Note: Descriptions are shown in the official language in which they were submitted.
CA 02883319 2015-02-27
Title: CANOLA VARIETY 43E03
FIELD
The discovery is in the field of Brassica napus breeding (i.e., canola
breeding),
specifically relating to the canola variety designated 43E03.
lo BACKGROUND
The present discovery relates to a novel rapeseed variety designated 43E03
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 43E03 is provided. This discovery
thus relates to the seeds of the 43E03 variety, to plants of the 43E03
variety, and to
methods for producing a canola plant by crossing the 43E03 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
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CA 02883319 2015-02-27
transgenes, and to transgenic plants produced by that method. This discovery
also
relates to canola seeds and plants produced by crossing the variety 43E03 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.
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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
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
to 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
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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.
Grain. Seed produced by the plant or a self or sib of the plant that is
intended
for food or feed use.
ro 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 Anthocyanin Coloration. The presence or absence of leaf anthocyanin
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.
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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
ro wrinkles when at least six leaves of the plant are completely developed.
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.
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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.
icr 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.
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.
Ploidv. This refers to the number of chromosomes exhibited by the line, for
example diploid or tetraploid.
Pod Anthocvanin 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).
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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
to
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-
's 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
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
20
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.
25
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
30
streaking within such coloration when the plant has reached at least the six-
leaf
stage.
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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
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.
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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 Anthocvanin 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
ro 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
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,
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selfing and mutagenesis. However, the breeder commonly has no direct control
at
the cellular level of the plant. Therefore, two breeders will never
independently
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
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
1 o 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
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
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
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
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.
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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
io 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 F4
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 selection has
been
used to incorporate blackleg resistance into certain cultivars of Brassica
napus.
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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
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
io 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
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
is combining ability or specific combining ability) of the inbred.
Various breeding procedures are also utilized with these breeding and
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
20 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
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
25 generation advance is completed.
In a multiple-seed procedure, canola breeders commonly harvest one or more
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
30 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 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
i o 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
is 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
20 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
25 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
30 inbreds in the breeding program. In Brassica napus, microspore culture
technique is
used in producing haploid embryos. The haploid embryos are then regenerated on
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appropriate media as haploid plantlets, doubling chromosomes of which results
in
doubled haploid plants. This can be advantageous because the process omits the
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
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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 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, et al.,
1987). A
fertility restorer for Ogura cytoplasmic male sterile plants has been
transferred from
Raphanus sativus (radish) to Brassica by lnstit. National de Recherche
Agricole
(INRA) in Rennes, France (Pelletier, et al., 1987). The OGU INRA restorer
gene, 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 F1 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.
CA 02883319 2015-02-27
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
to 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 F1 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.
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 NS6992 is designated NS6992MC.
The restorer was developed at Georgetown Research Centre of Pioneer Hi-Bred
Production Limited bi-parental cross (NS5870MC/NS5648MC). The last crossing
was completed in 2006. Doubled haploid lines were the produced from the cross
in
May 2007. The Double haploid lines were evaluated in Ontario nursery in 2008.
During nursery evaluation, the lines were selected for early maturity, high
oil, high
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CA 02883319 2015-02-27
protein, low glucosinolates, general vigor and uniformity. The doubled haploid
lines
from the selected rows were sent to Chile for hybrid seed production in small
cages
during 2008/09 and the hybrids were evaluated in 2009 preliminary yield
trials.
NS6992MC as a restorer line was advanced to next level of hybrid testing in
2010,
and in 2011 11N290R utilizing the male NS6992MC was created in Chile and
tested.
NS6992MC was increased in a cage in Chile (2010/2011) and this seed was bulked
for breeder seed in 2011.
Hybrid Development
43E03 (11N290R) is a fully restored spring Brassica napus hybrid with
glyphosate resistant gene, based on OGU INRA system. It was developed at
Edmonton Research Centre of Pioneer Hi-Bred Production LP (subsidiary of
DuPont
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
herbicide
resistant 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.
17
CA 02883319 2015-02-27
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
to 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
is 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
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)
20 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
25 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.
30 Hybrid seed production requires inactivation of pollen produced by the
female
parent. Incomplete inactivation of the pollen provides the potential for self-
pollination.
18
CA 02883319 2015-02-27
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
lo 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 43E03 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
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 43E03
43E03 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
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
19
CA 02883319 2015-02-27
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
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
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
to 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
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% or 10%
significance level, when measured in plants grown in the same environmental
conditions. For example, a locus conversion of 43E03 may be characterized as
having essentially the same phenotypic traits as 43E03. 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 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 43E03 will retain the genetic integrity of 43E03. A
locus
conversion of 43E03 will comprise at least 92%, 93%, 94%, 95%, 96%, 97%, 98%
or
99% of the base genetics of 43E03. For example, a locus conversion of 43E03
can
be developed when DNA sequences are introduced through backcrossing (Hallauer
CA 02883319 2015-02-27
et al., 1988), with a parent of 43E03 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 Canola
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
micromoles (limo') glucosinolates per gram of defatted (oil-free) meal. These
types
of rapeseed are distinguished by their edibility in comparison to more
traditional
varieties of the species.
Disease - Sclerotinia
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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
io
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, et al., 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
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
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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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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, et al., 1984). In addition
to partial
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, et al., 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, et al. 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
24
CA 02883319 2015-02-27
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
varieties)
avoid Sclerotinia infection to a greater extent (Okuyama, et al., 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
to 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 Sclerotinia-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/Lance TM 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-
CA 02883319 2015-02-27
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.
Characteristics of 43E03
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
ro
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
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 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) 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.
43E03 is an early maturing, high yielding glyphosate resistant Brassica napus
canola hybrid having moderately resistant (MR) rating for blackleg and
resistant (R)
rating for Fusarium wilt. Its oil content is 0.9% higher than WCC/RRC checks.
Its
26
CA 02883319 2015-02-27
protein is 1.0% higher than mean of the checks and chlorophyll is 2.0% lower
than
the checks.
Table 1 provides data on morphological, agronomic, and quality traits for
43E03 and canola variety 43E02.
When preparing the detailed phenotypic
information that follows, plants of the new 43E03 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.
Observations were recorded on various morphological traits for the hybrid
43E03 and comparative check cultivars. (See Table 1).
to
Hybrid 43E03 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 43E03. 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 43E03. Further, both first and second parent
canola
plants can come from the canola variety 43E03. Either the first or the second
parent
plant may be male sterile.
Still further, this discovery also is directed to methods for producing a
43E03-
derived canola plant by crossing canola variety 43E03 with a second canola
plant and
growing the progeny seed, and repeating the crossing and growing steps with
the
canola 43E03-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 43E03 are part of this
discovery: open pollination, selfing, backcrosses, hybrid production, crosses
to
populations, and the like. All plants produced using canola variety 43E03 as a
parent
are within the scope of this discovery, including plants derived from canola
variety
43E03. This includes canola lines derived from 43E03 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 43E03. A single-gene
conversion occurs when DNA sequences are introduced through traditional (non-
27
CA 02883319 2015-02-27
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
ro selection for a trait associated with a dominant allele. Selection
of progeny for 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 43E03 can be manipulated to be male sterile by any of a number of
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 43E03. The male sterility may be either partial or complete
male
sterility. This discovery is also directed to F1 hybrid seed and plants
produced by the
use of Canola variety 43E03. Canola variety 43E03 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 43E03 could then be used as the male plant in
hybrid
seed production.
This discovery is also directed to the use of 43E03 in tissue culture. As used
herein, the term plant includes plant protoplasts, plant cell tissue cultures
from which
28
CA 02883319 2015-02-27
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.,
to
(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;
"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 43E03 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
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
43E03.
29
CA 02883319 2015-02-27
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.
io
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
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
plurality of transgenic plants which are harvested in a conventional manner,
and a
CA 02883319 2015-02-27
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
to 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
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, et al., (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) Cell 78:1089 (Arabidopsis RSP2 gene for resistance to Pseudomonas
syringae); McDowell and Woffenden, (2003) Trends Biotechnol. 21(4):178-83 and
31
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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, et al., (1998) P1. PhysioL 117(1):33-
41).
(C) A
Bacillus thuringiensis (Bt) protein, a derivative thereof or a synthetic
polypeptide modeled thereon. See, for example, Geiser, et al., (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
to
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, et al., (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) J.
Biol. Chem. 269:9 (expression cloning yields DNA coding for insect diuretic
hormone
receptor) and Pratt, et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an
allostatin is identified in Diploptera puntata); Chattopadhyay, et al., (2004)
Critical
Reviews in Microbiology 30(1):33-54 2004; Zjawiony, (2004) J Nat Prod
67(2):300-
310; Carlini 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.
32
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(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
io
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, et al., (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.
(l) 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
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.
33
CA 02883319 2015-02-27
(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, et al., (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.
(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.
34
CA 02883319 2015-02-27
(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
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
pyridinoxy or phenoxy propionic acids and cycloshexones (ACCase inhibitor-
CA 02883319 2015-02-27
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 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908;
5,312,910;
5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;
4,535,060;
4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and
international publications EP1173580; WO 01/66704; EP1173581 and EP1173582.
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/rechnology 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 B1 and 5,879,903. Exemplary of genes conferring
resistance
to phenoxy propionic acids and cycloshexones, such as sethoxydim and
haloxyfop,
are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al.,
(1992)
Theor. Appl. 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
36
CA 02883319 2015-02-27
Patent Number 4,810,648 to Stalker, and DNA molecules containing these genes
are
available under ATCC Accession Numbers 53435, 67441 and 67442. Cloning and
expression of DNA coding for a glutathione S-transferase is described by
Hayes, et
al., (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 cytochronne P4507A1 and yeast NADPH-cytochrome P450
1 o 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.
Sci.
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),
37
CA 02883319 2015-02-27
(3) Altering conjugated linolenic or linoleic acid content, such as in
WO 01/12800,
(4) Altering LEC1, AGP, Dek1, Superalt mi1ps, 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,
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
io (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,
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,
et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide sequence of Bacillus
subtilis
levansucrase gene), Pen, et al., (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),
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Sogaard, et al., (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of
barley
alpha-amylase gene) and Fisher, et al., (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.
(D)
Altered antioxidant content or composition, such as alteration of
i o
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
39
CA 02883319 2015-02-27
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).
io 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,
et al.,
and chromosomal translocations as described by Patterson in US Patents Numbers
3,861,709 and 3,710,511. In addition to these methods, Albertsen, et al., 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).
CA 02883319 2015-02-27
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).
io 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 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, et al., 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
41
CA 02883319 2015-02-27
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
lo 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),
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 43E03. "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
42
CA 02883319 2015-02-27
directed to produce subsequent generations of seed from seed of variety 43E03,
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
to 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 43E03 variety, the plant produced from such seed, various
parts of the 43E03 hybrid canola plant or its progeny, a canola plant produced
from
the crossing of the 43E03 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.
43
CA 02883319 2015-04-17
DEPOSIT
Applicant(s) have made or will make a deposit of at least 2500 seeds of canola
variety 43E03 with the American Type Culture Collection (ATCC), 10801
University
Boulevard, Manassas, VA 20110-2209 USA, ATCC Deposit No. PTA-122028. The
seeds deposited with the ATCC on February 20, 2015 for PTA-122028 were taken
from the seed stock maintained by Pioneer Hi-Bred International, Inc., 7250 NW
62'd
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 determined by the
o 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 43E03
with
the American Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, VA 20110-2209. This deposit of seed of canola variety 43E03 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 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 43E03 under the Plant Variety Protection Act (7 USC 2321 et seq.).
30
44
CA 02883319 2015-02-27
Varietal Characteristics (See also Tables 1 through 4)
Seed yield 6.4% lower than the mean of the WCC/RRC checks.
Disease reaction Classified as Moderately Resistant (MR) to blackleg
(Leptospaera maculans)
according to WCC/RRC guidelines. Classified as resistant (R) to Fusarium wilt
according to trials.
Plant height Approximately 8 cm shorter than the mean of the WCC/RRC
checks.
Maturity Approximately 2 days earlier maturing than the WCC/RRC
checks.
Lodging Slightly inferior lodging rating compared to the mean of
the WCC/RRC checks.
Herbicide tolerance Tolerant to glyphosate herbicides. Field testing
confirms that 43E03 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 0.9% higher than the mean of the WCC/RRC checks.
Seed protein content 1.0% higher than the mean of the WCC/RRC checks.
Erucic acid Less than 0.5% (maximum allowable limit).
Total saturates 0.02% lower than the mean of the WCC/RRC checks.
Total glucosinolates Canola quality ¨ 3.2 umol/g lower than the WCC/RRC
checks.
Chlorophyll 2.0 ppm lower than the mean of the WCC/RRC checks.
CA 02883319 2015-02-27
Table 1. Variety Descriptions based on Morphological, Agronomic and Quality
Traits
43E02
43E03
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
1.2 Seasonal Type Spring
Cotyledon width
3=narrow
2.1 5 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
2.3 3 Weak 3 Weak
3=weak
5=medium
7=strong
9=very strong
Leaf type
2.4 1=petiolate 8 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=medium 4 Short/Medium 6 Medium/Long
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=medium green 2 Medium green 2 Medium green
3=dark green
4=blue-green
Leaf lobe
development
1=absent or very
weak 2 Absent or very 3
2.12 Weak
3=weak weak/ Weak
5=medium
7=strong
9=very strong
Number of leaf
2.13 2 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
46
CA 02883319 2015-02-27
43E02
43E03 (Check Variety)
Trait
Trait Mean DescriptiCode on Mean Description
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 clasping 2 Partial clasping
clasping
2=partial clasping
3=non-clasping
3.1 Flower date 50% 46.7
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 5 Medium 5 Medium
5=medium
7=long
Petal width
3=narrow
3.7 5 Medium 5 Medium
5=medium
7=wide
Petal spacing
1=open
3=not touching Touching/Slight 5
3.8 6 Touching
5=touching Overlap
7=slight overlap
9=strongly overlap
Anther fertility
1=sterile All anthers All anthers
9 9
3'11 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)
47
CA 02883319 2015-02-27
3E03 43E02
4
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
Pod (silique) width Medium (4
3=narrow (3mm)
3.13 5 Medium (4 mm) 6 mm)/Wide (5
5=medium (4 mm)
mm)
7=wide (5mm)
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
3.165 Medium 5 Medium
5=medium
7=long
Maturity (days from
3.17 ant 100.1
pling)
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 6.5 Fair/Good
5=fair
7=good
9=excellent
Blackleg resistance
0=not tested
1=resistant
3=moderately
6.3
resistant Moderately
3
5=moderately Resistant
susceptible
7=susceptible
9=highly
susceptible
48
CA 02883319 2015-02-27
4
43E03 3E02
(Check Variety)
Trait
Trait Mean Description Mean Description
Code
Fusarium wilt
resistance
0=not tested
1=resistant
3=moderately
6.7 resistant 1 Resistant
5=moderately
susceptible
7=susceptible
9=highly
susceptible
Oil content
8.1 48.88
percentage
Protein percentage
8.5 46.31
(whole dry seed)
Glucosinolates
(pmoles total
glucs/g whole seed) Very low
8.7 1= very low (<10) 1 (<10 pmol per
2= low (10-15) gram)
3=medium (15-20)
4=high (>20)
Chlorophyll content
(mg/kg seed)
1=low (<8 ppm)
8.8 1 Low (<8 ppm)
2=medium (8-15
ppm)
3=high (>15 ppm)
49
CA 02 88331 9 2015-02-27
Example 1. Herbicide Resistance
Appropriate field tests have shown that 43E03 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
43E03 in
herbicide tolerance trials in 2012 and 2013
2012 Vegreville, AB
% Stand Days Gluc's
Treat Yield Height Days to % % Oil +
Variety Reduction to @
Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
43E03 2X 22.8 0 51 87 99 45.5 51.4 96.9
16.1 4.2
43E02 2X 20.7 0 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
ment q/ha (cm) Maturity Oil Protein Protein @
Chlorophyll
(PCTSR) Flower 8.5%
_
43E03 2X 26.0 o 51 103 101 49.5 44.6 94.1
9.7 2.2
43E02 2X 24.8 o 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
CA 02 88331 9 2 015-02-2 7
Table 2, continued
2013 Saskatoon, SK
Gluc's
Treat Yield % Stand Days
Height Days to % % Oil +
Variety Reduction to @
Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8,5%
43E03 2X 15.4 3 46 85 98 48.3 45.9 94.2 9.4
5.0
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
% Stand DaysGluc's
Treat Yield Height Days to % % Oil +
Variety Reduction to @
Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
43E03 2X 20.6 2 49.0 94.0 100.0 48.9 45.3
94.2 9.6 3.6
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
Gluc's
Treat Yield % Stand Days
Height Days to % % Oil +
Variety Reduction to @
Chlorophyll
ment q/ha (cm) Maturity Oil Protein Protein
(PCTSR) Flower 8.5%
43E03 2X 15.4 1 49 92 99 47.8 47.3 95.1
11.7 3.8
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
51
CA 02883319 2015-02-27
Locations 3 3 3 3 3 3 3 3 3 3
52
CA 02883319 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
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
40
53
CA 02883319 2015-02-27
Table 3. Summary of Blackleg Ratings for 43E03
2012 2013
Plum2 Year %
Manitou Rosebank Alvena Boissevain Carman Vegreville
Class
Coulee
Average Westar
43E03 1.3 1.7 0.9 0.7 1.2 0.7 1.7 1.2 34.5
MR
Westar 2.6 3.7 4.1 3.3 2.9 3.4 3.8 3.4
Example 3: Summary of Performance of 43E03 in two years of Co-op Testing
Two years (2012 and 2013) of trials were conducted. WCC/RRC guidelines
were followed for conducting trials. Each trial had four 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 selected sites and were analyzed
for quality
traits such as oil and protein percent at 8.5% moisture, total whole seed
glucosinolates
at 8.5%, chlorophyll, total saturated fatty acid, 1000 seed weight etc.
WCC/RRC
guidelines were followed for analyzing quality parameters.
54
CA 02 88331 9 2015-02-27
Table 4. Summary of Performance of 43E03 in two years of Co-op Testing
E.) -a
. -z-6-
.>,
f =(-1i 92 z Li-
i µ,1) 77
(.7 o
o 1=
= 8 I'D - ? = E -a
= 0 0_ -a . 0
o _S)
>, 2 ..tin. a u) A .:E-= 0 d)
g (?) A
- 0
u) ,- - --5., 0
.a.) 'a-'c
LL > 0) .0) 2 _
-o ,,'' o a)
o >, ,_- c ,_- 41) a) < o =
<':'' _c (/)
= 16 --- o_ c
lo tc) = -
cp
> -cn o I
T
0 _ N C 3 n 2
a) >, , w 0. 7 ,
3 o_ c .) c a)
o 2 (1)
_0 0-
co c
a co
a ,- -I µ_ -
- - ci_ o_ o o -
3 1-o 1-0 _
2012
43E03 20.8 96.9 99.0 51.4 6.3 5.7 110.4 48.88 48.24 11.57 6.49 1.00 0.9 3.9
7.2
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 -0.7 -3.1 -2.2 -1.5 0.7 -0.7 -8.1 0.68 1.00 -2.99 0.03 -2.00 -0.6 0.0 -
0.7
Check
2013
43E03 32.8 91.6 100.8 43.9 6.9 116.8 49.84 43.89 7.59 6.53
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 -3.0 -8.4 -1.7 -1.0 -0.3 -7.4 0.90 -3.46 -0.09
Check
2 Year Average
43E03 28.3 93.6 100.1 46.7 6.3 6.5 114.4 49.31 46.31 9.80 6.51 1.00 0.9 3.9
7.2
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
i
CA 02 88331 9 2015-02-27
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
# Locs 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 -2.1 -6.4 -1.9 -1.2 0.7 -0.4 -7.6 0.92 0.96 -3.20 -0.02 -2.00 -0.6 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
to in the examples, but should be given the broadest interpretation
consistent with the
description as a whole.
56
