Note: Descriptions are shown in the official language in which they were submitted.
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Animal Feed with Low Phytic Acid, Oil Burdened and Protein
Laden Grain
The present invention is based on and claims benefit to U.S.
Provisional Application No.s 601051,854 and 601051,855, filed Juiy 7,
s 1997, the entire contents of which are incorporated herein by
reference.
Field of Invention
io Broadly, the present invention relates to grain having increased
oil and increased protein and amino acids, increased total phosphorus
bioavaiiability and decreased phytic acid. This grain can be used as
feedstuffs for animals. More particularly, this invention relates to grain
based feed that provide improved animal nutrition, and reduces the
is environmental impact of animal production.
Background
Over the last fifty years, approaches toward providing animal
2o nutrition have changed. No longer are the animals fed whatever grain
or forage may be available. Instead, the diets of animals are closely
monitored for total nutrition value, and for cost. The anima! on the diet
is monitored, for quality and performance characteristics, and for the
environmental impact of the waste from the animal. The information
2s gathered is employed to adjust the feed to increase nutrition value of
the feed and the animal performance characteristics while decreasing
the cost and environmental impact.
Cereals and fats are used in feeding programs for nonruminants
such as swine and poultry to provide a nutritional source of calories.
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The ratio of cereals to supplements, such as vitamins, minerals and
fats, have changed across years in an attempt to maximize feeding
efficiency of the animals. The feeding efficiency (the feed conversion
ratio) or how much feed is required to produce one pound of animal
s weight is determined by a combination of matching the genetic
potential of the animal, and the nutrients supplied to the animal. As
the feed conversion ratio has risen due to genetic enhancements, the
mineral nutrient requirements in the feed have risen to assure a
complete and heathy diet.
~o Since an animal's ability to feed limits the amount of nutrients
and calories it can consume, the feed industry has had to develop
ways to make feeds that are more highly caloric. To increase the
caloric density of the feed, producers have added fat to the feed. Fat
has often been added to the feed in the form of a liquid. Fat has the
~s advantage of supplying calories to each mouthful of feed. However,
adding fat to feed has some disadvantages such as costs, added labor
and technical difficulties with automatic feeding systems. Additionally,
the fat is often of poor quality, thus reducing the overall quality of the
feed.
2o To reduce the use of liquid fat in feeds, the industry has tried
increasing the oil content of the grain used in the feeds. The Dupont
company has developed and commercialized high oil corn as a method
for increasing the oil content of feed. Other companies have
developed corn that has more oil than no.2 yellow dent corn but less
2s than Dupont's high oii corn. High oil and elevated oil corn is herein
alternatively referred to as oil burdened corn. This extra oil in the corn
reduces and may eliminate the need for the addition of the liquid fat to
the feed.
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Traditionally, oil burdened corn has been thought to contain
increased level of phytic acid, as compared with levels in No. 2 yellow
dent corn. Raboy et al (Journal of Heredity 1989: 80: 311-315) have
reported however, that there is an apparent negative relationship
s between selection for oil and total phytic acid, phytic acid phosphorus
and phosphorus per kernel, per germ and per endosperm of Illinois
High Oil and Low Oil lines, as opposed to the previously expected
apparent positive relationship on a concentration basis (i.e., mg
constituent per g kernel, germ or endosperm). Raboy explains that the
io discrepancy between total contents per organ and concentration per
organ results from the large divergence in organ dry weights exhibited
between the Illinois High Oi1 {IHO) and Illinois Low Oil (1L0) seed used
in his study; IHO germ being about twice the dry weight of ILO germ
and ILO endosperm having nearly three times the dry weight of IHO
is endosperm. In contrast to this trend for high oil being linked to lower
phytic acid, Raboy also reports a consistent positive relationship
between increasing protein selection and increasing amounts of phytic
acid, phytic acid phosphorus and phosphorus. Thus, there is an
apparent positive relationship between selection for protein and total
2o phytic acid, phytic acid phosphorus and phosphorus per kernel, per
germ and per endosperm of Illinois high protein and low protein lines.
This was maintained even when the data are expressed on a
concentration basis (i.e., mg constituent per g kernel, germ or
endosperm}. Thus selection for protein and oil appears to divergently
2s affect phytate content in seed.
As reports suggest an average increase of 0.38% protein with
each 1 % increase in oil (Han Y. Et al., 1987 Poultry Science 6fi:103-
111; Keshararz, Poultry Pointers, pp6-7), it is uncertain from the art
whether grains containing high oil, high protein and low phytic acid
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could be produced (Brewer, "Optimum~ High Oil Corn Improves Poultry
Rations" Poultry Digest, FebruarylMarch 1998 pp30-31 ). Brewer
states that while high oil corn is available as of 1998, varieties which
are high in oil, high in protein and high in digestible phosphorus (i.e.,
s low in phytic acid phosphorus}, have yet to be developed.
The concentration of phytic acid in grain-based diets has long
been of concern to humans and animal nutritionists, because evidence
has shown that phytic acid acts to form insoluble salts with nutritionally
important minerals that subsequently are not absorbed in the intestine.
io Phytic acid (myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate))
is a form of phosphorus (P) in seeds which is stored in the form of
phytate salts. Phytate salts have a negative nutritional impact on the
animal because phosphorus bound to phytate is not available to the
animal as a source of nutrition. Moreover, the animal does not retain
is the minerals such as Ca, Zn and the like and these needed minerals
are excreted. Finally, the animal waste contain phytate P which then
contributes to the surface and ground water pollution. If the grain is
used for milling purposes then the milling by-products contain phytate
P which then contributes to the surface and ground water pollution.
2o Swine, for example, lack the digestive enzyme (phytase)
required to cleave the phosphorus from the phytate molecule and thus
can not readily use phytate-phosphorus. Increasing the availability of
phosphorus by elimination of the phytate salts binding the phosphorus
would enable a reduction in dietary total phosphorus content without
Zs jeopardizing the animal's health or production performance. Increasing
the bioavailability of phosphorus results in a lower phosphorus content
in the swine wastes, which is environmentally desirable.
In one attempt to release a portion of the phytate P present in
maize and soybean meal the feed industry has added microbial
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phytase to the feed of animals. This method of dealing with phytate in
the grain appears to partially decrease the phosphorus excreted by the
animal. This research apparently fed to further methods of degrading
phytate in feed. One method includes adding an enzymatic cocktail
s and AspergiUus niger mycelium to feed. These components function to
hydrolyze phytate present in the corn-soybean diet. Turkeys fed the
enzymatic cocktail and the fungal mycellium showed enhanced
performance and retention of P and Ca. These feed studies were
planned to dephosphorylate the corn and soybean based feeds prior to
to consumption by the animal and thus reduce the P excreted. This
method of dealing with phytate in the grain has the distinct
disadvantage of adding labor and cost to the feed.
Mogen, in US Patent No. 5,593,963, describes production of a
temperature stable phytase enzyme from Aspergillus in a corn or soy
Is seed through genetic engineering techniques. The genetically
produced phytase was designed to reduce the phytic acid content in
animal feed by degrading the phytic acid being released from the grain
and thus decrease the level of phosphorus excreted by the animal.
Low phytic acid mutant yellow dent corn seeds have been
2o produced by Raboy and described in U.S. Patent No. 5,689,054. This
patent describes the discovery of a single gene, nonlethal Ipa1 mutants
in maize that cause the reduction of kernel phytic acid phosphorus by
up to 95% over the wildtype phytic acid phosphorus levels. Raboy
notes that while the mutants of his invention are phenotypically very
2s similar to the wild-type, the mutants would need to be introduced in to
a breeding program in order to introduce the low phytic acid trait in to a
commercial line. Moreover, Raboy explains that the low phytic acid
maize mutants of his invention are characterized by a small kernel dry
weight reduction which could result in a reductionin productivity and
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that homozygous mutants may reduce or eliminate agronomically
important characteristics. As Raboy et al (Journal of Heredity 1989)
has indicated that divergent selection for high protein consistently
produces higher phytic acid lines, it is unclear how the Ipa1-R and
s Ipa2-R mutations described in the Raboy patent in yellow dent corn will
interact with genes for high-protein and oil-burdened corn seed. Thus
one could not have predicted with certainty whether it would have been
possible to maintain a high-protein oil burdened seed in combination
with a low phytic acid mutant.
to Although the feed industry has addressed both the need for
more energy in the feed and the need for less phytate-phosphorus, the
feed industry has not addressed the need for a method of providing, in
a cost efficient manner, both the high nutrient density (i.e., high protein
and high oil) and the tow phytic acid in feed. There is a need to reduce
1 s the amount of phytate salts formed in feed and increase the amount of
energy in feed without having to add phytase and liquid oil to feed.
There remains a need, which has not been addressed, for a grain
having a combination of increased protein and oil burden and low
phytic acid levels. To reduce feed costs in animal production requires
2o a nutritionally dense material that is cost-effective and environmentally
friendly. Additionally, there remains a need for a feed containing an oil
burdened, protein laden corn with tow phytic acid levels which can be
used for milling or for feed purposes.
2s Summary of the Invention
An object of the present invention is to provide a nutrient-dense
grain that contains both high levels of energy, through oil and improved
amino-acid content, through protein, and low levels of phytic acid.
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Another object of the present invention is to decrease the
phosphate andlor phosphorus excretion of animals consuming the feed
while increasing the energy levels per daily feed intake and
bioavailability of minerals and other nutrients.
s An object of the present invention is to provide an animal feed
that contains both high levels of energy through oil and protein and low
levels of phytic acid.
Still a further object of the present invention is to provide a
highly nutrient dense feed source to livestock which has less phytic
~o acid present then the same feed source when made with regular
commodity corn (i.e., no. 2 yellow dent corns).
Still a further object of the present invention is to provide a high
energy feed source to livestock containing sufficient supplies of any
rate limiting amino acid which has less phytic acid present then the
Is same feed source when made with regular commodity corn ( no. 2
yellow dent corns).
It is another object of the invention to provide a method of
reducing animal phosphorus waste andlor pollution, and subsequent
algal and microbial blooms caused therefrom, which method includes
2o feeding animals, such as pigs and chickens, the animal feed of the
present invention.
In one embodiment, the present invention provides a non-lethal,
mutant seed or grain of a cereal plant species, such as corn (maize),
rice, barley and soy, having at least about 5% by dry weight,
2s preferably at least about 6%, alternatively at least about 7%, oil; at
least 11 % by dry weight, preferably at least about 12%, alternatively at
least about 13%, protein; and at least about a one third (33%)
reduction in dry weight in the phytic acid amount (as measured by any
of total phosphorus, phytic acid or phytic acid phosphorus), preferably
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at feast about a one half (50%) reduction, alternatively at least about
60-70% reduction, relative to wild-type seed of said species. Where
the seed of the present invention is corn, the comparison in reduction
is preferably made relative to standard number (no.) 2 yellow dent
s corn.
In another embodiment, the present invention provides an
increase in phosphorus availability of from 28% for yellow dent corn to
greater than about 70%, preferably less than about 90%, alternatively
about 80% to about 84-85%. Availability being the amount of utilizable
~o phosphorus compared to total phosphorous from feed. The hybrid
grain of the present invention is preferably a cross between useful
inbreds and an inbred line ExSeed line U095 -Ipa1-E (alternatively
referred to as 0095-E or U095py; deposited as strain designation
EX1965py on July 7, 1998 with American Type Culture Collection,
~s 10801 University Blvd., Manassas, VA 20110-2209 USA, under
conditions of the Budapest Treaty, Accession No. . Source
U095-py 1656-W97 - Florida - 100) The "E" or "py" designation used
herein indicates the introduction of a ipa1 mutation by the present
inventors. A number of other crosses and inbreds can be employed.
2o For example, the following female inbreds BD68py, TR306py, WD22py
and TR329py were crossed with male inbreds U095py, UU01 py,
UE95py, TR335py and TR386py to make high-yielding hybrid
combinations. Crosses with U095py are particularly preferred and the
inbred U095py and hybrids made therefrom are specific embodiments
2s of the present invention. The hybrid grain of the present invention
characterized by having ~6% oil and 12% protein (or 3% mare oil and
3% more protein than yellow dent corn) and at least about 33%
reduction in phytic acid content.
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In another embodiment, the present invention provides a feed
containing a seed, as described herein, and at least one source of
vitamins or minerals, containing, for example, any one or a mixture of
at least two of calcium or phosphorus or salts thereof, vitamin A,
s vitamin D, vitamin E, B,2, riboflavin, pantothenic acid, niacin, biotin,
and trace minerals, such as iron, copper, manganese, zinc, iodine, and
selenium, and/or additional feed additives, such as antibiotics,
arsencials, chemotherapeutics, flavoring, antioxidants and plant
extracts; said feed providing a nutritionally balanced diet and a greater
~ o amount of biologically useful phosphorus to an animal consuming said
feed than does the same feed formed with wild-type seed of the
species. The feed of the present invention may also contain amino
acid additives, such as lysine and methionine.
in another embodiment, the present invention provides an
Is improved feed which is otherwise formulated for swine or poultry but
includes the seed, preferably corn seed, of the present invention.
In yet another embodiment, the present invention provides a
method of increasing bioavailability of phosphorus from products
containing wild-type seed of a species, said method including the steps
20 of providing a seed containing product, such as a feed as described
herein, for consumption, wherein the seed containing product contains
a seed of the present invention, and feeding the seed containing
product to an animal which will benefit from an increased bioavailability
of phosphorus.
zs In a further embodiment, the present invention provides
germplasm which will yield the seed of the present invention. In a
preferred embodiment, the present invention provides corn germplasm
which will yield the com seed described herein.
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In yet another embodiment, the present invention provides a
plant produced from a seed of the present invention.
In yet a further embodiment, the present invention provides a
seed of the present invention which is fully mature.
s Still, further objects and advantages will become apparent from
a consideration of the ensuing description.
Brief Description of the Figures
~o Figure 1 shows a frequency distribution of phytate contents of
screened M2 seed.
Detailed Description
is The present invention provide grain, feed made from the grain,
petfood made from the grain, and food products made from the grain.
The grain is preferably maize grain with the following characteristics: oil
burdened, elevated protein content, and low phytate levels. The
preferred grain has at least 5% oil, at least 11 % protein, and at least
2o about 20% to about 70%, preferably at least about to 33% to about
60% reduction in the phytic acid level relative to wild-type grain, such
as standard yellow dent corn. More preferably the grain has at least
6%, more preferably 7% oil, at least 12%, and more preferably 13%
protein, and at least a one half reduction in the phytic acid level relative
2s to standard yellow dent corn wherein the grain is low phytate.
Percentages are expressed on a dry weight basis as amount of a
constituent per kernel, unless described otherwise. The combination
of oil burdened, protein laden, decreased phytate characteristics in
grain makes a grain that provides more calories, protein and
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phosphorus and other nutrients to the feeding animal. Pet foods,
animal feeds and corn food products made of the present invention will
provide increased nutrition because of the increased bioavailability of
the components of the grain.
s In other words the present invention includes an animal feed for
a specific animal type. In one embodiment, the present invention
provides a feed having a gross energy and at least the same ratio of
performance limiting amino acids to gross energy as a nutritionally
balanced feed using no. 2 yellow dent corn formulated for a similar
Io type animal. The feed is formulated with an energy source including
elevated oil, protein and low phytic acid maize. Additionally, the feed
can contain at least one protein source including a potentially
performance limiting amino acid component in a ratio to said gross
energy such that the amino acid is not performance limiting, at least
is one source of vitamins and minerals; wherein the feed provides to the
animal a higher calorie and lower phytic acid grain than no. 2 yellow
dent corn in a nutritionally balanced feed. The limiting amino acid can
be various different amino acids according to the needs of the animal
species but it preferably includes lysine, tryptophan, threonine and
2o methionine. The protein source of the feed can also include soybeans
as a component. The feed of the present invention can also include
the vitamins and a mineral source such as calcium, phosphorus and
salt. In one embodiment, the feed of the present invention has
vitamins and mineral sources which include any one or a mixture of
2s vitamin A, E, D, B,2, riboflavin, pantothenic acid, niacin, biotin; trace
minerals, such as any one or a mixture of iron, copper, manganese,
zinc, iodine, selenium, and feed additives, such as are known in the
art and may include any one or a mixture of antibiotics, arsanicals,
chemotherapeutics, flavoring, antioxidants and plant extracts.
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The present invention provides a method of increasing
bioavailability of phosphorus from maize containing products
comprising the steps of: providing a maize containing product for
consumption, wherein said maize containing product is formed from
maize grain of the present invention, such as that characterized by
having at least 5% oil, at feast 11 % protein, and at least a one third
reduction in the phytic acid level relative to standard yellow dent corn,
wherein the grain of the present invention is lower in phytate
concentration than standard yellow dent corn; and consuming said
to maize containing product which contains less phytate in the maize
material than the same maize product made with yellow dent corn
wherein the bioavailability of the phosphorus in said maize is increased
over the same product made with yellow dent corn.
The present invention further provides a feed for any non
is ruminant animals. The feed of the present invention is particularly well-
suited as a constituent in the diets of swine or poultry.
In another embodiment, the present invention provides an
animal feed having a gross energy content for a specific animal type,
said animal requiring a certain level of an amino acid in the feed to
2o achieve good performance from said feed, the feed containing corn
having an elevated amount of oil compared to the average oil levels of
no.2 yellow dent corn and low phytic acid levels compared to the
average phytic acid levels of no. 2 yellow dent corn; a protein source,
preferably substantially provided from the grain of the present
2s invention; and having at least the same ratio of performance limiting
amino acid to gross energy as a nutritionally balanced feed using no. 2
yellow dent corn formulated for a similar type animal.
The present invention provides grain having increased energy,
protein and low phytic acid. This grain can be used as feedstuffs for
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animals or this grain can be milled. The present invention provides a
new maize seed, plant and grain that carry the oil burdened, protein
laden and the low phytic acid and the use for such new grain. The
grain-based feedstuffs provide improved animal nutrition, and reduce
s the environmental impact of animal production. Even more particularly
this invention provides an animal feed formulated using the grain of
the present invention.
Many crop plants are used for the production of food for human
or animal consumption, for commercial processes yielding products for
to human consumption, for the development of industrial products and far
other purposes. Traditionally, the improvement of crop plant species
involves the introduction of desired traits by genetic crosses. The
present invention likewise can be made repeatedly though the use of
standard crop, such as corn, breeding and mutation practices.
is Corn grain is considered to be a high quality grain for use in
foods and feeds. High oil corn is considered more energy dense then
other corn. High oil corn presently is commercially available from
Dupont. However, this corn like all corn contains phytic acid. Until the
present invention low phytic acid and oil burdened, protein laden corn
Zo plants and grain did not exist. The invention also provides an
improved flour from milling of the seed of the present invention. Thus,
low phytate, ail burdened, elevated protein maize grain should address
the need for energy, protein dense corn and mineral bioavailability
within the cereal grain.
2s The method of repeatedly making the grain of the present
invention is as follows. Generally, in the course of a maize breeding
program oil burdened, protein laden com plants are crossed with
maize plants carrying the low phytic acid allele. Oil burdened corn
plants can be developed by recurrent selection as evidenced by
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University of Illinois high oil corn (commercially available from Dupont),
or from transformation (methods of transforming corn are well known to
those having ordinary skill in the art). This is one part of the starting
material to make the present invention. The other part is a low phytic
s acid plant. Although this is not commercially available, experimental
material is available.
High oil grain is commercially available from Dupont as either a
Top Cross grain or a high oil hybrid corn (high oil is any grain of corn
having greater then 3.5% oil). Oil burdened corn is defined as corn
jo having on average a higher percentage of total oil levels than the
average standard yellow dent corn oil levels.
The following table shows the levels weight % of total oil of
various oils of a number of oilseed crops. The numbers under corn are
the levels in standard no.2 dent corn.
Table 1 Fatty
Acid Compositions
of Commercial
Crops
sun(hi0) canol IolinCanCrambe Corn Soy
(C16:0) 6 4 5 2 11 11
palmitic
(C16:1) 0.3 0.3 0.4 0.3
paimitoleic
(C18:0) 5 1.5 1.5 1 2 4
stearic
(C18:1) 20 (81) 62 62 17 28 22
oleic
(C18:2) 68 {9) 20 27 8 58 53
linoleic
(C18:3) 0.2 10 2 7 1 8
linolenic
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sun(hi0)-sunflower high oil
' canol-canoia
IolinCan-low linoleic canoia
s Crambe
com
soy-soybean
Thus the present invention encompasses grain that is oil
~o burdened,preferably with fatty acid compositions in the ratios listed
above. The present invention also encompasses corn that has
increased the fatty acid compositions such that there is not an overall
increase but there is a result increase in usefulness of the material to
the animal fed the altered corn. The present invention also
Is encompasses grain that is oil burdened and the fatty acid compositions
are in a different ratio than listed above.
The ratio of oils present in grain cereals can be varied though
genetic mutation, selective breeding, transformation and the like. The
essential grain for the feed can be produced by standard methods of
?o production of hybrid material such as the crossing of a oil burdened,
elevated protein inbred with a low phytic acid inbred to produce hybrids
having the grain characteristics including the low fatty acid and the
elevated oil.
As noted above, Raboy et al have reported a positive correlation
2s between protein selection and increased levels of phytic acid in corn.
The following table lists the value of the amino acids present in one
type of nutrient dense corn which can be used to form the grain of the
present invention. The protein in corn is controlled by many different
genes. The preferred high protein corn has an increased percentage
30 of most of the amino acids present in corn grain. One example of high
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protein material has been the high protein corn developed by recurrent
selection in the University of Illinois. Many of the other high protein
corn available have an increase in lysine and not necessarily an overall
increase in most amino acids. This type of high protein is also useful
s as this increased protein is needed by the feeding animal for overall
health and this protein is better utilized when combined with the other
traits of the present invention. To facilitate the breeding of hybrid seed
the high protein trait is additive or dominant. Quality protein maize
includes the high protein material available from Wilson seeds and
io from Crows and from public depositories and universities.
Alternatively, high protein material can be generated by recurrent
selection or by the use of certain mutation in corn like o2au2 which
have 49% more lysine then normal hybrids but have decreased protein
yields. In development of the present invention a high protein trait that
is was additive and increased most protein levels beyond average yellow
dent corn was used. Protein laden corn shall refer to corn that has
increased amino acid content when compared to average amino acid
content of yellow dent corn, andlor differing ratios of amino acid
contents when compared to average amino acid content of yellow dent
2o corn. High protein corn shall refer to corn that has increased amino
acid content when compared to average amino acid content of yellow
dent corn.
Table 2:Yeliow-dent vs. EX404
2s ( EX404 has ~1% wt% more oil and ~2-3% wt% more protein then
does standard yellow dent corn (average 3-3.5% by weight oil and 7-
9% by weight protein) EX404 and ES404 as used herein are the same)
Amino Acid Composition:
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Dent ES404 % change
TRYPTOPHAN .06 .06 100%
ASPARTIC ACID .57 .83 146%
THREONINE .29 .47 162%
s SERINE .41 .63 154%
GLUTAMIC ACID 1.52 1.89 124%
PROLINE .74 1.04 141
GLYCINE .33 .41 124%
ALANINE .63 .87 138%
o CYSTEINE .18 .24 133%
VALINE .38 .47 124%
METHIONINE .17 .26 152%
ISOLEUCINE .26 .36 138%
LEUCINE 1.01 1.48 146%
is HISTIDINE .24 .35 145%
LYSINE .24 .31 129%
ARGININE .29 .42 107%
This table clearly evidences that most of the the amino acid
2o values of the nutrient dense corn are increased over the yellow dent
material. This extra protein appears to be available for the animaNs
use when the phytic acid is substantially reduced. The ratio of proteins
present in grain cereals can be varied though genetic mutation,
selective breeding, transformation and the like. The present invention
2s provides a grain containing increased levels of bioavailable protein in
conjunction with increased levels of oil and reduced levels of phytic
acid.
Prior to the release by the USDA of the Low Phytic Acid mutants
B73 Ipa1-R, A632 Ipa1-R and selection thereof, there was only the
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conventional method of breeding for producing low phytic acid in corn
seed. Low phytic acid in corn seed developed by standard breeding
appeared to carry some undesirable agronomic traits. Due to the
recessive nature of the phytic acid gene the preferred method requires
s that this gene be fixed in both inbreds. The mutant containing low
phytic acid can be developed according to the following method.
Selection for phytic levels must be carefully performed as too low a
level of phytic acid may result in lethal seeds.
The low phytic acid plant of the present invention can be
Io developed by following the fisted steps which do not take undue
experimentation and can be done by the ordinarily skilled person in the
art of plant breeding. The best method for generating a low phytic acid
maize plant employs maize pollen mutagenesis. The induced mutation
in a haploid pollen grain would give rise to a heterozygous genotype in
is the seed. Since low phytic acid presently is known as a recessive
gene, the resultant mutant seed must be planted and selfed and the
resultant plants seeds assayed for the mutant phenotype. The
assaying of the seed should be done when the seed is in the mature
stage or harvesting stage.
2o Mutagenesis is effected by conventional means in the art such
as irradiation, chemical treatment, and transposable element insertion.
One standard procedure is taught by Neuffer et al. Maize for Biological
Research, W.F. Sheridan (Ed.), Plant Molecular Biology Association
{1982). This procedure uses ethyl methane sulfonate (EMS) applied to
2s pollen. The pollen is used for pollination and the resultant seeds are
planted and the seeds from the second generation can be tested for
phytic acid content. The test for P has been known in the art since
1990 when the HYPE method was published in Maydica 35:383 (1990)
by Raboy. The method relies on differential migration of phosphorus
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compounds. After electrophoretically fractionating the compounds a
chromatogram allows a semi quantitative assessment of the phytic acid
relative to other compounds. An alternative method involves screening
for higher levels of inorganic P in the grain. For example grain
s samples can be ground (to pass a 2 mm screen in a Wiley mill)
followed by addition of either 50 mg of grain germ or 1 gram of
endosperm in 15m1 of 0.4 M HCL in 0.7 Naz S04. Phytic acid
precipitates as an iron salt. Phosphorus in the ferric phytate
precipitates and total P are determined. Phytic acid P (mg) are
~o converted to phytic acid by use of a conversion factor of 3.5. These
results lead to the selection of the desired maize plants containing the
desired alleles. Other methods of testing for P are known and can be
used to select plants. The seed containing the desired phytic acid is
then increased. This process was employed in the present invention
is and inbred line were selected that carried new alleles at the Ipa1 locus.
These included EX404 (low phytic) which was crossed to one of the
inbreds of the present invention to form a hybrid that produced the
grain of the present invention. Additionally, the developed inbreds of
the present invention were from stiff stalk, Lancaster and another
2o versatile heterotic patterns so that the inbreds when crossed together
with the appropriate heterotic groups formed excellent hybrid material.
It was also discovered that a number of the developed mutations of
the present invention, though low in phytic acid were not the same
mutant as the Ipa1-R mutation as indicated by allelic testing.
2s The method used in the present invention can be used to form
two inbreds which would be crossed to form a high yielding hybrid.
Inbreds are commercially available from Universities and Foundation
Seed Companies and can be made by plant breeders skilled in the art
of breeding maize inbred lines. One or both of the inbreds fixed for the
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low phytic acid can be crossed to an inbred which carries the elevated
oil trait. Either inbred can cary the high protein trait if it was selected
as an additive trait. Thus allowing the hybrid combination to carry both
traits the recessive low phytic acid and the dominant or additive oil
s genes. A corn plant is repeatedly bred until the low phytic acid allele is
present in two inbreds that cross well to one another. Likewise at least
one of the inbreds in the hybrid combination must contain the oil
burdened trait andlor the protein laden corn alleles.
Alternatively this grain can be produced by using the crossing
io method of breeding taught in the Dupont patent application
W092108341 (US 615,839). This method requires that the male
pollinating plant carries the oil burdened trait. In Dupont's method the
oil pollinator which is an inbred is mixed in with hybrid seed that is male
sterile. The male pollinator then forms oil burdened grain on a sturdy
is hybrid plant.
In the present invention the male pollinator would preferably
have two traits, a fixed recessive gene for phytic acid and the oil
burdened, protein laden trait. Additionally, the male sterile hybrid
would have the low phytic acid trait also.
2o Methods of formulating an animal feed that contains additional
energy, protein and low phytic acid in one grain source are herein
described. The preferred method is to improve feeds or petfoods by
substituting the grain of the present invention for the corn grain
normally employed. Other ingredients in the feed can be milling
2s coproducts, protein ingredients, soybean meal, trace minerals and
vitamins, and other cereals and feed additives and flavorings. This
feed requires that grain be produced that is characterized as having
increased oil and increased protein/amino acid and low in phytic acid.
Surprisingly, the combination of the low phytic acid with the oil
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burdened and elevated protein appears to allow the animal to utilize
the extra protein that is in the oil burdened corn and the newly
available phosphorus more efficiently leading to increased feed
efficiencies. The grain resulting from this hybrid combination can be
s used in an animal feed. This feed should contain the combination of oil
burdened, elevated protein and low phytic acid and other feedstuffs
employed in the diets of either swine or poultry.
Preferably if other grains are used, these grains are selected to
have as low a phytic acid content as is possible in that type of grain.
to Though the percentage of the grain as a part of the overall diet would
determine how stringently the selection against phytic acid would have
to be.
The present invention is of particular use for feeding non
ruminant animals. The feed ingredients for the standard swine diet are
is provided below. The feed mixture of the present invention substitutes
the grain of the present invention for the no. 2 corn. The grain having
characteristics of low phytic acid and higher oil and increased amino
acids. This grain, when provided to the animal, results in a higher
caloric density available and a greater bioavailability of a number of
2o nutrients such as minerals. The use of this grain in feed reduces the
need for added animal tat or vegetable oils.
Swine are primarily fed a fortified corn-soybean meal diet.
Piglets, pre-weaning or early weaned, often have additives in the diets
such as dried milk products, and other high protein and fat sources
2s added to the diet. A complete swine diet provides all the nutritional
needs of the pig in one diet. These diets may be prepared by mixing a
balanced supplement with corn: soybeans meal and a vitamin-mineral
premix with corn. Conventional feeds were often supplemented with
sources of supplemental fat which are plant seed oils (extracted),
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grease and tallow, and commercial dry fat, corn oil, soybean oil or full
fatted cooked soybeans. Flowability of the diet in automated feeders
was compromised at 6 percent fat. Overall 1 percent fat produced
approximately 2 percent improvement in efficiency (additional weight
s gain per weight of feed consumed). Increased oil from the use of
the present feed (employing maize grain that is elevated oil and low
phytic acid) increases the caloric density of the diet bioavailability of
metals and reduces undesired waste. The feed of the present
invention employs the corn grain of the present invention as a
~o substitute for the grain presently employed in diets for animals. The
following two new feed combinations will employ such grain. Both of
these feed uses are for animals which are preferable non ruminant
animals. Such animals include swine, poultry, cats, dogs, horses,
sheep and the like.
is Dietary nutrient density, when increased, maximizes animal
performance. This includes animals such as sheep, swine, poultry,
dogs, cats and horses. This grain is primarily effective with most
monogastric animals. To formulate a diet containing the grain of the
present invention that contains higher levels of oil and amino acid than
2o standard corn requires the step of substituting the grain of the present
invention for the yellow dent corn (or even for the oil burdened corn) in
the diet. The invention then however, requires the step of adjusting the
protein to energy level of the diet according to the needs of the animal.
For example if the diet is for swine then the ratio of lysine should be
2s increased. If the diet is for poultry then the rate of methionine should
be increased and likewise for each animal.
The amino acid composition of protein specifically lysine which
acts as a growth limiting amino acid for swine is increased in fight of
increased energy levels to keep a good balance of energy verses
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protein when using the feed of the present invention. The invention
itself increases the lysine. In the example given in table 2 of one of the
maize grains containing increased oil and amino acid content the
_ increase in Lysine is 29% over the regular maize grain. If the increase
s in the rate limiting amino acid in the grain of the present invention is
sufficient, the diet may not require additional increases in this amino
acid. However, to the extent that the grain does not carry all of the
necessary amino acids if sufficient amounts to put the diet in balance,
they must be added to the diet.
io The following table includes ingredients that are used in a
standard feed for swine. In the present invention these ingredients can
likewise be used, however, the standard corn and corn products made
from standard corn should be substituted by using the grain of the
present invention or products made from the grain of the present
Is invention. Preferably added fats are not required in the feed.
Additionally use of low phytic legumes and other protein sources are
encouraged. These examples, as others herein, are illustrative of the
use of the present invention and are not intended to limit the scope of
the disclosed invention.
Energy, Fat, Protein
of Typical Ingredients
Used in Swine
Diets
Ingredient kcal metaboiizableprotein % fat
energyllb
Animal fat 3585 100
Alfalfa 775 17 3.0
Barley 1380 11 1.8
Blood meal 1060 86.0 1.0
Corn, yellow 1555 8.0 3.25
Corn, gluten (mi) 1760 60.0 1.0
cottonseed meal 1160 41.0 2.0
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distillers dried 1515 27.0 8
grain
wl solubles
fish meal 1500 60.0 6.0
Lysine 1100 48.0 5.0
hydrochloride,
limestone, dicalcium
phosphate
Milo 1490 9.0 2.0
Molasses 910 3.0
Oats 1243 12 3.0
Skimmed Miik 1630 33 1.0
(dried)
Soybean meal 1535 47.5 1.0
Soybeans (fat 1650 37 18.0
cooked)
Soybean Oil 3300 100.0
sunflower 1000 28.0 2.0
Wheat 1500 13 3.0
Whey 1405 14 .5
As is clearly evidenced in articles such as Adams, K.L. and
Jensen A.H. "High Fat Maize in Diets for Pigs and Sows in Animal
s Feed" in Science and Technology, 17 (1987) 201-212, hogs fed higher
energy diets using just high oil corn (and not the present invention of
oil burdened, elevated protein and low phytic acid) with an energy:
lysine ratio adjusted to the animal type (ie farrowing, starter pigs,
finishing pigs) showed better feed efficiency then the same type of pig
~o on a No.2 corn soybean diet. The present invention used on this type
of pig will provide a diet that is not only calorie dense and thus
increases feed efficiency but also is low in phytic acid which permits
the feeding animals to absorb from the grain the trace minerals, amino
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acids and macro minerals needed for increased efficiency and gain.
However, like the previous tests with high oil corn the pig on a diet
including grain of the present invention will require the increase in
nutrients from the oil burdened, elevated protein and low phytic acid
s grain to be balanced with an increase in the lysine level, if necessary.
Pig diets to be nutritionally balanced should have protein, amino
acids, such as lysine, threonine, tryptophan, and major minerals, such
as calcium, phosphorus, salt, and vitamins such as A, D, E, B,2,
Riboflavin, Pantothenic acid, Niacin, Biotin and trace minerals such as
to iron, copper, manganese zinc, iodine, selenium. Additionally feed diets
often include feed additives such as flavorings and may have some of
the following chemicals added Apramycin, bacitracin, chlortetracycline,
bambemycin, carbadox, lincomycin, penicillin, tylosin, virginiamycin
and the like.
~s Another potential use (other than as petfood and feed for swine)
of the feed described herein is for poultry feed. Poultry includes most
domesticated fowl including duck, geese, pheasant, turkeys and
chickens. The nutrient requirements of poultry and swine differ but the
basic ingredients in their diets are very similar. Poultry utilizes cereal
2o grains and grain co-products, fats and carbohydrates in their diets.
The largest part of the poultry diet is made up of cereal grain. These at
least include corn, wheat, oats, milo, barley. However, in accordance
with the present invention the preferred main grain ingredient is oil
burdened, protein laden and low phytic acid corn. The present
2s invention envisions that little to no corn is used in the feed of the
present invention that is not the grain or a product of the grain of the
present invention. In addition the feed of the present invention
includes proteins which may come from peanut meal, soybean meal,
cottonseed meal, fish byproducts, blood meal and poultry byproducts
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2fi
and the like. The feed of the present invention may also include
minerals and vitamins particularly calcium as limestone or oyster shells
and vitamins D3, choline, menadione, A, E, B,2 and feed additives such
as antioxidants, antibiotics. Like swine, poultry seem to have a specific
s amino acid that, if deficient, will reduce the animal's performance on
the feed. For poultry, the limiting amino acid is methionine while for
swine the limiting amino acid is lysine. Thus, when the present
invention feed is formulated there must be an increase in the amount
of methionine and general protein to keep the desired protein gross
to energy ratio in the diet as compared to the same ratio in the diet
formulated with no. 2 grain maize. Again the present invention
provides not only the nutrient dense feed but increases the
bioavaiiability of the trace metals due to the decrease in the chelating
agent, phytic acid, resulting in an increase in feed efficiency of poultry
Is on the present invention.
Although the description above contains many specificities,
these should not be construed as limiting the scope of the invention but
as merely providing illustrations of some of the presently preferred
embodiments of this invention. Various other embodiments and
2o ramifications are possible within its scope.
The Iowa State University extension office publication entitled
"Life cycle Swine Nutrition," PM489, revised 1996 on pages 12,15 and
20 show a number of diets for sows and boars under standard
production conditions. The following information shows that pig feeds
2s and poultry feeds for different animals life stages are know in the prior
art. The following is an example of one diet for a pig at a given
development stage.
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Example 1
A pig having a moderate, lean growth potential with the
following weights are defined by the following stage development
numbers. Pounds 6-8=stage 1, Pounds 8-13 =stage 2, Pounds 7 3-
s 18= stage 3, Pounds 18-26= stage 4, Pounds 26-37= stage 5, Pounds
37-51== stage 6, Pounds 51-69=stage 7, Pounds 69-91= stage 8,
Pounds 91-118= stage 9, Pounds 118-150 = stage 10, Pounds 150-
188 = stage 11, Pounds 188- 233 = stage 12, Pounds 233-283 =
stage 13.
~o The pig diet developed herein was formulated for a pig in stage
six. A stage six pig would require for every one thousand pounds of
feed: 648.4 Ibs of corn, 320.0 Ibs soybean meat, dehulled 8.50 Ibs of
limestone, 15.25 Ibs. dicalcium phosphate, 4.10 Ibs salt, 1.65 Ibs trace
mineral premix, 0.75 Ibs Fat soluble vitamin premix, 0.65 lbs vitamin B
~ s premix, 0.50 Ibs Biotin and Folic acid premix, 0.20 Ibs choline premix
and any feed additives approved by FDA. This feed is made according
to the present invention by substituting the corn of the present
invention into the feed instead of the no. 2 corn and increasing the
level of lysine to match the increased energy in the diet to avoid the
20 lack of lysine from limiting in the animal's growth. If the feed is
pelleted
then the vitamins should be increased approximately 20%.
Corn is the basic cereal grain used in animal feeds. Corn
contains many of the essential nutrients that are required by the
animal. The modern animal, with its tremendous genetic ability to
2s deposit lean tissue versus fat, has a tremendous requirement for
nutrients. As this demand for fat, has a tremendous requirement for
nutrients, as this demand for increased nutrition, we have at the same
time see a decline in feed intake, While animal genetics had and
continue to improve carcass qualities by reducing total body fat, the
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environmentalists have indicated that our concentrated animal feeding
operations are polluting the ground and subsequent groundwater.
Knowing that corn is the leading feeding ingredient used in animal
production the present invention is a corn that contains concentrated
s nutrients, and nutrients that had higher biological availability to the
animal. Such nutrients include protein, energy, amino acids and
minerals. The key mineral, phosphorus, is essential for growth, health
and production. Phosphorus in corn is only ~30% available to the
animal because of the phytic acid content of the corn. However, the
to nutrient dense corn of the present invention has a greater availability of
the phosphorus.
Example 2
This experiment determines the response of commercial broiler
is chickens to low phytic acid mutant yellow dent corn as compared to
yellow dent corn.
The trials use commercial broiler chickens in a 13-day feeding
study. The basal diet is an NRC (National Recommendation
Committee) based corn diet. The treatment is a replacement of
2o conventional yellow dent corn with low phytic acid hybrid grain
obtained from a cross between B73 Ipa1-R and an Exseed inbred line
UU01-Ipa1-E (alternatively referred to herein as UU01-E or UU01-py)
which is a low phytic mutant of yellow dent corn.
All experiments were managed under the Good Laboratory
2s Practices with animals welfare at the forefront. Feed and water is
supplied ad libitum, body weight and feed intake is measured
throughout the experiments, phosphorus availability is measured by
means known in the art. Carcass evaluations, total body weight gain,
feed conversion ratio, mortality, morbidity and intakes are reported.
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The expected results are improved phosphorus availability of low
phytic acid yellow dent corn versus yellow dent corn.
It is believed that the grain of the present invention in the diet
will result in less excreted phosphorus. This will result in less pollution.
s In this study, 8 day old New Hampshire x Columbian male
chicks with an average initial weight of 73.78 were used to compare
the phosphorus availability of low phytate yellow dent corn of the
present invention compared to conventional corn (yellow dent). This
experiment was a CRD; 7Tx4Rx5C, 13 day trial (7 treatments, 4
to replications, 5 chicks per treatment).
The basal diet was as follows: cornstarchldextrose (2:1 ratio) to
100%; soybean meal 47,4%; soybean oil 5%; Limestone 1.2 %; Salt
0.35%; vitamin mix (A, D3, E, K, scotin, riboflavin, pantothenic acid,
niacin, choline chloride, folic acid, thiamine, B6, B,2) 0.35%; mineral mix
Is (copper, iodine, iron, manganese, selenium, zinc) 0.15%; chofine 60,
0.1 %; DL-Met 0.25; flavomycin 0.05%. The basal diet contained 0.1
available phosphorus, 0.63% Ca and 23% CP. All percentages are on
a weight basis.
The treatment design for this trial was as follows 1: Basal diet; 2:
2o Basal plus 0.06% phosphorus (inorganic phosphorus) from potassium
phosphate (KHZP04); 3: Basal plus 0.12% phosphorus from potassium
phosphate (KH2P04); 4: Basal plus 20% conventional corn; 5: Basal
plus 40% conventional corn; fi: Basal plus 20% low phytate corn; 7:
Basal plus 40% low phytate corn.
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Weight
gain
-
8
-
20
d
(g)
1 2 3 4 5 6 7
R1 227.8 269.8 291.2 209.2 249.4 253.2 283.0
R2 216.8*267.6 295.4 224.0 258.8 249.4 273.0*
R3 237.0 285.6 294.4 229.8 259.6 244.2 255.0
R4 224.6 258.4 300.2 232.8 236.8 241.6 269.6
mean 226.54270.3b 295.3a223.9 251.1 247.1 270.1
' ' b
glcld 17.4 20.7 22.7 17.2 19.3 19.0 20.7
Pooled .5;
SEM *chick
= removed
4.6; or
LSD died;
= gJcld
13 =
gramslchick/day
Feed
Intake
-
8
-
20
d
(g)
1 2 3 4 5 6 7
R1 346.4 383.2 417.8 317.0 354.2 358.8 367.8
R2 325.9*397.6 428.4 332.2 368.6 350.6 365.6*
R3 353.0 415.4 414.6 337.6 371.0 338.4 355.6
R4 348.6 387.0 435.6 343.8 349.8 344.6 367.6
mean 343.4 395.8 424.1 332.8'360.9'4348.1 364.1
a e '
glcld 26.4 30.4 32.6 25.5 27.7 26.7 28.0
Pooled
SEM
=
5.3;
LSD
=
15.6;
*chick
removed
or
died
GainIFeed
-
8
-
20
d
(g)
1 2 3 4 5 6 7
R1 0.658 0.704 0.697 0.660 0.704 0.706 0.769
R2 0.665*0.673 0.690 0.674 0.702 0.711 0.747*
R3 0.671 0.688 0.710 0.681 0.700 0.722 0.717
R4 0.644 0.668 0.689 0.677 0.677 0.701 0.733
mean 0.659'0.683' 0.696b'0.673e 0.695' 0.710b0.7418
Pooled
SEM
=
0.006;
LSD
=
0.020;
*chick
removed
or
died
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Tibia
Bone
Ash
(%)
1 2 3 4 5 6 7
R1 30.7 35.8 37.1 29.4 34.2 31.5 36.5
R2 30.2' 33.9 38.5 29.0 31.7 28.9 34.9"
R3 28.1 33.3 36.9 28.4 32.5 31.1 34.1
R4 30.2 33.7 36.6 29.1 31.0 29.7 35.0
mean 29.84 34.1b 37.2 28.94 32.3 30.3 35.1b
PooledSEM d
=
0.5;
LSD
=
1.5;
*chick
removed
or
die
Tibia
Bone
Ash
(g)
1 2 3 4 5 6 7
R1 0.242 0.318 0.427 0.215 0.262 0.283 0.338
R2 0.230*0.312 0.418 0.232 0.296 0.250 0.345*
R3 0.239 0.327 0.394 0.233 0.270 0.253 0.327
R4 0.241 0.300 0.390 0.248 0.254 0.261 0.339
mean 0.238'0.314' 0.407a 0.232' 0.270 0.26140.337b
Pooled
SEM
=
0.006;
LSD
=
0.019;
*chick
removed
or
died
s Slope ratio and standard curve analysis from the above results
indicates that the low phytate corn contains 3 times more available
phosphorus than in conventional corn.
Example 3
Io The method of producing these elite, agronomically sound and
high yielding mutants is a known method called mutagenesis. The
process is outlined in the Neuffer paper Maize Genetic Newsletter
45:14fi. It should be noted that EMS is a mutation process. Like all
mutation processes the act of mutation can adversely effect the
is agronomic traits especially yields of the plant. However, the starting
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germplasm is superior to that in which the phytate mutant was
previously formed. Thus the overall agronomic traits of the plant of the
present invention are more easily preserved and selected for then the
industries approach of recurrent selection or backcrossing. Mutations
s were induced in the inbred line by treating pollen with ethyl methane
sulfonate in paraffin oil according to the procedure described by
Neuffer (1974). This treatment was performed on a number of inbreds
from the various plant genotypes of cereal. This example will focus on
the development of maize low-phytate mutants by this process. This
to mutagenesis process has been used to make a number of cereal
mutants.
The general steps of the process of the present invention
include treating inbred pollen (in this case maize) with ethyl methane
sulfonate hereinafter "EMS". Inbred pollen is placed in EMS in oil for
~s 45 minutes. A paint brush is used and the pollen is brushed on to the
silks of a receptive corn ear. This forms the Mutant-1 (M1 ) seed.
Such seed are grown and self-pollinated to produce the Mutant-2 (M2)
kernels. The resulting M2 kernels are tested for the low phytate
phenotype.
Example 4
The HVPE method is a common test for low phytate mutants.
(Raboy, Mydica 35:383 (1990)). The method relies on differential
migration of phosphorus compounds. After electrophoretically
2s fractionating the compounds a chromatogram allows a semi
quantitative assessment of the phytic acid relative to other
compounds. An alternative method is screening for higher levels of
inorganic P in the grain. For example grain samples can be ground
(to pass a 2 mm screen in A Wiiey mill) add either 50 mg of grain
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germ or 1 gram of endosperm in 15rn1 of 0.4 M HCL in 0.7 Na2 S04.
Phytic acid precipitates as an iron salt. Phosphorus in the ferric
phytate precipitates and total P are determined. Phytic acid P (mg)
are converted to phytic acid by conversion factor 3.5.
s The principles of phytate measurement are known. In the
method employed in the present study, a solution of 5-sulfosalicylic
acid and FeCi3 (Wade reagent) forms a pink chromophore. Phytic
acid binds iron in this solution decreasing .the level of the pink color.
The measurement of this loss of color can be used as an indication of
~o phytic acid levels. Since the blank contains no phytate, all readings of
samples that do contain phytate will be negative numbers. if there is
too much phytate, however, the iron-phytate complex can precipitate
as a milky white substance. In this case the pink color will not be
present but the milky white matter will absorb sight and result in falsely
~s high readings. Thus some visual observation may be necessary.
This may necessitate using a smaller aliquot (less than 25 microliters)
of the corn extract if the corn variety has high levels of phytate.
A rapid screening procedure, such as described as follows,
may be used to score for putative low phytate seeds. In this
2o procedure, a single edge razor is used to cut the kernel tip cap off just
behind the black layer. The cut should transect the scutellum at a
point at or near the radicle tip. Ususally, 8 representative kernels
were selected from each ear. The kernels are then placed, cut
surface up, on a microplate that has the surface covered with
2s cellophane tape (sticky side up). The staining procedure was
completed after dissection of at least 100 families.
Staining was done with the use of a repeating pipette to place a
microliter drop of Wade reagent (as described below) on the cut
surface of each kernel. After a few minutes the color disappeared as
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the phytic acid from the scutellum binds the iron in the Wade reagent.
Observations were made for families that segregate for slower
disappearance of the pink color relative to the others (perhaps 5 % of
the total). These slower families were re-analyzed by the quantitative
s procedure described herein.
Phytate was quantified as follows. Individual kernels (7 to 10
from each family) were crushed in steel plate of a Carver hand-pump
press (best results obtained when wells of plate lined with glycine
paper and crushed with about 5000 Ibs. pressure) and placed into 1.5
~o mi microcentrifuge tubes. 1 ml of 0.65 N hydrochloric acid was added
and allowed to stand overnight. The combination was mixed by
inversion the next day and allowed to settle for 5 minutes. A 15
microliter aliquot of the supernate was added to a microfuge tube with
100 microliters of buffer A (as described herein) and mixed. Low
is phytate mutants turn a very blue color due to the high phosphorus
levels of the seeds. Mutants were generally retested the following
day. The 0.65 N HCI extraction solution was made by adding 21fi ml
of 12.1 N HCI to 3784 ml water. Reagent A was made fresh daily and
included 2 parts (by volume) deionized water, 1 part (by volume)
2o ascorbic acid solution, 1 part (by volume) ammonium molybdate
solution and 1 part (by volume) H2S04 solution. Ammonium
molybdate solution was made by adding 25 g (NH4)s M0,024 x 4Hz0 to
make 1 L with water. H2S04 solution was made by adding 167 ml of
36 N sulfuric acid to 833 ml water. Ascorbic acid solution was made
2s by adding 100 g L-ascorbic acid to make 1 L with water. Ascorbic acid
solution was stable with refrigeration for about seven weeks but only
about two hours unrefrigerated.
Phytic Acid standards were prepared by means known in the
art.
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Maize kernel {M2} free phosphate was visually screened as
follows. Kernels were selected, the phenotypes noted and placed in a
multiple well crushing plate. Kernels were crushed in the multiple well
crushing plate using a hydraulic press. Crushed kernels were
s transferred into 1.5 ml eppendorf microcentrifuge tubes. 0.5 ml of
Reagent A was added. After allowing 2 hours reaction time, 0.5 ml
reagent B was added. The tubes were capped and mixed by inverting.
The reactions were scored visually for blueness after 1 hour, using a
light box where necessary, and the bluest samples were selected as
~o having highest phosphate. Often, the bluest samples from each
family (ear) were selected and compared for final selection. Reagent
A of this assay was prepared from 50 ml DMSO and 50 ml Reagent B.
Reagent B was made fresh daily and prepared from 60 ml distilled
water; 30 ml of 10% ascorbic solution (10 g ascorbic acid water to 700
is ml total volume; the ascorbic solution was refrigerated and stable for 1
week); 30 ml of 3.5% ammonium molybdate solution {2.5 g (NH4)6
Mo, 02,*4H20 add water to 100 ml total volume); 30 ml of 6N sulfuric
acid solution (170 ml water plus 25 ml concentrated HzS04, adjust to
200 ml total volume with water).
2o Phytate (Red Test) was quantitatively measured as follows.
Approximately 12 mature seeds were crushed in a steel crush plate of
a Carver hand-pump press. Best results were obtained when wells of
the plate were lined with weighing paper. Kernels were crushed with
5000 to 10000 Ibs. pressure and transferred to Eppendorf tubes. 1 ml
2s of 0.65 N HCI was added to same, allowed to sit overnight and mixed
the following day by tube inversion. To assay, 200 ~.1 of Wade-A
reagent (described below) was combined with 10 p,l of the above
obtained cornIHCI juice extract in individual wells of a microtiter plate.
Any change in color was noted and samples which remained red
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were noted as low in phytate since phytate binds with iron and turns
the solution white. Quantitation may be completed with a
spectrophotometer measuring at 490 nm. Wade-A reagent used
herein was prepared by adding 25.4 g of 5-sulfosalicylic acid and 350
s mg of FeC13.8H20 (ground with mortar and pestel if necessary) to 1.5
L deionized water. NaOH was used to adjust the pH to 3.05 and
volume adjusted with d.H20 to 2L. This reagent was stable in a
refrigerator for about 1 mo. 0.65N HCI was prepared by adding 216 ml
HCI (12.iN) to 3784 mf of d.HzO.
io These assays allow the selection of the desired maize plants
containing the desired alleles. Other methods of testing for
phosphorus are known and can be used to select plants.
Seed containing the desired levels of phytic acid are then
increased. This process was employed in the present invention and a
is number of inbred fines were selected that carried the low phytic acid
mutation. These included several low phytate inbred lines with good
combining ability which were crossed together to form a hybrid that
produced the grain of the present invention. Thus, the developed
inbreds of the present invention were produced from stiff stalk,
?o Lancaster and another versatile heterotic patterns so that the inbreds
when crossed together with the appropriate heterotic pattern formed
excellent hybrid material. It was also discovered that a number of the
developed mutations of the present invention though low in phytic acid
were not the same mutant as the Ipa1-R mutation. Additionally, the
2s seed were screened for germinability in standard seed germination
tests. It was found that some low phytate mutants were unable to
germinate whereas others would germinate normally. Only the seed
with good germination characteristics were maintained.
i
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Figure 1 shows an example of some data obtained form 3 such
inbred lines screened for phytate content. Plotted as a frequency
distribution curve it is clearly evident that there were a few samples
with very low phytate content (less than 0.5 units (weight percent)
s whereas the bulk of the samples were higher in phytate content. The
line known as U095 line was selected as a starting material for its
higher than average protein and oil levels. This line produces seed
which are oil burdened and protein laden and which germinate
normally. U095py retains these characteristics with the low phytate
~o levels described above. When crossed with certain other inbred lines,
the resulting hybrid produces grain which are oil burdened and protein
laden and contain low phytic levels.
The following table represents phytic acid contents (mglg of
seed) for mutant corn according to the present invention compared
t s with wild-type seed.
I No. Wild-typePhytic Mutant Phytic % reduction
Seeds Line acid Line acid
24 UU01 1.16 UU01-py 0.17 85.3
12 U095 1.85 U095-py 0.14 92.4
12 WD22 1.45 WD22-py 0.05 96.6
2o The following provides an example of an inbred line according
to the present invention.
Inbred Protein Oil Phytate % phytate
reduction
Wild-type
U095 13.4 606 1.85
UU01 12.7 2.9 1.16
B73 11.3 4.4 ---
WD22 -- --- 1.45
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Mutants
U095py 14.4 5.3 0.14 85.3
UU01 py 12.2 3.1 0.17 92.4
B731pa1-R 13.2 3.2 --- ---
WD22py --- --- 0.05 96.6
Protein and oil contents were measured by NIR analysis on a
Dickey-John Reflectance Near Infra Red Spectrometer.
s The grain of the present invention can also be used as a
substitute source for the corn grain or flour used to make corn tortilla,
corn meal, and cornfiakes by substituting the grain of the present
invention in the recipe and baking or processing as one would
normally.
to The grain of the present invention can also be used as a
substitute for the corn wet milling industry by substituting the grain of
the present invention in order to increase milling efficiency and
recoverable starch content. Animal feed made as a by-product of the
milling process is also substantially reduced in phytate content.
is
* * * * * *
The entire contents of references referred to herein are
incorporated in their entirety by reference.
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