[go: up one dir, main page]

US20080189801A1 - Selective Ablation of Diploid Embryos - Google Patents

Selective Ablation of Diploid Embryos Download PDF

Info

Publication number
US20080189801A1
US20080189801A1 US12/023,152 US2315208A US2008189801A1 US 20080189801 A1 US20080189801 A1 US 20080189801A1 US 2315208 A US2315208 A US 2315208A US 2008189801 A1 US2008189801 A1 US 2008189801A1
Authority
US
United States
Prior art keywords
polynucleotide
embryos
haploid
plant
embryo
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.)
Abandoned
Application number
US12/023,152
Other languages
English (en)
Inventor
Mark E. Williams
William J. Gordon-Kamm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pioneer Hi Bred International Inc
EIDP Inc
Original Assignee
Pioneer Hi Bred International Inc
EI Du Pont de Nemours and Co
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Pioneer Hi Bred International Inc, EI Du Pont de Nemours and Co filed Critical Pioneer Hi Bred International Inc
Priority to US12/023,152 priority Critical patent/US20080189801A1/en
Publication of US20080189801A1 publication Critical patent/US20080189801A1/en
Priority to US13/251,597 priority patent/US8269061B2/en
Priority to US13/586,893 priority patent/US9121032B2/en
Abandoned legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8263—Ablation; Apoptosis
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8217—Gene switch
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

  • the present invention relates to the field of plant breeding and plant biotechnology.
  • Homozygous plants are basic for product development and commercialization of plants. To obtain homozygous plants requires several generations of self-pollination and segregation analysis. This is an inefficient use of labor and time. It would therefore be useful to develop a method to reduce hand pollination steps normally required to obtain a homozygous plant and reduce the amount of time required to obtain a homozygous population of plants.
  • One way to obtain homozygous plants without the need to self-pollinate multiple generations is to produce haploids and then double the chromosomes to form doubled haploids. A process to assist in the selection of haploid embryos and elimination of diploid embryos would increase the efficiency of doubled haploid production.
  • Methods for identifying haploids by preventing the growth of diploid embryos are provided.
  • Methods for identifying haploid plants, seeds, embryos, and plant cells are provided.
  • Methods for producing haploid inducer lines and the haploid inducer lines are provided.
  • a haploid plant has a single set (genome) of chromosomes and the reduced number of chromosomes (n) in the haploid plant is equal to that in the gamete.
  • a diploid plant has two sets (genomes) of chromosomes and the chromosome number (2n) is equal to that in the zygote.
  • a haploid cell is one with a single genome, male or female.
  • a doubled haploid or doubled haploid plant or cell is one that is developed by the doubling of a haploid set of chromosomes.
  • a plant or seed that is obtained from a doubled haploid plant that is selfed any number of generations may still be identified as a doubled haploid plant.
  • a doubled haploid plant is considered a homozygous plant.
  • a plant is considered to be doubled haploid if it is fertile, even if the entire vegetative part of the plant does not consist of the cells with the doubled set of chromosomes.
  • a plant will be considered a doubled haploid plant if it contains viable gametes, even if it is chimeric.
  • a “haploid embryo” is defined as the embryo formed after one sperm nucleus from a pollen grain fuses with the polar nuclei in the embryo sac to create a triploid (3N) endosperm and the embryo forms without the contribution of the male genome.
  • An “immature haploid embryo” is defined as the embryo formed after one sperm nucleus from a pollen grain fuses with the polar nuclei in the embryo sac to create a triploid (3N) endosperm and before dry down. And the embryo forms without the contribution of the male genome.
  • a “doubled haploid embryo” is an embryo that has one or more cells that contain 2 sets of homozygous chromosomes.
  • Callus refers to a dedifferentiated proliferating mass of cells or tissue.
  • the phrases “contacting”, “comes in contact with” or “placed in contact with” can be used to mean “direct contact” or “indirect contact”.
  • the medium comprising a doubling agent may have direct contact with the haploid cell or the medium comprising the doubling agent may be separated from the haploid cell by filter paper, plant tissues, or other cells thus the doubling agent is transferred through the filter paper or cells to the haploid cell.
  • medium includes compounds in liquid, gas, or solid state.
  • plant includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same.
  • Plant cell includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • the haploid inducer line comprises; 1) a pollen lethality polynucleotide or a non-transmission pollen polynucleotide, 2) an embryo lethality polynucleotide, and 3) an embryo lethality repressor.
  • the pollen lethality polynucleotide or a non-transmission pollen polynucleotide indicates any polynucleotide that prevents the pollen from achieving fertilization.
  • the pollen non-transmission is most effective if it occurs at the post-meiotic or gametophytic stage.
  • the non-transmission pollen polynucleotide can work through various mechanisms. It may prevent the pollen from being viable for example by the expression of a toxic compound.
  • the embryo lethality polynucleotide is any polynucleotide that prevents proper development of the embryo or causes the embryo to be non-viable.
  • the embryo lethality polynucleotide may slow the growth of the embryo so that one may be able to distinguish an embryo that carries the embryo lethality polynucleotide from an embryo that does not carry the embryo lethality polynucleotide.
  • the embryo lethality repressor is any polynucleotide that when expressed causes the embryo lethality polynucleotide to be non-effective and allows a viable embryo to develop. This can be achieved through various mechanisms.
  • the embryo lethality repressor can detoxify the molecule expressed by the embryo lethality polynucleotide.
  • An embryo repressor protein may be expressed that inhibits the lethality of another protein for example through a protein-protein interaction.
  • Another way the embryo repressor may work is to deter or prevent the expression of the embryo lethality polynucleotide, for example by expressing a molecule that binds to the promoter of the lethality polynucleotide and blocks some or all expression.
  • Another example that could be used in the system is gene silencing.
  • the embryo repressor may be a polynucleotide that prevents the embryo lethal polynucleotide from functioning through gene silencing.
  • the embryo lethality polynucleotide and the embryo lethality repressor polynucleotide are not limited to being expressed only in the embryo.
  • the genes can be expressed in other tissues. It is more effective if the embryo lethality polynucleotide is not expressed in the endosperm so that the haploid seed can develop normally.
  • Another consideration for the method to work at its optimum is that the embryo lethality polynucleotide expression should to be matched by the expression of the embryo lethality repressor polynucleotide. Anytime the polynucleotides are inherited together the embryo lethality repressor polynucleotide should be expressed at a level to deter the negative effects of the embryo lethality polynucleotide.
  • the non-transmission pollen polynucleotide and the embryo lethality repressor are linked in the developed inducer line. They may be tightly linked. For the most convenience the non-transmission pollen polynucleotide and the embryo lethality repressor should be adjacent to each other, for example on the same construct. For efficient results the non-transmission pollen polynucleotide and the embryo lethality repressor will segregate together. Also typically the non-transmission pollen polynucleotide and the lethality repressor are linked, but are at a different location than the embryo lethality polynucleotide.
  • the embryo lethality polynucleotide will segregate from the non-transmission pollen polynucleotide and the embryo lethality repressor. For the greatest efficiency, the embryo lethality polynucleotide is not linked to the non-transmission pollen polynucleotide and the embryo lethality repressor.
  • the developed inducer line may or may not contain selectable markers. It may or may not be developed using transgenes that are selectable markers.
  • Construct A comprises a non-transmission pollen polynucleotide and an embryo lethality repressor, and a selectable marker gene.
  • Construct B comprises a selectable marker gene and an embryo lethality gene. To facilitate segregation upon pollination the two constructs can be located at two different locations in the genome. For further efficiency Construct A and Construct B should be unlinked.
  • Construct A and B can be co-transformed into the inducer line or the transformation process can be done sequentially, most conveniently with Construct A first and Construct B second.
  • the initial T 0 inducer plant will be heterozygous for both constructs, A and B.
  • the expected results of the self-pollination of this plant are given in Table 1.
  • Another way to introduce the constructs into the inducer line would be to cross or breed them into the inducer line.
  • Construct A and B could be co-transformed into a plant and then bred into an inducer line.
  • the embryo lethal repressor needs to present with the embryo lethal polynucleotide. Therefore, one may transform with construct containing the embryo lethal repressor polynucleotide to obtain stably transformed cells and then transform with construct comprising the embryo lethal polynucleotide.
  • T 0 plant production is possible because both the embryo lethal and the embryo lethal repressor are both expressed in somatic embryos and embryogenic callus.
  • Pollen containing Construct A is non-viable or non-transmissible.
  • Embryos containing Construct B but not Construct A are non-viable due to the expression of the embryo lethal gene without the embryo lethal repressor gene.
  • the A-BB genotype will have two doses of the embryo lethal gene and one dose of the embryo repressor gene.
  • the embryos or plants from embryos not containing both constructs can be selected against by contacting the tissue with the selectable agent. If the selectable marker is a visual marker, or some other type of marker, this can also be observed and the tissue not containing both markers can be selected against. Any type of selectable markers can be used.
  • the remaining plants will either be heterozygous for both constructs (A-B-) or heterozygous for construct A and homozygous for construct B, (A-BB).
  • A-BB homozygous for construct B
  • the result of selfing the A-B- genotype will result in the progeny as seen previously, Table 1.
  • the result of selfing the A-BB genotype will give you only genotypes with that are homozygous for Construct B.
  • progeny will have the selectable marker b.
  • This is the desired genotype, A-BB.
  • the plants that are heterozygous for both constructs will produce some progeny that do not contain selectable marker b.
  • the resultant progeny from selfing the A-BB genotypes are described in Table 2.
  • the desired genotype, A-BB produces only one viable pollen genotype, -B.
  • This transgenic inducer line is crossed as a male to a wildtype female, this will result in the ablation of all diploid embryos, Table 3.
  • maternal haploid embryos do not inherit the embryo lethal from the male parent and are therefore viable.
  • the maternal haploids do not contain the transgenes from Construct A or Construct B.
  • the pollen carrying the embryo lethal gene will have one sperm nucleus that fuses with the polar nuclei in the embryo sac to create a triploid (3N) endosperm. Therefore the embryo lethal gene either cannot be expressed in the endosperm or aleurone; or if it is expressed in these cells it will not kill the haploid embryo.
  • an inducer line could also be produced without any selectable markers. It could also be produced using only one selectable marker on either construct or with the same selectable marker on both constructs.
  • Another method of the invention includes co-transformation with the pollen non-transmission and embryo lethal repressor genes on one construct and a selectable marker gene on a second construct. After obtaining transformed tissue a second co-transformation can be conducted with the embryo lethal and a second selectable marker. After transgenic plants are developed the selectable markers can be segregated away from the other transgenes.
  • Another method may be utilized with chemical application or contact acting as the embryo lethal repressor
  • One may be able to obtain plants with only Construct B if a chemical can be used to repress the embryo lethal polynucleotide.
  • a chemical can be used to repress the embryo lethal polynucleotide.
  • one could transform the constructs into two different plants and then breed both constructs into the inducer line.
  • the chemical that represses the embryo lethality would have to be used for the plant transformed with Construct B until the both constructs are contained in the same plant.
  • One would then select for an inducer line containing the two segregating constructs, A and B. Trait integration through backcrossing is well known in the art.
  • Haploid induction systems have been developed for various plants to produce haploid tissues, plants and seeds.
  • the haploid induction system can produce haploid plants from any genotype by crossing a selected line (as female) with an inducer line.
  • inducer lines for maize include, but are not limited to, Stock 6 and Stock 6 derivatives (Coe, 1959, Am. Nat. 93:381-382; Sarkar and Coe, 1966, Genetics 54:453-464; Sarkar et al, 1972, Development of maternal-haploidy-inducer lines in maize ( Zea mays L.) Indian J. Agric. Sci.
  • haploid embryos are derived.
  • One sperm nucleus from the pollen fuses with the polar nuclei in the embryo sac to create a triploid (3N) endosperm.
  • the triploid endosperm will contain 2 sets of chromosomes from the female and 1 set of chromosomes from the male, which in this case is the inducer line.
  • the haploid embryo contains a single set of chromosomes, which are derived from the female plant.
  • Rnj is a commonly used allele for haploid/diploid screening of mature seeds (Nanda and Chase, 1966. An embryo marker for detecting monoploids of maize ( Zea mays L). Crop Sci. 6:213-215; Greenblatt and Bock, 1967. A commercially desirable procedure for detection of monoploids in maize. J. Hered. 58:9-13). R-nj expression levels have been shown to be correlated with parameters of kernel maturation (Alexander and Cross, 1983, Grain fill characteristics of early maize ( Zea mays L) strains selected for variable R-nj expression, Euphytica 32:839-844).
  • R-scm2 has also been used (Kato A., 2002, Chromosome doubling of haploid maize seedlings using nitrous oxide gas at the flower primordial stage. Plant breeding 121:370-377; and Kato A., 2003, Chromosome doubling method, U.S. Publication 2003/0005479).
  • the R-nj anthocyanin marker gene used to distinguish haploids and diploids at the mature seed stage is not expressed until late in embryo development.
  • haploid embryos can be identified using a transgenic marker, lec1-GFP, which is present in the inducer line.
  • lec1-GFP is valuable because the gene allows one to identify the non-haploid embryos at an early stage of embryo development (U.S. application Ser. No. 09/718,754, U.S. Pat. No. 6,486,382).
  • the absence of the GFP marker expression is used to identify haploid embryos.
  • the GFP system requires a labor intensive screening process to determine which embryos express the GFP marker and which embryos do not express the GFP marker.
  • the use of a lethal marker in an inducer line would only allow haploid embryo formation and thus eliminate the need for a less efficient screening process. This system would increase the efficiency of the doubled haploid process.
  • Various types of systems could be utilized in the inducer line in order to increase the efficiency of the doubled haploid process: 1) toxicity/antidote systems 2) transcriptional regulator systems 3) gene silencing systems.
  • Barnase is the name of the extracellular ribonuclease produced by Bacillus amyloliquefaciens .
  • the inhibitor of barnase is called barstar and is produced intracellularly by the same organism that secretes barnase.
  • the function of barstar is to protect the B. amyloliquefaciens from the toxic effects of intracellular barnase, rendering it inactive (Hartley, R. W. (1989) barnase and barstar: Two small proteins to fit and fold together. Trends Biochem. Sci. 14:450-454). Both of these genes have been cloned and sequenced (Hartley, R.
  • the transmission of a transgene through pollen can be prevented by linking the transgene to a pollen-lethality gene composed of a cytotoxic gene under the control of a pollen-specific (gametophyte) promoter (Twell (1995) Diptheria toxin-mediated cell ablation in developing pollen: vegetative cell ablation blocks generative cell migration. Protoplasma 187:144-154; Williams et al., (1997) Male sterility through recombinant DNA technology. In Pollen Biotechnology for Crop Production and Improvement, K. R. Shivanna and V. K. Sawhney, eds (Cambridge, UK: Cambridge University Press) pp 237-257). This type of construct can be maintained because it is transmitted through the female.
  • Barstar inhibits the function (RNase) of the barnase protein in the haploid-inducer line. Diploid embryos are ablated because the barstar PTU (plant transcription unit) is linked to the pollen non-transmission PTU, and thus is not present in the diploid embryos to inhibit barnase.
  • the barstar system can be improved by optimizing the coding region of the barstar gene. One may also optimize the system by increasing the affinity of barstar to barnase.
  • Lac1 Repressor Carbon Helix-turn-helix N-terminal source utilization ArsR Repressor Metal Helix-turn-helix Central resistance IclR Repressor/ Carbon Helix-turn-helix N-terminal activator metabolism, efflux pumps MerR Repressor Resistance Helix-turn-helix N-terminal and detoxification AsnC Activator/ Amino acid Helix-turn-helix N-terminal repressor biosynthesis MarR Activator/ Multiple Helix-turn-helix Central repressor antibiotic resistance NtrC Activator Nitrogen Helix-turn-helix C-terminal (EBP) assimilation, aromatic amino acid synthesis, flagella, catabolic pathways, phage response etc.
  • EBP Helix-turn-helix C-terminal
  • OmpR Activator Heavy metal Winged helix C-terminal and virulence DeoR Repressor Sugar Helix-turn-helix N-terminal metabolism Cold Activator Low- RNA binding Variable shock temperature domain (CSD) resistance GntR Repressor General Helix-turn-helix N-terminal metabolism Crp Activator/ Global Helix-turn-helix C-terminal repressor responses, catabolic repression and anaerobiosis
  • CSD Variable shock temperature domain
  • the tet repressor system comprises repressor and operator elements.
  • the operon system is controlled by the presence of tetracycline, and self-regulates the level of expression of tetA and tetR genes.
  • the product of tetA removes tetracycline from the cell.
  • the product of tetR is the repressor protein which binds to the operator elements with a K d of about 10 pM in the absence of tetracycline, thereby blocking expression or tetA and tetR.
  • the TET repressor can be optimized for maize and the promoter used to stop development of the embryo can be modified with TET operator sequences.
  • lac repressor system Ulmasov et al. (1997) Plant Mol Biol 35-417-424; Wilde et al. (1992) EMBO J 11:1251-1259.
  • This repressor/operator based-system is derived from the prokaryotic operon, E. coli lactose operon.
  • This system controls the activity of a promoter by placing operator sequences near the transcriptional start site of a gene such that gene expression from the operon is inhibited upon the binding of the repressor protein to its cognate operator sequence.
  • IPTG isopropyl-B-D-thiogalactopyranoside
  • tetracycline, and/or doxycyline are commonly used inducing agents for the tet system.
  • IPTG isopropyl-B-D-thiogalactopyranoside
  • the lac repressor has a high association constant for its operator, and IPTG reduces the affinity of repressor for the operator by 300-fold (Barkley & Bourgeois (1980) The Operon , Miller & Reznikoff, Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp 177-220), only 30-fold repression has been reported using the lactose repressor (Ulmasov et al. (1997) Plant Mol Biol 35-417-424).
  • the lac repressor can be maize optimized and the promoter used to stop development of the embryo can be modified with lac operator sequences.
  • Gene silencing may also be used in the selective ablation of diploid embryos system.
  • the repression of embryo lethality may be at the RNA or protein stage for example, anti-sense RNA, hair-pins, and other mechanisms for gene silencing.
  • This construct comprising the hairpin can be linked to a construct that does not allow pollen fertilization.
  • the other construct could comprise the barnase polynucleotide driven by the lec1 promoter. This would effectively turn off barnase until the hairpin locus was segregated away.
  • Any polynucleotide that prevents normal development of the embryo along with a corresponding gene silencing construct can be used in the system.
  • genes that may be driven by a pollen promoter and used to prevent fertile pollen in the invention include DAM (GenBank J01600, Nucleic Acids Res. 11:837-851 (1983); alpha-amylase (GenBank L25805, Plant Physiol. 105(2):759-760 (1994)); D8 (Physiol. Plant 100(3):550-560 (1997)); SacB (Plant Physiol. 110(2):355-363 (1996)), lipases and ribonucleases; tasselseed2 (ts2) (Calderon-Urrea M. and S. L. Dellaporta (1999) Development 126:435-441); diphtheria toxin A (DTA) (Greenfield et al. Proc. Natl. Acad. Sci. 80:6853 (1983); Palmiter et al., Cell 50:435 (1987)).
  • DAM GenBank J01600, Nucleic Acids Res. 11:837-851 (1983
  • pollen specific promoters examples include but are not limited to PG47 (Rogers et al. (1991), Pollen specific cDNA clones from Zea mays , Biochem. Biophys. Acta 1089, 411-413; Allen, R. L., and Lonsdale, D. M. (1992) Sequence analysis of three members of the maize polygalacturonase gene family expressed during pollen development, Plant Mol. Biol. 20, 343-345, Allen, R. L., and Lonsdale, D. M. (1993), Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development. The Plant Journal 3, 261-271, U.S. Pat. No.
  • Such promoters are known in the art or can be discovered by known techniques; see, e.g., Bhalla and Singh (1999) Molecular control of male fertility in Brassica Proc. 10 th Annual Rapeseed Congress, Canberra, Australia; van Tunen et al. (1990) Pollen-specific chi promoters from petunia: tandem promoter regulation of the chiA gene, Plant Cell 2:393-40; Jeon et al. (1999); and Twell et al. (1993) Activation and developmental regulation of an Arabidopsis anther-specific promoter in microspores and pollen of Nicotiana tabacum , Sex. Plant Reprod. 6:217-224.
  • Cereal genes whose promoters are associated with early seed and embryo development include lec1 (U.S. Pat. No. 7,122,658), rice glutelin (“GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol 37:107-11; “GluB-1,” Takaiwa et al., 1996, Plant Mol Biol 30:1207-21; Washida et al., 1999, Plant Mol Biol 40:1-12; “Gt3,” Leisy et al., 1990, Plant Mol Biol 14:41-50), rice prolamin (Zhou & Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack et al., 1993, EMBO J 12:545-54), maize zein (Z4, Matzke et al., 1990, Plant Mol Biol 14:323-32), and barley B-hordeiis (Entwistle et al., 1991, Plant Mol Biol 17:1217-31).
  • seed-preferred promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message), cZ19B1 (maize 19 kDa zein), mi1ps (myo-inositol-1-phosphate synthase); see WO 00/11177 and U.S. Pat. No. 6,225,529.
  • Gamma-zein is an endosperm-specific promoter.
  • Globulin-1 Glob-1 (Glob-1) is a representative embryo-specific promoter.
  • the maize Glb1 gene encodes globulin-1, a major embryo storage protein.
  • Glb1 is expressed in the developing maize seed during embryo development.
  • Belanger, F. C., et al. (1989) Plant Physiol. 91:636-643 The promoter region of Glb1 has been identified, cloned, and introduced into tobacco plants by Agrobacterium -mediated transformation. (Liu, S., et al.
  • the transformed plants demonstrate that the Glb1 promoter has desirable temporal and tissue specificity.
  • the Glb1 promoter is positively regulated by abscisic acid (ABA).
  • ABA abscisic acid
  • levels of the plant hormone ABA are known to fluctuate under conditions of cold or desiccation.
  • seed-specific promoters include, but are not limited to, bean ⁇ -phaseolin, napin, ⁇ -conglycinin, soybean lectin, cruciferin, and the like. More examples of seed-specific promoters from monocots include, but are not limited to, maize 15 kDa zein; 22 kDa zein; 27 kDa zein (Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P. and Palau, Jr.; Isolation and sequencing of a 28 kD glutelin-2 gene from maize: Common elements in the 5′ flanking regions among zein and glutelin genes; Plant Sci.
  • constitutive promoters examples include the 1′- or 2′-promoter of Agrobacterium tumefaciens (see, e.g., O'Grady (1995) Plant Mol. Biol. 29:99-108).
  • Other plant promoters include the ribulose-1,3-bisphosphate carboxylase small subunit promoter, the phaseolin promoter, alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar (1996) Plant Mol. Biol. 31:897-904), sucrose synthase promoters, ⁇ -tubulin promoters, actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang (1997) Plant Mol. Biol.
  • constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); PEMU (Last et al. (1991) Theor. Appl. Genet.
  • promoters of bacterial origin which operate in plants can be used in the invention. They include, e.g., the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids. See, Herrera-Estrella et al. (1983) Nature 303:209. Viral promoters can also be used. Examples of viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV). See, Odell et al., (1985) Nature 313:810; and, Dagless (1997) Arch. Virol. 142:183-191.
  • constitutive promoters from viruses which infect plants include the promoter of the tobacco mosaic virus; cauliflower mosaic virus (CaMV) 19S and 35S promoters or the promoter of Figwort mosaic virus, e.g., the figwort mosaic virus 35S promoter (see, e.g., Maiti (1997) Transgenic Res. 6:143-156), etc.
  • novel promoters with useful characteristics can be identified from any viral, bacterial, or plant source by methods, including sequence analysis, enhancer or promoter trapping, and the like, known in the art.
  • Tissue-preferred (tissue-specific) promoters and enhancers can be utilized to target enhanced gene expression within a particular plant tissue.
  • Tissue-preferred (tissue-specific) promoters include, e.g., those described in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.
  • tissue-specific promoter can drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue, but can also lead to some expression in other tissues as well.
  • leaf specific promoters can be used, e.g., pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize), cab-m1 Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS) promoters (e.g., the tomato RBCS1, RBCS2 and RBCS3A genes, which are expressed in leaves and light-grown seedlings, while RBCS1 and RBCS2 are expressed in developing tomato fruits, and/or a ribulose bisphosphate carboxylase promoter which is expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, etc.), and the like.
  • PPDK orthophosphate dikinase
  • Atmyb5 the Arabidopsis thaliana myb-related gene promoter
  • RBCS ribulose biphosphate carboxy
  • leaf-specific promoters include, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
  • senescence specific promoters can be used (e.g., a tomato promoter active during fruit ripening, senescence and abscission of leaves, a maize promoter of gene encoding a cysteine protease, and the like). See, e.g., Blume (1997) Plant J. 12:731-746; Griffiths et al., (1997) Sequencing, expression pattern and RFLP mapping of a senescence - enhanced cDNA from Zea Mays with high homology to oryzain gamma and aleurain, Plant Mol. Biol.
  • Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens ); and Miao et al.
  • MAS mannopine synthase
  • TR1′ gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics.
  • Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
  • promoters that act from 0-25 days after pollination (DAP), 4-21, 4-12, or 8-12 DAP can be selected, e.g., promoters such as cim1 and Itp2.
  • promoters that act from 0-14 days after pollination can also be used, such as SAG12 (See WO 96/29858, Richard M. Amasino, published 3 Oct. 1996) and ZAG1 or ZAG2 (See R. J.
  • ZAG 1 the Maize Homolog of the Arabidopsis Floral Homeotic Gene AGAMOUS , Plant-Cell 5(7):729-37 (July 1993)
  • Other useful promoters include maize zag2. 1, Zap (also known as ZmMADS; U.S. patent application Ser. No. 10/387,937; WO 03/078590); and the maize tb1 promoter (see also Hubbarda et al., Genetics 162:1927-1935, 2002).
  • Shoot-preferred promoters include, shoot meristem-preferred promoters such as promoters disclosed in Weigal et al. (1992) Cell 69:853-859; Accession No. AJ131822; Accession No. Z71981; Accession No. AF059870, the ZAP promoter (U.S. patent application Ser. No. 10/387,937), the maize promoter (Wang et al. (1999) Nature 398:236-239, and shoot-preferred promoters disclosed in McAvoy et al. (2003) Acta Hort . ( ISHS ) 625:379-385.
  • shoot meristem-preferred promoters such as promoters disclosed in Weigal et al. (1992) Cell 69:853-859; Accession No. AJ131822; Accession No. Z71981; Accession No. AF059870, the ZAP promoter (U.S. patent application Ser. No. 10/387,937)
  • An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed, or will be transcribed at a level lower than in an induced state.
  • the inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, drought, heat, salt, toxins. Plant promoters which are inducible upon exposure to plant hormones, such as auxins, can be used.
  • the invention can use the auxin-response elements E1 promoter subsequence (AuxREs) from the soybean ( Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10:955-966); the auxin-inducible parC promoter from tobacco; a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10:933-937); and the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
  • AuxREs E1 promoter subsequence
  • Plant promoters which are inducible upon exposure to chemical reagents which can be applied to the plant, such as herbicides or antibiotics, are also used to express polynucleotides.
  • the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem.
  • An ACC synthase coding sequence or RNA configuration can also be under the control of, e.g., tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; U.S. Pat. Nos. 5,814,618 and 5,789,156; and, Masgrau (1997) Plant J. 11:465-473 (describing transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene with a tetracycline-inducible promoter); or, a salicylic acid-responsive element (Stange (1997) Plant J.
  • tetracycline-inducible and tetracycline-repressible promoters see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; U.
  • Other chemical-inducible promoters are known in the art and include, but are not limited to, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257).
  • inducible regulatory elements include a metallothionein regulatory element, a copper-inducible regulatory element, or a tetracycline-inducible regulatory element, the transcription from which can be effected in response to divalent metal ions, copper or tetracycline, respectively (Furst et al., Cell 55:705-717, 1988; Mett et al., Proc. Natl. Acad. Sci., USA 90:4567-4571, 1993; Gatz et al., Plant J. 2:397-404, 1992; Roder et al., Mol. Gen. Genet. 243:32-38, 1994).
  • Inducible regulatory elements also include an ecdysone regulatory element or a glucocorticoid regulatory element, the transcription from which can be effected in response to ecdysone or other steroid (Christopherson et al., Proc. Natl. Acad. Sci., USA 89:6314-6318, 1992; Schena et al., Proc. Natl. Acad. Sci., USA 88:10421-10425, 1991; U.S. Pat. No. 6,504,082); a cold responsive regulatory element or a heat shock regulatory element, the transcription of which can be effected in response to exposure to cold or heat, respectively (Takahashi et al., Plant Physiol.
  • an inducible regulatory element also can be the promoter of the maize In2-1 or In2-2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen. Gene 227:229-237, 1991; Gatz et al., Mol. Gen.
  • Stress inducible promoters include salt/water stress-inducible promoters such as P5CS (Zang et al. (1997) Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a (Hajela et al. (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm et al. (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet et al. (1998) FEBS Lett.
  • salt/water stress-inducible promoters such as P5CS (Zang et al. (1997) Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a (Hajela et al. (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm et al. (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet et al.
  • rd29a Yamaguchi-Shinozaki et al. (1993) Mol. Gen. Genetics 236:331-340.
  • Certain promoters are inducible by wounding, including the Agrobacterium pmas promoter (Guevara-Garcia et al. (1993) Plant J. 4(3):495-505) and the Agrobacterium ORF13 promoter (Hansen et al., (1997) Mol. Gen. Genet. 254(3):337-343).
  • the expression cassette used in the invention can include, at the 3′ terminus of the heterologous nucleotide sequence of interest, a transcriptional and translational termination region functional in plants.
  • the termination region can be native with the promoter nucleotide sequence of the present invention, can be native with the DNA sequence of interest, or can be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens , such as the octopine synthase and nopaline synthase termination regions.
  • the 3′ terminus of the pinII-(potato proteinase inhibitor) can be used. See Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al.
  • the expression cassettes can additionally contain 5′ leader sequences.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region), Elroy-Stein et al. (1989) Proc. Nat Acad. Sci. USA 86:6126-6130; potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus), Virology 154:9-20; human immunoglobulin heavy-chain binding protein (BiP), Macejak et al.
  • EMCV leader Engelphalomyocarditis 5′ noncoding region
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus), Allison et al. (1986
  • the cassette can also contain sequences that enhance translation and/or mRNA stability such as introns.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium -mediated transformation (Townsend et al., U.S. Pat. No.
  • Transformation of maize can follow a well-established bombardment transformation protocol used for introducing DNA into the scutellum of immature maize embryos (See, e.g., Tomes et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G. C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995).
  • Cells are transformed by culturing maize immature embryos (approximately 1-1.5 mm in length) onto medium containing N6 salts, Erikkson's vitamins, 0.69 g/l proline, 2 mg/l 2,4-D and 3% sucrose.
  • embryos are removed from the first medium and cultured onto similar medium containing 12% sucrose. Embryos are allowed to acclimate to this medium for 3 h prior to transformation. The scutellar surface of the immature embryos is targeted using particle bombardment. Embryos are transformed using the PDS-1000 Helium Gun from Bio-Rad at one shot per sample using 650 PSI rupture disks. DNA delivered per shot averages at 0.1667 ⁇ g.
  • Transformation of maize can also be done using the Agrobacterium mediated DNA delivery method, as described by U.S. Pat. No. 5,981,840 with the following modifications.
  • Agrobacteria are grown to the log phase in liquid minimal A medium containing 100 ⁇ M spectinomycin. Embryos are immersed in a log phase suspension of Agrobacteria adjusted to obtain an effective concentration of 5 ⁇ 10 8 cfu/ml. Embryos are infected for 5 minutes and then co-cultured on culture medium containing acetosyringone for 7 days at 20° C. in the dark.
  • the embryos are transferred to standard culture medium (MS salts with N6 macronutrients, 1 mg/L 2,4-D, 1 mg/L Dicamba, 20 g/L sucrose, 0.6 g/L glucose, 1 mg/L silver nitrate, and 100 mg/L carbenicillin) with a selective agent. Plates are maintained at 28° C. in the dark and are observed for colony recovery with transfers to fresh medium every two to three weeks. Recovered colonies and plants are scored based on the selectable or screenable phenotype imparted by the marker gene(s) introduced (i.e. herbicide resistance, fluorescence or anthocyanin production), and by molecular characterization via PCR and Southern analysis.
  • a selectable marker can be utilized in the recovery of transformed cells.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium (the active ingredient in BASTATM), bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • NEO neomycin phosphotransferase II
  • HPT hygromycin phosphotransferase
  • genes conferring resistance to herbicidal compounds such as glufosinate ammonium (the active ingredient in BASTATM), bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • GAT herbicide resistance polynucleotide encoding glyphosate
  • PAT phosphinothricin acetyltransferase
  • the PAT (phosphinothricin acetyltransferase) polynucleotide could be used for resistance to phosphinothricin (DeBlock et al., 1987, Engineering herbicide resistance in plant by expression of a detoxifying enzyme, EMBO J. 6:2513-2518).
  • ALS acetolactate synthase
  • Another example is an ALS (acetolactate synthase) polynucleotide for resistance to imidazolines (Sathasivan et al., 1990, Nucleotide sequence of a mutant acetolactate synthase gene from an imidazolinone-resistant Arabidopsis thaliana var, Columbia, Nucleic Acids Res. 18:2188).
  • Additional selectable markers include phenotypic markers such as ⁇ -galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al.
  • GFP green fluorescent protein
  • haploid embryos Once haploid embryos have been identified the haploid cells, haploid embryos, haploid seeds, haploid seedlings or haploid plants can be treated with a chromosome doubling agent. Homozygous plants can be regenerated from haploid cells by contacting the haploid cells, such as haploid embryo cells, with chromosome doubling agents. The haploid cells may come in contact with the doubling agent at the time of pollination, anytime after pollination, typically 6 hours to 21 days after pollination, 6 hours to 15 days after pollination, at the mature seed stage, at the seedling stage, or at the plant stage.
  • the haploid embryo may come in contact with the doubling agent when one sperm nucleus from a pollen grain fuses with the polar nuclei in the embryo sac to create a triploid (3N) endosperm (when the haploid embryo is formed), anytime after the pollination, typically 6 hours to 21 days after pollination, 6 hours to 15 days after pollination, or at the mature seed stage.
  • the haploid embryo may be isolated. It may be contained within the kernel, ovule, or seed. It may also be on the ear in the case of corn, or on the spike as in the case of other grains such as wheat.
  • the ear comprising the haploid embryo may be on the plant or isolated from the plant. The ear also may be sectioned.
  • the doubled haploid embryo will contain 2 copies of maternally derived chromosomes.
  • the efficiency of the process for obtaining doubled haploid plants from haploid embryos may be greater than 10%, 20%, 30%, 50%, 60%, 70%, 80%, or 90%.
  • Typical methods involve contacting the cells with colchicine, anti-microtubule agents or anti-microtubule herbicides, pronamide, nitrous oxide, or any mitotic inhibitor to create homozygous doubled haploid cells.
  • the amount of colchicine used in medium is generally 0.01%-0.2% or approximately 0.05% or APM (5-225 ⁇ M).
  • the amount of pronamide in medium is approximately 0.5- ⁇ M.
  • mitotic inhibitors are included but not limited to those indicated in Table 5.
  • Other agents may be used with the mitotic inhibitors to improve doubling efficiency. Such agents may be dimethyl sulfoxide (DMSO), adjuvants, surfactants, and the like.
  • the chromosome doubling agent may come in contact with the embryo at various times. If the embryo is isolated the doubling agent may come in contact immediately after isolation and before germination. If the embryo is contained within the seed, it may come in contact with the doubling agent anytime after pollination and before dry down. The embryo whether it is isolated or not may come in contact with the doubling agent any time between 6 hours after pollination and 21 days after pollination. The duration of contact between the chromosomal doubling agent may vary. Contact may be from less than 24 hours to about a week. The duration of contact is generally from about 24 hours to 2 days.
  • Non-callus promoting medium refers to a medium that does not support proliferation of dedifferentiated masses of cells or tissue.
  • a preferred “non-callus promoting medium” is used for embryo rescue, containing typical salt and vitamin formulations well known in the art. Such embryo rescue, or embryo culture, media contain little or no auxin [for review see Raghaven, V., 1966. Biol. Rev. 41:1-58]. Embryo maturation medium also represents another preferred “non-callus promoting medium”.
  • Embryo maturation medium is used to promote development of in vitro cultured embryos, preventing precocious germination, and typically contain standard salt/vitamin formulations (depending on the species), increased sugar levels and/or exogenously added abscisic acid, with little or no auxin.
  • Another type of medium is used for shoot culture, or multiple shoot proliferation. This multiple-shoot medium can again contain little or reduced auxin, but instead contain elevated levels of cytokinin that promote meristem proliferation and growth.
  • auxin is defined as an endogenous plant hormone such as indole acetic acid (IAA), derivatives of IAA such as indole-3-buteric acid, as well as compounds with auxin-like activity such as 2,4-D; picloram; dicamba; 3,4-D; 2,4,5-T and naphthalene acetic acid (NAA).
  • IAA indole acetic acid
  • NAA naphthalene acetic acid
  • a cytokinin is defined as a naturally occurring plant hormone such as 2-isopentynel adenine (2iP), zeatin and dihydrozeatin, or a synthetic compound with cytokinin-like activity such as kinetin and BAP (beynzylaminopurine).
  • Haploid cells from embryos, seeds, plants, etc. can be identified by several methods, such as, by chromosomal counts, measuring the length of guard cells, or by use of a Flow Cytometer.
  • Molecular markers or quantitative PCR can be used to determine if a tissue or plant is made of doubled haploid cells or is made of diploid cells (cells obtained through normal pollination).
  • Haploid embryos which are derived by any of the above techniques can be cultured to regenerate a whole plant. Such techniques are called embryo rescue.
  • Embryo rescue media can comprise certain phytohormones and energy sources or just energy sources.
  • the growth medium may also contain a selection agent such as a biocide and/or herbicide. This selection agent can be used to indicate a marker which has been introduced through the transformation process. For transformation and regeneration of maize see, Gordon-Kamm et al., The Plant Cell 2:603-618 (1990).
  • Zea mays also identified as corn or maize
  • soybean oilseed Brassica
  • alfalfa also identified as corn or maize
  • rice rye
  • sorghum sunflower
  • tobacco potato
  • peanuts cotton
  • sweet potato cassaya
  • sugar beets tomato
  • oats barley, and wheat.
  • Embryo rescue techniques can be used to generate immature doubled haploid embryos into plants (Recent Research Developments in Genetics & Breeding. Vol. 1, Part II, 287-308 2004). The disclosure of which is herein incorporated by reference.
  • the temperature at which the methods can be performed can vary.
  • the methods provided can be practiced at any temperature that does not kill a plant cell or plant or from about 16 degrees Celsius to 32 degrees Celsius.
  • the method includes but is not limited to producing about 3% or greater, about 5% or greater, or about 10% or greater viable haploid embryos. For calculating the percentage, the total number of embryos is determined by adding the number of haploid embryos to the number of embryos that did not develop properly.
  • the non-viable diploid embryos may be selected against any time after pollination.
  • the method includes but is not limited to selection at 7-15 days, 10-21 days, or at the time the haploid seed is fully mature, for example during or after dry down.
  • the inducer line has a gene that is expressed in the embryo and can be lethal.
  • the inducer maize line has a gene that prevents pollen transmission.
  • the gene that prevents pollen transmission is closely linked to a gene that when expressed in the embryo inhibits the lethality of the gene at the first location.
  • haploid embryos will easily be selected at an early stage of development. Having the lethal gene and the inhibitor gene at different locations in the genome, for example having the genes unlinked, allows the genes to segregate so that an adequate amount of pollen comprising the lethal gene is produced.
  • the gene that prevents pollen transmission is closely linked to, or adjacent to, the gene that inhibits embryo ablation.
  • a maize inducer line can be transformed with two different expression cassettes.
  • a first expression cassette includes a polynucleotide that when expressed in the embryo is lethal to the embryo or prevents the embryo from developing normally.
  • a second expression cassette includes a polynucleotide that prevents viable or transmissible pollen from developing and a polynucleotide that when expressed in the embryo prevents death or abnormal growth that would be caused by the polynucleotide on the first expression cassette.
  • the transformation process can be by various methods for example particle bombardment or agrobacterium infection.
  • the transformation process with the two cassettes may be done simultaneously or sequentially with the cassette comprising the polynucleotide to prevent embryo lethality being introgressed into the plant cell first.
  • the expression cassettes need to segregate in the gamete, therefore it is preferred that the two expression cassettes not be tightly linked.
  • any maize line may be transformed with the first, or first and second expression cassette.
  • the first and/or second expression cassettes may be used to transform one or two maize plants.
  • the expression cassettes can then be transferred simultaneously or sequentially into a maize inducer line by crossing.
  • the method can include backcrossing one or all trangenes into a maize inducer line. The repressor could be replaced and segregated out.
  • the inhibition described can be by various mechanisms.
  • the transcription of the lethal polynucleotide may be prevented.
  • This type of inhibition could be achieved by using a tet repressor polynucleotide that expresses a protein that binds to the lethal gene and inhibits expression.
  • Other types of prevention of lethality may be at the RNA or protein stage for example, anti-sense RNA, hair-pins, and other mechanisms for gene silencing.
  • the gene that prevents pollen transmission can be a lethal gene with a pollen specific promoter.
  • the gene can express alpha-amylase (Gene Bank L25805), Plant Physiology 105(2):759-760 (1994)) and it can be controlled with a PG47 promoter (U.S. Pat. No. 5,412,085; U.S. Pat. No. 5,545,546; Plant J. 3(2): 261-271 (1993)).
  • the expression of the polynucleotide that causes ablation and the polynucleotide that inhibits ablation can be controlled by various types of promoters.
  • the promoter may be a constitutive promoter, an inducible promoter, or a tissue preferred promoter such as an embryo preferred promoter. It would be most efficient if the promoter that drives the polynucleotide that causes ablation of the embryo would not express in endosperm. Or if the promoter is expressed in the endosperm the expression would be low enough that one could still determine a difference in growth between the diploid seed (normal fertilization) and the haploid seed.
  • An example of embryo preferred promoter is lec1.
  • the invention disclosed includes a maize inducer line comprising the two expression cassettes as described.
  • gat genes encoding glyphosate N-acetyltransferase (GAT). See PCT publication WO02/36782 and U.S. application Ser. No. 10/427,692.
  • lec1 indicates a leafy cotyledon 1 transcriptional activator polynucleotide. See U.S. patent application Ser. No. 09/435,054. lec1 promoter is characterized in U.S. Pat. No. 7,122,658.
  • moCah is a maize optimized gene that encodes for the Myrothecium verrucaria cyanamide hydratase protein [CAH] that can hydrate cyanamide to non-toxic urea.
  • pinII indicates potato proteinase inhibitor. See Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498.
  • Pro indicates a promoter sequence.
  • Term indicates a terminator sequence.
  • Ubi Pro indicates a ubiquitin promoter. See Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689)
  • Ubi1ZM Pro indicates a ubiquitin maize promoter.
  • haploid inducer lines such as Stock 6, RWS, KEMS, KMS or ZMS
  • the ears are surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • Embryos (generally about 7-14 days after pollination) are isolated from a maize plant and the embryos are contacted with a suspension of Agrobacterium , where the bacteria are capable of transferring the nucleotide sequences of interest to at least one cell of at least one of the embryos.
  • the Agrobacterium comprises the following expression cassette.
  • the embryos are typically immersed in an Agrobacterium suspension for the initiation of inoculation.
  • the Agrobacterium suspension contains 100 ⁇ M acetosyringone.
  • the embryos are co-cultured for a time with the Agrobacterium.
  • the embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step lasting 6-7 days is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants. Next, inoculated embryos are cultured on solid medium containing a selective agent for GAT, which is the herbicide glyphosate (GAT—genes encoding glyphosate N-acetyltransferase (GAT). See PCT publication WO02/36782 and U.S. application Ser. No. 10/427,692).
  • GAT herbicide glyphosate
  • This plasmid DNA is precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 ⁇ l prepared tungsten particles in water, 10 ⁇ l (1 ⁇ g) DNA in TrisEDTA buffer (1 ⁇ g total), 100 ⁇ l 2.5 M CaC1 2 , 10 ⁇ l 0.1 M spermidine.
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer.
  • the final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes.
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ⁇ l 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 ⁇ l 100% ethanol is added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
  • the sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
  • the callus is kept on 560Y medium for 2 days, then transferred to selection medium containing the selection agent for the PAT gene, bialophos, and subcultured every 2 weeks.
  • selection-resistant callus clones are transferred to regeneration medium to initiate plants. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5′′ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for the genotype and/or phenotype of interest.
  • Bombardment medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000 ⁇ SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H20 following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H20); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Selection medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000 ⁇ SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H20 following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H20); and 0.85 mg/l silver nitrate and selection agent (both added after sterilizing the medium and cooling to room temperature).
  • the inducer line can be used to produce haploid embryos. This can be achieved through the growing of F1 corn seed. For example a corn breeder wanting to produce a superior inbred with the best characteristics from elite inbred line A and elite inbred line B crosses the two inbreds to form F1 hybrid seed. This F1 seed is grown along with the ablation inducer line. The timing of the planting is such that the pollen of the ablation inducer line is ready at the silking time of the F1 hybrid seed. The silks of the F1 plants are pollinated with the ablation inducer line pollen. This can be achieved by planting alternate rows of the F1 and the inducer line, and various combinations thereof with the F1, or female, plants being detasseled before pollination. The crosses can also be done by shoot bagging and controlling pollination by hand pollinating.
  • the seed After the seed has matured the seed can be harvested and the only viable seed will be the haploid seed.
  • the haploid seed is then planted and undergoes chromosome doubling using a chromosomal doubling agent such as pronamide.
  • Hi-II maize seeds are planted.
  • the ears at 9-12 days after pollination are harvested surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • the embryos are isolated from ears using a scalpel.
  • Embryos are contacted with a suspension of Agrobacterium , where the bacteria are capable of transferring the nucleotide sequences of interest to at least one cell of at least one of the embryos. In this example the embryos are co-transformed with 2 Agrobacteria.
  • One Agrobacterium comprises the following expression cassette.
  • the second Agrobacterium comprises the following expression cassette.
  • the barnase gene includes an intron for the purpose of preventing expression of barnase in the Agrobacterium.
  • tissue culture media and process is the same as described above with the selection agent, bialophos. Callus that only comprises Construct E will be selected against with the use of bialophos. Callus that only comprises Construct F will not regenerate because the barstar construct is not available to prevent the toxicity of the barnase.
  • Plants will be regenerated that comprise Constructs E and F. These plants will be used to backcross both constructs into a maize haploid inducer line. During the backcross process the co-transformed lines will evaluated to determine the linkage between the constructs. The closer the constructs are linked the less transmission of the barnase polynucleotide through the pollen will occur because it will segregate with the alpha-amylase polynucleotide expressed in the pollen making the pollen non-viable.
  • Barstar and barnase are both expressed in early embryogenesis on separate, unlinked transgenes in the haploid inducer line.
  • the haploid inducer line can be maintained.
  • One Agrobacterium comprises the following 2 expression cassettes.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
US12/023,152 2007-02-02 2008-01-31 Selective Ablation of Diploid Embryos Abandoned US20080189801A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US12/023,152 US20080189801A1 (en) 2007-02-02 2008-01-31 Selective Ablation of Diploid Embryos
US13/251,597 US8269061B2 (en) 2007-02-02 2011-10-03 Selective ablation of diploid embryos
US13/586,893 US9121032B2 (en) 2007-02-02 2012-08-16 Selective ablation of diploid embryos

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US88782807P 2007-02-02 2007-02-02
US12/023,152 US20080189801A1 (en) 2007-02-02 2008-01-31 Selective Ablation of Diploid Embryos

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/251,597 Division US8269061B2 (en) 2007-02-02 2011-10-03 Selective ablation of diploid embryos

Publications (1)

Publication Number Publication Date
US20080189801A1 true US20080189801A1 (en) 2008-08-07

Family

ID=39591375

Family Applications (3)

Application Number Title Priority Date Filing Date
US12/023,152 Abandoned US20080189801A1 (en) 2007-02-02 2008-01-31 Selective Ablation of Diploid Embryos
US13/251,597 Active US8269061B2 (en) 2007-02-02 2011-10-03 Selective ablation of diploid embryos
US13/586,893 Active 2028-11-25 US9121032B2 (en) 2007-02-02 2012-08-16 Selective ablation of diploid embryos

Family Applications After (2)

Application Number Title Priority Date Filing Date
US13/251,597 Active US8269061B2 (en) 2007-02-02 2011-10-03 Selective ablation of diploid embryos
US13/586,893 Active 2028-11-25 US9121032B2 (en) 2007-02-02 2012-08-16 Selective ablation of diploid embryos

Country Status (7)

Country Link
US (3) US20080189801A1 (es)
CN (1) CN101652481A (es)
AR (1) AR065103A1 (es)
AU (1) AU2008214051A1 (es)
BR (1) BRPI0806864A2 (es)
MX (1) MX2009008210A (es)
WO (1) WO2008097791A2 (es)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130180005A1 (en) * 2012-01-06 2013-07-11 Pioneer Hi Bred International Inc Method to Screen Plants for Genetic Elements Inducing Parthenogenesis in Plants
WO2014110274A3 (en) * 2013-01-09 2015-02-26 Regents Of The University Of California A California Corporation Generation of haploid plants
CN108476979A (zh) * 2018-03-13 2018-09-04 南京理想农业科技有限公司 一种不结球白菜四倍体育种方法
US11759476B2 (en) 2020-12-14 2023-09-19 Regeneron Pharmaceuticals, Inc. Methods of treating metabolic disorders and cardiovascular disease with Inhibin Subunit Beta E (INHBE) inhibitors

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103053414B (zh) * 2011-10-24 2015-07-08 中国农业大学 利用除草剂加倍玉米单倍体的方法及其专用除草剂
CN103053413A (zh) * 2012-12-16 2013-04-24 四川农业大学 一种化学加倍玉米单倍体幼胚的处理方法
WO2017004375A1 (en) * 2015-06-30 2017-01-05 Regents Of The University Of Minnesota Haploid inducer line for accelerated genome editing
JP2018530323A (ja) * 2015-10-02 2018-10-18 キージーン ナムローゼ フェンノートシャップ 半数体及びそれに続く倍加半数体植物の産生方法
US10519456B2 (en) 2016-12-02 2019-12-31 Syngenta Participations Ag Simultaneous gene editing and haploid induction
WO2018185320A1 (en) 2017-04-07 2018-10-11 Limagrain Europe Method for sorting corn kernels of a batch of corn kernels
CN113412333B (zh) 2019-03-11 2025-04-08 先锋国际良种公司 用于克隆植物生产的方法
CN111837944A (zh) * 2020-07-31 2020-10-30 湖北康农种业股份有限公司 玉米单倍体加倍的方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364780A (en) * 1989-03-17 1994-11-15 E. I. Du Pont De Nemours And Company External regulation of gene expression by inducible promoters
US5412085A (en) * 1992-07-09 1995-05-02 Pioneer Hi-Bred International Inc. Pollen-specific promoter from maize
US5639951A (en) * 1993-07-06 1997-06-17 Sandoz Ltd. Doubled haploids
US6235975B1 (en) * 1997-02-21 2001-05-22 The Regents Of The University Of California Leafy cotyledon1 genes and methods of modulating embryo development in transgenic plants
US20010007154A1 (en) * 1997-01-24 2001-07-05 Dna Plant Technology Corporation Materials and methods for hybrid seed production
US20020188965A1 (en) * 2001-04-20 2002-12-12 Zou-Yu Zhao Methods of transforming plants
US6743968B2 (en) * 2000-02-28 2004-06-01 Yale University Methods and compositions to reduce or eliminate transmission of a transgene
US20050229269A1 (en) * 1992-07-29 2005-10-13 Zeneca Limited Plant germplasm
US7250296B2 (en) * 2003-03-05 2007-07-31 University Of Florida Research Foundation, Inc. Nucleotide sequences of 2S albumin gene and its promoter from grape and uses thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5749169A (en) * 1995-06-07 1998-05-12 Pioneer Hi-Bred International, Inc. Use of the indeterminate gametophyte gene for maize improvement
US6392119B1 (en) * 1997-01-24 2002-05-21 Dna Plant Technology Corporation Two component plant cell lethality methods and compositions
DE69942650D1 (de) 1998-12-22 2010-09-16 Dow Agrosciences Llc Verfahren zum limitieren des herauskreuzens und unerwünschten genflusses in nutzpflanzen
US8859846B2 (en) * 2005-09-21 2014-10-14 E. I. Du Pont De Nemours And Company Doubling of chromosomes in haploid embryos

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364780A (en) * 1989-03-17 1994-11-15 E. I. Du Pont De Nemours And Company External regulation of gene expression by inducible promoters
US5412085A (en) * 1992-07-09 1995-05-02 Pioneer Hi-Bred International Inc. Pollen-specific promoter from maize
US5545546A (en) * 1992-07-09 1996-08-13 Pioneer Hi-Bred International, Inc. Pollen-specific promoter from maize
US20050229269A1 (en) * 1992-07-29 2005-10-13 Zeneca Limited Plant germplasm
US5639951A (en) * 1993-07-06 1997-06-17 Sandoz Ltd. Doubled haploids
US20010007154A1 (en) * 1997-01-24 2001-07-05 Dna Plant Technology Corporation Materials and methods for hybrid seed production
US6235975B1 (en) * 1997-02-21 2001-05-22 The Regents Of The University Of California Leafy cotyledon1 genes and methods of modulating embryo development in transgenic plants
US6743968B2 (en) * 2000-02-28 2004-06-01 Yale University Methods and compositions to reduce or eliminate transmission of a transgene
US20020188965A1 (en) * 2001-04-20 2002-12-12 Zou-Yu Zhao Methods of transforming plants
US7250296B2 (en) * 2003-03-05 2007-07-31 University Of Florida Research Foundation, Inc. Nucleotide sequences of 2S albumin gene and its promoter from grape and uses thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130180005A1 (en) * 2012-01-06 2013-07-11 Pioneer Hi Bred International Inc Method to Screen Plants for Genetic Elements Inducing Parthenogenesis in Plants
WO2014110274A3 (en) * 2013-01-09 2015-02-26 Regents Of The University Of California A California Corporation Generation of haploid plants
CN108476979A (zh) * 2018-03-13 2018-09-04 南京理想农业科技有限公司 一种不结球白菜四倍体育种方法
US11759476B2 (en) 2020-12-14 2023-09-19 Regeneron Pharmaceuticals, Inc. Methods of treating metabolic disorders and cardiovascular disease with Inhibin Subunit Beta E (INHBE) inhibitors
US11957704B2 (en) 2020-12-14 2024-04-16 Regeneron Pharmaceuticals, Inc. Methods of treating metabolic disorders and cardiovascular disease with inhibin subunit beta E (INHBE) inhibitors

Also Published As

Publication number Publication date
BRPI0806864A2 (pt) 2014-04-29
CN101652481A (zh) 2010-02-17
US20120311736A1 (en) 2012-12-06
MX2009008210A (es) 2009-08-12
AU2008214051A1 (en) 2008-08-14
WO2008097791A2 (en) 2008-08-14
AR065103A1 (es) 2009-05-13
US9121032B2 (en) 2015-09-01
WO2008097791A3 (en) 2008-10-02
US8269061B2 (en) 2012-09-18
US20120023612A1 (en) 2012-01-26

Similar Documents

Publication Publication Date Title
US9121032B2 (en) Selective ablation of diploid embryos
US20230203516A1 (en) Systems and methods for cellular reprogramming of a plant cell
US20230189734A1 (en) Embryogenesis factors for cellular reprogramming of a plant cell
US10870860B2 (en) Genetic reduction of male fertility in plants
PT1696721E (pt) Transgénicos com supressão de gene dominante e processos para a sua utilização
US20150240254A1 (en) Genetic reduction of male fertility in plants
MX2014011037A (es) Reduccion genetica de la fertilidad masculina en plantas.
US20250311689A1 (en) Seedling germination and growth conditions
CN104703998A (zh) 植物中雄性育性的遗传减少
WO2013138309A1 (en) Genetic reduction of male fertility in plants
US20230407324A1 (en) Doubled haploid inducer
MXPA05000401A (es) Gametofito 1 indeterminado (ig1), mutaciones de ig1, ortologos de ig1 y usos del mismo.
HK40034402A (en) Systems and methods for cellular reprogramming of a plant cell

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION