Study Of Ultrasound On The Growth And Metabolism Of Trifoliate Leaf Callus

The plant Akebia trifoliata is a defoliated vine,which belongs to Akebia Decne of Lardizabalaceae.It is the genuine in China Pharmacopoeia.The main medicinal position is rattan;Its fruits,seeds,roots and leave also have medicine value.For the lack of the Akebia trifoliata wild resource in domestic market,the extraction of its active ingredient damaged Akebia trifoliata wild resource seriously,and understanding the biological effort of ultrasound in theory further.This paper choose Akebia trifoliata leave as the research object and do some research as follows:research on the effect of the induction and subculture of Akebia trifoliata leave callus after ultrasonic treatments with different time;the effect of the activity of protective enzyme and the production of the secondary metabolites of Akebia trifoliata leave callus after ultrasonic treatments with different time; the effect of surface and microstructure of Akebia trifoliata leave callus after ultrasonic treatments with different time,with a view to explore a new way to improve the secondary metabolites of the medicinal plants,to provide the cytology evidence for the growth of the Akebia trifoliata leave callus and accumulate data for the biological effects of the ultrasound.The results of this experiment are as follows:1 Effect of the induction and subculture of Akebia trifoliata leave callus after ultrasonic treatments with different time.Ultrasonic treatment for 1min,cultured in initial medium MS+2,4-D2mg/L+KT0.5mg/L could induce Akebia trifoliata leave callus effectively.13d after inoculation,the white and light yellow callus appeared in the incision of the ultrasound treatment leave,the rate of callus reached highest,74.07%,significantly higher than other groups(p＜0.05 ). The optimum treated time of ultrasound could promote the subculture growth of the Akebia trifoliata leave callus:after ultrasound treatment for 6min and cultured in initial medium MS+2,4-D2mg/L+KT0.5mg/L,the logarithmic phase of Akebia trifoliata leave callus was 5-10d earlier than the control group.Its fresh weight and dry weight reached highest,2.42g and 0.191g, respectively,significantly higher than the control group(p＜0.05 ).2 Effect of the buds induction and anti-browning of Akebia trifoliata leave callus after ultrasonic treatments with different time.Ultrasonic treatment for 1min,cultured in differentiation medium MS+6-BA1.0mg/L+NAA0.5mg/L+KT0.3mg/L+PVPP0.6mg/L for 35d,the differentiation rate was 79.01%,significantly higher than the control group(p＜0.05),and the browning rate was 33.33%,significantly lower than the control group(p＜0.05 ).Study shows that,optimum treated time of ultrasound could promote the differentiation rate of Akebia trifoliata leave callus and reduce its browning rate. 3 Effect of activity of the protective enzyme of Akebia trifoliata leave callus after ultrasonic treatments with different time.After ultrasonic treatment for 4min and 6min,the SOD activity,POD activity and CAT activity of Akebia trifoliata leave callus reached highest value cultured for 8d.The SOD activity and CAT activity of Akebia trifoliata leave callus increased significantly compared with the control group after ultrasonic treatment for 4min and 6min(p＜0.05 ),while the POD activity increased significantly compared with the control group after ultrasonic treatment for 4min.It showed that ultrasonic treatment could active the activity of the protective enzyme of Akebia trifoliata leave callus.4 Effect of surface structure and microstructure of Akebia trifoliata leave callus after ultrasonic treatments with different time.Surface structure of Akebia trifoliata leave callus were observed by Environmental Scanning Electron Microscope.The surface structure of Akebia trifoliata leave callus was smooth and irregularities without ultrasonic treatment.While treated with ultrasound,part of the cells formed cell mass,the cells of callus were homogeneous,grain and loose in consistency.Judge from the observation results,treated with ultrasound for 4min and 6min could promote the differentiation of the calls.Paraffin section observation showed the prominent nucleus and the increase of the number of parenchyma cells.Based on the results,treated with ultrasound for 4min and 6min could promote the proliferation of Akebia trifoliata leave callus effectively.5 Effect of the differentiation growth and oleanolic acid content of Akebia trifoliata leave callus after ultrasonic treatments with different time.Cultured in differentiation medium MS+6-BA1.0mg/L+NAA0.5mg/L+KT0.3mg/L+PVPP0.6mg/L,the growth curve of fresh weight and dry weight of Akebia trifoliata leave callus were S-type.Cultured for 35d is favorable for the differentiation growth and oleanolic acid accumulation on kebia trifoliata leave callus.Without ultrasonic treatment and cultured in differentiation medium MS+6-BA 1.0mg/L+ NAA0.5mg/L +KT0.3mg/L+PVPP0.6mg/L for 35d,the growth of callus was slowly and the content of oleanolic acid was 0.012285%;treated with ultrasound for 4min,the growth of callus increased significantly compared with the control group(p＜0.05),the content of oleanolic acid content was 0.014565%,no significantly difference with the control group.The study shows that treated with ultrasound for 4min was favorable for the differentiation growth of Akebia trifoliata leave callus,while both treated with ultrasound for 4min and without ultrasonic treatment are favorable for the accumulation of the oleanolic acid in Akebia trifoliata leave callus.