| Pinellia ternata Breit is herbaceous monocots plant of Araceae Pinellia ..It is one of the Chinese tradition medicines with a long medical history about 2000 years.The self-propagation coefficient of Pinellia ternata Breit is low,and it can not meet the increasing demand of application and export.The wild resource of Pinellia ternata Breit is drying up .Man has cultivated it only for 30years.,and the growth of Pinellia ternata Breit is greatly affected by circumstance,in addition the cost of cultivation and disease affected the yield of Pinellia ternata Breit .So ,we have propagated it by tissue culture and hope it would offer information for manufactory yield tubercles and active component, then,established Suspension system.we also explored transformation system of Pinellia ternata Breit mediated by Agrabacterium tumefaciens,The main results of experiment as follows.: 1 .Regeneration system was established with young leaves and petiole as explants.Young leaves and petiole were both cultured on severial inducing MS medium ,which supplemented with different concentration of growth regulators,such as 2,4-D/BA,NAA/BA. Calli were produced from explants after 15 days culture.Then, calli were respectively transferred to severial MS medium which supplemented with growth regulators of NAA/BA,NAA/KT in order to induce bud initiation.Planlets wereobtainede after one month culture. We believe the better inducing medium is :MS+2.0mg/L2,4-D+0.5mg/LKT(MSl),the frequency of calli inducing surpassed 80%.The highest differentiation frequency reached 100% on MS medium supplemented with BAlmg/L and NAA0.2mg/L. 2.Analyzition of main chemical composition between wild and tissue culture tubers.HPLC was employed to analyzed the extracts of 80%methanol, 0.2 mg/L H2SO4and distilled water from wild and tissue culture tubers.The experiment showed that the total content of the component tested in the tissue culture tubers were higher than that in the wild, and the main chemical composition were similarity between them. 3. Suspension system was established.Bright yellow calli were suspened in liquid medium,after 3~4times subculture ,good dispesive calli were obtained. Calli growth process in suspension culture presented a S-shaped curve. It reach logarithmic growth phase from 12th to 16th days, the fresh weight of calli reach maximum at 18th day. PH value of suspension cultural medium according to the calli growth curve in the lograithmses. Then calli of suspension culture was transferred to solid MS medium with NAA0.2mg/L and BA1.0mg/L, they regenerated new plantlets after 40days culture. 4. Transformation system was explored with young petiole and calli as target material.Young petioles and calli were excised from sterile seedling or explant after sterilizing,cut into 0.5cm,dip into Agrobaterium suspention (OD600 adjusted to 0.2) for 15 min.Patted dry on the filter paper, transferred to MSI medium and co-cultured for 3days in darkness.After co-cultivation ,petioles were washed in liquid MS medium containing 500mg/L cef for 2hours, Subsequently ,petioles were transferred to MSI medium supplenmented with 500mg/L cef and 100mg/L Kan for selection culture, and resistant calli obtained. |
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