Note: Descriptions are shown in the official language in which they were submitted.
CA 02883471 2015-02-27
Title: CANOLA VARIETY PV 532 G
FIELD
The discovery is in the field of Brassica napus breeding (i.e., canola
breeding), specifically relating to the canola variety designated PV 532 G.
BACKGROUND
The present discovery relates to a novel rapeseed variety designated PV 532
G which is the result of years of careful breeding and selection. Since such
variety is
of high quality and possesses a relatively low level of erucic acid in the
vegetable oil
component and a relatively low level of glucosinolate content in the meal
component,
it can be termed "canola" in accordance with the terminology commonly used by
plant scientists.
The goal of plant breeding is to combine in a single variety or hybrid various
desirable traits. For field crops, these traits may include resistance to
diseases and
insects, tolerance to heat and drought, reducing the time to crop maturity,
greater
yield, and better agronomic quality. With mechanical harvesting of many crops,
uniformity of plant characteristics such as germination and stand
establishment,
growth rate, maturity, and plant and pod height, is important. The creation of
new
superior, agronomically sound, and stable high-yielding cultivars of many
plant types
including canola has posed an ongoing challenge to plant breeders. Therefore,
there is a continuing need in the field of agriculture for canola plants
having desirable
agronomic and industrial characteristics.
SUMMARY
A novel Brassica napus variety designated PV 532 G is provided. This
discovery thus relates to the seeds of the PV 532 G variety, to plants of the
PV 532
G variety, and to methods for producing a canola plant by crossing the PV 532
G
variety with itself or another canola plant (whether by use of male sterility
or open
pollination), and to methods for producing a canola plant containing in its
genetic
material one or more transgenes, and to transgenic plants produced by that
method.
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This discovery also relates to canola seeds and plants produced by crossing
the
variety PV 532 G with another line.
DEFINITIONS
In the description and tables which follow, a number of terms are used. In
order to aid in a clear and consistent understanding of the specification, the
following
definitions and evaluation criteria are provided.
Anther Fertility. The ability of a plant to produce pollen; measured by pollen
production. 1 = sterile, 9 = all anthers shedding pollen (vs. Pollen Formation
which is
amount of pollen produced).
Anther Arrangement. The general disposition of the anthers in typical fully
opened flowers is observed.
Chlorophyll Content. The typical chlorophyll content of the mature seeds is
determined by using methods recommended by the Western Canada
Canola/Rapeseed Recommending Committee (WCC/RRC). 1 = low (less than 8
ppm), 2 = medium (8 to 15 ppm), 3 = high (greater than 15 ppm). Also,
chlorophyll
could be analyzed using NIR (Near Infrared) spectroscopy as long as the
instrument
is calibrated according to the manufacturer's specifications.
CMS. Abbreviation for cytoplasmic male sterility.
Cotyledon. A cotyledon is a part of the embryo within the seed of a plant; it
is
also referred to as a seed leaf. Upon germination, the cotyledon may become
the
embryonic first leaf of a seedling.
Cotyledon Length. The distance between the indentation at the top of the
cotyledon and the point where the width of the petiole is approximately 4 mm.
Cotyledon Width. The width at the widest point of the cotyledon when the
plant is at the two to three-leaf stage of development. 3 = narrow, 5 =
medium, 7 =
wide.
CV%: Abbreviation for coefficient of variation.
Disease Resistance: Resistance to various diseases is evaluated and is
expressed on a scale of 0 = not tested, 1 = resistant, 3 = moderately
resistant, 5 =
moderately susceptible, 7 = susceptible, and 9 = highly susceptible.
Erucic Acid Content: The percentage of the fatty acids in the form of C22:1.as
determined by one of the methods recommended by the WCC/RRC, being AOCS
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Official Method Ce 2-66 Preparation of Methyl esters of Long-Chain Fatty Acids
or
AOCS Official Method Ce 1-66 Fatty Acid Composition by Gas Chromatography.
Fatty Acid Content: The typical percentages by weight of fatty acids present
in the endogenously formed oil of the mature whole dried seeds are determined.
During such determination the seeds are crushed and are extracted as fatty
acid
methyl esters following reaction with methanol and sodium methoxide. Next the
resulting ester is analyzed for fatty acid content by gas liquid
chromatography using
a capillary column which allows separation on the basis of the degree of
unsaturation
and fatty acid chain length. This procedure is described in the work of Daun,
et al.,
(1983) J. Amer. Oil Chem. Soc. 60:1751 to 1754.
Flower Bud Location. A determination is made whether typical buds are
disposed above or below the most recently opened flowers.
Flower Date 50%. (Same as Time to Flowering) The number of days from
planting until 50% of the plants in a planted area have at least one open
flower.
Flower Petal Coloration. The coloration of open exposed petals on the first
day of flowering is observed.
Frost Tolerance (Spring Type Only). The ability of young plants to withstand
late spring frosts at a typical growing area is evaluated and is expressed on
a scale
of 1 (poor) to 5 (excellent).
Gene Silencing. The interruption or suppression of the expression of a gene
at the level of transcription or translation.
Genotype. Refers to the genetic constitution of a cell or organism.
Glucosinolate Content. The total glucosinolates of seed at 8.5% moisture, as
measured by AOCS Official Method AK-1-92 (determination of glucosinolates
content in rapeseed ¨colza by HPLC), is expressed as micromoles per gram of
defatted, oil-free meal. Capillary gas chromatography of the trimethylsityl
derivatives
of extracted and purified desulfoglucosinolates with optimization to obtain
optimum
indole glucosinolate detection is described in "Procedures of the Western
Canada
Canola/Rapeseed Recommending Committee Incorporated for the Evaluation and
Recommendation for Registration of Canola/Rapeseed Candidate Cultivars in
Western Canada". Also, glucosinolates could be analyzed using NIR (Near
Infrared)
spectroscopy as long as the instrument is calibrated according to the
manufacturer's
specifications.
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Grain. Seed produced by the plant or a self or sib of the plant that is
intended
for food or feed use.
Green Seed. The number of seeds that are distinctly green throughout as
defined by the Canadian Grain Commission. Expressed as a percentage of seeds
tested.
Herbicide Resistance: Resistance to various herbicides when applied at
standard recommended application rates is expressed on a scale of 1
(resistant), 2
(tolerant), or 3 (susceptible).
Leaf Anthocvanin Coloration. The presence or absence of leaf anthocyanin
113 coloration, and the degree thereof if present, are observed when the
plant has
reached the 9- to 11-leaf stage.
Leaf Attachment to Stem. The presence or absence of clasping where the
leaf attaches to the stem, and when present the degree thereof, are observed.
Leaf Attitude. The disposition of typical leaves with respect to the petiole
is
observed when at least 6 leaves of the plant are formed.
Leaf Color. The leaf blade coloration is observed when at least six leaves of
the plant are completely developed.
Leaf Glaucosity. The presence or absence of a fine whitish powdery coating
on the surface of the leaves, and the degree thereof when present, are
observed.
Leaf Length. The length of the leaf blades and petioles are observed when at
least six leaves of the plant are completely developed.
Leaf Lobes. The fully developed upper stem leaves are observed for the
presence or absence of leaf lobes when at least 6 leaves of the plant are
completely
developed.
Leaf Margin Indentation. A rating of the depth of the indentations along the
upper third of the margin of the largest leaf. 1 = absent or very weak (very
shallow),
3 = weak (shallow), 5 = medium, 7 = strong (deep), 9 = very strong (very
deep).
Leaf Margin Hairiness. The leaf margins of the first leaf are observed for the
presence or absence of pubescence, and the degree thereof, when the plant is
at
the two leaf-stage.
Leaf Margin Shape. A visual rating of the indentations along the upper third
of
the margin of the largest leaf. 1 = undulating, 2 = rounded, 3 = sharp.
Leaf Surface. The leaf surface is observed for the presence or absence of
wrinkles when at least six leaves of the plant are completely developed.
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Leaf Tip Reflexion. The presence or absence of bending of typical leaf tips
and the degree thereof, if present, are observed at the six to eleven leaf-
stage.
Leaf Upper Side Hairiness. The upper surfaces of the leaves are observed
for the presence or absence of hairiness, and the degree thereof if present,
when at
least six leaves of the plant are formed.
Leaf Width. The width of the leaf blades is observed when at least six leaves
of the plant are completely developed.
Locus. A specific location on a chromosome.
Locus Conversion. A locus conversion refers to plants within a variety that
have been modified in a manner that retains the overall genetics of the
variety and
further comprises one or more loci with a specific desired trait, such as male
sterility,
insect, disease or herbicide resistance. Examples of single locus conversions
include mutant genes, transgenes and native traits finely mapped to a single
locus.
One or more locus conversion traits may be introduced into a single canola
variety.
Lodging Resistance. Resistance to lodging at maturity is observed. 1 = not
tested, 3 = poor, 5 = fair, 7 = good, 9 = excellent.
LSD. Abbreviation for least significant difference.
Maturity. The number of days from planting to maturity is observed, with
maturity being defined as the plant stage when pods with seed change color,
occurring from green to brown or black, on the bottom third of the pod-bearing
area
of the main stem.
NMS. Abbreviation for nuclear male sterility.
Number of Leaf Lobes. The frequency of leaf lobes, when present, is
observed when at least six leaves of the plant are completely developed.
Oil Content: The typical percentage by weight oil present in the mature whole
dried seeds is determined by ISO 10565:1993 Oilseeds Simultaneous
determination
of oil and water - Pulsed NMR method. Also, oil could be analyzed using NIR
(Near
Infrared) spectroscopy as long as the instrument is calibrated according to
the
manufacturer's specifications, reference AOCS Procedure Am 1-92 Determination
of
Oil, Moisture and Volatile Matter, and Protein by Near-Infrared Reflectance.
Pedicel Length. The typical length of the silique stem when mature is
observed. 3 = short, 5 = medium, 7 = long.
Petal Length. The lengths of typical petals of fully opened flowers are
observed. 3 = short, 5 = medium, 7 = long.
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Petal Width. The widths of typical petals of fully opened flowers are
observed.
3 = short, 5 = medium, 7 = long.
Petiole Length. The length of the petioles is observed, in a line forming
lobed
leaves, when at least six leaves of the plant are completely developed. 3 =
short, 5
= medium, 7 = long.
Plant Height. The overall plant height at the end of flowering is observed. 3
=
short, 5 = medium, 7 = tall.
Ploidy. This refers to the number of chromosomes exhibited by the line, for
example diploid or tetraploid.
Pod Anthocyanin Coloration. The presence or absence at maturity of silique
anthocyanin coloration, and the degree thereof if present, are observed.
Pod (Silique) Beak Length. The typical length of the silique beak when
mature is observed. 3 = short, 5 = medium, 7 = long.
Pod Habit. The typical manner in which the siliques are borne on the plant at
maturity is observed.
Pod (Silique) Length. The typical silique length is observed. 1 = short (less
than 7 cm), 5 = medium (7 to 10 cm), 9 = long (greater than 10 cm).
Pod (Silique) Attitude. A visual rating of the angle joining the pedicel to
the
pod at maturity. 1 = erect, 3 = semi-erect, 5 = horizontal, 7 = semi-drooping,
9 =
drooping.
Pod Type. The overall configuration of the silique is observed.
Pod (Silique) Width. The typical pod width when mature is observed. 3 =
narrow (3 mm), 5 = medium (4 mm), 7 = wide (5 mm).
Pollen Formation. The relative level of pollen formation is observed at the
time of dehiscence.
Protein Content: The typical percentage by weight of protein in the oil free
meal of the mature whole dried seeds is determined by AOCS Official Method Ba
4e-
93 Combustion Method for the Determination of Crude Protein. Also, protein
could
be analyzed using NIR (Near Infrared) spectroscopy as long as the instrument
is
calibrated according to the manufacturer's specifications, reference AOCS
Procedure Am 1-92 Determination of Oil, Moisture and Volatile Matter, and
Protein
by Near-Infrared Reflectance.
Resistance. The ability of a plant to withstand exposure to an insect,
disease,
herbicide, or other condition. A resistant plant variety or hybrid will have a
level of
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resistance higher than a comparable wild-type variety or hybrid. "Tolerance"
is a
term commonly used in crops such as canola, soybean, and sunflower affected by
an insect, disease, such as Sclerotinia, herbicide, or other condition and is
used to
describe an improved level of field resistance.
Root Anthocyanin Coloration. The presence or absence of anthocyanin
coloration in the skin at the top of the root is observed when the plant has
reached at
least the six- leaf stage.
Root Anthocyanin Expression. When anthocyanin coloration is present in skin
at the top of the root, it further is observed for the exhibition of a reddish
or bluish
cast within such coloration when the plant has reached at least the six-leaf
stage.
Root Anthocyanin Streaking. When anthocyanin coloration is present in the
skin at the top of the root, it further is observed for the presence or
absence of
streaking within such coloration when the plant has reached at least the six-
leaf
stage.
Root Chlorophyll Coloration. The presence or absence of chlorophyll
coloration in the skin at the top of the root is observed when the plant has
reached at
least the six-leaf stage.
Root Coloration Below Ground. The coloration of the root skin below ground
is observed when the plant has reached at least the six-leaf stage.
Root Depth in Soil. The typical root depth is observed when the plant has
reached at least the six-leaf stage.
Root Flesh Coloration. The internal coloration of the root flesh is observed
when the plant has reached at least the six-leaf stage.
SE. Abbreviation for standard error.
Seedling Growth Habit. The growth habit of young seedlings is observed for
the presence of a weak or strong rosette character. 1 = weak rosette, 9 =
strong
rosette.
Seeds Per Pod. The average number of seeds per pod is observed.
Seed Coat Color. The seed coat color of typical mature seeds is observed. 1
= black, 2 = brown, 3 = tan, 4 = yellow, 5 = mixed, 6 = other.
Seed Coat Mucilage. The presence or absence of mucilage on the seed coat
is determined and is expressed on a scale of 1 (absent) to 9 (present). During
such
determination a petri dish is filled to a depth of 0.3 cm. with water provided
at room
temperature. Seeds are added to the petri dish and are immersed in water where
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they are allowed to stand for five minutes. The contents of the petri dish
containing
the immersed seeds are then examined under a stereo microscope equipped with
transmitted light. The presence of mucilage and the level thereof is observed
as the
intensity of a halo surrounding each seed.
Seed Size. The weight in grams of 1,000 typical seeds is determined at
maturity while such seeds exhibit a moisture content of approximately 5 to 6
percent
by weight.
Shatter Resistance. Resistance to silique shattering is observed at seed
maturity. 1 = not tested, 3 = poor, 5 = fair, 7 = good, 9 = does not shatter.
SI. Abbreviation for self-incompatible.
Speed of Root Formation. The typical speed of root formation is observed
when the plant has reached the four to eleven-leaf stage.
SSFS. Abbreviation for Sclerotinia sclerotiorum Field Severity score, a rating
based on both percentage infection and disease severity.
Stem Anthocyanin Intensity. The presence or absence of leaf anthocyanin
coloration and the intensity thereof, if present, are observed when the plant
has
reached the nine to eleven-leaf stage. 1 = absent or very weak, 3 = weak, 5 =
medium, 7 = strong, 9 = very strong.
Stem Lodging at Maturity. A visual rating of a plant's ability to resist stem
lodging at maturity. 1 = very weak (lodged), 9 = very strong (erect).
Time to Flowering. A determination is made of the number of days when at
least 50 percent of the plants have one or more open buds on a terminal raceme
in
the year of sowing.
Seasonal Type. This refers to whether the new line is considered to be
primarily a Spring or Winter type of canola.
Winter Survival (Winter Type Only).
The ability to withstand winter
temperatures at a typical growing area is evaluated and is expressed on a
scale of 1
(poor) to 5 (excellent).
DETAILED DESCRIPTION
Field crops are bred through techniques that take advantage of the plant's
method of pollination.
A plant is self-pollinated if pollen from one flower is
transferred to the same or another flower of the same plant or a genetically
identical
plant. A plant is sib-pollinated when individuals within the same family or
line are
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used for pollination. A plant is cross-pollinated if the pollen comes from a
flower on a
genetically different plant from a different family or line. The term "cross-
pollination"
used herein does not include self-pollination or sib-pollination.
In the practical application of a chosen breeding program, the breeder often
initially selects and crosses two or more parental lines, followed by repeated
selfing
and selection, thereby producing many unique genetic combinations. The breeder
can theoretically generate billions of different genetic combinations via
crossing,
selfing and mutagenesis. However, the breeder commonly has no direct control
at
the cellular level of the plant. Therefore, two breeders will never
independently
lo develop the same variety having the same canola traits.
In each cycle of evaluation, the plant breeder selects the germplasm to
advance to the next generation. This germplasm is grown under chosen
geographical, climatic and soil conditions, and further selections are then
made
during and at the end of the growing season. The characteristics of the
varieties
is developed are incapable of prediction in advance. This unpredictability
is because
the selection occurs in unique environments, with no control at the DNA level
(using
conventional breeding procedures), and with millions of different possible
genetic
combinations being generated. A breeder of ordinary skill cannot predict in
advance
the final resulting varieties that are to be developed, except possibly in a
very gross
20 and general fashion. Even the same breeder is incapable of producing the
same
variety twice by using the same original parents and the same selection
techniques.
This unpredictability commonly results in the expenditure of large research
monies
and effort to develop a new and superior canola variety.
Canola breeding programs utilize techniques such as mass and recurrent
25 selection, backcrossing, pedigree breeding and haploidy. For a general
description
of rapeseed and Canola breeding, see, Downey and Rakow, (1987) "Rapeseed and
Mustard" In: Principles of Cultivar Development, Fehr, (ed.), pp 437-486; New
York;
Macmillan and Co.; Thompson, (1983) "Breeding winter oilseed rape Brassica
napus"; Advances in Applied Biology 7:1-104; and Ward, et. al., (1985) Oilseed
30 Rape, Farming Press Ltd., Wharfedale Road, Ipswich, Suffolk.
Recurrent selection is used to improve populations of either self- or cross-
pollinating Brassica. Through recurrent selection, a genetically variable
population of
heterozygous individuals is created by intercrossing several different
parents. The
best plants are selected based on individual superiority, outstanding progeny,
and/or
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excellent combining ability. The selected plants are intercrossed to produce a
new
population in which further cycles of selection are continued. Various
recurrent
selection techniques are used to improve quantitatively inherited traits
controlled by
numerous genes.
Breeding programs use backcross breeding to transfer genes for a simply
inherited, highly heritable trait into another line that serves as the
recurrent parent.
The source of the trait to be transferred is called the donor parent. After
the initial
cross, individual plants possessing the desired trait of the donor parent are
selected
and are crossed (backcrossed) to the recurrent parent for several generations.
The
resulting plant is expected to have the attributes of the recurrent parent and
the
desirable trait transferred from the donor parent. This approach has been used
for
breeding disease resistant phenotypes of many plant species, and has been used
to
transfer low erucic acid and low glucosinolate content into lines and breeding
populations of Brassica.
Pedigree breeding and recurrent selection breeding methods are used to
develop varieties from breeding populations. Pedigree breeding starts with the
crossing of two genotypes, each of which may have one or more desirable
characteristics that is lacking in the other or which complements the other.
If the two
original parents do not provide all of the desired characteristics, other
sources can
be included in the breeding population. In the pedigree method, superior
plants are
selfed and selected in successive generations. In the succeeding generations
the
heterozygous condition gives way to homogeneous lines as a result of self-
pollination and selection. Typically in the pedigree method of breeding, five
or more
generations of selfing and selection are practiced: F1 to F2; F2 to F3; F3 to
F4; F4 to
F5, etc. For example, two parents that are believed to possess favorable
complementary traits are crossed to produce an F1. An F2 population is
produced by
selfing one or several Fi's or by intercrossing two Fi's (i.e., sib mating).
Selection of
the best individuals may begin in the F2 population, and beginning in the F3
the best
individuals in the best families are selected. Replicated testing of families
can begin
in the Fit generation to improve the effectiveness of selection for traits
with low
heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best
lines or
mixtures of phenotypically similar lines commonly are tested for potential
release as
new cultivars. Backcrossing may be used in conjunction with pedigree breeding;
for
example, a combination of backcrossing and pedigree breeding with recurrent
CA 02883471 2015-02-27
selection has been used to incorporate blackleg resistance into certain
cultivars of
Brassica napus.
Plants that have been self-pollinated and selected for type for many
generations become homozygous at almost all gene loci and produce a uniform
population of true breeding progeny. If desired, double-haploid methods can
also be
used to extract homogeneous lines. A cross between two different homozygous
lines produces a uniform population of hybrid plants that may be heterozygous
for
many gene loci. A cross of two plants each heterozygous at a number of gene
loci
will produce a population of hybrid plants that differ genetically and will
not be
to uniform.
The 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, such as F1 hybrid variety or open pollinated variety. A
true
breeding homozygous line can also be used as a parental line (inbred line) in
a
is commercial hybrid. If the line is being developed as an inbred for use
in a hybrid, an
appropriate pollination control system should be incorporated in the line.
Suitability
of an inbred line in a hybrid combination will depend upon the combining
ability
(general combining ability or specific combining ability) of the inbred.
Various breeding procedures are also utilized with these breeding and
20 selection methods. The single-seed descent procedure in the strict sense
refers to
planting a segregating population, harvesting a sample of one seed per plant,
and
using the one-seed sample to plant the next generation. When the population
has
been advanced from the F2 to the desired level of inbreeding, the plants from
which
lines are derived will each trace to different F2 individuals. The number of
plants in a
25 population declines each generation due to failure of some seeds to
germinate or
some plants to produce at least one seed. As a result, not all of the F2
plants
originally sampled in the population will be represented by a progeny when
generation advance is completed.
In a multiple-seed procedure, canola breeders commonly harvest one or more
30 pods from each plant in a population and thresh them together to form a
bulk. Part
of the bulk is used to plant the next generation and part is put in reserve.
The
procedure has been referred to as modified single-seed descent or the pod-bulk
technique. The multiple-seed procedure has been used to save labor at harvest.
It
is considerably faster to thresh pods with a machine than to remove one seed
from
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each by hand for the single-seed procedure. The multiple-seed procedure also
makes it possible to plant the same number of seeds of a population each
generation of inbreeding. Enough seeds are harvested to make up for those
plants
that did not germinate or produce seed. If desired, doubled-haploid methods
can be
used to extract homogeneous lines.
Molecular markers, including 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. 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 in the plant's genome.
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 markers 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 or Marker
Assisted Selection (MAS).
The production of doubled haploids can also be used for the development of
inbreds in the breeding program. In Brassica napus, microspore culture
technique is
used in producing haploid embryos. The haploid embryos are then regenerated on
appropriate media as haploid plantlets, doubling chromosomes of which results
in
doubled haploid plants. This can be advantageous because the process omits the
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generations of selfing needed to obtain a homozygous plant from a heterozygous
source.
The development of a canola hybrid in a canola plant breeding program
involves three steps: (1) the selection of plants from various germplasm pools
for
initial breeding crosses; (2) the selfing of the selected plants from the
breeding
crosses for several generations to produce a series of inbred lines, which,
although
different from each other, breed true and are highly uniform; and (3) crossing
the
selected inbred lines with different inbred lines to produce the hybrids.
During the
inbreeding process in canola, the vigor of the lines decreases. Vigor is
restored
when two different inbred lines are crossed to produce the hybrid. An
important
consequence of the homozygosity and homogeneity of the inbred lines is that
the
hybrid between a defined pair of inbreds will always be the same. Once the
inbreds
that give a superior hybrid have been identified, the hybrid seed can be
reproduced
indefinitely as long as the homogeneity of the inbred parents is maintained.
Controlling Self-Pollination
Canola varieties are mainly self-pollinated; therefore, self-pollination of
the
parental varieties must be controlled to make hybrid development feasible. In
developing improved new Brassica hybrid varieties, breeders may use self-
incompatible (SI), cytoplasmic male sterile (CMS) or nuclear male sterile
(NMS)
Brassica plants as the female parent. In using these plants, breeders are
attempting
to improve the efficiency of seed production and the quality of the F1 hybrids
and to
reduce the breeding costs. When hybridization is conducted without using SI,
CMS
or NMS plants, it is more difficult to obtain and isolate the desired traits
in the
progeny (F1 generation) because the parents are capable of undergoing both
cross-
pollination and self-pollination. If one of the parents is a SI, CMS or NMS
plant that
is incapable of producing pollen, only cross pollination will occur. By
eliminating the
pollen of one parental variety in a cross, a plant breeder is assured of
obtaining
hybrid seed of uniform quality, provided that the parents are of uniform
quality and
the breeder conducts a single cross.
In one instance, production of F1 hybrids includes crossing a CMS Brassica
female parent with a pollen-producing male Brassica parent. To reproduce
effectively, however, the male parent of the F1 hybrid must have a fertility
restorer
gene (Rf gene). The presence of an Rf gene means that the F1 generation will
not
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be completely or partially sterile, so that either self-pollination or cross
pollination
may occur. Self-pollination of the F1 generation to produce several subsequent
generations is important to ensure that a desired trait is heritable and
stable and that
a new variety has been isolated.
An example of a Brassica plant which is cytoplasmic male sterile and used for
breeding is Ogura (OGU) cytoplasmic male sterile (Pellan-Delourme, etal.,
1987). A
fertility restorer for Ogura cytoplasmic male sterile plants has been
transferred from
Raphanus sativus (radish) to Brassica by Instit. National de Recherche
Agricole
(INRA) in Rennes, France (Pelletier, et al., 1987). The OGU INRA restorer
gene,
lo Rf1 originating from radish, is described in WO 92/05251 and in
Delourme, et al.,
(1991). Improved versions of this restorer have been developed. For example,
see
W098/27806, oilseed brassica containing an improved fertility restorer gene
for
Ogura cytoplasmic male sterility.
Other sources and refinements of CMS sterility in canola include the Polima
cytoplasmic male sterile plant, as well as those of US Patent Number
5,789,566,
DNA sequence imparting cytoplasmic male sterility, mitochondrial genome,
nuclear
genome, mitochondria and plant containing said sequence and process for the
preparation of hybrids; US Patent Number 5,973,233 Cytoplasmic male sterility
system production canola hybrids; and W097/02737 Cytoplasmic male sterility
system producing canola hybrids; EP Patent Application Number 0 599042A
Methods for introducing a fertility restorer gene and for producing Fl hybrids
of
Brassica plants thereby; US Patent Number 6,229,072 Cytoplasmic male sterility
system production canola hybrids; US Patent Number 4,658,085 Hybridization
using
cytoplasmic male sterility, cytoplasmic herbicide tolerance, and herbicide
tolerance
from nuclear genes.
Promising advanced breeding lines commonly are tested and compared to
appropriate standards in environments representative of the commercial target
area(s). The best lines are candidates for new commercial lines; and those
still
deficient in a few traits may be used as parents to produce new populations
for
further selection.
Inbred Development ¨ Female
The female parent is developed by crossing a male sterile version of variety
NS6882 (A-line) with a maintainer line of variety NS6882 (B-line). The A and B
lines
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CA 02883471 2015-02-27
are genetically alike except the A-line carries the OGU INRA cytoplasm, while
the B-
line carries the normal B. napus cytoplasm.
The B-line was developed from a three way cross ((NS5111BR x 43A56) x
NS5626BR). The last crossing was completed in 2006. During the fall of 2006,
the
three way Fl was selfed to produce F2 seed. The F2s were planted in winter of
2007
in the GH and the screened for Glyphosate and blackleg resistance in the GH.
The
surviving single plants were harvested individually to produce F3 lines which
were
then evaluated in the Edmonton field nursery in 2007 for glyphosate tolerance,
early
maturity, lodging resistance, high oil and protein, general vigor and
uniformity.
ro Remnant seed for the selected F3s was planted in the GH and two
generations were
advanced while screening out plants susceptible for glyphosate and blackleg.
The
F5 were again evaluated in 2008 2nd year Edmonton nursery using the same
selection criteria as 2007. The line 09ENB01459 was selected and was assigned
a
breeder code NS6882BR.
Inbred Development ¨ Male
A male parent or restorer (R line) of variety NS6646 is designated
NS6646MC. The restorer was developed at Edmonton Research Centre of Pioneer
Hi-Bred Production Limited using pedigree selection from a four parent cross
(((NS5295MC x NS4304MC) x Peace) x NS1822MC). The last crossing was
completed in 2005. F3 lines were evaluated at the Edmonton nursery in 2006.
During nursery evaluation, the lines were selected for early maturity, high
oil, high
protein, low glucosinolates, general vigor and uniformity. Lines were also
self-
pollinated in bags in the nursery and F4 lines sent to Chile for F5 line
production.
Lines were then evaluated in the Edmonton nursery again in 2007. Selected
lines
were sent to Chile for hybrid production for 2008 evaluation. Seed production
of
NS6646MC was conducted in small cages in Chile in 2007/2008. NS6646MC was
also increased in a cage in Chile (2009/2010) and this seed was bulked for
breeder
seed in 2010.
Hybrid Development
PV 532 G (11N212R) is a fully restored spring Brassica napus hybrid with a
glyphosate resistance gene, based on OGU INRA system. It was developed at
Edmonton Research Centre of Pioneer Hi-Bred Production LP (subsidiary of
DuPont
CA 02883471 2015-02-27
Pioneer). It is a single cross hybrid produced by crossing a female parent
(male
sterile inbred-A line x maintainer inbred-B line) carrying a glyphosate
resistance gene
by a restorer ¨ R male line, where A and B lines are genetically alike except
A line
carries the OGU INRA cytoplasm, while B line carries the normal B. napus
cytoplasm.
A pollination control system and effective transfer of pollen from one parent
to
the other offers improved plant breeding and an effective method for producing
hybrid canola seed and plants. For example, the Ogura cytoplasmic male
sterility
(CMS) system, developed via protoplast fusion between radish (Raphanus
sativus)
and rapeseed (Brassica napus), is one of the most frequently used methods of
hybrid production. It provides stable expression of the male sterility trait
(Ogura,
1968, Pelletier, et al., 1983) and an effective nuclear restorer gene (Heyn,
1976).
For most traits the true genotypic value may be masked by other confounding
plant traits or environmental factors. One method for identifying a superior
plant is to
observe its performance relative to other experimental plants and to one or
more
widely grown standard varieties. If a single observation is inconclusive,
replicated
observations provide a better estimate of the genetic worth.
Proper testing should detect any major faults and establish the level of
superiority or improvement over current varieties. In addition to showing
superior
performance, there must be a demand for a new variety that is compatible with
industry standards or which creates a new market. The introduction of a new
variety
commonly will incur additional costs to the seed producer, the grower, the
processor
and the consumer, for special advertising and marketing, altered seed and
commercial production practices, and new product utilization. The testing
preceding
release of a new variety should take into consideration research and
development
costs as well as technical superiority of the final variety. For seed-
propagated
varieties, it must be feasible to produce seed easily and economically.
These processes, which lead to the final step of marketing and distribution,
usually take from approximately six to twelve years from the time the first
cross is
made. Therefore, the development of new varieties is a time-consuming process
that requires precise forward planning, efficient use of resources, and a
minimum of
changes in direction.
Further, as a result of the advances in sterility systems, lines are developed
that can be used as an open pollinated variety (i.e., a pureline cultivar sold
to the
16
CA 02883471 2015-02-27
grower for planting) and/or as a sterile inbred (female) used in the
production of F1
hybrid seed. In the latter case, favorable combining ability with a restorer
(male)
would be desirable. The resulting hybrid seed would then be sold to the grower
for
planting.
Combining ability of a line, as well as the performance of the line per se, is
a
factor in the selection of improved canola lines that may be used as inbreds.
Combining ability refers to a line's contribution as a parent when crossed
with other
lines to form hybrids. The hybrids formed for the purpose of selecting
superior lines
are designated test crosses. One way of measuring combining ability is by
using
breeding values. Breeding values are based on the overall mean of a number of
test
crosses. This mean is then adjusted to remove environmental effects and it is
adjusted for known genetic relationships among the lines.
Hybrid seed production requires inactivation of pollen produced by the female
parent.
Incomplete inactivation of the pollen provides the potential for self-
pollination. This inadvertently self-pollinated seed may be unintentionally
harvested
and packaged with hybrid seed. Similarly, because the male parent is grown
next to
the female parent in the field, there is also the potential that the male
selfed seed
could be unintentionally harvested and packaged with the hybrid seed. Once the
seed from the hybrid bag is planted, it is possible to identify and select
these self-
pollinated plants. These self-pollinated plants will be genetically equivalent
to one of
the inbred lines used to produce the hybrid. Though the possibility of inbreds
being
included in hybrid seed bags exists, the occurrence is rare because much care
is
taken to avoid such inclusions. These self-pollinated plants can be identified
and
selected by one skilled in the art, through either visual or molecular
methods.
Brassica napus canola plants, absent the use of sterility systems, are
recognized to commonly be self-fertile with approximately 70 to 90 percent of
the
seed normally forming as the result of self-pollination. The percentage of
cross
pollination may be further enhanced when populations of recognized insect
pollinators at a given growing site are greater. Thus open pollination is
often used in
commercial canola production.
Since canola variety PV 532 G is a hybrid produced from substantially
homogeneous parents, it can be reproduced by planting seeds of such parents,
growing the resulting canola plants under controlled pollination conditions
with
17
CA 02883471 2015-02-27
adequate isolation so that cross-pollination occurs between the parents, and
harvesting the resulting hybrid seed using conventional agronomic practices.
Locus Conversions of Canola Variety PV 532 G
PV 532 G represents a new base genetic line into which a new locus or trait
may be introduced. Direct transformation and backcrossing represent two
important
methods that can be used to accomplish such an introgression. The term locus
conversion is used to designate the product of such an introgression.
To select and develop a superior hybrid, it is necessary to identify and
select
io genetically unique individuals that occur in a segregating population.
The
segregating population is the result of a combination of crossover events plus
the
independent assortment of specific combinations of alleles at many gene loci
that
results in specific and unique genotypes. Once such a variety is developed its
value
to society is substantial since it is important to advance the germplasm base
as a
is whole in order to maintain or improve traits such as yield, disease
resistance, pest
resistance and plant performance in extreme weather conditions. Locus
conversions
are routinely used to add or modify one or a few traits of such a line and
this further
enhances its value and usefulness to society.
Backcrossing can be used to improve inbred varieties and a hybrid variety
20 which is made using those inbreds. Backcrossing can be used to transfer
a specific
desirable trait from one variety, the donor parent, to an inbred called the
recurrent
parent which has overall good agronomic characteristics yet that lacks the
desirable
trait. This transfer of the desirable trait into an inbred with overall good
agronomic
characteristics can be accomplished by first crossing a recurrent parent to a
donor
25 parent (non-recurrent parent). The progeny of this cross is then mated
back to the
recurrent parent followed by selection in the resultant progeny for the
desired trait to
be transferred from the non-recurrent parent.
Traits may be used by those of ordinary skill in the art to characterize
progeny. Traits are commonly evaluated at a significance level, such as a 1%,
5%
30 or 10% significance level, when measured in plants grown in the same
environmental conditions. For example, a locus conversion of PV 532 G may be
characterized as having essentially the same phenotypic traits as PV 532 G.
The
traits used for comparison may be those traits shown in any of Tables 1-6.
Molecular markers can also be used during the breeding process for the
selection of
18
CA 02883471 2015-02-27
qualitative traits. For example, markers 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.
A locus conversion of PV 532 G will retain the genetic integrity of PV 532 G.
A locus conversion of PV 532 G will comprise at least 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% of the base genetics of PV 532 G. For example, a locus
conversion of PV 532 G can be developed when DNA sequences are introduced
through backcrossing (Hallauer et al., 1988), with a parent of PV 532 G
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 locus conversion in at least one 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, OR, where it is demonstrated
that
a backcross conversion can be made in as few as two backcrosses.
Uses of Can ola
Currently Brassica napus canola is being recognized as an increasingly
important oilseed crop and a source of meal in many parts of the world. The
oil as
removed from the seeds commonly contains a lesser concentration of
endogenously
formed saturated fatty acids than other vegetable oils and is well suited for
use in the
production of salad oil or other food products or in cooking or frying
applications.
The oil also finds utility in industrial applications. Additionally, the meal
component
of the seeds can be used as a nutritious protein concentrate for livestock.
Canola oil has the lowest level of saturated fatty acids of all vegetable
oils.
"Canola" refers to rapeseed (Brassica) which (1) has an erucic acid (C22.1)
content of
at most 2 percent by weight based on the total fatty acid content of a seed,
preferably at most 0.5 percent by weight and most preferably essentially 0
percent
by weight; and (2) produces, after crushing, an air-dried meal containing less
than 30
19
CA 02883471 2015-02-27
micromoles ( rnol) glucosinolates per gram of detailed (oil-free) meal. These
types
of rapeseed are distinguished by their edibility in comparison to more
traditional
varieties of the species.
Disease - Sclerotinia
Sclerotinia infects over 100 species of plants, including numerous
economically important crops such as Brassica species, sunflowers, dry beans,
soybeans, field peas, lentils, lettuce, and potatoes (Boland and Hall, 1994).
Sclerotinia sclerotiorum is responsible for over 99% of Sclerotinia disease,
while
Sclerotinia minor produces less than 1% of the disease. Sclerotinia produces
sclerotia, irregularly-shaped, dark overwintering bodies, which can endure in
soil for
four to five years. The sclerotia can germinate carpogenically or
myceliogenically,
depending on the environmental conditions and crop canopies. The two types of
germination cause two distinct types of diseases.
Sclerotia that germinate
carpogenically produce apothecia and ascospores that infect above-ground
tissues,
resulting in stem blight, stalk rot, head rot, pod rot, white mold and blossom
blight of
plants. Sclerotia that germinate myceliogenically produce mycelia that infect
root
tissues, causing crown rot, root rot and basal stalk rot.
Sclerotinia causes Sclerotinia stem rot, also known as white mold, in
Brassica, including canola. Canola is a type of Brassica having a low level of
glucosinolates and erucic acid in the seed. The sclerotia germinate
carpogenically in
the summer, producing apothecia. The apothecia release wind-borne ascospores
that travel up to one kilometer. The disease is favoured by moist soil
conditions (at
least 10 days at or near field capacity) and temperatures of 15-25 C, prior to
and
during canola flowering. The spores cannot infect leaves and stems directly;
they
must first land on flowers, fallen petals, and pollen on the stems and leaves.
Petal
age affects the efficiency of infection, with older petals more likely to
result in
infection (Heran, et al., 1999). The fungal spores use the flower parts as a
food
source as they germinate and infect the plant.
The severity of Sclerotinia in Brassica is variable, and is dependent on the
time of infection and climatic conditions (Heran, etal., 1999). The disease is
favored
by cool temperatures and prolonged periods of precipitation. Temperatures
between
20 and 25 C and relative humidities of greater than 80% are required for
optimal
CA 02883471 2015-02-27
plant infection (Heran, et al., 1999). Losses ranging from 5 to 100% have been
reported for individual fields (Manitoba Agriculture, Food and Rural
Initiatives, 2004).
On average, yield losses are estimated to be 0.4 to 0.5 times the Sclerotinia
sclerotiorum Field Severity score, a rating based on both percentage infection
and
disease severity. More information is provided herein at Example 4. For
example, if
a field has 20% infection (20/100 plants infected), then the yield loss would
be about
10% provided plants are dying prematurely due to the infection of the main
stem
(rating 5-SSFS=20%). If the plants are affected much less (rating 1-SSFS=4%),
yield
loss is reduced accordingly. Further, Sclerotinia can cause heavy losses in
wet
swaths. Sclerotinia sclerotiorum caused economic losses to canola growers in
Minnesota and North Dakota of 17.3, 20.8, and 16.8 million dollars in 1999,
2000
and 2001, respectively (Bradley, et al. 2006). In Canada, this disease is
extremely
important in Southern Manitoba, parts of South Central Alberta and also in
Eastern
areas of Saskatchewan. Since weather plays an important role in development of
this disease, its occurrence is irregular and unpredictable. Certain reports
estimate
about 0.8 to 1.3 million acres of canola being sprayed with fungicide in
Southern
Manitoba annually. The fungicide application costs about $25 per acre, which
represents a significant cost for canola producers. Moreover, producers may
decide
to apply fungicide based on the weather forecast, while later changes in the
weather
pattern discourage disease development, resulting in wasted product, time, and
fuel.
Creation of Sclerotinia tolerant canola cultivars has been an important goal
for many
of the Canadian canola breeding organizations.
The symptoms of Sclerotinia infection usually develop several weeks after
flowering begins. The plants develop pale-grey to white lesions, at or above
the soil
line and on upper branches and pods. The infections often develop where the
leaf
and the stem join because the infected petals lodge there. Once plants are
infected,
the mold continues to grow into the stem and invade healthy tissue. Infected
stems
appear bleached and tend to shred. Hard black fungal sclerotia develop within
the
infected stems, branches, or pods. Plants infected at flowering produce little
or no
seed. Plants with girdled stems wilt and ripen prematurely. Severely infected
crops
frequently lodge, shatter at swathing, and make swathing more time consuming.
Infections can occur in all above-ground plant parts, especially in dense or
lodged
stands, where plant-to-plant contact facilitates the spread of infection. New
sclerotia
carry the disease over to the next season.
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CA 02883471 2015-02-27
Conventional methods for control of Sclerotinia diseases include (a) chemical
control, (b) disease resistance and (c) cultural control, each of which is
described
below.
(a)
Fungicides such as benomyl, vinclozolin and iprodione remain the main
method of control of Sclerotinia disease (MoraII, et al., 1985; Tu, 1983).
Recently,
additional fungicidal formulations have been developed for use against
Sclerotinia,
including azoxystrobin, prothioconazole, and boscalid. (Johnson, 2005)
However,
use of fungicide is expensive and can be harmful to the user and environment.
Further, resistance to some fungicides has occurred due to repeated use.
(b) In
certain cultivars of bean, safflower, sunflower and soybean, some
progress has been made in developing partial (incomplete) resistance. Partial
resistance is often referred to as tolerance. However, success in developing
partial
resistance has been very limited, probably because partial physiological
resistance is
a multigene trait as demonstrated in bean (Fuller, etal., 1984). In addition
to partial
is physiological resistance, some progress has been made to breed for
morphological
traits to avoid Sclerotinia infection, such as upright growth habit, lodging
resistance
and narrow canopy. For example, bean plants with partial physiological
resistance
and with an upright stature, narrow canopy and indeterminate growth habit were
best
able to avoid Sclerotinia (Saindon, etal., 1993). Early maturing cultivars of
safflower
showed good field resistance to Sclerotinia.
Finally, in soybean, cultivar
characteristics such as height, early maturity and great lodging resistance
result in
less disease, primarily because of a reduction of favorable microclimate
conditions
for the disease. (Boland and Hall, 1987; Buzzell, etal. 1993)
(c)
Cultural practices, such as using pathogen-free or fungicide-treated
seed, increasing row spacing, decreasing seeding rate to reduce secondary
spread
of the disease, and burying sclerotia to prevent carpogenic germination, may
reduce
Sclerotinia disease but not effectively control the disease.
All Canadian canola genotypes are susceptible to Sclerotinia stem rot
(Manitoba Agriculture, Food and Rural Initiatives, 2004). This includes all
known
spring petalled genotypes of canola quality. There is also no resistance to
Sclerotinia in Australian canola varieties. (Hind-Lanoiselet, et al. 2004).
Some
varieties with certain morphological traits are better able to withstand
Sclerotinia
infection. For example, Polish varieties (Brassica rapa) have lighter canopies
and
seem to have much lower infection levels. In addition, petal-less varieties
(apetalous
22
CA 02883471 2015-02-27
varieties) avoid Sclerotinia infection to a greater extent (Okuyama, et at.,
1995; Fu,
1990). Other examples of morphological traits which confer a degree of reduced
field susceptibility in Brassica genotypes include increased standability,
reduced
petal retention, branching (less compact and/or higher), and early leaf
abscission.
Jurke and Fernando, (2003) screened eleven canola genotypes for Sclerotinia
disease incidence. Significant variation in disease incidence was explained by
plant
morphology, and the difference in petal retention was identified as the most
important factor. However, these morphological traits alone do not confer
resistance
to Sclerotinia, and all canola products in Canada are considered susceptible
to
Sclerotinia.
Winter canola genotypes are also susceptible to Sclerotinia. In Germany, for
example, no Scierotinia-resistant varieties are available. (Specht, 2005) The
widely-
grown German variety Express is considered susceptible to moderately
susceptible
and belongs to the group of less susceptible varieties/hybrids.
Spraying with fungicide is the only means of controlling Sclerotinia in canola
crops grown under disease-favorable conditions at flowering. Typical
fungicides
used for controlling Sclerotinia on Brassica include RovralTm/ProlineTm from
Bayer
and RonilanTm/LanceTm from BASF. The active ingredient in LanceTM is Boscalid,
and it is marketed as EnduraTM in the United States. The fungicide should be
applied before symptoms of stem rot are visible and usually at the 20-30%
bloom
stage of the crop. If infection is already evident, there is no use in
applying fungicide
as it is too late to have an effect. Accordingly, growers must assess their
fields for
disease risk to decide whether to apply a fungicide. This can be done by using
a
government provided checklist or by using a petal testing kit. Either method
is
cumbersome and prone to errors. (Hind-Lanoiselet, 2004; Johnson, 2005)
Numerous efforts have been made to develop Sclerotinia resistant Brassica
plants.
Built-in resistance would be more convenient, economical, and environmentally-
friendly than controlling Sclerotinia by application of fungicides. Since the
trait is
polygenic it would be stable and not prone to loss of efficacy, as fungicides
may be.
In some embodiments, PV 532 G can be modified to have resistance to
Sclerotinia.
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CA 02883471 2015-02-27
Characteristics of PV 532 G
Homogenous and reproducible canola hybrids are useful for the production of
a commercial crop on a reliable basis. There are a number of analytical
methods
available to determine the phenotypic stability of a canola hybrid.
The oldest and most traditional method of analysis is the observation of
phenotypic traits. The data are usually collected in field experiments over
the life of
the canola plants to be examined. Phenotypic characteristics most often are
observed for traits associated with seed yield, seed oil content, seed protein
content,
fatty acid composition of oil, glucosinolate content of meal, growth habit,
lodging
io resistance, plant height, shatter resistance, etc.
In addition to phenotypic observations, the genotype of a plant can also be
examined. A plant's genotype can be used to identify plants of the same
variety or a
related variety. For example, the genotype can be used to determine the
pedigree of
a plant. There are many laboratory-based techniques available for the
analysis,
comparison and characterization of plant genotype; among these are lsozyme
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) which are also referred
to as Microsatellites, and Single Nucleotide Polymorphisms (SNPs).
The variety of the present discovery has shown uniformity and stability for
all
traits, as described in the following variety description information. The
variety has
been increased with continued observation for uniformity.
PV 532 G is an early maturing, high yielding glyphosate resistant Brassica
napus canola hybrid having resistant (R) rating for Fusarium wilt. Its oil
content is
0.7% higher than WCC/RRC checks. Its protein is 1.0% lower than mean of the
checks and chlorophyll is 3.0% lower than the checks.
Table 1 provides data on morphological, agronomic, and quality traits for PV
532 G and canola variety 43E02. When preparing the detailed phenotypic
information that follows, plants of the new PV 532 G variety were observed
while
being grown using conventional agronomic practices. For comparative purposes,
canola plants of canola varieties, 43E02 was similarly grown in a replicated
experiment.
24
CA 02883471 2015-02-27
Observations were recorded on various morphological traits for the hybrid PV
532 G and comparative check cultivars. (See Table 1).
Hybrid PV 532 G can be advantageously used in accordance with the
breeding methods described herein and those known in the art to produce
hybrids
and other progeny plants retaining desired trait combinations of PV 532 G.
This
discovery is thus also directed to methods for producing a canola plant by
crossing a
first parent canola plant with a second parent canola plant wherein either the
first or
second parent canola plant is canola variety PV 532 G. Further, both first and
second parent canola plants can come from the canola variety PV 532 G. Either
the
first or the second parent plant may be male sterile.
Still further, this discovery also is directed to methods for producing a PV
532
G-derived canola plant by crossing canola variety PV 532 G with a second
canola
plant and growing the progeny seed, and repeating the crossing and growing
steps
with the canola PV 532 G-derived plant from 1 to 2 times, 1 to 3 times, 1 to 4
times,
or 1 to 5 times. Thus, any such methods using the canola variety PV 532 G are
part
of this discovery: open pollination, selfing, backcrosses, hybrid production,
crosses
to populations, and the like. All plants produced using canola variety PV 532
G as a
parent are within the scope of this discovery, including plants derived from
canola
variety PV 532 G. This includes canola lines derived from PV 532 G which
include
components for either male sterility or for restoration of fertility.
Advantageously, the
canola variety is used in crosses with other, different, canola plants to
produce first
generation (F1) canola hybrid seeds and plants with superior characteristics.
The discovery also includes a single-gene conversion of PV 532 G. A single-
gene conversion occurs when DNA sequences are introduced through traditional
(non-transformation) breeding techniques, such as backcrossing. DNA sequences,
whether naturally occurring or transgenes, may be introduced using these
traditional
breeding techniques. Desired traits transferred through this process include,
but are
not limited to, fertility restoration, fatty acid profile modification, other
nutritional
enhancements, industrial enhancements, disease resistance, insect resistance,
herbicide resistance and yield enhancements. The trait of interest is
transferred from
the donor parent to the recurrent parent, in this case, the canola plant
disclosed
herein. Single-gene traits may result from the transfer of either a dominant
allele or
a recessive allele. Selection of progeny containing the trait of interest is
done by
direct selection for a trait associated with a dominant allele. Selection of
progeny for
CA 02883471 2015-02-27
a trait that is transferred via a recessive allele will require growing and
selfing the first
backcross 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 gene of interest.
It should be understood that the canola variety of the discovery can, through
routine manipulation by cytoplasmic genes, nuclear genes, or other factors, be
produced in a male-sterile or restorer form as described in the references
discussed
earlier. Such embodiments are also within the scope of the present claims.
Canola
variety PV 532 G can be manipulated to be male sterile by any of a number of
io
methods known in the art, including by the use of mechanical methods, chemical
methods, self-incompatibility (SI), cytoplasmic male sterility (CMS) (either
Ogura or
another system), or nuclear male sterility (NMS). The term "manipulated to be
male
sterile" refers to the use of any available techniques to produce a male
sterile version
of canola variety PV 532 G. The male sterility may be either partial or
complete male
sterility. This discovery is also directed to Fl hybrid seed and plants
produced by
the use of Canola variety PV 532 G. Canola variety PV 532 G can also further
comprise a component for fertility restoration of a male sterile plant, such
as an Rf
restorer gene. In this case, canola variety PV 532 G could then be used as the
male
plant in hybrid seed production.
This discovery is also directed to the use of PV 532 G in tissue culture. As
used herein, the term plant includes plant protoplasts, plant cell tissue
cultures from
which canola plants can be regenerated, plant calli, plant clumps, and plant
cells that
are intact in plants or parts of plants, such as embryos, pollen, ovules,
seeds,
flowers, kernels, ears, cobs, leaves, husks, stalks, roots, root tips,
anthers, silk and
the like. PauIs, et al., (2006) (Canadian J of Botany 84(4):668-678) confirmed
that
tissue culture as well as microspore culture for regeneration of canola plants
can be
accomplished successfully. Chuong, et al., (1985) "A Simple Culture Method for
Brassica Hypocotyl Protoplasts", Plant Cell Reports 4:4-6; Barsby, et aL,
(Spring
1996) "A Rapid and Efficient Alternative Procedure for the Regeneration of
Plants
from Hypocotyl Protoplasts of Brassica napus", Plant Cell Reports; Kartha, et
al.,
(1974) "In vitro Plant Formation from Stem Explants of Rape", PhysioL Plant
31:217-
220; Narasimhulu, et al., (Spring 1988) "Species Specific Shoot Regeneration
Response of Cotyledonary Explants of Brassicas", Plant Cell Reports; Swanson,
(1990) "Microspore Culture in Brassica", Methods in Molecular Biology
6(17):159;
26
CA 02883471 2015-02-27
"Cell Culture techniques and Canola improvement" J. Am. Oil Chem. Soc.
66(4):455-
56 (1989). Thus, it is clear from the literature that the state of the art is
such that
these methods of obtaining plants are, and were, "conventional" in the sense
that
they are routinely used and have a very high rate of success.
The utility of canola variety PV 532 G also extends to crosses with other
species. Commonly, suitable species will be of the family Brassicae.
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
m
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 that are
inserted into the genome using transformation are referred to herein
collectively as
"transgenes". Over the last fifteen to twenty years several methods for
producing
transgenic plants have been developed, and the present discovery, in
particular
embodiments, also relates to transformed versions of the claimed canola
variety PV
532 G.
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, and Genetic Transformation for the
improvement
of Canola World Conf, Biotechnol. Fats and Oils Ind. 43-46 (1988). 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 and Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.
The most prevalent types of plant transformation involve the construction of
an expression vector. Such a vector comprises a DNA sequence that contains a
gene under the control of or operatively linked to a regulatory element, for
example a
promoter. The vector may contain one or more genes and one or more regulatory
elements.
A genetic trait which has been engineered into a particular canola plant using
transformation techniques could be moved into another line using traditional
27
CA 02883471 2015-02-27
breeding techniques that are well known in the plant breeding arts. For
example, a
backcrossing approach could be used to move a transgene from a transformed
canola plant to an elite inbred line and the resulting progeny would comprise
a
transgene. Also, if an inbred line was used for the transformation then the
transgenic plants could be crossed to a different line in order to produce a
transgenic
hybrid canola plant. As used herein, "crossing" can refer to a simple X by Y
cross, or
the process of backcrossing, depending on the context. 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. See, US Patent Number 6,222,101.
With transgenic plants according to the present discovery, 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
is
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, (1981) Anal. Biochem.
114:92-
96.
A genetic map can be generated, primarily via conventional Restriction
Fragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)
analysis, Simple Sequence Repeats (SSR), and Single Nucleotide Polymorphisms
(SNPs), which identifies the approximate chromosomal location of the
integrated
DNA molecule coding for the foreign protein. For exemplary methodologies in
this
regard, see, Glick and Thompson, METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY 269-284 (CRC Press, Boca Raton, 1993). 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,
SNP, and sequencing, all of which are conventional techniques.
Likewise, by means of the present discovery, plants can be genetically
engineered to express various phenotypes of agronomic interest. Exemplary
28
CA 02883471 2015-02-27
transgenes 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:
(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 cloned resistance gene to engineer plants that
are
resistant to specific pathogen strains. See, for example Jones, etal., (1994)
Science
266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporium
fulvum);
Martin, et al., (1993) Science 262:1432 (tomato Pto gene for resistance to
Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos, et al.,
(1994) Ce// 78:1089 (Arabidopsis RSP2 gene for resistance to Pseudomonas
syringae); McDowell and Woffenden, (2003) Trends BiotechnoL 21(4):178-83 and
Toyoda, et al., (2002) Transgenic Res. 11(6):567-82. A plant resistant to a
disease
is one that is more resistant to a pathogen as compared to the wild type
plant.
(B) A gene conferring resistance to fungal pathogens, such as oxalate
oxidase or oxalate decarboxylase (Zhou, etal., (1998) Pl. PhysioL 117(1):33-
41).
(C) A Bacillus thuringiensis (Bt) protein, a derivative thereof or a
synthetic
polypeptide modeled thereon. See, for example, Geiser, etal., (1986) Gene
48:109,
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 Numbers. 40098, 67136, 31995 and 31998. Other examples of Bacillus
thuringiensis transgenes being genetically engineered are given in the
following
patents and patent applications: 5,188,960; 5,689,052; 5,880,275; WO
91/114778;
WO 99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and US Application
Serial Numbers 10/032,717; 10/414,637; and 10/606,320.
(D) An insect-specific hormone or pheromone such as an ecdysteroid and
juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist
or
agonist thereof. See, for example, the disclosure by Hammock, etal., (1990)
Nature
344:458, of baculovirus expression of cloned juvenile hormone esterase, an
inactivator of juvenile hormone.
(E) An insect-specific peptide which, upon expression, disrupts the
physiology of the affected pest. For example, see the disclosures of Regan,
(1994)
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CA 02883471 2015-02-27
J. Biol. Chem. 269:9 (expression cloning yields DNA coding for insect diuretic
hormone receptor) and Pratt, etal., (1989) Biochem. Biophys. Res. Comm.
163:1243
(an allostatin is identified in Diploptera puntata); Chattopadhyay, etal.,
(2004) Critical
Reviews in Microbiology 30(1):33-54 2004; Zjawiony, (2004) J Nat Prod
67(2):300-
310; Carlini and Grossi-de-Sa, (2002) Toxicon 40(11):1515-1539; Ussuf, et al.,
(2001) Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira, (2004) Toxicon
44(4):385-403. See also, US Patent Number 5,266,317 to Tomalski, et al., who
disclose genes encoding insect-specific, paralytic neurotoxins.
(F) An enzyme responsible for a hyperaccumulation of a monterpene, a
sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or
another
non-protein molecule with insecticidal activity.
(G) 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 Number WO 93/02197 in the name of 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
Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem.
Molec.
Biol. 23:691, who teach the nucleotide sequence of a cDNA encoding tobacco
hookworm chitinase, and Kawalleck et al., (1993) Plant Molec. Biol. 21:673,
who
provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene, US
Patent
Application Serial Numbers 10/389,432, 10/692,367 and US Patent Number
6,563,020.
(H) A molecule that stimulates signal transduction. For example, see the
disclosure by Botella, et al., (1994) Plant Molec. Biol. 24:757, of nucleotide
sequences for mung bean calmodulin cDNA clones, and Griess, et al., (1994)
Plant
PhysioL 104:1467, who provide the nucleotide sequence of a maize calmodulin
cDNA clone.
(I) A hydrophobic moment peptide. See, PCT Application Number
W095/16776 and US Patent Number 5,580,852 (disclosure of peptide derivatives
of
Tachyplesin which inhibit fungal plant pathogens) and PCT Application Number
CA 02883471 2015-02-27
W095/18855 and US Patent Number 5,607,914 (teaches synthetic antimicrobial
peptides that confer disease resistance).
(J) A membrane permease, a channel former or a channel blocker. For
example, see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43, of
heterologous expression of a cecropin-beta lytic peptide analog to render
transgenic
tobacco plants resistant to Pseudomonas solanacearum.
(K) 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., (1990) Ann. Rev. PhytopathoL 28:451. Coat protein-mediated resistance has
been conferred upon transformed plants against alfalfa mosaic virus, cucumber
mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco
etch virus,
tobacco rattle virus and tobacco mosaic virus. Id.
(L) 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. Cf. 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).
(M) A virus-specific antibody. See, for example, Tavladoraki, etal., (1993)
Nature 366:469, who show that transgenic plants expressing recombinant
antibody
genes are protected from virus attack.
(N) 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., (1992) Bio/Technology 10:1436. The cloning
and characterization of a gene which encodes a bean endopolygalacturonase-
inhibiting protein is described by Toubart, et al., (1992) Plant J. 2:367.
(0) A
developmental-arrestive protein produced in nature by a plant. For
example, Logemann, et al., (1992) Bio/Technology 10:305, have shown that
transgenic plants expressing the barley ribosome-inactivating gene have an
increased resistance to fungal disease.
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(P) Genes involved in the Systemic Acquired Resistance (SAR)
Response
and/or the pathogenesis related genes. Briggs, (1995) Current Biology 5(2):128-
131, Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio 7(4):456-64 and
Somssich,
(2003) Cell 113(7):815-6.
(Q) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.
101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell, et
al.,
(1998) Can. J. of Plant Path. 20(2):137-149. Also see, US Patent Application
Number 09/950,933.
(R) Detoxification genes, such as for fumonisin, beauvericin, moniliformin
lo and zearalenone and their structurally related derivatives. For example,
see, US
Patent Number 5,792,931.
(S) Cystatin and cysteine proteinase inhibitors. See, US Patent
Application Serial Number 10/947,979.
(T) Defensin genes. See, W003/000863 and US Patent Application Serial
Number 10/178,213.
(U) Genes that confer resistance to Phytophthora Root Rot, such as the
Brassica equivalents of 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, (1995) Phytophthora Root Rot
Resistance Gene Mapping in Soybean, Plant Genome IV Conference, San Diego,
CA.
2. Genes that confer resistance to a herbicide, for example:
(A) A herbicide that inhibits the growing point or meristem, such as an
imidazalinone or a sulfonylurea. Exemplary genes in this category code for
mutant
ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.
7:1241, and Miki, et al., (1990) Theor. Appl.Genet. 80:449, respectively. See
also,
US Patent Numbers 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180;
5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; and international
publication WO 96/33270.
(B) Glyphosate (resistance imparted by mutant 5-enolpyruv1-3-
phosphikimate synthase (EPSP) and aroA genes, respectively) and other
phosphono
compounds such as glufosinate (phosphinothricin acetyl transferase, PAT) and
Streptomyces hygroscopicus phosphinothricin-acetyl transferase, bar, genes),
and
32
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pyridinoxy or phenoxy propionic acids and cycloshexones (ACCase inhibitor-
encoding genes). See, for example, US Patent Number 4,940,835 to Shah, et al.,
which discloses the nucleotide sequence of a form of EPSP which can confer
glyphosate resistance. See also, US Patent Number 7,405,074, and related
applications, which disclose compositions and means for providing glyphosate
resistance. US Patent Number 5,627,061 to Barry, et al., also describes genes
encoding EPSPS enzymes. See also, US Patent Numbers 6,566,587; 6,338,961;
6,248,876 BI; 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 BI; 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.
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 US Patent Number 4,769,061 to Comai. European Patent Application
Number 0 333 033 to Kumada, et al., and US Patent Number 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 phosphinothricin-acetyl-transferase gene is provided in European
Application Number 0 242 246 to Leemans, et al., De Greef, et al., (1989)
Bio/Technology 7:61, describe the production of transgenic plants that express
chimeric bar genes coding for phosphinothricin acetyl transferase activity.
See also,
US Patent Numbers 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675;
5,561,236; 5,648,477; 5,646,024; 6,177,616 Bland 5,879,903. Exemplary of genes
conferring resistance to phenoxy propionic acids and cycloshexones, such as
sethoxydim and haloxyfop, are the Accl-S1, Accl-S2 and Accl-S3 genes described
by Marshall, et al., (1992) Theor. App!. Genet. 83:435. See also, US Patent
Numbers 5,188,642; 5,352,605; 5,530,196; 5,633,435; 5,717,084; 5,728,925;
5,804,425 and Canadian Patent Number 1,313,830.
(C)
A herbicide that inhibits photosynthesis, such as a triazine (psbA and
gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991)
Plant Cell
3:169, describe the transformation of Chlamydomonas with plasmids encoding
mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in
US
Patent Number 4,810,648 to Stalker, and DNA molecules containing these genes
are available under ATCC Accession Numbers 53435, 67441 and 67442. Cloning
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and expression of DNA coding for a glutathione S-transferase is described by
Hayes,
etal., (1992) Biochem. J. 285:173.
(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, e.g., Hattori, et al., (1995) Mol Gen Genet
246:419).
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., (1994) Plant Physiol 106:17), genes for
glutathione
reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol
36:1687, and genes for various phosphotransferases (Datta, et al., (1992)
Plant Mol
Biol 20:619).
(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 US Patent Numbers 6,288,306 B1; 6,282,837 B1; and
5,767,373; and international publication WO 01/12825.
3. Transgenes that confer or contribute to an altered grain characteristic,
such
as:
(A) Altered fatty acids, for example, by
(1) Down-regulation of stearoyl-ACP desaturase to increase stearic
acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad.
Sc!.
USA 89:2624 and W099/64579 (Genes for Desaturases to Alter Lipid Profiles
in Corn),
(2) Elevating oleic acid via FAD-2 gene modification and/or
decreasing linolenic acid via FAD-3 gene modification (see, US Patent
Numbers 6,063,947; 6,323,392; 6,372,965 and WO 93/11245),
(3) Altering
conjugated linolenic or linoleic acid content, such as in
WO 01/12800,
(4)
Altering LEC1, AGP, Dek1, Superalt mil ps, various Ipa genes
such as Ipat Ipa3, hpt or hggt. For example, see WO 02/42424, WO
98/22604, WO 03/011015, US Patent Numbers 6,423,886, 6,197,561,
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CA 02883471 2015-02-27
6,825,397, US Patent Application Publication Numbers 2003/0079247,
2003/0204870, W002/057439, W003/011015 and Rivera-Madrid, et al.,
(1995) Proc. Natl. Acad. Sci. 92:5620-5624.
(B) Altered phosphate content, for example, by the
(1) Introduction of
a phytase-encoding gene would enhance
breakdown of phytate, adding more free phosphate to the transformed plant.
For example, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for a
disclosure of the nucleotide sequence of an Aspergillus niger phytase gene.
(2)
Up-regulation of a gene that reduces phytate content. In maize,
lo
this, for example, could be accomplished, by cloning and then re-introducing
DNA associated with one or more of the alleles, such as the LPA alleles,
identified in maize mutants characterized by low levels of phytic acid, such
as
in Raboy, et al., (1990) Maydica 35:383 and/or by altering inositol kinase
activity as in WO 02/059324, US Patent Application Publication Number
2003/0009011, WO 03/027243, US Patent Application Publication Number
2003/0079247, WO 99/05298, US Patent Numbers 6,197,561, 6,291,224,
6,391,348, W02002/059324, US Patent Application Publication Number
2003/0079247, W098/45448, W099/55882, W001/04147.
(C) Altered carbohydrates effected, for example, by altering a gene for an
enzyme that affects the branching pattern of starch, a gene altering
thioredoxin.
(See, US Patent Number 6,531,648). See, Shiroza, et al., (1988) J. Bacteriol
170:810 (nucleotide sequence of Streptococcus mutans fructosyltransferase
gene),
Steinmetz, etal., (1985) MoL Gen. Genet. 200:220 (nucleotide sequence of
Bacillus
subtilis levansucrase gene), Pen, etal., (1992) Bio/Technology 10:292
(production of
transgenic plants that express Bacillus licheniformis alpha-amylase), Elliot,
et aL,
(1993) Plant Molec Biol 21:515 (nucleotide sequences of tomato invertase
genes),
Sogaard, et al., (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of
barley alpha-amylase gene) and Fisher, etal., (1993) Plant Physiol 102:1045
(maize
endosperm starch branching enzyme II), WO 99/10498 (improved digestibility
and/or
starch extraction through modification of UDP-D-xylose 4-epimerase, Fragile 1
and
2, Ref1, HCHL, C4H), US Patent Number 6,232,529 (method of producing high oil
seed by modification of starch levels (AGP)). The fatty acid modification
genes
mentioned above may also be used to affect starch content and/or composition
through the interrelationship of the starch and oil pathways.
CA 02883471 2015-02-27
(D) Altered antioxidant content or composition, such as alteration of
tocopherol or tocotrienols. For example, see, US Patent Number 6,787,683, US
Patent Application Publication Number 2004/0034886 and WO 00/68393 involving
the manipulation of antioxidant levels through alteration of a phytl prenyl
transferase
(ppt), WO 03/082899 through alteration of a homogentisate geranyl geranyl
transferase (hggt).
(E) Altered essential seed amino acids. For example, see, US Patent
Number 6,127,600 (method of increasing accumulation of essential amino acids
in
seeds), US Patent Number 6,080,913 (binary methods of increasing accumulation
of
essential amino acids in seeds), US Patent Number 5,990,389 (high lysine),
W099/40209 (alteration of amino acid compositions in seeds), W099/29882
(methods for altering amino acid content of proteins), US Patent Number
5,850,016
(alteration of amino acid compositions in seeds), W098/20133 (proteins with
enhanced levels of essential amino acids), US Patent Number 5,885,802 (high
methionine), US Patent Number 5,885,801 (high threonine), US Patent Number
6,664,445 (plant amino acid biosynthetic enzymes), US Patent Number 6,459,019
(increased lysine and threonine), US Patent Number 6,441,274 (plant tryptophan
synthase beta subunit), US Patent Number 6,346,403 (methionine metabolic
enzymes), US Patent Number 5,939,599 (high sulfur), US Patent Number 5,912,414
(increased methionine), W098/56935 (plant amino acid biosynthetic enzymes),
W098/45458 (engineered seed protein having higher percentage of essential
amino
acids), W098/42831 (increased lysine), US Patent Number 5,633,436 (increasing
sulfur amino acid content), US Patent Number 5,559,223 (synthetic storage
proteins
with defined structure containing programmable levels of essential amino acids
for
improvement of the nutritional value of plants), W096/01905 (increased
threonine),
W095/15392 (increased lysine), US Patent Application Publication Number
2003/0163838, US Patent Application Publication Number 2003/0150014, US Patent
Application Publication Number 2004/0068767, US Patent Number 6,803,498,
W001/79516, and W000/09706 (Ces A: cellulose synthase), US Patent Number
6,194,638 (hemicellulose), US Patent Number 6,399,859 and US Patent
Application
Publication Number 2004/0025203 (UDPGdH), US Patent Number 6,194,638 (RGP).
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4. Genes that control pollination, hybrid seed production, or male-
sterility:
There are several methods of conferring genetic male sterility available, such
as multiple mutant genes at separate locations within the genome that confer
male
sterility, as disclosed in US Patent Numbers 4,654,465 and 4,727,219 to Brar,
etal.,
and chromosomal translocations as described by Patterson in US Patents Numbers
3,861,709 and 3,710,511. In addition to these methods, Albertsen, etal., US
Patent
Number 5,432,068, describe a system of nuclear male sterility which includes:
identifying a gene which is critical to male fertility; silencing this native
gene which is
critical to male fertility; removing the native promoter from the essential
male fertility
gene and replacing it with an inducible promoter; inserting this genetically
engineered gene back into the plant; and thus creating a plant that is male
sterile
because the inducible promoter is not "on" resulting in the male fertility
gene not
being transcribed. Fertility is restored by inducing, or turning "on", the
promoter,
which in turn allows the gene that confers male fertility to be transcribed.
(A) Introduction of a deacetylase gene under the control of a tapetum-
specific promoter and with the application of the chemical N-Ac-PPT (WO
01/29237).
(B) Introduction of various stamen-specific promoters (WO 92/13956, WO
92/13957).
(C) Introduction of the barnase and the barstar gene (Paul, et al., (1992)
Plant Mol. Biol. 19:611-622).
For additional examples of nuclear male and female sterility systems and
genes, see also, US Patent Numbers 5,859,341; 6,297,426; 5,478,369; 5,824,524;
5,850,014 and 6,265,640.
Also see, US Patent Number 5,426,041 (discovery relating to a method for the
preparation of a seed of a plant comprising crossing a male sterile plant and
a
second plant which is male fertile), US Patent Number 6,013,859 (molecular
methods of hybrid seed production) and US Patent Number 6,037,523 (use of male
tissue-preferred regulatory region in mediating fertility).
5. Genes that create a site for site specific DNA integration.
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., (2003) "Site-Specific Recombination for Genetic
Engineering in
Plants", Plant Cell Rep 21:925-932 and WO 99/25821. Other systems that may be
37
CA 02883471 2015-02-27
used include the Gin recombinase of phage Mu (Maeser, et al., 1991), the Pin
recombinase of E. coli (Enomoto, et al., 1983), and the R/RS system of the
pSR1
plasmid (Araki, etal., 1992).
6. Genes that affect abiotic stress resistance (including but not limited
to
flowering, ear 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; US Patent Numbers 5,892,009, 5,965,705, 5,929,305,
5,891,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034, 6,801,104,
W02000060089,
W02001026459, W02001035725, W02001034726,
W02001035727,
W02001036444, W02001036597, W02001036598,
W02002015675,
W02002017430, W02002077185, W02002079403,
W02003013227,
W02003013228, W02003014327, W02004031349, W02004076638, W09809521
and W09938977 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
Patent
Application Publication Number 2004/0148654 and W001/36596 where abscisic
acid is altered in plants resulting in improved plant phenotype such as
increased
yield and/or increased tolerance to abiotic stress; W02000/006341,
W004/090143,
US Patent Application Serial Numbers 10/817483 and 09/545,334 where cytokinin
expression is modified resulting in plants with increased stress tolerance,
such as
drought tolerance, and/or increased yield. Also see W00202776, W003052063,
JP2002281975, US Patent Number 6,084,153, W00164898, US Patent Number
6,177,275 and US Patent Number 6,107,547 (enhancement of nitrogen utilization
and altered nitrogen responsiveness). For ethylene alteration, see, US Patent
Application Publication Numbers 2004/0128719, 2003/0166197 and W0200032761.
For plant transcription factors or transcriptional regulators of abiotic
stress, see e.g.,
US Patent Application Publication Number 2004/0098764 or US Patent Application
Publication Number 2004/0078852.
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., W097/49811 (LHY), W098/56918 (ESD4),
38
CA 02883471 2015-02-27
W097/10339 and US6573430 (TFL), US6713663 (FT), W096/14414 (CON),
W096/38560, W001/21822 (VRN1), W000/44918 (VRN2), W099/49064 (GI),
W000/46358 (FRI), W097/29123, US Patent Numbers 6,794,560, 6,307,126 (GAI),
W099/09174 (D8 and Rht), and W02004076638 and W02004031349 (transcription
-- factors).
Seed Cleaning
This discovery is also directed to methods for producing cleaned canola seed
by cleaning seed of variety PV 532 G. "Cleaning a seed" or "seed cleaning"
refers to
-- the removal of foreign material from the surface of the seed. Foreign
material to be
removed from the surface of the seed includes but is not limited to fungi,
bacteria,
insect material, including insect eggs, larvae, and parts thereof, and any
other pests
that exist on the surface of the seed. The terms "cleaning a seed" or "seed
cleaning"
also refer to the removal of any debris or low quality, infested, or infected
seeds and
-- seeds of different species that are foreign to the sample. This discovery
is also
directed to produce subsequent generations of seed from seed of variety PV 532
G,
harvesting the subsequent generation of seed; and planting the subsequent
generation of seed.
-- Seed Treatment
"Treating a seed" or "applying a treatment to a seed" refers to the
application
of a composition to a seed as a coating or otherwise. The composition may be
applied to the seed in a seed treatment at any time from harvesting of the
seed to
sowing of the seed. The composition may be applied using methods including but
-- not limited to mixing in a container, mechanical application, tumbling,
spraying,
misting, and immersion. Thus, the composition may be applied as a slurry, a
mist, or
a soak. The composition to be used as a seed treatment can be a pesticide,
fungicide, insecticide, or antimicrobial. For a general discussion of
techniques used
to apply fungicides to seeds, see "Seed Treatment," 2d ed., (1986), edited by
K. A
-- Jeffs (chapter 9).
Industrial Applicability
The seed of the PV 532 G variety, the plant produced from such seed, various
parts of the PV 532 G hybrid canola plant or its progeny, a canola plant
produced
39
CA 02883471 2015-02-27
from the crossing of the PV 532 G variety, and the resulting seed, can be
utilized in
the production of an edible vegetable oil or other food products in accordance
with
known techniques. The remaining solid meal component derived from seeds can be
used as a nutritious livestock feed.
CA 02883471 2015-04-17
DEPOSIT
Applicant(s) have made or will make a deposit of at least 2500 seeds of
canola variety PV 532 G with the American Type Culture Collection (ATCC),
10801
University Boulevard, Manassas, VA 20110-2209 USA, ATCC Deposit No. PTA-
s 122027. The seeds deposited with the ATCC on February 20, 2015 for PTA-
122027
were taken from the seed stock maintained by Pioneer Hi-Bred International,
Inc.,
7250 NW 62nd Avenue, Johnston, Iowa 50131-1000 since prior to the filing date
of
this application. Access to this deposit will be available during the pendency
of the
application to the Commissioner of Patents and Trademarks and persons
to determined by the Commissioner to be entitled thereto upon request. Upon
allowance of any claims in the application, the Applicant will make available
to the
public, pursuant to 37 C.F.R. 1.808, sample(s) of the deposit of at least
2500
seeds of canola variety PV 532 G with the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, VA 20110-2209. This deposit of
15 seed of canola variety PV 532 G will be maintained in the ATCC
depository, which is
a public depository, for a period of 30 years, or 5 years after the most
recent request,
or for the enforceable life of the patent, whichever is longer, and will be
replaced if it
becomes nonviable during that period. Additionally, Applicant has satisfied
all the
requirements of 37 C.F.R. 1.801 - 1.809, including providing an indication
of the
20 viability of the sample upon deposit. Applicant has no authority to
waive any
restrictions imposed by law on the transfer of biological material or its
transportation
in commerce. Applicant(s) do not waive any infringement of their rights
granted
under this patent or rights applicable to canola hybrid PV 532 G under the
Plant
Variety Protection Act (7 USC 2321 et seq.).
41
CA 02883471 2015-02-27
Varietal Characteristics (See also Tables 1 through 11)
Seed yield 7.5% lower than the mean of the WCC/RRC checks (45H29 &
In Vigor 5440)
Disease reaction Classified as Resistant (R) to blackleg (Leptospaera
maculans) according to
WCC/RRC guidelines. Classified as resistant (R) to Fusarium wilt according to
trials.
Plant height Approximately 9 cm shorter than the mean of the WCC/RRC
checks.
Maturity Approximately 1.3 days earlier maturing than the WCC/RRC
checks.
Lodging Similar lodging rating to the mean of the WCC/RRC
checks.
Herbicide tolerance Tolerant to glyphosate herbicides. Field testing
confirms that PV 532 G
tolerates the recommended rate of glyphosate (1.5L/ha) without showing plant
injury or any significant negative effect on yield, agronomic, and quality
traits.
Variants Exhibits less than 1500/10,000 (<15%) glyphosate
susceptible plants.
Seed Characteristics
Seed color Dark brown
Grain size 1000 seed weight is similar to the mean of the WCC/RRC
checks.
Seed oil content 0.7% higher than the mean of the WCC/RRC checks.
Seed protein content 1.1% lower than the mean of the WCC/RRC checks.
Erucic acid Less than 0.5% (maximum allowable limit).
Total saturates 0.06% lower than the mean of the WCC/RRC checks.
Total glucosinolates Canola quality ¨ 2.7 umol/g lower than the WCC/RRC
checks.
Chlorophyll 3.0 ppm lower than the mean of the WCC/RRC checks.
42
CA 02883471 2015-02-27
Table 1. Variety Descriptions based on Morphological, Agronomic and Quality
Traits
PV 532 G 43E02
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
1.2 Seasonal Type Spring
Cotyledon width
3=narrow
2.15 Medium 5 Medium
5=medium
7=wide
Seedling growth
habit (leaf rosette)
2.2 5 5
1=weak rosette
9 = strong rosette
Stem anthocyanin
intensity
1=absent or very
weak Absent or very
2.3 2 3 Weak
3=weak weak to weak
5=medium
7=strong
9=very strong
Leaf type
2.4 1=petiolate 9 Lyrate 1 Petiolate
9=Iyrate
Leaf shape
2.5 3=narrow elliptic 3 Narrow elliptic 3 Narrow elliptic
7=orbicular
Leaf length
3=short
2.6 6 Medium/Long 6 Medium/Long
5=medium
7=long
Leaf width
3=narrow
2.7 4 Narrow/Medium 4 Narrow/Medium
5=medium
7=wide
Leaf color
1=light green
2.8 2=nnedium green 2 Medium green 2 Medium green
3=dark green
4=blue-green
Leaf lobe
development
1=absent or very
2.12 weak 3 Weak 3 Weak
3=weak
5=medium
7=strong
9=very strong
Number of leaf
2.13 3 4
lobes
Petiole length
3=short
2.15 2 Short 5 Medium
5=medium
7=long
Leaf margin shape
1=undulating
2.16 3 Sharp 3 Sharp
2=rounded
3=sharp
43
CA 02883471 2015-02-27
PV 532 G 43E02
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
Leaf margin
indentation
1=absent or very
weak (very shallow)
2.17 3=weak (shallow) 5 Medium 5 Medium
5=medium
7=strong (deep)
9=very strong (very
deep)
Leaf attachment to
stem
1=complete
2.18 2 Partial claspingclasping 2 Partial clasping
2=partial clasping
3=non-clasping
3.1 Flower date 50% 47.9
Plant height at
maturity
3.2 3=short 6 Medium/Tall
5=medium
7=tall
Flower bud location
1=buds above most
recently opened Buds above Buds above
3.4 flowers 1 most recently 1 most recently
9=buds below most opened flowers opened flowers
recently opened
flowers
Petal color
1=white
2=light yellow
3.5 3=medium yellow 3 Medium yellow 3 Medium
yellow
4=dark yellow
5=orange
6=other
Petal length
3=short
3.6 um 5 Medium 5 Medium
5=medi
7=long
Petal width
3=narrow
3.7 5 Medium 5 Medium
5=medium
7=wide
Petal spacing
1=open
3=not touching
3.8 5 Touching 5 Touching
5=touching
7=slight overlap
9=strongly overlap
Anther fertility
1=sterile All anthers All anthers
3.11 9 9
9=all anthers shedding pollen shedding pollen
shedding pollen
Pod (silique) length
1=short (<7cm)
3.12 5=medium (7- 5 Medium 5 Medium
10cm)
9=long (>10cm)
Pod (silique) width
3=narrow (3nnm) 5 M Medium (4 mm)
5=medium (4 mm)
3.13 edium (4 mm ) 6
to Wide (5 mm)
7=wide (5mm)
44
CA 02883471 2015-02-27
43E02
PV 532 G
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
Pod (silique)
attitude
1=erect
3.14 3=semi-erect 3 Semi-erect .. 3 .. Semi-erect
5=horizontal
7=slightly drooping
9=drooping
Pod (silique) beak
length
3.15 3=short 5 Medium 5 Medium
5=medium
7=long
Pedicel length
3=short
3.16 4 Short/Medium 5 Medium
5=medium
7=long
Mantuting)rity (days from
3.17 a 100.6
pl
Seed coat color
1=black
2=brown
4.1 3=tan 1.5 Black to brown 1.5 Black to brown
4=yellow
5=mixed
6=other
Seed
weight/Thousand
seed weight (5-6% 3.9
4.3
moisture content):
grams per 1,000
seeds
Shatter resistance
1 = Not tested
3 = Poor
5.1 5 = Fair 7.2
7 = Good
9 = Does not
shatter
Lodging resistance
1=not tested
3=poor
5.2 6.9 Fair/Good
5=fair
7=good
9=excellent
Blackleg resistance
0=not tested
1=resistant
3=moderately
resistant
6.3 1 Resistant
5=moderately
susceptible
7=susceptible
9=highly
susceptible
Fusarium wilt
resistance
0=not tested
1=resistant
3=moderately
6.7 resistant 1 Resistant
5=moderately
susceptible
7=susceptible
9=highly
susceptible
CA 02883471 2015-02-27
43E02
PV 532 G
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
Oil content
8.1 49.12
percentage
Protein percentage
44.25
8.5 (whole dry seed)
Glucosinolates
(pmoles total
glucs/g whole seed)
Low (10-15
8.7 1= very low (<10) 2
pmol per gram)
2= low (10-15)
3=medium (15-20)
4=high (>20)
Chlorophyll content
(mg/kg seed)
8.8 1=low (<8 ppm) 1 Low (<8 ppm)
2=medium (8-15
PPrn)
3=high (>15 ppm)
46
CA 02 88347 1 2015-02-27
Example 1. Herbicide Resistance
Appropriate field tests have shown that PV 532 G tolerates the recommended
rate (1.5L/ha) of glyphosate herbicide without showing plant injury or any
significant
negative effect on yield, agronomic, or quality traits. This hybrid exhibits
less than
1500/10,000 (<15%) glyphosate-susceptible plants.
Table 2. Effect of herbicide application on agronomic and quality traits of PV
532 G
in herbicide tolerance trials in 2012 and 2013
2012 Vegreville, AB
Stand Days
Treat Yield `)/0
Height Days to % % Oil +
Gluc's
Variety Reduction to @
Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
PV 532 G 2X 18.0 0 52 84 100 47.4 48.4 95.8
13.9 2.0
43E02 2X 20.7 o 51 86 99 47.4 50.6 97.9 14.5
5.7
CV% 10.5 142.5 1.9 6.6 1.3 3.0 2.2 0.8 10.8
57.1
LSD (0.05) 2.9 0.0 1.0 8.0 2.0 2.8 2.2 1.5
3.3 7.2
SE 1.06 0.00 0.71 2.83 0.71 0.99 0.78
0.50 1.20 2.55
2013 Vegreville, AB
Treat Yield % Stand Days Height Days to % % Oil +
Gluc's
Variety Reduction to @ Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
PV 532 G 2X 26.4 0 51 97 102 47.9 44.0 91.8
11.4 3.1
43E02 2X 24.8 0 51 95 99 48.5 46.0 94.5 10.5 3.2
CV% 9.3 404.5 1.1 5.6 1.1 2.9 3.3 0.7 9.6
26.7
LSD (0.05) 3.9 0.0 1.0 9.0 2.0 2.3 2.5 1.0
1.7 1.2
SE 1.34 0.00 0.00 3.54 0.71 0.85 0.85
0.35 0.57 0.42
47
CA 02 88347 1 2015-02-27
Table 2, continued
2013 Saskatoon, SK
Gluc's
Treat Yield % Stand Days
Variety Reduction to Height Days to % % Oil +
@ Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
PV 532 G 2X 13.7 0 46 78 96 47.6 43.0 90.6 10.7
3.1
43E02 2X 9.6 2 45 72 98 48.0 45.0 93.0 10.0
6.7
CV% 13.4 198.5 2.8 8.6 1.5 1.6 2.3 0.7 12.5
39.3
LSD (0.05) 4.6 3.0 2.0 11.0 2.0 1.2 1.7 1.1
2.2 3.2
SE 1.63 1.41 0.71 3.54 0.71 0.42 0.57
0.42 0.78 1.13
2013 Average
Gluc's
Treat Yield % Stand Days
Variety Reduction to Height Days to % % Oil +
@ Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
PV 532 G 2X 20.2 0 48.0 88.0 99.0 47.7 43.5 91.2
11.0 3.1
43E02 2X 17.6 1 48.0 83.0 99.0 48.3 45.5 93.7
10.2 5.0
CV% 11.4 267.4 2.0 6.9 1.3 2.4 2.9 0.7 11.2
38.2
LSD (0.05) 3.1 2.0 2.0 12.0 2.0 1.4 1.5 1.3
1.4 2.7
SE 1.13 0.71 0.71 4.24 0.71 0.50 0.50
0.42 0.50 0.92
2 Year Average
Glue's
Treat Yield % Stand Days Height Days to % % Oil +
Variety Reduction to @
Chlorophyll
ment q/ha (cm) Maturity Oil Protein
Protein 8.5%
(PCTSR) Flower
PV 532 G 2X 19.4 0 50 86 99 47.6 45.1 92.7 12.0
2.7
43E02 2X 18.4 1 49 84 99 48.0 47.2 95.1 11.7
5.2
CV% 11.1 248.5 1.9 6.9 1.3 2.5 2.6 0.7 11.0
41.0
LSD (0.05) 1.9 1.0 0.9 5.2 1.1 1.0 1.1 0.6 1.2
1.8
_
SE 0.70 0.35 0.31 1.88 0.41 0.38 0.40
0.21 0.42 0.64
Locations 3 3 3 3 3 3 3 3 3 3
48
CA 02883471 2015-02-27
Example 2. Miscellaneous Disease Resistance
Blackleg
Blackleg tolerance was measured following the standard procedure described
in the Procedures of the Western Canada Canola/Rapeseed Recommending
Committee (WCC/RRC) Incorporated for the Evaluation and Recommendation for
Registration of Canola/Rapeseed Candidate Cultivars in Western Canada.
Blackleg
was rated on a scale of 0 to 5: a plant with zero rating is completely immune
to
disease while a plant with "5" rating is dead due to blackleg infection.
Canola variety "Westar" was included as an entry/control in each blackleg
trial. Tests are considered valid when the mean rating for Westar is greater
than or
equal to 2.6 and less than or equal to 4.5. (In years when there is poor
disease
development in Western Canada the WCC/RRC may accept the use of data from
is trials with a rating for Westar exceeding 2Ø)
The ratings are converted to a percentage severity index for each line, and
the following scale is used to describe the level of resistance:
Classification Rating (% of Westar)
R (Resistant) <30
MR (Moderately Resistant) 30 ¨49
MS (Moderately Susceptible) 50 ¨69
S (Susceptible) 70 ¨ 89
HS (Highly Susceptible) 90 - 100
49
CA 02883471 2015-02-27
Table 3. Summary of Blackleg Ratings for PV 532 G
2012 2013
Plum
itou Rosebank Alvena Boissevain Carman
VegreviIle 2 Year %
Coulee Man
Average Westar Class
PV 532 G 1.0 1.1 0.6 0.6 1.5 1.0 1.1 1.0 28.8
R
Westar 2.6 3.7 4.1 3.3 2.9 3.4 3.8 3.4
Example 3: Summary of Performance of PV 532 G in two years of Co-op Testing
Two years (2012 and 2013) of trials were conducted. WCC/RRC guidelines
io were followed for conducting trials. Each trial had three replicates and
had a plot size
of 1.5m x 6m. Yield and agronomic traits were recorded and seed samples were
collected from two of the four replicates at almost all sites. Seed samples
were
analyzed using NIR (near infrared spectroscopy) for oil, protein, total
glucosinoaltes
and cholorophyll. Oil and protein were expressed at zero moisture while total
glucosinolates were expressed at 8.50 moisture. Fatty acid analysis was done
using
gas chromatography. WCC/RRC guidelines were followed for analyzing quality
parameters.
CA 02 88347 1 2 015-02-2 7
Table 4. Summary of Performance of PV 532 G in two years of Co-op Testing
2
S")
(I)- - -13) ro
= ID'
Lcri TA 22 V) :ro. e (7, LL
1 -.E. 92 t=
,o 2 0 2 -Y- ? s.- -g a. , i c.:.), 4g)
>, -c == 6 -3 2 gr) II cc/3 8 - .. E- =
z - Co - = 0
. 0 Tt -0 0 u _ 5, a) .0 2
(I)
CO v a) te o o) a) ,c 8 = -
ag _' co -0 µa-, LI
> a) ce 0 v. o - 0 z
co co 0 mo ..., N C = co
'=-=' O.
co 2 a
0 CD = 0
CD
ay 0
, >-. .tli
2 cn
6 >, -8 5L 2 0
0 _ _, _ _ 1-2. To'
'I:.
6
0 a co "
a -
- - o_
3 a. o 0
I- I-
o
,-
2012
PV
20.4 94.9 99.9 53.1 5.9 6.3 111.0 49.03 46.24 11.99 6.40 0.00 1.2 3.9 7.2
532 G
5440 21.7 101.1 101.5 52.7 5.4 6.6 118.6 47.57 46.73 13.19 6.37 3.00 1.0
4.0 7.8
45H29 21.2 98.9 100.8 53.0 5.8 6.2 118.4 48.83 47.75 15.92 6.56 3.00 2.1
3.7 8.0
# Locs 9 9 10 6 10 8 8 10 10 10 10
10 8 8 2
Check
21.5 100.0 101.2 52.9 5.6 6.4 118.5 48.20 47.24 14.56 6.47 3.00 1.6 3.8 7.9
Avg.
Diff.
from -1.1 -5.1 -1.3 0.3 0.3 -0.1 -7.5 0.83 -1.00 -2.57 -0.06 -3.00 -0.4 0.0 -
0.7
Check
2013
PV
32.7 91.1 101.1 44.7 7.2 114.7 49.24 41.76 8.15 6.57
532 G
5440 36.9 103.0 102.9 45.4 7.6 123.7 47.93 42.91 9.90 6.53
45H29 34.8 97.0 102.1 44.4 6.8 124.6 49.33 43.07 12.20 6.70
# Locs 15 15 14 10 15 13 8 8 8 8
Check 35.9 100.0 102.5 44.9 7.2 124.2 48.6 42.99 11.05 6.62
Avg.
Diff.
from -3.2 -8.9 -1.4 -0.2 0.0 -9.5 -1.23 -2.90 -0.04
Check
2 Year Average
PV
28.1 92.5 100.6 47.9 5.9 6.9 113.3 49.12 44.25 10.28
6.48 0.00 1.2 3.9 7.2
532 G
5440 31.2 102.3 102.3 48.1 5.4 7.3 121.8 47.73 45.03 11.73
6.44 3.00 1.0 4.0 7.8
45H29 29.7 97.7 101.6 47.6 5.8 6.6 122.2 49.05 45.67 14.27
6.62 3.00 2.1 3.7 8.0
51
CA 02883471 2015-02-27
# Lacs 24 24 24 16 10 23 21 18 18 18 18 10
8 8 2
Check
30.5 100.0 101.9 47.9 5.6 6.9 122.0 48.39 45.35 13.00 6.53 3.00 1.6
3.8 7.9
Avg.
Diff.
from -2.4 -7.5 -1.3 0.0 0.3 0.0 -8.7 0.73 -1.10 -2.71 -0.06 -3.00 -0.4 0.0 -
0.7
Check
The foregoing discovery has been described in detail by way of illustration
and
example for purposes of exemplification. However, it will be apparent that
changes
and modifications such as single gene modifications and mutations, somaclonal
variants, variant individuals selected from populations of the plants of the
instant
variety, and the like, are considered to be within the scope of the present
discovery.
The scope of the claims should not be limited by the preferred embodiments set
forth
in the examples, but should be given the broadest interpretation consistent
with the
description as a whole.
52
