**2.** *In vitro* **multiplication of plants from genus** *Thymus***,** *Mentha***,** *Calendula* **and** *Catharanthus*

The multiplication by *in vitro* culture, means micropropagation, is a very important methodology to obtain a great number of homogeneous plants in a short period of time. *In vitro* culture is an important system in order to optimize and increase the secondary metabolites production. With this technique, *explants* from different species could be micropropagated under optimized condition of culture media, temperature and photoperiod. In this study, different species of plants with antifungal activity were micropropagated, and the fungicide activity of *in vitro* plants compared with the field plants.

In all the studies presented, the culture media were solidified with 0.7% of agar, and pH was adjusted to 5.6-5.8. Culture media were autoclaved at 121ºC for 15 min. The cultures were maintained in a growth chamber at 24 ± 1 ºC on a 16/8-h photoperiod (73 mol m-2 s-1).

Data were subjected to analysis of variance (ANOVA) using STATVIEW 5.0 program, treatment means separated using Fisher´s Least Significant Difference (LSD) test at P = 0.05.

#### **2.1 Micropropagation of** *Thymus*

Thyme is a perennial herb, a 20 to 50 cm shrub, of the Lamiaceae family, an aromatic plant native to the Mediterranean region (Miguel et al., 1999). The genus *Thymus* is exceptionally rich in species, and due to the diversity and plasticity of these plants, their geographical range is very wide. In Portugal are known, at least, 11 *Thymus* species (Afonso & McMurtrie, 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 oil for delaying the autoxidation of food lipids (Youdim et al., 2002).

#### **2.1.1 Methodology**

120 Fungicides for Plant and Animal Diseases

level facilitate strategies for the effective manipulation of secondary products in plants

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

**name Therapeutic use**

(Pina-Vaz et al., 2004)

(Edris et al., 2003)

(Hänsel et al., 1992)

(Jaleel et al., 2009)

Antiseptic, antispasmodic, expectorant, vasoconstrictor

Skin problems, fevers, anti-inflammatory, anti-viral, anti-bacterial and fungicide

Anticancer, antidiabetic, laryngitis, rheumatism, dysmenorrhea

and *Thymus zygis* Lamiaceae Thyme Antiseptic, antispasmodic, antitussive

periwinkle

**2.** *In vitro* **multiplication of plants from genus** *Thymus***,** *Mentha***,** *Calendula*

The multiplication by *in vitro* culture, means micropropagation, is a very important methodology to obtain a great number of homogeneous plants in a short period of time. *In vitro* culture is an important system in order to optimize and increase the secondary metabolites production. With this technique, *explants* from different species could be micropropagated under optimized condition of culture media, temperature and photoperiod. In this study, different species of plants with antifungal activity were micropropagated, and

In all the studies presented, the culture media were solidified with 0.7% of agar, and pH was adjusted to 5.6-5.8. Culture media were autoclaved at 121ºC for 15 min. The cultures were maintained in a growth chamber at 24 ± 1 ºC on a 16/8-h photoperiod (73 mol m-2 s-1).

Data were subjected to analysis of variance (ANOVA) using STATVIEW 5.0 program, treatment means separated using Fisher´s Least Significant Difference (LSD) test at P = 0.05.

Thyme is a perennial herb, a 20 to 50 cm shrub, of the Lamiaceae family, an aromatic plant native to the Mediterranean region (Miguel et al., 1999). The genus *Thymus* is exceptionally rich in species, and due to the diversity and plasticity of these plants, their geographical range is very wide. In Portugal are known, at least, 11 *Thymus* species (Afonso & McMurtrie,

(Kumar & Gupta, 2007).

are given in Table 1.

*Thymus mastichina* 

**and** *Catharanthus*

**2.1 Micropropagation of** *Thymus* 

**Plant species Family Common** 

*Mentha rotundifolia* Lamiaceae Applemint

*Calendula sp.* Asteraceae Marigold

*Catharanthus roseus* Apocynaceae Madagascar

Table 1. Ethnomedical information of the studied species.

the fungicide activity of *in vitro* plants compared with the field plants.

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

#### **2.1.2 Results and discussion**

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, shoot number and length.

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

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 123

The genus *Mentha* belongs to the family Lamiaceae (Labiatae) consisting of about 25 to 30 species mainly found in temperate regions of Eurasia, Australia , South Africa and North America (Shinwari et al., 2011). Mints grow 10 - 120 centimetres tall and can spread over an indeterminate sized area. Mints are used either in the herb form or as an essential oil for flavouring, perfume production and medicinal purposes (Edris et al., 2003). The *Mentha* plant produces secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides. Terpens and quinines are used in food and pharmaceutical, cosmetics and pesticide industries (Khanuja et al., 2000). Some members of this genus are also used as herbal teas and condiments both in fresh and dried form due to their distinct aroma. Morphological markers (such as plant height, leaf shape, colour, etc.) are among the oldest markers used in the evaluation of genetic variability, namely in *Mentha*. However, they are not sufficiently specific and informative because different gene expression in different environments causes wide variability of phenotypic characters in individuals. In some cases congruence between morphology and molecular phylogenetics were reported (Shinwari et al., 2011 as cited in Shinwari, 1995). Genetic diversity refers to the variation at the level of individual gene and provides a mechanism for the plants to adapt in ever changing

The existence of different chemotypes, based on qualitative differences within a taxon, is a common feature in most *Mentha* species and hybrids. As a result, the mint plants produce a number of commercially valuable essential oils, viz. spearmint oil, peppermint oil,

Nodal segments of *Mentha rotundifolia* plants were collected in the greenhouse of Botanical Garden of UTAD (Vila Real, Portugal). The *explants* were disinfected first with ethanol 70% (v/v) then with commercial bleach 40% (v/v) and washed three times with sterile water, then were fragmented into nodal segments and placed in different culture media. MS culture media with different concentrations of BAP (1.0 or 2.0 mg/L) and NAA (0.1 or 0.2

The number and length of shoots and roots, and the presence and diameter of *calli* were evaluated during the eight weeks of *in vitro* culture. After that acclimatization was done in a

The micropropagation of Menthe had, among others, the purpose to evaluate the effect of cytokinin and auxin concentrations in the plant development. After four weeks of *in vitro* culture there was the development of plants in all culture media (Fig. 3). The results obtained in MS + 1 mg/L BAP medium with different concentrations of auxin (0.1 or 0.2 mg/L) revealed that the lowest concentration of auxin induces higher values of shoots length (27.0 mm), and the highest concentration causes higher values of the roots length (16.9 mm)(Table 2). These results are due to the fact that a low ratio of auxin / cytokinin stimulates the development of stem sprouts, and a higher ratio to promote root development (Torres et al., 1998). Pascal et al. (1991) found that the addition of a synthetic

**2.2 Micropropagation of** *Mentha*

pennyroyal oil, etc. (Edris et al., 2003).

environment.

**2.2.1 Methodology** 

mg/L) were used.

mixture of peat and perlite (1:1).

**2.2.2 Results and discussion** 

Fig. 1. Micropropagation of *Thymus* plants. A – *Explant* in the establishment medium culture; B – *In vitro* plants of *T. zygis*; C – *In vitro* plants of *T. mastichina*.

Fig. 2. Effect of culture media (MS) in shoot number and length in *T. zygis* (A and B) and *T. mastichina* (C and D). GLU- glucose, SUC- sucrose, 20- 2%, 30- 3% 40- 4%. Error bars show the 95% confidence interval.

#### **2.2 Micropropagation of** *Mentha*

122 Fungicides for Plant and Animal Diseases

Fig. 1. Micropropagation of *Thymus* plants. A – *Explant* in the establishment medium

A B C

Fig. 2. Effect of culture media (MS) in shoot number and length in *T. zygis* (A and B) and *T. mastichina* (C and D). GLU- glucose, SUC- sucrose, 20- 2%, 30- 3% 40- 4%. Error bars show

the 95% confidence interval.

culture; B – *In vitro* plants of *T. zygis*; C – *In vitro* plants of *T. mastichina*.

The genus *Mentha* belongs to the family Lamiaceae (Labiatae) consisting of about 25 to 30 species mainly found in temperate regions of Eurasia, Australia , South Africa and North America (Shinwari et al., 2011). Mints grow 10 - 120 centimetres tall and can spread over an indeterminate sized area. Mints are used either in the herb form or as an essential oil for flavouring, perfume production and medicinal purposes (Edris et al., 2003). The *Mentha* plant produces secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides. Terpens and quinines are used in food and pharmaceutical, cosmetics and pesticide industries (Khanuja et al., 2000). Some members of this genus are also used as herbal teas and condiments both in fresh and dried form due to their distinct aroma. Morphological markers (such as plant height, leaf shape, colour, etc.) are among the oldest markers used in the evaluation of genetic variability, namely in *Mentha*. However, they are not sufficiently specific and informative because different gene expression in different environments causes wide variability of phenotypic characters in individuals. In some cases congruence between morphology and molecular phylogenetics were reported (Shinwari et al., 2011 as cited in Shinwari, 1995). Genetic diversity refers to the variation at the level of individual gene and provides a mechanism for the plants to adapt in ever changing environment.

The existence of different chemotypes, based on qualitative differences within a taxon, is a common feature in most *Mentha* species and hybrids. As a result, the mint plants produce a number of commercially valuable essential oils, viz. spearmint oil, peppermint oil, pennyroyal oil, etc. (Edris et al., 2003).

#### **2.2.1 Methodology**

Nodal segments of *Mentha rotundifolia* plants were collected in the greenhouse of Botanical Garden of UTAD (Vila Real, Portugal). The *explants* were disinfected first with ethanol 70% (v/v) then with commercial bleach 40% (v/v) and washed three times with sterile water, then were fragmented into nodal segments and placed in different culture media. MS culture media with different concentrations of BAP (1.0 or 2.0 mg/L) and NAA (0.1 or 0.2 mg/L) were used.

The number and length of shoots and roots, and the presence and diameter of *calli* were evaluated during the eight weeks of *in vitro* culture. After that acclimatization was done in a mixture of peat and perlite (1:1).

#### **2.2.2 Results and discussion**

The micropropagation of Menthe had, among others, the purpose to evaluate the effect of cytokinin and auxin concentrations in the plant development. After four weeks of *in vitro* culture there was the development of plants in all culture media (Fig. 3). The results obtained in MS + 1 mg/L BAP medium with different concentrations of auxin (0.1 or 0.2 mg/L) revealed that the lowest concentration of auxin induces higher values of shoots length (27.0 mm), and the highest concentration causes higher values of the roots length (16.9 mm)(Table 2). These results are due to the fact that a low ratio of auxin / cytokinin stimulates the development of stem sprouts, and a higher ratio to promote root development (Torres et al., 1998). Pascal et al. (1991) found that the addition of a synthetic

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 125

as ornamental and medicinal plants. These plants are known to contain saponins, triterpenic alcohols and their fatty acid esters, carotenoids, flavonoids, coumarins, essential oils, hydrocarbons and fatty acids (Hänsel et al., 1992). *Calendula* is known by its medicinal properties - mainly anti-inflammatory, anti-viral, anti-bacterian and fungicide. Considering secondary metabolites, *C. arvensis* is very similar to *C. officinalis* and therefore, the majority of the traditional or folk medicinal uses are similar for both species, including for the cure of skin problems, fevers, chronic infections, wounds, bites and stings (Grieve, 1984; Chevallier, 1996). *Calendula* sp. micropropagation was achieved for the first time using several types of *explants* (Çoçu et al., 2004). Callogenesis can be useful to obtain suspension cell cultures and regeneration of plants *via* indirect organogenesis. *Calli* induction can be obtained with the addition of growth regulators, namely auxins. However, some genotypes are able to induce callus formation even in its absence. In *C. officinalis*, 2,4-D and IAA associated with cytokinins promoted an efficient callus development (Grzelak & Janiszowska, 2002). As secondary metabolites are accumulated in cell cultures from numerous species, much work is focused on their production in shoot or root cultures formed by dedifferentiation of callus (Banthorpe, 1994). Metabolites extracts can be obtained from *in vitro* material, namely *calli* (Grzelak & Janiszowska, 2002), plantlets (Schmeda-Hirschmann, 2005) and flowers

Nodal segments obtained from plants growing in greenhouse conditions were washed in running water, surface-sterilized with a 25% (v/v) sodium hypochlorite solution for 15 min. and finally rinsed 3 times with sterile distilled water (10 min./wash). The *explants* were inoculated in MS media supplemented with BAP (1 or 2 mg/L) alone or combined with 0.1 mg/L of NAA. All the media were supplemented with 30 g/L sucrose. After 4 weeks, the results were analyzed by several parameters like the number and length of shoots and induction and development of *calli*. The *calli* obtained were transferred to MS culture

In general *calli* induction was detected in all the media. Callogenesis occurred simultaneously with organogenesis and, after 4 weeks of *in vitro* culture, flower induction was obtained (Fig. 4). It was not observed a significant effect of the culture medium on the number and length of shoots. The highest number of shoots was obtained in the medium with 2 mg/L BAP and the larger length was obtained in the medium with 1

Considering all the media tested, the average rate of multiplication of *C. arvensis* was 4.21 shoots/*explant*. ÇoÇu et al*.* (2004), using different culture media to micropropagate *C. officinalis* obtained similar values. The best results were obtained in the culture medium with 2.0 mg/L of BAP (7.8 shoots/*explant*) (Fig. 5). The presence of the auxin NAA (in combination with the cytokinin BAP), did not induce a positive effect in the number of shoots (Fig. 5). The media with 2.0 mg/L BAP and 0.1 mg/L NAA registered 4.75 shoots/*explant*. The medium with 1 mg/L BAP produced a higher number of shoots/*explant* in the presence of NAA, but not statistically different (P>0.05) of the

medium added with 3 mg/L 2,4-D, for organogenic development.

(Hamburger et al., 2003).

**2.3.2 Results and discussion** 

mg/L BAP.

medium without it.

**2.3.1 Methodology** 

auxin (IBA) induces an increase in mulberry shoots. Analyzing the effect of BAP concentration, culture media MS + 1 mg/L BAP + 0.2 mg/L NAA and MS + 2 mg/L BAP + 0.2 mg/L NAA, it is clear that the medium with the lowest BAP concentration led to higher values for all parameters, except for *calli* induction that are similar in both medium (0.7 mm diameter). Similar results were obtained by Erig et al. (2002) with black mulberry tree by checking that an increase in BAP concentration resulted in a decrease in length of shoots.

The results showed that the composition of the culture medium influences the response of Menthe to micropropagation. Overall, MS medium without growth regulators showed better results in terms of the number of shoots (2.0 / *explant*), although shoots length was higher in MS medium with 1 mg/L BAP and 0.1 mg/L NAA (27.0 mm). The plants were successfully acclimatized with a rate of 100%.

Fig. 3. Micropropagation of *M. rotundifolia*, plants after four weeks in different culture media. A – MS; B – MS + 1 mg/L BAP + 0.1 mg/L NAA; C – MS + 1 mg/L BAP + 0.2 mg/L NAA; D – MS + 2 mg/L BAP + 0.2 mg/L NAA.


Table 2. Number and length of shoots and roots and percentage and diameter of *calli* per explant, during eight weeks of *in vitro* culture.

#### **2.3 Micropropagation of** *Calendula*

The genus *Calendula* belongs to Asteraceae family, also known as Compositae*,* and includes several species, namely *Calendula arvensis* and *Calendula officinalis*, which are commonly used

as ornamental and medicinal plants. These plants are known to contain saponins, triterpenic alcohols and their fatty acid esters, carotenoids, flavonoids, coumarins, essential oils, hydrocarbons and fatty acids (Hänsel et al., 1992). *Calendula* is known by its medicinal properties - mainly anti-inflammatory, anti-viral, anti-bacterian and fungicide. Considering secondary metabolites, *C. arvensis* is very similar to *C. officinalis* and therefore, the majority of the traditional or folk medicinal uses are similar for both species, including for the cure of skin problems, fevers, chronic infections, wounds, bites and stings (Grieve, 1984; Chevallier, 1996). *Calendula* sp. micropropagation was achieved for the first time using several types of *explants* (Çoçu et al., 2004). Callogenesis can be useful to obtain suspension cell cultures and regeneration of plants *via* indirect organogenesis. *Calli* induction can be obtained with the addition of growth regulators, namely auxins. However, some genotypes are able to induce callus formation even in its absence. In *C. officinalis*, 2,4-D and IAA associated with cytokinins promoted an efficient callus development (Grzelak & Janiszowska, 2002). As secondary metabolites are accumulated in cell cultures from numerous species, much work is focused on their production in shoot or root cultures formed by dedifferentiation of callus (Banthorpe, 1994). Metabolites extracts can be obtained from *in vitro* material, namely *calli* (Grzelak & Janiszowska, 2002), plantlets (Schmeda-Hirschmann, 2005) and flowers (Hamburger et al., 2003).

#### **2.3.1 Methodology**

124 Fungicides for Plant and Animal Diseases

auxin (IBA) induces an increase in mulberry shoots. Analyzing the effect of BAP concentration, culture media MS + 1 mg/L BAP + 0.2 mg/L NAA and MS + 2 mg/L BAP + 0.2 mg/L NAA, it is clear that the medium with the lowest BAP concentration led to higher values for all parameters, except for *calli* induction that are similar in both medium (0.7 mm diameter). Similar results were obtained by Erig et al. (2002) with black mulberry tree by checking that an increase in BAP concentration resulted in a decrease in length of shoots.

The results showed that the composition of the culture medium influences the response of Menthe to micropropagation. Overall, MS medium without growth regulators showed better results in terms of the number of shoots (2.0 / *explant*), although shoots length was higher in MS medium with 1 mg/L BAP and 0.1 mg/L NAA (27.0 mm). The plants were

Fig. 3. Micropropagation of *M. rotundifolia*, plants after four weeks in different culture media. A – MS; B – MS + 1 mg/L BAP + 0.1 mg/L NAA; C – MS + 1 mg/L BAP + 0.2 mg/L

**A B CD**

**MS + 1BAP+0.2NAA** 

**MS + 2BAP+0.2NAA** 

**1BAP+0.1NAA** 

Shoot number 2.0 1.9 1.8 1.5

Root number 3.2 2.6 2.5 0.4

(mm) 18.0 27.0 22.2 11.8

(mm) 18.9 14.5 16.9 4.3

*calli* (%) 0.0 15 15 6

(mm) 0.0 0.7 0.8 0.7

Table 2. Number and length of shoots and roots and percentage and diameter of *calli* per

The genus *Calendula* belongs to Asteraceae family, also known as Compositae*,* and includes several species, namely *Calendula arvensis* and *Calendula officinalis*, which are commonly used

successfully acclimatized with a rate of 100%.

NAA; D – MS + 2 mg/L BAP + 0.2 mg/L NAA.

**media MS MS +** 

explant, during eight weeks of *in vitro* culture.

**2.3 Micropropagation of** *Calendula*

**Culture** 

Shoot length

Root length

*Explants* with

*Calli* diameter

Nodal segments obtained from plants growing in greenhouse conditions were washed in running water, surface-sterilized with a 25% (v/v) sodium hypochlorite solution for 15 min. and finally rinsed 3 times with sterile distilled water (10 min./wash). The *explants* were inoculated in MS media supplemented with BAP (1 or 2 mg/L) alone or combined with 0.1 mg/L of NAA. All the media were supplemented with 30 g/L sucrose. After 4 weeks, the results were analyzed by several parameters like the number and length of shoots and induction and development of *calli*. The *calli* obtained were transferred to MS culture medium added with 3 mg/L 2,4-D, for organogenic development.

#### **2.3.2 Results and discussion**

In general *calli* induction was detected in all the media. Callogenesis occurred simultaneously with organogenesis and, after 4 weeks of *in vitro* culture, flower induction was obtained (Fig. 4). It was not observed a significant effect of the culture medium on the number and length of shoots. The highest number of shoots was obtained in the medium with 2 mg/L BAP and the larger length was obtained in the medium with 1 mg/L BAP.

Considering all the media tested, the average rate of multiplication of *C. arvensis* was 4.21 shoots/*explant*. ÇoÇu et al*.* (2004), using different culture media to micropropagate *C. officinalis* obtained similar values. The best results were obtained in the culture medium with 2.0 mg/L of BAP (7.8 shoots/*explant*) (Fig. 5). The presence of the auxin NAA (in combination with the cytokinin BAP), did not induce a positive effect in the number of shoots (Fig. 5). The media with 2.0 mg/L BAP and 0.1 mg/L NAA registered 4.75 shoots/*explant*. The medium with 1 mg/L BAP produced a higher number of shoots/*explant* in the presence of NAA, but not statistically different (P>0.05) of the medium without it.

*In Vitro* Multiplication of Aromatic and Medicinal Plants and Fungicide Activity 127

*roseus* (L.) G. Don is one of the most important medicinal plants due the accumulation in the leaves of two indolalcaloids vinblastine and vincristine, which were the first natural anticancer agents to be clinically used. The high pharmaceutical value of these secondary metabolites made of *C. roseus* one of the most studied medicinal species. A few protocols for micropropagation of *C. roseus* were reported in the last years (Junaid et al., 2007;

Nodal segments of *Catharanthus roseus* were collected from plants that were potted. *Explants* were disinfected with a 40% (v/v) sodium hypochlorite solution for 15 min. and finally rinsed 3 times with sterile distilled water (10 min./wash). After that, *explants* were inoculated in MS media alone or supplemented with BAP (1 or 1.5 mg/L) alone or combined with 0.2 mg/L of IBA (indole butyric acid) or NAA. All the media were supplemented with 30 g/L sucrose. To evaluate the effect of culture medium in the development of *explant*s was recorded, over eight weeks, the number and length of their

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)

**B** 

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*.

**C** 

shoots and roots, the number of internodes and the presence and diameter of *calli*.

**A** 

Dhandapani et al., 2008; Ilah et al., 2009).

**2.4.1 Methodology** 

**2.4.2 Results and discussion** 

described good results with this media.

*roseus* with flower, at eight weeks of *in vitro* culture.

Fig. 4. *C*. *arvensis* plants after four weeks of *in vitro* culture showing flower development.

The weak response of NAA was also observed by ÇoÇu et al. (2004) with hypocotyl and cotyledon *explants* of C. *officinalis* concluding that the addition of KIN and NAA to culture media reduced the frequency of shoot organogenesis. Considering the length of the shoots, they reached an average of 23.44 mm at the 4th week of *in vitro* culture.

Culture medium

Fig. 5. Effect of different culture media on the number and length of shoots of *C. arvensis*.

#### **2.4 Micropropagation of** *Catharanthus*

*Catharanthus roseus*, an important medicinal plant of Apocynaceae family, is an herbaceous sub-shrub, also known as Madagascar periwinkle and *Vinca rosea* (synonym). Worldwide has been extensively studied by different groups and has been identified to be a source of numerous active principles of therapeutic importance. It is cultivated mainly for its alkaloids (Taylor et al., 1975), with anticancer activities (Jaleel et al., 2009). *Catharanthus*  *roseus* (L.) G. Don is one of the most important medicinal plants due the accumulation in the leaves of two indolalcaloids vinblastine and vincristine, which were the first natural anticancer agents to be clinically used. The high pharmaceutical value of these secondary metabolites made of *C. roseus* one of the most studied medicinal species. A few protocols for micropropagation of *C. roseus* were reported in the last years (Junaid et al., 2007; Dhandapani et al., 2008; Ilah et al., 2009).
