Claims for Patent: 5,244,802

Summary for Patent: 5,244,802

Title:	Regeneration of cotton
Abstract:	There are provided methods for regenerating cotton by tissue and suspension culture starting with explants which are the hypocotyl, cotyledon or immature embryos. This also taught methods to transform cotton and improve cotton by selective growth.
Inventor(s):	Rangan; Thirumale S. (Pasadena, CA)
Assignee:	Phytogen (Pasadena, CA)
Application Number:	07/680,048
Patent Claims:	1. A method for the regeneration of a cotton plant from somatic cells which comprises the steps of: (a) providing a cotton explant which is an immature zygotic embryo or an explant selected from the group consisting of hypocotyl, cotyledon and mixtures thereof from a cotton seedling; (b) culturing the explant in a first solid callus growth medium which is a full or half-strength Murashige and Skoog growth medium containing glucose as the carbon source supplemented with thiamine hydrochloride, naphthaleneacetic acid and kinetin and inositol at a temperature of from about 25.degree. to about 35.degree. C. under a light-dark cycle of about 16 hours of light at a light intensity of about 2,000 to about 4,000 lux followed by about 8 hours of darkness, said light-dark cycle and changing of the medium being repeated for a period of time sufficient for phenolic secretion from the explant to end, to enable undifferentiated callus to form from the explant; (c) transferring the callus from the first solid callus growth medium to a second solid callus growth medium which is full or half strength Murashige and Skoog growth medium comprising sucrose as the carbon source and from about 1 to about 10 mg/1 of naphthaleneacetic acid and culturing the callus at a temperature from about 25.degree. to 35.degree. C. under a light-dark cycle of about 16 hours light at a light intensity of about 2,000 to about 4.000 lux followed by about 8 hours of darkness, said light-dark cycle being repeated for a time sufficient to form yellow and white embryogenic callus; (d) further subculturing the embryogenic callus to develop to callus containing somatic embryos; and (e) transferring somatic embryos to an embryo germination medium which is a Beasley and Ting's medium containing up to about 500 mg/l casein hydrolysate and up to about 1200 mg/l ammonium nitrate and growing the embryos in the embryo germination medium to plantlets sufficiently developed for transfer to soil; and (f) transferring the plantlets from the embryo germination medium to soil. 2. The method as claimed in claim 1, in which the explant is from a cotton seedling developed by: a) sterilizing cotton seed in a first sterilizing solution; b) rinsing the seed in sterile water; c) sterilizing the seed in a second sterilizing solution; f) rinsing the second sterilization solution from the seed with sterile water; g) transferring the seed to a seed germination medium; and h) growing the seed in the seed germination medium in the dark for a period of time sufficient to produce a seedling; and i) excising the explant from the seedling. 3. The method as defined in claim 2 in which the first sterilizing solution is an aqueous solution containing about 95% by volume ethanol and the second sterilizing solution is an aqueous solution containing about 15% by weight sodium hypochlorite. 4. A method as claimed in claim 2 in which the seed germination medium is a basal agar medium and growth of the seedling in the basal agar medium prior to excising the explant is up to about 4 weeks. 5. A method as claimed in claim 1 in which the light intensity during the hours of light is about 3,000 to about 4,000 lux. 6. A method as claimed in claim 1 wherein once phenolic secretions end the explant is transferred to the second solid callus growth medium and the second solid callus growth medium is changed to a fresh second solid callus growth medium at about 3 to about 4 week intervals. 7. A method as claimed in claim 1 further including the step of transferring the plantlet to soil under conditions of high humidity for a time sufficient for the plantlet to mature to a stage sufficient to enable its transfer to a hot house or field for growth to final maturity. 8. A method as claimed in claim 1 in which the first callus growth medium is a Murashige and Skoog growth medium containing about 0.4 mg/l thiamine hydrochloride, about 30 g/l glucose, about 2 mg/l naphthaleneacetic acid, about 1 mg/l kinetin and about 100 mg/l inositol. 9. A method as claimed in claim 1 in which the second callus growth medium contains from about 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinin. 10. A method as claimed in claim 8 in which the second callus growth medium contains from about 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinin. 11. A method as claimed in claim 1 in which the first callus growth medium is changed about every 10 days while phenolics are secreted from the explant, and thereafter including sucrose in the second solid callus growth medium as the primary carbon source. 12. A method as claimed in claim 1 in which the first callus growth medium is changed about every ten days while phenolics are secreted from explant, and thereafter including sucrose in the second solid callus growth medium as the primary carbon source. 13. A method for the regeneration of cotton plants from somatic cells, said method comprising the steps of: (a) sterilizing at least one cotton seed in a solution containing 95% by volume ethanol for a period of approximately 2-3 minutes; (b) rinsing the seed in sterile water; (c) soaking the seed in a solution of sodium hypochlorite containing about 15% by weight sodium hypochlorite for a period of from about 15 to about 20 minutes; (d) rinsing the seed in sterile water; (e) germinating the seed in a dark environment on modified basal agar medium selected from the group consisting of White's medium and half strength Murashige and Skoog medium for a period of up to about 14 days to produce a seedling; (f) excising at least one segment selected from the group consisting of hypocotyl, cotyledon or mixtures thereof from the seedling; (g) culturing the excised segment on first solid callus growth medium which is a Murashige and Skoog medium supplemented with about 0.4 mg/l thiamine hydrochloride, about 3 g/l glucose, about 2 mg/l naphthaleneacetic acid, about 1 mg/l kinetin and about 100 mg/l inositol in an environment of a temperature of from 25.degree. to 30.degree. C. under a light-dark cycle of 16 hours of light followed by about 8 hours of darkness, at about 3,000 to 4,000 lux light intensity during the hours of light during which the medium is replaced by fresh first solid callus growth medium about every 10 days, said light-dark cycle with changes of the medium being repeated until phenolic secretions from the excised segment end, to enable formation of undifferentiated callus from the explant; (h) transferring the callus onto a Murashige and Skoog medium comprising sucrose and about 2 mg/l naphthaleneacetic acid and about 1 mg/l cytokinetin; (i) culturing the callus over a period of about three to four months to produce at least one embryo; (j) transferring the embryo to a Beasley and Ting's medium comprising up to about 500 mg/l casein hydrolysate, and containing up to about 1,200 mg/l ammonium nitrate and culturing the embryo for a period of about 2 to about 3 months, to produce a plantlet; and (k) transferring the plantlet to soil and incubating the plantlet in high humidity to form a plant. 14. The method as defined in claim 13 including the steps of self-pollinating the plant formed from the plantlet to produce seeds and germinating the seeds to produce seedlings. 15. A method for the regeneration of a cotton plant from somatic cotton cells of Acala cotton which comprises steps of: (a) providing a cotton explant which is an immature zygotic embryo or an explant selected from the group consisting of hypocotyl, cotyledon and mixtures thereof from the Acala cotton seedling; (b) culturing the cotton explant in a first solid callus growth medium which is a full or half-strength Murashige and Skoog growth medium supplemented with thiamine hydrochloride, naphthaleneacetic acid and kinetin and inositol and containing glucose as a carbon source in an environment temperature of from about 25.degree. to about 35.degree. C. under a light-dark cycle of 16 hours of light at about 2,000 to 4,000 lux light intensity followed by about 8 hours of darkness, said light-dark cycle and changing of the medium being repeated for a period of time sufficient for phenolic secretions from the explant to end to enable undifferentiated callus to develop from the explant; (c) transferring the callus when phenolic secretions end to a second solid callus growth medium which is selected from group consisting of full strength Murashige and Skoog and half strength Murashige and Skoog growth medium comprising sucrose as the carbon source and from about 1 to about 10 mg/l of naphthaleneacetic acid; (d) culturing the callus on the second solid callus growth medium at a temperature of from 25.degree. to 35.degree. C. under a light-dark cycle of 16 hours of light at about 2,000 to 4,000 lux light intensity followed by about 8 hours of darkness, said light-dark cycle being repeated for a period of time sufficient to form yellow to white embryogenic callus providing at least one embryo; (e) further subculturing the embryogenic callus to develop callus containing somatic embryos; (f) transferring somatic embryos to a plant germination medium which is a Beasley and Ting's medium containing up to about 500 mg/l casein hydrolysate and up to about 1200 mg/l ammonium nitrate; (g) culturing the embryo on the plant germination medium for a period of time sufficient to develop a plantlet from the embryo; and (h) transferring the plantlets from the embryo germination medium to soil. 16. A method for the regeneration of a cotton plant from somatic cells which comprises the steps of: (a) providing an explant which is an immature zygotic embryo or an explant selected from the group consisting of hypocotyl, cotyledon and mixtures thereof derived from a cotton seedling; (b) culturing the explant in a first solid callus growth medium which is a Murashige and Skoog growth medium supplemented with thiamine hydrochloride, glucose, naphthaleneacetic acid, kinetin and inositol at a temperature of from about 25.degree. to about 35.degree. C. under a light-dark cycle of about 16 hours light at a light intensity of about 2,000 to about 4,000 lux followed by a darkness cycle about 8 hours of darkness, said light-dark cycle and changing of the medium being repeated for a period of time sufficient for phenolic secretion from the explant to end to enable undifferentiated callus to form from the explant; (c) transferring the callus from the first callus growth medium when phenolic secretions end to a second callus growth medium which is a Murashige and Skoog growth medium containing sucrose and from about 1 to about 10 mg/l of naphthaleneacetic acid and culturing the callus at a temperature from about 25.degree. to about 35.degree. C. under a light-dark cycle of about 16 hours of light at a light intensity of about 2,000 to about 4,000 lux followed by about 8 hours of darkness, said light-dark cycle being repeated for a time sufficient to develop embryogenic callus; (d) suspending parts of the embryogenic callus in fresh liquid second callus growth medium at a concentration from about 750 to about 1,000 mg of callus parts per 8 ml of said liquid second callus growth medium which is a Murashige and Skoog growth medium containing sucrose and from about 1 to about 10 mg/l of naphthaleneacetic acid and culturing the suspended callus at a temperature from about 25.degree. to 35.degree. C. under a light-dark cycle of about 16 hours of light at a light intensity of about 2,000 to about 4,000 lux followed by about 8 hours of darkness, said light-dark cycle being repeated for a time sufficient to develop embryogenic callus; and allowing the suspended parts to grow for a time sufficient to develop embryogenic clumps of a size greater than about 600 microns; (e) separating the embryogenic clumps greater than about 600 microns from clumps less than about 600 microns; and (f) transferring the embryogenic clumps of a size greater than 600 microns to a plant germination medium which is a Beasley and Ting's medium containing up to about 500 mg/l casein hydrolysate and up to about 1,200 mg/l ammonium nitrate and growing the embryogenic clumps to plantlets. 17. A method for the regeneration of a cotton plant from somatic cells of Acala cotton which comprises the steps of: (a) providing an explant which is an immature zygotic embryo or an explant selected from the group consisting of hypocotyl, cotyledon and mixtures thereof derived from an Acala cotton seedling; (b) culturing the explant in a first solid callus growth medium which is a Murashige and Skoog growth medium supplemented with thiamine hydrochloride, glucose, naphthaleneacetic acid, kinetin and inositol at a temperature of from about 25.degree. to about 35.degree. C. under a light-dark cycle of about 16 hours light at a light intensity of about 2,000 to about 4,000 lux followed by a darkness cycle about 8 hours of darkness, said light-dark cycle and changing of the medium being repeated for a period of time sufficient for phenolic secretion from the explant to end to enable undifferentiated callus to form from the explant; (c) transferring the callus from the first callus growth medium when phenolic secretions end to a second callus growth medium which is a Murashige and Skoog growth medium containing sucrose and from about 1 to about 10 mg/l of naphthaleneacetic acid and culturing the callus at a temperature from about 25.degree. to about 35.degree. C. under a light-dark cycle of about 16 hours of light at a light intensity of about 2,000 to about 4,000 lux followed by about 8 hours of darkness, said light-dark cycle being repeated for a time sufficient to develop embryogenic callus; (d) suspending parts of the embryogenic callus in fresh liquid second callus growth medium at a concentration from about 750 to about 1,000 mg of callus parts per 8 ml of said liquid second callus growth medium which is a Murashige and Skoog growth medium containing sucrose and from about 1 to about 10 mg/l of naphthaleneacetic acid and culturing the suspended callus at a temperature from about 25.degree. to 35.degree. C. under a light-dark cycle of about 16 hours of light at a light intensity of about 2,000 to about 4,000 lux followed by about 8 hours of darkness, said light-dark cycle being repeated for a time sufficient to develop embryogenic callus; and allowing the suspended parts to grow for a time sufficient to develop embryogenic clumps of a size greater than about 600 microns; (e) separating the embryogenic clumps greater than about 600 microns from clumps less than about 600 microns; and (f) transferring the embryogenic clumps of a size greater than 600 microns to a plant germination medium which is a Beasley and Ting's medium containing up to about 500 mg/l casein hydrolysate and up to about 1200 mg/l ammonium nitrate and growing the embryogenic clumps to plantlets. 18. A method as claimed in claim 16 in which the clumps of a size less than 600 microns are resuspended in fresh second callus growth medium for further growth of embryogenic clumps. 19. A method as claimed in claim 16 in which the explant is obtained by: a) sterilizing of the seed in a first-sterilized solution; b) rinsing the seed with sterile water; c) sterilizing the seed in a second sterilizing solution; d) rinsing the second sterilization solution from the seed with sterile water; e) transferring the sterilized seed to a seed germination medium; f) culturing the seed in the seed germination medium in the dark for a period of time sufficient to form cotton seedling; and g) excising the explant from the cotton seedling. 20. The method as claimed in claim 17 in which the explant is obtained by: a) sterilizing cotton seed in a first sterilizing solution; b) rinsing the seed with sterile water; c) sterilizing the seed in a second sterilizing solution; d) rinsing the second sterilization solution from the seed with water; e) transferring the sterilized seed to a seed germination medium; f) culturing the seed in the seed germination medium in the dark for a period of time sufficient to form a cotton seedling; and g) excising the explant from the cotton seedling. 21. The method of claim 20 in which the first sterilizing solution is an aqueous solution containing about 95% by volume ethanol; and the second sterilizing solution is an aqueous solution containing about 15% by weight sodium hypochlorite. 22. A method as claimed in claim 16 in which seed germination medium is a basal agar medium and growth of the seedling is for a period of up to about 14 days prior to excising the explant from the seedling. 23. A method as claimed in claim 16 in which clumps greater than about 800 microns are removed from suspension for plant growth. 24. A method as claimed in claim 16 in which the light intensity during the hours of light is about 3,000 to about 4,000 lux. 25. A method as claimed in claim 16 further including the step of transferring the plantlets from the plant germination medium to soil under conditions of high humidity for a time sufficient to allow the plantlets to mature to a stage sufficient to enable them to be transferred to a hot house and, after said further growth in the hot house, said matured plants are transferred to a field for growth to final maturity. 26. A method as claimed in claim 16 in which the embryogenic clumps less than 800 microns are resuspended in fresh second callus growth medium as per step d) and steps e) and f) are repeated. 27. A method as claimed in claim 16 in which the first callus growth medium is a Murashige and Skoog growth medium containing about 0.4 mg/l thiamine hydrochloride, about 30 g/l glucose, about 2 mg/l naphthaleneacetic acid, about 1 mg/l kinetin and about 100 mg/l inositol. 28. A method as claimed in claim 16 in which the second callus growth medium comprises sucrose and from 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinin. 29. A method as claimed in claim 26 in which the liquid callus growth medium contains from 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinin. 30. A method as claimed in claim 17 in which the liquid callus growth medium contains from 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinin. 31. The method as claimed in claim 15, in which the explant is from a cotton seedling developed by: (a) sterilizing cotton seed in a first sterilizing solution; (b) rinsing the seed in sterile water; (c) sterilizing the seed in a second sterilizing solution; (d) rinsing the second sterilization solution from the seed with sterile water; (e) transferring the seed to a seed germination medium; and (f) growing the seed in the seed germination medium in the dark for a period of time sufficient to produce a seedling; and (g) excising the explant from the seedling. 32. The method as defined in claim 31 in which the first sterilizing solution is an aqueous solution containing about 95% by volume ethanol and the second sterilizing solution is an aqueous solution containing about 15% by weight sodium hypochlorite. 33. A method as claimed in claim 31 in which the seed germination medium is a basal agar medium and growth of the seedling in the basal agar medium prior to excising the explant is up to about 4 weeks. 34. A method as claimed in claim 15 in which the light intensity during the hours of light is about 3,000 to about 4,000 lux. 35. The method as claimed in claim 15 further including the step of transferring the plantlet to soil under conditions of high humidity for a time sufficient for the plantlet to mature to a stage sufficient to enable its transfer to a hot house or field for growth to final maturity. 36. A method as claimed in claim 15 in which the first callus growth medium is changed about every ten days while phenolics are secreted from the explant, and thereafter including sucrose in the second solid callus growth medium as the primary carbon source. 37. A method as claimed in claim 16 in which the first callus growth medium is changed about every ten days while phenolics are secreted from the explant, and thereafter including sucrose in the second solid callus growth medium as the primary carbon source. 38. A method for the regeneration of cotton plants from somatic cells of Acala cotton, said method comprising the steps of: (a) sterilizing at least one Acala cotton seed in a solution containing 95% by volume ethanol for a period of approximately 2 to 3 minutes; (b) rinsing the seed in sterile water; (c) soaking the seed in a solution of sodium hypochlorite containing about 15% by weight sodium hypochlorite for a period of from about 15 to about 20 minutes; (d) rinsing the seed in sterile water; (e) germinating the seed in a dark environment on modified basal agar medium selected from the group consisting of White's medium and half strength Murashige and Skoog medium for a period up to about 14 days to produce a seedling; (f) excising at least one segment selected from the group consisting of hypocotyl, cotyledon or mixtures thereof from the seedling; (g) culturing the excised segment on first solid callus growth medium which is a Murashige and Skoog medium supplemented with about 0.4 mg/l thiamine hydrochloride, about 30 g/l glucose, about 2 mg/l naphthaleneacetic acid, about 1 mg/l kinetin and about 100 mg/l inositol in an environment of a temperature of from 25.degree. to 30.degree. C. under a light-dark cycle of 16 hours of light followed by about 8 hours of darkness, at about 3,000 to 4,000 lux light intensity during the hours of light during which the medium is replaced by fresh first solid callus growth medium about every 10 days, said light-dark cycle with changes of the medium being repeated until phenolic secretions from the excised segment end, to enable formation of undifferentiated callus from the explant; (h) transferring the callus onto Murashige and Skoog medium comprising sucrose and about 2 mg/l naphthaleneacetic acid and about 1 mg/l cytokinetin; (i) culturing the callus over a period of about 3 to 4 months to produce at least one embryo; (j) transferring the embryo to Beasley and Ting's medium comprising up to about 500 mg/l casein hydrolysate, and containing up to about 1200 mg/l ammonium nitrate and culturing the embryo for a period of about 2 to about 3 months, to produce a plantlet; and (k) transferring the plantlet to soil and incubating the plantlet in high humidity to form a plant. 39. The method as defined in claim 38 including the steps of self-pollinating the plant formed from the plantlet to produce seeds and germinating the seeds to produce seedlings. 40. The method of claim 17 in which the first sterilizing solution is an aqueous solution containing about 95% by volume ethanol; and the second sterilizing solution is an aqueous solution containing about 15% by weight sodium hypochlorite. 41. A method as claimed in claim 17 in which seed germination medium is a basal agar medium and growth of the seedling is for a period of up to about 14 days prior to excising the explant from the seedling. 42. A method as claimed in claim 17 in which the light intensity during the hours of light is about 3,000 to about 4,000 lux. 43. A method as claimed ion claim 17 further including the step of transferring the plantlets from the plant germination medium to soil under conditions of high humidity for a time sufficient to allow the plantlets to mature to a stage sufficient to enable them to be transferred to a hot house and, after said further growth in the hot house, said matured plants are transferred to a field for growth to final maturity. 44. A method as claimed in claim 17 in which the embryogenic clumps less than 600 microns are re-suspended in fresh second callus growth medium as per step (d) and steps (e) and (f) are repeated. 45. A method as claimed in claim 17 in which the second callus growth medium comprises sucrose and from 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinetin. 46. A method as claimed in claim 17 in which the liquid callus growth medium contains from 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinetin. 47. A method as claimed in claim 44 in which the liquid callus growth medium contains from 1 to about 5 mg/l naphthaleneacetic acid and from 0 to about 1 mg/l cytokinetin. 48. A method as claimed in claim 38 in which the first callus growth medium is changed about every 10 days while phenolics are secreted from the explant, and thereafter including sucrose in the second solid callus growth medium as the primary carbon source.

Details for Patent 5,244,802

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2010-09-14