Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll ...cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of
plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on
cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four
cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.
Sunflower, as one of the most important oil-producing crops, represents an important target for genetic improvement through gene transfer or somatic hybridization. Unfortunately, sunflower is ...recognized as recalcitrant to in vitro culture. The aim of our paper was to improve sunflower protoplast regeneration. Three cultivars (Romanian hybrids) and one inbred line were used for protoplast isolation from etiolated hypocotyls. Isolated protoplasts were embedded in alginate disks and cultured in two plating densities, using two culture regimes as indicated by previous authors. Plating efficiency, callus development and plant regeneration were evaluated as well as old callus histology. In cv. ‘Select’, the effects of 1:50 haemoglobin and 1 mM spermidine were assayed on asymmetric division and/or plating efficiency. Plant regeneration from hypocotyl protoplasts was achieved for two cvs., ‘Florom 328’ and ‘Turbo’, with the former proving once more its totipotency. The best culture regime proved to be as recommended by Krasnyanski and Menczel (1993), but the best density in the culture medium was the highest ever tested, 8 × 105 pp ml−1. Moreover, the histology of old green compact protoplast-derived callus revealed a very well organized structure suggesting senescence. In the non-responsive cv. ‘Select’, haemoglobin was found to stimulate protoplast asymmetric division and the development of heart-shaped embryo-like structures, while spermidine stimulated overall protoplast plating efficiency.
