Early studies on the effect of peptide growth factor phytosulfokine-α on Brassica oleracea var. capitata L. protoplasts

Abstract

Phytosulfokines (PSK) are peptidyl growth factors with the potential of inducing cell proliferation. We examined the effect of supplementation of liquid culture medium with 0.1 µM phytosulfokine-α (PSK-α) on protoplast viability and division frequencies in seven accessions of Brassica oleracea var. capitata L., including cultivars and breeding lines. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants and immobilized in calcium-alginate layers. Cabbage protoplast-derived cells cultured in medium supplemented with 0.1 µM of PSK-α had higher viability and division frequencies compared to cells cultured in PSK-α-free control medium. The effect of PSK-α was more pronounced in low-responding accessions ('Sława z Gołębiewa' , 'Ramkila F1' , LM, and LM98); however, in two cultivars with very low response ('Badger Shipper' and 'Oregon 123'), although the division frequencies in the media supplemented with PSK-α were increased over the control, the differences were not significant. Obtained callus colonies were subjected to regeneration. PSK-α supplemented into the liquid culture medium had an indirect effect on shoot regeneration by inducing sustained cell divisions leading to an increase in shoot regeneration in Sława z Gołębiewa and both breeding lines.

Peptide Growth Factor Phytosulfokine-α Stimulates Cell Divisions and Enhances Regeneration from B. oleracea var. capitata L. Protoplast Culture

“…In this study, the protoplast-derived cells were cultured in the presence of PSK-α for 10 days, as the media used for replacement was PSK-free. In our previous study, 0.1 µM PSK-α was present in the culture medium throughout whole liquid culture phase and was removed before regeneration; the division frequencies for selected accessions were as follows: Sława z Gołębiewa 38%, Oregon 123 4.5%, LM 33%, LM98 47% (Kiełkowska and Adamus 2017). Comparison of those results with the corresponding results from the present study (0.1 µM PSK-α and the same accessions) (Fig.…”

“…Interestingly, in low-responding Oregon 123, supplementation of culture medium with 0.1 µM PSK-α for 10 days stimulated division in a higher number of cells (14%, Fig. 1) in comparison with a 4-week culture in the presence of this peptide (4.5%) (Kiełkowska and Adamus 2017).…”

“…The results showed an approximate 3-5-fold increase in the frequency of regeneration from protoplastderived calluses cultured on the regeneration media containing PSK-α in comparison to PSK-free controls. In our previous study, we observed an indirect effect of PSK-α on regeneration, reflected in the higher number of shoots produced form cultures supplemented with PSK-α at the liquid culture stage and transferred for regeneration onto PSK-α free media (Kiełkowska and Adamus 2017). The results obtained in this study also support these observations, as the increase in regeneration from calluses developed from protoplast-derived cells cultured in the presence of PSK-α (2-4%) was also detectable in comparison to the PSK-free controls (0.7-0.8%).…”

“…Supplementing the culture medium with PSK (either chemically synthesized or isolated from conditioned medium) promoted proliferation of cells in suspension cultures (Matsubayashi et al , 1997Hanai et al 2000;Grzebelus et al 2012), and these results were the basis for performing our experiments. A preliminary study on the supplementation of the medium for culturing B. oleracea protoplasts with 0.1 µM PSK-α showed its beneficial effect on cell division, and the effect of PSK-α was more pronounced in low-responding accessions (Kiełkowska and Adamus 2017). In this study, we wanted to examine (i) whether the effect of PSK-α on cabbage protoplasts is dose-dependent, (ii) whether PSK-α plays a role in cell wall regeneration, which is a prerequisite for cell division, and (iii) whether PSK-α has a direct effect on organogenesis from protoplast-derived callus colonies.…”

“…Our preliminary results showed that PSK added to the culture medium stimulated protoplast proliferation especially in low-responding cabbage genotypes (Kiełkowska and Adamus 2017). This study aims to evaluate the effects of broader spectra of PSK-α concentration in the culture medium on cabbage protoplasts, as well as its effect on cell wall regeneration.…”