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.

苋菜是一种重要的药用植物，其根茎可用作蔬菜和药用食品。在本研究中，我们成功地从大花杨叶肉组织中分离出原生质体，以促进愈伤组织的发育。原生质体分离的理想条件已经确定，包括酶的组合、渗透压和酶消化的时间长度。为了获得最有效的结果，使用含有 1.0% Viscozyme® L + 3% Celluclast® 1.5 L + 0.5% Pectinex® XXL + 0.4 M 甘露醇的细胞原生质体洗涤 (CPW) 培养基并孵育 8 小时，从叶肉组织中分离原生质体。利用薄藻酸盐层（TAL）方法来包埋原生质体。随后，TAL 片段在 Murashige 和 Skoog (MS) 培养基中培养，添加 3% 蔗糖、2,4-二氯苯氧基乙酸 (2,4-D, 0, 0.56, 1.12 µM) 和植物磺因子 (PSK, 0, 0.05, 0.1 µM）。使用响应面法 (RSM) 和中心复合设计 (CCD) 预测启动原生质体分裂、多细胞发育和微愈伤组织形成的 2,4-D 和 PSK 的最佳浓度。根据 CCD 响应，确定 1.12 µM 2,4-D 和 0.065 µM PSK 是诱导原生质体分裂和微愈伤组织形成的最佳浓度。所得结果对大花大花原生质体分离培养具有参考价值；然而，利用原生质体产生的愈伤组织再生植物需要协调一致的努力。