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

Fernanda Leal1, Manuela Matos1,

Ana Cláudia Coelho2 and Olinda Pinto-Carnide1

*1IBB-Institute for Biotechnology and Bioengineering,* 

*Centre of Genomic and Biotechnology, University of Trás-os-Montes and Alto-Douro, Department of Genetics and Biotechnology 2CECAV- Center for Animal Science and Veterinary,* 

*University of Trás-os-Montes and Alto-Douro, Department of Veterinary Sciences, Portugal* 

#### **1. Introduction**

118 Fungicides for Plant and Animal Diseases

Zygmunt, J.B. & Namiesnik, J. (2003). Preparation of samples of plant material for

Aromatic and medicinal plants, widely used as folk medicine are, beyond fruits, vegetables grains and spices, the principal source of antioxidant compounds. Several studies demonstrated that antioxidants have also antifungal activity (Jayashree & Subramanyam, 2000; Rasooli & Abyaneh, 2004). More and more, humanity try to replace synthetic metabolites by natural metabolites. Therefore, studies in aromatic and medicinal plants with the capacity to produce a different range of secondary metabolites extremely increase in late years. On the other hand, chemical products, like pesticides, fungicides or bactericides are widely used in agriculture. However, they have disadvantages to the environment, due to contamination of the soils, the final consumers or the producers. Still, the indiscriminate and recurrent use of synthetic fungicides has been found to induce resistance in several fungi, the residual toxicity of these compounds result in human health hazards and requires caution in their use for plant disease control (Singh et al., 2009). Thus, some aromatic and medicinal plants, with antifungal capacity (Soliman & Badeaa, 2002; Goun et al. 2003; Sucharita & Padma, 2010), like genus *Thymus*, *Mentha*, *Calendula* and *Catharanthus* were micropropagated for antifungal activity evaluation.

Medicinal and aromatic plants are important sources for plant secondary metabolites that are involved in many other aspects of a plant's interaction with its immediate environment. The genetic manipulation of plants together with the establishment of *in vitro* plant regeneration systems facilitates efforts to engineer secondary product metabolic pathways (Kumar & Gupta, 2007). Improvement of the yield and quality of these natural plant products through conventional breeding is still a challenge. However, recent advances in plant genomics research has generated knowledge leading to a better understanding of the complex genetics and biochemistry involved in biosynthesis of these plant secondary metabolites (Gómez-Galer et al., 2008). Advances in the cloning of genes involved in relevant pathways, the development of high throughput screening systems for chemical and biological activity, genomics tools and resources, and the recognition of a higher order of regulation of secondary plant metabolism operating at the whole plant

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 121

1991). *Thymus* plants are of much interest owing to their use in different applications, in medicine because of their antiseptic properties, in the cosmetic industry or as a food additive for their organoleptic properties (Duke et al., 2002; Torras et al., 2007). *Thymus* species differ with regard to their morphological features and metabolism, which influences their chemical constitution. Within individual species there are chemical variations that are characterized by different plant oil compositions, usually without any morphological differences (Smolik et al., 2009). Increasingly, plant breeding has taken advantage of developments in molecular biology in order to genotype the species of interest in a way that considerably accelerates their selection. These types of approaches consist of choosing desired genotypes on the basis of molecular markers, or having prior knowledge of the genes that determine the formation of a particular trait in a plant (Pradeep-Reedy et al., 2002). There are many publications related to the antibacterial and antifungal activities of thyme essential oil (Urbanczyk et al., 2002; Priestley et al., 2003; Rasooli & Mirmostafa., 2003). It has been used as weed germination inhibitor (Angelini et al., 2003), and the different extracts from thyme leaves have shown the presence of a large number of flavonoids and vitamin E, compounds of great interest in the food industry due to their antioxidant activities (Sotomayor et al., 2004). There is also interest in using thyme essential

Branches of *Thymus* plants, species *Thymus mastichina* L. and *Thymus zygis* L., with 10 to 20 cm lenght were collected in University of Trás-os-Montes e Alto Douro (UTAD) Botanical Garden, in Vila Real, Portugal. The *explants* disinfected with commercial bleach 60% (v/v) and washed three times with sterile water, were fragmented into nodal segments and placed in different culture media. Basal culture media MS (Murashige & Skoog, 1962) were evaluated without growth regulators or supplemented with a cytokinin, 1 mg/L BAP (6-benzylaminopurine), alone or combined with an auxin, 0.2 mg/L NAA (α-naphthalene-acetic acid). Two carbon sources, sucrose and glucose, at three concentrations (2, 3 or 4 % w/v) were also tested.

The two *Thymus* species studied showed a good response to *in vitro* culture conditions (Fig. 1). After four weeks in culture, *T. mastichina* L. presented higher length of shoots (11.2 mm vs 6.0 mm), while *T. zygis* L. showed bigger shoot number per *explant* (1.9 vs 1.8). The culture media revealed a statistical significant effect (P<0.05) for the parameters evaluated,

In what concern to carbon source, sucrose seems to be the best carbon source to thyme micropropagation. For both species, MS medium containing 3 % sucrose produced the highest shoots length (Fig. 2), similar results were obtained by Bandeira et al. (2007) in *T. vulgaris* L.. With 3 % sucrose concentration the highest shoots length (15.9 mm) was observed in *T. mastichina* L., in MS medium supplied with 1 mg/L BAP. One of the explanations for this is the presence of endogen auxin that releases the addition NAA. The biggest shoot number (9.7) appeared in *T. zygis* L. in MS medium with 2 % sucrose and 1 mg/L BAP. However, considering all the media, the ones with glucose revealed higher values in shoot number when compare with the sucrose ones, 3.6 *vs* 3.3 (though, the difference was not statistically significant P>0.05). Cunha and Fernandes-Ferreira (1999) and Harada e Murai (1996) obtain

oil for delaying the autoxidation of food lipids (Youdim et al., 2002).

Number and length of shoots have been evaluated after four weeks in culture.

similar results with *Linum usitatissium* L. and *Prunus mume*, respectively.

**2.1.1 Methodology** 

**2.1.2 Results and discussion** 

shoot number and length.

level facilitate strategies for the effective manipulation of secondary products in plants (Kumar & Gupta, 2007).

To overcome the problem of antifungal resistance in human pathogens, plants with antimicrobial properties have been extensively studied for a possible application in food microbiology and as alternative treatments for diseases or to prevent bacterial and fungal growth. Many studies have proven very good fungicide effect of plants (Zabka et al., 2011).

The details of plants screened, their families, vernacular names and their therapeutic uses are given in Table 1.


Table 1. Ethnomedical information of the studied species.
