direct visualization of T-DNA sequences in the host
chromosomes, it provides direct information not otherwise
obtained from slot blot or Southern blot analyses.

Likewise, 1 situ hybridization eliminates the lengthy

 

process of location by genetic mapping such as the work of
Chyi et a1. (1986). A non-radioactive probe detection

system was used in In situ hybridization because it also

 

permitted rapid detection of hybridized probe sequences at
different chromosomal locations (Rayburn et a1. 1985).

The T-DNA sequence which is integrated into the host
plant chromosomes from pMON200 is about 7.8 kb (Rogers et

al. 1986; 1988). The i situ hybridization procedure

 

allowed to detect clearly and localize the sites of T-DNA,
present in the chromosomes of transgenic plants, with a
target size of around 7.8 kb with 900 bp NPTII fragment as
probe. T-DNA inserts have already been detected by In gInn
hybridization with the length of target from 17 kb (Ambros
et al. 1986a) to 1-2 kb (Mouras et al. 1989). Single-copy
target sequences as short as 0.9 kb in plant (Gustafson et

al. 1990) and as 0.5 kb have been localized on human

41
chromosomes by In sILn hybridization (Jhanwar et al. 1983).
The results in the study indicate that T-DNA can
integrate into numerous locations in PetunIg chromosomes.
In addition, two T-DNA inserts were found in the genome of
plant 9. Previous results obtained from Southern blot
experiments have shown that T-DNA inserts can integrate
into either unique or repetitive plant DNA sequences
(Fraley et al. 1985; Wallroth et al. 1986). Wallroth et al.
(1986) mapped the genetic locations of the T-DNA inserts in
nine independent transgenic VR hybrid Petunia, and reported
that all of which are in unique sites distributed over four
of the seven chromosomes. Of those, 7/9 plants had one T-
DNA insert, one plant had two T-DNA inserts, and another
plant had two possible tandemly repeated T-DNAs. Their data
on Southern hybridization and genetic analysis showed that
the T-DNA inserts were distributed among chromosomes I,
III, IV and V, and suggested that the integration was at
random, but not distributed equally over the different
PgtnnIa chromosomes. However, the locations of T-DNA
inserts obtained by our In sInn hybridization study
indicated not only that the T-DNA integrates randomly into
Pgtnnia chromosomes but also that each chromosome appears
equally likely to be a target. Similar results were
obtained in a study of the Ri T-DNA integrations in
transgenic Crepis plants (2n=6) by In §I§n hybridization

(Ambros et al. 1986b), whose results indicated that the T-

42
DNA inserted into all 3/3 chromosomes in four independent
transformed root lines. For the T-DNA position, some
signals were found near the telomere of chromosomes (three
of five), and the T-DNA also integrated into chromosomes
(two of five).

The hybridization intensity of slot blot analysis showed
that independent transgenic plants contained different T-
DNA copy numbers. The 10 plants which had a high intensity
hybridization (+++ or ++) with the T-DNA probe in slot blot
assay were chosen to localize the sites of T-DNA inserts by
In gIIn hybridization. However, the signal of T-DNA was not
observed in the plants which had low intensity
hybridization (+) of slot blot. Furthermore, most of
transgenic plants (9/10) revealed only one T-DNA insert
except plant 9 that had two T-DNA inserts present on
different chromosomes. These results suggested that usually
T-DNA integrates into a single site, 9 plants. The possibly
that two or more tandemly repeated T-DNAs inserted in
ngnnIn as has been reported (Fraley et al. 1985). Our
results do not excluded the possibility that two or more
tandemly repeated copies are present in a single site.
Tandemly repeat T-DNA sequences were also found at a single
position in chromosomes of transgenic tobacco plants
(Mouras et al. 1987). On the other hand, occasionally two
T-DNA inserts were present on separate chromosomes, in one

Petunia plant (plant 9). Ambros et al. (1986b) reported

43
that a transgenic Crepis plant contained two T-DNA inserts
on separate chromosomes. Thus, genetic analyses support
both insertion patterns which were found in Petunig (Fraley
et al. 1985; Wallroth et al. 1986).

The signals of T-DNA inserts were detected and
visualized by In sIgn hybridization procedure used in this
study not only on metaphase chromosomes but also in
interphase chromosomes. This result indicates that the
visualization of the T-DNA sequences is possible in
structurally well-preserved plant nuclei. These detections
give further confirmation of T-DNA fragment integrating
into the plant genome. In plants 1 and 2, while one signal
of T-DNA insert was detected on the metaphase chromosomes,
one hybridization signal was obtained in the interphase
nuclei. Furthermore, two separate signals of T-DNAs were
observed in a nucleus of transgenic plant 9 in which two T-
DNA inserts were present on different chromosomes. Le et
al. (1991) reported that detection of alien DNA of
interphase chromosomes may permit rapid decision-making and
efficiency in plant breeding.

The study of localization and distribution of T-DNA
sequences on plant chromosomes is only an initial step for
their use in plant breeding. The long term goal is to
improve crop species by transferring genes of agronomic
importance such as disease resistance and flower color

which are hard to isolate but have been genetically mapped.

44
The approach presented in this study has such potential
applications. Since T-DNA inserts were present in different
chromosomal location in each transgenic plant, and each
chromosome has been a target for T-DNA insertion at least
once, T-DNA inserts could be used as a selectable marker at
the chromosome level. The method described would make it
possible to identify individual chromosomes or chromosome
fragments containing the genes of interest with T-DNA as a
probe. Using a T-DNA marked donor species, genes of
interest could be transferred by asymmetric hybridization
with T-DNA as a selectable marker as already demonstrated

by Bates et al. (1987) and Toki et al. (1990).

suamcmucu some u++
huflmcmvcfl 304 "+
fluoHn uon

 

45

 

Bum unocm mo canvas HHH ++ + + OH
mumeoawu you: >H
sum mcoa no muscue a HHH +++ + + m
whosoamu >H ++ + + w
ouosoamu HH> ++ + + h
mafiaawumm HH +++ + + m
ouosouucmo H>\> +++ + + m
mumsoamu you: H +++ + + v
mumsoaop H>\> +++ + + m
Bum ecu mo maccwe H +++ + + m
onwaamumm HH ++ + + H
AmvmfiomOEouco co Hones: .auwmcmucfl ammmm
cowuflmom «zone geomoaouco uoan uon onwammoz omflciummq .oz ucmam

 

.MHmmMMN Ufiunxn m> oflcwmwcmuu Cw mwsomoeounu co mcowumuoa czols
can uoan uoam .COwuoscoum mafiammoc .mocmumflmmu swoafimcmx uo coflumuwufium> .H wanna

46

I! M M n I: r; 8:

tr Kit in In: 1: u» n
II n u u a: u n

It A). A!“ In M u

:1 K! n n u an: n
‘i n u u u n In

I II III N V 0' VI VII

__ “ _—_ -_ —__—___._.__‘_ .—

 

Figure 1. The karyotype of P; hybrida, the genotype VR

47

 

Figure 2. Leaf explant transformation of Petunia VR hybrid
co-cultivated with pMON200. (A) Leaf explants placed on MS
medium containing kanamycin 100 ug/ml, (B) Callus and
regenerated shoots, after three weeks in culture.

48

 

Figure 3. Transgenic Petunia plant 2 rooted in MS
containing 100 ug/ml kanamycin.

49

 

Figure 4. Leaf explants after three weeks in culture from
A) non-transformed and B) transgenic Petunia VR hybrid
plants on MS medium containing 300 ug/ml kanamycin.

50

 

Figure 5. Nopaline assay on non-transformed and transformed
leaf tissue. Arrow indicates spot representing nopaline.
Lane 1: Arginine marker. Lane 2: Untransformed plant. Lane
3-8, 10-13: Independent transgenic plants 9, 8, 2, 3, 5, 6,
1, 4, 7 and 10 in order. Lane 14-15: Another two transgenic
Petunia plant. Lane 9 and 16: Nopaline marker.

 

C) «D (D ‘4 CD (I 4}, gg hi .1

' r

d

dd
h) .1

Figure 6. Slot-blot analysis of Petunia transformants. The
blots were probed with NPTII gene fragment labeled with
dCTP 32P. Lane 1: Positive control. Lane 2: Negative
control. Lanes 3-12: independent transgenic plants 1 to 10.

 

 

 

Figure 7. In situ hybridization of NPTII fragment as probe
to metaphase chromosomes of Petunia plants. The signals
obtained in plants 1 to 10 are shown in photographs A to J.
Black arrows indicate the locations of T-DNA insert. White
arrow indicates background hybridization

1 of 3 pages

 

 

 

 

d , ,r 5‘. o .
I z
w \a

Figure 7
e
3“
C
3
b
l.

2 of 3 pages

54

Figure 7. cont'd

 

3 of 3 pages

 

55

 

Figure 8. In situ hybridization with NPTII probe in
untransformed P; hybrigg. No twin spots indicating on
chromosomes.

56

 

I II III IV V or VI VII

 

 

 

 

 

 

 

Figure 9. Localizations of T-DNA inserts by In situ
hybridization indicating on the karyogram of Petunia in 10
independent transgenic plants. The T-DNA locations labelled
with arrows. Arabic numbers indicated plants 1-10.

 

"‘;\

 

 

57

Figure 10. In situ hybridization with ribosomal DNA probe
on non-transformed P; hybrida chromosomes. The rDNA
fragment was detected in the nucleolar organizer region
associated with the satellites. Arrows indicate the
signals. '

LI ST OF REFERENCES

58

LITERATURE CITED

Ambros P, Matzke M and Matzke A. 1986a. Detection of a
17 kb unique sequence (T-DNA) in plant chromosomes by In
sign hybridization. Chromosoma 94:11-18.

Ambros P, Matzke A and Matzke M. 1986b. Localization of
A; anzogenes T-DNA in plant chromosome by In situ
hybridization. EMBO 5:2073-2077.

Andre D, Colau D, Schell J, Van Montagu M and Hernalsteens
J-P. 1986. Gene tagging in plants by a T-DNA insertion
mutagen that generates APH(3')II-plant gene fusions. Molec.
Gen. Genet. 204:512-518.

Bates G, Hasenkampf C, Contolini C and Piastuch W. 1987.
Asymmetric hybridization in Nicotiana by fusion of
irradiated protoplasts. Theor. Appl. Genet. 74:718-726.

Bergey D, Stelly D, Price J and McKnight T. 1989. In sitn
hybridization of biotinylated DNA probes to cotton meiotic
chromosomes. Stain Technol. 64:25-37.

Chyi Y-S, Jorgensen R, Goldstein D, Tanksley S and Loaiza-
Figueroa F. 1986. Locations and stability of Agrobacterium
mediated T-DNA insertions in the Lycopersion genome. Molec.
Gen. Genet. 204:46-49.

Dellaporta S, Wood J and Hicks J. 1985. Maize DNA miniprep.
In: Molecular biology of plants. p.36-37. Cold Spring
Harbor. New York.

Fraley R, Rogers 8, Horsch R, Eichholtz D, Flick J,
Fink C, Hoffmann N and Saders P. 1985. The SEV system:
a new disarmed Ti plasmid vector for plant
transformation. Bio/Technology 3:629-635.

Guri A and Sink K. C. 1988. Agrobactegium
transformation of eggplant. J. Plant Physiol. 133:52-
55.

Gustafson J, Butler E and McIntyre C. 1989. Physical
mapping of a low-copy DNA sequence in rye (Secale
ggrealg L;). Proc. Natl. Acad. Sci. USA. 87:1899-
1902.

Horsch R, Fry J, Hoffmann N, Eichholtz D, Rogers 8 and
Fraley R. 1985. A simple and general method for
transferring gene into plants. Science 227:1229-1231.

59

Huang P-L, Hahlbrock K and Somssich E. 1988. Detection
of a single-copy gene on plant chromosomes by In sItn
hybridization. Molec. Gen. Genet. 211:143-147.

Huang P-L, Hahlbrock K and Somssich E. 1989.
Chromosomal localization of parsley 4-coumarate: CoA
ligase genes by In situ hybridization with a
complementary DNA. Plant Cell Reports 8:59-62.

Jhanwar S, Neel B, Hayward W and Chaganti R. 1983.
Localization of c-ras oncogene family on human germ
line chromosomes. Proc. Natl. Acad. Sci. USA 80:4794-
4797.

Klee H, Horsch R and Rogers 8. 1987. Agrobacterium-
mediated plant transformation and its further
application to plant biology. Ann. Rev. Plant Physiol.
38:467-486.

Le H and Armstrong K. 1991. In situ hybridization as a
rapid means to assess meiotic pairing and detection of
alien DNA transfer in interphase cells of wide crosses
involving wheat and rye. Molec. Gen. Genet. 225:33-37.

Maizonnier D and Moessner A. 1979. Localization of the
linkage groups on the seven chromosomes of the Petunia
hybrida genome. Genetica 51,2:143-148.

Maizonnier D. 1984. Cytology. In: Sink K (Ed)
Petunia. Springer-Verlay Berlin Heidelberg. p21-33.

Matsumoto S, Ito Y, Hosoi T, Takahashi Y and Machida
Y. 1990. Integration of Agrobacterium T-DNA into a
tobacco chromosome: Possible involvement of DNA
homology between T-DNA and plant DNA. Molec. Gen.
Genet. 224:309-316.

Mouras A, Saul M, Essad S and Potrykus I. 1987.
Localization by in situ hybridization of a low copy
chimeric resistance gene introduced into plants by
direct gene transfer. Molec. Gen. Genet. 207:204-209.

Mouras A, Negrutiu I, Horth M and Jacobs M. 1989. From
repetitive DNA sequences to single-copy gene mapping
in plant chromosomes by In situ hybridization.

Plant Physiol. Biochem. 27:161-168.

Mukai Y, Endo T and Gill B. 1990. Physical mapping of
the 58 rRNA multigene family in common wheat. J
Hered. 81:290-295.

Mukai Y, Endo T and Gill B. 1991. Physical mapping of

60

the 188.268 rRNA multigene family in common wheat:
Identification of a new locus. Chromosoma 100:71-78.

Murashige T and Skoog F. 1962. A revised medium for
rapid growth and bioassays with tobacco tissue
cultures. Physiol. plant. 15:473-479. 373.

Otten L and Schiperoort R..1978. A rapid

microscale method for the detection of lysopine and
nopaline dehydrogenase activities. Biochem. Biophys.
Acta. 527:497-500.

Perani L, Radke S, Wilke-Douglas M and Bossert M.
1986. Gene transfer methods for crop improvement:
Introduction of foreign DNA into plants. Physiol.
Plant. 68:566-570.

Piastuch W and Bates G. 1990. Chromosomal analysis of
Nicotiana asymmetric somatic hybrids by dot blotting
and In situ hybridization. Molec. Gen. Genet. 222:97-
103.

Potrykus I. 1991. Gene transfer to plants: Assessment
of published approaches and results. Ann. Rev. Plant
Physiol. 42:205-225.

Rayburn A and Gill B. 1985. Use of biotin-labeled
probes to map specific DNA sequences on wheat
chromosomes. J Hered. 76:78-81.

Rivin, C. 1986. Analyzing genome variation in plants.
Methods Enzymol. 118:75-86.

Rogers 8, Horsch R and Fraley R. 1986. Gene transfer
in plants. Methods Enzymol. 118:627-640.

Rogers 8, Klee H, Horsch R and Fraley R. 1988. Use of
cointegrating Ti plasmid vectors. Plant Molec. Biol.
Manu. 2:1-12.

Shen D-L, Wang Z-F and Wu M. 1987. Gene mapping on
maize pachytene chromosomes by In situ hybridization.
Chromosoma. 95:311-314.

 

Simpson P, Newman M-A and Davies R. 1988. Detection of
legumin gene DNA sequences in pea by In situ
hybridization. Chromosoma 96:454-458.

Simpson P, Newman M-A, Davies R, Ellis N and Matthews
P. 1990. Identification of translocations in pea by In
sItu hybridization with chromosome-specific DNA
probes. Genome 33:745-749.

61

Smith F and Oud J. 1972. The possibility to
distinguish chromosomes of Petunia hybrida by
quinacrine fluorescence. Genetica 43:589-596.

Smith F, Oud J and Jong J. 1973. A standard karyogram
of Pgtunig hybrida. Hort. Genetica 44:474-484.

Toki S, Kameya T and Abe T. 1990. Production of a
triple mutant, chlorophyll-deficient, streptomycin-,
and kanamycin-resistant Nicotiana tabacum, and its use
in intergeneric somatic hybrid formation with SgIgnnm
nelongena. Theor. Appl. Genet. 80:588-592.

Wallroth M, Gerats A, Rogers 8, Fralay R and Horsch R.
1986. Chromosomal localization of foreign genes in
Petunia hybrida. Molec. Gen. Genet. 202:6-15.

APPENDIX

.wanmu many :fl 30cm no: can A+++V amcu .mzna .cowUMchwunxn
mwflw mfl you poms mums A+++v aufiwcmucw scan cmsocm now£3 mucmHQ m "muoz
szmcmucfl maccwau++ Ie
aufimcmDCw 30H "+ In
m>Hummmc "I IN
w>fluwmcom "m «mucmumwmou "m IH

 

62

 

+ + m 0v I + m on
I + m an I .. _m ma
I + m mm + + m ma
++ + m hm + + m SH
I + m cm ++ + m ma
+ + m mm I + m ma
I + m cm + + m «a
I + m mm + + m ma
++ + m mm I + m NH
I + m an + + m HH
+ + m on I + m OH
I + m an ++ + m m
I + m mm I + m m
++ + m hm v++ + m b
+ + m mm + + m o
I + m mm + + m m
I I m vm + + m w
+ + m mm + + m m
+ + m mm n+ + m m
+ + m Hm «I + R a
cachamcmx CAU>Emcmx.
huwmcmucfl ou hmmmm auwmcmucw on xmmmm

HOHQ UOHm OGMHMQOZ UmHOIumOA Hanan uOHQ UOHm OCHHMQOZ Umflfilumwd ucmam

 

.ummu uoHn uoam can :«uwamcmx on uncommon
.cowuuscoum unwamaoc you mucmsuoumcmuu muwmmNd 4N no muaammu >mmm< .H manna

GRN STRTE UNIV. L

“iijIIII

IBRQRIES
lil Will I llllllllililillilliiil
0 706

12 3 08978