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
Author
Filipović, Biljana K.Mentor
Subotić, AngelinaCommittee members
Dragićević, IvanaSimonović, Ana
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Show full item recordAbstract
Kičica (Centaurium erythraea Rafn.) je biljna vrsta širokog spektra lekovitih
dejstava, koja se odlikuje visokom regenerativnom sposobnošću u uslovima in vitro
kulture. Osnovni cilj disertacije bio je izučavanje pojedinih biohemijskih i molekularnih
aspekata in vitro morfogeneze kičice sa svrhom rasvetljavanja osnova razvojne
plastičnosti ove biljne vrste koja se ispoljava u uslovima in vitro kulture. U ovom
istraživanju je in vitro morfogeneza kičice indukovana iz odsečaka listova na svetlosti i
u mraku. Na hranljivoj podlozi bez regulatora rastenja i na svetlosti odvijao se proces
direktne organogeneze pupoljaka, a u mraku proces direktne rizogeneze. Regeneracija
biljaka putem indirektne somatske embriogeneze (SE) je postignuta na hranljivim
podlogama sa regulatorima rastenja 2,4-D i CPPU i na svetlosti i u mraku, dok je na
istim podlogama, za regeneraciju pupoljaka indirektnom organogenezom, svetlost bila
obligatorni faktor. Anatomska istraživanja su pokazala da je razvoj somatskih ...embriona
bio asinhron, a na svetlosti se odvijao simultano sa razvojem adventivnih pupoljaka. U
ovim istraživanjima je uspostavljen model-sistem pogodan za komparativnu analizu in
vitro morfogenetskih procesa kod kičice na molekularnom i biohemijskom nivou.
Ispitana je ukupna aktivnost antioksidativnih enzima, superoksid dismutaza (SOD),
katalaza (CAT) i peroksidaza (POX), kao i zastupljenost i aktivnost pojedinih izoformi
ispitivanih enzima tokom različitih morfogenetskih procesa. Analize su pokazale da su
SOD i CAT uključene u antioksidativnu zaštitu listova kičice od oštećenja izazvanih
povredom, do koje dolazi prilikom izolovanja eksplantata, dok je totalna aktivnost POX
bila snažno indukovana tokom morfogeneze na svim ispitivanim hranljivim podlogama.
Uloga arabinogalaktanskih proteina (AGP), proteina ćelijskog zida bogatih
hidroksiprolinom, tokom različitih morfogenetskih procesa in vitro kičice istražena je
primenom nekoliko pristupa. Dodavanje β-D-glukozil Yariv reagensa (βGlcY), koji
specifično vezuje i precipitira AGP i na taj način sprečava AGP da obave svoju
funkciju, u hranljive podloge dovelo je do smanjenja broja somatskih embriona i
adventivnih pupoljaka po eksplantatu. Uporednom analizom je nađeno da se ispitivani morfogenetski procesi razlikuju po svojoj osetljivosti na βGlcY, te da se po rastućoj
senzitivnosti mogu poređati kao: direktna rizogeneza (neosetljiva) < indirektna
organogeneza pupoljaka < direktna organogeneza pupoljaka << indirektna SE (veoma
osetljiva)...
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 as...ynchronous 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)...