Pioneering In vitro Studies for Callus Formation of Colchicum chalcedonicum

Colchicum calcedonicum Azn, pek cok endemik bitki turunun goruldugu Turkiye'de yayilis gosteren endemik turlerden biridir. Toprak altinda uzun-oval sekilli sogani ile genellikle 3-4 yapraklidir. Kallus kulturu kullanarak endemik turlerin in vitro uretimi, bu turlerin korunmasinda umut verici bir calisma haline gelmistir. Bu calismanin amaci, in vitro C. chalcedonicum uretimi icin verimli kallus protokolunun olusturulabilmesidir. Explantlarin sterilizasyonunda, 20 dk %0.25 (w/v) civa klorur (HgCl2) kullanilmistir. Civa klorure ilaveten, yuzey sterilizasyonunda 30 dk boyunca Tween 80, %6,5 NaCl ile birlikte kullanilmistir. Bu calismada, 19 farkli besiyeri kullanilmis olup primer kallus olusumu 2,4-D (2 mg L-1), 2IP (0,5 mg L-1), %3 sukroz ve %0,05 aktif karbon iceren Murashige & Skoog bazal besiyerinde elde edilmistir. Calismamiz, aktif karbon kullaniminin primer kallus olusumunda etkili oldugunu gostermistir. Bu calisma, C. chalcedonicum’un primer kallus olusumu icin ilk rapordur. Bununla birlikte, calismamiz endemik tur olan C. chalcedonicum'un in vitro korunmasi ve kallus olusum protokolunun gelistirilmesinde oncu bir calismadir.


Introduction
The genus Colchicum L. within the family Colchicaceae have been known for more than 2000 years for their marked beneficial and poisonous effects (Brickell 1984). In Turkey, 50 Colchicum species were described of which 15 are endemic. Their limited distribution at a very high altitude and the restricted period Colchicum chalcedonicum Azn., which is also known as Kadıköy (Chalcedon) crocus, is one of the endemic species of Colchicaceae in Turkey. Colchicum chalcedonicum was collected in Kadıköy in İstanbul by Aznavour in 1897 (Aznavour 1897). It has usually 4 leaves and long-oval shaped corms under the soil. Their chromosome number is 2n=50. The plant grows in rich red soils in dry stony and rocky places. They are also found on rare peaks and shrubs. Colchicum e2 E. Karlık et al. chalcedonium flowers from August to September, being out of leaves and fruits from February to April. Additionally, the plants only live underground for a part of the year, thus in vivo and in vitro micro-propagation and in vitro culture of this species are difficult making the species to be known as calcitrant (Brickell 1984, Persson 1988, 1999a, 1999b, 1999c, 2000, 2001, 2007, Akan & Eker 2005. Several studies reported on production of colchicine alkaloid by plant tissue cultures ). Callus tissues were first induced from flowering shoots of C. autumnale L. by using MS (Murashige & Skoog 1962) containing 2,4-Dichlorophenoxyacetic acid (2,4-D), while colchicine from callus tissue was produced by MS with indole butyric acid (IBA) and kinetin ). Daradkeh et al. (2012) used C. hierosolymitanum Feinbrun for callus production on MS supplemented with 0.45 μM 2, 4-dichlorophenoxyacetic acid under dark conditions. To induce colchicine production, callus was sub-cultured every 27 days on the same liquid media supplemented with 0.54 μM 1naphthaleneacetic acid. The researchers observed that higher cell fresh weight was resulted with 9 μM 6-benzyladenine with 0.45 μM 2, 4dichlorophenoxyacetic acid. Additionally, the highest colchicine alkaloid (0.090 mg g -1 DW) was obtained at 0.1 M sucrose after 4 weeks incubation (Daradkeh et al. 2012). Different parts of C. chalcedonicum and C. micranthum Boiss., which are endemic for Turkey, were also investigated for cytotoxic activities for future medical approaches. Daradkeh et al. (2012) managed to isolate colchicine, colchifoline, 2-demethylcolchicine, demecolcine, 4-hydroxycolchicine and N-deacetyl-Nformylcolchicine which showed high cytotoxicity. The main alkaloids of these two Colchicum species were found as colchicine and colchifoline. According to the results of this study, the greatest diversity in tropolone alkaloids were found in the seed of C. chalcedonicum (Gulsoy-Toplan et al. 2018). Despite its importance, no systematic attempt has been performed for mass propagation of C. chalcedonicum. The restricted distribution of this endemic plant has endangered its survival. Therefore, tissue culture approaches are required to get rapid propagation as in vitro protocol for micro-propagation, but there is no a practical protocol available for in vitro mass propagation of C. chalcedonicum. In the present study, we report the first and efficient protocol for callus generation of C. chalcedonicum using corms.

Field Studies and the Plant Material
Colchicum chalcedonicum corms used in this study were kindly provided by Erdal Uzen from Kadıköy. Field studies were performed from April 2017 to November 2017 at Çamlıca Hill (Ferah neighbourhood) (Fig. 1). The specimen was obtained from seed, leave, and corm during seeding, blooming and fruit time of C. chalcedonicum (Fig. 2). The specimens are deposited in the Istanbul University Alfred Heilbronn Botanical Garden.
Corms were long-ovaloid like egg-shaped, and corm shells mostly with several layers, the outer coriaceous and dark brown, the inner thin and reddish-brown. Leaves were 3-4, hysteranthous, patent, and oblong-lanceolate, light or dark-green hereinbefore mentioned by Küçüker (1984).

Explant Preparation
The fresh corms, leaves and corm shells were used as explants. The explants were excised aseptically with sterile scissors and washed with running tap water for 30 minutes, washed with dH2O for three times for 5 min. The explants were then sterilized with 0.25% (w/v) mercuric chloride (HgCl2) solution for 20 min. followed by surface sterilization with 6.5% NaCl with Tween 80 for 30 min. Then, the explants were rinsed thoroughly with sterile dH2O for three times for 5 min. followed by 70% ethanol for 10 min. The explants were again washed with dH2O three times and blotted dry on sterilized filter paper. Finally, ten sterilized explants were aseptically placed on tissue culture mediums.

Results
Various explant types, PGRs, different sugar concentrations, and chemicals were tested for callus induction in C. chalcedonicum. Callus were only formed from corms (see Fig. 3 . In our sterilization protocol, we observed 0.25% (w/v) mercuric chloride (HgCl2) and 6.5% NaCl with Tween 80 were required to sterile C. chalcedonicum corms. However, NaOCl is not necessary for sterilization of C. chalcedonicum corms.
After determination of the sterilization protocol, different sucrose concentrations were tested. At the first step, two sucrose concentrations (3% and 8%) were tested for all different PGRs combinations. Corms on 8% sucrose were formed as green and soft callus. Callus formation in 3% sucrose was found to be slower than 8% sucrose, thus corms in 3% sucrose were transferred to e4 E. Karlık et al.
mediums containing 10% sucrose to enhance callus formation.

Discussion
The genus Colchicum in Turkey has a wide distributional range with 50 species of which 15 are endemic for the country (Dahlgren et al. 1985, Persson 1999a, Akan & Eker 2005. In addition to the relatively wide distribution range for the genus, Turkey is the richest country in terms of the species diversity. However, in the present study, only 61 C. chalcedonicum, known to be grown on rich red soils at dry stony and rocky places, were found only at Çamlıca Hill which is formed from the red, iron-rich Terra Rossa soil (see Fig. 2).

Limited number of experiments were performed about
Colchicum tissue culture , Khan et al. 2011, Daradkeh et al. 2012, Wagh et al. 2015. In our sterilization protocol, we used 0.25% (w/v) HgCl2 for surface sterilization, while Wagh et al. (2015) used 0.15% (w/v) HgCl2 for surface sterilization of C. luteum Baker corms. The reason for the use of 0.25% (w/v) HgCl2 in our study was that the soil where the corm explants obtained from Çamlıca Hill is composed of mineral particles, organic matter, water, air and living organisms.
In our study, we tested different sucrose concentrations -(3, 8 and 10%) and 8% and 10% resulted with better callus formation. Nagaraju et al. (2002) tested 30, 60, 90 and 120 g L -1 sucrose concentrations and observed that sucrose showed a significant effect on corm size and weight, leaf e5 Trakya Univ J Nat Sci, Online First for 21 (2): X-X, 2020 weight and root length. Additionally, studies revealed that Colchicum corms were more desirable for callus induction (Daradkeh et al. 2012, Wagh et al. 2015. Our study demonstrated that corm was better than leaves and corm shells as an explant for callus development. Callus formation rates for corm were observed around 15% and 75% for MS supplemented with NAA (1 mg ml -1 ), ZEA (1 mg ml -1 ) containing 10% sucrose-and 2 -1 MS supplemented with 2,4 D (2 mg ml -1 ), 2IP (0.5 mg ml -1 ), 3% sucrose and 0.05% active carbon medium, respectively (see Figs 3,4). However, no callus formation was observed from leaves and corm shells, indicating that they are not suitable for callus formation as explant. Colchicum chalcedonicum is known as a calcitrant species. Because of its advantages, micro-propagation of corm plants could be an alternative to the conventional techniques for vegetative propagation, increasing many times the multiplication level, enabling the plant materials to be freed from diseases especially for medicinal plants (Shibli & Ajlouni 2000, Chang et al. 2000. Our efforts are continuing for developing callus formation protocol. Yalcin Mendi et al. (2017) reported micro-propagation of some endemic Colchicum species, but not on tissue culture for C. chalcedonicum. However, they used to active carbon to induce callus formation in Colchicum cultures. Active carbon is frequently used in tissue cultures to improve micro-propagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, corm formation etc (Thomas 2008). Studies with activated carbon in tissue culture demonstrated that activated carbon may be provide irreversible adsorption of inhibitory compounds in the culture medium significantly reducing the toxic metabolites, phenolic exudation and brown exudate accumulation (Thomas 2008). In our study, active carbon may enhance callus formation by limiting the brown exudate accumulation. We observed that green and soft callus formation resulted in medium containing active carbon. Also, callus formation in medium containing active carbon was faster than other mediums we tested.
Colchicum species comprise flavonoids, phenolic acids, tannin, fatty acids and colchicine is the major alkaloid isolated from Colchicum species such as C. autumnale and C. luteum (Kapadia et al. 1972, Levy et al. 1991, Evans 2002. Studies demonstrated that colchicine possesses antitumoral and anti-inflammatory activity and that it has a great potential for cancer treatment, making derivatives of colchicine, especially demecolcine and trimehyl colchicine acid methyl ester, to be evaluated as anti-cancer agent (Cocco et al. 2010, Bisi et al. 2015. However, no systematic attempt has been performed on micro-propagation for elite genotypes such as "Medicinal Plants" including Colchicum species. Our callus formation protocol has served promising results for future. Hayashi et al. (1988) used IBA and kinetin as PGRs in C. autumnale tissue culture for callus formation, and they managed to obtain colchicine by callus tissue culture system. However, production of secondary metabolites can be achieved by two main groups of in vitro cultures: organized cultures of differentiated tissues (i.e., organ cultures as root, shoot and embryo cultures) and unorganized cultures of undifferentiated cells (i.e., callus and cell suspension cultures). Although organized cultures of differentiated tissues produce the same secondary metabolites as the plant itself, which are relatively more stable than the undifferentiated cells, especially non-embryogenic plant callus cultures are mostly used for production of valuable secondary metabolites, including such as tropane alkaloids, hyoscyamine and scopolamine (Verpoorte et al. 2002, Filova 2014). Our present study is the pioneering study for tissue culture of C. chalcedonicum., and tissue culture studies of C. chalcedonicum may also be applied for callus formation of important Colchicum species possessing colchicine. An organism is identified as "endemic" which is native and has a restricted geographical region. Endemic species may be restricted due to physical barriers to dispersion, as in the case of many island faunas and flora, the barriers surround its area of origin, and consequently, they evolve within their limited distributional ranges (Masseti 2009). The extinction of plant and animal species, particularly with the ongoing climate change effects, has become an important issue, especially for endemic species. An alternative method of protection of endemic plant species is producing them via multiplying and conservation of plants by using in vitro culture techniques. The producing of endemic plants using tissue culture systems via multiplying is termed micropropagation which has lots of advantages including high coefficient of multiplying, small needs on number of initial plants, small needs on space and reproducing of plants regardless seasons of the year, through multiplying intervals between subcultures in slow growing species (Kováč 1995, Engelmann 1997. We aimed to find the best nutrient media, PGRs and explants for micropropagation and in vitro conservation of C. chalcedonicum. However, we were able to establish only efficient callus protocol were the pioneering of micropropagation. This callus formation protocol will serve the improvement of tissue culture techniques to obtain more efficient callus formation protocols and to lead the micropropagation of C. chalcedonicum in the future.

Conclusion
The success of efficient callus protocol improvement for in vitro conservation of C. chalcedonicum relies on the optimal choice of the explants, on the efficiency of the sterilization method, and on the establishment of an in vitro culture protocol for these calcitrant species for the beginning of aseptic proliferative cultures and on the optimal choice of nutrient media and PGRs. According to our experimental data, the optimal media for efficient callus formation of C. chalcedonicum were MS supplemented with NAA (1 mg ml-1), ZEA (1 mg ml-1) containing 10% sucrose-and 2-1 MS supplemented with e6 E. Karlık et al.
2,4 D (2 mg ml-1), 2IP (0.5 mg ml-1), 3% sucrose and 0.05% active carbon medium. Additionally, supplying of active carbon in the media induced the callus formation. Our work is the pioneering study to obtain sophisticated callus formation protocol for in vitro conservation of C. chalcedonicum. Our study may help to save C. chalcedonicum which is endemic in İstanbul, Turkey.