Phytosulfokine promotes cell division in protoplast culture and adventitious shoot formation in protoplast-derived calluses of Nicotiana benthamiana

Abstract

Phytosulfokines (PSK) belong to a new class of peptide growth factors involved in intercellular signaling in plants. In this study, we examined the effect of PSK on cell division and subsequent adventitious shoot formation in protoplast-derived callus culture of Nicotiana benthamiana. Protoplast cultures and protoplast-derived calluses of N. benthamiana were treated with different concentrations of PSK (0, 0.01, 0.03, 0.1, 0.3, and 1 μM). Cell division efficiency was the highest at 32% in the 0.1 μM PSK treatment, which was about two times higher than that of control treatment. PSK concentrations？<？0.1 μM were more effective in inducing cell division in the protoplasts than those？>？0.3 μM. In contrast, higher concentrations of PSK were more effective in inducing adventitious shoot formation in protoplast-derived calluses. The frequency of adventitious shoot formation was the highest at 8.5% when the calluses were treated with 1 μM PSK. qRT-PCR analysis showed an increase in the expression of genes involved in cell division and differentiation: G1/S specific cyclin (CYCD3-1), cytokinin dependent kinase (CDK), and WUSCHEL (WUS) at 0.1 μM PSK treatment. These results indicate that PSK plays a significant role in promoting cell division and adventitious shoot formation in N. benthamiana. The results obtained in this study could be applied to promote cell division and plant regeneration in protoplast cultures of diverse recalcitrant crop plant species.