Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation

KLENOTICOVA HELENA, SMÝKALOVA IVA, SVABOVA LENKA, GRIGA MIROSLAV: Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 2013, LXI, No. 5, pp. 1279–1288 Optimisation of in vitro regeneration systems of two explant types for low-tannine cultivars of faba bean based on culturing of shoot apices and cotyledonary nodes were provided by usage of various antioxidants ascorbic acid, citric acid, glutathione and activated charcoal. In subsequent testing, the combined eff ects of antioxidants with transformation co-cultivation compounds acetosyringone and L-cysteine was studied. The application of antioxidants lead to decreased callogenesis, citric acids treatments (50 mg.l−1) dramatically decreased necrotic response of explants. However, citric acid, used together with ascorbic acid completely inhibited shoot growth in shoot apex cultures. Glutathion evoked hyperhydricity of explants. Activated charcoal induced rooting on media which are commonly used for shoot proliferation. Combination of acetosyringone with antioxidants infl uenced shoot proliferation, except of variant with ascorbic acid. Citric acid was the best and universal antioxidant in faba bean in vitro cultures and its use is recommended for faba bean genetic transformation experiments. acetosyringone, antioxidants, citric acid, co-cultivation agents, faba bean, organogenesis The effi cient protocols for in vitro regeneration are necessary for the application of biotechnological methods, including transformation methodology into plant breeding. While the establishment of in vitro culture in pea or soybean is relatively easy, faba bean represents one of in vitro recalcitrant legume due to frequent deterioration of explant material and cultivated tissues as a result of the action of phenolic compounds (Pickardt, 2003; Griga et al., 1986, 1987; Griga, 1988). In faba bean, organogenic in vitro systems are the most frequently used, such as multiple shoot culture initiated from meristems of cotyledon explants with embryonal axis (Anwar et al., 2011), and/or apices (Abdelwahd et al., 2008; Fakhrai et al., 1989; Griga, 1988; Griga and Klenoticova, 1994; Selva et al., 1989; Tejklova et al., 1984). Transformation of plant genome using Agrobacterium tumefaciens is the exploitation of the process of pathogen infection (Dan, 2008). The defence strategy of legumes, starts with rapid accumulation of phenols at the infection site to slow growth rate of pathogen and activation of specifi c defence mechanisms consisting of synthesis molecules related to the stress (Mohamed et al., 1992). Initial response of plants to pathogen attack is an oxidative burst with rapid and transient production of reactive oxygen species (ROS) (Wojtaszek, 1997 in Dan, 2008). In faba bean, even germination of seeds under dark conditions induce increased activity of antioxidant enzymes (Younis et al., 2010) and further, faba bean cuttings of explants lead to rapid strong phenol production, mainly on callus zones. During normal cellular metabolism plants continuously produce ROS but they also simultaneously scavenge these ROS with the help of antioxidant enzymes 1280 Helena Klenoticova, Iva Smýkalova, Lenka Svabova, Miroslav Griga and/or metabolites (Watanabe et al., 2006 in Younis et al., 2010). When explants are cut they exude phenolic compounds that readily oxidize. Oxidized phenolic compounds inhibit enzyme activity and darken the culture medium. Several antioxidants such as ascorbate, cysteine, dithiothreitol, glutathione and tocopherol were successfully used in plants to overcome tissue browning during Agrobacterium-mediated transformation (AMT). Dan (2008) recognised two groups of antioxidants used in plant transformation based on their function. The fi rst group consisting of ascorbic acid (AA), cysteine (CYS), dithiothreitol (DTT), lipoic acid, and polyvinylpyrrolidone (PVP), functions to reduce explant necrosis, increase viability of explants, and improve transformation effi ciency while the other one, which include glutathione (G), selenite and -tocopherol, reduces hyperhydricity and ROS and increases transformation effi ciency. Successful AMT methodology for diff erent varieties of Vicia faba L. was developed by Jelenic et al., 2000; Bottinger et al., 2001 and Hanafy et al., 2005, but with quite low transformation effi ciency (Atif et al., 2013). Although, tested cultivars of broad bean were very susceptible to AMT, the regeneration of transgenic broad bean plants still remains the main problem of the practical use of Agrobacterium-mediated gene delivery in the broad bean (Jelenic et al., 2000). A range of chemical substances with a diff erent nature and action were used for cocultivation during AMT with the aim of enhancing the fi nal effi ciency of the transformation protocol (Svabova and Griga, 2008). Generally, these substances work through induction of virulence – acetosyringone (AS), reduction of resistance of the recipient cell (thiol compounds, antioxidants), or a facilitation to penetrate through the cell wall (mechanically or chemically) (Dandekar and Fisk, 2005; Opabode, 2006; Vrbova et al., 2013). The aim of our study was to elaborate effi cient and highly reproducible regeneration protocol for faba bean as a basis for application of AMT methodology. Several antioxidants were examined to solve the problems connected with occurrence of phenolic compounds and to prevent browning during the whole regeneration period and transient expression studies a er AMT. For the fi rst time in Vicia faba L. , antioxidants were used in combination with co-cultivation substances (AS, CYS) to test their capability of joint application in genetic