U.S. patent application number 14/246157 was filed with the patent office on 2014-08-07 for genomically multiplied rapeseed plants, compositions derived therefrom and uses of same.
This patent application is currently assigned to Kaiima Bio Agritech Ltd.. The applicant listed for this patent is Kaiima Bio Agritech Ltd.. Invention is credited to Amit Avidov, Alon Lerner.
Application Number | 20140223594 14/246157 |
Document ID | / |
Family ID | 40293763 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140223594 |
Kind Code |
A1 |
Avidov; Amit ; et
al. |
August 7, 2014 |
GENOMICALLY MULTIPLIED RAPESEED PLANTS, COMPOSITIONS DERIVED
THEREFROM AND USES OF SAME
Abstract
A rapeseed plant having a multiplied genome being at least as
fertile as a euploid rapeseed plant isogenic to the genomically
multiplied plant grown under similar conditions.
Inventors: |
Avidov; Amit; (Kiryat-Tivon,
IL) ; Lerner; Alon; (Moshav Sharona, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kaiima Bio Agritech Ltd. |
Kfar-Tavor |
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IL |
|
|
Assignee: |
Kaiima Bio Agritech Ltd.
Kfar-Tavor
IL
|
Family ID: |
40293763 |
Appl. No.: |
14/246157 |
Filed: |
April 7, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12741376 |
May 5, 2010 |
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PCT/IL2008/001468 |
Nov 6, 2008 |
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14246157 |
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60996213 |
Nov 6, 2007 |
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Current U.S.
Class: |
800/260 ;
435/410; 554/9; 800/306 |
Current CPC
Class: |
A01H 5/10 20130101; A01H
1/08 20130101 |
Class at
Publication: |
800/260 ;
800/306; 435/410; 554/9 |
International
Class: |
A01H 5/10 20060101
A01H005/10; A01H 1/08 20060101 A01H001/08 |
Claims
1. A Brassica napus rapeseed plant having a doubled amphidiploid
genome being at least as fertile as a euploid Brassica napus
rapeseed plant isogenic to the genomically doubled plant grown
under similar conditions, wherein fertility is determined by number
of seeds per plant.
2. The plant of claim 1, exhibiting genomic stability for at least
2 generations.
3. The plant of claim 1, having a seed weight per 40 seeds
exceeding that of said euploid rapeseed plant.
4. The plant of claim 1, having seed yield or seed weight at least
as similar to that of said isogenic euploid plant grown under the
same conditions and being of the same developmental age.
5. The plant of claim 1, having photosynthetic efficiency at least
as similar to that of said isogenic euploid plant grown under the
same conditions and being of the same developmental age.
6. A plant part of the rapeseed plant of claim 1.
7. The plant part of claim 6, being a seed.
8. The plant part of claim 7, wherein said seed is a hybrid
seed.
9. Rapeseed meal produced from the plant of claim 1.
10. An isolated regenerable cell of the rapeseed plant of claim
1.
11. The cell of claim 10, exhibiting genomic stability for at least
2 generations in culture.
12. The cell of claim 10 being from a meristem, pollen, a leaf, a
root, a root tip, an anther, a pistil, a flower, a seed or a
stem.
13. A tissue culture comprising the regenerable cell of claim
10.
14. A method of producing seeds of rapeseed, comprising
self-breeding or cross-breeding the plant of claim 1.
15. A method of producing rapeseed oil, the method comprising: (a)
harvesting seeds of the rapeseed plant of claim 1; and (b)
processing said seeds so as to produce the rapeseed oil.
16. A tissue culture comprising the regenerable cell of claim 12.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/741,376 filed on May 5, 2010, which is a
National Phase of PCT Patent Application No. PCT/IL2008/001468
having International filing date of Nov. 6, 2008, which claims the
benefit of priority of U.S. Provisional Patent Application No.
60/996,213 filed on Nov. 6, 2007. The contents of the above
applications are all incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention, in some embodiments thereof, relates
to genomically multiplied rapeseed plants, compositions derived
therefrom and uses of same.
[0003] Rapeseed (Brassica napus), also known as rape, oilseed rape,
rapa, and (in the case of one particular group of cultivars)
canola, is a bright yellow flowering member of the family
Brassicaceae (mustard or cabbage family).
[0004] Rapeseed is grown for the production of animal feed,
vegetable oil for human consumption, and biodiesel; leading
producers include the European Union, Canada, the United States,
Australia, China and India. In India, it is grown on 13% of cropped
land. According to the United States Department of Agriculture,
rapeseed was the third leading source of vegetable oil in the world
in 2000, after soybean and oil palm, and also the world's second
leading source of protein meal, although only one-fifth of the
production of the leading soybean meal. World production is growing
rapidly, with FAO reporting that 36 million tonnes of rapeseed was
produced in the 2003-4 season, and 46 million tons in 2004-5.
[0005] The rapeseed is the valuable, harvested component of the
crop. The crop is also grown as a winter-cover crop. It provides
good coverage of the soil in winter, and limits nitrogen run-off.
The plant is ploughed back in the soil or used as bedding.
Processing of rapeseed for oil production provides rapeseed animal
meal as a by-product. The by-product is a high-protein animal feed,
competitive with soya. The feed is mostly employed for cattle
feeding, but also for pigs and chickens (though less valuable for
these). The meal has a very low content of the glucosinolates
responsible for metabolism disruption in cattle and pigs. Rapeseed
"oil cake" is also used as a fertilizer in China, and may be used
for ornamentals, such as Bonsai, as well. Rapeseed leaves and stems
are also edible, similar to those of the related bok choy or kale.
Rapeseed is a heavy nectar producer, and honeybees produce a light
colored, but peppery honey from it. Canola oil (or rapeseed oil)
contains both omega-6 and omega-3 fatty acids in a ratio of 2:1 and
is second only to flax oil in omega-3 fatty acid. Canola oil's
proponents claim that it is one of the most heart-healthy oils and
has been reported to reduce cholesterol levels, lower serum
tryglyceride levels, and prevent hypercoagulation.
[0006] Rapeseed oil is used in the manufacture of biodiesel for
powering motor vehicles. Biodiesel may be used in pure form in
newer engines without engine damage, and is frequently combined
with fossil-fuel diesel in ratios varying from 2% to 20% biodiesel.
Formerly, owing to the costs of growing, crushing, and refining
rapeseed biodiesel, rapeseed derived biodiesel cost more to produce
than standard diesel fuel. Prices of rapeseed oil are at very high
levels presently (start November 2005) owing to increased demand on
rapeseed oil for this purpose. Rapeseed oil is the preferred oil
stock for biodiesel production in most of Europe, partly because
rapeseed produces more oil per unit of land area compared to other
oil sources, such as soy beans.
[0007] Thus, rapeseed, is an important and valuable field crop.
Therefore, a continuing goal of plant breeders is to develop
stable, high yielding rapeseed cultivars that are agronomically
sound. The reasons for this goal are obviously to maximize the
amount of grain produced on the land used and to supply food for
both animals and humans. To accomplish this goal, the rapeseed
breeder must select and develop rapeseed plants that have the
traits that result in superior cultivars.
[0008] To date, there are no tetraploid rapeseed varieties and the
main obstacles seem to be genetic instability and absence of
sufficient sexual fertility to achieve a commercially valuable
hybrid.
[0009] Additional background art includes:
[0010] C. Mollers, M. C. M. Iqbal and G. Robbelen; Efficient
production of doubled haploid Brassica napus plants by colchicine
treatment of microspores, Euphytica, Vol. 75, Numbers 1-2/January,
1994, Springer, Netherlands.
[0011] Zhang, G. Q.; Resynthesizing Brassica napus from
interspecific hybridization between Brassica rapa and B. oleracea
through ovary culture. Euphytica 140(3), 2004.
[0012] Robert T. Gaeta, J. Chris Pires, Federico Iniguez-Luy,
Enrique Leon and Thomas C. Osborn; Genomic Changes in Resynthesized
Brassica napus and Their Effect on Gene Expression and Phenotype;
The Plant Cell 19:3403-3417. 2007.
SUMMARY OF THE INVENTION
[0013] According to an aspect of some embodiments of the present
invention there is provided a rapeseed plant having a multiplied
genome being at least as fertile as a euploid rapeseed plant
isogenic to the genomically multiplied plant grown under similar
conditions.
[0014] According to some embodiments of the invention, the
fertility is determined by at least one of:
[0015] number of seeds per plant;
[0016] gamete fertility assay; and
[0017] acetocarmine staining.
[0018] According to some embodiments of the invention, the plant
exhibits genomic stability for at least 2 passages.
[0019] According to some embodiments of the invention, the plant
has a seed weight exceeding that of the euploid rapeseed plant.
[0020] According to an aspect of some embodiments of the present
invention there is provided a rapeseed plant as deposited under the
Budapest treaty in NCIMB Ltd. and having Accession No. NCIMB 41592
Brassica napus 187-2-4N.
[0021] According to an aspect of some embodiments of the present
invention there is provided a plant part of the rapeseed plant.
[0022] According to an aspect of some embodiments of the present
invention there is provided a rapeseed oil produced from the plant
or plant part.
[0023] According to an aspect of some embodiments of the present
invention there is provided a rapeseed meal produced from the plant
or plant part.
[0024] According to some embodiments of the invention, the plant
part is a seed.
[0025] According to an aspect of some embodiments of the present
invention there is provided an isolated regenerable cell of the
rapeseed plant.
[0026] According to some embodiments of the invention, the cell
exhibits genomic stability for at least 2 passages in culture.
[0027] According to some embodiments of the invention, the cell is
from a mertistem, pollen, a leaf, a root, a root tip, an anther, a
pistil, a flower, a seed or a stem.
[0028] According to an aspect of some embodiments of the present
invention there is provided a tissue culture comprising the
regenerable cells.
[0029] According to an aspect of some embodiments of the present
invention there is provided a method of producing seeds of
rapeseed, comprising self-breeding or cross-breeding the plant.
[0030] According to an aspect of some embodiments of the present
invention there is provided a method of producing rapeseed oil, the
method comprising:
[0031] (a) harvesting seeds of the rapeseed plant or plant part;
and
[0032] (b) processing the seeds so as to produce the rapeseed
oil.
[0033] According to an aspect of some embodiments of the present
invention there is provided a method of generating a genomically
multiplied rapeseed, the method comprising contacting seeds of the
rapeseed with a G2/M cell cycle inhibitor under a magnetic field
thereby generating the genetically multiplied rapeseed seeds.
[0034] According to some embodiments of the invention, the G2/M
cell cycle inhibitor comprises a microtubule polymerization
inhibitor.
[0035] According to some embodiments of the invention, the
microtubule polymerization inhibitor is selected from the group
consisting of colchicine, nocodazole, oryzaline, trifluraline and
vinblastine sulphate.
[0036] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0038] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0039] In the drawings:
[0040] FIGS. 1A-D are FACS output images showing propidium iodide
staining in a multiplied rapeseed plant (FIGS. 1C-D) vs control
euploid plant (FIGS. 1A-B). An FL2 laser was used to detect the
Propidium Iodide dye. Flow cytometer analysis displayed by
histograms: one-parameter histogram that displays the distribution
of cells according to their DNA content. The G1 phase of the
non-multiplied plant positioned on channel 300 (M1). Second dot
plot histogram shows that all the cells that belong to channel 300
are of the same size (FL2W). S phase of the cell cycle is marked as
M2. On channel 600 appears the G2 of the cell cycle of the
non-multiplied plant (M3). FIGS. 1C and 1D panels, the M3 region
shows the G1 of the multiplied plant.
As is evident from the dot plot, the cells moved to 600 and show a
bigger phenotype.
[0041] FIG. 2 is a table the statistics of the sterility trait in a
hybrid between a multiplied male-sterile plant and a multiplied
fully fertile plant.
[0042] FIGS. 3A-F are photographs showing the difference between
the multiplied rapeseed generated according to the present
teachings and the euploid plants in terms of pod size, number of
seeds, seed size and flower size.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0043] The present invention, in some embodiments thereof, relates
genomically multiplied rapeseed plants, compositions derived
therefrom and uses of same.
[0044] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0045] Rapeseed is grown for the production of animal feed,
vegetable oil for human consumption, and biodiesel. Leading
producers include the European Union, Canada, the United States,
Australia, China and India. In India, it is grown on 13% of cropped
land. Over the years scientists have attempted to increase the
overall oil content of the seed without compromising agronomic
performance.
[0046] In order to meet these needs, the present inventors have
identified conditions for genome multiplication in rapeseed plant
seeds. Genomically multiplied rapeseed generated according to the
present teachings provide for progeny plants characterized by as
high a yield (e.g., seed yield, oil yield) and fertility as their
isogenic euploid plants.
[0047] Thus, according to an aspect of the present invention there
is provided a rapeseed plant having a multiplied genome being at
least as fertile as a euploid rapeseed plant isogenic to the
genomically multiplied plant grown under similar conditions.
[0048] As used herein the phrase "rapeseed plant" is the bright
yellow flowering member of the family Brassicaceae (mustard or
cabbage family) also termed as rape, oilseed rape, rapa, rapaseed
and in the case of one particular group of cultivars, canola.
[0049] Rapeseed is a dibasic allotetraploid (i.e., amphidiploid)
formed of two genomes (i.e., the A-genome and C-genome) and has a
total of 38 chromosomes. The A-genome component is derived from
Brassica campestris and consists of 20 chromosomes. The C-genome
component is derived from Brassica oleracea and consists of 18
chromosomes. A rapeseed plant having the 38 chromosomes as
described hereinabove is referred to herein as being euploid (i.e.,
non-multiplied). In some embodiments of the present invention the
euploid plant is isogenic. The euploid plant, as used herein is
isogenic to the multiplied plant i.e., the sets of chromosomes
contain essentially identical alleles in all locations. The euploid
plant may be naturally occurring, genetically modified or a
breeding product.
[0050] The rapeseed plant of some embodiments of the present
invention refers to a whole plant or portions thereof, processed or
non-processed (e.g., seeds, oil, dry tissue, meal, cake etc.),
regenerable tissue culture and cells isolated therefrom.
[0051] As used herein the term "multiplied genome" refers to a
plant in which there is a higher (e.g., double) ploidy than in the
isogenic euploid progenitor, that is a higher chromosome copy
number than that of the euploid plant (e.g., 5N, 6N, 7N, 8N, 10N).
According to some embodiments of the present invention, the
genomically multiplied plant is an autopolyploid i.e., the result
of chromosome duplication.
[0052] As used herein the term "fertile" refers to the ability to
reproduce sexually. Fertility can be assayed using methods which
are well known in the art. The following parameters may be assayed
in order to determine fertility: the number of seeds; gamete
fertility may be determined by pollen germination such as on a
sucrose substrate; and pollen fertility such as assayed
microscopically using acetocarmine, whereby a fertile pollen is
stained.
[0053] According to some embodiments of the present invention, a
mature multiplied rapeseed plant has at least about the same
(+/-10%) number of seeds as its isogenic progenitor when grown
under the same conditions; and optionally further has at least 90%
fertile pollen that are stained by acetocarmine; and alternatively
or additionally at least 90% of seeds germinate on sucrose.
[0054] Assays done for characterizing traits (e.g., fertility,
yield, biomass and vigor) of the multiplied plants of the present
invention, are typically effected in comparison to the isogenic
progenitor (i.e., the euploid plant) being of the same
developmental age as the tested plant and under similar growth
conditions.
[0055] Thus, according to some embodiments of the present
invention, the genomically multiplied plant has a larger surface
area of a leaf than that of the euploid rapeseed plant. In
exemplary embodiments leaf area: 30%-100% larger than that of the
euploid plant and leaf thickness is at least 1.5-2.5 greater than
that of the euploid plant.
[0056] According to some embodiments of the present invention, the
genomically multiplied plant has a larger stomata surface than that
of the euploid rapeseed plant. In an exemplary embodiment the
stomata surface area is at least 1.5-2.5 greater than that of the
euploid plant.
[0057] According to some embodiments of the present invention, the
genomically multiplied plant is capable of cross-breeding with a
euploid plant.
[0058] According to some embodiments of the present invention, the
genomically multiplied has higher photosynthetic efficiency that
the of the euploid plant.
[0059] According to some embodiments of the present invention, the
genomically multiplied plant is stable for at least 4, 5, 7, 9 or
10 generations.
As used herein the term "stable" refers to the number of
chromosomes or chromosome copies, which remains constant through
several generations, while the plant exhibits no substantial
decline in at least one of the following parameters: yield,
fertility, biomass and vigor.
[0060] According to some embodiments of the present invention, the
genomically multiplied plant has seed yield (as determined by at
least one of: seed number, seed dimensions and volumetric oil
content) at least as similar to an isogenic euploid plant grown
under the same conditions and being of the same developmental age.
According to further embodiments of the present invention, the seed
yield exceeds that of the euploid plant by at least about 1.25,
1.5, 1.75, 2, 2.5 3 or 5 folds.
[0061] According to some embodiments of the present invention, the
polyploid plant has seed yield (as determined by at least one of:
seed number, seed dimensions and volumetric oil content) at least
as similar to the isogenic euploid plant. According to further
embodiments of the present invention, the seed yield exceeds that
of the euploid plant by at least about 1.15, 1.25, 1.5, 1.75, 2,
2.5, 3 or 5 folds.
[0062] According to further embodiments of the present invention,
the seed weight exceeds that of the euploid plant by at least about
1.15, 1.25, 1.5, 1.75, 2, 2.5, 3 or 5 folds.
[0063] The plants of this aspect of the present invention can be
generated using an improved method of colchicination, as
follows.
[0064] Polyploid plants of the present invention can be generated
using an improved method of colchicination, as described infra.
[0065] Germinating the seeds for 8 hours at a temperature of
27.degree. C. in distilled water. Thereafter soaking the seeds in a
multiplication solution comprising: 0.5% colchicine 0.5% DMSO,
0.03% Triton x 100 for 20 hours. Finally, the seeds are washed and
seeded in an appropriate germination bed in 27.degree. C.
[0066] Additionally or alternatively, multiplied rapeseed plants of
the present invention can be generated using colchicine or any
other cell cycle inhibitor (e.g., G2/M phase inhibitors, such as
microtubule assembly inhibitors e.g., colchicine, vinblastine,
nocodazole, oryzaline and trifluraline), whereby the targeting
agent is a magnetic field for targeted delivery of the inhibitor to
the chromatin fibers.
[0067] A specific embodiment of such a method is provided
hereinbelow. Of note, measures are taken to maintain the indicated
pH values each phase (such as with HCL or NaOH).
[0068] Stage One--3 Hours:
[0069] Seeds are incubated in a Petri dish at a temperature of
26.degree. C. in the dark in a vinblastine sulphate (0.1% v/v)
solution comprising 0.5% DMSO titrated to pH 5.6. pH conditions are
monitored so as to maintain constant pH (5.6) throughout this
phase.
[0070] The vessel is positioned in a magnetic field of 1300 Gauss,
whereby the magnets are located 10.5 cm from each other.
[0071] Stage Two--3 Hours:
[0072] The seeds are incubated in the above solution in day-light
conditions 4.degree. C. and pH is titrated to 6.
[0073] Stage Three--6 Hours
[0074] The seeds are incubated in day-light conditions 20.degree.
C. and pH is titrated to 5.4.
[0075] Stage Four--12 Hours
[0076] The seeds are incubated in day-light conditions 26.degree.
C. and pH is titrated to 6. The magnetic field is removed and
Nocodazole is added to a concentration of 5 m/ml.
[0077] Stage Five--12 Hours:
[0078] The seeds are incubated at day light under constant
temperature conditions (26.degree. C.).
[0079] The seeds are washed well in water so as to increase pH to
7. Thereafter, the seeds are seeded on appropriate growth beds
under long-day light conditions (16 hours) 26.degree. C.
[0080] Using the above teachings, the present inventors have
established genetically multiplied rapeseed plants such as that
deposited under the Budapest treaty in NCIMB Ltd. and having
Accession No. NCIMB 41592 Brassica napus 187-2-4N.
[0081] Once established, the rapeseed plants of the present
invention can be propagated sexually or asexually such as by using
tissue culturing techniques.
[0082] As used herein the phrase "tissue culture" refers to plant
cells or plant parts from which rapeseed plants can be generated,
including plant protoplasts, plant cali, plant clumps, and plant
cells that are intact in plants, or part of plants, such as seeds,
leaves, stems, pollens, roots, root tips, anthers, ovules, petals,
flowers, embryos, fibers and bolls.
[0083] According to some embodiments of the present invention, the
cultured cells exhibit genomic stability for at least 2, 3, 4, 5,
7, 9 or 10 passages in culture.
[0084] Techniques of generating plant tissue culture and
regenerating plants from tissue culture are well known in the art.
For example, such techniques are set forth by Vasil., 1984. Cell
Culture and Somatic Cell Genetics of Plants, Vol I, II, III,
Laboratory Procedures and Their Applications, Academic Press, New
York; Green et al., 1987. Plant Tissue and Cell Culture, Academic
Press, New York; Weissbach and Weissbach. 1989. Methods for Plant
Molecular Biology, Academic Press; Gelvin et al., 1990, Plant
Molecular Biology Manual, Kluwer Academic Publishers; Evans et al.,
1983, Handbook of Plant Cell Culture, MacMillian Publishing
Company, New York; and Klee et al., 1987. Ann. Rev. of Plant Phys.
38:467 486.
[0085] The tissue culture can be generated from cells or
protoplasts of a tissue selected from the group consisting of
seeds, leaves, stems, pollens, roots, root tips, anthers, ovules,
petals, flowers, embryos, fibers and bolls.
[0086] It will be appreciated that the plants of the present
invention can also be used in plant breeding along with other
rapeseed plants (i.e., self-breeding or cross breeding) in order to
generate novel plants or plant lines which exhibit at least some of
the characteristics of the rapeseed plants of the present
invention.
[0087] Plants resultant from crossing any of these with another
plant can be utilized in pedigree breeding, transformation and/or
backcrossing to generate additional cultivars which exhibit the
characteristics of the rapeseed plants of the present invention and
any other desired traits. Screening techniques employing molecular
or biochemical procedures well known in the art can be used to
ensure that the important commercial characteristics sought after
are preserved in each breeding generation.
[0088] The goal of backcrossing is to alter or substitute a single
trait or characteristic in a recurrent parental line. To accomplish
this, a single gene of the recurrent parental line is substituted
or supplemented with the desired gene from the nonrecurrent line,
while retaining essentially all of the rest of the desired genes,
and therefore the desired physiological and morphological
constitution of the original line. The choice of the particular
nonrecurrent parent will depend on the purpose of the backcross.
One of the major purposes is to add some commercially desirable,
agronomically important trait to the plant. The exact backcrossing
protocol will depend on the characteristic or trait being altered
or added to determine an appropriate testing protocol. Although
backcrossing methods are simplified when the characteristic being
transferred is a dominant allele, a recessive allele may also be
transferred. In this instance, it may be necessary to introduce a
test of the progeny to determine if the desired characteristic has
been successfully transferred. Likewise, transgenes can be
introduced into the plant using any of a variety of established
transformation methods well-known to persons skilled in the art,
such as: Gressel., 1985. Biotechnologically Conferring Herbicide
Resistance in Crops: The Present Realities, In: Molecular Form and
Function of the plant Genome, L van Vloten-Doting, (ed.), Plenum
Press, New York; Huftner, S. L., et al., 1992, Revising Oversight
of Genetically Modified Plants, Bio/Technology; Klee, H., et al.,
1989, Plant Gene Vectors and Genetic Transformation: Plant
Transformation Systems Based on the use of Agrobacterium
tumefaciens, Cell Culture and Somatic Cell Genetics of Plants; and
Koncz, C., et al. 1986, Molecular and General Genetics.
[0089] It will be appreciated that rapeseed plants (progenitor or
multiplied) of the present invention can be genetically modified
such as in order to introduce traits of interest e.g. Improved oil
composition and enhanced resistance to stress (e.g., biotic or
abiotic). Non-limiting examples of nucleic acid sequences useful
for altering oil composition of rapeseed plants and methods of
rapeseed transformation, as well as nucleic acid constructs useful
for same are described in U.S. Pat. No. 6,974,893, which is hereby
incorporated by reference in it's entirety.
[0090] According to some embodiments of the present invention the
fatty acid composition of the multiplied rapeseed is about the same
as that of the euploid rapeseed plant, although the level of the
different components may vary.
[0091] Thus, the present invention provides novel rapeseed plants
and cultivars, and seeds and tissue culture for generating
same.
[0092] Rapeseed plants generated based on the present teachings can
be further processed to generate rapeseed plant products which are
commonly used in for numerous industrial applications, including
animal feed, vegetable oil for human consumption, and
biodiesel.
[0093] U.S. Pat. No. 6,441,278 provides exemplary methods for
processing rapeseed and is hereby incorporated by reference in its
entirety. Following is a non-limiting description. Rapeseed seed is
collected and crushed by techniques known in the art. The seed
typically is tempered by spraying the seed with water to raise the
moisture to, for example, 8.5%. The tempered seed is flaked using
smooth roller with, for example, a gap setting of 0.23 to 0.27 mm.
Heat may be applied to the flakes to deactivate enzymes, facilitate
further cell rupturing, coalesce the oil droplets and agglomerate
protein particles in order to ease the extraction process.
[0094] Typically, oil is removed from the heated rapeseed flakes by
a screw press to press out a major fraction of the oil from the
flakes. The resulting press cake contains some residual oil.
[0095] Crude oil produced from the pressing operation typically is
passed through a settling tank with a slotted wire drainage top to
remove the solids expressed out with the oil in the screw pressing
operation. The clarified oil can be passed through a plate and
frame filter to remove the remaining fine solid particles.
[0096] Rapeseed press cake produced from the screw pressing
operation can be extracted with commercial n-Hexane. The rapeseed
oil recovered from the extraction process is combined with the
clarified oil from the screw pressing operation, resulting in a
blended crude oil.
[0097] Free fatty acids and gums typically are removed from the
crude oil by heating in a batch refining tank to which food grade
phosphoric acid has been added. The acid serves to convert the
non-hydratable phosphatides to a hydratable form, and to chelate
minor metals that are present in the crude oil. The phosphatides
and the metal salts are removed from the oil along with the
soapstock. The oil-acid mixture is treated with sodium hydroxide
solution to neutralize the free fatty acids and the phosphoric acid
in the acid-oil mixture. The neutralized free fatty acids,
phosphatides and the like (soapstock) are drained off from the
neutralized oil. A water wash may be done to further reduce the
soap content of the oil. The oil may be bleached and deodorized
before use, if desired, by techniques known in the art.
[0098] It is expected that during the life of a patent maturing
from this application many relevant rapeseed products will be
developed and the scope of the patent is intended to include all
such new technologies a priori.
[0099] As used herein the term "about" refers to .+-.10%.
[0100] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to". This term encompasses the terms "consisting of" and
"consisting essentially of".
[0101] The phrase "consisting essentially of" means that the
composition or method may include additional ingredients and/or
steps, but only if the additional ingredients and/or steps do not
materially alter the basic and novel characteristics of the claimed
composition or method.
[0102] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0103] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0104] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0105] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0106] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0107] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0108] Reference is now made to the following examples, which
together with the above descriptions, illustrate some embodiments
of the invention in a non limiting fashion.
Example 1
Generation of Polyploid Rapeseed Plants
[0109] The Agricultural Academy of Anhui Province in China provided
the two male parent lines (CHARO 1 and CHARO 8) and one female line
with genetic sterility (CHAO 1). These parental lines underwent
genome multiplication treatment using the mutation free genome
multiplication (MFGM) technology according to any of the protocols
described herein.
[0110] The treated plants underwent preliminary selection at the
seedling stage and later were planted in a section protected by an
insect net.
[0111] When the plants developed to the level of 5.sup.th real
leaf, they were all tested in a FACS machine (Fluorescence
Activated Cell Sorter) for genome multiplication. Briefly, nuclei
were released from 2 cm.times.2 cm leaf tissue by immersion in
chopping buffer for 30 seconds.
[0112] Chopping buffer consisted of 4.575 gram MgCl.sub.2, 2.095
gr.--MOPS, 4.4 gr--Sodium Citrate, 1 gr--DTT, 1.65 gr--Triton x 100
per 500 ml of distilled water. Chopping was done with a razor
blade, in one direction. The chopped tissue was transferred to
Petri dish and placed on ice.
[0113] The sample was filtered before use (20 mesh). Nuclei samples
(2 cc-6 cc) were removed to a FACS tube and 15 .mu.l propidium
iodide (PI) was added to each sample. Following 15 min, the samples
were analysed in a flow cytometer fitted with a Cyonics argon laser
(488 nm) operating at 15 mW.
[0114] Fluorescence that exceeded 635 nm is gated and results
displayed as single parameter histograms of number of nuclei in
each of 1024 channels. Control was fixed to channel 300.
[0115] FIGS. 1A-D show the output of a FACS machine for a control
euploid plant versus a polyploid plant. All the genomically
multiplied plants were tested for pollen fertility by germinating
the pollen on a background containing sucrose solution.
[0116] Only the plants which pollen fertility was intact and the
pollen grains appeared unharmed in a visual microscopic examination
were left in the field for self pollination and for producing F1
hybrids. When the seeds matured, self-pollination seeds were
collected from 50 multiplied plants from each of the two Chinese
male varieties, the CHARO 1 and CHARO 8. These seeds were planted
to test stability 2 generations and only the stable lines were kept
for the remainder of the program. Hybridizations between all the
fertile multiplied plants and between all the multiplied sterile
plants from the CHAO 1 female line were effected.
[0117] Only the plants with the Aaaa genotype are capable of
maintaining the sterile line and the distribution of the gametes of
the Aaaa X aaaa cross is shown in FIG. 2.
[0118] All the hybridization offspring were planted for offspring
tests in order to locate populations that divide 1:1 fertile and
sterile.
[0119] In any such population the sterile plants are fertilized
with pollen that are collected from the fertile plants in order to
maintain the sterile line.
[0120] The hybrid tetraploid seeds were planted for a comparative
yield test that included 4 repeats.
Example 2
Polyploid Fertility as Determined by Pollen Germination and Number
of Pods
[0121] Rapeseed polyploids are known to have lower pollen fertility
and therefore lower number of seeds in the pods. To test the
fertility of polyploids generated according to the present
teachings, the germination percentage and the number of seeds per
pod in 2N canola populations were compared to the polyploids
isogenic 4N line created by using the "MFGM" technology.
[0122] In the flowering period pollen was collected and germinated
on sugar solution. Briefly, seeds were incubated on sucrose beds
(2% sucrose and 2 mM H.sub.3BO.sub.3) for 12 hours in 26.degree. C.
Germination was evaluated thereafter. The germination percentage
was calculated by counting the germinated pollen grains under a
microscope. After full pods set up the seeds of all the 10 upper
pods from the same plants were collected and counted.
[0123] Results:
TABLE-US-00001 TABLE 1 Pollen germination and number of seeds per
pod (2N-non-multiplied; 4N- multiplied) Pollen germi- Seeds Pollen
Seeds nation Per germination Per 2N Line % Pods 4N line % Pods
CHARO 1-1 92 17 CHARO 1-1-2 94 19 CHARO 1-2 94 15 CHARO 1-1-3 94 18
CHARO 1-3 93 20 CHARO 1-1-4 97 16 CHARO 1-4 94 19 CHARO 1-1-5 96 17
CHARO 1-5 95 17 CHARO 1-1-6 96 18 CHARO 1-6 97 18 CHARO 1-11-1 93
19 CHARO 1-7 95 16 CHARO 1-11-2 95 17 CHARO 1-8 93 19 CHARO 1-11-3
94 15 CHARO 1-9 94 18 CHARO 1-11-4 94 15 CHARO 1-10 94 17 CHARO
1-11-5 93 20 CHARO 1-11 95 15 CHARO 1-11-6 95 21 CHARO 1-12 96 18
CHARO 1-11-7 95 17 CHARO 1-13 95 19 CHARO 1-11-8 94 18 CHARO 1-14
94 20 CHARO 3-5-8 92 18
[0124] Matched pair analyses showed that there was no significant
difference between the number of seeds per pod and pollen
germination percentage in the euploid plants and the isogenic
multiplied plants.
[0125] Seed weight comparison between the multiplied plant and the
isogenic euploid progenitor (CHARO1, Chao1) is provided in Table 2
below.
TABLE-US-00002 TABLE 2 Number Number Field of of Weight Weight
Number repeats seeds (gr) 1000 (gr) 3 1 40 0.274 2 40 0.259 3 40
0.263 4 40 0.255 6.56875 0.26275 5 1 40 0.195 2 40 0.187 3 40 0.183
4 40 0.183 4.675 0.187 23 1 40 0.251 2 40 0.252 3 40 0.25 4 40
0.242 6.21875 0.24875 49 1 40 0.248 2 40 0.247 3 40 0.252 4 40
0.247 6.2125 0.2485 81 1 40 0.237 2 40 0.23 3 40 0.219 4 40 0.227
5.70625 0.22825 82 1 40 0.25 2 40 0.225 3 40 0.244 4 40 0.229 5.925
0.237 98 1 40 0.235 1 40 0.233 1 40 0.224 1 40 0.237 5.80625
0.23225 99 1 40 0.225 2 40 0.224 3 40 0.208 4 40 0.221 5.4875
0.2195 289 1 40 0.224 2 40 0.235 3 40 0.238 4 40 0.237 5.8375
0.2335 290 1 40 0.248 2 40 0.239 3 40 0.244 4 40 0.237 6.05 0.242
291 1 40 0.222 2 40 0.217 3 40 0.218 4 40 0.197 5.3375 0.2135 292 1
40 0.235 2 40 0.24 3 40 0.232 4 40 0.252 5.99375 0.23975 293 1 40
0.222 2 40 0.225 3 40 0.217 4 40 0.245 5.68125 0.22725 294 1 40
0.228 2 40 0.233 3 40 0.238 4 40 0.24 5.86875 0.23475 295 1 40
0.244 2 40 0.228 3 40 0.253 4 40 0.235 6 0.24 296 1 40 0.162 2 40
0.168 3 40 0.154 4 40 0.161 4.03125 0.16125 CHARO1 2n 1 40 0.133 2
40 0.131 3 40 0.127 4 40 0.135 3.2875 0.1315 Rapeseeds Chao1 2009 1
40 0.146 2 40 0.138 3 40 0.142 4 40 0.141 3.54375 0.14175
[0126] These results indicate that the multiplied lines are as
fertile as the euploid lines.
[0127] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0128] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
REFERENCES
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