EP0759076A1 - Genes regulant la reponse des mais au deficit hydrique - Google Patents

Genes regulant la reponse des mais au deficit hydrique

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Publication number
EP0759076A1
EP0759076A1 EP95917777A EP95917777A EP0759076A1 EP 0759076 A1 EP0759076 A1 EP 0759076A1 EP 95917777 A EP95917777 A EP 95917777A EP 95917777 A EP95917777 A EP 95917777A EP 0759076 A1 EP0759076 A1 EP 0759076A1
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EP
European Patent Office
Prior art keywords
wilt
gene
maize
plant
plants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95917777A
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German (de)
English (en)
Inventor
Paul Chomet
Stephen L. Dellaporta
Peter Orr
Roger W. Krueger
Brenda A. Lowe
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Yale University
DeKalb Genetics Corp
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Yale University
DeKalb Genetics Corp
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Publication of EP0759076A1 publication Critical patent/EP0759076A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates generally to the field of maize growth and the response to varying conditions of water availability, the use of water, and resistance to water deficit.
  • the invention also concerns the genes involved in plant resistance to water deficit and availability of water. More particularly, it concerns the identification and various uses of genes involved in the process of wilting during periods of drought.
  • Quaternary ammonium compounds such as glycine-betaine
  • glycine-betaine have been implicated (McCue & Hanson, 1990) , as has proline which is thought to play an important role in osmoregulation in plants (Delauney & Verma, 1993) .
  • drought-resistant maize lines have been shown to contain thicker veins, larger epidermal cells, and a greater vessel cross sectional area per vascular bundle than drought sensitive lines (Ristic & Cass, 1991) . These differences at the cellular level may indicate that resistant plants have a greater potential to transport water which may allow faster adaptation to, and recovery from, water deficits. The molecular biology of osmoregulation is less understood.
  • LEA early embryogenic abundant
  • RAB responsive to abscisic acid
  • ABA abscisic acid
  • the LEA and RAB proteins are highly conserved and have been found in several plant species. These proteins contain conserved amino acid sequence motifs that are thought to form amphiphilic alpha helical structures which may have a functional role in preventing cellular damage during desiccation.
  • Cis-acting sequence motifs, ABREs (ABA responsive elements) have been identified in RAB promoters which are involved in transcriptional activation in response to ABA (Mundy et al . , 1990). While these studies suggest an association of specific genes with drought and desiccation, further direct evidence is needed.
  • a mutational approach has a distinct advantage over conventional physiological and molecular studies. Rather than a descriptive approach, mutations are used to define genes that are functionally implicated in this process based on mutant phenotypes. By relying on phenotypes. characterizing genes or gene products that are simply correlated with the process can be avoided. Moreover, by characterizing mutant phenotypes at the physiological and cellular levels, it is possible select those that are potentially more interesting. This provides a direct link between genes and biological responses at the cellular and whole plant levels.
  • Mutations are only one component of an approach to understanding wilt-related genes. Besides the requisite physiological and biochemical techniques, cloned genes are needed for functional studies of genes and the products they encode. In complex genomes, such as maize, chemical mutagens can be effective at defining loci but these alleles have little value in obtaining molecular probes. Nevertheless, both goals (mutant alleles and cloned genes) can be achieved using non-conventional mutagenesis techniques such as gene tagging methods. Maize, in particular, is well suited for such studies given the dearth of well-characterized transposable element systems and genetic strategies for gene tagging (Chomet, 1994; Dellaporta & Moreno, 1994; Cone, 1994) .
  • Transposable elements have enabled the isolation and characterization of numerous genes in several biosynthetic pathways, including, anthocyanins (Fedoroff et al . , 1984; Cone et al . , 1986; Dellaporta et al . , 1988; O'Reilly et al . , 1985; Wienand et al . , 1986; McLaughlin & Walbot, 1987), carotenoids (Buckner et al . , 1990), carbohydrate metabolism (Sullivan et al . , 1991), and the regulation seed storage protein synthesis (Schmidt et al . , 1987) to name a few.
  • anthocyanins Fedoroff et al . , 1984; Cone et al . , 1986; Dellaporta et al . , 1988; O'Reilly et al . , 1985; Wienand et al . , 1986; McLaughlin & Walbot, 1987
  • Maize is an ideal organism for these studies for many reasons. Maize is a model genetic system for plant studies because of well developed technologies for formal genetic and molecular analyses (Freeling & Walbot, 1994) . Because maize is an important agricultural plant, the results obtained will have direct relevance to an important agricultural problem.
  • the present invention in a general and overall sense, concerns plant genes, and particularly maize genes, that are involved in the maintenance of water relations in a plant.
  • Such genes are herein termed wilt genes (wilt) , due to the fact that disruption of these genes impairs a plant's normal balance of turgor potential and hence causes the plant to wilt.
  • a wilt gene in accordance with the invention is a gene that, when present within a plant, allows the plant to maintain proper internal water balance under normal conditions or under low water availability or contributes to such a response and a drought-resistance phenotype. It is envisioned that a wilt gene will encode one or more proteins or polypeptides that function in the mechanisms of drought-resistance or that stimulate the production of, or regulate, other proteins involved in drought- resistance.
  • a ready means to assess whether a gene or cDNA falls within the wilt gene group is to disrupt, inactivate or otherwise mutate the gene, and to assess whether plants bearing such a disrupted gene wilt under normal water level conditions. The precise mechanisms by which a wilt gene achieves its phenotypic effect are not relevant to the practice of the invention, given that the isolation and various uses of are wilt genes is disclosed herein.
  • the invention provides, in certain embodiments, purified DNA segments that include a maize wilt gene.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a given plant species, particularly maize. Therefore, a purified maize wilt gene DNA segment refers to a DNA segment that contains maize wilt gene coding sequences yet is isolated away from, or purified free from, total genomic DNA of Zea mays . Included within the term "DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • gene is used herein for simplicity to refer to DNA coding unit that encodes a functional protein, polypeptide or peptide, which coding unit is isolated substantially away from other genes or protein encoding sequences.
  • this functional term includes both genomic sequences and cDNA sequences.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case a wilt gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions (i.e., around 40-50 kb or larger) of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions.
  • Wilt-1256 is a 18 kilobase Sal I fragment isolatable by the methods disclosed herein in Examples I through III.
  • Recombinant vectors that include a wilt gene DNA segment form important further aspects of the present invention.
  • Particularly useful vectors are contemplated to be those vectors in which the wilt DNA segment is positioned under the control of a promoter.
  • the promoter will be one that is operable in maize plants.
  • the term "operable in maize plants" is used to describe a promoter that is capable of directing the expression of a gene or transcription unit in a maize cell or maize plant.
  • other promoters not operable in maize is also contemplated, such as may be employed in directing the expression of a recombinant protein in a bacterial host cell, as is commonly performed in the art.
  • a preferred embodiment of the present invention is the use of a promoter that is operable in maize plants, with the promoter that is naturally associated with a wilt gene in Zea may ⁇ being particularly preferred.
  • regions such as enhancers, silencers and transcriptional activation sequences that may also be used, and are encompassed by the present invention.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a wilt gene in its natural environment.
  • Such promoters may include promoters normally associated with other maize genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or even a mammalian cell.
  • promoters such as the Cauliflower Mosaic Virus 35S, rice actin, rbcS, ⁇ -tubulin and oc ⁇ promoters.
  • the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al . (1989).
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous for inducing an increased resistance to water deprivation in a plant or simply to provide high level production of recombinant protein.
  • Further embodiments of the invention concern methods for preparing a wilt gene DNA segment. Certain preferred methods are those involving a technique known in the art as transposon tagging. To obtain a wilt gene DNA segment in this manner one would generally, first, obtain a collection of maize plants bearing transposon induced mutations and screen the group of plants to identify one or more plants that exhibit a wilt phenotype. The process of obtaining or preparing a collection of maize plants bearing transposon induced mutations involved identifying plants that harbor an active transposon system.
  • transposon systems i.e., activator
  • activator it is possible to select individual plants in which the transposon has moved to different locations in the genome (Chomet, 1994; Dellaporta & Moreno, 1994, Cone, 1994, incorporated herein by reference) .
  • Such plants are then either selfed or crossed to plants carrying a previously identified wilt mutation.
  • the selfed progeny or test crossed progeny are then screened for the wilt phenotype.
  • Plants that exhibit the wilt phenotype are readily identifiable by simply growing the collection, or population, of plants under normal water levels and detecting those plants that exhibit decreased turgor, i.e., visibly wilt, leaf roll, or droop, under such conditions or by identifying those plants that show significantly decreased turgor in comparison to other plants in the collection.
  • the precise mechanisms by which the mutations that affect water balance achieves the identified phenotypic effects are not relevant to the practice of the invention, it is clear, however, that leaf roll precedes wilting.
  • Leaf roll was effective in providing an early indication of wilting, for example, as early as the third leaf stage.
  • a genomic library from such a plant or plants. That is, one would obtain DNA from the nuclei of cells from such a plant and formulate the DNA so that it contains smaller, distinct pieces of DNA that can identified as distinct from other pieces - using any one of a variety of structural and functional characteristics.
  • the library of DNA thus formed would than be screened to identify a genomic clone that comprises the wilt gene in association with a transposon. This screening process will generally involve using a probe directed to a transposon sequence to identify the transposon in combination with the wilt gene.
  • a number of transposons that induce mutations may be used with the present invention, with the Ac and D ⁇ transposons being particularly preferred.
  • Such methods generally comprise identifying a DNA sequence that flanks the transposable element located within the genomic clone first isolated and using the flanking sequence in another round of molecular screening.
  • the flanking sequence would be employed to screen a cDNA library prepared from a wild type plant and, by identifying sequences that hybridize, to thus identify one or cDNA clones that include a wilt gene.
  • Suitable methods for preparing and screening cDNA libraries will be generally known to those of skill in the art of molecular biology in light of the present disclosure and published references such as, for example, Sambrook et al . (1989; incorporated herein by reference).
  • Yet further aspects of the present invention concern plants, particularly maize plants, that have been stably transformed with a purified wilt gene DNA segment, such as the wilt-1256 18 kb DNA segment.
  • the stably transformed plants will include a recombinant vector that comprises a wilt gene that is positioned under the control of a promoter that is operable in maize plants and will thus direct the expression of the wilt gene in the plant.
  • Exemplary promoters that may be used in this regard include the
  • the present invention thus also provides methods for preparing plants, particularly maize plants, that have increased turgor, i.e., exhibit an increased resistance to water deprivation or drought.
  • Such methods involve stably introducing a purified wilt gene DNA segment, such as the 18 kb wilt-1256 DNA segment, into the genome of a host plant in a manner effective to result in expression of the wilt gene.
  • the gene will be introduced in the form of a recombinant vector and will be under the control of a promoter operable in maize plants. Any means of introducing the DNA segment into the host plant may be employed including, for example, electroporation and microprojectile bombardment.
  • Another embodiment of the present invention is a method for the selective breeding of plants that are resistant to water deprivation.
  • the method includes, generally, obtaining a collection of maize plants and screening for increased expression, copy number, or message stability of a wilt gene DNA segment.
  • Those isolates having a favorable genotype, that is an increase in expression, copy number or message stability are tested for increased resistance to water deprivation followed by breeding of those isolates having increased resistance to water deprivation.
  • the progeny are then screened with a wilt gene DNA segment in accordance with the present invention, and the favorable genotypes are tested resistance to water deprivation and once again bred.
  • DNA segments of the present invention encompass biologically functional equivalent wilt genes and their corresponding proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e . g. , to introduce improvements to the stability or specific expression of the resultant protein or to test wilt mutants in order to examine the mechanisms underlying wilting activity at the molecular level.
  • Transposon tagging has several benefits over conventional gene cloning strategies. No prior knowledge of the gene product or even previous identification of the gene is a prerequisite for transposon tagging. It can be used to clone and study genes whose products are rarely expressed. Insertional mutations often completely or partially block gene function, resulting in phenotypic alterations. This phenotypic alteration often implicates the gene or its product in a particular developmental or biochemical pathway. Transposon tagging strategies can operate efficiently in organisms with large genomes, even with great amounts of repetitive DNA. Genes for disease resistance, nutritional qualities, and developmental processes can thus be tagged and cloned. Mutant alleles can also serve as the basis for saturation mutagenesis and fine structure analysis of complex loci.
  • An essential feature for developing an efficient transposon tagging system is the ability to efficiently recover transposition events.
  • Two genetic features of Ac transposition were exploited to develop an efficient recovery strategy.
  • the inventors' strategy relies on detecting the lineage containing the filled donor site and tr-Ac element. This lineage contains two Ac elements (the original donor Ac and the transposed Ac element) .
  • the increase in Ac copy number results in a dosage effect - transposition is developmentally delayed.
  • any Ds reporter allele will suffice, however.
  • the developmental pattern of Ds transposition e.g. early verses late
  • the inventors recovered approximately 10,000 kernels containing tr-Ac elements (Ml kernels) by using the Ds reporter system to recover high dose Ac kernel progeny.
  • the efficiency of recover was greater than 75%.
  • control populations coarse variegated sib kernels
  • the efficiency of transposed Ac recover was less than 10%.
  • the Ml kernel population was screened four times. First, Ml kernel populations and control siblings were field grown and screened for dominant mutations. Mutations involved in the flowering process such as early or late flowering phenotypes, floral morphological transformations, male and female sterility and mutations affecting vegetative characters such as ligule and leaf development, or tillering were sought. All Ml and control plants were self-pollinated to generate M2 and F2 control progeny populations. Mutants were also testcrossed to Ac tester stocks to identify Ac-linked phenotypes.
  • the second screen was for mutations affecting seed characteristics such as endosperm morphology and defective kernels. This screen was done directly on the M2 and F2 ears. For instance, mutants in starch, protein, and carotenoid pathways were detected in endosperm cells. From this screen, mutations affecting kernel development (defective and embryoless kernels) were recovered. Several mutations affecting starch biosynthesis (shrunken (sh) and brittle (bt) ) were also recovered.
  • the third screen involved the growing the first seed set in sand benches to score for percent germination and aberrant seedling phenotypes such as chlorophyll pigmentation changes and disease susceptibility.
  • Two sets of twenty kernels were selected randomly from each M2 and F2 family. From this screen many mutations affecting seedling development (lethals, pale-greens, albinos, yellow-greens, dwarfs, high chlorophyll fluorescence, disease lesion mimics, and the like) were recovered.
  • the fourth screen involved growing the second seed set in field plots and scoring for segregation of vegetative and floral phenotypes. From this screen mutations affecting leaf morphology (liguleless, wilts, knotted, etc.) and inflorescence morphology (tasselseeds) were recovered.
  • Transposable elements have been useful in the identification and cloning of genes that were previously inaccessible by other cloning methods.
  • three transposable element systems have been utilized for transposon tagging: Activator /Dissociation, Suppre ⁇ or- mutator (Enhancer /Inhibitor) , and Robertson's Mutator.
  • Activator /Dissociation Suppre ⁇ or- mutator (Enhancer /Inhibitor)
  • Robertson's Mutator Each system has inherent advantages and disadvantages,and the choice of system depends on the tagging approach taken, the genomic position and the expression of the gene sought, as well as the stocks available for tagging.
  • the Mutator transposable element family was originally identified in lines that exhibited an unusually high frequency of forward mutation (Robertson, 1978) . Extensive genetic and molecular analyses have demonstrated that the increase in mutation frequency is caused by a family of transposable elements, designated Mutator (Mu) elements.
  • the Mutator system consists of more than eight different classes of Mu transposable elements each of which can be found in multiple copies. Each element class is defined by a unique internal sequence flanked by inverted repeats about 200 bp long common to all Mu elements. Germinal transposition (and presumably forward mutation) and somatic excision of the non-autonomous Mu elements are under the control of the autonomous MuRl element (Chomet et al . , 1991), now designated MuDR-1.
  • MuA2 Qin et al. , 1991
  • Mu9 Hershberger et al . , 1991
  • Lines harboring the autonomous element(s) are referred to as Active Mutator lines.
  • the Mu system has been useful in cloning a number of genes, including al (O'Reilly et al . , 1985), bz2 (McLaughlin & Walbot, 1987), vpl (Mccarty et al . , 1989), hcfl06 (Martienssen et al . , 1989), yl (Buckner et al . , 1990), and hml .
  • At least 10 s gametes should be screened for a directed tagging study. Furthermore, it is important to have prior knowledge of the spontaneous mutation rate for the targeted locus. Some loci can be highly unstable in the absence of a transposable element system (Pryor, 1987; Stadler, 1948). In the case of directed tagging of dominant loci, it is possible to "knock out" the dominant mutant allele with Mu (Hake, 1992) . In this case, a Mutator line homozygous for the dominant allele is crossed (as a female) to a wild-type tester. The F, is screened for phenotypically wild-type plants. This presumes a deletion heterozygote for the given locus is phenotypically wild-type. Such an assumption may not always be the case and tests should be undertaken to determine the phenotype of the hemizygote before proceeding with the screening of the F, population.
  • the nontargeted approach allows the investigator to identify new, uncharacterized mutations as well as to identify new mutations with previously characterized phenotypes.
  • Active Mutator lines should be crossed by a non-Mutator line (inbred) or a tester line carrying a Mu- induced unstable marker allele. (See non-autonomou ⁇ genetic marker, below) .
  • the Fj seed is selfed to produce F 2 populations; 20-40 seeds from each F 2 ear are then screened for the mutant phenotype(s) .
  • lethal or sterile phenotypes can be identified as homozygotes and recovered as heterozygotes in the population.
  • Robertson devised an assay for Mutator activity that give an estimate of the forward mutation frequency of Mutator plants (Robertson, 1978) .
  • the test is performed by selfing and crossing the Mutator plant as a male to a hybrid non-Mutator line to produce Fj seed.
  • the second ear on the non-Mutator plant is selfed as well.
  • F, seed, from plants which did not segregate for a visible mutant (as determined by the parental selfs) are sown and plants are selfed to produce F 2 ears. Kernels from these F 2 ears are planted in a sand bench and observed for new seedling traits (i.e., albino, yellow, yellow-green, etc.). With active Mutator lines, Robertson reported approximately 10% of the F 2 families exhibited a new seedling mutation (1980) .
  • Non-autonomous Mu insertion alleles report on the presence or absence of MuDR, the regulator element (Chomet et al . , 1991).
  • the presence or absence of spotting of a Mu-induced insertion allele reports only on the somatic reversion of one Mu element (such as Mul as bzlmum ⁇ , or Mul at al-mum2) .
  • This is in contrast to the Robertson test, which can measure th forward mutation or insertion of diverse Mu into many different loci. Since these two assays do not measure the same event, use of one assay is not always a substitute for another. However, there is a correlation between an increase in forward mutation rate with lines containing multiple regulator elements that exhibit a high frequency of spotting.
  • Plants that segregate for one MuDR-1 usually do not exhibit a high forward mutation rate (Robertson & Stinard, 1989) .
  • a mutable Mu allele as a marker for Mu activity, it is best to propagate the high spotting pattern. This will likely select for an increased number of MuDR-1 elements in the stock (Chomet et al . , 1991).
  • a homozygous stock can be utilized to screen for germinal reversion events; revertants are a useful tool in molecular identification of a tagged locus.
  • non- Mutator lines will be useful for subsequent molecular analyses. Identifying the original, tagged locus of interest will be important and can be accomplished because RFLPs associated with the locus can be associated within a given line. Crossing to different lines will also allow the production of F 2 populations segregating for the mutant allele. These populations are necessary for subsequent molecular analyses. Furthermore, introduction of the mutant allele into a number of different lines will also insure expression and subsequent recovery of the mutant in later generations because expressivity or penetrance of the mutant can be affected by genetic background.
  • Mutator line is recessive for Al , CI, Bzl or Bz2 (or other anthocyanin loci) , it is advantageous to outcross the mutant to a line lacking Mutator activity and homozygous for a Mu-induced allele of the same gene (such as bz-mum9, or aal-mum2 ) .
  • This allows selection in the Fj generation of seed that carried or lacked Mutator activity. These seed can then be grown, selfed, and screened for the mutant phenotype. Production of populations segregating for the mutation of interest and lacking Mutator activity facilitates molecular analyses. New Mu fragments are not generated, and existing, unliked Mu fragments segregate out of the line with subsequent outcrosses.
  • Identifying the gene responsible for the mutant phenotype involves screening for a Mu-homologous fragment that cosegregates with the mutant phenotype. This is done by examining the DNA of the segregating population(s) (as produced above) by Southern blot analyses. The preliminary screen is expedited by examining a small population first. As many different outcrossed segregating lines should be examined as possible (See, Walbot, 1992) . It is also useful to examine the population utilizing a number of different restriction enzymes, since segregating fragments may be obscured by other Mu homologous bands. The population should also be screened with probes to all known Mu elements. Inclusion of DNA from t he parent lines on these blots is also important.
  • a cosegregating fragment should not be present in the parental plant. Once a cosegregating fragment is identified, additional analyses with different populations and a larger population set should be performed. Furthermore, as noted above, Mu- induced suppressible alleles can confound the cosegregation analysis (Martienssen et al . , 1989; 1990). For this reason, it is important to emphasize linkage of a fragment with mutant individuals may be genotypically mutant.
  • flanking probe is then used to prove the locus is responsible for the mutant phenotype. This can be accomplished in a variety of ways:
  • RNA hybridization can facilitate identification of the correct clone in special cases where the expression of the locus is well understood, as was done for Bz2 (McLaughlin & Walbot, 1987) . Hybridization were performed with the putative clones to RNA isolated from various tissues or allelic variants that showed a predicted pattern of expression.
  • Independent transpositions of Ac from a donor locus are selected by excision of Ac and its reinsertion elsewhere in the genome.
  • Recovery of the transposed Ac element in the heterozygous condition is possible in kernel progeny using Ds-induced reporter genes.
  • Ds-induced reporter genes Using this strategy, and a slightly modified version, over 10,000 independent transpositions of Ac have been recovered and screened for mutations.
  • Plants carrying transposed Ac elements are field-grown, screened for dominant mutations, and self-pollinated to uncover recessive Ac-induced mutations.
  • the F2 progeny are screened for recessive mutations. By this approach, several hundred mutations have been identified in transposed Ac families.
  • wilt-1256 and wilt-6945 were shown to be linked to the donor P locus (source of Ac) on chromosome IS. Linkage of the wilt mutants to the donor P locus is expected, in some cases, since Ac usually transposes to nearby chromosomal sites. The genetic location of the other two mutations is unknown.
  • the wilt-1256 mutant showed the first signs of leaf roll at the third leaf stage. Young expanding leaves were severely leaf rolled, yet older leaves appeared to recover and support growth of the plant. In contrast, wilt-1269 mutants did not show any visible signs of leaf roll until just prior to flowering.
  • the wilt-1 mutant (obtainable from the Maize Genetics Cooperation Stock Center, E.B. Patterson, S-118 Turner Hall, Agronomy Department, University of Illinois, 1102 S. Goodwin Avenue, Urbana, IL 61801) exhibited wilting in late vegetative growth. Plants homozygous for wilt-1 reached maturity and produced seed, although plants were generally reduced in stature.
  • wilt-1270 and wilt-6945 are seedling lethals, i.e., mutant plants grew to the 4-5 leaf stage, leaves wilted, growth ceased, and cell necrosis eventually killed the plant.
  • this characteristic is not linked to the early wilting effect.
  • wilt-1270 and wilt- 6945 were seedling lethals while wilt-1256 wilted during this same developmental period, yet recovered.
  • additional wilt mutations may be employed to define additional genes involved in the water stress response pathway. Fifteen additional wilt mutations were identified in Ac-Ds and Robertson's Mutator screens. All mutants are regrown to confirm Mendelian inheritance patterns and dominant, codominant, recessive relationships to wild-type. Pairwise complementation tests between each mutations is performed. In addition, a directed tagging study of wiltl-1 using the transposable element Mutator is performed. The testcross population of over 100,000 kernels is screened to identify wilt-1 alleles.
  • exogenous ABA application may be used to identify mutants that can be rescued or identify mutants that show differential sensitivity.
  • Standard genetic tests can be used to define potential mutants that are linked to transposed Ac or Mutator transposable elements (Dellaporta & Moreno, 1994) .
  • Complementation analysis determines the number of independent loci that mutate to give the wilt phenotype. Based on this information, selected mutations are characterized further and cloned.
  • the loci are mapped by bulk segregant analysis (Michelmore et al . , 1991).
  • Wilt-1270 another Ac-derived wilt phenotype mutant is mapped in this manner. Briefly, the mutant is outcrossed to a different genetic background (e.g. B73) and Fl plants are self-pollinated. DNA from normal and mutant F2 plants are pooled and bulked DNA is hybridized with probes from known linkage groups. Unlinked probes hybridize with equal intensity (linkage equilibrium) to both bulked DNA samples while probes linked to the mutation show differential hybridization signals (linkage disequilibrium) . These studies help map genes on the maize genome involved in the response to water stress which may be important for future studies. EXAMPLE II MOLECULAR CHARACTERIZATION OF WILT MUTATIONS
  • the wilt-1256 gene is cloned according to the procedures disclosed by Federoff (Federoff et al . , 1984; Federoff, 1988; both references incorporated herein by reference) .
  • a Sal I genomic library was constructed in EMBL3 using DNA isolated from a mutant plant.
  • One clone was isolated which contained the A ⁇ -hybridizing 18 kb Sal I genomic fragment ( ⁇ 5Sal, FIG. 1) .
  • This clone was shown by restriction mapping to contain a transposed Ac element.
  • Unique probes were isolated from DNA flanking the Ac element of this clone (shown in FIG. 1 as 0.4 B/S) and used to probe genomic blots.
  • the second criteria used to establish identity is the analysis of revertant alleles detected as Ac excision events from the wilt-1256 allele. Phenotypic reversion is accompanied by excision of Ac if the cloned sequence is indeed the wilt-1256 gene. Either criteria provides the necessary data to confirm the identity of the clone.
  • cDNA clones of the wilt-1256 locus are analyzed. Clones that hybridize to DNA probes adjacent to the Ac insertion are isolated from a cDNA library made from maize shoot apices (leaves and shoots) . These clones are restriction mapped and hybridized back to genomic DNA to confirm their identity as wilt-1256 sequences. Part of this genomic DNA has been sequenced and determined to be an open reading frame as shown below:
  • sequence data of the wilt-1256 gene and its predicted protein product are analyzed via computer algorithms such as BLAST (Altschul et al., 1990) and BLOCKS (Henikoff & Henikoff, 1991) to search for the presence of functional domains in the wilt-1256 protein.
  • BLAST Altschul et al., 1990
  • BLOCKS Henikoff & Henikoff, 1991
  • This information may be useful in the design of future biochemical and physiological studies. For example, it is contemplated that this analysis may suggest a regulatory (e.g. DNA binding protein) or biochemical (e.g. enzymatic activity) role of the wilt-1256 protein by its similarity to known proteins.
  • the wilt-1256 coding region is subcloned into E. coli expression vectors for production of protein.
  • the protein is tagged with a peptide (polyhistidine or maltose binding protein) for affinity purification.
  • Protein is purified by metal chelation chromatography using commercially available resins (Le Grice & Grueninger-Leitch, 1990) or by maltose affinity techniques (Kellermann & Ferenci, 1982; Guan et al . , 1988) . Polyclonal antibodies to the fusion protein are produced and used in immunolocalization studies.
  • the temporal and spatial expression pattern of the wilt-1256 gene is important in understanding the function of this gene. It is contemplated that one may want to know whether gene expression is constitutive or induced in leaf cells.
  • Northern hybridization analysis (Ausebel et al . , 1992)
  • tissue-specific expression of mRNA (leaf, root, stem, inflorescence, etc.)
  • Polyclonal antibodies and Western analysis (Harlow & Lane, 1988) are used to determine protein expression patterns.
  • Both water-stressed, unstressed, and ABA-treated tissue are analyzed by Northern hybridization and Western analysis to determine whether induction occurs (RNA or protein) .
  • RNA or protein Detailed characterization of the regulation of expression of wilt-1256 regulation is the result of these studies.
  • Northern analysis will indicate whether steady-state mRNA levels are affected by water stress, yet Western analysis may indicate water deficits affect protein levels. This result would suggest regulation is post-transcriptional and would be further investigated. It is further contemplated that regulation of wilt-1256 activity may be at the level of RNA transcription or protein processing.
  • riboprobes sense and antisense
  • antibodies pre-immune and post-immune
  • mRNA and proteins are used to localize mRNA and proteins, respectively, in particular cell types. For example, if Northern and Western results indicate that wilt-1256 is only expressed in leaves, the cell-type specificity (mesophyll, bundle sheath, epidermal, veins, etc.) of wilt-1256 mRNA and protein localization are determined.
  • wilt mutations allow the water stress response pathway to be defined using formal genetic studies. For example, complementation studies with existing and forthcoming wilt mutants determines the number of genes that contribute to the wilt phenotype. Each available wilt mutant is crossed systematically to all other mutants. For viable mutants, homozygous lines are crossed reciprocally. For lethal mutations, heterozygous lines are employed. Complementation groups are assigned. Double mutant lines are constructed with complementing mutations to determine genetic epistasis. To follow each allele, maize RFLPs or microsatellite markers are employed for genotyping purposes (Hosington, 1987) . Homozygous double mutants are examined to determine whether an interaction between gene exists. For example, it is contemplated that mutations may be additive, synergistic, antagonistic, or epistatic to one another. Mutations that define independent pathways are additive while those that lie in a shared signal transduction pathway show epistasis.
  • mutant phenotypes may preclude a formal epistasis analysis. Nevertheless, single and double mutant lines are useful for defining molecular interactions. For example, it is contemplated that one can determine whether gene or protein expression is affected by mutation(s) at other loci using wilt-1256 probes. This result indicates that certain genes lie upstream (e.g. regulate) of wilt-1256 function.
  • the molecular characterization of wilt-1256 will determine the temporal and spatial pattern of gene expression. This information indicates which tissues and cells contain the primary defect.
  • tissue and cellular morphology of wilt-1256 mutants and wild type siblings under normal and water deficit conditions are examined. Histological analysis of stressed and unstressed tissue from mutant and wild-type plants are performed (Sylvester & Ruzin, 1994) . Cross sections through the appropriate tissues (e.g. leaves or roots) reveal any structural aberrations. Transmission and scanning electron microscopy are used to characterize mutant and wild-type cell structure and epidermal surfaces. Leaf surfaces are also examined for normal stomate structure and density using epidermal peels (Ristic & Cass, 1991) .
  • the relationship between the wilt phenotype and the phytohormone ABA is investigated. Mutant rescue studies using ABA are performed on wilt-1256 mutants to determine whether ABA can rescue the phenotype (wilting and/or lethality) or whether the mutant is hypo- or hyper-sensitive to ABA when compared to its normal sibs. If wilt-1256 reveals an increased or decreased ABA sensitivity, this suggests that an important step in ABA synthesis or reception is affected. To further investigate this relationship, the ABA content of seedling leaves and roots is measured using a radioimmuno-based assay (Quarrie et al . , 1988). This assay helps to identify whether the defect is in ABA synthesis or reception. For example mutations in the ABA biosynthetic pathway are expected to have reduced levels of ABA in the leaves and/or roots, whereas, ABA receptor mutants have normal or elevated levels.
  • Vectors are constructed that drive expression of a wilt gene in Zea may ⁇ cells.
  • a vector is constructed to direct constitutive expression.
  • the Cauliflower Mosaic Virus 35S promoter (Odell et al . , 1985) is placed 5' of the wilt gene.
  • the rice actin gene promoter (Wang et al . , 1992) is placed 5' of the wilt gene. It is anticipated that all promoters which direct constitutive gene expression in maize are useful when operably linked to a wilt gene. Sequences which direct polyadenylation are linked 3' to the wilt gene.
  • tissue specific expression of a wilt gene will enhance the agronomic performance of a maize plant.
  • Vectors for use in tissue- specific targeting of wilt genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences. Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure.
  • ricS promoter specific for green tissue
  • oc ⁇ no ⁇ and ma ⁇ promoters which have higher activity in roots or wounded leaf tissue
  • a truncated (-90 to +8) 35S promoter which directs enhanced expression in roots
  • an ⁇ -tubulin gene that directs expression in roots and promoters derived from zein storage protein genes which direct expression in endosperm.
  • oc ⁇ octopine synthase
  • wilt genes in transgenic plants may be desired under specified conditions.
  • the expression of wilt genes may be desired only under actual stress conditions.
  • rbcS encoding the small subunit of ribulose bisphosphate carboxylase
  • Other genes are induced by secondary stimuli.
  • ABA abscisic acid
  • a number of genes have been shown to be induced by ABA (Skriver & Mundy, 1990) . Promoter regions that regulate expression of these genes will be useful when operably linked to a wilt gene.
  • tissue-specific promoter sequences for use in accordance with the present invention.
  • one may first isolate cDNA clones from the tissue concerned and identify those clones which are expressed specifically in that tissue, for example, using Northern blotting.
  • tissue concerned a tissue concerned
  • identify those clones which are expressed specifically in that tissue for example, using Northern blotting.
  • the promoter and control elements of corresponding genomic clones may then be localized using the techniques of molecular biology known to those of skill in the art.
  • a wilt gene in a transgenic plant will be desired only in a certain time period during the development of the plant. Developmental timing is frequently correlated with tissue specific gene expression. For example, expression of zein storage proteins is initiated in the endosperm about 15 days after pollination.
  • Wilt genes may be introduced into maize cells including, but not limited to, cultured cells or immature embryos.
  • Transformable cell lines of maize are developed using the protocols familiar to one of skill in the art, including, but not limited to the following. The composition of each culture medium is listed in Table 2.
  • the first suspension cultures of AT824 were initiated 31 months after culture initiation. Suspension cultures were initiated in a variety of culture media including media containing 2,4-D as well as dicamba as the auxin source, e.g., media designated 210, 401, 409, 279 (see Table 2) . Cultures were maintained by transfer of approximately 2 ml packed cell volume to 20 ml fresh culture medium at 3 % day intervals. AT824 can be routinely transferred between liquid and solid culture media with no effect on growth or morphology. Suspension cultures of AT824 were initially cryopreserved 33-37 months after culture initiation. The survival rate of this culture is improved when it is cryopreserved following three months in suspension culture. AT824 suspension cultures have been cryopreserved and re-initiated from cryopreserved cells at regular intervals since the initial date of freezing. Repeated cycles of freezing have not affected the growth or transformability of this culture.
  • Table 2 Illustrative Embodiments of Tissue Culture Media Which are Used for Type II Callus Development, Development of Suspension Cultures and Regeneration of Plant Cells (Specifically Maize Cells)
  • Transformable cell cultures are routinely developed from the genotype Hi-II using the following protocol.
  • the Hi-II genotype of corn was developed from an A188 x B73 cross. This genotype was developed specifically for a high frequency of initiation of type II cultures (100% response rate, Armstrong et al . , 1991).
  • Immature embryos (8-12 days post-pollination, 1 to 1.2 mm) are excised and cultured embryonic axis down on N6 medium containing 1 mg/L 2,4-D, 25 mM L-proline (201, see Table 2) or N6 medium containing 1.5 mg/L 2,4-D, 6mM L-proline (734, see Table 2) .
  • Type II callus can be initiated either with or without the presence of 100 ⁇ M.
  • AgN0 3 Cultures initiated in the presence of AgNo 3 are transferred to medium lacking this compound 14-28 days after culture initiation.
  • Hi-II type II callus is maintained by manual selection of callus at each transfer.
  • callus can be resuspended in liquid culture medium, passed through a 1.9 mm sieve and replated on solid culture medium at the time of transfer. It is believed that this sequence of manipulations is one way to enrich for recipient cell types.
  • Regenerable type II callus that is suitable for transformation can be routinely developed from the Hi-II genotype and hence new cultures are developed every 6-9 months. Routine generation of new cultures reduces the period of time over which each culture is maintained and hence insures reproducible, highly regenerable, cultures that routinely produce fertile plants.
  • DNA is introduced into cultured cells as follows. Cultured cells are subcultured to fresh medium 409 (see Table 2) two days prior to particle bombardment. If grown in liquid medium cells are plated on solid 409 (Table 2) medium 16-24 hours before bombardment (about 0.5 ml packed cell volume per filter). Tissue is treated with 409 (Table 2) medium containing 200 mOsm sorbitol (medium 431, see Table 2) for 1 hour prior to bombardment.
  • DNA is introduced into cells using the DuPont Biolistics PDSlOOOHe particle bombardment device.
  • DNA is precipitated onto gold particles as follows.
  • a stock solution of gold particles is prepared by adding 60 mg of 1 ⁇ m gold particles to 1000 ⁇ l absolute ethanol and incubating for at least 3 hours at room temperature followed by storage at -20°C. Twenty to thirty five ⁇ l sterile gold particles are centrifuged in a microcentrifuge for 1 min. The supernatant is removed and one ml sterile water is added to the tube, followed by centrifugation at 2000 rpm for 5 minutes.
  • Microprojectile particles are resuspended in 30 ⁇ l of DNA solution (30 ⁇ g total DNA) .
  • the DNA solution contains a vector containing a chimeric wilt gene and a vector containing a selectable marker gene, e.g., the bar gene which is used for selection of transformants based on resistance to the herbicide bialaphos (Gordon-Kamm et al . , 1990).
  • Two hundred twenty microliters sterile water, 250 ⁇ l 2.5 M CaCl 2 and 50 ⁇ l spermidine are added. The mixture is thoroughly mixed and placed on ice, followed by vortexing at 4°C for 10 minutes and centrifugation at 500 rpm for 5 minutes. The supernatant is removed and the pellet resuspended in 600 ⁇ l absolute ethanol. Following centrifugation at 500 rpm for 5 minutes the pellet is resuspended in 36 ⁇ l of absolute ethanol.
  • immature embryos may be directly subjected to microprojectile bombardment.
  • Immature embryos (1.2 - 2.0 mm in length) are excised from surface-sterilized, greenhouse-grown ears of Hi-II 11-12 days post-pollination.
  • the Hi-II genotype was developed from an A188 x B73 cross for high frequency development of type II callus from immature embryos (Armstrong et al . , 1991).
  • Embryos are cultured in the dark for two days at 24°C.
  • embryos are transferred to the above culture medium with the sucrose concentration increased from 3% to 12%.
  • sucrose concentration increased from 3% to 12%.
  • embryos are transferred to the high osmoticum medium they are arranged in concentric circles on the plate, starting 2 cm from the center of the dish, positioned such that their coleorhizal end is orientated toward the center of the dish.
  • two concentric circles are formed with 25-35 embryos per plate.
  • the plates containing embryos are placed on the third shelf from the bottom, 5 cm below the stopping screen.
  • the 1100 psi rupture discs are used. Each plate of embryos is bombarded once. Embryos are allowed to recover overnight on high osmotic strength medium prior to initiation of selection.
  • DNA will be introduced into cells using any one of a number of techniques from which it is possible to recover fertile transgenic plants.
  • fertile transgenic plants are produced following electroporation of cells as described in Krzyzek et al, incorporated herein by reference.
  • Transformants are selected based on resistance to a toxic compound for which the introduced gene confers resistance. For example cells transformed with a gene encoding a glyphosate resistant EPSPS protein are resistant to the herbicide glyphosate. Similarly, cells transformed with a gene encoding neomycin phosphotransferase II are resistant to the antibiotics kanamycin or G418. It is anticipated that other selectable marker genes will be useful for identification of transformants. In this example transformants are identified based on resistance to the herbicide bialaphos conferred by the introduced jbar gene. Following one week culture in liquid medium 409 (Table 2) without selection pressure, particle bombarded tissue is transferred to liquid medium 409 (Table 2) containing 1 mg/L bialaphos.
  • transformants are recovered. Embryos are allowed to recover on high osmoticum medium (735, 12% sucrose, see Table 2) overnight (16 - 24 hours) and are then transferred to selection medium containing 1 mg/1 bialaphos (739 or 750, see Table 2) . Embryos are maintained in the dark at 24° C. After three to four weeks on the initial selection plates about 90% of the embryos form Type II callus and are transferred to selective medium containing 3 mg/1 bialaphos (758, Table 2) . Responding tissue is subcultured about every two weeks onto fresh selection medium (758, Table 2). Transformants are identified six to eight weeks after bombardment.
  • Plants are regenerated from transformants.
  • the following protocol describes method for plant regeneration, but one skilled in the art will be familiar with other equally efficient protocols.
  • tissue is first transferred to solid medium 223 (Table 2) and incubated for two weeks.
  • Transformants may be initially subcultured on any solid culture that supports callus growth. Subsequently transformants are subcultured one to three times, but usually twice on 189 medium (Table 2; first passage in the dark and second passage in low light) and once or twice on 101 medium (Table 2) in petri dishes before being transferred to 607 medium (Table 2) in Plant Cons®. Variations in the regeneration protocol are normal based on the progress of plant regeneration.
  • Plantlets in soil are incubated in an illuminated growth chamber and conditions was slowly adjusted to adapt or condition the plantlets to the drier and more illuminated conditions of the greenhouse. After adaptation/conditioning in the growth chamber, plants are transplanted individually to 5 gallon pots of soil in the greenhouse. Transformed plants in soil are cultivated in the greenhouse following standard greenhouse protocols and pollinated using standard plant breeding techniques. It is the experience of the inventors that seed is recovered from most transgenic plants generated in accordance with these procedures.
  • Progeny and subsequent generations are grown in the field and assayed for their performance under a range of water availability conditions. Both qualitative and quantitative measures of the plant's ability to withstand water stress are made. Seeds are germinated in the greenhouses, growth chambers and field conditions under ample water supply. At one or more times during the plant's life cycle, water availability is reduced in order to identify plants expressing the wilt gene(s) . Visual signs of wilting or the reduction of turgor are noted. In addition to the visual signs of wilting, which may only be observed under more pronounced drought stress, measures of plant water relations are made.
  • Total water potential, osmotic potential and turgor potential are quantitatively measured and detection of differences in turgor or the ability of the plants not to wilt can be made even when no signs of plant stress are visible to the eye. Plants expressing the most favorable water status result in superior growth under water stress. Different measures of growth are used to document this superior performance. Measures of cell and leaf area expansion are used to identify superior plant growth under stress.
  • the physiological and biochemical activity of the transformed plant tissue is indicative of its improved stress tolerance.
  • Such screening of plants with the measurement of photosynthetic activity or transpirational activity are examples, but not all of the types of measurement that can be done to identify the superiority of the wilt expressing plants compared to non-transformed plants.
  • Measurements of reproductive capacity including, but not limited to the synchrony of pollen shed and silk emergence are indicators of improved stress tolerance when the wilt gene is expressed.
  • the expression of the wilt gene will minimize kernel abortion during times of stress thereby increasing the amount of grain to reach maturity.
  • the expression of the wilt gene allows for superior late season plant health and development of full ears. It is contemplated that barrenness will not be a problem.
  • testcrosses are made and hybrid seed is produced.
  • the testcross hybrids and breeding populations are planted in several different fashions in the field.
  • One scheme of evaluation is to grow populations of hybrid plants containing the wilt gene in many different locations and measure the performance of the plants at these different locations. Given the variability of rainfall distribution, the different locations receive different quantities of rainfall and in some locations, the plants will receive stress. Yield information as well as measures which quantify plant response to stress as described earlier, are made. The information regarding the performance of these hybrids along with that of the performance of non- transformed or non-wilt containing hybrids is compared. It is anticipated that the hybrids expressing the wilt gene will be higher in yield performance at a given level of water availability than the controls.
  • the parent selections are advanced and inbred lines are produced through conventional breeding techniques.
  • Hybrid plants having one or more parents containing the wilt gene are tested in commercial testing and evaluation programs and performance documented. This testing include performances trials over a wide geographical area as well as dedicated trials where water availability is varied to reveal performance advantage and hence value.
  • An additional advantage of the expression of the wilt gene is the superior performance of the parental inbred lines in production of hybrids. Less stress related parent yield loss is associated with higher green seed yield and thereby higher economic margins.
  • nucleic acids are isolated from potential Fl progeny and screened for the overexpression of mRNA or protein, amplification of the gene locus or increased stability of the gene product.
  • nucleic acids and proteins are well known to those of skill in the art (Maniatis et al . , 1991), and may be used in conjunction with the gene of the present invention to selectively segregate plants that have increased resistance to water deprivation, with or without, genetic alteration of the plant.
  • Natural variants of maize may be isolated and selected for breeding following a screen for altered wilt resistance gene message, product or product stability.
  • wilt genes will be useful as DNA probes for marker assisted breeding.
  • DNA sequences are used to follow desirable agronomic traits (Tanksley et al . , 1989) in the process of plant breeding.
  • a wilt gene probe will be useful for identification of plants with enhanced ability to utilize water resources.
  • wilt gene probe it will be possible to combine genes that enhance the ability to utilize water. It is contemplated that such a combination of genes would be difficult to identify without marker assisted breeding, unless the plant is grown under conditions of limited water availability.
  • Marker assisted breeding using the wilt gene is undertaken as follows. Seed of plants with the desired ability to utilize water resources are planted in soil in the greenhouse or in the field. Leaf tissue is harvested from the plant for preparation of DNA at any point in growth at which approximately one gram of leaf tissue can be removed from the plant without compromising the viability of the plant. Genomic DNA is isolated using a procedure modified from Shure et al . (1983). Approximately one gram of leaf tissue from a seedling is lyophilized overnight in 15 ml polypropylene tubes. Freeze-dried tissue is ground to a powder in the tube using a glass rod. Powdered tissue is mixed thoroughly with 3 ml extraction buffer (7.0 M urea, 0.35 M NaCI, 0.05 M Tris-HCI ph 8.0, 0.01 M EDTA, 1% sarcosine) .
  • Tissue/buffer homogenate is extracted with 3 ml phenol/ chloroform.
  • the aqueous phase is separated by centrifugation, and precipitated twice using 1/10 volume of 4.4 M ammonium acetate pH 5.2, and an equal volume of isopropanol.
  • the precipitate is washed with 75% ethanol and resuspended in 100-500 ⁇ l TE (0.01 M Tris-HCI, 0.001 M EDTA, pH 8.0).
  • Genomic DNA is digested with a 3-fold excess of restriction enzymes, electrophoresed through 0.8% agarose (FMC) , and transferred (Southern, 1975) to Nytran using 10X SCP (2OX SCP: 2 M NaCI, 0.6 M disodium phosphate, 0.02 M disodium EDTA). - 55 -
  • restriction enzymes will be useful and the choice of restriction enzyme will depend on the DNA sequence of the wilt gene that is used as a probe and the DNA sequence in the maize genome surrounding the wilt gene. One will select a restriction enzyme that produces a DNA fragment following hybridization that is identifiable as that wilt gene. It is anticipated that one or more restriction enzymes will be used to digest genomic DNA either singly or in combinations.
  • Filters are prehybridized in 6X SCP, 10% dextran sulfate, 2% sarcosine, and 500 ⁇ g/ml denatured salmon sperm DNA and 32 P-labelled wilt gene probe generated by random priming (Feinberg & Vogelstein, 1983; Boehringer-Mannhelm) .
  • Hybridized filters are washed in 2X SCP, 1% SDS at 65° for 30 minutes and visualized by autoradiography using Kodak XAR5 film.
  • Each lane of the Southern blot represents DNA isolated from one plant.
  • the wilt gene composition of each plant is determined. Correlations are established between the contributions of particular wilt genes to the ability of the plant to adapt to conditions of decreased water availability. Only those plants that contain the desired combination of wilt genes are advanced to maturity and used for pollination. DNA probes corresponding to wilt genes are useful markers during the course of plant breeding to identify and combine particular wilt genes without having to grow the plants under conditions of decreased water availability and assay the plants for agronomic performance under these stressed conditions.
  • 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 composition, methods and in the steps or in the sequence of steps of the method 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.
  • Sex Determination gene Tassleseed2 of Maize encodes a short-chain alcohol dehydrogenase required for stage-specific floral organ abortion. Cell 74: 757-768. Dooner & Belachew. 1989. Transposition pattern of the maize element Ac from the bz-m2 (Ac ) allele, Genetic ⁇ , 122:447-457.
  • ADDRESSEE ARNOLD, WHITE & DURKEE
  • B STREET: P.O. BOX 4433
  • MOLECULE TYPE other nucleic acid

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Abstract

La présente invention concerne l'identification et la caractérisation de gènes jouant un rôle dans la réponse des maïs au stress hydrique. Un gène allèle mutant du gène de flétrissement a été identifié par marquage de transposons. Cela a permis d'observer une corrélation avec un phénotype de flétrissement de mutants dans des conditions hydriques normales.
EP95917777A 1994-04-29 1995-04-28 Genes regulant la reponse des mais au deficit hydrique Withdrawn EP0759076A1 (fr)

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FR2734838B1 (fr) * 1995-05-31 1997-08-22 Agronomique Inst Nat Rech Fragment d'acide nucleique codant pour un enzyme implique dans la voie de biosynthese de l'acide abscisique (aba) chez les plantes
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