Subcellular Changes in Asplenium During Culture and Senescence

Abstract

The use of tissue culture for the study of the control of development in plants has long been known. There has however been little investigation of fern sporophyte callus induction and subsequent organogenesis using tissue culture. Preliminary investigations into the tissue culture of leaf tissue of three Asplenium species; A. bulbiferum, A. oblongifolium and A. flaccidum using a variety of growth regulators and culture media showed that callus could reliably be produced only from A. bulbiferum on media containing 5 mg 1-1 2,4-D, when grown in the dark. Using the calli produced from A. bulbiferum in this manner to investigate organogenesis, it was found that the calli readily produced roots but never shoots, even when grown on media containing no auxin. However, necrotic callus left in low light for several months occasionally formed aposporous gametophytes. Much of the tissue necrosed on the culture plates rather than undergoing senescence. To see how these two processes, necrosis and senescence, were different an ultrastructural investigation of A. bulbiferum was carried out. Ultrastructurally the young and mature leaves of the three Asplenium species were not significantly different. A study of the young, mature and senescing leaves of A. bulbiferum was undertaken and compared with the ultrastructure of necrosing leaf explant material, and callus tissue. This work was supplemented by histological investigations of these tissues using Sudan and PAS stains. The early stages of senescence and necrosis were found to be similar though later stages were profoundly different, especially with regard to the fate of the chloroplasts. In necrosis the chloroplasts become electron opaque, with the membranes becoming swirled. These remnant chloroplasts then coagulate against the cell walls, which have an electron dense lining. In senescence the chloroplasts become chromoplasts, which then empty of contents, and the membranes disappear, and the cells become empty except for spherical, bodies. The protein changes occurring during senescence and necrosis were investigated using one- and two- dimensional polyacrylamide gel electrophoresis. The differences between immature and mature leaves were so numerous that a close comparison between necrotic cultured tissue and mature senescent tissue was not possible. However different stages of senescence were studied using two-dimensional PAGE and five proteins wore chosen for further investigation using MALDI.-TOF mass spectrometry. One of these proteins could be related to a cytochrome C1 heme protein precursor of potato, but only extremely tentatively.