Somatic embryogenesis and direct shoot regeneration of rice (Oryza sativa L.) using thin cell layer culture of apical meristematic tissue

A new and simple method was designed to directly produce shoot regeneration of rice (Oryza sativa L.), within a short time-span, with a high yield, and without the need for subculture. Transverse thin cell layers (tTCLs) excised from the shoot apical meristems of two-week old seedlings, which received pretreatment in darkness with 2,4-D from 1–20 μmol/L, were placed on Murashige and Skoog. (1962) medium supplemented with 3% (w/v) sucrose and a combination of 2,4-D varying from 1–20 μmol/L and BAP varying from 1–10 μmol/L. Somatic embryos and the embryo-like structures which developed into shoots were kept in the dark for 2 weeks after the culture of tTCL explants, and produced a maximum of 6 somatic embryos and 16 shoots per tTCL explant. Also important for shoot regeneration was the effect of the 2-week period of exposure to darkness followed by light exposure. The regenerated shoots were transferred to a medium with no hormone for rooting. More than 95% of plantlets developed successfully when they were transferred to the greenhouse.