Process for transforming plant protoplast
AssigneeCiba-Geigy Corporation
InventorsIoan Negrutiu, Ingo Potrykus
Life of this patent
Estimating the term…
Abstract
The present invention relates to an improved process for transforming plant protoplasts using simple, purely chemical, process steps, and to the plant material obtainable by this method. The transfer of the genetic material into the plant cell is carried out directly, without the use of a natural plant-infective system such as a plant bacterium or plant virus, and without transfer by insects or phytopathogenic fungi, by joint incubation in a suitable incubation medium of the DNA to be transformed and plant protoplasts. In this manner, desired genes can very simply and efficiently be transferred to plant material, resulting in plants with improved properties.
Claims34
A process for direct gene transfer into plant protoplasts that are amenable to transformation via direct gene transfer, which process consisting:
- isolating plant protoplasts from plant tissue and culturing said protoplasts in a nutrient media customarily used for culturing plant protoplasts;
- pre-incubating said protoplasts at a temperature of from 4.degree. to 10.degree. C. for from 20 minutes to 6 hours in a pre-incubation medium containing Ca.sup.2+, K.sup.+ and Na.sup.+ ions and a suitable carbon source before the actual transformation;
- separating said protoplasts from the pre-incubation medium and resuspending said protoplasts in a transformation medium which contains as an essential component from 6.0 to 60 mM Mg.sup.2+ ions in the presence or absence of Ca.sup.2+ ions;
- directly thereafter, adding a DNA sample to said transformation medium;
- from 0.1 to 10 minutes thereafter, adding polyethylene glycol (PEG) to said transformation medium;
- and
- incubating said protoplasts and DNA sample in said transformation medium for a period that is sufficient for the incorporation of said DNA into said protoplasts.
A process for direct gene transfer into plant protoplasts that are amenable to transformation via direct gene transfer, which process comprising:
- isolating plant protoplasts from plant tissue and culturing said protoplasts in a nutrient media customarily used for culturing plant protoplasts;
- pre-incubating said protoplasts at a temperature of from 4.degree. to 10.degree. C. for from 20 minutes to 6 hours in a pre-incubation medium containing Ca.sup.2+, K.sup.+ and Na.sup.+ ions and a suitable carbon source before the actual transformation;
- separating said protoplasts from the pre-incubation medium and resuspending said protoplasts in a transformation medium which contains as an essential component from 10 to 30 mM Mg.sup.2+ ions in the presence or absence of Ca.sup.2+ ions;
- directly thereafter, adding a DNA sample to said transformation medium;
- from 0.1 to 10 minutes thereafter, adding polyethylene glycol (PEG) to said transformation medium;
- and
- incubating said protoplasts and DNA sample in said transformation medium for a period that is sufficient for the incorporation of said DNA into said protoplasts.
Dependencies are inferred from the claim text.