Transformation method for obtaining marker-free plants and plants obtained therewith
The invention relates to a transformation method for obtaining transgenic plants and plants obtained with said method. The invention provides a method for transforming a plant cell comprising providing a plant cell with a recombinant nucleic acid comprising a T-DNA construct allowing for transfer of said construct into the genome of a plant cell, said construct provided with a foreign nucleic acid that is free of nucleic acid encoding a selective marker.
1 . A method for obtaining a market-free, uniform transgenic plant, comprising
transforming a plant cell with a recombinant nucleic acid comprising a T-DNA construct wherein said T-DNA is provided with a foreign nucleic acid that is free of a nucleic acid encoding a selective marker
regeneration of said cell under no selective pressure
testing said cell or progeny thereof for the presence or absence of at least a functional part of said foreign nucleic acid and identify transformed plant cells or progeny thereof
growing a plant from said identified cell or progeny thereof,
testing the obtained plant for uniformness and selecting a uniform plant.
2 . A method according to claim 1 , wherein said nucleic acid that comprises a foreign nucleic acid that is free of nucleic acid encoding a selective marker is delivered to said plant cell by an Agrobacterium strain.
3 . A method according to claim 3 , wherein said Agrobacterium strain is a virulent Agrobacterium strain.
4 . A method according to claim 2 wherein said Agrobacterium strain carries a DNA region originating from the virulence region of the Ti plasmid pTiBo542.
5 . A method according to claim 2 , wherein said Agrobacterium strain is selected from EHA 101, AGL-0, AGL-1.
6 . A method according to claim 1 wherein said foreign nucleic acid allows for regulation of the expression of a target gene in the genome of said plant cell.
7 . A method according to claim 6 wherein said regulation comprises downregulation.
8 . A method according to claim 6 wherein said foreign nucleic acid includes an inverted repeat of at least part of a polynucleotide region of said target gene.
9 . A method according to claim 6 , wherein said target gene encodes a granule-bound starch synthase (GBSSI) enzyme.
10 . A method according to claim 1 , wherein said foreign nucleic acid allows for expression of a heterologous polypeptide in the genome of said plant cell.
11 . A method according to claim 10 , wherein said heterologous polypeptide comprises an enzyme.
12 . A method according to claim 11 , wherein said enzyme comprises a-, preferably feedback-insensitive, dihydrodipicolinate synthase (DHPS).
13 . A method according to claim 1 , wherein said testing for the presence or absence of at least a functional part of said foreign nucleic acid is performed by nucleic acid techniques, ELISA, bioassay or chemical analytical methods.
14 . A method according to claim 13 , wherein said testing is performed by PCR.
15 . A method according to claim 1 , further comprising testing said cell or progeny thereof for the presence or absence of undesired vector material such as vector backbone sequences.
16 . A method according to claim 1 , further comprising determining the amount of copies of said foreign nucleic acid and selecting the one that contains only one insert of said T-DNA.
17 . A plant cell obtainable with a method according to claim 1 .
18 . A plant, or part thereof, derived from a plant cell according to claim 17 .
19 . A tuberous plant, or part thereof, according to claim 18 .
20 . A plant, or part thereof, according to claim 19 which is selected from potato or cassava plants.