Increased regeneration capacity in spinach lines obtained by in vitro self-fertilisation

Somatic embryos (SEs) were induced from apical sections of the lateral roots of spinach seedlings (1 cm), which were cultivated on solid Murashige and Skoog (MS) medium with 20 μM α-naphthaleneacetic acid and 5 μM gibberellic acid. Apical shoots of the same lines were isolated and cultivated on plant growth regulator-free medium. The regeneration capacities of seedlings randomly chosen from a population were quite low and variable, and only 4 out of 30 lines responded at the frequency of 85–100%, with 6.96–9.96 SEs per explant and up to 347 SEs per seedling over a 12-week period. These SEs were isolated and maintained on medium with 5 μM kinetin. Plants derived from seedlings’ apical shoot and SEs self-fertilised in vitro and set seeds, and these seedlings (S₁) were used to induce regeneration. Similarly, S₂–S₄ seedlings were obtained and the regeneration capacities of 23 S₁, 23 S₂, 17 S₃ and 5 S₄-seedlings were compared to parental lines. Of these, four S₃ and S₄ lines with extremely high regeneration capacities were selected. These lines exhibited 78–139 fold higher embryo-forming capacities than the mother plant, and produced 38.9–68.3 SEs per explant and 1339–2181 SEs per seedling during the same time period. In addition, the process of somatic embryogenesis began 2–4 weeks earlier in these lines, and root explants taken from SE-derived plants of these lines retained high and stable regeneration capacities, and therefore may be ideal material for genetic transformation.