U.S. patent application number 14/776498 was filed with the patent office on 2016-02-04 for turfgrass varieties having desirable looking turf when mowed infrequently.
This patent application is currently assigned to J.R. SIMPLOT COMPANY. The applicant listed for this patent is J.R. SIMPLOT COMPANY. Invention is credited to ANDREW DOUGLAS BREDE.
Application Number | 20160029585 14/776498 |
Document ID | / |
Family ID | 51625218 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160029585 |
Kind Code |
A1 |
BREDE; ANDREW DOUGLAS |
February 4, 2016 |
TURFGRASS VARIETIES HAVING DESIRABLE LOOKING TURF WHEN MOWED
INFREQUENTLY
Abstract
Turfgrass varieties with desirable looking turf when mowed
infrequently are provided. Also provided is a method of producing a
turfgrass plant having desirable looking turf when mowed
infrequently. The turfgrass plants of the present invention display
green leaf chlorophyll concentrations above 1.8 mg/g and a field
insitu CM-1000 chlorophyll meter reading of 341.7 or higher, as
well as high general turfgrass quality ratings. The turfgrass
plants of the present invention retain a dark green color even when
scalped back by infrequent mowing.
Inventors: |
BREDE; ANDREW DOUGLAS;
(VERADALE, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
J.R. SIMPLOT COMPANY |
Boise |
ID |
US |
|
|
Assignee: |
J.R. SIMPLOT COMPANY
Boise
ID
|
Family ID: |
51625218 |
Appl. No.: |
14/776498 |
Filed: |
March 12, 2014 |
PCT Filed: |
March 12, 2014 |
PCT NO: |
PCT/US14/24563 |
371 Date: |
September 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61782611 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
530/370 ; 554/9;
800/260; 800/278; 800/279; 800/298; 800/300; 800/301; 800/302 |
Current CPC
Class: |
C12N 15/8275 20130101;
C12N 15/8279 20130101; C12N 15/8277 20130101; A01G 20/20 20180201;
C12N 15/8274 20130101; A01H 5/12 20130101; C12N 15/8278 20130101;
C12N 15/8286 20130101; A01H 1/02 20130101; C12N 15/8241
20130101 |
International
Class: |
A01H 5/12 20060101
A01H005/12; C12N 15/82 20060101 C12N015/82; A01H 1/02 20060101
A01H001/02 |
Claims
1. A method of producing a turfgrass plant having desirable looking
turf when mowed infrequently, said method comprising: a. Planting
turfgrass genotypes; b. Selfing said turfgrass genotypes for one or
more generations to produce progeny plants; c. Selecting progeny
plants having short, basal growth of leaves, adequate production of
seed heads, and an acceptable level of apomixis; d. Further
selecting said progeny plants to identify plants with one or more
characteristics different from the maternal parental line of said
turfgrass genotype and having desirable looking turf when mowed
infrequently; and e. Harvesting seed or vegetative propagating
material from said selected progeny plants.
2. A turfgrass plant produced by the method of claim 1.
3. A turfgrass plant having desirable looking turf when mowed
infrequently, wherein said turfgrass plant has a green leaf
chlorophyll concentration above 1.8 mg/g.
4. A method of producing a bluegrass plant having desirable looking
turf when mowed infrequently, said method comprising: a. Planting
bluegrass genotypes; b. Selfing said bluegrass genotypes for one or
more generations to produce progeny plants; c. Selecting progeny
plants having short, basal growth of leaves, adequate production of
seed heads, and an acceptable level of apomixis; d. Further
selecting said progeny plants to identify plants with one or more
characteristics different from the maternal parental line of said
bluegrass genotype and having desirable looking turf when mowed
infrequently; and e. Harvesting seed or vegetative propagating
material from said selected progeny plants.
5. A bluegrass plant produced by the method of claim 4.
6. A bluegrass plant having desirable looking turf when mowed
infrequently, wherein said bluegrass plant has a green leaf
chlorophyll concentration above 1.8 mg/g.
7. The bluegrass plant of claim 6, wherein said bluegrass plant has
a field CM-1000 chlorophyll meter reading of between 341.7 and
620.0.
8. A grass seed produced by the plant of claim 6, wherein
representative samples of seed of said grass were deposited under
NCIMB Nos. 42110, 42111, and 42112.
9. Sod, comprising the grass plant of claim 6.
10. The grass plant of claim 6, wherein said grass is planted in a
lawn.
11. A vegetative sprig or clone of the grass plant of claim 6.
12. A method for producing a bluegrass seed, comprising crossing
two bluegrass plants and harvesting the resultant bluegrass seed,
wherein at least one bluegrass plant is the bluegrass plant of
claim 6.
13. A bluegrass seed produced by the method of claim 12.
14. A bluegrass plant, or a part thereof, produced by growing said
seed of claim 13.
15. The method of claim 12, wherein at least one of said bluegrass
plants is transgenic.
16. A method of producing an herbicide resistant bluegrass plant,
wherein said method comprises introducing a gene conferring
herbicide resistance into the plant of claim 6.
17. A herbicide resistant bluegrass plant produced by the method of
claim 16, wherein the gene confers resistance to a herbicide
selected from the group consisting of glyphosate, sulfonylurea,
imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,
L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, and
benzonitrile.
18. A method of producing a pest or insect resistant bluegrass
plant, wherein said method comprises introducing a gene conferring
pest or insect resistance into the bluegrass plant of claim 6.
19. A pest or insect resistant bluegrass plant produced by the
method of claim 18.
20. The bluegrass plant of claim 19, wherein the gene encodes a
Bacillus thuringiensis (Bt) endotoxin.
21. A method of producing a disease resistant bluegrass plant,
wherein said method comprises introducing a gene which confers
disease resistance into the bluegrass plant of claim 6.
22. A disease resistant bluegrass plant produced by the method of
claim 21.
23. A method of introducing a desired trait into the plant of claim
6, wherein the method comprises: a. crossing the plant of claim 6
with a plant selected from the group consisting of another
bluegrass variety, a species of Poa, and another plant genus that
comprises a desired trait to produce progeny plants; b. selecting
one or more progeny plants that have the desired trait to produce
selected progeny plants; c. backcrossing the selected progeny
plants with a plant of claim 6 to produce backcross progeny plants;
d. selecting for backcross progeny plants that have the desired
trait; and e. repeating steps c. and d. two or more times in
succession to produce selected third or higher backcross progeny
plants that comprise the desired trait.
24. The method of claim 23, wherein the desired trait is a
phenotypic trait, a gene, or a molecular marker.
25. A bluegrass plant produced by the method of claim 24, wherein
the plant has the desired trait and otherwise all the physiological
and morphological characteristics of the bluegrass plant of claim
6.
26. A method of producing a commodity plant product, said method
comprising obtaining the plant of claim 6, or a part thereof, and
producing the commodity plant product from said plant or plant part
thereof, wherein said commodity plant product is selected from the
group consisting of protein concentrate, protein isolate and
oil.
27. A bluegrass variety having desirable looking turf when mowed
infrequently selected from the group consisting of bluegrass
varieties `03-0582`, `03-0441` and `99-2495`, wherein
representative samples of seed of said varieties were deposited
under NCIMB Nos. 42111, 42110, and 42112, respectively.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is the U.S. National Phase under 35 U.S.C.
371 of International Application PCT/US2014/024563, filed Mar. 12,
2014, published in English, which claims the benefit of priority
from U.S. provisional patent application Ser. No. 61/782,611, filed
on Mar. 14, 2013. The disclosure of each of the above-listed
priority applications is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to turfgrass varieties having
desirable looking turf when mowed infrequently. All publications
cited in this application are herein incorporated by reference.
[0003] Turfgrass plays a major role in our daily life. Turfgrass,
from a beautification standpoint, provides a canvas for landscaped
areas contributing to aesthetic appeal and adding to economic
value. Recreational facilities include an array of sports fields,
golf courses, parks and lawns. Turfgrass also provides functional
value including dust control, erosion control, and glare
reduction.
[0004] Use and appearance are prime considerations for turfgrass.
To best serve a particular function, the turf should be suitable
for the use for which it is intended and aesthetically appealing.
It should also be well-adapted to the environment where it will be
planted. Based on climatic adaptation, turfgrass species have been
placed into four categories: adapted for cool humid regions, warm
humid regions, cool arid regions, and warm arid regions. The major
turfgrasses adapted to the cool humid regions, and irrigated areas
of the cool arid regions, are species of Agrostis, Poa, Festuca,
and Lolium. In the warm humid and irrigated areas of the warm arid
regions, the major adapted turfgrasses are species of Cynodon,
Zoysia, Stenotaphrum, Eremochloa, Paspalum, Festuca, and Agropyron.
In the non-irrigated warm arid regions, species of Buchloe and
Bouteloua are adapted.
[0005] Kentucky bluegrass (Poa pratensis), also called smooth
meadow grass, spear grass and June grass, is a perennial species of
grass native to Europe, northern Asia and the mountains of Algeria
and Morocco. Although the species is spread over all of the cool,
humid parts of the United States, Kentucky bluegrass is native only
to portions of North America. Kentucky bluegrass forms a valuable
pasture plant, characteristic of well-drained, fertile soil, and is
a popular sod-forming grass that is used on golf courses, ski
slopes, campsites, gardens and lawns. Kentucky bluegrass is also an
important forage species for sheep and cattle. The name Kentucky
bluegrass derives from its flower heads, which are blue when the
plant is allowed to grow to its natural height of two to three
feet.
[0006] Over 100 varieties of Kentucky bluegrass have been developed
during the past 25 years. Some varieties tolerate southern climates
better than others, some have moderate shade tolerance, and some
tolerate closer mowing. Many of these grasses also differ in their
degree of susceptibility to diseases. Kentucky bluegrass is
distinguished from Canada bluegrass (Poa compressus) by its darker
green foliage, longer leaves, and pubescence at the bases of the
leaves. Kentucky bluegrass can also be compared to Annual
Meadowgrass (Poa annua) and Rough Meadowgrass (Poa trivialis),
which have a ligule that is silvery and pointed, whereas Kentucky
bluegrass has a ligule that is extremely short and square
ended.
[0007] Kentucky bluegrass is often included in seed mixes that are
used to revegetate roadbanks Kentucky bluegrass is a slow-growing
plant that establishes in 2 to 3 years and forms a dense sod.
Kentucky bluegrass grows best on well-drained loams or clay loams
rich in humus and on soils with limestone parent material. Kentucky
bluegrass needs large amounts of nitrogen during active growth
stages and has an optimal soil pH of between 5.8 and 8.2.
Additionally, Kentucky bluegrass is intolerant of drought,
excessive flooding high water tables, and poorly drained soils, and
is sometimes vulnerable to fungal infections including Fusarium,
Helminthosporium, leaf spot, rust and powdery mildew.
[0008] Kentucky bluegrass typically grows 18 to 24 inches tall and
is readily identified by its boat-shaped leaf tip. Kentucky
bluegrass spreads by rhizomes and tillers and forms a dense sod.
New shoots (rhizomes and tillers) are produced primarily in the
spring and late summer. Most shoots produced in the spring remain
vegetative, while shoots produced in late summer often terminate in
an inflorescence the following spring. The lifetime of a Kentucky
bluegrass shoot that terminates in an inflorescence ends soon after
the seeds mature.
[0009] Because use and appearance are prime considerations for
turfgrass, it is desirable to have turfgrass varieties that can be
less frequently mowed (defoliated) and still produce an attractive,
green lawn turf.
[0010] The foregoing examples of the related art and limitations
related therewith are intended to be illustrative and not
exclusive. Other limitations of the related art will become
apparent to those of skill in the art upon a reading of the
specification.
SUMMARY OF THE INVENTION
[0011] The following embodiments and aspects thereof are described
in conjunction with systems, tools and methods which are meant to
be exemplary, not limiting in scope. In various embodiments, one or
more of the above-described problems have been reduced or
eliminated, while other embodiments are directed to other
improvements.
[0012] In one aspect of the present invention there is provided a
method of producing a turfgrass plant having desirable looking turf
when mowed infrequently.
[0013] In one aspect of the invention there are provided turfgrass
varieties that produce a desirable looking turf when mowed
infrequently, such as mowed once every 4 weeks or more.
[0014] In one aspect of the present invention there are provided
turfgrass varieties that have chlorophyll concentrations above 1.8
mg/g.
[0015] In one aspect of the present invention there are provided
turfgrass varieties that have a CM-1000.TM. field chlorophyll
reading of 341.7 or higher resulting in a darker green color and
less brown when mowed.
[0016] In another aspect of the present invention there are
provided turfgrass varieties that have low-growing
characteristics.
[0017] In another aspect of the present invention there are
provided turfgrass varieties that have high general turfgrass
quality ratings.
[0018] In a further aspect of the present invention there are
provided Kentucky bluegrass plants of varieties `03-0582`,
`03-0441`, and `99-2495`. Also provided are seeds and progeny of
these varieties.
[0019] According to the invention, there are provided novel
turfgrass varieties having desirable looking turf when mowed
infrequently, including Kentucky bluegrass varieties designated
`03-0582`, `03-0441`, and `99-2495`. This invention thus relates to
the seeds of novel turfgrass varieties, such as `03-0582`,
`03-0441`, and `99-2495`, to the plants or part(s) thereof of novel
turfgrass varieties, such as Kentucky bluegrass varieties
`03-0582`, `03-0441`, and `99-2495`, to plants or part(s) thereof
having all the phenotypic and morphological characteristics of
novel turfgrass varieties, such as Kentucky bluegrass varieties
`03-0582`, `03-0441`, and `99-2495`. Plant parts of the Kentucky
bluegrass varieties of the present invention are also provided.
[0020] In another aspect, the present invention provides
regenerable cells for use in tissue culture of turfgrass varieties
having desirable looking turf when mowed infrequently, including
Kentucky bluegrass varieties `03-0582`, `03-0441`, and `99-2495`.
The tissue culture will preferably be capable of regenerating
plants having the physiological and morphological characteristics
of the turfgrass of the present invention, such as Kentucky
bluegrass varieties `03-0582`, `03-0441`, and `99-2495`.
Preferably, the cells of such tissue culture will be embryos,
meristematic cells, seeds, callus, pollen, leaves, anthers,
pistils, roots, root tips, pods, flowers and stems. Protoplasts
produced from such tissue culture are also included in the present
invention. The turfgrass plants regenerated from the tissue culture
are also part of the invention.
[0021] Also included in the invention are methods for producing a
turfgrass plant produced by crossing turfgrass varieties having
desirable looking turf when mowed infrequently, such as Kentucky
bluegrass variety `03-0582`, `03-0441`, or `99-2495` with itself or
another turfgrass or Kentucky bluegrass variety. When crossed with
itself, i.e., when crossed with another Kentucky bluegrass variety
`03-0582`, `03-0441`, or `99-2495` plant or self-pollinated,
Kentucky bluegrass variety `03-0582`, `03-0441`, or `99-2495` will
be conserved. When crossed with another, different bluegrass plant,
an F.sub.1 hybrid seed is produced. F.sub.1 hybrid seeds and plants
produced by growing said hybrid seeds are included in the present
invention. A method for producing an F.sub.1 hybrid seed comprising
crossing a turfgrass plant of the present invention having
desirable looking turf when mowed infrequently, such as Kentucky
bluegrass variety `03-0582`, `03-0441`, or `99-2495` plant with a
different bluegrass plant and harvesting the resultant hybrid
bluegrass seed are also part of the invention. The hybrid bluegrass
seed produced by the method comprising crossing a turfgrass plant
having desirable looking turf when mowed infrequently, such as
Kentucky bluegrass variety `03-0582`, `03-0441`, or `99-2495` plant
with a different bluegrass plant and harvesting the resultant
hybrid bluegrass seed, are included in the invention, as are the
hybrid bluegrass plant or part(s) thereof, and seeds produced by
growing said hybrid bluegrass seed.
[0022] In another aspect, the present invention provides
transformed turfgrass varieties having desirable looking turf when
mowed infrequently, including `03-0582`, `03-0441`, or `99-2495`
Kentucky bluegrass variety plants or part(s) thereof that have been
transformed so that its genetic material contains one or more
transgenes, preferably operably linked to one or more regulatory
elements. Also, the invention provides methods for producing a
turfgrass plant having desirable looking turf when mowed
infrequently containing in its genetic material one or more
transgenes, preferably operably linked to one or more regulatory
elements, by crossing transformed turfgrass plants, such as
`03-0582`, `03-0441`, or `99-2495` Kentucky bluegrass variety
plants with either a second plant of another turfgrass or bluegrass
variety, or a non-transformed `03-0582`, `03-0441`, or `99-2495`
Kentucky bluegrass variety, so that the genetic material of the
progeny that results from the cross contains the transgene(s),
preferably operably linked to one or more regulatory elements. The
invention also provides methods for producing a turfgrass plant
having desirable looking turf when mowed infrequently that contains
in its genetic material one or more transgene(s), wherein the
method comprises crossing the turfgrass variety, such as `03-0582`,
`03-0441`, or `99-2495` with a second turfgrass or bluegrass
variety of another bluegrass variety which contains one or more
transgene(s) operably linked to one or more regulatory element(s)
so that the genetic material of the progeny that results from the
cross contains the transgene(s) operably linked to one or more
regulatory element(s). Transgenic turfgrass cultivars, or part(s)
thereof produced by the methods are in the scope of the present
invention.
[0023] More specifically, the invention comprises methods for
producing a male sterile turfgrass plant, an herbicide resistant
turfgrass plant, an insect resistant turfgrass plant, a disease
resistant turfgrass plant, a water stress tolerant turfgrass plant,
a heat stress tolerant turfgrass plant, and a turfgrass plant with
improved shelf-life. Said methods comprise transforming a turfgrass
variety of the present invention, such as `03-0582`, `03-0441`, or
`99-2495` plant with a nucleic acid molecule that confers male
sterility, herbicide resistance, insect resistance, disease
resistance, water stress tolerance, heat stress tolerance, or
improved shelf life, respectively. The transformed turfgrass
plants, or part(s) thereof, obtained from the provided methods,
including a male sterile turfgrass plant, an herbicide resistant
turfgrass plant, an insect resistant turfgrass plant, a disease
resistant turfgrass plant, a turfgrass plant tolerant to water
stress, a turfgrass plant tolerant to heat stress or a turfgrass
plant with improved shelf-life are included in the present
invention. For the present invention and the skilled artisan,
disease is understood to be fungal diseases, viral diseases,
bacterial diseases or other plant pathogenic diseases and a disease
resistant plant will encompass a plant resistant to fungal, viral,
bacterial and other plant pathogens.
[0024] In another aspect, the present invention provides for
methods of introducing one or more desired trait(s) into turfgrass
varieties having desirable looking turf when mowed infrequently,
such as bluegrass varieties `03-0582`, `03-0441`, or `99-2495` and
plants obtained from such methods. The desired trait(s) may be, but
not exclusively, a single gene, preferably a dominant but also a
recessive allele. Preferably, the transferred gene or genes will
confer such traits as male sterility, herbicide resistance, insect
resistance, resistance to bacterial, fungal, or viral disease,
increased leaf number, improved shelf-life, and tolerance to water
stress or heat stress. The gene or genes may be naturally occurring
gene(s) or transgene(s) introduced through genetic engineering
techniques. The method for introducing the desired trait(s) is
preferably a backcrossing process making use of a series of
backcrosses to turfgrass plants having desirable looking turf when
mowed infrequently, such as bluegrass variety `03-0582`, `03-0441`,
or `99-2495` during which the desired trait(s) is maintained by
selection.
[0025] In a preferred embodiment, the present invention provides
methods for increasing and producing turfgrass varieties having
desirable looking turf when mowed infrequently, such as bluegrass
varieties `03-0582`, `03-0441`, or `99-2495` seed, whether by
crossing a first parent bluegrass variety plant with a second
parent bluegrass variety plant and harvesting the resultant
bluegrass seed, wherein both said first and second parent bluegrass
variety plant are the bluegrass variety `03-0582`, `03-0441`, or
`99-2495` or by planting a bluegrass seed of the bluegrass variety
`03-0582`, `03-0441`, or `99-2495`, growing a bluegrass variety
`03-0582`, `03-0441`, or `99-2495` plant from said seed,
controlling a self pollination of the plant where the pollen
produced by a grown bluegrass variety `03-0582`, `03-0441`, or
`99-2495` plant pollinates the ovules produced by the very same
bluegrass variety `03-0582`, `03-0441`, or `99-2495` grown plant,
and harvesting the resultant seed.
[0026] The invention further provides methods for developing
turfgrass and Kentucky bluegrass cultivars having desirable looking
turf when mowed infrequently in a turfgrass breeding program using
plant breeding techniques including recurrent selection,
backcrossing, pedigree breeding, molecular markers (Isozyme
Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),
Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed
Polymerase Chain Reaction (AP-PCR), DNA Amplification
Fingerprinting (DAF), Sequence Characterized Amplified Regions
(SCARs). Amplified Fragment Length Polymorphisms (AFLPs), and
Simple Sequence Repeats (SSRs) which are also referred to as
Microsatellites, etc.) enhanced selection, genetic marker enhanced
selection, and transformation. Seeds, turfgrass plants, and part(s)
thereof produced by such breeding methods are also part of the
invention.
[0027] In addition to the exemplary aspects and embodiments
described above, further aspects and embodiments will become
apparent by study of the following descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows the results of regression analysis that was
performed to correlate visual turfgrass quality ratings with Field
Scout CM 1000.TM. scan results for test plots mowed three times per
year in Idaho. Regression analysis indicated that a visual quality
estimate of 5 corresponds to a CM 1000.TM. meter reading of 341.7,
with an unexpectedly high correlation co-efficient of 0.86, and an
R.sup.2 value of 0.73, which was significant at the 0.0001 level.
In FIG. 1, circles represent individual observations, a solid line
indicates regression fit to the data, and dashed lines indicate 95%
confidence intervals.
DETAILED DESCRIPTION OF THE INVENTION
[0029] In the description and examples that follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided. If
no definition is provided, all other technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the invention belongs.
[0030] Acceptable level of apomixes. Refers to 80% or higher level
of apomixes.
[0031] Adequate production of seed heads. Refers to 15 grams or
more of clean seed recovered from one individual spaced plant.
[0032] Allele. An allele is any of one or more alternative forms of
a gene which relate to one trait or characteristic. In a diploid
cell or organism, the two alleles of a given gene occupy
corresponding loci on a pair of homologous chromosomes.
[0033] Alter. Alter refers to the utilization of up-regulation,
down-regulation, or gene silencing.
[0034] Apomictic. As used herein, "apomictic" describes a plant
that reproduces using apomixis
[0035] Apomixis. Asexual reproduction in organisms that are also
able to reproduce sexually, in which embryos are formed without
fertilization or the creation of specialized reproductive
cells.
[0036] Backcrossing. Backcrossing is a process in which a breeder
repeatedly crosses hybrid progeny back to one of the parents, for
example, a first generation hybrid F.sub.1 with one of the parental
genotypes of the F.sub.1 hybrid.
[0037] Cell. Cell as used herein includes a plant cell, whether
isolated, in tissue culture or incorporated in a plant or plant
part.
[0038] Characteristics different from the maternal parental line.
As used herein, refers to characteristics that are different from
the maternal parental line, including but not limited to color and
width of the leaves prior to seedhead expression and a different
date when the seedhead emerges from the sheath of the
turfgrass.
[0039] Chlorophyll concentration. The milligrams per gram of
chlorophyll contained in plant tissue weight. Also referred to as
"chlorophyll content".
[0040] Chlorophyll meter reading. The digital readout of the CM
1000.TM. chlorophyll field scanner.
[0041] Cisgenesis. The genetic modification of a recipient plant
with a natural gene from a sexually compatible plant. Such a gene
includes its introns and is flanked by it native promoter and
terminator in the normal sense orientation. Cisgenic plants can
harbor one or more cisgenes, but they do not contain any
trangenes.
[0042] Cisgenic plant. A plant that contains no foreign genes.
[0043] Commercial perennial bluegrass. A commercial perennial
bluegrass is one which has been sold commercially.
[0044] Cotyledon. A cotyledon is a seed leaf.
[0045] Crossbreeding. As used herein, "crossbreeding" refers to the
act of mating (crossing) individuals of different species or
varieties of plants to produce hybrids.
[0046] Crown. The crown in grass is the area at which top growth
and root growth originate.
[0047] Culm. The culm is the main aerial shoot to which leaves and
inflorescences are attached. The culm is a rounded or slightly
flattened stem with one or more solid joints known as nodes. The
leaves are attached at the nodes and if the stem is not simple but
branched, branches arise only at nodes. Roots may also develop from
a node where the node comes into contact with the ground (as in
decumbent and prostrate stems).
[0048] Desirable looking turf As used herein, "desirable looking
turf" is quantified by an increased chlorophyll concentration of
greater than 1.8 mg/g indicating the turf retention of green color
even when scalped back (severely defoliated) by infrequent mowing.
The chlorophyll concentration is additionally quantified by a
CM-1000.TM. field chlorophyll reading of 341.7 or higher. "Turf"
refers to any type of turfgrass, including bluegrass.
[0049] Embryo. The embryo is the small plant contained within a
mature seed.
[0050] Endophyte. The term endophyte is applied to fungi which live
within plant tissues for all or part of their lifecycle and cause
no apparent infections.
[0051] Famigenic plant. A transformed plant developed by
transferring at least some DNA from one plant to a sexually
incompatible plant that belongs to the same family.
[0052] Field hybridization nursery. A spaced plant field breeding
nursery having access to plants that grew from one seed, wherein a
mother plant is surrounded by clones of itself (selfing) or a
mother plant is surrounded by clones that are different than the
mother plant (intraspecific hybridization).
[0053] Field Scout CM 1000.TM. chlorophyll meter. A
"point-and-shoot" device to instantly measure relative chlorophyll
content. The CM 1000.TM. senses light at wavelengths of 700 nm and
840 nm to estimate the quantity of chlorophyll in leaves. The
ambient and reflected light at each wavelength is measured. Made by
Spectrum Technologies, Inc., Plainfield, Ill.
[0054] Gene. As used herein, "gene" refers to a DNA segment that
contributes to phenotype/function, including associated regulatory
elements such as promoters. A gene can be introduced into a genome
of a species, whether from a different species or from the same
species, using transformation or various breeding methods.
[0055] Gene silencing. The interruption or suppression of the
expression of a gene at the level of transcription or
translation.
[0056] Genotype. Refers to the genetic constitution of a cell or
organism.
[0057] Grass flower or inflorescence. Flowers of grasses are borne
in an inflorescence or flower head which terminates the culm and
other branches of the stem. Smaller units of the inflorescence are
called spikelets and these are arranged on one or more branches in
a wide variety of different ways to which the standard terminology
for inflorescences can be applied, but using the spikelet instead
of the individual flower.
[0058] Growing season. As used herein, "growing season" refers to
the time of year during which turfgrass is actively growing, which
is typically spring through fall in the U.S. and Europe.
[0059] Hybrid. Heterozygous offspring of two parents that differ in
one or more inheritable characteristics.
[0060] Hypocotyl. A hypocotyl is the portion of an embryo or
seedling between the cotyledons and the root. Therefore, it can be
considered a transition zone between shoot and root.
[0061] Intragenic plant. A transformed plant that only contains
genetic elements derived from within the sexual compatibility
group.
[0062] Intergenic DNA. Any of the DNA in between gene-coding DNA,
including untranslated regions, 5' and 3' flanking regions,
introns, non-functional pseudogenes, and non-functional repetitive
sequences. This DNA may or may not encode regulatory functions.
[0063] Internode. The internodes act as spacers that distance one
node from another.
[0064] Intercalary meristem. Intercalary meristem is a meristem at
the base of the internode in monocot stems (particularly grass
stems).
[0065] Lawn. A plot of grass, usually tended or mowed, such as one
near a house, on an estate, in a yard, garden or park, or a golf
course, or any other such area covered with grass.
[0066] Linkage. Refers to a phenomenon wherein alleles on the same
chromosome tend to segregate together more often than expected by
chance if their transmission was independent.
[0067] Linkage disequilibrium. Refers to a phenomenon wherein
alleles tend to remain together in linkage groups when segregating
from parents to offspring, with a greater frequency than expected
from their individual frequencies.
[0068] Mature sod. Means sod that is 8 to 14 months old after
seeding, wherein sod is mature at 8 to 11 months old after a fall
seeding and sod is mature at 12 to 14 months old after a spring
seeding.
[0069] Mowed infrequently. As used herein, "mowed infrequently" or
"infrequently mowed" refers to grass that has been subjected to
mowing with a conventional lawnmower once every four weeks or
longer, such as once every 30, 32, 34, 36, 38, 39, 40 or more days,
or any integer or fraction thereof.
[0070] Node. A node in a grass stem is a solid point at which the
intercalary meristem is located. The node also contains the bud
that is capable of producing a new shoot. The terminal node
contains the bud that produces the inflorescence.
[0071] Pedigree distance. Pedigree distance refers to the
relationship among generations based on their ancestral links as
evidenced in pedigrees. Pedigree distance may be measured by the
distance of the pedigree from a given starting point in the
ancestry.
[0072] Percent identity. Percent identity as used herein refers to
the comparison of the homozygous alleles of two perennial bluegrass
varieties. Percent identity is determined by comparing a
statistically significant number of the homozygous alleles of two
developed varieties. For example, a percent identity of 90% between
perennial bluegrass variety 1 and perennial bluegrass variety 2
means that the two varieties have the same allele at 90% of their
loci.
[0073] Percent similarity. Percent similarity as used herein refers
to the comparison of the homozygous alleles of one perennial
bluegrass variety with another bluegrass plant, and if the
homozygous allele of the first bluegrass matches at least one of
the alleles from the other plant then they are scored as similar.
Percent similarity is determined by comparing a statistically
significant number of loci and recording the number of loci with
similar alleles as a percentage. A percent similarity of 90%
between the first bluegrass and another plant means that the first
bluegrass matches at least one of the alleles of the other plant at
90% of the loci.
[0074] Plant. As used herein, the term "plant" includes reference
to an immature or mature whole plant, including a plant from which
seed, roots or leaves have been removed. Seed or embryo that will
produce the plant is also considered to be the plant.
[0075] Plant height. The length of the grass leaf blade measured
from the rhizome to the tip of the blade.
[0076] Plant parts. As used herein, the term "plant parts" (or a
perennial bluegrass plant, or a part thereof) includes protoplasts,
leaves, stems, roots, root tips, anthers, pistils, seed, embryo,
pollen, ovules, cotyledon, hypocotyl, flower, shoot, tissue,
petiole, cells, meristematic cells and the like.
[0077] Polynucleotide. A polymeric compound, usually DNA or RNA,
consisting of a number of nucleotides.
[0078] Primary tillers. Primary tillers are shoots arising at the
crown.
[0079] Progeny. As used herein, includes an F.sub.1 turfgrass plant
produced from the cross of two turfgrass plants where at least one
plant includes a turfgrass plant of the present invention and
progeny further includes, but is not limited to, subsequent
F.sub.2, F.sub.3, F.sub.4, F.sub.5, F.sub.6, F.sub.7, F.sub.8,
F.sub.9, and F.sub.10 generational crosses with the recurrent
parental line. As used herein, progeny also refers to plants
produced by selfing a turfgrass plant produced by the present
invention.
[0080] Promoter. A segment of DNA usually occurring upstream from a
gene coding region and acting as a controlling element in the
expression of that gene.
[0081] Quantitative Trait Loci (QTL). Quantitative trait loci (QTL)
refer to genetic loci that control to some degree numerically
representable traits that are usually continuously distributed.
[0082] Regeneration. Regeneration refers to the development of a
plant from tissue culture.
[0083] Rhizome. A rhizome is a modified stem that grows
underground. Rhizomes are jointed (thus distinguishable from roots)
with bladeless leaves (scales) arising from the joints Rhizomes
enable a grass plant to spread horizontally as new culms develop
vertically from the joints. Thus, grasses with extensive rhizome
development will form a turf rather than distinct tufts or
bunches.
[0084] Scalped back (severely defoliated). Refers to mowing of
turfgrass wherein greater than one third of the leaf area is
removed.
[0085] Secondary tillers. Secondary tillers are tillers arising as
branches of the primary tillers.
[0086] Seedhead. The flowering (reproductive) part of the grass
plant.
[0087] Seedhead expression. Refers to the emergence, full
expression and maturation of the seedhead.
[0088] Selfing. Pollinating a plant and hybridizing it with its own
pollen or pollen from a clone of that plant.
[0089] Short, basal growth of leaves. Refers to a preponderance of
green leaf material in the 0 to 4 inch zone above the soil
surface.
[0090] Single Gene Converted (Conversion). Single gene converted
(conversion) plants refers to plants which are developed by a plant
breeding technique called backcrossing wherein essentially all of
the desired morphological and physiological characteristics of a
variety are recovered in addition to the single gene transferred
into the variety via the backcrossing technique or via genetic
engineering.
[0091] Sod. A section of grass-covered surface soil held together
by matted roots; turf
[0092] Stolon. A stolon is a stem that creeps across the surface of
the ground, and is really a basal branch of the culm that will
develop roots and shoots from some or all of its nodes. Like a
rhizome, a stolon results in a spreading or turf forming grass
plant.
[0093] Tensile strength. Means the amount of force in pounds
required to tear a piece of sod in two. Tensile strength is
determined with a mechanical sod stretcher coupled to a device to
measure force in pounds. Tensile strength, tear point and sod
strength are used interchangeably.
[0094] Tiller. A tiller is another name for a grass stem.
[0095] Tiller length. Tiller length is measured in centimeters from
the lowest node to the last node subtending the green foliage.
[0096] Transgene. A gene that is transferred from an organism of
one species to an organism of another species by genetic
engineering.
[0097] Variant. As used herein, refers to offspring that occur in a
naturally apomictic species by means of hybridization of pollen and
ovule, resulting in offspring with some but not all characteristics
of the maternal parent plant.
[0098] Variety. A taxonomic subdivision of a species consisting of
naturally occurring or selectively bred populations or individuals
that differ from the remainder of the species in certain minor
characteristics.
[0099] Vernalization. Vernalization induces plants to begin the
reproductive cycle after exposure to cold temperatures and short
day length. The amount of cold exposure and short day lengths
required varies with the species.
[0100] Xenogenic modification. Introduction of powerful new traits
that might outperform native traits by transforming plants with
synthetic genes.
[0101] The following detailed description is of the currently
contemplated modes of carrying out the invention. The description
is not to be taken in a limiting sense, but is made merely for the
purpose of illustrating the general principles of the invention,
since the scope of the invention is best defined by the appended
claims.
[0102] Broadly, the present invention is directed toward turfgrass
varieties, including both plants and seeds, having desirable
looking turf when mowed infrequently. The turfgrass varieties of
the present invention were developed to retain a dark green color
even when scalped back by infrequent mowing. Part of this tolerance
traces to the method of breeding, which stresses low growing
plants, with foliage tight to the ground. However, there are also
other traits that are less quantitative that convey infrequent
mowing tolerance, such as the physiological ability to withstand
defoliation and recover without discoloration or objectionable
stunting. In essence, these plants do not just grow vertically
slower than other varieties, they also possess growth
characteristics that allow them to endure on a infrequently mowed
lawn while maintaining a desirable looking turf.
[0103] In the field hybridization nursery, individual spaced plants
are identified with promising characteristics such as the short
growth of leaves (defined as a preponderance of green leaf material
in the 0 to 4 inch zone above the soil surface), adequate seed
heads (defined as 15 g or more of clean seed recovered from one
individual spaced plant), and acceptable apomixes (defined as 80%
or higher). These promising plants are individually hand harvested
and cleaned of chaff and seed are planted in replicated turf
trials, subject to mowing once every 4 weeks or longer. Each plot
in the experiment is evaluated monthly during the growing season,
using a visual rating scale of 1 to 9, where 9 is highly desirable
turf and 5 is minimal acceptable quality, and 1 is totally brown or
dead. During one or more evaluation dates, the chlorophyll meter is
used to evaluate plots to impartially differentiate green tissue
from lifeless brown tissue created from the scalping process.
[0104] Most turfgrass managers practice the "one third rule" for
clipping lawns and golf courses. The "one third rule" states that a
maximum of one third of the leaf area should be removed during any
one mowing. Deviating from this "one third rule" could lead to an
unacceptable quantity of the plant's photosynthetic surface being
removed. As a result, the plant goes into a shock mode and
sacrifices leaves in exchange for survival. Plant varieties that
grow vertically very tall or extremely fast are more subject to
damage from defoliation. The visual result is that the plant
rapidly turns brown within 24 hours and may take days or weeks to
recover. An additional result of this shock is the loss of shoot
density of the plant, defined as the number of live vegetative
shoots per square centimeter of ground surface. The present
invention described herein deals with plants that do not exhibit
this browning shock when they are subject to infrequent mowing.
[0105] The present invention provides a method of producing a
turfgrass plant having desirable-looking turf when mowed
infrequently, said method comprises selfing (pollinating a plant
and hybridizing it with its own pollen) for one or more generations
to create a plant with short growth of leaves (defined as a
preponderance of green leaf material in the 0 to 4 inch zone above
the soil surface), adequate seed heads (defined as 15 g or more of
clean seed recovered from one individual spaced plant), and
acceptable apomixes (defined as 80% or higher). The hybridization
can take place in a confined greenhouse using pollen restricting
bags, or take place in the field surrounded by clones of the same
genotype, or any combination of these two methods. The progeny of
said hybridizations are then field screened for approximately 15
months to identify plants with characteristics different than the
maternal parent line; such differences are most commonly identified
during the vegetative or pre-seedhead phase, but also may be
identified during seedhead expression. The method of the present
invention unexpectedly produced turfgrass plants having very basal
growth habit, with leaves growing close to the ground rather than
growing tall, which have desirable-looking turf when mowed
infrequently.
[0106] It should be understood, of course, that the foregoing
relates to exemplary embodiments of the invention and that
modifications may be made without departing from the spirit and
scope of the invention as set forth in the following claims.
FURTHER EMBODIMENTS OF THE INVENTION
[0107] Choice of breeding or selection methods depends on the mode
of plant reproduction, the heritability of the trait(s) being
improved, and the type of cultivar used commercially (e.g., F.sub.1
hybrid cultivar, pureline cultivar, etc.). Popular selection
methods commonly include population formation by hybridization,
genomic selection, marker assisted selection, recurrent selection,
mutation breeding, single-seed descent, bulk selection, pedigree
selection, modified pedigree selection, and mass selection.
Breeding Methods
[0108] The following describes breeding methods that may be used
with the turfgrass varieties of the present invention having
desirable looking turf when mowed infrequently, such as bluegrass
varieties `03-0582`, `03-0441`, or `99-2495` in the development of
further bluegrass plants. One such embodiment is a method for
developing a cultivar `03-0582`, `03-0441`, or `99-2495` progeny
bluegrass plant in a bluegrass plant breeding program comprising:
obtaining the bluegrass plant, or a part thereof, of cultivar
`03-0582`, `03-0441`, or `99-2495` utilizing said plant or plant
part as a source of breeding material and selecting a bluegrass
cultivar `03-0582`, `03-0441`, or `99-2495` progeny plant with
molecular markers in common with variety `03-0582`, `03-0441`, or
`99-2495` and/or with morphological and/or physiological
characteristics described herein.
[0109] Another method involves producing a population of turfgrass
varieties of the present invention having desirable looking turf
when mowed infrequently, such as bluegrass varieties `03-0582`,
`03-0441`, or `99-2495` progeny bluegrass plants, comprising
crossing cultivar `03-0582`, `03-0441`, or `99-2495` with another
bluegrass plant, thereby producing a population of bluegrass
plants, which, on average, derive 50% of their alleles from
bluegrass variety `03-0582`, `03-0441`, or `99-2495`. A plant of
this population may be selected and repeatedly selfed or sibbed
with a bluegrass cultivar resulting from these successive filial
generations. In some embodiments, the bluegrass cultivar produced
by this method and that has obtained at least 50% of its alleles
from bluegrass variety `03-0582`, `03-0441`, or `99-2495`.
[0110] One of ordinary skill in the art of plant breeding would
know how to evaluate the traits of two plant varieties to determine
if there is no significant difference between the two traits
expressed by those varieties. For example, see Fehr and Walt,
Principles of Cultivar Development, p 261-286 (1987). Thus the
methods and variety described herein includes turfgrass plants
having desirable looking turf when mowed infrequently, such as
bluegrass cultivar `03-0582`, `03-0441`, or `99-2495` progeny
bluegrass plants comprising a combination of at least two cultivar
`03-0582`, `03-0441`, or `99-2495` traits or the cultivar
`03-0582`, `03-0441`, or `99-2495` combination of traits listed in
the Examples, so that said progeny bluegrass plant is not
significantly different for said traits than bluegrass variety
`03-0582`, `03-0441`, or `99-2495` as determined at the 5%
significance level when grown in the same environmental conditions.
Using techniques described herein, molecular markers may be used to
identify said progeny plant as a bluegrass variety `03-0582`,
`03-0441`, or `99-2495` progeny plant. Mean trait values may be
used to determine whether trait differences are significant, and
preferably the traits are measured on plants grown under the same
environmental conditions. Once such a variety is developed its
value is substantial since it is important to advance the germplasm
base as a whole in order to maintain or improve traits such as
yield, disease resistance, pest resistance, and plant performance
in extreme environmental conditions.
[0111] Progeny of turfgrass plants of the present invention having
desirable looking turf when mowed infrequently, such as bluegrass
variety `03-0582`, `03-0441`, or `99-2495` may also be
characterized through their filial relationship with bluegrass
variety `03-0582`, `03-0441`, or `99-2495`, as for example, being
within a certain number of breeding crosses of bluegrass variety
`03-0582`, `03-0441`, or `99-2495`. A breeding cross is a cross
made to introduce new genetics into the progeny, and is
distinguished from a cross, such as a self or a sib cross, made to
select among existing genetic alleles. The lower the number of
breeding crosses in the pedigree, the closer the relationship
between bluegrass variety `03-0582`, `03-0441`, or `99-2495` and
its progeny. For example, progeny produced by the methods described
herein may be within 1, 2, 3, 4 or 5 breeding crosses of bluegrass
variety `03-0582`, `03-0441`, or `99-2495`.
[0112] Recurrent selection is a method used in a plant breeding
program to improve a population of plants. Turfgrass having
desirable looking turf when mowed infrequently, such as `03-0582`,
`03-0441`, or `99-2495` is suitable for use in a recurrent
selection program. The method entails individual plants cross
pollinating with each other to form progeny. The progeny are grown
and the superior progeny selected by any number of selection
methods, which include individual plant, half-sib progeny, full-sib
progeny and selfed progeny. The selected progeny are cross
pollinated with each other to form progeny for another population.
This population is planted and again superior plants are selected
to cross pollinate with each other. Recurrent selection is a
cyclical process and therefore can be repeated as many times as
desired. The objective of recurrent selection is to improve the
traits of a population. The improved population can then be used as
a source of breeding material to obtain new varieties for
commercial or breeding use, including the production of a synthetic
cultivar. A synthetic cultivar is the resultant progeny formed by
the intercrossing of several selected varieties. The number of
parental plant varieties, populations, wild accessions, ecotypes,
etc., that are used to generate a synthetic can vary from as little
as 10 to as much as 500. Typically, about 100 to 300 varieties,
populations, etc., are used a parents for the synthetic variety.
Seed from the parental seed production plot of a synthetic variety
can be sold to the farmer. Alternatively, seed from the parental
seed production plot can subsequently undergo one or two
generations of multiplication, depending on the amount of seed
produced in the parental plot and the demand for seed.
[0113] Mass selection is a useful technique when used in
conjunction with molecular marker enhanced selection. In mass
selection seeds from individuals are selected based on phenotype or
genotype. These selected seeds are then bulked and used to grow the
next generation. Bulk selection requires growing a population of
plants in a bulk plot, allowing the plants to self-pollinate,
harvesting the seed in bulk and then using a sample of the seed
harvested in bulk to plant the next generation. Also, instead of
self pollination, directed pollination could be used as part of the
breeding program.
[0114] Mutation breeding is another method of introducing new
traits into turfgrass plants having desirable looking turf when
mowed infrequently, such as bluegrass variety `03-0582`, `03-0441`,
or `99-2495`. Mutations that occur spontaneously or are
artificially induced can be useful sources of variability for a
plant breeder. The goal of artificial mutagenesis is to increase
the rate of mutation for a desired characteristic. Mutation rates
can be increased by many different means including temperature,
long-term seed storage, tissue culture conditions, radiation; such
as X-rays, Gamma rays (e.g. cobalt 60 or cesium 137), neutrons,
(product of nuclear fission by uranium 235 in an atomic reactor),
Beta radiation (emitted from radioisotopes such as phosphorus 32 or
carbon 14), or ultraviolet radiation (such as from 2500 to 2900
nm), or chemical mutagens (such as base analogues (5-bromo-uracil),
related compounds (8-ethoxy caffeine), antibiotics (streptonigrin),
alkylating agents (sulfur mustards, nitrogen mustards, epoxides,
ethylenamines, sulfates, sulfonates, sulfones, lactones), azide,
hydroxylamine, nitrous acid, or acridines. Once a desired trait is
observed through mutagenesis the trait may then be incorporated
into existing germplasm by traditional breeding techniques. Details
of mutation breeding can be found in Fehr, 1993. Principles of
Cultivar Development, Macmillan Publishing Company. In addition,
mutations created in other bluegrass plants may be used to produce
a backcross conversion of bluegrass variety `03-0582`, `03-0441`,
or `99-2495` that comprises such mutation.
Breeding with Molecular Markers
[0115] Molecular markers, which include markers identified through
the use of techniques such as Isozyme Electrophoresis, Restriction
Fragment Length Polymorphisms (RFLPs), Randomly Amplified
Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain
Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence
Characterized Amplified Regions (SCARs), Amplified Fragment Length
Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) and Single
Nucleotide Polymorphisms (SNPs), may be used in plant breeding
methods utilizing bluegrass variety `03-0582`, `03-0441`, or
`99-2495`.
[0116] Isozyme Electrophoresis and RFLPs have been widely used to
determine genetic composition. Shoemaker and Olsen, (O'Brien, S.
J., (ed.) 1993. Genetic Maps: Locus Maps of Complex Genomes. Cold
Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.),
developed a molecular genetic linkage map that consisted of 25
linkage groups with about 365 RFLP, 11 RAPD (random amplified
polymorphic DNA), three classical markers, and four isozyme loci.
See also, Shoemaker R. C. 1994. "RFLP Map of Soybean" p 299-309 In
R. L. Phillips and I. K. Vasil (ed.) DNA-Based Markers in Plants.
Kluwer Academic Press Dordrecht, the Netherlands. In switchgrass,
Missaoui also described RFLP markers (Missaoui et al., 2006,
"Molecular markers for the classification of switchgrass (Panicum
virgatum L.) germplasm and to assess genetic diversity in three
synthetic switchgrass populations" Genetic Resources and Crop
Evolution 53:1291-1302).
[0117] SSR technology is currently the most efficient and practical
marker technology; more marker loci can be routinely used and more
alleles per marker locus can be found using SSRs in comparison to
RFLPs. For example Diwan and Cregan, described a highly polymorphic
microsatellite loci in soybean with as many as 26 alleles. (Diwan,
N., and P. B. Cregan. 1997 "Automated sizing of fluorescent-labeled
simple sequence repeat (SSR) markers to assay genetic variation in
soybean". Theor. Appl. Genet. 95:220-225). Single Nucleotide
Polymorphisms (SNPs) may also be used to identify the unique
genetic composition of `03-0582`, `03-0441`, or `99-2495` and
progeny varieties retaining that unique genetic composition.
Various molecular marker techniques may be used in combination to
enhance overall resolution.
[0118] One use of molecular markers is Quantitative Trait Loci
(QTL) mapping. QTL mapping is the use of markers, which are known
to be closely linked to alleles that have measurable effects on a
quantitative trait. Selection in the breeding process is based upon
the accumulation of markers linked to the positive effecting
alleles and/or the elimination of the markers linked to the
negative effecting alleles from the plant's genome.
[0119] Molecular markers can also be used during the breeding
process for the selection of qualitative traits. For example,
markers closely linked to alleles or markers containing sequences
within the actual alleles of interest can be used to select plants
that contain the alleles of interest during a backcrossing breeding
program. The markers can also be used to select for the genome of
the recurrent parent and against the genome of the donor parent.
Using this procedure can minimize the amount of genome from the
donor parent that remains in the selected plants. It can also be
used to reduce the number of crosses back to the recurrent parent
needed in a backcrossing program. The use of molecular markers in
the selection process is often called genetic marker enhanced
selection. Molecular markers may also be used to identify and
exclude certain sources of germplasm as parental varieties or
ancestors of a plant by providing a means of tracking genetic
profiles through crosses.
Gene Conversions
[0120] When the term "turfgrass plant" or "bluegrass plant" is used
in the context of the methods and varieties described herein, this
also includes any gene conversions of that variety. The term gene
converted plant as used herein refers to those turfgrass plants
which are developed by a plant breeding technique called
backcrossing wherein essentially all of the desired morphological
and physiological characteristics of a variety are recovered in
addition to the one or more genes transferred into the variety via
the backcrossing technique. Backcrossing methods can be used with
the methods and variety described herein to improve or introduce
one or more characteristics into the variety. The term
"backcrossing" as used herein refers to the repeated crossing of a
hybrid progeny back to the recurrent parent, i.e., backcrossing 1,
2, 3, 4, 5, 6, 7, 8 or more times to the recurrent parent. The
parental turfgrass plant that contributes the gene(s) for the
desired characteristic is termed the nonrecurrent or donor parent.
This terminology refers to the fact that the nonrecurrent parent is
used one time in the backcross protocol and therefore does not
recur. The parental turfgrass plant to which the gene or genes from
the nonrecurrent parent are transferred is known as the recurrent
parent as it is used for several rounds in the backcrossing
protocol (Poehlman & Sleper, 1994; Fehr, Principles of Cultivar
Development pp. 261-286 (1987)). In a typical backcross protocol,
the original variety of interest (recurrent parent) is crossed to a
second variety (nonrecurrent parent) that carries the gene(s) of
interest to be transferred. The resulting progeny from this cross
are then crossed again to the recurrent parent and the process is
repeated until a turfgrass plant is obtained wherein essentially
all of the desired morphological and physiological characteristics
of the recurrent parent are recovered in the converted plant, in
addition to the transferred gene(s) from the nonrecurrent
parent.
[0121] The selection of a suitable recurrent parent is an important
step for a successful backcrossing procedure. The goal of a
backcross protocol is to alter or substitute one or more traits or
characteristics in the original variety. To accomplish this, one or
more genes of the recurrent variety is/are modified or substituted
with the desired gene(s) from the nonrecurrent parent, while
retaining essentially all of the rest of the desired genetic, and
therefore the desired physiological and morphological, constitution
of the original variety. The choice of the particular nonrecurrent
parent will depend on the purpose of the backcross; one of the
major purposes is to add some agronomically important trait to the
plant. The exact backcrossing protocol will depend on the
characteristic(s) or trait(s) being altered to determine an
appropriate testing protocol. Although backcrossing methods are
simplified when the characteristic being transferred is a dominant
allele, a recessive allele may also be transferred. In this
instance it may be necessary to introduce a test of the progeny to
determine if the desired characteristic(s) has been successfully
transferred.
[0122] Many traits have been identified that are not regularly
selected for in the development of a new variety but that can be
improved by backcrossing techniques. Traits may or may not be
transgenic; examples of these traits include but are not limited
to, male sterility, herbicide resistance, resistance for bacterial,
fungal, or viral disease, insect resistance, male fertility,
enhanced nutritional quality, modified oil content, industrial
usage, yield stability and yield enhancement. These genes are
generally inherited through the nucleus. Several of these traits
are described in U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445;
the disclosures of which are specifically hereby incorporated by
reference for this purpose.
Introduction of a New Trait or Locus into Turfgrass Plants Having
Desirable Looking Turf when Mowed Infrequently, Such as `03-0582`,
`03-0441`, or `99-2495`
[0123] Turfgrass plants of the present invention having desirable
looking turf when mowed infrequently, such as variety `03-0582`,
`03-0441`, or `99-2495` represent a new base genetic variety into
which a new locus or trait may be introgressed. Direct
transformation and backcrossing represent two important methods
that can be used to accomplish such an introgression. The term
backcross conversion and locus conversion are used interchangeably
to designate the product of a backcrossing program.
Backcross Conversions of Turfgrass Plants Having Desirable Looking
Turf when Mowed Infrequently, Such as `03-0582`, `03-0441`, or
`99-2495`
[0124] A backcross conversion of turfgrass plants of the present
invention having desirable looking turf when mowed infrequently,
such as `03-0582`, `03-0441`, or `99-2495` occurs when DNA
sequences are introduced through backcrossing (Poehlman, Breeding
Field Crops, p. 204 (1987), with `03-0582`, `03-0441`, or `99-2495`
utilized as the recurrent parent. Both naturally occurring and
transgenic DNA sequences may be introduced through backcrossing
techniques. A backcross conversion may produce a plant with a trait
or locus conversion in at least two or more backcrosses, including
at least 2 crosses, at least 3 crosses, at least 4 crosses, at
least 5 crosses and the like. Molecular marker assisted breeding or
selection may be utilized to reduce the number of backcrosses
necessary to achieve the backcross conversion. For example, see
Openshaw, S. J. et al., "Marker-assisted Selection in Backcross
Breeding" In: Proceedings Symposium of the Analysis of Molecular
Data, August 1994, Crop Science Society of America, Corvallis,
Oreg., where it is demonstrated that a backcross conversion can be
made in as few as two backcrosses.
[0125] The complexity of the backcross conversion method depends on
the type of trait being transferred (single genes or closely linked
genes vs unlinked genes), the level of expression of the trait, the
type of inheritance (cytoplasmic or nuclear) and the types of
parents included in the cross. It is understood by those of
ordinary skill in the art that for single gene traits that are
relatively easy to classify, the backcross method is effective and
relatively easy to manage. (See Hallauer et al. in Corn and Corn
Improvement, Sprague and Dudley, Third Ed. 1998). Desired traits
that may be transferred through backcross conversion include, but
are not limited to, sterility (nuclear and cytoplasmic), fertility
restoration, nutritional enhancements, drought tolerance, nitrogen
utilization, altered fatty acid profile, altered carbohydrate
profile, modified oil production, industrial enhancements, disease
resistance (bacterial, fungal or viral), insect resistance and
herbicide resistance. In addition, an introgression site itself,
such as an FRT site, Lox site or other site-specific integration
site, may be inserted by backcrossing and utilized for direct
insertion of one or more genes of interest into a specific plant
variety. In some embodiments, the number of loci that may be
backcrossed into `03-0582`, `03-0441`, or `99-2495` is at least 1,
2, 3, 4, or 5 and/or no more than 6, 5, 4, 3, or 2. A single locus
may contain several transgenes, such as a transgene for disease
resistance that, in the same expression vector, also contains a
transgene for herbicide resistance. The gene for herbicide
resistance may be used as a selectable marker and/or as a
phenotypic trait. A single locus conversion of a site-specific
integration system allows for the integration of multiple genes at
the converted loci.
[0126] The backcross conversion may result from either the transfer
of a dominant allele or a recessive allele. Selection of progeny
containing the trait of interest is accomplished by direct
selection for a trait associated with a dominant allele. Transgenes
transferred via backcrossing typically function as a dominant
single gene trait and are relatively easy to classify. Selection of
progeny for a trait that is transferred via a recessive allele
requires growing and selfing the first backcross generation to
determine which plants carry the recessive alleles. Recessive
traits may require additional progeny testing in successive
backcross generations to determine the presence of the locus of
interest. The last backcross generation is usually selfed to give
pure breeding progeny for the gene(s) being transferred, although a
backcross conversion with a stably introgressed trait may also be
maintained by further backcrossing to the recurrent parent with
selection for the converted trait.
[0127] Along with selection for the trait of interest, progeny are
selected for the phenotype of the recurrent parent. The backcross
is a form of inbreeding, and the features of the recurrent parent
are automatically recovered after successive backcrosses. Poehlman,
Breeding Field Crops, p. 204 (1987). Poehlman suggests from one to
four or more backcrosses, but as noted above, the number of
backcrosses necessary can be reduced with the use of molecular
markers. Other factors, such as a genetically similar donor parent,
may also reduce the number of backcrosses necessary. As noted by
Poehlman, backcrossing is easiest for simply inherited, dominant
and easily recognized traits.
[0128] One process for adding or modifying a trait or locus in
turfgrass plants of the present invention having desirable looking
turf when mowed infrequently, such as bluegrass variety `03-0582`,
`03-0441`, or `99-2495` comprises crossing `03-0582`, `03-0441`, or
`99-2495` plants grown from `03-0582`, `03-0441`, or `99-2495` seed
with plants of another bluegrass variety that comprise the desired
trait or locus, selecting F.sub.1 progeny plants that comprise the
desired trait or locus to produce selected F.sub.1 progeny plants,
crossing the selected progeny plants with the `03-0582`, `03-0441`,
or `99-2495` plants to produce backcross progeny plants, selecting
for backcross progeny plants that have the desired trait or locus
and the morphological characteristics of bluegrass variety
`03-0582`, `03-0441`, or `99-2495` to produce selected backcross
progeny plants; and backcrossing to `03-0582`, `03-0441`, or
`99-2495` three or more times in succession to produce selected
fourth or higher backcross progeny plants that comprise said trait
or locus. The modified `03-0582`, `03-0441`, or `99-2495` may be
further characterized as having the physiological and morphological
characteristics of bluegrass variety `03-0582`, `03-0441`, or
`99-2495` and/or may be characterized by percent similarity or
identity to `03-0582`, `03-0441`, or `99-2495` as determined by SSR
markers. The above method may be utilized with fewer backcrosses in
appropriate situations, such as when the donor parent is highly
related or markers are used in the selection step. Desired traits
that may be used include those nucleic acids known in the art, some
of which are mentioned herein, that will affect traits through
nucleic acid expression or inhibition. Desired loci include the
introgression of FRT, Lox and other sites for site specific
integration, which may also affect a desired trait if a functional
nucleic acid is inserted at the integration site.
Pedigree Breeding
[0129] Pedigree breeding starts with the crossing of two genotypes,
such as turfgrass plants of the present invention having desirable
looking turf when mowed infrequently, such as `03-0582`, `03-0441`,
or `99-2495` and another bluegrass variety having one or more
desirable characteristics that is lacking or which complements
`03-0582`, `03-0441`, or `99-2495`. If the two original parents do
not provide all the desired characteristics, other sources can be
included in the breeding population. In the pedigree method,
superior plants are selfed and selected in successive filial
generations. In the succeeding filial generations the heterozygous
condition gives way to homogeneous varieties as a result of
self-pollination and selection. Typically in the pedigree method of
breeding, five or more successive filial generations of selfing and
selection is practiced: F.sub.1 to F.sub.2; F.sub.2 to F.sub.3;
F.sub.3 to F.sub.4; F.sub.4 to F.sub.5, etc. After a sufficient
amount of inbreeding, successive filial generations will serve to
increase seed of the developed variety. In some embodiments, the
developed variety comprises homozygous alleles at about 95% or more
of its loci.
[0130] In addition to being used to create a backcross conversion,
backcrossing can also be used in combination with pedigree
breeding. As discussed previously, backcrossing can be used to
transfer one or more specifically desirable traits from one
variety, the donor parent, to a developed variety called the
recurrent parent, which has overall good agronomic characteristics
yet lacks that desirable trait or traits. However, the same
procedure can be used to move the progeny toward the genotype of
the recurrent parent but at the same time retain many components of
the non-recurrent parent by stopping the backcrossing at an early
stage and proceeding with selfing and selection. For example, a
turfgrass variety may be crossed with another variety to produce a
first generation progeny plant. The first generation progeny plant
may then be backcrossed to one of its parent varieties to create a
BC.sub.1 or BC.sub.2. Progeny are selfed and selected so that the
newly developed variety has many of the attributes of the recurrent
parent and yet several of the desired attributes of the
non-recurrent parent. This approach leverages the value and
strengths of the recurrent parent for use in new turfgrass
varieties.
[0131] Therefore, an embodiment is a method of making a backcross
conversion of turfgrass plants of the present invention having
desirable looking turf when mowed infrequently, such as bluegrass
variety `03-0582`, `03-0441`, or `99-2495`, comprising the steps of
crossing a plant of bluegrass variety `03-0582`, `03-0441`, or
`99-2495` with a donor plant comprising a desired trait, selecting
an F.sub.1 progeny plant comprising the desired trait, and
backcrossing the selected F.sub.1 progeny plant to a plant of
bluegrass variety `03-0582`, `03-0441`, or `99-2495`. This method
may further comprise the step of obtaining a molecular marker
profile of bluegrass variety `03-0582`, `03-0441`, or `99-2495` and
using the molecular marker profile to select for a progeny plant
with the desired trait and the molecular marker profile of
`03-0582`, `03-0441`, or `99-2495`. In one embodiment the desired
trait is a mutant gene or transgene present in the donor
parent.
[0132] In addition, the above process and other similar processes
described herein may be used to produce first generation progeny
bluegrass seed by adding a step at the end of the process that
comprises crossing turfgrass plants of the present invention having
desirable looking turf when mowed infrequently, such as `03-0582`,
`03-0441`, or `99-2495` with the introgressed trait or locus with a
different bluegrass plant and harvesting the resultant first
generation progeny bluegrass seed.
Transgenic Turfgrass
[0133] The advent of new molecular biological techniques has
allowed the isolation and characterization of genetic elements with
specific functions, such as encoding specific protein products.
Scientists in the field of plant biology developed a strong
interest in engineering the genome of plants to contain and express
foreign genetic elements, or additional, or modified versions of
native or endogenous genetic elements in order to alter the traits
of a plant in a specific manner. Any DNA sequences, whether from a
different species or from the same species, which are inserted into
the genome using transformation are referred to herein collectively
as "transgenes". In some embodiments of the invention, transgenic
variants of the turfgrass varieties of the present invention may
contain at least one transgene but could contain at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 and/or no more than 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, 4, 3, or 2. Over the last fifteen to twenty years
several methods for producing transgenic plants have been
developed, and the present invention also relates to transgenic
variants of the claimed turfgrass varieties of the present
invention.
[0134] Genetic engineering of the plants of the present invention
includes various methods for crop improvement, including transgenic
modification, xenogenic modification, intragenic modification,
famigenic modification and cisgenic modification.
[0135] One embodiment of the invention is a process for producing
turfgrass varieties further comprising a desired trait, said
process comprising transforming a turfgrass plant with a transgene
that confers a desired trait. Another embodiment is the product
produced by this process. In one embodiment the desired trait may
be one or more of herbicide resistance, insect resistance, disease
resistance or modified fatty acid or carbohydrate metabolism. In
one embodiment the desired trait may be increased or modified oil
content. The specific gene may be any known in the art or listed
herein, including: a polynucleotide conferring resistance to
imidazolinone, sulfonylurea, glyphosate, glufosinate, triazine,
benzonitrile, cyclohexanedione, phenoxy proprionic acid and
L-phosphinothricin; a polynucleotide encoding a Bacillus
thuringiensis polypeptide, or a polynucleotide conferring
resistance to one or more nematodes, Phytophthora root rot, or
other fungi, or one or more viruses.
[0136] Numerous methods for plant transformation have been
developed, including biological and physical plant transformation
protocols. See, for example, Miki et al., "Procedures for
Introducing Foreign DNA into Plants" in Methods in Plant Molecular
Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds.
(CRC Press, Inc., Boca Raton, 1993) pages 67-88 and Armstrong, "The
First Decade of Maize Transformation: A Review and Future
Perspective" (Maydica 44:101-109, 1999). In addition, expression
vectors and in vitro culture methods for plant cell or tissue
transformation and regeneration of plants are available. See, for
example, Gruber et al., "Vectors for Plant Transformation" in
Methods in Plant Molecular Biology and Biotechnology, Glick, B. R.
and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages
89-119.
[0137] A genetic trait which has been engineered into the genome of
a particular turfgrass plant may then be moved into the genome of
another turfgrass variety using traditional breeding techniques
that are well known in the plant breeding arts. For example, a
backcrossing approach is commonly used to move a transgene from a
transformed bluegrass variety into an already developed bluegrass
variety, and the resulting backcross conversion plant would then
comprise the transgene(s).
[0138] Various genetic elements can be introduced into the plant
genome using transformation. These elements include, but are not
limited to genes, coding sequences, inducible, constitutive, and
tissue specific promoters, enhancing sequences, and signal and
targeting sequences. For example, see the traits, genes and
transformation methods listed in U.S. Pat. No. 6,118,055.
[0139] Plant transformation involves the construction of an
expression vector which will function in plant cells. Such a vector
comprises DNA comprising a gene under control of, or operatively
linked to, a regulatory element (for example, a promoter). The
expression vector may contain one or more such operably linked
gene/regulatory element combinations. The vector(s) may be in the
form of a plasmid and can be used alone or in combination with
other plasmids to provide transformed turfgrass plants using
transformation methods as described below to incorporate transgenes
into the genetic material of the turfgrass plant(s).
[0140] With transgenic plants according to the methods and variety
described herein, a foreign or endogenous protein or oil can be
produced in commercial quantities. Thus, techniques for the
selection and propagation of transformed plants, which are well
understood in the art, yield a plurality of transgenic plants which
are harvested in a conventional manner, and a foreign or endogenous
protein or oil then can be extracted from a tissue of interest or
from total biomass. Protein extraction from plant biomass can be
accomplished by known methods which are discussed, for example, by
Heney and Orr, Anal. Biochem. 114:92-6 (1981).
[0141] According to a one embodiment, the transgenic plant provided
for commercial production of foreign or endogenous protein or oil
is a turfgrass plant. In another embodiment, the biomass of
interest is leaves, stems, or other plant parts. For the relatively
small number of transgenic plants that show higher levels of
expression, a genetic map can be generated, primarily via
conventional RFLP, PCR and SSR analysis, which identifies the
approximate chromosomal location of the integrated DNA molecule.
For exemplary methodologies in this regard, see Glick and Thompson,
Methods in Plant Molecular Biology and Biotechnology, CRC Press,
Boca Raton 269:284 (1993). Map information concerning chromosomal
location is useful for proprietary protection of a subject
transgenic plant.
Expression Vectors for Turfgrass Transformation: Marker Genes
[0142] Expression vectors include at least one genetic marker
operably linked to a regulatory element (a promoter, for example)
that allows transformed cells containing the marker to be either
recovered by negative selection, i.e., inhibiting growth of cells
that do not contain the selectable marker gene, or by positive
selection, i.e., screening for the product encoded by the genetic
marker. Many commonly used selectable marker genes for plant
transformation are well known in the transformation arts, and
include, for example, genes that code for enzymes that
metabolically detoxify a selective chemical agent which may be an
antibiotic or an herbicide, or genes that encode an altered target
which is insensitive to the inhibitor. A few positive selection
methods are also known in the art.
[0143] One commonly used selectable marker gene for plant
transformation is the neomycin phosphotransferase II (nptII) gene
which, when under the control of plant regulatory signals, confers
resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. USA,
80:4803 (1983). Another commonly used selectable marker gene is the
hygromycin phosphotransferase gene which confers resistance to the
antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299
(1985).
[0144] Additional selectable marker genes of bacterial origin that
confer resistance to antibiotics include gentamycin acetyl
transferase, streptomycin phosphotransferase and
aminoglycoside-3'-adenyl transferase, the bleomycin resistance
determinant (Hayford et al., Plant Physiol. 86:1216 (1988), Jones
et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant Mol.
Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986)).
Other selectable marker genes confer resistance to herbicides such
as glyphosate, glufosinate or bromoxynil (Comai et al., Nature
317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990)
and Stalker et al., Science 242:419-423 (1988)).
[0145] Selectable marker genes for plant transformation not of
bacterial origin include, for example, mouse dihydrofolate
reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and
plant acetolactate synthase (Eichholtz et al., Somatic Cell Mol.
Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest
et al., Plant Cell Rep. 8:643 (1990)).
[0146] Another class of marker genes for plant transformation
requires screening of presumptively transformed plant cells rather
than direct genetic selection of transformed cells for resistance
to a toxic substance such as an antibiotic. These genes are
particularly useful to quantify or visualize the spatial pattern of
expression of a gene in specific tissues and are frequently
referred to as reporter genes because they can be fused to a gene
or gene regulatory sequence for the investigation of gene
expression. Commonly used genes for screening presumptively
transformed cells include .beta.-glucuronidase (GUS),
.beta.-galactosidase, luciferase and chloramphenicol
acetyltransferase (Jefferson, R. A., Plant Mol. Biol. Rep. 5:387
(1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc.
Natl. Acad. Sci. USA 84:131 (1987), DeBlock et al., EMBO J. 3:1681
(1984)).
[0147] In vivo methods for visualizing GUS activity that do not
require destruction of plant tissue are available (Molecular Probes
publication 2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al.,
J. Cell Biol. 115:151a (1991)). However, these in vivo methods for
visualizing GUS activity have not proven useful for recovery of
transformed cells because of low sensitivity, high fluorescent
backgrounds and limitations associated with the use of luciferase
genes as selectable markers.
[0148] More recently, a gene encoding Green Fluorescent Protein
(GFP) has been utilized as a marker for gene expression in
prokaryotic and eukaryotic cells (Chalfie et al., Science 263:802
(1994)). GFP and mutants of GFP may be used as screenable
markers.
Expression Vectors for Turfgrass Transformation: Promoters
[0149] Genes included in expression vectors must be driven by a
nucleotide sequence comprising a regulatory element, for example, a
promoter. Several types of promoters are well known in the
transformation arts as are other regulatory elements that can be
used alone or in combination with promoters.
[0150] As used herein, "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells. Examples of promoters
under developmental control include promoters that preferentially
initiate transcription in certain tissues, such as leaves, roots,
seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such
promoters are referred to as "tissue-preferred". Promoters that
initiate transcription only in a certain tissue are referred to as
"tissue-specific". A "cell-type" specific promoter primarily drives
expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" promoter
is a promoter which is under environmental control. Examples of
environmental conditions that may effect transcription by inducible
promoters include anaerobic conditions or the presence of light.
Tissue-specific, tissue-preferred, cell type specific, and
inducible promoters constitute the class of "non-constitutive"
promoters. A "constitutive" promoter is a promoter that is active
under most environmental conditions.
[0151] The choice of regulatory regions to be included in a
recombinant construct depends upon several factors, including, but
not limited to, efficiency, selectability, inducibility, desired
expression level, and cell- or tissue-preferential expression. It
is a routine matter for one of skill in the art to modulate the
expression of a coding sequence by appropriately selecting and
positioning regulatory regions relative to the coding sequence.
Transcription of a nucleic acid can be modulated in a similar
manner. Some suitable regulatory regions initiate transcription
only, or predominantly, in certain cell types. Methods for
identifying and characterizing regulatory regions in plant genomic
DNA are known, including, for example, those described in the
following references: Jordano et al., Plant Cell, 1:855-866 (1989);
Bustos et al., Plant Cell, 1:839-854 (1989); Green et al., EMBO J.,
7:4035-4044 (1988); Meier et al., Plant Cell, 3:309-316 (1991); and
Zhang et al., Plant Physiology, 110:1069-1079 (1996). Examples of
various regulatory regions are described in more detail in U.S.
Patent Application Ser. Nos. US20080072340, US20080044898,
US20070277269, US20070226830, US20070136839, US 20070124834, and US
20060107346. It will be appreciated that a regulatory region may
meet criteria for one classification based on its activity in one
plant species, and yet meet criteria for a different classification
based on its activity in another plant species. Examples of
regulatory regions include broadly expressing promoters, root
promoters, maturing endosperm promoters, ovary tissue promoters,
embryo sac/early endosperm promoters, embryo promoters,
photosynthetic tissue promoters, vascular tissue promoters,
inducible promoters, basal promoters, or other regulatory
regions.
[0152] A. Inducible Promoters--An inducible promoter is operably
linked to a gene for expression in turfgrass. Optionally, the
inducible promoter is operably linked to a nucleotide sequence
encoding a signal sequence which is operably linked to a gene for
expression in perennial turfgrass. With an inducible promoter the
rate of transcription increases in response to an inducing
agent.
[0153] Any inducible promoter can be used in the instant invention.
See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary
inducible promoters include, but are not limited to, that from the
ACEI system which responds to copper (Mett et al., Proc. Natl.
Acad. Sci. USA 90:4567-4571 (1993)); In2 gene from maize which
responds to benzenesulfonamide herbicide safeners (Hershey et al.,
Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen.
Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al.,
Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferred
inducible promoter is a promoter that responds to an inducing agent
to which plants do not normally respond. An exemplary inducible
promoter is the inducible promoter from a steroid hormone gene, the
transcriptional activity of which is induced by a
glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci.
USA 88:0421 (1991)).
[0154] B. Constitutive Promoters--A constitutive promoter is
operably linked to a gene for expression in turfgrass or the
constitutive promoter is operably linked to a nucleotide sequence
encoding a signal sequence which is operably linked to a gene for
expression in turfgrass.
[0155] Many different constitutive promoters can be utilized in the
instant invention. Exemplary constitutive promoters include, but
are not limited to, the promoters from plant viruses such as the
35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985))
and the promoters from such genes as rice actin (McElroy et al.,
Plant Cell 2: 163-171 (1990)); ubiquitin (Christensen et al., Plant
Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol.
Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet.
81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984))
and maize H3 histone (Lepetit et al., Mol. Gen. Genetics
231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):
291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5' to the
Brassica napus ALS3 structural gene (or a nucleotide sequence
similarity to said Xbal/Ncol fragment), represents a particularly
useful constitutive promoter. See PCT application WO 96/30530.
[0156] C. Tissue-specific or Tissue-preferred Promoters--A
tissue-specific promoter is operably linked to a gene for
expression in turfgrass. Optionally, the tissue-specific promoter
is operably linked to a nucleotide sequence encoding a signal
sequence which is operably linked to a gene for expression in
turfgrass. Plants transformed with a gene of interest operably
linked to a tissue-specific promoter produce the protein product of
the transgene exclusively, or preferentially, in a specific
tissue.
[0157] Any tissue-specific or tissue-preferred promoter can be
utilized in the instant invention. Exemplary tissue-specific or
tissue-preferred promoters include, but are not limited to, a
root-preferred promoter such as that from the phaseolin gene (Murai
et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al.,
Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a leaf-specific
and light-induced promoter such as that from cab or rubisco
(Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al.,
Nature 318:579-582 (1985)); an anther-specific promoter such as
that from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245
(1989)); a pollen-specific promoter such as that from Zm13
(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a
microspore-preferred promoter such as that from apg (Twell et al.,
Sex. Plant Reprod. 6:217-224 (1993)).
Signal Sequences for Targeting Proteins to Subcellular
Compartments
[0158] Transport of a protein produced by transgenes to a
subcellular compartment such as the chloroplast, vacuole,
peroxisome, glyoxysome, cell wall or mitochondrion or for secretion
into the apoplast, is accomplished by means of operably linking the
nucleotide sequence encoding a signal sequence to the 5' and/or 3'
region of a gene encoding the protein of interest. Targeting
sequences at the 5' and/or 3' end of the structural gene may
determine during protein synthesis and processing where the encoded
protein is ultimately compartmentalized.
[0159] The presence of a signal sequence directs a polypeptide to
either an intracellular organelle or subcellular compartment or for
secretion to the apoplast. Many signal sequences are known in the
art. See, for example, Becker et al., Plant Mol. Biol. 20:49
(1992); Knox, C., et al., Plant Mol. Biol. 9:3-17 (1987); Lerner et
al., Plant Physiol. 91:124-129 (1989); Frontes et al., Plant Cell
3:483-496 (1991); Matsuoka et al., Proc. Natl. Acad. Sci. 88:834
(1991); Gould et al., J. Cell. Biol. 108:1657 (1989); Creissen et
al., Plant J. 2:129 (1991); Kalderon, et al., Cell 39:499-509
(1984); Steifel, et al., Plant Cell 2:785-793 (1990).
Foreign Protein Genes and Agronomic Genes
[0160] With transgenic plants according to the present invention, a
foreign protein can be produced in commercial quantities. Thus,
techniques for the selection and propagation of transformed plants,
which are well understood in the art, yield a plurality of
transgenic plants which are harvested in a conventional manner, and
a foreign protein then can be extracted from a tissue of interest
or from total biomass. Protein extraction from plant biomass can be
accomplished by known methods which are discussed, for example, by
Heney and Orr, Anal. Biochem. 114:92-6 (1981).
[0161] Wang et al. discuss "Large Scale Identification, Mapping and
Genotyping of Single-Nucleotide Polymorphisms in the Human Genome",
Science, 280:1077-1082, 1998, and similar capabilities are becoming
available for the bluegrass genome. Map information concerning
chromosomal location is useful for proprietary protection of a
subject transgenic plant. If unauthorized propagation is undertaken
and crosses made with other germplasm, the map of the integration
region can be compared to similar maps for suspect plants to
determine if the latter have a common parentage with the subject
plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR
and sequencing, all of which are conventional techniques. SNPs may
also be used alone or in combination with other techniques.
[0162] Likewise, by means of the present invention, plants can be
genetically engineered to express various phenotypes of interest.
Through the transformation of turfgrass the expression of genes can
be altered to enhance disease resistance, insect resistance,
herbicide resistance and other traits, such as oil content. DNA
sequences native to turfgrass as well as non-native DNA sequences
can be transformed into turfgrass and used to alter levels of
native or non-native proteins. Various promoters, targeting
sequences, enhancing sequences, and other DNA sequences can be
inserted into the genome for the purpose of altering the expression
of proteins. Reduction of the activity of specific genes (also
known as gene silencing, or gene suppression) is desirable for
several aspects of genetic engineering in plants.
[0163] Many techniques for gene silencing are well known to one of
skill in the art, including but not limited to knock-outs (such as
by insertion of a transposable element such as mu (Vicki Chandler,
The Maize Handbook ch. 118 (Springer-Verlag 1994) or other genetic
elements such as a FRT, Lox or other site specific integration
site, antisense technology (see, e.g., Sheehy et al. (1988) PNAS
USA 85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566; and
5,759,829); co-suppression (e.g., Taylor (1997) Plant Cell 9:1245;
Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS
USA 91:3490-3496; Finnegan et al. (1994) Bio/Technology 12:
883-888; and Neuhuber et al. (1994) Mol. Gen. Genet. 244:230-241);
RNA interference (Napoli et al. (1990) Plant Cell 2:279-289; U.S.
Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141; Zamore et
al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNAS USA
95:15502-15507), virus-induced gene silencing (Burton, et al.
(2000) Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant
Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff et al.
(1988) Nature 334: 585-591); hairpin structures (Smith et al.
(2000) Nature 407:319-320; WO 99/53050; and WO 98/53083); MicroRNA
(Aukerman & Sakai (2003) Plant Cell 15:2730-2741); ribozymes
(Steinecke et al. (1992) EMBO J. 11:1525; and Perriman et al.
(1993) Antisense Res. Dev. 3:253); oligonucleotide mediated
targeted modification (e.g., WO 03/076574 and WO 99/25853);
Zn-finger targeted molecules (e.g., WO 01/52620; WO 03/048345; and
WO 00/42219); and other methods or combinations of the above
methods known to those of skill in the art.
[0164] Likewise, by means of the present invention, additional
genes of interest can be expressed in transformed plants. Exemplary
genes implicated in this regard include, but are not limited to,
those categorized below:
1. Genes that Confer Resistance to Pests or Disease and that
Encode:
[0165] A. Plant disease resistance genes. Plant defenses are often
activated by specific interaction between the product of a disease
resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr) gene in the pathogen. A plant variety can be
transformed with one or more cloned resistance genes to engineer
plants that are resistant to specific pathogen strains. See, for
example Jones et al., Science 266:789 (1994) (cloning of the tomato
Cf-9 gene for resistance to Cladosporium fulvum); Martin et al.,
Science 262:1432 (1993) (tomato Pto gene for resistance to
Pseudomonas syringae pv. tomato encodes a protein kinase);
Mindrinos et al. Cell 78:1089 (1994) (Arabidopsis RSP2 gene for
resistance to Pseudomonas syringae), McDowell & Woffenden,
(2003) Trends Biotechnol. 21(4): 178-83 and Toyoda et al., (2002)
Transgenic Res. 11 (6):567-82.
[0166] B. A gene conferring resistance to a pest, such as a
nematode. See e.g., PCT Application WO 96/30517; PCT Application WO
93/19181.
[0167] C. A Bacillus thuringiensis protein, a derivative thereof or
a synthetic polypeptide modeled thereon. See, for example, Geiser
et al., Gene 48:109 (1986), who disclose the cloning and nucleotide
sequence of a Bt .delta.-endotoxin gene. Moreover, DNA molecules
encoding .delta.-endotoxin genes can be purchased from American
Type Culture Collection, Manassas, Va., for example, under ATCC
Accession Nos. 40098, 67136, 31995 and 31998.
[0168] D. A lectin. See, for example, Van Damme et al., Plant
Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of
several Clivia miniata mannose-binding lectin genes.
[0169] E. A vitamin-binding protein such as avidin. See PCT
application US 93/06487 which teaches the use of avidin and avidin
homologues as larvicides against insect pests.
[0170] F. An enzyme inhibitor, for example, a protease or
proteinase inhibitor or an amylase inhibitor. See, for example, Abe
et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of
rice cysteine proteinase inhibitor), Huub et al., Plant Molec.
Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco
proteinase inhibitor I), Sumitani et al., Biosci. Biotech. Biochem.
57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus
.alpha.-amylase inhibitor) and U.S. Pat. No. 5,494,813 (Hepher and
Atkinson, issued Feb. 27, 1996).
[0171] G. An insect-specific hormone or pheromone such as an
ecdysteroid or juvenile hormone, a variant thereof, a mimetic based
thereon, or an antagonist or agonist thereof. See, for example, the
disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus
expression of cloned juvenile hormone esterase, an inactivator of
juvenile hormone.
[0172] H. An insect-specific peptide or neuropeptide which, upon
expression, disrupts the physiology of the affected pest. For
example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994)
(expression cloning yields DNA coding for insect diuretic hormone
receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243
(1989) (an allostatin is identified in Diploptera puntata);
Chattopadhyay et al. (2004) Critical Reviews in Microbiology 30
(1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2): 300-310;
Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539;
Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and Vasconcelos
& Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat.
No. 5,266,317 to Tomalski et al., which discloses genes encoding
insect-specific, paralytic neurotoxins.
[0173] I. An insect-specific venom produced in nature by a snake, a
wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for
disclosure of heterologous expression in plants of a gene coding
for a scorpion insectotoxic peptide.
[0174] J. An enzyme responsible for a hyperaccumulation of a
monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a
phenylpropanoid derivative or another non-protein molecule with
insecticidal activity.
[0175] K. An enzyme involved in the modification, including the
post-translational modification, of a biologically active molecule;
for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic
enzyme, a nuclease, a cyclase, a transaminase, an esterase, a
hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase,
an elastase, a chitinase and a glucanase, whether natural or
synthetic. See PCT application WO 93/02197 (Scott et al.), which
discloses the nucleotide sequence of a callase gene. DNA molecules
which contain chitinase-encoding sequences can be obtained, for
example, from the ATCC under Accession Nos. 39637 and 67152. See
also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who
teach the nucleotide sequence of a cDNA encoding tobacco hornworm
chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673 (1993),
who provide the nucleotide sequence of the parsley ubi4-2
polyubiquitin gene, U.S. Pat. Nos. 7,145,060, 7,087,810 and
6,563,020.
[0176] L. A molecule that stimulates signal transduction. For
example, see the disclosure by Botella et al., Plant Molec. Biol.
24:757 (1994), of nucleotide sequences for mung bean calmodulin
cDNA clones, and Griess et al., Plant Physiol. 104:1467 (1994), who
provide the nucleotide sequence of a maize calmodulin cDNA
clone.
[0177] M. A hydrophobic moment peptide. See PCT application WO
95/16776 and U.S. Pat. No. 5,580,852, which disclose peptide
derivatives of tachyplesin which inhibit fungal plant pathogens,
and PCT application WO 95/18855 and U.S. Pat. No. 5,607,914 which
teaches synthetic antimicrobial peptides that confer disease
resistance.
[0178] N. A membrane permease, a channel former or a channel
blocker. For example, see the disclosure of Jaynes et al., Plant
Sci 89:43 (1993), of heterologous expression of a cecropin-.beta.
lytic peptide analog to render transgenic tobacco plants resistant
to Pseudomonas solanacearum.
[0179] O. A viral-invasive protein or a complex toxin derived
therefrom. For example, the accumulation of viral coat proteins in
transformed plant cells imparts resistance to viral infection
and/or disease development effected by the virus from which the
coat protein gene is derived, as well as by related viruses. See
Beachy et al., Ann. Rev. Phytopathol. 28:451 (1990). Coat
protein-mediated resistance has been conferred upon transformed
plants against alfalfa mosaic virus, cucumber mosaic virus and
tobacco mosaic virus.
[0180] P. An insect-specific antibody or an immunotoxin derived
therefrom. Thus, an antibody targeted to a critical metabolic
function in the insect gut would inactivate an affected enzyme,
killing the insect. See Taylor et al., Abstract #497, Seventh Int'l
Symposium on Molecular Plant-Microbe Interactions (Edinburgh,
Scotland) (1994) (enzymatic inactivation in transgenic tobacco via
production of single-chain antibody fragments).
[0181] Q. A virus-specific antibody. See, for example, Tavladoraki
et al., Nature 366:469 (1993), who show that transgenic plants
expressing recombinant antibody genes are protected from virus
attack.
[0182] R. A developmental-arrestive protein produced in nature by a
pathogen or a parasite. Thus, fungal
endo-.alpha.-1,4-D-polygalacturonases facilitate fungal
colonization and plant nutrient release by solubilizing plant cell
wall homo-.alpha.-1,4-D-galacturonase. See Lamb et al.,
Bio/Technology 10:1436 (1992). The cloning and characterization of
a gene which encodes a bean endopolygalacturonase-inhibiting
protein is described by Toubart et al., Plant J. 2:367 (1992).
[0183] S. A developmental-arrestive protein produced in nature by a
plant. For example, Logemann et al., Bio/Technology 10:305 (1992),
have shown that transgenic plants expressing the barley
ribosome-inactivating gene have an increased resistance to fungal
disease.
[0184] T. Genes involved in the Systemic Acquired Resistance (SAR)
Response and/or the pathogenesis-related genes. Briggs, S., Current
Biology, 5(2) (1995); Pieterse & Van Loon (2004) Curr. Opin.
Plant Bio. 7(4):456-64 and Somssich (2003) Cell 113(7):815-6.
[0185] U. Antifungal genes. See Cornelissen and Melchers, Plant
Physiol., 101:709-712 (1993); Parijs et al., Planta 183:258-264
(1991) and Bushnell et al., Can. J. of Plant Path. 20(2):137-149
(1998). Also see U.S. Pat. No. 6,875,907.
[0186] V. Detoxification genes, such as for fumonisin, beauvericin,
moniliformin and zearalenone and their structurally related
derivatives. For example, see U.S. Pat. No. 5,792,931.
[0187] W. Cystatin and cysteine proteinase inhibitors. See U.S.
Pat. No. 7,205,453.
[0188] X. Defensin genes. See WO 03/000863 and U.S. Pat. No.
6,911,577.
[0189] Y. Genes that confer resistance to Phytophthora root rot,
such as the Rps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps
1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7
and other Rps genes. See, for example, Shoemaker et al.,
Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant
Genome IV Conference, San Diego, Calif. (1995).
2. Genes that Confer Resistance to an Herbicide, for Example:
[0190] A. An herbicide that inhibits the growing point or meristem,
such as an imidazolinone or a sulfonylurea. Exemplary genes in this
category code for mutant ALS and AHAS enzyme as described, for
example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al.,
Theor. Appl. Genet. 80:449 (1990), respectively.
[0191] B. Glyphosate (resistance conferred by mutant
5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,
respectively) and other phosphono compounds such as glufosinate
(phosphinothricin acetyl transferase (PAT) and Streptomyces
hygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic
acids and cyclohexanediones (ACCase inhibitor-encoding genes). See,
for example, U.S. Pat. No. 4,940,835 to Shah, et al., which
discloses the nucleotide sequence of a form of EPSPS which can
confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry et
al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.
Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;
5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;
5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060;
4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and
5,491,288; and international publications EP1173580; WO 01/66704;
EP1173581 and EP1173582, which are incorporated herein by reference
for this purpose. Glyphosate resistance is also imparted to plants
that express a gene that encodes a glyphosate oxido-reductase
enzyme as described more fully in U.S. Pat. Nos. 5,776,760 and
5,463,175, which are incorporated herein by reference for this
purpose. In addition glyphosate resistance can be imparted to
plants by the over expression of genes encoding glyphosate
N-acetyltransferase. See, for example, U.S. application Ser. No.
10/427,692. A DNA molecule encoding a mutant aroA gene can be
obtained under ATCC accession number 39256, and the nucleotide
sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061
to Comai. European patent application No. 0 333 033 to Kumada et
al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclose
nucleotide sequences of glutamine synthetase genes which confer
resistance to herbicides such as L-phosphinothricin. The nucleotide
sequence of a PAT gene is provided in European application No. 0
242 246 to Leemans et al. DeGreef et al., Bio/Technology 7:61
(1989) describe the production of transgenic plants that express
chimeric bar genes coding for phosphinothricin acetyl transferase
activity. Exemplary of genes conferring resistance to phenoxy
proprionic acids and cyclohexones, such as sethoxydim and haloxyfop
are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described by Marshall
et al., Theor. Appl. Genet. 83:435 (1992).
[0192] C. An herbicide that inhibits photosynthesis, such as a
triazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene).
Przibila et al., Plant Cell 3:169 (1991), describe the
transformation of Chlamydomonas with plasmids encoding mutant psbA
genes. Nucleotide sequences for nitrilase genes are disclosed in
U.S. Pat. No. 4,810,648 to Stalker and DNA molecules containing
these genes are available under ATCC Accession Nos. 53435, 67441
and 67442. Cloning and expression of DNA coding for a glutathione
S-transferase is described by Hayes et al., Biochem. J. 285:173
(1992).
[0193] D. Acetohydroxy acid synthase, which has been found to make
plants that express this enzyme resistant to multiple types of
herbicides, has been introduced into a variety of plants. See
Hattori et al., Mol. Gen. Genet. 246:419, 1995. Other genes that
confer tolerance to herbicides include a gene encoding a chimeric
protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450
oxidoreductase (Shiota et al., Plant Physiol., 106:17, 1994), genes
for glutathione reductase and superoxide dismutase (Aono et al.,
Plant Cell Physiol. 36:1687, 1995), and genes for various
phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,
1992).
[0194] E. Protoporphyrinogen oxidase (protox) is necessary for the
production of chlorophyll, which is necessary for all plant
survival. The protox enzyme serves as the target for a variety of
herbicidal compounds. These herbicides also inhibit growth of all
the different species of plants present, causing their total
destruction. The development of plants containing altered protox
activity which are resistant to these herbicides are described in
U.S. Pat. Nos. 6,288,306; 6,282,837; 5,767,373; and international
publication WO 01/12825.
5. Genes that Create a Site for Site Specific DNA Integration.
[0195] This includes the introduction of FRT sites that may be used
in the FLP/FRT system and/or Lox sites that may be used in the
Cre/Loxp system. For example, see Lyznik, et al., Site-Specific
Recombination for Genetic Engineering in Plants, Plant Cell Rep
(2003) 21:925-932 and WO 99/25821, which are hereby incorporated by
reference. Other systems that may be used include the Gin
recombinase of phage Mu (Maeser et al., 1991; Vicki Chandler, The
Maize Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase
of E. coli (Enomoto et al., 1983), and the R/RS system of the pSR1
plasmid (Araki et al., 1992).
6. Genes that Affect Abiotic Stress Resistance.
[0196] Genes that affect abiotic stress resistance (including but
not limited to flowering, pod and seed development, enhancement of
nitrogen utilization efficiency, altered nitrogen responsiveness,
drought resistance or tolerance, cold resistance or tolerance, and
salt resistance or tolerance) and increased yield under stress. For
example, see: WO 00/73475 where water use efficiency is altered
through alteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat.
No. 5,965,705, U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859,
U.S. Pat. No. 6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No.
6,706,866, U.S. Pat. No. 6,717,034, U.S. Pat. No. 6,801,104, WO
2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO
2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO
2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO
2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO
2004/076638, WO 98/09521, and WO 99/38977 describing genes,
including CBF genes and transcription factors effective in
mitigating the negative effects of freezing, high salinity, and
drought on plants, as well as conferring other positive effects on
plant phenotype; US 2004/0148654 and WO 01/36596 where abscisic
acid is altered in plants resulting in improved plant phenotype
such as increased yield and/or increased tolerance to abiotic
stress; WO 2000/006341, WO 04/090143, U.S. application Ser. No.
10/817,483 and U.S. Pat. No. 6,992,237 where cytokinin expression
is modified resulting in plants with increased stress tolerance,
such as drought tolerance, and/or increased yield. Also see WO
02/02776, WO 2003/052063, JP2002281975, U.S. Pat. No. 6,084,153, WO
01/64898, U.S. Pat. Nos. 6,177,275 and 6,107,547 (enhancement of
nitrogen utilization and altered nitrogen responsiveness). For
ethylene alteration, see US 20040128719, US 20030166197 and WO
2000/32761. For plant transcription factors or transcriptional
regulators of abiotic stress, see e.g. US 20040098764 or US
20040078852.
[0197] Other genes and transcription factors that affect plant
growth and agronomic traits such as yield, flowering, plant growth
and/or plant structure, can be introduced or introgressed into
plants, see e.g. WO 97/49811 (LHY), WO 98/56918 (ESD4), WO 97/10339
and U.S. Pat No. 6,573,430 (TFL), U.S. Pat No. 6713663 (FT), WO
96/14414 (CON), WO 96/38560, WO 01/21822 (VRN1), WO 00/44918
(VRN2), WO 99/49064 (GI), WO 00/46358 (FRI), WO 97/29123, U.S. Pat.
No. 6,794,560, U.S. Pat. No. 6307126 (GAI), WO 99/09174 (D8 and
Rht), and WO 2004/076638 and WO 2004/031349 (transcription
factors).
Methods for Turfgrass Transformation
[0198] Numerous methods for plant transformation have been
developed including biological and physical plant transformation
protocols. See, for example, Miki et al., "Procedures for
Introducing Foreign DNA into Plants" in Methods in Plant Molecular
Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds.
(CRC Press, Inc. Boca Raton, 1993) pages 67-88. In addition,
expression vectors and in-vitro culture methods for plant cell or
tissue transformation and regeneration of plants are available.
See, for example, Gruber et al., "Vectors for Plant Transformation"
in Methods in Plant Molecular Biology and Biotechnology, Glick, B.
R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)
pages 89-119.
[0199] A. Agrobacterium-mediated Transformation--One method for
introducing an expression vector into plants is based on the
natural transformation system of Agrobacterium. See, for example,
Horsch et al., Science 227:1229 (1985). A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of the plant. See, for example, Kado, C. I., Crit.
Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector
systems and methods for Agrobacterium-mediated gene transfer are
provided by Gruber et al., supra, Miki et al., supra and Moloney et
al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.
5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.
[0200] B. Direct Gene Transfer--Several methods of plant
transformation, collectively referred to as direct gene transfer,
have been developed as an alternative to Agrobacterium-mediated
transformation. A generally applicable method of plant
transformation is microprojectile-mediated transformation where DNA
is carried on the surface of microprojectiles measuring 1 to 4
.mu.m. The expression vector is introduced into plant tissues with
a biolistic device that accelerates the microprojectiles to speeds
of 300 to 600 m/s which is sufficient to penetrate plant cell walls
and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987);
Sanford, J. C., Trends Biotech. 6:299 (1988); Klein et al.,
Bio/Tech. 6:559-563 (1988); Sanford, J. C. Physiol Plant 7:206
(1990); Klein et al., Biotechnology 10:268 (1992). See also U.S.
Pat. No. 5,015,580 (Christou, et al.), issued May 14, 1991 and U.S.
Pat. No. 5,322,783 (Tomes, et al.), issued Jun. 21, 1994.
[0201] Another method for physical delivery of DNA to plants is
sonication of target cells. Zhang et al., Bio/Technology 9:996
(1991). Alternatively, liposome and spheroplast fusion have been
used to introduce expression vectors into plants. Deshayes et al.,
EMBO J., 4:2731 (1985); Christou et al., Proc Natl. Acad. Sci. USA
84:3962 (1987). Direct uptake of DNA into protoplasts using
CaCl.sub.2 precipitation, polyvinyl alcohol or poly-L-ornithine
have also been reported. Hain et al., Mol. Gen. Genet. 199:161
(1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).
Electroporation of protoplasts and whole cells and tissues have
also been described (Donn et al., In Abstracts of VIIth
International Congress on Plant Cell and Tissue Culture IAPTC,
A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992)
and Spencer et al., Plant Mol. Biol. 24:51-61 (1994)).
[0202] Following transformation of bluegrass target tissues,
expression of the above-described selectable marker genes allows
for preferential selection of transformed cells, tissues and/or
plants, using regeneration and selection methods well known in the
art.
Genetic Marker Profile through SSR and First Generation Progeny
[0203] In addition to phenotypic observations, a plant can also be
identified by its genotype. The genotype of a plant can be
characterized through a genetic marker profile which can identify
plants of the same variety or a related variety or be used to
determine or validate a pedigree. Genetic marker profiles can be
obtained by techniques such as Restriction Fragment Length
Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),
Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA
Amplification Fingerprinting (DAF), Sequence Characterized
Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms
(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to
as Microsatellites, and Single Nucleotide Polymorphisms (SNPs). For
example, see Cregan et. al,
[0204] "An Integrated Genetic Linkage Map of the Soybean Genome"
Crop Science 39:1464-1490 (1999), and Berry et al., Assessing
Probability of Ancestry Using Simple Sequence Repeat Profiles:
Applications to Maize Inbred Lines and Soybean Varieties" Genetics
165:331-342 (2003).
[0205] Particular markers used for these purposes are not limited
to any particular set of markers, but are envisioned to include any
type of marker and marker profile which provides a means of
distinguishing varieties.
[0206] Means of performing genetic marker profiles using SSR
polymorphisms are well known in the art. SSRs are genetic markers
based on polymorphisms in repeated nucleotide sequences, such as
microsatellites. A marker system based on SSRs can be highly
informative in linkage analysis relative to other marker systems in
that multiple alleles may be present. Another advantage of this
type of marker is that, through use of flanking primers, detection
of SSRs can be achieved, for example, by the polymerase chain
reaction (PCR), thereby eliminating the need for labor-intensive
Southern hybridization. The PCR detection is done by use of two
oligonucleotide primers flanking the polymorphic segment of
repetitive DNA. Repeated cycles of heat denaturation of the DNA
followed by annealing of the primers to their complementary
sequences at low temperatures, and extension of the annealed
primers with DNA polymerase, comprise the major part of the
methodology.
[0207] Following amplification, markers can be scored by
electrophoresis of the amplification products. Scoring of marker
genotype is based on the size of the amplified fragment, which may
be measured by the number of base pairs of the fragment. While
variation in the primer used or in laboratory procedures can affect
the reported fragment size, relative values should remain constant
regardless of the specific primer or laboratory used. When
comparing varieties it is preferable if all SSR profiles are
performed in the same lab.
Tissue Culture
[0208] Further reproduction of the turfgrass varieties of the
present invention can occur by tissue culture and regeneration.
Tissue culture of various tissues of turfgrass and regeneration of
plants therefrom is well known and widely published. For example,
reference may be had to Bradley, D. E. et al. 2001. Effects of
cultivar, explant treatment, and medium supplements on callus
induction and plantlet regeneration in perennial bluegrass. Int.
Turfgrass Soc. Res. J. 9:152-156; Cao, M. X., et al. 2006.
Transformation of recalcitrant turfgrass cultivars through
improvement of tissue culture and selection regime. Plant, Cell,
Tissue Organ Culture. 85:307-316; WenZhen, L. et al. Factors
effecting on tissue culture of perennial ryegrass (Lolium perenne
L.), Forest Res. 2004. 17:95-101 Thus, another aspect of this
invention is to provide cells which upon growth and differentiation
produce turfgrass plants having the physiological and morphological
characteristics of the turfgrass plants of the present
invention.
[0209] As used herein, the term "tissue culture" indicates a
composition comprising isolated cells of the same or a different
type or a collection of such cells organized into parts of a plant.
Exemplary types of tissue cultures are protoplasts, calli, plant
clumps, and plant cells that can generate tissue culture that are
intact in plants or parts of plants, such as embryos, pollen,
flowers, seeds, culms, leaves, stems, roots, root tips, anthers,
pistils and the like. Means for preparing and maintaining plant
tissue culture are well known in the art. By way of example, a
tissue culture comprising organs has been used to produce
regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and
5,977,445 describe certain techniques, the disclosures of which are
incorporated herein by reference.
EXAMPLES
[0210] The following examples are provided to further illustrate
the present invention and are not intended to limit the invention
beyond the limitations set forth in the appended claims. The three
cultivars listed below are the first in a series of new turfgrass
varieties. The following descriptions characterize their breeding
history, development, and the seed production characteristics of
these cultivars.
Example 1
Method of Producing a Turfgrass Plant having Desirable Looking Turf
when Mowed Infrequently
[0211] In the field hybridization nursery, individual spaced plants
are identified with promising characteristics such as the short
growth of leaves (defined as a preponderance of green leaf material
in the 0 to 4 inch zone above the soil surface), adequate seed
heads (defined as 15 g or more of clean seed recovered from one
individual spaced plant), and acceptable apomixes (defined as 80%
or higher). These promising plants are individually hand harvested
and cleaned of chaff and seed are planted in replicated turf
trials, subject to mowing once every 4 weeks or less frequent. Each
plot in the experiment is evaluated monthly during the growing
season, using a visual rating scale of 1 to 9, where 9 is highly
desirable turf and 5 is minimal acceptable quality, and 1 is
totally brown or dead. During one or more evaluation dates, the
chlorophyll meter is used to evaluate plots to impartially
differentiate green tissue from lifeless brown tissue created from
the scalping process.
[0212] Most turfgrass managers practice the "one third rule" for
clipping lawns and golf courses. The "one third rule" states that a
maximum of one third of the leaf area should be removed during any
one mowing. Deviating from this "one third rule" could lead to an
unacceptable quantity of the plant's photosynthetic surface being
removed. As a result, the plant goes into a shock mode and
sacrifices leaves in exchange for survival. Plant varieties that
grow vertically very tall or extremely fast are more subject to
damage from defoliation. The visual result is that the plant
rapidly turns brown within 24 hours and may take days or weeks to
recover. An additional result of this shock is the loss of shoot
density of the plant, defined as the number of live vegetative
shoots per square centimeter of ground surface. The present
invention described herein deals with plants that do not exhibit
this browning shock when they are subject to infrequent mowing.
[0213] The present invention provides a method of producing a
turfgrass plant having desirable-looking turf when mowed
infrequently, said method comprises selfing (pollinating a plant
and hybridizing it with its own pollen) for one or more generations
to create a plant with short growth of leaves (defined as a
preponderance of green leaf material in the 0 to 4 inch zone above
the soil surface), adequate seed heads (defined as 15 g or more of
clean seed recovered from one individual spaced plant), and
acceptable apomixes (defined as 80% or higher). The hybridization
can take place in a confined greenhouse using pollen restricting
bags, or take place in the field surrounded by clones of the same
genotype, or any combination of these two methods. The progeny of
said hybridizations are then field screened for approximately 15
months to identify plants with characteristics different than the
maternal parent line; such differences are most commonly identified
during the vegetative or pre-seedhead phase, but also may be
identified during seedhead expression, and include differences in
color and width of the leaves prior to seedhead expression and a
different date when the seedhead emerges from the sheath of said
turfgrass genotype. The method of the present invention
unexpectedly produced turfgrass plants having very basal growth
habit, with leaves growing close to the ground rather than growing
tall, which have desirable-looking turf when mowed
infrequently.
Example 2
Development and Characteristics of Kentucky Bluegrass Plant
`03-0582`
[0214] `03-0582` Kentucky bluegrass, also known by the designation
`J-0582`, originated as a low-growing, apomictic, single-plant
selection from the progeny of Jacklin Seed breeding line `98-2111`.
Breeding line `98-2111` was self pollinated to produce `03-0582`.
Breeding line `98-2111` originated as a selected low-growing
progeny of breeding line `96-0305`, first identified and harvested
in the field in May 1996. Breeding line `96-0305` had a medium seed
yield potential, 50% apomixis, medium-early reproductive maturity,
and an average culm length of 45 cm. Based on the prior art, it was
totally unexpected that a variety with low apomixes would give rise
to a variety with acceptable or high levels of apomixes. Breeding
line `96-0305` was a selection from the progeny of a hybrid cross
between Absolute Kentucky bluegrass pollinated by BlueChip, created
as a paired field cross.
[0215] Seed harvested from `03-0582` was used to establish
infrequently mowed trials in Idaho in 2006, and in Maryland and
Ohio in 2007. `03-0582` was selected for release based on its turf
quality performance in all months of the year under twice yearly
mowing, as well as its seed production characteristics in
Washington State.
[0216] `03-0582` is an ideal plant form for infrequent mowing with
low foliage and an unexpected proliferation of large dense seed
heads on short strawed culms. At anthesis the stand and panicles
have a yellowish green appearance with very little purplish
coloration with the exception of the anthers. The panicles take on
an almost whitish cast when seen from a distance; however, up close
several of the florets and sometimes entire panicles have an
appearance like they were dipped in diffuse purple dye. Spaced
nursery plants are shaped like a cupcake with vertical, uniformly
length straw and a proliferation of seed heads at the top. There is
a difference in culm length by about 50% from the center to the
perimeter of the plant. The foliage of the space plants is dark
green in color and broad in width. Heading is considered medium in
maturity however anthesis is occurring on the same day as with the
later 5-Steps Above type varieties (e.g., NuGlade and Award).
[0217] Culms are smooth to the touch in both directions and medium
green in color. Flag leaves are deep bluish green in color and
relatively short. They are smooth to the touch in the upward
direction and rough to the touch when felt in the direction toward
the base. The flag leaf node junction is vertical with very little
to no bend and little to no coloration difference from the culm
itself. Panicles are small in size with a good number of florets
that are medium to small in size. The panicles have numerous
branches, more than some varieties and the branches are mostly
oriented in the horizontal to downward direction with little to no
waviness. The tips are mostly drooping, as are the second or third
nodes from the tip; the rest are somewhat ascending. There is
little to no influence of soil on seed head productivity except on
the area of the field that received little to no water.
[0218] Progeny trials were conducted in spaced-plant nurseries,
established June 2010, to determine the level of apomixis. A survey
of 579 plants of `03-0582` showed that 4.2% of plants were variants
in the vegetative (pre-flowering) stage, 1.7% were heading maturity
variants, 0.3% were seedhead variants, 0% were variants appearing
in the dry-down phase, and 2.5% were miniature plants. Many of the
variants have shorter culms than the majority plant form with less
purple panicle coloration. Variants may not be obvious in
commercial seed production due to the masking effect of bulk
populations. The spaced-plant apomixis rate of `03-0582` averages
91%, but it varies from year to year depending on growing
conditions.
[0219] Variants in this variety appear primarily during the
vegetative growth stage before heading. The majority of the
variants have culm lengths equal to or shorter than the majority
plant form and as such will be relatively inconspicuous in seed
production. Soil variations make the variety appear non-uniform in
places however this is an adjustment of the variety to soil and not
a difference in genetics. Most of the variants have a similar
vegetative form to the majority with the exception of more vertical
or less vertical culm orientation. Approximately 2.5% of plants are
miniature plants which will not be apparent in seed production.
[0220] `03-0582` Kentucky bluegrass is a stable and uniform
variety. All seedlots evaluated have produced turf of comparable
quality and acceptable uniformity. Aberrant progeny are rogued from
Breeders, Foundation, and Registered fields to insure continued
uniformity and stability, but they will continue to occur in every
generation.
[0221] `03-0582` is a versatile Kentucky bluegrass variety, with
applications on golf courses, sod farms, sports fields, home lawns,
roadsides, cemeteries, and other turf areas, where bluegrass is
well adapted. `03-0582` performs well in full sun or partial
shade.
Example 3
Development and Characteristics of Kentucky Bluegrass Plant
`03-0441`
[0222] `03-0441` Kentucky bluegrass, also known by the designation
`J-0441`, originated as a low-growing, apomictic, single-plant
selection from the progeny of Jacklin Seed breeding line `01-0307`.
Breeding line `01-0307` was self pollinated to produce `03-0441`.
Breeding line `01-0307` originated as the low-growing progeny of a
hybrid field cross of Chicago II Kentucky bluegrass pollinated by
breeding line `95-2986`. Breeding line `95-2986` had an excellent
record in a 1997 turf trial in Idaho and a medium record in a 1999
trial in Maryland. It was first identified and harvested in the
field in May 1995. Breeding line `95-2986` was a selection from the
hybrid progeny of Midnight Kentucky bluegrass pollinated by
Limousine, created as a paired field cross Jun. 3, 1991.
[0223] Seed harvested from `03-0441` was used to establish
infrequently mowed trials in Idaho in 2006, and in Maryland and
Ohio in 2007. `03-0441` was selected for release based on its turf
quality performance in all months of the year under twice yearly
mowing, as well as its seed production characteristics in
Washington State.
[0224] `03-0441` is a classic low-growing plant with medium
reproductive maturity, stocky culms and a dense proliferation of
seed heads, featuring unexpected excellent powdery mildew disease
resistance. Space plants average 35 cm across after one year's
growth from rhizomes. Color of the stand at anthesis is light green
with almost no purple coloration whatsoever. Plants are strongly
blocky in shape with little to no lodging towards the perimeter of
space plants. There is little to no influence of soil from plant to
plant on productivity.
[0225] Panicles are small in size, however florets are large and
panicle numbers are high. There is some tapering of culm length
towards the perimeter of space plants giving the top of the space
plant a rounded appearance. The center culms are approximately 10
cm taller than the culms at the perimeter of the plant. Culms are
smooth to the touch in the upward direction and very lightly rough
in the downward direction. Panicles are drooping at the tip and
florets are drooping across most of the panicle but with the two
lower nodes ascending. There is no waviness to the branching. Flag
leaf margins are rough when felt in the downward direction and
slightly rough in the upward direction. Flag leaf node is olive
green in color and about 2 mm wide and slightly broader than the
culm itself. Foliage color of space plants is medium dark green and
flag leaves are distinctly bluish. Culms are medium green in
color.
[0226] Progeny trials were conducted in spaced-plant nurseries,
established June 2010, to determine the level of apomixis. A survey
of 974 plants of showed that 12.8% of plants were variants in the
vegetative (pre-flowering) stage, 1.4% were heading maturity
variants, 0.1% were seedhead variants, 0.9% were variants appearing
in the dry-down phase, and 2.1% were miniature plants. Many of the
variants have shorter culms than the majority plant form with less
purple panicle coloration. Variants may not be obvious in
commercial seed production due to the masking effect of bulk
populations. The spaced-plant apomixis rate of `03-0441` averages
83%, but it varies depending on growing conditions.
[0227] Variants are distributed across all phases of maturation.
The primary variant has a more yellow panicle and lighter colored
foliage than the majority plant form. About 1% of variants show a
strongly drooping seed head with a longer panicle and lighter
foliage. Most variants seem to share the stocky upright growth
habit of the majority plant form.
[0228] `03-0441` Kentucky bluegrass is a stable and uniform
variety. All seedlots evaluated have produced turf of comparable
quality and acceptable uniformity. Aberrant progeny are rogued from
Breeders, Foundation, and Registered fields to insure continued
uniformity and stability, but they will continue to occur in every
generation.
[0229] `03-0441` is a versatile Kentucky bluegrass variety, with
applications on golf courses, sod farms, sports fields, home lawns,
roadsides, cemeteries, and other turf areas, where bluegrass is
well adapted. `03-0441` performs well in full sun or partial
shade.
Example 4
Development and Characteristics of Kentucky Bluegrass Plant
`99-2495`
[0230] `99-2495` Kentucky bluegrass, also known by the designation
`J-2495`, originated as a low-growing, apomictic, single-plant
selection from the progeny of Jacklin Seed breeding line `97-0429`.
Breeding line `97-0429` was self pollinated to produce `99-2495`.
Breeding line `97-0429` had a medium leaf color and reproductive
maturity, a culm length of 54 cm, a high level of apomixis, and was
susceptible to ergot and powdery mildew. Breeding line `97-0429`
originated as a low-growing progeny of a hybrid cross of BlueChip
Kentucky bluegrass pollinated by Blacksburg, created as a paired
field cross Jun. 3, 1999.
[0231] Seed harvested from `99-2495` was used to establish
infrequently mowed trials in Idaho in 2006, and in Maryland and
Ohio in 2007. `99-2495` was selected for release based on its turf
quality performance in all months of the year under twice yearly
mowing, as well as its seed production characteristics in
Washington State.
[0232] `99-2495` is a late maturing, shorter growing, fine textured
version of a standard 5-Steps Above variety. `99-2495` has
susceptibility to powdery mildew. Space plants average 45 cm across
after one year's growth from rhizomes. The most unexpected and
distinctive characteristic of this variety is its extremely fine,
extremely dark green foliage as spaced plants. Of all the varieties
in the Breeder blocks, this one has the most fine, dark leaves.
Seed head productivity is excellent with some influence of
plant-to-plant yield variation due to soil.
[0233] Panicle color at anthesis is light green with a diffusion of
purplish color on the tips of florets. However, it does not appear
as a distinct speckling as it does in some cultivars. Culms are
medium green in color and are mostly smooth with only a bit of
roughness. Flag leafs are smooth when felt in the upward direction
and rough in the downward direction. Attachment node of flag leaves
is the same color as the culm with only a slight knob-like
appearance, with little to no bend at the node. Spaced plant form
is mostly blocky with a uniform culm length with little tapering
(maybe 5 cm) towards the perimeter of the plant. Panicles are
medium in size and florets are medium in size. Internode length
gets shorter towards the tip of the panicle. There is a minor
amount of waviness to the branching in the panicle, mostly towards
the tip. Tips are mostly upright to slightly nodding. Most panicle
branching is ascending however the lowest node is horizontal to
descending. The attachment of the florets is horizontal to slightly
downward.
[0234] Progeny trials were conducted in spaced-plant nurseries,
established June 2010, to determine the level of apomixis. A survey
of 1044 plants of `99-2495` showed that 11.7% of plants were
variants in the vegetative (pre-flowering) stage, 1.8% were heading
maturity variants, 0.2% were seedhead variants, 0.3% were variants
appearing in the dry-down phase, and 0.8% were miniature plants.
Many of the variants have shorter culms than the majority plant
form with less purple panicle coloration. Variants may not be
obvious in commercial seed production due to the masking effect of
bulk populations. The spaced-plant apomixis rate of `99-2495`
averages 85%, but it varies depending on growing conditions.
[0235] Variants in this cultivar appear during outbreaks of powdery
mildew which shows resistant types. Most of the vegetative variants
are slightly taller than the majority plant form. About 0.2% of
plants are an open, easily lodged plant with drooping seed heads.
The heading maturity variants differ from the majority plant form
by a slightly taller and more yellow panicles.
[0236] `99-2495` Kentucky bluegrass is a stable and uniform
variety. All seedlots evaluated have produced turf of comparable
quality and acceptable uniformity. Aberrant progeny are rogued from
Breeders, Foundation, and Registered fields to insure continued
uniformity and stability, but they will continue to occur in every
generation.
[0237] `99-2495` is a versatile Kentucky bluegrass variety, with
applications on golf courses, sod farms, sports fields, home lawns,
roadsides, cemeteries, and other turf areas, where bluegrass is
well adapted. `99-2495` performs well in full sun or partial
shade.
Example 5
Physiological and Morphological Characteristics of New Kentucky
Bluegrass Varieties
[0238] Table 1 shows a summary of the data obtained from trials
held in 2010. The most promising varieties were planted into a
spaced plant breeder blocks in Connell, Washington, in the heart of
Kentucky bluegrass seed production country. The goal was to see how
they performed in seed production, to pick the best of the best.
After a year of observation and data collection, the three best
entries were chosen; they appear at the top of Table 1. The
remaining varieties in the table were not chosen due to one or more
undesirable traits.
[0239] In Table 1 below, apomixis is the percentage of apomictic
seed reproduction in the varieties. A value of 100 would indicate
all offspring are identical genetically. Data in the remaining
columns were visually rated on a 1 to 9 scale, with 9 equal to most
low-growing characteristics, greatest powdery mildew resistance,
highest seed yield, least soil variability from one plant to the
next, and highest general turfgrass quality ratings in Ohio and
Maryland averaged across monthly readings taken over three growing
seasons. Mean values over 5 would be considered good and values of
8 or more could be considered outstanding. Commercial standards
(i.e. varieties that are being sold on the market today) were
tested in the initial Idaho studies. All commercial standards
performed poorly compared to the promising experimentals of the
present invention.
TABLE-US-00001 TABLE 1 Dwarf Dwarf Mildew Yield Soil var 2007 OH
2007 OH 2007 MD 2007 MD ID 28apr11 16jun11 28apr11 16jun11 9 = none
Rating 2008 Rating 2009 Rating 2008 Rating 2009 Apomixis 03-0582 8
7 8 8 8 6.00 5.21 6.00 5.21 91 03-0441 8 6.5 9 7 6 6.00 6.57 6.67
4.00 83 99-2495 5.5 5.5 6 5.5 6 5.67 5.98 6.33 6.17 85 02-2139 6 7
2 4 5 6.33 6.33 6.67 4.50 91 02-2217 6 5 7 5 3 5.00 4.76 7.33 5.71
89 93-1436 6 3 7 8 8 4.33 6.50 7.33 5.17 87 93-1897 4 6 4 5 6 6.33
6.86 5.00 4.33 93 97-0428 6 6.5 4.5 6.5 5.5 5.00 6.55 6.00 5.50 91
99-2304 4 4 5 6 4 6.67 6.83 90 99-2891 6 7 7 6 7 4.67 5.61 6.67
5.67 95
[0240] As shown in Table 1, Kentucky bluegrass varieties designated
`03-0582`, `03-0441`, and `99-2495` have the necessary attributes
to provide a beautiful lawn with substantially reduced mowing
requirements.
Example 6
Chlorophyll Concentration of new Kentucky Bluegrass Varieties
[0241] A Field Scout CM 1000.TM. chlorophyll meter was used for
documenting the chlorophyll in field plots of infrequently mowed
turf. In August 2009, 1000+ experimental plots were planted in Post
Falls, Id. These plots were maintained under three-times-per-year
mowing at 2.5-inch cut. Some plots did well under this mowing
regime but most did not. A typical plot in the latter contained a
majority of brown, lifeless stems and very little green leafy
material. The chlorophyll meter picked up these differences in the
good and bad plots and put a number to it.
[0242] This procedure is different than the lab procedure described
elsewhere in that the CM 1000.TM. was detecting differences between
the brown lifeless material and green leafy material in the plots
maintained under infrequent mowing. In the lab procedure, only
green tissue was used for chlorophyll concentration analysis.
Moreover, the CM 1000.TM. did not produce chlorophyll readings in
terms of parts per million of chlorophyll in leaf tissue. Instead,
it produced a reading of 0 to 999 based on what the sensors
detected in certain narrow wave bands (described below) which are
sensitive to the spectral nature of the chlorophyll molecule. In
the test plots, readings fell between 150 and 600 on that
scale.
[0243] Visual turfgrass quality ratings and the CM 1000.TM.
chlorophyll scans were reconciled as shown in FIG. 1. Forty-two
readings were taken on the plots described above on Oct. 29, 2012
using both visual rating and CM 1000.TM. meter readings. These
observations were graphed and were analyzed using linear
regression. Solving the regression equation for a turf quality
rating of `5` (the minimum acceptable number used in plant breeding
to develop the cultivars) the equivalent CM 1000.TM. reading would
be 341.7. The turfgrass plants of the present invention have a CM
1000.TM. meter reading of 341.7 and above on turfgrass mowed once
every 4 weeks or fewer, combined with a previously specified
chlorophyll concentration in green tissue--in other words, the
ability to produce acceptable lawn turf while being mowed
infrequently.
[0244] Regression analysis was performed to correlate the visual
turfgrass quality ratings with Field Scout CM 1000.TM. scan results
for test plots mowed three times per year in Idaho and the results
are shown in FIG. 1. Regression analysis indicated that a visual
quality estimate of 5 corresponds to a CM 1000.TM. meter reading of
341.7, with an unexpectedly high correlation co-efficient of 0.86,
and an R.sup.2 value of 0.73, which was significant at the 0.0001
level. In FIG. 1, circles represent individual observations, a
solid line indicates regression fit to the data, and dashed lines
indicate 95% confidence intervals.
[0245] Table 2 shows the chlorophyll concentration of Kentucky
bluegrass varieties designated `03-0582`, `03-0441`, and `99-2495`
compared to similar varieties; varieties that are under development
are listed with their experimental numbers, while commercial
varieties are shown with their cultivar names. Clippings were
weighed (0.1g) and placed in a glass test tube (1.0 cm in width and
14.8 cm in length) with 10 mL of dimethyl sulfoxide (DMSO) (Hiscox
and Israelstam, 1979). Samples were incubated in 65.degree. C.
water for 1.5 hr. Upon completion, samples were passed through
filter paper (Whatman 41, Whatman, England) and remaining extract
(1 mL) transferred into cuvettes. Absorbance values were recorded
at 663 nm and 645 nm wavelengths using a Spectrophotometer. Blanks
were initially run and also after every sixth sample. The following
formula was used to calculate total shoot chlorophyll: (mg
g.sup.-1)=(8.02*D663+20.2 * D645)*0.1 (Amon, 1949). Column 1 shows
the sample name and column 2 shows the total shoot chlorophyll in
mg/g.
TABLE-US-00002 TABLE 2 Sample Total Shoot Chlorophyll (mg/g)
03-0582 2.57 99-2495 1.91 03-0441 3.21 O2-2139 2.22 O2-2217 2.61
93-1897 2.02 99-2304 2.42 97-0428 2.82 99-2891 3.46 J-1770 2.35
Troy 1.18 Camas 1.40 Nublue 1.75 Merit 1.23 Action 0.89 Thermal
1.54
[0246] As shown in Table 2, bluegrass varieties designated
`03-0582`, `03-0441`, and `99-2495` all have chlorophyll
concentrations above 1.8 mg/g, whereas the commercial varieties
Troy, Camas, Nublue, Merit, Action and Thermal all have chlorophyll
concentrations below 1.8 mg/g.
[0247] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. For example, if the range 10-15 is disclosed, then
11, 12, 13, and 14 are also disclosed. All methods described herein
can be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and
does not pose a limitation on the scope of the invention unless
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the invention.
Deposit Information
[0248] A deposit of the bluegrass varieties `03-0582`, `03-0441`
and `99-2495` developed by the method of the present invention
disclosed above and recited in the appended claims has been made
with the National Collections of Industrial, Food and Marine
Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB24 3RY,
United Kingdom. The date of deposit was Feb. 12, 2013. The deposit
of 2,500 seeds was taken from the same deposit maintained by
Jacklin Seed since prior to the filing date of this application.
Upon allowance of any claims in this application, all restrictions
on the availability to the public of the variety will be
irrevocably removed by affording access to a deposit of at least
2,500 seeds of the same variety with the National Collections of
Industrial, Food and Marine Bacteria (NCIMB), Aberdeen, Scotland,
and the deposit is intended to meet all of the requirements of 37
C.F.R. 1.801-1.809. The NCIMB numbers are 42111, 42110, and 42112,
respectively. The deposit will be maintained in the depository for
a period of 30 years, or 5 years after the last request, or for the
effective life of the patent, whichever is longer, and will be
replaced as necessary during that period.
[0249] While a number of exemplary aspects and embodiments have
been discussed above, those of skill in the art will recognize
certain modifications, permutations, additions and sub-combinations
thereof. It is therefore intended that the following appended
claims and claims hereafter introduced are interpreted to include
all such modifications, permutations, additions and
sub-combinations as are within their true spirit and scope.
* * * * *