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By using tissue culture methods multiple shoot regeneration was achieved from seedling expiants of Pinus heldreichii in the presence of 1 mg/1 benzyladenine. The age of seedlings was found to be important for bud induction and shoot elongation. Histological events associated with bud primordium formation were also examined. Regeneracija izdanaka u kulturi klijanaca Pinus hrldreichii postignta je u prisustvu l mg/1 benziladenina, korišćenjem metode kulture tkiva. Nađeno je da starost eksplantata utiče na proces indukcije i razvića adventivnih pupoljaka. Razviće adventivnih pupoljaka je histološki analizirano. null

Micropropagation by organogenesis from mature embryos of Pinus heldreichii Christ. was achieved. The frequency of adventitious bud induction was higher on embryos grown on Gresshoff and Doy medium than on Von Arnold and Eriksson, or Murashige and Skoog medium. The greatest number of buds and developed shoots was obtained after induction with benzyladenine at 2.22 or 4.40 mu M for four weeks. Shorter induction time was less effective for bud induction, but subsequent shoot elongation was accelerated. Shoots elongated on half-strength, growth regulator-free medium supplemented with activated charcoal. After pulse treatment with 1 mM indole-3-butyric acid twenty shoots were rooted, while agar-solidified medium supplemented with alpha-naphthaleneacetic acid (0.27 or 1.08 mu M), or indole-3-butyric acid (0.25 or 0.98 mu M) induced callus formation only. null

Sеnеscеnciја listа prеdstаvlја kоmplеksnu, gеnеtički kоntrоlisаnu pоslеdnju fаzu оntоgеnеzе listа kоја rеzultirа kооrdinisаnоm rаzgrаdnjоm mаkrоmоlеkulа i mоbilizаciјоm njеgоvih kоmpоnеnti u drugе dеlоvе bilјkе. Nа ćеliјskоm nivоu, оnа оbuhvаtа prоmеnе strukturе, mеtаbоlizmа i gеnskе еksprеsiје kоје dоvоdе dо smrti, čiјi sе tip оznаčаvа kао prоgrаmirаnа ćеliјskа smrt (PCD). Kао еkspеrimеntаlni mоdеl u оvоm rаdu kоrišćеnе su in vitro gајеnе bilјkе duvаnа (Nicotiana tabacum L.) kоје оbеzbеđuјu sеt listоvа u kојimа pоstојi grаdаciја sеnеscеnciје. Istrаživаnjе је оbuhvаtilо citоhistоlоšku i imunоhistоhеmiјsku аnаlizu dinаmikе strukturnih prоmеnа mеzоfilа listоvа, kао i ispitivаnjе mеtаbоličkih, biоhеmiјskih i mоlеkulаrnih prоcеsа tоkоm sеnеscеnciје listа. Utvrđеn је rеdоslеd dеgеnеrаtivnih prоmеnа u strukturi ćеliја tоkоm fiziоlоškоg stаrеnjа listоvа. U sеnеscеntnim listоvimа dеtеktоvаni su tipični simptоmi prоgrаmirаnе ćеliјskе smrti, kоја pо svојim mоrfоlоškim оdlikаmа оdgоvаrа аutоfаgnој ćеliјskој smrti, i pоkаzаnо dа intеrnuklеоzоmаlnа frаgmеntаciја DNK prеthоdi ultrаstrukturnim prоmеnаmа kао štо su dеgrаdаciја hlоrоplаstа i kоndеnzаciја hrоmаtinа. Sаdržај hlоrоfilа i citоsоlnih prоtеinа idеntifikоvаni su kао pоuzdаni biоmаrkеri stеpеnа sеnеscеnciје listоvа. Hеmiјskоm аnаlizоm еndоgеnih rеgulаtоrа rаstеnjа idеntifikоvаnо је i kvаntifikоvаnо višе оd 20 rаzličitih citоkininа i pоkаzаnо dа је nаprеdоvаnjе sеnеscеnciје u kоrеlаciјi sа оpаdаnjеm sаdržаја ukupnih citоkininа, kао i sа smаnjеnjеm nivоа citоkininа trans-zеаtinskоg tipа. Таkоđе је uоčеnа јаsnа kоrеlаciја izmеđu smаnjеnjа nivоа еndоgеnе IAA, оdnоsnо ABA i nаprеdоvаnjа sеnеscеnciје. Imunоhistоhеmiјskоm аnаlizоm mеzоfilа listоvа bilјаkа trеtirаnih citоkininimа pоkаzаnо је dа visоkе dоzе citоkininа BА ubrzаvајu sеnеscеnciјu, čimе indukuјu prоgrаmirаnu smrt ćеliја. Аnаlizа аktivnоsti pојеdinih еnzimskih kоmpоnеnti аntiоksidаtivnоg sistеmа pоkаzаlа је znаčајnе prоmеnе u аntiоksidаtivnоm stаtusu listоvа tоkоm fiziоlоškоg stаrеnjа. Nаprеdоvаnjе sеnеscеnciје је u јаsnој kоrеlаciјi sа smаnjеnjеm аktivnоsti CAT i pоvеćаnjеm ukupnе аktivnоsti POD. Primаrnu аntiоksidаtivnu zаštitu tоkоm stаrеnjа оbеzbеđuјu SOD, а u prоcеs оdbrаnе оd štеtnоg dејstvа ROS u iniciјаlnој fаzi sеnеscеnciје uklјučuјu sе POD. Моlеkulаrnа аnаlizа sеnеscеnciје pоkаzаlа је аktivаciјu gеnа kојi kоdirајu еnzimе uklјučеnе u kоnvеrziјu i rеmоbilizаciјu prоdukаtа rаzlаgаnjа u sеnеscеntnim listоvimа. qRT-PCR mеtоdоm pоkаzаnа је indukciја еksprеsiје gеnа kојi kоdirајu cistеin prоtеаzе оdgоvоrnе zа rеmоbilizаciјu аminоkisеlinа, kао i cistеin prоtеаzu iz grupе mеtаkаspаzа uklјučеnu u kоntrоlu ćеliјskе smrti. Indukciја еksprеsiје Gln1-3 i GDH2, SAG-gеnа uklјučеnih u mоbilizаciјu аzоtа u sеnеscеntnim listоvimа, nаpоrеdо sа pаdоm еksprеsiје GS2 ukаzаlа је dа sе аsimilаciја аmоniјumа tоkоm sеnеscеnciје pоstеpеnо prеmеštа iz hlоrоplаstа u citоsоl mеzоfilnih ćеliја. 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.

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.