Somatic embryogenesis of Gentiana cruciata (L.): Histological and ultrastructural changes in seedling hypocotyl explant

Abstract

Structure and ultrastructure changes that occurred during tissue culture of upper explants of hypocotyl (adjacent to cotyledons) of 10-d-old seedlings of Gentiana cruciata were studied. The explants were cultured on Murashige and Skoog induction medium supplemented with 1.0 mg l−1 dicamba +0.1 mg l−1 naphthaleneacetic acid +2.0 mg l−1 benzyladenine +80.0 mg l−1 adenine sulfate. The initial response of the explant and callus formation were ultrastructurally analyzed during the first 11 d of culture. After 6–8 wk, various methods were employed to collect evidence of indirect somatic embryogenesis. After 48 h of culture, the earliest cell response was cell division of epidermis and primary cortex. There were numerous disturbances of karyo- and cytokinesis, leading to formation of multinuclear cells. With time, the divisions ceased, and cortex cells underwent strong expansion, vacuolization and degradation. About the 6th day of culture, callus tissue proliferated and the initial divisions of vascular cylinder cells were observed. Their division appeared normal. Cells originating from that tissue were small, weakly vacuolated, with dense cytoplasm containing active-looking cell organelles. Numerous divisions occurred in the vascular cylinder, which led to its expansion and the formation of embryogenic callus tissue. During the 6–8th wk of culture, in the proximal end of the explant, masses of somatic embryos were formed from outer parts of intensively proliferating tissue.