Analysis Of Oxidative Stress During Protoplast Regeneration Of Ponkan

Protoplast regeneration is a complicated developmental process, including cell wall regeneration, cell elongation and division, and then re-entry into the cell cycle to form embryoids. In response to exogenous stimulus during the process, active oxygen species (AOS) will accumulate in the cell, causing oxidative stress, which may constitute a vital role in protoplast regeneration. Polyamines (PAs) can reduce the accumulation of active oxygen species (AOS), enhance the activity of antioxidative enzymes and affect cell development. In this work, differences in cell wall regeneration among protoplasts isolated from different materials was compared. In addition, the oxidative stress responses of protoplasts during cell wall regeneration by exogenously adding PAs and PAs inhibitor are discussed. The main results were as follows:1. Protoplasts from Ponkan callus and mesophyll were isolated after enzymolysis. After thin layer liquid culture, these protoplasts were subjected to Calcofluor White M2R staining for the observation of cell wall regeneration. The results showed that protoplasts from callus initiated cell wall regeneration after 3 days'culturing and finished at day 6; but the those from mesophyll could not initiate the regeneration and died gradually.2. Hydrogen peroxide levels and the activities of several antioxidants were analyzed in the protoplasts from callus and mesophyll. Although hydrogen peroxide level was increased gradually in both callus and mesophyll protoplasts, the former accumulated less. As for antioxidants, protoplasts from callus had higher activities during the culture, but the activities in protoplasts from mesophyll maintained undetectable change even declined.3. Addition of 10 mM D-Arg resulted in increased hydrogen peroxide accumulation and lowered antioxidase activities in the protoplasts from callus; but by adding 10 mM putrescine in the culture medium of protoplasts from mesophyll, hydrogen peroxide still continued to accumulate with unchanged antioxidase activities.4. Protoplasts from Ponkan callus were cultured in the medium with exogenous addition of PAs and PAs inhibitor, respectively. Calcofluor White M2R staining suggested that PAs can advance cell wall regeneration of the protoplasts while PAs inhibitor postpone. 5. Fluorescein diacetate (FDA) staining results indicated that the vitality of protoplasts isolated from callus was higher than that of the ones from mesophyll throughout the culturing period.6. Solid semi-embedding culture was employed to check cell division of the protoplasts from callus. The observation demonstrated that cell division of nearly all the protoplasts was initiated on day 7, indicating the end of cell wall regeneration.