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The potential of somatic embryogenesis induction was examined by in vitro growing of isolated ovules of Pinus heldreichii (Christ.) on nutrient media. The induction of embryogenic tissue was stimulated by Gresshoff and Doy (GD) medium with nitrogen compounds reduced to one half and with 0.5 mg/l BA and 2.0 mg/l 2.4-D. Still, the most important factor in this process was the development stage of embryos in the ovule. An induction frequency of 10% was obtained on the ovules in which embryos were in the pre-cotyledonary stage. When the dominant embryo in the ovule was in the cotyledonary stage, the induction frequency was lower or absent. Proliferation of embryogenic tissue was achieved on the media with reduced concentration of growth regulators, and the initial stages of embryo maturation were obtained on the media with ABA and 5% sucrose. Izolovane ovule Pinus heldreichii (Christ.) gajene su in vitro na hranljivim podlogama radi ispitivanja mogućnosti indukcije somatske embriogeneze. Hranljiva podloga Gresshoff and Doy (GD) sa azotnim jedinjenjima, smanjenim na polovinu i BA 0.5 mg/L i 2.4-D 2.0 mg/L, stimulisala je indukciju embriogenog tkiva. Ipak, najvažniji faktor u ovom procesu bio je stadijum razvića embriona u ovuli. Frekvencija indukcije od 10% dobijena je na ovulama u kojima su embrioni bili u prekotiledonarnom stadijumu razvića. Kada je dominantni embrion u ovuli bio u kotiledonarnom stadijumu razvića, frekvencija indukcije bila je manja ili je nije ni bilo. Proliferacija embriogenog tkiva dobijena je na podlogama sa smanjenom koncentracijom regulatora rastenja, a početni stadijumi sazrevanja embriona na podlogama sa ABA i 5% saharoze. Projekat ministarstva br. 143026B

Senescencija lista predstavlja kompleksnu, genetički kontrolisanu poslednju fazu ontogeneze lista koja rezultira koordinisanom razgradnjom makromolekula i mobilizacijom njegovih komponenti u druge delove biljke. Na ćelijskom nivou, ona obuhvata promene strukture, metabolizma i genske ekspresije koje dovode do smrti, čiji se tip označava kao programirana ćelijska smrt (PCD). Kao eksperimentalni model u ovom radu korišćene su in vitro gajene biljke duvana (Nicotiana tabacum L.) koje obezbeđuju set listova u kojima postoji gradacija senescencije. Istraživanje je obuhvatilo citohistološku i imunohistohemijsku analizu dinamike strukturnih promena mezofila listova, kao i ispitivanje metaboličkih, biohemijskih i molekularnih procesa tokom senescencije lista. Utvrđen je redosled degenerativnih promena u strukturi ćelija tokom fiziološkog starenja listova. U senescentnim listovima detektovani su tipični simptomi programirane ćelijske smrti, koja po svojim morfološkim odlikama odgovara autofagnoj ćelijskoj smrti, i pokazano da internukleozomalna fragmentacija DNK prethodi ultrastrukturnim promenama kao što su degradacija hloroplasta i kondenzacija hromatina. Sadržaj hlorofila i citosolnih proteina identifikovani su kao pouzdani biomarkeri stepena senescencije listova. Hemijskom analizom endogenih regulatora rastenja identifikovano je i kvantifikovano više od 20 različitih citokinina i pokazano da je napredovanje senescencije u korelaciji sa opadanjem sadržaja ukupnih citokinina, kao i sa smanjenjem nivoa citokinina trans-zeatinskog tipa. Takođe je uočena jasna korelacija između smanjenja nivoa endogene IAA, odnosno ABA i napredovanja senescencije. Imunohistohemijskom analizom mezofila listova biljaka tretiranih citokininima pokazano je da visoke doze citokinina BA ubrzavaju senescenciju, čime indukuju programiranu smrt ćelija. Analiza aktivnosti pojedinih enzimskih komponenti antioksidativnog sistema pokazala je značajne promene u antioksidativnom statusu listova tokom fiziološkog starenja. Napredovanje senescencije je u jasnoj korelaciji sa smanjenjem aktivnosti CAT i povećanjem ukupne aktivnosti POD. Primarnu antioksidativnu zaštitu tokom starenja obezbeđuju SOD, a u proces odbrane od štetnog dejstva ROS u inicijalnoj fazi senescencije uključuju se POD. Molekularna analiza senescencije pokazala je aktivaciju gena koji kodiraju enzime uključene u konverziju i remobilizaciju produkata razlaganja u senescentnim listovima. qRT-PCR metodom pokazana je indukcija ekspresije gena koji kodiraju cistein proteaze odgovorne za remobilizaciju aminokiselina, kao i cistein proteazu iz grupe metakaspaza uključenu u kontrolu ćelijske smrti. Indukcija ekspresije Gln1-3 i GDH2, SAG-gena uključenih u mobilizaciju azota u senescentnim listovima, naporedo sa padom ekspresije GS2 ukazala je da se asimilacija amonijuma tokom senescencije postepeno premešta iz hloroplasta u citosol mezofilnih ćelija. Leaf senescence constitutes the final stage of leaf development that results in the coordinated degradation of macromolcules and the subsequent nutrient relocation to other plant parts. Leaf senescence involves an integrated action at the cellular, tissue, organ and organism levels under the control of a highly regulated genetic program. During senescence, leaf cells undergo orderly changes in cell structure, metabolism and gene expression leading to cell death, recognized as a form of programmed cell death (PCD). In vitro grown Nicotiana tabacum L. has been chosen as a model plant system, since a single plant provides a large set of leaves in which there is a gradation of senescence. The dynamics of ultrastructural changes in leaf mesophyll cells, as well as metabolic, biochemical and molecular changes during leaf senescence were investigated. We determined the time sequence of senescence-related events in mesophyll cells during physiological ageing of leaves. Typical symptoms of autophagic PCD were detected in senescing leaves using cytological analysis and TUNEL assay. We showed that PCD-specific nuclear DNA fragmentation occurs during natural tobacco leaf senescence but that it starts early in leaf development, thus preceeding ultrastructural changes such as chloroplast degradation and chromatin condensation. Chlorophyll and soluble protein content were identified as reliable biomarkers for estimating the relative age of a particular leaf along the main plant axis. Using HPLC/MS more than 20 different cytokinins were identified and quantified in tobacco leaves. The correlation of senescence with decline in total cytokinin content and the levels of trans-Z was found. The progression of leaf senescence correlated with a gradual decrease in endogenous levels of both IAA and ABA. Immunohistological analysis showed that high levels of cytokinin benzyladenine accelerate senescence thus inducing PCD. The activities of different antioxidative enzymes were analyzed during physiological ageing of leaves. Progression of senescence is correlated with decrease in CAT activity and a concomitant increase in POD activity. Primary antioxidative protection in the course of ageing is provided by SOD, whereas POD are implicated in defense against detrimental ROS action in an initial phase of senescence. Molecular analysis of leaf senescence has revealed activation of genes encoding enzymes involved in conversion and remobilization of degradation products in senescing leaves. Quantitative RT-PCR showed that the expression of cysteine proteases responsible for amino acid remobilization, typically associated with leaf senescence and not found in other forms of PCD, was induced in senescent leaves. Induced expression of SAGs involved in nitrogen mobilization in senescing leaves, Gln1-3 и GDH2, contiguous with decrease in GS2 expression indicated that, during senescence, ammonia assimilation is progressively shifted from the chloroplasts to the cytosol of leaf mesophyll cells.