Isolation, culture of Platycodon grandiflorus protoplasts: factors affecting protoplast yield, cell division, and micro-callus formation

Abstract

Plyatocodon grandiflorus is an important medicinal herb, its rhizomes are utilized as vegetables and as medicinal food. In this investigation, we were able to successfully isolate protoplasts from P. grandiflorus leaf mesophyll tissues in order to promote the development of calli. It has been determined what are the ideal conditions for protoplast isolation, involving the combination of enzymes, the osmoticum, and the length of enzyme digestion. For the most effective results, cell protoplast washing (CPW) medium with 1.0% Viscozyme® L + 3% Celluclast® 1.5 L + 0.5% Pectinex® XXL + 0.4 M mannitol and 8 h of incubation was used to isolate protoplasts from leaf mesophyll tissues. The thin alginate layer (TAL) approach was utilized to embed the protoplasts. Subsequently, TAL segments were cultured in Murashige and Skoog (MS) media supplemented with 3% sucrose, 2,4-dichlorophenoxyacetic acid (2,4-D, 0, 0.56, 1.12 µM), and phytosulfokine (PSK, 0, 0.05, 0.1 µM). The optimal concentration of 2,4-D and PSK for initiation of protoplast division, multicell development, and micro-callus formation was predicted using response surface methodology (RSM) with central composite design (CCD). Based on the CCD responses, it was determined that 1.12 µM 2,4-D and 0.065 µM PSK were the best concentrations to induce protoplast division and the formation of micro-calluses. The acquired results are valuable for P. grandiflorus protoplast isolation and culture; however, coordinated efforts are required for plant regeneration using callus produced from protoplasts.