Studies On The Continuous Cultivation Of Glycyrrhiza Inflate Cells In Perfusion Bioreactor

Licorice,for medicinal use,has a long history in the world.Especially under the conduction of Traditional Chinese Medicine,Licorice plays a significant role.However,it is facing to terribly insufficient resource of Licorice in nature.So,to utilize modern biotechnology for enlarging resource of Licorice,such as plant tissue,plant cell culture,is indispensable.An enormous potential has showed for a large-scale industries manufacture in bioreactors,which results from affluent Secondary metabolites produced by plant cell culture.Based on the previous researches,we designed a perfusion bioreactor system for medicinal plant cell continuous culture.In this work,interception efficiency,settlement efficiency and volumetric oxygen mass transfer coefficient were tested.Consequently,we provided a comprehensive performance description of the bioreactor system we designed.Meanwhile,we induced the callus,established suspension cell line of Glycyrrhiza inflata.And cultured them in three different ways,shake-flask culture,bioreactor batch culture and bioreactor perfusion culture.It was found that,the best optimal was the hypocotyls for callus induction of Glycyrrhiza inflata and the best appropriate medium is MS +BA 1.0mg/L + 2,4-D 2.0mg/L.the medium for initial Licorice cell culture is MS + BA 2.0mg/L +NAA 0.4mg/L +IAA 0.4mg/L +Sucrose 30g/L.We have tackled the problem that cells often browning during plant cell culture by the method of adding 30g/L citric acid into the cell culture medium.Meanwhile,we found it was good for cell growth by adding 500mg/L Hydrolyzed Casein Protein into the liquid medium.By comparison among three different culture system for Glycyrrhiza inflata cell,we measured the cell growth kinetics curves and nutrition consumption curve respectively.The results shows that,in shaking culture,lag period of cell growth was 6d,and 10-20d was logarithmic growth phase then it would step into stable phase after 25 days of culture process.In the whole process,the highest growth(fresh weight) was 106.3g/L,which was more than 13 times to the vaccination.And in the later period of culture course,nearly all the nutrients have been consumed.For bioreactor culture of Glycyrrhiza inflata cells,the phenomena of growth stagnation or growth inhibition were not obvious.In the bioreactor Batch culture,4~6days later,the cells entered the index growing period.The maximum biomass appeared after 21 days and the fresh weight and the dry weight were 191.3g/L and 15.8g/L respectively.The cell growth rate and cell growth ratio achieved the peaks at 15th day and 12th day respectively.Nevertheless,after 18 days,the cell growth began to slow down and cell growth rate and growth ratio were less than 0,which results from nutrition consumption and biomass and toxic substance accumulation in the culture medium.However,bioreactor Perfusion culture makes a contrast with the Batch culture,it has more advantages not only that fresh weight and dry weight reached 288.5g/L and 18.4g/L respectively,but also that the cell growth inhibition phenomenon by the feedback has markedly ameliorated,cell growth rate has not slowed noticeably.It is because that nutrition inflow and waste outflow can keep balanced and continuous.The highest cell growth rate and highest cell growth ratio appears at the 12th day and 9th day respectively. Moreover,both cell growth rate and cell growth ratio were stabilized after the 21 days.In addition,through the comparison of highest growth rate among three different culture methods,perfusion bioreactor was higher 20 percentage point than the culture course of batch bioreactor and was higher 33 percentage point than shake-flask culture respectively.It was indicated that the continuous cultivation of plant cells was realized preliminarily.The characteristics and Innovations of this paper was that a perfusion bioreactor system was designed, which used for medicinal plant cell continuous culture.Based on this system,we cultured suspension cells of Glycyrrhiza inflate in three different culture system and through the comparison,we illustrate that the advantages of the perfusion bioreactor system is used for medicinal plant cell continuous culture.Meanwhile, the research we do provides reference and foundation for enlarging bioreactor culture of licorice cell and large-scale industry manufacture.