Development Of The Key Techniques For In Vitro Industrial Culture Of Sedum Aizoon L. Organs

Sedum aizoon L.is often used as medicine with root or whole plant for clinical applications in stopping bleeding and disperse phlegm,soothing the nerves and so on.The culture system of S.aizoon adventitious roots is developed with the method of plant tissue culture,which would be a feasible approach to produce alternatives to field cultivation or wild resources,consequently,and help us overcome the resource problems besetting this species.In this paper,the S.aizoon was used as explants to optimize the induction and culture conditions of callus and adventitious roots,and a stratified combined culture system was established for efficient induction of S.aizoon adventitious roots.What's more,the liquid culture and scaling experiment culture in bioreactor of adventitious roots were carried out.Meanwhile,the accumulation of anthocyanin,antioxidant activity and antibacterial activity of S.aizoon cultures were studied.The main results are as follows:?1?The induction of S.aizoon adventitious roots was mainly of dedifferentiation and redifferentiation,which was callus formation and adventitious roots formation,respectively.An effective callus induction system was established based on leaves as explants supplemented with 2,4-D?2,4-dichlorophenoxyacetic acid?2.0 mg/L and 6-BA?6-benzylaminopurine?0.5 mg/L in medium.During the proliferation of callus,combination of NAA?1-Naphthylacetic acid?0.1 mg/L+2,4-D 2.0 mg/L+6-BA 0.1 mg/L was tested to be the best with features of high net weight of callus,tight texture,relative growth rate of 0.0631,which was beneficial to the induction of adventitious roots.At the same time,it was found that the callus showed an"S"growth curve during the growth process,and its optimal growth period was about 28-30 days.?2?The combination of phytohormones and ingredients of nutrients in the medium exerted the most significant influence on the induction rate of adventitious root.The results showed that when MS?Murashige&Skoog?with more diversified nutrient composition was selected as the basic medium and IBA?indole butyric acid?4.0 mg/L+KT?excitin?1.0mg/L+NAA 1.0 mg/L,the ratio of NO₃^-/NH₄⁺was 5:1?the total content was 60 mmol/L?,the growth demand of adventitious roots could be satisfied and the induction rate was 98.32%.At the same time,the stratified combination medium had the advantages of controlled release of plant hormones,omitting of subculture,the operation links was reduced to some extent,and led to the short growth duration.?3?Under the condition of liquid shaker culture,the adventitious root grew well at 110rpm,at which large adventitious roots density was gained because of improved growth with a13.66 times of multiplication ratio.In the experiment of scale-up culture in bioreactor,rapid growth of adventitious root was obtained at the aeration rate of 0.167 m³/?m³·min?.?4?Results through assay of anthocyanin ingredients from callus collected at various growth period showed that the peak concentrations of anthocyanin,carotenoid,and flavonoid were?584.4±8.12??g/g,?9.98±0.21?mg/g,and?3.66±0.25?mg/g,respectively.?5?Our data indicated that extracts of both adventitious roots and callus exhibited higher scavenging capacity of hydroxyl free radicals and DPPH free radicals,while extracts of both cultures had similar scavenging capacity of hydroxyl free radicals?77.35±2.05%and83.14±2.34%?.In terms of DPPH radical scavenging ability,the removal rate of adventitious root extract was up to?91.09±2.12?%,and the callus clearance rate was?72.15±2.14?%.?6?By using bacteriostasis test we conculed that extract of S.aizoon adventitious roots had inhibitory ability against all five kinds of test bacteria.The MIC of the adventitious root extract against Escherichia coli and Vibrio splendidus was 0.0625 g/mL,Staphylococcus aureus,Sarcina luted and Pseudomonas aeruginosa was 0.125 g/mL.In conclusion,a technological process of S.aizoon adventitious root culture was developed being composed of callus induction,adventitious root induction and solid propagation,liquid culture of adventitious root,and pilot culture of 3 L bioreactor,etc.Meanwhile,the accumulation and bacteriostasis test of callus and adventitious root derived anthocyanin were also studied in this text.And realization of efficient induction and culture of S.aizoon adventitious root would lay foundation for the subsequent large scale culture and complete commercial production of this species.