Plantlets grown in vitro might be easily impaired by sudden changes in environmental conditions after ex vitro transfer. They usually need several weeks under shade and gradually decreasing air humidity to acclimate to the new conditions and to correct all abnormalities in their anatomy and physiology induced by special conditions of in vitro culture. For plant survival, the most important changes include development of cuticle, epicuticular waxes, and effective stomatal regulation of transpiration leading to stabilization of water status. For plant growth, changes in photosynthetic parameters (chlorophyll content, chloroplast ultrastructure, efficiency of photosystem 2, net photosynthetic rate) ensuring fully autotrophic growth with the rate corresponding to naturally grown plants are the most important. Acclimation can be speeded up by hardening of plantlets in vitro or after transplantation by decreasing the transpiration rate by antitranspirants including abscisic acid, or by increasing photosynthetic rate by elevated CO 2 concentration. INTRODUCTION Within the last four decades, plant micropropagation has developed from a laboratory curiosity to a real industry. Its use in horticulture, agriculture and forestry is currently expanding worldwide. Micropropagation of many species can be achieved through the establishment of explants, their initial growth in vitro being followed by transplanting into the greenhouse or field. During in vitro cultivation, plantlets grow under constant temperature, very high air humidity, low irradiance, very low air turbulence, variable and often insufficient CO 2 concentration, water potential dependent on medium composition, sugars as carbon source, growth regulators in nutrient medium, ethylene and other volatiles, etc. The conditions are very dependent on the vessel and closure types (e.g., Solárová et al., 1996). Acclimation to these conditions leads to formation of plantlets with morphology, anatomy and physiology different from naturally grown plants (for review see, e.g.,

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Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.Additional key words: ascorbate peroxidase, catalase, glutathione reductase, guaiacol peroxidases, superoxide dismutase, syringaldazine peroxidase.⎯⎯⎯⎯ sequence of the gene coding for small subunit of Rubisco; SPOD -syringaldazine peroxidase; SOD -superoxide dismutase; T -transgenic rooted plants; T/C -transgenic grafts; TEM -transmission electron microscopy.

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions. enzyme; Pssupromoter sequence of the gene coding for small subunit of RuBPCO; SE -transgenic rooted plants; SPOD -syringaldazine peroxidase; SOD -superoxide dismutase; SR1 -non-transgenic wild type rooted plants; SRG -non-transgenic grafted plants