Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
1999-01-15
2001-08-14
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C800S278000, C435S468000, C435S419000, C435S430000
Reexamination Certificate
active
06274791
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods of genetically transforming plants and more specifically to genetically transforming strawberry plants. In particular,
Agrobacterium tumefaciens
is used in two protocols to improve transformation efficiencies.
BACKGROUND OF THE INVENTION
Strawberries (
Fragaria X ananassa
) are an important component of the U.S. specialty fruit crop. The fruit is grown in nearly every state, both commercially and in home gardens. The U.S. market value for strawberries was approximately $519 million in 1995, with a value of between $10,000 and $13,000 per acre harvested. Worldwide production is estimated at 2.4 million tons.
Strawberries are prone to multiple diseases including viruses, rots, leaf spots, and root and crown disease. Strawberry viruses are spread by aphids, nematodes, leafhoppers, and pollen. Viruses may also be transmitted from mother plants during planting. Pre- and post-harvest rotting of strawberries reduces yields by up to 15% annually. Gray mold caused by
Botrytis cinerea
is responsible for the majority of losses due to rot. Common leaf spot and leaf scorch reduce the vigor of infected leaves, lowering the robustness, yield, and quality of the fruit. Parasitic nematodes, bacteria, and fungi act to cause root and crown disease, lowering the yield of the crop.
In addition to traditional breeding techniques, incorporation of disease resistance, improvements to flavor and color, increased or modified sugar content, and other desirable traits can be envisioned using the modem tools of molecular biology.
Matthews, H. V. et al. (
In Vitro Cell. Dev. Biol.
31: 36-43 (1995) and WO 95/35388, Dec. 28, 1995) describe the transformation of strawberries using
Agrobacterium tumefaciens
containing a binary vector. Explants of leaf, meristem, and petiole were co-cultured with Agrobacterium for 1-3 days, followed by an stepwise selection process in media containing 3% sucrose and increasing concentrations of kanamycin. All experimental plant cultures and tissues were maintained in a 16:8 light: dark photoperiod. Strawberry plants were regenerated from pure transgenic explants.
Nehra, N. S. et al. (
J. Amer. Soc. Hort. Sci.
114: 1014-1018 (1989)) reported shoot regeneration from strawberry leaf disks. Efficient regeneration of Redcoat strawberries was achieved in media containing sucrose, benzyladenine, and indoleacetic acid. Low light intensities were found to be conducive to explant regeneration.
Nehra, N. S. et al. (
Plant Cell Rep.
9: 10-13 (1990)) describe the transformation of strawberry via callus culture with
Agrobacterium tumefaciens
as the DNA delivery agent. Leaf explants were inoculated with bacterial suspension, and co-cultured on callus induction media containing 3% sucrose. Selection was performed for four weeks on media containing kanamycin, carbenicillin, and cefotaxime. Shoots were regenerated from selected calli, and rooted on medium containing kanamycin, benzyladenine, and indolebutyric acid.
James, D. J. and Barbara, D. J. (
Acta Horticulturae
280: 495-502 (1990)) describe the transformation of apple and strawberry leaf disks and petioles. The method of Horsch, R. B. et al. (
Science
227: 1229-1231 (1989), for tobacco and petunia) was used, varying the co-cultivation period, the type and quality of agar, plant growth regulators, and the length of the kanamycin selection step. All media used sucrose as a carbohydrate source.
James, D. J. et al. (
Plant Science
69: 79-94 (1990)) describe low efficiency transformation of strawberry leaf disks with
Agrobacterium tumefaciens.
Plants were regenerated on MS medium supplemented with benzylaminopurine and 2,4-dichlorophenoxyacetic acid. Cefotaxime and kanamycin were used as selection agents, and sucrose was used as the carbohydrate source.
Nehra, N. S. et al. (
Plant Cell Rep.
9: 293-298 (1990)) describes the transformation of strawberry via a leaf disk regeneration system. Leaf disks were inoculated with a non-oncogenic
Agrobacterium tumefaciens
strain harboring a binary vector plasmid. Disks were pre-cultured for 10 days on non-selective shoot regeneration medium containing 3% sucrose, and transferred to selective medium containing kanamycin. Selected shoots were multiplied on selective shoot proliferation media. Shoots were rooted and regenerated into strawberry plants.
Nyman, M. and Wallin, A. (
Plant Cell Rep.
11: 105-108 (1992)) presented transient gene expression in strawberry protoplasts via electroporation. Purified protoplasts were suspended in an electroporation buffer containing 10 mM MES, 1 mM calcium chloride, and 0.5 M glucose as an osmotic support.
There exists a need for improved strawberry transformation methods to promote the engineering of desirable traits into this agronomically important crop.
SUMMARY OF THE INVENTION
The present invention relates to methods of preparing transgenic strawberry plants. The substitution of glucose or fructose for sucrose was found to have a pronounced positive effect on transformation efficiencies. The use of folded, immature leaves, and thidiazuron were also found to be beneficial for the preparation of transgenic strawberry explants, shoots, and plants.
In a preferred embodiment, the invention describes a method for the preparation of transgenic strawberry explants comprising contacting strawberry explants with
Agrobacterium tumefaciens
in a co-cultivation medium containing glucose or fructose.
The
Agrobacterium tumefaciens
may generally contain a nucleic acid sequence endogenous to
Agrobacterium tumefaciens,
a nucleic acid sequence endogenous to strawberry, or a nucleic acid sequence from another organism. Alternatively, the
Agrobacterium tumefaciens
contains a nucleic acid sequence exogenous to strawberry, exogenous to
Agrobacterium tumefaciens,
or exogenous to both strawberry and
Agrobacterium tumefaciens.
The nucleic acid sequence may comprise a selectable marker. The selectable marker may generally be any selectable marker suitable for use in
Agrobacterium tumefaciens
or strawberry, and preferably is NPT II, HPT, or EPSPS.
The method may further comprise an incubation step for incubating the transformed strawberry explants for a delay period in delay media under low light conditions. The light conditions are generally any light conditions suitable for the transformation of strawberries, preferably are between about 0 &mgr;Einsteins m
−2
sec
−3
and about 40 &mgr;Einsteins m
−2
sec
−1
, and more preferably are between about 0 &mgr;Einsteins m
−2
sec
−1
and about 20 &mgr;Einsteins m
−2
sec
−1
. The delay period may be about 0 to about 5 days, preferably about 1 to about 4 days, and more preferably is about 3 days.
The delay medium preferably contains glucose. The glucose concentration may generally be about 0.1% (w/v) to about 20% (w/v), preferably about 1% (w/v) to about 4% (w/v), and more preferably about 2% (w/v) to about 3% (w/v). The explants may generally be prepared from any strawberry tissue, and preferably is prepared from either micropropagated strawberry cultures or strawberry sheath leaves. The sheath leaves are preferably runner leaves, node leaves, or crown leaves. The strawberry sheath leaves are preferably folded leaves.
The co-cultivation medium preferably contains glucose. The glucose concentration may generally be about 0.1% (w/v) to about 20% (w/v), preferably about 1% (w/v) to about 4% (w/v), and more preferably about 2% (w/v) to about 3% (w/v). The co-cultivation medium preferably contains thidiazuron. The concentration of thidiazuron may generally be about 0 &mgr;M to about 20 &mgr;M.
In an alternative embodiment, the invention describes a method for the preparation of transgenic strawberry shoots comprising culturing transformed strawberry explants in selection medium containing glucose or fructose. The selection medium may contain an auxin, a cytokinin, an antibiotic, or a plant selection agent.
The selection medium preferably contains glucose. The glucose concentration may generally be about 0.1% (w/v) to a
Dhir Seema
Hinchee Maud A. W.
Layton Jeanne G
Oakes Janette V.
(VPP Corporation) DNA Plant Technology Corporation
Benzion Gary
Kimball Melissa L.
Townsend & Townsend & Crew LLP
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