WO2001013707A1 - Procede perfectionne servant a la transformation et a la regeneration des plantes - Google Patents

Procede perfectionne servant a la transformation et a la regeneration des plantes Download PDF

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Publication number
WO2001013707A1
WO2001013707A1 PCT/CA2000/000965 CA0000965W WO0113707A1 WO 2001013707 A1 WO2001013707 A1 WO 2001013707A1 CA 0000965 W CA0000965 W CA 0000965W WO 0113707 A1 WO0113707 A1 WO 0113707A1
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Prior art keywords
plant
transformation
concentration
cytokinin
agrobacterium
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PCT/CA2000/000965
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English (en)
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Massimo Sanago
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University Of Guelph
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    • 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
    • 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
    • A01H4/008—Methods for regeneration to complete plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—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)
    • C12N15/8201—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

  • Plants FIELD OF THE INVENTION The present invention is in the field of plant transformation and regeneration.
  • an improved method for the transformation and regeneration of transgenic plants using either high velocity microprojectiles or Agrobacterium-mediated transformation BACKGROUND OF THE INVENTION: Development of procedures to regenerate plants from single cells and organized tissue and the discovery of novel techniques to transfer genes into plant cells has provided the prerequisite for the practical use of genetic engineering in crop improvement. These advances have allowed the creation, characterization and selection of plant cultivars which could not be obtained by traditional breeding methods.
  • transformation has been difficult to achieve.
  • very few efficient protocols are presently available for the transformation of plants such as legumes (for example, pea and soybean), alfalfa and canola.
  • methods are available for the transformation of these plant species, transformation rates are typically less than 1% and the time required to obtain transgenic plants is about 1 year.
  • Characteristics of an ideal efficient transformation method include: 1) a rapid recovery of large numbers of fertile transgenic plants; 2) little or no callus which might induce genetic variation; and 3) little dependence on genotype.
  • the present invention provides an improved method for the transformation and regeneration of transgenic plants using either high velocity microprojectiles or Agrobacterium-mediated transformation.
  • the present invention provides a highly efficient method for the transformation and regeneration of plants.
  • a method (A) for the transformation and regeneration of a plant comprising the steps of:
  • the invention is also directed to the method (A) as defined above, wherein the insertion of a heterologous gene of interest within a vector into the explant tissue is performed using any suitable transformation technique, for example high velocity microprojectiles and Agrobacterium-mediated transformation.
  • the invention is also related to the method (A) as defined above, wherein the seeds are germinated on culture medium comprising a high concentration of one or more cytokinins for a period of about 3 days to about 10 days.
  • the present invention also relates to a method (A) as defined above, wherein the plant may be any plant of interest, in particular legumes such as pea (Pisum s ⁇ tivum) and soybean (Glycine max), alfalfa (Medicago sativa) and canola (Brassica napus).
  • the plant is a dicotyledon.
  • the present invention also involves a method (A) as defined above, wherein the explant tissue from the germinated seed comprises tissue selected from the group consisting of cotyledons, hypocotyls and epicotyls.
  • a method (B) for the transformation and regeneration of a plant using Agrobacterium-mediated transformation comprising the steps of:
  • the present invention also relates to a method (B) as defined above wherein the phenolic compound is acetosyringone.
  • the invention is also related to the method (B) as defined above, wherein the seeds are germinated on tissue culture medium comprising a high concentration of one or more cytokinins for a period of about 3 days to about 10 days.
  • the present invention also relates to a method (B) as defined above, wherein the plant may be any plant of interest, in particular legumes such pea (Pisum sativum) and soybean (Glycine max), alfalfa (Medicago sativa) and canola (Brassica napus).
  • the plant is a dicotyledon.
  • the present invention also involves a method (B) as defined above, wherein the explant tissue from the germinated seed comprises tissue selected from the group consisting of cotyledons, hypocotyls and epicotyls.
  • the method of the invention involves the use of plant material derived from mature seed. Use of different explants such as cotyledons, epicotyls, leaves and hypocotyls as the plant material to be transformed is advantageous because these tissues are highly regenerable and produce a large number of shoots when placed on regeneration medium. The highly regenerable nature of these plant materials increases the probability of transformed cells regenerating into transformed plants.
  • the method also involves the use of high concentrations of a cytokinin in the culture medium during germination of the seed.
  • cytokinins such as thidiazuron and benzylaminopurine
  • the present invention also provides a method for transforming and regenerating plants wherein the transformed regenerated shoot is obtained from non-callus material. Avoiding the callus stage decreases the time needed to obtain a transformed plant and reduces the risk of somaclonal variation due to mutations accumulating in the callus due to extended culture time. The method is also genotype independent.
  • the present inventin also includes plants prepared by the methods (A) and (B) of the invention.
  • Figure 1 is a schematic of the pAHC25 vector used in soybean transformation using microprojectile bombardment.
  • Figure 2 is a schematic of the pCAM1301 vector used in canola transformation.
  • Figure 3 is a schematic of the pIG121-HM vector used in soybean Agrobacterium-mediated transformation.
  • Figure 4 is a schematic of the pBISNl vector used in alfalfa transformation with the P02D gene.
  • Figure 5 is a schematic of the pBSINl vector used in alfalfa transformation with the P02 gene.
  • Figure 6 is a schematic of the pBSINl vector used in alfalfa transformation with the GUS gene.
  • Figure 7 is a Western blot showing the expression of the P02D protein in transgenic alfalfa.
  • Lane A is the standard low range molecular weight marker;
  • Lanes B-F are protein isolates from 5 transgenic plants;
  • Lane I is an alfalfa tissue culture control.
  • the present invention provides an improved method for plant transformation and regeneration.
  • the present invention provides an improved method for the transformation and regeneration of transgenic plants using either high velocity microprojectiles or Agrobacterium-mediated transformation.
  • the method of the present invention is directed to all plants of interest, in particular legumes such as pea (Pisum s ⁇ tivum) and soybean (Glycine max), alfalfa (Medicago sativa) and canola (Brassica napus) .
  • the plant is a dicotyledon.
  • Transformation efficieny is increased compared to known literature methods. Increased transformation efficiency is evidenced by two parameters: increased transformation rate and a shorter period of time to obtain primary transformants. Transformation rate refers to the percentage of explants transformed with the heterologous gene of interest.
  • One of the reasons for increased transformation efficiency is that regeneration of plants using the method of the invention does not proceed through a callus stage. Avoiding the callus stage decreases the time needed to obtain a transformed plant and reduces the risk of somaclonal variation due to mutations accumulating in the callus due to extended culture time.
  • plants are transformed and regenerated from mature seeds.
  • Mature seeds are seeds that have developed on the plant to full size, have dried and are capable of germinating independently.
  • the method initially involves the germination of sterile seeds on a germination medium comprising a high concentration of one or more cytokinins.
  • the cytokinin may be selected from any natural or synthetic cytokinin or any growth hormone having cytokinin-like activity, including, but not limited to thidiazuron (TDZ), benzylaminopurine (BAP), zeatin, N-(2-chloro-4-pyridyl)- N'-phenylurea (CPPU) and N6-isopentyladenine (2-z-P).
  • cytokinin is selected from TDZ and BAP.
  • high concentration of cytokinin means a concentration of cytokinin sufficient to result in increased transformation efficiencies. This number will vary depending on the cytokinin and the plant. The concentration of cytokinin is greater than what has been previously used in literature procedures. For example, when transforming and regenerating soybean, a suitable high concentration of cytokinin is about 5-50 ⁇ M TDZ or about 50-70 ⁇ M BAP. When alfalfa is the plant to be transformed and regenerated, a suitable high concentration of cytokinin is about 10-75 ⁇ M TDZ or about 20-70 ⁇ M BAP.
  • cytokinin must be sufficient to increase degradation of plant proteins thereby providing amino acids to the developing embryo.
  • Analysis of the endogenous amino acid pools showed major changes in contents of asparagine, aspartic acid, homoserine and proline in the developing seedling.
  • a higher concentration of cytokinin will result in more degradation and therefore more available amino acids.
  • the concentration of cytokinin must not be so high as to irreversibly harm the plant.
  • a person skilled in the art would be able to determine an optimum concentration of cytokinin for a plant by varying the concentration of cytokinin in the germination medium and observing the effect on transformation efficiency.
  • the germination medium may comprise any incubation medium suitable for the culture of the seed.
  • a medium may include, but is not limited to, basal medium, for example MS
  • sucrose from about 5 g/L to about 50 g/L;
  • At least one amino acid such as, but not limited to, asparagine, aspartic acid, homoserine and proline, from about 5 mg/L to about 300 mg/L;
  • the seeds are cultured on a germination medium comprising a high concentration of cytokinin for a period of about 3 days to about 10 days, preferably about 5 days to about 7 days.
  • a germination medium comprising a high concentration of cytokinin for a period of about 3 days to about 10 days, preferably about 5 days to about 7 days.
  • an explant is obtained from the embryonic axis of the germinated seed.
  • the explant may be selected from the cotyledons, epicotyls, leaves and hypocotyls or, depending on the size of the explant may include all of these tissues.
  • the epicotyl and hypocotyl sections are cut below and above the cotyledons to provide explants approximately 7-9 mm in length with the cotyledons intact. If necessary, the ends of the cotyledons may be cut off.
  • a heterologous gene of interest within a vector is then inserted into the explant using any known technique. Numerous methods have been developed for transferring genes into plant tissues; for example, Agrobacterium-mediated transfer, direct DNA uptake, microinjection, high velocity microprojectiles and electroporation. Preferably the heterologous gene of interest within a vector is inserted into the explant using either high velocity microprojectiles or Agrobacterium-mediated transformation.
  • microprojectile bombardment Procedures for transforming plant cells using high velocity microprojectiles (microprojectile bombardment) are well known. The general process has been described by Klein, et ⁇ l. in Proc. Natl. Acad. Sci. USA, 1988, 85:4305-4309, which is incorporated herein by reference. The basic process includes coating DNA onto small high density particles (microprojectiles) which are then placed into a particle gun or helium gun apparatus and accelerated to a high velocity to penetrate plant cell walls and membranes and carry the DNA or other substance into the interior of the bombarded cell. Procedures have been developed for the transformation of many plants from tissue explants using microprojectile bombardment, including alfalfa (Buising, CM.
  • the explants may be transformed with a heterologous gene of interest in a vector using standard high velocity microprojectiles techniques as described in the above references.
  • Agrobacterium-mediated gene transfer is by far the most widely used gene transfer technique. This method was first described in Murai, N. et ⁇ l. Science, 1983, 222:476-482 and Fraley R.T. et ⁇ l, Proc. Natl. Acad.
  • the explants may be transformed with a heterologous gene of interest within a vector using any of the Agrobacterium-mediated transformation techniques described in the above references. It is another aspect of the present invention to provide a modified method for the insertion of a heterologous gene of interest within a vector into plants using Agrobacterium-mediated transformation. According to this modified method, the explant tissue is exposed to or inoculated with a suspension of Agrobacterium cells comprising a gene of interest (within an optional marker gene) within a vector. According to the method of the invention, the Agrobacterium suspension is also supplemented with a phenolic compound, for example, but not limited to acetosyringone, at a concentration in the range of about
  • the exposure or inoculation of the explant tissue may last from about 1 minute to about 120 minutes, preferably from about 5 minutes to about 60 minutes. Most preferably the inoculation is from about 10 minutes to about 15 minutes, followed by the addition of more bacterial suspension and further incubation for about 10 minutes to about 15 minutes.
  • the Agrobacterium suspension comprises any incubation medium suitable for the culture of the explant.
  • such a medium may include, but is not limited to, basal medium, for example, MS (Murashige and Skoog basal medium, Physiol. Plant, 1962, 15:473-497) further comprising:
  • sucrose from about 5 g/L to about 50 g/L; • at least one amino acid, such as, but not limited to, asparagine, aspartic acid, homoserine and proline, from about 5 mg/L to about 300 mg/L;
  • growth hormones for example, 2,4-D, or its equivalent, from about 1 mg/L to about 50 mg/L, BAP or TDZ, or their equivalent, from about 1 mg/L to about 50 mg/L, or 2,4-D and BAP or TDZ, from about 1 mg/L to about 50 mg/L; and optionally,
  • casein hydrolysate from about 5 mg/L to about 1 g/L, gellan from about 1 g/L to about 3 g/L and dicamba, from about 0.5 mg/L to about 20 mg/L.
  • the explant may be cultivated on co-cultivation medium for a period of about 1 day to about 5 days, suitably about 2 days to about 3 days.
  • Co-cultivation medium comprises any incubation medium suitable for the culture of the explant.
  • a medium may include, but is not limited to, basal medium, for example, MS (Murashige and Skoog basal medium, Physiol. Plant, 1962, 15:473-497) further comprising:
  • sucrose from about 5 g/L to about 50 g/L;
  • At least one amino acid such as, but not limited to, asparagine, aspartic acid, homoserine and proline, from about 5 mg/L to about 300 mg/L;
  • growth hormones for example, TDZ, from about 1 ⁇ M to about 75 ⁇ M or BAP, from about 1 ⁇ M to about 75 ⁇ M.
  • the explants may then be transferred to regeneration medium for regeneration and selection for the occurrence of the selectable marker.
  • the explants may be left on selection medium for a sufficient period of time to allow direct shoots or somatic embryos to form.
  • direct shoots develop within about 1 week to about 4 weeks, preferably about 1 to about 2 weeks.
  • Regeneration medium comprises any incubation medium suitable for the culture of the explant.
  • basal medium for example, MS (Murashige and Skoog basal medium, Physiol. Plant, 1962, 15:473-497) further comprising:
  • sucrose from about 5 g/L to about 50 g/L
  • At least one amino acid such as, but not limited to, a asparagine, aspartic acid, homoserine and proline, from about 5 mg/L to about 300 mg/L;
  • growth hormones for example, TDZ, from about 1 ⁇ M to about 25 ⁇ M or BAP, from about 1 ⁇ M to about 50 ⁇ M;
  • selection agent for example hygromycin, from about 10 mg/L to about 200 mg/L, kanamycin, from about 10 mg/L to about
  • Rooting medium comprises any incubation medium suitable for the culture of the explant.
  • a medium may include, but is not limited to, basal medium, for example, full or 1/2 MS (Murashige and Skoog basal medium, Physiol. Plant, 1962, 15:473-497) further comprising:
  • sucrose from about 5 g/L to about 50 g/L; and • gellan from about 1 g/L to about 3 g/L; • selection agent, for example hygromycin, from about 10 mg/L to about 200 mg/L, kanamycin, from about 10 mg/L to about 500 m/L or basta , from about 10 mg/L to about 400 mg/L;
  • selection agent for example hygromycin, from about 10 mg/L to about 200 mg/L, kanamycin, from about 10 mg/L to about 500 m/L or basta , from about 10 mg/L to about 400 mg/L;
  • antibiotic against Agrobacterium for example claforan, from about 100 mg/L to about 700 mg/L; and optionally
  • growth hormones for example, NAA or its equivalent, from about 1 ⁇ M to about 5 ⁇ M;
  • the shoots may be transferred to an elongation medium, containing no growth hormones after the regeneration medium and before the rooting medium.
  • the shoots may be left on the elongation medium for a period of about 0.5 weeks to about 4 weeks, preferably about 1 to about 2 weeks.
  • Elongation medium comprises any incubation medium suitable for the culture of the explant.
  • basal medium for example, MS (Murashige and Skoog basal medium, Physiol. Plant, 1962, 15:473-497) further comprising:
  • sucrose from about 5 g/L to about 50 g/L
  • gellan from about 1 g/L to about 3 g/L
  • selection agent for example hygromycin, from about 10 mg/L to about 200 mg/L, kanamycin, from about 10 mg/L to about 500 m/L or basta , from about 10 mg/L to about 400 mg/L; and
  • antibiotic against Agrobacterium for Agrobacterium-mediated transformations only, for example claforan, from about 100 mg/L to about 700 mg/L.
  • plants with well developed roots and shoots When plantlets with well developed roots and shoots are obtained, they may be transferred into pots with soil. Using the method of the invention, seeds may be obtained from primary transformants within about 3 to about 6 months, making it possible to obtain R 2 plants in less than a year.
  • Seeds were surface sterilized by immersing them in 95% ethanol for 1 min., then in 1.5% sodium hypochlorite solution with 2 drops of Tween-20 per 100 ml for 20 min. followed by 10 min. rinse in sterile distilled water repeated 3 times. Seedling Culture
  • Seeds were cultured on different germination media at the rate of 5-10 seeds per petri dish, or Magenta box, containing 25 ml to 50 mL of appropriate media. All germination media were supplemented with B-5 vitamins (Gamborg et al 1968) and 3% sucrose and were solidified with 0.3% Gelrite (w/V). The pH was adjusted to 5.7 before autoclaving at 121°C for 20 min. The petri dishes were sealed with parafilm and were incubated in growth room, at 25° 2°C, and a 16 h photoperiod. See Tables 1 and 2 for specific germination conditions for soybean, pea and alfalfa respectively. Preparation of Explants Seeds were germinated on germination media for 3 to 7 days.
  • Seedlings were excised in three sections, epicotyl and hypocotyl sections were cut below and above the cotyledons into 7 or fewer 8 mm long segments and cotyledons remained intact.
  • Bacterial strains and plasmids Agrobacterium tumefaciens, strains EHA105, EHA101 and C58, were used as host for the plasmids. The following plasmids were used:
  • Plasmid pAHC25 containing the uidA and bar genes, each under the control of a maize Ubil promoter see Figure 1 and Thompson, et al. EMBO J. 1987, 6: 2519-2523; Jefferson, et al. EMBO J. 1987, 6:3901-3907; Christensen et al. Plant Mol. Biol. 1992, 18:675-689; Bevan, et al.
  • Plasmid pCAMBIA 1301 containing the uidA and hygromycin genes, each under the control of a CaMV35S promoter (see Figure 2). Plasmid pIG121-Hmcontaining the uidA and hygromycin genes, each under the control of a CaMV35S promoter (see Figure 3 and Akama, K. et al. Plant Cell Rep. 1992, 12:7-11; Hiei, Y. et al. Plant J. 1994, 6:271-282).
  • Plasmid pBISNl-P02D (see Figure 4) containing the P02D [fusion between P02 (Qui, X.. et al.
  • Plasmid pBISNl-P02 (see Figure 5) containing the P02 (Qui, X.. et al. Plant Science, 1997, 124:41-47) and kanamycin genes, each under a control of the polyA-nos promoter.
  • Plasmid pBISNl (see Figure 6) Ni, Min et al. The Plant Journal, 1995, 7:661-676) containing the uidA and kanamycin genes, each under the control of a polyA-nos promoter.
  • Agrobacterium was grown overnight in LB (Luria-Bertani) medium containing the appropriate antibiotics. Bacteria were pelleted at 7000 g for 10 min., washed twice with equal volumes of LB medium, resuspended in liquid MS basal medium and the OD 60 o nm determined.
  • DNA (1 g/L), 20 L spermidine (0.1 ⁇ M), and 50 L CaCl 2 (2.5 ⁇ M) were added and mixed by continuous vortexing during each addition. The final mixture was vortexed for 2-3 min. at room temperature and centrifuged in a microfuge for 30 s. The supernatant was removed and the pellet washed briefly with 70 and then 100% ethanol without disturbing the pellet. The DNA coated particles were resuspended in 46 L of 100% ethanol and 5 L of the suspension was pipetted onto macrocarriers. Bombardment Method
  • the petri plate containing the explants was positioned 6 cm below the microprojectile stopping plate. It was bombarded with the DNA coated particles under 70 cm (3.1 kpa) partial vacuum at a rupture pressure of 900 psi using a PDS-1000 He Biolistic device. After bombardment, all explants were left on the co-cultivation medium without selection for 2 to 3 days. Selection of Transformants
  • Table 5 provides a summary of the 3 separate transformation experiments conducted with three different genotypes. For each experiment 100 seeds were used and 3 different plasmids.

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Abstract

La présente invention concerne un nouveau procédé servant à la transformation et à la régénération de plantes transgéniques par l'intermédiaire de microprojectiles à grande vitesse ou une transformation par Agrobacterium. Le procédé de l'invention ne procède pas par la phase de cal, ce qui permet de réduire le temps nécessaire pour obtenir une plante transformée et réduire le risque de variation somaclonale due à des mutations accumulées dans le cal durant un temps de culture prolongé. Le procédé s'applique à toutes les plantes et permet l'utilisation de tissus tels que des cotylédons, des épicotyles, des feuilles ou des hypocotyles comme explants.
PCT/CA2000/000965 1999-08-20 2000-08-18 Procede perfectionne servant a la transformation et a la regeneration des plantes WO2001013707A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8653326B2 (en) 2003-11-18 2014-02-18 Bayer Cropscience N.V. Targeted DNA insertion in plants

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US5015580A (en) 1987-07-29 1991-05-14 Agracetus Particle-mediated transformation of soybean plants and lines
EP0430511A1 (fr) 1989-11-17 1991-06-05 Agracetus, Inc. Plantes de soja résistantes aux inhibiteurs de la synthétase de glutamine
US5024944A (en) 1986-08-04 1991-06-18 Lubrizol Genetics, Inc. Transformation, somatic embryogenesis and whole plant regeneration method for Glycine species
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5324646A (en) 1992-01-06 1994-06-28 Pioneer Hi-Bred International, Inc. Methods of regeneration of Medicago sativa and expressing foreign DNA in same
US5503998A (en) 1990-02-26 1996-04-02 Agracetus, Inc. Plant transformation process with early identification of germ line transformation events
US5565346A (en) 1988-07-27 1996-10-15 Calgene, Inc. Transformation and regeneration system for legumes

Patent Citations (8)

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Publication number Priority date Publication date Assignee Title
US5024944A (en) 1986-08-04 1991-06-18 Lubrizol Genetics, Inc. Transformation, somatic embryogenesis and whole plant regeneration method for Glycine species
US5015580A (en) 1987-07-29 1991-05-14 Agracetus Particle-mediated transformation of soybean plants and lines
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