*In vitro* **Propagation of Critically Endangered Endemic** *Rhaponticoides mykalea* **(Hub.-Mor.) by Axillary Shoot Proliferation**

Yelda Emek and Bengi Erdag

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55599

### **1. Introduction**

Turkey is one of the richest countries in variability of flora. It has nearly 9000 plant species about 3000 of which are endemic [1]. *Asteraceae*, is represented by 50 species in Turkey with an endemism of nearly 54% [2]. *Rhaponticoides mykalea* (Hub.-Mor.) M.V. Agab. & Greuter which belongs to the *Asteraceae* family, falls within the CR (Critically Endangered) category in the Red Data Book of Turkey [1]. While *R. mykalea* (Hub.-Mor.) was classified under the section *Centaurea* as *Centaurea mykalea* (Hub.-Mor.) before now. Today it has been separated from the section Centaurea [3]. It spreads very scarce in Kuşadası (Aydın), Muğla and Isparta, and faces with the danger of extinction. *R. mykalea* that has very limited number of individuals is under strong anthropogenic pressure such as the gradually increase in ongoing urbanization due to rapid developments of tourism sector, the conversion of natural habitats into human domi‐ nated lands, the over-grazing and collecting capitula of *R. mykalea* by local people for food. The species has already been under the threat of extinction and the situation above will increase the risk of extinction of this species even more [4]. For this reason, local protection measures and global conservation strategies are necessary [5].

Nowadays, the conservation of wild plant genetic resources is very important for preventing a decrease in genetic variability. Conservation of the endemic or threatened plants is carried out using different strategies. *In vitro* culture is an efficient method for *ex situ* conservation of plant diversity [6,7], because many endangered species can be quickly propagated and preserved from a minimum of plant material with low impact on wild populations with this technology [8]. In recent years, there has been an increased interest in *in vitro* techniques that offer powerful tools for germplasm conservation and the mass multiplication of many

© 2013 Emek and Erdag; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

threatened plant species [9]. Especially *in vitro* propagation of endangered plants can offer considerable benefits for the rapid cultivation of at risk species that have a limited reproductive capacity and exist in threatened habitats [5].

Micropropagation constitutes a powerful tool for *ex situ* conservation programs of threatened plants, especially for species with very reduced populations or low seed production [6,7]. This technique facilitates the rapid establishment of a large number of stock plants, from a minimum of original plant material, thus imposing minimum impact on the endangered wild popula‐ tions. Axillary shoot proliferation typically results in average tenfold increase in shoot number per monthly culture passage. In a period of 6 months, it is feasible to obtain as many as 1 000 000 propagulesor plants, starting from a single explant [10].With this technology various endemic and endangered species have been successfully propagated; such as and *Centaurea paui* [8], *Anthemis xylopoda* O.Schwarz [11], *Centaurea spachii* [12], *Centaurea zeybek*ii [13], *Centaurea junoniana* [14], *Astragalus chrysochlorus* [15], *Centaurium rigualii* [16] and *Syzygium alternifolium* [17].

However, during our literature search, no report concerning *in vitro* regeneration of *R.myka‐ lea* by axillary shoot proliferation was found.

**2.2. Achene viability**

**Figure 1.** Achene containing mature embryo (before dormancy).

seed is considered as viable or not [20].

embryos were evaluated as alive.

Achene viability was subjected to tetrazolium test.Tetrazolium test is based on reduction of colourless solution 2,3,5–tripheniltetrazolim chloride or bromide into insoluble 2,3,5– triphe‐ nilformazan red in colour. This solution acts as an indicator for detection of reduction processes that take place in living parts of the seed. Inside the seed, tetrazolium intakes hydrogen from dehydrogenase. By hidrogenization of tetrazolium a red, stable substance called formazan, which dyes living parts of the seed, is formed in the living cells.Tissue of many plant species must be removed to introduce the dye into the tissue. Tissue removal can be done by pilling the seed coat off, punching, and longitudinal or cross-cutting of unessential seed parts. Prepared seed is submerged into 0,5 – 1% tetrazolium solution. Seed must be completely covered with solution, and not exposed to direct light. After the time needed for dyeing expires (it depends on plant species) the estimation of dyeing is approached. All tissue (necessary for normal seedling development) of a viable seed should be dyed. Except completely dyed, viable seeds, and completely undyed, unviable seeds, a partly dyed seeds may also be found. Depending on the species, small undyed spots of some parts of these tissues may be accepted. Location, size of undyed areas, and sometimes intensity of dyeing, determine whether some

*In vitro* Propagation of Critically Endangered Endemic *Rhaponticoides mykalea* (Hub.-Mor.) by Axillary Shoot

Proliferation

205

http://dx.doi.org/10.5772/55599

To determine achene viability of *R. mykalea*, longitudinally-halved seeds were treated in tetrazolium solution (TTC, 1%) for 2 h at room temperature. After that time, red staining

In order to determine proper sterilisation procedure, achenes isolated from capitula were washed thoroughly under running tap water for 30 mins. Subsequently at various times, achenes were put in 70% (w/v) ethanol and 4.5% (w/v) sodium hypochlorite containing 2 drops

**2.3. Seed sterilisation, media preparation and culture conditions**

The objective of the present study was to establish an efficient *in vitro* method for the rapid propagation via axillary shoot propagation of *R.mykalea,* a critically endangared endemic plant species. The shoots that were obtained from *in vitro* germinated mature embryos were used for axillary shoot proliferation. For that reason, the most appropriate cytokinin type and concentration were determined.

### **2. Material and methods**

#### **2.1. Plant material and explant source**

Capitula of *Rhaponticoides mykalea* were collected from a wild population in Aydın-Turkey (Samsun mountain, localities: N 37 º 47.01 " ; E 027º 19.16 ") during summer period (July and August -2008) before seed dormancy period (Figure 1).

*R. mykalea* has been propagated from seed in the past [18]. However, researchers have explained that the seed is not suitable explant for *in vitro* propagation of *R. mykalea* due to strong seed dormancy and low germination frequency even after dormancy period. Therefore, embryos isolated from achenes which have not yet crossed dormancy periods were used as initial explant.

The achenes isolated from capitula were sterilised, and mature zygotic embryos that were dissected out from achene were used as initial explant. Mature zygotic embryos were dissected out from achenes and cultured on Murashige and Skoog (MS) [19] basal medium for germi‐ nation. The shoots that were obtained from *in vitro* germinated mature embryos were used for axillary shoot proliferation.

*In vitro* Propagation of Critically Endangered Endemic *Rhaponticoides mykalea* (Hub.-Mor.) by Axillary Shoot Proliferation http://dx.doi.org/10.5772/55599 205

**Figure 1.** Achene containing mature embryo (before dormancy).

#### **2.2. Achene viability**

threatened plant species [9]. Especially *in vitro* propagation of endangered plants can offer considerable benefits for the rapid cultivation of at risk species that have a limited reproductive

Micropropagation constitutes a powerful tool for *ex situ* conservation programs of threatened plants, especially for species with very reduced populations or low seed production [6,7]. This technique facilitates the rapid establishment of a large number of stock plants, from a minimum of original plant material, thus imposing minimum impact on the endangered wild popula‐ tions. Axillary shoot proliferation typically results in average tenfold increase in shoot number per monthly culture passage. In a period of 6 months, it is feasible to obtain as many as 1 000 000 propagulesor plants, starting from a single explant [10].With this technology various endemic and endangered species have been successfully propagated; such as and *Centaurea paui* [8], *Anthemis xylopoda* O.Schwarz [11], *Centaurea spachii* [12], *Centaurea zeybek*ii [13], *Centaurea junoniana* [14], *Astragalus chrysochlorus* [15], *Centaurium rigualii* [16] and *Syzygium*

However, during our literature search, no report concerning *in vitro* regeneration of *R.myka‐*

The objective of the present study was to establish an efficient *in vitro* method for the rapid propagation via axillary shoot propagation of *R.mykalea,* a critically endangared endemic plant species. The shoots that were obtained from *in vitro* germinated mature embryos were used for axillary shoot proliferation. For that reason, the most appropriate cytokinin type and

Capitula of *Rhaponticoides mykalea* were collected from a wild population in Aydın-Turkey (Samsun mountain, localities: N 37 º 47.01 " ; E 027º 19.16 ") during summer period (July and

*R. mykalea* has been propagated from seed in the past [18]. However, researchers have explained that the seed is not suitable explant for *in vitro* propagation of *R. mykalea* due to strong seed dormancy and low germination frequency even after dormancy period. Therefore, embryos isolated from achenes which have not yet crossed dormancy periods were used as

The achenes isolated from capitula were sterilised, and mature zygotic embryos that were dissected out from achene were used as initial explant. Mature zygotic embryos were dissected out from achenes and cultured on Murashige and Skoog (MS) [19] basal medium for germi‐ nation. The shoots that were obtained from *in vitro* germinated mature embryos were used for

capacity and exist in threatened habitats [5].

204 Current Progress in Biological Research

*lea* by axillary shoot proliferation was found.

concentration were determined.

**2. Material and methods**

initial explant.

axillary shoot proliferation.

**2.1. Plant material and explant source**

August -2008) before seed dormancy period (Figure 1).

*alternifolium* [17].

Achene viability was subjected to tetrazolium test.Tetrazolium test is based on reduction of colourless solution 2,3,5–tripheniltetrazolim chloride or bromide into insoluble 2,3,5– triphe‐ nilformazan red in colour. This solution acts as an indicator for detection of reduction processes that take place in living parts of the seed. Inside the seed, tetrazolium intakes hydrogen from dehydrogenase. By hidrogenization of tetrazolium a red, stable substance called formazan, which dyes living parts of the seed, is formed in the living cells.Tissue of many plant species must be removed to introduce the dye into the tissue. Tissue removal can be done by pilling the seed coat off, punching, and longitudinal or cross-cutting of unessential seed parts. Prepared seed is submerged into 0,5 – 1% tetrazolium solution. Seed must be completely covered with solution, and not exposed to direct light. After the time needed for dyeing expires (it depends on plant species) the estimation of dyeing is approached. All tissue (necessary for normal seedling development) of a viable seed should be dyed. Except completely dyed, viable seeds, and completely undyed, unviable seeds, a partly dyed seeds may also be found. Depending on the species, small undyed spots of some parts of these tissues may be accepted. Location, size of undyed areas, and sometimes intensity of dyeing, determine whether some seed is considered as viable or not [20].

To determine achene viability of *R. mykalea*, longitudinally-halved seeds were treated in tetrazolium solution (TTC, 1%) for 2 h at room temperature. After that time, red staining embryos were evaluated as alive.

#### **2.3. Seed sterilisation, media preparation and culture conditions**

In order to determine proper sterilisation procedure, achenes isolated from capitula were washed thoroughly under running tap water for 30 mins. Subsequently at various times, achenes were put in 70% (w/v) ethanol and 4.5% (w/v) sodium hypochlorite containing 2 drops of wetting agent (Tween-80); afterwards, the achenes were rinsed three times (5 mins each) with sterile distilled water in a laminar flow hood. After sterilisation, zygotic embryos were isolated from achenes and cultured on PDA (Potato Dextrose Agar) to determine early contamination. PDA cultures were maintained at 24 ± 2 ºC for 3 days. At the end of this period, observations were made in order to determine the appropriate sterilisation time.

under 24 ± 2°C and 16-h light photoperiod. After 4 weeks the plantlets kept at normal labora‐

*In vitro* Propagation of Critically Endangered Endemic *Rhaponticoides mykalea* (Hub.-Mor.) by Axillary Shoot

Proliferation

207

http://dx.doi.org/10.5772/55599

Means of shoot number per explant, shoot lenght and frequency of rooting were ana‐ lyzed by one-way analysis of variance (ANOVA, SPSS for Windows v.9., SPSS, USA). Differences were analyzed by analysis of variance and the means compared using Duncan's multiple range test at p< 0.05. Data giving in percentages were subjected to x´ = arcsine √

The viability percentage of achenes was 80% according to Tetrazolium test. According to our results, the most suitable sterilisation procedure of achenes is as follows: The achenes are washed thoroughly under running tap water for 30 mins. After this process, seeds must be exposed to 70% (w/v) ethanol for five mins and then to 4.5% (w/v) sodium hypochlorite containing 2 drops wetting agent (Tween-80) for eight mins. Finally, seeds are rinsed three times with sterilised distilled water (5 mins each). Sterile cultures are than obtained in high

Four-week-old sterile seedlings obtained from mature zygotic embryos were used as explant for axillary shoot proliferation experiments. At the end of the experiments, the most appro‐ priate cytokinin type and concentration were determined (Figure 2). Axillary shoot propaga‐ tion of *R. mykalea* was obtained in all media without or with cytokinin. Cytokinins are generally recognized as critical for the production of shoot primordia under *in vitro* conditions. Both cytokinins induced healthy shoots in our study. However, it is shown that BA is more effective cytokinin than KIN. The maximum shoot number per explant were obtained in 0.5 mgl-1 BA

A decrease in the number of shoots were observed at both higher (1 and 2 mgl-1) and lower concentrations of BA (0.1 mgl-1). Similar results were also reported for axillary shoot prolifer‐ ation of *Centaurea spachii* [12] and *Centaurea zeybekii* [13]. BA was also reported as an effective cytokinin for other endemic and threatened *Centaurea* species [14, 16]. However, BA was evaulated as an effective cytokinin for shoot multiplication in many species of *Asteraceae*; such as *Centaurea junoniana* [14], *Gerbera jamesonii* hybrida [22], *Centaurium rigualii* [16], *Syzygium*

MS medium supplemented with 0.1 mgl-1 Kinetin (KIN) was determined as the most suitable medium for the maximum shoot length (7.35 cm) (Figure 4). While BA is more effective cytokinin on shoot multiplication, KIN is more effective on shoot lenght. In spite of the increased number of shoots on media containing cytokinin, the shoot length is decreased. A negative correlation between the shoot number and their length has been observed. This kind of negative correlation was reported in *Centaurea paui* by using inflorescence stalk as explant

tory conditions.

**2.6. Statistical analyses**

(x/100) transformation [21].

proportion (100%).

**3. Results and discussion**

added MS medium (5.8 shoot/explant) (Figure 2 and 3).

*alternifolium* [17] and *Anthemis xylopoda* [11].

[8] and *C. zeybekii* by axillary shoot proliferation [13].

All the experiments were maintained on semi-solid basal medium supplemented with or without various concentration of plant growth regulators. Basal medium contained Murashige and Skoog (MS) [19] mineral salts, 100 mgl-1 myo-inositol, 2 mgl-1 glycine, 0.5 mgl-1 nicotinic acid, 0.5 mgl-1 pyridoxine-HCl, 0.1 mgl-1 thiamine-HCL, 3% (w/v) sucrose, 8 gl-1 agar (Agaragar), various concentration of plant growth regulators 1 N6 - Benzyladenine (BA) and Kinetin (KIN), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) were used in experiments depending on experimental objectives.

The pH of media was adjusted to 5.8 with 1M NaOH or HCl prior to autoclaving at 105 kPa and 121° C for 15 min. Culture vessels were 190 ml glass jars containing 30 ml of medium.

#### **2.4. Axillary shoot proliferation**

Mature embryos that were isolated from achenes were cultured on MS basal medium to obtain sterile seedlings (unpublished data). After eight weeks, seedlings (~2-3 cm), were separated from primary roots and transferred to MS medium containing different concentrations of BA or KIN (0.1, 0.5, 1.0 and 2.0 mgl-1) for axillary shoot propagation. A control treatment without cytokinins was also included. At the end of the 3 subculture, the number of shoots per explant and average shoot length was evaluated for each cytokinin type and concentration.

Axillary shoot proliferation experiments were conducted with 15 replications consisting of one explant per jar and were repeated three times. Cultures were incubated at 24 ± 2 °C under a light regime of 16 h photoperiod by cool-white fluorescent lamps. The cultures were subcul‐ tured to fresh medium of the same composition at an interval of 4 weeks.

#### **2.5. Shoot rooting and acclimatization of plantlets**

After three subcultures, elongated shoots (~4 cm) were excised stock cultures and transferred to MS and half strength MS medium (½ MS) with or without different concentrations (0.5, 1.0, 2.0 and 5.0 mgl-1) of auxins (IBA, IAA or NAA) for rooting. The results of rooting experiments were expressed in percentage after 6 weeks of culture initiation.

Rooting experiments were conducted with 15 replications consisting of one explant per jar and were repeated three times. Cultures were incubated at 24 ± 2 °C under a light regime of 16 h photoperiod by cool-white fluorescent lamps. The cultures were subcultured to fresh medium of the same composition at an interval of 4 weeks.

After 8 weeks of rooting *in vitro*, the plantlets were removed from the culture jars then the agar was carefully washed off the rooted plantlets to minimize pathogen attack. The plantlets were planted into 10 cm diameter plastic pots containing garden soil and kept in the growth chamber under 24 ± 2°C and 16-h light photoperiod. After 4 weeks the plantlets kept at normal labora‐ tory conditions.

#### **2.6. Statistical analyses**

of wetting agent (Tween-80); afterwards, the achenes were rinsed three times (5 mins each) with sterile distilled water in a laminar flow hood. After sterilisation, zygotic embryos were isolated from achenes and cultured on PDA (Potato Dextrose Agar) to determine early contamination. PDA cultures were maintained at 24 ± 2 ºC for 3 days. At the end of this period,

All the experiments were maintained on semi-solid basal medium supplemented with or without various concentration of plant growth regulators. Basal medium contained Murashige and Skoog (MS) [19] mineral salts, 100 mgl-1 myo-inositol, 2 mgl-1 glycine, 0.5 mgl-1 nicotinic acid, 0.5 mgl-1 pyridoxine-HCl, 0.1 mgl-1 thiamine-HCL, 3% (w/v) sucrose, 8 gl-1 agar (Agar-

(KIN), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA)

The pH of media was adjusted to 5.8 with 1M NaOH or HCl prior to autoclaving at 105 kPa and 121° C for 15 min. Culture vessels were 190 ml glass jars containing 30 ml of medium.

Mature embryos that were isolated from achenes were cultured on MS basal medium to obtain sterile seedlings (unpublished data). After eight weeks, seedlings (~2-3 cm), were separated from primary roots and transferred to MS medium containing different concentrations of BA or KIN (0.1, 0.5, 1.0 and 2.0 mgl-1) for axillary shoot propagation. A control treatment without cytokinins was also included. At the end of the 3 subculture, the number of shoots per explant

Axillary shoot proliferation experiments were conducted with 15 replications consisting of one explant per jar and were repeated three times. Cultures were incubated at 24 ± 2 °C under a light regime of 16 h photoperiod by cool-white fluorescent lamps. The cultures were subcul‐

After three subcultures, elongated shoots (~4 cm) were excised stock cultures and transferred to MS and half strength MS medium (½ MS) with or without different concentrations (0.5, 1.0, 2.0 and 5.0 mgl-1) of auxins (IBA, IAA or NAA) for rooting. The results of rooting experiments

Rooting experiments were conducted with 15 replications consisting of one explant per jar and were repeated three times. Cultures were incubated at 24 ± 2 °C under a light regime of 16 h photoperiod by cool-white fluorescent lamps. The cultures were subcultured to fresh medium

After 8 weeks of rooting *in vitro*, the plantlets were removed from the culture jars then the agar was carefully washed off the rooted plantlets to minimize pathogen attack. The plantlets were planted into 10 cm diameter plastic pots containing garden soil and kept in the growth chamber

and average shoot length was evaluated for each cytokinin type and concentration.

tured to fresh medium of the same composition at an interval of 4 weeks.

were expressed in percentage after 6 weeks of culture initiation.

**2.5. Shoot rooting and acclimatization of plantlets**

of the same composition at an interval of 4 weeks.

N6


observations were made in order to determine the appropriate sterilisation time.

agar), various concentration of plant growth regulators 1

**2.4. Axillary shoot proliferation**

206 Current Progress in Biological Research

were used in experiments depending on experimental objectives.

Means of shoot number per explant, shoot lenght and frequency of rooting were ana‐ lyzed by one-way analysis of variance (ANOVA, SPSS for Windows v.9., SPSS, USA). Differences were analyzed by analysis of variance and the means compared using Duncan's multiple range test at p< 0.05. Data giving in percentages were subjected to x´ = arcsine √ (x/100) transformation [21].

### **3. Results and discussion**

The viability percentage of achenes was 80% according to Tetrazolium test. According to our results, the most suitable sterilisation procedure of achenes is as follows: The achenes are washed thoroughly under running tap water for 30 mins. After this process, seeds must be exposed to 70% (w/v) ethanol for five mins and then to 4.5% (w/v) sodium hypochlorite containing 2 drops wetting agent (Tween-80) for eight mins. Finally, seeds are rinsed three times with sterilised distilled water (5 mins each). Sterile cultures are than obtained in high proportion (100%).

Four-week-old sterile seedlings obtained from mature zygotic embryos were used as explant for axillary shoot proliferation experiments. At the end of the experiments, the most appro‐ priate cytokinin type and concentration were determined (Figure 2). Axillary shoot propaga‐ tion of *R. mykalea* was obtained in all media without or with cytokinin. Cytokinins are generally recognized as critical for the production of shoot primordia under *in vitro* conditions. Both cytokinins induced healthy shoots in our study. However, it is shown that BA is more effective cytokinin than KIN. The maximum shoot number per explant were obtained in 0.5 mgl-1 BA added MS medium (5.8 shoot/explant) (Figure 2 and 3).

A decrease in the number of shoots were observed at both higher (1 and 2 mgl-1) and lower concentrations of BA (0.1 mgl-1). Similar results were also reported for axillary shoot prolifer‐ ation of *Centaurea spachii* [12] and *Centaurea zeybekii* [13]. BA was also reported as an effective cytokinin for other endemic and threatened *Centaurea* species [14, 16]. However, BA was evaulated as an effective cytokinin for shoot multiplication in many species of *Asteraceae*; such as *Centaurea junoniana* [14], *Gerbera jamesonii* hybrida [22], *Centaurium rigualii* [16], *Syzygium alternifolium* [17] and *Anthemis xylopoda* [11].

MS medium supplemented with 0.1 mgl-1 Kinetin (KIN) was determined as the most suitable medium for the maximum shoot length (7.35 cm) (Figure 4). While BA is more effective cytokinin on shoot multiplication, KIN is more effective on shoot lenght. In spite of the increased number of shoots on media containing cytokinin, the shoot length is decreased. A negative correlation between the shoot number and their length has been observed. This kind of negative correlation was reported in *Centaurea paui* by using inflorescence stalk as explant [8] and *C. zeybekii* by axillary shoot proliferation [13].

**Figure 2.** Axillary shoot proliferation on MS medium added 0.5 mgl-1BA.

Figure 2. Axillary shoot proliferation on MS medium added 0.5 mgl-1BA.

Figure 3. Cytokinin effects on axillary shoot multiplication of *R.mykalea.* Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according to Duncan Multiple Range Test.

Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according to Duncan

more effective than other auxins on rooting for Asteraceae such as *Anthemis xylopoda* [11, 23] , *Centaurea spachii* [12], *Centaurea ragusina* [24], *Centaurea zeybekii* [25] and *Saussurea*

Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each col‐ umn and capital letters in each row are significantly different (p< 0.05), according to Duncan Multiple Range Test.

Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p< 0.05), according to Duncan

**Plant growth regulators (mg/L)**

**Plant growth regulators (mg/L)**

Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p< 0.05), according to Duncan

<sup>d</sup> <sup>e</sup>

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**7** 6-7-8 Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p<0.05), according to Duncan

**7** 6-7-8 Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p<0.05), according to Duncan

This information is a figure (5) information, Could you please place the bottom of the figure 5?

*In vitro* Propagation of Critically Endangered Endemic *Rhaponticoides mykalea* (Hub.-Mor.) by Axillary Shoot

This information is a figure (5) information, Could you please place the bottom of the figure 5?

cd

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Control 0,1 BA 0,5 BA 1 BA 2 BA 0,1 KIN 0,5 KIN 1 KIN 2 KIN

Control 0,1 BA 0,5 BA 1 BA 2 BA 0,1 KIN 0,5 KIN 1 KIN 2 KIN

Figure 4. Average shoot lengths of axillary shoots dependent on cytokinin type and concentration. Means by different letters in each column and capital letters in each row are significantly different (*p<*

Figure 4. Average shoot lengths of axillary shoots dependent on cytokinin type and concentration. Means by different letters in each column and capital letters in each row are significantly different (*p<*

Means by different letters in each column and capital letters in each row are significantly different (*p*< 0.05), according

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**Plant growth regulators(mg/L)**

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http://dx.doi.org/10.5772/55599

Proliferation

209

Multiple Range Test.

Multiple Range Test.

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**Avarage shoot lenght (cm)**

**Avarage shoot lenght (cm)**

to Duncan Multiple Range Test.

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0.05), according to Duncan Multiple Range Test.

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Figure 5. Rooting of *R.mykalea* axillary shoots.

Figure 5. Rooting of *R.mykalea* axillary shoots.

<sup>h</sup> <sup>g</sup>

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**Figure 4.** Average shoot lengths of axillary shoots dependent on cytokinin type and concentration.

**8** 1 technique techniques

**8** 1 technique techniques

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> There was a statistically significant difference between MS and ½ MS medium on root‐ ing. ½ MS medium is more effective than MS medium on rooting in all experiments. Also, there was a statistically significant difference on rooting of *R. mykalea* between auxin type

> > 2

2

*obvallata* [26].

Multiple Range Test.

Multiple Range Test.

**Figure 5.** Rooting of *R.mykalea* axillary shoots.

0 10 20 30 40 50 60 **R** 70 **o o t i n g ( %)**

0 10 20 30 40 50 60 **R** 70 **o o t i n g ( %)**

and concentration.

Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according to Duncan

Multiple Range Test. **Figure 3.** Cytokinin effects on axillary shoot multiplication of *R.mykalea.*

Multiple Range Test.

h de g f cd a ef c b 0 1 2 3 4 5 6 7 8 9 **Avarage shoot lenght (cm)** After three subculturing, solitary shoots excised from multiple shoot cultures were transferred to MS and ½ MS media containing IAA, IBA and NAA at various concentra‐ tions for rooting. Rhizogenezis was not occured MS and ½ MS medium without plant growth regulators. Auxin is necessary for *in vitro* rooting of *R.mykalea* axillary shoots. Generally, ½ MS medium added auxin is more effective than MS medium added auxin for rooting. The maximum rooting rate was obtained with half-strength MS medium supple‐ mented with 0.5 mgl-1 IBA (55%) (Figure 5 and 6).There are many of reports about IBA is

Control 0,1 BA 0,5 BA 1 BA 2 BA 0,1 KIN 0,5 KIN 1 KIN 2 KIN

**Plant growth regulators (mg/L)**

Figure 4. Average shoot lengths of axillary shoots dependent on cytokinin type and concentration.

This information is a figure (5) information, Could you please place the bottom of the figure 5? a 9 *In vitro* Propagation of Critically Endangered Endemic *Rhaponticoides mykalea* (Hub.-Mor.) by Axillary Shoot Proliferation http://dx.doi.org/10.5772/55599 209

**7** 6-7-8 Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p<0.05), according to Duncan

This information is a figure (5) information, Could you please place the bottom of the figure 5?

**7** 6-7-8 Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p<0.05), according to Duncan

Figure 4. Average shoot lengths of axillary shoots dependent on cytokinin type and concentration. Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according to Duncan Multiple Range Test. Means by different letters in each column and capital letters in each row are significantly different (*p*< 0.05), according to Duncan Multiple Range Test. Could you please replace Figure 4?

**Plant growth regulators (mg/L)** Figure 5. Rooting of *R.mykalea* axillary shoots. Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each col‐ umn and capital letters in each row are significantly different (p< 0.05), according to Duncan Multiple Range Test.

 each column and capital letters in each row are significantly different (p< 0.05), according to Duncan Multiple Range Test. **Figure 5.** Rooting of *R.mykalea* axillary shoots.

**)**

Figure 5. Rooting of *R.mykalea* axillary shoots.

Could you please replace Figure 4?

Multiple Range Test.

8

Multiple Range Test.

**7** Figur e 4

**7** Figur e 4

**7** Figur e 5

**7** Figur e 5

**Figure 2.** Axillary shoot proliferation on MS medium added 0.5 mgl-1BA.

cd

Figure 2. Axillary shoot proliferation on MS medium added 0.5 mgl-1BA.

a

b

bc

Control 0,1 BA 0,5 BA 1 BA 2 BA 0,1 KIN 0,5 KIN 1 KIN 2 KIN

**Plant growth regulators (mg/L)**

Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according

After three subculturing, solitary shoots excised from multiple shoot cultures were transferred to MS and ½ MS media containing IAA, IBA and NAA at various concentra‐ tions for rooting. Rhizogenezis was not occured MS and ½ MS medium without plant growth regulators. Auxin is necessary for *in vitro* rooting of *R.mykalea* axillary shoots. Generally, ½ MS medium added auxin is more effective than MS medium added auxin for rooting. The maximum rooting rate was obtained with half-strength MS medium supple‐ mented with 0.5 mgl-1 IBA (55%) (Figure 5 and 6).There are many of reports about IBA is

cd

Control 0,1 BA 0,5 BA 1 BA 2 BA 0,1 KIN 0,5 KIN 1 KIN 2 KIN

**Plant growth regulators (mg/L)**

a

de

b

bc

d

Figure 3. Cytokinin effects on axillary shoot multiplication of *R.mykalea.*

g

f

Figure 4. Average shoot lengths of axillary shoots dependent on cytokinin type and concentration.

Multiple Range Test.

h

de

**Figure 3.** Cytokinin effects on axillary shoot multiplication of *R.mykalea.*

**Shoot**

to Duncan Multiple Range Test.

**number/ explant**

208 Current Progress in Biological Research

e

Multiple Range Test.

**Avarage shoot lenght (cm)**

Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according to Duncan

ef

c

b

Means by different letters in each column and capital letters in each row are significantly different (*p<* 0.05), according to Duncan

more effective than other auxins on rooting for Asteraceae such as *Anthemis xylopoda* [11, 23] , *Centaurea spachii* [12], *Centaurea ragusina* [24], *Centaurea zeybekii* [25] and *Saussurea obvallata* [26]. Data were subjected to x´ = arcsine √(x/100) transformation and used analysis. Means by different letters in each column and capital letters in each row are significantly different (p< 0.05), according to Duncan Multiple Range Test. Could you please replace Figure 5? Could you please replace Figure 5? **8** 1 technique techniques

There was a statistically significant difference between MS and ½ MS medium on root‐ ing. ½ MS medium is more effective than MS medium on rooting in all experiments. Also, there was a statistically significant difference on rooting of *R. mykalea* between auxin type and concentration. **8** 1 technique techniques 2

2

**4. Conclusions**

endemic plant.

FEF-07012)

**Author details**

and Bengi Erdag

\*Address all correspondence to: yelda@adu.edu.tr

ciation for the Conservation of Nature; (2000).

Yelda Emek\*

**References**

Press; (1975).

**Acknowledgements**

gered endemic plant species.

In conclusion, the present work presents a simple and successful procedure for the *in vitro* propagation of *Rhaponticoides mykalea* (Hub.-Mor.) M. V. Agab. & Greuter, a critically endan‐

*In vitro* Propagation of Critically Endangered Endemic *Rhaponticoides mykalea* (Hub.-Mor.) by Axillary Shoot

Proliferation

211

http://dx.doi.org/10.5772/55599

To date there is no report on micropropagation of *R. mykalea*. This study is the first report on micropropagation of this species using seedlings from *in vitro* germinated embryos and aims to contrubute ongoing *ex situ* conservation programs. Additionally, this outlined protocol can be utilized as an aid in the local conservation programs to preserve this species, and it will lead for further studies on conservation and propagation of this rare and critically endangered

The authors are thankful to University of Adnan Menderes for financial support (Project no:

Department of Biology, Faculty of Arts & Science, Adnan Menderes University, Aydın, Turkey

[1] Ekim, T, Koyuncu, M, Vural, M, Duman, H, Aytaç, Z, & Adigüzel, N. Red Data Book of Turkish Plants (Pteridophyta and Spermatophyta). Ankara-Turkey: Turkish Asso‐

[2] Davis, P. H. Flora of Turkey and East Aegean Islands. Edinburg, U.K.: University

[3] Hellwig, F. H. Centaureinae (Asteraceae) in the Mediterranean-history of ecogeo‐

[4] Emek, Y, & Erdag, B. Observations on Kuşadası Population of *Rhaponticoides mykalea*,

graphical radiation. Plant Systematics and Evolution (2004). , 246-137.

Resarch Journal of Biology Sciences (2010). , 3(2), 169-174.

**Figure 6.** Rooting plantlets on ½ MS medium added 0.5 mgl-1 IBA.

In this study, we described a successful and rapid propagation techniques to regenerate critically endangered *R.mykalea* the first time by *in vitro* tissue culture techniques. Mature zygotic embryos isolated from achenes were used as starting material. The shoots that were obtained from *in vitro* germinated mature embryos were separated from primary roots and used for axillary shoot propagation. The highest axillary shoot number per explant was obtained on MS medium supplemented with 0.5 mgl-1 BA (5.8 shoot/explant). MS medium supplemented with 0.1 mgl-1 KIN was determined as the most suitable medium for the maximum shoot length (7.35 cm). Solitary shoots, removed from stock cultures, were trans‐ ferred onto half-strength MS (½ MS) or MS media supplemented with various concentrations of auxins. The maximum rooting rate was obtained with half-strength MS medium supple‐ mented with 0.5 mgl-1 IBA (55%). Rooted plantlets were transferred to external environment step by step.

The plantlets with well devoloped root were transferred to *ex vitro* conditions (Figure 7). Percentage of survival of shoots was approximately 60%. The appearance and growth of these plantlet were also normal.

**Figure 7.** Acclimatized plantlets.

### **4. Conclusions**

In conclusion, the present work presents a simple and successful procedure for the *in vitro* propagation of *Rhaponticoides mykalea* (Hub.-Mor.) M. V. Agab. & Greuter, a critically endan‐ gered endemic plant species.

To date there is no report on micropropagation of *R. mykalea*. This study is the first report on micropropagation of this species using seedlings from *in vitro* germinated embryos and aims to contrubute ongoing *ex situ* conservation programs. Additionally, this outlined protocol can be utilized as an aid in the local conservation programs to preserve this species, and it will lead for further studies on conservation and propagation of this rare and critically endangered endemic plant.

### **Acknowledgements**

In this study, we described a successful and rapid propagation techniques to regenerate critically endangered *R.mykalea* the first time by *in vitro* tissue culture techniques. Mature zygotic embryos isolated from achenes were used as starting material. The shoots that were obtained from *in vitro* germinated mature embryos were separated from primary roots and used for axillary shoot propagation. The highest axillary shoot number per explant was obtained on MS medium supplemented with 0.5 mgl-1 BA (5.8 shoot/explant). MS medium supplemented with 0.1 mgl-1 KIN was determined as the most suitable medium for the maximum shoot length (7.35 cm). Solitary shoots, removed from stock cultures, were trans‐ ferred onto half-strength MS (½ MS) or MS media supplemented with various concentrations of auxins. The maximum rooting rate was obtained with half-strength MS medium supple‐ mented with 0.5 mgl-1 IBA (55%). Rooted plantlets were transferred to external environment

**Figure 6.** Rooting plantlets on ½ MS medium added 0.5 mgl-1 IBA.

The plantlets with well devoloped root were transferred to *ex vitro* conditions (Figure 7). Percentage of survival of shoots was approximately 60%. The appearance and growth of these

step by step.

plantlet were also normal.

210 Current Progress in Biological Research

**Figure 7.** Acclimatized plantlets.

The authors are thankful to University of Adnan Menderes for financial support (Project no: FEF-07012)

### **Author details**

Yelda Emek\* and Bengi Erdag

\*Address all correspondence to: yelda@adu.edu.tr

Department of Biology, Faculty of Arts & Science, Adnan Menderes University, Aydın, Turkey

### **References**


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[20] Miloševic, M, Vujakovic, M, & Karagic, D. Vigour Tests as Indıcators of Seed Viabili‐

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[21] Fowler, J, & Cohen, L. Practical Statistics for Field Biology. Buckingham: Open Uni‐

[22] Ruffoni, B, & Massabo, F. Tissue Culture in *Gerbera jamesonii* hybrida. Acta Horticul‐

[23] Erdag, B, & Emek, Y. Adventitious Shoot Regeneration and *In Vitro* Flowering of *An‐ themis xylopoda* O. Schwarz, a Critically Endangered Turkish Endemic.Turkish Jour‐

[24] Pevalek, K. B. *In vitro* germination of *Centaurea ragusina* L., a Croatian Endemic Spe‐

[25] Kurt, S. Researchs on *In Vitro* Seed Germination of The *Centaurea zeybekii* Wagenitz.

[26] Dhar, U, & Joshi, M. Efficient Plant Regeneration Protocol Through Callus for *Saus‐ surea obvallata* (DC.) Edgew. (Asteraceae): Effect of Explant Type, Age and Plant

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**Section 2**

**Biosciences, Genetics and Health**

**Biosciences, Genetics and Health**

**Chapter 10**

**Some Observations on Plant Karyology and**

Karyology deals with the structure of cell nuclei, especially chromosomes. Cytology dealing with the study of cells in terms of structure also, function is known nowadays to mean only the study of chromosomes or nucleus and made synonymous with karyology wrongly. Cytotaxonomy means the application of cytological data to taxonomy. Cytotaxonomy studies the morphological and cytological characteristics of the organism along with their chromo‐ some numbers and structures (karyotype)[1, 2]. It is a secondary discipline that reinforces the principles of plant and animal taxonomy by abiding to the phylogenetic kinships. In classical taxonomy, the plants are categorized through determining their natural kinships in accordance with their morphological characteristics. Especially, the taxonomists are advisable in connec‐ tion with chromosomes. So, often chromosome number is assumed to be the all important, if not the only, chromosome character of interest to taxonomists, but size, shape, and behavior of chromosomes may throw more light on a taxonomic problem than their number alone.

F. Ehrendorfer, in an erudite essay on Cytologie, Taxonomie und Evolution bei Samenpflan‐ zen [Cytology, taxonomy and evolution in seed plants], gives a detailed outline of these developments in cytogenetics since 1900 that have had a bearing on problems of taxono‐ my. Examples of taxonomic corrections based on chromosome studies include the remov‐ al of *Yucca* and *Agave* from the Amaryllidaceae to the Agavaceae and a number of rearrangements of species and genera in the Gramineae, Liliaceae, Compositae and other families; warnings are sounded against an uncritical use of chromosome pairing as a criterion of affinity, secondary pairing being dismissed altogether. A concrete example of the use of morphological and cytological considerations in deciding questions of relation‐ ships and evolution is given for the Dipsacaceae, where two distinct lines of phylogenetic development are traced: one of bushy species with clear polyploid series and the other of

> © 2013 Candan; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Investigation Methods**

Additional information is available at the end of the chapter

Feyza Candan

**1. Introduction**

http://dx.doi.org/10.5772/56081
