Hygromycin promotes somatic embryogenesis in spinach

Jelena Milojević, Ljiljana Tubić, Vladimir Nolić, Nevena Mitić, Dušica Ćalić-Dragosavac, Branka Vinterhalter, Snežana Zdravković-Korać

June 2012, Volume 109, Issue 3, pp 573-579
Abstract:

Hygromycin (hyg) at low doses (0.5–1.0 mg l⁻¹) promoted somatic embryogenesis from apical sections of spinach lateral roots. The highest promoting effect on both the frequency of regeneration and the mean number of somatic embryos (SE) per explant was achieved at 0.5 mg l⁻¹ hyg. With increasing the concentration of hyg to 1 mg l⁻¹, the regeneration frequency decreased, while the mean SE number remained significantly higher than in control (hyg-free medium). Complete inhibition of SE regeneration started at 7.5 mg l⁻¹ hyg. Moreover, hyg efficiently promoted the process of secondary somatic embryogenesis. Compared to control, a 2.75-fold increase in the secondary somatic embryo (SSE) mean number was obtained at 0.5 mg l⁻¹ hyg, and the increment was still discernible at 1.0 and 2.5 mg l⁻¹ hyg. Both primary SE and SSE explants became completely necrotic at 12.5 mg l⁻¹ hyg. Since attempts with direct selection at 20 mg l⁻¹ hyg proved unsuccessful, the results obtained in this study suggest that a stepwise selection procedure is suitable, starting with selection at 0.5 mg l⁻¹ hyg, to exploit the promoting effect of low hyg doses on SE regeneration from transformed cells, then gradually increasing the hyg concentration to 20 mg l⁻¹ for final selection. Complete SE and SSE explant mortality at hyg above 12.5 mg l⁻¹guarantees a low possibility of escape during the selection process. This study will be useful for increasing the efficiency of transgenic plant regeneration following genetic transformation in spinach.

Keywords: Hygromycin, Somatic embryogenesis, Secondary somatic embryogenesis, Spinacia oleracea, Tissue culture