## 4 Discussion  
Disinfection of plant material is a crucial step in plants' micropropagation, as only contamination-free explants may be used for tissue culture initiation. It is especially important in the case of plants endangered by extinction, for which only a limited amount of source explants are available. A reliable method of surface sterilization guarantees the success of tissue culture initiation, with no harm to donor specimens. The presented paper concerns a disinfection method for _Aldrovanda vesiculosa_ tissue culture. The species is critically endangered by extinction and present in only a few stands worldwide; therefore, it is important not to reduce existing populations. There is little information regarding surface sterilization methods for _A. vesiculosa_, and none of them concern the European races. [PERSON] et al. [6] initiated a tissue culture of the Japanese Hanyu race of aldrovanda from seeds, which were collected and surface sterilized with 1% benzalkonium chloride for 5 min, then 1-2% of sodium hypochlorite solution for 5 min, and then 70% ethanol for a few seconds. Next, the seeds were rinsed in distilled water 3-4 times and placed in a B5 Gamborg liquid medium supplemented with 2% sucrose, 20-50 mg\(\cdot\)dm\({}^{-3}\) of gibberellic acid, and some drops of penicillin and streptomycin, with the pH set up to 5.5 for a day. The next day, each seed was placed individually in the same medium in tubes and shaken under the light. After a week, 60% of the seeds germinated without any symptoms of contamination. The authors tried to initiate the cultures from shoot tips, but they failed, and stated that it was too difficult, as they contained many aquatic microorganisms in the traps. The authors managed to initiate the cultures from winter buds with the same method as seeds. At the same time, the authors stated that the growth of the Polish race is different. The studies on the Polish race of _A. vesiculosa_ were conducted by [PERSON] et al. [7]. However, the authors used the plant material obtained from Kondo. They tried to obtain a disinfected in vitro culture of aldrovanda from seeds, and they obtained over 44% germinated seedlings and over 52% contaminations. The authors stated that it was not sufficient, and recommended the use of defoliated shoot pieces and mild surface disinfection. However, the method was not described. As the disinfection of aldrovanda is difficult and there is little plant material available, we decided to use silver nanoparticles to surface-disinfect the explants. Unfortunately, the use of AgNPs was disadvantageous, as it led to the obtaining of a high number of necrotic  
Figure 4: Subsequent influence of the AgNPs 1/5 MS media supplementation on the _Aldrovanda vesiculosa_ morphological features of shoots after the third 8-week passage in the AgNPs-free media. explants and inhibited regeneration of shoots. One of the latest ideas is the application of nanomaterials for the surface sterilization of plants. There have been many experiments carried out to study the influence of AgNPs as a disinfection agent for plant material, and the harmful effects of nano-silver were proved on more than 600 microorganisms. [PERSON] et al. [37] applied AgNPs for the first time to control bacterial contaminations in _Valeriana officinalis_. They obtained single node explants from plants grown in a greenhouse and surface disinfected with 70% EtOH for 1 min, 10% Clorox for 1 min, and then applied AgNPs at a concentration of 100 mg\(\cdot\)dm\({}^{-3}\) for 180 min They obtained 89% contamination-free cultures and nano-silver did not affect either regeneration or rooting of plants. Similarly, [PERSON] and [PERSON] [38] noted a positive effect of AgNPs application in _Cynodon dactylon \"_Teefgreen\". They treated explants with 30% Clorox for 15 min, then rinsed them in distilled water and soaked for 30, 60, or 120 min in 100 or 200 mg\(\cdot\)dm\({}^{-3}\) AgNPs.