The early stages of graft union, when male branch was grafted onto female branch in Ginkgo biloba L. by cleft graft, have been observed under light microscope in order to determine the origin of callus cells between the stock and scion. Pith parenchyma cells near the graft interface were the earliest cells in response to such method of grafting. These cells de differentiated and then divided within 7～12 days after grafting. A large number of callus cells extended from the pith into the space between the graft interface linking the stock with the scion about 18～20 days after grafting; and then continued to proliferate and extend outwards along the space. Cambium cells and immature vascular tissue near the graft interface dedifferentiated into callus rather late. The over all link between the stock and scion was completed in the sites 30 days after grafting. Callus cells were also produced from cortical parenchyma cells, but they were much limited in quantity. In conclusion, the graft interface may be considered as a “natural culture bed” after grafting, in which all undamaged, living cells are capable of dedifferentiation and producing callus cells for compatible graft union. In the case of G. biloba (male/female) it were the pith parenchyma cells that appeared first to form the callus cells and later extend to link the stock with the scion.
The early stage of pollen chamber development in ovule and the cytological mechanism of nucellar cell death were studied in Ginkgo biloba L. DNA ladder appearance and TUNEL assay demonstrated that the nucellar cell death, doomed to bring about pollen chamber formation, was a process of programmed cell death (PCD). A spatial distribution of PCD was visualized during the development of pollen chamber. Together with the observation under the scanning electron microscope, these results have revealed that the early developmental pattern of pollen chamber consists of four phases. Firstly, several layers of the nucellar cells at the micropylar end elongate longitudinally. Thereafter, the uppermost layer of the nucellar cells at the micropylar end initiate PCD; and the nucellar cell death extends in a basally and laterally oriented direction to form a cavity. Finally, the epidermal cells at the micropylar end detach from the other epidermis by dehiscence, bringing about the opening of the pollen chamber. The early development of pollen chamber begins sometime after the stage of megasporocyte and continues by the time of the formation of megaspore tetrad, and finally completes at the stage of development of female gametophyte. This shows a synchronous development of megaspore and pollen chamber.
Programmed cell death (PCD) of the nucellar cells at the micropylar end is involved in pollen chamber morphogenesis in Ginkgo biloba L. A development-course observation of the morphological changes in the nucellar cells undergoing PCD to form pollen chamber was performed. During the PCD, the nucellar cells degraded their cellular components through an orderly progression. Through the vactiolation, the cytosol was engulfed by the enlarging vacuole, leaving out various organelles, which remained morphologically integrated. As the vacuolation continued, the vacuole collapsed with the breakage of the tonoplast and the cytosol disappeared completely. Organelles were subsequently destroyed. Ultimately, nucellar cells digested away all of their cytoplasm, leaving with cell walls. They became collapsed as the nucellus developed. Intracellular membranes were strikingly changed, playing a role in leading to cell death. Some of these noticeable changes were the appearance of multivesicular body, multicycle-like membranes, membrane-bounded bodies containing some organelles, tonoplast rupture and numerous vesicles. The dehiscence of the apical epidermis, resulting in the opening, appeared to have followed two different pathways with one involving a specific epidermal cell autolysis and the other by detachment from middle lamella of two neighboring epidermal cells without cell autolysis. The specific epidermal cells had been dead prior to the dehiscence of the apical epidermis, which marked the sites of the dehiscence followed. In view of the changes in the cellular morphology, a process of nucellar cell PCD in the course of the pollen chamber formation was demonstrated.
