Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.