EP1945774A1 - Transformation de brassica par bombardement de microprojectiles - Google Patents

Transformation de brassica par bombardement de microprojectiles

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Publication number
EP1945774A1
EP1945774A1 EP05851482A EP05851482A EP1945774A1 EP 1945774 A1 EP1945774 A1 EP 1945774A1 EP 05851482 A EP05851482 A EP 05851482A EP 05851482 A EP05851482 A EP 05851482A EP 1945774 A1 EP1945774 A1 EP 1945774A1
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EP
European Patent Office
Prior art keywords
plant
microspore
brassica
embryo
bombardment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05851482A
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German (de)
English (en)
Inventor
Wenpin Chen
Lomas Tulsieram
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Pioneer Hi Bred International Inc
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Pioneer Hi Bred International Inc
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Publication date
Application filed by Pioneer Hi Bred International Inc filed Critical Pioneer Hi Bred International Inc
Publication of EP1945774A1 publication Critical patent/EP1945774A1/fr
Withdrawn legal-status Critical Current

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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

Definitions

  • the field of the invention relates to the genetic engineering of plants, particularly methods for genetically transforming Brassica plants.
  • Brassica species are used as a source of vegetable oil, animal feeds, vegetables and condiments. Brassica plants that are used for vegetable production include cabbage, cauliflower, broccoli, kale, kohlrabi, leaf mustard and rutabaga. Seeds of B. hirta are used to produce the popular American condiment, yellow mustard. However, on a world-wide basis, the most economically important use of Brassica species is for the production of seed-derived, vegetable oils. The predominant Brassica species grown for oil production is B. napus, followed by B. juncea and S. rapa. Seeds of ⁇ . napus, B. juncea and B. rapa are referred to as rapeseed.
  • oilseed rape Brassica species that are grown primarily for oil production are often called oilseed rape.
  • canola a type of oilseed rape that has been selected for low levels of erucic acid and glucosinolates in seeds, is the predominant Brassica plant grown for the production of vegetable oil for human consumption. While low-erucic-acid rapeseed oils, such as canola oil, may be favored for human consumption, high-erucic-acid rapeseed oils are desirable for a variety of industrial applications including the production of cosmetics, lubricants, plasticizers and surfactants.
  • Novel, recombinant DNA-based strategies for incorporating new traits, such as disease and insect resistance, modified seed oil composition and modified seed protein composition, are being developed for canola and other Brassica species. All of these strategies depend on genetic transformation methods to introduce the recombinant DNA into the genomes of Brassica plants.
  • the most favored methods for transforming Brassica species involve the use of Agrobacterium.
  • the Agrobacterium-based transformation methods provide a reliable means for introducing foreign DNA into plants, there are a number of disadvantages to methods of plant transformation that involve the use of Agrobacterium.
  • an undesired consequence of all Agrobacterium-based methods is the introduction of at least one T-DNA border into the genome of the recipient plant.
  • T-DNA border is an essential element of the genetic mechanism by which Agrobacterium transfers DNA to a plant cell
  • the T-DNA border is not essential for the expression of foreign genes in the recipient plant. Additionally, the accumulation of multiple T-DNA borders throughout the genome of a plant may have deleterious effects on the plant or its progeny.
  • the co- cultivation of plant tissues with Agrobacterium may slow the regeneration of a transformed plant from a transformed cell. After the co-cultivation phase, Agrobacterium must be eliminated from cultures of the plant tissues. High levels of bactericidal agents must be applied to the plant cultures to kill the Agrobacterium.
  • US 6,297,056 describes the transformation of cotyledonary petioles.
  • US 6,515,206 and US 2003/0200568 describe the use of transformation of plastids in true leaves.
  • Chen and Beversdorf Theor. Appl. Genet. 88: 187-192 (1994) describe a biolistic transformation procedure of microspore-derived hypocotyls involving DNA imbibition.
  • Fukuoka et al. Plant Cell Reports 17: 323-328 (1998) describe biolistic transformation of fresh microspores.
  • Nehlin et al., Plant Physiol. Vol. 156: 175-183 (2000) describe transient biolistic transformation of pre-incubated microspores, but no stable transgenics were reported.
  • Methods are provided for producing transgenic Brassica plants.
  • the methods comprise introducing DNA constructs by microprojectile bombardment.
  • the introduced DNA constructs can encode proteins or can suppress endogenous genes.
  • the methods find use in agriculture, particularly in the development of improved varieties of Brassica plants through the incorporation of desirable agronomic traits.
  • the methods involve introducing a DNA construct by microprojectile bombardment into a Brassica cell that is capable of regenerating into a stably transformed Brassica plant and regenerating such a Brassica plant from the cell.
  • An aspect of the invention is to provide a method of producing a transformed
  • Brassica cell by particle bombardment comprising: (a) culturing a pre-incubated microspore-derived explant comprising a cell under a condition of plasmolysis for a period of about half an hour to about 4 hours prior to bombardment; (b) introducing a DNA construct by microprojectile bombardment into an exposed cell on a surface of the pre-incubated microspore-derived explant, wherein the explant is under the condition of plasmolysis; and (c) continuing to culture the bombarded pre-incubated microspore-derived explant under the condition of plasmolysis for a period of about 4 hours to about 20 hours, to produce a transformed Brassica cell.
  • a pre-incubated microspore-derived explant is a microspore or any tissue derived from the microspore (for example a microspore-derived embryo or a microspore-derived hypocotyl) that has been cultured for a period of time of between 1 day and 30 days prior to bombardment.
  • the condition of plasmolysis can be selected from the group consisting of (a) culturing the explant on osmotic medium and (b) culturing the explant on wetted filter paper.
  • the explant can be a pre-incubated microspore, a pre-incubated microspore-derived embryo, or a pre-incubated microspore-derived hypocotyl.
  • the method can further comprise the steps of regenerating a transformed plant from the transformed cell, comprising: (a) culturing said microspore-derived explant on a regeneration medium to produce a regenerated embryo or tissue; and (b) regenerating a fertilestably transformed Brassica plant from said embryo or tissue
  • An aspect of the present invention is to provide a method for producing a stably transformed Brassica plant, comprising: (a) introducing a DNA construct by microprojectile bombardment into a pre-incubated explant, which may be a microspore, or a microspore-derived embryo or portion of a microspore-derived embryo; (b) culturing the pre-incubated explant to produce an embryo or tissue; and (c) regenerating a stably transformed Brassica plant from the embryo or tissue.
  • a pre-incubated microspore can be produced by culturing a microspore in a culture medium for a period of about two to ten days prior to bombardment.
  • the period can be from about four to eight days, or from seven to eight days.
  • the method can further comprise a step of inducing plasmolysis of the pre-incubated microspore prior to, during and after bombardment.
  • plasmolysis can be induced by (a) culturing the pre-incubated microspore on osmotic medium prior to, during and after bombardment, or (b) culturing the pre-incubated microspores on wetted filter paper prior to, during and after bombardment.
  • the osmotic medium can comprise between about 17 and 19% sucrose and between about 0.8 and 1.6% Phytagel l M agar (w/v).
  • the pre-incubated microspore can be cultured on osmotic medium for about between half an hour and four hours prior to bombardment and for about between four hours and twenty hours after bombardment.
  • the method can further comprise a selection step after bombardment comprising culturing the bombarded pre-incubated microspore on a medium comprising a selection agent against a gene encoded by the DNA construct.
  • the selection agent can be selected from the group consisting of kanamycin, G418 and glyphosate.
  • the concentration of G418 can be between about 5 and 10 mg/l.
  • the concentration of glyphosate in the medium can be between about 0.1 mM and 0.2 mM.
  • the method may further comprise a step of orientating the pre-incubated microspore during bombardment so that a surface of the microspore is exposed during the bombardment.
  • the method may further comprise a step of collecting the pre-incubated microspore such that it is viable and embryogenic prior to bombardment.
  • the step of collecting the pre-incubated microspore can be a filtration step or a step of Percoll ® gradient centrifugation (Percoll ® concentration is 35-45%).
  • the filtration step can be done using a sieve having a pore size of about 15 to 48 ⁇ m.
  • the microprojectile bombardment can be conducted using bombardment factors comprising about 12.5 ng to 5 ⁇ g of said DNA construct, about 15 ⁇ g to 100 ⁇ g gold particles per shot at the size of 0.4 micron to 0.6 micron, about 2.5 M CaCl 2 and a 650 to 900 psi rupture disk.
  • the step of regenerating a stably transformed plant can comprise culturing the bombarded pre-incubated microspore on a first liquid selection medium for a first period of time, a second liquid selection medium for a second period of time, and then transferring the resistant embryo or tissue derived from the pre-incubated microspore to solid medium for a third period of time.
  • the first period of time can be about 7 days and the pre-incubated microspore can be cultured in darkness.
  • the pre-incubated microspore, or tissue or embryo derived from the pre-incubated microspore can be cultured in the second liquid selection medium for approximately 14 days in dim light of approximately 240 foot candles or 2,583 Lux.
  • the second liquid medium can be liquid NLN-6.5S and further comprise growth regulators.
  • the growth regulators in the second liquid selection medium can comprise 0.5 mg/L NAA and 0.05 mg/l BAP.
  • the first, the second, or both the first and the second liquid selection media can comprise G418 or glyphosate.
  • the solid medium may comprise growth regulators to induce regeneration, and optionally further comprise a selection agent against a gene encoded by the DNA construct.
  • the solid medium can be MMW medium with indoleacetic acid (IAA), thidiazuron (TDZ) and silver nitrate (AgNOs).
  • the solid medium can further comprise a selection agent against a gene encoded by the DNA construct.
  • the method can further comprise use of a chromosome doubling agent to produce a doubled haploid transgenic plant. The doubling agent can be administered within one day after bombardment and can be administered for approximately 7 days.
  • the method may comprise: (a) culturing a microspore-derived embryo on osmotic medium for a period of about half an hour to about 4 hours prior to bombardment; (b) introducing a DNA construct by microprojectile bombardment into an exposed surface of the microspore-derived embryo on osmotic medium; (c) continuing to culture the bombarded microspore-derived embryo on osmotic medium for a period of about 4 hours to about 20 hours; (d) culturing said bombarded microspore-derived embryo on regeneration media to produce a regenerated embryo or tissue; and (e) regenerating a stably transformed Brassica plant from the regenerated embryo or tissue.
  • the microspore-derived embryo can be between about 11 and 20 days old.
  • the microspore-derived embryo can be between about 11 and 14 days old.
  • the method can further comprise the step of collecting the embryo using a pipette and transferring the embryo onto filter paper prior to step (a).
  • Step (d) can comprise culturing the embryo on liquid medium for a first period of time and then on solid medium for a second period of time.
  • the first period of time can be about 7 to 14 days.
  • the method can further comprise an optional step of excising a hypocotyl from the regenerated embryo and culturing the hypocotyl on regeneration media.
  • the method can further comprise a step of selecting for a transformed embryo comprising culturing the embryo on media supplemented with a selection agent against a gene encoded by the DNA construct.
  • the method can comprise use of a chromosome doubling agent to produce a double haploid transgenic plant.
  • the method may comprise: (a) culturing a hypocotyl excised from a microspore-derived embryo on osmotic medium for a period of about half an hour to about 4 hours prior to bombardment; (b) introducing a DNA construct by microprojectile bombardment into an exposed surface of the microspore-derived hypocotyl on osmotic medium; (c) continuing to culture the bombarded microspore- derived hypocotyl on osmotic medium for a period of about 4 hours to 20 hours; (d) culturing said microspore-derived hypocotyl on a regeneration medium to produce regenerated embryos or tissues; and (e) regenerating a stably transformed Brassica plant from said embryo or tissue.
  • the microspore-derived hypocotyl can be excised from a microspore-derived embryo of between about 21 and 26 days.
  • the method can further comprise a step of culturing the microspore-derived hypocotyl on a cell division induction medium comprising plant growth regulators for between about 1 to 20 hours prior to bombardment.
  • Step (d) can comprise culturing the embryo on a first solid medium for a first period of time and then on a second solid medium for a second period of time.
  • the first solid medium can comprise plant growth regulators for bud induction.
  • the second solid medium can be free of plant growth regulators or comprises plant growth regulators for shoot formation.
  • the method can further comprise a step of selecting a transformed embryo or tissue comprising culturing the embryo or tissue on media supplemented with a selection agent against a gene encoded by the DNA construct.
  • the method can further comprise use of a chromosome doubling agent to produce a doubled haploid transgenic plant.
  • Another aspect of the invention is to provide a Brassica cell or a stably transformed Brassica plant produced by any one of the methods described above.
  • the plant or cell can be selected from Brassica napus, Brassica rapa, Brassica juncea, Brassica oleracea, Brassica carinata and Brassica nigra. Progeny of the plant and cell are also provided.
  • the invention is drawn to methods for transforming Brassica plants.
  • the methods find use in agriculture in the development of transgenic crop plants with improved agronomic characteristics.
  • the methods find particular use in introducing desirable traits into a Brassica plant.
  • Such new traits may be, for example, resistance to an herbicide, resistance to pathogens and insects, modified seed oil composition and the like.
  • the methods involve introducing a DNA construct into the genome of a cell of a Brassica plant and regenerating a stably transformed plant from the cell.
  • Brassica cell is intended a cell from a Brassica plant or a cell that is produced by in vitro culture methods and is descended from a cell from a Brassica plant.
  • somatic embryo an embryo that develops from a somatic cell.
  • the developmental process by which a somatic embryo develops from a cell is known as “somatic embryogenesis.”
  • Such a “somatic embryo” is distinct from a “zygotic embryo” which develops from a zygote.
  • microspore-derived embryo is intended an embryo that develops from a microspore. Because it develops from a germ cell, such a microspore-derived embryo” is distinct from both somatic and zygotic embryos which develop from somatic cells and zygotes, respectively.
  • microspore-derived hypocotyl is intended a hypocotyl of an embryo that develops from a microspore.
  • adventitious is intended an organ or other structure of a plant that does not originate in the usual location on the plant body.
  • a shoot that originated from a hypocotyl of a microspore-derived embryo is an “adventitious shoot.”
  • canola is intended a Brassica plant or oil from a Brassica plant wherein the oil must contain less than 2% erucic acid and the solid component of the seed must contain less than 30 micromoles of any one or any mixture of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, and 2- hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil free solid.
  • organogenesis is intended the developmental process wherein a cell or group of cells gives rise to an organ such as, for example, a shoot, a bud or a root.
  • chromosome doubling is intended that each of the chromosomes in a cell is duplicated resulting in a doubling of the number of chromosomes in the cell.
  • ploidy is intended the number of complete sets of chromosomes in the nucleus of a cell.
  • a "haploid” cell has one set of chromosomes, and a “diploid” cell has two sets.
  • effective amount is intended an amount of an agent, compound or plant growth regulator that is capable of causing the desired effect on an organism. It is recognized that an “effective amount” may vary depending on factors, such as, for example, the organism, the target tissue of the organism, the method of administration, temperature, light, relative humidity and the like. Further, it is recognized that an “effective amount” of a particular agent may be determined by administering a range of amounts of the agent to an organism and then determining which amount or amounts cause the desired effect.
  • pre-incubated microspore-derived explant is intended a microspore or any tissue derived from the microspore (for example a microspore-derived embryo or a microspore-derived hypocotyl) that has been cultured for a period of time of between about 1 day and 30 days prior to bombardment.
  • a pre- incubated microspore may be cultured for about two to ten days from the time of isolation of the microspore from a donor plant.
  • a pre-incubated microspore-derived embryo may be cultured for a period of about 11 days to 20 days from the time of isolation of the microspore from a donor plant.
  • a pre-incubated microspore-derived hypocotyl may be cultured for a period of about 21 to 26 days from the time of isolation of the microspore from a donor plant.
  • progeny is intended descendents of a particular cell or plant which comprise at least a portion of the transgene inserted at the locus of the genome of the TO plant cell.
  • progeny can be seeds developed on a plant and plants derived from such seeds.
  • progeny of a plant include seeds formed on TO, T1 , T2 and subsequent generation plants, and plants derived from such seeds.
  • Progeny also includes seeds formed by cross pollination using pollen of a TO, T1 , T2, T3, etc. plant.
  • the progeny can be the result of selfing, outcrossing or backcrossing.
  • the progeny can also include asexually propagated plants or cells derived from TO, T1 , T2, etc plants or cells that include at least a portion of the transgene inserted at the locus of the genome in the TO plant cell.
  • plants produced via cuttings, tissue culture, microspore culture, etc. that comprise at least a portion of the original transgene inserted at the locus of the genome of the TO plant cell are also considered progeny.
  • Methods are provided for transforming a Brassica plant.
  • the methods involve transforming a Brassica cell with a DNA construct by microprojectile bombardment.
  • the methods further involve regenerating the transformed cell into a transformed Brassica plant.
  • Such a transformed Brassica plant possesses at least one copy of the DNA construct, or portion thereof, incorporated into its genome.
  • the transformed Brassica plants of the invention may be stably transformed Brassica plants.
  • Such transformed Brassica plants are capable of producing at least one offspring that possesses at least one copy of the DNA construct of the invention, or portion thereof, stably incorporated within its genome.
  • Cells of the present invention may originate from (1) pre-incubated microspores, (2) microspore-derived embryos or (3) microspore-derived hypocotyls. It is recognized that the cells of these tissues are most likely haploid.
  • the cells may be diploid if the cells undergo spontaneous chromosome doubling, or if the cells are subjected to chromosome doubling agents. Transformation of haploid cells is advantageous because the resulting chromosome doubled transgenic plant is homozygous.
  • a DNA construct of interest is introduced into the cell by microprojectile bombardment. Microprojectile bombardment is also known by other terms, including particle bombardment, microparticle bombardment, ballistic particle acceleration and biolistic transformation.
  • such methods involve applying to or coating the surface of microprojectiles with the DNA construct of interest, and then delivering the DNA-coated microprojectiles to the target tissue at a velocity sufficient to allow the particles to pass through cell walls and membranes and thus, enter plant cells.
  • a velocity sufficient to allow the particles to pass through cell walls and membranes and thus, enter plant cells.
  • DNA constructs of the invention may comprise at least one nucleotide sequence of interest operably linked to a promoter that drives expression in a plant cell.
  • DNA constructs may comprise a selectable marker gene and at least one additional nucleotide sequence of interest operably linked to a promoter that drives expression in a plant cell.
  • DNA constructs may comprise a selectable marker gene and at least one additional nucleotide sequence that is capable of conferring a desired trait on a Brassica plant.
  • the methods of the present invention additionally comprise regenerating the transformed cell of the invention into a stably transformed Brassica plant.
  • Regeneration of the transformed plant involves culturing the transformed cell under conditions that result in the growth and development of the transformed cell into a transformed plant.
  • the transformed cell or descendents thereof may develop into a transformed embryo, particularly a transformed microspore-derived embryo or somatic embryo which then develops into a transformed plant.
  • the transformed cell and descendents thereof may develop into a transformed organ, such as, for example, an adventitious shoot. It is recognized that regenerating a transformed Brassica plant from a transformed cell via an adventitious shoot may additionally involve the formation of callus before adventitious shoot formation. Such an adventitious shoot may be used to produce the stably transformed Brassica plant by methods known in the art.
  • Such methods generally involve culturing an adventitious shoot in a medium and environment which favors the formation of adventitious roots on the adventitious shoot.
  • Methods for rooting adventitious shoots are known in the art. The methods of the present invention do not depend on a particular method for rooting transformed Brassica shoots. Any method known in the art for rooting adventitious shoots may be employed in the methods of the present invention.
  • rooting adventitious shoots will involve incubating shoots, for a period of time, on a medium that contains an effective amount of an auxin, such as, for example, indolebutyric acid, to induce root formation. See, for example, Moloney et al.
  • Rooted shoots may then be removed from culture, transferred to soil or potting medium and subjected to environmental conditions that favour growth, maturation and seed production.
  • transformed embryos, transformed adventitious organs, and transformed plants of the invention may be chimeric. That is, such transformed embryos, organs and plants may be comprised of both transformed and non-transformed cells, or may be comprised of two or more differentially transformed cells. It is further recognized that such chimeric plants may give rise to progeny plants that comprise a DNA construct of interest, or portion thereof, stably incorporated into the genomes of all of their somatic and germ line cells.
  • the methods of the invention involve the transformation of cells from
  • the cells may be haploid cells. While haploid cells generally do not give rise to diploid plants, it is recognized that occasionally a haploid cell may spontaneously give rise to a diploid cell that is capable of developing into a fertile plant. If necessary, chromosome-doubling agents may be employed in the methods of the invention to increase the ploidy of a haploid cell two fold. That is, a haploid cell becomes a diploid cell. Such a diploid cell may give rise to a fertile, stably transformed Brassica plant.
  • the methods of the present invention do not depend on a particular genetic mechanism of chromosome doubling. It is likely, however, that chromosome doubling results from chromosome duplication as would occur for example, during mitosis, but in the absence of cytokinesis.
  • Induced chromosome doubling of the invention involves administering an effective amount of a chromosome-doubling agent to a cell, preferably a haploid cell.
  • a chromosome-doubling agent include, but are not limited to, trifluralin, colchicine, oryzalin, amiprophosmethyl and pronamide.
  • a chromosome-doubling agent may be administered to a tissue, or a cell thereof, before, after, or both before and after, introducing a DNA construct into a cell by microprojectile bombardment.
  • an effective amount of a chromosome-doubling agent is administered after bombardment.
  • the plants regenerated from transformed Brassica cells are referred to as the TO generation or TO plants.
  • the seeds produced by various sexual crosses of TO generation plants are referred to as T1 progeny or T1 generations.
  • T1 seeds When T1 seeds are germinated, the resulting plants are also referred to as T1 generation.
  • T2 seeds Seeds produced on the T1 plant or from crosses using T1 pollen, are referred to as T2 seeds, which give rise to T2 plants.
  • Seeds produced on the T2 plant or from crosses using T2 pollen are referred to as T3 seeds.
  • T3 seeds give rise to T3 plants. Accordingly, the generations progress through T4, T5, T6, etc.
  • the seeds and plants of the T1 , T2, T3, T4, etc. can be analyzed to ensure successful transmission of the transgene.
  • the plants can be selfed, outcrossed or backcrossed.
  • the transgenic plants (TO, T1 , T2, etc) can be propagated asexually, for example by cloning, tissue culture, cuttings, microspore culture, etc. as is known to those skilled in the art.
  • methods are provided for transforming Brassica pre-incubated microspores and regenerating stably transformed plants therefrom.
  • the methods of the first embodiment involve bombarding pre-incubated microspores with microprojectiles coated with a DNA construct of interest.
  • Microspores are isolated by methods that are known to those skilled in the art. For example, see Fukuoka et al. (1996) Plant Physiol. 111:39-47; Keller et al. (1987) Proc. 7 th Int. Rapeseed Congr. (Plant Breeding and Acclimatization Institute, Poznan, Tru) pp. 152-157, Swanson et al. (1987) Plant Cell Reports 6: 94-97 and Baillie et al. (1992) Plant Cell Reports 11 : 234-237.
  • the microspores are haploid.
  • the microspores may be isolated and cultured in a medium with a high level of sucrose, for example 17% sucrose, for 2 to 3 days.
  • the high level of sucrose is recommended to ensure the integrity of the microspores immediately after isolation. Further, high osmotic stress would have a positive effect on embryogenesis induction (Maraschin et al., 2005 J Exp Bot 56: 1711-1726 and Prem et al. 2005 In Vitro Cell. Dev. Biol. - Plant 41 :266-273).
  • the microspores are then cultured for 4 to 8 days in medium containing a reduced level of sucrose, for example in the range of 10% sucrose to promote microspore division.
  • a pre-incubation period of 2 to 10 days is within the scope of the invention.
  • the pre-incubated microspores are collected in a manner to enrich for viable and embryogenic microspores. This can be done, for example, by using a NitexTM sieve of 15 to 48 ⁇ m in pore size. The pore size may be between 15 and 25 ⁇ m.
  • the embryogenic microspores can also be enriched by Percoll ® gradient centrifugation (Touraev 1996 Sex Plant Reprod 9: 209-215). Percoll ® concentration is between 35 to 45%.
  • the NitexTM sieve holding the pre-incubated microspores is then placed on an osmotic treatment medium prior to bombardment, during bombardment and for a period of time after bombardment.
  • the osmotic treatment induces slight plasmolysis of the microspores to ensure they will not burst due to the bombardment procedure.
  • a surface of the microspores may be exposed to the path of the bombarding particles coated with DNA to facilitate entry of the particles and also to prevent any sudden influx of medium (i.e. the surface of the microspores that is exposed is not embedded in the medium). The osmotic treatment facilitates this.
  • the pre-incubated microspores may be subjected to the osmotic treatment for 1 to 2 hours prior to bombardment, during the bombardment, and for 1 to 24 hours after bombardment.
  • the osmotic treatment may comprise placing the pre-incubated microspores on medium containing between 0.8 to 1.6% PhytagelTM or agar, between 17 and 19% sucrose and 1g/l MES (2-[N- Morpholino] ethanesulfonic acid).
  • the osmotic treatment can also be done by placing microspores (optionally on sieves) in a petri dish (3.5 cm in diameter) prior to bombardment, during the bombardment and after the bombardment.
  • a piece of filter paper (3.2 cm in diameter) wet with NLN-13S medium is placed in the petri dish to prevent microspore dehydration.
  • the microspores and sieve can be cultured in medium containing a doubling agent and a high level of sucrose, for example 13% sucrose, for about 7 days.
  • the bombarded pre-incubated microspores may be transferred to medium containing an appropriate selective agent for that particular selectable marker gene. Such a transfer may occur immediately after bombardment or after a period of time. For example, the transfer may occur between 0 and about 30 days after bombardment.
  • the pre-incubated microspores may be sub- cultured in selection NLN medium, which may contain a reduced sucrose content, for example 6.5% and growth regulators, for example cytokinins and auxins. Selection may be conducted in the light.
  • the pre-incubated microspores may then be monitored for the appearance of transformed embryos and/or adventitious shoots.
  • Such transformed embryos and/or adventitious shoots may then be cultured in shoot regeneration medium that may contain MS or B5 components and further, may also contain selection agents (for example, kanamycin or glyphosate) with or without plant growth regulators.
  • the regenerated shoots are rooted in B5 medium containing 0.1 mg/l GA 3 .
  • methods are provided for transforming cells from microspore-derived embryos with microprojectiles coated with a DNA construct of interest.
  • Methods are known in the art for producing embryos from Brassica microspores. See Fukuoka et al. (1996) Plant Physiol. 111:39-47 and Keller et al. (1987) Proc. 7 th Int. Rapeseed Congr. (Plant Breeding and Acclimatization Institute, Poznan, Tru) pp. 152-157.
  • the cells comprising such microspore-derived embryos are haploid.
  • whole microspore-derived embryos are bombarded with DNA-coated microprojectiles.
  • the microspore-derived embryos may be greater than 10 days old and approximately greater than 1.5 mm in size.
  • the microspore-derived embryos can be between 11 and 20 days old.
  • the embryos may be globular or heart shaped.
  • the embryos are placed on osmotic medium prior to, during and after bombardment for the same reasons as discussed above. At least one surface of the embryos should be exposed to the path of the bombarded particles (i.e. not in the medium) during bombardment to facilitate entry of the particle and to avoid any sudden influx of medium into the cell.
  • the embryos may be subjected to the osmotic treatment for approximately 4 hours prior to bombardment and for approximately 20 hours (for example, overnight) after bombardment.
  • the osmotic treatment may comprise, for example, a medium containing 17 to 19% sucrose and 1.5% agar, and acts to prevent the cells of the embryos from bursting during and after bombardment.
  • the osmotic treatment may comprise placing the embryos on a petri dish having a wet filter paper. The embryos are then transferred to regeneration medium.
  • the regeneration media may include, but are not limited to, B5 media, MS-based media (MS salts with organics, 2% (w/v) sucrose, 0.6% (w/v) Sigma agar, pH 5.8).
  • Embryo-derived hypocotyls may be excised and cultured in selection medium to induce transgenic shoots.
  • a microspore-derived embryo gives rise to a single or a few adventitious shoots as a result of growth from the apical meristem or hypocotyl area.
  • Methods of the second and third embodiments can involve adventitious shoot regeneration of the microspore-derived embryos and microspore-derived hypocotyls. Such methods find use in increasing the number of transformed plants recovered from a transformation attempt.
  • adventitious shoot regeneration involves the formation of multiple shoots arising from a microspore-derived embryo.
  • a single microspore-derived embryo can yield multiple transformed shoots from a transformation.
  • all of the transformed shoots that arise from a single microspore-derived embryo are thought to be an independent transformant. That is, each transformed shoot is derived from an independently transformed cell and thus, is genetically distinct. For the purposes of this investigation, however, all multiple events from each embryo were combined.
  • Methods of adventitious shoot regeneration are known in the art. While the methods of the present invention do not depend on a particular method of adventitious shoot regeneration, the methods may involve subjecting the microspore-derived embryos to an effective amount of cytokinin to induce adventitious shoots.
  • adventitious shoot regeneration may be accomplished within less than about 30 days after administering a cytokinin to the microspore-derived embryos.
  • adventitious shoot regeneration may be accomplished within less than about 10 days after administering the cytokinin.
  • the methods of secondary regeneration of the present invention may additionally involve subjecting the microspore-derived embryos to an effective amount of an auxin.
  • an effective amount of a cytokinin is administered, with or without an effective amount of an auxin, to the microspore-derived embryos following bombardment to induce adventitious shoot regeneration.
  • the DNA construct utilized in methods of the second embodiment comprises a selectable marker gene
  • selection may be applied immediately after bombardment or after a period of time of less than 2 day to about 30 days.
  • Selection may be applied by subjecting the microspore-derived embryos to an effective amount of an appropriate selective agent for the selectable marker gene of the DNA construct of interest.
  • An effective amount of the selective agent may be added to the medium on which the microspore-derived embryo is cultured.
  • the selective agent may be administered alone or in combination with one or more other compounds such as a chromosome-doubling agent or a plant growth regulator.
  • microspores are isolated and cultured as is known to those skilled in the art. Approximately 21 days after culture, when the embryos are generally torpedo shaped, the culture medium is diluted with fresh culture medium and the microspores are allowed to culture for approximately 5 more days. The majority of the embryos are generally at the cotyledon stage after 26 days in culture. Hypocotyl sections from the embryos are excised and cultured for a period of time on medium supplemented with plant growth regulators to induce cell division. For example, the excised hypocotyls may be cultured overnight on MMW + 4 mg/l BAP + 0.25 mg/l NAA.
  • the excised hypocotyls Prior to bombardment, during bombardment and after bombardment, the excised hypocotyls are subjected to osmotic treatment as described above.
  • a surface of the hypocotyls in direct line with the bombardment route is exposed to the path of the bombarding particle (i.e. the surface of the hypocotyls that is in the direct line with the bombardment route is not embedded in the medium) to facilitate entry of the particle coated with DNA and to avoid any sudden influx of medium.
  • the osmotic medium may comprise 17 to 19% sucrose, 1.5% agar and 1g/l MES.
  • the hypocotyls may be cultured on the osmotic medium for 4 hours prior to bombardment and overnight after bombardment.
  • hypocotyls may be transferred to bud induction medium (for example, MMW + 4 mg/l BAP + 25-100 mg/l KAN).
  • a bud can be an immature shoot, leaf, embryo or flower.
  • Hypocotyls comprising adventitious buds may then be transferred to shoot regeneration medium (for example, MMW without hormones, or MMW + 0.2 mg/l BAP).
  • a selection agent may be added to any of the media after bombardment.
  • the methods of the first, second, and third embodiments may comprise administering an effective amount of a chromosome doubling agent to the culture medium before, or optionally after, bombardment.
  • a chromosome doubling agent is not necessary in all cases, because the rate of spontaneous doubling can be high, especially in the embodiments employing microspore-derived embryos and microspore-derived hypocotyls.
  • Such chromosome-doubling agents and methods of use are known to those skilled in the art and were discussed above.
  • the methods of the present invention involve the use of plant growth regulators such as, for example, auxins and cytokinins.
  • plant growth regulators of the invention include, but are not limited to, both free and conjugated forms of naturally occurring plant growth regulators. Additionally, the plant growth regulators of the invention encompass synthetic analogues and precursors of such naturally occurring plant growth regulators.
  • auxins include, but are not limited to, indoleacetic acid (IAA), 3-indolebutyric acid (IBA), a-napthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy) butyric acid, 2,4,5-trichlorophenoxyacetic acid (2,4, 5-T), (4-chloro-2-methylphenoxy) acetic acid (MCPA), 4-(4-chloro-2-methylphenoxy) butanoic acid (MCPB), mecoprop, dicloprop, quinclorac, picloram, triclopyr, clopyralid, fluroxypyr and dicamba.
  • IAA indoleacetic acid
  • IBA 3-indolebutyric acid
  • NAA a-napthaleneacetic acid
  • 2,4-D 2,4-dichlorophenoxyacetic acid
  • MCPA 4-(2,4-dichlorophenoxy
  • Naturally occurring and synthetic analogues of cytokinins include, but are not limited to, kinetin, thidiazuron (TDZ), zeatin, zeatin riboside, zeatin riboside phosphate, dihydrozeatin, isopentyl adenine and 6-benzyladenine (BAP).
  • the methods of the present invention may include use of G418 disulfate salt (GibcoTM), also sold as GeneticinTM from Fluka as a selection agent or glyphosate as a selection agent. After bombardment of pre-incubated microspores, selection may be done in liquid medium in dark first and then under low light intensity (for example, approximately 240 foot-candles or 2,583 lux). However, other selection agents, as is known to those skilled in the art, can be used. For example, kanamycin (Beck et al. (1982) Gene 19:327-336; Mazodier et al. (1985) Nucleic
  • Stable transgenic plants may be confirmed by polymerase chain reaction (PCR) analysis and Southern blot hybridization analysis.
  • PCR polymerase chain reaction
  • DNA constructs are not intended to limit the present invention to nucleotide constructs comprising DNA.
  • nucleotide constructs particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein.
  • the DNA constructs of the present invention encompass all nucleotide constructs which can be employed in the methods of the present invention for transforming Brassica plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • the DNA constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single- stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
  • the methods of the invention may employ a DNA construct that is capable of directing, in a transformed plant, the expression of at least one protein, or at least one RNA, such as, for example, an rRNA, a tRNA or an antisense RNA that is complementary to at least a portion of an mRNA.
  • a DNA construct is comprised of a coding sequence for a protein or an RNA operably linked to 5' and 3' transcriptional regulatory regions.
  • the methods of the invention may employ a DNA construct "that is not capable of directing, in a transformed plant, the expression of a protein or RNA.
  • methods of the present invention do not depend on the incorporation of the entire DNA construct into the genome, only that the genome of the Brassica plant is altered as a result of the introduction of the DNA construct into a Brassica cell.
  • alterations to the genome include additions, deletions and substitution of nucleotides in the genome. While the methods of the present invention do not depend on additions, deletions, or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions or substitutions comprise at least one nucleotide.
  • the DNA constructs of the invention also encompass nucleotide constructs, that may be employed in methods for altering or mutating a genomic nucleotide sequence in an organism, including, but not limited to, chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self- complementary chimeric oligonucleotides and recombinogenic oligonucleobases.
  • nucleotide constructs and methods of use such as, for example, chimeraplasty, are known in the art.
  • Chimeraplasty involves the use of such nucleotide constructs to introduce site-specific changes into the sequence of genomic DNA within an organism. See, U.S. Patent Nos.
  • DNA-coated microprojectiles used herein is not intended to limit the methods of the present invention to microprojectiles coated with DNA. Rather, the term “DNA-coated microprojectiles” as used herein encompasses microprojectiles coated with any one or more of the DNA constructs of the invention as described supra.
  • the DNA constructs of the invention may be comprised of expression cassettes for expression in the Brassica plant of interest.
  • the expression cassette may include 5' and 3' regulatory sequences operably linked to a gene of interest.
  • operably linked is intended a functional linkage between a regulatory sequence and a second sequence, wherein the regulatory sequence affects initiation and mediation of transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette may include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a gene of interest and a transcriptional and translational termination region functional in plants.
  • the transcriptional initiation region, the promoter may be native (or analogous) or foreign (or heterologous) to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the native promoter sequences may be used. Such constructs would change expression levels of the gene of the interest in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) MoI. Gen. Genet. 262:141-144; Proudfoot (1991) Ce// 64:671 -674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Ce// 2:1261-1272; Munroe et al. (1990) Gene 91 :151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant- preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831 , and 5,436,391 , and Murray et al. (1989) Nucleic Acids Res. 17:477-498.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the gene can undergo gene shuffling to enhance expression. For example, the glyphosate acetyl transferase gene used in the examples underwent gene shuffling (Castle et al.
  • the expression cassettes may additionally contain 5'-leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5'-noncoding region) (Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al.
  • Virology 154: 9-20 MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.
  • AMV RNA 4 alfalfa mosaic virus
  • TMV tobacco mosaic virus leader
  • MCMV maize chlorotic mottle virus leader
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in Brassica plants.
  • Such constitutive promoters include, for example, the core promoter of the Rsyn7 (U.S. Patent No. 6,072,050); the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Ce// 2:163-171); ubiquitin (Christensen et al. (1989) Plant MoI. Biol. 12:619- 25 632 and Christensen et al. (1992) Plant MoI. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.
  • MAS Velten et al. (1984) EMBO J. 3:2723-2730
  • ALS promoter U.S. Patent No. 5,659,026), SCP (WO 97/47756A1 , WO 99/438380); H2b (Rasco-Gaunt et al. (2003) Plant Cell Rep. 21 :569-576); SCP1 (US Patent No. 6,677,503 B1) and the like.
  • Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
  • Tissue-preferred promoters can be utilized to target enhanced expression of the gene of interest within a particular plant tissue.
  • Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hanson et a1. (1997) MoI. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331 -1341 ; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.
  • seed-germinating promoters examples include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-l-phosphate synthase); and celA (cellulose synthase) (see U.S. Patent No. 6,225,529).
  • seed-specific promoters include, but are not limited to, bean ⁇ -phaseolin (Chandrasekharan et al., 2003 Plant J. 33: 853-866), napin, ⁇ -conglycinin (Chamberland et al. 1992 Plant MoI. Biol. 19: 937-949), soybean lectin, cruciferin, and the like.
  • Various changes in phenotype are of interest including modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, enhancing tolerance to abiotic stress, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant. These changes result in a change in phenotype of the transformed plant. Genes or nucleotide sequences of interest are reflective of the commercial markets and interests of those involved in the development of the crop.
  • genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products.
  • Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like.
  • Agronomically important traits such as oil, starch, and protein content can be genetically altered by methods of the invention in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch.
  • Hordothionin protein modifications are described in U.S. Patent Nos. 5,990,389, 5,885,801 , 5,885,802, and 5,703,409. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Patent No. 5,850,801 , and the chymotrypsin inhibitor from barley, described in Williamson et al. (1987) Bur. J. Biochem. 165:99-106
  • Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide.
  • the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, WO 98/20133.
  • Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Illinois), pp. 497-502); corn (Pedersen et al. (1986) J. Biol. Chem.
  • Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European corn borer, and the like.
  • Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881 ; and Geiser et a1. (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant MoI. Biol. 24:825; Ahman et al. 2000 WO0001223); and the like.
  • Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Patent No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); and the like.
  • Genes conferring resistance to Sclerotica have been introduced into sunflower and Brassica (U.S. Patent No. 6,441 ,275 B1).
  • An endochitinase gene under a constitutive promoter was introduced into canola ⁇ Brassica napus var. oleifera) inbred line.
  • Progeny from the transformed plants were challenged using three different fungal pathogens (Cylindrosporium concentricum, Phoma lingam, Sclerotinia sclerotiorum) in field trials.
  • the plants exhibited an increased tolerance to disease as compared with the nontransgenic parental plants (Grison et al. (1996) Nature Biotechnology 14: 643-646). Additional disease resistance genes are discussed in Stewart and Broadway, 2005 (US6927322); Salmeron et al. 2003 (US6528702) and Chye and Zhao 2002 (US20030097682).
  • Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or Basta ® (e.g., the bar gene), or other such genes known in the art.
  • ALS acetolactate synthase
  • ALS sulfonylurea-type herbicides
  • glutamine synthase such as phosphinothricin or Basta ® (e.g., the bar gene)
  • the ALS-gene mutants encode resistance to the herbicide chlorsulfuron (Swanson et al (1989) Theor Appl Genet 78:525-530, EP0257993 B1).
  • the glyphosate acetyl transferase (GAT) gene confers resistance to glyphosate (Castle et al. (2004) Science 304:1151-1154).
  • Sterility genes can also be encoded in an expression cassette and provide an alternative to physical emasculation. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as avidin and streptavidin, described in U.S. Patent No.
  • the quality of seed is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose.
  • traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose.
  • U.S. Patent Nos. 5,990,389, 5,885,801 , 5,885,802, and 5,703,409 provide descriptions of modifications of proteins for desired purposes.
  • Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like.
  • the level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
  • a DNA construct of the present invention may comprise an antisense construction complementary to at least a portion of a messenger RNA (mRNA) of a gene of interest. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA.
  • mRNA messenger RNA
  • antisense constructions having 70%, 80%, or 85% or more sequence identity to the complementary sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used. Typically, such antisense constructions will be operably linked to a promoter that drives expression in a plant.
  • the DNA constructs of the invention may also be employed in sense suppression methods to suppress the expression of endogenous genes in plants.
  • Methods for suppressing gene expression in plants using nucleotide sequences in the sense orientation are known in the art.
  • the methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene.
  • a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, for example, greater than about 65%, 85% or 95% sequence identity. See, U.S. Patent Nos. 5,283,184 and 5,034,323.
  • the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase Il (NPTII), and hygromycin phosphotransferase (HPT). Genes conferring resistance to herbicidal compounds may also be used, such as glyphosate acetyl transferase, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech.
  • the expression cassette may comprise a screenable marker gene, for example the gene encoding ⁇ -glucuronidase (GUS) (Jefferson et al. (1986) Proc. Natl. Acad. Sci. USA 83:8447-8451 ; Jefferson et al. (1987) EMBO J. 6:3901-3907) or the gene encoding the green fluorescent protein (GFP) (Chalfie et al. (1994) Science 263: 802-805).
  • GUS ⁇ -glucuronidase
  • GFP green fluorescent protein
  • Brassica plants of the invention include, but are not limited to, Brassica carnata (Ethiopian mustard), Brassica juncea (leaf mustard), Brassica napus (rape), Brassica napus var. rapifera (Swedish turnip), Brassica nigra (black mustard), Brassica oleracea, Brassica oleracea var. acephala (kale), Brassica oleracea var. alboglabra (Chinese kale), Brassica oleracea var.
  • the Brassica plants of the invention are oilseed Brassica plants.
  • oilseed Brassica plants are used for oil production and include but are not limited to, Brassica juncea, Brassica napus and Brassica rapa.
  • the Brassica plants may be canola plants.
  • Such canola plants are selections of oilseed Brassica plants (Brassica rapa, Brassica napus and Brassica juncea) that contain low levels of both erucic acid and glucosinolates in their seeds.
  • Canola oil must contain less than 2% erucic acid and the solid component of the seed must contain less than 30 micromoles of any one or any mixture of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, and 2- hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil free solid.
  • the seeds of such canola plants are favored for the extraction of edible oils.
  • microspore-derived embryos were isolated as is known to those skilled in the art, for example, see Swanson et al. 1987 and cultured in NLN medium (see Section entitled "Media Recipes" for components of all media used in this invention) for approximately 11 to 14 days. However, the microspore-derived embryos can be cultured for between 11 to 20 days. The embryos were produced from the microspores and were detectable with the naked eye. The size of the microspore-derived embryos was generally smaller than 1 mm and the embryos were globular or heart shaped. The microspore-derived embryos were collected to enrich for viable embryos.
  • this may be done using a pipette and transferring the embryos to a filter paper or membrane, for example GelmanTM membrane (Prod. No. 60110).
  • the filter paper or membrane can have a O. ⁇ m pore size.
  • the embryos and membrane were cultured on osmotic medium for example, the medium may contain 17 to 19% sucrose + 0.8 to 1 % agar + 1 g/l MES, pH 6.0.
  • the embryos were subjected to the osmotic treatment before, during and after bombardment. For example, the embryos were subjected to the osmotic treatment for 4 hours prior to bombardment.
  • the DNA construct used in bombardments was PHP18644. PHP18644, and other vectors used are described in the Table 9.
  • the bombardment was done as is known to those skilled in the art, using approximately 10 ng to 5 ⁇ g of DNA per preparation, 15 ⁇ g to 300 ⁇ g of gold particles of approximately 0.4-1.0 micron per shot, CaCb at 0.5M to 2.5M and a rupture disk of 650, 900 or 1100 psi. After bombardment, the embryos were cultured on the osmotic medium for 4 hours to 20 hours (approximately overnight) and then transferred to liquid NLN medium. The bombarded embryos were subsequently cultured for 7 to 14 days.
  • the bombarded embryos or hypocotyls excised from the embryos at age 3 to 4 weeks were transferred from liquid NLN medium to solid selection medium MMW + IAA + TDZ + K25-50 (embryos) or MMW + BAP + K25- 50 (hypocotyls) for bud induction.
  • the resistant regenerated buds were excised and cultured on MMW-H + K50-100 for plant regeneration.
  • the objective was to screen different rupture disks to determine those that would result in the highest transformation efficiency.
  • Part A Fourteen day old microspore-derived embryos were pre-incubated for 4 hours on osmotic medium containing 17% sucrose, 1 g/l MES and 10 g/l agar with pH 6.0. The microspore-derived embryos were bombarded with PHP18644 precipitated on gold particles (100 ⁇ g/shot). The bombarded embryos were cultured for 4 hours after bombardment on the osmotic medium containing 17% sucrose, 1 g/l MES and 10 g/l agar with pH 6.0 and then cultured in NLN medium.
  • Rupture disks of 450 psi, 650 psi and 900 psi were tested.
  • Transient transformation efficiency was determined by analyzing the bombarded embryos using the GUS assay, as is known to those skilled in the art.
  • Table 1 shows the results of the GUS assay.
  • Rupture disk 900 psi produced the greatest number of cells expressing GUS in this experiment.
  • Part B The construct used was PHP18644. Bombarded small embryos were cultured in NLN-13S for 2-3 weeks. The embryos or excised hypocotyls from the embryos were cultured in bud regeneration medium MMW + IAA +TDZ + kanamycin (50 mg/l) (embryos) or MMW + BAP + Kanamycin (50 mg/l) (for hypocotyls).
  • Table 2 shows the results of resistant shoot formation using different rupture disk strengths. In part B of the experiment, no significant difference was found using the 650 psi, 900 psi or 1100 psi rupture disks. The 650 and 900 psi rupture disks were easiest to use because less time was required to achieve the pressure to rupture the disks. Accordingly, the 650 psi and 900 psi rupture disks were used in later experiments. Table 2 also shows that excising hypocotyl segments from the bombarded embryos results in resistant shoot formation.
  • the objective was to demonstrate that kanamycin resistant plants were produced from bombarded microspore-derived embryos.
  • the construct used was PHPi 8644 and the cultivar was 46A65.
  • Kanamycin resistant shoots were produced by bombardment of microspore-derived embryos. The shoots were regenerated into kanamycin resistant plants.
  • the kanamycin resistant plants were analyzed using the REDExtract-N-AMPTM plant PCR kit from Sigma as is known to those skilled in the art. DNA was extracted from 14 kanamycin resistant plants and analyzed by PCR for the nptll gene. Table 3 indicates that the nptll gene was found in 12 of the 14 plants. Accordingly, stable transgenic plants were obtained by bombarding microspore-derived embryos.
  • Microspore-derived hypocotyls were isolated as is known to those skilled in the art and cultured in NLN medium for 21 to 28 days (Swanson et al., 1987). Hypocotyls were excised from the embryos produced from the microspores when the embryos were approximately 3-5 mm in size.
  • Microspores of cultivar 46A65 were cultured in NLN medium for approximately 21 days. On the 21st day, the NLN medium was changed and diluted (1 :20) with fresh NLN medium and the embryos were cultured for 5 more days. The hypocotyls were excised from the embryos and preconditioned overnight on MMW + BAP (4 mg/l) + NAA (0.25 mg/l). The hypocotyls were transferred to osmotic medium (for example, 17% sucrose + 1 % agar + 1 g/l MES, pH 6.0) for 4 hours and then bombarded. The DNA construct used in bombardments was PHP18644.
  • hypocotyls were cultured on the osmotic medium for approximately between 4 and 20 hours (for example, overnight).
  • the bombarded hypocotyis were then cultured on MMVV + BAP (4 mg/l) + kanamycin (25-50 mg/l) for bud induction.
  • Regenerated buds were cultured on MMW + kanamycin (50-100 mg/l) or MMW + BAP (0.2 mg/l) + kanamycin (50-100 mg/l) for plant regeneration.
  • the objective was to find the appropriate concentration of sucrose in the osmotic medium (sucrose + 1 % agar + 1 g/l MES, pH 6.0) in order to produce the highest transformation efficiency.
  • sucrose concentrations of 15, 17, 19 and 21 % were tested as shown in Table 4. The results indicated that using 19% sucrose produced the greatest number of transiently transformed cells.
  • the objective was to determine the optimal amount of gold particles per bombardment.
  • the construct used was PHP18644.
  • Table 5 shows the results using 100, 200 and 300 ⁇ g of gold per shot. The results were not consistent in three experiments. 100 ⁇ g gold particles per shot were used in the remainder of the experiments. Table 5. Effect of the amount of gold particle per bombardment on GUS transient expression using microspore-derived hypocotyls
  • the objective was to determine which of four rupture disk strengths tested produced the greatest number of cells expressing GUS.
  • the construct used was PHP18644.
  • Table 6 shows the results of GUS transient expression using a rupture disk strength of 450 psi, 650 psi, 900 psi or 1100 psi. Results indicate that using rupture disks 650 psi and 900 psi produced the highest number of cells transiently expressing GUS.
  • the objective was to screen rupture disks to determine those that produced the greatest number of resistant buds on 50 mg/l kanamycin.
  • Tabie 8 shows that seven kanamycin resistant plants were analyzed by GUS assay. Six plants were positive. This confirms that stable transgenic plants were produced by bombarding microspore-derived hypocotyls.
  • Experiments 8 to 17 describe the work done using pre-incubated microspores.
  • the microspores were cultured in NLN-17S/10S for 2-10 days. Optimally, the microspores are cultured for 5-7 days. Embryos could not be detected with the naked eye.
  • the embryogenic microspores were collected so that they remain viable and embryogenic with a NitexTM sieve (15-36 urn in pore size) or Percoll ® (35-45%) gradient centrifugation.
  • Pre-incubated microspores were used as bombarded materials. Any Brassica line that is capable of regenerating by microspore culture can be used. The microspores were cultured for 1-3 days in NLN-17S at 31.5 0 C, and then in NLN-10S for 4-5 days at 25 0 C.
  • the constructs used in the bombardments were PHP18644, PHP21965, PHP22024, PHP22021 , and PHP23560 (see Table 9).
  • the constructs can be the full plasmid or the expression cassette only. For example, PHP22024 can be either the expression cassette or the full plasmid.
  • the pre-incubated microspores were filtrated with sieves of pore size of 15 ⁇ m to 36 ⁇ m.
  • microspores were used for bombardment.
  • the microspores were loaded on a sieve 15 ⁇ m or 20 ⁇ m on two layers of filter-paper and dried for less than one minute.
  • the microspores and sieves were transferred to osmotic medium that contained B5 components, 1 g/l MES and 0.8-1.6% gelrite, 15-21 % sucrose (pH S. C).
  • Th ⁇ microspores v B 6 ⁇ 6 treated for at least OU ⁇ S hour on the osmotic medium before bombardment.
  • the pre-incubated microspores were bombarded with 12.5 ng to 5 ⁇ g DNA per preparation, 15-100 ⁇ g Au particles per shot, 2.5 M CaCI2, and 650-900 psi rupture disk.
  • a surface of the pre- incubated microspores was exposed to the path of the bombarded particles coated with DNA (i.e. the surface was not embedded in the medium).
  • the bombarded microspores were cultured at least four hours in the osmotic medium after bombardment.
  • One to two sieves holding the microspores were cultured in 5 ml of NLN-13S with or without glyphosate per plate for approximately 7 days in the dark. After the 7 days, the spent medium was replaced with 10 ml of NLN-6.5S in each plate or the spent medium was diluted to obtain a sucrose concentration 6.5% in each plate. If the selectable marker gene was the NPTII gene, NAA, BAP and G418 were added to the medium.
  • the selectable marker gene was GAT
  • glyphosate was added in the medium.
  • the embryos were cultured under dim light of approximately 240 foot-candles or 2,583 lux.
  • the final concentration of G418 was 10 mg/l.
  • the concentration of glyphosate was 0.1 mM or 0.2 mM.
  • a doubling agent was optionally added to the medium. Resistant embryos were recorded after two to three weeks of culture.
  • PHP18644 and GAT (glyphosate acetyltransferase) constructs were used in transformation experiments using pre-incubated microspores.
  • the GAT gene was isolated from a bacterium as described by Castle et al. (2004) Science 304: 1151-1154. The gene was shuffled 11 rounds for increasing expression level of glyphosate acetyltransferase. There was one to several variants in each shuffling round.
  • PHP18644 also contains the GUS marker gene. The constructs are described in Table 9.
  • the objective was to determine the concentration of G418 that kills non- transgenic microspores. Prior to this experiment, selection was done on solid medium, not in liquid medium. Using liquid selection for Brassica transgenic cells is novel. Selection in liquid medium is advantageous for at least the following reasons: (a) it allows selection at an early stage, thereby eliminating the need for subsequent transfers of explants, (b) it allows for a cleaner selection because the explants are generally smaller when they are in liquid medium and the liquid allows all the surfaces of the explant to be exposed to the selection agent, (c) it may reduce the frequency of chimeras, and (d) a lower amount of the selection agent is needed therefore reducing the toxicity to the researcher and the environment.
  • Experiment 9 Production of G418 and glyphosate resistant tissue and buds after bombardment of pre-incubated microspores followed by selection in liquid medium containing G418 or glyphosate.
  • the objective was to obtain transgenic plants by bombarding pre-incubated microspores that were cultured for up to 11 days and selecting resistant tissue and buds on liquid medium supplemented with 10 mg/l G418 or glyphosate at 0.1mM and 0.2mM.
  • seven, eight, nine and eleven day old pre- incubated microspores were bombarded with PHP18644.
  • NAA 0.5 mg/l
  • BAP 0.05 mg/l
  • Resistant microspores were transferred and cultured in the selection medium MMW+IAA+TDZ+AgNO 3 5+C+K100 to induce resistant buds and confirm resistance. Accordingly, selection was initiated in the liquid culture medium with G418 and completed in the solid culture medium using kanamycin.
  • Table 11 shows the results of the experiments. The data indicate that bombardment of pre-incubated microspores followed by selection in liquid medium using G418 or glyphosate produces resistant buds and PCR positive shoots. Table 11. Effect of the pre-incubation period on resistant bud production
  • the objective was to determine whether culturing bombarded tissue on osmotic medium after bombardment would increase transformation efficiency.
  • Table 12 indicates that bombarded pre-incubated microspores cultured on osmotic medium for 4 hours after bombardment produced a greater number of transgenic sectors than bombarded pre-incubated microspores that were not cultured on osmotic medium soon after bombardment. After the osmotic treatment, the bombarded pre-incubated microspores were cultured on liquid NLN medium.
  • the objective was to confirm that selection in liquid medium produces resistant buds.
  • nptll gene was confirmed in resistant plants using PCR analysis. Eight plants were selected at random and analyzed, six were found to have the nptll gene (Table 14).
  • the purpose of this experiment was to find the optimal concentration of sucrose in the osmotic medium.
  • Microspores were cultured for 4-7 days using 17S/10S protocol.
  • the construct used was PHP23560 and the concentration of DNA was 28 ng/preparation.
  • the rupture disk was 900 psi.
  • the bombarded microspores were cultured in the dark for 7-10 days in NLN-13S containing a doubling agent and 0.2 mM Glyphosate.
  • the culture was diluted with NLN-OS without glyphosate to NLN- 6.5S containing 0.1 mM glyphosate.
  • the diluted cultures were incubated in the light for 2-3 weeks. There was no significant difference in the number of green embryos (normal and abnormal) produced using 15%, 17% and 19% sucrose.
  • the objective was to determine whether a 4 hour or 20 hour culture in osmotic medium results in a greater number of transgenic events.
  • the construct used was PHP21965. Microspores were pre-incubated for 6 to 7 days, and then collected with 25 uM NitexTM sieve. The collected microspores were subsequently loaded onto NitexTM sieves with pore size of 20 uM. The bombarded microspores and NitexTM sieves were cultured for 4 hours or 20 hours on osmotic medium B5 + 1 g/l MES + 190 g/l sucrose + 12 g/l gelrite (pH 5.8-6.0) after bombardments. Table 17 shows that treating microspores for 4 hours produced a comparable number of resistant embryos to treating microspores for 20 hours.
  • Transgenic (T1) plants identified by glyphosate resistance were analyzed using Southern blot hybridization analysis.
  • Plant genomic DNA was extracted using cetyltrimethylammonium bromide (CTAB) buffer (Rogers et al., (1994) Plant Molecular Biology Manual, 2 nd Ed. 1 :1-8.).
  • CTAB cetyltrimethylammonium bromide
  • the DNA samples were digested with Bam HI or Pst I.
  • the hybridization probe was the GAT gene.
  • the hybridization was made following Rajasekaran et al. (2000) Plant Cell Rep. 19: 539-545.
  • the GAT gene copy number was determined by the highest number of bands from the hybridization blots for each digest.
  • Microspores are cultured as is known to those skilled in the art, for example see Fukuoka et al. (1996) Plant Physiol. 111:39-47; Keller et al. (1987) Proc. 7 th Int.
  • Rapeseed Congr. Plant Breeding and Acclimatization Institute, Poznan, Tru) pp. 152-157, Swanson et al. (1987) Plant Cell Reports 6: 94-97 and Baillie et al.
  • Decant B5-W add 45 ml B5-W, centrifuge, decant, add 45 ml B5-W, centrifuge and repeat for a total of 4 washes.
  • adjust microspore density to 100,000 microspores per ml with NLN-17S using a heamocytometer.
  • Gold particle amount per shot is 100 ⁇ g/shot (3 mg/30 shots).
  • 3 pg DNA/bp/prep plasmid DNA 1 50 ⁇ l CaCI2 (2.5 M, aliquoted into small volumes), 20 ⁇ l spermidine (0.1 M, base-free, aliquoted into small volumes) to a tube of gold particle aliquot. Pipette 30-50 times after each addition. Shake for 3 minutes on vortex shaker. Centrifuge for 10 seconds at 10,000 rpm and discard supernatant. Gently add 200 ⁇ l 100% EtOH and set in ice for 10 minutes and discard supernatant. Wash gently with 200 ⁇ l 100% EtOH twice and discard washes. Add 150 ⁇ l 100% EtOH.
  • the microcarrier launch assembly parts, the macrocarrier holders, the rupture retaining cap, macroccarrier, petri dish holder and the stopping screens can be sterilized either by soaking (or spraying) in 70% EtOH for 15 minutes and drying in the laminar flow cabinet, or by autoclaving.
  • the rupture disks should be sterilized in 50% iso-propanol for 10-30 seconds. Sterilize the chamber by spraying 70% EtOH. Build up helium pressure higher than rupture disk.
  • Medium contains no selection agent (nptll selection) or 0.1-0.2 mM glyphosate (GAT selection). Each plate (9 cm) contains 5 ml medium and one piece of microspores/Nitex. Culture the bombarded microspores for 7 days at 25C in dark. Replace medium with 10 ml of NLN-6.5S with final concentration of NAA0.5BAP0.05 + G418 (10 mg/l) if using the nptll gene as selectable marker or add 5 ml of NLN-OS with Glyphosate 0.1-0.2 mM if using the gat gene as selectable marker. Culture under light for 2 weeks.
  • Plant regeneration Culture green embryos or tissue in MMW+IAA2+TDZ0.5+STS6 with 25 mg/l kanamycin or 0.1 mM glyphosate for 4 weeks (STS6 is silver thiosulfate at concentration of 6 ⁇ M). Isolate regenerated buds and culture in MMW+BAP0.2 or B5+GA with 50 mg/l kanamycin or 0.1 mM glyphosate. Excise shoots and transfer to rooting medium 1/2MMW+1 %sucrose+IBA2 with 25 mg/l kanamycin or B5+GA with 0.1 mM glyphosate.
  • GUS assay GUS analysis is known to those skilled in the art.
  • the protocol can be found in numerous references, for example Wu H, McCormac AC, Elliott MC, Chen DF (1998) Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare). Plant Cell Tissue and Organ Culture 54:161-171. PCR analysis of the GAT and NPTII genes
  • PCR analysis is known to those skilled in the art. The protocol can be found in numerous references. For example, PCR analysis of the nptll gene was done according to Broothaerts W, Wiersma PA, Lane WD (2001) Multiplex PCR combining transgene and S-allele control primers to simultaneously confirm cultivar identity and transformation in apple. Plant Cell Rep 20:349-353.
  • Plant DNA was extracted following SigmaTM Technical Bulletin Code MB-850 and using REDExtract-N-AmpTM Plant PCR kit.
  • the temperature cycle was 95 0 C, 2 min; (94 0 C, 30 s; 64 0 C, 30 s; 72 0 C, 30 s) for 35 cycles; 75 0 C, 5' for the GAT gene amplification, or 95 0 C, 2 min; (95 0 C, 15 s; 6O 0 C, 30 s; 72 0 C, 30 s) for 35 cycles; 75 0 C, 5' for the nptll gene.
  • GAT4604 and 4618 317 bp; GAT4621 : 255 bp; NPTII: 700 bp.
  • Southern blot hybridization analysis Southern blot hybridization analysis is known to those skilled in the art. See for example, ( see Rajasekarran et al. (2002) Plant Cell Rep. 19:539-545. Media recipes
  • Components are as Lichter (1982) Z convincedphysiol 105:427-434 without potato broth and plant growth regulators.
  • Medium pH is 6.0.
  • NLN contains 17%, 10% or 6.5% sucrose
  • Rooting medium (1/2MMW +1% sucrose+2 IBA) Half strength MMW Sucrose (10 g/l) IBA (2 mg/l)

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Abstract

L'invention concerne la manipulation génétique de plantes, particulièrement de plantes du genre Brassica. Des procédés sont mis à disposition pour la production de plantes de Brassica transgéniques impliquant l'introduction d'un ADN construit par un bombardement de microprojectiles dans des microspores incubées par avance, des embryons dérivés de microspores et des hypocotyles dérivés de microspores. Les procédés trouvent une utilisation dans le développement de variétés agricoles améliorées de plantes de Brassica grâce à l'incorporation de caractéristiques agronomiques souhaitables.
EP05851482A 2005-11-10 2005-11-10 Transformation de brassica par bombardement de microprojectiles Withdrawn EP1945774A1 (fr)

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CA2629284A1 (fr) 2007-05-18
EA200801303A1 (ru) 2009-02-27
WO2007055687A1 (fr) 2007-05-18

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