Uloga arabinogalaktanskih proteina u procesu morfogeneze in vitro kod kičice (Centaurium erythraea Rafn.)
The role of arabinogalactan proteins in centaury (Centaurium erythraea Rafn.) morphogenesis in vitro
Doctoral thesis (Published version)
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Common centaury (Centaurium erythraea Rafn.) is a plant species with a broad spectrum of medicinal effects, characterized with high regenerative potential under conditions of in vitro culture. The main objective of the current dissertation was the study of some biochemical and molecular aspects of in vitro morphogenesis of centaury, with an aim to elucidate the basis of its developmental plasticity that is manifested in in vitro culture. In the current study the in vitro morphogenesis was induced on light and in darkness. Direct shoot organogenesis was induced on growth regulator-free culture media on light, whereas on the same media in darkness, direct rhizogenesis occurred. Plant regeneration via indirect somatic embryogenesis (SE) was accomplished on growth media with growth regulators 2,4-D and CPPU on both light regimes, while for shoot regeneration by indirect organogenesis, light was an obligatory factor. Anatomical studies revealed that the development of somatic embryos was asynchronous and proceeded simultaneously with the development of adventitious buds on light. Thus, this work established a model-system for comparative analysis of in vitro morphogenetic processes in centaury on molecular and biochemical level. Total activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), as well as the presence and activities of their isoforms during different morphogenetic processes, were also determined. It was shown that SOD and CAT were involved in antioxidative protection of centaury leaves wounded during explant isolation, whereas total POX activity was strongly induced during morphogenesis on all growth media. The role of arabinogalactan proteins (AGPs), plant cell-wall hydroxyproline-rich proteins, during different morphogenetic processes in vitro was investigated using several approaches. The addition of β-D- glucosyl Yariv reagent (βGlcY) that specifically binds to, precipitates and thus inactivates AGPs, to the culture media, caused a reduction of the number of somatic embryos and adventitious buds formed per explant. Comparative analysis revealed that the investigated morphogenetic processes differ in sensitivity to βGlcY, so that they can be arranged in order of increasing sensitivity to bGlcY as: direct rhizogenesis (insensitive) < indirect shoot organogenesis < direct shoot organogenesis << indirect SE (very sensitive). The AGP content, as determined by radial diffusion method, increased during induction of different morphogenetic processes. Using crossed electrophoresis method it was shown that different morphogenetic processes are accompanied by synthesis of different AGP sets. Using monoclonal antibodies LM2, JIM4 and JIM13, specific for carbohydrate moieties of AGP molecules, the specific AGP epitopes were immunohistochemically localized to the cell wall and their developmental regulation during SE was demonstrated. Starting from sequenced and assembled leaf and root centaury transcriptome, four AGP genes were identified and named CeAGP1 through 4. The transcript sequences were verified by direct sequencing, and the bioinformatics analysis revealed that CeAGP1, CeAGP2 and CeAGP4 encode AGPs with fasciclin (FAS) domain, whereas CeAGP3 is an AG peptide with a conserved DUF1070 domain. The expression analysis of these genes by qRT-PCR amplification showed that CeAGP1 is specifically induced during indirect SE, while CeAGP3 is expressed during morphogenesis on all growth media. In the current work, centaury is for the first time proposed as an excellent model organism in developmental plant biology, suitable for study of biochemical and molecular aspects of in vitro morphogenesis. The data obtained on the role of antioxidative enzymes and AGPs during in vitro morphogenesis in centaury represent the first study of morphogenesis in this species on biochemical and molecular level.
Keywords:Centaurium erythraea Rafn.; In vitro culture; Organogenesis; Somatic embryogenesis; Antioxidative enzymes; Arabinogalactan proteins; Β-D- glucosyl Yariv (βglcy) reagent; Immunohistochemistry; FLA; DUF1070
Source:University of Belgrade, Faculty of Biology, 2015, 1-181
- 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)