Effects of 6-benzylaminopurine and Activated Carbon on Indirect Organogenesis of Fritillaria Imperialis

Fritillaria imperialis needs 2-3 years to regenerate from seeds, which makes its production quite challenging. In this study, effects of three concentrations of 6-Benzylaminopurine (BAP) (0.00, 0.10 and 0.15 mg/L) and three different amounts (0.0, 3.0 and 3.5 g/L) of activated carbon (AC) on the growth of F. imperialis plantlets in vitro were determined. The auxins were at similar concentration (0.01 mg/L of NAA and 0.01 mg/L of IAA) for all treatments. It has been found that the size and number of bulblets, as well as root and shoot numbers in regenerated plantlets were increased significantly (p<0.05) by adding 3.5 g/L of AC in culture medium supplemented with 0.10 mg/L of BAP. Moreover, addition of AC significantly reduced the length of roots in all doses studied (p<0.05). The supreme concentration of BAP (0.15 mg/L) decreased the bulblet number in all treatments, however, when supplemented with 3.5 g/L of AC, it was found to raise all the values measured (p<0.05). Overall, the best growth of the plant parts from callus were achieved with the highest concentration of AC and 1.5 mg/L of BAP. Research Article Article History Received : 30.09.2019 Accepted : 11.11.2019

In vitro culture methods are essential components of plant genetic resources management and they are becoming more important for the conservation of endangered and rare plant species (Bhatia et al., 2002;Almeida et al., 2005). Micropropagation techniques can allow fast and large-scale propagation of plants.
Regeneration without crossing callus is named as direct organogenesis. Indirect organogenesis, which includes the crossing of callus, has been applied for overcoming geophyte dependency and keeping long period to totipotency in Cuminum cyminum (Ebrahimie et al., 2006).
Under in vitro culture conditions, the growth of the bulblets of F. imperialis can be stimulated by a combination of plant growth hormones (Li et al., 2011). Suitable bulblet size for acclimatization is also achieved by transferring the plantlets to a growth medium containing no plant growth regulator (PGR) but an elevated level of carbohydrate contained with activated carbon (Staikkidou et al., 2000).
In in vitro culture, AC is widely used to promote the rooting of micropropagated plantlets since it helps the uptake of vitamins, ferritin, hormones, and darkens the medium (Dumas and Monteuuis, 1995). Besides, the detrimental substances generated by the explant or culture medium are adsorbed by AC (Fridborg and Eriksson, 1975;Fridborg et al., 1978). Cytokinin hormone is an important component of the medium for micropropagation, particularly at the proliferation stage. It has been widely known that cytokinin plays various roles in plant growth such as protein synthesis, simulation of division and cell expansion (George et al., 2008). This study investigates the impact of varying amounts of AC alone and combined with different concentrations of BAP. on the development of micropropagated F. imperialis grown in the media containing the same amount of Naphthaleneacetic acid (NAA) and Indole-3-acetic acid (IAA).

Plant materials
The plant materials were kindly provided by Prof. Dr. Mehmet Nuri Nas of Horticulture Department of Kahramanmaras Sutcu Imam University. The fresh bulbs before sterilization are seen in Figure 1. Soil particles, dead parts and insects were removed from the bulb surface by washing them thoroughly by tap water and rinsing with sterile water three times ( In our experiment, we used Murashige and Skoog (MS) basal medium to grow the explants. The medium pH was set to 5.5±0.9 and it was autoclaved at 121 º C for 15 minutes before adding 5.5 g/L of agar plant (Duchefa Biochemie, Netherland) into the medium in order to provide it with a gel-like texture. As the energy and carbon source, 30 g/L of sucrose was added to the KSU J. Agric Nat 23 (2): 297-303, 2020 Araştırma Makalesi Research Article medium. The plant materials were cultured in a growth room with a constant 25±2°C temperature and 66±4% humidity and 16/8 hours light/dark period (sustained by enlightening with white fluorescence).

Callus Formation
The clean bulbs were cut into smaller pieces and soaked in 1% (w/v) NaClO (sodium hypochlorite) (Hypo, Turkey) with Tween-20 (1%) (OGH, Germany) for fifteen minutes (Figure 3). Then, the surfacesterilized pieces were prepared washed extensively with distilled water for four times and cultured ( Figure  4) on MS supplemented with 0.125 mg/L of TDZ (Thidiazuron) (Sigma-Aldrich, Germany). Statistical analysis IBM SPSS Statistics software was used to perform statistical analyses. In order to distinguish the effect of activated charcoal and hormones, and to find out their interaction, variance analyses were made by Two Way ANOVA. Groups with a p value lower than 0.05 were considered as significantly different (p<0.05). For the purpose of confirmation of the results of Two-Way ANOVA and to label the experimental and control groups depending on their similarities, Tukey's HSD post hoc test was applied.

RESULTS and DISCUSSION
We evaluated the impact of three different doses (0.0, 3.0 and 3.5 g/L) of AC by itself or in combination with three different amounts (0.00, 0.10, 0.15 mg/L) of BAP on the growth of the plantlets of F. imperialis, cultured via indirect organogenesis. When we made the first measurements on the 9 th week, we found that the moderate amount (0.10 mg/L) of BAP along with the maximum dose of AC (3.5 g/L) produced more bulblets, roots and shoots (13.00±3.1, 23.40±5.6 and 16.60±1.9) compared to the control group (1.20±0.4; 14.60±2.0; 14.20±3.30) to which no AC and no BAP was applied (Table 1). When the plantlets were grown in the media containing no AC but with moderate concentration (0.10 mg/L) of BAP, the lowest number of roots and shoots (6.80±1.9 and 8.20±1.9) were generated. Our results showed that adding AC to the growth media stimulated and fed plants tissue more effectively and that helped the explants in modification and differentiation. These observations were in agreement with the reports authored by Sipayung and co-workers in 2018. Similarly, Olah (2017) demonstrated that AC could improve bulblet growth by possibly prohibiting exudates that cause tissue browning. Moreover, according to Cheng et al. (2013), AC could act as a promoter of embryogenesis in the microspore culture of different Capsicum annuum L genotypes.
After 13 weeks of treatment, number of roots, shoots and the bulblets were determined ( Despite the fact that they worked on another species (Narcissus tazetta), Steinits and Yahel (1982) also showed that AC promoted the growth of bulblets, which was in line with our findings. The presence of AC in the growth media was likely to increase the efficiency of cytokinin hormone which in turn improves the growth of the explants (Nhut et al., 2001). Besides, activated charcoal was also known to remove the effect of inhibitory compounds from the medium (Anagnostakis, 1974). Expectedly, we have observed the lowest number of shoots and bulblets (2.20±0.4 and 12±1.9) in the culture medium containing neither AC nor BAP (Table 1). As for the roots, the fewest numbers (19.00±3.8) were determined in the medium containing the highest concentration of BAP (0.15mg/L) without AC ( Table 2).
The inhibition observed in embryogenesis and morphogenesis of the plants grown in no AC containing media is most likely to be related to the presence of phenolic compounds in the growth medium, as it was previously reported by Nayanakantha et al. (2010) Although the concentration of BAP was more than ten folds less compared to our previous (Saeed and Cömertpay, 2017) work (1.75 mg/L), but the negative impact of the phenolic compounds was observed on root numbers in the absence of AC in the growth medium.
When the measurements obtained for both the 9 th and the 13 th week were compared, it was revealed the best growth performance of bulblets (number and diameter) were determined when moderate hormone concentration (0.10 mg/L) BAP and the highest dose of AC (3.5 g/L) were added to the media ( Figure 5). These results were in accordance with of Staikkidou and coworkers (2000), who reported that the bulblets were grown better when PGR levels were reduced during the multiplication phase, which counts for the role of activated charcoal in adsorbing PGR. The highest amount (3.5g/L) of AC and moderate concentration (0.10 mg/L) of BAP increased the diameter of bulblets to 0.46±0.1 and 0.75±0.1 after 9 and 13 weeks of growth respectively ( Figure 5).
On the contrary, and regardless to the duration of the treatment, shorter roots were produced when 0.10 mg/L of BAP was used in the growth media with no AC. Similar to what we have observed, Staikkidou et al. (2000) stated that charcoal effected the development of the plantlets by inducing the formation of large number of roots and stimulating root elongation.
Some representative figures of plantlet development are presented in Figure 7.

CONCLUSION
Results of our study indicate the positive role of AC in the growth media on the development of the F. imperialis plantlets, especially on bulblet, root and shoot formation. The positive effects of AC is more pronounced at low concentrations of BAP with an adverse tendency at higher BAP levels. More studies are needed to address the interaction between nutrients and the AC in the growth media to standardize the application of the active carbon.