Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.) |
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Authors: | Mihaljević Snježana Radić Sandra Bauer Nataša Garić Rade Mihaljević Branka Horvat Gordana Leljak-Levanić Dunja Jelaska Sibila |
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Affiliation: | a Institute Ru?er Boškovi?, Bijeni?ka Cesta 54, 10000 Zagreb, Croatia b Faculty of Science, 10000 Zagreb, Croatia c Croatian National Institute of Public Health, 10000 Zagreb, Croatia |
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Abstract: | Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH4+) as the sole source of nitrogen. Growth of NH4+-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH4+ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH4+ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH4+ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH4+ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin. |
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Keywords: | 2,4-D, 2,4-dichlorophenoxyacetic acid EST, esterases FDA, fluorescein diacetate Gln, smallcaps" >l-glutamine GS, glutamine synthetase MES, 2-(N-morpholino)-ethane-sulfonic acid NH4+, ammonium NO3&minus , nitrate PAL, phenylalanine ammonia-lyase POD, soluble peroxidase SOD, superoxide dismutase |
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