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Vacuum Infiltration of flowering plants

Patent application filed by The Samuel Roberts Noble Foundation

Actual pending claims

EP 1171618 A2
Claim 1A method for direct plant transformation using plants and Agrobacterium comprising: A) contacting the aerial portions of at least one plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.
Claim 9A method for direct transformation of a plant comprising: A) vernalizing and germinating initial seed to form said plant contacting the aerial portions of said plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.
Claim 17A method for direct plant transformation using plants at the time of flowering and Agrobacterium comprising:
A) contacting aerial portions of at least one plant at the time of flowering with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector with a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.
Claim 25A method for direct transformation of a plant at the time of flowering comprising: A) vernalizing and germinating initial seed to form said plant contacting aerial portions of said plant at the time of flowering with a mixture of Agrobacterium cells, said mixture comprising cells from a Agrobacterium strain harboring a vector with a DNA fragment and cells from said Agrobacterium strain harboring said vector with a second DNA fragment, said vector enabling said Agrobacterium cells to transfer said T-DNA to said plant; and
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different.
Claim 33A method for direct plant transformation using plants at the time of flowering and Agrobacterium comprising: A) contacting aerial portions of at least one plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacterium cells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said plant;
B) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different;
C) allowing said transformed plant to grow to maturity and set seed;
D) germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression; and
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.
Claim 36A method for direct transformation of a plant at the time of flowering comprising: A) vernalizing and germinating initial seed to form said plant;
B) contacting aerial portions of said plant at the time of flowering with Agrobacterium cells, said Agrobacterium cells harboring a vector, said vector enabling said Agrobacteriumcells to transfer T-DNA containing at least one gene or gene fragment and a selectable marker gene to said plant;
C) applying a vacuum to said plant portions in contact with said Agrobacterium cells at a first time, said vacuum of sufficient strength to force said Agrobacterium cells into intimate contact with said plant such that said Agrobacterium cells transfer said T-DNA to cells of said plant at a second time to form a transformed plant, wherein said first time and said second time are the same or different;
D) allowing said transformed plant to grow to maturity and set seed; germinating said seed to form progeny;
E) exposing said progeny to an agent enabling detection of selectable marker gene expression; and
F) selecting for progeny expressing said selectable marker gene and at least one gene, said expression of said selectable marker gene and at least one gene indicating gene transfer.