Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells
AssigneeCiba-Geigy Corporation
InventorsRay Shillito, Gleta Carswell, Christian Harms, Cindy Boyce, Yin-Fu Chang
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Abstract
Methods of regenerating fertile Zea mays plants from protoplasts or protoplast-derived cells are described. The protoplasts or cells may be derived from embryogenic cell cultures or callus cultures. The protoplasts, cells and resulting plants may be transgenic, containing, for example, chimeric genes coding for a polypeptide having substantially the insect toxicity properties of the crystal protein produced by Bacillus thuringiensis.
Claims31
A Zea mays protoplast or a protoplast-derived plant cell of Zea mays, wherein said protoplast or protoplast-derived cell is capable of regenerating fertile Zea mays plants, and wherein said protoplast or protoplast-derived cell is obtained by a process comprising the steps of:
- (a) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by the culture of said callus on a callus-maintaining medium comprising 2,4-D,
- (b) transferring the callus to a liquid medium to form a suspension of cells or cell aggregates,
- (c) subculturing the suspension under conditions sufficient to maintain the cells and cell aggregates in a viable state,
- (d) selecting and retaining those cultures from the subcultured suspension of step (c) that contain aggregates of dense, cytoplasmic, dividing cells, sufficient to obtain, in a viable, dividing stage, Zea mays protoplasts capable of being regenerated into fertile plants, and
- (e) removing the cell walls with suitable enzymes, and isolating Zea mays protoplasts.
An embryogenic callus culture of Zea mays from which Zea mays protoplasts can be isolated, wherein the Zea mays protoplasts regenerate cell walls, divide and form callus capable of regenerating fertile Zea mays plants, and wherein said embryogenic callus culture is obtained from embryogenic Zea mays callus, wherein said embryogenic callus culture is obtained by a process comprising the steps of:
- (a1) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by culture of said callus on a callus-maintaining medium comprising 2,4-D and
- (a2) growing the callus of step (a1) on a callus maintaining medium, wherein Zea mays protoplasts can be isolated from said callus culture, wherein the Zea mays protoplasts regenerate cell walls, divide and form callus capable of regenerating fertile Zea mays plants.
An embryogenic suspension culture of Zea mays from which Zea mays protoplasts can be isolated, wherein the Zea mays protoplasts regenerate cell walls, divide and form callus capable of regenerating fertile Zea mays plants, and wherein said embryogenic suspension culture is obtained by a process comprising the steps of:
- (a) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by the culture of said callus on a callus-maintaining medium comprising 2,4-D,
- (b) transferring the callus to a liquid medium comprising 2,4-D to form a suspension of cells or cell aggregates,
- (c) subculturing the suspension (i) for a period of time and (ii) with a frequency sufficient to maintain any cells and cell aggregates in a viable state,
- (d) selecting and retaining those cultures from the subcultured suspension of step (c) that contain aggregates of dense, cytoplasmic, dividing cells, sufficient to obtain, in a viable, dividing stage, Zea mays protoplasts capable of being regenerated into fertile plants.
Callus formed from cultured Zea mays protoplasts, wherein said callus is capable of regenerating fertile Zea mays plants, wherein said protoplasts are obtained from embryogenic Zea mays callus, wherein said callus is produced by a process comprising the steps of:
- (a) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by the culture of said callus on a callus-maintaining medium comprising 2,4-D,
- (b) transferring the callus to a liquid medium to form a suspension of cells or cell aggregates,
- (c) subculturing the suspension under conditions sufficient to maintain the cells and cell aggregates in a viable state,
- (d) selecting and retaining those cultures from the subcultured suspension of step (c) that contain aggregates of dense, cytoplasmic, dividing cells, sufficient to obtain, in a viable, dividing stage, Zea mays protoplasts capable of being regenerated into fertile plants,
- (e) removing the cell walls with suitable enzymes, and isolating Zea mays protoplasts, and
- (f) forming callus from said protoplasts, wherein said callus is capable of regenerating fertile Zea mays plants.
A method for producing a callus culture of Zea mays from which protoplasts can be isolated, wherein the protoplasts are capable of being regenerated into fertile plants, which method comprises the steps of:
- (a1) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by culture of said callus on a callus-maintaining medium comprising 2,4-D and
- (a2) growing the callus of step (a1) on a callus maintaining medium, wherein protoplasts can be isolated from said callus culture, wherein the protoplasts are capable of being regenerated into fertile plants.
A method for producing cell suspension cultures of Zea mays cells and cell aggregates from which protoplasts can be isolated, wherein the protoplasts are capable of regenerating fertile plants, which method comprises the steps of:
- (a) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by the culture of said callus on a callus-maintaining medium comprising 2,4-D,
- (b) transferring the callus to a liquid medium comprising 2,4-D to form a suspension of cells or cell aggregates,
- (c) subculturing the suspension (i) for a period of time and (ii) with a frequency sufficient to maintain any cells and cell aggregates in a viable state,
- (d) selecting and retaining those cultures from the subcultured suspension of step (c) that contain aggregates of dense, cytoplasmic, dividing cells, sufficient to obtain, in a viable, dividing stage, Zea mays protoplasts capable of being regenerated into fertile plants.
A method for producing Zea mays protoplasts capable of regenerating fertile plants, which method comprises the steps of:
- (a) obtaining embryogenic Zea mays callus that is relatively nonmucilaginous, and is granular and friable, said callus being obtained by culturing an immature Zea mays embryo on a callus-inducing medium comprising 2,4-D followed by the culture of said callus on a callus-maintaining medium comprising 2,4-D,
- (b) transferring the callus to a liquid medium to form a suspension of cells or cell aggregates,
- (c) subculturing the suspension under conditions sufficient to maintain the cells and cell aggregates in a viable state,
- (d) selecting and retaining those cultures from the subcultured suspension of step (c) that contain aggregates of dense, cytoplasmic, dividing cells, sufficient to obtain, in a viable, dividing stage, Zea mays protoplasts capable of being regenerated into fertile plants, and
- (e) removing the cell walls with suitable enzymes, and isolating Zea mays protoplasts.
A method for increasing division of, or colony formation from, Zea mays plant protoplasts or Zea mays plant cells, comprising culturing the protoplasts or cells in the presence of an amount of an O-lower alkanoyl-salicylic acid selected from the group consisting of acetyl-salicylic acid and O-propionyl-salicylic acid sufficient to promote cell division or colony formation.
Dependencies are inferred from the claim text.