Cytoskeletal and biochemical events during early imbibition of maize embryonic axes and the impact of cryostorage
Seed Science and Technology
School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa
The caryopses of a Zea mays hybrid grown in South Africa could not withstand direct immersion in liquid nitrogen for cryopreservation. This was due to the size of the seed and the consequent uneven freezing and thawing of the component tissues. Isolation of the embryonic axes and their culture in vitro after cryopreservation overcame this problem. However, isolation of the axes from the air-dry seed was both damaging and difficult. Short imbibition periods were therefore used to soften the testa of the caryopses. Axes isolated from seed which had been imbibed for 5.5 and 6.5 hours although viable after cryopreservation did not produce roots (shoots were formed), whereas axes from seeds imbibed for shorter periods (0.5 - 4.5 hours) produced fully functional in vitro plantlets albeit at a slower rate than the unfrozen controls. Measurement of selected biomolecules in the embryonic axes and in the leachate obtained during imbibition revealed that the combined concentrations of the amino acids (including proline) and glucose were sufficiently high to act as cryoprotectants. The microfilament and microtubule components of the cytoskeleton became progressively more organised during imbibition however, they were disrupted by cryopreservation. The requirement for the repair and re-assembly of the cytoskeleton is thus considered to be, in part, related to the reduced vigour of the cryopreserved material. At and after 5.5 hours of imbibition, specific cytoskeletal arrays were identifiable (e.g. the actin nuclear basket) but these were completely disintegrated by the cryo-treatment. Since that material did not develop roots in vitro, it is suggested that cryopreservation had compromised the repair processes including those associated with the re-assembly of the cytoskeleton. The transition from desiccation tolerance to sensitivity has been linked with the resumption of metabolic activity. The present data imply that the root meristematic region of the 0.5 - 4.5 hours-imbibed material still retained desiccation tolerance (and hence cryostorability) but this was lost at 5.5 hours imbibition. It also suggests that in maize the root meristem loses desiccation tolerance before the shoot meristem.