Research On The Cell Cultures Of Cistanche Deserticols Ma. And Original Study On The Induction Of Hairy Root Of Them

Cistanche deserticols Ma, family compositae, is a famous Chinese traditional drug, in which phenylethanoid glycosides (PeG) were its main bioactive components. Because of the poor natural resources, the feasibility of PeG production by cell cultures of C. deserticols was demonstrated in this paper, and the work on the regulation of phenylethanoid glycosides biosynthesis in cell suspension culture of C. deserticols were investigated, and the induction of hairy roots of them was originally studied. The callus lines were selected from the original callus of C. deserticols through cell-aggregate cloning method. The callus lines could grow rapidly on solidified B5 medium supplemented with 500mg casein hydrolysted l-1, 2 mg IAA l-1, 1 mg BA l-1. After 25d culture, the biomass reached 13.2 g dry wt callus l-1 medium and its PeG content was 12.9%(w/w). The HPLC assay results showed that the bioactive components in the callus were identical with those in explants. Cell suspension cultures of C. deserticols were established. The MS medium supplemented with 800mg casein hydrolysted l-1, 10mg l-1 GA3, 0.5mg BA l-1 were suitable for cell growth and PeG formation. The total culture period was 36d,which the growth curve was assumed "S model". When the cell was harvested on the day 36d, the biomass reached 15.4 g dry wt callus l-1 medium and its PeG content was 2.4%(w/w), which was equal with those in explants. The HPLC assay results showed that the bioactive components in suspension cultures of C. deserticols were different from those in explants. The PeG biosynthesis in cell suspension cultures of C.deserticols were stimulated by treating with two abiotic elicitors, which were methyl jasmonate and salicylic acid. The affection of the elicitation was dependent on the elicitor concentration and the addition time of the elicitor. The yields of PeG were significantly increased in the presence of 5μmol/L methyl jasmonate on day 14(up to 2.59 fold). At the same time, the final biomass (15.2 g dry wt l-1) was not affected. Treated with 50μmol/L salicylic acid on day 28, the maximum content of them were 2.71 fold higher than the untreated cell cultures. However, the final biomass (14.8 g dry wt l-1) was inhibited by the salicylic acid. Both of PAL activity in the cell treated with the two elicitors were significantly promoted Hairy root clones of C.deserticols transformed with transformed with Agrobacterium rhizogenes strains A4.However, the transformation frequency of hairy root from callus was low, and the hairy root with little lateral branches grew slowly .PCR analysis confirmed the integration of the T-DNA fragment of Ri plasmid from A.rhizogenes strain A4 into the transformed root genome. The result showed that there is a problem by using the hairy root of C.deserticols to produce the PeG.