U.S. patent application number 09/756468 was filed with the patent office on 2002-05-23 for inbred corn plant 94ink1b and seeds thereof.
Invention is credited to Innes, Robert, Popi, Jon.
Application Number | 20020062505 09/756468 |
Document ID | / |
Family ID | 26882452 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020062505 |
Kind Code |
A1 |
Innes, Robert ; et
al. |
May 23, 2002 |
Inbred corn plant 94INK1B and seeds thereof
Abstract
According to the invention, there is provided an inbred corn
plant designated 94INK1B. This invention thus relates to the
plants, seeds and tissue cultures of the inbred corn plant 94INK1B,
and to methods for producing a corn plant produced by crossing the
inbred corn plant 94INK1B with itself or with another corn plant,
such as another inbred. This invention further relates to corn
seeds and plants produced by crossing the inbred plant 94INK1B with
another corn plant, such as another inbred, and to crosses with
related species. This invention further relates to the inbred and
hybrid genetic complements of the inbred corn plant 94INK1B, and
also to the SSR and genetic isozyme typing profiles of inbred corn
plant 94INK1B
Inventors: |
Innes, Robert; (West Lorne,
CA) ; Popi, Jon; (London, CA) |
Correspondence
Address: |
Robert E. Hanson
Fulbright & Jaworski L.L.P.
Suite 2400
600 Congress Avenue
Austin
TX
78701
US
|
Family ID: |
26882452 |
Appl. No.: |
09/756468 |
Filed: |
January 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60186822 |
Mar 3, 2000 |
|
|
|
Current U.S.
Class: |
800/320.1 ;
800/275 |
Current CPC
Class: |
A01H 5/10 20130101; A01H
6/4684 20180501 |
Class at
Publication: |
800/320.1 ;
800/275 |
International
Class: |
A01H 005/00; A01H
001/02 |
Claims
What is claimed is:
1. Inbred corn seed of the corn plant 94INK1B, a sample of said
seed having been deposited under ATCC Accession No. ______.
2. The inbred corn seed of claim 1, further defined as an
essentially homogeneous population of inbred corn seed.
3. The inbred corn seed of claim 1, further defined as essentially
free from hybrid seed.
4. An inbred corn plant produced by growing the seed of the inbred
corn plant 94INK1B, a sample of said seed having been deposited
under ATCC Accession No. ______.
5. Pollen of the plant of claim 4.
6. An ovule of the plant of claim 4.
7. An essentially homogeneous population of corn plants produced by
growing the seed of the inbred corn plant 94INK1B, a sample of said
seed having been deposited under ATCC Accession No. ______.
8. A corn plant capable of expressing all the physiological and
morphological characteristics of the inbred corn plant 94INK1B, a
sample of the seed of said inbred corn plant 94INK1B having been
deposited under ATCC Accession No. ______.
9. The corn plant of claim 8, further comprising a factor
conferring male sterility.
10. A tissue culture of regenerable cells of inbred corn plant
94INK1B, wherein the tissue regenerates plants capable of
expressing all the physiological and morphological characteristics
of the inbred corn plant 94NK 1B, a sample of the seed of said
inbred corn plant 94INK1B having been deposited under ATCC
Accession No. ______.
11. The tissue culture of claim 10, wherein the regenerable cells
comprise cells derived from embryos, immature embryos, meristematic
cells, immature tassels, microspores, pollen, leaves, anthers,
roots, root tips, silk, flowers, kernels, ears, cobs, husks, or
stalks.
12. The tissue culture of claim 11, wherein the regenerable cells
comprise protoplasts or callus.
13. A corn plant regenerated from the tissue culture of claim 10,
wherein said corn plant is capable of expressing all of the
physiological and morphological characteristics of the inbred corn
plant designated 94INK1B, a sample of the seed of said inbred corn
plant designated 94INK1B having been deposited under ATCC Accession
No. ______.
14. An inbred corn plant cell of the corn plant of claim 8, said
cell comprising: (a) an SSR genetic marker profile in accordance
with the profile shown in Table 6; or (b) a genetic isozyme typing
profile in accordance with the profile shown in Table 7.
15. A corn seed comprising the inbred corn plant cell of claim
14.
16. A tissue culture comprising the inbred corn plant cell of claim
14.
17. The inbred corn plant of claim 8, comprising: (a) an SSR
genetic marker profile in accordance with the profile shown in
Table 6; or (b) a genetic isozyme typing profile in accordance with
the profile shown in Table 7.
18. A process of producing corn seed, comprising crossing a first
parent corn plant with a second parent corn plant, wherein said
first or second corn plant is the inbred corn plant 94INK1B, a
sample of the seed of said inbred corn plant 94INK1B having been
deposited under ATCC Accession No. ______, wherein seed is allowed
to form.
19. The process of claim 18, further defined as a process of
producing hybrid corn seed, comprising crossing a first inbred corn
plant with a second, distinct inbred corn plant, wherein said first
or second inbred corn plant is the inbred corn plant 94INK1B, a
sample of the seed of said inbred corn plant 94INK1B having been
deposited under ATCC Accession No. ______.
20. The process of claim 19, wherein crossing comprises the steps
of: (a) planting in pollinating proximity seeds of said first and
second inbred corn plants; (b) cultivating the seeds of said first
and second inbred corn plants into plants that bear flowers; (c)
emasculating the male flowers of said first or second inbred corn
plant to produce an emasculated corn plant; (d) allowing
cross-pollination to occur between said first and second inbred
corn plants; and (e) harvesting seeds produced on said emasculated
corn plant.
21. The process of claim 20, further comprising growing said
harvested seed to produce a hybrid corn plant.
22. Hybrid corn seed produced by the process of claim 20.
23. A hybrid corn plant produced by the process of claim 21.
24. The hybrid corn plant of claim 23, wherein the plant is a first
generation (F.sub.1) hybrid corn plant.
25. The corn plant of claim 4, further comprising a single locus
conversion.
26. The corn plant of claim 25, wherein the single locus was stably
inserted into a corn genome by transformation.
27. The corn plant of claim 25, wherein the locus is selected from
the group consisting of a dominant allele and a recessive
allele.
28. The corn plant of claim 25, wherein the locus confers a trait
selected from the group consisting of herbicide resistance, insect
resistance, resistance to bacterial, fungal, nematode or viral
disease, yield enhancement, waxy starch, improved nutritional
quality, enhanced yield stability, male sterility and restoration
of male fertility.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims the priority of U.S. Provisional
Application No. 60/186,822, filed Mar. 3, 2000.
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the field of corn
breeding. In particular, the invention relates to inbred corn seed
and plants designated 94INK1B, and derivatives and tissue cultures
thereof.
[0004] 2. Description of Related Art
[0005] The goal of field crop breeding is to combine various
desirable traits in a single variety/hybrid. Such desirable traits
include greater yield, better stalks, better roots, resistance to
insecticides, herbicides, pests, and disease, tolerance to heat and
drought, reduced time to crop maturity, better agronomic quality,
higher nutritional value, and uniformity in germination times,
stand establishment, growth rate, maturity, and fruit size.
[0006] Breeding techniques take advantage of a plant's method of
pollination. There are two general methods of pollination: a plant
self-pollinates if pollen from one flower is transferred to the
same or another flower of the same plant. A plant cross-pollinates
if pollen comes to it from a flower on a different plant.
[0007] Corn plants (Zea mays L.) can be bred by both
self-pollination and cross-pollination. Both types of pollination
involve the corn plant's flowers. Corn has separate male and female
flowers on the same plant, located on the tassel and the ear,
respectively. Natural pollination occurs in corn when wind blows
pollen from the tassels to the silks that protrude from the tops of
the ear shoot.
[0008] Plants that have been self-pollinated and selected for type
over many generations become homozygous at almost all gene loci and
produce a uniform population of true breeding progeny, a homozygous
plant. A cross between two such homozygous plants produces a
uniform population of hybrid plants that are heterozygous for many
gene loci. Conversely, a cross of two plants each heterozygous at a
number of loci produces a population of hybrid plants that differ
genetically and are not uniform. The resulting non-uniformity makes
performance unpredictable.
[0009] The development of uniform corn plant hybrids requires the
development of homozygous inbred plants, the crossing of these
inbred plants, and the evaluation of the crosses. Pedigree breeding
and recurrent selection are examples of breeding methods used to
develop inbred plants from breeding populations. Those breeding
methods combine the genetic backgrounds from two or more inbred
plants or various other broad-based sources into breeding pools
from which new inbred plants are developed by selfing and selection
of desired phenotypes. The new inbreds are crossed with other
inbred plants and the hybrids from these crosses are evaluated to
determine which of those have commercial potential.
[0010] The pedigree breeding method involves crossing two
genotypes. Each genotype can have one or more desirable
characteristics lacking in the other; or, each genotype can
complement the other. If the two original parental genotypes do not
provide all of the desired characteristics, other genotypes can be
included in the breeding population. Superior plants that are the
products of these crosses are selfed and selected in successive
generations. Each succeeding generation becomes more homogeneous as
a result of self-pollination and selection. Typically, this method
of breeding involves five or more generations of selfing and
selection: S.sub.1.fwdarw.S.sub.2; S.sub.2.fwdarw.S.sub.3;
S.sub.3.fwdarw.S.sub.4; S.sub.4.fwdarw.S.sub.5, etc. After at least
five generations, the inbred plant is considered genetically
pure.
[0011] Backcrossing can also be used to improve an inbred plant.
Backcrossing transfers a specific desirable trait from one inbred
or non-inbred source to an inbred that lacks that trait. This can
be accomplished, for example, by first crossing a superior inbred
(A) (recurrent parent) to a donor inbred (non-recurrent parent),
which carries the appropriate locus or loci for the trait in
question. The progeny of this cross are then mated back to the
superior recurrent parent (A) followed by selection in the
resultant progeny for the desired trait to be transferred from the
non-recurrent parent. After five or more backcross generations with
selection for the desired trait, the progeny are heterozygous for
loci controlling the characteristic being transferred, but are like
the superior parent for most or almost all other loci. The last
backcross generation would be selfed to give pure breeding progeny
for the trait being transferred.
[0012] A single cross hybrid corn variety is the cross of two
inbred plants, each of which has a genotype which complements the
genotype of the other. The hybrid progeny of the first generation
is designated F.sub.1. Typically, F.sub.1 hybrids are more vigorous
than their inbred parents. This hybrid vigor, or heterosis, is
manifested in many polygenic traits, including markedly improved
yields, better stalks, better roots, better uniformity and better
insect and disease resistance. In the development of hybrids only
the F.sub.1 hybrid plants are typically sought. An F.sub.1 single
cross hybrid is produced when two inbred plants are crossed. A
double cross hybrid is produced from four inbred plants crossed in
pairs (A.times.B and C.times.D) and then the two F.sub.1 hybrids
are crossed again (A.times.B).times.(C.times.D).
[0013] The development of a hybrid corn variety involves three
steps: (1) the selection of plants from various germplasm pools;
(2) the selfing of the selected plants for several generations to
produce a series of inbred plants, which, although different from
each other, each breed true and are highly uniform; and (3)
crossing the selected inbred plants with unrelated inbred plants to
produce the hybrid progeny (F.sub.1). During the inbreeding process
in corn, the vigor of the plants decreases. Vigor is restored when
two unrelated inbred plants are crossed to produce the hybrid
progeny (F.sub.1). An important consequence of the homozygosity and
homogeneity of the inbred plants is that the hybrid between any two
inbreds is always the same. Once the inbreds that give a superior
hybrid have been identified, hybrid seed can be reproduced
indefinitely as long as the homogeneity of the inbred parents is
maintained. Conversely, much of the hybrid vigor exhibited by
F.sub.1 hybrids is lost in the next generation (F.sub.2).
Consequently, seed from hybrid varieties is not used for planting
stock. It is not generally beneficial for farmers to save seed of
F.sub.1 hybrids. Rather, farmers purchase F.sub.1 hybrid seed for
planting every year.
[0014] North American farmers plant tens of millions of acres of
corn at the present time and there are extensive national and
international commercial corn breeding programs. A continuing goal
of these corn breeding programs is to develop corn hybrids that are
based on stable inbred plants and have one or more desirable
characteristics. To accomplish this goal, the corn breeder must
select and develop superior inbred parental plants.
SUMMARY OF THE INVENTION
[0015] In one aspect, the present invention provides a corn plant
designated 94INK1B. Also provided are corn plants having all the
physiological and morphological characteristics of corn plant
94INK1B. The inbred corn plant of the invention may further
comprise, or have, a cytoplasmic or nuclear factor that is capable
of conferring male sterility. Parts of the corn plant of the
present invention are also provided, for example, pollen obtained
from an inbred plant and an ovule of the inbred plant.
[0016] The invention also concerns seed of the corn plant 94INK1B.
A sample of this seed has been deposited under ATCC Accession No.
______. The inbred corn seed of the invention may be provided as an
essentially homogeneous population of inbred corn seed of the corn
plant designated 94INK1B. Essentially homogeneous populations of
inbred seed are those that consist essentially of the particular
inbred seed, and are generally free from substantial numbers of
other seed, so that the inbred seed forms between about 90% and
about 100% of the total seed, and preferably, between about 95% and
about 100% of the total seed. Most preferably, an essentially
homogeneous population of inbred corn seed will contain between
about 98.5%, 99%, 99.5% and about 99.9% of inbred seed, as measured
by seed grow outs.
[0017] Therefore, in the practice of the present invention, inbred
seed generally forms at least about 97% of the total seed. However,
even if a population of inbred corn seed was found, for some
reason, to contain about 50%, or even about 20% or 15% of inbred
seed, this would still be distinguished from the small fraction of
inbred seed that may be found within a population of hybrid seed,
e.g., within a bag of hybrid seed. In such a bag of hybrid seed
offered for sale, the Governmental regulations require that the
hybrid seed be at least about 95% of the total seed. In the most
preferred practice of the invention, the female inbred seed that
may be found within a bag of hybrid seed will be about 1% of the
total seed, or less, and the male inbred seed that may be found
within a bag of hybrid seed will be negligible, i.e., will be on
the order of about a maximum of 1 per 100,000, and usually less
than this value.
[0018] The population of inbred corn seed of the invention can
further be particularly defined as being essentially free from
hybrid seed. The inbred seed population may be separately grown to
provide an essentially homogeneous population of inbred corn plants
designated 94INK1B.
[0019] In another aspect of the invention, single locus converted
plants of 94INK1B are provided. The single transferred locus may
preferably be a dominant or recessive allele. Preferably, the
single transferred locus will confer such traits as male sterility,
yield stability, waxy starch, yield enhancement, industrial usage,
herbicide resistance, insect resistance, resistance to bacterial,
fungal, nematode or viral disease, male fertility, and enhanced
nutritional quality. The single locus may be a naturally occurring
maize gene or a transgene introduced through genetic transformation
techniques. When introduced through transformation, a single locus
may comprise one or more transgenes integrated at a single
chromosomal location.
[0020] In yet another aspect of the invention, an inbred corn plant
designated 94INK1B is provided, wherein a cytoplasmically-inherited
trait has been introduced into said inbred plant. Such
cytoplasmically-inherite- d traits are passed to progeny through
the female parent in a particular cross. An exemplary
cytoplasmically-inherited trait is the male sterility trait. A
cytoplasmically inherited trait may be a naturally occurring maize
trait or a trait introduced through genetic transformation
techniques.
[0021] In another aspect of the invention, a tissue culture of
regenerable cells of inbred corn plant 94INK1B is provided. The
tissue culture will preferably be capable of regenerating plants
capable of expressing all of the physiological and morphological
characteristics of the foregoing inbred corn plant, and of
regenerating plants having substantially the same genotype as the
foregoing inbred corn plant. Examples of some of the physiological
and morphological characteristics of the inbred corn plant 94INK1B
include characteristics related to yield, maturity, and kernel
quality, each of which are specifically disclosed herein. The
regenerable cells in such tissue cultures will preferably be
derived from embryos, meristematic cells, immature tassels,
microspores, pollen, leaves, anthers, roots, root tips, silk,
flowers, kernels, ears, cobs, husks, or stalks, or callus or
protoplasts derived from these tissues. Still further, the present
invention provides corn plants regenerated from the tissue cultures
of the invention, the plants having all the physiological and
morphological characteristics of corn plant 94INK1B.
[0022] In yet another aspect of the invention, processes are
provided for producing corn seeds or plants, which processes
generally comprise crossing a first parent corn plant with a second
parent corn plant, wherein at least one of the first or second
parent corn plants is the inbred corn plant designated 94INK1B.
These processes may be further exemplified as processes for
preparing hybrid corn seed or plants, wherein a first inbred corn
plant is crossed with a second, distinct inbred corn plant to
provide a hybrid that has, as one of its parents, the inbred corn
plant 94INK1B. In these processes, the step of crossing will result
in the production of seed. The seed production occurs regardless of
whether the seed is collected or not.
[0023] In a preferred embodiment of the invention, crossing
comprises planting in pollinating proximity seeds of a first and
second parent corn plant, and preferably, seeds of a first inbred
corn plant and a second, distinct inbred corn plant; cultivating or
growing the seeds of said first and second parent corn plants into
plants that bear flowers; emasculating the male flowers of the
first or second parent corn plant, (i.e., treating or manipulating
the flowers so as to prevent pollen production, in order to produce
an emasculated parent corn plant) allowing natural
cross-pollination to occur between the first and second parent corn
plants; and harvesting the seeds from the emasculated parent corn
plant. Where desired, the harvested seed is grown to produce a corn
plant or hybrid corn plant.
[0024] The present invention also provides corn seed and plants
produced by a process that comprises crossing a first parent corn
plant with a second parent corn plant, wherein at least one of the
first or second parent corn plants is the inbred corn plant
designated 94INK1B. In one embodiment of the invention, corn plants
produced by the process are first generation (F.sub.1) hybrid corn
plants produced by crossing an inbred in accordance with the
invention with another, distinct inbred. The present invention
further contemplates seed of an F.sub.1 hybrid corn plant.
Therefore, certain exemplary embodiments of the invention provide
an F.sub.1 hybrid corn plant and seed thereof. An example of such a
hybrid which can be produced with the inbred designated 94INK1B is
the hybrid corn plant designated 8002161.
[0025] In still yet another aspect of the invention, an inbred
genetic complement of the corn plant designated 94INK1B is
provided. The phrase "genetic complement" is used to refer to the
aggregate of nucleotide sequences, the expression of which
sequences defines the phenotype of, in the present case, a corn
plant, or a cell or tissue of that plant. An inbred genetic
complement thus represents the genetic make up of an inbred cell,
tissue or plant, and a hybrid genetic complement represents the
genetic make up of a hybrid cell, tissue or plant. The invention
thus provides corn plant cells that have a genetic complement in
accordance with the inbred corn plant cells disclosed herein, and
plants, seeds and diploid plants containing such cells.
[0026] Plant genetic complements may be assessed by genetic marker
profiles, and by the expression of phenotypic traits that are
characteristic of the expression of the genetic complement, e.g.,
isozyme typing profiles. Thus, such corn plant cells may be defined
as having an SSR genetic marker profile in accordance with the
profile shown in Table 6, or a genetic isozyme typing profile in
accordance with the profile shown in Table 7, or having both an SSR
genetic marker profile and a genetic isozyme typing profile in
accordance with the profiles shown in Table 6 and Table 7. It is
understood that 94INK1B could also be identified by other types of
genetic markers such as, for example, Simple Sequence Length
Polymorphisms (SSLPS) (Williams et al., 1990), Randomly Amplified
Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),
Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed
Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length
Polymorphisms (AFLPs) (EP 534 858, specifically incorporated herein
by reference in its entirety), and Single Nucleotide Polymorphisms
(SNPs) (Wang et al., 1998).
[0027] In still yet another aspect, the present invention provides
hybrid genetic complements, as represented by corn plant cells,
tissues, plants, and seeds, formed by the combination of a haploid
genetic complement of an inbred corn plant of the invention with a
haploid genetic complement of a second corn plant, preferably,
another, distinct inbred corn plant. In another aspect, the present
invention provides a corn plant regenerated from a tissue culture
that comprises a hybrid genetic complement of this invention.
DETAILED DESCRIPTION OF THE INVENTION
[0028] I. Definitions of Plant Characteristics
[0029] Barren Plants: Plants that are barren, i.e., lack an ear
with grain, or have an ear with only a few scattered kernels.
[0030] Cg: Colletotrichum graminicola rating. Rating times 10 is
approximately equal to percent total plant infection.
[0031] CLN: Corn Lethal Necrosis (combination of Maize Chlorotic
Mottle Virus and Maize Dwarf Mosaic virus) rating: numerical
ratings are based on a severity scale where 1=most resistant to
9=susceptible.
[0032] Cn: Corynebacterium nebraskense rating. Rating times 10 is
approximately equal to percent total plant infection.
[0033] Cz: Cercospora zeae-maydis rating. Rating times 10 is
approximately equal to percent total plant infection.
[0034] Dgg: Diatraea grandiosella girdling rating (values are
percent plants girdled and stalk lodged).
[0035] Dropped Ears: Ears that have fallen from the plant to the
ground.
[0036] Dsp: Diabrotica species root ratings (1=least affected to
9=severe pruning).
[0037] Ear-Attitude: The attitude or position of the ear at harvest
scored as upright, horizontal, or pendant.
[0038] Ear-Cob Color: The color of the cob, scored as white, pink,
red, or brown.
[0039] Ear-Cob Diameter: The average diameter of the cob measured
at the midpoint.
[0040] Ear-Cob Strength: A measure of mechanical strength of the
cobs to breakage, scored as strong or weak.
[0041] Ear-Diameter: The average diameter of the ear at its
midpoint.
[0042] Ear-Dry Husk Color: The color of the husks at harvest scored
as buff, red, or purple.
[0043] Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks
after pollination scored as green, red, or purple.
[0044] Ear-Husk Bract: The length of an average husk leaf scored as
short, medium, or long.
[0045] Ear-Husk Cover: The average distance from the tip of the ear
to the tip of the husks. Minimum value no less than zero.
[0046] Ear-Husk Opening: An evaluation of husk tightness at harvest
scored as tight, intermediate, or open.
[0047] Ear-Length: The average length of the ear.
[0048] Ear-Number Per Stalk: The average number of ears per
plant.
[0049] Ear-Shank Internodes: The average number of internodes on
the ear shank.
[0050] Ear-Shank Length: The average length of the ear shank.
Ear-Shelling Percent: The average of the shelled grain weight
divided by the sum of the shelled grain weight and cob weight for a
single ear.
[0051] Ear-Silk Color: The color of the silk observed 2 to 3 days
after silk emergence scored as green-yellow, yellow, pink, red, or
purple.
[0052] Ear-Taper (Shape): The taper or shape of the ear scored as
conical, semi-conical, or cylindrical.
[0053] Ear-Weight: The average weight of an ear.
[0054] Early Stand: The percent of plants that emerge from the
ground as determined in the early spring.
[0055] ER: Ear rot rating (values approximate percent ear
rotted).
[0056] Final Stand Count: The number of plants just prior to
harvest.
[0057] GDUs: Growing degree units which are calculated by the
Barger Method, where the heat units for a 24-h period are
calculated as GDUs=[(Maximum daily temperature+Minimum daily
temperature)/2]-50. The highest maximum daily temperature used is
86.degree. F. and the lowest minimum temperature used is 50.degree.
F.
[0058] GDUs to Shed: The number of growing degree units (GDUs) or
heat units required for an inbred line or hybrid to have
approximately 50% of the plants shedding pollen as measured from
time of planting. GDUs to shed is determined by summing the
individual GDU daily values from planting date to the date of 50%
pollen shed. GDUs to Silk: The number of growing degree units for
an inbred line or hybrid to have approximately 50% of the plants
with silk emergence as measured from time of planting. GDUs to silk
is determined by summing the individual GDU daily values from
planting date to the date of 50% silking.
[0059] Hc2: Helminthosporium carbonum race 2 rating. Rating times
10 is approximately equal to percent total plant infection.
[0060] Hc3: Helminthosporium carbonum race 3 rating. Rating times
is approximately equal to percent total plant infection.
[0061] Hm: Helminthosporium maydis race 0 rating. Rating times 10
is approximately equal to percent total plant infection.
[0062] Ht1: Helminthosporium turcicum race 1 rating. Rating times
10 is approximately equal to percent total plant infection.
[0063] Ht2: Helminthosporium turcicum race 2 rating. Rating times
10 is approximately equal to percent total plant infection.
[0064] HtG: Chlorotic-lesion type resistance. +=indicates the
presence of Ht chlorotic-lesion type resistance; -=indicates
absence of Ht chlorotic-lesion type resistance; and +/- indicates
segregation of Ht chlorotic-lesion type resistance. Rating times 10
is approximately equal to percent total plant infection.
[0065] Kernel-Aleurone Color: The color of the aleurone scored as
white, pink, tan, brown, bronze, red, purple, pale purple,
colorless, or variegated.
[0066] Kernel-Cap Color: The color of the kernel cap observed at
dry stage, scored as white, lemon-yellow, yellow, or orange.
[0067] Kernel-Endosperm Color: The color of the endosperm scored as
white, pale yellow, or yellow.
[0068] Kernel-Endosperm Type: The type of endosperm scored as
normal, waxy, or opaque.
[0069] Kernel-Grade: The percent of kernels that are classified as
rounds.
[0070] Kernel-Length: The average distance from the cap of the
kernel to the pedicel.
[0071] Kernel-Number Per Row: The average number of kernels in a
single row.
[0072] Kernel-Pericarp Color: The color of the pericarp scored as
colorless, red-white crown, tan, bronze, brown, light red, cherry
red, or variegated.
[0073] Kernel-Row Direction: The direction of the kernel rows on
the ear scored as straight, slightly curved, spiral, or indistinct
(scattered).
[0074] Kernel-Row Number: The average number of rows of kernels on
a single ear.
[0075] Kernel-Side Color: The color of the kernel side observed at
the dry stage, scored as white, pale yellow, yellow, orange, red,
or brown.
[0076] Kernel-Thickness: The distance across the narrow side of the
kernel.
[0077] Kernel-Type: The type of kernel scored as dent, flint, or
intermediate.
[0078] Kernel-Weight: The average weight of a predetermined number
of kernels.
[0079] Kernel-Width: The distance across the flat side of the
kernel.
[0080] Kz: Kabatiella zeae rating. Rating times 10 is approximately
equal to percent total plant infection.
[0081] Leaf-Angle: Angle of the upper leaves to the stalk scored as
upright (0 to 30 degrees), intermediate (30 to 60 degrees), or lax
(60 to 90 degrees).
[0082] Leaf-Color: The color of the leaves 1 to 2 weeks after
pollination scored as light green, medium green, dark green, or
very dark green.
[0083] Leaf-Length: The average length of the primary ear leaf
[0084] Leaf-Longitudinal Creases: A rating of the number of
longitudinal creases on the leaf surface 1 to 2 weeks after
pollination. Creases are scored as absent, few, or many.
[0085] Leaf-Marginal Waves: A rating of the waviness of the leaf
margin 1 to 2 weeks after pollination. Rated as none, few, or
many.
[0086] Leaf-Number: The average number of leaves of a mature plant.
Counting begins with the cotyledonary leaf and ends with the flag
leaf.
[0087] Leaf-Sheath Anthocyanin: A rating of the level of
anthocyanin in the leaf sheath 1 to 2 weeks after pollination,
scored as absent, basal-weak, basal-strong, weak or strong.
[0088] Leaf-Sheath Pubescence: A rating of the pubescence of the
leaf sheath. Ratings are taken 1 to 2 weeks after pollination and
scored as light, medium, or heavy.
[0089] Leaf-Width: The average width of the primary ear leaf
measured at its widest point.
[0090] LSS: Late season standability (values times 10 approximate
percent plants lodged in disease evaluation plots).
[0091] Moisture: The moisture of the grain at harvest.
[0092] On1: Ostrinia nubilalis 1st brood rating (1=resistant to
9=susceptible).
[0093] On2: Ostrinia nubilalis 2nd brood rating (1=resistant to
9susceptible).
[0094] Relative Maturity: A maturity rating based on regression
analysis. The regression analysis is developed by utilizing check
hybrids and their previously established day rating versus actual
harvest moistures. Harvest moisture on the hybrid in question is
determined and that moisture value is inserted into the regression
equation to yield a relative maturity.
[0095] Root Lodging: Root lodging is the percentage of plants that
root lodge. A plant is counted as root lodged if a portion of the
plant leans from the vertical axis by approximately 30 degrees or
more.
[0096] Seedling Color: Color of leaves at the 6 to 8 leaf
stage.
[0097] Seedling Height: Plant height at the 6 to 8 leaf stage.
[0098] Seedling Vigor: A visual rating of the amount of vegetative
growth on a 1 to 9 scale, where 1 equals best. The score is taken
when the average entry in a trial is at the fifth leaf stage.
[0099] Selection Index: The selection index gives a single measure
of hybrid's worth based on information from multiple traits. One of
the traits that is almost always included is yield. Traits may be
weighted according to the level of importance assigned to them.
[0100] Sr: Sphacelotheca reiliana rating is actual percent
infection.
[0101] Stalk-Anthocyanin: A rating of the amount of anthocyanin
pigmentation in the stalk. The stalk is rated 1 to 2 weeks after
pollination as absent, basal-weak, basal-strong, weak, or
strong.
[0102] Stalk-Brace Root Color: The color of the brace roots
observed 1 to 2 weeks after pollination as green, red, or
purple.
[0103] Stalk-Diameter: The average diameter of the lowest visible
internode of the stalk.
[0104] Stalk-Ear Height: The average height of the ear measured
from the ground to the point of attachment of the ear shank of the
top developed ear to the stalk. Stalk-Internode Direction: The
direction of the stalk internode observed after pollination as
straight or zigzag.
[0105] Stalk-Internode Length: The average length of the internode
above the primary ear.
[0106] Stalk Lodging: The percentage of plants that did stalk
lodge. Plants are counted as stalk lodged if the plant is broken
over or off below the ear.
[0107] Stalk-Nodes With Brace Roots: The average number of nodes
having brace roots per plant.
[0108] Stalk-Plant Height: The average height of the plant as
measured from the soil to the tip of the tassel. Stalk-Tillers: The
percent of plants that have tillers. A tiller is defined as a
secondary shoot that has developed as a tassel capable of shedding
pollen.
[0109] Staygreen: Staygreen is a measure of general plant health
near the time of black layer formation (physiological maturity). It
is usually recorded at the time the ear husks of most entries
within a trial have turned a mature color. Scoring is on a 1 to 9
basis where 1 equals best.
[0110] STR: Stalk rot rating (values represent severity rating of
1=25% of inoculated internode rotted to 9=entire stalk rotted and
collapsed).
[0111] SVC: Southeastern Virus Complex (combination of Maize
Chlorotic Dwarf Virus and Maize Dwarf Mosaic Virus) rating;
numerical ratings are based on a severity scale where 1=most
resistant to 9 susceptible (1988 reactions are largely Maize Dwarf
Mosaic Virus reactions).
[0112] Tassel-Anther Color: The color of the anthers at 50% pollen
shed scored as green-yellow, yellow, pink, red, or purple.
[0113] Tassel-Attitude: The attitude of the tassel after
pollination scored as open or compact.
[0114] Tassel-Branch Angle: The angle of an average tassel branch
to the main stem of the tassel scored as upright (less than 30
degrees), intermediate (30 to 45 degrees), or lax (greater than 45
degrees).
[0115] Tassel-Branch Number: The average number of primary tassel
branches.
[0116] Tassel-Glume Band: The closed anthocyanin band at the base
of the glume scored as present or absent.
[0117] Tassel-Glume Color: The color of the glumes at 50% shed
scored as green, red, or purple.
[0118] Tassel-Length: The length of the tassel measured from the
base of the bottom informative in linkage analysis relative to
other marker systems in that multiple alleles may be present.
Another advantage of this type of marker is that, through use of
flanking primers, detection of SSRs can be achieved, for example,
by the polymerase chain reaction (PCR.TM.), thereby eliminating the
need for labor-intensive Southern hybridization. The PCR.TM.
detection is done by use of two oligonucleotide primers flanking
the polymorphic segment of repetitive DNA. Repeated cycles of heat
denaturation of the DNA followed by annealing of the primers to
their complementary sequences at low temperatures, and extension of
the annealed primers with DNA polymerase, comprise the major part
of the methodology. Following amplification, markers can be scored
by gel electrophoresis of the amplification products. Scoring of
marker genotype is based on the size (number of base pairs) of the
amplified segment.
[0119] Means for performing genetic analyses using SSR
polymorphisms are well known in the art. The SSR analyses reported
herein were conducted by Celera AgGen in Davis, Calif. This service
is available to the public on a contractual basis. This analysis
was carried out by amplification of simple repeats followed by
detection of marker genotypes using gel electrophoresis. Markers
were scored based on the size of the amplified fragment.
[0120] The SSR genetic marker profile of the parental inbreds and
exemplary resultant hybrid described herein were determined.
Because an inbred is essentially homozygous at all relevant loci,
an inbred should, in almost all cases, have only one allele at each
locus. In contrast, a diploid genetic marker profile of a hybrid
should be the sum of those parents, e.g., if one inbred parent had
the allele 168 (base pairs) at a particular locus, and the other
inbred parent had 172, the hybrid is 168.172 by inference.
Subsequent generations of progeny produced by selection and
breeding are expected to be of genotype 168, 172, or 168.172 for
that locus position. When the F.sub.1 plant is used to produce an
inbred, the locus should be either 168 or 172 for that position.
Surprisingly, it has been observed that in certain instances, novel
SSR genotypes arise during the breeding process. For example, a
genotype of 170 may be observed at a particular locus position from
the selective elution.
[0121] Crossing: The pollination of a female flower of a corn
plant, thereby resulting in the production of seed from the
flower.
[0122] Cross-pollination: Fertilization by the union of two gametes
from different plants.
[0123] Diploid: A cell or organism having two sets of
chromosomes.
[0124] Electrophoresis: A process by which particles suspended in a
fluid or a gel matrix are moved under the action of an electrical
field, and thereby separated according to their charge and
molecular weight. This method of separation is well known to those
skilled in the art and is typically applied to separating various
forms of enzymes and of DNA fragments produced by restriction
endonucleases.
[0125] Emasculate: The removal of plant male sex organs or the
inactivation of the organs with a chemical agent or a cytoplasmic
or nuclear genetic factor conferring male sterility.
[0126] Enzymes: Molecules which can act as catalysts in biological
reactions.
[0127] F.sub.1 Hybrid: The first generation progeny of the cross of
two plants.
[0128] Genetic Complement: An aggregate of nucleotide sequences,
the expression of which sequences defines the phenotype in corn
plants, or components of plants including cells or tissue.
[0129] Genotype: The genetic constitution of a cell or
organism.
[0130] Haploid: A cell or organism having one set of the two sets
of chromosomes in a diploid.
[0131] Isozymes: Detectable variants of an enzyme, the variants
catalyzing the same reaction(s) but differing from each other,
e.g., in primary structure and/or electrophoretic mobility. The
differences between isozymes are under single gene, codominant
control. Consequently, electrophoretic separation to produce band
patterns can be equated to different alleles at the DNA level.
Structural differences that do not alter charge cannot be detected
by this method.
[0132] Isozyme typing profile: A profile of band patterns of
isozymes separated by electrophoresis that can be equated to
different alleles at the DNA level.
[0133] Linkage: A phenomenon wherein alleles on the same chromosome
tend to segregate together more often than expected by chance if
their transmission was independent.
[0134] Marker: A readily detectable phenotype, preferably inherited
in codominant fashion (both alleles at a locus in a diploid
heterozygote are readily detectable), with no environmental
variance component, i.e., heritability of 1.
[0135] 94INK1B: The corn plant from which seeds having ATCC
Accession No. ______ were obtained, as well as plants grown from
those seeds.
[0136] Phenotype: The detectable characteristics of a cell or
organism, which characteristics are the manifestation of gene
expression.
[0137] Quantitative Trait Loci (QTL): Genetic loci that contribute,
at least in part, certain numerically representable traits that are
usually continuously distributed.
[0138] Regeneration: The development of a plant from tissue
culture.
[0139] SSR genetic marker profile: A profile of simple sequence
repeats scored by gel electrophoresis following PCRTM amplification
using flanking oligonucleotide primers.
[0140] Self-pollination: The transfer of pollen from the anther to
the stigma of the same plant.
[0141] Single Locus Converted (Conversion) Plant: Plants which are
developed by a plant breeding technique called backcrossing wherein
essentially all of the desired morphological and physiological
characteristics of an inbred are recovered in addition to the
characteristics conferred by the single locus transferred into the
inbred via the backcrossing technique. A single locus may comprise
one gene, or in the case of transgenic plants, one or more
transgenes integrated into the host genome at a single site
(locus).
[0142] Tissue Culture: A composition comprising isolated cells of
the same or a different type or a collection of such cells
organized into parts of a plant.
[0143] Transgene: A genetic sequence which has been introduced into
the nuclear or chloroplast genome of a maize plant by a genetic
transformation technique.
[0144] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventor to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
[0145] III. Inbred Corn Plant 94INK1B
[0146] In accordance with one aspect of the present invention,
there is provided a novel inbred corn plant, designated 94INK1B.
Inbred corn plant 94INK1B can be compared to inbred corn plants
3IIH6 and 94KBZ1. 94INK1B differs significantly (at the 1%, 5%, or
10% level) from these inbred lines in several aspects (Table 1 and
Table 2).
1TABLE 1 Comparison of 94INK1B with 3IIH6 94INK1B 3IIH6 DIFF P
VALUE BARREN % 2.9 1.9 1.0 0.196 EHT INCH 29.3 26.6 2.7 0.461 FINAL
61.0 62.0 -1.0 0.247 MST % 15.0 16.4 -1.4 0.907 PHT INCH 84.9 65.2
19.7 0.310 RTL % 9.1 0.6 8.5 0.000** SHED GDU 1367.7 1399.7 -32.0
0.643 SILK GDU 1364.8 1423.3 -58.5 1.000 STL % 3.6 4.0 -0.4 0.041+
YLD BU/A 54.1 72.2 -18.1 0.447 Significance Levels are indicated
as: + = 10%, * = 5%, ** = 1%. Legend Abbreviations: BARREN % =
Barren Plants (percent) EHT INCH = Ear Height (inches) FINAL =
Final Stand MST % = Moisture (percent) PHT INCH = Plant Height
(inches) RTL % = Root Lodging (percent) SHED GDU = GDUs to Shed
SILK GDU = GDUs to Silk STL % = Stalk Lodging (percent) YLD BU/A =
Yield (bushels/acre)
[0147]
2TABLE 2 Comparison of 94INK1B with 94KBZ1 94INK1B 94KBZ1 DIFF P
VALUE BARREN % 2.9 7.6 -4.7 0.036+ EHT INCH 29.3 22.8 6.5 0.225
FINAL 61.0 63.5 -2.5 0.285 MST % 15.0 13.4 1.6 0.964 PHT INCH 84.9
64.9 20.0 0.461 RTL % 9.1 9.0 0.1 0.657 SHED GDU 1367.7 1232.2
135.5 0.224 SILK GDU 1364.8 1241.8 123.0 0.159 STL % 3.6 5.4 -1.8
0.016+ YLD BU/A 54.1 36.3 17.8 0.161 Significance Levels are
indicated as: + = 10%, * = 5%, ** = 1%. Legend Abbreviations:
BARREN % = Barren Plants (percent) EHT INCH = Ear Height (inches)
FINAL = Final Stand MST % = Moisture (percent) PHT INCH = Plant
Height (inches) RTL % = Root Lodging (percent) SHED GDU = GDUs to
Shed SILK GDU = GDUs to Silk STL % = Stalk Lodging (percent) YLD
BU/A = Yield (bushels/acre)
[0148] A. Origin and Breeding History
[0149] Inbred plant 94INK1B was derived from a cross between the
lines 3IIH6 and 94KBZ1. The origin and breeding history of inbred
plant 94INK1B can be summarized as follows:
3 Summer 1992 The inbred line 3IIH6 (a proprietary DEKALB Genetics
Corporation inbred) was crossed to the inbred line 94KBZ1 (a
proprietary DEKALB Genetics Corporation inbred). Winter 1992-93 The
S0 seed was grown and self-pollinated in nursery row C92:1936.
Summer 1993 The S1 seed was grown and self-pollinated in nursery
rows G93:19-98 through 19-100 and 20-100 through 20-54.
Thirty-three ears were selected. Summer 1994 S2 ears were grown
ear-to-row and self-pollinated. Two ears were selected in nursery
row G94:59-71. Summer 1995 S3 ears were grown ear-to-row and
self-pollinated. Three ears were selected in nursery row G95:64-65.
Summer 1996 S4 ears were grown ear-to-row and self-pollinated. Four
ears were selected in nursery row G96:401-78. Summer 1997 S5 ears
were grown ear-to-row and self-pollinated. Two ears from nursery
row G97:502-71 were selected and designated as coded inbred
94INK1B. Winter 1997-98 S6 ears were grown ear-to-row and
self-pollinated. Twenty-seven ears from nursery rows H97 11Y:3-19
through 3-22 were selected. Summer 1998 S7 ears were grown
ear-to-row and self-pollinated. Fifteen ears were selected from
nursery row G98:35-52 through 35-54. Winter 1998-99 S8 ears were
grown ear-to-row and self-pollinated. Final selection was completed
in nursery rows PR98:7-20 through 7-36. This selection consisted of
bulking S9 ears.
[0150] 94INK1B shows uniformity and stability within the limits of
environmental influence for the traits described hereinafter in
Table 3. 94INK1B has been self-pollinated and ear-rowed a
sufficient number of generations with careful attention paid to
uniformity of plant type to ensure homozygosity and phenotypic
stability. No variant traits have been observed or are expected in
94INK1B.
[0151] Inbred corn plants can be reproduced by planting the seeds
of the inbred corn plant 94INK1B, growing the resulting corn plants
under self-pollinating or sib-pollinating conditions with adequate
isolation using standard techniques well known to an artisan
skilled in the agricultural arts. Seeds can be harvested from such
a plant using standard, well known procedures.
[0152] B. Phenotypic Description
[0153] In accordance with another aspect of the present invention,
there is provided a corn plant having the physiological and
morphological characteristics of corn plant 94INK1B. A description
of the physiological and morphological characteristics of corn
plant 94INK1B is presented in Table 3.
4TABLE 3 Morphological Traits for the 94INK1B Phenotype VALUE
CHARACTERISTIC 94INK1B 3IIH6 94KBZ1 1. STALK Diameter (width) cm.
2.2 2.5 2.0 Anthocyanin Basel- Absent Absent Weak Brace Root Color
Moderate Absent -- Nodes With Brace Roots 0.9 1.3 1.3 Internode
Direction Straight Straight Straight Internode Length cm. 14.8 13.9
15.2 2. LEAF Color Green Green Green Length cm. 66.5 36.2 46.4
Width cm. 9.3 4.3 6.7 Sheath Anthocyanin Basel- -- -- Weak Sheath
Pubescence Light Moderate Light Marginal Waves Few Few Few
Longitudinal Creases Moderate Moderate Moderate 3. TASSEL Length
cm. 43.1 41.0 38.3 Spike Length cm. 21.1 24.4 19.1 Peduncle Length
cm. 9.7 7.5 9.3 Branch Number 5.3 13.1 8.0 Anther Color Green- Red
Green- Yellow Yellow Glume Color Green Green Green Glume Band
Absent Absent Absent 4. EAR Silk Color Green- Red Red Yellow Number
Per Stalk 1.0 1.2 1.1 Position (attitude) Pendant -- -- Length cm.
14.0 15.8 14.0 Shape Semi- Semi- Semi- Conical Conical Conical
Diameter cm. 3.8 3.7 3.3 Weight gm. -- 103.1 72.9 Shank Length cm.
11.0 12.2 12.3 Husk Bract Short Short Short Husk Cover cm. 5.4 3.4
2.6 Husk Opening Very Inter- Inter- Loose mediate mediate Husk
Color Fresh Green Green Green Husk Color Dry Buff Buff Buff Cob
Diameter cm. 2.2 2.1 1.8 Cob Color Red Red Pink Shelling Percent
83.9 85.3 86.5 5. KERNEL Row Number 14.4 15.7 13.2 Number Per Row
23.6 30.1 27.0 Row Direction Straight Slightly Slightly Curved
Curved Type Semi-Dent Dent Dent Cap Color Yellow Yellow Yellow Side
Color Yellow Deep- Orange Yellow Length (depth) mm. 10.0 10.1 9.7
Width mm. 8.6 7.4 7.5 Thickness 3.8 4.5 4.5 Weight of l000K gm.
291.0 223.1 220.6 Endosperm Type Normal Normal Normal Endosperm
Color Yellow Yellow Yellow *These are typical values. Values may
vary due to environment. Other values that are substantially
equivalent are also within the scope of the invention.
Substantially equivalent refers to quantitative traits that when
compared do not show statistical differences of their means.
[0154] C. Deposit Information
[0155] A deposit of 2500 seeds of the inbred corn plant designated
94INK1B has been made with the American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va. on (______). Those
deposited seeds have been assigned ATCC Accession No. ______. The
deposit was made in accordance with the terms and provisions of the
Budapest Treaty relating to deposit of microorganisms and was made
for a term of at least thirty (30) years and at least five (05)
years after the most recent request for the furnishing of a sample
of the deposit is received by the depository, or for the effective
term of the patent, whichever is longer, and will be replaced if it
becomes non-viable during that period.
[0156] IV. Single Locus Conversions
[0157] When the term inbred corn plant is used in the context of
the present invention, this also includes any single locus
conversions of that inbred. The term single locus converted plant
as used herein refers to those corn plants which are developed by a
plant breeding technique called backcrossing wherein essentially
all of the desired morphological and physiological characteristics
of an inbred are recovered in addition to the single locus
transferred into the inbred via the backcrossing technique.
Backcrossing methods can be used with the present invention to
improve or introduce a characteristic into the inbred. The term
backcrossing as used herein refers to the repeated crossing of a
hybrid progeny back to one of the parental corn plants for that
inbred. The parental corn plant which contributes the locus or loci
for the desired characteristic is termed the nonrecurrent or donor
parent. This terminology refers to the fact that the nonrecurrent
parent is used one time in the backcross protocol and therefore
does not recur. The parental corn plant to which the locus or loci
from the nonrecurrent parent are transferred is known as the
recurrent parent as it is used for several rounds in the
backcrossing protocol (Poehlman et aL, 1995; Fehr, 1987; Sprague
and Dudley, 1988). In a typical backcross protocol, the original
inbred of interest (recurrent parent) is crossed to a second inbred
(nonrecurrent parent) that carries the single locus of interest to
be transferred. The resulting progeny from this cross are then
crossed again to the recurrent parent and the process is repeated
until a corn plant is obtained wherein essentially all of the
desired morphological and physiological characteristics of the
recurrent parent are recovered in the converted plant, in addition
to the single transferred locus from the nonrecurrent parent. The
backcross process may be accelerated by the use of genetic markers,
such as SSR, RFLP, SNP or AFLP markers to identify plants with the
greatest genetic complement from the recurrent parent.
[0158] The selection of a suitable recurrent parent is an important
step for a successful backcrossing procedure. The goal of a
backcross protocol is to alter or substitute a single trait or
characteristic in the original inbred. To accomplish this, a single
locus of the recurrent inbred is modified or substituted with the
desired locus from the nonrecurrent parent, while retaining
essentially all of the rest of the desired genetic, and therefore
the desired physiological and morphological constitution of the
original inbred. 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 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.
[0159] Many single locus traits have been identified that are not
regularly selected for in the development of a new inbred but that
can be improved by backcrossing techniques. Single locus traits may
or may not be transgenic; examples of these traits include, but are
not limited to, male sterility, waxy starch, herbicide resistance,
resistance for bacterial, fungal, or viral disease, insect
resistance, male fertility, enhanced nutritional quality,
industrial usage, yield stability, and yield enhancement. These
genes are generally inherited through the nucleus, but may be
inherited through the cytoplasm. Some known exceptions to this are
genes for male sterility, some of which are inherited
cytoplasmically, but still act as single locus traits. A number of
exemplary single locus traits are described in, for example, PCT
Application WO 95/06128, the disclosure of which is specifically
incorporated herein by reference.
[0160] Examples of genes conferring male sterility include those
disclosed in U.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S.
Pat. No. 4,654,465, U.S. Patent No 5,625,132, and U.S. Pat. No.
4,727,219, each of the disclosures of which are specifically
incorporated herein by reference in their entirety. A particularly
useful type of male sterility gene is one which can be induced by
exposure to a chemical agent, for example, a herbicide (U.S. patent
Ser. No. 08/927,368, filed Sep. 11, 1997, the disclosure of which
is specifically incorporated herein by reference in its entirety).
Both inducible and non-inducible male sterility genes can increase
the efficiency with which hybrids are made, in that they eliminate
the need to physically emasculate the corn plant used as a female
in a given cross.
[0161] Where one desires to employ male-sterility systems with a
corn plant in accordance with the invention, it may be beneficial
to also utilize one or more male-fertility restorer genes. For
example, where cytoplasmic male sterility (CMS) is used, hybrid
seed production requires three inbred lines: (1) a cytoplasmically
male-sterile line having a CMS cytoplasm; (2) a fertile inbred with
normal cytoplasm, which is isogenic with the CMS line for nuclear
genes ("maintainer line"), and (3) a distinct, fertile inbred with
normal cytoplasm, carrying a fertility restoring gene ("restorer"
line). The CMS line is propagated by pollination with the
maintainer line, with all of the progeny being male sterile, as the
CMS cytoplasm is derived from the female parent. These male sterile
plants can then be efficiently employed as the female parent in
hybrid crosses with the restorer line, without the need for
physical emasculation of the male reproductive parts of the female
parent.
[0162] The presence of a male-fertility restorer gene results in
the production of fully fertile F.sub.1 hybrid progeny. If no
restorer gene is present in the male parent, male-sterile hybrids
are obtained. Such hybrids are useful where the vegetative tissue
of the corn plant is utilized, e.g., for silage, but in most cases,
the seeds will be deemed the most valuable portion of the crop, so
fertility of the hybrids in these crops must be restored.
Therefore, one aspect of the current invention concerns the inbred
corn plant 94INK1B comprising a single gene capable of restoring
male fertility in an otherwise male-sterile inbred or hybrid plant.
Examples of male-sterility genes and corresponding restorers which
could be employed with the inbred of the invention are well known
to those of skill in the art of plant breeding and are disclosed
in, for instance, U.S. Pat. No. 5,530,191; U.S. Pat. No. 5,689,041;
U.S. Pat. No. 5,741,684; and U.S. Pat. No. 5,684,242, the
disclosures of which are each specifically incorporated herein by
reference in their entirety.
[0163] Direct selection may be applied where a single locus acts as
a dominant trait. An example of a dominant trait is the herbicide
resistance trait. For this selection process, the progeny of the
initial cross are sprayed with the herbicide prior to the
backcrossing. The spraying eliminates any plants which do not have
the desired herbicide resistance characteristic, and only those
plants which have the herbicide resistance gene are used in the
subsequent backcross. This process is then repeated for all
additional backcross generations.
[0164] Many useful single locus traits are those which are
introduced by genetic transformation techniques. Methods for the
genetic transformation of maize are known to those of skill in the
art. For example, methods which have been described for the genetic
transformation of maize include electroporation (U.S. Pat. No.
5,384,253), electrotransformation (U.S. Pat. No. 5,371,003),
microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No.
5,736,369, U.S. Pat. No. 5,538,880; and PCT Publication WO
95/06128), Agrobacterium-mediated transformation (U.S. Pat. No.
5,591,616 and E.P. Publication EP672752), direct DNA uptake
transformation of protoplasts (Omirulleh et al., 1993) and silicon
carbide fiber-mediated transformation (U.S. Pat. No. 5,302,532 and
U.S. Pat. No. 5,464,765).
[0165] A type of single locus trait which can be introduced by
genetic transformation (U.S. Pat. No. 5,554,798) and has particular
utility is a gene which confers resistance to the herbicide
glyphosate. Glyphosate inhibits the action of the enzyme EPSPS,
which is active in the biosynthetic pathway of aromatic amino
acids. Inhibition of this enzyme leads to starvation for the amino
acids phenylalanine, tyrosine, and tryptophan and secondary
metabolites derived therefrom. Mutants of this enzyme are available
which are resistant to glyphosate. For example, U.S. Pat. No.
4,535,060 describes the isolation of EPSPS mutations which confer
glyphosate resistance upon organisms having the Salmonella
typhimurium gene for EPSPS, aroA. A mutant EPSPS gene having
similar mutations has also been cloned from Zea mays. The mutant
gene encodes a protein with amino acid changes at residues 102 and
106 (PCT Publication WO 97/04103). When a plant comprises such a
gene, a herbicide resistant phenotype results.
[0166] Plants having inherited a transgene comprising a mutated
EPSPS gene may, therefore, be directly treated with the herbicide
glyphosate without the result of significant damage to the plant.
This phenotype provides farmers with the benefit of controlling
weed growth in a field of plants having the herbicide resistance
trait by application of the broad spectrum herbicide glyphosate.
For example, one could apply the herbicide ROUNDUP.TM., a
commercial formulation of glyphosate manufactured and sold by the
Monsanto Company, over the top in fields where glyphosate resistant
corn plants are grown. The herbicide application rates may
typically range from 4 ounces of ROUNDUP.TM. to 256 ounces
ROUNDUP.TM. per acre. More preferably, about 16 ounces to about 64
ounces per acre of ROUNDUP.TM. may be applied to the field.
However, the application rate may be increased or decreased as
needed, based on the abundance and/or type of weeds being treated.
Additionally, depending on the location of the field and weather
conditions, which will influence weed growth and the type of weed
infestation, it may be desirable to conduct further glyphosate
treatments. The second glyphosate application will also typically
comprise an application rate of about 16 ounces to about 64 ounces
of ROUNDUP.TM. per acre treated. Again, the treatment rate may be
adjusted based on field conditions. Such methods of application of
herbicides to agricultural crops are well known in the art and are
summarized in general in Anderson (1983).
[0167] Alternatively, more than one single locus trait may be
introgressed into an elite inbred by the method of backcross
conversion. A selectable marker gene and a gene encoding a protein
which confers a trait of interest may be simultaneously introduced
into a maize plant as a result of genetic transformation. Usually
one or more introduced genes will integrate into a single
chromosome site in the host cell's genome. For example, a
selectable marker gene encoding phosphinothricin acetyl transferase
(PPT) (e.g., a bar gene) and conferring resistance to the active
ingredient in some herbicides by inhibiting glutamine synthetase,
and a gene encoding an endotoxin from Bacillus thuringiensis (Bt)
and conferring resistance to particular classes of insects, e.g.,
lepidopteran insects, in particular the European Corn Borer, may be
simultaneously introduced into a host genome. Furthermore, through
the process of backcross conversion more than one transgenic trait
may be transferred into an elite inbred.
[0168] The waxy characteristic is an example of a recessive trait.
In this example, the progeny resulting from the first backcross
generation (BC 1) must be grown and selfed. A test is then run on
the selfed seed from the BC1 plant to determine which BC1 plants
carried the recessive gene for the waxy trait. In other recessive
traits additional progeny testing, for example growing additional
generations such as the BC 1S1, may be required to determine which
plants carry the recessive gene.
[0169] V. Origin and Breeding History of an Exemplary Single Locus
Converted Plant
[0170] 85DGD1 MLms is a single locus conversion of 85DGD1 to
cytoplasmic male sterility. 85DGD1 MLms was derived using backcross
methods. 85DGD1 (a proprietary inbred of DEKALB Genetics
Corporation) was used as the recurrent parent and MLms, a germplasm
source carrying ML cytoplasmic sterility, was used as the
nonrecurrent parent. The breeding history of the single locus
converted inbred 85DGD1 MLms can be summarized as follows:
5 Hawaii Nurseries Planting Date 04-02-1992 Made up S-O: Female row
585 male row 500 Hawaii Nurseries Planting Date 07-15-1992 S-O was
grown and plants were backcrossed times 85DGD1 (rows 444 ' 443)
Hawaii Nurseries Planting Date 11-18-1992 Bulked seed of the BC1
was grown and backcrossed times 85DGD1 (rows V3- 27 ' V3-26) Hawaii
Nurseries Planting Date 04-02-1993 Bulked seed of the BC2 was grown
and backcrossed times 85DGD1 (rows 37 ' 36) Hawaii Nurseries
Planting Date 07-14-1993 Bulked seed of the BC3 was grown and
backcrossed times 85DGD1 (rows 99 ' 98) Hawaii Nurseries Planting
Date 10-28-1993 Bulked seed of BC4 was grown and backcrossed times
85DGD1 (rows KS- 63 ' KS-62) Summer 1994 A single ear of the BC5
was grown and backcrossed times 85DGD1 (MC94-822 ' MC94-822-7)
Winter 1994 Bulked seed of the BC6 was grown and backcrossed times
85DGD1 (3Q-1 ' 3Q- 2) Summer 1995 Seed of the BC7 was bulked and
named 85DGD1 MLms.
[0171] VI. Tissue Cultures and in vitro Regeneration of Corn
Plants
[0172] A further aspect of the invention relates to tissue cultures
of the corn plant designated 94INK1B. As used herein, the term
"tissue culture" indicates a composition comprising isolated cells
of the same or a different type or a collection of such cells
organized into parts of a plant. Exemplary types of tissue cultures
are protoplasts, calli and plant cells that are intact in plants or
parts of plants, such as embryos, pollen, flowers, kernels, ears,
cobs, leaves, husks, stalks, roots, root tips, anthers, silk, and
the like. In a preferred embodiment, the tissue culture comprises
embryos, protoplasts, meristematic cells, pollen, leaves or anthers
derived from immature tissues of these plant parts. Means for
preparing and maintaining plant tissue cultures are well known in
the art (U.S. Pat. No. 5,538,880; and U.S. Pat. No. 5,550,318, each
incorporated herein by reference in their entirety). By way of
example, a tissue culture comprising organs such as tassels or
anthers has been used to produce regenerated plants (U.S. Pat. No.
5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of which are
incorporated herein by reference).
[0173] VII. Tassel/Anther Culture
[0174] Tassels contain anthers which in turn enclose microspores.
Microspores develop into pollen. For anther/microspore culture, if
tassels are the plant composition, they are preferably selected at
a stage when the microspores are uninucleate, that is, include only
one, rather than 2 or 3 nuclei. Methods to determine the correct
stage are well known to those skilled in the art and include
mitramycin fluorescent staining (Pace et al., 1987), trypan blue
(preferred) and acetocarmine squashing. The mid-uninucleate
microspore stage has been found to be the developmental stage most
responsive to the subsequent methods disclosed to ultimately
produce plants.
[0175] Although microspore-containing plant organs such as tassels
can generally be pretreated at any cold temperature below about
25.degree. C., a range of 4 to 25.degree. C. is preferred, and a
range of 8 to 14.degree. C. is particularly preferred. Although
other temperatures yield embryoids and regenerated plants, cold
temperatures produce optimum response rates compared to
pretreatment at temperatures outside the preferred range. Response
rate is measured as either the number of embryoids or the number of
regenerated plants per number of microspores initiated in culture.
Exemplary methods of microspore culture are disclosed in, for
example, U.S. Pat. No. 5,322,789 and U.S. Pat. No 5,445,961, the
disclosures of which are specifically incorporated herein by
reference.
[0176] Although not required, when tassels are employed as the
plant organ, it is generally preferred to sterilize their surface.
Following surface sterilization of the tassels, for example, with a
solution of calcium hypochloride, the anthers are removed from
about 70 to 150 spikelets (small portions of the tassels) and
placed in a preculture or pretreatment medium. Larger or smaller
amounts can be used depending on the number of anthers.
[0177] When one elects to employ tassels directly, tassels are
preferably pretreated at a cold temperature for a predefined time,
preferably at 1 0C for about 4 days. After pretreatment of a whole
tassel at a cold temperature, dissected anthers are further
pretreated in an environment that diverts microspores from their
developmental pathway. The function of the preculture medium is to
switch the developmental program from one of pollen development to
that of embryoid/callus development. An embodiment of such an
environment in the form of a preculture medium includes a sugar
alcohol, for example mannitol or sorbitol, inositol or the like. An
exemplary synergistic combination is the use of mannitol at a
temperature of about 10.degree. C. for a period ranging from about
10 to 14 days. In a preferred embodiment, 3 ml of 0.3 M mannitol
combined with 50 mg/l of ascorbic acid, silver nitrate, and
colchicine is used for incubation of anthers at 10.degree. C. for
between 10 and 14 days. Another embodiment is to substitute
sorbitol for mannitol. The colchicine produces chromosome doubling
at this early stage. The chromosome doubling agent is preferably
only present at the preculture stage.
[0178] It is believed that the mannitol or other similar carbon
structure or environmental stress induces starvation and functions
to force microspores to focus their energies on entering
developmental stages. The cells are unable to use, for example,
mannitol as a carbon source at this stage. It is believed that
these treatments confuse the cells causing them to develop as
embryoids and plants from microspores. Dramatic increases in
development from these haploid cells, as high as 25 embryoids in
104 microspores, have resulted from using these methods.
[0179] In embodiments where microspores are obtained from anthers,
microspores can be released from the anthers into an isolation
medium following the mannitol preculture step. One method of
release is by disruption of the anthers, for example, by chopping
the anthers into pieces with a sharp instrument, such as a razor
blade, scalpel, or Waring blender. The resulting mixture of
released microspores, anther fragments, and isolation medium are
then passed through a filter to separate microspores from anther
wall fragments. An embodiment of a filter is a mesh, more
specifically, a nylon mesh of about 112 mm pore size. The filtrate
which results from filtering the microspore-containing solution is
preferably relatively free of anther fragments, cell walls, and
other debris.
[0180] In a preferred embodiment, isolation of microspores is
accomplished at a temperature below about 25.degree. C. and
preferably, at a temperature of less than about 15.degree. C.
Preferably, the isolation media, dispersing tool (e.g., razor
blade), funnels, centrifuge tubes, and dispersing container (e.g.,
petri dish) are all maintained at the reduced temperature during
isolation. The use of a precooled dispersing tool to isolate maize
microspores has been reported (Gaillard et al., 1991).
[0181] Where appropriate and desired, the anther filtrate is then
washed several times in isolation medium. The purpose of the
washing and centrifugation is to eliminate any toxic compounds
which are contained in the non-microspore part of the filtrate and
are created by the chopping process. The centrifugation is usually
done at decreasing spin speeds, for example, 1000, 750, and finally
500 rpms. The result of the foregoing steps is the preparation of a
relatively pure tissue culture suspension of microspores that are
relatively free of debris and anther remnants.
[0182] To isolate microspores, an isolation media is preferred. An
isolation media is used to separate microspores from the anther
walls while maintaining their viability and embryogenic potential.
An illustrative embodiment of an isolation media includes a 6%
sucrose or maltose solution combined with an antioxidant such as 50
mg/l of ascorbic acid, 0.1 mg/l biotin, and 400 mg/l of proline,
combined with 10 mg/l of nicotinic acid and 0.5 mg/l AgNO.sub.3. In
another embodiment, the biotin and proline are omitted.
[0183] An isolation media preferably has a higher antioxidant level
where it is used to isolate microspores from a donor plant (a plant
from which a plant composition containing a microspore is obtained)
that is field grown in contrast to greenhouse grown. A preferred
level of ascorbic acid in an isolation medium is from about 50 mg/l
to about 125 mg/l and, more preferably, from about 50 mg/l to about
100 mg/l.
[0184] One can find particular benefit in employing a support for
the microspores during culturing and subculturing. Any support that
maintains the cells near the surface can be used. The microspore
suspension is layered onto a support, for example by pipetting.
There are several types of supports which are suitable and are
within the scope of the invention. An illustrative embodiment of a
solid support is a TRANSWELL.RTM. culture dish. Another embodiment
of a solid support for development of the microspores is a bilayer
plate wherein liquid media is on top of a solid base. Other
embodiments include a mesh or a millipore filter. Preferably, a
solid support is a nylon mesh in the shape of a raft. A raft is
defined as an approximately circular support material which is
capable of floating slightly above the bottom of a tissue culture
vessel, for example, a petri dish, of about a 60 or 100 mm size,
although any other laboratory tissue culture vessel will suffice.
In an illustrative embodiment, a raft is about 55 mm in
diameter.
[0185] Culturing isolated microspores on a solid support, for
example, on a 10 mm pore nylon raft floating on 2.2 ml of medium in
a 60 mm petri dish, prevents microspores from sinking into the
liquid medium and thus avoiding low oxygen tension. These types of
cell supports enable the serial transfer of the nylon raft with its
associated microspore/embryoids ultimately to full strength medium
containing activated charcoal and solidified with, for example,
GELRITE.TM. (solidifying agent). The charcoal is believed to absorb
toxic wastes and intermediaries. The solid medium allows embryoids
to mature.
[0186] The liquid medium passes through the mesh while the
microspores are retained and supported at the medium-air interface.
The surface tension of the liquid medium in the petri dish causes
the raft to float. The liquid is able to pass through the mesh;
consequently, the microspores stay on top. The mesh remains on top
of the total volume of liquid medium. An advantage of the raft is
to permit diffusion of nutrients to the microspores. Use of a raft
also permits transfer of the microspores from dish to dish during
subsequent subculture with minimal loss, disruption, or disturbance
of the induced embryoids that are developing. The rafts represent
an advantage over the multi-welled TRANSWELL.RTM. plates, which are
commercially available from COSTAR, in that the commercial plates
are expensive. Another disadvantage of these plates is that to
achieve the serial transfer of microspores to subsequent media, the
membrane support with cells must be peeled off the insert in the
wells. This procedure does not produce as good a yield nor as
efficient transfers, as when a mesh is used as a vehicle for cell
transfer.
[0187] The culture vessels can be further defined as either (1) a
bilayer 60 mm petri plate wherein the bottom 2 ml of medium are
solidified with 0.7% agarose overlaid with 1 mm of liquid
containing the microspores; (2) a nylon mesh raft wherein a wafer
of nylon is floated on 1.2 ml of medium and 1 ml of isolated
microspores is pipetted on top; or (3) TRANSWELL.RTM. plates
wherein isolated microspores are pipetted onto membrane inserts
which support the microspores at the surface of 2 ml of medium.
[0188] After the microspores have been isolated, they are cultured
in a low strength anther culture medium until about the 50 cell
stage when they are subcultured onto an embryoid/callus maturation
medium. Medium is defined at this stage as any combination of
nutrients that permit the microspores to develop into embryoids or
callus. Many examples of suitable embryoid/callus promoting media
are well known to those skilled in the art. These media will
typically comprise mineral salts, a carbon source, vitamins, and
growth regulators. A solidifying agent is optional. A preferred
embodiment of such a media is referred to as "D medium," which
typically includes 6N1 salts, AgNO.sub.3 and sucrose or
maltose.
[0189] In an illustrative embodiment, 1 ml of isolated microspores
are pipetted onto a 10 mm nylon raft and the raft is floated on 1.2
ml of medium "D," containing sucrose or preferably maltose. Both
calli and embryoids can develop. Calli are undifferentiated
aggregates of cells. Type I is a relatively compact, organized, and
slow growing callus. Type II is a soft, friable, and fast-growing
one. Embryoids are aggregates exhibiting some embryo-like
structures. The embryoids are preferred for subsequent steps to
regenerating plants. Culture medium "D" is an embodiment of medium
that follows the isolation medium and replaces it. Medium "D"
promotes growth to an embryoid/callus. This medium comprises 6N1
salts at 1/8 the strength of a basic stock solution (major
components) and minor components, plus 12% sucrose, or preferably
12% maltose, 0.1 mg/l B1, 0.5 mg/l nicotinic acid, 400 mg/l proline
and 0.5 mg/l silver nitrate. Silver nitrate is believed to act as
an inhibitor to the action of ethylene. Multi-cellular structures
of approximately 50 cells each generally arise during a period of
12 days to 3 weeks. Serial transfer after a two week incubation
period is preferred.
[0190] After the petri dish has been incubated for an appropriate
period of time, preferably two weeks in the dark at a predefined
temperature, a raft bearing the dividing microspores is transferred
serially to solid based media which promote embryo maturation. In
an illustrative embodiment, the incubation temperature is
30.degree. C. and the mesh raft supporting the embryoids is
transferred to a 100 mm petri dish containing the 6N1-TGR-4P
medium, an "anther culture medium." This medium contains 6N1 salts,
supplemented with 0.1 mg/l TIBA, 12% sugar (sucrose, maltose, or a
combination thereof), 0.5% activated charcoal, 400 mg/l proline,
0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2 percent GELRITE.TM.
(solidifying agent) and is capable of promoting the maturation of
the embryoids. Higher quality embryoids, that is, embryoids which
exhibit more organized development, such as better shoot meristem
formation without precocious germination, were typically obtained
with the transfer to full strength medium compared to those
resulting from continuous culture using only, for example, the
isolated microspore culture (IMC) Medium "D." The maturation
process permits the pollen embryoids to develop further in route
toward the eventual regeneration of plants. Serial transfer occurs
to full strength solidified 6N1 medium using either the nylon raft,
the TRANSWELL.RTM. membrane, or bilayer plates, each one requiring
the movement of developing embryoids to permit further development
into physiologically more mature structures. In an especially
preferred embodiment, microspores are isolated in an isolation
media comprising about 6% maltose, cultured for about two weeks in
an embryoid/calli induction medium comprising about 12% maltose and
then transferred to a solid medium comprising about 12%
sucrose.
[0191] At the point of transfer of the raft, after about two weeks
of incubation, embryoids exist on a nylon support. The purpose of
transferring the raft with the embryoids to a solidified medium
after the incubation is to facilitate embryo maturation. Mature
embryoids at this point are selected by visual inspection indicated
by zygotic embryo-like dimensions and structures and are
transferred to the shoot initiation medium. It is preferred that
shoots develop before roots, or that shoots and roots develop
concurrently. If roots develop before shoots, plant regeneration
can be impaired. To produce solidified media, the bottom of a petri
dish of approximately 100 mm is covered with about 30 ml of 0.2%
GELRITE.TM. solidified medium. A sequence of regeneration media are
used for whole plant formation from the embryoids.
[0192] During the regeneration process, individual embryoids are
induced to form plantlets. The number of different media in the
sequence can vary depending on the specific protocol used. Finally,
a rooting medium is used as a prelude to transplanting to soil.
When plantlets reach a height of about 5 cm, they are then
transferred to pots for further growth into flowering plants in a
greenhouse by methods well known to those skilled in the art.
[0193] Plants have been produced from isolated microspore cultures
by the methods disclosed herein, including self-pollinated plants.
The rate of embryoid induction was much higher with the synergistic
preculture treatment consisting of a combination of stress factors,
including a carbon source which can be capable of inducing
starvation, a cold temperature, and colchicine, than has previously
been reported. An illustrative embodiment of the synergistic
combination of treatments leading to the dramatically improved
response rate compared to prior methods, is a temperature of about
10C, mannitol as a carbon source, and 0.05% colchicine.
[0194] The inclusion of ascorbic acid, an anti-oxidant, in the
isolation medium is preferred for maintaining good microspore
viability. However, there seems to be no advantage to including
mineral salts in the isolation medium. The osmotic potential of the
isolation medium was maintained optimally with about 6% sucrose,
although a range of 2% to 12% is within the scope of this
invention.
[0195] In an embodiment of the embryoid/callus organizing media,
mineral salts concentration in IMC Culture Media "D" is
(1/8.times.), the concentration which is used also in anther
culture medium. The 6N1 salts major components have been modified
to remove ammonium nitrogen. Osmotic potential in the culture
medium is maintained with about 12% sucrose and about 400 mg/l
proline. Silver nitrate (0.5 mg/l) was included in the medium to
modify ethylene activity. The preculture media is further
characterized by having a pH of about 5.7 to 6 0. Silver nitrate
and vitamins do not appear to be crucial to this medium but do
improve the efficiency of the response.
[0196] Whole anther cultures can also be used in the production of
monocotyledonous plants from a plant culture system. There are some
basic similarities of anther culture methods and microspore culture
methods with regard to the media used. A difference from isolated
microspore cultures is that undisrupted anthers are cultured, so
that a support, e.g., a nylon mesh support, is not needed. The
first step in developing the anther cultures is to incubate tassels
at a cold temperature. A cold temperature is defined as less than
about 25.degree. C. More specifically, the incubation of the
tassels is preferably performed at about 10.degree. C. A range of 8
to 14.degree. C. is also within the scope of the invention. The
anthers are then dissected from the tassels, preferably after
surface sterilization using forceps, and placed on solidified
medium. An example of such a medium is designated 6N1 -TGR-P4.
[0197] The anthers are then treated with environmental conditions
that are combinations of stresses that are capable of diverting
microspores from gametogenesis to embryogenesis. It is believed
that the stress effect of sugar alcohols in the preculture medium,
for example, mannitol, is produced by inducing starvation at the
predefined temperature. In one embodiment, the incubation
pretreatment is for about 14 days at 10.degree. C. It was found
that treating the anthers in addition with a carbon structure, an
illustrative embodiment being a sugar alcohol, preferably mannitol,
produces dramatically higher anther culture response rates as
measured by the number of eventually regenerated plants, than by
treatment with either cold treatment or mannitol alone. These
results are particularly surprising in light of teachings that cold
is better than mannitol for these purposes, and that warmer
temperatures interact with mannitol better.
[0198] To incubate the anthers, they are floated on a preculture
medium which diverts the microspores from gametogenesis, preferably
on a mannitol carbon structure, more specifically, 0.3 M of
mannitol plus 50 mg/l of ascorbic acid. Three milliliters is about
the total amount in a dish, for example, a tissue culture dish,
more specifically, a 60 mm petri dish. Anthers are isolated from
about 120 spikelets for one dish yields about 360 anthers.
[0199] Chromosome doubling agents can be used in the preculture
media for anther cultures. Several techniques for doubling
chromosome number (Jensen, 1974; Wan et al., 1989) have been
described. Colchicine is one of the doubling agents. However,
developmental abnormalities arising from in vitro cloning are
further enhanced by colchicine treatments, and previous reports
indicated that colchicine is toxic to microspores. The addition of
colchicine in increasing concentrations during mannitol
pretreatment prior to anther culture and microspore culture has
achieved improved percentages.
[0200] An illustrative embodiment of the combination of a
chromosome doubling agent and preculture medium is one which
contains colchicine. In a specific embodiment, the colchicine level
is preferably about 0.05%. The anthers remain in the mannitol
preculture medium with the additives for about 10 days at
10.degree. C. Anthers are then placed on maturation media, for
example, that designated 6N1-TGR-P4, for 3 to 6 weeks to induce
embryoids. If the plants are to be regenerated from the embryoids,
shoot regeneration medium is employed, as in the isolated
microspore procedure described in the previous sections. Other
regeneration media can be used sequentially to complete
regeneration of whole plants.
[0201] The anthers are then exposed to embryoid/callus promoting
medium, for example, that designated 6N1-TGR-P4, to obtain callus
or embryoids. The embryoids are recognized visually by
identification of embryonic-like structures. At this stage, the
embryoids are transferred progressively through a series of
regeneration media. In an illustrative embodiment, the shoot
initiation medium comprises BAP (6-benzyl-amino-purine) and NAA
(naphthalene acetic acid). Regeneration protocols for isolated
microspore cultures and anther cultures are similar.
[0202] VIII. Additional Tissue Cultures and Regeneration
[0203] The present invention contemplates a corn plant regenerated
from a tissue culture of the inbred maize plant 94INK1B, or of a
hybrid maize plant produced by crossing 94INK1B. As is well known
in the art, tissue culture of corn can be used for the in vitro
regeneration of a corn plant. By way of example, a process of
tissue culturing and regeneration of corn is described in European
Patent Application 0 160 390, the disclosure of which is
incorporated herein by reference. Corn tissue culture procedures
are also described in Green and Rhodes (1982) and Duncan et al.
(1985). The study by Duncan et aL (1985) indicates that 97 percent
of cultured plants produced calli capable of regenerating plants.
Subsequent studies have shown that both inbreds and hybrids
produced 91% regenerable calli that produced plants.
[0204] Other studies indicate that non-traditional tissues are
capable of producing somatic embryogenesis and plant regeneration
(Songstad et al., 1988; Rao et al., 1986; Conger et al., 1987; the
disclosures of which are incorporated herein by reference).
Regenerable cultures, including Type I and Type II cultures, may be
initiated from immature embryos using methods described in, for
example, PCT Application WO 95/06128, the disclosure of which is
incorporated herein by reference in its entirety.
[0205] Briefly, by way of example, to regenerate a plant of this
invention, cells are selected following growth in culture. Where
employed, cultured cells are preferably grown either on solid
supports or in the form of liquid suspensions as set forth above.
In either instance, nutrients are provided to the cells in the form
of media, and environmental conditions are controlled. There are
many types of tissue culture media comprising amino acids, salts,
sugars, hormones, and vitamins. Most of the media employed to
regenerate inbred and hybrid plants have some similar components;
the media differ in the composition and proportions of their
ingredients depending on the particular application envisioned. For
example, various cell types usually grow in more than one type of
media, but exhibit different growth rates and different
morphologies, depending on the growth media. In some media, cells
survive but do not divide. Various types of media suitable for
culture of plant cells have been previously described and discussed
above.
[0206] An exemplary embodiment for culturing recipient corn cells
in suspension cultures includes using embryogenic cells in Type II
(Armstrong and Green, 1985; Gordon-Kamm et al., 1990) callus,
selecting for small (10 to 30 mm) isodiametric, cytoplasmically
dense cells, growing the cells in suspension cultures with hormone
containing media, subculturing into a progression of media to
facilitate development of shoots and roots, and finally, hardening
the plant and readying it metabolically for growth in soil.
[0207] Meristematic cells (i.e., plant cells capable of continual
cell division and characterized by an undifferentiated cytological
appearance, normally found at growing points or tissues in plants
such as root tips, stem apices, lateral buds, etc.) can be cultured
(U.S. Pat. No. 5,736,369, the disclosure of which is specifically
incorporated herein by reference).
[0208] Embryogenic calli are produced essentially as described in
PCT Application WO 95/06128. Specifically, inbred plants or plants
from hybrids produced from crossing an inbred of the present
invention with another inbred are grown to flowering in a
greenhouse. Explants from at least one of the following F.sub.1
tissues: the immature tassel tissue, intercalary meristems and leaf
bases, apical meristems, immature ears and immature embryos are
placed in an initiation medium which contain MS salts, supplemented
with thiamine, agar, and sucrose. Cultures are incubated in the
dark at about 23.degree. C. All culture manipulations and
selections are performed with the aid of a dissecting
microscope.
[0209] After about 5 to 7 days, cellular outgrowths are observed
from the surface of the explants. After about 7 to 21 days, the
outgrowths are subcultured by placing them into fresh medium of the
same composition. Some of the intact immature embryo explants are
placed on fresh medium. Several subcultures later (after about 2 to
3 months) enough material is present from explants for subdivision
of these embryogenic calli into two or more pieces.
[0210] Callus pieces from different explants are not mixed. After
further growth and subculture (about 6 months after embryogenic
callus initiation), there are usually between 1 and 100 pieces
derived ultimately from each selected explant. During this time of
culture expansion, a characteristic embryogenic culture morphology
develops as a result of careful selection at each subculture. Any
organized structures resembling roots or root primordia are
discarded. Material known from experience to lack the capacity for
sustained growth is also discarded (translucent, watery,
embryogenic structures). Structures with a firm consistency
resembling at least in part the scutelum of the in vivo embryo are
selected.
[0211] The callus is maintained on agar-solidified MS or N6-type
media. A preferred hormone is 2,4-D. A second preferred hormone is
dicamba. Visual selection of embryo-like structures is done to
obtain subcultures. Transfer of material other than that displaying
embryogenic morphology results in loss of the ability to recover
whole plants from the callus.
[0212] Cell suspensions are prepared from the calli by selecting
cell populations that appear homogeneous macroscopically. A portion
of the friable, rapidly growing embryogenic calli is inoculated
into MS or N6 Medium containing 2,4-D or dicamba. The calli in
medium are incubated at about 27.degree. C. on a gyrotary shaker in
the dark or in the presence of low light. The resultant suspension
culture is transferred about once every three to seven days,
preferably every three to four days, by taking about 5 to 10 ml of
the culture and introducing this inoculum into fresh medium of the
composition listed above (PCT Application WO 95/06128).
[0213] For regeneration of type I or type II callus, callus is
transferred to a solidified culture medium which includes a lower
concentration of 2,4-D or other auxins than is present in culture
medium used for callus maintenance (PCT Application WO 95/06128,
specifically incorporated herein by reference). Other hormones
which can be used in regeneration media include dicamba, NAA, ABA,
BAP, and 2-NCA. Regeneration of plants is completed by the transfer
of mature and germinating embryos to a hormone-free medium,
followed by the transfer of developed plantlets to soil and growth
to maturity. Plant regeneration is described in PCT Application WO
95/06128.
[0214] Cells from the meristem or cells fated to contribute to the
meristem of a cereal plant embryo at the early proembryo, mid
proembryo, late proembryo, transitional or early coleoptilar stage
may be cultured so as to produce a proliferation of shoots or
multiple meristems from which fertile plants may be regenerated.
Alternatively, cells from the meristem or cells fated to contribute
to the meristem of a cereal plant immature ear or tassel may be
cultured so as to produce a proliferation of shoots or multiple
meristems from which fertile plants may be regenerated (U.S. Pat.
No. 5,736,369).
[0215] Progeny of any generation are produced by taking pollen and
selfing, backcrossing, or sibling crossing regenerated plants by
methods well known to those skilled in the arts. Seeds are
collected from the regenerated plants. Alternatively, progeny of
any generation may be produced by pollinating a regenerated plant
with its own pollen or pollen of a second maize plant. Using the
methods described herein, tissue cultures and immature or mature
plant tissues may be used as recipient cell cultures for the
process of genetic transformation.
[0216] IX. Processes of Preparing Corn Plants and the Corn Plants
Produced by Such Crosses
[0217] The present invention also provides a process of preparing a
novel corn plant and a corn plant produced by such a process. In
accordance with such a process, a first parent corn plant is
crossed with a second parent corn plant wherein at least one of the
first and second corn plants is the inbred corn plant 94INK1B. An
important aspect of this process is that it can be used for the
development of novel inbred lines. For example, the inbred corn
plant 94INK1B could be crossed to any second plant, and the
resulting hybrid progeny each selfed for about 5 to 7 or more
generations, thereby providing a large number of distinct,
pure-breeding inbred lines. These inbred lines could then be
crossed with other inbred or non-inbred lines and the resulting
hybrid progeny analyzed for beneficial characteristics. In this
way, novel inbred lines conferring desirable characteristics could
be identified.
[0218] In selecting a second plant to cross with 94INK1B for the
purpose of developing novel inbred lines, it will typically be
desired choose those plants which either themselves exhibit one or
more selected desirable characteristics or which exhibit the
desired characteristic(s) when in hybrid combination. Examples of
potentially desired characteristics include greater yield, better
stalks, better roots, resistance to insecticides, herbicides,
pests, and disease, tolerance to heat and drought, reduced time to
crop maturity, better agronomic quality, higher nutritional value,
and uniformity in germination times, stand establishment, growth
rate, maturity, and fruit size. Alternatively, the inbred 94INK1B
may be crossed with a second, different inbred plant for the
purpose of producing hybrid seed which is sold to farmers for
planting in commercial production fields. In this case, a second
inbred variety is selected which confers desirable characteristics
when in hybrid combination with the first inbred line.
[0219] Corn plants (Zea mays L.) can be crossed by either natural
or mechanical techniques. Natural pollination occurs in corn when
wind blows pollen from the tassels to the silks that protrude from
the tops of the recipient ears. Mechanical pollination can be
effected either by controlling the types of pollen that can blow
onto the silks or by pollinating by hand.
[0220] In a preferred embodiment, crossing comprises the steps
of:
[0221] (a) planting in pollinating proximity seeds of a first and a
second parent corn plant, and preferably, seeds of a first inbred
corn plant and a second, distinct inbred corn plant;
[0222] (b) cultivating or growing the seeds of the first and second
parent corn plants into plants that bear flowers;
[0223] (c) emasculating flowers of either the first or second
parent corn plant, i.e., treating the flowers so as to prevent
pollen production, or alternatively, using as the female parent a
male sterile plant, thereby providing an emasculated parent corn
plant;
[0224] (d) allowing natural cross-pollination to occur between the
first and second parent corn plants;
[0225] (e) harvesting seeds produced on the emasculated parent corn
plant; and, where desired,
[0226] (f) growing the harvested seed into a corn plant,
preferably, a hybrid corn plant.
[0227] Parental plants are typically planted in pollinating
proximity to each other by planting the parental plants in
alternating rows, in blocks or in any other convenient planting
pattern. Where the parental plants differ in timing of sexual
maturity, it may be desired to plant the slower maturing plant
first, thereby ensuring the availability of pollen from the male
parent during the time at which silks on the female parent are
receptive to pollen. Plants of both parental parents are cultivated
and allowed to grow until the time of flowering. Advantageously,
during this growth stage, plants are in general treated with
fertilizer and/or other agricultural chemicals as considered
appropriate by the grower.
[0228] At the time of flowering, in the event that plant 94INK1B is
employed as the male parent, the tassels of the other parental
plant are removed from all plants employed as the female parental
plant to avoid self-pollination. The detasseling can be achieved
manually but also can be done by machine, if desired.
Alternatively, when the female parent corn plant comprises a
cytoplasmic or nuclear gene conferring male sterility, detasseling
may not be required. Additionally, a chemical gametocide may be
used to sterilize the male flowers of the female plant. In this
case, the parent plants used as the male may either not be treated
with the chemical agent or may comprise a genetic factor which
causes resistance to the emasculating effects of the chemical
agent. Gametocides affect processes or cells involved in the
development, maturation or release of pollen. Plants treated with
such gametocides are rendered male sterile, but typically remain
female fertile. The use of chemical gametocides is described, for
example, in U.S. Pat. No. 4,936,904, the disclosure of which is
specifically incorporated herein by reference in its entirety.
Furthermore, the use of Roundup herbicide in combination with
glyphosate tolerant maize plants to produce male sterile corn
plants is disclosed in U.S. patent application Ser. No. 08/927,368
and PCT Publication WO 98/44140.
[0229] Following emasculation, the plants are then typically
allowed to continue to grow and natural cross-pollination occurs as
a result of the action of wind, which is normal in the pollination
of grasses, including corn. As a result of the emasculation of the
female parent plant, all the pollen from the male parent plant is
available for pollination because tassels, and thereby pollen
bearing flowering parts, have been previously removed from all
plants of the inbred plant being used as the female in the
hybridization. Of course, during this hybridization procedure, the
parental varieties are grown such that they are isolated from other
corn fields to minimize or prevent any accidental contamination of
pollen from foreign sources. These isolation techniques are well
within the skill of those skilled in this art.
[0230] Both parental inbred plants of corn may be allowed to
continue to grow until maturity or the male rows may be destroyed
after flowering is complete. Only the ears from the female inbred
parental plants are harvested to obtain seeds of a novel F.sub.1
hybrid. The novel F.sub.1 hybrid seed produced can then be planted
in a subsequent growing season in commercial fields or,
alternatively, advanced in breeding protocols for purposes of
developing novel inbred lines.
[0231] Alternatively, in another embodiment of the invention, both
first and second parent corn plants can come from the same inbred
corn plant, i.e., from the inbred designated 94INK1B. Thus, any
corn plant produced using a process of the present invention and
inbred corn plant 94NK1B, is contemplated by the current inventor.
As used herein, crossing can mean selfing, backcrossing, crossing
to another or the same inbred, crossing to populations, and the
like. All corn plants produced using the inbred corn plant 94INK1B
as a parent are, therefore, within the scope of this invention.
[0232] The utility of the inbred plant 94INK1B also extends to
crosses with other species. Commonly, suitable species will be of
the family Graminaceae, and especially of the genera Zea,
Tripsacum, Coix, Schlerachne, Polytoca, Chionachne, and
Trilobachne, of the tribe Maydeae. Of these, Zea and Tripsacum, are
most preferred. Potentially suitable for crosses with 94INK1B can
also be the various varieties of grain sorghum, Sorghum bicolor
(L.) Moench.
[0233] A. F.sub.1 Hybrid Corn Plant and Seed Production
[0234] Any time the inbred corn plant 94INK1B is crossed with
another, different, corn inbred, a first generation (F.sub.1) corn
hybrid plant is produced. As such, an F.sub.1 hybrid corn plant may
be produced by crossing 94INK1B with any second inbred maize plant.
Therefore, any F.sub.1 hybrid corn plant or corn seed which is
produced with 94INK1B as a parent is part of the present invention.
An example of such an F.sub.1 hybrid which has been produced with
94INK1B as a parent is the hybrid 8002161. Hybrid 8002161 was
produced by crossing inbred corn plant 94INK1B with the inbred corn
plant designated 3AZA1 (U.S. Pat. No. 5,910,625, the disclosure of
which is specifically incorporated herein by reference in its
entirety).
[0235] The goal of the process of producing an F.sub.1 hybrid is to
manipulate the genetic complement of corn to generate new
combinations of genes which interact to yield new or improved
traits (phenotypic characteristics). A process of producing an
F.sub.1 hybrid typically begins with the production of one or more
inbred plants. Those plants are produced by repeated crossing of
ancestrally related corn plants to try to combine certain genes
within the inbred plants.
[0236] Corn has a diploid phase which means two conditions of a
gene (two alleles) occupy each locus (position on a chromosome). If
the alleles are the same at a locus, there is said to be
homozygosity. If they are different, there is said to be
heterozygosity. In a completely inbred plant, all loci are
homozygous. Because many loci when homozygous are deleterious to
the plant, in particular leading to reduced vigor, less kernels,
weak and/or poor growth, production of inbred plants is an
unpredictable and arduous process. Under some conditions,
heterozygous advantage at some loci effectively bars perpetuation
of homozygosity.
[0237] Inbreeding requires sophisticated manipulation by human
breeders. Even in the extremely unlikely event inbreeding rather
than crossbreeding occurred in natural corn, achievement of
complete inbreeding cannot be expected in nature due to well known
deleterious effects of homozygosity and the large number of
generations the plant would have to breed in isolation. The reason
for the breeder to create inbred plants is to have a known
reservoir of genes whose gametic transmission is predictable.
[0238] The development of inbred plants generally requires at least
about 5 to 7 generations of selfing. Inbred plants are then
cross-bred in an attempt to develop improved F.sub.1 hybrids.
Hybrids are then screened and evaluated in small scale field
trials. Typically, about 10 to 15 phenotypic traits, selected for
their potential commercial value, are measured. A selection index
of the most commercially important traits is used to help evaluate
hybrids. FACT, an acronym for Field Analysis Comparison Trial
(strip trials), is an on-farm experimental testing program employed
by DEKALB Genetics Corporation to perform the final evaluation of
the commercial potential of a product.
[0239] During the next several years, a progressive elimination of
hybrids occurs based on more detailed evaluation of their
phenotype. Eventually, strip trials (FACT) are conducted to
formally compare the experimental hybrids being developed with
other hybrids, some of which were previously developed and
generally are commercially successful. That is, comparisons of
experimental hybrids are made to competitive hybrids to determine
if there was any advantage to further development of the
experimental hybrids. Examples of such comparisons are presented
hereinbelow. After FACT testing is complete, determinations may be
made whether commercial development should proceed for a given
hybrid.
[0240] When the inbred corn plant 94INK1B is crossed with another
inbred plant to yield a hybrid, the original inbred can serve as
either the maternal or paternal plant. For many crosses, the
outcome is the same regardless of the assigned sex of the parental
plants.
[0241] However, there is often one of the parental plants that is
preferred as the maternal plant because of increased seed yield and
production characteristics. Some plants produce tighter ear husks
leading to more loss, for example due to rot. There can be delays
in silk formation which deleteriously affect timing of the
reproductive cycle for a pair of parental inbreds. Seed coat
characteristics can be preferable in one plant. Pollen can be shed
better by one plant. Other variables can also affect preferred
sexual assignment of a particular cross. In the case of the instant
inbred, it was generally preferable to use 94INK1B as the male
parent.
[0242] B. F.sub.1 Hybrid Comparisons
[0243]
[0244] As mentioned above, hybrids are progressively eliminated
following detailed evaluations of their phenotype, including formal
comparisons with other commercially successful hybrids. Strip
trials are used to compare the phenotypes of hybrids grown in as
many environments as possible. They are performed in many
environments to assess overall performance of the new hybrids and
to select optimum growing conditions. Because the corn is grown in
close proximity, environmental factors that affect gene expression,
such as moisture, temperature, sunlight, and pests, are minimized.
For a decision to be made to commercialize a hybrid, it is not
necessary that the hybrid be better than all other hybrids. Rather,
significant improvements must be shown in at least some traits that
would create improvements in some niches.
[0245] Examples of such comparative data are set forth hereinbelow
in Table 4, which presents a comparison of performance data for the
hybrid 8002161, a hybrid made with 94INK1B as one parent, versus
selected hybrids of commercial value.
[0246] All the data in Table 4 represents results across years and
locations for research and/or strip trials. The "NTEST" represents
the number of paired observations in designated tests at locations
around the United States.
6TABLE 4 Comparative Data of 8002161 SI YLD MST STL RTL DRP FLSTD
SV ELSTD PHT EHT BAR SG TST ESTR HYBRID NTEST % C. BU PTS % % % % M
RAT % M INCH INCH % RAT LBS FGDU DAYS 8002161 F 94 105.8 172.2 19.5
1.6 2.0 0.0 97.5 4.6 93.6 43.4 4.2 56.2 1410 116.6 DK679 97.4 165.3
19.6 3.7 5.3 0.1 97.6 4.5 100.3 51.0 4.8 57.8 1475 116.6 DIFF 8.4
6.8 -0.1 -2.1 -3.3 -0.1 -0.1 0.1 -6.6 -7.6 -0.6 -1.6 65 0.0 SIG **
** ** .dagger. ** ** ** .dagger. 8002161 R 126 100.0 180.2 21.1 6.4
4.7 0.0 65.2 57.7 DK679 100.0 170.6 21.3 9.5 11.4 0.7 64.3 59.5
DIFF 0.0 9.7 -0.2 -3.1 -6.7 -0.7 0.9 -1.9 SIG ** * ** * ** 8002161
F 70 107.1 173.6 19.8 1.7 3.2 0.0 98.3 4.6 93.6 43.4 4.1 55.8 1410
116.3 DK668 95.7 163.5 19.9 4.0 6.6 0.0 100.1 4.3 96.4 50.5 4.7
56.2 1475 116.6 DIFF 11.4 10.1 -0.1 -2.3 -3.5 0.0 -1.8 0.4 -2.7
-7.1 -0.6 -0.4 -65 0.3 SIG ** ** .dagger. * .dagger. ** .dagger.
.dagger. 8002161 R 140 100.0 183.9 21.0 6.1 4.9 0.0 65.2 57.8 DK668
100.0 165.6 20.6 7.0 7.9 0.1 64.2 58.4 DIFF 0.0 18.3 0.4 -1.0 -3.0
-0.1 1.1 -0.6 SIG ** ** ** Significance levels are indicated as:
.dagger. = 10%, * = 5%, ** = 1% LEGEND ABBREVIATIONS: HYBD = Hybrid
NTEST = Research/FACT SI % C. = Selection Index (percent of check)
YLD BU/A = Yield (bushels/acre) MST PTS = Moisture STL % = Stalk
Lodging (percent) RTL % = Root Lodging (percent) DRP % = Dropped
Ears (percent) FLSTD % M = Final Stand (percent of test mean) SV
RAT = Seedling Vigor Rating ELSTD % M = Early Stand (percent of
test mean) PHT INCH = Plant Height (inches) EHT INCH = Ear Height
(inches) BAR % = Barren Plants (percent) SG RAT = Staygreen Rating
TST LBS = Test Weight (pounds) FGDU = GDUs to Shed ESTR DAYS =
Estimated Relative Maturity (days)
[0247] C. Physical Description of F.sub.1 Hybrids
[0248] The present invention provides F.sub.1 hybrid corn plants
derived from the corn plant 94INKIB. The physical characteristics
of an exemplary hybrid produced using 94INK1B as one inbred parent
are set forth in Table 5, which concerns 8002161. An explanation of
terms used in Table 5 can be found in the Definitions, set forth
hereinabove.
7TABLE 5 Morphological Traits for the 8002161 Phenotype
CHARACTERISTIC VALUE 1. STALK Diameter (width) cm. 2.0 Anthocyanin
Absent Nodes With Brace Roots 1.1 Brace Root Color Faint Internode
Direction Straight Internode Length cm. 15.9 2. LEAF Color Green
Length cm. 80.3 Width cm. 9.6 Sheath Anthocyanin Absent Sheath
Pubescence Moderate Marginal Waves Moderate Longitudinal Creases
Few 3. TASSEL Length cm. 46.6 Spike Length cm. 25.3 Peduncle Length
cm. 9.3 Branch Number 6.7 Anther Color Green-Yellow Glume Color
Green Glume Band Absent 4. EAR Silk Color Mix Number Per Stalk 1.0
Position (attitude) Pendant Length cm. 17.5 Shape Semi-Conical
Diameter cm. 4.1 Weight gm. 160.2 Shank Length cm. 17.0 Husk Bract
Short Husk Cover cm. 0.7 Husk Opening Very Loose Husk Color Fresh
Green Husk Color Dry Buff Cob Diameter cm. 1.9 Cob Color Red
Shelling Percent 88.0 5 KERNEL Row Number 13.6 Number Per Row 38.0
Row Direction Straight Type Dent Cap Color Yellow Side Color Orange
Length (depth) mm. 11.4 Width mm. 7.7 Thickness 4.5 Weight of 1000K
gm. 363.5 Endosperm Type Normal Endosperm Color Yellow * These are
typical values. Values may vary due to environment. Other values
that are substantially equivalent are also within the scope of the
invention. Substantially equivalent refers to quantitative traits
that when compared do not show statistical differences of their
means.
[0249] X. Genetic Complements
[0250] The present invention provides a genetic complement of the
inbred corn plant designated 94INK1B. Further provided by the
invention is a hybrid genetic complement, wherein the complement is
formed by the combination of a haploid genetic complement from
94INK1B and another haploid genetic complement. Means for
determining such a genetic complement are well-known in the
art.
[0251] As used herein, the phrase "genetic complement" means an
aggregate of nucleotide sequences, the expression of which defines
the phenotype of a corn plant or a cell or tissue of that plant. By
way of example, a corn plant is genotyped to determine a
representative sample of the inherited markers it possesses.
Markers are alleles at a single locus. They are preferably
inherited in codominant fashion so that the presence of both
alleles at a diploid locus is readily detectable, and they are free
of environmental variation, i.e., their heritability is 1. This
genotyping is preferably performed on at least one generation of
the descendant plant for which the numerical value of the
quantitative trait or traits of interest are also determined. The
array of single locus genotypes is expressed as a profile of marker
alleles, two at each locus. The marker allelic composition of each
locus can be either homozygous or heterozygous. Homozygosity is a
condition where both alleles at a locus are characterized by the
same nucleotide sequence or size of a repeated sequence.
Heterozygosity refers to different conditions of the gene at a
locus. A preferred type of genetic marker for use with the
invention is simple sequence repeats (SSRs), although potentially
any other type of genetic marker could be used, for example,
restriction fragment length polymorphisms (RFLPs), amplified
fragment length polymorphisms (AFLPs), single nucleotide
polymorphisms (SNPs), and isozymes.
[0252] A genetic marker profile of an inbred may be predictive of
the agronomic traits of a hybrid produced using that inbred. For
example, if an inbred of known genetic marker profile and phenotype
is crossed with a second inbred of known genetic marker profile and
phenotype it is possible to predict the phenotype of the F.sub.1
hybrid based on the combined genetic marker profiles of the parent
inbreds. Methods for prediction of hybrid performance from genetic
marker data is disclosed in U.S. Pat. No. 5,492,547, the disclosure
of which is specifically incorporated herein by reference in its
entirety. Such predictions may be made using any suitable genetic
marker, for example, SSRs, RFLPs, AFLPs, SNPs, or isozymes.
[0253] SSRs are genetic markers based on polymorphisms in repeated
nucleotide sequences, such as microsatellites. A marker system
based on SSRs can be highly informative in linkage analysis
relative to other marker systems in that multiple alleles may be
present. Another advantage of this type of marker is that, through
use of flanking primers, detection of SSRs can be achieved, for
example, by the polymerase chain reaction (PCRT.TM.), thereby
eliminating the need for labor-intensive Southern hybridization.
The PCR.TM. detection is done by use of two oligonucleotide primers
flanking the polymorphic segment of repetitive DNA. Repeated cycles
of heat denaturation of the DNA followed by annealing of the
primers to their complementary sequences at low temperatures, and
extension of the annealed primers with DNA polymerase, comprise the
major part of the methodology. Following amplification, markers can
be scored by gel electrophoresis of the amplification products.
Scoring of marker genotype is based on the size (number of base
pairs) of the amplified segment.
[0254] Means for performing genetic analyses using SSR
polymorphisms are well known in the art. The SSR analyses reported
herein were conducted by Celera AgGen in Davis, Calif. This service
is available to the public on a contractual basis. This analysis
was carried out by amplification of simple repeats followed by
detection of marker genotypes using gel electrophoresis. Markers
were scored based on the size of the amplified fragment.
[0255] The SSR genetic marker profile of the parental inbreds and
exemplary resultant hybrid described herein were determined.
Because an inbred is essentially homozygous at all relevant loci,
an inbred should, in almost all cases, have only one allele at each
locus. In contrast, a diploid genetic marker profile of a hybrid
should be the sum of those parents, e.g., if one inbred parent had
the allele 168 (base pairs) at a particular locus, and the other
inbred parent had 172, the hybrid is 168.172 by inference.
Subsequent generations of progeny produced by selection and
breeding are expected to be of genotype 168, 172, or 168.172 for
that locus position. When the F.sub.1 plant is used to produce an
inbred, the locus should be either 168 or 172 for that position.
Surprisingly, it has been observed that in certain instances, novel
SSR genotypes arise during the breeding process. For example, a
genotype of 170 may be observed at a particular locus position from
the cross of parental inbreds with 168 and 172 at that locus. Such
a novel SSR genotype may further define an inbred from the parental
inbreds from which it was derived. An SSR genetic marker profile of
94INK1B is presented in Table 6.
8TABLE 6 SSR Profile of 94INK1B and Comparative Inbreds LOCUS
94INK1B 3IIH6 94KBZ1 BNGL105 92 92 96 BNGL118 110 110 119 BNGL149
183 183 165 BNGL244 202 -- 158 BNGL252 164 164 158 BNGL426 115 115
123 BNGL589 175 175 -- BNGL615 231 231 -- BNGL619 273 275 -- DUP14
105 -- 105 DUP28 133 133 123 MC1014 161 161 169 MC1017 196 196 187
MC1018 138 138 138 MC1022 123 116 123 MC1028 161 -- 161 MC1043 189
189 175 MC1046 181 -- -- MC1065 241 -- 230 MC1070 248 254 248
MC1079 169 173 -- MC1094 178 170 178 MC1108 138 -- -- MC1129 204
204 200 MC1131 117 -- 117 MC1138 188 190 188 MC1176 220 220 --
MC1182 82 82 104 MC1189 219 222 -- MC1194 143 143 -- MC1208 111 111
-- MC1209 184 184 178 MC1237 159 159 159 MC1257 195 -- 180 MC1265
220 220 204 MC1287 160 160 160 MC1288 113 113 -- MC1302 155 147 155
MC1305 160 200 160 MC1325 179 171 179 MC1329 95 -- 109 MC1360 143
143 151 MC1429 199 191 199 MC1484 117 124 117 MC1520 275 275 284
MC1523 200 199 200 MC1538 237 237 223 MC1605 134 110 -- MC1662 161
161 136 MC1720 245 245 -- MC1732 102 100 102 MC1740 167 167 129
MC1782 228 228 228 MC1784 254 254 248 MC1808 147 131 147 MC1831 178
184 186 MC1834 208 208 207 MC1839 194 194 -- MC1866 133 123 133
MC1890 195 -- 167 MC1904 183 191 191 MC1917 169 169 141 MC1931 174
-- -- MC2047 144 144 144 MC2086 229 247 229 MC2132 223 223 223
MC2238 191 195 191 MC2259 180 180 -- MC2305 190 216 180 NC004 185
148 185 PHI017 110 -- 113 PHI024 177 -- 177 PHI031 198 198 194
PHI033 257 257 257 PHI037 139 139 137 PHI050 92 -- 90 PHI051 149
149 149 PHI061 85 85 -- PHI064 90 90 90 PH1065 138 -- 138 PHI072
149 149 -- PHI078 129 133 129 PHI089 92 92 93 PHI093 293 293 293
PHI101 99 99 -- PHI119 168 168 168 PHI120 76 -- -- Primers used to
detect SSRs are from Celera AgGen, Inc., 1756 Picasso Ave., Davis,
CA 95616
[0256] Another aspect of this invention is a plant genetic
complement characterized by a genetic isozyme typing profile.
Isozymes are forms of proteins that are distinguishable, for
example, on starch gel electrophoresis, usually by charge and/or
molecular weight. The techniques and nomenclature for isozyme
analysis are described in, for example, Stuber et al (1988), which
is incorporated by reference.
[0257] A standard set of loci can be used as a reference set.
Comparative analysis of these loci is used to compare the purity of
hybrid seeds, to assess the increased variability in hybrids
compared to inbreds, and to determine the identity of seeds,
plants, and plant parts. In this respect, an isozyme reference set
can be used to develop genotypic "fingerprints."
[0258] Table 7 lists the identifying numbers of the alleles at
isozyme loci types, and represents the exemplary genetic isozyme
typing profile for 94INK1B.
9TABLE 7 Isozyme Profile of 94INK1B and Comparative Inbreds ISOZYME
ALLELE LOCI 94INK1B 3IIH6 94KBZ1 Acph1 2 2 3 Adh1 4 4 4 Cat3 9 9 9
Got3 -- 4 4 Got2 -- 4 4 Got1 -- 4 4 Idh1 4 4 4 Idh2 6 6 4 Mdh1 6 6
6 Mdh2 3.5 3.5 6 Mdh3 16 16 16 Mdh4 12 12 12 Mdh5 12 12 12 Pgm1 9 9
9 Pgm2 4 4 4 6Pgd1 3.8 3.8 2 6Pgd2 5 5 5 Phi1 4 4 4
[0259] The present invention also provides a hybrid genetic
complement formed by the combination of a haploid genetic
complement of the corn plant 94INK1B with a haploid genetic
complement of a second corn plant. Means for combining a haploid
genetic complement from the foregoing inbred with another haploid
genetic complement can comprise any method for producing a hybrid
plant from 94INK1B. It is contemplated that such a hybrid genetic
complement can be prepared using in vitro regeneration of a tissue
culture of a hybrid plant of this invention.
[0260] A hybrid genetic complement contained in the seed of a
hybrid derived from 94INK1B is a further aspect of this invention.
An exemplary hybrid genetic complement is that of the hybrid
8002161.
[0261] Table 8 shows the identifying numbers of the alleles for the
hybrid 8002161, which constitutes an exemplary SSR genetic marker
profile for hybrids derived from the inbred of the present
invention. Table 8 concerns 8002161, which has 94INK1B as one
inbred parent.
10TABLE 8 SSR Profile of 8002161 LOCUS Hybrid 8002161 BNGL105 92.94
BNGL118 110.119 BNGL149 183.183 BNGL244 202.145 BNGL252 164.164
BNGL426 115.119 BNGL589 175.175 BNGL615 231.227 BNGL619 273.277
DUP14 105.112 DUP28 133.123 MC1014 161.161 MC1017 196.196 MC1018
138.138 MC1022 123.67 MC1028 161.159 MC1043 189.175 MC1046 181.218
MC1065 241.230 MC1070 248.239 MC1079 169.182 MC1094 178.184 MC1108
138.122 MC1129 204.204 MC1131 117.127 MC1138 188.188 MC1176 220.218
MC1182 82.106 MC1189 219.223 MC1194 143.143 MC1208 111.111 MC1209
184.184 MC1257 195.187 MC1265 220.214 MC1287 160.160 MC1288 113.124
MC1302 155.147 MC1305 160.204 MC1325 179.175 MC1329 95.107 MC1360
143.147 MC1429 199.202 MC1484 117.136 MC1520 275.288 MC1523 200.199
MC1538 237.237 MC1605 134.128 MC1662 161.173 MC1720 245.241 MC1732
102.112 MC1784 254.250 MC1808 147.137 MC1831 178.186 MC1834 208.208
MC1839 194.194 MC1866 133.123 MC1890 195.136 MC1917 169.109 MC1931
174.174 MC2047 144.145 MC2086 229.240 MC2132 223.223 MC2238 191.191
MC2259 180.173 MC2305 190.180 NC004 185.148 PHI017 110.105 PHI024
177.171 PHI031 198.231 PHI033 257.257 PHI037 139.161 PHI050 92.92
PHI061 85.85 PHI064 90.84 PHI065 138.148 PHI072 149.150 PHI078
129.129 PHI093 293.293 PHI101 99.102 PHI119 168.174 PHI120 76.75
Primers used to detect SSRs are from Celera AgGen, Inc., 1756
Picasso Ave., Davis, CA 95616
[0262] The exemplary hybrid genetic complements of hybrid 8002161
may also be assessed by genetic isozyme typing profiles using a
standard set of loci as a reference set, using, e.g., the same, or
a different, set of loci to those described above. Table 9 lists
the identifying numbers of the alleles at isozyme loci types and
presents the exemplary genetic isozyme typing profile for the
hybrid 8002161, which is an exemplary hybrid derived from the
inbred of the present invention. Table 9 concerns 8002161, which
has 94INK1B as one inbred parent.
11TABLE 9 Isozyme Profile for Hybrid 8002161 Loci Isozyme Allele
Acph1 2/4 Adh1 4/4 Cat3 9/9 Idh1 4/4 Idh2 6/6 Mdh1 6/6 Mdh2 3.5/6
Mdh3 16/16 Mdh4 12/12 Mdh5 12/12 Pgm1 9/9 Pgm2 4/4 6-Pdg1 3.8/3.8
6-Pgd2 5/5 Phi1 4/4
[0263] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of the foregoing
illustrative embodiments, it will be apparent to those of skill in
the art that variations, changes, modifications, and alterations
may be applied to the composition, methods, and in the steps or in
the sequence of steps of the methods described herein, without
departing from the true concept, spirit, and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope, and concept of the invention as defined
by the appended claims.
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