Tissue Culture And Telomerase Activity Assay For Ancient Trees In Beijing Summer Palace

Old trees are the precious legacy of the long history,leaving to humanity with important scientific, cultural and economic value.In order to inherit the historical culture and reserve the valuable plant genetic resources of the old national garden plants,in vitro propagation of the ancient trees from the Summer Palace in Beijing was studied.The result indicated that clustering plants can be gained by using stem with lateral bud of the ancient willows grown on the west dyke from the Summer Palace as explants,taking MS as basal medium,supplement with 6-BA and NAA at proper doses.MS(2.0% sucrose) culture medium with no growth regulators was the best medium for initiating culture;the optimal medium for adventitious buds differentiation was MS+6-BA 0.8 mg/L+NAA 0.2 mg/L+3.0% sucrose;and the optimal medium for adventitious root formation was 1/2MS +NAA 0.2mg/L+1.5% sucrose.Using the stem of the ancient Catalpa bungei with lateral buds as explants in the tissue culturing system,the optimal medium for initiating culture was MS+6-BA 0.8 mg/L+NAA 0.1 mg/L. The optimal medium for differentiating culture was MS+6-BA 1.8 mg/L+NAA 0.3 mg/L,the optimal medium for adventitious root inducing was 1/2MS+IBA 0.3 mg/L+NAA 0.05 mg/L.Taking the new-sprouted stem cutting segment of Malus micromalus as explants in the tissue culturing system, the optimal medium for adventitious buds initiating culture was 1/2MS+IBA 0.3 mg/L+NAA 0.05 mg/L.To plant the sterile seedlings into differentiating alternate media of MS+6-BA1.0 mg/L +NAA 0.1 mg/L and MS+6-BA1.5 mg/L +NAA 0.4 mg/L,the seedlings were strong and had a high differentiating ratioThe optimal medium for adventitious root inducing was 1/2MS + IBA0.8mg/L和1/2MS +NAA1.0mg/L.The ancient trees were aging with time going on,and the aging process was closely relevant to the length of the telomere and telomerase activity.We constructed an optimized assaying method for plant telomerase activity based on the assaying method of telomerase activity for Hela cells.The reacting conditions were as follows:21bp primers were optimal and the best dosage was 0.1μg.The optimal dosage of templates were 50ng-500ng.The reacting condition of telomere DNA elongation was 45 min under 26℃.The circulating conditions of PCR were 94℃/30s,65℃/30 s,72℃/90 s with 33 circles. The designation of primers and the experiment results indicated that those parts with high fission ability showed telomerase activity.The sensibility of assaying was enhanced by refined experiments excluding the false negative results,and the modified argentation rendered the results more accurate.The relativity between aging and the ages can be acquired by the comparison of the telomerase activity among new-born apical buds,tissue-cultured seedlings and callus of ancient Catalpa bungei.