Influence of Medium Composition on In vitro Propagation of Amsonia orientalis Decne.

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1 Research Article Influence of Medium Composition on In vitro Propagation of Amsonia orientalis Decne. Arda ACEMİ *, Ruhiye KIRAN, Fazıl ÖZEN, Sinem BALCI, Sibel BAYDEMİR, Çağla İLMAZ Kocaeli University, Faculty of Sciences and Arts, Department of Biology, 41380, İzmit, Kocaeli, Turkey Abstract: Amsonia orientalis is a critically endangered plant which has natural distribution only in northwest Turkey and northeast Greece. In order to contribute to the conservation studies on this species, media with different compositions were tested in in vitro propagation of the plant. SH medium suggested the best results in axillary shoot induction phase (3.17 ± 0.28 cm mean shoot length with 1.51 ± 0.09 mean shoot number per explant at ± 3.85% frequency) while MS medium gave better results in rooting phase (2.65 ± 0.09 cm mean root length with 7.27 ± 0.32 roots per explant at ± 2.22% frequency). Our findings indicated that in culture medium, components such as pyridoxine, myo-inositol and glycine are required for developing an efficient in vitro propagation protocol for A. orientalis. Also, in culture medium, higher nitrogen levels could promote the in vitro development of the plant. In rooting phase, increasing sucrose levels made positive effect on both mean root length and number. Plantlets were successfully acclimatized and 85% of them survived. Keywords: Apocynaceae, Blue Star, In vitro rooting, Medium composition, Shoot multiplication Besi Ortamı Bileşiminin Amsonia orientalis Decne. nin In vitro Çoğaltımına Etkisi Özet: Amsonia orientalis tükenme tehlikesi altında bulunan ve sadece kuzeybatı Türkiye ve kuzeydoğu Yunanistan da doğal yayılışa sahip olan bir bitkidir. Bu tür üzerindeki koruma çalışmalarına katkıda bulunmak için bitkinin in vitro çoğaltımında farklı bileşimlerdeki besi ortamlarının etkileri denenmiştir. MS besi ortamı köklendirme aşamasında daha iyi sonuçlar verirken (%97.78 ± 2.22 sıklıkta 2.65 ± 0.09 cm ortalama kök uzunluğu ile eksplant başına ortalama 7.27 ± 0.32 kök), SH besi ortamı aksillar sürgün uyarımı aşamasında en iyi sonuçları önermiştir (%97.78 ± 2.22 sıklıkta 3.17 ± 0.28 cm ortlama sürgün uzunluğu ile 1.51 ± 0.09 eksplant başına ortalama sürgün sayısı). Bulgularımız, A. orientalis için etkin bir in vitro çoğaltım protokolünün geliştirilmesinde besi ortamında piridoksin, myo-inositol ve glisin gibi bileşenlerin gerekliliğini göstermiştir. Ayrıca, besi ortamındaki yüksek azot seviyeleri bitkinin in vitro gelişimini teşvik etmektedir. Köklendirme aşamasında, artan sükroz seviyeleri hem ortalama kök uzunluğu hem de sayısı üzerine olumlu etki yapmıştır. Bitkicikler başarılı bir şekilde dış ortama alıştırılmış ve %85 i hayatta kalmıştır. Anahtar Kelimeler: Apocynaceae, Mavi Yıldız, In vitro köklendirme, Besi ortamı bileşimi, Sürgün çoğaltımı Introduction Biodiversity hotspots are biologically richest places which face extreme threats. There are totally 34 hotspots on Earth. Turkey covers three of these hotspots, namely Anatolian, Caucasus and Mediterranean Basin (Myers et al., 2000). Ekim et al. (2000) declared that in Turkey, 4600 plant taxa are listed in a Red List Category of International Union for Conservation of Nature (IUCN). Amsonia orientalis Decne. (syn. Rhazya orientalis (Decne.) A. DC.) is a member of Apocynaceae family and listed in critically endangered (CR) category of Red Data Book of Turkish Plants (Ekim et al., 2000). While some other species of genus Amsonia, such as A. tabernaemontana, A. illustris and A. hubrichtii occur in a wide range of habitats throughout central, southern and eastern North America, a few others, such as A. orientalis and A. elliptica are native to the southern Europe, Turkey, Japan, Korea and China (Davis, 1978; Darke, 2005). Nevertheless, natural distribution of A. orientalis is limited only within northwest Turkey and northeast Greece. All Amsonia species are known as Blue stars because of their star shaped and pale blue colored flowers. The plant grows best in well-drained soil and also needs full sun or partial shade. It can be found along margins of lakes and streams. Field studies have indicated that plant is now very rare and near extinction (Özen, 2006). In the Bern Convention (1979), The European Council placed the plant on the list of the plant species that must be conserved on European scale

2 A.orientalis draws attention with its ornamental and medicinal properties. In addition to its extinction risk, several researchers have declared that the plant has a strong antimicrobial activity and contains flavonoid glycosides and some indole alkaloids known for anti-cancer and anti-tumour activities (Rahman et al., 1989; Itoh et al., 2002; Akyalçın et al., 2006). In vitro propagation studies on A. orientails were successfully conducted by Öz et al. (2008) and Acemi et al. (2012; 2013). In all previous in vitro propagation studies including our previous reports, only Murashige & Skoog s (MS) medium (1962) was employed. To develop a medium that fully provides the plant s macro and micro element requirements in in vitro culture, different medium compositions should be tested. The aims of this study are to reveal medium requirements for micropropagation of the plant and to supply plant material for ornamental purposes and potential medicinal studies. Materials and Methods Shoots, cm in length, were collected from field-grown individuals of Amsonia orientalis before flowering in May 2012 (Figure 1a). For the sake of easier manageability these shoots were cut into 10 cm long pieces and washed under tap water for 15 min. All leaves were cut off before washing step. These shoots were cut into 1-2 cm long pieces which have a single node at least. These nodal segments were surface sterilized by dipping in 70% ethyl alcohol (EtOH) for 2 min and 1% sodium hypochlorite (NaOCl) for min, respectively. A few drops of Tween 80 were added into NaOCl solution before surface sterilization. Traces of NaOCl were removed by two or three rinses with distilled sterile water. Surface sterilized single node explants were inoculated vertically in culture vessels containing 40 ml of MS medium supplemented with 1.0 mg l benzylaminopurine (BAP) for shoot induction and multiplication. After the culture period of 4 weeks, number and length of shoot were recorded. Induced shoots were cut into segments with at least one node. Shoot multiplication was continued on same fresh medium by subculturing axillary shoots under the same conditions. The shoots were subcultured at an interval of 4 weeks until desired shoot material was reached. In the following experiments, besides MS medium, De Greef & Jacobs (1979) (GJ), Linsmaier & Skoog (1965) (LS), Nitsch (1969) (NN), Schenk & Hildebrandt (1972) (SH) and Gamborg (1968) (B5) media were used to test influence of media composition on axillary shoot induction and growth. In order to compare only the effects of media composition, these media were not supplemented with any plant growth regulator (PGR) in this phase. Nodal segments obtained from multiplication phase were inoculated vertically in culture vessels containing 40 ml of medium indicated above. Sucrose compositions were applied as described in relevant literature. MS, LS and GJ media were supplemented with 30 g l -1 sucrose while B5 and NN were supplemented with 20 g l -1 sucrose. SH medium was supplemented with 10 g l -1 sucrose. In rooting phase, shoots with fully expanded leaves were inoculated on the media which showed most positive effect on the shoot induction and growth. Rooting media were supplemented with 1.0 mg l -1 indole-3-acetic acid (IAA). The rooting was evaluated after 4 weeks. All variants of media were solidified with 7 g l -1 of Plant agar (Duchefa). Their ph was adjusted to 5.7 with 1 N NaOH or 1 N HCl prior to autoclaving at 121 C under pressure of 118 kpa for 20 min. All treatments such as disinfection and inoculations were carried out aseptically in a laminar air flow cabinet. All cultures were maintained at 23 ± 1 C in a plant growth chamber with a 16/8 h light/dark photoperiod under of 80 μmol m -2 s -1 photosynthetic photon flux density provided by cool-white fluorescent lights. Rooted plantlets were planted in pots containing sterile soil and covered with transparent plastic bags with small holes for 3-4 days. After one week, plastic bags were removed and well acclimatized plants were maintained at room temperature under natural light conditions. Each treatment for the axillary shoots induction, multiplication, and rooting contained 15 explants. All treatments were repeated thrice and measurements were done at the end of the incubation periods. The software IBM SPSS Statistics 19 was used for statistical analysis

3 Results The axillary buds on nodal explants incubated on MS medium fortified with 1.0 mg l -1 BAP started breaking 5-7 days after inoculation. After 4 weeks on medium multiple shoots emerged directly from axillary buds of the cultured explants (Figure 1b). The mean number of shoots was found to be 8.9 ± 0.36 per explant at ± 3.85% frequency while the mean length of shoots from axillary buds was 4.28 ± 0.26 cm. A few explants had produced adventitious shoots, which appeared to arise from the calli at basal cut ends of explants. After three subcultures adequate number of nodal explants was obtained for following experiments. In the experiments about the influence of medium compositions on shoot induction and elongation, frequencies of shoot inductions were observed between a broad range ( %). Of all the six media tested, B5 medium was found to be superior for shoot induction (100 ± 0.00%). Also, MS medium promoted the frequency of shoot induction (97.78 ± 2.22%). There is no statistical difference between B5 and MS medium in terms of frequency of shoot induction. The lowest value for the frequency of shoot induction was found from the LS media (51.11 ± 4.45%). Mean shoot lengths were also greatly influenced by medium composition. Figure 1. In vitro propagation of Amsonia orientalis. a. General appearance of plant in garden, b. Shoot proliferation on MS medium with 1.0 mg l -1 BAP, c. Axillary shoots formation from nodal segments of in vitro regenerants on SH medium, d. Stunted growth of shoots on SH medium, e. Rooting of in vitro derived shoots in MS medium with 1.0 mg l -1 IAA, f. Acclimatized plant

4 Amongst the tested media, SH medium gave the best mean shoot length result (3.17 ± 0.28 cm) whereas GJ and NN media did not support shoot elongation well (Table 1). Table 1. Response of nodal explants excised from in vitro-raised shoots of Amsonia orientalis in different media. Medium Frequency of Shoot Induction (%) Mean Shoot Length (cm) Mean Number of Shoots MS ± 2.22 a 2.82 ± 0.26 a 1.00 ± 0.10 b LS ± 4.45 d 2.53 ± 0.31 a 0.51 ± 0.05 d SH ± 3.85 b 3.17 ± 0.28 a 1.51 ± 0.09 a NN ± 2.22 c 1.69 ± 0.33 b 1.17 ± 0.04 b B5 100 ± 0.00 a 3.05 ± 0.23 a 1.00 ± 0.00 b GJ ± 4.44 b 1.26 ± 0.15 b 0.77 ± 0.04 c Data represent means ± SE. Means having the same letter in a column were not significantly different by Duncan s multiple range test (p < 0.05). As compared to the SH medium, relatively lower responses were observed in B5 and MS media. SH medium was found to be the best for shoot elongation and multiplication while it gave a relatively lower shoot induction frequency (Figure 1c). Furthermore, in SH medium a few shoots showed stunted growth and they could not further elongate (Figure 1d). However, SH medium supplemented with the lowest sucrose level (10 g l -1 ) gave the highest mean shoot length. Higher sucrose content caused a decrease on the mean shoot lengths. In all media, shoot numbers per explant were observed at low numbers. Maximum mean number of shoots per explant was recorded from SH medium (1.51 ± 0.09) while LS medium suggested the minimum value (0.51 ± 0.05). The mean shoot number results obtained from MS, NN and B5 media were not statistically different. When the induction frequencies and mean shoot lengths and numbers were evaluated, SH, B5 and MS media were found to be better than NN and GJ media. These three media were also used in rooting experiments and were supplemented with 1.0 mg l -1 IAA. Before rhizogenesis, callus formation appeared in all medium at the basal parts of the shoots. All media induced callus formation in 1-2 weeks. The superiority of MS on rooting of axillary shoots was observed at the end of the 4 week of incubation (Table 2). Table 2. Effects of media supplemented with 1.0 mg l -1 IAA on in vitro rooting of axillary shoots Medium Frequency of Root Induction (%) Mean Root Length (cm) Mean Number of Roots MS ± 2.22 a 2.65 ± 0.09 a 7.27 ± 0.32 a SH ± 3.85 b 2.05 ± 0.07 b 5.63 ± 0.24 b B ± 2.22 ab 2.23 ± 0.09 b 6.37 ± 0.29 ab Data represent means ± SE. Means having the same letter in a column were not significantly different by Duncan s multiple range test (p < 0.05). MS medium gave better rhizogenic response (97.78 ± 2.22%) while the response given to SH (86.66 ± 3.85%) and B5 (91.11 ± 2.22%) media were relatively low. Media supplemented with higher sucrose levels promoted mean number of roots. Especially, MS medium gave the highest mean number of root (7.27 ± 0.32) (Figure 1e). Furthermore, MS media showed positive effect on mean root length. The optimal medium for shoot regeneration was found to be SH, whereas MS medium was the best for in vitro rooting. After the acclimatization phase the percentage of survival plantlets was found to be 85% under natural light conditions. In vitro propagated A. orientalis plantlets did not show any phenotypic variation (Figure 1f). Discussion The experimental part of this study was started using MS medium supplemented with 1.0 mg l -1 BAP for shoot induction. Because, in our previous report (Acemi et al., 2013), it was found to be the optimal concentration for shoot induction from mature nodal explants. Like in our previous study, MS medium with 1.0 mg l -1 BAP suggested a fairly good number of shoots per explant. LS medium includes only thiamine hydrochloride (Vitamin B1; 0.40 mg l -1 ) as vitamin while all other tested media contain pyridoxine hydrochloride (Vitamin B6) and nicotinic acid as vitamins, except thiamine hydrochloride. This difference between media compositions made significant differences on shoot induction frequencies and mean shoot - 4 -

5 numbers whereas mean shoot lengths were not affected significantly. The role of pyridoxine is to promote the growth of excised organs of plants. It has been shown that in the culture medium it promotes the uptake of glucose, nitrate and phosphate in excised roots of grasses (Chandra and Das, 2007). Additionally, Chen and Xiong (2005) declared that pyridoxine has a critical role in plant development and stress tolerance and suggested the pyridoxine as a new class of antioxidant in plants. Amino acids are usually added to plant media to satisfy the requirement of cultures for reduced nitrogen (George and De Klerk, 2008). As amino acid supplement, glycine was included only in MS and NN media. Nevertheless, MS medium gave better results than NN medium. This result could be attributed to relatively higher nitrogen level in MS medium. GJ medium which suggested the lower results like LS medium lacks both glycine and ammonium nitrate. Instead of ammonium nitrate, in GJ medium ammonium sulphate was employed. There are several reports in the literature indicating that ammonium sulphate does not provide such a good source of NH 4 + as ammonium nitrate or ammonium chloride (Steward and Hsu, 1977; Singh, 1978). Possibly the reason is that a medium containing ammonium sulphate has a greater tendency to become acid (Harris, 1956), than one containing less sulphate ions. Nevertheless, B5 medium which also contains ammonium sulphate gave the highest shoot induction frequency and promoted mean shoot length. Relatively higher potassium nitrate level (2500 mg l -1 ) in B5 medium could be the cause of this response. The other difference among media is myo-inositol content. Among the tested media, SH contains the highest amount of myo-inositol (1000 mg l -1 ). Myo-inositol takes a significant role in biosynthetic pathways of the formation of the pectin and hemicelluloses needed in cell walls (Loewus and Loewus, 1980). This property could be the reason of better results observed from SH medium. The optimum results were observed when potassium nitrate, myo-inositol and glycine levels were at their highest levels in culture medium. Like in our previous report, MS medium supplemented with 1.0 mg l -1 IAA was the best for adventitious root formation in Amsonia orientalis (Acemi et al., 2013). Additionally, media with higher sucrose level increased all the rooting frequency, mean root length and number. Hyndman et al. (1982) stated that an increase in the sucrose to nitrogen ratio can lead to improve in vitro rooting of Rosa cultivar Improved Blaze. Orlikowska (1992) tested the effects of various amino acids on in vitro rooting of apple dwarf rootstocks and found that some amino acids including glycine enhanced the root number. Fairly good result of root number from MS medium could be attributed not only to high sucrose but also the glycine content of the medium. Conclusion Our findings indicated that in culture medium, components such as pyridoxine, myo-inositol and glycine are required for developing an efficient in vitro propagation protocol for Amsonia orientalis. Also, higher nitrogen levels could promote the in vitro development of the plant. Consequently, the formulation of SH medium supports the development of the plant. However, in rooting phase, SH medium should be supplemented with higher sucrose levels to obtain an efficient rooting. Findings in this study will contribute to further studies on in vitro conservation of the plant. References Acemi, A., Özen, F., Kıran, R Development of An Efficient Callus Production Protocol for Amsonia orientalis: A Critically Endangered Medicinal Plant. Eurasian Journal of Biosciences. 6: Acemi, A., Özen, F., Kıran, R In vitro Propagation of Amsonia orientalis Decne. from Nodal Segments of Adult Plants. Propagation of Ornamental Plants. 13(1): Akyalçın, H., Özen, F., Dülger, B Anatomy, Morphology, Palynology and Antimicrobial Activity of Amsonia orientalis Decne. (Apocynaceae) Growing in Turkey. International Journal of Botany. 2(1): Bern Convention (1979). Convention on The Conservation of European Wildlife and Natural Habitats. Convention Relative à la Conservation de la vie Sauvage et du Milieu de l Europe. Appendix II, Bern/Berne, 19.IX Chandra, K., Das, M.R Effect of KNO3, Vitamin B6 and Salicylic Acid on Growth, False Siliqua Formation and Seed Yield in Toria. Indian Journal of Plant Physiology.12(1):

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