ABSTRACT
Rosmarinic acid is one of the main active components of Coleus blumei and is known to have numerous health benefits. The pharmacological significance of rosmarinic acid and its production through in vitro culture has been the subject of numerous studies. Here, the ability of different tissues to accumulate rosmarinic acid and sustainability in production over long cultivation have been tested. Calli, tumours, normal roots and hairy roots were established routinely by application of plant growth regulators or by transformation with agrobacteria. The differences among the established tumour lines were highly heterogeneous. Hairy root lines showed the highest mean growth rate and consistency in rosmarinic acid production. Although some tumour lines produced more rosmarinic acid than the hairy root lines, over a long cultivation period their productivity was unstable and decreased. Further, the effects of plant growth regulators on growth and rosmarinic acid accumulation were tested. 2,4-Dichlorophenoxyacetic acid significantly reduced tumour growth and rosmarinic acid production. 1-Naphthaleneacetic acid strongly stimulated hairy root growth whilst abscisic acid strongly enhanced rosmarinic acid production. Hairy roots cultured in an airlift bioreactor exhibited the highest potential for mass production of rosmarinic acid.
ABSTRACT
Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-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 NH(4)(+) medium with 25mM 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 NH(4)(+) 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 NH(4)(+) 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 (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), 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.
Subject(s)
Cucurbita/metabolism , Nitrates/pharmacology , Plant Somatic Embryogenesis Techniques , Quaternary Ammonium Compounds/pharmacology , Cucurbita/drug effects , Cucurbita/embryology , Cucurbita/enzymology , Esterases/metabolism , Glucans/metabolism , Glutamate-Ammonia Ligase/drug effects , Glutamate-Ammonia Ligase/metabolism , Hydrogen-Ion Concentration , Indoleacetic Acids/pharmacology , Nitrates/metabolism , Nitrogen/metabolism , Nitrogen/pharmacology , Oxidative Stress , Peroxidase/metabolism , Phenylalanine Ammonia-Lyase/drug effects , Phenylalanine Ammonia-Lyase/metabolism , Stress, Physiological , Superoxide Dismutase/metabolism , Time FactorsABSTRACT
Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 m M NH(4)Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 micro M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.
Subject(s)
Cucurbita/embryology , Cucurbita/metabolism , DNA Methylation/drug effects , DNA, Plant/metabolism , Seeds/embryology , Seeds/metabolism , 2,4-Dichlorophenoxyacetic Acid/pharmacology , Ammonium Chloride/pharmacology , Azacitidine/pharmacology , Cucurbita/genetics , Culture Media/pharmacology , DNA, Plant/genetics , Enzyme Inhibitors/pharmacology , Herbicides/pharmacology , Indoleacetic Acids/pharmacology , Nitrogen/metabolism , Nitrogen/pharmacology , Nitrogen Fixation/drug effects , Nitrogen Fixation/physiology , Regeneration/drug effects , Regeneration/physiology , Seeds/geneticsABSTRACT
Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.
Subject(s)
Cucurbita/growth & development , Nitrogen Compounds/pharmacology , Cell Culture Techniques/methods , Cucurbita/cytology , Cucurbita/drug effects , Cucurbita/embryology , Culture Media , Hydrogen-Ion Concentration , Nitrogen Compounds/metabolism , ZygoteABSTRACT
Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.
