SOYBEAN TRANSFORMATION USING HPPD INHIBITORS AS SELECTION AGENTS
The invention relates to methods for Agrobacterium -mediated transformation of soybean organogenic tissue using a gene or genes for tolerance to HPPD inhibitors as selection marker. The invention also relates to methods for regenerating transgenic soybean plants from said transformed soybean cells or tissue, and to transgenic soybean plants and seeds obtained by such methods.
1 . A method for transforming a soybean plant cell using an Agrobacterium -mediated process and a gene or genes for tolerance to HPPD inhibitors as selection marker, said method comprising the step of:
a) preparing a soybean organogenic explant comprising at least a plant cell capable of being transformed by an Agrobacterium strain,
b) exposing at least the region containing said plant cell in said explant to an Agrobacterium strain containing at least a first genetic construct comprising at least a gene for tolerance to an HPPD inhibitor,
c) culturing the explant in the presence of an HPPD inhibitor as selection agent,
d) optionally selecting the transformed plant cells.
2 . The method according to claim 1 wherein the soybean plant cell is from a cotyledonous explant, a half-seed explant or a half-embryo-seed explant.
3 . The method according to claim 1 wherein the Agrobacterium strain is Agrobacterium tumefaciens or Agrobacterium rhizogenes.
4 . A method for transforming a soybean plant cell according to claim 1 , characterized in that said Agrobacterium vector comprises further at least a second heterologous genetic construct which is introduced into said soybean plant cell conjointly with said first heterologous genetic construct comprising at least a gene for tolerance to HPPD inhibitors.
5 . The method according to claim 4 , characterized in that the different heterologous genetic constructs are included in one or several T-DNA(s).
6 . The method according to claim 4 wherein the heterologous genetic constructs comprises a gene conferring resistance to an herbicide, a gene conferring resistance to nematodes, a gene conferring resistance to insect.
7 . A method for preparing a transgenic soybean plant comprising at least a heterologous gene integrated into its genome, comprising the method of claim 1 , supplemented with the following steps of:
e) regenerating plants and seeds comprising said heterolous gene from the transformed cell or transformed tissue.
8 . A method for preparing a transgenic soybean plant using an Agrobacterium -mediated process and a gene or genes for tolerance to HPPD inhibitors as selection marker gene, said method comprising the steps of:
a) preparing a soybean organogenic explant comprising at least a plant cell capable of being transformed by an Agrobacterium strain,
b) exposing at least the region containing said plant cell in said explant to an Agrobacterium strain containing a heterologous DNA comprising at least a gene for tolerance to HPPD inhibitors,
c) culturing the explant in the presence of an HPPD inhibitors as selection agent,
d) allowing plants to be regenerated from cells of said explant,
e) selecting transformed plants.
9 . The method according to claim 1 , characterized in that the HPPD inhibitor is selected from the group consisting of isoxazoles, diketonitriles, triketones, and pyrazolinates.
10 . The method according to claim 1 , characterized in that the soybean plant cell or plant is further transformed, simultaneously or successively, with a gene functional in this plant allowing overexpression of a PDH (prephenate dehydrogenase) enzyme.
11 . The method according to claim 4 , characterized in that the selection marker gene for tolerance to HPPD inhibitors is eliminated by crossing the transformed plants comprising at least a second heterologous genetic construct and the selection marker gene with a non-transformed variety of the same plant.
12 . A transgenic soybean plant cell suitable to be obtained by the method of claim 1 .
13 . (canceled)
14 . The method according to claim 8 , characterized in that the HPPD inhibitor is selected from the group consisting of isoxazoles, diketonitriles, triketones, and pyrazolinates.
15 . The method according to claim 8 , characterized in that the soybean plant cell or plant is further transformed, simultaneously or successively, with a gene functional in this plant allowing overexpression of a PDH (prephenate dehydrogenase) enzyme.