Note: Descriptions are shown in the official language in which they were submitted.
WO 93/25695 ~ ~ ~ ~ ~ PCT/EP93/01489
1
MAINTENANCE OF MAhE-BTERIhE PI~INTB
This invention relates to a process for
maintaining male-sterile plant lines that can be used
for the production of hybrid seed of a crop, to
maintainer plants that can be used in such a process,
and to maintainer genes that can be used to produce
such maintainer plants.
Backcround of the Invention
In many, if not most, plant species, the
development of hybrid cultivars is highly desired
because of their generally increased productivity due
to heterosis: the superior performance of hybrid
individuals compared with their parents (see, e.g.,
Fehr (1987) "Principles of Cultivar Development,
Volume 1: Theory and Technique", MacMillan Publishing
Company, New York: Allard (1960) °Principles of Plant
Breeding", John Wiley and Sons, Inc., New York).
The development of hybrid cultivars of various
plant species depends upon the capability to achieve
almost complete cross-pollination between parents.
This is most simply achieved by rendering one of the
parent lines male-sterile (i.e., with pollen being
absent or nonfunctional), for example, by manually
removing the one parent's anthers or by providing the
one parent with naturally occurring cytoplasmic or
nuclear genes that prevent anther and/or pollen
development and/or function, using classical breeding
techniques (for a review of the genetics of male-
sterility in plants, see Kaul (1988) "Male Sterility
in Higher Plants", Springer Verlag, New York),
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2
For hybrid plants where the seed is the
harvested product (e.g., corn and oilseed rape), it
is, in most cases, also necessary to ensure that
fertility of the hybrid plants is fully restored. In
plants in which the male-sterility is under genetic
control, this requires the use of genes that can
restore male-fertility. Hence, the development of
hybrid cultivars is mainly dependent on the
availability of suitable and effective sterility and
restorer genes.
Endogenous nuclear loci are known for most plant
species that contain genotypes which effect male-
sterility, and generally, such loci need to be
homozygous for particular recessive alleles in order
to result in a male-sterile phenotype. The presence
of a dominant male-fertile allele at such loci
results in male-fertility.
Recently, it has been shown that male-sterility
can be induced in a plant by providing the plant with
a nuclear male-sterility genotype that includes a
chimaeric male-sterility gene comprising a DNA
sequence (or male-sterility DNA) coding, for example,
for a cytotoxic product (such as an RNase) and under
the control of a promoter which is predominantly
active in selected tissue of the plant's male
reproductive organs. In this regard, tapetum-specific
promoters, such as the promoter of the TA29 gene of
Nicotiana tabacum, have been shown to be particularly
useful for this purpose (Mariani et al (1990) Nature
347:737; European patent publication ("EP")
0,344,029). By providing the nuclear genome of the
plant with such a male-sterility gene, an artificial
nuclear male-sterility locus is created containing
the artifical male-sterility genotype that results in
a male-sterile plant.
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Examples of male-sterility DNAs include DNA
encoding: RNases such as RNase T1 (which degrades RNA
molecules by hydrolyzing the bond after any guanine residue)
and Barnase; DNases such as an endonuclease (e.g., EcoRI); or
proteases such as a papain (e. g., papain zymogen and papain
active protein); enzymes which catalyse the synthesis of
phytohormones, such as: isopentenyl transferase which is an
enzyme that catalyzes the first step in cytokinin
biosynthesis and is encoded by gene 4 of Agrobacterium T-DNA;
and the enzymes involved in the synthesis of auxin and
encoded by gene 1 and gene 2 of Agrobacterium T-DNA;
glucanases; lipases such as phospholipase AZ (Verheij et al
(1981) Rev. Biochem. Pharmacol. 91, 92-203); lipid
peroxidases; or plant cell wall inhibitors; proteins toxic to
plants cells, such as a bacterial toxin (e. g., the B-fragment
of diphtheria toxin or botulin). Still another example of a
male-sterility DNA is an antisense DNA which encodes a strand
of DNA complementary to a strand of DNA that is naturally
transcribed in the plant's stamen cells under the control of
an endogenous promoter. Such an antisense DNA can be
transcribed into an RNA sequence capable of binding to the
coding and/or non-coding portion of an RNA, naturally
produced in the stamen cell, so as to inhibit the translation
of the naturally produced RNA. An example of such an
antisense DNA is the antisense DNA of the TA29 gene which is
naturally expressed, under the control of the TA29 promoter,
in tapetum cells of the anthers of plants. A further example
of a male-sterility DNA encodes a specific RNA enzyme (i.e.,
a so-called "ribozyme"), capable of highly specific cleavage
against a given target sequence, as described by Haseloff and
Gerlach (1988) Nature 334, 585-591. Such a ribozyme is, for
example, the ribozyme targeted against the RNA encoded by the
TA29 gene. Still other examples of male-sterility DNAs
encode products which can render the stamen cells susceptible
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to specific diseases, such as fungus infections. Such a
male-sterility DNA can be used in a plant wherein all other
cells, in which the male-sterility DNA is not expressed, are
resistant to the specific disease.
WO 93/25695 PCT/EP93/01489
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3
In addition, it has been recently shown that
male-fertility can be restored to such a nuclear
male-sterile plant with a chimaeric fertility-
restorer gene comprising another DNA sequence (or
fertility-restorer DNA) that codes, for example, for
a protein that inhibits the activity of the cytotoxic
product or otherwise prevents the cytotoxic product
from being active at least in the selected tissue of
the plant's male reproductive organs (EP 0,412,911).
For example, the barnase gene of Bacillus
amyloliquefaciens codes for an RNase (Barnase) which
can be inhibited by a protein (Barstar) that is
encoded by the barstar gene of B. amyloliauefaciens.
Hence, the barnase gene can be used for the
construction of a chimaeric male-sterility gene while
the barstar gene can be used for the construction of
a chimaeric fertility-restorer gene. Experiments in
different plant species (e. g., oilseed rape) have
shown that such a chimaeric barstar gene can fully
restore the male-fertility of male-sterile lines in
which the male-sterility was due to the presence of
such a chimaeric barnase gene (EP 0,412,911: Mariani
et al (1991) Proceedings of the CCIRC Rapeseed
Congress, July 9-11, 1991 Saskatoon, Saskatchewan,
Canada: Mariani et al (1992) Nature 357:384). By
coupling a marker gene, such as a dominant herbicide
resistance gene (for example, the bar gene coding for
phosphinothricin acetyl transferase (PAT) that
converts herbicidal phosphinothricin to a non-toxic
compound [De Block et al (1987) EMBO J. 6:2513]), to
the chimaeric male-sterility and/or fertility
restorer gene, breeding systems can be implemented to
select for uniform populations of male-sterile plants
(EP 0,344,029: EP 0,412,911).
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4
The production of hybrid seed of any particular
cultivar of a plant species requires the: 1)
maintenance of small quantities of pure seed of each
inbred parent and 2) the preparation of larger
quantities of seed of each inbred parent. Such larger
quantities of seed would normally be obtained by
several (usually two) seed-multiplication rounds,
starting from a small quantity of pure seed ("basic
seed") and leading, in each multiplication round, to
a larger quantity of seed of the inbred parent and
finally to a stock of seed of the inbred parent
("parent seed" or "foundation seed") which is of
sufficient quantity to be planted to produce the
desired quantities of hybrid seed. Of course, in
each seed-multiplication round, larger planting areas
(fields) are required.
In order to maintain and enlarge a small stock
of seeds of male-sterile plants, it has been
necessary to cross the parent male-sterile plants
with normal pollen-producing parent plants. The
offspring of such a cross will, in all cases, be a
mixture of male-sterile and male-fertile plants, and
the latter have to be removed from the former. With
male-sterile plants containing an artificial male-
sterility locus as described above, such removal can
be facilitated by genetically linking the chimaeric
male-sterility gene to a suitable marker gene, such
as the bar gene, which allows the easy identification
and removal of the male-fertile plants. EP 0,198,288
and US Patent 4,717,219, by comparison, describe
methods for linking such marker genes (which can be
visible markers or dominant conditional markers) to
endogenous nuclear loci containing male-sterility
genotypes.
WO 93/25695 PCT/EP93/01489
:. . .
However, even when suitable marker genes are
linked to male-sterility genotypes, the maintenance
of parent male-sterile plants still requires the
removal from the field of a substantial number of
plants. For instance, in systems using a herbicide
resistance gene (e.g., the bar gene) linked to a
chimaeric male-sterility gene, only half of the
parent stock will result in male-sterile plants, thus
requiring the removal of the male-fertile plants by
herbicide spraying prior to flowering. In any given
field, the removal of male-fertile plants effectively
reduces the potential yield of hybrid seed or the
potential yield of male-sterile plants during each
round of seed multiplication for producing of parent
seed. This is economically unattractive for many
important crop species such as corn and oilseed rape.
In order to minimize the number of male-fertile
plants which have to be removed, male-fertile
maintainer plants have been sought which, when
crossed with a male-sterile parent plant, produce a
minimum, preferably no, male-fertile offspring,
thereby minimizing or avoiding altogether the need to
remove such male-fertile offspring. To solve an
analogous problem, US Patents 3,710,511 and 3,861,079
have described procedures for producing and
maintaining a homogenous population of male-sterile
plants by using specific chromosomal abnormalities
that are differentially transmitted to the egg and
the sperm in the plants.
Summary of the Invention
In accordance with this invention, a cell of a
transgenic plant ("the maintainer plant") is
provided, in which the nuclear genome contains stably
integrated therein: 1) at a first locus or male-
sterility locus, a male-sterility genotype in
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homozygous condition: and 2) at a second locus or
maintainer locus, a maintainer gene in heterozygous
condition; the male-sterility locus and the
maintainer locus preferably being unlinked; the
maintainer gene being a foreign DNA sequence,
preferably a foreign chimaeric DNA sequence,
containing:
a) a fertility-restorer gene that comprises at least:
i) a fertility-restorer DNA encoding a restorer RNA
and/or protein or polypeptide which, when
produced or overproduced in some or all of the
cells, preferably stamen cells, of the plant,
prevents phenotypic expression of the nuclear
male-sterility genotype that would render the
plant male-sterile in the absence of expression
of the fertility-restorer DNA in the some or all
stamen cells and
ii) a restorer promoter capable of directing
expression of the fertility-restorer DNA at
least in the some or all of the cells,
preferably stamen cells, of the plant, so that
the phenotypic expression of the nuclear male-
sterility genotype is prevented, the fertility-
restorer DNA being in the same transcriptional
unit as, and under the control of, the restorer
promoter and
b) a pollen-lethality gene that is selectively
expressed in microspores and/or pollen of the plant
to produce nonfunctional pollen and that comprises
at least:
iii) a pollen-lethality DNA coding for a pollen-
lethality RNA and/or protein or polypeptide
that, when produced or overproduced in the
microspores and/or pollen, significantly
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disrupts their metabolism, functioning and/or
development and
iv) a pollen-specific promoter capable of directing
expression of the pollen-lethality DNA
selectively in the microspores and/or pollen of
the plant, the pollen-lethality DNA being in the
same transcriptional unit as, and under the
control of, the pollen promoter.
The cell of the maintainer plant of this
invention preferably also comprises, especially in
the maintainer locus, at least one first marker gene
which comprises at least:
v) a first marker DNA encoding a first marker RNA
and/or protein or polypeptide which, when
present at least in a first specific tissue or
specific cells of the plant, renders the plant
easily separable from other plants which do not
contain the first marker RNA, protein or
polypeptide encoded by the first marker DNA at
least in the first specific tissue or specific
cells and
vi) a first marker promoter capable of directing
expression of the first marker DNA at least in
the first specific tissue or specific cells, the
first marker DNA being in the same
transcriptional unit as, and under the control
of, the first marker promoter.
The male-sterility genotype in the cell of the
maintainer plant of this invention can be foreign or
endogenous but is preferably a foreign, especially
chimaeric, male-sterility gene which comprises:
1) a male-sterility DNA encoding a sterility RNA
and/or protein or polypeptide which, when produced or
overproduced in a stamen cell of the plant in the
absence of the restorer RNA, protein or polypeptide,
WO 93/25695 PCT/EP93/01489
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8
significantly disturbs the metabolism, functioning
and/or development of the stamen cell and
2) a sterility promoter capable of directing
expression of the male-sterility DNA selectively in
stamen cells of the plant, the male-sterility DNA
being in the same transcriptional unit as, and under
the control of, the sterility promoter.
The male-sterility genotype in the maintainer
plant cell of this invention preferably comprises,
especially in the male-sterility locus, at least one
second marker gene which comprises at least:
3) a second marker DNA encoding a second marker RNA
and/or protein or polypeptide which, when present at
least in the second specific tissue or specific cells
of the plant, renders the plant easily separable from
other plants which do not contain the second marker
RNA, protein or polypeptide encoded by the second
marker DNA at least in the second specific tissue or
specific cells and
4) a second marker promoter capable of directing
expression of the second marker DNA at least in the
second specific tissue or specific cells, the second
marker DNA being in the same transcriptional unit as,
and under the control of, the second marker promoter.
Also in accordance with this invention are
provided the maintainer plants, the seeds of such
plants, and plant cell cultures, all of which consist
essentially of the cells of this invention.
Further in accordance with this invention are
provided the maintainer gene and plasmids containing
the maintainer gene, as well as bacterial host cells
(e. g., E. coli or Agrobacterium) containing such
plasmids.
Still further in accordance with this invention
is provided a process for producing, preferably
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enlarging, a homogeneous population of male-sterile plants
and their seed that contain a nuclear male-sterility gene in
homozygous condition, the process comprising the step of
crossing the male-sterile plants with the maintainer plants
of this invention. The seed from the resulting male-sterile
plants can be harvested and grown into the male-sterile
plants. Hybrid seed can then be produced by crossing the
male-sterile plants with male-fertile plants of another
inbred parent line used as pollinators.
According to another aspect of the present
invention, there is provided a process for maintaining a
male-sterile line of a plant species, said process
comprising the steps of: 1) crossing: a) a male-sterile line
to be maintained comprising male-sterile parent plants which
comprise a male-sterility gene, at a first genetic locus;
wherein said male-sterility gene is homozygous at said first
genetic locus; with b) a maintainer line comprising male-
fertile parent plants which comprise said homozygous male-
sterility gene at said first genetic locus, and which
further comprise, at a second genetic locus which segregates
independently from said first genetic locus, a foreign DNA
comprising: i) a restorer gene which, upon expression,
inhibits or prevents the phenotypic expression of said male-
sterility gene; and ii) a pollen-lethality gene comprising,
under the control of a first promoter that directs
expression selectively in a microspore or a pollen cell of
said male-fertile parent plants, a first DNA encoding a
first RNA, protein or polypeptide which, when produced in
said microspore or pollen cell of said male-fertile parent
plants, significantly disrupts the metabolism, functioning
or development of said microspore or pollen cell; wherein
said foreign DNA is heterozygous at said second genetic
locus; and 2) harvesting seeds from said male-sterile parent
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plants, wherein said seeds grow into a new generation of
male-sterile parent plants.
According to another aspect, there is provided a
process for maintaining a male-sterile line of a plant
species, said process comprising the steps of: 1) obtaining
a maintainer line comprising male-fertile parent plants
which comprise a male-sterility gene at a first genetic
locus, wherein said male-sterility gene is homozygous at
said first genetic locus, and which further comprise, at a
second genetic locus which segregates independently from
said first genetic locus, a foreign DNA comprising: a) a
restorer gene which, upon expression, inhibits or prevents
the phenotypic expression of said male-sterility gene; and
b) a pollen-lethality gene comprising, under the control of
a first promoter that directs expression selectively in a
microspore or a pollen cell of said male-fertile parent
plants, a first DNA encoding a first RNA, protein or
polypeptide which, when produced in said microspore or
pollen cell of said male-fertile parent plants,
significantly disrupts the metabolism, functioning or
development of said microspore or pollen cell; wherein said
foreign DNA is heterozygous at said second genetic locus;
and 2) crossing said maintainer line with a male-sterile
line to be maintained comprising male-sterile parent plants
which comprise said homozygous male-sterility gene at said
first genetic locus; and 3) harvesting seeds from said male-
sterile parent plants, wherein said seeds grow into a new
generation of male-sterile parent plants.
According to another aspect, there is provided a
process for obtaining seed of male-sterile parent plants
which comprise a homozygous male-sterility gene at a first
genetic locus, said process comprising the steps of:
1) crossing said male-sterile line with a maintainer line
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comprising male-fertile parent plants which comprise said
homozygous male-sterility gene at said first genetic locus,
and which further comprise, at a second genetic locus which
segregates independently from said first genetic locus, a
foreign DNA comprising: a) a restorer gene which, upon
expression, inhibits or prevents the phenotypic expression
of said male-sterility gene; and b) a pollen-lethality gene
comprising, under the control of a first promoter that
directs expression selectively in a microspore or a pollen
cell of said male-fertile parent plants, a first DNA
encoding a first RNA, protein or polypeptide which, when
produced in said microspore or pollen cell of said male-
fertile parent plants, significantly disrupts the
metabolism, functioning or development of said microspore or
pollen cell; and 2) harvesting seeds from said male-sterile
parent plants.
According to still another aspect of the present
invention, there is provided a cell of a male-fertile parent
plant for use in maintaining male-sterile parent plants
which comprise a homozygous male-sterility gene at a first
genetic locus, wherein said male-fertile parent plant
comprises said homozygous male-sterility gene at said first
genetic locus and further comprises, at a second genetic
locus which segregates independently from said first genetic
locus, a foreign DNA comprising: (i) a restorer gene which,
upon expression, inhibits or prevents the phenotypic
expression of said male-sterility gene; and (ii) a pollen-
lethality gene comprising, under the control of a first
promoter that directs expression selectively in a microspore
and/or pollen cell of said male-fertile parent plant, a
first DNA encoding a first RNA, protein or polypeptide which
when produced in said microspore or pollen cell of said
male-fertile parent plant significantly disrupts the
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metabolism, functioning or development of said microspore or
pollen cell; wherein said foreign DNA is heterozygous at
said second genetic locus, and wherein said male-fertile
parent plant can be crossed with said male-sterile parent
plants to produce, on said male-sterile parent plants, seeds
which grow into a new generation of male-sterile parent
plants.
According to yet another aspect of the present
invention, there is provided use of a male-fertile parent
plant for maintaining male-sterile parent plants which
comprise a homozygous male-sterility gene at a first genetic
locus, wherein said male-fertile parent plant comprises said
homozygous male-sterility gene at said first genetic locus
and further comprises, at a second genetic locus which
segregates independently from said first genetic locus, a
foreign DNA comprising: (i) a restorer gene which, upon
expression, inhibits or prevents the phenotypic expression
of said male-sterility gene; and (ii) a pollen-lethality
gene comprising, under the control of a first promoter that
directs expression selectively in a microspore or pollen
cell of said male-fertile parent plant, a first DNA encoding
a first RNA, protein or polypeptide which when produced in
said microspore or pollen cell of said male-fertile parent
plant significantly disrupts the metabolism, functioning or
development of said microspore or pollen cell; wherein said
foreign DNA is heterozygous at said second genetic locus,
and wherein said male-fertile parent plant can be crossed
with said male-sterile parent plants to produce, on said
male-sterile parent plants, seeds which grow into a new
generation of male-sterile parent plants.
According to a further aspect of the present
invention, there is provided use of a kit for maintaining a
male-sterile line of a plant species, said kit comprising:
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9d
a) a male-sterile line comprising male-sterile parent plants
which comprise a male-sterility gene at a first genetic
locus, wherein said male-sterility gene is homozygous at
said first genetic locus; and, b) a maintainer line
comprising male-fertile parent plants which comprise said
homozygous male-sterility gene at said first genetic locus,
and which further comprise, at a second genetic locus which
segregates independently from said first genetic locus, a
foreign DNA comprising: (i) a restorer gene which, upon
expression, inhibits or prevents the phenotypic expression
of said male-sterility gene; and (ii) a pollen-lethality
gene comprising, under the control of a first promoter that
directs expression selectively in a microspore and/or pollen
cell of said male-fertile parent plants, a first DNA
encoding a first RNA, protein or polypeptide which, when
produced in said microspore or pollen cell of said male-
fertile parent plants significantly disrupts the metabolism,
functioning or development of said microspore or pollen
cell; wherein said foreign DNA is heterozygous at said
second genetic locus; and wherein said male-sterile and
male-fertile parent plants can be crossed to produce, on
said male-sterile plants, seed which grow into a new
generation of male-sterile parent plants.
Yet further in accordance with this invention is
provided a process for producing, preferably enlarging, a
population of the maintainer plants, comprising the step of
selfing the maintainer plants.
Detailed Description of the Invention
A male-sterile plant of this invention is a plant
of a given species with a nuclear male-sterility genotype.
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9e
A restorer plant of this invention is a plant of
the same plant species containing, within its nuclear
genome, a fertility-restorer gene that is able to restore
the male-fertility in offspring which are obtained from a
cross between the male-sterile plant and the restorer plant
and which contain both the male-sterility genotype and the
fertility-restorer gene.
A restored plant of this invention is a plant of
the same species that is male-fertile and that contains,
within its nuclear genome, the male-sterility genotype and
the fertility-restorer gene.
A parent plant or parent of this invention is a
plant that can be used for the production of hybrid seed.
The female or seed parent plant is the parent from which the
hybrid seed is harvested. For the purposes of this
invention, the female parent will
WO 93/25695 PCT/EP93/01489
always be a male-sterile plant. The male or pollen
parent is the parent that is used to fertilize the
female parent. In many cases, the male parent will
also be a restorer plant.
A line is the progeny of a given individual
plant.
The male-sterility genotype of this invention is
the genotype of at least one locus, preferably only
one locus, in the nuclear genome of a plant (i.e.,
the male-sterility locus), the allelic composition of
which can result in male-sterility in the plant. A
male-sterility genotype can be endogenous to the
plant, but it is generally preferred that it be
foreign to the plant. Preferre3 foreign male-
sterility genotypes are those in which the allele
responsible for male-sterility contains a foreign
male-sterility gene that comprises:
1) a male-sterility DNA encoding a sterility RNA
and/or protein or polypeptide which, when
produced or overproduced in a stamen cell of the
plant, significantly disturbs the metabolism,
functioning and/or development of the stamen
cell and
2) a sterility promoter capable of directing
expression of the male-sterility DNA selectively
in stamen cells of the plant, the male-sterility
DNA being in the same transcriptional unit as,
and under the control of, the sterility
promoter.
Such a male-sterility gene is always a dominant
allele at a foreign male-sterility locus. The
recessive allele corresponds to the absence of the
male-sterility gene in the nuclear genome of the
plant.
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Preferred foreign male-sterility DNAs and
sterility promoters that can be used in the male-
sterility genes in female parent plants and
maintainer plants of this invention have been
described in EP 0,344,029. A particularly useful
male-sterility DNA codes for Barnase (Hartley (1988)
J.Mol. Biol. 202:913). Particularly useful sterility
promoters are tapetum-specific promoters such as: the
promoter of the TA29 gene of Nicotiana tabacum
(EP 0,344,029) which can be used in tobacco, oilseed
rape, lettuce, chicory, corn and other plant species;
the PT72, the PT42 and PEl promoters from rice, the
sequences of which are given in SEQ ID no. 7, SEQ ID
no. 8, and SEQ ID no. 9, respectively, of the
Sequence Listing and which can be used in rice and
other plant species (PCT application
PCT/EP 92/00274): and the PCA55 promoter from corn,
the sequence of which is given in SEQ ID No. 10,
which can be used in corn and other plant species
(PCT application PCT/EP 92/00275).
A preferred endogenous male-sterility genotype
is one in which a recessive allele ("m") in
homozygous condition (m/m) at a male-sterility locus
produces male-sterility. At a male-sterility locus,
male-fertility would otherwise be encoded by a
corresponding dominant allele ("M"). Such a male-
sterility genotype is known in many plant species
(see Kaul (1988) supra; and 1992 issues of Maize
Genetics Cooperation Newsletter, published by the
Department of Agronomy and U.S. Department of
Agriculture, University Of Missouri, Columbia,
Missouri, U.S.A.). The DNA sequences in the nuclear
genome of a plant corresponding to m and M alleles
can be identified by gene tagging, i.e., by
insertional mutagenesis using transposons, or by
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12
means of T-DNA integration (see, e.g., Wienand and
Saedler (1987) In "Plant DNA Infectious Agents", Ed.
by T.H.Hohn and J.Schell, Springer Verlag, New York,
p. 205; Shepherd (1988) In "Plant Molecular Biology:
a Practical Approach", IRL Press, p. 187; Teeri et al
(1986) EMBO J. 5:1755).
Fertility-restorer DNAs and restorer promoters
that can be used in the maintainer genes of this
invention with a foreign male-sterility genotype have
been described in EP 0,412,911. In this regard,
fertility-restorer genes in which the fertility-
restorer DNA encodes Barstar (Hartley (1988) J.Mol.
Biol. 202:913) and is under control of tapetum-
specific promoters, such as those described above as
sterility promoters, are of particular use. In
particular, it is believed that a fertility-restorer
DNA coding for a mutant of Barstar, in which the
cysteine residue at its position 40 is replaced by
serine (Hartley (1989) TIBS 14:450), functions better
in restoring the fertility in the restored plants of
some species.
When an endogenous male-sterility genotype is
homozygous for a recessive allele m, it is preferred
that the fertility-restorer gene be the dominant
allele M of that male-sterility genotype, preferably
under the control of its own promoter. The DNA
corresponding to such a dominant allele, including
its natural promoter, can be isolated from the
nuclear genome of the plant by means of gene tagging
as described above.
The pollen-lethality DNAs that are used in the
pollen-lethality genes of this invention preferably
encode an RNA and/or a protein or polypeptide that,
when expressed in microspores or pollen,
significantly disrupts their metabolism, functioning
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13
and/or development. In this regard, the pollen-
lethality DNAs can encode RNAs, proteins or
polypeptides such as are encoded by the male-
sterility DNAs described in EP 0,344,029. Of
particular interest are male-sterility DNAs that
encode ribonucleases (EP 0,344,029) such as RNase T1
from AsperQillus oryzae (Quaas et al (1988) Eur. J.
Biochem. 173:617) or Barnase from Bacillus
amyloliquefaciens (Hartley (1988) J.Mol.Biol.
202:913).
So that the pollen-lethality DNA is expressed
selectively in microspores or pollen of the
maintainer plant, it is preferred that the pollen-
specific promoter, which controls the pollen-
lethality DNA in the pollen-lethality gene, be a
promoter capable of directing gene expression
selectively in the microspores and/or pollen of the
plant. Such a pollen-specific promoter can be an
endogeneous promoter or a foreign promoter and can be
from the nuclear genome or from the mitochondrial or
chloroplast genome of a plant cell, but in any event,
the pollen-specific promoter is foreign in the
nuclear genome of the plant being transformed.
Preferably the pollen-specific promoter causes the
pollen-lethality DNA to be expressed only in the
microspores and/or pollen, i.e., after meiosis of the
microsporocytes in the anthers. The pollen-specific
promoter can be selected and isolated in a well known
manner from a plant species, preferably the plant
species to be rendered male-sterile, so that the
pollen-specific promoter directs expression of the
pollen-lethality DNA selectively in the microspores
and/or pollen so as to kill or disable the
microspores and/or pollen in which the pollen-
lethality gene is expressed. The pollen-specific
WO 93/25695 PCT/EP93/01489
14
promoter is preferably also selected and isolated so
that it is effective to prevent expression of the
pollen-lethality DNA in other tissues of the plant.
For example, a suitable endogeneous pollen-specific
promoter can be identified and isolated in a plant,
to be rendered male-sterile, by .
1. searching for an mRNA which is only present in
the plant during the development of its
microspores and/or pollen;
2. optionally isolating the microspore- and/c
pollen-specific mRNA;
3. preparing and isolating a cDNA from the
microspore- and/or pollen-specific mRNA;
4. using this cDNA as a probe to identify regions
in the plant genome which contain DNA coding for
the corresponding microspore- and/or pollen-
specific DNA or alternatively using inverse
polymerase chain reactions for the geometric
amplification of the DNA sequences which flank,
upstream and downstream, a chosen core region of
the genomic DNA corresponding to the sequence of
the microspore- and/or pollen-specific cDNA: and
5. identifying the portion of the plant genome that
is upstream (i.e. 5') from the DNA coding for
the microspore- and/or pollen-specific mRNA and
that contains the promoter of this DNA.
Examples of such pollen-specific promoters are well
known (see MacCormick (1991) TIG 7:298). In this
regard, Hamilton et al (1989) Sex. Plant Reprod.
2:208 describes a pollen-specific clone ("Zmgl3")
from maize inbred line W-22, and the use of the
promoter sequences of the clone to direct pollen-
specif~c expression in tobacco has been described by
Guerrero et al (1990) Mol.Gen.Genet. 224:161). Other
pollen-specific promoters that are likewise believed
WO 93/25695 PCT/EP93/01489
- ~~~~~~~
to be useful are: the promoter of the gene
corresponding to the Nicotiana tabacum pollen-
specific cDNA NTPc303 described by Weterings et al
(1992) Plant Mol. Biol. 18:1101; and the promoter of
the gene corresponding to the Brassica napus pollen-
specific cDNA B54 described by Shen and Hsu (1992)
Mol. Gen. Genet. 234:379.
If the fertility-restorer DNA in the fertility-
restorer gene of the maintainer gene is also
expressed in microspores and/or pollen at the same
time as the pollen-lethality DNA is expressed (due
for instance to the activity of the restorer promoter
in microspores and/or pollen), it is preferred that
the pollen-lethality DNA be different from the male-
sterility DNA (the expression of which is intended to
be prevented by expression of the fertility-restorer
DNA of the maintainer gene). For example, if the
male-sterility DNA encodes Barnase in the male-
sterile plants to be maintained, the fertility-
restorer DNA in the maintainer gene should encode
Barstar. Thus, if the restorer promoter in the
maintainer gene also directs expression of the
fertility-restorer DNA in microspores and/or pollen
and at the same time as the pollen-lethality DNA is
expressed, the pollen-lethality DNA preferably
should not encode Barnase but rather, for example,
another RNAse such as RNAse Tl.
First and second marker DNAs and first and
second marker promoters that can be used in the first
and second marker genes of this invention are also
well known (EP 0,344,029; EP 0,412,911). In this
regard, it is preferred that the first and second
marker DNAs be different, although the first and
second marker promoters may be the same.
WO 93/25695 PCT/EP93/01489
16
The fertility-restorer gene, the male-sterility
gene, the pollen-lethality gene, and the first and
second marker genes in accordance with this invention
are generally foreign DNA sequences, preferably
foreign chimaeric DNA sequences. Such foreign DNA
sequences are preferably provided with suitable 3'
transcription regulation . sequences and
polyadenylation signals, downstream (i.e. 3') from
their respective fertility-restorer DNA, male-
sterility DNA, pollen-lethality DNA, and first and
second marker DNAs. In this regard, either foreign or
endogenous, transcription termination and
polyadenylation signals suitable for obtaining
expression of such DNA sequences can be used. For
example, the foreign 3' untranslated ends of genes,
such as gene 7 (Velten and Schell (1985) Nucl. Acids
Res. 13:6998), the octopine synthase gene (De Greve
et al (1982) J.Mol. Appl. Genet. _1:499: Gielen et al
(1983) E.':BO J. 3:835; Ingelbrecht et al (1989) The
Plant Cell 1:671) the nopaline synthase gene of
the T-JNA region of Aarobacterium tumefaciens Ti-
piasmid (De Picker et al (1982) J.Mol. Appl. Genet.
1:561), the chalcone synthase gene (Sommer and Saedler
(1986) Mol. Gen. Genet. 202: 429-434), and the CaMV
19Si35S transcription unit (Mogen et al (1990) The Plant
Cell _2:?25i-1272), can be used.
BY "foreign" with regard to a gene or genotype
of this invention is meant that the gene or genotype
contains a foreign DNA sea_uence such as a male-
sterility DNA, a fertility-restorer DNA, a pollen-
lethality DNA, or a marker DNA and/or a foreign
promoter such as a sterility promoter, a restorer
promoter, a pollen-specific promoter or a marker
promoter. By "foreign" with regard to any DNA
sequence, such as a coding sequence or a promoter, in
a gene or genotype of this invention is meant that
such a DNA is not in the same genomic environment in
plant cell, transformed with such a DNA in
WO 93/25695 PCT/EP93/01489
~I3'~55~
accordance with this invention, as is such a DNA when
it is naturally found in the cell of the plant,
bacteria, animal, fungus, virus or the like, from
which such a DNA originates. This means, for example,
that a foreign fertility-restorer DNA, male-sterility
DNA, pollen-lethality DNA, or marker DNA can be: 1) a
nuclear DNA in a plant of origin; 2) endogenous to
the transformed plant cell (i.e., from a plant of
origin with the same genotype as the plant being
transformed): and 3) within the same transcriptional
unit as its own endogenous promoter and 3' end
transcription regulation signals (from the plant of
origin) in the foreign gene or genotype in the
transformed plant cell; but 4) inserted in a
different place in the nuclear genome of the
transformed plant cell than it was in the plant of
origin so that it is not surrounded in the
transformed plant cell by the genes which surrounded
it naturally in the plant of origin. Likewise, a
foreign fertility-restorer DNA, male-sterility DNA,
pollen-lethality DNA, or marker DNA can also, for
example, be: 1) a nuclear DNA in a plant of origin;
and 2) endogenous to the transformed plant cell; but
3) in the same transcriptional unit as a different
(i.e., not its own) endogenous promoter and/or 3' end
transcription regulation signals in a foreign
chimaeric gene or genotype of this invention in a
transformed plant cell. A foreign fertility-restorer
DNA, male-sterility DNA, pollen-lethality DNA, or
marker DNA can also, for example, be: 1) a nuclear
DNA in a plant of origin; and 2) endogenous to the
transformed plant cell; but 3) in the same
transcriptional unit as a heterologous promoter
and/or 3' end transcription regulation signals in a
foreign chimaeric gene or genotype of this invention
WO 93/25695 ~'~~ ~~~ PCT/EP93/01489
s , 18
in a transformed plant cell. A foreign fertility-
restorer DNA, a male-sterility DNA, pollen-lethality
DNA, or marker DNA can also, for example, be
heterologous to the transformed plant cell and in the
same transcriptional unit as an endogenous promoter
and/or 3' transcription regulation signals (e. g.,
from the nuclear genome of a plant with the same
genotype as the plant being transformed) in a foreign
chimaeric DNA sequence of this invention in a
transformed plant cell. Preferably, each fertility-
restorer DNA, male-sterility DNA, pollen-lethality
DNA, and marker DNA of this invention is heterologous
to the plant cell being transformed.
By "heterologous" with regard to a DNA, such as
a fertility-restorer DNA, a male-sterility DNA, a
pollen-lethality DNA, a marker DNA, a fertility-
restorer promoter, a sterility promoter, a pollen-
specific promoter or a marker promoter or any other
DNA sequence in a gene or a genotype of this
invention is meant that such a DNA is not naturally
found in the nuclear genome of cells of a plant with
the same genotype as the plant being transformed.
Examples of heterologous DNAs include chloroplast and
mitochondrial DNAs obtained from a plant with the
same genotype as the plant being transformed, but
preferred examples are chloroplast, mitochondrial,
and nuclear DNAs from plants having a different
genotype than the plant being transformed, DNAs from
animal and bacterial genomes, and chromosomal and
plasmidial DNAs from fungal, bacterial and viral
genomes.
By "chimaeric" with regard to a foreign DNA
sequence of this invention is meant that at least one
of its coding sequences : 1) is not naturally found
under the control of the promoter present in the
WO 93/25695 PGT/EP93/01489
_ 1'~~~~
19
foreign DNA sequence; and/or 2) is not naturally
found in the same genetic locus as at least one of
its associated marker DNAs. Examples of foreign
chimaeric DNA sequences of this invention comprise: a
pollen-lethality DNA of bacterial origin under the
control of a pollen-specific promoter of plant
origin; and a pollen-lethality DNA of plant origin
under the control of a pollen-specific promoter of
plant origin and in the same genetic locus as a
marker DNA of bacterial origin.
By "endogenous" with respect to a gene or
genotype of this invention is meant that it is not
foreign .
The foreign genes and genotypes of this
invention, such as the male-sterility gene and
genotype, the fertility-restorer gene and the
pollen-lethality gene, can be described like any
other genotype: capital letters denote the presence
of the foreign genes and genotypes (the dominant
allele) while small letters denote their absence (the
recessive allele). Hence, in this description of the
invention, "S" and "s" will denote the respective
presence and absence of the male-sterility gene, "R"
and "r" will denote the respective presence and
absence of the fertility-restorer gene, and "P" and
"p" will denote the respective presence and absence
of the maintainer gene.
For an endogeneous male-sterility genotype of
this invention, "m" will denote the recessive allele,
and "M" will denote the dominant allele. Thus, the
recessive allele m in homozygous condition (m/m) at a
male-sterility locus would result in male-sterility,
and the dominant allele M, when present at a male-
sterility locus either in homozygous or heterozygous
condition, results in male-fertility.
WO 93/25695 PGT/EP93/01489
,.
The cell of a plant, particularly a plant
capable of being infected with Agrobacterium such as
most dicotyledonous plants (e.g. Brassica napus), can
be transformed using a vector that is a disarmed Ti-
plasmid containing the male-sterility gene and/or the
fertility-restorer gene and/or the pollen-lethality
gene and/or the maintainer gene and/or the marker
genes) of this invention and carried by
Aqrobacterium. This transformation can be carried out
using the procedures described, for example, in EP
0,116,718 and EP 0,270,822. Preferred Ti-plasmid
vectors contain a foreign DNA sequence of this
invention between the border sequences, or at least
located to the left of the right border sequence, of
the T-DNA of the Ti-plasmid. Of course, other types
of vectors can be used to transform the plant cell,
using procedures such as direct gene transfer (as
described, for example, in EP 0, 233,247), pollen
mediated transformation (as described, for example,
in EP 0,270,356, PCT publication WO 85/01856, and US
patent 4,684,611), plant RNA virus-mediated
transformation (as described, for example, in
EP 0,067,553 and US patent 4,407,956) and liposome-
mediated transformation (as described, for example,
in US patent 4,536,475). Cells of monocotyledonous
plants, such as the major cereals including corn,
rice, wheat, barley and rye, can be transformed as
described in PCT application PCT/EP 91/02198. In case
the plant to be transformed is corn, other recently
developed methods can also be used such as, for
example, the methods described for certain lines of
corn by Fromm et al (1990) Bio/Technology 8:833,
Gordon-Kamm et al (1990) Bio/Technology 2:603 and
Gould et al (1991) Plant Physiol. 95:426. In case the
plant to be transformed is rice, recently developed
WO 93/25695 PCT/EP93/01489
__ 2 ~~~ .,
21
methods can also be used such as, for example, the
method described for certain lines of rice by
Shimamoto et al (1989) Nature 338:274, Datta et al
(1990) Bio/Technology 8:736 and Hayashimoto et al
(1990) Plant Physiol. 93:857.
The so-transformed cell can be regenerated into
a mature plant, and the resulting transformed plant
can be used in a conventional breeding scheme to
produce more transformed plants with the same
characteristics or to introduce the male-sterility
gene, the fertility-restorer gene, the pollen-
lethality gene, the marker genes and/or the
maintainer gene of this invention in other varieties
of the same or related plant species. Seeds obtained
from such plants contain the genes) of this
invention as a stable genomic insert.
The maintainer plant of this invention is of the
same species as a male-sterile plant line and can be
used for the maintenance of the male-sterile line,
i.e. to maintain a homogeneous population of male-
sterile plants and a stock of pure seed of the female
parent. The maintainer plant of this invention is
itself a plant in which male-fertility has been
restored and the genome of which contains both a
male-sterility genotype and, in the maintainer locus,
a fertility-restorer gene of this invention.
If a plant line with a homozygous male-sterility
genotype (A"'~'" or Asis) is available, a maintainer
plant for the male-sterile line can be directly
obtained by transforming a male-sterile plant of the
line with the maintainer gene of this invention and
then selecting those transgenic plants which are
male-fertility restored plants and in which the
maintainer gene is stably integrated in the nuclear
genome so that the genetic locus of the male-
WO 93/25695 PGT/EP93/01489
, .
22
sterility genotype and of the maintainer gene are
unlinked and segregate independently.
If the male-sterility genotype is foreign to the
plant line, alternative strategies can be followed.
For example, the maintainer plant of the present
invention can be obtained by: transforming a plant
cell of the plant line (A) with the maintainer gene
of this invention (P): and then regenerating, from
the so-transformed plant cell, a transgenic plant
containing, stably integrated in its genome, the
maintainer gene. Such a transgenic plant (APP) can
then be crossed as a female parent with a plant
AS/s,R/r of the same line, which contains at separate
loci in its genome a male-sterility gene (S) and a
corresponding fertility-restorer gene (R), both in
heterozygous condition, but which lacks the
maintainer gene. Thus, the cross is in fact: AsiS.Rir,pip
(male) x As~s,~~'',P~p (female) , and the offspring with
the genotype As~s,r/r,P/p (or hereinafter "As~S~P~P" fir
convenience) are selected and selfed. One eighth of
the offspring that have the desired genotype (Asis,Pip)
for a maintainer plant of this invention can then be
selected. Another eighth of the offspring with the
genotype (Asis'p~P) can be used as male-sterile plants
to be maintained.
Isolation of plants with desired genotypes can
be achieved by means of conventional testcrosses
(see, e.g., Fehr (198?) supra), preferably
supplemented by detection of the presence of specific
genes at the DNA level, e.g., by means of
amplification of DNA fragments by the polymerase
chain reaction, by Southern blot analysis and/or by
phenotypic analysis for the presence and expression
of first or second marker genes of this invention.
WO 93/25695 PCT/EP93/01489
23
The cross of a male-sterile plant containing a
male-sterility genotype in homozygous condition (Asis
or A"'~"') with a maintainer plant of this invention
(Asis,Pip or A'"~"',P~p, respectively) results in a
population of seeds that all contain the male-
sterility genotype in homozygous condition (Asps or
A'"~'", respectively) because the maintainer gene is not
transmitted through the pollen. This property can be used
to advantage in maintaining the basic seed and in the
multiplication of basic seed for the final production
of parent seed.
The maintainer plants of this invention (Asis,PiP
or Am~m,Plp) can themselves be maintained by selfing.
The offspring of such selfing will consist of 50%
male-fertile maintainer plants (As~s'P~p Or A'N'",P~p,
respectively) and 50% male-sterile plants containing
the male-sterility genotype in homozygous condition
(Asps or A"'~"', respectively ) . If desired, the male-
sterile plants can be removed either manually on the
basis of the male-sterile phenotype or, if the
maintainer gene comprises a suitable first marker
gene, preferably a first marker gene whose expression
confers herbicide resistance to the plant, by using
the phenotypic expression of the first marker gene
(e. g, by applying herbicide to the offspring so that
male-sterile plants that lack the herbicide-
resistance gene are killed while maintainer plants
with the herbicide-resistance gene survive).
Thus, the maintainer plant of this invention can
be easily used to maintain a homogeneous population
of male-sterile plants. In this regard, basic seed of
a female parent of a given plant species can be
crossed with an appropriate male parent to produce
hybrid seed. Also, the maintainer plant of this
invention can be used economically to multiply the
WO 93/25695 PCT/EP93/01489
24
basic seed of a female parent of a given plant
species, so as to obtain sufficient quantities of
female parent seed that can be crossed with an
appropriate male parent to produce desired
quantities of hybrid seed.
A male-sterile line, that is maintained and
multiplied by the use of the maintainer plants of
this invention, can be used for the production of
hybrid seed. In principle, the male-sterile line
(ASKS) can be crossed directly with another male
parent line (BSS) to produce hybrid seed (ABS~S) .
However, as all hybrid plants are male-sterile, no
reproduction and no seed set will occur. This is not
a problem if the seed is not the harvested product
(e.g., with lettuce), but where seed is the
harvested product (e. g., with corn and oilseed rape),
male-fertility in the hybrid plants should be at
least partially restored. This can be accomplished by
crossing the male-sterile line with a male-fertile
parent line (e. g:, BR~R) that is also a restorer line,
i.e. that also contains a fertility-restorer gene
(R) . The hybrids produced (ABSis,R/~) will be fully
male-fertile. Alternatively the male-sterile-line
(ASKS) can first be crossed with the male-fertile line
(ASKS) just prior to hybrid seed productions. This has
the advantage of giving a further multiplication of
the female parent line. The offspring (ASKS) can then
be crossed with a suitable male-fertile parent line
(Bs/s,r/r) to produce hybrid seed that is 50% male-
fertile. If hybrid seed with 100% male fertility is
desired, the offspring can be crossed with a suitable
restorer male parent line (BS~S.Ria) ,
In the case of a male-sterile line in which
male-sterility is due to an endogeneous male-
sterility genotype (A"~'") at a male-sterility locus,
WO 93/25695 PGT/EP93/01489
_ ~,
hybrid seed can easily be produced by crossing the
male-sterile line (A"'~"') with a line that is
homozygous with respect to the endogenous dominant
(male-fertility) allele at that male-sterility locus
(B"~"). All hybrid offspring of this cross will have
the genotype AB"i"' and will be fertile.
The maintainer plants of this invention can also
be used as pollinator (i.e., male-fertile) plants in
a cross with wild-type plants (As~s~p~P) of the same
inbred line. The progeny of this cross will all be
male-sterile and heterozygous for the male-sterility
genotype (As~s~pip) . The progeny can therefore be used
directly for hybrid seed production by crossing with
a pollinator plant line B (BS~$'p~P) . This scheme only
requires a male-sterilization of the wild-type
plants, for example by manually removing the anthers
(e. g., in corn) or by using a male gametocide.
Of course, by using the maintainer plants of
this invention to maintain a homogeneous population
of plants that are homozygous with respect to a
male-sterility allele (whether dominant or recessive)
that is encoded in the nuclear genome, the maintainer
plants acquire many of the characteristics of plants
of a cytoplasmic male-sterile line. However, such
plants do not have one of the major disadvantages of
cytoplasmic male-sterile plants, namely the
cytoplasmic uniformity of the various male-sterile
lines which, in corn, has led to serious problems
(see Craig (1977) In "Corn and Corn Improvement",
G.F. Sprague, ed., American Society of Agronomy,
Inc., Publisher, p. 671).
Thus, the maintainer gene of this invention,
when introduced into a particular line of a plant
species, can always be introduced into any other line
by backcrossing, but since the maintainer gene can
WO 93/25695 PCT/EP93/01489
~~;~~g
26
only be transmitted through an egg, it will always be
associated with the cytoplasm of the line in which it
was initially introduced. However, since a maintainer
plant line is only used for maintenance of a male-
sterile line and not as a female parent for hybrid
seed production, the hybrid seed will always contain
the cytoplasm of the female parent, as desired.
The following Examples illustrate this
invention. Unless otherwise indicated, all
experimental procedures for manipulating recombinant
DNA were carried out by the standardized procedures
described in Sambrook et al (1989) "Molecular
Cloning: A Laboratory Manual, Second Edition", Cold
Spring Harbor Laboratory Press, N.Y. USA. All
polymerase chain reactions ("PCR") were performed
under conventional conditions, using the Ventt"
polymerase (Cat. No. 254L - Biolabs New England,
Beverley, MA 01915, U.S.A.) isolated from
Thermococcus litoralis (Neuner et al (1990) Arch.
Microbiol. 153:205-207). Oligonucleotides were
designed by the methods described by Kramer and
Fritz (1968) Methods in Enzymology 154:350 and
synthesized by the phosphoramidite method (Beaucage
and Caruthers (1981) Tetrahedron Letters 22:1859) on
an applied Biosystems 380A DNA synthesizer (Applied
Biosystems B.V., Maarssen, Netherlands).
The following bacterial strains and plasmids,
used in the Examples, are available from the Deutsche
Sammlung fur Mikroorganismen and Zellkulturen
("DSM"), Mascheroder Weg 1B, Braunschweig, Germany:
WO 93/25695 PCT/EP93/01489
27
Bacterial strain plasmid DSM No Date of
Deposit
E. coli WK6 pMaS-8 DSM 4567 May 3, 1988
E. coli WK6 pMcS-8 DSM 4566 May 3, 1988
In the Examples, reference will be made to the
following Figure and Sequence Listing:
Figure
Figure 1: Ten-step procedure to obtain corn (e. g.
H99) maintainer plants of the invention
Seguence Listincr
SEQ ID no. 1: genomic DNA comprising the promoter of
the Zml3 gene from Zea mats
SEQ ID no. 2: sequence of plasmid "pVE144"
SEQ ID no. 3: sequence of plasmid "pVE108"
SEQ ID no. 4: sequence of oligonucleotide "MDB80"
SEQ ID no. 5: sequence of oligonucleotide "MDB81"
SEQ ID no. 6: sequence of oligonucleotide "MDB82"
SEQ ID No. 7: genomic DNA comprising the anther
specific promoter "PT72" from rice
SEQ ID No. 8: genomic DNA comprising the anther
specific promoter "PT42" from rice
SEQ ID No. 9: genomic DNA comprising the anther
specific promoter "PE1" from rice
SEQ ID No. 10: genomic DNA comprising the anther
specific promoter "PCA55" from corn
SEQ ID No. 11: Oligonucleotide Zm130LI2
SEQ ID No. 12: Oligonucleotide Zm130LI1
SEQ ID No. 13: Oligonucleotide Zm130LI5
SEQ ID No. 14: Oligonucleotide BXOL2
SEQ ID No. 15: Oligonucleotide TA29SBXOL2
SEQ ID No. 16: Oligonucleotide PTA290L5
SEQ ID No. 17: EcoRI-HindIII fragment of pTS218 carrying
the maintainer gene.
CA 02137559 2002-10-18
27620-13
28
Examples
Example 1 Isolation of the pollen-specific promoter
of the Zml3 gene from maize.
A pollen-specific cDNA from Zea mans inbred line
W-22, designated as "Zmcl3", has been isolated and
characterized by Hanson et al (1989) The Plant Cell
1:173. The corresponding genomic clone, designated as
"Zmgl3", containing substantial portions of the 5'
flanking region has been isolated and characterized
by Hamilton et al (1989) Sex. Plant Reprod. 2:208
(see also Hamilton et al (1992) Plant Mol. Biol.
18:211). The complete sequence of Zmgl3 is shown in
SEQ ID no. 1, and its promoter region will
hereinafter be referred to as the "Zml3 promoter".
A corresponding promoter region from corn inbred
line H99 was isolated as follows. Genomic DNA of
inbred line H99 was prepared as described by
Dellaporta et al (1983) Plant Mol. Biol. Repozts
1:19-21. Using the genome as a substrate, a 1471 by
fragment was' amplified by PCR using the
oligonucleotides MDB80 and MDB82, the sequences of
which are shown in SEQ ID no. 4 and SEQ ID no. 6,
respectively. MDB80 corresponds to nucleotides 8 to
28 of Zmgl3, while MDB82 is complementary to
nucleotides 1458 to 1478 of Zmgl3. Then, the
purified amplified 1471 by fragment was used as a
substrate for the amplification by PCR of a 1422 by
fragment, using the oligonucleotides MDB80 and MDB81.
MD881 is complementary to nucleotides 1409 to 1429 of
Zmgl3, and its sequence is shown in SEQ ID no. 'S. By
using MD881, a NcoI site is created in the amplified
1422 by fragment at the ATG translation initiation
codon.
The 1422 by fragment is then ligated in an SmaI
site of pGEM2* (Promega Corporation, Madison,
*Trade-mark
WO 93/25695 PCT/EP93/01489
29
Wisconsin 53711, U.S.A.), yielding plasmid pMDBl3,
and the fragment is sequenced (Maxam and Gilbert
(1980) Meth. Enzymol. 65:499). The pollen-specific
promoter of the Zml3 gene of corn inbred line H99 is
obtained from pMDBl3 as a EcoRV-NcoI fragment.
The Zml3 promoter is also cloned as follows.
Genomic DNA of Zea mat's line H99 is prepared as
described above. Using the genomic DNA as a
substrate, the following two fragments are amplified
by means of PCR: 1) a 875 by fragment is amplified
using the oligonucleotides MDB80 (SEQ ID No. 4) and
ZM130LI2 (which is complementary to nucleotides 859
to 882 of Zmgl3 and which sequence is given in SEQ ID
No. 11); and 2) a 661 by fragment is amplified using
the oligonucleotides Zm130LI1 (which corresponds to
nucleotides 767 to 791 of Zmgl3 and which sequence is
given in SEQ ID No. 12) and Zm130LI5 (which is
partially complementary to nucleotides 1397 to 1423
of Zmgl3 and which sequence is given in SEQ ID No.
13). The 875 by fragment, corresponding to the
upstream region of the Zml3 promoter, is cloned into
the SmaI site of pGEM2, yielding plasmid pTS204. The
661 by fragment, corresponding to the downstream
region of the Zml3 promoter, is digested with NcoI
and cloned into plasmid pJB66 (Botterman and Zabeau
(1987) DNA 6:583) digested with EcoRV and NcoI,
yielding plasmid pTS203. Both fragments partly
overlap and share a BstXI site in the region of
overlap. Ligation of the 567 by EcoRV-BstXI fragment
of pTS204 and the 638 by BstXI-NcoI fragment of
pTS203 results in a 1205 by fragment corresponding to
the Zml3 promoter. This 1205 by fragment, as cloned
from line H99, is sequenced, and its sequence is
found to be identical to the corresponding fragment
of Zmgl3 from line W-22 as given in SEQ ID No.l
WO 93/25695 PCT/EP93/01489
.. -
except at position 276 (G in W-22 is T in H99), 410 (G in)
W-22 is A in H99), and 1205-1206 (GC in W-22 is GGC
in H99, thus corresponding to a 1 nucleotide
insertion), numberings being as in SEQ ID No. 1.
Example 2 Construction of plant transformation
vectors comprising a maintainer ctene that contain DNA
encoding Barstar under the control of the TA29
promoter and DNA encoding Barnase under the control
of the Zml3 promoter.
The 1205 by EcoRV-NcoI fragment of pMDBl3 is
ligated to the large EcoRI-SmaI fragment of plasmid
pVE144 and to the 739 by EcoRI-NcoI fragment of
pVE108, yielding plasmid pGSJVRl. Plasmid pVE144, the
sequence of which is shown in SEQ ID no. 2, is a
plasmid derived from plasmid pUCl8 (Yanisch-Perron et
al (1985) Gene 33:103) and containing DNA encoding
neomycin phosphotranferase (neo) under the control of
the 3553 promoter (EP 0,359,617) from Cauliflower
Mosaic Virus isolate CabbB-JI (Hull and Howell (1978)
Virology 86:482) and DNA encoding the Barstar
(Hartley (1988) J.Mol.Biol. 202:913) under the
control of the tapetum-specific promoter of the TA29
gene of Nicotiana tabacum (EP 0,344,029; Seurinck et
al (1990) Nucleic Acids Res. 18:3403). Plasmid
pVE108 , the sequence of which is shown in SEQ ID no .
3, is a plasmid derived from pUCl8 and containing DNA
encoding phosphinothricin acetyl transferase (bar)
(EP 0,242,236) under the control of the 3553 promoter
and DNA encoding Barnase (Hartley (1980) supra) under
the control of the TA29 promoter. The resulting
plasmid, pGSJVRl (which is subsequently renamed
"pTS210"), is a pUCl8-derived plasmid that contains a
maintainer gene of this invention comprising: DNA
encoding Barnase as the pollen-lethality DNA, the
Zml3 promoter as the pollen-specific promoter, DNA
WO 93/25695 PCT/EP93/01489
_ ~ ~-~ .~ ,
31
encoding Barstar as the fertility-restorer DNA, the
TA29 promoter as the restorer promoter, neo as the
first marker DNA and the 3553 promoter as the first
marker promoter.
pTS210 is also obtained as follows. The 0.9 kb
HstXI-SacI fragment of pTS204 is ligated to the large
BstXI-Sacl fragment of pTS203, yielding plasmid
pTS206. The 1.47 Bgl II-tvcol kb fragment ofpTS206 i s then
ligated to the large NcoI-BglII fragment of pVE108,
yielding plasmid pTS207. Finally, the 1.9 kb EcoRV-
Eco-RI fragment of pTS207 is ligated to the large
Eco-RI-SmaI fragment of pVE144, yielding plasmid
pTS210.
A plasmid pTS218, which differs from pTS210 by
carrying the bar gene as a selectable marker gene, is
also obtained as follows:
- a 255 by DNA fragment, designated as bxx and
carrying the translation initiation site of the
PTA29-barstar gene, is obtained by PCR using
pvE144 as a template and oligonucleotides BXOL2
(SEQ ID No. 14) and TA29SBXOL2 (SEQ ID No. 15)
as primers.
- a 4 9 2 by DNA fragment is prepared by PCR us ing
pVE108 and bxx as a template and
oligonucleotides PTA290L5 (SEQ ID No. 16) and
BXOL2 as primers. This 492 by fragment is
digested with AsnI and BspEI, and a 274 by
fragment is purified on gel and ligated to the
6.28 kb fragment of pVEl44 which was digested
with BspEI and partially digested with AsnI. The
resulting plasmid is designated as pVEKl44 and
carries the PTA29-barstar-3'nos chimeric gene
WO 93/25695 PCT/EP93/01489
32
with an optimized translational initiation
context.
- pVEK144 is digested with MunI and HindIII, and
the 3.7 kbp fragment is isolated and ligated to
the 1.7 kbp MunI-HindIII fragment of pVE108,
yielding plasmid pVEB144 which carries the
PTA29-barstar-3'nos and the P35S-bar-3'nos
chimeric genes.
- theEcoRI-HindIII fragment of pVEB144, containing
the two chimeric genes, ~5 ligated to the large
EcoRI-HindIII fragment of pUCNew2, yielding
plasmid pVEC144. pUCNew2 is derived from pUCl9
as described in WO 92/13956.
- finally, the large EcoRI-SmaI fragment of
pVECl44 is ligated to the 1.9 by EcoRV-EcoRI
fragment of pTS207, yielding plasmid pTS218.
Plasmid pTS218 carries three chimeric genes, i.e.,
PTA29-barstar-3'nos (with optimized translational
initiation context), P35S-bar-3'nos, and
PZM13-barnase-3'nos. The EcoRI-HindIII fragment of
pTS218 carrying these three chimeric genes is
presented in the sequence listing as SEQ ID No. 17.
All steps of vector construction involving
fragments of the barnase DNA, such as pVE108, pVE144,
and ptS210, are carried out in E. coli strain MC1061
containing the cointegrate plasmid R702::pMcSBS which
is obtained as follows. Plasmid pMcSBS, containing
the barstar gene (encoding an inhibitor of barnase)
under the control of the tac promoter (De Boer et al
(1983) Proc. Natl. Acad. Sci. USA 80:21), is
constructed by: cloning the EcoRI-HindIII fragment of
plasmid pMT416 (Hartley (1988) supra) into the EcoRI
and HindIII sites of plasmid pMcS-8 (DSM 4566): and
then deleting the sequence starting with the
initiation codon of the phoA signal sequence and
ending with the last nucleotide before the
WO 93/25695 PCT/EP93/01489
translation initiation codon of the barstar-coding
region by means of a looping-out mutagenesis
procedure as generally described by Sollazo et al
(1985) Gene 37:199.
Plasmid 8702 is from Proteus mirabilis and can
replicate in E.coli (Villarroel et al (1983) Mol.
Gen. Genet. 189:390). Plasmid R702::pMcSBS is
obtained by cointegration through illegitimate
recombination between pMc58S and 8702, mediated by
transposable elements present on 8702 (Leemans (1982)
"Technieken voor het gebruik van Ti-plasmieden van
Agrobacterium tumefaciens als vectoren voor de
genetic engineering van planten", Ph.D. Thesis Vrije
Universiteit Brussel, Brussels, Belgium) and checked
for induced expression of Barstar.
The use of E.coli (R702::pMcSHS) allows the
construction, maintenance, amplification, and
purification of plasmids containing the barnase DNA,
such as pGSJVRl, without any lethal effect on the
host due to accidental expression of the barnase DNA.
However, because the Zml3 promoter is not expressed
in E. coli, all steps of vector construction
involving this promoter are also carried out in _E.
coli strain MC1061.
Example 3 Transformation of corn with the
maintainer Qene of Example 2.
Zygotic immature embryos of about 0.5 to 1 mm
are isolated from developing seeds of corn inbred
line H99. The freshly isolated embryos are
enzymatically treated for 1-2 minutes with an enzyme
solution II (0.3% macerozyme (Kinki Yakult,
Nishinomiya, Japan) in CPW salts (Powell & Chapman
(1985) "Plant Cell Culture, A Practical Approach",
R.A. Dixon ed., Chapter 3) with 10% mannitol and 5 mM
2-[N-Morpholino] ethane sulfonic acid (MES), pH 5.6).
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34
After 1-2 minutes incubation in this enzyme solution,
the embryos are carefully washed with N6aph solution
(macro- and micro-elements of N6 medium (Chu et al
(1975) Sci. Sin. Peking 18:659) supplemented with 6mM
asparagine, 12 mM proline, 1 mg/1 thiamine-HC1, 0.5
mg/i nicotinic acid, 100 mg/1 casein hydrolysate, 100
mg/1 inositol, 3o g/1 sucrose and 54 g/1 mannitol).
After washing, the embryos are incubated in the maize
electroporation buffer, EPM-RC1 (80 mM RC1, 5 mM
CaCl2, 10 ~ mM HEPES (N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid) and 0.425 M mannitol, pH
7.2). Approximately 100 embryos in 200 ~C1 EPM-KC1 are
loaded in each electroporation cuvette. About 20 ~cg
of a plasmid DNA, pPGSJYRl (of Example 2) linearized
with EcoRI, is added per cuvette.
After 1 hour DNA incubation with the explants,
the cuvettes are transferred to an ice bath. After 10
minutes incubation on ice, the electroporation is
carried out: one pulse with a field strength of 375
V/cm is discharged from a 900 ~cF capacitor. The
electroporation apparatus is as desczibed by Dekeyser
et al (1990) The Plant Cell 2:591. Immediately after
electroporation, fresh liquid N6aph substrate is
added to the explants in the cuvette, after which the
explants are incubated for a further 10 minute period
on ice.
Afterwards, the embryos are transferred to Mahl
VII substrate (macro- and micro-elements and vitamins -
of N6 medium supplemented with 100 mg/1 casein
hydrolysate, 6 mM proline, 0.5 g/1 MES, 1 mg/1 2,4-
dichlorophenoxyacetic acid (2,4-D) and 2% sucrose
solidified with 0.75 g/1 MgCl2 and 1.6 g/1 Phytagel*
(Sigma Chemical Company, St Louis, Mo. U.S.A.), pH
5.8) and supplemented with 0.2M mannitol. After 3
days, the embryos are transferred to the same
substrate supplemented with 200 mg/1 kanamycin. After
*Trade-mark
WO 93/25695 PCT/EP93/01489
approximately 14 days, the embryos are transferred to
Mahl VII substrate without mannitol, supplemented
with kanamycin. The embryos are further subcultured
on this selective substrate for approximately 2
months with subculturing intervals of about 3 weeks.
The induced embryogenic tissue is carefully isolated
and transferred to MS medium (Murashige and Skoog
(1962) Physiol. Plant 15:473) supplemented with 5
mg/1 6-benzylaminopurine for line H99. The
embryogenic tissue is maintained on this medium for
approximately 14 days and subsequently transferred to
MS medium without hormones and 6% sucrose for line
H99. Developing shoots are transferred to 1/2 MS
medium with 1.5% sucrose for further development to
normal plantlets. These plantlets are transferred to
soil and cultivated in the greenhouse.
In an analogous way; corn embryos are transformed
with a fragment of pTS218 DNA which contains the
maintainer gene and the chimeric P35S-bar-3'nos and
which is obtained by digestion of the plasmid with
EcoRI, XhoI and PstI and by purifying the longest
fragment. Transformation and plant regeneration is as
described in Example 5.
WO 93/25695 PGT/EP93/01489
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36
Example 4 Analysis of the transQenic corn plants of
Example 3.
Plants form Example 3 transformed with pGSJURl are analysed for the
presence of the maintainer gene by means of PCR. DNA
is prepared according to the protocol described by
Dellaporta et al (1983) Plant Mol. Biol. Reporter
1:19, adapted for application to tissue amounts of
about 10 to 2o mg. For each plant, such an amount of
tissue is macerated in extraction buffer in a
microfuge tube. Representative fragments of the
maintainer gene are amplified using appropriate
oligonucleotide probes.
Activity of the expression product of the first
marker gene (i.e., neomycin phosphotransferase II
(NPTIZ)) is assayed in plants as follows. Crude
extracts are prepared by grinding plant tissue in
extraction buffer (McDonnell et al (1987) Plant
Molecular Biol. Reporter 5:380). The extracts are
then subjected to non-denaturing polyacrylamide gel
electrophoresis according to the procedure described
by Reiss et al (1984) Gene 30:211. NPTII activity is
then assayed by in situ phosphorylation of kanamycin
using [gamma-32P]ATP as a substrate (McDonnell et al
(1987) supra).
The plants that are found to be positive on both
the PCR and NPTII assay are further analyzed by means
CA 02137559 2002-10-18
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37
of Southern hybridization. Genomic DNA is prepared
from plant tissue according to the protocol described
by Dellaporta et al (1983) supra, supplemented by a
treatment with RNase to remove remaining RNA. A non-
transformed H99 plant is used as a control. Samples
of the DNA are digested with appropriate restriction
enzymes and subjected to horizontal agarose
electrophoresis. Southern tzansfer to Hybond~' N+
(Amersham International PLC, Amersham, United
Kingdom) membranes by means of the "alkali blotting
of DNA" protocol and the subsequent hybridization are
performed as recommended by the manufacturer
(Amersham Hybond-N+ leaflet). Suitable radioactive
probes are prepared with the multi-prime DNA
labelling kit (Amersham) according to the protocol
supplied by the manufacturer which is derived from
published procedures (Feinberg and Vogelstein (1983)
Anal. Biochem. 132:6). The banding patterns show that
at least the maintainer gene is integrated into the
plant genomic DNA.
The PCR assays show that the maintainer gene is
present. The NPTII assays show that the first marker
DNA is expressed. The mature transformed plants can
then be analyzed phenotypically to see whether the
barstar DNA is expressed in tapetum cells and the
barnase gene is expressed in pollen cells. Expression
of barstar is determined by northern blotting of
anther mRNA and by making testcrosses to determine
the restoration in the progeny. Expression of the
pollen-lethality gene is determined by cytological
examination of the anther. In this regard, viable and
nonviable mature pollen is determined by analyzing
the staining of isolated pollen upon incubation for
3o minutes at 24'C in the following reaction mixture:
loo mM phosphate buffer pH 7.8, 100 mM
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WO 93/25695 ~ PCT/EP93/01489
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Sodiumsuccinate and 1 mM NitroBlue Tetrazolium,
followed by visual inspection of formazan
precipitation in viable pollen. Alternative
techniques for the differentiation between viable and
nonviable mature pollen are those described for
example by Alexander (1969) Stain Technology 44:117,
and by Heslop-Harrison and Heslop-Harrison (1970)
Stain Technology 45:115. The viability of microspores
is determined by embedding flower buds in plastic at
different developmental stages and subjecting the
buds to histochemical staining with the succinate
dehydrogenase assay, both as described by De Block
and Debrouwer (1992) The Plant Journal 2:261.
Ultimately, the progeny of the plant transformed
with the pollen-lethality gene is determined. None of
the offspring obtained from a cross using this plant
as a male parent have this gene, while 50% of the
offspring obtained from a cross using this plant as a
female parent possess the gene.
Plants from Example 3, transformed with pTS218
DNA, are analyzed in the same way, except that the
expression product of the first marker gene, i.e.,
phosphinothricine acetyltransferase, is assayed by
means of a PAT assay as described in Example 5.
Example 5 Production of male-sterile corn plants.
Zygotic embryos of corn inbred line H99 were
isolated, enzymatically treated, washed, and loaded
in electroporation buffer as described in Example 3.
Approximately 100 embryos in 200 ul EPM-KC1 were
loaded in each electroporation cuvette. About 20 y,g
of a plasmid DNA, pVE108 linearized with HindIII, was
added per cuvette. pvE108 is a 5620 by plasmid which
contains: a chimaeric gene comprising the bar DNA (EP
242236), encoding phosphinothricin acetyl transferase
(PAT) and conferring resistance to an herbicidal
glutamine synthetase inhibitor such as
phosphinothricin (PPT), under the control of the 3553
promoter; and another chimaeric gene comprising the
DNA coding for barnase (Hartley (1988) supra) under
the control of the tapetum-specific promoter of the
WO 93/25695 PCT/EP93/01489
TA29 gene (EP 344029) of N. tabacum. The complete
sequence of plasmid pVE108 is given in SEQ ID no. 4.
All vector constructions involving DNA fragments
comprising the barnase gene were carried out in E.
coli strain MC1061 containing the plasmid
R702::pMcSBS of Example 3. After a 1 hour DNA
incubation with the explants, the cuvettes were
transferred to an ice bath. After 10 minutes
incubation on ice, the electroporation was carried
out as described in Example 3. Immediately after
electroporation, fresh liquid N6aph substrate was
added to the explants in the cuvette, after which the
explants were incubated for a further 10 minute
period on ice.
Afterwards, the embryos from one electroporation
experiment were transferred to Mahl VII substrate
supplemented with 0.2 M mannitol and 2 mg/1 PPT.
After approximately 14 days, the embryos were
transferred to Mhl VII substrate (Mahl VII substrate
of Example 3 but without proline and casein
hydrolysate) supplemented with 2 mg/1 PPT but without
mannitol. After approximately 4 weeks, the embryos
were subcultured for another month on MhI VII
substrate supplemented with 10 mg/1 PPT. The induced
embryogenic tissue was carefully isolated and
transferred to MS medium supplemented with 5 mg/1 6-
benzylaminopurine. The embryogenic tissue was
maintained on this medium for approximately 14 days
and subsequently transferred to MS medium without
hormones and sucrose. Developing shoots were
transferred to 1/2 MS medium with 1.5% sucrose for
further development to normal plantlets. These
plantlets survived an in vitro spraying with doses of
BASTAR (Hoechst AG, Frankfurt am Main, Germany)
corresponding to 2 1/ha. These plantlets were then
WO 93/25695 PCT/EP93/01489
transferred to soil and cultivated in the greenhouse,
and two of the transformed plantlets, designated
RZM35-1 and RZM35-18, were further characterized.
The embryos from a second electroporation
experiment were transferred to Mhl VII substrate
supplemented with 2 mg/1 PPT and 0.2 M mannitol.
After about 14 days, the embryos were transferred to
Mhl VII substrate supplemented with 2 mg/1 PPT but
without mannitol. After approximately another three
weeks, the embryos were transferred to Mhl VII
substrate supplemented with 10 mg/1 PPT but without
mannitol. After another three weeks, the induced
embryogenic tissue was carefully isolated and
transferred to MS medium supplemented with 2 mg/1 PPT
and 5 mg/1 6-benzylaminopurine. The embryogenic
tissue was maintained on this medium for
approximately 14 days and subsequently transferred to
MS medium without hormones, sucrose or PPT.
Developing shoots were transferred to 1/2 MS medium
with 1.5% sucrose for further development to normal
plantlets. The resulting plantlets were transferred
to soil and cultivated in the greenhouse, and three
of the transformed plantlets, designated RZM34-1,
RZM34-12, and RZM34-14, were further characterized.
RZM34-1, RZM34-12, RZM34-14, RZM35-1, and
RZM35-18 were grown in the greenhouse. Activity of
the expression product of the bar gene in leaves of
the plants was assayed as follows in a "PAT assay".
100 mg of leaf tissue from each plant, together with
mg of acid-treated sea sand (Merck, Darmstadt,
Germany) and 5 mg polyvinylpolypyrrolidone (PVPP),
were ground in an Eppendorf tube with a glass rod in
50 ~,1 of extraction buffer (25 mM Tris-HCL pH 7.5, 1
mM Na2-EDTA (ethylenediaminetetraacetic acid disodium
salt), 0.15 mg/ml phenylmethylsulfonylfluoride
CA 02137559 2002-10-18
27620-13
41
(PMSF), 0.3 mg/ml dithiothreitol (DTT), and 0.3 mg/ml
bovine serum albumin). The extract was centrifuged in
a microfuge for 5 minutes at 16000 rpm. The
supernatant was recovered and diluted ten times with
TE 25/1 (25 mM Tris-HCL pH 7.5, 1 mM Na2-EDTA. To
twelve ul of the diluted extract was then added: lul
of 1 mM PPT in TE 25/1, 1 ~1 of 2 mM AcetylCoenzyme A
in TE 25/1, and 2 ~1 of [14C]AcetylCoenzym A (60
mCi/mmol, 0.02 mCi/ml, [NEN Research Products,
Dupont, Wilmington, Delaware, USA). The reaction
mixture was incubated for 30 minutes at 37'C and
spotted on a aluminium sheet silicagel* 60 t.l.c.
plate with concentrating zone (Merck). Ascending
chromatography was carried out in a 3 to 2 mixture of
1-propanol and NFi~OH (25~ NH3) . 14C was visualized by
.overnight autoradiography (XAR-5 Rodak film). The
tolerance to the herbicide BASTA~ was tested by
brushing a small area near the top of one leaf per
plant with a 1% solution of the herbicide and
obsezving the damage symptoms at and near the brushed
sites. While RZM34-1, RZM35-1 and RZM35-Z8 showed no
damage symptoms at all, RZM34-12 and RZM34-14
displayed slight browning and drying-out of the
brushed site. RZM34-1, RZM34-12, RZM34-14, RZM35-1
and RZM35-18 were also shown to be male-sterile but
otherwise phenotypically completely normal: female
fertility, for instance, was normal. The spikelets of
the male flowers were of about normal length but were
very thin and appeared to be empty, and they never
opened. A detailed analysis showed that the anthers
were reduced to almost .microscopic structures. This
phenotype indicates not only that at least one copy
of the barnase gene was expressed but also that it
was selectively expressed in some or all of the
tissues of the anthers.
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42
Southern analysis showed RZM35-1 and RZM35-18 to
have an identical integration pattern, with only one
copy of plasmid pVE108 being present in the genome of
each plant. A small part of the plasmid DNA sequence
adjacent to the HindIII site (used for linearization
prior to electroporation) seemed to be absent in the
integrated copy. Southern analysis of RZM34-1,
RZM34-12 and RZM34-14 showed that each of these
plants probably has two or three copies of part or
all of pVE108 integrated into its genome. The copies
are most likely not inserted in a concatemer
configuration.
Transformants RZM35-1 and RZM34-1 were
pollinated with pollen from an untransformed H99
plant, and progeny plantlets were recovered. From the
35 plantlets recovered from RZM35-1, 16 (46%) scored
positive in a PAT assay, while 19 (54%) were PAT
negative. This proportion in the F1 progeny does not
differ significantly from the 1:1 ratio expected
under normal Mendelian segregation of one active copy
of the chimaeric bar gene (X2 = 0.26).
From the 34 plantlets recovered from RZM34-1, 19
(56%) scored positive in a PAT assay, while 15 (44%)
were PAT negative. This proportion in the F1 progeny
does not differ significantly from the 1:1 ratio
expected under normal Mendelian segregation, assuming
that the transformed female parent had one active
copy, or alternatively multiple active, but closely
linked copies, of the chimaeric bar gene (X2 = 0.47).
Example 6 : Production of restorer corn plants.
Zygotic. embryos of corn inbred line H99 were
isolated, enzymatically treated, washed and loaded in
electroporation buffer as described in Example 5.
Approximately 100 embryos in 200 ~C1 EPM-RC1 were
loaded in each eleetroporation cuvette. About 20
WO 93/25695 PCT/EP93/01489
_ ~ ~' ~~~ y
43 '
of a plasmid DNA, pVE144 linearized with HindIII, was
added per cuvette. pVE144 is a 6555 by plasmid which
was described in Example 2.
The embryos were electroporated, and the
transformed cells were selected, grown into callus,
and regenerated as described in Example 3. Transgenic
plants were analyzed for the presence of the
fertility-restorer gene and the marker gene by means
of Southern hybridization and PCR. The expression of
the fertility-restorer gene is assayed by means of
Northern blotting, and the expression of the marker
gene is determined by NPTII assay as described in
Example 3.
Example 7 Production of maintainer corn plants and
a male-sterile corn line and maintenance of the
male-sterile corn line
Maintainer plants of this invention of corn line
H99 are obtained as outlined in Figure 1. A plant of
corn inbred line H99 with the male-sterility genotype
H99s~s,rir,pip, transformed with the male-sterility gene
of Example 5, is crossed with plants with the
genotype H99s~s.air,pip., transformed with the
fertility-restorer gene of Example 6. The progeny
that have the genotype H99s~s,Rir,piP are identified by
PCR analysis for the presence of the S and R genes.
These plants are selfed, yielding progeny with nine
different genotypes. Two of these genotypes
~H99sis,rir and H99s~s,rir) will develop into male-sterile
plants, while all the other genotypes will develop
into male-fertile plants. When these male-fertile
plants are selfed, progeny analysis allows the
identification of their genotype. Thus: a) the
progeny of selfings of H99s~s,aiR ~ H99sis.aia ~ H99s~s.a~a ~
H9gsis.air and H99sis.rir would all develop into male-
fertile plants; b) selfings of H99sis.air plants would
WO 93/25695 PCT/EP93/01489
44
produce progeny, of which 13 out of 16 would be
male-fertile, and since the male-sterility gene is
linked to the herbicide resistance gene, bar, 4 out
of the 13 male-fertile plants would be sensitive to
the herbicide BASTAR : and c) selfings of Hggsis,Rir
plants would produce progeny, of which 12 out 16
would be fertile (4 out of 16 would have the genotype
Hggsis,Ria and 8 out of 16 would have the genotype
Hggsis,eir) ~ all of which would be resistant to the
herbicide, and the male-sterile progeny of which (4
out of 16) would all be homozygous for the male-
sterility gene (Hggsis,rir) .
The homozygous male-sterile progeny (Hggsis,rir)
of selling (c) are then crossed with their male-
fertile siblings, and only when the cross is with
plants with the genotype H99s~s,Rir are the resulting
plants 50% male-sterile (all with the genotype
Hggs/s,r/r) and 50% male-fertile (all with the genotype
Hggs/S,R/r. Indeed, the alternative cross between
H99s~s,rir and Hggsis,Ria would result in 100 % male-
fertile progeny plants.
Maintainer plants are selected by crossing the
plant with the genotype H99s~s,R/r,p/p with a plant that
is heterozygous for the maintainer gene of Example 2,
i. e. , (Hggs/s,r/r,P/p) ~ using the latter plant as the
female parent. The offspring with the genotype
Hggsis,rir,vip are selected by means of testcrosses
supplemented with PCR analysis of the progeny (which
can be easily identified by PCR and Southern
blotting for the presence of the S and P genes and
the absence of the R gene). The selected fertile
offspring are then selfed. One out of eight offspring
have the desired genotype for a maintainer plant of
this invention (H99s~s~P~p) and can be further selected
by means of testcrosses and PCR analysis of the
WO 93/25695 ~ ~, ~ ~ ~ PCT/EP93/01489
progeny. Indeed, only plants with this genotype will
produce 50% male-sterile offspring (all H99s~s,p~p) and
50% male-fertile offspring (all H99S~s,P~p) , thus
growing at once both the desired homozygous male-
sterile line and the maintainer line of this
invention. Testcrosses also include the
pollination of wild type H99 plants with pollen of the
progeny plants obtained from the selfing of H99s~s,PiP
plants.
Homozygous male-sterile plants with the genotype
H99sis,rir,piP are then pollinated by maintainer plants
(Hg9S/s,r/r,P/p) o f his invention. All progeny have the
genotype H99s~s,r~r~pip, so that the male-sterile line is
maintained, as desired.
Example 8 : Introduction of the male-sterility 4ene
and the maintainer 4ene in inbred corn lines through
classical breeding.
The male-sterility gene of Example 5 and the
maintainer gene of Example 2 are transferred from
corn inbred line H99 to another corn inbred line (A)
by repeated backcrossings as follows. The maintainer
plant H99s~s,P~p is crossed as a female parent with an
untransformed plant of line A (A'~'~~p) . The offspring
with the genotype A-H99s~',P~p are selected by
screening, using PCR, for the presence of both the
maintainer gene (P) and the male-sterility gene (S).
These plants are then crossed again as female parents
Wlth As~s,P~p plants, and the offspring that are
heterozygous for both the P and S genes are again
selected by PCR. This process of backcrossing is
repeated until finally plants with the genotype
Asis,Pip are obtained. These plants are then selfed,
and the progeny are analyzed in the same way as
WO 93/25695 . PCT/EP93/01489
described in Example 7. In this way, male-sterile
plants with the genotype AS~s'p~p and maintainer plants
of this invention with the genotype AS~s~P~p are
obtained.
WO 93/25695 PCT/EP93/01489
2~37~~~
4~ ,
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: PLANT GENETIC SYSTEMS N.V.
(B) STREET: Jozef Plateaustraat 22
(C) CITY: Ghent
(E) COUNTRY: Belgium
(F) POSTAL CODE (ZIP): 9000
(G) TELEPHONE: 32 91 358411
(H) TELEFAX: 32 91 240694
(I) TELEX: 11.361 Pgsgen
(ii) TITLE OF INVENTION: Maintenance of male-sterile plants
(iii) NUMBER OF SEQUENCES: 17
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/899,072
(B) FILING DATE: 12-JUN-1992
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/970,849
(B) FILING DATE: 03-NOV-1992
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2661 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays
(B) STRAIN: inbred line W-22
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Hamilton et al.,
(C) JOURNAL: Sex Plant Reprod.
(D) VOLUME: 2
(F) PAGES: 208-
(G) DATE: 1989
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: l:
AAAGACCCCG CTTGTCAGTG AATGTTGCTA TTCTAGCAAA GGGAAGGTAT TTTTTCGGAC 60
WO 93/25695 PCT/EP93/01489
~'... ~
. ~
. '_
48
CGTA AA
C CCTTCG
CTT G CCAGATCGCAATCTAAATTTATTATTTTGAACAAATTAAT 120
GG T
ATTGCGAGGGGCTACTGTTG GGGACCTTCGGCATCCGAAGGTCCTCAAAAACAGGATTTA 180
ATAGTGTTTCTGGAGTATAA TGTGTGAACAGATATCTTCGGACTCAAGTCAGGCATCACA 240
GTAGACCAGAATAATACGAA GGTTGGTGAAGCGCCGAAGGTGTAAGCAGGAAAGCTTCGG 300
CAAGACAGCAGCAGTTGAAA CCGACTTAAAGATGAAAAGGCTATTTAGACCTCAACAGAT 360
TACTATAGGTTTATTATTAA GTGTAAAGGGCATTAATGTAATTTTGCACGGGCTACGTCC 420
CGTGCCTATAAATAGGTGAA CAGTATTCCCGTACTGTTCACGCTGACTTGGCATTCGCTT 480
TTTGCGTCACGCTTGTACTG TCATCTCATTCCTATTGAAGGTACACTTGTAATTCAACGA 540
TATTTCTGTTTGTACCTAAT AATAATATATAATTGTTCATGTTGTCTTTTATATTCTTTA 600
TATTTCATCCTTCGTCATTG TTTAATGAATTTATGAAGGTACGTCCTTCATAACCTTCGT 660
CCGTAAACCATTATATCCTA AGGGAAATAATGCTTCGAAGGACGAAGGACCTTAACGATT 720
AATATTTTCTATGTTGCCTT GTTCTTAACTCATAGCACTTGAGAACAAGTCTCCAACAGT 780
TTGGTTATTCCTATTCCACG TGGATTAGATGAGATTTAGATAAAATTAGAAATAATTTTG 840
ACTTACTAGGGATTTAAACC AACTCAGTCCCGTTCAATCCACATGGATTGAGATTAAAAC 900
AACTATTGAGATTTTATTGT ATCAACACTCAACACCGATGTGTTTTTATAATACATCTTG 960
CGTGACATTTGTCCAAGTAC TATGCTAAATATGAGAAGCTGCCATTTAGTGATTCTATAT 1020
ACTATTCACTTATGGATACA TTTAACTGATACCGTTTTGTTGAGCGCGTCTTATTTAGTT 1080
TTACATAGCAGCATAGAAGA TTAGAAGTCGCAAATCCAACTTTTGTGGACCGCTGAAAAA 1140
CTCAACCAAATTCGACATAT TTTTCACCTCCCCATGCCACAAAACTAGGTCAAAACGGCT 1200
TTCTGCCGTCGGCCACTATT TCTACGGGCAGCCAGACAAATCTTCGGGTCTCGCAGATTA 1260
TTTAAGGACACCACAGGCTG CGTTACGAAACCAGGCCAGATTTGCCACCCTCGTCTCACC 1320
CTCCCTCCCTCACACAAATA ATAAGGAAAGGTCCCGCCCTTTTCCTCCGACATCCACAAG 1380
GGGGGAGGGGAAAACACGTA CATTCACCCGGCGGCAATAATGGCCTCGGTTCCGGCTCCG 1440
GCGACGACGACCGCCGCCGT CATCCTATGCCTATGCGTCGTCCTCTCCTGTGCCGCGGCT 1500
GACGACCCGAACCTCCCCGA CTACGTCATCCAGGGCCGCGTGTACTGCGACACCTGCCGC 1560
GCCGGGTTCGTGACCAACGT CACCGAGTACATCGCGGGCGCCAAGGTGAGGCTGGAGTGC 1620
AAGCACTTCGGCACCGGCAA GCTCGAGCGCGCCATCGACGGGGTCACCGACGCGACCGGC 1680
ACCTACACGATCGAGCTCAA GGACAGCCACGAGGAGGACATCTGCCAGGTGGTGCTGGTG 1740
GCCAGCCCGCGCAAGGACTG CGACGAGGTCCAGGCGCTCAGGGACCGCGCCGGCGTCCTG 1800
CTCACCAGGAACGTTGGCAT CTCCGACAGCCTGCGCCCCGCCAACCCGCTAGGCTACTTC 1860
AAGGACGTGCCGCTCCCCGT CTGCGCCGCGCTGCTCAAGCAGCTGGACTCGGACGACGAC 1920
WO 93/25695 PCT/EP93/01489
~ ~. 3 ?.~ ~ 9
GACGACCAGTAAACTATACCACGGCGGCGTCGCGGACATG CTGCACAAAACTACAACGAT1980
ACAGAGCGAACGCATGGCATGGATAGCAGTATCTACGGAA AGAAAAGGAAGAAAAGGAAA2040
ATAAP.AAP.TGTATCAGAGTGCTTGATTCACTTGCTGCTGT CACCCATTCCCCGTTCTTAA2100
CATAACATGTGGGCCGGCTTGGCCCAGGCACAAGCCCATC TACGCATGGCCTACGGTCCG2160
CTAAAATATAGCCCTAATTATGAGCCGTGTTGTGCCGTCA CATGGATCGATCCAGCGGCA2220
TACGATACAACCCACAATTACTTATGTGTGATGGGCCGGC CAAAAAAGCCTAAGATGTCG2280
TAGTGTGCTAGACCGACTCATATATATAAAACATTAAAAC ATATTGTCGGGGACCATAAT2340
TAGGGGTACCCTTAAGGCTCCTAATTCTCAGCTGGTAACC CTCATCAGCGTAAAGCTGCA2400
AAGGCCTGATGGGTGCGATTAAGTCAGGGATCAGTCCATT CGAGGGACTCGATCACGCCT2460
CGCCCGAGCCTAGCCTCGGACAAGGGCAGCCGACCCCGGA GGATCTCCGTCTCGCCCGAG2520
GCCCTCCTCCAGCGGCGAACATATTTCCGGCTCGCCCGAG GCCCTGTCTTCGCCAAGAAG2580
CAACCCTGACCAAATCGCCGCACCGACCGACCAAATCGCA GGAGCATTTAATGCAAAGGT2640
GGCCTGACACATTTATCCTGA 2661
(2) INFORMATION FOR SEO ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6555 base pairs
(B) TYPE: nucleic acid
IC) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: plasmid pVE144 (replicable in E.coli)
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..396
(D) OTHER INFORMATION: /label= pUClB
/note= 'pUClB derived sequence"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (397..751)
(D) OTHER INFORMATION: /label= 3'nos
/note= ~3' regulatory sequence containing the
polyadenylation site derived from Agrobacterium
T-DNA nopaline synthase gene"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (752..1024)
(D) OTHER INFORMATION: /label= barstar
WO 93/25695 PCT/EP93/01489
.
~,~ 50
/note= "coding region of the barstar gene of
Bacillus amyloliquefaciens"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (1025..1607)
(D) OTHER INFORMATION: /label= TA29
/note= "promoter derived from the TA29 gene of
Nicotiana tabacum"'
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1608..2440
(D) OTHER INFORMATION: /label= 3553
/note= "3553 promoter sequence derived from
cauliflower mosaic virus isolate CabbB-JI"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 2441..3256
(D) OTHER INFORMATION: /label= neo
/note= "coding region of the neomycine
phosphotransferase gene of Tn5"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 3257..4315
(D) OTHER INFORMATION: /label= 3'ocs
/note= "3' regulatory sequence containing the
polyadenylation site derived from Agrobacterium
T-DNA octopine synthase gene"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 4316..6555
(D) OTHER INFORMATION: /label= pUClB
/note= "pUClB derived sequence"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 60
GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG 120
TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA 180
CTGAGAGTGC
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC 240
ATCAGGCGCC
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC 300
TCTTCGCTAT
TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA 360
ACGCCAGGGT
TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT 420
ACCCGGGGAT
CTTCCCGATC TAGTAACATA GATGACACCG CGCGCGATAA TTTATCCTAG 480
TTTGCGCGCT
ATATTTTGTT TTCTATCGCG TATTAAATGT ATAATTGCGG GACTCTAATC 540
ATAAAAACCC
ATCTCATAAA TAACGTCATG CATTACATGT TAATTATTAC ATGCTTAACG 600
TAATTCAACA
GAAATTATAT GATAATCATC GCAAGACCGG CAACAGGATT CAATCTTAAG 660
AAACTTTATT
WO 93/25695 ~ ~ ~ ~~ ~ ~ : [ . . ' PCT/EP93/01489
51
GCCAAATGTTTGAACGATCTGCTTCGGATCCTCTAGACCAAGCTAGCTTGCGGGTTTGTG 720
TTTCCATATTGTTCATCTCCCATTGATCGTATTAAGAAAGTATGATGGTGATGTCGCAGC 780
CTTCCGCTTTCGCTTCACGGAAAACCTGAAGCACACTCTCGGCGCCATTTTCAGTCAGCT 890
GCTTGCTTTGTTCAAACTGCCTCCATTCCAAAACGAGCGGGTACTCCACCCATCCGGTCA 900
GACAATCCCATAAAGCGTCCAGGTTTTCACCGTAGTATTCCGGAAGGGCAAGCTCCTTTT 960
TCAATGTCTGGTGGAGGTCGCTGATACTTCTGATTTGTTCCCCGTTAATGACTGCTTTTT 1020
TCATCGGTAGCTAATTTCTTTAAGTAAAAACTTTGATTTGAGTGATGATGTTGTACTGTT 1080
ACACTTGCACCACAAGGGCATATATAGAGCACAAGACATACACAACAACTTGCAAAACTA 1140
ACTTTTGTTGGAGCATTTCGAGGAAAATGGGGAGTAGCAGGCTAATCTGAGGGTAACATT 1200
AAGGTTTCATGTATTAATTTGTTGCAAACATGGACTTAGTGTGAGGAAAAAGTACCAAAA 1260
TTTTGTCTCACCCTGATTTCAGTTATGGAAATTACATTATGAAGCTGTGCTAGAGAAGAT 1320
GTTTATTCTAGTCCAGCCACCCACCTTATGCAAGTCTGCTTTTAGCTTGATTCAAAAACT 1380
GATTTAATTTACATTGCTAAATGTGCATACTTCGAGCCTATGTCGCTTTAATTCGAGTAG 1440
GATGTATATATTAGTACATAAAAAATCATGTTTGAATCATCTTTCATAAAGTGACAAGTC 1500
AATTGTCCCTTCTTGTTTGGCACTATATTCAATCTGTTAATGCAAATTATCCAGTTATAC 1560
TTAGCTAGATGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTCCTACGCAGCAGG 1620
TCTCATCAAGACGATCTACCCGAGTAACAATCTCCAGGAGATCAAATACCTTCCCAAGAA 1680
GGTTAAAGATGCAGTCAAAAGATTCAGGACTAATTGCATCAAGAACACAGAGAAAGACAT 1740
ATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCATAAACC 1800
AAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCTACTGAATCTAAGGCCAT 1860
GCATGGAGTCTAAGATTCAAATCGAGGATCTAACAGAACTCGCCGTGAAGACTGGCGAAC 1920
AGTTCATACAGAGTCTTTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGG 1980
AGCACGACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGG 2040
CTATTGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAG 2100
CTATCTGTCACTTCATCGAAAGGAC~AGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATC 2160
ATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATG 2220
GACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGC 2280
AAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTT 2340
CGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATC 2400
ACCAGTCTCTCTCTATAAATCTATCTCTCTCTCTATAACCATGGATCCGGCCAAGCTAGC 2460
TTGGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATT 2520
WO 93/25695 PGT/EP93/01489
....
52
CGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTC 2580
AGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACT 2640
GCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGT 2700
GCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCA 2760
GGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAAT 2820
GCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCG 2880
CATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGA 2940
AGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGA 3000
CGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAA 3060
TGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGA 3120
CATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTT 3180
CCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT 3240
TGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAAC 3300
CTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATC 3360
GTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTC 3920
GCCCACCCCCTGCTTTAATGAGATATGCGAGACGCCTATGATCGCATGATATTTGCTTTC 3480
AATTCTGTTGTGCACGTTGTAAAAAACCTGAGCATGTGTAGCTCAGATCCTTACCGCCGG 3540
TTTCGGTTCATTCTAATGAATATATCACCCGTTACTATCGTATTTTTATGAATAATATTC 3600
TCCGTTCAATTTACTGATTGTACCCTACTACTTATATGTACAATATTAAAATGAAAACAA 3660
TATATTGTGCTGAATAGGTTTATAGCGACATCTATGATAGAGCGCCACAATAACAAACAA 3720
TTGCGTTTTATTATTACAAATCCAATTTTAAAAAAAGCGGCAGAACCGGTCAAACCTAAA 3780
AGACTGATTACATAAATCTTATTCAAATTTCAAAAGGCCCCAGGGGCTAGTATCTACGAC 3840
ACACCGAGCGGCGAACTAATAACGTTCACTGAAGGGAACTCCGGTTCCCCGCCGGCGCGC 3900
ATGGGTGAGATTCCTTGAAGTTGAGTATTGGCCGTCCGCTCTACCGAAAGTTACGGGCAC 3960
CATTCAACCCGGTCCAGCACGGCGGCCGGGTAACCGACTTGCTGCCCCGAGAATTATGCA 4020
GCATTTTTTTGGTGTATGTGGGCCCCAAATGAAGTGCAGGTCAAACCTTGACAGTGACGA 4080
CAAATCGTTGGGCGGGTCCAGGGCGAATTTTGCGACAACATGTCGAGGCTCAGCAGGGGC 4140
TCGATCCCCTCGCGAGTTGGTTCAGCTGCTGCCTGAGGCTGGACGACCTCGCGGAGTTCT 4200
ACCGGCAGTGCAAATCCGTCGGCATCCAGGAAACCAGCAGCGGCTATCCGCGCATCCATG 4260
CCCCCGAACTGCAGGAGTGGGGAGGCACGATGGCCGCTTTGGTCGACCTGCAGCCAAGCT 4320
TGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC 4380
WO 93/25695 PCT/EP93/01489
5s
ACAACATACGAGCCGGAAGCATAAAGTGTA TGCCTAATGAGTGAGCTAAC 4440
AAGCCTGGGG
TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC 4500
TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCG 4560
CT.TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTC 4620
ACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT 4680
GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC 4740
ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA 4800
ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC 4860
CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG 4920
CGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC 4980
TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC 5040
GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA 5100
GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT 5160
ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG 5220
GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT 5280
TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT 5340
T.TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA 5400
GATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAA 5460
TCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCAC 5520
CTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA 5580
TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC 5640
CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA 5700
GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA 5760
GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCG 5820
TGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGC 5880
GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG 5940
TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATT 6000
CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT 6060
CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATA 6120
ATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC 6180
GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC 6240
WO 93/25695 PGT/EP93/01489
.
.
~ . .. 54 -
CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAA6300
GGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCT6360
TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATAT6420
TTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGC6480
CACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCA6540
CGAGGCCCTTTCGTC 6555
(2) INFORMATION FOR SE ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5620 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) AIv~I-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: plasmid pVE108 (replicable in E.coli)
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..395
(D) OTHER INFORMATION: /label= pUClB
/note= "pUClB derived sequence"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (396..802)
(D) OTHER INFORMATION: /label= 3'nos
/note= "3' regulatory sequence containing the
polyadenylation site derived from the nopaline
synthase gene from Agrobacterium T-DNA"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (803..1138)
(D) OTHER INFORMATION: /label= barnase
/note= "coding region of the barnase gene of
Bacillus amyloliquefaciens"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (1139..1683)
(D) OTHER INFORMATION: /label= TA29
/note= "sequence derived from tapetum specific
promoter of Nicotiana tabacum"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1684..2516
(D) OTHER INFORMATION: /label= 35S3
WO 93/25695 ~ ~ ~ ~ PCT/EP93/01489
/note= ""3553" promoter sequence derived from
cauliflower mosaic virus isolate CabbB-JI"
(ix) FEATURE:
1A) NAME/KEY: -
(B) LOCATION: 2517..3068
(D) OTHER INFORMATION: /label= bar
/note= "coding sequence of phosphinotricin
acetyltransferase gene"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 3069..3356
(D) OTHER INFORMATION: /label= 3'nos
lnote= "3' regulatory sequence containing the
polyadenylation site derived from Agrobacterium
T-DNA nopaline synthase gene"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 3357..5620
(D) OTHER INFORMATION: /label= pUClB
/note= "pUClB derived sequence"
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 3:
TCGCGCGTTTCGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA60
CAGCTTGTCTGTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG120
TTGGCGGGTGTCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC180
ACCATATGCGGTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC240
ATTCGCCATTCAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT300
TACGCCAGCTGGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT360
TTTCCCAGTCACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCGGGGAT420
CTTCCCGATCTAGTAACATA GATGACACCG CGCGCGATAA TTTATCCTAG TTTGCGCGCT480
ATATTTTGTTTTCTATCGCG TATTAAATGT ATAATTGCGG GACTCTAATC ATAAAAACCC540
ATCTCATAAATAACGTCATG CATTACATGT TAATTATTAC ATGCTTAACG TAATTCAACA600
GAAATTATATGATAATCATC GCAAGACCGG CAACAGGATT CAATCTTAAG AAACTTTATT660
GCCAAATGTTTGAACGATCT GCTTCGGATC CTCTAGAGNN NNCCGGAAAG TGAAATTGAC720
CGATCAGAGTTTGAAGAAAA ATTTATTACA CACTTTATGT AAAGCTGAAA AAAACGGCCT780
CCGCAGGAAGCCGTTTTTTT CGTTATCTGA TTTTTGTAAA GGTCTGATAA TGGTCCGTTG840
TTTTGTAAATCAGCCAGTCG CTTGAGTAAA GAATCCGGTC TGAATTTCTG AAGCCTGATG900
TATAGTTAATATCCGCTTCA CGCCATGTTC GTCCGCTTTT GCCCGGGAGT TTGCCTTCCC960
TGTTTGAGAAGATGTCTCCG CCGATGCTTT TCCCCGGAGC GACGTCTGCA AGGTTCCCTT1020
TTGATGCCACCCAGCCGAGG GCTTGTGCTT CTGATTTTGT AATGTAATTA TCAGGTAGCT1080
WO 93/25695 PCT/EP93/01489
56 -
TATGATATGT CTGAA~ATAA TCCGCAACCC CGTCAAACGT GTTGATAACC GGTACCATGG 1140
TAGCTAATTT CTTTAAGTAA AAACTTTGAT TTGAGTGATG ATGTTGTACT GTTACACTTG 1200
CACCACAAGG GCATATATAG AGCACAAGAC ATACACAACA ACTTGCAAAA CTAACTTTTG 1260
TTGGAGCATT TCGAGGAAAA TGGGGAGTAG CAGGCTAATC TGAGGGTAAC ATTAAGGTTT 1320
CATGTATTAA TTTGTTGCAA ACATGGACTT AGTGTGAGGA AAAAGTACCA AAATTTTGTC 1380
TCACCCTGAT TTCAGTTATG GAAATTACAT TATGAAGCTG TGCTAGAGAA GATGTTTATT 1440
CTAGTCCAGC CACCCACCTT ATGCAAGTCT GCTTTTAGCT TGATTCAAAA ACTGATTTAA 1500
TTTACATTGC TAAATGTGCA TACTTCGAGC CTATGTCGCT TTAATTCGAG TAGGATGTAT 1560
ATATTAGTAC ATAAAAAATC ATGTTTGAAT CATCTTTCAT AAAGTGACAA GTCAATTGTC 1620
CCTTCTTGTT TGGCACTATA TTCAATCTGT TAATGCAAAT TATCCAGTTA TACTTAGCTA 1680
GATCCTACGC AGCAGGTCTC ATCAAGACGA TCTACCCGAG TAACAATCTC CAGGAGATCA 1740
AATACCTTCC CAAGAAGGTT AAAGATGCAG TCAAAAGATT CAGGACTAAT TGCATCAAGA 1800
ACACAGAGAA AGACATATTT CTCAAGATCA GAAGTACTAT TCCAGTATGG ACGATTCAAG 1860
GCTTGCTTCA TAAACCAAGG CAAGTAATAG AGATTGGAGT CTCTAAAAAG GTAGTTCCTA 1920
CTGAATCTAA GGCCATGCAT GGAGTCTAAG ATTCAAATCG AGGATCTAAC AGAACTCGCC 1980
GTGAAGACTG GCGAACAGTT CATACAGAGT CTTTTACGAC TCAATGACAA GAAGAAAATC 2040
TTCGTCAACA TGGTGGAGCA CGACACTCTG GTCTACTCCA AAAATGTCAA AGATACAGTC 2100
TCAGAAGACC AAAGGGCTAT TGAGACTTTT CAACAAAGGA TAATTTCGGG AAACCTCCTC 2160
GGATTCCATT GCCCAGCTAT CTGTCACTTC ATCGAAAGGA CAGTAGAAAA GGAAGGTGGC 2220
TCCTACAAAT GCCATCATTG CGATAAAGGA AAGGCTATCA TTCAAGATGC CTCTGCCGAC 2280
AGTGGTCCCA AAGATGGACC CCCACCCACG AGGAGCATCG TGGAAAAAGA AGACGTTCCA 2340
ACCACGTCTT CAAAGCAAGT GGATTGATGT GACATCTCCA CTGACGTAAG GGATGACGCA 2400
CAATCCCACT ATCCTTCGCA AGACCCTTCC TCTATATAAG GAAGTTCATT TCATTTGGAG 2460
AGGACACGCT GAAATCACCA GTCTCTCTCT ATAAATCTAT CTCTCTCTCT ATAACCATGG 2520
ACCCAGAACG ACGCCCGGCC GACATCCGCC GTGCCACCGA GGCGGACATG CCGGCGGTCT 2580
GCACCATCGT CAACCACTAC ATCGAGACAA GCACGGTCAA CTTCCGTACC GAGCCGCAGG 2640
AACCGCAGGA GTGGACGGAC GACCTCGTCC GTCTGCGGGA GCGCTATCCC TGGCTCGTCG 2700
CCGAGGTGGA CGGCGAGGTC GCCGGCATCG CCTACGCGGG CCCCTGGAAG GCACGCAACG 2760
CCTACGACTG GACGGCCGAG TCGACCGTGT ACGTCTCCCC CCGCCACCAG CGGACGGGAC 2820
TGGGCTCCAC GCTCTACACC CACCTGCTGA AGTCCCTGGA GGCACAGGGC TTCAAGAGCG 2880
TGGTCGCTGT CATCGGGCTG CCCAACGACC CGAGCGTGCG CATGCACGAG GCGCTCGGAT 2940
WO 93/25695 ~ ' PGT/EP93/01489
- 57 ~~~~~~~ ""~ .
ATGCCCCCCGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGG3000
GTTTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCG3060
AGATCTGATCTCACGCGTCTAGGATCCGAAGCAGATCGTTCAAACATTTGGCAATAAAGT3120
TTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAAT3180
TACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTT3240
ATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCA3300
AACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAAGATCCTCTA3360
GAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGA3420
AATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC3480
TGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTC3540
CAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC3600
GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT3660
CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA3?20
GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAA3?80
AAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT3840
CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC3900
CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCC3960
GCCTT_TCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGT4020
TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC4080
CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG4140
CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA4200
GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGC4260
GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA4320
ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA4380
GGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAC4440
TCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA4500
AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT4560
TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATA4620
GTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCC4680
AGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAAC4?40
CAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG4800
WO 93/25695 PCT/EP93/01489
~'~
~
z ,, 58
TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAC4860
GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC9920
AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG4980
GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTC5040
ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCT5100
GTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC5160
TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTC5220
ATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC5280
AGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC5340
GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACA5400
CGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGT5460
TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT5520
CCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACA5580
TTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC 5620
(2) INFORMATION FOR SEO ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
lii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide MDB80
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /label= MDB80
/note= 'oligonucleotide designated as MDB80'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CCGCTTGTCA GTGAATGTTG C 21
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
WO 93/25695 ~ ~ ~ ~ PGT/EP93/01489
._ 59 ; . .
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide MDB81
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /label= MDB81
/note= "oligonucleotide designated as 1~B81"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
CCGAGGCCAT GGTTGCCGCC G 21
(2) INFORMATION FOR SEO ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide MDB82
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /label= MDB82
/note= "oligonucleotide designated as MDB82"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ACGCATAGGC ATAGGATGAC G 21
(2) INFORMATION FOR SEO ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3627 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
WO 93/25695 . , t . PCT/EP93/01489
2~~'~~~9 ~ 60 _.
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
fA) ORGANISM: Oryza sativa
(B) STRAIN: Akihikari
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..2845
(D) OTHER INFORMATION: /label= PT72
/note= "sequence comprising anther specific
promoter PT72"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 2733..2739
(D) OTHER INFORMATION: /label= TATA
/note= "TATA Box"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 2765
(D) OTHER INFORMATION: /note= "transcription initiation
determined by primer extension"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 2846..2848
(D) OTHER INFORMATION: /label= ATG
/note= "ATG start of translation of rice T72 gene"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GACAATACAT CAAGTAAATC AAACATTACA AATCAGAACC TGTCTAAGAA 60
TCCATCTTAA
TTCAGAAAAA AACTCAGATT AGATGTTCAT GCTTCCACCA GAAGCAGGAA 120
TGTGCAACCT
ACACTTCCTG TAATTTCCAT ACTACAATGT CCCCACTGAC CACTGTGCCT 180
GATGCTCTAT
TAGAATACCA CATCCTCCAT GGCTCCATGT AAATGCATAT AAATTTGACT 240
CTTTAAATTA
GTAACTACAA TTTAAAATTT ATCGAACATT GTTCAAATTT ATAAACAGTT 300
TCCCCAAATT
TAGATGCTCC CAAATGTACA CAGCTACTAG TAAAGCACCA TCCAGTTTCA 360
CCTGAACAGG
ACTGACATAA ATGTGTGAAA AGGGGACGTC ATTCCCCCAA ATACAACTGA 420
ACAATCCTCC
ATCAGAACAT TCATTTGATT GACATTACTC GGAGAGATAC AGCTCGCAGG 480
CACACGAGAT
TCTTCTGCCT TTCCAATTGC CACGAACCCA CATGTCACAC GACCAACCAA 540
AAAGAGAGAA
TTTTTCTTTG CACAAACAAA AAGTGAGATT TTTTTTTCGC CACAAAGGTG 600
CGAACTTTCT
TCTCTCTCCC ACTTTCCAAT CAAGAAACGA AGCACTCAAA CCAAGAACAA 660
ACCAAGGAAG
GAGAGATCGC TCCCTCTCCC AGAGCAAACG AAAGGAGAGA ACTCAGATGG 720
ATGCGAACTA
CTACCTTGCC TCTTTCCCCG GAGAAGCAGC GAAGGAGAAG AGCGCGATGC 780
CGCCGCCGCC
GCCGCCTCCG GCAACCTCCG GCTCCGGCGA GTCCGCCTCC TCCTCCTCTC 840
TCACCTCTCT
WO 93/25695 ~ PCT/EP93/01489
r
--
CTTCCCAACCGTGTGGTGTTCGAGAAGCTTTTATGCGAGCGACGTGCAGTGGAAGCGGTT 900
GCTCCCAAGTCAAACTGATGGAGACCACCTACTATCTTCCTCTTGTTTTCTTCTGCTTTT 960
CTTTTCTTTATCTTTTTTCTTTCATTTTATTTTGAGCGATGAACTTGAGAACAGTTTGGT 1020
TGTGGGTTAAATTAAACGGTGCAGAATTGCAAAGCTACGTCCTTTTCGTCTGATTAAGGT 1080
GGTATCAGAATCCTAATCTGTTAGCTCAGCATTTGTTTTTGTGTGTTTAATTGGCCATGA 1140
CATCAGATGGTTCAGACCGGTGGCAGGTCTTCATCGGAGAGGAGAATGAGAGCAATGCAA 1200
GTTGCAAACAACAAACAGGTCCTTCCAAACGGGTTGGTTTCATTCCACAGAACAGGATAG 1260
CAACCAGAGCACAAACCGTTCAACAATATATATATATATATATATATATATATATATATA 1320
TATATATATATATATATATGATTTAAAATTATATTACTATTTTTAGGATACGGAACTCTT 1380
AACACATGAAAATCTAAACATTTTCAACCAATCAGAACTACTAGAAAGATAATCTAACTA 1440
CTTCAAAATTTAAAATTTGACAAATAAAATAACTAGTTTTTTCTAAAGCTATCTTCACTG 1500
GACAACTTATGAATATTTATATTTATGAAGCGAGTACTCTCCTAGTACATATTACATATA 1560
T~:TTCTTCTTCTCATGAAAAATTAACTTCTCGCTATAAATCCGAACATATATTATGCGTA 1620
GCAAGTTGTTTTTTTTAACGGGTGGAGTAATATTAGAGTATTTAAATTCCTTCAAATTGC 1680
CATCCCTCTGGGACTTTGCTGCTGTTGTTCTTCCACGGTTGCTGTCAGTGTCACCCAGAT 1740
TTGCATCCTTTCCAGCTCGTAGCTACTGTTCTGCATGTATTGGACTTGGATTAAGATCAA 1800
ATGCAGTTGCTATTGTAACTGCACAATAGCAACTGCACACAATCATGTCCATTCGTTTTC 1860
AGATCCAACGGCTCTAGATGACTGCTACAGTACATGCATAATAGTACATCTCTGCTACAG 1920
TGTTTTTGCTGCAGTACCACTTCATATCCTGGCCTTCCGTTCTAGATCATGTGATGTACA 1980
TGTTTTTTTGAAACAACCCGCACAAGACATTGATAGAGTAGGAAATGTGATGTACATGTT 2040
AACGGCTTAAGTTACAGTTACAATAACAACTGCACAGGATCTTGATCCATTGGACTTGTA 2100
TAATATCTCATCTCGTCGTTCCATTATCGTGGTAACAGTTGGCAACTTGGCATCCAGTGC 2160
TGGAAACTATGCCGTGTGTACATCAGGATCGTCCTTTTTGTTCAGTTCCAAGATAGAACA 2220
AGTCCAAAAGATGGCCGTAGTTTTTTTAGTCACAGTGGAAGCTGACATAGCCGTGGAATA 2280
AGTTCTGCACAAAAGTTGCCATTCGAGATCAACTACTGGTAGTAGTAGTCATCTTCTACC 2340
ACTGCGAATATTCGAAGGGACACAAAAAGATCAACGAGTAAATTAGTTCACCGGAAGACG 2400
ACACATTATCACCACAAAAAGACTAAAAACAAAAAGAAATTGCCAGGCCAAAAAAGGCAA 2460
AAAAGAAAAAAAAAGATGGCACGAGGCCCAGGGCTACGGCCCATCTTGTCGCCGGCCCAA 2520
CCGCGCGCGCGAAACGCTCTCGTCGGCTCTCGGCTCGCCGCGACGCGATGGAGAGTTCGC 2580
GCCGCGGCGCGCGCGCGCGTTCGGTGGCTCACACGCTTGCGCCCTCGTCCTCCCGGCCGG 2640
CGCGGGCGCCGACCGCGCGTCCGCCGCATGCGCGCGGCGTAGGTGAGCAACGCGGGCCTC 2700
WO 93/25695 _ PCT/EP93/01489
62
GCCGCGCGCGCTCCCCTCCTTCGATCCCCTCCTATAAATCGAGCTCGCGTCGCGTATCGC2760
CACCACCACCACGACACACACGCACGCACCGTGCAGGCATCGACGACGAGCGAGAGCCCC2820
TCGGCGGCAGAAGACACTCACGGCGATGGCGGTGACGAGGACGGCGCTGCTGGTGGTGTT2880
GGTAGCGGGGGCGATGACGATGACGATGCGCGGGGCGGAGGCGCAGCAGCCGAGCTGCGC2940
GGCGCAGCTCACGCAGCTGGCGCCGTGCGCGCGAGTCGGCGTGGCGCCGGCGCCGGGGCA3000
GCCGCTGCCGGCGCCCCCGGCGGAGTGCTGCTCGGCGCTGGGCGCCGTGTCGCACGACTG3060
CGCCTGCGGCACGCTCGACATCATCAACAGCCTCCCCGCCAAGTGCGGCCTCCCGCGCGT3120
CACCTGCCGTAAGAAAACGAATAAAATCGATTTGCTATCTATCGATGATTGTGTTTTTGT3180
AGACTAAACTAAACCCCTATTAATAATCAACTAACCGATGAACTGATCGTTGCAGAGTGA3240
TGGAGATGGTGTGCCAAGGTAATTGCGTTTGCTCGTGCGAGGATGAGAAGAGAAGATTGA3300
ATAAGATGTTTGATGGCAACAAGTCATCAGGCGATCCGATCCCTGCAGCTATGAATGGGA3360
GTATACGTAGTAGTGGTCTCGTTAGCATCTGTGTGTCGCATATGCACGCCGTGCGTGCCG3420
TGTCTGTCCTGCTTGCTCTGCTGATCGTTCAATGAACGACAAATTAATCTAACTCTGr'~AG3480
TGACAAGTCGTTCGAGATATACTAATACTACCATGTGCAGGGTCTTTCAACCAAGGTTCA3540
TGTTTTCCACGAAAGCCGATTGAAACGAAACCGCGAAATTTTGATGCGAGATGAAAGCAG3600
ATTCCGAGTGAAATTTTAAATGGTTTT 3627
(2) INFORMATION FOR SEO ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2370 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(B) STRAIN: Akihikari
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..1808
(D) OTHER INFORMATION: /label= PT42
/note= 'sequence comprising anther specific
promoter PT92'
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1748..1755
(D) OTHER INFORMATION: /label= TATA
WO 93/25695 PGT/EP93/01489
'' 6 3
/note= "TATA Box"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1780
(D) OTHER INFORMATION: /note= "transcription initiation
site detezznined by primer extension"
(ix) FEATURE:
(A) NAME/KEY:
-
(B) LOCATION:
1809
(D) OTHER /label=
INFORMATION: ATG
/note=
"ATG
start
of translation
of rice
T42 gene"
(xi) S EQUENCE
DESCRIPTION:
SEQ ID
N0: 8:
GGCCATCACTGTCGGGTGCTGCGCCATGGACATCACCGTCTCCTTCCTGCGCCGCCGTCG 60
CCGGTGAGCTCCAAGGCCGAAGCCTTCTTCCCCTCACGCCACTACCTCTCTCTTCCCCAA 120
TTCCGGCCAACGCCGTCCGTTGCCACAGCGCCACCTCCACGCCATCCCAGAGCCCCGTGC 180
CGTGCCACCGGGTTCGCCTCCATCTCCTCTTGCCAACGCCGACGCTCGTCGCGGCAGCCA 240
TGCGCTGTCACCGATGAACACCGCCGCGCCACAGCCATGGCAGAGCACGGCCAGGGAGCC 300
ATGGCTGCTCTGCCTCCTCCTCCTTCTCTCACATCTGGTTGCAGCCGGACCTAGTCGGCT 360
TATACAAATGGCCCATGGGCAAAATTGTCTTTTATGAAAGTTTCTCTCACCGTTTCAGTC 420
GGAAATAATAAAATAATGGGAGGATTGTCCGCCAGCAAATTACCATATTTTTTCGGTGTC 480
CAAGAGCAAATACACGATCTTCGGGTGTTTCACAGCAAAGACCACAATTTCTAAGTGTCC 540
TGTAACAAATTTTGCCAATAAAAATTTAAAACCAAAGGAGAAGACTGTACATGAAGAAAA 600
ACAAAGAGAATGAAATTACATAAGCTCAGGGGTTATAAAGTTGATTTATTTTTAGGATGA 660
AGGAAGTGTGTGAAAACAATGGCCAATTGGGTGTCGGAAAATATAACGTGCTTGCTAAAA 720
TGTCGTCCCCATATCCTGTAGCTGATTATAGATAGACCCTGATGGTCAAGATGCCCTGTA 780
CTGGATCGTGTTTCCATGCTTCATCTCCGCTTCTCTCAAGTACTCCCCGAACTCACATAT 840
CTGGTGGGCTGGATCCACAGTAAGAAACAGTCAAACAACACTCACTTCATAGATAACCAA 900
TTGTTTAATTATTCTTAGTCCCTTATCTTATACTCCTAGTAAGTGCTTAAAAACTTGGTA 960
TAAATATCAAATTTATCGTACAATTACAATATAATTATAACGTATACCATGTAATTTTTA 1020
AAACTATTTTTAGATAAAAAAAATATGGTGATGAGCAGCCGCAGCAGCGGACGCCGAACC 1080
ACCTGCCGAACATCACCAAGATAGCGAGTCCTAAAAATTTTTAGTGTTCGTTTGCTGGGT 1140
TGGTAACTAATTAAAAAAAAAGAGCGACTCATTAGCTCATAAATAATTACGTATTAGCTA 1200
ATTTTTTTAAAAAATAAATTAATATAACTTATAAAGCAGCTTTTGTATAATTTTTTTTTT 1260
AAAAAAGTGTTGTTTAGCAGTTTTGGGAAGTGTGCCGAGGGAAAACGATGAGATGGGTTG 1320
GGGAAGGAGGGGGAAGAAGTGAAGAACACAGCAAATATAGGCAGCATCGTCCCGTACAGA 1380
WO 93/25695 PCT/EP93/01489
64
TCAGGCTGCAA~~CGCC
CC GCGGAGATAGTTAACGCGGCCCACGTTGTGCTATAGCCCG 1440
TCACTCTCGCGGGCCTCTCCAACCTCCAGTTTTTTTTCTAGCCCATCAGCTGATACGGGG 1500
CCTTCCCCCCATGCAGGAGGATGGCCCGCCACGCGGTGTTTTGGGCCGTTCTCGCCGCGC 1560
GCGCCCGTGCCGATCCGGGACTCATCCCACGTGCCGCCTCGCCACCGCCGCCGCCGCCGC 1620
TGCTGCTCCGGCTGCCGGCTGGACCTTCACGCTCACGCGCTCTCCCCTGCCCAACCACCA 1680
CGCAAACAAACACGAAGTTCGCGCCGTCGACCGGCTCCCCTCCTCCCCCGCGCGCATCGG 1740
ATCCCCCTACATAAACCCTCTCGCTCGCCATCGCCATGGCAGCAACTCCCCTCCTCCACT 1800
AGACCACCATGCACAGATCGATGGCCTCTCAGGCGGTGGCGCCCCTCCTCCTCATCCTCA 1860
TGCTCGCGGCGGCGGCGGGGGGCGCGTCGGCGGCGGTGCAGTGCGGGCAGGTGATGCAGC 1920
TGATGGCGCCGTGCATGCCGTACCTCGCCGGCGCCCCCGGGATGACGCCCTACGGCATCT 1980
GCTGCGACAGCCTCGGCGTGCTCAACCGGATGGCCCCGGCCCCCGCCGACCGCGTCGCCG 2040
TCTGCAACTGCGTCAAGGACGCCGCCGCCGGCTTCCCCGCCGTCGACTTCTCCCGCGCCT 2100
CCGCCCTCCCCGCCGCCTGCGGCCTCTCCATCAGCTTCACCATCGCCCCCAACATGGACT 2160
GCAACCAGTAAGTTCATTCATTCTTTCTTAACTCCAATTCAATTTATCCATCACCTCGAC 2220
TTAAGCCTGATTAAACTTAACTTGTTCTTTGCATGCTTGCACTATTGCAGGGTTACAGAG 2280
GAACTGAGAATCTGAGAGCGTGAGGAATCGAGTTCATGTTGCATTTATCATCAATCATCA 2340
TCGACTAGATCAATAAATCGAGCAAAGCTT 2370
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2407 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE "'YPE: DNA (genomic)
(iii) HYPOTHET.AL: NO
liii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(B)- STRAIN: Akihikari
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..2263
(D) OTHER INFORMATION: /label= PE1
/note= 'sequence comprising anther specific
promoter PE1"
lix) FEATURE:
(A) NAME/KEY: -
WO 93/25695 PCT/EP93/01489
-- 65
2~37~~9
(B) LOCATION: 2181..2187
(D) OTHER INFORMATION: /label= TATA
/note= "TATA Box"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 2211
(D) OTHER INFORMATION: /note= "transcription initiation
site determined by primer extension'
tix) FEATURE:
(A) NAME/KEY:
-
(B) LOCATION:
2264..2266
(D) OTHER /label=
INFORMATION: ATG
/note=
"ATG start
of translation
of E1
gene"
(xi) S EQUENCE
DESCRIPTION:
SEQ ID
NO: 9:
TGATAGTGACATACTCACATGCTTTGTCAATTCAAGTATCAGTTCTTTTCATATTGATTT 60
CTTAGTTGATGAAAGTATACATATTTCTTGCCATCAATTCTTTTAGTAGGTACATTTGGA 120
CACTAGTGGTCAGGGTTGAACTCTTAACTGGAGTCTCATCTGATTTGCTTATCTGAGACT 180
GGGTTTGTGCAAATCCTGTCATGAGGCAAGGTGGACTGTCAGTCCATGACACTTTGCTAC 240
TTCTATTAAGTTCTCGAAATCTTTTCCAGTGTATGTCCGTTCTCTTTCAAATGAATTATT 300
TATATGTTCTGACAGCCTCGCGGTGTACATTTCATTTAACTTTTGTCTTCACAGGGCCTC 360
TTGGTATTTTGTTGAGCAGATTGGAATCAACCTTCTTGTAGAACTTCTTGATGTCGTCGC 420
TACCCTTTGCAACTAGATGGTCAACTTCTGTCTTATATCTTTGGTACAACACTGGCAAAG 480
TGTGCGCGCACAAGAATCCTGTGAAGTAAGAAATACAAACTTGTCATTGTGAAAGTTTAG 540
CTTTATATGATCTTGACTCTAAATTGTTTCTCCTCAGATCCTTCTGTGTGATTGTTTTAT 600
TAAAATTTAATATTTATCTGGAATACCTACCAATATATAGTAGACTTGTCAAGCTGCAAG 660
AACTTCCAATCGCCGACAATACCAATAGAGATCCAACCACCTTAATATCATAAACAATCT 720
GATTGTTAGTCCAGAACTATATTGAGTAGTGAACAACAATAGCACATTAACATTATGAGG 780
ATTATTGGCTAACTCTGCAATTCAATATTCTGATGCGTCTAATCTGGTCAATTTTAGCGC 840
TCCAGAAAGAATTGCACAATCCTTGGACAATGTTGGCACTGGAACTGTTGCATGTTTTTA 900
CATCTCTTATTAACGTAGCAAAGGAGTAGATTATTATGTACCAGGAGAAATCTCTTCAGA 960
TCCTTTCCACATGCAATGTCGTAAAGAACAGATACAGTGTACGTTAGTTTGTAATGGACG 1020
GTCAATGCCATTTCTCTGAAGGCATGTTCAGAGATGATGATTTCTGGGATCCTTGGAGGG 1080
GCCCTGAAATTCGGAAACAGTTAGTTGAGTTTTAGTACCTAATGTCTTGCGTTATACTAC 1140
GTGAAATGCCATTTCTGTAAGCTGAGTTTTCTACCATCTCCACAGGAAATAAAGCTAATA 1200
CCTGTCCAAGAGTGGTGCGGCATTTGACCAAATGAAGATCACAAGCATGGCAAGAATGGC 1260
AATCTGGCAAAGGAGCGGAATTATATTGTATTCTACTACATCGAACAGGAACCATATCAA 1320
WO 93/25695 PCT/EP93/01489
66 '
y
TGTTGCCCCAGCAAGGACCCCCGCAGATAAGTTCCTGTTCTTCCACAGCAGAATATCCGC1380
AACTGCATAGCTCCCAACAATGAAATCCAAAACCACATCGGCTCAGAGAGAAGTTATGAT1440
AAAAGGCACTAATTCTGAATAATTTCCTAGAAAGCGAATAATAATAGCACACCTTGACCT1500
CCACCAAGAAGCTTGTGGATCGACTTGTGCCCATGAAATGGCATTCTGACATTCTGGTCA1560
CTGTCAGAATCTCTCGGAAAATGAGGAGGCATAGCTTCGTGTGTGTATGTGTGTGGGATA1620
TTACGCTGCTAAAACTTTGTGTTTCTGATCGATCTGGTTAGAGAGCATCGTCTTTATAAG1680
CACTTAAAAATGGTAGTATAATCTCTCAAGGAGCCTATACTGCCAAGGAAAGGATAGCTT1740
GGCCTGTGGGGATTGAGCCGTTGAAGGGAACAAACGAATACAGTTACCTTACCAGATGTT1800
TGCCACGACATGGGCAACGTCATTGCTAGACCAAGAAGGCAAGAAGCAAAGTTTAGCTGT1860
CAAAAAAGATATGCTAGAGGCTTTCCAGAATATGTTCTATCTCAGCCAGACCAATGGGGG1920
CAAAATTTACTACTATTTGCCATACATTAACCACGTAAAAGTCCTACACTCAACCTAACT1980
GTTGAACGGTCCTGTTCTGGCCAACGGTGAGAATGCACCTAATGGACGGGACAACACTTC2040
TTTCACCGTGCTACTGCTACATCCTGTAGACGGTGGACGCGTGAGGTGCTTTCGCCATGA2100
CCGTCCTTGGTTGTTGCAGTCACTTGCGCACGCTTGCACCGTGACTCACCTGCCACATTG2160
CCCCCGCCGTCGCCGGCGCCTACAAAAGCCACACACGCACGCCGGCCACGATAACCCATC2220
CTAGCATCCCGGTGTCCAGCAAGAGATCCATCAAGCCGTCGCGATGACGACGAGGCCTTC2280
TGTTTTTTCCACCGTTGTCGCGGCGATCGCCATCGCCGCGCTGCTGAGCAGCCTCCTCCT2340
CCTGCAGGCTACCCCGGCCGCGGCCAGCGCGAGGGCCTCGAAGAAGGCTTCGTGCGACCT2400
GATGCAG 2407
(2) INFORMATION FOR SEO ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2784 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..1179
(D) OTHER INFORMATION: /label= PCA55
/note= "region comprising the anther specific
promoter and the leader sequence, PCA55"
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-- 67
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1072
(D) OTHER INFORMATION: /label= TATA
/note= "TATA Box"
(ix) FEATURE:
(A) NAME/KEY:
-
(B) LOCATION:
1180..1596
(D) OTHER /note=
INFORMATION: "presumed
coding
sequence
of
corn CA55 gene"
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 10:
TGGTATGCATCAATAGAGCCGGAAGATGGTCTGGAGTAAGGACCTGGCAGTGTGATACGG 60
GAACTTGACATCTGAATAGATATTCTCCCTTGTCCCTCTGGTF~AAAAAAACTGTTGTCAC 120
ATTTGCCTTCGCTGTGACTTGGATGTATCATGTATATCTTTGACCATTGATATCTTGGTT 180
AATCAGACGGTGCATTACAATCATGGCCTCATTCATATAGGGTTTAGGGTTACCACGATT 240
GGTTTGCATAAGTAGTACCCCTCCGTTTCAAATTATGTCGTATTTTGATTTTTTAGATAC 300
ACTTTTTATATAATTTTTTATTTTAAATTAGGTGTTTTATATAATACGTATCTAAGTGTA 360
TAATAAAATATATGTATCTAAAAGCTGTAATTTAGTATAAATTAGAATGGTGTATATCTT 420
CAATGTATGACAAATAATTTGAAATGGAGGAGGGTATGAAAAGCCAAAACCTCCTAGAAT 480
ATGGAATGGAGGGAATACATACAAATTCTTTGCTTCAGTTAAAAGAAACGAGAAAAGGAG 540
GGGAATGGGGAATCGTACTTCAGTTTTTACGAGTTTTCATCAAACATGTATGCACGTCTT 600
CCCTTGGTTGATGCATCTTTTTGGCAAATCTTCGTTTAATTGCGGCTTCTTTTTTATACC 660
G'~TCGAAGGTTTTCGTCGTCAATGCTGAAACTCCACTTTCACCACCTTCGGTTGCATCTG 720
CTTGCTT_TCAATTCACCTCTAATTAGTCCAAGTGTTTCATTGGACGAAGGTCCAAGTCCT 780
TCAGATCATCTCAATTTTCTTTGATCTGAAACAACAATTTAAAACTGATTTTGTTACCTT 840
GACCTGTCGAAGACCTTCGAACGAACGGTACTGTAAAAATACTGTACCTCAGATTTGTGA 900
TTTCAATTCGATTCGGGTCTCCTGGCTGGATGAAACCAATGCGAGAGAAGAAGP~AAAAAT 960
GTTGCATTACGCTCACTCGATCGGTTACGAGCACGTAGTTGGCGCCTGTCACCCAACCAA 1020
ACCAGTAGTTGAGGCACGCCCTGTTTGCTCACGATCACGAACGTACAGCACTATAAAACA 1080
CGCAGGGACTGGAAAGCGAGATTTCACAGCTCAAAGCAGCCAAAACGCAGAAGCTGCACT 1140
GCATATACAGAAGATACATCGAGCTAACTAGCTGCAGCGATGTCTCGCTCCTGCTGCGTC 1200
GCCGTGTCGGTGCTTCTCGCTGTCGCCGCGACAGCCAGCGCCACCGCGCCGGCATGGCTG 1260
CACGAGGAGCAGCACCTCGAGGAGGCCATGGCCACGGGCCCGCTGGTCGCAGAGGGTGCG 1320
AGGGTGGCGCCCTCCGCGTCCACCTGGGCTGCCGACAAGGCGTCGCCGGCGAGGCCGAGC 1380
GGCGGCATGGCCACGCAGGGCGACGACCAGAGCTCGTCGGGCGGCAGTGGCAGCAGCGGT 1440
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..
68
GAGCACGGCAAGGCGGAGGGCGAGAAGCAGGGCAAGAGCTGCCTCACCAA 1500
GGAGGAGTGC
CACAAGAAGAAGATGATCTGTGGCAAGGGCTGCACGCTCTCGGCGCACAGCAAGTGCGCC 1560
GCCAAGTGCACCAAGTCCTGTGTCCCCACCTGCTAGGAGCCGAGGCCGGAGCTTGCCGGC 1620
GGCGAGACCTCGATCGATCGAGTGCTTCACTTCACTTCTTTGTTATAGTTCTTGTGTGTT 1680
GCCGTTGCGTTGCGTTGCGTAGACGAAGGGAATAAGGAAGGGTAATTGGATTACCTGTTC 1740
CAGATCTCTGTGTAAGCGTGTTGTCGTGACAAGTCTTTTGATCCAGAGCGAGGGATGGAT 1800
AGATCGCGCTCGCAGTTTTAATTGCAATGCTAGTTCAATATGTGTGCATCATGTTGGCAA 1860
CTACATAGTCCAGATTCAAACCGAGATCGCTGTTTAGCATGCCAGCACAATAATAACGGT 1920
ACAATCATATTATATTTTATACAAATGCACAATTTATCTCTAGAGATGTCAATGGGAAAT 1980
TCCTCATCGGGTTATATCATCTCAGACTCATCCCCATCATATTTGATTCATCCTCATACT 2040
CATCCTCATATCTATCATGAGTGCAAAACTCATTTCATACCCATCTCTATTTTGGTTTAG 2100
GGTCTCCATCCCTAATTAAGGGATAACTAGTACTAACAACTAGCACAAACTATCTAGATT 2160
TCAGATATCACCACATTGACAAACAATCATCCATGAACTATGATCCATTCATCCATCCAT 2220
CAAAAAATAAATCGGTATTTCGAGAACGATAGAAGAAATGAAGTCGGCTCACCTTTCTTG 2280
GTCACCATTTGAGTTTGTTGGTGCCTGAGAATCCATGGTCGTCATCGTCGTCCTAGGGAT 2340
CGGCGGTGCTCCTCGTTGTTGGTAAAGTCGCCAGTGTGTAGTGCTAGCGCAACTGTCCAG 2400
GCGTGCAACGGTTGGCCGGCTGGAAAGGGCATAGCGTATGGCTGGTTATTTTTAGGGTTT 2460
TGTTTTTTTACTAATCTGCTAGTTGCCTTGCCATGTTGTCTTATTGGGCTAGGATCTAGG 2520
GCT.TGTTACGCTGCTGTGTTGGGCTTGGTGTCCGGTTCAGCCTCAACTCATTCATACAAA 2580
TCAGATTCATACAAAACAGGTATACACGTATGAAATATCCATGGATAATCAGGTTCGAAT 2640
TATTGTCCCCTAAACCCATACACGTTTACCCAATGGATGGATATTTTGTCTCATATCCAT 2700
ACACATGAGACGATTTTTGTCCCATACCTGTGCTCTAATAGGAGAATTTCTCTCGGGATA 2760
GCGAGTATCGGATCCTCTAGAGTC 2784
(2) INFORMATION FOR SEO ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
WO 93/25695 ~ ~ ~ ~ r~ PCT/EP93/01489
_ 69
(A) ORGANISM: oligonucleotide Zm1301i2
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /label= Zm1301i2
/note= "oligonucleotide designated as Zm1301i2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GTGGATTGAA CGGGACTGAG TTGG 24
(2) INFORMATION FOR SEO ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide Zm1301i1
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..25
(D) OTHER INFORMATION: /label= Zm1301i1
/note= 'oligonucleotide designated as Zm1301i1'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
AAGTCTCCAA GACTTTGGTT ATTCC 25
(2) INFORMATION FOR SEO ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: lineaz
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oligonucleotide Zm1301i5
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..31
(D) OTHER INFORMATION: /label= Zm1301i5
WO 93/25695 - PCT/EP93/01489
/note= "oligonucleotide designated as Zm1301i5"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 13:
GGATCCATGG TTGCCGCCGG GTGAATGTAC G 31
(2) INFORMATION FOR SEO ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide BXOL2
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /label= BXOL2
/note= "oligonucleotide designated as BXOL2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
ACGGAAAACC TGAAGCACAC TCTC 24
(2) INFORMATION FOR SEO ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide TA29SBXOL2
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..49
(D) OTHER INFORMATION: /label= TA29SBXOL2
/note= "oligonucleotide designated as TA29SBXOL2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GTTTTTACTT AAAGAAATTA GCTACCATGA AAAAAGCAGT CATTAACGG 49
WO 93/25695 . PGT/EP93/01489
(~
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(i
i) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: oligonucleotide PTA290L5
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..27
(D) OTHER INFORMATION: /label= PTA290L5
/note= "oligonucleotide designated as PTA290L5"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 16:
TGGCCATAAC TGAAATCAGG GTGAGAC 27
(2) INFORMATION FOR SEO ID N0: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4808 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
liii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: EcoRI-HindIII fragment of plasmid pTS218
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (18..401)
(D) OTHER INFORMATION: /label= 3'nos
/note= "3' regulatory sequence containing the
polyadenylation site derived from Agrobacterium
T-DNA nopaline synthase gene'
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (402..737)
(D) OTHER INFORMATION: /label= barnase
/note= 'coding region of the barnase gene of
Bacillus amyloliquefaciens"
(ix) FEATURE:
WO 93/25695 ~ PCT/EP93/01489
~2 -
(A) NAME/KEY: -
(B) LOCATION: complement (738..1944)
(D) OTHER INFORMATION: /label= PZM13
/note= "promoter region of the Zml3 gene of Zea
mat's"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (1945..2281)
(D) OTHER INFORMATION: /label= 3'nos
lix)FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (2282..2554)
(D) OTHER INFORMATION: /label= barstar
/note= "coding region of the barstar gene
of
Bacillus amyloliquefaciens"
(ix)FEATURE:
(A) NAME/KEY: -
(B) LOCATION: complement (2555..3099)
(D) OTHER INFORMATION: /label= PTA29
/note= "promoter region of the TA29 gene
of
Nicotiana tabacum"
(ix)FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 3100..3932
(D) OTHER INFORMATION: /label= 3553
/note= ""3553" promoter sequence derived
from
cauliflower mosaic virus isolate CabbB-JI"
(ix)FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 3933..4484
(D) OTHER INFORMATION: /label= bar
/note= "coding region of the phosphinothricin
acetyltransferase gene"
(ix)FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 4485..4763
(D) OTHER INFORMATION: /label= 3'nos
(ix)FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 2333..2356
(D) OTHER INFORMATION: /label= BXOL2
/note= "region corresponding to oligonucleotide
BXOL2"
(ix)FEATURE:
(A) NAM/KEY: -
(B) LOCATION: complement (2538..2586)
(D) OTHER INFORMATION: /label= TA29SBXOL2
/note= 'region complementary to oligonucleotide
TA29SBXOL2"
(ix)FEATURE:
(A) NAME/KEY: -
(H) LOCATION: complement (2800..2823)
(D) OTHER INFORMATION: /label= PTA290L5
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_ 73
/note=
"region
complementary
to part
of
oligonucleotide PTA290L5"
(xi) SEQUENCE
DESCRIPTION:
SEQ ID N0: 17:
GAATTCGAGC TCGGTACCCGGGGATCTTCCCGATCTAGTAACATAGATGACACCGCGCGC60
GATAATTTAT CCTAGTTTGCGCGCTATATTTTGTTTTCTATCGCGTATTAAATGTATAAT120
TGCGGGACTC TAATCATAAAAACCCATCTCATAAATAACGTCATGCATTACATGTTAATT180
ATTACATGCT TAACGTAATTCAACAGAAATTATATGATAATCATCGCAAGACCGGCAACA240
GGATTCAATC TTAAGAAACTTTATTGCCAAATGTTTGAACGATCTGCTTCGGATCCTCTA300
GAGNNNNCCG GAAAGTGAAATTGACCGATCAGAGTTTGAAGAAAAATTTATTACACACTT360
TATGTAAAGC TGAAAAAAACGGCCTCCGCAGGAAGCCGTTTTTTTCGTTATCTGATTTTT420
GTAAAGGTCT GATAATGGTCCGTTGTTTTGTAAATCAGCCAGTCGCTTGAGTAAAGAATC480
CGGTCTGAAT TTCTGAAGCCTGATGTATAGTTAATATCCGCTTCACGCCATGTTCGTCCG540
CTTTTGCCCG GGAGTTTGCCTTCCCTGTTTGAGAAGATGTCTCCGCCGATGCTTTTCCCC600
GGAGCGACGT CTGCAAGGTTCCCTTTTGATGCCACCCAGCCGAGGGCTTGTGCTTCTGAT660
TTTGTAATGT AATTATCAGGTAGCTTATGATATGTCTGAAGATAATCCGCAACCCCGTCA720
AACGTGTTGA TAACCGGTACCATGGTTGCCGCCGGGTGAATGTACGTGTTTTCCCCTCCC780
CCCT_TGTGGA TGTCGGAGGAAAAGGGCGGGACCTTTCCTTATTATTTGTGTGAGGGAGGG840
AGGGTGAGAC GAGGGTGGCAAATCTGGCCTGGTTTCGTAACGCAGCCTGTGGTGTCCTTA900
AATAATCTGC GAGACCCGAAGATTTGTCTGGCTGCCCGTAGAAATAGTGGCCGACGGCCA960
GAAAGCCGTT TTGACCTAGTTTTGTGGCATGGGGAGGTGAAAAATATGTCGAATTTGGTT1020
GAGTTTTTCA GCGGTCCACAAAAGTTGGATTTGCGACTTCTAATCTTCTATGCTGCTATG1080
TAAAACTAAA TAAGACGCGCTCAACAAAACGGTATCAGTTAAATGTATCCATAAGTGAAT1140
AGTATATAGA ATCACTAAATGGCAGCTTCTCATATTTAGCATAGTACTTGGACAAATGTC1200
ACGCAAGATG TATTATAAAAACACATCGGTGTTGAGTGTTGATACAATAAAATCTCAATA1260
GTTGTTTTAA TCTCAATCCATGTGGATTGAACGGGACTGAGTTGGTTTAAATCCCTAGTA1320
AGTCAAAATT ATTTCTAATTTTATCTAAATCTCATCTAATCCACGTGGAATAGGAATAAC1380
CAAACTGTTG GAGACTTGTTCTCAAGTGCTATGAGTTAAGAACAAGGCAACATAGAAAAT1440
ATTAATCGTT AAGGTCCTTCGTCCTTCGAAGCATTATTTCCCTTAGGATATAATGGTTTA1500
CGGACGAAGG TTATGAAGGACGTACCTTCATAAATTCATTAAACAATGACGAAGGATGAA1560
ATATAAAGAA TATAAAAGACAACATGAACAATTATATATTATTATTAGGTACAAACAGAA1620
ATATCGTTGA ATTACAAGTGTACCTTCAATAGGAATGAGATGACAGTACAAGCGTGACGC1680
AAAAAGCGAA TGCCAAGTCAGCGTGAACAGTACGGGAATACTGTTCACCTATTTATAGGC1740
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74
ACGGGACGTAGCCTGTGCAA ATGCCCTTTACACTTAATAA 1800
AATTACATTA TAAACCTATA
GTAATCTGTTGAGGTCTAAATAGCCTTTTCATCTTTAAGTCGGTTTCAACTGCTGCTGTC 1860
TTGCCGAAGCTTTCCTGCTTACACCTTAGGCGCTTCACCAACCTTCGTATTATTCTGGTC 1920
TACTGTGATGCCTGACTTGAGTCCGAAGATGGGGATCTTCCCGATCTAGTAACATAGATG 1980
ACACCGCGCGCGATAATTTATCCTAGTTTGCGCGCTATATTTTGTTTTCTATCGCGTATT 2040
AAATGTATAATTGCGGGACTCTAATCATAAAAACCCATCTCATAAATAACGTCATGCATT 2100
ACATGTTAATTATTACATGCTTAACGTAATTCAACAGAAATTATATGATAATCATCGCAA 2160
GACCGGCAACAGGATTCAATCTTAAGAAACTTTATTGCCAAATGTTTGAACGATCTGCTT 2220
CGGATCCTCTAGACCAAGCTAGCTTGCGGGTTTGTGTTTCCATATTGTTCATCTCCCATT 2280
GATCGTATTAAGAAAGTATGATGGTGATGTCGCAGCCTTCCGCTTTCGCTTCACGGAAAA 2340
CCTGAAGCACACTCTCGGCGCCATTTTCAGTCAGCTGCTTGCTTTGTTCAAACTGCCTCC 2400
ATTCCAAAACGAGCGGGTACTCCACCCATCCGGTCAGACAATCCCATAAAGCGTCCAGGT 2960
TTTCACCGTAGTATTCCGGAAGGGCAAGCTCCTTTTTCAATGTCTGGTGGAGGTCGCTGA 2520
TACTTCTGATTTGTTCCCCGTTAATGACTGCTTTTTTCATGGTAGCTAATTTCTTTAAGT 2580
AAAAACTTTGATTTGAGTGATGATGTTGTACTGTTACACTTGCACCACAAGGGCATATAT 2640
AGAGCACAAGACATACACAACAACTTGCAAAACTAACTTTTGTTGGAGCATTTCGAGGAA 2700
AATGGGGAGTAGCAGGCTAATCTGAGGGTAACATTAAGGTTTCATGTATTAATTTGTTGC 2760
AAACATGGACTTAGTGTGAGGAAAAAGTACCAAAATTTTGTCTCACCCTGATTTCAGTTA 2820
TGGAAATTACATTATGAAGCTGTGCTAGAGAAGATGTTTATTCTAGTCCAGCCACCCACC 2880
TTATGCAAGTCTGCTTTTAGCTTGATTCAAAAACTGATTTAATTTACATTGCTAAATGTG 2940
CATACTTCGAGCCTATGTCGCTTTAATTCGAGTAGGATGTATATATTAGTACATAAAAAA 3000
TCATGTTTGAATCATCTTTCATAAAGTGACAAGTCAATTGTCCCTTCTTGTTTGGCACTA 3060
TATTCAATCTGTTAATGCAAATTATCCAGTTATACTTAGCTAGATCCTACGCAGCAGGTC 3120
TCATCAAGACGATCTACCCGAGTAACAATCTCCAGGAGATCAAATACCTTCCCAAGAAGG 3180
TTAAAGATGCAGTCAAAAGATTCAGGACTAATTGCATCAAGAACACAGAGAAAGACATAT 3240
.TTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCATAAACCAA 3300
GGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCTACTGAATCTAAGGCCATGC 3360
ATGGAGTCTAAGATTCAAATCGAGGATCTAACAGAACTCGCCGTGAAGACTGGCGAACAG 3420
TTCATACAGAGTCTTTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAG 3480
CACGACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCT 3540
ATTGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCT 3600
WO 93/25695 PCT/EP93/01489
/5 ,~~ ~ ~~~~
ATCTGTCACT TCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCAT 3660
TGCGATAAAG GAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGA 3720
CCCCCACCCA CGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAA 3780
GTGGATTGAT GTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCG 3840
CAAGACCCTT CCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCAC 3900
CAGTCTCTCT CTATAAATCTATCTCTCTCTCTATAACCATGGACCCAGAACGACGCCCGG 3960
CCGACATCCG CCGTGCCACCGAGGCGGACATGCCGGCGGTCTGCACCATCGTCAACCACT 4020
ACATCGAGAC AAGCACGGTCAACTTCCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGG 4080
ACGACCTCGT CCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGG 4140
TCGCCGGCAT CGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCG 4200
AGTCGACCGT GTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACA 4260
CCCACCTGCT GAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGC 4320
TGCCCAACGA CCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCATGC 4380
TGCGGGCGGC CGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGCTGGACT 4440
TCAGCCTGCC GGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATCTGATCTCACGCGT 4500
CTAGGATCCG AAGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCT 4560
GTTGCCGGTC TTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATA 4620
ATTAACATGT AATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAA 4680
TTATaCATTT AATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCG 4740
CGCGCGGTGT CATCTATGTTACTAGATCGGGAAGATCCTCTAGAGTCGACCTGCAGGCAT 4800
GCnAGCTT 4808
WO 93/25695 PGT/EP93/01489
76
FIGURE 1 ~
1. Action . Transform corn embryos (e. g. Ii99) with male-
sterility gene S, linked to herbicide resistance gene bar
(Example S)
Result . transformed plants with genotype S/s
2. Action . Transform corn embryos (e. g. Ii99) with fertility-
restorer gene R (Example 6)
Result . transformed plants with genotype R/r
3. Action . Transform corn embryos (e. g; H99) with maintainer
gene P (Example 3)
Result . transformed plants with genotype P/p
4. Action . Cross S/s,r/r x s/s,R/r.
Select offspring for presence of both S and R genes by
means of PCR.
Result . plants with genotype S/s,R/r
5. Action . Self selected plants of 4 (optional)
Result . Progeny plants with 9 different genotypes
gamete S,R S,r s,R s,r
d-~
S,R S/S,R/R S/S,R/r S/s,R/R S/s,R/r
S,r S/S,R/r S/S,r/r~' S/s,R/r S/s,r/r"
s,R S/s,R/R S/s,R/r s/s,R/R s/s,R/r
s,r S/s,R/r S/s,r/r" s/s,R/r s/s,r/r
" male-sterile plants
6. Action . self male-fertile progeny plants of 5 (Optional)
Result .
6.1. Self of S/S,R/R . 100 % male-fertile plants
Self of S/s,R/R . 100 % male-fertile plants
Self of s/s,R/R . 100 % male-fertile plants
Self of s/s,R/r . 100 % male-fertile plants
Self of s/s,r/r . 100 % male-fertile plants
6.2 Self of S/s,R/r . Same progeny as 5
in
13/16 male-fertile
plants
with 4/13 herbicide
sensitive
WO 93/25695 77~ ~ ~ 7 ~~ ~ PCT/EP93/01489
FIGURE 1 (continued 1)
6.3 Self of S/S,R/r . Progeny as follows .
gamete S,R S,r
d~
S,R S/S,R/R S/S,R/r
S,r S/S,R/r S/S,r/r"
" male-sterile plants
Thus . 3/4 male-fertile plants, 0$ herbicide sensitive
All male-sterile plants are of genotype S/S,r/r
7. Action . Cross
. P/p (from 3) x d . S/s,R/r (from 4)
- this equals in fact
. s/s,r/r,P/p x d' . S/s,R/r,p/p
Result . Progeny with the following genotypes
gamete S,R,p S,r,p s,R,p s,r,p
d--~
1
s,r,P S/s,R/r,P/p S/s,r/r,P/p s/s,R/r,P/p s/s,r/r,P/p
s,r,p S/s,R/r,p/p S/s,r/r,p/p s/s,R/r,p/p s/s,r/r,p/p
8. Action . From offspring of 7, select plants with genotype
S/s,r/r,P/p by screening, by means of PCR and/or Southern
blotting, for presence of S and P gene and absence of R
gene.
Result . plants with genotype S/s,P/p
WO 93/Z5695 ~ ~ ~ ~ ~'~ ~ ' 7 PCT/EP93/01489
b~ 8
FIGURE 1 (continued 2)
9. Action . Self plants with genotype S/s,P/p (from 8)
Result . progeny with the following genotypes
" male-sterile plants
Shaded genotypes cannot develop because male
gametes (pollen) are killed off by expression of
the maintainer gene P.
10. Action . self male fertile plants of 9.
Result
10.1. Self of s/s,P/p . 100 % male-fertile plants
Self of s/s,p/p . 100 % male-fertile plants
10.2 Self of S/s,P/p . Same progeny as in 9
5/8 male-fertile plants with
2/5 herbicide sensitive
10.3 Self of S/S,P/p . Progeny as follows .
" male-sterile plants
Shaded genotypes cannot develop because male
gametes (pollen) are killed off by
expression of the maintainer gene P.
FINAL RESULT
- 1/2 male-fertile plants, 0% herbicide sensitive. All
these plants are maintainer plants -
- 1/2 male sterile plants. All homozygous for the male-
sterility gene S.
