Ultrastructural changes in zygotic embryos of Gentiana punctata L. during callus formation and somatic embryogenesis

Imbibed immature zygotic embryos of Gentiana punctata L. were cultured on MS (Murashige and Skoog) medium consisting of 4.5 µM dicamba, 0.54 µM NAA (naphtaleneacetic acid), 8.88 µM BAP (6-benzylaminopurine) and 0.43 mM AS (adenine hemisulfate). The primary response of expiants consisted in thickening of the subcotyledon and hypocotyl root (HR) zone. Cotyledons and the seminal root did not show any response. Ultrastructural analysis of the initial stages of callus formation revealed numerous changes in cells of expiants. Dedifferentiation of the explant tissues was associated with separation of cells resulting from thickening and folding of walls, destruction of plasmodesmata, and enlargement of intercellular spaces. At the same time, the number of lipid bodies decreased and starch appeared. Indicative of changes in 3-day cultures, the first cell divisions were observed to occur in the HR zone, including cells of the primary cortex, endodermis and pericycle. The dividing cells contained small vacuoles, large, centrally located, layered nuclei with vacuolated nucleoli, amyloplasts with starch, lipid bodies, numerous active Golgi structures, mitochondria and rough endoplasmic reticulum. Actively dividing cells formed callus tissue in which three zones of cells could be distinguished after 14 days of culture: (I) outer (starch) layer, (II) middle layer with actively at dividing small cells, and (III) inner layer containing large vacuolated cells. As the result of cell divisions, at about the fourth week of culture the starch zone formed meristematically active centers of small cells, with dense cytoplasm and large amounts of starch. Among them were small cellular complexes consisting of three cells, with the cell wall structure typical for pre-embryos. By the fifth week of culture, numerous globular and early heart-shaped somatic embryos which formed cotyledons were observed, and further mature somatic embryos showing conversion ability.