Regeneration of cotton

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.

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.

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.

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.

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.