WO2004053055A2 - Transgenic maize with enhanced phenotype - Google Patents

Abstract

Transgenic maize with an enhanced phenotype from insertion of heterologous DNA. Enhanced phenotypes include yield such as yield under stress conditions, quality of plant morphology, physiology or seed composition, metabolic function, cell growth, and the like. Such transgenic maize is produced by generating a plurality of transgenic events for a plurality of unique transgenic DNA constructs where each of the transgenic events comprises introducing into the genome of a parental maize line a single transgenic DNA construct comprising a promoter operably linked to heterologous DNA in sufficient quantity to produce transgenic cells which can be cultured into plants of transgenic maize having the enhanced phenotype.

Description

Transgenic Maize With Enhanced Phenotype

Related Applications

This application claims priority to provisional application 60/337,358 filed December 4, 2001, the specification and sequence listing of which is incorporated herein by reference. Incorporation of Sequence Listing

The sequence listing is contained in the file named "pa_0043 l.rpt" which is 3,661 kilobytes (measured in MS-Windows) and was created on 26 November 2002 and is located on a CD-ROM, which is filed herewith and herein incoφorated by reference. Field of the Invention Disclosed herein is maize with enhanced phenotype, and methods of producing such maize. Background of the invention

Maize (also known as Zea mays and corn) is one of the major crops grown worldwide as a primary source for animal feed, human food and industrial resource. Maize plants with improved agronomic traits such as yield, pest resistance, herbicide resistance, higher seed component, and the like are desired by both farmers and consumers of maize. Considerable effort in breeding maize, e.g. to produce hybrids, has provided significant gains in desired phenotypes.

The ability to introduce specific transgenic DNA into the genome of corn has been used to enhance transgenic maize plants with a desired trait. Researchers have utilized the genetic transformation technology to test and prove the preconceived effects of a gene for plant phenotype enhancement. In many cases, much effort has been placed on the selection of the gene to introduce into the plant as a means to increase the overall success of the experiment to produce a more desirable plant. Nonetheless, the frequency of success of enhancing the transgenic plant is low due to a number of factors including the low predictability of the effects of a specific gene on the plant's growth, development and environmental response, the low frequency of maize transformation, the lack of highly predictable control of the gene once introduced into the genome, and other undesirable effects of the transformation event and tissue culture process. Even with all these problems, transformation is still practiced with persistence and diligence to identify those transgenic plants with the expected, predetermined phenotype.

Occasionally the unexpected phenotype is observed. See U.S. Patent 6, 395,966 which discloses transgenic maize with enhanced yield resulting unexpectedly from the introduction of a gene intended to confer herbicide resistance. Other enhanced traits have been achieved by mutation, e.g. induced by a transposon or chemical or physical mutagen. See for instance, U.S. Patent 6,410,831 which discloses the production of sunflower seed with enhanced stearic acid content by random mutagenesis. Summary of the Invention

This invention relates to the discovery that transformation by random insertion into the corn genome of genes, for the transcription of which there is no known phenotype in corn, can be used as a reliable generator of modification of the corn genome to produce unexpected but yet desired phenotypes. One aspect of the invention provides transgenic maize seed for a maize line which exhibits enhanced yield as compared to yield for a parental maize line; in another aspect the invention provide transgenic maize seed for a maize line characterized by enhanced yield under stress conditions. In another aspect the invention provides transgenic maize seed for maize lines characterized by other enhanced traits, e.g. an enhanced quality in a plant morphology, plant physiology or seed component phenotype as compared to a corresponding phenotype of a parental maize line.

Such transgenic maize seed characterized by enhanced phenotype is produced by introducing into the genome of parental maize a transgenic DNA construct comprising a promoter operably linked to heterologous DNA, where the heterologous DNA encodes a protein having an amino acid sequence with at least 60% identity to a sequence selected from the group consisting of SEQ ID NO:369 through SEQ ID NO:736. In a preferred aspect of the invention the transgenic maize is produced by introducing a transgenic DNA construct where the heterologous DNA comprises a protein coding segment of DNA having at least 60% identity to a sequence selected from the group consisting of SEQ ID NO:l through SEQ ID NO: 368. Other aspects of the invention provide transgenic maize seed for a maize line characterized by unique enhanced phenotype resulting from introduction of a specific heterologous DNA, e.g. shorter plants from decreased internode length, taller plants from increased internode length, early leaf senescence, sterility and elongated tassel central axis. Transgenic maize seed for shorter plants from decreased internode length can result from insertion of heterologous DNA coding for: (a) a TOCl-like receiver domain 3 having an amino acid sequence which is at least 60% identical to SEQ ID NO:436,

(b) a HY5-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO:565, or (c) a proline permease having an amino acid sequence which is at least 60% similar to

SEQ ID NO:371. Transgenic maize seed for taller plants from increased internode length can result from introduction of heterologous DNA coding for:

(a) a myb related transcription factor having an amino acid sequence which is at least 60% identical to SEQ ID NO:717, or

(b) an SVP-like protein having an amino acid sequence which is at least 60% identical to

SEQ ID NO: 609. Transgenic maize seed for plants with early leaf senescence can result from insertion of heterologous DNA coding for a Cytochrome P450 having an amino acid sequence which is at least 60% identical to SEQ ID NO:382.

Transgenic maize seed for sterile plants can result from insertion of heterologous DNA coding for:

(a) an RR3-like receiver domain 8 having an amino acid sequence which is at least 60% identical to SEQ ID NO:439, (b) an ARR2-like receiver domain having an amino acid sequence which is at least 60% identical to SEQ ID NO:434,

(c) an HSF protein having an amino acid sequence which is at least 60%) identical to SEQ ID NO:487, or

(d) an SVP-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO:609.

Transgenic maize seed for plants with elongated tassel central axis can result from insertion of heterologous DNA coding for an SVP-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO:609.

This invention also provides methods for introducing into a maize line an enhanced phenotype as compared to a phenotype in parental units of said maize line. The method comprises generating a population of transgenic plants comprising a variety of heterologous DNA for the transcription of which there is no known phenotype in maize. In one aspect of the invention the population is generated for a plurality of transgenic events for a plurality of unique transgenic DNA constructs. Each transgenic event comprises introducing into the genome of a parental units a single transgenic DNA construct comprising a promoter operably linked to heterologous DNA for the transcription of which there is no known phenotype in corn. The transgenic DNA construct is introduced into the parental genome in sufficient quantity to produce transgenic cells which can be cultured into plants of transgenic maize having said enhanced phenotype. The transgenic cells are cultured into transgenic plants producing progeny transgenic seed. The population of transgenic plants are screened for observable phenotypes. And, seed is collected from transgenic plants which are selected as having an unexpected enhanced phenotype. Optionally, the method comprises repeating a cycle of germinating transgenic seed, growing subsequent generation plants from said transgenic seed, observing phenotypes of said subsequent generation plants and collecting seeds from subsequent generation plants having an enhanced phenotype. In preferred aspects the method a large population is screened by employing at least 2 transgenic events for at least 20 unique transgenic DNA constructs, more preferably upwards of 10 or more transgenic events, say up to 100 or more transgenic events for upwards of 50 or more unique transgenic DNA constructs, say 100 or more or even 500 or more unique transgenic constructs. Other preferred aspects of the method employ DNA construct where the heterologous

DNA is operably linked to a selected promoter, e.g. the 5' end of a promoter region comprising a rice actin promoter and rice actin intron. The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

Yet another aspect of the invention provides a method comprising crossing transgenic plants from the population of transgenic plants with at least one other maize line to produce a hybrid population of transgenic plants, observing phenotypes in the hybrid population and selecting seed from transgenic plants in the hybrid population having unexpected enhanced phenotypes. Brief Description of Drawings Figure 1 illustrates a vector comprising a DNA construct useful in the practice of this invention. Detailed Description of Preferred Embodiments

Definitions - As used herein the following terms are specifically defined:

"Maize" means a variety of Zea mays also commonly known in some parts of the world as corn. Maize is cultivated as a crop. "Seed" means the reproductive tissue of a plant which is formed from a fertilized ovule and from which a new plant develops. Seed contains an embryo and discrete food store (cotyledon or endosperm) surrounded by an outer covering (testa). The measure of maize seed produced is reported as yield. Maize seed contains useful industrial and food resources of protein, oil and starch. "Phenotype" means a measurable crop trait and includes, but is not limited to, yield as compared to a parental maize line such as overall yield and yield under stress conditions such as drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability or high plant density. Other useful phenotypes include yield as manifested by increase number of kernels per unit planted area, number of ears per unit planted area and average weight of kernels; yield as manifested by increase in biomass per unit planted area or an increase in the root/shoot ratio; or yield as manifested by increased efficiency in water use, nitrogen use or phosphate use. Other useful phenotypes include enhanced quality as compared to a parental maize line in plant morphology, physiology or seed composition such as increased internode length, shortened internode length, sterility, elongated tassel central axis, earlier leaf senescence, setting a second ear at high planting density, earlier time of germination, increased production of kernel oil and increased production of kernel protein. Still other useful phenotypes include enhanced metabolic function such as increased amino acid production, increased amino acid transport, increased protein production and increased enzyme activity; enhanced cell growth, modified cell regulation and early cell senescence; early time of germination and early flowering.

"Parental maize line" means any maize variety that provides tissue for transformation and baseline phenotype.

"Heterologous" refers to a segment of DNA that is imported into a non-natural DNA construct, e.g. regulatory DNA as well as DNA coding for a protein. "Transgenic DNA construct" means a segment of DNA which is introduced into the genome of a parental maize line. While a transgenic DNA construct can comprise any segment of DNA that is heterologous to the insertion site, in preferred aspects of the invention the transgenic DNA construct will be designed to provide a specific function, e.g. suppress or over express a selected protein. Useful transgenic DNA constructs comprise gene regulatory segment operably linked to a protein coding segment. A gene regulatory segment can more specifically comprise promoter elements, enhancers, silencers, introns and untranslated regions. An especially useful gene regulatory segment for use in maize comprises a rice actin promoter with a rice actin intron as described more specifically below. Protein coding segment can be any coding segment that may be of interest for investigation into its effect in a transgenic plant.

Exemplary protein coding segments include DNA segments encoding all or a part of any protein such as a cytochrome p450, a transporter, a lipase, a kinase, a receiver domain, a synthase, a transcription factor, a reductase, a phosphatase, a ribonuclease, an anhydrase and the like. It is also useful to use DNA segments encoding protein of unknown function. In cases where over expression of heterologous DNA may not be satisfactory, effective or desirable in producing an observed enhanced phenotype, it is contemplated that a person or ordinary skill in the art would look to protein pathways for an alternate route to the desired enhanced phenotype. Such alternate route may include insertion of heterologous DNA coding for a protein which is upstream or downstream of the protein originally associated with the observed enhanced phenotype. Another alternate route may include insertion of heterologous DNA which is effective in suppression of a competitive protein. When suppression of protein expression is the intended objective, the heterologous DNA can be designed to produce a gene silencing effect, e.g. by an antisense or RNAi mechanism. Anti-sense suppression of genes in plants by introducing by transformation of a construct comprising DNA of the gene of interest in an anti-sense orientation is disclosed in U.S. Patents 5,107,065; 5,453,566; 5,759,829; 5,874,269; 5,922,602; 5,973,226; 6,005,167; WO 99/32619; WO 99/61631; WO 00/49035; WO 02/02798; all of which are incorporated herein by reference. Interfering RNA suppression of genes in a plant by introducing by transformation of a construct comprising DNA encoding a small (commonly less than 30 base pairs) double-stranded piece of RNA matching the RNA encoded by the gene of interest is disclosed in U.S. Patents 5,190,931; 5,272,065; 5,268,149; WO 99/61631; WO 01/75164; WO 01/92513, all of which are incorporated herein by reference. A "non-predetermined location in genomic DNA" means a random locus in a maize chromosome in which a transgenic DNA construct is inserted by chance.

"Transformation" means a method of introducing a transgenic DNA construct into a genome and can include any of the well-known and demonstrated methods including electroporation as illustrated in U.S. Patent 5,384,253, microprojectile bombardment as illustrated in U.S. Patents 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861and 6,403,865, Agrobacterium mediated transformation as illustrated in U.S. Patents 5,635,055; 5,824,877; 5,591,616; 5,981,840 and 6,384,301, and protoplast transformation as illustrated in U.S. Patents 5,508,184, all of which are incorporated herein by reference. "Tissue from a parental maize line" means tissue which is specifically adapted for a selected method of transformation and can include cell culture or embryonic callus.

"Yield" as used herein means the production of shelled corn kernels per unit of production area, e.g. in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g. at 15.5 %> moisture. As a bushel of corn is defined by law in the State of Iowa as 56 pounds by weight, a useful conversion factor for corn yield is: 100 bushels per acre is equivalent to 6.272 metric tons per hectare.

The maize seed provided by this invention is characterized by an enhanced phenotype as compared to its parental maize line. Such maize seed is preferably obtainable from a massive screening program by observing transformed plants for serendipitously imparted phenotype resulting from the introduction of a transgenic DNA construct into a non-predetermined location in the genomic DNA of tissue from a parental maize line. The transgenic DNA construct is introduced into the genome in sufficient quantity to produce transgenic cells which can be cultured into plants of transgenic maize having an enhanced phenotype as compared to the parental maize line. Such transgenic maize cells are cultured into transgenic plants which produce progeny transgenic seed. Preferably, the screening program is designed to evaluate multiple events of a plurality of distinct transgenic DNA constructs, e.g. from 2 to 20 or more transgenic events of each of from 2 to 20 or more transgenic DNA constructs, e.g. at least 50 or more or up to 100 or more transgenic DNA constructs. Although the design of a transgenic DNA construct can be based on a rational expectation of a phenotype modification, the method of the invention requires observation of an unexpected, yet desired enhanced phenotype. A useful population for screening for unexpected enhanced phenotypes may comprise 40 or more unique transgenic plants, e.g. at least 100 transgenic plants or even up to 1000 or more unique transgenic plants. Even larger populations can be provided by crossing transgenic plants with other plant lines to provide hybrid populations of transgenic plants, such populations can comprises tens of thousands of transgenic plants for screening. In methods of this invention transgenic plants and seeds are evaluated for desired phenotypes allowing the selection of seeds. Methods of this invention can be practiced with an optional repeating of a cycle of germinating transgenic seed, growing subsequent generation plants from said transgenic seed, observing phenotypes of said subsequent generation plants and collecting seeds from subsequent generation plants having a desirable enhanced phenotype. Heterologous DNA

The following Table 1 describes the heterologous DNA used to produce the transgenic maize of this invention including reference to nucleic acid and polypeptide sequences which are provided in the Sequence Listing. It is contemplated tiiat transgenic maize seed of this invention characterized by an enhanced phenotype will result from use of not only the heterologous DNA listed in Table 1 but also homolgs, orthologs and/or paralogs of such heterologous DNA or similar DNA which has been artificially modified to avoid or minimize an undesired effect but yet still produce the originally observed enhanced phenotype associated with the heterologous DNA listed in Table 1. Thus, heterologous DNA for use in this invention comprises not only DNA coding for a protein of a polypeptide listed in Table 1, e.g. with an amino acid sequence of SEQ ID NO: 369 to SEQ ID NO:738, but also DNA coding for a protein with an amino acid sequence which is at least 60% identical, e.g. at least 65%, 70% or 75% identical, in some cases more preferably at least 80%, 85%, 90% or 95% identical, to a sequence of SEQ ID NO: 369 to SEQ ID NO: 739. In another aspect of this invention the transgenic maize with an enhanced trait is provided by using heterologous DNA with a nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO:368 or a homologous DNA coding for a protein of similar function but with a nucleic acid sequence which is at least 70% identical, e.g. at least 75%, 80%>, 85%>, 90% or 95%> identical, to a sequence of SEQ ID NO:l to SEQ ID NO:368. Sequence identity is determined over a sequence of substantially the full length of a sequence listed in Table 1. More particularly, the headings for Table 1 have the following meanings: "NUCLEIC ACID SEQ ID NO" refers to a particular nucleic acid sequence in the Sequence Listing which defines a heterologous DNA used in a transgenic DNA construct of this invention. "PHE ID" refers to an arbitrary number used to identify experiments using a particular heterologous DNA.

"AMINO ACID SEQ ID NO" refers to a particular amino acid sequence in the Sequence Listing corresponding to the translated protein encoded by the heterologous DNA. "GENE NAME" refers to a common name for the heterologous DNA.

"DONOR ORGANISM" refers to the organism from which the heterologous DNA was derived. "CODING COORDINATES" refer to peptide coding segments of the heterologous DNA.

Table 1

NUCLEIC ACID PHE ID AMINO ACID GENE NAME DONOR CODING

SEQ ID NO SEQ ID NO ORGANISM COORDINATES

354 PHE0000415 722 soy Glycine max 160-1899 monosaccharide ] transporter 1

355 PHE0000416 723 corn Zea mays 74-1690 j monosaccharide

|_ piffibbbb™—. transporter 6 5

724 com Zea mays 146-1744 monosaccharide transporter 4

357 PHE0000419 725 soy Glycine max 63-1505 monosaccharide I transporter 2 I

358 PHE0000420 726 soy sucrose Glycine max 63-1595 ^{" '} transporter

359 PHE0000421 727 com sucrose Zea mays 76-1599 transporter 2

360 PHE0000422 728 com Zea mays 201-1763 monosaccharide i transporter 8

361 ⁽PHE0000423 729 com (Zea mays 93-1634 j monosaccharide |

,__ transporter 7 '

362 iPHE0000390 rice j Oryza sativa 136-1311

! chloroplastic j

1 sedoheptulose- 1

!

1,7- j . bisphosphatase (

(363 ιPHEθδbb39i 731 rice cytosolic j Oryza sativa 171-1187 i 1 fructose-1,6-

1 1 bisphosphatase 1

364 PHE0000392 ■732 Wheat ( Triticum 14-1192

1 sedoheptulose- j aestivum 1 7-

( bisphosphatase j

365 [PHE0000393 733 dual function 1 Ralstonia 80-1399 SBPase/FBPase (eutropha

366 ,PHE0000394 ^!734 sedoheptulose- ^■ Chlorella ^' 904238

1 1,7- Ssorokiniana bisphosphatase j

,367 1PHE0000425 1735 j soy isoflavone ' Glycine max l4^"5-1607

I 1 synthase j

[368 (PHE0000426 (736 _ ;soy ttgl-like 2 Glycine max 52-1059__

Plant Transformation Constructs

The construction of vectors which may be used in the invention will be known to those of skill of the art in light of the present disclosure. The techniques of the current invention are thus not limited to any particular DNA or method of plant transformation. In preparing populations of transgenic plants for phenotype screening the GATEWAY™ cloning technology (available from Invitrogen Life Technologies, Carlsbad, California) is useful for construction of vectors for transgenic DNA constructs that can be used in transformation. GATEWAY™ vector construction technology uses the site specific recombinase LR cloning reaction of the Integrase/αtt system from bacterophage lambda vector construction, instead of restriction endonucleases and ligases. The LR cloning reaction is disclosed in U.S. Patents 5,888,732 and 6,277,608, U.S. published patent application 20020007051 and International Patent Publication WO 02/081711 Al, all of which are incorporated herein by reference, and in the GATEWAY™ Cloning Technology Instruction Manual. The GATEWAY™ technology produces a high frequency of inserts in a plasmid in the correct orientation relative to other elements in the plasmid such as promoters, enhancers, and the such. Routine clomng of any desired DNA sequence into a vector comprising operable plant expression elements is thereby facilitated. Using the GATEWAY™ cloning technology, a desired DNA sequence, such as a coding sequence, may be amplified by PCR with the phage lambda αttBl sequence added to the 5' primer and the αttB2 sequence added to the 3' primer. Alternatively, nested primers comprising a set of αttBl and αttB2 specific primers and a second set of primers specific for the selected DNA sequence can be used. Sequences, such as coding sequences, flanked by αttBl and αttB2 sequences can be readily inserted into plant expression vectors using GATEWAY™ methods. In a more direct route a construct of interest flanked by αttLl and αttL2 sequences can be incorporated by recombination into a plasmid destination vector comprising a bacterial negative marker flanked by αttRl and αttR2 sites using LR clonase.

It is also contemplated that one may employ multiple genes on either the same or different vectors for transformation. In the latter case, the different vectors may be delivered concurrently to recipient cells if co-transformation into a single chromosomal location is desired. Transgenic DNA constructs used for transforming plant cells will comprise the heterologous DNA which one desires to introduced into and a promoter to express the heterologous DNA in the host maize cells. As is well known in the art such constructs can further include elements such as regulatory elements, 3 ' untranslated regions (such as polyadenylation sites), transit or signal peptides and marker genes elements as desired. 1. Regulatory Elements A number of promoters that are active in plant cells have been described in the literature both constitutive and tissue specific promoters and inducible promoters. See the background section of U.S. Patent 6,437,217 for a description of a wide variety of promoters that are functional in plants. Such promoters include the nopaline synthase (NOS) and octopine synthase (OCS) promoters that are carried on tumor-inducing plasmids of Agrobacterium tumefaciens, the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters and the figwort mosaic virus (FMV) 35S promoter, the enlianced CaMV35S promoter (e35S), the light-inducible promoter from the small subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide). For instance, see U.S. Patents 6,437,217 which discloses a maize RS81 promoter, 5,641,876 which discloses a rice actin promoter, 6,426,446 which discloses a maize RS324 promoter, 6,429,362 which discloses a maize PR-1 promoter, 6,232,526 which discloses a maize A3 promoter and 6,177,611 which discloses constitutive maize promoters, all of which are incorporated herein by reference. The rice actin 1 promoter with a rice actin intron is especially useful in the practice of the present invention.

It is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of the heterologous DNA of interest. The promoters used in the transgenic DNA constructs of the present invention may be modified, if desired, to affect their control characteristics. Promoters can be derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5' to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5' or 3' to the coding sequence. In some instances, these 5' enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5' introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers which could be used in accordance with the invention include elements from the

CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.

Where an enhancer is used in conjunction with a promoter for the expression of a selected protein, it is believed that it will be preferred to place the enhancer between the promoter and the start codon of the selected coding region. However, one also could use a different arrangement of the enhancer relative to other sequences and still realize the beneficial properties conferred by the enhancer. For example, the enhancer could be placed 5' of the promoter region, within the promoter region, within the coding sequence (including within any other intron sequences which may be present), or 3' of the coding region.

In addition to introns with enhancing activity, other types of elements can influence gene expression. For example, untranslated leader sequences predicted to enhance gene expression as well as "consensus" and preferred leader sequences have been identified. Preferred leader sequences are contemplated to include those which have sequences predicted to direct optimum expression of the attached coding region, i.e., to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants, and in maize in particular, will be most preferred, for example, sequences derived from the small subunit of ribulose bisphosphate carboxylase (RUBISCO).

In general it is preferred to introduce heterologous DNA randomly, i.e. at a non-specific location, in the genome of a parental maize line. In special cases it may be useful to target heterologous DNA insertion in order to achieve site specific integration, e.g. to replace an existing gene in the genome. In some other cases it may be useful to target a heterologous DNA integration into the genome at a predetermined site from which it is known that gene expression occurs. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Patent 4,959,317 and FLP-FRT as disclosed in U.S. Patent 5,527,695, both incorporated herein by reference. 2. 3 ' Untranslated Regions (3 ' UTR)

Transformation constructs prepared in accordance with the invention will typically include a 3' end untranslated sequence DNA sequence that follows the coding sequence and typically contains a polyadenylation sequence. One type of 3 ' untranslated sequence which may be used is a 3' UTR from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3' end). Where a 3 ' end other than a nos 3 ' UTR is used in accordance with the invention, the most preferred 3' ends are contemplated to be those from a gene encoding the small subunit of a ribulose- 1,5-bisphosphate carboxylase-oxygenase (rbcS), and more specifically, from a rice rbcS gene (see PCT Publication WO 00/70066), the 3 ' UTR for the T7 transcript of Agrobacterium tumefaciens, the 3' end of the protease inhibitor I or II genes from potato or tomato, and the 3' region isolated from Cauliflower Mosaic Virus. Alternatively, one also could use a gamma coixin, oleosin 3 or other 3' UTRs from the genus Coix (see PCT Publication WO 99/58659). 3. Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene, which are removed post-translationally from the initial translation product and which facilitate the transport of the protein into or through intracellular or extracellular membranes, are termed transit sequences (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, golgi apparatus, peroxisomes or glyoxysomes, and outside of the cellular membrane). By facilitating the transport of the protein into compartments inside and outside the cell, these sequences may increase the accumulation of a gene product protecting the protein from intracellular proteol tic degradation. These sequences also allow for additional mRNA sequences from highly expressed genes to be attached to the coding sequence of the genes. Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA 5' of the gene of interest may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear on the final polypeptide. It further is contemplated that targeting of certain proteins may be desirable in order to enhance the efficacy or stability of the protein as disclosed in U.S. Patent 5,545,818, incorporated herein by reference.

A particular example of such a use concerns the direction of a protein conferring herbicide resistance, such as a glyphosate resistant EPSPS protein, to a particular organelle such as the chloroplast, rather than to the cytoplasm. This is exemplified by the use of the rbcS transit peptide, the chloroplast transit peptide described in U.S. Patent 5,728,925, or the optimized transit peptide described in U.S. Patent 5,510,471, which confer plastid-specific targeting of proteins, both of which are incorporated herein by reference. In addition, it may be desirable to target certain genes responsible for male sterility to the mitochondria, or to target certain genes for resistance to phytopathogenic organisms to the extracellular spaces, or to target proteins to the vacuole. A further use concerns the direction of enzymes involved in amino acid biosynthesis or oil synthesis to the plastid. Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This generally will be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. An intracellular targeting DNA sequence may be operably linked 5 ' or 3' to the coding sequence depending on the particular targeting sequence. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post-translationally removed. 4. Marker Genes In practice DNA is introduced into only a small percentage of target cells in any one experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptIT), hygromycm B (aph TV) and gentamycin (aac3 and aacC ) or resistance to herbicides such as glufosinate (bar ox pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Patents 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a όetα-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g. an NPTII gene and a GFP gene. Culturing Transgenic Cells

Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. Well known cell culture media, e.g. designated as MS and N6, may be modified by including further substances such as growth regulators. Preferred growth regulators for plant regeneration include cytokins such as 6-benzylamino pierine, zeahin or the like, and abscisic acid which facilitate the growth of cells at specific developmental stages. Tissue may be maintained on a media with auxin type growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, then transferred to media conducive to maturation of embryoids. Cultures are transferred every 1-4 weeks, preferably every 2-3 weeks on this medium. Shoot development will signal the time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, are allowed to mature into plants. Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins

9 1 m^" s^" of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28 °C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced. Progeny may be recovered from transformed plants and tested for expression of the exogenous expressible gene. Note however, that seeds on transformed plants may occasionally require embryo rescue due to cessation of seed development and premature senescence of plants. To rescue developing embryos, they are excised from surface-disinfected seeds 10-20 days post- pollination and cultured. An embodiment of media used for culture at this stage comprises MS salts, 2% sucrose, and 5.5 g/1 agarose. In embryo rescue, large embryos (defined as greater than 3 mm in length) are germinated directly on an appropriate media. Embryos smaller than that may be cultured for 1 wk on media containing the above ingredients along with 10^"5M abscisic acid and then transferred to growth regulator-free medium for germination.

Characterization To confirm the presence of heterologous exogenous DNA or other exogenous

"transgene(s)" in the regenerating plants or transformed callus a variety of assays may be performed. Such assays include, for example, "molecular biological" assays, such as Southern and Northern blotting and PCR; "biochemical" assays, such as detecting the presence of RNA, e.g. double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

1. DNA Integration, RNA Expression and Inheritance

The presence of DNA elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant, but does not necessarily prove integration of the introduced gene into the host cell genome. Typically, DNA has been integrated into the genome of all transformants that demonstrate the presence of the DNA through PCR analysis. In addition, it is possible using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin by using PCR techniques to clone fragments of the host genomic DNA adjacent to an introduced DNA.

Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition, it is possible tlirough Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, /^'. e. , confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.

While DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCR techniques, referred to as RT- PCR, also may be used for detection and quantitation of RNA produced from introduced genes. In this RT-PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques will not demonstrate integrity of the RNA product. Northern blotting will demonstrate the presence of an RNA species and give information about the integrity of that RNA. It is further contemplated that TAQMAN® technology (available from Applied Biosystems, Foster City, CA) may be used to quantitate both DNA and RNA in a transgenic cell.

Methods of Evaluating Phenotype

Expression, and in some cases suppression, of the various genes embodied by heterologous DNA used in the present invention leads to improved phenotypes in transformed plants. Phenotypic data is collected during the transformation process in callus as well as during plant regeneration, as well as in plant tissue. Phenotypic data can also be collected in transformed callus relating to the morphological appearance as well as growth of the callus, e.g., shooty, rooty, starchy, mucoid, non-embryo genie, increased growth rate, decreased growth rate, dead. It is expected that one of skill in the art may recognize other phenotypic characteristics in transformed callus and plants and select transformed plants having enhanced traits with minimal drag on other key traits, e.g. yield. Phenotypic data is also collected during the process of plant regeneration as well as in regenerated plants transferred to soil. It is expected that one of skill in the art may recognize other phenotypic characteristics in transformed plants.

Although a wide variety of phenotypes are monitored during the process of plant breeding and testing in both inbred and hybrid plants. For example, in R0 plants (plants directly regenerated from callus) and Rl plants (the direct progeny of R0 plants), plant characteristic phenotypes and plant seed characteristic phenotypes can be observed. In R2 and R3 plants, days to pollen shed, days to silking, and plant type can be observed. Metabolite profiling of R2 plants can be conducted. A variety of phenotypes can also be assayed in hybrids of transgenic maize of this invention. For example, yield, moisture, test weight, nutritional composition, chlorophyll content, leaf temperature, stand, seedling vigor, plant height, leaf number, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, pest resistance (including diseases, viruses and insects) and metabolic profiles can be recorded. In addition, phenotypic characteristics of grain harvested from hybrids will be recorded, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality. Furthermore, characteristics such as photosynthesis, leaf area, husk structure, kernel dry down rate and internode length may be measured in hybrids or inbreds. It is expected that transcriptional profiling may be performed on transgenic plants expressing genes of the present invention.

In a further embodiment of the method of the invention, the transformation and selection steps may be followed by conventional plant improvement techniques thus leading to seeds having an even further improvement in the enhanced phenotype. In still another embodiment the seeds of the invention may be subjected to one or more further transformation treatments.

The maize plants with enhanced phenotype may be used in breeding programs for the development of elite maize lines or hybrids, which programs are aimed at the production of varieties meeting the requirements of farming practice regarding yield, disease resistance and other agronomically important traits in major maize growing areas in the world. Seeds resulting from these programs may be used in the growing of commercial maize crops.

To confirm hybrid yield in transgenic plants expressing genes of the present invention, it may be desirable that hybrids be tested over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g. in Iowa, Illinois or other locations in the midwestern United States, under "normal" field conditions as well as under stress conditions, e.g. under drought or population density stress. One of skill in the art knows how to design a yield trial such that a statistically significant yield difference can be detected between two hybrids at the desired rate of precision.

Plant Breeding Backcrossing can be used to improve a starting plant. Backcrossing transfers a specific desirable trait from one source to an inbred or other plant 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 gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent, then the selected progeny are mated back to the superior recurrent parent (A). After five or more backcross generations with selection for the desired trait, the progeny are hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give progeny which are pure breeding for the gene(s) being transferred, i.e. one or more transformation events .

Therefore, through a series a breeding manipulations, a selected transgene may be moved from one line into an entirely different line without the need for further recombinant manipulation. Transgenes are valuable in that they typically behave genetically as any other gene and can be manipulated by breeding techniques in a manner identical to any other corn gene. Therefore, one may produce inbred plants which are true breeding for one or more transgenes. By crossing different inbred plants, one may produce a large number of different hybrids with different combinations of transgenes. In this way, plants may be produced which have the desirable agronomic properties frequently associated with hybrids ("hybrid vigor"), as well as the desirable characteristics imparted by one or more transgene(s). It is desirable to introgress the genes of the present invention into maize hybrids for characterization of the phenotype conferred by each gene in a transformed plant. The host genotype into which the transgene was introduced, preferably LH59, is an elite inbred and therefore only limited breeding is necessary in order to produce high yielding maize hybrids. The transformed plant, regenerated from callus is crossed, to the same genotype, e.g., LH59. The progeny are self pollinated twice and plants homozygous for the transgene are identified.

Homozygous transgenic plants are crossed to a testcross parent in order to produce hybrids. The test cross parent is an inbred belonging to a heterotic group which is different from that of the transgenic parent and for which it is known that high yielding hybrids can be generated, for example hybrids are produced from crosses of LH59 to either LH195 or LH200. The present invention will be further illustrated by means of the following examples which are given for illustration purposes only and are in no way intended to limit the scope of the invention.

Materials used in the Examples

DNA constructs for use in this invention can be fabricated using Gateway® technology as described above. Figure 1 shows the elements of a plasmid, designated as pMON72472, which is useful as a destination vector into which the transgenic DNA construct can be cloned to provide a transformation vector for use in an Agrobacterium-mediated transformation. Figure 1 further illustrates restriction sites on plasmid pMON72472 which are useful for modification of the plasmid. The elements of die plasmid are summarized in Table 2. The plasmid comprises left and right T-DNA border sequences from Agrobacterium. The right border sequence is located 5' to the rice actin 1 promoter and the left border sequence is located 3' to the pinϊl 3' sequence situated 3' to the nptϊl gene. Furthermore the original pSK- backbone of pMON65164 is replaced by a plasmid backbone to facilitate replication of the plasmid in both E. coli and Agrobacterium tumefaciens. The backbone comprises an oriV wide host range origin of DNA replication functional in Agrobacterium, the rop sequence, a pBR322 origin of DNA replication functional in E. coli and a spectinomycin/streptomycin resistance gene for selection for the presence of the plasmid in both E. coli and Agrobacterium.

Table 2 Genetic Elements of Plasmid Vector pMON72472

Protein coding segments are amplified by PCR prior to insertion in a destination vector such as pMON72472. Primers for PCR amplification can be designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5' and 3' untranslated regions. PCR products are tailed with αttBl and αttB2 sequences, purified then recombined into a destination vectors to produce an expression vector which can be used in transformation.

All PCR amplification products are sequenced prior to introduction into a plant. PCR inserts in destination vectors are sequenced to confirm that the inserted sequenced encoded the expected amino acid sequence. See Table 1 for identification of protein coding sequence which was placed in transgenic DNA constructs in expression vectors.

Example 1 This example illustrates the preparation of recipient cells from a parental maize line. Parental maize plants of line LH59 were grown in the greenhouse. Ears are harvested from plants when the embryos were 1.5 to 2.0 mm in length, usually 10 to 15 days after pollination, and most frequently 11 to 12 days after pollination. Ears were surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Alternatively, ears were surface sterilized by immersion in 50% CLOROX™ containing 10% SDS for 20 minutes, followed by three rinses with sterile water.

Immature embryos were isolated from individual kernels using methods known to those of skill in the art. Immature embryos were cultured on medium 211 (N6 salts, 2% sucrose, 1 mg/L 2,4-D, 0.5 mg/L niacin, 1.0 mg/L thiamine-HCl, 0.91 g/L L-asparagine, 100 mg/L myo- inositiol, 0.5 g/L MES, 100 mg/L casein hydrolysate, 1.6 g/L MgCl₂, 0.69 g/L L-proline, 2 g/L GELGRO™, pH 5.8) containing 16.9 mg/L AgNO^ (designated medium 21 IV) for 3-6 days prior to Agrobacterium-mediated transformation, preferably 3-4 days prior to microprojectile bombardment. Example 2 This example illustrates the transformation of maize immature embryos using Agrobacterium tumefaciens, strain ABI. The ABI strain of Agrobacterium is derived from strain A208, a C58 nopaline type strain. The ABI strain of Agrobacterium is derived from strain A208, a C58 nopaline type strain, from which the Ti plasmid was eliminated by culture at 37°C, and further containing the modified Ti plasmid pMP90RK. An Agrobacterium tumefaciens binary vector system is preferably used to transform maize. See Klee et al, Agrobacterium-mediatcd plant transformation and its further applications to plant biology. Annu. Rev. Plant Physiol. Plant Mol Biol. 1987;38:467-486. Prior to co-culture of maize cells, Agrobacterium cells may be grown at 28°C in LB

(DIFCO) liquid medium comprising appropriate antibiotics to select for maintenance of the modified Ti plasmid and binary vector. It is well known to those skilled in the art to use appropriate selection agents to maintain plasmids in the host Agrobacterium strain. For example, ABI/expression vector may be grown in LB medium containing 50 ug/ml kanamycin to select for maintenance of the pMP90RK modified Ti plasmid and 100 ug/ml spectinomycin to select for maintenance of the expression vector. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature in AB medium comprising appropriate antibiotics for plasmid maintenance and 200 uM acetosyringone. Immediately prior to inoculation of maize cells, Agrobacterium are preferably pelleted by centrifugation, washed in Vi MSVI medium (2.2 g/L GIBCO (Carlsbad, CA) MS salts, 2 mg/L glycine, 0.5 g/L niacin, 0.5 g/L L-pyridoxine-HCl, 0.1 mg/L thiamine, 115 g/L L-proline, 10 g/L D-glucose, and 10 g/L sucrose, pH 5.4) containing 200 uM acetosyringone, and resuspended at 0.1 to 1.0 x 10⁹ cells/ml in Vi MSPL medium (2.2 g/L GIBCO MS salts, 2 mg/L glycine, 0.5 g/L niacin, 0.5 g/L L-pyridoxine-HCl, 0.1 mg/L thiamine, 115 g/L L-proline, 26 g/L D-glucose, 68.5 g/L sucrose, pH 5.4) containing 200 uM acetosyringone. One of skill in the art may substitute other media for Vz MSVI or ^lA MSPL.

Immature maize embryos are isolated as described previously. Embryos are inoculated with Agrobacterium 0-7 days after excision, preferably immediately after excision. Alternatively, immature embryos may be cultured for more than 7 days. For example, embryogenic callus may be initiated as described above and co-cultured with Agrobacterium. Preferably, immature maize embryos are excised, immersed in an Agrobacterium suspension in ^lA MSPL medium prepared as described above and incubated at room temperature with Agrobacterium for 5-20 minutes.

Following inoculation embryos are transferred to one-half strength MS medium containing 3.0 mg/L 2,4-dichlorophenyoxyacetic acid (2,4-D), 1% D-glucose, 2% sucrose, 0.115 g/L L-proline, 0.5 mg/L thiamine-HCl, 200 uM acetosyringone, and 20 uM silver nitrate or silver thiosulfate. Immature embryos are co-cultured with Agrobacterium for 1 to 3 days at 23 °C in the dark. One of skill in the art may substitute other media for the described media.

Co-cultured embryos are transferred to medium 15AA (462 mg/L (NH4)SO4, 400 mg/L KH2PO4, 186 mg/L MgSO4-7H20, 166 mg/L CaC12-2H20, 10 mg/L MnSO4-H2O, 3 mg/L H3B03, 2 mg/L ZnSO4-7H20, 0.25 mg/L NaMoO4-2H20, 0.025 mg/L CuSO4-5H20, 0.025 mg/L CoC12-6H20, 0.75 mg/L KI, 2.83 g/L KNO3, 0.2 mg/L niacin, 0.1 mg/L thiamine-HCl, 0.2 mg/L pyridoxine-HCl, 0.1 mg/L D-biotin, 0.1 mg/L choline chloride, 0.1 mg/L calcium pantothenate, 0.05 mg/L folic acid, 0.05 mg/L p-aminobenzoic acid, 0.05 mg/L riboflavin, 0.015 mg/L vitamin B12, 0.5 g/L casamino acids, 33.5 mg/L Na2EDTA, 1.38 g/L L-proline, 20 g/L sucrose, 10 g/L D-glucose), or MS medium containing 1.5 mg/L 2,4-D, 500 mg/L carbenicillin, 3% sucrose, 1.38 g/L L-proline and 20 uM silver nitrate or silver thiosulfate and cultured for 0 to 8 days in the dark at 27°C without selection. Culture media used for selection of transformants and regeneration of plants preferably contains 500 mg/L carbenicillin. One of skill in the art may substitute other antibiotics that control growth of Agrobacterium. Other culture media that support cell culture may be used alternatively. In the absence of a delay of selection (0 day culture), selection may be initiated on 25 mg/L paromomycin. Selection medium may comprise medium 211 (described above) or a variant of medium 211 in which N6 salts are replaced by MS salts. After two weeks, embryogenic callus are transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. When selection is delayed following co-culture, embryos are initially cultured on medium containing 50 mg/L paromomycin followed by subsequent culture of embryogenic callus on medium containing 100- 200 mg/L paromomycin. One of skill in the art will culture tissue on concentrations of paromomycin which inhibit growth of cells lacking the selectable marker gene, but a concentration on which transformed callus will proliferate. Alternatively, one may use other selectable markers to identify transformed cells. It is believed that initial culture on 25 to 50 mg/L paromocyin for about two weeks, followed by culture on 50-200 mg/L paromoycin will result in recovery of transformed callus. Transformants are recovered 6 to 8 weeks after initiation of selection. Plants are regenerated from transformed embryogenic callus, e.g. as described in Example 5.

Example 3 Agrobacterium Mediated Transformation of Maize Callus

This example describes methods for transformation of maize callus using Agrobacterium. The method is exemplified using an nptll selectable marker gene and paromomycin selective agent. One of skill in the art will be aware of other selectable marker and selective agent combinations that could be used alternatively. Callus was initiated from immature embryos using methods known to those of skill in the art. For example, 1.5 mm to 2.0 mm immature embryos were excised from developing maize seed of a genotype such as LH59 and cultured with the embryonic axis side down on medium 211V (described in Example 1 above), usually for 8-21 days after excision. Alternatively, established an established callus culture may be initiated and maintained by methods known to those of skill in the art.

Agrobacterium was prepared for inoculation of plant tissue according to the methods described in Example 10. Fifty to 100 pieces of callus was transferred to a 60 mm X 20 mm petri dish containing about 15 ml of Agrobacterium suspension at 0.1 to 1.0 x 10⁹ cfu/ml. A piece of callus was usually all of the callus produced by an immature embryo in up to 21 days of culture or a piece of established callus of 2 mm to 8 mm in diameter. Callus was incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration.

About 50 mL of sterile distilled water was added to a Whatman #1 filter paper in a 60 mm x 20 mm petri dish. After 1-5 minutes, 15 to 20 pieces of callus were transferred to each filter paper and the plate sealed with PARAFILM®, for example. The callus and Agrobacterium were co-cultured for about 3 days at 23 °C in the dark.

Calli were transferred from filter paper to medium 211 with 20 mM silver nitrate and 500 mg/L carbenicillin and cultured in the dark at 27°C to 28°C for 2-5 days, preferably 3 days. Selection was initiated by transferring callus to medium 211 containing 20 mM silver nitrate, 500 mg/L carbenicillin and 25 mg/L paromomycin. After 2 weeks culture in the dark at 27°C to 28°C, callus was transferred to medium 211 with 20 mM silver nitrate, 500 mg/L carbenicillin and 50 mg/L paromomycin (medium 211QRG). Callus was subcultured after two weeks to fresh medium 211 QRG and further cultured for two weeks in the dark at 27°C to 28°C. Callus was then transferred to medium 211 with 20mM silver nitrate, 500 mg/L carbenicillin and 75 mg/L paromomycin. After 2-3 weeks culture in the dark at 27°C to 28°C, paromomycin resistant callus was identified. One of skill in the art would recognize that times between subcultures of callus are approximate and one may be able to accelerate the selection process by transferring tissue at more frequent intervals, e.g., weekly rather than biweekly.

Plants were regenerated from transformed callus, transferred to soil and grown in the greenhouse. Following Agrobacterium mediated transformation, medium 217 further contained 500 mg/L carbenicillin and medium 127T further contained 250 mg/L carbenicillin.

Example 4 Methods of microprojectile bombardment

Approximately four hours prior to microprojectile bombardment, immature embryos were transferred to medium 211SV (medium 21 IV with the addition of sucrose to 12%). Twenty five immature embryos were preferably placed in a 60 x 15 mm petri dish, arranged in a 5 x 5 grid with the coleoptilar end of the scutellum pressed slightly into the culture medium at a 20 degree angle. Tissue was maintained in the dark prior to bombardment.

Prior to microprojectile bombardment, a suspension of gold particles was prepared onto which the desired transgenic DNA construct was precipitated. Ten milligrams of 0.6 mm gold particles (BioRad) were suspended in 50 mL buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0). Twenty five mL of a 2.4 nM solution of the desired DNA was added to the suspension of gold particles and gently vortexed for about five seconds. Seventy five mL of 0.1M spermidine was added and the solution vortexed gently for about 5 seconds. Seventy five mL of a 25% solution of polyethylene glycol (3000-4000 molecular weight, American Type Culture Collection) was added and the solution was gently vortexed for five seconds. Seventy five mL of 2.5 M CaCl₂ was added and the solution vortexed for five seconds. Following the addition of CaCl , the solution was incubated at room temperature for 10 to 15 minutes. The suspension was subsequently centrifuged for 20 seconds at 12,000 rpm (Sorval MC-12V centrifuge) and the supernatant discarded. The gold particle/DNA pellet was washed twice with 100% ethanol and resuspended in 10 mL 100% ethanol. The gold particle/DNA preparation was stored at -20°C for up to two weeks.

The transgenic DNA construct was introduced into maize cells using the electric discharge particle acceleration gene delivery device (US Patent No. 5,015,580). The gold particle/DNA suspension was coated on Mylar® polyester sheets (Du Pont Mylar® polyester film type SMMC2, aluminum coated on one side, over coated with PVDC co-polymer on both sides, cut to 18 mm square) by dispersion of 310 to 320 mL of the gold particle/DNA suspension on a sheet. After the gold particle suspension settled for one to three minutes, excess ethanol was removed and the sheets were air dried. Microprojectile bombardment of maize tissue was conducted as described in U.S. Patent No. 5,015,580. AC voltage may be varied in the electric discharge particle delivery device. For microprojectile bombardment of LH59 pre-cultured immature embryos, 35% to 45% of maximum voltage was preferably used. Following microprojectile bombardment, tissue was cultured in the dark at 27°C.

Example 5 Selection of transformed cells

Transformants were selected on culture medium comprising paromomycin, based on expression of a transgenic neomycin phosphotransferase II (nptll) gene. Twenty four hours after DNA delivery, tissue was transferred to 21 IV medium containing 25 mg/L paromomycin (medium 211HV). After three weeks incubation in the dark at 27°C, tissue was transferred to medium 211 containing 50 mg/L paromomycin (medium 211G). Tissue was transferred to medium 211 containing 75 mg/L paromomycin (medium 211XX) after three weeks. Transformants were isolated following 9 weeks of selection.

Example 6 Regeneration of fertile transgenic plants Fertile transgenic plants are produced from transformed maize cells. Transformed callus was transferred to medium 217 (N6 salts, 1 mg/L thiamine-HCl, 0.5 mg/L niacin, 3.52 mg/L benzylaminopurine, 0.91 mg/L L-asparagine monohydrate, 100 mg/L myo-inositol, 0.5 g/L MES, 1.6 g/L MgCl₂-6H₂O, 100 mg/L casein hydrolysate, 0.69 g/L L-proline, 20 g/L sucrose, 2 g/L GELGRO™, pH 5.8) for five to seven days in the dark at 27°C. Somatic embryos mature and shoot regeneration began on medium 217. Tissue was transferred to medium 127T (MS salts, 0.65 mg/L niacin, 0.125 mg/L pyridoxine-HCl, 0.125 mg/L thiamine-HCl, 0.125 mg/L Ca pantothenate, 150 mg/L L-asparagine, 100 mg/L myo-inositol, 10 g/L glucose, 20 g/L L-maltose, 100 mg/L paromomycin, 5.5 g PHYTAGAR™, pH 5.8) for shoot development. Tissue on medium 127T was cultured in the light at 400-600 lux at 26°C. Plantlets are transferred to soil, preferable 3 inch pots, about four to 6 weeks after transfer to 127T medium when the plantlets are about 3 inches tall and have roots. Plants were maintained for two weeks in a growth chamber at 26°C, followed by two weeks on a mist bench in a greenhouse before transplanting to 5 gallon pots for greenhouse growth. R0 plants were grown in the greenhouse to maturity and reciprocal pollinations were made with the inbred LH59. Seed was collected from the R0 plants and used for further breeding activities. For each plant representing a transgenic event, Fl seed was planted in a field producing plants which were assayed for phenotype and for the selectable kanamycin resistant marker. Each of the plants were self pollinated to produce F2 seed. Seed from nptll-positive plants, e.g. a few ears from each transgenic event, was planted and grown to produce F2 plants which were assayed for phenotype and kanamycin resistance. Kanarnycin- resistant F2 plants were self pollinated to produce F3 seed. F3 seed was screened for complete resistance to kanamycin indicating a homozygous transgene. Seeds from homozygous F3 ears were planted in the field to produce F3 plants which were self pollinated to produce F4 seed. F3 plants were also crossed to tester inbred lines to produce Fl hybrid transgenic seed. Phenotypes such as yield are determined from Fl hybrid transgenic seed; other phenotypes can be determined from either Fl hybrid transgenic lines or Fl, F2, F3 or F4 inbred transgenic lines. A variety of transgenic plants were grown in field conditions allowing observation of multiple events of the unexpected phenotypes listed in Table 3.

Table 3

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 preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which 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.

All publications and patent applications cited herein are incorporated by reference in their entirely to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. Transgenic maize seed characterized by enhanced yield (as measured in weight of crop per area ) as compared to a corresponding yield of its parental maize line, obtained by introduction into the genome of said parental line of a transgenic DNA construct comprising a promoter operably linked to heterologous DNA, wherein said heterologous DNA encodes a protein having an amino acid sequence which is at least 60% identical to a sequence selected from the group consisting of SEQ ID NO:368 through SEQ ID NO:736.

2. Transgenic maize seed according to claim lwherein said heterologous DNA comprises a protein coding segment of DNA having at least 60% identity with a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 368.

3. Transgenic maize seed according to claim lwherein said enhanced yield is the result of improved plant growth under one or more stress conditions in the group consisting of drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density.

4. Transgenic maize seed according to claim lwherein said enlianced yield is also manifested by increase in number of kernels per ear, number of ears per unit planted area, or average weight of kernels.

5. Transgenic maize seed according to claim lwherein said enhanced yield is also manifested by increase in biomass per unit planted area or an increase in the root/shoot ratio.

6. Transgenic maize seed according to claim lwherein said enhanced yield is also manifested by increased efficiency by said transgenic plant in water use, nitrogen use or phosphorus use.

7. Transgenic maize seed characterized by enhanced quality in the plant morphology, physiology or seed as compared to a corresponding phenotype of a parental maize line, obtained by introduction into the genome of said parental line a transgenic DNA construct comprising a promoter operably linked to heterologous DNA, wherein said heterologous DNA encodes a protein having an amino acid sequence which is at least 80%) identical to a sequence selected from die group consisting of SEQ ID NO:369 tlirough SEQ ID NO:738.

8. Transgenic maize seed according to claim 7 wherein said heterologous DNA comprises a protein coding segment of DNA having at least 70% identity with a sequence selected from the group consisting of SEQ ID NO:l through SEQ ID NO: 368.

9. Transgenic maize seed according to claim 7 wherein said enhanced quality is shortened internode length, increased internode length, early leaf senescence, sterility, elongated tassel central axis, setting a second ear at high planting density, earlier time of germination, increased production of kernel oil or increased production of kernel protein.

10. Transgenic maize seed according to claim 9 having an enhanced phenotype of decreased internode length resulting from introduction of heterologous DNA coding for: (d) a TOCl-like receiver domain 3 having an amino acid sequence which is at least 60% identical to SEQ ID NO:436,

(e) a HY5-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO:565, or

(f) a proline permease having an amino acid sequence which is at least 60%> identical to SEQ ID NO-.371.

11. Transgenic maize seed according to claim 9 having an enhanced phenotype of increased internode length resulting from introduction of heterologous DNA coding for:

(c) a myb transcription factor having an amino acid sequence which is at least 60% identical to SEQ ID NO:717, or (d) an SVP-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO: 609.

12. Transgenic maize seed according to claim 9 having an enhanced phenotype of early leaf senescence resulting from insertion of heterologous DNA coding for a Cytochrome P450 having an amino acid sequence which is at least 60%) identical to SEQ ID NO:382.

13. Transgenic maize seed according to claim 9 having an enhanced phenotype of sterility resulting from insertion of heterologous DNA coding for:

(e) an RR3-like receiver domain 8 having an amino acid sequence which is at least 60% identical to SEQ ID NO:439,

(f) an ARR2-like receiver domain having an amino acid sequence which is at least 60% identical to SEQ ID NO:434, (g) an HSF protein having an amino acid sequence which is at least 60% identical to

SEQ ID NO.-487, or (h) an SVP-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO:609.

14. Transgenic maize seed according to claim 9 having an enhanced phenotype of elongated tassel central axis resulting from insertion of heterologous DNA coding for an SVP-like protein having an amino acid sequence which is at least 60% identical to SEQ ID NO:609.

15. A method for introducing into a maize line an enhanced phenotype as compared to a phenotype in parental units of said maize line, said method comprising (a) generating a population of transgenic plants comprising a variety of heterologous DNA for the transcription of which there is no known phenotype in corn,

(b) observing phenotypes for said transgenic plants,

(c) selecting seeds from transgenic plants having an unexpected enhanced phenotype, and

(d) optionally, repeating a cycle of germinating transgenic seed, growing subsequent generation plants from said transgenic seed, observing phenotypes of said subsequent generation plants and collecting seeds from subsequent generation plants having an enlianced phenotype.

16. A method according to claim 15 wherein said population of transgenic plants is produced by generating a plurality of transgenic events for a plurality of unique transgenic DNA constructs wherein each of said transgenic events comprises introducing into the genome of a parental maize line a single transgenic DNA construct comprising a promoter operably linked to heterologous DNA, wherein said transgenic DNA construct is introduced into said genome in sufficient quantity to produce transgenic cells which can be cultured into plants of transgenic maize comprising said population, culturing said transgenic cells into a population of transgenic plants producing progeny transgenic seed,

17. A method according to claim 16 wherein said plurality of transgenic events is at least 2 and said plurality of unique transgenic DNA constructs is at least 20.

18. A method according to claim 16 wherein said plurality of transgenic events is at least 2 and said plurality of unique transgenic DNA constructs is at least 50.

19. A method according to claim 16 wherein said DNA construct comprises heterologous DNA operably linked to the 5' end of a promoter region comprising a rice actin promoter and rice actin intron.

20. A method according to claim 16 further comprising crossing transgenic plants from said population with at least one other maize line to produce a hybrid population, observing phenotypes in said hybrid population and selecting seed from plants having an unexpected phenotype.