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WO1999058644A1 - Compositions et procedes destines a la modification genetique de l'ananas - Google Patents

Compositions et procedes destines a la modification genetique de l'ananas Download PDF

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Publication number
WO1999058644A1
WO1999058644A1 PCT/US1999/010576 US9910576W WO9958644A1 WO 1999058644 A1 WO1999058644 A1 WO 1999058644A1 US 9910576 W US9910576 W US 9910576W WO 9958644 A1 WO9958644 A1 WO 9958644A1
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pineapple
totipotent
plant
transgenic
plants
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PCT/US1999/010576
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English (en)
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Guo-Ling Nan
Chifumi Nagai
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University Of Hawaii
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Priority to AU39027/99A priority Critical patent/AU3902799A/en
Publication of WO1999058644A1 publication Critical patent/WO1999058644A1/fr

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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
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    • 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
    • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—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 biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to improved methods for in vitro culture of pineapple-like protocorm-like bodies and to methods for the production of transgenic pineapple-like plants.
  • this invention relates to the in vitro culture of pineapple-like protocorm like bodies, and to the genetic transformation of pineapplelike protocorm-like bodies and of pineapple-like callus to produce transgenic pineapple-like plants.
  • Bromeliads are a family of plants which includes a wide variety of ornamental plants as well as plants valued for their edible fruit.
  • Foremost among the members of the Bromeliaceae family is pineapple which bears a fruit that is valuable both to the commercial producer and to the consumer, both of whom have come to expect certain characteristic in the fruit produced and consumed.
  • characteristics which are valued by the commercial producer include pest and disease resistance of both the plant and fruit, a high yield of fruit, high brix, and extended shelf-life
  • those valued by the consumer include low acidity (particularly in winter fruit), low fiber, and uniform fruit shape and color.
  • the present invention provides a stably transformed transgenic pineapple-like totipotent body. While not intending to limit the invention to a particular totipotent body, in a preferred embodiment, the totipotent body is a protocorm-like body. In an alternative preferred embodiment, the totipotent body is a callus. In yet another embodiment, the totipotent body comprises undifferentiated cells. In a further preferred embodiment, the pineapple-like totipotent body is a pineapple totipotent body. In another embodiment, the invention provides a stably transformed transgenic pineapple-like plant. Without limiting the type of tissue from which the transgenic pineapple-like plant is derived, in one preferred embodiment, the plant is derived from a transgenic pineapple-like totipotent body.
  • the totipotent body is a protocorm-like body. In yet a further preferred embodiment, the totipotent body is a callus. Without intending to limit the type of plant, in one preferred embodiment, the plant is a pineapple plant.
  • Yet another embodiment of the present invention is a method for producing a stably transformed transgenic pineapple-like totipotent body, comprising: a) providing: i) a pineapple-like totipotent body; and ii) a heterologous nucleic acid comprising an oligonucleotide sequence of interest; and b) introducing the oligonucleotide sequence of interest into the totipotent body under conditions such that a stably transformed transgenic pineapple-like totipotent body is produced.
  • the totipotent body is a callus.
  • the totipotent body is a protocorm-like body.
  • the method further comprises c) selecting the transgenic totipotent body.
  • the selecting is in liquid medium.
  • the selecting comprises detecting the oligonucleotide in the genome of the totipotent body.
  • the pineapple-like totipotent body is a pineapple totipotent body.
  • the oligonucleotide is introduced by bombarding the totipotent body with the oligonucleotide sequence of interest.
  • the oligonucleotide is introduced by infecting the totipotent body with Agrobacterium comprising the oligonucleotide sequence of interest.
  • the infecting comprises microwounding the totipotent body to produce a microwounded totipotent body, and infecting the microwounded totipotent body with the Agrobacterium.
  • the Agrobacterium is agropine-type.
  • the Agrobacterium is nopaline-type.
  • the Agrobacterium is octopine-type.
  • the present invention provides a method for producing a stably transformed transgenic pineapple-like plant, comprising: a) providing: i) a pineapple-like totipotent body; and ii) a heterologous nucleic acid comprising an oligonucleotide sequence of interest; b) introducing the oligonucleotide sequence of interest into the totipotent body under conditions such that a transgenic pineapple-like totipotent body is produced; and c) culturing the transgenic pineapple-like totipotent body under conditions such that a stably transformed transgenic pineapple-like plant is produced.
  • the oligonucleotide is introduced by bombarding the pineapple-like totipotent body with the oligonucleotide sequence of interest.
  • the oligonucleotide is introduced by infecting the pineapple-like totipotent body with Agrobacterium comprising the oligonucleotide sequence of interest.
  • the method further comprises prior to step c) selecting the transgenic totipotent body.
  • the selecting is in liquid medium.
  • the selecting comprises detecting the oligonucleotide in the genome of the pineapple-like totipotent body.
  • the pineapple-like plant is a pineapple plant.
  • the sequence of interest is selected from the group consisting of oligonucleotides encoding sucrose phosphate synthase, CpTi, thaumatin, and ACC deaminase.
  • the sequence of interest is selected from the group consisting of antisense polyphenol oxidase and ACC oxidase.
  • the invention provides a method for producing a pineapple-like protocorm-like body comprising maintaining the pineapple-like protocorm-like body in liquid medium.
  • the liquid medium substantially comprises PI medium.
  • the pineapple-like plant is a pineapple plant.
  • Figure 1 schematically shows the structure of pBI426.
  • Figure 2 schematically shows the structure of pBI121.
  • Figure 3 schematically shows the nucleotide sequence of the 5' transcribed region of the rice Actl-Gus gene fusion in pActl-F between the Actl TATA box and Gus translation initiation codon.
  • Figure 4 schematically shows the structure pAHC27.
  • Figure 5 schematically shows the structure of pHA9.
  • Figure 6 schematically shows the structure of pEmuGN.
  • Figure 7 schematically shows the structure of pWDl.
  • Figure 8 schematically shows the structure of pWD3.
  • Figure 9 schematically shows the structure of pML72.
  • Figure 10 schematically shows the structure of pML75.
  • Figure 11 shows transient expression of GUS protein as measured by histochemical staining in pineapple (A) leaf, (B) lead and fruit, and (C) flower petal used as target tissue for biolistic bombardment of expression constructs containing the Gus gene.
  • Figure 12 shows an antibiotic growth inhibition curve for F153 (A and C) and for D10 (B and D) protocorm-like bodies (plbs) treated for one month (open circles) or two months (filled squares) with different G418 concentrations in liquid PI media (A and B) and agar- solidified shoot regeneration PM2 media (C and D).
  • Figure 13 shows the nucleotide sequence (SEQ ID NO:l) encoding polyphenol oxidase.
  • Figure 14 shows the nucleotide sequence (SEQ ID NO:2) encoding maize sucrose phosphate synthase enzyme.
  • Figure 15 shows a Southern hybridization blot of genomic DNA isolated from pineapple leaves transfected with pBI426 and PBI121 using biolistic bombardment.
  • totipotent body refers to a collection of cells (e.g., a cell aggregate) comprising undifferentiated plant cells capable of differentiation into a plant; an totipotent body may also contain some differentiated cells.
  • An "totipotent body” includes, but is not limited to, a protocorm-like body, a callus, and the like.
  • the ability of an totipotent body to differentiate into a plant is determined using methods known in the art as well as methods described herein. For example, differentiation into shoots may be accomplished by culturing an totipotent body on agar-solidifled hormone-free modified MS medium, or on agar-solidified PM2 medium. Differentiation into roots may be accomplished by culture of an totipotent body in liquid modified MS medium containing 1 mg/L NAA.
  • plb when made in reference to pineapple refer to an totipotent body which is generally, though not necessarily, creamy yellow and globular shaped.
  • a plb derived form pineapple tissue is morphologically distinguishable from a callus derived from pineapple.
  • a plb which is derived from pineapple tissue is characterized by having a partially organized morphology with a pre-determined apical meristematic region covered by a distinctive epidermal layer but lacking vascular tissue; a callus derived from pineapple tissue is a disorganized mass of undifferentiated plant cells lacking apical meristem, epidermis, and vascular tissue.
  • pineapple plbs may be generated as previously described (Wakasa et al. (1978) Japan. J. Breed. 28:113-121; Mapes (1973) Proc. Intl. Plant Propagation Soc.
  • Pineapple callus may be produced as described by (Mathews et al. (1981) Scientia Horiculturae 14:227-234).
  • plant refers to a plurality of plant cells which are largely differentiated into a structure that is present at any stage of a plant's development.
  • Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.
  • the term "pineapple” refers to a member of the genera Ananas and Pseudoananas of the Bromeliaceae family.
  • the genus Pseudoananas is monotypic, i.e., consists of P.bianarius.
  • the genus Ananas consists of five species, namely, A. bracteatus, A. fritzmuelleri, A. comosus, A. erectifolius, and A. ananassoides.
  • pineapple-like refers to a member of the Bromeliaceae family. Genera comprised within the Bromeliaceae family include Tillansia, Aechmea, Neoregrelia, etc.
  • PI medium refers to supplemented modified MS medium. Modified
  • MS medium refers to the medium described by Murashige & Skoog (Murashige & Skoog, (1962) Physiol. Plant 15::473-497) (Gibco BRL) which is modified by the addition of 100 mg/L myo-inositol (Sigma), 1 mg/L thiamine-HCl (Sigma), and 30 g sucrose (Sigma), pH 5.6-5.8, and supplemented by the addition either of 2 mg/L ⁇ - naphtaleneacetic acid (NAA) (Sigma) and 1 mgL 6-benzyl aminopurine (BAP)
  • NAA ⁇ - naphtaleneacetic acid
  • BAP 6-benzyl aminopurine
  • transgenic when used in reference to a protocorm-like body refers to a protocorm-like body which comprises one or more cells that contain a transgene, or whose genome has been altered by the introduction of a transgene.
  • transgenic when used in reference to a plant refers to a plant which comprises one or more cells which contain a transgene, or whose genome has been altered by the introduction of a transgene.
  • transgenic plbs and transgenic plants may be produced by several methods including the introduction of a "transgene” comprising nucleic acid (usually DNA) into a target cell or integration into a chromosome of a target cell by way of human intervention, such as by the methods described herein.
  • transgene refers to any nucleic acid sequence which is introduced into the genome of a plant cell by experimental manipulations.
  • a transgene may be an "endogenous DNA sequence," or a “heterologous DNA sequence” (i.e., “foreign DNA”).
  • endogenous DNA sequence refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.
  • heterologous DNA sequence refers to a nucleotide sequence which is Hgated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not Hgated in nature, or to which it is ligated at a different location in nature.
  • Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell.
  • Heterologous DNA also includes an endogenous DNA sequence which contains some modification.
  • heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.
  • gene refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or its precursor.
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence.
  • transformation refers to the introduction of foreign DNA into cells. Transformation of a plant cell may be accomplished by a variety of means known in the art including particle mediated gene transfer (see, e.g., U.S. Patent
  • Transformation of a cell may be stable or transient.
  • transient transformation or “transiently transformed” refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA) which detects the presence of a polypeptide encoded by one or more of the transgenes.
  • ELISA enzyme-linked immunosorbent assay
  • transient transformant refers to a cell which has transiently integrated one or more transgenes.
  • stable transformation or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell.
  • Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the transgenes.
  • stable transformant refers to a cell which has stably integrated one or more transgenes into the genomic DNA.
  • a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene.
  • nucleic acid sequence of interest refers to any nucleic acid sequence the manipulation of which may be deemed desirable for any reason by one of ordinary skill in the art (e.g., confer improved qualities).
  • wild-type when made in reference to a gene refers to a gene which has the characteristics of a gene isolated from a naturally occurring source.
  • wild-type when made in reference to a gene product refers to a gene product which has the characteristics of a gene product isolated from a naturally occurring source.
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.
  • the term “modified” or “mutant” when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • antisense refers to a deoxyribonucleotide sequence whose sequence of deoxyribonucleotide residues is in reverse 5' to 3' orientation in relation to the sequence of deoxyribonucleotide residues in a sense strand of a DNA duplex.
  • a "sense strand" of a DNA duplex refers to a strand in a DNA duplex which is transcribed by a cell in its natural state into a "sense mRNA.”
  • an "antisense” sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex.
  • antisense mRNA refers to a ribonucleotide sequence whose sequence is complementary to an "antisense” sequence.
  • recombinant when made in reference to a DNA molecule refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
  • recombinant when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant DNA molecule.
  • vector and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
  • expression vector or “expression cassette” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • targeting vector or “targeting construct” refer to oligonucleotide sequences comprising a gene of interest flanked on either side by a recognition sequence which is capable of homologous recombination of the DNA sequence located between the flanking recognition sequences.
  • in operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • Southern blot refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane.
  • the immobilized DNA is then probed with a labeled oligo- deoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used.
  • the DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support.
  • Southern blots are a standard tool of molecular biologists [J. Sambrook et al. (1989) Molecular Cloning: A
  • infectious and “infection” with a bacterium refer to co-incubation of a target biological sample, (e.g., cell, tissue, etc.) with the bacterium under conditions such that nucleic acid sequences contained within the bacterium are introduced into one or more cells of the target biological sample.
  • a target biological sample e.g., cell, tissue, etc.
  • Agrobacterium refers to a soil-borne, Gram-neggative, rod-shaped phytopathogenic bacterium which causes crown gall.
  • Agrobacterium includes, but is not limited to, the strains Agrobacterium tumefaciens, (which typically causes crown gall in infected plants), and Agrobacterium rhizogens (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium tumefaciens, (which typically causes crown gall in infected plants), and Agrobacterium rhizogens (which causes hairy root disease in infected host plants). Infection of a plant cell with
  • Agrobacterium generally results in the production of opines (e.g., nopaline, agropine, octopine etc.) by the infected cell.
  • opines e.g., nopaline, agropine, octopine etc.
  • nopaline e.g., strain LBA4301, C58, A208
  • octopine-type e.g., strain LBA4404, Ach5, B6
  • octopine-type e.g., strain EHA105, EHA101, A281
  • agropine-type agropine-type
  • biolistic bombardment refers to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample.
  • a target biological sample e.g., cell, tissue, etc.
  • Methods for biolistic bombardment are known in the art (e.g., U.S. Patent No. 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He)
  • microwounding when made in reference to plant tissue refers to the introduction of microscopic wounds in that tissue. Microwounding may be achieved by, for example, particle bombardment as described herein.
  • probe when made in reference to an oligonucleotide
  • a sequence of nucleotides refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest.
  • a probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. Oligonucleotide probes may be labelled with a "reporter molecule," so that the probe is detectable using a detection system. Detection systems include, but are not limited to, enzyme, fluorescent, radioactive, and luminescent systems.
  • selectable marker refers to a gene which encodes an enzyme having an activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
  • Selectable markers may be "positive” or “negative.” Examples of positive selectable markers include the neomycin phosphotrasferase (NPTII) gene which confers resistance to G418 and to kanamycin, and the bacterial hygromycin phosphotransferase gene (hyg), which confers resistance to the antibiotic hygromycin.
  • Negative selectable markers encode an enzymatic activity whose expression is cytotoxic to the cell when grown in an appropriate selective medium. For example, the HSV-t gene is commonly used as a negative selectable marker.
  • HSV-tA expression of the HSV-tA; gene in cells grown in the presence of gancyclovir or acyclovir is cytotoxic; thus, growth of cells in selective medium containing gancyclovir or acyclovir selects against cells capable of expressing a functional HSV TK enzyme.
  • promoter element refers to a DNA sequence that is located at the 5' end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
  • PCR polymerase chain reaction
  • Mullis disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188, all of which are hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification.
  • This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.
  • the two primers are complementary to their respective strands of the double stranded target sequence.
  • the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous “cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • the method is referred to as the "polymerase chain reaction” (hereinafter "PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”
  • PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; and/or incorporation of 32 P-labeled deoxyribonucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
  • any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • Amplified target sequences may be used to obtain segments of DNA (e.g., genes) for the construction of targeting vectors, transgenes, etc.
  • hybridization refers to any process by which a strand of nucleic acid joins with a complementary strand through base pairing [Coombs J
  • the terms "complementary” or “complementarity” when used in reference to polynucleotides refer to polynucleotides which are related by the base- pairing rules. For example, for the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
  • the term "homology" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (i. e. , identity).
  • a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i. e. , the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency.
  • low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (/. e. , selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non- complementary target.
  • Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent
  • High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions.
  • factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions
  • Stringency when used in reference to nucleic acid hybridization typically occurs in a range from about T m -5°C (5°C below the T m of the probe) to about 20°C to 25°C below T m .
  • a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
  • stringent conditions a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences.
  • the present invention provides methods for the production of transgenic pineapple-like totipotent bodies.
  • this invention provides methods for producing transgenic pineapple-like callus and transgenic pineapple-like protocorm-like bodies.
  • transgenic plants from transgenic totipotent bodies. More particularly, the invention discloses methods for generating transgenic pineapple-like plants from transgenic pineapple-like protocorm-like bodies and from transgenic pineapple-like callus. Yet more particularly, the present invention provides methods for generating transgenic pineapple-like plants from plbs and callus.
  • the invention additionally provides transgenic pineapple-like plants.
  • the pineapple-like plants provided by this invention may be genetically engineered to exhibit resistance to pests and disease (e.g., mealybugs, nematodes, root-rot and heart- rot disease, wilt virus, etc.) and to environmental stress (e.g., water-logging, drought, heat, cold, light-intensity, day-length, chemicals, etc.) and to exhibit improved qualities (e.g., longer storage life, higher yield, higher sugar content, higher vitamins C and A content, lower acidity, non-translucent flesh color, longer storage, etc.).
  • pests and disease e.g., mealybugs, nematodes, root-rot and heart- rot disease, wilt virus, etc.
  • environmental stress e.g., water-logging, drought, heat, cold, light-intensity, day-length, chemicals, etc.
  • improved qualities e.g., longer storage life, higher yield, higher sugar content, higher vitamins
  • the invention further provides improved methods for the maintenance of pineapple-like protocorm-like bodies in culture. These methods are useful for the selection of stably transformed pineapple-like protocorm-like bodies. Such selection reduces the time, cost, and labor involved in screening for the presence of transgenic plants regenerated directly from protocorm-like bodies which are subjected to transformation protocols in the absence of a selection step to enrich for stably transformed protocorm-like bodies. These methods are also useful for the amplification of the number of transformed protocorm-like bodies, thus providing a larger amount of starting material for the regeneration of a greater number of cloned plants.
  • the description of the invention is divided into (a) generation of pineapple totipotent bodies, (b) maintenance in in vitro culture of pineapple protocorm- like bodies, (c) generation of transgenic pineapple totipotent bodies, and (d) generation of transgenic pineapple plants.
  • Totipotent bodies derived from pineapple comprise protocorm-like bodies and callus. Pineapple protocorm-like bodies are generated by in vitro culture of pineapple explants from different tissues as previously described (Wakasa et al. (1978) supra; Mapes (1973), supra) as well as using methods described herein.
  • explants from the crown and vegetative shoots may be used to initiate plb cultures by placing the explants in media containing growth regulators.
  • the generation of plbs is not limited to explants from crown tissue or vegetative shoot tissue. Any pineapple tissue which is capable of generating plbs in in vitro culture is contemplated to be within the scope of this invention.
  • the media used to initiate plb formation is liquid modified MS medium containing a combination of NAA ( ⁇ -naphtaleneacetic acid) and BAP (6- benzyl aminopurine).
  • the liquid medium used for the initiation of plbs is not limited to the media or to the media components and additives disclosed herein. Any media containing any combination of components which is capable of initiating plb formation is included within the scope of the invention.
  • the concentration of NAA is 2 mg/L and of BAP is 1 mg/L. In another embodiment, the concentration of NAA is 10 mg/L and of BAP is 1 mg/L. It is not intended that the present invention be limited by the concentration of NAA and/or BAP. The invention is also not limited to the use of NAA and/or BAP. Where NAA and BAP are used in the media, the concentrations of NAA and BAP may be varied such that the formation of differentiated structures (e.g., shoots) is minimized, while the formation of plbs is maximized.
  • differentiated structures e.g., shoots
  • plbs are initiated in liquid medium. It is preferred that plbs are initiated in liquid medium rather than on solidified (e.g., agar- solidified) medium since plbs on agar-solidified medium typically turn brown or differentiate into plants. It is also preferred, though not required, that liquid cultures be mixed gently by shaking on a gyrotory shaker in order to reduce the probability of re-orientation of cells of the largely undifferentiated totipotent body into a more differentiated structure. Pineapple callus may be generated using methods known in the art such as those described by Mathews et al. (1981), supra.
  • pineapple callus is initiated from the basal region of in vitro-obtained shoot buds on MS medium supplemented with 4.5 mg/L NAA, 5.2 mg/L IAA (indoleacetic acid), 2.1 mg/L kinetin and maintained on MS medium with 400 mg/L casein hydrolysate, 15% (v) coconut water, 10 mg/L NAA.
  • MS medium supplemented with 4.5 mg/L NAA, 5.2 mg/L IAA (indoleacetic acid), 2.1 mg/L kinetin and maintained on MS medium with 400 mg/L casein hydrolysate, 15% (v) coconut water, 10 mg/L NAA.
  • Such callus cultures when grown on MS medium devoid of any growth regulators, regenerated shoot buds and optimum regeneration was obtained on MS medium containing 5% coconut water.
  • the invention provides methods for the maintenance of pineapple totipotent bodies in in vitro culture.
  • the terms "maintained” and “maintenance” refer to the retention, following generation as described supra, of viable and dividing cells of totipotent bodies.
  • the methods of the invention are not limited to the maintenance of totipotent bodies which are generated in liquid culture.
  • the methods of this invention also contemplate the maintenance of totipotent bodies (e.g., plbs and callus) which are generated on solidified (e.g., agar solidified) medium.
  • the continued presence of viable and dividing cells in the totipotent body may be determined, for example, visually by observing an increase in size, measuring the volume of the totipotent body, observing incorporation of dyes such as tetrazolium chloride, etc.
  • Pineapple plbs and callus may be maintained in in vitro culture prior to and/or subsequent to transformation with a nucleic acid sequence of interest.
  • Data provided herein demonstrates that pineapple plbs may be maintained in liquid medium for at least one year.
  • plbs were maintained in liquid PI medium (i.e., modified MS medium containing 2 mg/L NAA and 1 mg/L BAP).
  • liquid PI medium i.e., modified MS medium containing 2 mg/L NAA and 1 mg/L BAP.
  • the nature of the components of the medium used for the maintenance of plbs is not a critical aspect of this invention so long as viable and dividing cells of totipotent bodies are maintained in the medium and so long as the medium is a liquid medium.
  • the term "liquid medium” as used herein refers to a medium which is in a state other than a solid state.
  • a liquid medium may include a semi-solid medium
  • Media components may be natural (e.g., enzymatic digest, coconut milk, etc.) or artificial (e.g., salts and growth regulators of defined chemical composition and amount).
  • a suitable medium furnishes minerals, nitrogen sources, vitamins, growth factors, and the like.
  • Components and methods for the preparation of plant culture media are commercially available, and are well within the ordinary skill in the art [see, e.g., "Handbook of Cell Culture,” Vol 2 and Vol. 3, W.R. Sharp, D.A. Evans, P.V. Ammirato, Y. Yamada (Eds.) (1983) MacMillan Publishing Co., New York].
  • the liquid culture used for the maintenance of pineapple totipotent bodies be shaken gently, e.g., on a gyrotory shaker; mixing of the liquid culture minimizes the re-orientation of cells of the undifferentiated totipotent body into a differentiated structure thus allowing the maintenance of undifferentiated structures over a longer period of time.
  • plbs were maintained by subculturing to fresh PI liquid medium every 2 to 3 weeks and by selecting vigorous growing globular shape bodies.
  • a transgenic pineapple totipotent body may be generated by introducing any nucleic acid sequence of interest into a pineapple totipotent body.
  • the transgenic pineapple totipotent bodies of the invention are not limited to totipotent bodies in which each and every cell expresses the nucleic acid sequence of interest. Rather, the invention includes within its scope any pineapple totipotent body which contains at least one cell which contains and is capable of expressing (or which expresses) the nucleic acid sequence of interest. Where it is desirable to express the nucleic acid sequence of interest in cells of an totipotent body, the nucleic acid sequence of interest is introduced into the cells of the totipotent body as part of an expression construct.
  • the methods of the invention involve transformation of pineapple plbs with expression constmcts in which the ⁇ -glucuronidase (GUS) gene is under the transcriptional control of a 35S promoter, double 35S promoter, rice actin-1 promoter, maize ubiquitin promoter, Emu promoter, scpepcdl promoter, scpecd2 promoter, scrbcsl promoter, scrbcs3 promoter, tarin gene promoter, or ⁇ -amylase promoter.
  • expression of the cowpea trypsin inhibitor (CpTi) gene is under the control of a cauliflower mosaic virus (CaMV) 35S RNA promoter sequence.
  • nucleic acid sequences encoding thaumatin protein are controlled by a double CaMV 35S promoter sequence.
  • the 1-aminocyclopropane-l-carboxylic acid (ACC) deaminase gene sequence is under the transcriptional control of a CaMV 35 S promoter sequence.
  • the methods of the invention are not limited to the expression constructs disclosed herein. Any expression construct which is capable of introducing a nucleic acid sequence of interest into a pineapple plb cell is contemplated to be within the scope of this invention.
  • expression constructs comprise the nucleic acid sequence of interest as well as companion sequences which allow the transcription of this sequence, and which allow cloning of the construct into a bacterial or phage host.
  • the construct preferably, though not necessarily, contains an origin of replication which is functional in a broad range of prokaryotic hosts.
  • a selectable marker is generally, but not necessarily, included to allow selection of cells bearing the desired construct.
  • the invention is not limited to the promoter sequences used herein. Any promoter sequence which is capable of directing expression of an operably linked nucleic acid sequence of interest in a pineapple plb cell is contemplated to be within the scope of the invention. Promoters include, but are not limited to, promoter sequences of bacterial, viral and plant origins. Promoters of bacterial origin include, but are not limited to, the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. Viral promoters include, but are not limited to, the 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), and T-DNA promoters from Agrobacterium.
  • CaMV cauliflower mosaic virus
  • Plant promoters include, but are not limited to, the ribulose-l,3-bisphosphate carboxylase small subunit promoter, maize ubiquitin promoters, the phaseolin promoter, the E8 promoter, and the Tob7 promoter.
  • the invention is not limited to the number of promoters used to control expression of a nucleic acid sequence of interest. Any number of promoters may be used so long as expression of the nucleic acid sequence of interest is controlled in a desired manner.
  • the use of two or more promoters for the control of gene expression is known in art, and is exemplified herein by the double 35S RNA promoter of CaMV (Table 4; Figure 1). Chimeric promoters which are derived from different genes are also included within the scope of the invention.
  • a chimeric promoter containing a trimer of the octopine synthase (ocs) upstream activating sequence ligated to the mannopine synthase (mas2 ') promoter/activator has been previously described (Ni et al. (1995) The Plant J. 7:661-676). Promoters may control constitutive or inducible expression. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Examples of promoters under developmental control include promoters that initiate transcription only in response to ethylene, such as E8 and Tob7.
  • promoter may be governed by the desirability that expression be over the whole plant, or localized to selected tissues of the plant, e.g., root, leaves, fruit, etc.
  • promoters active in flowers are known (Benfy et al. (1990) Plant Cell 2:849-856).
  • the promoter activity of any nucleic acid sequence in pineapple plb cells may be determined (i.e., measured or assessed) using methods well known in the art and exemplified herein.
  • a candidate promoter sequence may be tested by ligating it in-frame to a reporter gene sequence to generate a reporter construct, introducing the reporter construct into pineapple plbs using methods described herein, and detecting the expression of the reporter gene (e.g., detecting the presence of encoded mRNA or encoded protein, or the activity of a protein encoded by the reporter gene).
  • the reporter gene may confer antibiotic or herbicide resistance.
  • reporter genes include, but are not limited to, dhfr which confers resistance to methotrexate [Wigler M et al, (1980) Proc Natl Acad Sci 77:3567-70]; npt, which confers resistance to the aminoglycosides neomycin and G-418 [Colbere-Garapin F et al, (1981) J. Mol. Biol. 150:1-14] and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyl transferase, respectively.
  • the reporter gene is a GUS gene.
  • the expression construct preferably contains a transcription termination sequence downstream of the nucleic acid sequence of interest to provide for efficient termination.
  • the termination sequence is the nopaline synthase (NOS) sequence.
  • the termination region comprises different fragments of sugarcane ribulose-l,5-biphosphate carboxylase/oxygenase (rubisco) small subunit (scrbcs) gene.
  • the termination sequences of the expression constructs are not critical to the invention. The termination sequence may be obtained from the same gene as the promoter sequence or may be obtained form different genes.
  • polyadenylation sequences are also commonly added to the expression construct.
  • the polyadenylation sequences include, but are not limited to, the Agrobacterium octopine synthase signal, or the nopaline synthase signal.
  • the nucleic acid sequence of interest is not translated into a polypeptide, e.g., where the nucleic acid sequence of interest encodes an antisense RNA, polyadenylation signals are not necessary.
  • Constructs for the transformation of pineapple plbs are not limited to the type or nature of the expressed genes disclosed herein.
  • nucleic acid sequences of interest include sequences which encode a polypeptide sequence whose expression may be deemed useful by one of skill in the art.
  • an enzyme is the ACC deaminase enzyme which metabolizes ACC in plant tissue thereby lowering the level of ethylene which is responsible for fruit ripening (U.S. Patent No. 5,512,466, the contents of which are hereby incorporated by reference).
  • sucrose phosphate synthase enzyme which increases the level of sucrose in the fruit.
  • the nucleic acid sequence of the gene encoding this enzyme is known [e.g., in maize; Worrell et al. (1991) Plant Cell 3:1121-1130] ( Figure 14) (SEQ ID NO:2) and has been assigned GenBank accession number m97550.
  • Yet another example of a protein whose expression in pineapple may be desirable is the sweetness protein.
  • the nucleic acid sequence for the gene encoding the sweetness protein is known in the art (see, e.g., U.S. Patent Application No. 08/670,186, the contents of which are herein incorporated by reference). Transformation of pineapple plants with the sweetness protein is useful in, for example, providing a base level of sweetness in the fruit, thus reducing the effects of differences in fruit maturity by providing more uniform sweetness from the bottom to the top of the fruit.
  • nucleic acid sequence which encodes an antisense RNA that hybridizes with a genomic plant DNA sequence it may be of advantage to express antisense RNA which is specific for genomic plant DNA sequences that encode an enzyme whose activity is sought to be decreased.
  • DNA sequences whose reduced expression may be desirable are known in the art including, but not limited to, the ethylene inducible sequences in fruit (U.S. Patent No. 5,545,815, the entire contents of which are herein incorporated by reference). Expression of antisense RNA which is homologous with these ethylene inducible sequences is useful in delaying fruit ripening and in increasing fruit firmness.
  • DNA sequences whose expression may be desirably reduced include the ACC synthase gene which encodes the ACC synthetase enzyme that is the first and rate limiting step in ethylene biosynthesis. Nucleic acid sequences for this gene have been described from a number of plant sources (e.g., Picton et al. (1993) The Plant J.
  • ACC synthase genomic sequences in pineapple plants may be desirable to delay fruit ripening.
  • Yet another sequence whose expression may be advantageously reduced is the genomic sequence encoding the enzyme polyphenol oxidase. This enzyme is involved in the browning reaction that occurs during chilling injury. Nucleic acid sequences encoding this enzyme have been previously described in the art (e.g., Shahar et al. (1992) Plant Cell 4:135-147], is shown in Figure 13 (SEQ ID NO:2), and has been assigned GenBank accession number s40548. The use of antisense polyphenol antisense sequences has been reported to inhibit polyphenol oxidase (PPO) gene expression and to inhibit browning [Bachem et al.
  • PPO polyphenol oxidase
  • the antisense DNA segment to be introduced into the plant may include the full length coding region of the targeted gene or a portion thereof. Complete homology between the nucleotide sequences of the antisense RNA and the targeted genomic DNA is not required. Rather, antisense DNA sequences which encode antisense RNA sequences that are partially homologous to a targeted genomic DNA sequence are contemplated to be within the scope of the invention so long as the antisense RNA sequences are capable of repressing expression of the target genomic DNA sequence.
  • the invention is not limited to constructs which express a single nucleic acid sequence of interest.
  • Constructs which contain a plurality of (i.e., two or more) nucleic acid sequences under the transcriptional control of the same promoter sequence are expressly contemplated to be within the scope of the invention. Such constructs may be desirable, for example, where the expression products of the plurality of nucleic acid sequences contained within the construct provide protection against different pathogens, and where simultaneous protection against these different pathogens is deemed advantageous.
  • constructs which contain the same or different nucleic acid sequences under the transcriptional control of different promoters Such constructs may be desirable to, for example, target expression of the same or different nucleic acid sequences of interest to selected plant tissues.
  • Expression constructs may be introduced into plbs using methods known in the art.
  • the expression constructs are introduced into pineapple plbs by particle mediated gene transfer.
  • Particle mediated gene transfer methods are known in the art, are commercially available, and include, but are not limited to, the gas driven gene delivery instrument descried in McCabe, U.S. Patent No. 5,584,807, the entire contents of which are herein incorporated by reference. This method involves coating the nucleic acid sequence of interest onto heavy metal particles, and accelerating the coated particles under the pressure of compressed gas for delivery to the target tissue. Other particle bombardment methods are also available for the introduction of heterologous nucleic acid sequences into plbs.
  • these methods involve depositing the nucleic acid sequence of interest upon the surface of small, dense particles of a material such as gold, platinum, or tungsten.
  • the coated particles are themselves then coated onto either a rigid surface, such as a metal plate, or onto a carrier sheet made of a fragile material such as mylar.
  • the coated sheet is then accelerated toward the target biological tissue.
  • the use of the flat sheet generates a uniform spread of accelerated particles which maximizes the number of cells receiving particles under uniform conditions, resulting in the introduction of the nucleic acid sample into the target tissue.
  • an expression construct may be inserted into the genome of pineapple plb cells by infecting plbs with a bacterium, including but not limited to an Agrobacterium strain previously transformed with the nucleic acid sequence of interest.
  • a bacterium including but not limited to an Agrobacterium strain previously transformed with the nucleic acid sequence of interest.
  • disarmed Agrobacterium cells are transformed with recombinant Ti plasmids of Agrobacterium tumefaciens or Ri plasmids of Agrobacterium rhizogenes
  • nucleic acid sequence of interest is then stably integrated into the plant genome by infection with the transformed Agrobacterium strain.
  • heterologous nucleic acid sequences have been introduced into plant tissues using the natural DNA transfer system of Agrobacterium tumefaciens and Agrobacterium rhizogenes bacteria (for review, see Klee et al. (1987) Ann. Rev. Plant Phys. 38:467- 486).
  • Agrobacterium may be enhanced by using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens [Shahla et al. (1987) Plant Molec. Biol. 8:291-298]. Alternatively, transformation efficiency may be enhanced by wounding the target tissue to be transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles, etc. [see, e.g., Bidney et al. (1992) Plant Molec. Biol. 18:301-313].
  • a natural wound response molecule such as acetosyringone (AS)
  • AS acetosyringone
  • nucleic acid sequence of interest may be desirable to target the nucleic acid sequence of interest to a particular locus on the plant genome.
  • Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium- ⁇ oxxwe ⁇ sequences.
  • plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (Offringa et al, (1996), U.S. Patent No. 5,501,967, the entire contents of which are herein incorporated by reference).
  • T-DNA Agrobacterium transfer-DNA
  • homologous recombination may be achieved using targeting vectors which contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.
  • the targeting vector used may be of the replacement- or insertion-type (Offringa et al. (1996), supra).
  • Replacement-type vectors generally contain two regions which are homologous with the targeted genomic sequence and which flank a heterologous nucleic acid sequence, e.g., a selectable marker gene sequence.
  • Replacement type vectors result in the insertion of the selectable marker gene which thereby disrupts the targeted gene.
  • Insertion-type vectors contain a single region of homology with the targeted gene and result in the insertion of the entire targeting vector into the targeted gene.
  • Pineapple totipotent bodies transformed with a heterologous nucleic acid sequence of interest are readily detected using methods known in the art including, but not limited to, restriction mapping of the genomic DNA, PCR-analysis, DNA-DNA hybridization, DNA-RNA hybridization, DNA sequence analysis and the like.
  • selection of transformed totipotent bodies may be accomplished using a selection marker gene. It is preferred, though not necessary, that a selection marker gene be used to select transformed plbs.
  • a selection marker gene may confer positive or negative selection.
  • a positive selection marker gene may be used in constructs for random integration and site-directed integration. Positive selection marker genes include antibiotic resistance genes, and herbicide resistance genes and the like.
  • the positive selection marker gene is the NPTII gene which confers resistance to geneticin (G418) or kanamycin.
  • the positive selection marker gene is the HPT gene which confers resistance to hygromycin. The choice of the positive selection marker gene is not critical to the invention as long as it encodes a functional polypeptide product.
  • Positive selection genes known in the art include, but are not limited to, the ALS gene (chlorsulphuron resistance), and the DHFR-gene (methothrexate resistance).
  • a negative selection marker gene may also be included in the constructs.
  • the use of one or more negative selection marker genes in combination with a positive selection marker gene is preferred in constructs used for homologous recombination.
  • Negative selection marker genes are generally placed outside the regions involved in the homologous recombination event.
  • the negative selection marker gene serves to provide a disadvantage (preferably lethality) to cells that have integrated these genes into their genome in an expressible manner. Cells in which the targeting vectors for homologous recombination are randomly integrated in the genome will be harmed or killed due the presence of the negative selection marker gene. Where a positive selection marker gene is included in the construct, only those cells having the positive selection marker gene integrated in their genome will survive.
  • the choice of the negative selection marker gene is not critical to the invention as long as it encodes a functional polypeptide in the transformed pineapple plbs.
  • the negative selection gene may for instance be chosen from the aux-2 gene from the Ti- plasmid of Agrobacterium, the t£-gene from SV40, cytochrome P450 from Streptomyces griseolus, the Adh-gene from Maize or Arabidopsis, etc. Any gene encoding an enzyme capable of converting a substance which is otherwise harmless to pineapple plb cells into a substance which is harmful to pineapple plb cells may be used.
  • the present invention provide transgenic pineapple plants.
  • the transgenic pineapple plants of the invention are not limited to plants in which each and every cell expresses the nucleic acid sequence of interest. Included within the scope of this invention is any pineapple plant which contains at least one cell which expresses the nucleic acid sequence of interest. It is preferred, though not necessary, that the transgenic plant express the nucleic acid sequence of interest in more than one cell, and more preferably in one or more tissue.
  • Transgenic pineapple plants may be generated from transformed totipotent bodies using, for example, methods described herein. For example, where transgenic plbs are used to regenerate transgenic plants, shoots are first generated by transferring plbs onto the surface of agar solidified (e.g., 0.8% Bacto agar) hormone-free medium or PM2 (i.e., 2 mg/L BAP) medium. Roots are then generated by transferring the structures containing shoots onto the surface of agar-solidified (e.g., 0.8% Bacto agar) medium containing 1 mg/L NAA. The resulting structures are potted in soil in pots and placed in a greenhouse. Once the plants reach a size suitable for transfer, they may be transferred to the field for further growth and development
  • agar solidified e.g. 0.8% Bacto agar
  • PM2 i.e., 2 mg/L BAP
  • Roots are then generated by transferring the structures containing shoots onto the surface of agar-solidified (e.g.,
  • transgenic callus is used to regenerate transgenic plants
  • plants are regenerated by the method described by Mathews et al. (1981) as described supra.
  • Transformation of pineapple plants with the nucleic acid sequence of interest may be determined using any number of methods known in the art, e.g., restriction mapping of genomic DNA, PCR analysis, DNA-DNA hybridization, DNA-RNA hybridization, DNA sequence analysis, etc. Additionally, transformation may be determined by visual inspection and comparison of transgenic and wild-type plants, the phenotype inspected depends on the phenotypic characteristics expected to be conferred by the nucleic acid sequence of interest used in the transformation of the plants. Examples of phenotypic characteristics may include fruit flesh color, fruit tissue firmness, leaf color, fruit yield, plant vigor, etc.
  • microMolar microMolar
  • nm nanom
  • L liter
  • ml milliliter
  • ⁇ l microliter
  • °C degrees Centigrade
  • m meter
  • sec. second
  • DNA deoxyribonucleic acid
  • cDNA complementary DNA
  • RNA ribonucleic acid
  • mRNA messenger ribonucleic acid
  • X-gal 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside
  • LB Lia Broth
  • PAGE polyacrylamide gel electrophoresis
  • NAA ⁇ -naphtaleneacetic acid
  • BAP 6-benzyl aminopurine
  • Tris tris(hydroxymethyl)-aminomethane
  • PBS phosphate buffered saline
  • 2 X SSC 0.3 M NaCl, 0.03 M Na 3 citrate, pH 7.0
  • Pineapple plbs were initiated from crown and vegetative shoot explants, maintained for several months and used to generate pineapple plants which showed low phenotypic variation as compared to pineapples generated by traditional shoot regeneration methods from callus.
  • plb generation vegetative shoots from the D10 cultivar were used to determine whether the phenotypic variation of plb-derived plants was affected by the tissue source from which plbs were initiated. Vegetative shoot in vitro cultures were initiated to generate plants from D10 since this method is the traditionally used in vitro method for regeneration of pineapple plants.
  • Explants were obtained by removing all the leaves except the inner 3 to 4 leaves from crowns of 16- to 17-month-old crop plants, or from 3- to 6-month-old vegetative shoots.
  • the suitability of different "CLOROX” (5.25% sodium hypochlorite solution) concentrations and durations of treatment for surface sterilization of crowns and vegetative shoots from F153 was investigated. Generally, sterilization with 20 or 50% "CLOROX" for 30 min gave 22.9% success rate on sectioned explants from F153 crowns and 14.7% success rate on sectioned explants from F153 vegetative shoots.
  • CLOROX 5.25% sodium hypochlorite solution
  • Plbs whether derived from crowns or shoots, were initiated in liquid culture without the use of agar solidified medium. Without limiting the invention to any particular theory, liquid culture and shaking is advantageous for the initiation and maintenance of pineapple plbs since these conditions are believed to reduce the orientation of cells. Plbs are a group of polarized cells predetermined as apical meristems which will become shoots. Shaking in liquid culture permits initiation and maintenance of plb cells which are not fully differentiated as leaf primodia.
  • BRL 100 mg/L myo-inositol (Sigma), 1 mg/L thiamine-HCl (Sigma), 30 g sucrose (Sigma), pH 5.6-5.8: supplemented with 2 mg/L ⁇ -naphtaleneacetic acid (NAA) (Sigma) and 1 mg/L 6-benzyl aminopurine (BAP) (Sigma)] or in liquid modified MS media with 10 mg/L NAA and 1 mg/L BAP.
  • the culture was maintained on a gyratory shaker at 80-90 rpm at 27°C with a 16 h photoperiod with 35 ⁇ mol.m "2 .sec " '
  • PAR Photosynthetically Active Radiation
  • ⁇ Einstein or ⁇ E provided by 40 watt white fluorescent and Sylvania Gro-Lux lamps (GTE Corp.). New growth of shoots and globular cell structure was observed 2 to 4 weeks after culture initiation. Uniform protocorm-like bodies (plbs) were obtained by transferring the globular structures to fresh PI medium (i.e., modified MS medium containing 2 mg/L NAA and 1 mg/L
  • Plb cultures from D10 vegetative shoots were initiated through shoot multiplication in PM2 (2 mg/L BAP in liquid modified MS medium). This culture resulted in plbs which were more organized than the plbs derived from F153 crowns, and which also contained shoots. Transfer of these plbs (herein referred to as "shoot plbs") to PI medium (i.e., modified MS medium containing 2 mg/L NAA and 1 mg/L BAP) resulted within a few weeks in dedifferentiated plbs which were similar to the plbs obtained from F153 crowns. These plbs were maintained (i.e., shoot plbs were not produced) in PI by transferring to fresh medium every 3 weeks.
  • PI medium i.e., modified MS medium containing 2 mg/L NAA and 1 mg/L BAP
  • Root initiation in liquid culture was preferred since roots developed on agar-modified MS medium were often injured or broken when the plant was transferred from agar-solidified medium to soil. These conditions resulted in rooting of approximately 97-99% of the shoots and the generation of plants
  • plants which were regenerated from plbs of each of the F153 (150 plants) and D10 (150 plants) pineapple cultivars and from F153 (154 plants) and D10 (30,000 plants) in vitro shoot multiplication were planted in pots containing soil in a greenhouse and grown for about 3 months until the resulting plants reached 3 inches in height. Leaves of the potted plants were morphologically evaluated for leaf variegation, spininess, and malformations.
  • plbs derived from the F153 and D10 cultivars were transformed with plasmid constructs containing the ⁇ -glucuronidase (GUS) reporter gene and optionally contained either the neomycin phosphotransferase (NPTII) selection gene, or the hygromycin phosphotransferase (HPT) selection gene, under the transcriptional control of different promoters. Transformation of plb tissue was accomplished by using either a biolistic transformation method (also known as gene gun bombardment method), or by a Agrobacterium-mediated transformation method. Stable transformation was determined by selection in liquid PI medium of plants regenerated from transfected plbs on geneticin (G418) for expression of NPTII, or on hygromycin for expression of HPT.
  • GUS ⁇ -glucuronidase
  • NPTII neomycin phosphotransferase
  • HPT hygromycin phosphotransferase
  • Figures 1-10 show the structures of plasmids pBI426, pBI 121, pActl-F, pAHC27, pHA9, pEmuGN, pWDl, pWD3, pML72, and pML75.
  • the promoters in plasmids pBI426, and pBI121 are generally used for expression in dicotyledous plants, while the promoters in plasmids, pActl-F, pAHC27, pHA9, pEmuGN, pWDl, pWD3, pML72, and pML75 are generally used for expression in monocotyledous plants.
  • Plbs were transformed simultaneously with pHA9 and pAHC27 in order to simultaneously introduce into the plbs the NPTII selection marker gene of pHA9 and the GUS reporter gene of pAHC27.
  • Transient transformation was determined by GUS staining of plbs 48 h after transformation. Stable transformation was determined by ELISA analysis of plant tissue for NPTII gene product.
  • Figure 3 shows the nucleotide sequence of the 5' transcribed region of the rice Actl-Gus gene fusion in pActl-F between the Actl TATA box and Gus translation initiation codon. Nucleotides are numbered with the A of the Actl transcription initiation site designated as +1. Restriction sites are indicated and underlined. The different regions of the Actl 5 '-transcribed sequences are: Upper case italic letters,
  • Actl 5 '-flanking region Upper case letters, Actl exon sequence; lower case letters, Actl 5'-intron sequence; slashed lines, exon-intron splice sites, bold letters, Actl (5'- most) and Gus (3 '-most) translation initiation codons.
  • the translation product of the Actl-Gus junction region is shown below their respective codons.
  • Pineapple plb tissue was used as target tissue for transformation using two approaches, i. e. , a gene gun bombardment method, and an Agrobacterium-mediated transformation method as described below.
  • Biolistic transformation and transient expression of GUS Pineapple plbs were bombarded using the helium gas-driven microprojectile accelerator (PDS-1000/He) (Bio-Rad). Two to three days before bombardment, 20-30 plbs of 3-5 mm in diameter were placed in the 2.5-cm center of a 10-cm diameter petri plate containing modified MS medium supplemented with 0.8% Difco Bacto agar and 3% sucrose. Gold microcarriers (1.6 ⁇ m diameter) (Bio-Rad) were prepared by washing 3 times in absolute ethanol by vortexing for 1-2 min. and centrifuged at
  • GUS GUS staining buffer
  • the gus gene product in GUS-expressing cells, the gus gene product, ⁇ -glucuronidase, can cleave the colorless X-gluc to produce a blue precipitate.
  • chlorophyll was cleared by destaining the tissue with several changes of 70% ethanol. Transient expression was generally scored as the number of foci showing blue staining.
  • the optimized conditions for transient GUS gene expression were (a) using 10 ⁇ g of each DNA per bombardment; i.e. where more than one construct was used (e.g., pHA9 and pAHC27), 10 ⁇ g of each construct was employed per bombardment; (b) positioning plb samples 7.5 cm below the stopping screen in the Biolistics device; (c) using helium gas at a pressure of 1 100 psi or 1550 psi; and (d) using two shots per plate of plbs.
  • more than one construct e.g., pHA9 and pAHC27
  • Strain LBA4404 (Life Technologies Inc., GIBCO/BRL), is a disarmed octopine-type Agrobacterium, which has been previously described by Ooms et al.
  • Plasmid 7:15-29 contains the disarmed Ti plasmid pBI121 which contains the selection marker NPTII under the control of the NOS promoter, as well as the GUS reporter gene under the control of the CaMV double 35S promoter.
  • Strain LBA4301 is a nopaline-type Agrobacterium strain which has been previously described by Klapwijk et al. (1979) Mol. Gen. Genet. 173:171-175 and Rogowsky et al. (1990) Plasmid 23:85-106] is derived from the C58 strain contains plasmid pUCD2716 which contains the HPT selection marker gene as well as the GUS reporter gene under the transcriptional control of the CaMV 35S promoter.
  • the inoculation process included microwounding by PDS-1000/He at 1 100 psi, with 1.6 ⁇ m gold (Bio-Rad) and addition of Agrobacterium virulence inducer (100 ⁇ M acetosyringone) (Sigma) to the overnight grown bacterial culture two to three hours prior to the actual inoculation.
  • Overnight bacterial suspension was 2X diluted with bacterial culture medium (LB) before inoculation. Plbs were immersed in diluted bacterial suspension for 5—10 min and were blotted dry on sterilized paper towels before co-cultivation.
  • Co-cultivation was performed on 0.8% agar PM2 medium or in liquid PI medium for 2-3 days before transfer to media containing 200 mg/L cefotaxime (Agri-Bio Inc.) to kill Agrobacterium.
  • the plbs were transferred to liquid PI medium or 0.8% agar PM2 medium containing selection antibiotics.
  • the antibiotic G418 (Sigma) was included to select for cells transformed with constructs containing the NPTII gene, while the antibiotic hygromycin (Sigma) was used to select cells transformed with constructs containing the HPT gene.
  • Figure 12 shows a representative antibiotic growth inhibition curve for F153 (Figure 12A and 12C) and for D10 ( Figure 12B and 12D) plbs treated for one month (open circles) or two months (filled squares) with different G418 concentrations
  • Agrobacterium transformation were transferred to 0.8% agar PM2 medium containing selection antibiotics to allow differentiation into plants.
  • Transformed plbs selected in liquid culture regenerated numerous (approximately 20-30) plants compared to the few individual plants (generally fewer than 5) regenerated during selection of transformed plbs on agar media immediately after bombardment.
  • NPTII protein For example, after 3 months of liquid media selection, randomly selected plbs bombarded separately with pBI426 and pAHC27 showed 2 to 5 times greater expression of NPTII protein than the unbombarded controls as measured by NPTII ELISA.
  • plants regenerated from pBI426- and pBI121-transformed plbs expressed high levels of the antibiotic resistance gene products as determined by ELISA.
  • Expressed levels of NPTII in plb-derived plants transformed with the double 35S promoter-driver fused GUS::NPTII gene in pBI426 were double the levels of expressed NPTII in plb-derived plants transformed with the NOS promoter-driven
  • NPTII gene in pBI121 Stable transformation was also observed in plants generated from plbs transformed separately with pAHC27 and pHA9.
  • Genomic DNA was isolated from leaves (approximately 15 cm. long) from approximately 4 month-old pineapple plants grown in light boxes according to the method of Doyle et al. [Doyle et al. (1990) Focus 12:13-15]. The final DNA pellet was dissolved in H 2 O or TE buffer (10 mM Tris, 1 mM EDTA, pH 8) and quantified on a 0.7-1.0% agarose gel following electrophoresis. Isolated genomic DNA was digested with EcoRl and Hindlll and hybridized to a probe obtained by digesting pBI426 with EcoRl and Hindlll.
  • Probes used in hybridization were either labeled with radioactive ⁇ -P 32 -dCTP (Amersham) or non-radioactive digoxygenin-1 1- dUTP (DIG High Prime DNA Labeling and Detection Kit, Boehringer Mannheim).
  • Figure 15 shows a Southern hybridization blot of genomic DNA isolated from pineapple leaves transfected with pBI426 and PBI121 using biolistic bombardment; lane 1 : 750 pg digested pBII21, equivalent to 10 copies of gene insertion into the pineapple genome; lane 2: 75 pg digested pBI121, equivalent to 1 copy of gene insertion; lane 3: 8 ⁇ g digested genomic DNA extracted from one transgenic pineapple plant line, transformed with pBI121 using particle bombardment; lane 4: 300 pg digested pBI426, equivalent to 10 copies of gene insertion; lane 5: 30 pg digested pBI426, equivalent to 1 copy of gene insertion; lane 6: 8 ⁇ g digested genomic DNA extracted from an untransformed pineapple plant; lane 7: 8 ⁇ g digested genomic DNA extracted from a transgenic pineapple plant line, transformed with pBI426 particle bombardment.
  • the results in Figure 15 demonstrate the integration of pBI121 (3.0 Kb
  • Nematode control of the pineapple crop is a major problem for several reasons. Pineapple lacks natural resistance to nematodes. This necessitates the use of nematicides which are expensive and add to a grower's input costs. Additionally, even when nematicides are used to control nematode infestation of pineapple, nematodes can impose up to 40% loss of yield in the 30 to 36 month growing period of the crop that follows nematicide application.
  • methyl bromide and 1,3-dichloropropene are environmentally unacceptable; methyl bromide is a carcinogen, causes bromide buildup in soils, and is believed to be a primary cause of the depletion of the ozone layer; telone also causes halogen accumulation in soils and presents a major health risk for agricultural workers using this pesticide.
  • methyl bromide is a carcinogen, causes bromide buildup in soils, and is believed to be a primary cause of the depletion of the ozone layer
  • telone also causes halogen accumulation in soils and presents a major health risk for agricultural workers using this pesticide.
  • Proteinase inhibitors e.g., trypsin inhibitors
  • trypsin inhibitors are known to be potent anti- nematode agents (see, e.g., Hepher et al., U.S. Patent No. 5,494,813, the entire contents of which are herein incorporated by reference).
  • transformation with the cowpea trypsin inhibitor (CpTi) has been shown to confer nematode resistance to potatoes (U.S. Patent No. 5,494,813, hereby incorporated by reference).
  • delivery of proteinase inhibitor genes to pineapples by transformation of pineapple plants with a DNA sequence encoding the CpTi could confer nematode resistance on transformed pineapple plants.
  • This Example is divided into (a) generation of pineapple plants transgenic for CpTi, and (b) screening transgenic pineapple plants for nematode resistance.
  • Transgenic pineapple plants are produced by bombarding pineapple plbs generated as described above (see, Example 1) with pROK CpTi+5 using a helium gas-driven apparatus (PDS-1000/He) (BioRad).
  • DNA carriers e.g., tungsten (Sylvania M10, 1.1 - ⁇ m-diameter) and gold (1.6- ⁇ m-diameter, BioRad) are used to precipitate DNA onto the mircroparticles according to the manufacturer's directions.
  • Flight distance of the particles is 7.5 cm
  • target plbs are placed in 2.5-cm- diameter areas in the center of 10-cm-diameter petri dish with modified MS media containing 0.8% Difco Bacto agar and 3% sucrose.
  • Bombardment with two shots is carried out at different helium pressures (e.g., 1100 or 1550 psi). Controls are bombarded with uncoated particles.
  • Transformed plbs are selected by culturing for one month in 20-30 mg/ml G418. Transformed plbs, i.e., plbs which survive the G418 selection, are used for the generation of plants as described in Example 1, and the regenerated G418-resistant plants screened for stable transformation by determining (a) NPTII expression as described supra, (b) expression of CpTi by Western blotting as described in U.S. Patent No. 5,494,813, or (c)
  • soluble protein is extracted in PBS, electrophoresed on a 17% polyacrylamide gel, transferred to nitrocellulose and CpTi detected using rabbit anti-CpTi primary antibodies followed by horseradish peroxidase-conjugated donkey anti-rabbit secondary antibodies (Amersham) and autoradiography.
  • genomic DNA is extracted from different plant tissues or from the entire plant, digested with Hindlll, and the digest separated on agarose gel, blotted onto filters and the filter-immobilized DNA hybridized with one of two labeled probes (i.e., a 564 bp Alul/Scal fragment from pUSSR c3/2 which contains the CpTi coding sequence, and the 800 bp Pstl/BamHI fragment from pNEO which contains the coding sequence for the NPTII gene). Since a unique Hindlll restriction site is present between the CpTi and NPTII genes, a Hindlll digest of genomic DNA containing a single copy of the T-DNA will generate two junction fragments.
  • two labeled probes i.e., a 564 bp Alul/Scal fragment from pUSSR c3/2 which contains the CpTi coding sequence, and the 800 bp Pstl/BamHI fragment from pNEO which contains the
  • the size of the junction fragments also differs. Multiple copies of the T-DNA integrated at different sites within the same genome produces a commensurate number of bands when probed with a sequence specific to one of the junctions, thus allowing unambiguous determination of gene copy number.
  • Nematode resistance of pineapple plants transgenic for the CpTi gene is determined by the infestation of transgenic and control wild-type plants with nematodes and the determination following different periods of infestation of a reduction in the number and size of nematodes, and in the proportion of female nematodes.
  • Pineapple plants of different ages i.e., newly planted, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-month-old
  • Thaumatin is a protein originating from the arils of the fruit of Tahumatococcus daiellii. On a weight basis, thaumatin is 1600 times sweeter than sucrose. The nucleic acid sequence encoding thaumatin protein and constructs for expression of the protein in prokaryotes have been previously reported (U.S.
  • Pineapple plbs are transformed with constructs which express the thaumatin gene. This results in the production of the sweet thaumatin protein and the generation of pineapple fruit of increased sweetness.
  • the nucleic acid sequence which encodes thaumatin is inserted using methods well known in the art into the pBI426 plasmid described supra such that expression of the thaumatin nucleic acid sequence is under the control of the double 35 S promoter sequence.
  • Transformed plbs are selected for G418 resistance, and the G418-selected plbs allowed to regenerate pineapple plants. Transformed plants are allowed to produce pineapple fruit in the field, and the fruit is assessed for the expression of thaumatin and for sweetness.
  • Stable transformation of thaumatin- encoding nucleic acid sequences in fruit is detected by Southern hybridization of genomic DNA from the fruit with probes specific for the thaumatin nucleic acid sequence.
  • the presence of the thaumatin protein in fruit is detected by Western blot analysis using a monoclonal or polyclonal anti-thaumatin antibody generated using methods well known in the art.
  • sweetness is subjectively compared between wild-type fruit and fruit which are stably transformed with thaumatin nucleic acid sequences. It is expected that fruit transformed with thaumatin-encoding nucleic acid sequences express thaumatin and have increased sweetness as compared with wild type fruit. The increased expression of thaumatin and/or of sweetness demonstrates expression of thaumatin protein in the transformed pineapple fruit.
  • ethylene production stimulates pineapple flowering and fruit ripening.
  • Ethylene is produced by the conversion of 1-aminocyclopropane-l-carboxylic acid (ACC) to ethylene by the ethylene forming enzyme ACC oxidase.
  • ACC 1-aminocyclopropane-l-carboxylic acid
  • ACC oxidase ethylene forming enzyme
  • the expression of an ACC metabolizing enzyme (e.g., ACC deaminase) in the desired plant tissue has been shown to reduce the level of ethylene in that tissue and to delay ripening.
  • pineapple fruit with an extended harvesting season and an extended shelf life are produced by transformation of pineapple plbs with DNA encoding ACC deaminase and regeneration of transformed pineapple plants from these plbs.
  • the nucleotide sequence of ACC deaminase and constructs (e.g., pMON977) capable of expression of ACC deaminase in plants have been previously described (U.S. Patent No. 5,512,466 the contents of which are herein incorporated by reference).
  • the pMON977 expression plasmid which is capable of expressing ACC deaminase protein under the transcriptional control of a CaMV35S promoter sequence, and which contains a NPTII selection gene sequence is used to transform pineapple plbs by biolistic bombardment as described above. Transformed plbs are screened for G418 resistance as described supra. Plbs which survive G418 selection are used for the generation of plants as described in Example 1 , supra.
  • Plants are transferred to the field to produce fruit. Stable transformation of the ACC deaminase gene into plbs, plants, and fruit is determined by screening for the presence of NPTII gene and measuring ACC deaminase activity in converting ⁇ -ketobutyrate acid to ethylene as previously described (U.S. Patent No. 5,512,466). Plants which express the ACC deaminase gene are examined for inhibition of fruit ripening by scoring the timing when fruit begin to ripen, and the number of fruit entering the ripening stage. Wild- type pineapple plants are included as controls. Delay in the initiation of ripening and/or the time it takes to reach a fully ripe state is expected. Such delay demonstrates that expression of the AC deaminase gene delays pineapple fruit ripening.
  • transgenic pineapple plants which express antisense RNA that is specific for mRNA which is encoded by pineapple ACC oxidase.
  • Pineapple ACC oxidase sequences are isolated from a cDNA library is prepared from pineapple fruit tissue using methods known in the art and screened by hybridization with cDNA sequences derived from pTOM13.
  • pTOM13 contains DNA which is believed to code for tomato ACC oxidase (Grierson et al.,
  • Tomato plants which were transgenic for a DNA sequence that encoded RNA which was antisense to the tomato ACC oxidase mRNA showed at least 80% reduction in the level of ethylene present during fruit ripening (U.S. Patent No. 5,365,015).
  • This example involves (a) construction of a pineapple cDNA library, (b) cloning and characterization of pineapple fruit-specific ACC oxidase oligonucleotides homologous to tomato ACC oxidase cDNA, (c) construction of vectors for expression of anti-sense pineapple ACC oxidase transcripts, and (d) transformation of pineapple callus and regeneration of transgenic pineapple plants.
  • RNA Total RNA is isolated from fruit tissue of Ananas comosus (L.) cultivar (F153) using the method of Su and Gibor [Anal. Biochem. 174:650 (1988)] as modified by Lopez-Gomez and Gomez-Lim [J. Plant Physiol. 141 :82 (1992)].
  • Messenger RNA (polyA + RNA) is isolated using the PolyATtract® mRNA isolation system IV (Promega).
  • First and second strand cDNA is synthesized using the ZAP-cDNA synthesis kit (Stratagene).
  • the double-stranded cDNA is recovered and blunt ended using Klenow enzyme (Stratagene).
  • EcoRI linkers [5'-AATTCGGCACGAG-3' (S ⁇ Q ID NO:3) and 5'-CTCGTGCCG-3'] are added to the cDNA.
  • Xhol "sticky ends" are generated at the end of the cDN A which corresponds to the 3 ' end of the mRNA by digestion of the Xhol site in the linker-primer.
  • cDNA with EcoRI sticky ends at the 5' end and Xhol sticky ends at the 3' end is electrophoresed on a 1% agarose gel to determine the size distribution of the cDNA. Fractions containing the largest cDNA molecules are ligated into Uni-ZAPTM, a lambda ZAP vector digested with EeoRI and Xhol (Stratagene).
  • Pineapple cDNA clones are probed with a 1.3 kb cDNA insert cut by Pstl from pTOM13 (U.S. Patent No. 5,365,015).
  • the 1.3 kb cDNA pTOM insert is radioactively labeled with Prime-a-Gene® random labeling system (Promega).
  • Recombinant phages are screened by plaque-lifting onto Magna nylon or NitroPure nitrocellulose membranes (MSI Corp.) by fixing recombinant phage DNA to the membranes.
  • the membranes are placed sequentially on filter paper saturated with 0.5 M NaOH and 1.5 M NaCl for five minutes, then on filter paper containing 0.5 M Tris- HC1 (pH 8.0) and 1.5 M NaCl and finally on paper containing 2 X SSC and 0.2 M Tris-HCl for 30 seconds.
  • the DNA is cross linked to the nylon membranes using UV light (12,000 ⁇ Joules) using a UV Stratalinker 1800 (Stratagene) and baking at 80°C for 1 hour under vacuum.
  • the membranes are prehybridized in 50 ml of 5X SSP ⁇ , 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml sonicated salmon sperm DNA for 1 hour.
  • Hybridization is carried out in the same buffer containing the labelled probe at 65°C in a Hybaid Mark II hybridization oven. Clones which hybridize to the pineapple ACC oxidase probe are expected to be obtained. Clones are subjected to DNA sequencing using the Sequenase® version 2.0 DNA sequencing kit (USB) with either the T3 or T7 promoter primers.
  • An expression vector containing pineapple ACC oxidase cDNA is constructed by releasing the cDNA from the lambda ZAP vector plasmid by restriction enzyme digestion and insertion into a pBI121 expression vector. The digestion products are separated by electrophoresis on a 1% SeaPlaque agarose gel, the cDNA is recovered and purified using the Genclean II kit (Bio 101, Vista, CA) and inserted into pBI121 in-frame with the promoter. The resulting plasmid is electroporated into E. coli XL1- Blue cells (Stratagene) and transformants are expected to be obtained.
  • Example 1 for plb culture except that initiation is on agar-MS medium supplemented with 5.4 mg/L NAA, 5.2 mg/L IAA and 2.1 mg/L kinetin as described by Mathews et al (1981) Scientia Hor. 11 :319-328. Once callus cultures are initiated, they are maintained on agar-MS medium with 400 mg/L casein hydrolysate and 10 mg/L NAA with 15% (v/v) coconut water. 2. Bombardment conditions
  • Pineapple calli are bombarded using helium gas-driven microprojectile accelerator (PDS 1000/ ⁇ e). Preparation of calli for bombardment is the same as that for plb preparation for bombardment as described in Example 2, supra. Bombardment conditions are determined by changing helium pressure between about 1 100 psi and about 1500 psi, and the optimum conditions are used for callus bombardment in further experiments. Each plate of calli is bombarded with about 2 ⁇ g to about 6 ⁇ g of plasmid pBI121 which expresses antisense pineapple ACC oxidase.
  • Selection of transformed callus Bombarded calli are selected on liquid or agar-MS medium with 400 mg/L casein hydrolysate and 10 mg/L NAA with 15% (v/v) coconut water containing the selection antibiotic G418 to select cells which express the NPTII gene.
  • the amount of G418 used for selection of F153 callus is 20-30 mg/L.
  • Transformation of callus selected for neomycin resistance is confirmed by Southern analysis using pineapple ACC oxidase cDNA as probe.
  • Transformed callus is used to regenerate pineapple plants.
  • Fruit derived from transformed and from control callus i.e., unmodified pineapple or pineapple transformed with pBI121
  • ACC oxidase mRNA expression and accumulation is measured by Northern blotting using pineapple ACC oxidase cDNA as probe.
  • Ethylene production is measured in control and transgenic (i.e., which express anti- sense sequences) pineapple fruit by enclosing individual fruits in sealed jars and detecting ethylene in the head space of the jar by gas chromatography.
  • fruit from transformed pineapple plants which express ACC oxidase antisense will produce a reduced level of ethylene, and will ripen more slowly, as compared to control pineapple fruit.
  • Pineapple plants may be vegetatively propagated. Plants which are homozygous for the transgene may be produced from seeds and used for further breeding.
  • Fruit produced by transgenic plants are expected to have the characteristics of delayed ripening (due to inhibition of endogenous ethylene production). It is also expected that ripening will be inducible by exposure of the transgenic fruit to exogenous ethylene.
  • the present invention provides methods for producing pineapple-like plants with desirable characteristics.
  • the invention provides methods for producing transgenic pineapple-like totipotent bodies which are useful in generating pineapple-like plants.
  • the present invention provides methods for producing transgenic pineapple-like totipotent bodies using biolistic bombardment alone or in combination with Agrobacterium transformation. Also provided by this invention are methods for culturing pineapple- like totipotent bodies which are useful in long-term culture and selection of transgenic totipotent bodies of pineapple-like plants.
  • ATTORNEY/AGENT INFORMATION (A) NAME: Carroll, Peter G. ⁇ B) REGISTRATION NUMBER: 32,837 (C) REFERENCE/DOCKET NUMBER: UH-03321
  • ATTAGTATAA TTATGTGTGG TTTGAGTTAT GTTTTATGTT AAAATGTATC AGCTCGATCG 1980
  • GGCGCGCACA TGAACTTCAA CCCCTCGCAC TACTTCGTCG AGGAGGTGGT CAAGGGCGTC 300
  • GACGAGAGCG ACCTCCACCG GACGTGGATC AAGGTCGTCG CCACCCGCAA CGCCCGCGAG 360
  • GAGATGTTAT GCGCCGGTTC CAATGATGGA GAGGGGATGG GTGAGAGTGG CGGAGCCTAC 900
  • TACTCATGGC CGGAGCACTG CCGCACTTAC CTCACCAGGG TGGCCGGGTG CCGGTTAAGG 2160
  • GCTGTGGACT GCTACCAGGA CGATGGCCGT GCTAGCAAGA AGATGCTGCA GGTGATCCAG 2520
  • GAAGTTTTCA GAGCAGTCCG ATCGGACTCC CAGATGTTCA AGATCTCAGG GTTCACGCTG 2580
  • CTGCTAGCAT CAAGGTCACA GGCACTCAGG TATCTTTCCG TGCGCTGGGG CGTATCTGTG 3060
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • GCCATGCTAA ACAAGATCAG GAAGAGGGGA AAAGGGCACT ATGGTTTATA TTTTTATATA 420 TTTCTGCTGC TGCTCGTCAG GCTTAGATGT GCTAGATCTT TCTTTCTTCT TTTTGTGGGT 480

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Abstract

L'invention concerne des procédés pour fabriquer des corps totipotents semblables à ceux d'un ananas transgénique et, notamment, le cal semblable à celui d'un ananas transgénique et des corps de type protocormes semblables à ceux d'un ananas transgénique. L'invention concerne également des procédés pour produire des plantes transgéniques à partir de corps totipotents transgéniques qui comprennent le cal et les corps de type protocormes semblables à ceux d'un ananas transgénique. L'invention concerne en outre des plantes semblables à un ananas transgénique auxquelles on peut appliquer le génie génétique pour leur conférer une résistance aux maladies et insectes et des qualités améliorées. De plus, l'invention concerne des procédés améliorés pour maintenir des corps de type protocormes semblables à ceux d'un ananas transgénique dans une culture, ainsi que des procédés utiles pour réduire l'investissement en termes de temps, de coût et de main d'oeuvre nécessaires à la sélection de corps de type protocormes semblables à un ananas et transformés de manière stable.
PCT/US1999/010576 1998-05-14 1999-05-13 Compositions et procedes destines a la modification genetique de l'ananas WO1999058644A1 (fr)

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AU39027/99A AU3902799A (en) 1998-05-14 1999-05-13 Compositions and methods for genetic transformation of pineapple

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US7886298A 1998-05-14 1998-05-14
US09/078,862 1998-05-14

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Cited By (6)

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JP2006508681A (ja) * 2002-12-06 2006-03-16 デル・モンテ・フレッシュ・プロデュース・カンパニー カロテノイドレベルを改変したトランスジェニックパイナップル植物体及びその作製方法
US7169597B2 (en) 2001-04-13 2007-01-30 Michigan State University Compositions and methods for the production of betaine lipids
US7663021B2 (en) 2002-12-06 2010-02-16 Del Monte Fresh Produce Company Transgenic pineapple plants with modified carotenoid levels and methods of their production
WO2011083076A1 (fr) * 2010-01-08 2011-07-14 Roche Diagnostics Gmbh Récupération améliorée d'acides nucléiques à partir de particules de verre magnétiques
CN110628778A (zh) * 2019-09-18 2019-12-31 天津农学院 一种加快水果成熟的基因、蛋白质、载体、重组基因工程菌及其应用
US12221647B2 (en) 2015-09-11 2025-02-11 Bacteria Detection Ltd Methods for isolating microbial cells from a blood sample

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7169597B2 (en) 2001-04-13 2007-01-30 Michigan State University Compositions and methods for the production of betaine lipids
JP2006508681A (ja) * 2002-12-06 2006-03-16 デル・モンテ・フレッシュ・プロデュース・カンパニー カロテノイドレベルを改変したトランスジェニックパイナップル植物体及びその作製方法
EP1571899A4 (fr) * 2002-12-06 2006-08-30 Del Monte Fresh Produce Compan Transformation et regeneration organogenique
US7663021B2 (en) 2002-12-06 2010-02-16 Del Monte Fresh Produce Company Transgenic pineapple plants with modified carotenoid levels and methods of their production
US8049067B2 (en) 2002-12-06 2011-11-01 Del Monte Fresh Produce Company Organogenic transformation and regeneration
WO2011083076A1 (fr) * 2010-01-08 2011-07-14 Roche Diagnostics Gmbh Récupération améliorée d'acides nucléiques à partir de particules de verre magnétiques
US8420801B2 (en) 2010-01-08 2013-04-16 Roche Molecular Systems, Inc. Recovery of nucleic acids from magnetic glass particles
US12221647B2 (en) 2015-09-11 2025-02-11 Bacteria Detection Ltd Methods for isolating microbial cells from a blood sample
CN110628778A (zh) * 2019-09-18 2019-12-31 天津农学院 一种加快水果成熟的基因、蛋白质、载体、重组基因工程菌及其应用

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