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Preliminary Study on Bioreactor of Pelargonium Graveolens L’Hér

Author: XuZhiMin
Tutor: ZuoWeiZuo
School: East China Normal University
Course: Botany
Keywords: Pelargonium Bioreactor Geranium oil Cell suspension culture Hairy root culture Tissue culture Anti-tumor Gas Chromatography Mass Spectrometry
CLC: Q943.1
Type: Master's thesis
Year: 2007
Downloads: 27
Quote: 0
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Abstract


In this paper, based on the establishment of Pelargonium (Pelargonium graveolens L'Hér) bioreactor system, study the endophyte pollution control, suspension cells and hairy root culture of the establishment of the system, and tissue culture material and field material essential oil extraction method and influencing factors were compared. On this basis, the analysis of the chemical composition of essential oils tissue culture, nasopharyngeal carcinoma cells in vitro growth inhibition test, tissue culture to study the efficacy of the material basis of geranium oil. The endophyte pollution of Pelargonium in tissue culture proliferation experimental system to establish the technical barriers to the study, we tested different inhibitory method, the results show: Amp150 mg / L and the Amp100mg / L Km50mg / L plant growth exuberant the rooting rate higher than 90%, the plant essential oil content was slightly lower than the negative control, indicating that the antibacterial agents have little influence on the content of essential oil components, but there are still some combination of the two contamination rate. In the process to create a cell suspension culture system, we screened the callus 28 solid culture medium, sucrose concentration; suspension cells in a liquid medium. The results showed that 1% sucrose concentration the MS BAP1.0mg-/ L 2,4-D0 1.7 mg / L and MS BAP1.0mg / L 2,4-D1 .0 mg / L culture medium suitable for the generation of loose nor callus differentiation organization. Culture 31 best effect of suspended cells, the cells proliferate faster, good dispersity. Suspension cell culture medium observed by light microscopy, the suspension does have a single cell and small cell groups. Scanning electron microscopy found that a single cell has a spherical, spindle various forms the spherical diameter 60.100μm, the fusiform size (25-80) × (25-80) × (200-300) gm < sup> 3 ranging from basic cell differentiation. Suspension cell culture conditions screening results show that the amount of 50 ml of liquid, 4% sucrose concentration, 120rpm dark culture suitable for suspension cell cultures. Bioreactor system for the establishment of hairy roots of Pelargonium hair root the Agrobacterium A4 and the C58C1 transfection plants, the hair root induction rate: leaves as explants in the the C58C1 bacteria dope soak for 5 minutes, light 16h / d conditions, do not add mannitol, hormone-free MS 0 medium cultured 2d is an ideal conversion. PCR results showed that the hair root gene RO1B, RO1C gene has been integrated into the genome of Pelargonium hairy root. Hairy root growth cycle tests showed that the 40 day biomass reached the maximum wet weight increase of 10.2 times the dry weight increase of 11.8 times; statistical analysis of the results of the activity of glucose-6 - phosphate dehydrogenase (G-6-PDH), G-6-PDH activity in the 36th reached the maximum value of 40.50 ± 1.926, compared with vaccination was significantly increased (P <0.01). Hairy root extracted with n-hexane GC-MS composition analysis results show that inoculation identified 14 essential oil components, content accounted for only 0.95% of the total extract; 44 day identification of components up to $ 26 species, accounting for 6.93% of the total content; the of citronellol, formic acid ester lemongrass main ingredient in 48 Telsda to the highest value, accounting for 1.53% of the extract. Furthermore different media filter, the hairy root culture conditions. The results showed that, compared with the hairy root culture on the basis of 48 days, MS 0 Add NAA 0.1 mg / L hairy root culture only biomass was significantly lower (P <0.05), glucose -6 - phosphate dehydrogenase activity (P <0.01) and menthol, citronellol, formic acid ester lemongrass content were higher. Hairy roots induced regeneration of seedlings geranium oil component analysis, the MS and MS of 2 -induced regeneration of seedlings with the control group in the medium Pei Pu rooted the essential oils The ingredients are similar, slightly different content. To MS 0 the regenerated plants citronellyl medium-induced, formic acid ester lemongrass content, accounting for 39.33% of the total, indicating that the MS 0 suitable for hairy root regeneration seedlings induction and growth. Essential oil content less tissue culture material further the field of tissue culture material culture time, the quality of steam distillation time or n-hexane extraction of essential oil content factors, the results show that, 30g stems distillation 12min 50g field materials ideal conditions of the distillation of essential oils; tissue culture roots and leaves July more suitable for distillation and extraction field of stem and leaf geranium oil; cultured for 50 days and 40 days of roots and leaves than for steam distillation and n-hexane extraction; July Daejeon root and liquid culture 60-day group Bacon applies to n-hexane extraction. Study the efficacy material basis for the analysis of the chemical composition of the essential oils of Pelargonium tissue culture, this paper, the tissue culture geraniol oil, geranium oil and its major monomer component in vitro human nasopharyngeal carcinoma cell growth inhibition test. The results showed that the treatment of nasopharyngeal carcinoma cells after 24 h of geranium oil and monomer, resulting in varying degrees of proliferation, enhanced with the increase in drug concentration, showing a dose-effect relationship. Strong tissue culture geranium oil and acetic acid the lemongrass ester tumor suppressor role, the IC 50 value reached 94.25 nL / mL and 43.98 nL / mL. Geranium oil and terpineol citronellyl acetate treated cell morphology change greatly, most of the cells became round narrow, budding with formation of apoptotic bodies; flow cytometry with PI fluorescence probe The cell cycle was detected, geranium oil and terpineol may improve the rate of nasopharyngeal carcinoma cell apoptosis inhibition of tumor cell growth.

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