Establishment Of High Efficient Regeneration System Of Lavandula And Genetic Transformation Of Terpene Synthase Genes

Lavender,a plant of the genus（Lavandula）in the（Lamiaceae）family,is widely planted in Xinjiang Province of China.Lavandula essential oil has the function of antioxidant,anti-inflammatory,sedative,sleep-promoting effects,resulting in widespread use in cosmetics,medical and health and other fields.In this study,the related research in Lavandula variety"Zahua"as the object of experimental material was carried out.The main results are following:1.The regeneration system of"Zahua"Lavandula was established by different plant hormones with different concentration.There are three main mediums in the regeneration system,which include the callus induction medium with MS+6-BA 1.0 mg/L+NAA 0.3mg/L+sugar 30 g/L+agar 7 g/L;the induction medium of bud induction made of MS+6-BA2.0 mg/L+NAA 0.4 mg/L+sugar 30 g/L+agar 7 g/L;and the induction medium of rooting comprised of 1/2MS+NAA 0.2 mg/L+sugar 30 g/L+agar 7 g/L.The results of cytological observation of the callus showed that under plant hormone treatment the cell growth was so fast that the vascular bundles began to be lignified after culture of 21 days,and the vascular bundles formed primary xylem after culture of 35 days.2.The overexpression vectors of p Cambia3301-DXS,p Cambia3301-DXR,p Cambia3301-LIS1 and p Cambia3301-LIS2 were constructed and transformed into Agrobacterium tumefaciens strain GV3101 by freeze-thaw method.Arabidopsis thaliana was transformed by flower soaking method,which was screened by the methods of spraying glyphosate and PCR.Some transgenic strains of Arabidopsis thaliana were obtained,which include 4 with DXS gene,8 with DXR gene,7 with LIS1 gene,and 3 with LIS2 gene in T₁generation,respectively.The terpenoid components of transgenic Arabidopsis thaliana leaves with LIS1 gene were determined,the results showed that the contents of isophorone and ethyl2-furoate were significantly up-regulated,while the content of 3-ethyl-benzaldehyde was significantly down-regulated.The results of fluorescence quantitative and semi-quantitative analysis showed that the expression of DXS,DXR,LIS1 and LIS2 genes were significantly higher than that of the control group.Compared with wild-type plants,there were not morphological difference between transgenic Arabidopsis thaliana and the control.3.A genetic transformation system of Lavandula leaves was established:the time of pre-cultured was 7 days and the OD₆₀₀concentration of Agrobacterium tumefaciens solution is 0.5-0.7 for 7 minutes.The explant were transferred to the screening medium with Cef concentration of 200 mg/m L and Kana concentration of 30 mg/m L after co-culture for 2 days,and the resistant seedlings were obtained after subculture for 3 to 4 cycles.The Lavandula resistant seedlings were detected by PCR,the target gene is identified in the transgenic Lavandula.