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LSU Historical Dissertations and Theses
Graduate School
1992
Transgenic Rice Plants Containing a Chlorsulfuron
Resistance Gene, a Hygromycin Resistance Gene
and the Maizeactivator Transposable Element.
Zhijian Li
Louisiana State University and Agricultural & Mechanical College
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Li, Zhijian, "Transgenic Rice Plants Containing a Chlorsulfuron Resistance Gene, a Hygromycin Resistance Gene and the
Maizeactivator Transposable Element." (1992). LSU Historical Dissertations and Theses. 5329.
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Transgenic rice plan ts containing a chlorsulfuron resistan ce
gene, a hygrom ycin resistan ce gene and th e m aize activator
tran sp osab le elem ent
Li, Zhijian, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1992
300 N. Zeeb Rd.
Ann Arbor, MI 48106
TRANSGENIC RICE PLANTS CONTAINING A CHLORSULFURON
RESISTANCE GENE, A HYGROMYCIN RESISTANCE GENE AND THE
MAIZE ACTIVATOR TRANSPOSABLE ELEMENT
A Dissertation
Submitted to the Graduate Faculty o f the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Plant Pathology and Crop Physiology
by
Zhijian Li
B.S., South China Agricultural University, 1981
M.S., Louisiana State University, 1988
May 1992
ACKNOW LEDGEM ENTS
I wish to express my sincere appreciation to my major advisor Dr. N. Murai
for his invaluable guidance, support and encouragement throughout my entire graduate
training program. I acknowledge the support of this research in part by the Louisiana
State Educational Quality Support Fund.
I also wish to acknowledge gratefully the members of my advisory committee,
Drs. M. E. Musgrave, M. C. Rush, J. B. Baker, P. T. Evans and C. W. Kennedy for
their persistent interest and constructive advise. I am especially grateful to Drs. M. E.
Musgrave and P. T. Evans for their perceptive and helpful comments and criticisms
on manuscripts related to this research. Also, I sincerely thank Drs. M. C. Rush and
J. B. Baker for their generous assistance in experiments involving the use of
greenhouse and field space and facilities.
I am grateful to my friends and colleagues, Drs. A. Hayashimoto, K.
Sathasivan, W. S. Lee, M. D. Burow, Q. J. Xie, Messrs. S. Footit, Z. W. Zheng, and
Y. Kawagoe for their cheerful help and encouragement.
I am thankful to the staff in the Department of Plant Pathology and Crop
Physiology, LSU, for their kind assistance during my graduate study.
I am especially indebted to my late mother S. Y. Lang for her love, care and
sacrifice. I am thankful to my father D. X. Li for the love and encouragement I
received. Finally, I would like to express my sincerest gratitude to my wife D. Li and
my daughter B. Li for their true love, sacrifice and encouragement that were
indispensable to the completion of my graduate study in the United States.
TABLE O F CONTENTS
Page
ACKNOWLEDGEMENTS
ii
LIST OF TABLES
v
LIST OF FIGURES
vi
ABSTRACT
vii
CHAPTER
I.
II.
INTRODUCTION
1
A NOVEL SELECTABLE MARKER FOR RICE
TRANSFORMATION
5
PHENOTYPIC AND MOLECULAR EVALUATION OF
RICE PLANTS CONTAINING THE HPH GENE
AND THE MAIZE AC TRANSPOSABLE ELEMENT
7
LITERATURE CITED
9
A SULFONYLUREA HERBICIDE RESISTANCE GENE
FROM ARABIDOPSIS THALIANA AS A NEW
SELECTABLE MARKER FOR TRANSGENIC RICE
13
INTRODUCTION
14
MATERIALS AND METHODS
15
RESULTS
21
DISCUSSION
32
CHAPTER SUMMARY
36
LITERATURE CITED
37
iii
III.
IV.
PHENOTYPIC EFFECTS ON RICE PROTOPLAST
REGENERATION AND TRANSFORMATION WITH
THE HPH GENE AND THE MAIZE ACTIVATOR ELEMENT
40
INTRODUCTION
41
MATERIALS AND METHODS
42
RESULTS
45
DISCUSSION
64
CHAPTER SUMMARY
67
LITERATURE CITED
68
SUMMARY AND CONCLUSION
70
APPENDIX A.
PERMISSION ACKNOWLEDGEMENT
74
APPENDIX B.
REPRINT PERMISSION FROM THE PUBLISHER 75
APPENDIX C.
A PROTOCOL FOR RICE TRANSFORMATION
VITA
76
93
iv
L IST O F TABLES
Effect of chlorsulfuron selection on colony recovery
after transformation of Nipponbare protoplasts
with plasmids pTRA132, pTRA133 and pTRA153
Major agronomic traits of field-grown seed-derived
plants and progeny of protoplast-derived non transformed
plants and transgenic plants {Nipponbare) containing
the hph gene and the maize Ac element
Inheritance of hygromycin resistance in progeny of
transgenic rice plants containing the hph gene
Summary of six major agronomic traits of field-grown
progeny of nontransformed controls and transgenic
rice plants containing the hph gene and
the maize Ac element
L IST O F FIGURES
Figure
Page
2.1
Schematic structure of pTRA133 and pTRA153 plasmids
17
2.2
Hybridization probes and predicted hybridization
fragments based on the structure of pTRA133 and pTRA153
20
Southern blot hybridization of total DNA from ALS
gene-containing cell lines with the Arabidopsis
ALS gene probe
22
Southern blot hybridization of total DNA from ALS
gene-containing cell lines with the hph gene probe
24
Callus growth of cell lines transformed with and
without the mutant Arabidopsis ALS gene in the
presence of chlorsulfuron
26
Effect of amino acids on the restoration o f callus
growth in the presence of chlorsulfuron
27
Northern blot analysis of the mutant Arabidopsis
ALS transcripts in transformed rice cells
29
Inhibition o f the growth of 14-day-old protoplastderived microcalli by chlorsulfuron
30
Protoplast-derived calli after selection with hygromycin B
or chlorsulfuron
31
Hybridization probes and predicted hybridization
fragments based on the structure of pTRA137R before
and after Ac transposition
46
Southern blot hybridization of total DNA from transgenic
plants containing the hph gene (pTRA132)
55
Southern blot hybridization of total DNA from transgenic
rice plants containing the maize Ac element (pTRA137R)
60
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.1
3.2
3.3
vi
ABSTRACT
A mutant Arabidopsis acetolactate synthase (ALS) gene csr-1-1, conferring
chlorsulfuron resistance, was tested in transgenic rice to develop an alternative
selectable marker for rice transformation. Up to a 200-fold increase in chlorsulfuron
resistance was detected in transgenic rice calli expressing the mutant ALS gene under
control of the CaMV 35S promoter. Efficient selection of resistant protoplast colonies
was achieved by using 10 to 50 nM of chlorsulfuron after transformation with the
35S/ALS gene.
Fertile transgenic rice plants were successfully recovered from
35S/ALS-containing transformants. These results demonstrated that the 35S/ALS gene
could be used as a selectable marker gene in rice transformation.
Progeny of protoplast-derived nontransformed rice plants and transgenic rice
plants containing the hygromycin phosphotransferase (hph) gene and the maize
activator (Ac) transposable element were investigated in a field experiment.
Comparison with seed-derived plants for six major agronomic traits indicated that
nontransformed protoplast-derived plants were essentially indistinguishable from the
parent cultivar, suggesting that somaclonal variation in these plants was not significant.
Hph-containing transgenic plants were relatively normal. The generally low fertility
in these plants may have been caused by the hygromycin selection procedure. In the
majority of transgenic rice plants, the hph gene was inherited as a single dominant
locus. Transferred DNA was maintained stably in the rice genome through the third
generation of selfing. Ac-containing transgenic plants produced a significantly higher
frequency of phenotypic variations than protoplast-derived nontransformed plants and
transgenic plants containing the hph gene. In some varied traits such as sterility and
albinism were segregated as a single locus among transgenic progeny and may have
been associated with the presence of an active Ac element. This field experiment
enabled us to assess the traits of transgenic rice plants for transposon tagging and
obtain useful mutant materials.
CHAPTER I
INTRODUCTION
1
In recent years, transgenic plant systems have greatly facilitated the investigation
of gene expression and regulation. However, the use of transgenic dicotyledonous plants
such as tobacco, sunflower and petunia frequently generated difficulties in the
interpretation of monocotyledonous gene expression. For example, a wheat gene for the
ribulose 1,5-bisphosphate carboxylase small subunit (rbcS) did not express in transgenic
tobacco (Keith and Chua, 1986). In a similar case, an oat phytochrome gene did not
show any transcription activity in transgenic tobacco and Nicotiana plumbaginifolia
(Keller et al. 1989). More recently, Schaffner and Sheen (1991) showed that the presence
of at least six constitutive and light sensitive upstream regulatory elements was required
for the expression of the maize rbcS gene in monocot cells, while the evolutionarily
conserved GT and G boxes of dicot rbcS promoters in pea, tobacco, and Arabidopsis were
not essential for light responsive expression in monocot leaf cells. Moreover, unexpected
patterns of transcript splicing and tissue-specific expression of monocot genes in
transgenic dicot plants were frequently observed (Lloyd et al. 1991, Ueng et al. 1988,
Egeland et al. 1989). These lines of evidence demonstrate that substantial differences in
gene regulation may exist between dicot and monocot plant species.
We are interested in developing a transgenic rice system for the study of monocot
genes. Rice (Oryza sativa L.) has a number of characteristics advantageous for molecular
genetic studies.
Rice is diploid (2n=24) with a relatively small genome size. Over 50% of the rice
DNA sequences are single copy DNA. Recently, by using flow cytometry measurement,
Arumuganathan and Earle (1991) revealed that rice cells had a diploid nuclear DNA
3
content ranging from 0.86 to 0.96 pg, while the diploid DNA content of Arabidopsis was
0.30 pg. Thus, the small genome, only about three times as large as Arabidopsis, should
facilitate gene cloning experiments and subsequent expression analysis in rice.
Due to its importance as a major food crop in the world, rice has been the subject
of extensive genetic and cytological investigations. A large collection of rice germplasm
(^200,000 accessions) has been made.
Chromosomal linkage map and restriction
fragment length polymorphism (RFLP) maps with various morphological, isozyme and
genomic DNA sequence markers have become available for this crop (Kinoshita 1986,
Khush and Singh, 1986, Wu et al. 1988, Ranjhan et al. 1988, McCouch et al. 1988). The
diversity of germplasm materials and extensive genetic information will be beneficial for
studies aiming at understanding plant genes.
A prerequisite for molecular genetic studies using transgenic plants is an efficient
transformation system. Although the transformation of rice has been achieved through
infection with Agrobacterium tumefaciens (Raineri et al. 1990), DNA application to pollen
tubes (Luo and Wu, 1989), electroporation (Toriyama et al. 1988, Shimamoto et al. 1989),
and polyethylene glycol (PEG) treatment of protoplasts (Zhang and Wu, 1988, Yang et
al. 1988), the transformation efficiencies reported have been relatively low (up to about
4 x 10'5). Only a few groups have been able to regenerate a limited number of fertile
transgenic plants (Shimamoto et al. 1989). Thus, these reported procedures are hardly
applicable for the generation of sufficient numbers of transgenic plants for gene
expression studies.
We had previously developed an efficient and reproducible rice protoplast
regeneration system based on the use of General medium and the nurse culture method
(Li and Murai, 1990). By using this regeneration system, a PEG-mediated protoplast
transformation protocol was defined (see Appendix C). Important parameters such as
PEG concentrations, DNA concentrations, gene promoters, and hygromycin selection after
transformation were optimized (Hayashimoto et al. 1990). This transformation system
enabled us to obtain transformation frequencies ten times higher than those reported by
other groups and to regenerate a large number of fertile transgenic rice plants
(Hayashimoto et al. 1990).
In spite of the significant improvement, the use of this transgenic rice system for
gene analysis was still hampered by a number of problems. The availability of only a
few selectable markers suitable for rice transformation generated limitations in further
improvement of the selection and regeneration of transgenic rice plants and restricted the
development of more complicated designs of expression. Furthermore, no genetic study
has been conducted with transgenic rice plants. The usefulness of transgenic rice plants
for molecular genetic studies involving foreign genes remains undetermined.
This
dissertation research was developed to address these problems with the goal of improving
the established transgenic rice system.
5
A NOVEL SELECTABLE MARKER FOR RICE TRANSFORMATION
In transformation experiments, transformants with foreign genes are often
identified by using marker genes. These marker genes provide a selectable phenotype
necessary for transformed cells to outgrow a large number of nontransformed cells under
selection conditions. Thus far, a number of marker genes have been developed for plant
transformation, including those conferring resistance to kanamycin (Bevan et al. 1983,
Herrera-Estrella et al. 1983); hygromycin (Gritz et al. 1983, Waldron et al. 1985);
gentamicin (Hayford et al. 1988); bleomycin (Hille et al. 1986); methotrexate (Simonsen
and Levinson, 1983); and phosphinothricin (DeBlock et al. 1987).
However, most marker genes successfully used in dicot plants can not be readily
adapted to rice. The use of the kanamycin resistance gene has been unreliable due to a
high level of endogenous resistance to kanamycin (up to 500 mg/1) in rice cells (Dekeyser
et al. 1989). Rice calli transformed with a mutant mouse dihydrofolate reductase (dhfr)
gene displayed poor growth after selection with methotrexate (Dekeyser et al. 1989).
Until now, the hygromycin phosphotransferase (hph) gene has been the only useful
selectable marker gene for rice transformation. Hygromycin selection has resulted in
efficient recovery of transformed rice calli and regeneration of normal transgenic plants
(Shimamoto et al. 1989, Hayashimoto et al. 1990), even though sterility problems in
transgenic rice plants might be related to the use of hygromycin (Li et al. 1990, and see
Chapter 3).
To improve the selection process and to increase the number of selectable markers,
it is necessary to identify new selectable genes and develop novel selection systems useful
for rice transformation. Here we tested a mutant Arabidopsis acetolactate synthase (ALS)
gene conferring resistance to chlorsulfuron for its potential as a selectable marker for rice
transformation.
The chlorsulfuron-resistant mutant Arabidopsis ALS gene, designated as csrl-1,
was first isolated and characterized by Haughn and Somerville (1986). It was found that
the mutant ALS gene contained a C to T conversion at nucleotide 870 relative to the
initiation codon of the ALS gene, leading to an amino acid substitution from proline to
serine within a conserved region of the ALS protein (Haughn et al. 1988, Mazur et al.
1987). The mutant ALS gene conferred a high level of resistance by producing an altered
form of the ALS enzyme with a lower affinity to chlorsulfuron, a competitive inhibitor
for the ALS enzyme (Haughn et al. 1988).
The objective of this study was to evaluate the chlorsulfuron-resistance conferred
by the mutant Arabidopsis ALS gene under the native Arabidopsis promoter, or a
cauliflower mosaic virus 35S promoter, in transgenic rice and to determine the effective
concentrations of chlorsulfuron for efficient selection of transformed rice protoplasts.
PHENOTYPIC AND MOLECULAR EVALUATION OF
TRANSGENIC RICE PLANTS CONTAINING THE HPH
GENE AND THE MAIZE AC TRANSPOSABLE ELEMENT
A large number of transgenic rice plants have been produced through the
established transformation procedures (Hayashimoto et al. 1990, Li et al. 1990).
However, transgenic rice plants have not yet been characterized to determine whether the
transformation procedures employed are capable of generating normal transgenic rice
plants.
It has been well documented that somaclonal variations occur frequently during
in vitro culture of rice cells (Oono 1981). Nineteen percent of the plants derived from
protoplasts of five cultivars produced abnormal phenotypes (Ogura et al. 1987).
Somaclonal variations were caused by chromosomal changes, DNA rearrangement and
modification, and other unknown processes occurred during the culture period (Lee and
Phillips, 1988, Ogura et al. 1987, Muller et al. 1990, Brown et al. 1990, LoSchiavo et al.
1989). The presence of somaclonal variation could be detrimental to the subsequent use
of transgenic rice plants.
The genetic stability of transgenic rice plants becomes more important in
transposon tagging experiments using introduced transposable elements (Baker et al. 1986,
Yoder et al. 1988, Knapp et al. 1988, VanSluys et al. 1987). In our effort to establish a
transposon mutagenesis system in rice, we introduced the maize activator (Ac)
transposable element into transgenic rice plants to overcome the lack of defined
endogenous transposable elements.
DNA analysis of primary transgenic rice plants
demonstrated the transposition of the maize Ac element in the rice genome (Murai et al.
1991). However, the successful isolation of unknown genes from transgenic rice plants
depends on the identification of unique phenotypes associated with the transposition and
reintegration of the Ac element.
In this study, by comparing the phenotypes of progeny of transgenic rice plants
containing the hygromycin phosphotransferase (hph) gene or the maize Ac element and
seed- and protoplast-derived plants under field conditions, we wish to determine the level
of somaclonal variation associated with the protoplast regeneration procedures, mutations
caused by insertion of the transferred DNA after transformation and phenotypic diversity
derived from transposition of the maize Ac element in transgenic rice plants.
9
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12
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CHAPTER II
A SULFONYLUREA HERBICIDE RESISTANCE GENE FROM
ARABIDOPSIS THALIANA AS A NEW SELECTABLE MARKER
FOR TRANSGENIC RICE
13
14
INTRODUCTION
Selectable marker genes facilitate selection of transformed cells from a larger
number of nontransformed counterparts. Several selectable marker genes have been tested
for the transformation of rice (Dekeyser et al. 1989, Hauptmann et al. 1988). However,
the hygromycin phosphotransferase {hph) gene has been the only selectable marker
capable of supporting consistent recovery of transformed cells and efficient production
of fertile transgenic rice plants (Shimamoto et al. 1989, Hayashimoto et al. 1990).
Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic
pathway of branched-chain amino acids leucine, isoleucine and valine.
Sulfonylurea
herbicides inhibit ALS as a competitive inhibitor. A mutant ALS gene (csr-1-1) was
isolated from a sulfonylurea-resistant mutant line GH50 Arabidopsis thaliana (Haughn
and Somerville 1986). The mutation was caused by a single base pair conversion from
C to T at nucleotide 870 relative to the initiation codon in the ALS coding sequence and
leads to a proline to serine substitution in a conserved region of the amino acid sequence
of ALS (Haughn et al. 1988).
The mutant enzyme had much lower affinity to
sulfonylurea herbicides in Arabidopsis (Haughn et al. 1988, Mazur et al. 1987).
To increase the number of alternative selectable markers, the mutant Arabidopsis
ALS gene was introduced into transgenic rice. Our objectives were to determine the
chlorsulfuron resistance in transgenic rice conferred by the mutant ALS gene under
control of either the native Arabidopsis promoter or the CaMV 35S promoter and to
identify the effective concentrations of chlorsulfuron for efficient selection of transformed
rice protoplasts.
15
MATERIALS AND METHODS
Plant M aterials, Plasmids, and Bacterial Strains
Callus induction from scutella of mature seeds of cv. Nipponbare (Oryza sativa
L.) and subsequent suspension culture using General medium have been previously
described (Li et al. 1990, Li and Murai 1990).
Nurse cells used for protoplast
propagation were derived from a long-term suspension culture of cv. Labelle, kindly
provided by Dr. MC Rush in this department.
The mutant Arabidopsis ALS gene, csrl-1 in pGHI, conferring resistance to
sulfonylureas was kindly provided by Drs. GW Haughn and CR Somerville, Michigan
State University. Hygromycin resistance genes in pTRA131, pTRA132 and pTRA141
were described elsewhere (Hayashimoto et al. 1990, Zheng et al. 1991).
Transform ation Plasmid Construction
Two plasmids with the mutant Arabidopsis ALS gene were constructed for the
transformation of rice protoplasts (Fig. 2.1). A 5.8 kbp Xbal fragment containing the
entire cloned Arabidopsis sequence in pGHI was isolated and inserted into a Xbal site of
pTRA131, forming pTRA133 (Haughn et al. 1988, Hayashimoto et al. 1990). The
fragment from pGHI encompasses the 2.0 kbp coding, 2.7 kpb 5 ’-upstream, and 1.1 kbp
3*-downstream sequences of the mutant ALS gene. Since transcription-controlling
elements of the ALS gene promoter were previously identified within the 5 ’-upstream
sequence (Mazur et al. 1987), we called the inserted fragment in pTRA133 a native ALS
gene, or the ALS gene under control of the native Arabidopsis promoter. Alternatively,
the entire 2.7 kbp 5’-upstream of the mutant Arabidopsis ALS gene was replaced by a
16
350 bp CaMV 35S promoter in pGHl. The subsequent introduction of the hygromycin
gene cassette from pTRA141 resulted in plasmid pTRA153. We called the mutant gene
in pTRA153 "35S/ALS", the ALS gene under control of the 35S promoter.
Both
pTRA133 and pTRA153 contain a hygromycin resistance gene, the former under control
of a nopaline synthase (nos) promoter and the latter under control of the CaMV 35S
promoter.
Protoplast Transformation and Development of Transformed Cell Lines
Rice protoplasts of cv. Nipponbare were transformed with plasmids pTRA132,
pTRA133 and pTRA153 following the method described previously (Li et al. 1990 and
APPENDIX C). Protoplasts isolated from 4-day-old suspension cells were transformed
with plasmid DNA in the presence of 25% (w/v) PEG and regenerated by co-culturing
with Labelle nurse cells. Since all three plasmids have the hph gene for hygromycin
resistance (Fig. 2,1 and Hayashimoto et al. 1990), 14-day-old transformed protoplastderived microcalli were first identified by selection with 95 pM hygromycin B. Resistant
colonies including one containing pTRA132, five containing pTRA133 and four
containing pTRA153 were developed into cell lines for subsequent DNA and
chlorsulfuron resistance analyses.
All cell lines were maintained in liquid General
medium supplemented with 4.5 pM 2,4-D and subcultured weekly. Subsequent analysis
with these cell lines were conducted within a 2-month period. Transgenic rice plants
were regenerated from transformed calli as described previously (Cao e t . 1989, Li et al.
1990).
17
E
E
E
tml hph nos
N
Arabidopsis
E
ALS
pUC12
pTRA133
P N
E
35S ALS
KE
KE
tml hph 35S
pUC19
pTRA153
Figure 2.1. Schematic structure of pTRA133 and pTRA153 plasmids. ALS
indicates the mutant Arabidopsis acetolactate synthase gene coding sequence. Arabidopsis
represents the 5 ’ upstream genomic sequence of the ALS gene. The hygromycin
phosphotransferase gene is identified as hph. Nos and tml indicate the promoter of the
nopaline synthase gene and the transcription termination and polyadenylation sites of the
tumor morphology large gene of Agrobacterium, respectively. A cauliflower mosaic virus
35S promoter is marked by 35S. pUC12 and pUC19 are cloning vectors. Abbreviations
for restriction sites are E: EcoRI, N: Ncol, P: PstI, and K: KpnI. Arrow lines indicate the
transcriptional direction of the specified gene.
18
DNA Analysis of Transformed Cell Lines Containing pTRA133 and pTRA153
Total DNA was isolated from actively growing suspension cells of one
nontransformed cell line and eight transformed cell lines containing pTRA133 and
pTRA153, respectively, following the method described previously (Hayashimoto et al.
1990). Ten pg DNA from each sample was digested with appropriate restriction enzymes
and then deproteinized by phenol/chloroform extraction.
After fractionation by
electrophoresis in a 0.8% agarose gel, DNA fragments were denatured and transferred to
a Nytran nylon membrane. The DNA blot was hybridized to appropriate radioactive
probe prepared by oligolabeling (Hayashimoto et al. 1990).
Total DNA of four pTRA133-containing cell lines (N133-2 to -5) was digested
with EcoRI and probed with the radioactively labeled 0.92 kbp NcoI-EcoRI fragments
corresponding to the 5* half of the mutant ALS coding sequence (Fig. 2.2, PI). EcoRI
digestion of pTRA133 should release a 2.1 kbp EcoRI signal representing a 1.2 kbp 5'upstream sequence and a 0.92 kbp ALS 5’-coding sequence (Fig. 2.2).
Alternative
hybridization with the probe corresponding to the 1.1 kbp hph coding sequence (Fig. 2.2,
P2) should produce two hybridization signals, one 1.2 kbp fragment representing 35S
promoter and 5’ region of the hph gene, and another 1.8 kbp fragment representing 3’
region of the hph gene, nos terminator and upstream region of the Arabidopsis ALS
promoter (Fig. 2.2).
Total DNA of four pTRA153-containing cell lines was digested with PstI and
EcoRI. After hybridization with the ALS gene probe PI, a 1.3 kbp signal representing
a 0.36 kbp 35S promoter and a 0.92 kbp ALS 5’-coding sequence should be recognized
19
(Fig. 2.2). Alternative hybridization with the hph gene probe P2 should generate a 1.7
kbp signal representing the entire hph gene cassette (Fig. 2.2).
Analysis of Chlorsulfuron Resistance
Callus clumps of 10 mg in size were selected from a nontransformed cell line and
transformed cell lines N132-1, N133-1, N133-2, N153-1 and N153-4 were cultured on a
MS callus growth medium supplemented with 9 pM 2,4-D and 95 pM hygromycin B or
chlorsulfuron in various concentrations ranging from 10 to 10,000 nM. After 20 d of
culture, fresh weight of each callus clump was determined.
Callus Culture in the Presence of Chlorsulfuron and Amino Acids
Callus clumps of 10 mg in size from three cell lines N 132-1, N133-1 and N 153-4
were grown on a MS callus growth medium with or without the addition of 100 pM each
of three amino acids valine, leucine and isoleucine in the presence of 0.2, 1 and 10 pM
chlorsulfuron, respectively.
Northern Blot Hybridization
Total RNA was isolated from 4-day-old suspension cells using a modification of
the method of Wadsworth et al. (1988).
Approximately 150 pg of total RNA was
obtained from one g of suspension cells from each cell line. Five and ten pg of total
RNA were fractionated in a 1% agarose gel containing formaldehyde. Fractionated RNAs
were transferred to a Nytran nylon membrane. The RNA blot was hybridized to the 32Plabeled Arabidopsis ALS DNA probe. The size of hybridized RNA species was estimated
based on the positions of 18S and 25S rRNA separated in the same agarose gel. In the
20
pTR A 133
E
Ji
E
E
4,
X
N
E
I
tml hph nos Arabidopsis
P2 H H IH
PI
ALS
1.8 kb
1.2 kb
2.1 kb
pTR A 153
P N
E
K E
1
K E
m
-!r
- k
35 S
PI 1.3 kb
ALS
tml hph 35 S
P 2 - _
1.7 kb
Figure 2.2. Hybridization probes and predicted hybridization fragments based on
the structure of pTRA133 and pTRA153. Symbols are the same as in Fig. 2.1. Two
DNA probes are marked by bold bars and aligned to corresponding regions of plasmid
DNA. PI stands for a 32P-labeled 0.92 kbp NcoI/EcoRI fragment representing the 5 ’
coding sequence of the Arabidopsis ALS gene. P2 indicates a 32P-labeled 1.1 kbp BamHI
fragment corresponding to the entire hph gene coding sequence. Expected hybridization
fragments and their sizes are indicated by aligned lines.
21
above
procedures
all
the
aqueous
solutions
were
treated
with
1%
(v/v)
diethylpyrocarbonate and the glassware was baked at 250°C overnight prior to use.
Chlorsulfuron Selection after Transform ation
Rice protoplasts were transformed with pTRA132, pTRA133 and pTRA153 and
regenerated as described above.
Fourteen-day-old protoplast-derived microcalli were
selected by 95 |iM hygromycin B or from 1 to 100 nM chlorsulfuron for 10 d. Protoplast
colonies were propagated on soft agarose General medium at 27°C in the dark for 4 to
6 d.
Growing colonies were counted to determine the transformation frequency,
representing the number of resistant colonies per million treated protoplasts.
RESULTS
Integration of Plasmid DNA in Transform ed Rice Cells
Hygromycin-resistant protoplast-derived colonies were obtained after PEGmediated transformation with pTRA133 and pTRA153. To confirm the integration of the
transferred DNA, Southern blot hybridization was conducted with total DNA from one
nontransformed cell line and four each transformed cell lines containing pTRA133 and
pTRA153, respectively.
After hybridization with the mutant ALS gene probe PI as
shown in Fig. 2.2, no positive signal was detected from the nontransformed cell line NB
(Fig. 2.3, lane 1). The expected 2.1 kbp signal was detected in three pTRA133-containing
cell lines N133-2, N133-3, and N133-5 (lanes 2, 3 and 5). The remaining line N133-4
produced a 6.7 kbp hybridization signal (lane 4).
The expected 1.3 kbp signal was
demonstrated in all four pTRA153-containing cell lines N153-1, N153-2, N153-3 and
N153-4 (lanes 6 to 9). Densitometric analysis of hybridization intensity with copy
22
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rr »r> ?h
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i -M
tH
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PQ
iH
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(A
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oo ou
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,*7*w1 A JS <v?'
y*i. t ••■>t % . » ->■
*j* *? *1 *v*
sy *'»>'
w # i* w
2. 3
2.0
..
-
1 2 3
4 5
9. 4
6.6
6 7
8 9
0.6
10 11
Figure 2.3. Southern blot hybridization of total DNA from ALS gene-containing
cell lines with the Arabidopsis ALS gene probe. Total DNA from the nontransformed
cell line and cell lines containing pTRA133 was digested with restriction enzyme EcoRI.
Total DNA from cell lines with pTRA153 was digested with restriction enzymes EcoRI
and Pstl. Digested DNA was separated on a 0.8% agarose gel by electrophoresis,
transferred to a nylon membrane and hybridized to the probe PI. Arrowheads indicate
the expected 2.1 kbp hybridized fragments (top) released from pTRA133 transformants
and the 1.3 kbp fragments (bottom) from pTRA153 transformants. Lanes 10 and 11 are
gene copy number standards corresponding to 1 and 5 copies, respectively, per diploid
rice genome.
23
number controls (lane 10, one copy; lane 11, five copies) revealed that one copy of the
ALS gene was present in cell lines N133-2, N 133-3 and N133-4; 2 copies in N 133-5 and
N153-3; and up to 3 copies in N153-1.
An alternative hybridization was conducted to determine the presence of the hph
gene. The same DNA blot was reprobed with the hph gene probe P2. No hybridization
signal was detected from the nontransformed cell line (Fig. 2.4, lane 1). Both expected
1.8 kbp and 1.2 kbp fragments were detected from three pTRA133-transformed cell lines
N133-2, N 133-3 and N 133-5 (lanes 2, 3 and 5). The remaining line N 133-4 produced a
1.2 kbp and a 7 kbp signals (lane 4). The expected 1.7 kbp hybridized fragment was
shown in EcoRI/Pstl-digested DNA of all four pTRA153-transformed cell lines (lanes 6
to 9). Based on densitometric analysis with copy number controls (lane 10, one copy;
lane 11, five copies), the copy number of the hph gene in these transformants was similar
to that of the mutant ALS gene detected previously. Thus these results indicated that all
the 8 transformed cell lines contain not only the mutant ALS gene but also the intact hph
gene.
Chlorsulfuron Resistance in Transformed Callus
In the resistance test all calli from transformed cell lines N 132-1, N133-1 and -2
and N 153-1 and -4 increased callus fresh weights in the presence of 95 |iM hygromycin
B while calli from the nontransformed line did not (data not shown). In the presence of
chlorsulfuron similar increase in callus fresh weight was found between the
nontransformed cell line and transformed cell line N 132-1; two transformed cell lines
N133-1 and N133-2; transformed cell lines N153-1 and N153-4. Average values of callus
24
r* <*)
■
•
W fO
W
i-H
^
Z
Z
E
10 i—i r t ro
tt
■
■
•
■
rt
I
■
fO fO W M
»H
Z
fH
Z
tH
Z
fH
Z
Z
*•■<
E
(A
•Si
Cl ‘S.
o 0
V L>
1n
kbp
- 23. 1
- 9. 4
-
6.6
- 4. 4
/
_ 2. 3
-
00 f #
2.0
00
‘■m*
m
** W h*
-
1 2 3
4 5
6 7
89
0. 6
10 11
Figure 2.4. Southern blot hybridization of total DNA from ALS gene-containing
cell lines with the hph gene probe. After complete removal of the PI probe by stringent
wash, the same DNA blot was hybridized to P2 probe. The top and bottom arrowheads
indicate the expected 1.8 kbp and 1.2 kbp hybridized fragments generated from pTRA133
transformants. The middle one shows the expected 1.7 kbp hybridized fragment released
from pTRA153 transformants.
25
fresh weights from three representative lines N 132-1, N 133-1 and N 153-4 are presented
in Fig. 2.5. Cell line N132-1 containing only the hph gene did not grow on higher than
50 nM of chlorsulfuron. Poor growth of N 133-1 calli containing the native ALS gene
was found on higher than 200 nM of chlorsulfuron. In contrast, N153-4 calli containing
the 35S/ALS gene grew on up to 10 pM of chlorsulfuron without significant reduction
in callus fresh weight, compared to the control culture in the absence of chlorsulfuron.
Based on these observations, the chlorsulfuron resistance in calli of pTRA133-containing
and pTRA153-containing transformants was about 4-fold and at least 200-fold,
respectively, higher than that of control calli without the mutant ALS gene.
Restoration of Chlorsulfuron Inhibition by Amino Acids
With the addition of the three amino acids leucine, isoleucine, and valine, callus
growth of all three tested cell lines (N132-1, N 133-1 and N 153-4) was not increased in
the presence of 0.2 pM chlorsulfuron (Fig. 2.6).
However, in selection with higher
concentrations of chlorsulfuron, callus growth of all cell lines was slightly increased. In
the presence of 10 pM chlorsulfuron callus fresh weights of cell lines N132-1 and N 133-1
were increased up to 100% after the addition of the three amino acids. There was a 15%
increase in callus fresh weight was found in cell line N153-4 under the similar conditions
(Fig. 2.6).
The mutant ALS Gene Transcript in Transformed Rice Cells
To understand the difference in the level of chlorsulfuron resistance between N133
and N153 transformants, a northern blot hybridization analysis was conducted using the
DNA probe P2 representing the 5’ region of the mutant Arabidopsis ALS gene. No
26
160
cn
3
*3
■§»
e,
02
M
□
0
N132-1
N133-1
N153-4
4- *
• »—«
2n
u
Chlorsulfuron Concentration (|±M)
Figure 2.5. Callus growth of cell lines transformed with and without the mutant
Arabidopsis ALS gene in the presence of chlorsulfuron. Callus clumps of about 10 mg
in size were selected from cell lines N 132-1, N133-1 and N 153-4 and cultured on a MS
callus growth medium supplemented with 9 pM 2,4-D and a serial concentrations of
chlorsulfuron. The final callus fresh weight was determined after 20 d. Bars represent
the average values of a total of 9 callus clumps in three replicate plates. Standard error
for each value is indicated.
27
160
□
^
bh
120
N132-1
N133-1
N153-4
Selection Treatment
Figure 2.6. Effect of amino acids on the restoration of callus growth in the
presence of chlorsulfuron. Three amino acids, leucine, isoleucine and valine, were added
to a MS callus growth medium containing 2 mg/L 2,4-D and 0.2, 1, and 10 |iM of
chlorsulfuron, respectively. Callus fresh weight was determined after 20 d and listed
along side with values obtained from control culture without the addition of amino acids.
Bars indicate the average values o f a total of 9 callus clumps in three replicate plates.
Standard error for each value was indicated.
28
hybridization signal was detected from 5 pg and 10 fig RNA samples of the
nontransformed cell line and the two transformed cell lines N132-1 and N133-4 (Fig. 2.7,
lanes 1 to 3 and 5 to 7). A single hybridization RNA signal was found in N153-4 RNA
samples (lanes 4 and 8). The size of the hybridizing RNA fragment in N 153-4 was about
2.2 kb, corresponding to the expected size of the mature Arabidopsis ALS transcripts
(Mazur et al. 1987).
Inhibition of the Growth of Rice Protoplast Colonies by Chlorsulfuron
In the presence of chlorsulfuron, significant growth inhibition was observed in 14day-old colonies derived from nontransformed protoplasts at the concentrations higher
than 3 nM (Fig. 2.8). Under these conditions the number of growing protoplast colonies
per million protoplasts plated was reduced from the initial of 135,000 to less than 100.
No growing protoplast colonies were found on medium with 10 nM chlorsulfuron.
The morphology of selected protoplast-derived colonies was compared after
selection with 95 pM hygromycin B and 10 nM chlorsulfuron (Fig. 2.9). In contact with
hygromycin, protoplast colonies immediately stopped growing and were totally collapsed
at the end of the selection period (Fig. 2.9A). In contact with chlorsulfuron, protoplast
colonies apparently survived, but stopped dividing at an early stage. Subsequently, their
cells developed large vacuoles and cellular volumes about five times larger than normal
cells. These giant cells usually burst by the end of the selection period (Fig. 2.9B).
The Mutant ALS Gene in Rice as a Selectable Marker
Rice protoplasts transformed with the pTRA 132, pTRA133 and pTRA153 plasmids
were selected in the presence of 95 pM hygromycin B or chlorsulfuron at 1 to 100 nM
29
5 Jig
lO jig
1-*
I
1
rr
•
•n
z z
z
Z
cc
I
r*
m
Z
Z
tj-
•
z
Tf
•
m
m
rH
z
kb
2.2
1 2
3
4
5
6
7
8
Figure 2.7. Northern blot analysis of the mutant Arabidopsis ALS transcripts in
transformed rice cells. Total RNA was extracted from rice suspension cells. Ten and
five p.g of total RNA were fractionated on a 1% agarose gel containing formaldehyde,
transferred to a nylon membrane, and probed with a 32P-labeled 0.93 kb NcoI-EcoRI DNA
fragment covering 5’ coding sequence of the Arabidopsis ALS gene (PI). The size of the
putative ALS transcript was indicated by an arrowhead.
0
2
4
6
8
10
Chlorsulfuron Concentration (nM)
Figure 2.8. Inhibition of the growth of 14-day-old protoplast-derived microcalli
by chlorsulfuron. Fourteen-day-old protoplast-derived microcalli, after nurse culture, were
transferred to a liquid General protoplast medium containing various concentrations of
chlorsulfuron. After 10 days of culture, protoplast-derived calli were propagated onto a
soft agarose medium. The average of growing calli per million treated protoplasts was
determined at the end of the forth week by counting 10 agarose blocks for each
concentration.
31
W
, • > *
a
j
*.
Figure 2.9. Protoplast-derived calli after selection with hygromycin B or
chlorsulfuron. Fourteen-day-old protoplast-derived microcalli regenerated from
nontransformed protoplasts were selected for 10 d with either 95 |iM of hygromycin B
(A) or 100 nM of chlorsulfuron (B). Selected calli were cultured on soft agarose medium
for two weeks before microscopic observation.
32
amounts. The average number of hygromycin resistant colonies per million treated
protoplasts after four experiments were 160.5 for pTRA132, 51.0 for pTRA133 and 176.3
for pTRA153.
Protoplasts transformed with pTRA132 and pTRA133 grew at
concentrations up to 5 nM chlorsulfuron (Table 2.1).
pTRA153 grew on up to 100 nM chlorsulfuron.
Protoplasts transformed with
Efficient selection of pTRA153-
containing resistant colonies was achieved by using 10 nM chlorsulfuron (Table 2.1).
Under these conditions the transformation frequency obtained after chlorsulfuron selection
was comparable to that with hygromycin selection.
DISCUSSION
Close similarity of amino acid sequence (85%) was found in ALS proteins from
many plant species, including Arabidopsis thaliana (Haughn et al. 1988), tobacco (Mazur
et al. 1987) and Brassica napus (Wiersma et al. 1989). However, the mutant ALS gene
was not expressed effectively under control of the native Arabidopsis promoter in
transformed rice cells. Replacement of the native Arabidopsis promoter by the CaMV
35S promoter led to a high level of expression of ALS transcripts, conferring more than
200-fold higher resistance to chlorsulfuron. The results indicate that the mutant form of
the Arabidopsis ALS enzyme remains functional in rice cells, substituting for the rice
ALS enzyme, which should be susceptible to chlorsulfuron inhibition. Thus, in spite of
possible differences in the regulation of expression, the functional similarity of the ALS
enzymes allows the use of the herbicide-resistant Arabidopsis ALS gene as a selectable
marker in rice.
33
Table 2.1.
Effect of chlorsulfuron selection on colony recovery after
transformation of Nipponbare protoplasts with plasmids pTRA132, pTRA133 and
pTRA153. The number of resistant colonies per million protoplasts is given as the
average of two independent experiments. SE represents standard error.
Number of resistant colonies per million protoplasts
Mean (SE)
Chlorsulfuron (nM)
Plasmid
DNA
1
5
10
50
100
No DNA
22.5(7.5)
0
0
0
0
pTRA132
30.0(4.1)
2.8(1.8)
0
0
0
pTRA133
110.0(9.1)
20.0(4.1)
0
0
0
pTRA153
342.5(33.0) 317.5(18.9)
111.2(17.5)
33.3(3.3)
7.5(4.8)
34
Direct selection of transformants using chlorsulfuron has been demonstrated in
tobacco (Gabard et al. 1989, Haughn et al. 1988, Lee et al. 1988, Olszewski et al. 1988)
and maize (Fromm et al. 1990). In all these cases selection was performed on calli with
chlorsulfuron concentrations ranging from 50 nM to 200 nM. In our study, we found that
protoplast-derived rice microcalli were highly sensitive to chlorsulfuron.
Significant
inhibition of cell growth by chlorsulfuron was found on higher than 3 nM.
These
effective concentrations are almost 10,000-fold less than that of hygromycin for inhibition
of rice protoplasts (Hayashimoto et al. 1990).
The growth inhibition by such low
concentrations of chlorsulfuron suggests that the chlorsulfuron selection is rather target
specific. Target-specific inhibition by chlorsulfuron may reduce the detrimental effects
on the selected cells observed in other selection systems using aminoglycoside antibiotics.
Successful selection of transformants by chlorsulfuron and subsequent regeneration of
fertile transgenic maize plants has been reported (Fromm et al. 1990).
Efficient recovery of resistant colonies after protoplast transformation with the
mutant ALS gene in pTRA153 was achieved by chlorsulfuron selection in concentrations
ranging from 5 to 10 nM. Selection on higher than 50 nM of chlorsulfuron dramatically
reduced the transformation frequency. This is probably due to the fact that the resistance
was caused by a mutated form of enzyme with lower affinity to chlorsulfuron. Previous
studies indicate that marker genes such as nptll and hph using detoxification mechanism
are able to confer wider selection windows at higher concentrations of selection agents
(Dekeyser et al. 1989, Hauptmann et al. 1988). Marker genes such as the mouse dhfr
with lower affinity to the selection agent conferred resistance at extremely low
35
concentration (Dekeyser et al. 1989, Hauptmann et al. 1988, Simonsen and Levinson
1983). Results of this study suggest that an alternative approach can be used to select
among larger transformed calli by chlorsulfuron. For example, transformed rice calli with
the 35S/ALS gene in pTRA153 were selected by 50 to 10,000 nM of chlorsulfuron for
20 d.
This selection of callus should be very useful for microprojectile-mediated
transformation of multiple cell clumps (Fromm et al. 1990).
Studies using the same mutant Arabidopsis ALS gene in transgenic tobacco
(Gabard et al. 1989) and Brassica napus (Miki et al. 1990) demonstrated that
chlorsulfuron-resistant transformants were also resistant to other sulfonylurea herbicides
including low residual ones with degradation rates 20 to 50 times faster than chlorsulfuron
(Beyer et al. 1987). It remains to be seen whether chlorsulfuron-resistant transgenic rice
will show similar cross-resistance to other sulfonylurea herbicides.
Thus far, 32 transgenic rice plants have been regenerated from two independent
chlorsulfuron-resistant cell lines transformed with pTRA153. These plants grown in the
greenhouse showed normal phenotype and set viable seeds. We have not determined the
chlorsulfuron-resistance at the plant level. However, previous studies with the same
mutant ALS gene (Charest et al. 1990, Chaleff and Mauvais 1984, Haughn and
Somerville 1986, Haughn et al. 1988) or a chlorsulfuron-resistant ALS gene isolated from
a maize mutant (Fromm et al. 1990) indicated that the resistance was stably present in
most plant tissues including callus, embryo, plantlets and mature leaves at all
developmental stages.
Chlorsulfuron has a low residual rate and short half life, and
serves as one of the ideal low toxicity herbicides in weed control in cereal crops. The
36
use of chlorsulfuron in rice requires the addition of an appropriate herbicide safener
(Barrett 1989). The herbicide resistance phenotype of transgenic rice should eliminate the
need for the safener and increase the utility of chlorsulfuron or other sulfonylurea
herbicides in rice weed control. Similar attempts have been made recently to examine
the potential of newly released cultivars of transgenic Brassica napus with genetically
engineered resistance to sulfonylureas for improved weed control and enhancement of
crop production (Miki et al. 1990).
CHA PTER SUMMARY
We report a new selectable marker gene for rice protoplast transformation. A
mutant acetolactate synthase (ALS) gene csr-1-1 isolated from a chlorsulfuron-resistant
Arabidopsis thaliana was placed under control of a CaMV 35S promoter and the
35S/ALS gene was introduced into rice protoplasts. The 35S/ALS gene was expressed
effectively as demonstrated by northern hybridization analysis, and conferred to
transformed rice calli a greater than 200-fold increase in resistance to chlorsulfuron over
the nontransformed control.
Effective selection of protoplasts transformed with the
35S/ALS gene was achieved at chlorsulfuron concentrations from 10 to 50 nM. Thus,
these results indicated that the 35S/ALS gene is an alternative selectable marker for rice
protoplast transformation.
37
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Beyer, E. M., Brown, H. M., and Duffy, M. J. (1987) Sulfonylurea herbicide soil
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Chaleff, R. W., and M auvais, C. J. (1984) Acetolactate synthase is the site of action of
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Charest, P. J., H atorri, J., DeMoor, J., Iyer, V. N., and Miki, B. L. (1990) In vitro
study of transgenic tobacco expressing Arabidopsis wild type and mutant
acetohydroxyacid synthase genes. Plant Cell Rep 8: 643-646.
Dekeyser, R., Claes, B., M arichal, M., Van M ontagu, M., and Caplan, A. (1989)
Evaluation of selectable markers for rice transformation. Plant Physiol 90: 217223.
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M. (1990) Inheritance and expression of chimeric genes in the progeny of
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H auptm ann, R. M., Vasil, V., Ozias-Skins, P., T abaeizadeh, Z., Rogers, S. G.,
Fraley, R. T., Horsch, R. B., and Vasil, I. K. (1988) Evaluation of selectable
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rice (Oryza sativa L.) protoplasts. Plant Cell Rep 9: 216-220.
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plant genes coding for acetolactate synthase, the target enzyme for two classes of
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Miki, B. L., Labbe, H., H attori, J ., O uellet, T., G abard, J ., Suncohara, G.,
C harest, P. J., and Iyer, V. N. (1990) Transformation of Brassica napus
canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and
analysis of herbicide resistance. Theor Appl Genet 80: 449-458.
Olszewski, N. E., M artin, F. B., an d Ausubel, F. M. (1988) Specialized binary vector
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39
W adsworth, G. J., Redinbaugh, M. C., and Scandalios, J. G. (1988) A procedure for
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Malcolm, S. K. (1985) Resistance to hygromycin B: a new marker for plant
transformation studies. Plant Mol Biol 5: 103-108.
Wiersma, P. A., Schiemann, M. G., Condie, M. G., Crosby, W. L., and Maloney, M.
M. (1989) Isolation, expression and phylogenetic inheritance of an acetolactate
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CH A PTER III
PH EN OTYPIC EFFECTS ON R IC E PRO TO PLA ST
REGENERATIO N AND TRANSFORM ATION W ITH
TH E H PH GENE AND TH E M AIZE A C TIV ATO R ELEM EN T.
40
41
INTRODUCTION
The successful production of transgenic rice plants in recent years provides not
only a promising avenue for the genetic manipulation of this world’s most important food
crop but also an important system for the study of monocotyledonous genes (Shimamoto
et al. 1989, Hayashimoto et al. 1990). A prerequisite for such prospective applications
of transgenic rice is the demonstration that genetically stable and phenotypically normal
plants are recovered after transformation treatments.
However, this demonstration
requires a large population of transgenic rice plants grown under uniform growth
conditions for meaningful comparison. This would be difficult to accomplish in currentlyrestricted small scale greenhouse experiments.
The maize activator (Ac) element has been introduced into transgenic rice plants
(Murai et al. 1991, Izawa et al. 1991) with the goal of establishing transposon tagging in
rice for gene isolation. A successful transposon tagging experiment depends greatly on
the identification of phenotypic changes caused by transposition. However, this is often
compromised by variations induced during in vitro culture and transformation.
For
example, rice callus culture was associated frequently with somaclonal variations (Oono,
1981; Fukui, 1983; Muller et al. 1990; Cao et al. 1991). Nineteen percent of protoplastderived plants from five japonica cultivars yielded phenotypically abnormal progeny
(Ogura et al. 1987). Somaclonal variations and possible DNA insertion mutation make
it difficult to identify transposition-induced phenotypic changes in protoplast-derived
transgenic rice plants.
42
To assess the contribution of somaclonal variation in transgenic rice plants as well
as to identify transposition-induced mutation from ^-containing transgenic plants, we
grew progeny of protoplast-derived nontransformed plants and transgenic plants
containing the hph gene and the Ac element under field conditions and investigated six
major agronomic traits. Here we present evidence that protoplast-derived rice plants and
transgenic plants with the hph gene were relatively normal and that Ac-containing
transgenic plants had a significantly higher frequency of phenotypic variation, among
which one sterile and one albino phenotype may be associated with active Ac
transposition.
MATERIALS AND METHODS
Plant Materials
Transgenic rice plants (Oryza sativa L., cultivar Nipponbare or Taipei 309)
containing a hygromycin phosphotransferase gene (hph) (pTRA131 and pTRA132), and
the maize Ac element (pTRA137 and pTRA137R) were generated after PEG-mediated
protoplast transformation as described previously (Hayashimoto et al. 1990, Murai et al.
1991). Primary transgenic plants and nontransformed protoplast-derived control plants
were grown to maturity in the greenhouse. All T1 seeds derived from self-pollinated
primary transgenic plants and R1 seeds from protoplast-derived plants were harvested at
the mature stage and dried in an oven at 50°C for 6 hr.
Germination Test for Hygromycin-resistance
T1 seeds of transgenic plants containing pTRA131 and pTRA132 were dehulled
and surface-sterilized as described previously (Li et al. 1990). Seeds were placed on
43
filter paper in 0.1 x MS liquid medium containing 190 jiM hygromycin B and allowed
to germinate at 27°C under light (56 |iE. m'2 s'1) for 10 d. The hygromycin-resistance of
transgenic progeny was determined by comparing seedling size with seedlings of
nontransformed control seeds germinated in the absence of hygromycin as a positive
control, or in the presence of hygromycin as the negative control.
Field Trial with Transgenic Plants
The field trial experiment was performed during the period from May to October
1990 at the LSU Rice Research Station in southwest Louisiana. Rice seeds were soaked
in sterile water for one d and then germinated on wet filter paper at 27°C under light (56
jlE. m'2 s'1) for 3 d. Germinated seeds were then transferred to 20 cm diameter plastic
pots containing a soil mixture of sterilized Crowley silt loam, peatmoss and fine sand
(2:1:1) and grown in a greenhouse bench flooded with tap water (Li et al. 1990). After
3 weeks, seedlings were removed from the pots and transplanted to a paddy field that had
been treated with 3 lb per acre propanil |7V-(3,4-dichlorophenyl) propionamide] and then
flooded with water to about 4 cm deep for 2 d. Seedlings were singly planted in rows 90
cm apart with 30 cm between plants. Plants were fertilized once with fertilizer 20/10/10
(N, P20 2, K20 ) at a rate of 600 lb per acre the second week after transplanting. According
to the experiment permit (No. 90-029-01) granted by the U.S. Department of Agriculture
and Forestry for genetically engineered plants, flowering panicles were covered by
pollination bags during the entire flowering stage to prevent dissemination of viable
pollen to the adjacent environment. The experiment plot was enclosed with aerial netting
to prevent spread of seeds by birds.
44
Analysis of Major Agronomic Traits
Six major agronomic traits including plant height, tiller number, panicle length,
panicle spikelet number, fertility and 1000-seed weight were determined from mature
plants. Plant height was measured from the soil surface to the tip of the panicle on the
longest tiller of each plant. Panicle length and spikelet number were averaged from five
randomly-chosen panicles from each plant. Fertility was expressed as the percentage of
mature seeds over total spikelets of five randomly chosen panicles from each plant. Up
to one hundred dried mature seeds from each plant were weighed and the one thousand
grain weight was calculated. Means of each group of protoplast-derived nontransformed
and transformed plants were compared by Tukey’s test with statistical significance at the
0.05 probability level.
Southern Blot Hybridization Analysis
Total DNA was isolated from leaf tissue of rice plants using a modified method
of Rogers and Bendich (1985) and purified by CsCl density gradient centrifugation. DNA
was digested to completion with appropriate restriction enzymes, electrophoretically
fractionated in 0.8% agarose gels, and blotted to Nytran nylon membranes (BioRad).
DNA blots were hybridized with 3ZP-labeled probes as specified in the text.
The analysis of DNA isolated from /zp/i-containing plants was performed as
previously described (Hayashimoto et al. 1990).
After enzymatic digestion and gel
electrophoretic separation, DNA blots were hybridized with a 1.1 kb radioactive probe
representing the entire hph gene sequence.
45
The rearrangement of the reconstituted empty donor site and Ac elements in
pTRA137R-containing plants was determined according to methods described previously
(Murai et al. 1991). Expected hybridizing fragments after hybridization with different
probes were shown in Fig. 3.1. Hindlll/EcoRI digests hybridized to a DNA probe PI
representing the 35S promoter sequences should generate a 0.6 kb hybridization signal
corresponding to the empty donor site after Ac excision. BamHI digests hybridized to a
DNA probe P2 corresponding to center region of Ac sequences should produce intact Accontaining hybridization fragment with sizes larger than 4.6 kb. PvuII digests hybridized
to the P2 probe should display a 2.5 kb internal fragment from non-methylated active Ac
elements.
RESULTS
We have grown a large number of primary protoplast-derived plants and transgenic
plants in the greenhouse. These plants produced agronomic traits distinct from the parent
cultivar, including shorter or longer culm length, lower tiller number, smaller panicle size,
and lower fertility (data not shown). However, only field-grown progeny of transgenic
plants were analyzed due to the unreliability of greenhouse data that may have been
biased by the artificial conditions and micro-climatic variation in the greenhouse.
Phenotype of Field-grown Protoplast-derived Plants
Protoplast-derived plants were produced through essentially the same in vitro
culture conditions as transgenic plants, only without DNA treatments and hygromycin
selection. Under field conditions, six major agronomic traits of 251 progeny plants from
7 R1 lines and 2 R2 lines were comparable to that of seed-derived parental plants (Table
46
PI
H 1.5 kb
P2
q
tH dX b
Pv
8.8 kb
h
A 2.5 kb
Hd
Ec
Hd
Pv
B hB hE c
Ac
35S
Bh
hph
Ec
tml
p T R A 137R
E x c is io n
Hd Xb
Ec
Ec
j
R e -in te g r a tio n
Pv
i
PI
Hd
i
Ec
i
Hd
i
Pv
i
Bh
i
P2
H 0.6 kb
> 8 .8 kb
H 2.5 kb
Figure 3.1. Hybridization probes and predicted hybridization fragments based on
the structure o f pTRA137R before and after Ac transposition. PI indicates the 0.3 kb 32Plabeled plasmid DNA fragments covering the CaMV 35S promoter sequence. P2
represents radioactively labeled DNA fragments homologous to the 1.6 kb HindlH region
of the Ac element. Sizes of hybridized fragments to DNA probes were labeled.
Abbreviations for restriction sites are: Bh for BamHI, Ec for EcoRI, Hd for Hindlll, Pv
for PvuII, and Xb for Xbal.
47
3.1).
One line, NBP2, showed generally low plant height and tiller numbers.
No
segregation of compared traits was found in any protoplast-derived progeny lines
(Table 3.1). However, protoplast-derived progeny plants displayed slightly lower values
in major agronomic traits compared to seed-derived control plants. This generally lower
performance of protoplast-derived rice plants of the same cultivar was also reported
previously (Ogura et al. 1990).
Analysis of Transgenic Plants C ontaining the Hph Gene
A. Inheritance of hygromycin resistance
Two fcp/t-containing plasmids,
pTRA131 and pTRA132, were introduced into transgenic rice plants. The coding sequence
of the hph gene was under control of the nopaline synthase (nos) promoter in pTRA131
or under control of the CaMV 35S promoter in pTRA132. To determine the transmission
of the hph gene in these transgenic plants, we first analyzed the inheritance of
hygromycin resistance in the T1 progeny. Nontransformed Nipponbare seeds failed to
germinate in the presence of 190 |iM hygromycin B (Table 3.2). However, T1 seeds
derived from self-fertilized transgenic plants with pTRA131 or pTRA132 germinated at
different ratios, indicating the segregation of hygromycin-resistance.
Among 24
transgenic plants tested, 13 showed a segregation equivalent to a 3:1 ratio, and 10 had a
less than 3:1 ratio (Table 3.2). These results suggest that the hygromycin resistance trait
was in a single locus or two closely-linked loci in the genome o f nearly all plants and
transmitted to progeny in a Mendelian manner.
B. Phenotypic analysis Sixty T1 plants from 4 lines containing pTRA131 were
examined for their phenotype. Progeny of three lines (N131-7, -15 and -18) had a plant
48
Table 3.1. Major agronomic traits of field-grown seed-derived plants and progeny
of protoplast-derived nontransformed plants and transgenic plants (Nipponbare) containing
the hph gene and the maize Ac element. Data of major agronomic traits were collected
from mature plants as described in the text. Thousand seed weight of seed- and
protoplast-derived plants and /aph-containing plants was determined from 10 representative
plants and listed in Table 3.3. Mean of individual line and standard error are listed.
Transferred genes are indicated in parentheses.
Plant Type
No. of Plants
Examined
Plant Height
(cm)
No. of Tillers
per Plant
Panicle Length
(cm)
No. of Spikelets
per Panicle
Seed Fertility
(%)
Mean (SE)
75.1 (0.24)
46.14 (0.99)
19.0 (0.24)
92.0 (2.66)
69.1 (1.89)
29
73.0 (0.94)
39.5 (1.60)
18.6 (0.25)
97.9 (2.13)
68.6 (2.83)
NBP2
Al
64.4 (1.58)
31.0 (1.75)
18.7 (0.31)
96.4 (1.29)
65.8 (2.18)
NBP2-1
25
73.3 (0.57)
49.3 (2.11)
18.8 (0.20)
94.2 (2.42)
73.6 (2.74)
NBP2-2
25
73.9 (0.82)
40.3 (1.49)
18.7 (0.28)
95J5 (3.39)
69.5 (1.69)
NBP3
NBP4
25
25
77.0 (0.69)
48.5 (1.70)
19.2 (0.24)
94.9 (1.41)
61.0 (3.10)
73.3 (1.49)
43.1 (1.97)
18.3 (0.29)
81.7 (7.75)
58.9 (3.85)
NBP5
25
69.7 (0.77)
18.4 (0.22)
101.1 (2.94)
61.4 (3.93)
NBP7
25
75.3 (0.86)
40.6 (1.68)
39.9 (2.02)
18.4 (0.20)
84.7 (2.88)
60.3 (3.10)
NBP8
25
76.4 (0.65)
48.5 (2.03)
19.2 (0.21)
96.4 (3.10)
58.4 (3.02)
55.3 (2.38)
69.7 (5.36)
18.3 (0.54)
83.4 (2.61)
48.2 (4.23)
70.4 (0.70)
51.0 (2.53)
17.5 (0.17)
70.0 (1.42)
56.4 (2.56)
Seed-derived Plants
110
Protoplast-derived Plants
NBP1
pTRAl 31 Plants ( nos-hph )
N131-6
6
N131-7
N131-15
13
70.8 (1.69)
62.9 (4.35)
18.1 (0.20)
85.1 (3.11)
43.5 (6.13)
N131-18
7
68.1 (0.99)
54.3 (4.41)
16.9 (0.45)
87.9 (5.38)
37.2 (5.15)
pTRA132 Plants (35S-hph )
N132-1
13
78.3 (0.71)
56.1 (3.67)
20.1 (0.30)
124.5 (3.21)
45.0 (4.17)
N132-5
15
68.3 (1.15)
57.0 (1.20)
18.0 (0.33)
88.0 (3.49)
57.8 (5.06)
N132-13
24
74.4 (0.84)
38.5 (1.72)
18.0 (0.23)
82.0 (2.50)
62.3 (3.50)
N132-23
5
73.6 (0.75)
41.3 (1.20)
18.0 (0.21)
107.3 (2.88)
42.5 (4.38)
'O
Plant Type No. of Plants Plant Height No. of Tillers Panicle Length No. of Spikelets Seed Fertility 1000 Seed
Examined
(cm)
per Plant
(cm)
per Panicle
(%)
Weight (g)
Mean (SE)
pTRA137 Plants
(m -h p h , Ac )
N137-l(a)
9
69.9 (1.02)
51.0 (1.07)
18.1 (0.17)
101.3 (1.94)
35.6 (4.78)
20.9 (0.07)
N137-l(b)
2
48.0 (10.00)
37.0 (19.00)
14.7 (2.60)
58.5 (26.3)
0.5
25.2 (0.29)
N137-6
6
69.0 (2.45)
53.3 (2.08)
17.4 (0.32)
91.9 (4.96)
46.7 (4.03)
22.1 (0.06)
23.6 (0.06)
pTRA137R Plants QSS-hph , reversed Ac )
N137R-l(a)
31
72.9 (0.73)
44.6 (2.13)
17.4 (0.15)
84.6 (1.50)
37.1 (2.79)
N137R-l(b)
6
48.7 (2.08)
49.2 (3.00)
13.1 (0.51)
29.7 (3.82)
0
—
N137R-2
5
54.6 (1.60)
37.0 (1.14)
12.8 (0.34)
23.8 (3.62)
0
—
N137R-4
26
60.6 (2.37)
40.2 (2.67)
15.4 (0.44)
48.5 (4.25)
18.0 (4.75)
20.6 (0.13)
N137R-6(a)
162
71.5 (0.42)
43.7 (0.86)
16.4 (0.19)
74.2 (5.30)
65.2 (2.31)
22.3 (0.02)
N137R-6(b)
2
52.5 (8.50)
35.5 (5.50)
14.6 (2.20)
34.4 (13.60)
0
N137R-7
81
67.4 (1.27)
54.3 (2.29)
17.7 (0.48)
86.9 (3.70)
54.8 (3.35)
22.7 (0.02)
N137R-8
69
66.5 (0.49)
72.7 (2.99)
17.6(0.11)
75.2 (1.23)
25.7 (2.78)
21.1 (0.03)
N137R-9
27
50.4 (1.58)
50.6 (0.46)
17.0 (0.68)
48.6 (3.52)
0
—
39.0
30.0
15.4
42
0
—
N137R-10
1
—
51
Table 3.2. Inheritance of hygromycin resistance in progeny of transgenic rice
plants containing the hph gene. T1 Seeds of self-fertilized primary transgenic rice plants
of Nipponbare containing the hph gene were germinated in the presence of 190 |iM
hygromycin B. At the end of 10 days, seedling size of transgenic progeny was compared
to that of nontransformed seeds germinated in the presence and absence of hygromycin
to determine the hygromycin resistance. The ratio of hygromycin resistant to sensitive
progeny was determined according to Chi square test. Values of the 95% and 99%
confidence level in the Chi square test are 3.84 and 6.63, respectively.
Rice
plants
Total number Hygromycin
o f seeds
resistant
Hygromycin
sensitive
R:S
ratio
X2
Nontrans.
control
20
0
20
—-
—
N131-1
N131-4
N131-6
N131-7
N131-15
N131-18
N132-1
N 132-2
N 132-4
N 132-5
N 132-7
N 132-8
N 132-9
N132-13
N132-14
N 132-20
N 132-23
N132-24
N 132-27
N 132-28
N 132-31
N132-32
N132-34
N132-35
9
7
18
40
28
29
50
15
7
48
39
13
8
50
9
45
20
20
40
58
59
48
40
30
7
5
11
34
26
22
22
5
5
39
14
5
6
40
7
22
16
15
34
33
38
27
20
17
2
2
7
6
2
7
28
10
2
9
25
8
2
10
2
23
4
5
6
25
21
21
20
13
3 1
3 1
3 1
3 1
15:1
3 1
11
11
3 1
3 1
11
11
3 1
3 1
3 1
11
3 1
3 1
3 1
11
3 1
11
11
11
0.037
0.048
1.852
2.133
0.038
0.011
0.768
1.667
0.048
1.000
3.103
0.692
0
0.667
0.037
0.022
0.267
0
2.133
1.103
3.531
0.750
0
0.533
52
height similar to protoplast-derived plants (Table 3.1). One line (N131-6) was much
shorter than the other three and contributed the most to the statistical difference of plant
height in this group (Table 3.3). However, all the progeny plants of this line produced
more tillers with fewer spikelets per panicle than the protoplast control. Even though no
sterile plants were found, the fertility of pTRA131-containing plants was only 70% o f that
of protoplast-derived progeny.
Fifty seven T1 plants of 4 lines containing pTRA132 showed similar phenotypes
to that of protoplast-derived plants (Tables 3.2). Progeny of lines N132-1 and -5 had
more tillers (Table 3.1). Progeny of line N132-1 also generated longer panicles with
more spikelets than protoplast-derived plants. However, at least in two lines (N132-1 and
-23) the fertility was considerably lowered. Two sterile progeny plants were found in line
N132-13.
C.
DNA analysis of /ip/i-containing plants with d w arf tra it Since phenotypic
variation may also be caused by insertion of transferred DNA after transformation or
DNA rearrangement during sexual propagation, we analyzed the DNA hybridization
pattern of T1 progeny of a pTRA132-containing Taipei 309 plant, in which a dwarf trait
was segregating consistently with a 3 to 1 ratio in one T1 population (45 normal and 17
dwarf) and one normal T1 plant-derived T2 line (72 normal and 24 dwarf). All the dwarf
plants had a uniform plant height only 60% of the normal siblings and 2.5-fold more
tillers than normal plants with unique chlorophyll-deficiency symptom (data not shown).
Thus, this dwarfism seems to be a single locus mutation.
Table 3.3. Summary of six major agronomic traits of field-grown progeny of nontransformed controls and transgenic rice
plants containing the hph gene and the Ac element.
Means of each group of plants were compared by Tukey’s test.
Means within a column followed by the same letter are not significantly different at the 0.05 probability level.
Plant Type
No. of lines
Plant Height No. of Tillers Panicle Length No. of Spikelets Seed Fertility
(cm)
per Plant
(cm)
per Panicle
(%)
1000-seed
Weight (g)
Mean
ProtoDlast-derived Plants
9
72.9 a
42.2 a
18.7 a
93.6 a
64.2 a
22.2
4
66.2 ab
59.5 a
17.7 a
81.6 ab
46.3 a
22.7
4
73.7 a
48.2 a
18.5 ab
51.9 a
21.7
3
62.3 ab
47.1 a
16.7 ab
83.9 ab
27.6 ab
22.7
10
58.4 b
45.8 a
15.7 b
54.8 b
40.2 b
22.1
(nontransformed)
pTRA131 Plants
( nos-hph )
pTRA132 Plants
100.5 a
(35S- hph )
pTRA137 Plants
Q5S-hph, Ac)
pTRA137R Plants
(35S- h p h , reversed A c )
54
DNA from one normal T1 plant (T132-3-1), one normal T2 plant (T132-3-1-19)
and one dwarf T2 plant (T132-3-1-28) was analyzed by Southern hybridization with hph
probe as described before (Hayashimoto et al. 1990). All three progeny plants, regardless
of different phenotypes, generated an identical hybridization profile as the primary parent
/ip/i-containing plant after BamHI (Fig. 3.2A) or Hindlll digestion (Fig. 3.2B)
(Hayashimoto et al. 1990). Thus, we concluded that no DNA rearrangement occurred
with the stably integrated hph gene in the genome of these plants after at least two rounds
of sexual propagation. In other words, the dwarf phenotype observed in the progenies of
transgenic T132-3 plant was not correlated with any physical change of inserted DNA.
Dwarfism may have been derived from a single recessive somaclonal mutation (Larkin
and Scowcroft 1981).
Analysis of Transgenic Plants Containing the A c Element
A.
Phenotypic analysis Among 3 T1 lines examined, 2, N137-l(a) and N137-6,
produced agronomic traits comparable to that of protoplast-derived plants (Table 3.1).
However, all the progeny plants had an extremely low fertility.
Line N137-1 was
separated into two sub-lines based on two morphologically different primary transgenic
plants regenerated presumably from the same transformant. In the greenhouse N 137-la
was phenotypically normal compared to seed-derived plants, whereas the N 137-lb plant
had wider and thicker leaves, a shorter culm, large seeds with long awns and lower
fertility (data not shown). Under field conditions these traits of the T1 generation
remained unchanged (Table 3.1). Thus, the distinct phenotype in N137-lb may have
been caused by somaclonal mutation or DNA insertion.
55
Figure 3.2. Southern blot hybridization of total DNA from transgenic plants
containing the hph gene (pTRA132). Total DNA was isolated from transgenic progeny
plants of Taipei 309 and digested with BamHI (A) and Hindlll (B), respectively.
Digested DNA was separated in a 0.8% agarose gel by electrophoresis and transferred to
a nylon membrane. DNA blot was hybridized with 32P-labeled 1.1 kb BamHI fragments
corresponding to the hph coding sequence.
56
00
fS
■
rr>
p*•
m
•
m
■
m
OS
I
t*i
r*
tn
2
H
^>»
—
a.
g<
O
©
«
v>
v
•=
a
g*
O
_
o
U
kbp
iH W)
-2 3 .1
— 9.4
—
ss
6.6
— 4.4
— 2.3
m
1
m
m
2
3
4
—
2.0
—
0.6
5
Figure 3.2A. Southern blot of BamHI-digested DNA from /zp/z-containing plants.
57
i
*
m
H
00
r*i
i
•
rq
•
i
tn•
H
H
►»
a
yo
m
IA
4)
ex
©
o
in
kbp
9 .4
6 .6
4 .4
2 .3
2.0
1
2
3
4
5
Figure 3.2B. Southern blot of HindlH-digested DNA from /jpA-containing plants.
58
Much greater phenotypic variations were evident with a larger number of
pTRA137R-containing transgenic plants tested. Four out of six agronomic traits o f the
T1 progeny of these plants were significantly different from protoplast-derived plants
(Table 3.3). Only 2, N137R-l(a) and N137R-6(a), out of 10 progeny lines generated a
relatively normal plant height with other traits similar to protoplast-derived control (Table
3.1). The above-mentioned separation of two plant phenotypes was also observed in lines
N137R-1 and -6 (Table 3.1). Among 81 plants in the line N137R-7, 6 plants were less
than 30 cm high, only 45% of the average plant height of other siblings. Progeny of line
N137R-8 generated many more tillers than seed- and protoplast-derived control plants.
Between different lines, the spikelet number per panicle varied from 84.6 (N137R-la) to
23.8 (N137R-2).
A major characteristic of N137R plants was the presence of sterile
plants. Five out of ten lines produced only sterile progeny (Table 3.1). Progeny of the
other lines set seeds at extremely low frequencies. In line N137R-7 five out of abovementioned six dwarf plants failed to produce any mature seeds. A 3 to 1 segregation of
fertile (53) to sterile (15) progeny plants was found in line N137R-8, indicating that a
single locus mutation may be responsible for the sterility. Only one line (N137R-6a) had
an average fertility rate similar to that of protoplast-derived plants (Table 3.1).
B.
DNA analysis of Ac-containing plants with sterility o r albinism phenotype
To investigate the possibility that increased phenotypic variation in Ac-containing
transgenic plants may have been caused by Ac transposition, DNA was analyzed in five
N137R-containing plants: a primary transgenic plant (N137R-7) and one T1 progeny plant
(N137R-7-6), and three T1 progeny plants (N137R-8-1, -50 and -49) derived from another
59
primary transgenic plant (N137R-8) that was analyzed previously (Murai et al. 1991). In
contrast to its normal parental N137R-7 plant, the T1 progeny N137R-7-6 plant was dwarf
and sterile. All three T1 progeny plants of N137R-8 were morphologically normal, except
that N137R-8-49 was sterile. A T2 population derived from N137R-8-1 generated a near
3:1 segregation for normal (52) to albino/yellow (12) seedlings in a subsequent
experiment, indicating that a single nuclear gene mutation in the T1 plant may be
responsible for the albinism.
One 0.6 kb hybridization fragment corresponding to the empty donor site was
detected in the primary transgenic N137R-7 plant (Fig. 3.3A, line 1), while no
hybridization signal was detected in the T1 progeny N137R-7-6 plant (lane 2), indicating
that the empty donor site in the progeny was lost during sexual propagation. The empty
donor site was present in three T1 progeny plants of N137R-8 with the essentially same
hybridization pattern (lanes 3-5) as found in parent N137R-8 plant (Murai et al. 1991).
All five transgenic plants analyzed with Ac probe after BamHI digestion produced
hybridization fragments larger than 4.6 kb (Fig. 3.3B, lanes 1 to 5). Notably, one unique
19 kb Ac-containing fragment was detected in the progeny N137R-7-6 plant which did
not contain any empty donor molecule (lane 2). The expected 2.5 kb fragment released
after PvuII digestion was detected in both parent and progeny plants of N137R-7 (Fig.
3.3C, lanes 1 and 2). Based on the fact that only one copy of the 2.5 kb Ac-containing
fragment was present in the progeny, the 19 kb BamHI fragment might actually contain
an intact and active Ac element. Accordingly, a single active Ac molecule in transgenic
N137R-7 plant was probably transmitted to the progeny N137R-7-6 plant through
60
Figure 3.3. Southern blot hybridization of total DNA from transgenic rice plants
containing the maize Ac element (pTRA137R). A: Southern blot hybridization of
HindiII/EcoRI-digested DNA with the CaMV 35S promoter probe (PI). Total DNA was
isolated from leaf tissues of transgenic rice plants and digested to completion with Hindlll
and EcoRI enzymes. Digested DNA was separated by electrophoresis in 0.8% (w/v)
agarose and transferred onto Nytran nylon membrane. DNA blot was then hybridized to
PI probe as indicated in Fig. 3.1. Standards for 1 and 5 copies of plasmid DNA per
diploid rice genome were reconstructed using pTRA137R DNA. B: Southern blot
hybridization of BamHI-digested DNA with the Ac internal probe (P2). Hybridization
procedures were the same as described except that the total DNA was digested with
BamHI and the probe used was the 1.6 kb Hindlll fragment of Ac internal sequence (P2).
C: Southern blot hybridization of PvuII-digested DNA with the Ac internal probe (P2).
Total DNA was digested with PvuII to completion and subjected to Southern blot
hybridization the probe P2 representing the 1.6 kb Ac internal Hindlll fragment.
61
vo
tH
00
P6
z
Pi
m
w
Z
9
ID
00
Pi
t>
Pi
w
Z
Z
©V
Tf
00
a;
fz
to
►
> .Si
'a
a.
o
o
w u
-N in
kbp
23.1
9.4
6.6
4.4
2.3
2.0
0.6
Figure 3.3A. Southern blot hybridization of Hindlll/EcoRI-digested DNA with the
CaMV 35S promoter probe (PI).
62
IT)
00
06
r-~
oe•
OS 06
r-~ t"
Z
Z
Z
Z
•
I
C
ti
r-
Ov
•«t■
00
■
t-
^
Q<
Z
rH
2 2 s
M
«»
tA
CJ
QO
U
tfl
• art
-
kbp
-
23.1
-
9.4
-
6.6
-
4.4
-
2.3
2.0
-
0 .6
-
1
2
3
4
5
6
7
Figure 3.3B. Southern blot hybridization of BamHI-digested total DNA with the
Ac internal probe (P2).
63
voa
i
1 ■ 00I
pa
pa pa r-~
ro
i-H tH tH
Z Z Z
o
■
00■
t"ro
Z
O
Tt1n
001
pa
•m m
a.
o
tH
kbp
Z
- 2 3 .1
-
9.4
-
6.6
-
4.4
4 |i h -4
-
1
2
3
2 .3
2 .0
4 ' 5 6 7
Figure 3.3C. Southern blot hybridization of PvuII-digested DNA with the Ac
internal probe (P2).
64
germinal transposition and integrated into a different location. Alternatively, this change
might have been caused by selective DNA rearrangement.
Three progeny plants of N137R-8 showed slightly different hybridization patterns
after BamHI digestion, although all of them contained larger than 4.6 kb Ac-containing
fragments (Fig. 3.3B, lanes 3 to 5). With the appearance of a new 6.6 kb fragment, the
intensity of a 5 kb common fragment in the N137R-8-1 progeny was proportionally
reduced (lane 3). Progeny N137R-8-50 plant retained exactly the same hybridization
pattern as detected previously from the parent plant (lane 4) (Murai et al. 1991). We
found that only two progeny plants (N137R-8-1 and -49) had active Ac elements by
showing the expected 2.5 kb PvuII fragments (Fig. 3.3C, lane 3 and 5). Corresponding
to the unchanged hybridization pattern after BamHI digestion, progeny N137R-8-50 plant
did not contain any active Ac element with the absence of the 2.5 kb PvuII fragment (lane
4), probably derived from methylation of Ac sequence.
At this point, it would be
interesting to further determine the relationship between the presence of unique active Ac
element and the segregation of sterility in T1 generation and albinism in T2 population
of N137R-8-1.
DISCUSSION
In rice in vitro culture the frequency of somaclonal variations is often associated
with the type of tissue cultured, the genetic heterozygosity of cultured materials, and in
particular, the culture conditions, reflecting the degree of stress on cultured cells. The
length of culture period has been found to have a pronounced effect on somaclonal
variation (Fukui 1983). The results indicated that progeny of protoplast-derived rice
65
plants were comparable to those of seed-derived plants. The phenotypic similarity of
protoplast-derived plants suggests that somaclonal variation may not be significant in the
plant regeneration system used (Li et al. 1990).
Comparison analysis with transgenic rice plants containing the hph gene showed
that we produced relatively normal transgenic plants. Progeny of these transgenic plants
displayed a small number of phenotypic variations, among which the most significant was
the generally low fertility.
However, based on the results of inheritance of the
hygromycin resistance gene and DNA analysis, the phenotypic variations observed in
these transgenic plants was probably not caused by DNA insertion and rearrangement, but
came from mutations induced during the hygromycin selection process.
Kanamycin
selection of rice and maize transformants frequently resulted in sterility (Rhodes et al.
1988), while selection with other selective agents such as chlorsulfuron successfully
produced fertile transgenic maize plants (Fromm et al. 1990). It would be interesting to
see whether the genetic stability of transgenic rice plants can be improved by using other
alternative selection systems.
Progeny of transgenic rice plants containing the maize Ac element showed a
significantly higher frequency of phenotypic variations.
A number of single-locus
mutations for dwarfism, sterility and albinism were observed in progeny of Ac-containing
transgenic plants.
These variations seem associated with the presence of active Ac
elements and their reintegration in new genomic locations. Independent segregation of
donor molecule and active Ac element was evident in the DNA analysis of the transgenic
plant N137R-7 and its progeny, probably reflecting active germinal transposition of the
66
Ac element in the rice genome.
Independent assortment of donor molecule and Ac
element as a result of active transposition was also demonstrated previously in transgenic
tomato (Belzile et al. 1989).
The active transposition of Ac element may increase
insertion frequency and facilitate identification of transposon-induced variations for gene
isolation.
The Ac element has been known for the phenomenon of changing in phase, in
which the element undergoes reversible cycling from active to inactive state and vice
versa (McClintock, 1963, 1964).
Inactivation of the Ac element in this case was
associated with a high degree of methylation, while revertants with restored transposition
activity were correlated with a progressive demethylation (Schwartz and Dennis, 1986,
Chomet et al. 1987). In these cases methylation sensitive PvuII digestion was often used
to determine the degree of methylation along the Ac sequence. The results of DNA
analysis of transgenic rice plants containing the maize Ac element indicated that the
translocation of Ac elements was associated with the availability of both internal PvuII
sites for complete digestion. Hybridization intensity of the moved Ac-containing DNA
fragments found in four transgenic plants was consistent with that of Ac-containing
fragments released after PvuII digestion. Accordingly, the translocation of Ac elements
in these plants may have been derived from active Ac transposition. It will be interesting
to see whether the transposition activity of the maize Ac element in transgenic rice plants
is also controlled by the DNA methylation mechanism as found in maize.
67
CHAPTER SUMMARY
To determine the extent of somaclonal variation, DNA insertion mutagenesis and
transposition-induced mutation in transgenic rice plants, field-grown progeny of
protoplast-derived plants and transgenic rice plants (Oryza sativa L.) containing a hph
gene and the maize Ac element were analyzed by comparing six major agronomic traits
with seed-derived controls. Lines from rice plants regenerated from protoplasts were
phenotypically comparable to their parent cultivar.
Seven out of eight progeny lines
containing the hph gene showed relatively normal phenotype compared to protoplastderived plants.
However, these transgenic plants displayed a generally low fertility.
Hygromycin resistance test and DNA analysis indicated that the hph gene and the
resistance were largely transmitted to the progeny as a single locus trait. DNA analysis
also demonstrated that a dwarf phenotype in a /ip/j-containing plant did not correspond
to the unchanged integration pattern of transferred DNA in the rice genome. In contrast,
transgenic plants containing the Ac element produced a significantly higher frequency of
phenotypic variations. Among 13 progeny lines, only 4 produced phenotypes resembled
to that of protoplast-derived controls. One sterility and one albinism phenotypic variation
in Ac-containing progeny were associated with the presence of active Ac element that had
been integrated into different genomic location. Thus, these analyses allow us to identify
transposition-induced mutants and provide meaningful starting materials for transposon
tagging in rice.
68
LITERATURE CITED
Belzile, F., Lassner, M. W., Tong. Y., Khush, R. and Yoder, J. I. (1989) Sexual
transmission of transposed Activator elements in transgenic tomato. Genetics
123:181-189.
Cao, J., Rush, M. C., Nabors, M., Xie, Q. J., Croughan, T. P., and Nowick, T. P.
(1991) Development and inheritance of somaclonal variation in rice. In Biological
Nitrogen Fixation Associated with Rice Production. Eds. S. K. Dutta and X.
Sloger. Oxford and IBH Publishing. New Deli, India, pp 385-419.
Chomet, P. S., Wessler, S. and Dellaporta, S. L. (1987) Inactivation of the maize
transposable elements is associated with its DNA modification. EMBO J.
6:295-302.
Fromm, M., Fionnuala, M., Arm strong, C., Williams, R., Thomas, J. and Klein, T.
M. (1990) Inheritance and expression of chimeric genes in the progeny of
transgenic maize plants. Biotechn. 8:833-839.
Fukui, K. (1983) Sequential occurrence of mutations in a growing rice callus. Theor.
Appl. Genet. 65:225-230.
Hayashimoi , A., Li, Z. J. and M urai, N. (1990) A polyethylene glycol-mediated
protoplast transformation system for production of fertile transgenic rice plants.
Plant Physiol. 93:857-863.
Izawa, T., Miyazaki, C., Yamamoto, M., T erada, R., Iida, S. and Shimamoto, K.
(1991) Introduction and transposition of the maize transposable element Ac in
rice (Oryza sativa L.). Mol. Gen. Genet. 227:391-396.
Larkin, P. J. and Scowcroft, W. R. (1981) Somaclonal variation- a novel source of
variability from cell cultures for plant improvement. Theor. Appl. Genet.
60:197-214.
Li, Z. J., Burow, M. d. and M urai, N. (1990) High frequency generation of fertile
transgenic rice plants after PEG-mediated protoplast transformation. Plant Mol.
Biol. Rep. 8:276-291.
McClintock, B. (1963) Further studies of gene-control systems in maize. Carnegie
Inst. Washington Year Book 62:486-493.
McClintock, B. (1964) Aspects of gene regulation in maize. Carnegie Inst.
Washington Year Book 63:592-602.
69
M uller, E., Brown, P. T. H., H artke, S. and Lorz, H. (1990) DNA variation in
tissue-culture-derived rice plants. Theor. Appl. Genet. 80:673-679.
M urai, N., Li, Z. J., Kawagoe, Y. and Hayashimoto, A. (1991) Transposition of the
maize activator element in transgenic rice plants. Nucl. Acids Res. 19:617-622.
O gura, H., Kyozuka, J., Hayashi, Y., Koba, T. and Shimamoto, K. (1987) Field
performance and cytology of protoplast-derived rice (Oryza sativa): high yield
and low degree of variation of four japonica cultivars. Theor. Appl. Genet.
74:670-676.
Oono, K. (1981) In vitro method applied to rice. In: Theope, T. A. (ed) Plant tissue
culture methods and applications in agriculture. Academic Press, New York, pp
273-298.
Rhodes, C. A., Pierce, D. A., M ettler, I. J., M ascarenhas, D., and Detm er, J.
(1988) Genetically transformed maize plants from protoplasts. Science 240:204207.
Rogers, S. O. and Bendich, A. J. (1985) Extraction of DNA from milligram amounts
of fresh, herbarium and mummified plant tissues. Plant Mol. Biol. 5:69-76.
Schwartz, D. and Dennis, E. (1986) Transposase activity of the Ac controlling
element in maize is regulated by its degree of methylation. Mol. Gen. Genet.
205:476-482.
Shimamoto, K., T erada, R., Izawa, T. and Fujim oto, H. (1989) Fertile transgenic
rice plants regenerated from transformed protoplasts. Nature 338:274-276.
CHA PTER IV
SUMMARY AND CONCLUSION
70
71
In recent years, regeneration of a large number of transgenic rice plants became
feasible through efficient transformation systems using protoplasts.
However, the
successful selection of rice transformants was, until now, only achieved by using the
hygromycin resistance gene as a selectable maker. In order to develop alternative marker
genes suitable for rice transformation, a mutant Arabidopsis acetolactate synthase (ALS)
gene csr-1-1 conferring chlorsulfuron resistance was transferred to transgenic rice. More
than 200-fold chlorsulfuron resistance was detected in rice calli after transformation with
the mutant ALS gene driven by CaMV 35S promoter (35S/ALS).
The attained
chlorsulfuron resistance was correlated with the expression of the mutant ALS gene.
Transformation of rice protoplasts with the mutant ALS gene resulted in efficient isolation
of resistant protoplast colonies after selection with chlorsulfuron at 5 to 10 nM levels.
Accordingly, the concentration of chlorsulfuron effective for transformant selection with
35S/ALS gene was several thousand-fold less than that of hygromycin. Fertile transgenic
rice plants with the mutant Arabidopsis ALS gene were subsequently recovered. The
results suggest that the 35S/ALS gene can be used as an alternative selection marker to
hygromycin resistance gene in rice transformation. However, besides the effectiveness
and the sensitivity in selection, other advantages such as improvement of plant
regeneration for the use of the 35S/ALS marker gene in rice transformation have to be
experimentally determined.
Even though transgenic rice plants were routinely produced, little was known
about the genetic stability of transgenic rice plants. The lack of understanding in this
72
aspect may generate uncertainty about the usefulness of transgenic rice in subsequent
applications, particularly in transposon tagging experiments using transgenic rice plants.
To investigate somaclonal variation, DNA insertion- and transposition-induced
mutation after transformation, progeny of transgenic rice plants containing the hph gene
and the maize Ac element and nontransformed seed- and protoplast-derived controls were
grown in the field conditions and compared for six major agronomic traits. Analysis of
progeny from protoplast-derived plants indicated that protoplast-derived rice plants were
phenotypically indistinguishable from seed-derived plants, indicating that the somaclonal
variation is insignificant after plant regeneration through the established procedures.
Similar phenotypic analysis
demonstrated that the majority of transgenic plants
containing the hph gene were relatively normal except that the low fertility was observed
/
in these transgenic plants, probably induced by hygromycin selection. The hygromycin
resistance test and DNA analysis indicated that the hph gene was inherited stably as a
single locus or closely-linked loci in the great majority of transgenic rice plants.
The relatively normal phenotype and stable transmission of transferred gene
provided an opportunity to identify transposition-induced mutation in transgenic rice
plants.
A high frequency of phenotypic variations was evident in Ac-containing
transgenic plants.
Up to 90% of progeny lines tested had phenotypic variation in
compared traits.
In accordance with the high frequency of phenotypic variation,
independent transmission of intact and active Ac elements was detected in transgenic
progeny. This might be explained by novel insertion of Ac elements into new genomic
locations in transgenic progeny as a result of germinal transposition.
Single-locus
73
segregation for sterility and albino/yellow traits were also identified in Ac-containing
progeny. In both cases the segregation was found in association with the presence of
active Ac element. However, further evidence is still needed to determine the relationship
between observed phenotypic changes and Ac insertion. The identified mutation materials
will be valuable for subsequent gene isolation experiments.
In conclusion, rice is a promising system for the study of monocotyledonous
genes. Great progress has been made to establish an efficient rice transformation system.
In this study, the development of an alternative selectable marker gene based on the use
of a mutant Arabidopsis ALS gene will provide more flexibility for disposal of selection
systems in rice transformation. The investigation on transgenic rice plants containing a
hph gene demonstrated the phenotypic normality after transformation and stable
inheritance of transferred genes and thus ensured future applications of transgenic rice
plants. Our primary findings in the analysis of transgenic progeny containing the maize
Ac transposable element also shed a promising light on the use of maize Ac transposable
element and provide a novel avenue for isolation of unknown rice genes via transposon
tagging.
APPENDIX A. PERMISSION ACKNOWLEDGEMENT
The following protocol has been reproduced from Plant Molecular Biology
Reporter volume 8(4) pages from 276 to 291 by kind permission of Transaction
Periodicals Consortium, Rutgers University, NJ and International Society for Plant
Molecular Biology.
74
APPENDIX B. LETTER OF PERMISSION FROM THE PUBLISHER
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Dept, of Plant Pathology
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75
APPENDIX C. A PROTOCOL FOR RICE TRANSFORMATION
76
PLEASE NOTE
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77-92,
Appendix C
University Microfilms International
VITA
Zhijian Li was born on September 29, 1958 in Guangdong province, the People’s
Republic of China. After primary and secondary education, he worked as a technician
in the Shunde Rice Experiment Station for three years. In 1978 he attended Southern
China Agricultural University for undergraduate study in the major of Plant Genetics and
Breeding. After receiving his B. S. degree in Agronomy in 1981, he entered the Institute
of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences in Beijing
as a research associate in the Department of Rice Breeding. There he assisted with
research work on rice anther culture for cultivar improvement and genetic analysis of
disease resistance in rice. In 1986, he joined the Department of Plant Sciences at Texas
Tech University in Lubbock, Texas for his graduate study. From Spring of 1987, he
continued his graduate training under the supervision of Dr. N. Murai in the Department
of Plant Pathology and Crop Physiology at LSU. His graduate research involved rice
protoplast regeneration, genetic transformation and subsequent applications to molecular
studies. He received his M. S. degree in Plant Health in 1988. He is now a candidate
for the degree of Doctor of Philosophy in Plant Health. During his graduate study, he
authored and co-authored the following publications from his research programs:
Li, Z. J., and Murai, N. (1990) General medium for efficient plant regeneration from
rice (Oryza sativa L.) protoplast. Plant Cell Reports 9:216-220.
Li, Z. J., Burow, M. D., and Murai, N. (1990) High frequency generation of fertile
transgenic rice plants after PEG-mediated transformation. Plant Molecular
Biology Reporter 8:276-291.
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Li, Z. J., Xie, Q. J., Rush, M. C., and Murai, N. (1992) Fertile transgenic rice plants
generated via protoplasts from the U.S. cultivar Labelle. Crop Science (in
press).
Murai, N., Li, Z. J., Kawagoe, Y., and Hayashimoto, A. (1991) Transposition of maize
activator element in transgenic rice plants. Nucleic Acids Research 19:617-622.
Hayashimoto, A., Li, Z. J., and Murai, N. (1990) A polyethylene glycol-mediated
protoplast transformation system for production of fertile transgenic rice
plants. Plant Physiology 93:857-863.
Zheng, Z. W., Hayashimoto, A., Li, Z. J., and Murai, N. (1991) Hygromycin resistance
gene cassettes for vector construction and selection of transformed rice
protoplasts. Plant Physiology 97:832-835.
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
Zhijian Li
Major Field:
Plant Health
Title o f Dissertation:
Transgenic rice plants containing a chlorsulfuron
resistance gene, a hygromycin resistance gene and the maize
activator transposable element.
A pproved:
Major Professor and Chairman
EXAMINING COMMITTEE:
A
01. C .
Date of Examination:
April 3, 1992