Materials and Methods
Induction of somatic embryogenesis and maintenance of stock cultures of embryos
Young leaves (2 cm in length) of E. senticosus were collected from in vitro grown plants and cut into 5 x 5 mm pieces, cultured on Murashige and Skoog medium (MS, Murashige and Skoog, 1962; pH 5.8; Duchefa, Haarlem, Netherlands) with 1 mg L-1 2,4-dichlorophenoxy acetic acid (2,4-D), 3% (w/v) sucrose and 0.2% (w/v) gel rite and cultures were maintained in dark at 25ºC. Embryogenic callus was developed from the leaves within twelve weeks after culture. Embryogenic callus was maintained on MS liquid medium supplemented with 1 mg L-1 2,4-D, 3% (w/v) sucrose and 0.2% (w/v) gel rite by sub-culturing once in four weeks.
Embryogenic cell suspension culture
Embryogenic cells of E. senticosus were transferred to MS liquid medium supplemented with 1 mg L-1 2,4-D and suspension cultures were sub-cultured at every two weeks interval. To induce somatic embryos, 2 weeks old embryogenic cell clumps were filtered through a sterile 212 µm stainless steel sieve to remove the larger clumps. The suspension was allowed to settle for 5 min for easier removal of the used medium. About 500 mg of cell clumps was transferred to 100 mL MS liquid medium without 2,4-D in 300 mL Erlenmeyer flasks. The cultures were incubated at 100 rpm on a gyratory shaker (SI-600R, Jeio Tech, Seoul, South Korea) in dark at 25ºC. At the end of four weeks of culture, the content of flask was passed through different stainless steel sieves to separate different stages of embryos (>800 µm = cotyledonary; 600 µm = torpedo; 420 µm = heart;
Establishment of large scale suspension cultures in bioreactors
Ten grams of cotyledonary somatic embryos were transferred to 3 L balloon type bubble bioreactor with 2 L MS liquid medium with 3% (w/v) sucrose and 4 mg L-1 GA3. The pH of the medium was adjusted to 5.8 before autoclaving by using 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. The volume of input air was adjusted to 0.1 v/v (air volume/culture volume) per min. Cultures were kept under a 16 hrs photoperiod at 35 µmol m-2 s-1 photosynthetic photon flux. In an elicitation experiment different concentrations of MJ (0, 50, 100, 150, 200, 300 or 400 µM) was added to the cultures on the day of inoculation. The fresh and dry weights were recorded after 6 weeks of culture. Dry weight was determined after drying the biomass for 24 hrs at 60ºC. Data were subjected to Duncan's multiple range tests using SAS program (Version 6.12, SAS Institute Inc., Cary, USA).
Determination of eleutherosides
Germinated somatic embryos were dried and powdered (2 g) with a blender and extracted with 60% aqueous methanol (2 x 50 mL) for 30 min. each at 60ºC, and filtered through filter paper (No. 2, 90 mm, Advantec, Toyo, Japan). The combined extract, was evaporated to dryness in vacuum and washed with 50 ml of ether. The insoluble fraction was dissolved in water and extracted with n-butanol (water saturated). The organic phase was evaporated to dryness, dissolved in (10 mL) high performance liquid chromatography (HPLC) grade methanol and filtered through 0.45 µm membrane filter (Polyvinylidene difluoride, Whatman, USA) filter. Eleutherosides were quantified by HPLC (Waters 2690 separation modules, Waters, USA) equipped with a Symmetry® C 18 column (4.6 mm x 250 mm, Waters, USA) by following the procedure described previously (Apers et al. 2005). Eleutherosides and chlorogenic acid were separated using a flow rate 0.8 mL min-1 with water (solvent A) and acetonitrile (solvent B) as the mobile phase. The elution programme was: initially 90:10 (A:B) with isocratic elution for 5 min followed by linear gradient to 80:20 in 22 min, linear gradient to 60:40 in 15 min, isocratic for 5 min, linear gradient to the starting conditions (90:10) in 5 min and isocratic for 5 min (equilibration time). Quantification was based on ultraviolet absorption at 216 nm. The peak areas corresponding to eleutherosides from the samples, with same retention time as authentic eleutherosides and chlorogenic acid. Retention times were 9.86, 13.10, 21.90 and 31.20 min for eleutheroside B, chlorogenic acid, eleutheroside E and E1 respectively.
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