**2.4.2 Results and discussion**

In this study a protocol was established for the micropropagation of *Catharantus roseus* (Fig. 6), MS culture medium was used because Pietrosiuk *et al* (2007) and Dhandapani *et al* (2008) described good results with this media.

Fig. 6. Different phases of the micropropagation of *C*. *roseus*. A – *Explants* after two weeks of in vitro culture; B - Plantlet of *C*. *roseus*, at six weeks of *in vitro* culture; C – Plantlet of *C*. *roseus* with flower, at eight weeks of *in vitro* culture.

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 129

*Aspergillus terreus* or *Aspergillus nidulans* are quantitatively less prevalent (Karthaus, 2010). *A. fumigatus* is one of the most ubiquitous of the airborne saprophytic fungi that is pathogenic to plants, animals and humans. Inhalation of conidia by immunocompetent individuals rarely has any adverse effect (Latgé, 1999). However, apart from the production of mycotoxins, *A. fumigatus* is a dangerous human pathogenic, which is able to cause very serious human and animal mycoses with a high frequency of resistance to chemical antifungal drugs (Verweij et al., 2009; Lass-Florl et al., 2010; Xu et al., 2010; Zabka et al., 2011). Dramatic increases in the incidence of aspergillosis caused primarily by *A. fumigatus* have occurred in recent years. A high infection-associated death rate of up to and over 50% is attributed even today to these fungi (Karthaus, 2010). *A. fumigatus* has become the most important airborne pathogen in developed countries, causing a significant mortality in invasive aspergillosis (Latgé, 1999; Chamilos & Kontoyiannis, 2005). Patients who have been treated with steroid therapy or those with chronic obstructive pulmonary disease or severe hepatic failure are at high risk for developing pulmonary aspergillosis (Meersseman et al.,

The treatment of aspergillosis is most problematic and questionable owing to toxicity and side effects of the used medicines on the base of synthetic fungicides (Karthaus, 2010). Fungal infections remain a significant cause of morbidity and mortality despite advances in medicine and the emergence of new antifungal agents. Drug resistance in fungi is increasing and is becoming a serious concern and the high use and misuse of antifungal as probably the main cause of this situation. Therefore, there is an urgent need to search for effective new antifungal agents in treatment of infectious diseases at present (Xing et al., 2011).

Currently, there are four classes of antifungal agents with activity against *Aspergillus*: the polyenes, such as amphotericin B its lipid formulations, and nystatin (including liposomal nystatin); the triazoles, including itraconazole, the voriconazole and the investigational posaconazole, the echinocandins, such as caspofungin, the micafungin, and the anidulafungin; and the allylamines such as terbinafine (Groll & Kolve, 2004; Chamilos & Kontoyiannis, 2005; Meersseman et al., 2007; Shi et al., 2010). Clinical resistance of invasive aspergillosis to amphotericin B based therapy is observed frequently in clinical practice (Chamilos & Kontoyiannis, 2005). However, they are intrinsically resistant to fluconazole

In other way, pathogenic and toxinogenic fungi are one of the major economic problems of crop and food production (Zabka et al., 2011). In terms of food safety, species of *Fusarium, Aspergillus* and *Penicillium* genera are considered the most significant because they produce the great majority of known mycotoxins (Palumbo et al., 2008; Zabka et al., 2011). There has been increasing concern of the consumers about foods free or with lower level of chemical preservatives because these could be toxic for humans (Bedin et al., 1999; Souza et al., 2005). This resulted in increasing search for new technologies for use in food conservation systems

The spread of multidrug-resistant strains of fungus and the reduced number of drugs available led to a search for therapeutic alternatives, namely among medicinal plants and compounds isolated from them used for their antifungal properties. In these natural sources, a series of molecules with antifungal activity have been found, which are of great importance to

which include alternatives antimicrobials (Brull & Coote, 1999; Souza et al., 2005).

2007; Morace & Bhorgi, 2010).

(Moghaddam et al., 2010).

**3.2 Aromatic plants and antifungal activity** 

Regarding the addition of growth regulators and their effect on the development of the *explant*s, it was found that, in general, the number of shoots was favored in medium supplemented with BAP (MS medium + 1 mg/L BAP) and without auxins (2.23 shoots/*explant*) (Table 3). The length of shoots was higher in the media with the highest concentration of BAP but with the addition of the auxin NAA, MS + 1.5 mg/L BAP + 0.2 mg/L NAA (16.07 mm). It is explained, among other things, by the presence of auxin which promotes cell elongation.


Table 3. Number and length of shoots and roots, number of internodes and percentage and diameter of *calli per explant,* during eight weeks of *in vitro* culture.

In rooting, the results were not as expected because, as has been said, the auxin IBA, is related to the root development, so was expect that MS medium + BAP + 0.2 IBA had had a higher rate of rooting, as results obtained by Dhandapani et al. (2008). However, in our study, MS medium supplied with 1.5 BAP + 0.2 NAA revealed the largest number and length of roots, 11.3 roots/*explant* with 35.4 mm. Similar results were verified by Echeverrigaray and colleagues (2005) for thyme cultivars. With regard to the number of internodes, the medium with the addition of IBA (MS + 1 mg/L BAP + 0.2 mg/L IBA) originated the highest values.

This result was not expected since the auxin IBA is more related with rooting, as reported by Dhandapani et al. (2008) also in *Catharanthus roseus*. The culture media with the lowest values for the internodes was MS medium supplemented with NAA (MS + 1 mg/L BAP + 0.2 mg/L NAA). The culture medium MS + 1.5 mg/L BAP + 0.2 mg/L NAA proved to be the best in the number and diameter of *calli* (3.41 mm), this result was expected, since Paramageetham et al. (2007) in *Centella asiática* L. had found that the formation of *calli* occurred in response to an interaction between auxin, its concentration and type of *explant*.
