Screening And Scale-up Culture Of Stevia Rebaudiana Hairy Roots To Produce Chlorogenic Acids

Stevia rebaudiana Bertoni is a perennial herb of compositae genus.Its leaves are mainly used to produce steviosides(low-calorie,high-sweet natural sweetener-stevioside).Furthermore,these leaves contain some bioactive components such as chlorogenic acids.But these components have not yet attracted researchers and producers attention because of its low content.In this study,the leaves of Stevia rebaudiana were used as experimental materials to induce hairy root,and the fine monoclonal roots were screened from the induced hairy roots.The production technology of chlorogenic acids by the culture of Stevia rebaudiana hairy roots was established.Moreover,to provide reference for industrial production of chlorogenic acids by hairy roots,the scale-up culture experiment of Stevia rebaudiana hairy roots was carried out.The main results and conclusions were as follows:1.Hairy roots of Stevia rebaudiana were induced by four Agrobacterium rhizogenes of R1601,ACCC10060,A4 and ATCC15834.Except Agrobacterium rhizogenes ATCC15834,hairy root was induced by other Agrobacterium rhizogenes successfully.The induced roots showed many branches,non-directional and rapid growth.The PCR analysis results indicated that rolB and rolC gene of Ri plasmid in Agrobacterium rhizogenes had been successfully transferred into hairy root genome.Among Agrobacterium rhizogenes,the induction ability of R1601 was strongest.Under this Agrobacterium rhizogenes induction,the white tubers were grown after 7 days,and hairy roots induced from the leaves wound after 2 weeks.The hairy root induction rate was 35.7%.2.30 monoclonal hairy roots were screened from hairy roots induced by three Agrobacterium rhizogenes.The results showed that the content of chlorogenic acids of R2 was highest in the 10 monoclonal roots induced by R1601,the content of chlorogenic acids of R2 root was 4.12%.10 monoclonal roots induced by ACCC10060,the content of chlorogenic acids of A7 root was highest,and its content was 3.38%.10 monoclonal roots induced by A4,the chlorogenic acids content of T1 root was highest,and its content was 3.38%.The content of chlorogenic acids in the root system of R2 root induced by R1601 was highest,and the content of chlorogenic acids was 4.12%,and it was higher than other roots.So,R2 root can be chose for subsequent scale-up culture experiment.3.The "swing oscillation","aeration and mixing","bubble" of three culture methods of hairy root was studied.The results showed that the bubble cultivating mode can be more adapted to the scale-up culture of hairy roots of Stevia rebaudiana,after 28 d culture,biomass and production of chlorogenic acids of hairy root reached 2.41 g/L and 24.58 mg/L.4.The key technical parameters of chlorogenic acids by the culture of Stevia rebaudiana hairy roots was established,the results showed that cylindrical incubator was more suitable for culture of Stevia rebaudiana hairy roots.And the ―bottom circular distributed‖ ventilation,ventilation pipe diameter of 7 mm and ventilation was 30~50 m L/min.Under this condition,hairy roots grew faster,after 28 d culture,biomass and the content of chlorogenic acids of hairy roots were 2.21 g/L and 40.44 mg/L respectively.5.The 10 L bubble type culture experiment of Stevia rebaudiana hairy roots was studied.Under this optimization of culture conditions,biomass and content of chlorogenic acids of hairy roots were 4.72 g/L and 159.07 mg/L respectively.6.Nutrient consumption regular of Stevia rebaudiana hairy roots in 10 bubble type culture was studied,and result showed that hairy root growth status was close relationship with nutrient solution of sucrose,ammonium nitrogen,nitrate nitrogen and phosphate ions consumption rate.The experiment results showed that medium of carbon,nitrogen and phosphate ion consumped fast,when hairy roots were from the adaptation period to the growing stage.And consumption rate of iron ion showed that consumption rate of iron ions was fast in the early stage of culture,and the consumption rate decreased significantly after 7 days,and the concentration of iron ions decreased slowly.The medium of pH value and dissolved oxygen showed a trend of decrease and then increase as a whole in culture period.The results showed that hairy roots consume most of the nutrients of mediun at grew period of 21 d.Therefore,the supplementary culture experiment was carried out.According to nutrient consumption regular of hairy roots culture in bubble type,and the supplementary culture experiment was studied.After hairy root culture in a 5 L bubbling incubator at 21 d.Adding to 1/2V original medium,hairy root growth cycle may extend approximately four weeks.Biomass of hairy roots was 4.77 g/L,and the content of chlorogenic acids was 189.84 mg/L.From the above,this study aimed to screen the fine monoclonal hairy root and increased the content of chlorogenic acids.The production technology of chlorogenic acids by scale-up culture of hairy roots in10 L bubbling was established and optimized.The targe production was further improved by supplementary culture.