Ultrastructural Observations on Proliferating Storage Cells of Mature Cotyledons of Phaseolus vulgaris L. Cultured in vitro

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Abstract
Explants of mature cotyledons of Phaseolus vulgaris form callus rapidly when cultured in vitro with their adaxial surfaces embedded in a solidified nutrient medium containing coconut milk, kinetin and 2,4-D. Proliferation is confined to the highly polyploid storage cells and commences near the adaxial epidermis, which is soon ruptured by the callus developing internally. Callus formation progresses to the abaxial tissue and within 3–4 weeks sub-culturing is possible. The in vitro grown storage cells undergo thinning of their walls, loss of food reserves, hypertrophy, development of various new-walls and nuclear activation leading to division. The induction of nuclear and cell divisions within this mature storage tissue contrasts with normal germination in which these cells undergo rapid senescence after depletion of their food reserves. Nuclear division in early callus growth is apparently mainly amitotic. It is preceded by the development of multiple nucleoli. The nuclear envelope also becomes more complex and deeply lobed; leading to formation of a nuclear isthmus and final separation into two nuclei. No chromosomes are visible during nuclear fragmentation. Amitosis is accompanied by freely-forming walls, which may develop adjacent to a nuclear isthmus and perhaps participate in nuclear fragmentation. Large labyrinthine wall bodies frequently occur on these walls. Mitoses are only observed in already divided storage cells. A cell plate forms between the two daughter nuclei, and microtubules are present at its margins in contrast to freely-forming walls where none are evident.

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