Bioreactor Culture Of Mutiple Buds And Enhancement Of Effective Compound Accumulation In Anoectochilus Roxburghii(Wall.)Lindl

Anoectochilus roxburghii(Wall.)Lindl is a rare plant with high medicinal value,however,the plant material of A.roxburghii is difficult to meet the market demand because of the deficient wild resource and low yield of artificial cultivation.To provide multiple buds as alternative raw meterials using in the product production,the present study cultured multiple buds(MBs)in bioreactors to investigate factors affecting biomass and effective compounds accumulation;to improve the effective compounds production,the effects of elictors were studied.To select a suitable mudium during the MBs culture,the uniform design was used to design two sets of experiments.In the first experiment,effects of combination groups of MS medium strength and concentration of plant growth regulators were investigated and found that 3/4MS mudium supplemented with 2.0 mg·L-1 BA,0.2 mg·L-1 KT,and 0.5 mg·L"1 NAA was benefit for production of four effective compounds(kinsenoside,polysaccharides,phenolics and flavonoids)in MBs;in the second experiment,effects of combination groups of sucrose,phosphate and nitrogen concentration was determined,the group of 35 g·L-1 sucrose,0.93 mM phosphate,and 45 mM was the more favorable than other groups.Furthermore,the effect of NO3-and NH4+concentrations in 3/4MS mudium were examined,30 mM of NO3-and 15 mM of NH4+were optimal for MB growth and four effective compound accumulation.MBs growth was better in a bioreactor with a net at the bottom of the sphere in the bioreactor compared with other bioreactors.During MBs bioreactor culture,the kinetic study of MBs growth showed that 0-20 d was the lag phase,20-30 d was the exponential phase,and 30-40 d was the stationary phase.The maximum accumulation of all effective compounds was found at 30 d.Inoculation density,air volume,and light intensity were significantly affected MBs biomass and effective compound accumulation,the optimal inoculation density was the 12.5 g·L-1 and air volume was the 500 mL·min-1 for all effective compound production.However,the maximum productivies of varied compounds were determined under diffenrent light intensities,namely,phenolics and flavonoids at 1600 Lux but kinsenoside and polysaccharide at 2400 Lux.Salicylic acid(SA),methyl jasmonate(MeJA),and yeast extract(YE)were used as elicitors to promote effective compound accumulation.Three kinds of elictors were added to culture medium after 30 d of MBs bioreactor culture to select the optimum treatment days and concentrations of an elicitor.The maximum kinsenoside and flavonoid production were separately determined when MBs were treated with 500μM SA for 10 d and 550 μM MeJA for 14 d.The elicitors of SA,MeJA and YE equally promoted polysaccharide and phenolic production.Addintionlly,MBs extracts possessed an antioxidant activity,the DPPH radical scarvenging activity exerted in a dose-dependent manner.