# Patent

## Title
Inbred corn line G06-NP2546

## Abstract

Broadly, this invention provides for an inbred corn line designated G06-NP2546, methods for producing a corn plant by crossing plants of the inbred line G06-NP2546 with plants of another corn plants. The invention relates to the various parts of inbred G06-NP2546 including culturable cells. This invention also relates to methods for introducing transgenic transgenes into inbred corn line G06-NP2546 and plants produced by said methods.

## Background
FIELD OF THE INVENTION
This invention is in the field of corn breeding, specifically relating to an inbred corn line designated G06-NP2546. This invention also is in the field of hybrid maize production employing the present inbred.
BACKGROUND OF THE INVENTION
The original maize plant was indigenous to the Western Hemisphere. The plants were weedlike and only through the efforts of early breeders were cultivated crop species developed. The crop cultivated by early breeders, like the crop today, could be wind pollinated. The physical traits of maize are such that wind pollination results in self-pollination or cross-pollination between plants. Each maize plant has a separate male and female flower that contributes to pollination, the tassel and ear, respectively. Natural pollination occurs when wind transfers pollen from tassel to the silks on the corn ears. This type of pollination has contributed to the wide variation of maize varieties present in the Western Hemisphere.
The development of a planned breeding program for maize only occurred in the last century. A large part of the development of the maize product into a profitable agricultural crop was due to the work done by land grant colleges. Originally, maize was an open pollinated variety having heterogeneous genotypes. The maize farmer selected uniform ears from the yield of these genotypes and preserved them for planting the next season. The result was a field of maize plants that were segregating for a variety of traits. This type of maize selection led to, at most, incremental increases in seed yield.
Large increases in seed yield were due to the work done by land grant colleges that resulted in the development of numerous hybrid corn varieties in planned breeding programs. Hybrids were developed by selecting corn lines and selfing these lines for several generations to develop homozygous pure inbred lines. One selected inbred line was crossed with another selected inbred line to produce hybrid progeny (F1). The resulting hybrids, due to heterosis, are robust and vigorous plants. Inbreds on the other hand are mostly homozygous. This homozygosity renders the inbred lines less vigorous. Inbred seed can be difficult to produce since the inbreeding process in corn lines decreases the vigor. However, when two inbred lines are crossed, the hybrid plant evidences greatly increased vigor and seed yield compared to open pollinated, segregating maize plants. An important consequence of the homozygosity and the homogenity of the inbred maize lines is that all hybrid seed produced from any cross of two such elite lines will be the same hybrid seed and make the same hybrid plant. Thus the use of inbreds makes hybrid seed which can be reproduced readily. The hybrid plant in contrast does not produce hybrid seed that is readily reproducible. The seed on a hybrid plant is segregating for traits.
The ultimate objective of the commercial maize seed companies is to produce high yielding, agronomically sound plants that perform well in certain regions or areas of the Corn Belt. To produce these types of hybrids, the companies must develop inbreds, which carry needed traits into the hybrid combination. Hybrids are not often uniformly adapted for the entire Corn Belt, but most often are specifically adapted for regions of the Corn Belt. Northern regions of the Corn Belt require shorter season hybrids than do southern regions of the Corn Belt. Hybrids that grow well in Colorado and Nebraska soils may not flourish in richer Illinois and Iowa soil. Thus, a variety of major agronomic traits is important in hybrid combination for the various Corn Belt regions, and has an impact on hybrid performance.
Inbred line development and hybrid testing have been emphasized in the past half-century in commercial maize production as a means to increase hybrid performance. Inbred development is usually done by pedigree selection. Pedigree selection can be selection in an F2 population produced from a planned cross of two genotypes (often elite inbred lines), or selection of progeny of synthetic varieties, open pollinated, composite, or backcrossed populations. This type of selection is effective for highly inheritable traits, but other traits, for example, yield requires replicated test crosses at a variety of stages for accurate selection.
Maize breeders select for a variety of traits in inbreds that impact hybrid performance along with selecting for acceptable parental traits. Such traits include: yield potential in hybrid combination; dry down; maturity; grain moisture at harvest; greensnap; resistance to root lodging; resistance to stalk lodging; grain quality; disease and insect resistance; ear and plant height. Additionally, Hybrid performance will differ in different soil types such as low levels of organic matter, clay, sand, black, high pH, low pH; or in different environments such as wet environments, drought environments, and no tillage conditions. These traits appear to be governed by a complex genetic system that makes selection and breeding of an inbred line extremely difficult. Even if an inbred in hybrid combination has excellent yield (a desired characteristic), it may not be useful because it fails to have acceptable parental traits such as seed yield, seed size, pollen production, good silks, plant height, etc.
To illustrate the difficulty of breeding and developing inbred lines, the following example is given. Two inbreds compared for similarity of 29 traits differed significantly for 18 traits between the two lines. If 18 simply inherited single gene traits were polymorphic with gene frequencies of 0.5 in the parental lines, and assuming independent segregation (as would essentially be the case if each trait resided on a different chromosome arm), then the specific combination of these traits as embodied in an inbred would only be expected to become fixed at a rate of one in 262,144 possible homozygous genetic combinations. Selection of the specific inbred combination is also influenced by the specific selection environment on many of these 18 traits which makes the probability of obtaining this one inbred even more remote. In addition, most traits in the corn genome are regrettably not single dominant genes but are multi-genetic with additive gene action not dominant gene action. Thus, the general procedure of producing a non segregating F1 generation and self pollinating to produce a F2 generation that segregates for traits and selecting progeny with the visual traits desired does not easily lead to a useful inbred. Great care and breeder expertise must be used in selection of breeding material to continue to increase yield and the agronomics of inbreds and resultant commercial hybrids.
Certain regions of the Corn Belt have specific difficulties that other regions may not have. Thus the hybrids developed from the inbreds have to have traits that overcome or at least minimize these regional growing problems. Examples of these problems include in the eastern corn belt Gray Leaf Spot, in the north cool temperatures during seedling emergence, in the Nebraska region CLN (corn Lethal necrosis and in the west soil that has excessively high pH levels. The industry often targets inbreds that address these issues specifically forming niche products. However, the aim of most large seed producers is to provide a number of traits to each inbred so that the corresponding hybrid can useful in a broader regions of the Corn Belt. The new biotechnology techniques such as Microsatellites, RFLPs, RAPDs and the like have provided breeders with additional tools to accomplish these goals.
SUMMARY OF THE INVENTION
The present invention relates to an inbred corn line G06-NP2546. Specifically, this invention relates to plants and seeds of this line. Additionally, this relates to a method of producing from this inbred, hybrid seed corn and hybrid plants with seeds from such hybrid seed. More particularly, this invention relates to the unique combination of traits that combine in corn line G06-NP2546.
Generally then, broadly the present invention includes an inbred corn seed designated G06-NP2546. This seed produces a corn plant.
The invention also includes the tissue culture of regenerable cells of G06-NP2546 wherein the cells of the tissue culture regenerates plants capable of expressing the genotype of G06-NP2546. The tissue culture is selected from the group consisting of leaf, pollen, embryo, root, root tip, guard cell, ovule, seed, anther, silk, flower, kernel, ear, cob, husk and stalk, cell and protoplast thereof. The corn plant regenerated from G06-NP2546 or any part thereof is included in the present invention. The present invention includes regenerated corn plants that are capable of expressing G06-NP2546's genotype, phenotype or mutants or variants thereof.
The invention extends to hybrid seed produced by planting, in pollinating proximity which includes using preserved maize pollen as explained in U.S. Pat. No. 5,596,838 to Greaves, seeds of corn inbred lines G06-NP2546 and another inbred line if preserved pollen is not used; cultivating corn plants resulting from said planting; preventing pollen production by the plants of one of the inbred lines if two are employed; allowing cross pollination to occur between said inbred lines; and harvesting seeds produced on plants of the selected inbred. The hybrid seed produced by hybrid combination of plants of inbred corn seed designated G06-NP2546 and plants of another inbred line are apart of the present invention. This inventions scope covers hybrid plants and the plant parts including the grain and pollen grown from this hybrid seed.
The invention further includes a method of hybrid F1 production. A first generation (F1) hybrid corn plant produced by the process of planting seeds of corn inbred line G06-NP2546; cultivating corn plants resulting from said planting; permitting pollen from another inbred line to cross pollinate inbred line G06-NP2546; harvesting seeds produced on plants of the inbred; and growing a harvested seed are part of the method of this invention.
Likewise included is a first generation (F1) hybrid corn plant produced by the process of planting seeds of corn inbred line G06-NP2546; cultivating corn plants resulting from said planting; permitting pollen from inbred line G06-NP2546 to cross pollinate another inbred line; harvesting seeds produced on plants of the inbred; and growing a plant from such a harvested seed.
The inbred corn line G06-NP2546 and at least one transgenic gene adapted to give G06-NP2546 additional and/or altered phenotypic traits are within the scope of the invention. Such transgenes are usually associated with regulatory elements (promoters, enhancers, terminators and the like). Presently, trangenes provide the invention with traits such as insect resistance, herbicide resistance, disease resistance increased or deceased starch or sugars or oils, increased or decreased life cycle or other altered trait.
The present invention includes inbred corn line G06-NP2546 and at least one transgenic gene adapted to give G06-NP2546 modified starch traits. Furthermore this invention includes the inbred corn line G06-NP2546 and at least one mutant gene adapted to give modified starch, acid or oil traits. The present invention includes the inbred corn line G06-NP2546 and at least one transgenic gene for example: VIP, bacillus thuringiensis, the bar or pat gene encoding Phosphinothricin acetyl Transferase, Gdha gene, GOX, EPSP synthase gene, low phytic acid producing gene, and zein. The inbred corn line G06-NP2546 and at least one transgenic gene useful as a selectable marker or a screenable marker is covered by the present invention.
A tissue culture of the regenerable cells of hybrid plants produced with use of G06-NP2546 genetic material is covered by this invention. A tissue culture of the regenerable cells of the corn plant produced by the method described above is also included.
Definitions
In the description and examples, which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specifications and claims, including the scope to be given such terms, the following definitions are provided.










Color Choices:

















 1. light green



 2. medium green



 3. dark green



 4. very dark green



 5. green-yellow



 6. pale yellow



 7. yellow



 8. yelow-orange



 9. salmon



10. pink-orange



11. pink



12. light red



13. cherry red



14. red



15. red and white



16. pale purple (describe)



17. purple



18. colorless



19. white



20. white capped



21. buff



22. tan



23. brown



24. bronze



25. variegated



26. other (describe)























Form


Input


#
ABR.
Description
Value







A1
EMRGN
Final number of plants per plot
#


A2
REGNN
Region Developed: 1. Northwest
#




2. Northcentral 3. Northeast 4. Southeast




5. Southcentral 6. Southwest 7. Other


A3
CRTYN
Cross type: 1. sc 2. dc 3. 3w 4. msc
#




5. m3w 6. inbred 7. rel. line 8. other


A4
KRTPN
Kernel type: 1. sweet 2. dent 3. flint
#




4. flour 5. pop 6. ornamental 7. pipecorn




8. other


A5
EMERN
Days to Emergence EMERN
# Days


B1
ERTLP
% Root lodging: (before anthesis):
# %


B2
GRSNP
% Brittle snapping: (before anthesis):
# %


C1
TBANN
Tassel branch angle of 2nd primary lateral
degree




branch (at anthesis):


C10
HUPSN
Heat units to 50% pollen shed: (from
# HU




emergence)


C11
SLKCN
Silk color:
#/Munsell





value


C12
HU5SN
Heat units to 50% silk: (from emergence)
# HU


C13
DSAZN
Days to 50% silk in adapted zone:
# Days


C14
HU9PN
Heat units to 90% pollen shed: (from
# HU




emergence)


C15
HU19N
Heat units from 10% to 90% pollen shed:
# HU


C16
DA19N
Days from 10% to 90% pollen shed:
# Days


C2
LSPUR
Leaf sheath pubescence of second leaf
#




above the ear (at anthesis) 1-9 (1 = none):


C3
ANGBN
Angle between stalk and 2nd leaf above
degree




the ear (at anthesis):


C4
CR2LN
Color of 2nd leaf above the ear (at
#/Munsell




anthesis):
value


C5
GLCRN
Glume Color:
#/Munsell





value


C6
GLCBN
Glume color bars perpendicular to their
#




veins (glume bands): 1. absent 2. present


C7
ANTCN
Anther color:
#/Munsell





value


C8
PLQUR
Pollen Shed: 1-9 (0 = male sterile)
#


C9
HU1PN
Heat units to 10% pollen shed:
# HU




(from emergence)


D1
LAERN
Number of leaves above the top ear node:
#


D10
LTBRN
Number of lateral tassel branches that
#




originate from the central spike:


D11
EARPN
Number of ears per stalk:
#


D12
APBRR
Anthocyanin pigment of brace roots:
#




1. absent 2. faint 3. moderate 4. dark


D13
TILLN
Number of tillers:
#


D14
HSKCN
Husk color 25 days after 50% silk: (fresh)
#/Munsell





value


D2
MLWVR
Leaf marginal waves: 1-9 (1 = none)
#


D3
LFLCR
Leaf longitudinal creases: 1-9 (1 = none)
#


D4
ERLLN
Length of ear leaf at the top ear node:
# cm


D5
ERLWN
Width of ear leaf at the top ear node at the
# cm




widest point:


D6
PLHTN
Plant height to tassel tip:
# cm


D7
ERHCN
Plant height to the top ear node:
# cm


D8
LTEIN
Length of the internode between the ear
# cm




node and the node above:


D9
LTASN
Length of the tassel from top leaf collar to
# cm




tassel tip:


E1
HSKDN
Husk color 65 days after 50% silk: (dry)
#/Munsell





value


E10
DSGMN
Days from 50% silk to 25% grain moisture
# Days




in adapted zone:


E11
SHLNN
Shank length:
# cm


E12
ERLNN
Ear length:
# cm


E13
ERDIN
Diameter of the ear at the midpoint:
# mm


E14
EWGTN
Weight of a husked ear:
# gm


E15
KRRWR
Kernel rows: 1. indistinct 2. distinct


E16
KRNAR
Kernel row alignment: 1. straight
#




2. slightly curved 3. curved


E17
ETAPR
Ear taper: 1. slight 2. average 3. extreme
#


E18
KRRWN
Number of kernel rows:
#


E19
COBCN
Cob color:
#/Munsell





value


E2
HSKTR
Husk tightness 65 days after 50% silk: 1-9
#




(1 = loose)


E20
COBDN
Diameter of the cob at the midpoint:
# mm


E21
YBUAN
Yield:
# kg/ha


E22
KRTEN
Endosperm type: 1. sweet 2. extra sweet
3




3. normal 4. high amylose 5. waxy 6. high




protein 7. high lysine 8. super sweet




9. high oil 10. other


E23
KRCLN
Hard endosperm color:
#/Munsell





value


E24
ALECN
Aleurone color:
#/Munsell





value


E25
ALCPR
Aleurone color pattern: 1. homozygous
#




2. segregating


E26
KRLNN
Kernel length:
# mm


E27
KRWDN
Kernel width:
# mm


E28
KRDPN
Kernel thickness:
# mm


E29
K1KHN
100 kernel weight:
# gm


E3
HSKCR
Husk extension: 1. short (ear exposed)
#




2. medium (8 cm) 3. long (8-10 cm)




4. very long (>10 cm)


E30
KRPRN
% round kernels on 13/64 slotted screen:
# %


E4
HEPSR
Position of ear 65 days after 50% silk:
#




1. upright 2. horizontal 3. pendent


E5
STGRP
Staygreen 65 days after anthesis: 1-9
#




(1 = worst)


E6
DPOPP
% dropped ears 65 days after anthesis:
%


E7
LRTRP
% root lodging 65 days after anthesis:
%


E8
HU25N
Heat units to 25% grain moisture: (from
# HU




emergence)


E9
HUSGN
Heat units from 50% silk to 25% grain
# HU




moisture in adapted zone:











DETAILED DESCRIPTION OF THE INVENTION
G06-NP2546 is shown in comparison with Mo17 a standard used for comparison by the US PVP office.
The inbred provides uniformity and stability within the limits of environmental influence for traits as described in the Variety Description Information (Table 1) that follows.
The inbred has been self-pollinated for a sufficient number of generations to give inbred uniformity. During plant selection in each generation, the uniformity of plant type was selected to ensure homozygosity and phenotypic stability. The line has been increased in isolated farmland environments with data on uniformity and agronomic traits being observed to assure uniformity and stability. No variant traits have been observed or are expected in G06-NP2546.
The best method of producing the invention, G06-NP2546 which is substantially homozygous, is by planting the seed of G06-NP2546 which is substantially homozygous and self-pollinating or sib pollinating the resultant plant in an isolated environment, and harvesting the resultant seed.







TABLE 1







G06-NP2546


VARIETY DESCRIPTION INFORMATION












#1
Type: corn - Dent



#2
Region where developed in the USA



Southcentral


#3
Maturity



Days
Heat Units



59
1222.5 from emergence to 50% of plants in silk



59
1221.4 from emergence to 50% plants in pollen




   81 from 10% to 90% of pollen shed


#4
Plant Traits



Plant height
210.8 cm.



Ear height
 74.5 cm



Length of top ear internode
 16.1 cm



Average no. Tillers
0.1



Average no. of ears per stalk
1.0



Anthocyanin of brace roots
faint


#5
Leaf Traits



Width of ear node leaf
 9.2 cm



Length of ear node leaf
 79.0 cm



No. of leaves above top ear
005



Degrees of leaf angle
 18



Leaf color
04 (Very Dark green) (Munsell Code 5GY 3/4)



Leaf sheath pubescence
 6*



Marginal waves
 5**



Longitudinal creases
 6**


#6
Tassel Traits




No. of primary lateral branches
03



Branch angle from central spike
20



Tassel length
 40.4 cm



Pollen shed
 5***



Anther Color
14 (red) (Munsell Code 5R 4/4)



Glume Color
26 medium green (Munsell Code 5GY 6/6)



Bar glumes
present


#7a
Ear (unhusked) Traits




Silk Color
 5 Green-yellow (Munsell Code 2.5GY 8/8)



Fresh husk color
 5 Green-yellow (Munsell Code 5GY 7/6)



Dry husk color
22 tan (Munsell Code 2.5Y 8/4)



Position of ear at dry husk stage
horizontal



Husk tightness
6****



Husk extension
 2 short (meduim)


#7b
Ear (husked) Traits




Ear length
 14.3 cm



Ear diameter at midpoint
 44.4 mm



Ear weight
127.5 gm



No. of kernel rows
13



Kernel rows
Distinct



Row alignment
slightly curved



Shank length
 6.4 cm



Ear taper
average


#8
Kernel (dried)



Kernel length
 11.2 mm



Kernel width
 9.2 mm



Kernel thickness
 4.0 mm



% round kernels
34.1



Aleruone color pattern
Homozygous



Aleurone color
white



Hard endosperm color
7 yellow (Munsell Code 2.5Y 8/10)



Endosperm type
Normal starch



Weight per 100 kernels
 28.1 gm


#9
Cob



Diameter at mid-point
 28.2 mm



Cob color
13 cherry red (Munsell Code 10R 5/8)





*scale from 1 = none to 9 = like peach fuzz


**scale from 1-none to 9-many


***scale from 0 = male sterile to 9 = heavy shed


****scale from 1 to 9; 1 = loose 9 = tight






Heat Units per day were calculated: HU=[MaxTemp (86)=Min Temp (50)]/2-50. Large standard deviations are probable due to environmental factors at the locations of observation. Inbred's response to environment is often more pronounced than a hybrid.

The comparable inbred to G06-NP2546 is MO17. The present invention has differing ear leaf length and a significantly smaller ear size than does A619. Other traits that differ are the cob diameter and the ear diameter and the ear length which is shorter for the present invention. Additionally, the present invention is also an earlier flowering inbred than is A619.
The data provided above is often a color. The Munsell code is a reference book of color, which is known and used in the industry and by persons with ordinary skill in the art of plant breeding.
Inbred Performance of G06-NP2546
The traits and characteristics of inbred corn line G06-NP2546 are listed to compare with other inbreds. The G06-NP2546 data shows the characteristics and traits of importance, giving a snapshot of G06-NP2546 in these specific environments.







TABLE 2







PAIRED INBRED COMPARISON DATA






















Heat
Heat Units





Cob
Ear
Ear Leaf
Ear
Heat Units
Units
to 90%
Leaf
Tassel



Diameter
Diameter
Length
Length
to 10%
to 50%
Pollen
Sheath
Length



(mm)
(mm)
(cm)
(cm)
Pollen Shed
Silk
Shed
Pubscence
(cm)




















G06-
24
39
81
12
986
1009
1073
7
33


NP2536


A619
26
44
70
15
1032
1095
1156
2
45


Mean
25
42
76
13
1009
1052
1110
5
39


General


Trials
5
5
5
5
5
5
5
5
5


w/data


Entries
2
2
2
2
2
2
2
2
2


w/data


LSD
1
2
6
2
40
73
67
2
5


General


(95%


confidence


level)


CV
2
2
4
8
2
4
3
26
8


(Effective)


%


Probability
1
0
1
2
3
3
3
0
0


%









The cob and ear of the present invention and that of Mo17 are different. The cob girth of the present invention is larger than Mo17. Likewise the ear girth of the present invention is larger than Mo17. The Ear leaf Length is also different as the present inventions is shorter.

The present invention is earlier to silk and is faster to 90% pollen shed than is Mo17. The anther colors show a strong difference between the two lines also.

The present invention is shown in Table 2 is the mean from individual data gathered for two years at two separate locations. This data was gathered to produce a snapshot of the inbred across a number of physical and environmental conditions. This data shows an inbred that has a smaller cob size than does the comparison inbred.

This invention also is directed to methods for producing a corn plant by crossing a first parent corn plant with a second parent corn plant wherein the first or second parent corn plant is an inbred corn plant from the line G06-NP2546. Further, both first and second parent corn plants can come from the inbred corn line G06-NP2546 which produces a self of the inbred invention. The present invention can be employed in a variety of breeding methods which can be selected depending on the mode of reproduction, the trait, and the condition of the germplasm. Thus, any breeding methods using the inbred corn line G06-NP2546 are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, haploid by such old and known methods of using stock six material that induces haploids and anther culturing and the like.
All plants and plant cells produced using inbred corn line G06-NP2546 are within the scope of this invention. The invention encompasses the inbred corn line used in crosses with other, different, corn inbreds to produce (F1) corn hybrid seeds and hybrid plants and the grain produced on the hybrid plant. This invention includes plant and plant cells, which upon growth and differentiation produce corn plants having the physiological and morphological characteristics of the inbred line G06-NP2546.
Additionally, this maize can, within the scope of the invention, contain: a mutant gene such as, but not limited to, the amylose, amylase, sugary 1, shrunken 1, waxy, AE or imazethapyr tolerant (IT or IRTM) mutant gene; or transgenic genes such as but not limited to insect resistant genes such as Corn Rootworm genes, Bacillus thuringiensis (Cry genes), or herbicide resistant genes such as Pat gene or Bar gene, EPSP, GOX, Glyphosate resistant genes, or disease resistant genes such as the Mosaic virus resistant gene, etc., or trait altering genes such as flowering genes, oil modifying genes, senescence genes and the like. The methods and techniques for inserting, or producing and/or identifying a mutation or a transgene into the present invention through breeding, transformation, or mutating are well known and understood by those of ordinary skill in the art.
Various techniques for breeding and moving or altering genetic material within or into the present invention (whether it is an inbred or in hybrid combination) are also known to those skilled in the art. These techniques to list only a few are anther culturing, haploid production, (stock six is a method that has been in use for thirty years and is well known to those with skill in the art), transformation, irradiation to produce mutations, chemical or biological mutation agents and a host of other methods are within the scope of the invention. All parts of the G06-NP2546 plant including its plant cells produced using the inbred corn line is within the scope of this invention. The term transgenic plant refers to plants having genetic sequences, which are introduced into the genome of a plant by a transformation method and the progeny thereof. Transformation methods are means for integrating new genetic coding sequences into the plant's genome by the incorporation of these sequences into a plant through man's assistance, but not by breeding practices. The transgene once introduced into plant material and integrated stably can be moved into other germplasm by standard breeding practices.
Though there are a large number of known methods to transform plants, certain types of plants are more amenable to transformation than are others. Transformation of dicots is usually achievable for example, tobacco is a readily transformable plant. Monocots can present some transformation challenges, however, the basic steps of transforming plants monocots have been known in the art for about 15 years. The most common method of maize transformation is referred to as gunning or microprojectile bombardment though other methods can be used. The process employs small gold-coated particles coated with DNA which are shot into the transformable material. Detailed techniques for gunning DNA into cells, tissue, callus, embryos, and the like are well known in the prior art. One example of steps that can be involved in monocot transformation are concisely outlined in U.S. Pat. No. 5,484,956 “Fertile Transgenic Zea mays Plants Comprising Heterologous DNA Encoding Bacillus Thuringiensis Endotoxin” issued Jan. 16, 1996 and also in U.S. Pat. No. 5,489,520 “Process of Producing Fertile Zea mays Plants and Progeny Comprising a Gene Encoding Phosphinothricin Acetyl Transferase” issued Feb. 6, 1996.
Plant cells such as maize can be transformed not only by the use of a gunning device but also by a number of different techniques. Some of these techniques include maize pollen transformation (See University of Toledo 1993 U.S. Pat. No. 5,177,010); Whiskers technology (See U.S. Pat. Nos. 5,464,765 and 5,302,523); electroporation; PEG on Maize; Agrobacterium (See 1996 article on transformation of maize cells in Nature Biotechnology, Volume 14, June 1996) along with numerous other methods which may have slightly lower efficiency rates. Some of these methods require specific types of cells and other methods can be practiced on any number of cell types.
The use of pollen, cotyledons, zygotic embryos, meristems and ovum as the target issue can eliminate the need for extensive tissue culture work. Generally, cells derived from meristematic tissue are useful. The method of transformation of meristematic cells of cereal is taught in the PCT application WO96/04392. Any number of various cell lines, tissues, calli and plant parts can and have been transformed by those having knowledge in the art. Methods of preparing callus or protoplasts from various plants are well known in the art and specific methods are detailed in patents and references used by those skilled in the art. Cultures can be initiated from most of the above-identified tissue. The only true requirement of the transforming plant material is that it can form a transformed plant.
The DNA used for transformation of these plants clearly may be circular, linear, and double or single stranded. Usually, the DNA is in the form of a plasmid. The plasmid usually contains regulatory and/or targeting sequences which assists the expression of the gene in the plant. The methods of forming plasmids for transformation are known in the art. Plasmid components can include such items as: leader sequences, transit polypeptides, promoters, terminators, genes, introns, marker genes, etc. The structures of the gene orientations can be sense, antisense, partial antisense, or partial sense: multiple gene copies can be used. The transgenic gene can come from various non-plant genes (such as; bacteria, yeast, animals, and viruses) along with being from plants.
The regulatory promoters employed can be constitutive such as CaMv35S (usually for dicots) and polyubiquitin for monocots or tissue specific promoters such as CAB promoters, MR7 described in U.S. Pat. No. 5,837,848, etc. The prior art promoters, includes but is not limited to, octopine synthase, nopaline synthase, CaMv19S, mannopine synthase. These regulatory sequences can be combined with introns, terminators, enhancers, leader sequences and the like in the material used for transformation.
The isolated DNA is then transformed into the plant. After the transformation of the plant material is complete, the next step is identifying the cells or material, which has been transformed. In some cases, a screenable marker is employed such as the beta-glucuronidase gene of the uidA locus of E. coli. Then, the transformed cells expressing the colored protein are selected. In many cases, a selectable marker identifies the transformed material. The putatively transformed material is exposed to a toxic agent at varying concentrations. The cells not transformed with the selectable marker, which provides resistance to this toxic agent, die. Cells or tissues containing the resistant selectable marker generally proliferate. It has been noted that although selectable markers protect the cells from some of the toxic affects of the herbicide or antibiotic, the cells may still be slightly affected by the toxic agent by having slower growth rates. If the transformed material was cell lines then these lines are regenerated into plants. The cells' lines are treated to induce tissue differentiation. Methods of regeneration of cellular maize material are well known in the art.
A deposit of at least 2500 seeds of this invention will be maintained by Syngenta Seed Inc. Access to this deposit will be available during the pendency of this application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. All restrictions on availability to the public of such material will be removed upon issuance of a granted patent of this application by depositing at least 2500 seeds of this invention at the American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, Va. 20110. The date of deposit was Feb. 8, 2008. The ATCC number of the deposit is PTA-8934 and on Feb. 29, 2008 the deposit was found viable when tested. The deposit of at least 2500 seeds will be from the same inbred seed taken from the deposit maintained by Syngenta Seed Inc. The ATCC deposit will be maintained in that depository, which is a public depository, for a period of 30 years, or 5 years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period.
Additional public information on the present invention may be available from the PVP Office, a division of the US Government.
Accordingly, the present invention has been described with some degree of particularity directed to the preferred embodiment of the present invention. It should be appreciated, though, that the present invention is defined by the following claims construed in light of the prior art so that modifications or changes may be made to the preferred embodiment of the present invention without departing from the inventive concepts contained herein.

## Claims

1. Seed of maize inbred line designated G06-NP2546, representative seed of said line having been deposited under ATCC Accession No. PTA-8934.

2. A maize plant, or a part thereof, produced by growing the seed of claim 1.

3. The maize plant of claim 2, wherein said plant has been detasseled.

4. A tissue culture of regenerable cells produced from the plant of claim 2.

5. Protoplasts produced from the tissue culture of claim 4.

6. The tissue culture of claim 4, wherein cells of the tissue culture are from a plant part selected from the group consisting of leaf, pollen, embryo, root, root tip, anther, silk, flower, kernel, ear, cob, husk and stalk.

7. A maize plant regenerated from the tissue culture of claim 4, said plant having all the morphological and physiological characteristics of inbred line G06-NP2546, representative seed of said line having been deposited under ATCC Accession No. PTA-8934.

8. A method for producing an F1 hybrid maize seed, said method comprising crossing the plant of claim 2 with a different maize plant and harvesting the resultant F1 hybrid maize seed.

9. A method of producing a male sterile maize plant, said method comprising transforming the maize plant of claim 2 with a nucleic acid molecule that confers male sterility.

10. A male sterile maize plant produced by the method of claim 9.

11. A method of producing an herbicide resistant maize plant, said method comprising transforming the maize plant of claim 2 with a transgene that confers herbicide resistance.

12. An herbicide resistant maize plant produced by the method of claim 11.

13. The maize plant of claim 12, wherein the transgene confers resistance to an herbicide selected from the group consisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.

14. A method of producing an insect resistant maize plant, said method comprising transforming the maize plant of claim 2 with a transgene that confers insect resistance.

15. An insect resistant maize plant produced by the method of claim 14.

16. The maize plant of claim 15, wherein the transgene encodes a Bacillus thuringiensis endotoxin.

17. A method of producing a disease resistant maize plant, said method comprising transforming the maize plant of claim 2 with a transgene that confers disease resistance.

18. A disease resistant maize plant produced by the method of claim 17.

19. A method of producing a maize plant with decreased phytate content, said method comprising transforming the maize plant of claim 2 with a transgene encoding phytase.

20. A maize plant with decreased phytate content produced by the method of claim 19.

21. A method of producing a maize plant with modified fatty acid metabolism or modified carbohydrate metabolism, said method comprising transforming the maize plant of claim 2 with a transgene encoding a protein selected from the group consisting of stearyl-ACP desaturase, fructosyltransferase, levansucrase, alpha-amylase, invertase and starch branching enzyme.

22. A maize plant produced by the method of claim 21.

23. The maize plant of claim 22, wherein the transgene confers a trait selected from the group consisting of waxy starch and increased amylose starch.

24. A maize plant, or part thereof, having all the physiological and morphological characteristics of the inbred line G06-NP2546, representative seed of said line having been deposited under ATCC Accession No. PTA-8934.

25. A method of introducing a desired trait into maize inbred line G06-NP2546, said method comprising:
(a) crossing G06-NP2546 plants grown from G06-NP2546 seed, representative seed of which has been deposited under ATCC Accession No. PTA-8934, with plants of another maize line that comprise a desired trait to produce F1 progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, disease resistance and waxy starch;
(b) selecting F1 progeny plants that have the desired trait to produce selected F1 progeny plants;
(c) crossing the selected progeny plants with the G06-NP2546 plants to produce backcross progeny plants;
(d) selecting for backcross progeny plants that have the desired trait and physiological and morphological characteristics of maize inbred line G06-NP2546 listed in Table 1 to produce selected backcross progeny plants; and
(e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of maize inbred line G06-NP2546 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.

26. A plant produced by the method of claim 25, wherein the plant has the desired trait and all of the physiological and morphological characteristics of maize inbred line G06-NP2546 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.

27. The plant of claim 26, wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.

28. The plant of claim 26, wherein the desired trait is insect resistance and the insect resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin.

29. The plant of claim 26, wherein the desired trait is male sterility and the trait is conferred by a cytoplasmic nucleic acid molecule that confers male sterility.

30. A method of modifying fatty acid metabolism, modified phytic acid metabolism or modified carbohydrate metabolism into maize inbred line G06-NP2546, said method comprising:
(a) crossing G06-NP2546 plants grown from G06-NP2546 seed, representative seed of which has been deposited under ATCC Accession No. PTA-8934, with plants of another maize line that comprise a nucleic acid molecule encoding an enzyme selected from the group consisting of phytase, stearyl-ACP desaturase, fructosyltransferase, levansucrase, alphaamylase, invertase and starch branching enzyme;
(b) selecting F1 progeny plants that have said nucleic acid molecule to produce selected F1 progeny plants;
(c) crossing the selected progeny plants with the G06-NP2546 plants to produce backcross progeny plants;
(d) selecting for backcross progeny plants that have said nucleic acid molecule and physiological and morphological characteristics of maize inbred line G06-NP2546 listed in Table 1 to produce selected backcross progeny plants; and
(e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise said nucleic acid molecule and have all of the physiological and morphological characteristics of maize inbred line G06-NP2546 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.

31. A plant produced by the method of claim 30, wherein the plant comprises the nucleic acid molecule and has all of the physiological and morphological characteristics of maize inbred line G06-NP2546 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.