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ŠUMARSKI LIST 5-6/2016 str. 73     <-- 73 -->        PDF

(Rifaud and Cornu 1981; Tricoli et al. 1985; Druart 1992; Dolcet-Sanjuan et al. 2004).
There have been various studies on the effect of different compositions of culture media for in vitro production of root stock for grafting wild cherry and cultivated cherry. Culture media MS, ½ MS, QL (Quorin and Lepoivre 1977), ½ QL, Knop, Heller, commonly fortified with agar 7 g/L and pH 5.8 are cited in the works of: Ivanička and Pretova (1986), Pevalek-Kozlina and Jelaska (1987), Hammatt (1999), Fotopoulos and Sotiropoulos (2005), Sedlak et al. (2008), Scaltsoyiannes et al. (2009) and Tančeva Crmarić (2011). Drawing on different experience, including our own, and on the results from literature, new culture media were formed for this research for all the stages of in vitro cultivation of wild cherry.
Protocols developed for micropropagation of wild cherry and other species of the genus Prunus (Yang and Schmidt 1994; Al-Sabbagh et al. 1999; Pruski et al. 2000; Ruzič et al. 2003; Ruzič and Vujovič 2008) are not broadly used; rather, the protocols are determined by the genotype. In this research, the selected culture medium and a combination of growth regulators resulted in acceptable multiplication of morphologically well formed plants, with no vitrification, for all the 23 genotypes of wild cherry under study.
Although past literature mentions only one cytokinin for multiplication and only one auxin for rooting, in this research the quality of multiplied shoots was improved with a combination of cytokinin with the auxin IAA through all the subcultures of multiplications, which had a favourable effect on plant rooting later on.The use of BAP in the multiplication process of both wild and cultivated cherry is common. Concentrations range from 0.2 mg/L to 2.0 mg/L (Garin et al. 1997; Hammatt and Grant 1997; Grant and Hammatt 1999, 2000; Fidanci et al. 2008; Sedlak et al. 2008; Scaltsoyiannes et al. 2009).
The following cytokinins have so far been used in wild cherry multiplication: BAP, kinetin, Thidiazuron. However, there are no data on the use of 2iP. For this reason, multiplication with two concentrations was attempted in this work in order to study its effects. This cytokinin did not prove suitable for wild cherry multiplication because it did not stimulate the formation of axillary shoots, unlike black chokeberry (Aronia melanocarpa L.), where it proved excellent. In view of the above, experiments with concentrations above 10 mg/L are required since the use of 2iP in the multiplication of black chokeberry resulted in the development of tall plants (unpublished data), taller than 4 cm, which is very important in the rooting stage, and later on for survival in the acclimatisation stage. Numerous investigations of in vitro plant production involved the use of antibiotics in the process for the purpose of eliminating bacterial contamination and preserving healthy planting stock. Sedlak et al. (2008) reports on the application of the antibiotic Cefotaxime 200 mg/L to overcome bacterial contamination during multiplication. In this research, 3 mg/L of a broad-spectrum antibiotic Rifampicin was successfully used in the multiplication phase and no bacterial contamination was recorded.
Zilkah et al. (1992) supplemented the medium with GA3 for elongation. In order to achieve elongation, Scaltsoyiannes et al. (2009) put a red cover over the pots with microplantlets during the multiplication stage.
In vitro rooting is estimated to account for up to 75% of production costs in laboratories (Debergh and Maene 1981). For this reason, a number of authors used in vivo rooting or concentrated on obtaining tall plants of normal appearance, whose survival was high during the acclimatisation stage.
An increase in sugar concentrations in the culture medium led to more resistant plants capable of better adaptation to the transfer since an increase in plant size and total dry matter content was confirmed (Wainwright and Scarce 1989). However, there are opposite views which argue that sugar quantities should be lowered if CO2 sources and photon flux density are increased (Kozai and Iwanami 1988). In order to obtain plants with closed stomata, tests aimed at reducing humidity in apple microplantlets were carried out. The procedure involved the use of desiccants in the elongation and rooting stage, mechanical cooling of glass walls or opening the tops of glass containers (Brainerd and Fuchigami 1981).
There were also experiments involving the addition of growth retardants to reduce the leaf area. The most commonly used retardant for apple was paclobutrazol (Swietlik and Miller 1983). In their experiments, Wardle et al. (1983) added abscisic acid to force plants to lose their leaves and form new leaves in the acclimatisation stage.
Plant acclimatisation is the key step to successful production of in vitro plants. Although extensive research has been dedicated to in vitro production, efforts in overcoming production difficulties in the laboratory will be futile if the problem of transferring the plants from in vitro to in vivo conditions is not solved. Plant acclimatisation is always the bottleneck of this technology (Hazarika 2003). In order to sustain successful plant survival in the acclimatisation substrate, adequate conditions should be ensured, including air and substrate moisture by means of fog and mist irrigation systems and substrate temperature by means of winter heating and summer cooling systems. Additional light should also be provided during short winter days, as well as shade during summer months when light intensity is very high. Moreover, plant nutrition and protection during the entire acclimatisation period is of outstanding importance.