Turkey is one of the richest countries in variability of flora. It has nearly 9000 plant species about 3000 of which are endemic .
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.
Micropropagation constitutes a powerful tool for
However, during our literature search, no report concerning
The objective of the present study was to establish an efficient
2. Material and methods
2.1. Plant material and explant source
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)  basal medium for germination. The shoots that were obtained from
2.2. Achene viability
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– triphenilformazan 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 .
To determine achene viability of
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.
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)  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-agar), various concentration of plant growth regulators 1N6- 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 subcultured 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
2.6. Statistical analyses
Means of shoot number per explant, shoot lenght and frequency of rooting were analyzed 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 .
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 appropriate cytokinin type and concentration were determined (Figure 2). Axillary shoot propagation of
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 proliferation of
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
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 concentrations for rooting. Rhizogenezis was not occured MS and ½ MS medium without plant growth regulators. Auxin is necessary for
There was a statistically significant difference between MS and ½ MS medium on rooting. ½ MS medium is more effective than MS medium on rooting in all experiments. Also, there was a statistically significant difference on rooting of
In this study, we described a successful and rapid propagation techniques to regenerate critically endangered
The plantlets with well devoloped root were transferred to
In conclusion, the present work presents a simple and successful procedure for the
To date there is no report on micropropagation of