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ŠUMARSKI LIST 5-6/2016 str. 72     <-- 72 -->        PDF

The plant material is later stored in open sites where, protected with a net from weather conditions, it remains until delivery. The material can be delivered in the growth or dormant stage. The material is characterized by excellent health (regularly controlled by the Plant Protection Institute) and by well developed roots.
A certain number of wild cherry plants, one plant from each clone produced in vitro and acclimatized to in vivo conditions during the winter of 2009/2010 were pot planted in the spring and monitored. Intensive growth was recorded during spring, and most of the young plants reached a height of over 1 meter (Figure 2).
The disinfection procedure was established for all the 24 clones of wild cherry in all the four seasons of the year. Research was initiated in July 2007. To isolate the explants, 30 cm long twigs were collected from the ramets with vegetative axillary buds in the dormant stage. A total of 23 clones were successfully introduced over the four seasons. Clone L3 was not successfully disinfected and was not introduced into the initial culture.
In several of the published papers on micropropagation of wild cherry (Harrington et al. 1994; Pevalek et al. 1994; Hammatt and Grant 1997; Hammatt 1999; Osterc et al. 2004; Ďurković 2006; Mansseri-Lamrioui et al. 2009), the initial explants were established by means of axillary or terminal buds. Initial explants are generally established in the initial culture during active growth of the mother plant in early spring. Fidanci et al. (2008) reports on the introduction of explants from the end of April to the beginning of June, since later collection of explants leads to a high degree of contamination, browning of clumps and reduced growth accompanied by a low multiplication rate. The introduction of woody plant material of wild cherry into the initial micropropagation culture may also present a problem due to overmature ortets or the infected initial material growing in natural forest habitats.
The need to produce wild cherry in vitro may also occur during different seasons, particularly if a certain genotype should be preserved. For this reason, it is of utmost importance to disinfect the initial material of wild cherry throughout the year.
According to the results of different research (Pevalek-Kozlina and Jelaska 1987; Sedlak et al. 2008; Scaltsoyiannes et al. 2009), the best time of collection and establishment is at the beginning of vegetation and intensive growth in the spring. Such material is healthy and is hardly ever contaminated with pathogens or external agents. The research resulted in a high survival rate of the initial explants (70-80%), and all the clones were introduced into the initial culture.
In vitro research with woody plant material frequently reports on the use of mercury chloride for disinfection. To disinfect axillary buds from 10-year-old material, Sedlak et al. (2008) used 0.15% solution of mercury chloride for one minute, while Mansseri-Lamrioui et al. (2009) used 2.5 g/L for 20 minutes. In the works of Szczygieł and Wojda (2008), disinfection with calcium hypochlorite was unsuccessful, but disinfection with 0.1% mercury chloride was successful. Pevalek-Kozlina and Jelaska (1987) and Scaltsoyiannes et al. (2009) successfully disinfected wild cherry with mercury chloride, applied over a shorter time interval and in lower concentrations. Apart from hypochlorite solutions and mercury chloride, some newer disinfectants have been used more recently, such as DICA (dichloroisocyanuric acid sodium salt) in combination with 70% alcohol (Osterch et al. 2004).
The success of micropropagation through in vitro cultures depends on a number of factors. The mineral and hormonal composition of the culture medium, as well as the age of initial explants is of high importance (Chikh 2000). Different mineral systems are used for micropropagation of the selected species, and formulations vary greatly depending on the cultivar, genotype or source of plant material