The Culture In vitro of Immature Cotton Embryos

1. A method which proved successful in culturing proembryo and early heart-stage cotton embryos to maturity in vitro has been developed. The culture medium used was composed of White's nutrient mixture with all ingredients at five times the usual concentration and supplements of 40 mg/l adenine sulfate, 250 mg/l casein hydrolysate, 150 ml/l coconut milk, and 7 gm/l NaCl. The medium was hardened with 8 gm/l Bacto-agar and 20 gm/l sucrose was included as carbohydrate source. 2. The principal feature of this medium in the success of the culture method was the adjustment of the osmotic pressure to a high level by the inclusion of the 7 gm/l NaCl. After the embryos had grown on this high-osmotic-pressure medium for 3-4 weeks, they were transferred to a medium of intermediate osmotic pressure (3 gm/l NaCl replacing the 7 gm/l) and then, after a further growth period of 2 weeks, to a medium containing no NaCl. The successfully cultured embryos germinated on the last medium and were potted in soil. 3. While this technique has been at best successful only with a limited number of embryos in any population, it emphasizes the function of osmotic pressure as a developmental control for embryonic growth in cotton. With the use of appropriate osmotic pressures cotton embryos were successfully cultured to maturity when excised 7 days after pollination. These embryos were 1-2 weeks younger than were any cultured by previous methods.