The Development Of Siraitia Grosvenorii Cell Suspension Culture System And It's Cropreservation Method

Mogrosides V is one kind of natural sweeteners with high market value,and it was mostly extracted from the Mogrosides fruits.However,the extraction yield was very low and the quality was always affected by the natural conditions such as season,region,weather and so on.These problems could be solved by Siraitia grosvenorii cell suspension culture.In this research,the cell suspension culture technology of S.grosvenorii was developed and the process condition was optimized.The scale up of S.grosvenorii cell suspension culture was achieved in a 5 L bioreactor.Besides,the cryopreservation method for S.grosvenorii cell was developed.This research provided some effective and useful methods for mogroside V production through large-scale plant cell suspension culture.1.Optimization for embryo-callus induction of S.grosvenoriiThe embryo-callus induction of S.grosvenorii was investigated.Results showed that the seed embryo is the best explants for embryogenic callus induction,and the highest embryogenic callus induction rate was achieved with the hormone concentration at 6-benzyl adenine(6-BA)1.0 mg/L and naphthylacetic acid(NAA)0.5 mg/L.When the inoculation size was 2 g fresh weight in the solid medium,the callus exponential phase was from 15 d to 30 d.So the optimum subculture time of callus was 25-30 d in solid culture.However the hormone concentration for callus growth and callus induction were different.The optimized concentrations for 6-BA,NAA and casein hydrolysate were 1.0 mg/L,2.0 mg/L and 100 mg/L,respectively.2.Development of Sgrosvenorii cell suspension culture system and scale-up in a 5 L bioreactorTo develop a stable suspension culture system,the effect of hormone concentration,amino acids,carbon source,initial pH,and culture volume on cell growth and activity were tested.The optimum of culture conditions were showed as follows:1.0 mg/L 6-BA,2.0 mg/L NAA,3%sucrose concentration,10 mg/L proline,initial pH 5.0 and 30%broth volume.The precursor(phenylalanine)and inducer(methyl jasmonate,salicylic acid)also affected the cell growth and the biosynthesis of mogroside V.Compared with the control,500 mg/L of phenylalanine improved the content of mogroside V in cells and increased the yield of mogroside V by 25.58%,The experiment of scale up culture of S.grosvenorii cell in a 5 L bioreactor showed that the suspension cell could be cultured in 5 L bioreactor,which has significant contribution to the industrial production of mogroside V.3.Development of a rapid cryopreservation method for S.grosvenorii cells based on the detection of cells'capacitance and cell activityThe cryopreservation of cell lines is a very good way to preserve the high-yielding cells.But the traditional optimization method for cryopreservation is both expensive and effort costly.A rapid quantitative method for S.grosvenorii cells was successfully developed based on an online viable cell mass monitor.And the cryopreservation method for S.grosvenorii suspension cells was also optimized.The results showed that the suspension cells treated with 0.2 M sucrose for 9h was the best method for the cryopreservation of the cell.And the optimized cryoprotectant was 10%DMSO and 10%sucrose.