This research was conducted to establish the optimal in vitro culture conditions for proliferation and accumulation of bioactive compounds of R. australis shoot clumps. A 400 mL bottle and 3 L bioreactor were used to find out the optimal physical and chemical conditions of proliferation of shoot clumps and accumulation of bioactive compounds. Also, at the finishing part, an elicitor experiment was done to increase accumulation of bioactive compounds of shoot clumps.

1. Effect of types and concentrations of plant growth regulator on shoot clumps proliferation

A 4-week experiment was conducted to find out the most effective type and concentration of cytokinin (TDZ, BA, kinetin) on the proliferation of R. australis, proliferation of the shoot clumps was best in the medium supplemented with 0.5 mg/L kinetin. But in TDZ and BA treatments, shoot clumps hardened and showed browning reaction .
The shoot clumps biomass and rooting rate were significantly affected by auxin types and concentrations, resulted in the highest biomass and rooting rate on 0.5 mg/L IBA containing medium. But the fresh weight and dry weight in 0.5 mg/L IBA treatment were not as good as those in 0.5 mg/L kinetin treatment.
To study the combination effect of cytokinins and auxins, 0.5 mg/L TDZ, kinetin and BA were tested with different concentrations of IBA, NAA, IAA, respectively. Considering the stiffened shoot and browning phenomenon, combination of kinetin and NAA 0.1 mg/L was seen as the most effective. But all treatments did not show rooting.

2. Optimization physical and chemical factors for production of shoot clumps and bioactive compounds in bioreactor cultures

When shoot clumps of R. australis were cultured in a 3 L bioreactor, the fresh weight and dry weight were highest at 3/4 MS medium, but the contents of bioactive compounds (total polyphenolics, total flavonoids, coumarin) gradually declined with increasing MS medium salt strength. At 3/4MS medium, the biomass was approximately 3 times higher than that of the dry weight at 1/4 MS medium. Therefore, 3/4 MS medium was most effective for further production of biomass and bioactive compounds.
Both shoot clumps biomass and bioactive compounds increased with raising the inoculum density, but highest growth ratio was obtained at a 10 g/L inoculum.
An experiment to figure out what effect the supply of air supply rate has on the proliferation and accumulation of bioactive compounds of R. australis shoot clumps. Conclusively, aeration of 0.1 vvm showed the best biomass and bioactive compound accumulation.
Comparative studies on medium supply in bioreactors (raft, immersion, ebb and flood) revealed that immersion method was most satisfactory for shoot clumps biomass and accumulation of bioactive compounds due to constant supply of nutrients.

3. Enhancement of bioactive compound production by elicitors

To increase accumulation of bioactive compounds in R. australis shoot clumps using 3 L bioreactors, different concentrations of methyl jasmonate(MJ) and salicylic acid(SA) were added after 4 weeks of culture, and harvested after 5 days. The highest amount of bioactive compounds was obtained at 100 μmol MJ without decreasing biomass of shoot clumps. 50 μmol SA resulted in the highest accumulation of bioactive compounds but the effect was less significant than that of MJ.
In addition, identification of raoulic acid in R. australis shoot clumps was confirmed by LC-MS and MJ and SA additions significantly enhanced raoulic acid accumulation.