In vitro regeneration protocol for synthetic seed production in upland cotton (Gossypium hirsutum L.)

Synthetic seeds were obtained in vitro through the encapsulation of axillary buds of Gossypium hirsutum in calcium alginate beads. The optimal combination for the production of uniform, easy to handle, firm and clear beads was found to be 3 % sodium alginate in liquid Murashige and Skoog (MS) medium supplemented with 100 mM CaCl2. The optimal combination for the synthetic endosperm contained 0.5 mg L−1 indole-3-butyric acid and 0.5 mg L−1 α-naphthalene acetic acid, which was used in alginate beads in the MS basal medium. On this synthetic endosperm medium, a 95 % synthetic seed germination rate was observed and 35.8 % of synthetic seeds rooted, with an average length of 2.2 cm after 30 days of culturing. The optimal synthetic seed coat was obtained using a solution containing 3 % sodium alginate and 1 % chitosan dissolved in 85 mM acetic acid, and solidified liquid containing 200 mM CaCl2 and 0.02 % NaOH. Here, 96.1 % of synthetic seeds germinated on MS medium, and the synthetic seeds were stored in the open for 24 h at 25 °C with only an 8 % water loss rate. The percentage of seedling emergence from the synthetic seeds was 35.2 % in the aseptic mixture. This is the first report on synthetic G. hirsutum seeds.