In vitro Callus Induction of Catunaregam spinosa Using Leaves as Explant

Aims: C. spinosa (Family Rubiaceae) is a valuable medicinal since years ago. A protocol was developed including best sterilization and best medium for callus induction of C. spinosa using leaf with 1.0 mg L -1 BAP and 3.0 mg L -1 NAA produced green friable calli with 0.0969±0.01 g mean dry weight after 3 months. Some traits were found to be depended on synergetic effect of growth regulators and genotypic characteristics of explant. Conclusion: The study provides a better sterilization protocol and medium for in vitro calli induction and growth of C. spinosa .


INTRODUCTION
Catunaregam spinosa (Thunb.) Tirveng is well recognized medicinal plant grows in tropical and sub-tropical countries. C. spinosa contains diverse range of phytochemicals including alkaloids, saponins, triterpenoids, flavonoids, cardiac and cyanogenic glycosides, tannins and volatile oils. These compounds employ in different pharmacological activities such as cytotoxicity, piscicidal, anti-bacterial, antiinflammatory, hepatoprotective, insecticidal and anthelmintic activities. Alkaloids are important in treatments of cancer, malaria and diabetes [1]. Further it is used 25-75% in drug development [2]. Saponins have hypolipidemic, cytotoxic and anti-diabatic properties [3,4,5]. Jangwan and Singh, [6] studied in vitro cytotoxicity of stem bark of C. spinosa on Breast cancer cell line (MDA-MB-231), Human Neuronale Glioblastoma (Astrozytom) cell-Line (U87MG), Human Skin Melanoma cell line (SK-MEL-2) and Neuroblastoma cell line derived from human bone marrow (BE(2)C). It revealed significant effect of triterpene isolated from stem bark on SK-MEL-2 cell line as 93.33 μg mL -1 (IC50) [6]. Ragib et al. [7] studied cytotoxicity of methanolic and aqueous leaf extracts of C. spinosa using brine shrimp lethality assay and reported 0.94 and 0.23 µg mL -1 as LC50 compared to Dimethyl sulfoxide (LC50, 1.07 µg mL -1 ) [7]. Traditional people used crushed parts of C. spinosa for fish harvesting and several literatures mentioned about its piscicidal activity [8][9][10][11]. Anthelmintic activity of fruit pulp of C. spinosa has reported against tapeworms and earthworms as folklore remedy [12]. Ethanol extract of fruits of C. spinosa paralyzed and killed Pheretima posthuma in a short period of time (8.47± 0.42 min.) compared to ethyl acetate and petroleum ether extracts (14.26 ± 0.61 min. and 26.20 ± 0.67 min. respectively) [13]. Numerous studies mentioned the antioxidant activity of different parts of C. spinosa. Satpute et al. [14] found high nitric oxide scavenging activity of chloroform (350.0 µg mL -1 ) and ethyl acetate (480.0 µg mL -1 ) fractions of fruits of C. spinosa [14]. Superior antioxidant activity of leaves was recorded as 85% and 2.5 µg mL -1 using DPPH and Fluorescence recovery after photobleaching (FRAP) assays respectively [15]. Hepatoprotective activity of C. spinosa was revealed by Kandimalla et al. [16] using carbon tetrachloride (CCl 4 ) induced hepatically damaged wistar rats. It found reversal of abnormal parameters related to hepatic damages after diagnosed with ethanol leaf and bark extracts of C. spinosa [16]. High anti-inflammatory activity was revealed in both methanol and ethyl acetate extracts of fruit of C. spinosa on induced rat paw inflammation at 200.0 mg/kg [17]. Emesis activity is mostly reputed pharmacological activity of C. spinosa where its extreme bitterness induces the vomiting sensation [18]. Anoopkumar, [19] studied insecticidal activity of seeds of C. spinosa on 4 th instar larvae of Aedes agypti and found LC50 as 210.212 mg L -1 [19].
Plant tissue culture is a rapid method of producing plantlets under controlled conditions. It overcomes the limitations incurred in conventional propagation such as long term seed dormancy, lack of seed viability, delayed rooting of seedlings, constraints due to seasonal changes while expanding the potential of mass propagation. Conventional propagation methods such as seed germination, rooting of cuttings and grafting show low growth rate. This can be expected due to extended time duration taken by plants to produce high content of secondary metabolites. Root suckers are also natural asexual reproduction method reported in C. spinosa [20]. There are many types of cultures such as protoplast, embryo, anther, ovule, ovary cell and callus cultures [21]. In present study callus cultures were used due to its common use in tissue culture and high possibility of working on most of the plant species. Thus it will be advantageous to get an idea about In vitro growth of C. spinosa using leaf discs as explants which is not previously studied. Moreover, callus culture is an easy method with basic steps compared to protoplast cultures where it undergoes enzymatic cell wall degradation, callus formation and later production of plantlets [22,23]. Diploid plantlets are produced through callus cultures unlike ovary or anther cultures which produce haploid plantlets. Leaves, nodes and internodes are readily available for callus cultures where maternal tissues of ovules, seeds or capsules isolated in aseptic conditions are used for embryo cultures. [24]. Calli are the prerequisite element for cell cultures that can be obtained from callus cultures. Calli are potent sources of valuable secondary metabolites which are produced in a significantly short period of time [25,26]. Thus callus culture is a better alternative method compared to other types of cultures in direct production of calli for isolation of important secondary metabolites. Ramos et al. [27] used callus culture of Ageratina pichinchensis to produce secondary metabolites with anti-inflammatory activity [27]. Jain et al. [28] reported callus culture of Alternanthera brasiliana in producing antinociceptive compounds [28]. Based on these factors callus culture was used as the type of culture in producing calli of C. spinosa using leaf explants. Pharmaceutical industry shows raised demand over secondary metabolites. It is not sufficiently addressed due to low production rate of metabolites in plants up to the desired level [29,30]. However, literatures on in vitro growth of C. spinosa were fairly low. Further no reports were found regarding in vitro callus induction of C. spinosa using leaf explants. Begum et al. [31] studied in vitro seed germination of C. spinosa. Results mentioned maximum number of shoots (12.7) obtained from seedlings grown in MS medium supplemented with 1.0 mg L -1 each of BAP and NAA [31].
Nevertheless, each species demands unique plant growth regulators and concentrations for an optimum in vitro growth [32]. Thus, it is prerequisite to optimize particular level of concentrations of growth regulators in callus induction of C. spinosa whereas a sterilization protocol needs to be optimized beforehand. Present study intended to develop a successful sterilization protocol and to determine the best medium for in vitro callus induction of C. spinosa using leaf explants.

Optimization of Surface Sterilization Protocol
Immature leave were collected from plants grown in an open area of University of Sri Jayewardenepura, Sri Lanka. Leaves were washed with disinfectants (Dettol), 5% teepol (v/v, liquid detergent) under running tap water for 1 h. After that leaves were washed with few drops of Tween 20 for 5 mins. Under aseptic conditions sterilization protocol was tested with 0.2 and 0.3% of Carbendazim ® for 10 and 15 min. and Sodium hypochlorite (Clorox) with 10 and 15% for 10, 15 mins. Finally the explants were washed with 70% ethanol for 30 sec. Each step was followed with two successive washings in sterile distilled water. Sterilized leaves were cut into 1.0 cm 2 pieces along midrib. Leaf discs were cultured on half MS medium [32]. Wellsealed culture media were incubated under 25°C and 60% humidity. They were provided with 50 μmol m −2 s −1 white fluorescent light in 16/8 h light/ dark photoperiod. There were 15 replicates in each treatment. Percentage contamination and percentage survival were determined after three months of incubation.

Determination of Best Medium for Callus Induction from Leaf Explants
Immature leaves from in vivo grown plants were used as explants. Leaf cuttings were sterilized following best surface sterilization protocol determined. Half MS medium [33] containing macro and micro nutrients, vitamins, Ethylenediamine tetraacetic acid (EDTA) ferric sodium salt, sugar and agar was supplemented with different concentration of NAA and BAP (1.0 -6.0 mg L -1 ). Nitrogen as a macro nutrient plays a vital role in production of amino acids, nucleic acids and proteins in new cells development. Micro nutrients which need in small quantities important for healthy growth of calli especially iron for chlorophyll synthesis. Proper functioning of enzymes in developing calli is assisted by vitamins such as Thiamine and Myo-inositol found in MS medium which act as coenzymes [34,35]. Sugar was used as carbohydrate and energy source and also to balance the osmotic pressure [36]. EDTA acts as chelating agent to make iron available in nutrient solution otherwise it precipitates and becomes unavailable to plantlets [37]. Different concentrations of BAP and NAA were examined which produce signals either to stimulate, inhibit or regulate the optimum growth and development of calli [38]. Medium pH was adjusted to 5.78± 0.02. Culture media were sterilized by autoclaving. Growth regulators free media were used as controls. Completely randomized design was followed with 10 replicates in each concentration. Color and texture of calli were determined by visual observations. Dry weights of calli were measured after three months of growth. Data were statistically analyzed using ANOVA at significant level of p =0.05. Medium which produced healthy flourished calli within a minimum number of days with a significantly high mean dry weight was selected as the best medium for obtaining calli of C. spinosa from leaf discs explants.

Optimization of Surface Sterilization Protocol
Protocol which exhibited lowest percentage contamination and highest percentage survival selected as the best sterilization protocol. Best sterilization protocol found to be the leaf discs washing with 0.3% carbendazim for 10 minutes, 10% Clorox for 10 minutes and 70% ethanol for 30 sec followed by successive washing twice with sterile distilled water. It exhibited lowest percentage contamination (13.34%) and highest percentage survival (86.66%) after eight weeks of incubation (Table 1). At 15% concentration of Clorox soft tissues of leaf discs tended to damage by necrosis in first week of incubation. Explants disinfected with 0.3% carbendazim for 15 mins recorded lowest contamination percentages of 13.33%. However, exposure to a high concentrations of fungicides and bleaching agents for a long period of time can lead for growing mutations, affect the regeneration capacity, cell viability and bleaching of cells causing them to die and to reduce the survival rate [39]. This trait was observed when leaf discs exposed to15% carbendazim and 15% Clorox each for 15 min. The combination significantly reduced the percentage contamination (13.33%) and concurrently decreased the percentage survival (46.66%) (T12). However, about 40.01% of explants were died following browning in this medium. Surprisingly, all replicates of best combination of carbendazim and Clorox produced calli with same percentage contamination (13.34%) with highest survival rate (86.66%). Thus 0.3% carbendazim for 10 mins and 10% Clorox for 10 min were selected as a part of best sterilization protocol for In vitro culturing of C. spinosa using leaf explants.

Determination of Best Medium for Callus Induction from Leaf Explants
Callus induction and growth rate varied among the tested concentrations (   [40]. BAP started to form new cells by stimulating cell division at the presence of optimal concentration of NAA. New cell formation expanded the leaf explants to form calli with optimum weights. Based on that we can predict the possible reason days (13.2±1.03 days) to produce pronounced, healthy, green and friable calli. Ikeuchi et al. [41] mentioned intermediate ratio of auxin to cytokinin promotes callus formation and high ratio of auxin to cytokinin and vice versa promote root and shoots respectively [41]. However according to Neibaur et al. [42] high concentration of auxin to low concentration of cytokinin is typical combination to promote mass cell proliferation [42]. The same scenario has proven in a study of callus induction of Coffea arabica L. using leaf cultures. The prominent friable calli have formed in 2.5 mg L -1 2,4-D and 0.5 mg L -1 BAP concentrations [40]. In our study also best medium was contained with high concentration of auxin (3.0 mg L -1 ) to low concentration of cytokinin (1.0 mg L -1 ) ratio.

CONCLUSION
Callus production is possible in C. spinosa as a plant with significant phytochemicals. Best sterilization protocol for explants grown in open environment is disinfecting with 0.3% carbendazim for 10 mins, 10% Clorox for 10 mins. and 70% ethanol for 30 sec. which recorded lowest contamination and highest survival percentages compared to other combinations. Half MS medium supplemented with 3.0 mg L -1 of BAP and 1.0 mg L -1 NAA was best medium for callus induction of C. spinosa from leaf discs. Callus morphology and mean dry weights of calli were depended on the type of plant growth regulator and their concentrations. Present study provides better sterilization protocol and medium for in vitro callus induction and growth of C. spinosa using leaves as explants which would assist any future studies regarding secondary metabolites isolation or other applications calli of C. spinosa are involved.

DISCLAIMER
Commonly and predominantly used products in Sri Lanka have been used for this research. This research was conducted solely for advancement of knowledge. Thus there is no conflict of interest between authors and companies of products supplied. Further, research is completely funded by University of Sri Jayewardenepura under the grant no: ASP/01/RE/2019/15 not by any other product producing company.

SIGNIFICANCE OF THE STUDY
This study provides a better protocol for in vitro callus induction of C. spinosa which is a reputed medicinal plant since years ago. Mass propagation of C. spinoa plantlets can be ensured via indirect organogenesis if the callus production protocol was developed. Further calli can be used as the source of secondary metabolites which would stamp the value of C. spinosa as a medicinal plant more in the pharmaceutical industry.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.