Study On Extraction Of Essential Oil In Thymus Vulgaris L. Of Tissue Culture

Thyme (Thymus vulgaris L.),a Labiatae (Lamiaceae) is a thyme (Thymus) perennial aromtic plant, the whole plant can be used as medicine, is the great development value of the spices, medicinal and ornamental plants. High value of thyme essential oil, with "liquid gold" in the world, but the essential oil was very low, and the natural plant growth cycle is too long, growth vulnerable to geographic, environmental and climate factors, it directly extract secon-dary metabolites Product has great limitations. How to use plant tissue culture technology to produce useful secondary metabolites, and increaseproduction of metabolites has been the focus of scientific research.The screening of adventitious bud proliferation condition was studied in the paper, The results of which showed the optimum medium was as follow:MS+NAA 0.25mg·L-1+6-BA 0.25mg·L-1,and the propagation coefficient reached 5.97.Simultaneously,to establish the optimum propagation system,the proliferation rate of adventitious bud was studied in differe-nt culture conditions, including solid media, liquid media with light and liquid media without light Moreover, the conductance and pH value of the medium were detected in the liquid culture condition. The results showed:the optimum culture condition for propagation was liquid media with light,which with shorter cycle compared with other conditions, and the propagation coefficient reached to the maximum on 21d. The culture medium should be changed or the nutrients was added on 21d～28d so as to the adventitious bud could grow well with adapatived conductance and pH value.Thymus vulgaris adventitious buds in vitro culture were used to study the effect of NaCl and chitosan in propagation of T. vulgaris adventitious buds and physiological indexes. The results showed:0.25～1.00 g·L-1NaCl and 0.1-0.2 g·L-1 chitosan concentration could help to adventitious buds proliferation,and the best proliferation was found when adding 0.5 g·L-1 NaCl and 0.2 g·L-1 chitosan.Proliferation coefficient increased significantly compared with the control 39.78% and 45.01%. During the process of proliferation in 0.25～1.00 g·L-1 NaCl and 0.1-0.2 g-L-1 chitosan concentration,the content of soluble protein content increased and then decreased,its peak value were increased 38.31 and 22.78μg/g,while the soluble sugar content is first decreased and then increased,the peak higher than the control,respectively 0.16 and 1.29 mg/g;adventitious buds of SOD activity first increased then decreased,while in contrary for the POD activity. CAT activity was decreased slowly with the time,their were improved compared with the control.Our studies had shown that 0.5 g·L-1NaCl and 0.2 g·L-1 chitosan can enhance the proliferation of adventitious buds,they reached significant difference compared with control.Both 0.5 g·L-1NaCl and 0.2 g·L-1 chitosan can enhance their material contents and the activities of protective enzyme in adventitious buds.maintenance the normal physiological metabolism of adventitious buds,showed strong proliferation.The essential oils from the Seedlings, the Aseptic Seedlings and the Tissue Culture Seed-Lings of Thymus vulgaris L. have been extracted by steam distillation, the chemical compone-Nts and the relative contents from the three samples were identified and analyzed by gas chro-matography-mass spectrometry (GC/MS) and peak area normalization method. The result showed that the main chemical components of essential oil in this three samples have no sign-ificant difference, they all contained the main compents of essential oil in Thymus vulgaris L. Thymol、o-Cymene、y-Terpinene、Caryophyllene et. al and only have a slight difference in the relative content. This study provided important theoretical foundation and data reference for further study on production of essential oil in thyme by tissue culture technolgy.Verify the 0.5 g·L-1NaCl and 0.2 g·L-1 chitosan treated essential oil content and composi-tion of the shoot, the results show that:essential oil content of 0.2% and 0.21%, respectively, compared with no added 0.05% increase and 0.06%. Respectively 23 and 22 components, respectively, compared with no added increase of 5 and 4 components, of which 0.5 g·L-1 NaCl the most abundant component of treatment caryophyllene (39.894%), thymol (14.337%) and eucalyptus leaf.delta.-Selinene (8.516%); 0.2 g·L-1 chitosan was the most abundant constituents thymol (36.063%),.delta.-Selinene eucalyptus leaves (14.600%) and caryophyllene (11.155%). Under the stimulus of thyme essential oil content increased by tissue culture, and 0.5 g·L-1 NaCl and 0.2 g·L-1 under the effect of chitosan thyme essential oil components of tissue culture has changed.