Alelopatski efekti transformisanih korenova Chenopodium murale L. in vitro
Allelopathic effects of Chenopodium murale L. hairy roots in vitro
Doctoral thesis (Published version)
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Root system produces and releases chemical compounds with allelopathic activity. Isolation and characterization of these metabolites, as well as investigation of their effects on other organisms, are very complicated processes. The aim of the present study was to initiate and establish Chenopodium murale L. transgenic hairy root in vitro culture system, as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established 13 hairy root clones differed in their morphology and growth potential. The liquid culture system of characterized hairy root clones was maintained for over two years. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene presence were observed. We investigated allelopathic effect of wild-type and hairy root growth media (phytotoxic media) against germination and seedling development of Arabidopsis thaliana (L.) Heynh, wheat (Triticum aestivum L.) and lettuce (Lactuca sativa L.) test plants. The inhibitory potential varied depending on the test plant and hairy root clone. R1 and R8 clones had strong allelopathic effects. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. In test plants A. thaliana and wheat, after treatment with phytotoxic media, activity of different antioxidative enzymes (catalase, peroxidase and superoxide dismutase) were analyzed spectrophotometrically and electrophoretically. Activities of these antioxidative enzymes were higher in comparison to the control, except the catalase activity in A. thaliana, which was the same or lower. Phytotoxic media from different hairy root clones cause different changes in activities of catalases, peroxidases and superoxide dismutases in test plants. Allelopathic interactions implicate the inhibition of cell division by allelochemicals. We investigated expression of cell cycle-related genes (CDK and CYC) in test plant A. thaliana and wheat, after phytotoxic medium treatment of clone R8. We demonstrated, by q-PCR method, that expression of genes involved in G1-to-S and G2-to-M transition were significantly down-regulated by phytotoxic medium of clone R8 in root and shoot apexes of test plants, indicating inhibition of DNK synthesis and mitosis. Homologous genes of A. thaliana and wheat (CDKA;1/CDC2 and CYCD2;1/CYCD2) had similar expression profiles, after the treatment with phytotoxic medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07 – 2.85 μmol/l) identified and quantified by HPLC analyses in the growth media, were significantly lower than the inhibitory active concentration of pure grade phenolic acids when applied to A. thaliana (0.5 mmol/l) or wheat and lettuce (5.0 mmol/l, both), suggesting that phenolic acids have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.
Keywords:Agrobacterium rhizogenes; Allelopathy; Antioxidant enzyme; Chenopodium murale L.; Phenolic acids; Genetic transformation; Cell cycle-related genes; Hairy roots; qPCR; Rol genes
Source:University of Belgrade, Faculty of Biology, 2012, 1-183
- Physiological, chemical and molecular analysis of the diversity of selected rare and endangered plant species and application of biotechnology for ex situ conservation and production of biologically active compounds (RS-173024)