Studies On Cell Culture Of Gentiana Straminea Maxim And Gentiana Macrophylla Pall

Gentiana straminea Maxim and Gentiana macrophylla Pall, are perennial herb inthe family Gentianaceae. They distributed mainly in Tibet, Qinghai, Shaanxi, Gansu,Sichuan, and are the key-protected wild medicinal materials in China. Demand for G.straminea and G. macrophylla in the pharmaceutical industry is met from the wild. Asa result of over harvesting and lack of organized cultivation, the wild resources of G.straminea and G. macrophylla have reduced rapidly. Therefore, it is imperative todevelop appropriate tissue culture techniques to safeguard these species. In this study,G. straminea and G. macrophylla were chosen as experimental materials. The callusculture and cell suspension culture of the two species, as well as the changes ofgentiopicroside contents during culture were studied. These would provide technicalbasis for the further researches on the production of secondary metabolites throughcell culture in G. straminea and G. macrophylla.The results obtained were as follows:1. Taking the leaf of G. straminea as explants, the highest frequency (90.6%) ofcallus induction was obtained on MS medium supplemented with2.0mg·L-12,4-Dand0.5mg·L-16-BA, and0.85%(w/v) agar.2. Taking the leaf of G. macrophylla as explants, the highest frequency (87.7%)of callus induction was obtained on MS medium supplemented with1.5mg·L-12,4-Dand0.5mg·L-16-BA, and0.85%(w/v) agar.3. The callus culture of G. straminea was studied. The growth curve of calli was“S” type. The growth period was33days and composed of four stages, namely lagphase, exponential phase, stationary phase and decline phase. The mitotic indexes inthe corresponding periods were22.18,54.23,67.55and26.97%, respectively. Thecontents of gentiopicroside in the corresponding periods were7.92,15.01,25.02and14.04mg·g-1DW, respectively. The accumulated gentiopicroside increased along withthe callus proliferation. The gentiopicroside contents reached the highest level in thestationary phase.4. The callus culture of G. macrophylla was studied. The growth curve of calliwas “S” type. The growth period was33days and composed of four stages, namelylag phase, exponential phase, stationary phase and decline phase. The mitotic indexesin the corresponding periods were25.54,47.09,63.56and34.15%, respectively. The contents of gentiopicroside in the corresponding periods were11.80,16.38,22.49and8.79mg·g-1DW, respectively. The accumulated gentiopicroside increased along withthe callus proliferation. The gentiopicroside contents reached the highest level in thestationary phase.5. The morphological structures of different growth stages of callus cells of G.straminea and G. macrophylla were studied. The cells were small, nearly sphericalshape in lag phase; Cells were nearly equilateral shape in exponential phase with richcytoplasm and strong division activities; Cells began to elongate in stationary phase;Cells were long tube shape in decline phase. With the growth of cells, the plastidswere gradually mature and began to disintegrate in decline phase.6. The effects of different inoculum densities, concentration of sucrose, rotationspeed, temperature, medium, and elicitors on cell suspension culture of G. stramineawere studied. MS medium, inoculum densities of30g·L-1,3%(w/v) sucrose, rotationspeed of110r·min-1, temperature of22℃,100mg·L-1YE or5g·L-1L-fructose werethe optimum conditions for cell growth and gentiopicroside synthesis in G. straminea.7. The effects of different inoculum densities, concentration of sucrose, rotationspeed, temperature, medium, and elicitors on cell suspension culture of G.macrophylla were studied. MS medium, inoculum densities of20g·L-1,3.5%(w/v)sucrose, rotation speed of110r·min-1, temperature of22℃,100mg·L-1CH or5g·L-1L-fructose were the optimum conditions for cell growth and gentiopicroside synthesisin G. macrophylla.8. Based on the optimum conditions of cell suspension culture, the changes ofgentiopicroside contents during cell suspension culture in G. straminea were studied.The growth curve of suspension cell was “S” type. The growth period was30daysand composed of four stages, namely lag phase, exponential phase, stationary phaseand decline phase. The gentiopicroside contents of intracellular in the correspondingperiods were16.53,20.14,23.86and14.63mg·g-1DW, respectively. Thegentiopicroside contents of extracellular in the corresponding periods were5.22,7.97,9.11and11.10mg·g-1DW, respectively. The gentiopicroside contents reached thehighest level (32.97mg·g-1DW) in the stationary phase. The accumulatedgentiopicroside increased along with the suspension cell proliferation.9. Based on the optimum conditions of cell suspension culture, the changes ofgentiopicroside contents during cell suspension culture in G. macrophylla werestudied. The growth curve of suspension cell was “S” type. The growth period was30 days and composed of four stages, namely lag phase, exponential phase, stationaryphase and decline phase. The gentiopicroside contents of intracellular in thecorresponding periods were8.69,18.82,23.22and11.44mg·g-1DW, respectively.The gentiopicroside contents of extracellular in the corresponding periods were5.92,6.81,7.82and8.94mg·g-1DW, respectively. The gentiopicroside contents reached thehighest level (31.04mg·g-1DW) in the stationary phase. The accumulatedgentiopicroside increased along with the suspension cell proliferation.