Optimization of callus, cell suspension and adventitions root cultures for the production of active components in Stevia rebaudiana

Abstract

Stevia rebaudiana Bertoni is an important plant known for antidiabetic steviol glycosides and several other bioactive compounds. Owing to the importance of Stevia plant and demand of natural secondary metabolites, the current research was conducted at Plant Tissue Culture Lab., Departmnet of Plant Breeding and Genetics, The University of Agriculture Peshawar, during the year of 2014-15. The objectives of the study were to optimize/evaluate the effect of sucrose (05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1), pH (5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0) and inoculum sizes (0.5, 1.0, 1.5 and 2.0 g) on biomass yield and valuable secondary metabolites accumulation in callus, cell and adventitious root cultures. Preliminary experiments were also carried out to optimize the effect of various monochromatic spectral lights (white, blue, green, yellow and red) on callus culture growth and secondary metabolites production. To check the effect of sucrose, media pH and inoculum sizes on cultures productivity, research work was conducted in Complete Randomized Design (CRD). Each culture was planned for a period of 30 days and 3 days intervals were kept to develop growth curves. The control light (16/8 hr) resulted in the maximum callogenic response (92.73%) along with the accumulation of maximum biomass (5.78 g l-1) during prolong log phase at 18th day of culture. Blue light was found the most effective for the production of total phenolics content (TPC), total flavonoids content (TFC) along with total antioxidant capicity (TAC) (102.32 μg/g-DW) (22.07 μg/g-DW) and (11.63 μg/g DW), respectively. On the other hand, reducing power assay (RPA; 0.71 Fe (II) g -1 DW) and DPPH-radical scavenging activity (DRSA; 80%) were considerably enhanced as a result of green and red lights, respectively. Sucrose concentrations also affected the biomass accumulation, growth kinetics and secondary metabolites production. The lag phase of 9 days was followed by log phase till 27th day of culture was recorded in callus cultures on 05, 10, 15 and 20 g l-1 sucrose. The rest of the cultures did not show a lag phase. All cultures, except a few displayed nonviability after 27 days of inoculation. The cultures initiated in media having 20, 25 and 30 g l-1 sucrose were found in stationary phase after 27 days of log phase. Similarly, cell cultures grown on 05-30 g l-1 sucrose concentrations displayed relatively shorter lag phase of 3 days as compared to 12 days lag phase in cultures with 35-50 g l-1 sucrose concentrations. The Lag phase in each cell culture was preceded by log phase till 18th day. Most cultures showed a stationery phases with or without decline phases. However, growth curve of adventitious root cultures was characterized with direct log phase till 18th day. The lag phase of 15 days was observed in cultures developed in media having 5 and 10 g l-1 sucrose, followed by very short log phase. The fresh and dry biomass of callus, cell suspension and adventitious root cultures was significantly affected by sucrose concentrations. The highest fresh and dry biomass (142.38 g l-1and 11.71 g l-1) in callus culture was with 40 g l-1 and 50 g l-1 sucrose, respectively. However, the maximum total phenolics content (TPC; 124.20 mg/g-DW), total flavonoids content (TFC; 49.36 mg/g-DW), rebaudioside contents (6.56 mg/g-DW) and antioxidant activity (92.82 %) in callus cultures was observed at sucrose concentration of 30 g l-1. By contrast, the highest stevioside (42.34 mg/g-DW) and rebaudioside (22.67 mg/g-DW) contents were recorded in callus culture grown at 15 and 20 g l-1 sucrose, respectively. In cell suspension culture, media having 20 g l-1 sucrose resulted in the maximum fresh (97.71 g l-1) and dry (8.57 g l-1) but the highest TPC (139.20 mg/g-DW) and TFC (41.46 mg/g-DW) was at 40 g l-1 sucrose. The highest antioxidant activity (83.87%) was observed at 30 g l-1 sucrose. While the stevioside content (40.32 mg/g-DW) was the maximum on media supplemented with 10 g l-1 sucrose, the highest rebaudioside (27.64 mg/g-DW) and dulcoside (6.43 mg/g-DW) contents were observed with 20 g l-1 sucrose. In case of adventitious root culture, the maximum fresh (175.43 g l-1) and dry (11.14 g l-1) biomass was accumulated in cultures having 50 g l-1 sucrose but the highest TPC (155.00 mg/g-DW) and TFC (94.78 mg/g-DW) were recorded with 30 g l-1 sucrose. While the highest antioxidant activity (94.43 %) was recorded in culture, established in media augmented with 20 g l-1 sucrose. The stevioside (73.97 mg/g-DW) and rebaudioside (24.57 mg/g-DW) content were the highest in media containing 10 g l-1 sucrose. By contrast, the dulcoside content (12.24 mg/g-DW) was the maximum at 40 g l-1 sucrose. It is suggested that sucrose concentration modulates biomass and metabolites of interest in callus, cell suspension and adventitious root cultures of Stevia rebaudiana. The media pH significantly influenced fresh and dry biomass of callus, cell suspension and adventitious root cultures. The biomass accumulation revealed a short lag phase of 3 days in callus cultures on media pH 4.0, 5.9 and 6.0; and cell suspension culture on pH 5.6 and 5.7; while the adventitious roots culture expressed a lag phase of 3 days on media pH 5.5-6.0. The log phase was followed by decline phases in callus and cell suspension cultures. However, root growth was not restricted in cultures having pH 5.2 and 5.4 even after 27 days of the culture. pH 5.6 was optimized for the maximum fresh (130.57 g l-1) and dry biomass (12.10 g l-1) of callus cultures. However, the highest TPC (43.38 mg/g-DW), TFC (37.55 mg/g-DW) and antioxidant activities (87.68 %) in callus cultures were recorded on media pH 5.6. Media pH 5.6 was also found optimum for the maximum stevioside (62.20 mg/g-DW) and rebaudioside (22.79 mg/g-DW), while 5.1 for dulcoside (5.92 mg/g-DW) production in callus cultures. Similarly, the maximum fresh and dry biomass (85.81 g l-1; 8.84 g l-1) of cell suspensions were observed on media pH 5.6 and 5.5, respectively. The highest TPC (72.13 mg/g-DW), TFC (57.32 mg/g-DW), DRSA (93.99%), rebaudioside (7.01 mg/g-DW) and dulcoside (4.72 mg/g-DW) contents were observed in cell suspension cultures established in media having pH 5.8. However, stevioside contents were induced to their maximal level (41.47 mg/g-DW) at pH level 5.2 in cell culture. In contrast, the highest fresh (112.86 g l-1) and dry (8.29 g l-1) biomass were accumulated in adventitious root culture on media pH 6.0. The maximum TPC (70.06 mg/g-DW), TFC (50.19 mg/g-DW), DRSA (92.67 %) and dulcoside contents (2.57 mg/g-DW) in adventitious root cultures were recorded on media pH 5.8. However, the highest stevioside (79.48 mg/g-DW) and rebaudioside (13.10 mg/g-DW) contents in adventitious root cultures were accumulated at 5.1 media pH. Various inoculum sizes also had significant influence on growth kinetics, biomass and secondary metabolites production in callus, cell suspension and adventitious root cultures. Relatively short lag phase of 3 days in callus cultures, while elongated lag phases from day 3rd to 12th day of the cultures developed from inoculum sizes (0.5-2.0g) was recorded. However, adventitious root cultures did not display lag phases. An increase in biomass with elongated log phases from day 3rd to 27th day of the culture was observed in callus cultures. Among all inoculum sizes, 2.0 g started sudden increased in biomass accumulation up to 15 days and increments in growth was further continued till 27th day of culture. Log phase was followed by sudden decline phase without having any stationery phase in all cultures. Similarly, the highest fresh and dry biomass (112.29 g l-1; 7.71 g l-1) in callus cultures was observed at 2.0 g inoculum. However, cultures developed from smaller inoculum (0.5 g) resulted the maximum TPC (28.54 mg/g-DW), TFC (24.78 mg/g-DW), DRSA (77.57 %), stevioside (43.89 mg/g-DW) and rebaudioside (36.54 mg/g-DW) contents in callus cultures, while the highest dulcoside contents (2.57 mg/g-DW) were observed at 1.0 g inoculum. On the other hand, Cell suspension also accumulated the maximum fresh (102.71 g l-1) and dry biomass (5.38 g l-1) at 1.5 g inoculum. Moreover, the highest TPC (45.36 mg/g-DW), TFC (36.50 mg/g-DW), stevioside (59.89 mg/g-DW), rebaudioside (24.41 mg/g-DW) and dulcoside (1.85 mg/g-DW) contents were found in cell cultures established from 0.5 g inoculum. However, the maximum DRSA (78.30%) was found in cell cultures having initial inoculum size 2.0 g. Similarly, adventitious root cultures accumulated the maximum fresh biomass (106.86 g l-1), dry biomass (5.05 g l-1) and dulcoside contents (0.71 mg/g-DW) at 1.5 g inoculum. Inoculum size 2.0 g was optimized for the maximum TPC (41.46 mg/g-DW), TFC (33.44 mg/g-DW) and DRSA (98.82 %). However, stevioside (64.75 mg/g-DW) and rebaudioside (29.67 mg/g-DW) contents were significantly increased to their maximal level using initial inoculum size (1.0 g). Herein, we concluded that the utilization of various colored spectral lights, concentrations, pH levels and inoculum sizes are promising strategies for enhanced biomass yield and secondary metabolites production in callus, cell suspension and adventitious root cultures of Stevia rebaudiana.