Previous reports of plant regeneration of natural tetraploid T. pratense L. 'Elci' could be realized only through the apical meristem calli. In order to proceed to the production stage, other regeneration methods need to be tried. Aseptic seedlings were used for the production of somatic embryos through various 2,4-D and kinetin trials. Nonuniform external callus cells with translucent cytoplasm were observed in various developmental stages of somatic embryos. Beneath these cells, there were uniformly aligned, dark-stained embryo cells with dense cytoplasm. Despite the similar developmental stages and cell characteristics of zygotic and somatic embryos, the walls of somatic embryo cells revealed a highly wavy pattern. The nucleus generally contained only one nucleolus, which was spherical, dark stained, and electron-dense. Electron-dense droplets were seen in vacuoles. The cytoplasm consisted of starch-containing amyloplasts, mitochondria, plastids, ribosomes, endoplasmic reticulum, dictyosomes, lipid, and protein bodies. In some of the somatic embryos at the globular and heart stages, vacuole or electron-translucent zones were observed in the nucleolus. Additionally, a few embryo degenerations were recorded during developmental stages of the zygotic embryo. For the first time, the somatic embryos of natural tetraploid T. pratense were produced from hypocotyl (85%), cotyledon (75%), and apical meristem (60%) explants in 0.3 mg/L 2,4-D and 2 mg/L kinetin-containing MS medium. Our study developed an effective and efficient in vitro production method for using natural tetraploid T. pratense in biotechnological studies.
