# US 5,350,689 A · Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,350,689 A - Document ID: US5350689A - Status: Granted - Kind: A - Application number: 08/024,875 - Filed: 1993-03-01 - Granted: 1994-09-27 - Earliest claimed: 1987-05-20 - Assignees: Ciba-Geigy Corporation - Inventors: Ray Shillito; Gleta Carswell; Christian Harms; Cindy Boyce; Yin-Fu Chang - Web page: https://patentgrep.com/patent/US5350689A ## CPC - C12N 5/0025: {Culture media for plant cell or plant tissue culture} - A01H 4/00: Plant reproduction by tissue culture techniques {; Tissue culture techniques therefor} - A01H 4/008: {Methods for regeneration to complete plants} - C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins - C12N 9/2442: {Chitinase (3.2.1.14)} - C12N 15/52: Genes encoding for enzymes or proenzymes - C12N 15/8206: {by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated} - C12N 15/8278: {Sulfonylurea} - C12N 15/8286: {for insect resistance} - C12Y 302/01014: Chitinase (3.2.1.14) - Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants - Y10S 47/01: Methods of plant-breeding and including chromosome multiplication - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Y: ENZYMES - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The protoplast or the cell according to claim 1 wherein the protoplast or cell is of a Zea mays inbred line. ### Claim 3 (depends on 1) The protoplast or the cell according to claim 1 wherein the protoplast or cell is of an elite Zea mays inbred line. ### Claim 4 (depends on 3) The protoplast or the cell according to claim 3 wherein the protoplast or cell is from the Zea mays line which is on deposit at the American Type Culture Collection having the ATCC accession number 40326. ### Claim 5 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. ### Claim 6 (depends on 5) A Zea mays protoplast obtained from the embryogenic callus culture of claim 5. ### Claim 7 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. ### Claim 8 (depends on 7) A Zea mays protoplast obtained from the embryogenic suspension culture of claim 7. ### Claim 9 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. ### Claim 10 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. ### Claim 11 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. ### Claim 12 (depends on 11) The method according to claim 11 wherein the subculturing of step (c) is carried out for a period of about 7 days and with a frequency sufficient to maintain the cells and cell aggregates in a viable state. ### Claim 13 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. ### Claim 14 (depends on 13) The method according to claim 13 wherein the subculturing of step (c) is carried out for a period of about 7 days and with a frequency sufficient to maintain the cells and cell aggregates in a viable state. ### Claim 15 (depends on 13) A method for regenerating Zea mays plants from protoplasts according to claim 13 further comprising the steps of: (f) plating the protoplasts in a suitable medium comprising a growth regulator selected from the group consisting of 2,4-D, dicamba, picloram and pCPA in which the protoplasts divide and form cells, (g1) first, subculturing the cells resulting from step (f) in a suitable medium comprising 2,4-D so as to multiply the cells, (g2) second, further culturing the cells from step (g1) in a suitable medium optionally comprising a cytokinin selected from the group consisting of kinetin, BAP, and zeatin, (g3) third, further culturing the cells from step (g2) in a suitable medium without growth regulators so as to regenerate Zea mays plantlets, and (h) culturing the Zea mays plantlets on a growing medium to obtain fertile plants. ### Claim 16 (depends on 15) The method according to claim 15 wherein the Zea mays protoplasts are cultured in the presence of a growth regulatingly effective non-toxic amount of a plant growth regulator selected from the group consisting of 2,4-D, dicamba, picloram and pCPA and in the presence of an amount of a O-lower alkanoyl-salicylic acid selected from the group consisting of O-acetyl-salicylic acid and O-propionyl-salicylic acid sufficient to promote cell division or cell colony formation. ### Claim 17 (depends on 16) The method according to claim 16, wherein the amount of the plant growth regulator is in the range of about 0.01 to about 100 mg/liter. ### Claim 18 (depends on 16) The method according to claim 16, wherein the plant growth regulator is 2,4-D. ### Claim 19 (depends on 16) The method according to claim 16, comprising the further presence of an amount of DMSO sufficient to promote cell division or colony formation. ### Claim 20 (depends on 15) The method of claim 15 wherein the protoplasts in step (f) divide so as to produce callus. ### Claim 21 (depends on 20) The method of claim 20 wherein the callus forms embryos or proembryos before they regenerate plantlets. ### Claim 22 (depends on 15) The method of claim 15 wherein the cells in step (g3) form embryos before they regenerate plantlets. ### Claim 23 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. ### Claim 24 (depends on 23) The method according to claim 23 wherein the O-lower alkanoyl-salicylic acid is O-acetyl-salicylic acid. ### Claim 25 (depends on 23) The method according to claim 23 further comprising the presence of a growth regulatingly effective non-toxic amount of a plant growth regulator. ### Claim 26 (depends on 25) The method according to claim 25 wherein the amount of the plant growth regulator in the medium is in the range of about 0.01 to about 100 mg/liter. ### Claim 27 (depends on 26) The method according to claim 26 wherein the amount of the plant growth regulator in the medium is in the range of about 0.1 to about 100 mg/liter. ### Claim 28 (depends on 27) The method according to claim 27 wherein the amount of the plant growth regulator in the medium is in the range of about 0.1 to about 10 mg/liter. ### Claim 29 (depends on 23) The method according to claim 23 further comprising the presence of an amount of DMSO sufficient to promote cell division or colony formation. ### Claim 30 (depends on 5) A protoplast-derived plant cell of Zea mays obtained from the embryogenic callus culture of claim 5. ### Claim 31 (depends on 7) A protoplast-derived plant cell of Zea mays obtained from the embryogenic suspension culture of claim 7.