Despite the complexity of their culturing, protoplasts are extensively used in research of plant development, physiology, molecular biology, production of somatic hybrids and cybrids, genetic transformation and more recently genome editing. Especially for the last three a high cell division frequency and successful plant regeneration is required. To achieve that with four cultivars of the species Brassica oleracea, we first tested two different protocols of culturing protoplasts, then we tested the effect of the mitogenic peptide PSK-α on protoplast regeneration and finally the effect of PSK-α or a suspension of untransformed feeder protoplasts on the regeneration of transformed protoplasts. We cultured mesophyll protoplasts of two cauliflower and two red cabbage varieties embedded in alginate matrix at lower densities than normally used for culturing protoplasts of this species which is also the case with transformed protoplasts as selection decreases the number of viable cells. The first protocol with changing media composition proved to be successful at regenerating all four cultivars while the second with constant media composition proved to be successful at regenerating only one cultivar. In the second part of the experiment, PSK-α increased microcallus regeneration frequency of only cauliflower cultivars while it had no effect on cabbage protoplasts. Finally, PSK-α exerted a stimulatory effect on regeneration of transformed protoplasts but it was of a smaller magnitude compared to the effect of feeder protoplast suspension. This finding together with the smallest effect of PSK-α at the lowest protoplast density indicated insufficient amounts of added PSK-α and/or that other yet unknown growth factors beside PSK-α are also crucial for protoplast regeneration.
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