Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8206—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
  • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—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
  • C12N15/8205—Agrobacterium mediated transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology

Definitions

• the present invention relates generally to transgenic grass and to a method of producing same. More particularly, the present invention is directed to transgenic grass of the group Monocotyledoneae.
• the transgenic grass of the present invention exhibits the potential to express a range of beneficial traits such as reduced allergenicity, enhanced nutritional content and increased disease resistance.
• Grasses are one of the most important agricultural plants in the world.
• Plants of the group Monocotyledoneae are particularly important and include members of the family Poaceae (Gramineae) such as food crops (for example maize, wheat, rice) and forage grasses (for example, Lolitan, Festuca and Dactylis). They are, however, very difficult to manipulate in vitro and to genetically transform (Potrykus, 1990; 1991). Direct DNA injection into young floral tillers and systems involving a mixture of pollen and exogenous DNA to obtain genetic transformation (Ohta, 1986) have not proven generally applicable in monocots.
• Poaceae Gramae
• Food crops for example maize, wheat, rice
• forage grasses for example, Lolitan, Festuca and Dactylis
• Regeneration of plants from single cells or protoplasts is essential in the genetic manipulation of plants using direct gene transfer technology.
• Microprojectile bombardment allows recovery of transgenic plants without the constraints of protoplast culture and Agrobacterium host-specificity.
• Microprojectile bombardment employs high velocity metal particles to deliver biologically active DNA into plant cells. The concept was first described by Klein et al (1987) and has become a successful DNA delivery method in a number of plants.
• Grass pollen is a significant contributor of respiratory disorders such as hayfever and other allergies with concomitant major downstream health costs including lost production time.
• one aspect of the present invention relates to a transgenic monocot grass exhibiting at least one altered characteristic when compared to a non-transgenic monocot grass of the same species.
• the present invention is directed to a transgenic monocot grass exhibiting at least one altered characteristic when compared to a non-transgenic monocot grass of the same species wherein said transgenic monocot grass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrobacterium- cdia ed transfer of genetic material responsible for said altered characteristic.
• the present invention provides a transgenic monocot grass exhibiting at least one altered characteristic when compared to a non-transgenic monocot grass of the same species wherein said transgenic monocot grass is regenerated from a callus wherein cells of said callus are subjected to microparticle bombardment and/or Agrob ⁇ cte ⁇ um-mediated transfer of genetic material responsible for said altered characteristic and wherein the callus is subjected to genetic transformation and regeneration on a solid support.
• preferred altered characteristics include but are not limited to altered biochemical or physiological properties, altered nutritional properties of the grass or seeds thereof and altered allergenicity of the grass or its seeds or pollen.
• characteristics contemplated herein include, but are not limited to, the following:
• An "altered characteristic'' is readily determined by comparing a transgenic monocot grass with a non-transgenic grass of the same species. The comparison may be at the biochemical, physiological or visual level.
• a monocot grass of the present invention includes any grass falling within the major subfamilies, namely the Bambosoideae which includes bamboo, Arundinoideae which includes pampas grass and reeds, Pooideae which includes oats, barley, rye, wheat, corn and the forage grasses fescue (Festuca) and ryegrass (Lolium), Chloridoideae which includes millet, t'ef, Mitchell grass, Rhodes grass, Bermuda grass and Kallar grass and Panicoideae which includes sorghum, maize, Surinam grass, Pangola grass, Buffel grass, Bahia grass, sugar cane and Vetiver grass.
• the present invention is described and exemplified herein in relation to ryegrass, the techniques of transformation and regeneration are broadly applicable to any of the grasses as contemplated above. In a preferred embodiment, the present invention does not extend to barley, wheat or corn.
• the present invention contemplates a transgenic plant having altered characteristics by the introduction of non-indigenous DNA.
• a "non- indigenous" DNA is one not normally resident in the plant before transformation or is not normally present in more than one copy.
• a further copy of an indigenous gene or genetic sequence may be introduced for the purposes of co-suppression.
• a non- indigenous gene is also referred to as a foreign gene and includes genetic sequences classically corresponding to a "gene" or a part thereof or may be a nucleic acid molecule which in some way effects a change in the transgenic plant. Examples of foreign genes include:
• a bloat resistance gene (c) a bloat resistance gene; (d) an antibody gene (also referred to as a "plantabody”);
• an insect resistance gene including BT toxin gene and proteinase inhibitor gene from Nicotiana alata;
• a selectable marker gene such as those conferring resistance to kanamycin, phosphinothricin, spectinomycin and hygromycin;
• a reporter gene such as GUS, CAT and pigment genes
• Lol proteins including but not limited to Lolpl, Lolpll, Lolp ⁇ ll, Lolp ⁇ W,
• LolpV LolplX or LolpXL.
• transgenic in relation to monocot grasses.
• Such a transgenic monocot grass includes grasses having reduced or modified allergenic proteins such as Lolpl, Lolpll, Lolplll, LolplV, LolpV, LolplX or LolpXl.
• Another aspect of the present invention contemplates a method for producing a transgenic monocot grass, said method comprising deriving callus from a monocot grass and subjecting same to microparticle bombardment and/or Agrobacterium-mediated genetic transfer and then placing said callus under conditions sufficient to permit regeneration of said callus into plantlets.
• the present invention is directed to a method for producing a transgenic monocot grass having at least one altered characteristic compared to a non- transgenic monocot grass of the same species, said method comprising obtaining callus from a monocot grass on a solid support, introducing into cells of said callus on said solid support genetic material effecting said altered characteristic via microparticle bombardment and/or Agrobacterium mediated transfer, subjecting said callus to selection conditions and then subjecting callus to regeneration conditions to form plantlets.
• the selection conditions comprises antibiotic or chemical resistance where a gene encoding resistance is on the introduced genetic sequence or vector carrying same. Most preferably, the selection conditions comprise resistance to at least two antibiotics or at least one antibiotic and at least one chemical agent.
• the callus is prepared from mature and/or developing embryos obtained from seeds. More preferably the callus is grown on a callus induction medium which is based on that of Murashige and Skoog (1992) [MS medium] but contains 2,4-dichlorophenoxy acetic acid (2,4D) in a range from about 1 to about 10 mg L, preferably about Smg/L, L-asparagine (at about 150mg/L) or equivalent amino acid or chemical entity and preferably thiamine HC1 (at about 0.5 mg L) or equivalent.
• the medium may also contain MS medium minerals and/or, in a particularly preferred embodiment, potassium iodide or equivalent or functional analogue at 0.83 mg/L.
• the cells selected for transformation are the healthy cells on the callus and this is facilitated by excision of the embryo and adjacent cells. More preferably, the healthy cells are selected for transformation at an age of between 1 to 10 weeks, more preferably 3 to 6 weeks and most preferably at about 4 weeks old.
• a particular advantage of the present invention is that the genetic transfer and regeneration is conducted with the callus on solid medium such as agar and/or filter paper. A suspension culture is not first prepared as commonly occurs in the prior art.
• the callus is generally from cells which have been either directly or indirectly obtained from the monocot grass tissue. This may be from embryo tissue, root, shoot including leaf tissue and inflorescence tissues.
• the callus is derived from mature and/or developing embryos or seeds.
• Preferred monocot grasses include forage grass such as but not limited to ryegrass Lolium (ryegrass), Agaropyron, Phalaris, Poa, Festuca, Dactylis, Aleopecuris and Phleum.
• Genetic material contemplated herein include vectors suitable for introduction into cells by microparticle bombardment and/or Agrobacterium mediated transformation.
• Agrobacterium mediated transformation on its own has yet to be widely used in monocots, in accordance with the present invention, the combination of microparticle bombardment and Agrobacterium mediated transformation is contemplated to be particularly useful as well as microparticle bombardment on its own.
• Suitable vectors will be well known by those skilled in the art and are described, for example in Croy R.R.D. (Ed) Plant Molecular Biology, Bios Scientific Publications, Blackwell Scientific Publication (1993).
• a particularly useful vector for microparticle bombardment is pBIN19 or related vector or a pUC-based vector such as pUC18 or pUC19 or equivalent or functional analogue.
• the genetic material effects a reduction in levels of ryegrass pollen allergens such as Lolpl, Lolpll, Lolplll, LolplV, LolpV, LolplX or LolpXl (see, for example, International Patent Application No. PCT/AU89/00123).
• the selectable marker encoded by the construct confers resistance to an antibiotic it preferably encodes resistance to a class of antibiotics.
• This feature allows selection with two or more antibiotics which is a particularly preferred feature of the present invention. This provides a way of overcoming the problem of untransformed plant cells being able to grow on the selected medium.
• the construct may encode the gene for neomycin phosphotransferase, npt II,or other antibiotic resistance genes such as hygromycin resistance, aph 3 * 11 or aph IV amongst others.
• the gene is coupled to an appropriate promoter such as pEmu, Ubi I, Act I or the CaMV 35S promoter.
• kanamycin and geneticin are used in the selection medium.
• these antibiotics are present in the amount 15 to 50 mg/L kanamycin and 15 to 50 mg/L geneticin.
• a further aspect of the present invention contemplates a method of regenerating monocot grass plants from transformed plant cells, said method comprising contacting cells on a solid support upon which said cells are subjected to transformation means on a medium and under appropriate conditions and time wherein said medium contains shooting and rooting hormones and then culturing the resultant shoots on a medium in the absence of biologically active hormones.
• the medium which has an absence of hormones may be any suitable medium and includes soil or potting mix.
• a rooting hormone is a class of compounds known as auxins which include indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and indolebutyric acid (IBA).
• auxins include indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and indolebutyric acid (IBA).
• a shooting hormone is a class of compounds known as cytokinins which includes synthetic shooting hormones such as 6-benzylaminopurine (BAP), kinetin, zeatin, 2iP (N ⁇ ( ⁇ 2 -isopentenyl)-adenine fr ⁇ ns-6-(4-hydroxy-3-me ⁇ ylbut-2-enyl) amino purine.
• BAP 6-benzylaminopurine
• kinetin zeatin
• 2iP N ⁇ ( ⁇ 2 -isopentenyl)-adenine fr ⁇ ns-6-(4-hydroxy-3-me ⁇ ylbut-2-enyl) amino purine.
• callus cultures are particularly suitable for regeneration because green plantlets or plants can be regenerated efficiently from callus. This is in contrast to the albino or weak, non-viable plants which have been reported in the prior art.
• This aspect of the present invention avoids the use of protoplasts or cell suspensions, and hence, genetically uniform cells can be used as a starting material. In addition, this avoids the problem of using physiologically fragile and nutritionally fastidious protoplasts or cell suspensions of the prior art.
• the cells are from forage grasses, more preferably from the genus Lolium, most preferably ryegrass.
• the plant cells may be transformed by any appropriate method. Most preferably the cells are transformed by the microprojectile bombardment technique or a combination of this technique with Agrobacterium-mediated transformation.
• the transformed cells are callus cells produced from mature and/or developing embryos.
• the callus is prepared from mature and/or developing embryos obtained from seeds. More preferably the callus is grown on a callus induction medium which is based on that of Murashige and Skoog (1962) but contains 2,4-D in a range from about 1 to about 10 mg/L and more preferably about 5mg/L, L-asparagine at approximately 150 mg/L and thiamine HC1 at approximately 0.5 mg/L.
• the cells chosen for transformation are the healthy cells on the callus and this is facilitated by excision of the embryo and adjacent cells.
• the healthy cells are selected for transformation at an age of between about 1 to about 10 weeks, more preferably about 3 to about 6 weeks such as at about 4 weeks old from cultures grown in the absence of light.
• the present invention extends to cultures earlier than 3 weeks (e.g. 1 week) or older than 6 weeks (e.g. 10 weeks).
• the regeneration medium contains a shooting:rooting hormone ratio of from about 1:0 to about 10:1 and is preferably about 2:1.
• the medium may contain from about 0.05 to 2.0 mg/L shooting hormone and from about zero to about 0.075 mg/L rooting hormone.
• the medium contains about 0.5 mg/L shooting hormone and 0.025 mg/L rooting hormone.
• An example of suitable shooting hormone is BAP and of a rooting hormone is IAA. If only a shooting hormone is used, then BAP at approximately 0.2 mg/L to approximately 4.0 mg/L and more preferably at about 2 mg/L is appropriate.
• the regeneration medium preferably also contains L-asparagine at about 50 to about 750 mg/L and thiamine HC1 at about 0.05 to about 5 mg/L.
• the shoots may be transferred directly to soil or potting mix and grown under appropriate conditions.
• the plantlets are grown at a temperature of about 15- 30°C although 25°C is preferred and the cells are allowed to regenerate in light.
• a particularly preferred aspect of the present invention contemplates a method of producing a transformed monocot grass comprising bombarding callus cells derived from a mature embryo of a monocot grass with a microprojectile containing a nucleic acid construct encoding a desired trait under the control of an appropriate promoter and carrying a selectable marker, under appropriate conditions to allow transformation, optionally further subjecting cells to Agrobacterium-media!Led transfer of a similar nucleic acid construct, selecting transformed cells on a selection medium under conditions and for a time sufficient to permit regeneration of plants from the transformed cells wherein said regeneration conditions comprise shooting hormone and rooting hormone or shooting hormone alone and then culturing the resultant shoots on a medium in the absence of biologically active hormones.
• the above conditions lead to regeneration of green plantlets and plants with photosynthetic ability.
• the invention also extends to transgenic plants made by the methods described above.
• the methods of the present invention permit for the first time, the production of monocot grasses exhibiting altered characteristics such that the modified plants have desired properties.
• transgenic ryegrass can now be constructed having pollen with reduced allergenic properties.
• antisense, cosuppression, ribozymes, regulatory genes or other suitable means may be employed to reduce expression of allergenic proteins such as Lolpl, Lolpll, Lolplll, LolplV, LolpV, LolplX or LolpXl or isoforms thereof.
• the present invention extends, therefore, in a particularly preferred embodiment, to pollen from transgenic monocot grasses, such as ryegrass, exhibiting useful properties, such as exhibiting reduced allergenicity.
• another aspect of the present invention provides a seed or seeds or pollen from a transgenic monocot grass wherein said transgenic monocot grass exhibits at least one altered characteristic when compared to a non-transgenic monocot grass of the same species.
• the transgenic monocot grass exhibits altered biochemical or physiological properties, altered nutritional properties of the grass or seeds there of altered allergenicity of the grass or its seeds or pollen.
• the transgenic monocot grass exhibits at least one of the following characteristics:
• the seed or seeds or pollen which exhibits reduced allergenic properties.
• the seed or seeds or pollen which exhibits reduced levels of one or more of Lolpl, Lolpll, Lolplll, LolplV, LolpV, LolplX or LolpXl are particularly preferred.
• the seed or seeds or pollen is from forage grass such as ryegrass.
• Figure 1 is a photographic representation showing cell cultures and plantlets.
• Panel A shows embryos on callus induction medium.
• Panel B shows cultured callus from embryos and shoot initiation on callus.
• Panel C shows plantlets forming shoots; these plantlets are derived from transformed callus cells.
• Panel D shows a regenerated plant derived from transformed callus.
• FIG. 2 is a photographic representation of DNA gel blot analysis of ryegrass transformants. Each lane contains 20 ⁇ g of total leaf DNA which was digested with EcoRI and Bam ⁇ U and hybridized to a 32 P labelled NptJI gene probe. Lanes 1 and 2 contain digested DNA from non-transformed control plants. Lanes 3 to 9 contain DNA from transformed plants derived from single callus. EXAMPLE 1 Embryo production
• Embryos from Lolium rigidum and or Lolium perenne seeds were used to induce callus.
• Mature seed is surface sterilized in 5% v/v hypochlorite and soaked from 8 hours to 48 hours in sterile water.
• Mature embryos are dissected and placed on MS medium (Murashige and Skoog basal salt and minerals supplement with L-asparagine 150 mg/L, i-hiamine HC1 0.5 mg/L, 5mg/L 2,4D,2% w/v sucrose and 0.8% w/v agar at pH5.8) [Murashige and Skoog, 1962].
• Potassium iodide may also be used at approximately 0.83 mg L.
• Embryonic callus cultures were found to be ideal targets from microprojectile-mediated transformation because regenerable cells are not excessively shielded, and can be arranged to occupy most of the target area.
• Compact embryonic callus is transferred to MS medium containing 2% w/v sucrose, 0.5 mg/1 BAP, 0.025 mg/1 IAA and 0.8% w/v agar at 25°C in light. Roots were regenerated from shoots on MS medium with no hormones and containing 0.6% w/v agar.
• Figure 1 shows shoots regenerated from callus of ryegrass. These shoots are green.
• Microprojectile bombardment conditions were optimised on the basis of frequency of transient expression in callus tissues. Parameters like microprojectile velocity, multiple bombardments and age and size of embryogenic callus were evaluated in order to achieve high efficiency of transient expressing cells. Pressure used was 24 to 29 inches Hg, distance from stopping screen was 6-13 cm and rupture disk strength was 1100 psi. The strong moncot promoter pEmu, coupled to the GUS reporter gene (Last et al, 1991) was used to optimise bombardment conditions in transient assays. The GUS histochemical assay for callus tissue is performed according to Klein et al (1988).
• npt-II neomycin phosphotransferase
• kanamycin and neomycin and anther specific promoter Bgp 1 coupled to GUS in addition to the 35S promoter coupled to npt II.
• This plasmid also has the CaMv 35S promoter coupled to the npt-II (neomycin phosphtransferase) gene in order to allow selection on an antibiotic.
• the concentration of the antibiotics, geneticin, and kanamycin required to inhibit the growth and to kill control untransformed callus was determined. There were found to be in the range of 15 to 50 mg/L geneticin and 15 mg/L to 50 mg/L kanamycin. Plants were regenerated from the callus lines which grown in the presence of a selection medium containing geneticin. Concentration of geneticin was the amount required to kill control (untransformed) tissue-cultured. The regenerated shoots were again exposed to medium containing geneticin and kanamycin. Surviving shoots were considered as the putative transformant shoots.
• NPT-II in tissue of transformants was measured using an NPT-II ELISA assay kit (5 prime-3 prime Inc.) Leaf tissue of transformed tissue-culture plants was used. Leaf tissue from transformed tissue-culture plants showed significant increase of NPT-II levels over untransformed control plants. PCR analysis:
• the gemonic DNA from leaf tissues of transformed plants were used from RAPD PCR analysis.
• Npt-II gene coding region segment was chosen from PCR amplification.
• Transformed plants showed the presence of amplified band of DNA which untransformed plants fail to show corresponding DNA fragment.
• Genomic DNA from leaf tissue was extract using a method by Dellaporta et al (1983).
• Genomic DNA was digested with EcoRI and/or BamYQ. The digested DNA resolved and was examined for completeness of digestion by electrophoresis on a 0.7% w/v agarose gel. After transferring to a nylon membrane it was be probed with npt-II coding sequence labelled with 32 P. The membrane was then analysed using autoradiography. DNA from transformed plants displayed hybridisation with a single approximately 3.1- 3.5 kb band while no signal was obtained from the DNA of untransformed plants ( Figure 2).
• the construct pAHC25 which encodes the bar gene conferring phosphinothricin resistance and GUS is used to bombard 4 week old callus cells as described above.
• the callus cells are then selected on a suitable medium containing Basta (glufosinate ammonia) between 0.1 to 5.0 mg/L, preferably 0.5 to 5.0 mg/L and most preferably 1.0 to 3.0 mg/L.
• Basta glufosinate ammonia
• the callus cells are transferred to fresh Basta containing medium at two week intervals over a period of six weeks.
• Shoots are regenerated on suitable medium without herbicide or suitable medium containing 1 mg/L Basta. Regenerated shoots are then exposed to Basta to confirm tolerance. Roots are then regenerated from the shoots. Presence of the introduced DNA is then confirmed by Southern analysis.

Applications Claiming Priority (3)

Publications (1)

Family

ID=3784951

Family Applications (1)

Country Status (5)

Cited By (2)

Families Citing this family (6)

Family Cites Families (1)

• 1995
  • 1995-01-16 AU AUPN0563A patent/AUPN056395A0/en not_active Abandoned
• 1996
  • 1996-01-15 WO PCT/AU1996/000016 patent/WO1996022015A1/en not_active Application Discontinuation
  • 1996-01-15 EP EP96900470A patent/EP0809432A1/de not_active Withdrawn
  • 1996-01-15 CA CA002210526A patent/CA2210526A1/en active Pending
  • 1996-01-15 NZ NZ298713A patent/NZ298713A/en unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events