Effect of subculture times on genetic fidelity, endogenous hormone level and pharmaceutical potential of Tetrastigma hemsleyanum callus

The effects of subculture frequency on genetic variations of Tetrastigma hemsleyanum callus under a long-term tissue culture were evaluated by means of flow cytometry (FCM), inter simple sequence repeat (ISSR), and sequence-related amplified polymorphism (SRAP) markers to clarify the variation pattern and frequency of somatic variation in the course of the subculture. The effect of subculture frequency on endogenous hormone content was also assessed by Enzyme-linked immunosorbent assay to explore the correlation within the differentiation rate, somaclonal variation frequency of callus and the endogenous hormone content. Moreover, the flavonoid content in the callus of different subculture times was compared and the antitumor activity of them was evaluated by using MTT cell proliferation and cytotoxicity assay. FCM confirmed similar ploidy level in mother plant and all callus, but some changes were observed in the ISSR and SRAP patterns, of which 10.3 and 6.06 % were polymorphic, respectively. The somatic variation frequency tended to increase with the increasing subculture time in the early stage of subculture, with a peak in the 4–6 times and then kept nearly at a constant after 8 times. High IAA content may be one reason for this variation by this period. The flavonoid content and antitumor potential increased and remained at high level during the 2nd and 6th subculture. Though there was a significant decline after the 6th subculture, they still had about the same flavonoid content and antitumor potential as those of mother plant. The results suggested that the in vitro callus of T.hemsleyanum is potentially useful for pharmaceutical uses. For supplying true-to-true seedlings, germplasm conservation and further genetic transformation, subculture frequency of callus should be controlled within 4 times to obtain clonally identical plantlets. However, for the development of germplasm improvement of the species, subculture frequency should be controlled in 6–8 times to make better use of somatic variations.