Mechanisms And Control Of Browning In Taxus Cell Cultures

Plant cell cultures for the production of valuable secondary metabolites is one of the promising alternatives of plant resources, and this technology has been widely extended in production of heterologous protein. However, there are many obstacles in the appliocation and study of plant cell cultures. Browning phenomenon, occurring in the whole scale-up process of plant cell cultures, is one of the problems, and should be resolved as the first problem. Nevertheless, the browning mechanisms are still unclear due to the characteristics of plant cells and the complicated environments of plant cell cultures. Up to now, there still have been no more targeted, effective and applicable approaches for controle of this browning problem. Therefore, the Taxus chinensis cells, with application promising with severe browning problem, are selected as typical materials fot the study of browning mechanisms in plant cell cultures, and the more targeted, effective and applicable approaches for controle of this browning problem are presented. The main study results are as follows:(1) Presented the browning characteristics in plant cell cultures after a systematic study. Browning phenomenon occurs during the whole scale-up process of plant cell cultures at room temperature without heat treatment. And the browning phenomenon still occurred when the media only contain components of sucrose that added by filtration sterilization. Sodium thiosulfate could prevent browning via inhibiting polyphenol oxidase (PPO) activites.(2) Study and analyzed the relationships between factors in Taxus chinensis cell culture environments (media components, osmotic pressure, fluid shear stress, dissoloved oxygen, sucose concentration, carbohydrates) and enzymatic browning. The resuls confirmed that although all the factors can change the browning degree in Taxus chinensis cell cultures, sucrose in media got the biggest effects, and when there was no sucrose in media, there wo no browning occurrence, and when the sucrose content in media elevated, the browning degree was also promoted; Over-expression of the key enzyme NINV gene in sucrose metabolism could promote browning degree via enhancing phenolic biosynthesis. Furthermore, glucose and fructose as carbohydrates in meida could also lead to occurrence of enzymatic browning, but non-metabolizable sugar mannitol and sorbitol couldn’t lead to occurrence of enzymatic browning. Under the premise condition of metabolic sugar existence in media, osmotic pressure or fluid shear stress mainly promote the leakage and contact of PPOs and phenolics to induce the reaction of enzymatic bowning; Dissolved osxygen mainly enhance the phenolic biosynthesis to elevate the browning degree.(3) Flavonoids are the main phenolic compounds that result in enzymatic browning in Taxus chinensis cell cultures. Two cell lines, new induced6-months cell lines with severe browning problem and long subcultred10-years cell lines without browning were compared, and flavonoids were primarily deduced to be the main phenolic compounds leading to enzymatic browning. Two peaks of the UV scanning atlas of the browning compounds extracted fom media confirmed that flavonoids are the main compounds participated in the enzymatic brownin. HPLV-MS/MS analysis of the browning conpounds further deduced that myricetin and quercetin are the specific flavonoids that contribute to the enzymatic browning. GA3, as an inhibitor, could prevent browning occurrence of enzymatic browning in Taxus chinensis cell cultures via inhibiting flavonoid biosynthesis. After study and analysis of transcriptional and flavonoid profiles with GA3treatment, it was found that although majority of enzemy genes in phenylpropanoid pathway and flavonoid biosynthesis pathway were up-regulated after GA3treatments,4CL gene expression level in phenylpropanoid pathway and FLS gene expression level in flavonoid biosynthesis pathway were all surppressed, and total flavonoid biosynthesis, and myricetin and quercetin biosynthesis were significantly inhibited,leading to prevention of enzymatic browning.(4) The browning mechanisms in Taxus chinensis cell cultures were revealed according to above results, and3approaches for the controle of enzymatic browning according to the mechanisms were presented.①Sucrose perfusion culture:Decrease the sucrose content in the initial cultured media to decrease the phenolic biosynthesis and browning degree.②Small cell aggregates cultures: Small cell aggragates cultures could decrase the injury of fluid sheare oo cells and prevent the browning phenomena. In addition, small cell aggragates cultures could also promote biomass and taxane zccumulations in Taxus chinensis cell cultures.③Application of gibberellic acid (GA3): GA3could prevent browning occurrence of enzymatic browning in Taxus chinensis cell cultures via inhibiting flavonoid biosynthesis, and also could promote biomass accumulation.