Expanding from remote areas of Mexico to a worldwide scale, the ten-striped insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), has risen from being an innocuous beetle to a prominent global pest. A diverse life cycle, phenotypic plasticity, adaptation to adverse conditions, and capability to detoxify or tolerate toxins make this insect appear to be virtually “indestructible”. With increasing advances in molecular biology, tools of biotechnological warfare were deployed to combat CPB. In the last three decades, genetically modified potato has created a new challenge for the beetle. After reviewing hundreds of scientific papers dealing with CPB control, it became clear that even biotechnological means of control, if used alone, would not defeat the Colorado potato beetle. This control measure once again appears to be provoking the potato beetle to exhibit its remarkable adaptability. Nonetheless, the potential for adaptation to these techniques has increased our knowledge of this pest and thus opened possibilities for devising more sustainable CPB management programs.

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Cross-talk between phytohormones and sugars is intensely involved in plant metabolism, growth and regeneration. We documented alterations in cytokinin (CK) homeostasis in four developmental stages during de novo shoot organogenesis (DNSO) of kohlrabi (Brassica oleracea var. gongylodes cv. Vienna Purple) seedlings induced by exogenous CKs, trans-zeatin (transZ) and thidiazuron (TDZ), added together with elevated sucrose concentration (6% and 9%). Significant impact of CK and sucrose treatment and their interaction was recorded in all investigated stages, including plantlet development before calli formation (T1 and T2), calli formation (T3) and shoot regeneration (T4). Results showed remarkable increase in total CK levels for transZ treatment, particularly with 9% sucrose. This trend was observed for all physiological and structural groups of CKs. Application of TDZ contributed to little or no increase in CK levels regardless of sucrose concentration. Analysis of expression profiles of organogenesis-related genes involved in auxin transport, CK response, shoot apical meristem formation and cell division revealed that higher sugar concentration significantly downregulated the analysed genes, particularly in T3. This continued on TDZ, but transZ induced an opposite effect with 9% sucrose in T4, increasing gene activity. Our results demonstrated that phytohormone metabolism might be triggered by sucrose signalling in kohlrabi DNSO.

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.Additional key words: GUS activity, hairy roots, shoot regeneration, St. John's wort, sucrose. ⎯⎯⎯⎯ Hypericum perforatum L. (St. John's wort) is an important medicinal plant rich in secondary metabolites considered as an interesting drug source for the pharmaceutical industry. Most of the in vitro culture studies on this species are usually dedicated to cell and tissue cultures and their ability to produce various secondary metabolites. The aim of our investigation was to obtain and study hairy root cultures of H. perforatum. For this purpose we selected A. rhizogenes strain A4M70GUS, previously used in our laboratory to obtain hairy root cultures in several species of Gentiana (Momčilović et al. 1997, Vinterhalter et al. 1999), Lotus corniculatus (Nikolić et al. 2003/04), Centaurium erythrea (Subotić et al. 2003/04), Aesculus hippocastanum (Zdravković-Korać et al. 2004) and Blackstonia perfoliata (Bjelović et al. 2004). So far there are no papers published on Agrobacterium mediated transformation of H. perforatum.Hypericum perforatum L. seeds collected on locations around Belgrade were washed with 70 % ethanol for 1 min and than surface sterilized in 15 % commercial bleach (4 -5 % NaOCl) for 20 min. Seeds were rinsed 3 × 5 min in autoclaved water and aseptically germinated. Skoog (1962; MS) microsalts, iron and vitamins. Media were supplemented with 0.62 % agar and 2 % sucrose. Media pH was adjusted to 5.8 prior to autoclaving performed 20 min at 114 °C. Conditions in the growth room were: temperature of 25 ± 2 °C, 16-h photoperiod and irradiance 46.5 μmol m -2 s -1 . Basal media contained woody plant medium (Lloyd and McCown 1981; WPM) macrosalts and Murashige andEpicotyls were excised from seedling and cultured on basal media supplemented with 0.2 -0.5 mg dm -3 kinetin. Shoot cultures derived from epicotyls were subculture in 5 -6 weeks intervals.A. rhizogenes strain A4M70GUS contains a GUS construct integrated into the TL region of the cointegrative plasmid pRiA4 (Tepfer and Casse-Delbart 1987). GUS construct contains uidA sequence under the 70S promoter (enhancer-doubled 35S Ca MV promoter), followed by NOS polyadenylation sequence. Bacterial strains were maintained according to Van Larebake et al. (1977).Shoots comprising 3 -4 internodes were inoculated by wounding with a needle dipped in bacterial suspension. Wounding was done at the first node above ⎯⎯⎯⎯

De novo shoot organogenesis (DNSO) is a procedure commonly used for the in vitro regeneration of shoots from a variety of plant tissues. Shoot regeneration occurs on nutrient media supplemented with the plant hormones cytokinin (CK) and auxin, which play essential roles in this process, and genes involved in their signaling cascades act as master regulators of the different phases of shoot regeneration. In the last 20 years, the genetic regulation of DNSO has been characterized in detail. However, as of today, the CK and auxin signaling events associated with shoot regeneration are often interpreted as a consequence of these hormones simply being present in the regeneration media, whereas the roles for their prior uptake and transport into the cultivated plant tissues are generally overlooked. Additionally, sucrose, commonly added to the regeneration media as a carbon source, plays a signaling role and has been recently shown to interact with CK and auxin and to affect the efficiency of shoot regeneration. In this review, we provide an integrative interpretation of the roles for CK and auxin in the process of DNSO, adding emphasis on their uptake from the regeneration media and their interaction with sucrose present in the media to their complex signaling outputs that mediate shoot regeneration.