Optimization of culture conditions for callus proliferation of Curculigo orchioides Gaertn

Curculigo orchioides Gaertn is a herbaceous plant that has long been used as a tonic in Vietnam with noticeable health benefits. However, the demand for rhizomes of this species could not be met due to their decreasing number in natural habitats. Despite its vulnerability, there are still not enough researches on producing calli of C. orchioides, which is a method having the capacity of creating a large source of cell biomass for bioactive compounds’ extractions. Thus, this study was conducted to figure out the best conditions for C. orchioides’s callus proliferation. Different light regimes, mineral media, and concentrations of some factors like kinetin (KIN), α-naphthaleneacetic acid (NAA), yeast extract (YE), activated charcoal (AC), and silver nitrate (AgNO3) were examined. It is shown by the results that half-strength MS medium (1⁄2 MS) given 0.5 mg/L KIN and MS medium supplemented with 0.5 mg/L KIN and put in the dark (0 light hour : 24 dark hours) are the optimal conditions for callus proliferation, with the highest fresh weights (FWs), dry weights (DWs), and growth indices (GIs) of 3.89 g / 0.45 g / 7.78, and 4.10 g / 0.47 g / 8.20, respectively. Additionally, the inhibitory effects of AgNO3, YE, and AC were demonstrated since there was no observed heavy callus in the media containing those factors.

Callus induction is the most suitable procedure for satisfying the mentioned need since it can greatly increase cell biomass to produce large amounts of secondary metabolites without depending on the traditionally cultivated plants (Ahn, 1996). Though callus cells are genetically unstable, this problem can be solved by continuously subculturing calli for a long period (from several weeks to several years) until their growth parameters remain unchanged after three consecutive subculture cycles to create stable cell lines (Fett Neto, 1994;Miura, 1998;Bourgaud, 2001;Miguel-Chavez, 2007;Jin, Keng, 2013;Biswas, 2013). However, there are few published studies on callus induction of C. orchioides. In particular, Nagesh et al. (2010) indicated that the highest callus formation level was obtained in the medium supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 3 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid). Moreover, as it was proven in Dhenuka et al.'s study (1999), proliferating green callus was given rise from leaf explants by using MS (Murashige, Skoog, 1962) medium added with 9 μM BAP.
Because of C. orchioides's low number in their natural habitats and the lack of studies on callus proliferation of this valuable plant, we investigated the impacts of different factors on callus growth of C. orchioides to create a sufficient source of biomass for extracting bioactive compounds. Several factors (mineral media, photoperiods, yeast extract (YE), activated charcoal (AC), and silver nitrate) were tested for the first time to find new stimuli and inhibitors of C. orchioides's callus formation, which could then be applied in the cell culture stage of secondary metabolites' productions.

Plant materials
Primary calli provided by the Department of Biology, University of Sciences (Thua Thien Hue, Vietnam) were utilized as materials in this study.

Effects of several factors on callus proliferation
To optimize the condition for callus growth, the following experiments were conducted; each experiment lasted for four weeks: 1. Different concentrations of kinetin (KIN) (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L) were supplemented into the MS medium to determine the best KIN amount, which was then used in further experiments.
3. Basal MS medium, half-strength MS medium (1/2 MS), and quarter-strength MS medium (1/4 MS) were compared to point out the most suitable mineral medium.
4. The proper photoperiod was figured out by comparing two conditions namely 0 hour and 16 hours of light per day. 5. To examine the effect of silver nitrate (AgNO 3 ) on callus proliferation, 0, 0.5, 1.0, and 1.5 mg/L AgNO 3 were added into the MS medium.
6. The impact of YE on multiplying C. orchioides callus was investigated by supplementing different quantities of YE (0, 100, 200, 300, and 400 mg/L) into the MS medium.
7. To indicate how calli grow in medium with AC, 0, 0.5, 1.0, and 1.5 g/L of AC were supplemented into the MS medium.
The conclusion of the optimal condition for callus multiplication was made by statistically comparing the best concentrations of the above factors.

Culture conditions
In each experiment, 0.5 g callus was cultured on different media, containing 30 g/L sucrose and 8 g/L agar.
All media (pH = 5.8-6.0) were autoclaved at 121 o C, 1 atm for 20 minutes, after which 20 mL was poured into 100 mL glass bottles covered by 2 layers of aluminum foils.
Cultures were incubated at the temperature of 24-26 o C, under white LED tubes (1.2 m), with each tube's photon flux density of 34 μmol/m 2 s.

Data analysis
The growth index (GI) of callus after four weeks of culturing was calculated as following: Where FW is the average callus fresh weight after four weeks, IW is the initial weight of the primary callus.
All experiments were repeated three times so that the sample size for each treatment was 30 bottles (10 bottles per replicate).

Effects of plant growth regulators (PGRs) on callus proliferation
As it is highlighted in Table 1, low KIN concentration has a positive impact on callus growth. Specifically, FW, dry weight (DW), and GI of calli reached the peaks of 2.31, 0.36, and 4.62 respectively on MS medium given 0.5 mg/L KIN. Low levels of callus growth were observed in media with higher KIN concentrations (1.0-3.0 mg/L) (Fig. 1a).
Being similar to KIN, the greatest stimulatory effect of NAA was obtained at low concentration. Particularly, 0.5 mg/L is the optimal NAA amount, with maximum FW and GI of 3.8 and 7.6 respectively. However, many calli growing on media containing NAA are black, watery, and have the average DW values being lower than the ones on medium supplemented with 0.5 mg/L KIN alone ( Table 2, Fig. 1b). Table 1 Effect of KIN concentrations on callus proliferation after 4 weeks of culturing

Effects of different mineral media on callus proliferation
Lowering compounds' quantities in MS medium stimulates the formation of large calli. However, callus proliferation is inhibited on a medium whose compounds are over-eliminated. In particular, ½ MS is the optimal mineral medium for callus growth (with the greatest FW, DW, and GI of 3.89, 0.45, and 7.78 respectively) while ¼ MS is the most unsuitable medium of all examined ones since no callus with high FW, DW and GI was observed (Table 3, Fig.  2a). Additionally, ½ MS medium with 0.5 mg/L KIN is between the two best culture conditions for the proliferation of C. orchioides callus (Fig. 4). All calli are ivory (some of the calli are black). The number of black calli on media with NAA is higher than that on media with KIN alone. The calli separate from one another with no large cluster formed. Scale bar: 1 cm.

Effect of photoperiod on callus proliferation
Both the light hours of 0 and 16 have positive impacts on callus growth, with high GI values of 8.20 and 5.16 respectively. However, the average FW (4.10) and DW (0.47) of calli cultured in dark conditions are larger than those of calli growing in bottles which were illuminated 16 hours per day (Table 4, Fig. 2b). Moreover, putting calli in the dark is considered to be one of the best conditions for growth of C. orchioides callus (Fig. 4).  concentrations, namely 0-400 mg/L, 0-1.5 g/L and 0-1.5 mg/L, respectively (in a figure section, factor's concentrations were ordered from top to bottom). There are inverse correlations between callus growth and the amounts of these 3 factors. Specifically, callus proliferation falls when the 3 factors' quantities increase. High numbers of black calli were observed on media with YE or AC. Additionally, no large callus cluster was found on culture media supplemented with AC, YE or AgNO 3 . Scale bar: 1 cm.

Effects of YE, AC and AgNO 3 on callus proliferation
It is demonstrated in Table 5 that YE, AC, and AgNO 3 are all inhibitors of the growth of C. orchioides callus. Specifically, no examined concentration of these 3 factors could produce calli with high FW, DW, and GI. Additionally, calli cultured on medium with AC and YE tend to turn black (Fig. 3). Therefore, YE, AC, and AgNO 3 should not be used as supplementary compounds for callus multiplication of C. orchioides.

Discussion
KIN is a PGR with the ability to enhance callus proliferation and regeneration by influencing mitosis, cytokinesis, total protein synthesis, lignin biosynthesis, vascular differentiation, and the differentiation of mature chloroplasts from protoplastids (Wan, 1988). However, it is indicated by the results in Table 1 that only a low quantity of KIN has these positive effects, which is in line Optimizing Curculigo orchioides's callus proliferation with the work of Farzinebrahimi et al. (2014), who concluded that raising KIN concentration from 1 mg/L to 5 mg/L gradually lowers callus induction rate of Gardenia jasminoides Ellis. The same phenomenon was also observed in using BAP (6-benzylaminopurine, which is also a cytokinin) for callus induction of Pogostemon mollis Benth. found the stimulatory effect of 2 mg/L KIN + 1 mg/L NAA combination, which is enhancing the formation of calli having the greatest average FW (1.477 g). Being similar to these findings, the average FW of C. orchioides calli treated with the optimal NAA + KIN combination in our research is significantly high (3.8 g). This result was different from that of Nagesh et al. (2010) claiming that C. orchioides calli grown on MS media given NAA and KIN only had the low average FW of 0.713 g. However, the quality of the produced C. orchioides calli was low, with the black color and low DW. Therefore, combining NAA and KIN seems to be inappropriate for C. orchioides's callus proliferation and we then only used 0.5 mg/l KIN in further experiments. The found negative impact might be caused by ethylene, a growth inhibitor, which is synthesized when NAA presents in culture media (George, 2008).
As it is mentioned above, there are only two published studies on callus proliferation of C. orchioides (Dhenuka, 1999;Nagesh, 2010). However, those studies only focused on examining the impacts of PGRs on callus induction of this plant without considering other factors, which have been reported to be able to affect the formation of calli, such as photoperiod, mineral media, AC, and YE (Vasil, Hildebrandt, 1966;Pan, Staden, 1998;Ma, 2009;Wani, 2014). Thus, we examined those new factors to find out how C. orchioides calli responded.
As it is highlighted in Table 3, lowering nutrients in MS medium in half created the optimal medium for callus proliferation of C. orchioides. There are also researches on different species providing a similar conclusion about the strong growth of calli on ½ MS medium. First of all, after testing MS, ½ MS, and ¼ MS medium, Wani et al. (2014) reported that maximum callus growth of Costus pictus was obtained when ½ MS and ¼ MS were utilized while no callus formation was detected on full strength MS medium. Additionally, MS medium is proven to be not effective as ½ MS medium in inducing callus of Boerhaavia paniculata Rich (Souza, 2014). Finally, in a study on Dendrocalamus hamiltonii, an insignificant difference in terms of promoting callus formation between the two most efficient media, namely MS and ½ MS, was illustrated by Zang et al. (2016).
Illuminating callus cultures for 16 hours per day is the best photoperiod for callus growth of some species such as Litchi chinensis Sonn. and Nicotiana tabacum L. In particular, a high of 16.67 g callus was produced when L. chinensis leaf explants were incubated under the light regime of 16 light hours (L) : 8 dark hours (D) (Ma, 2009). Additionally, the results of comparing 16 L : 8 D and 0 L : 24 D shown by Siddique and Islam (2015) demonstrated that the optimal amount of N. tabacum L. callus (97.20 %) was obtained under light conditions while dark conditions could only produce a low quantity of watery, glossy silver calli with few embryogenic features. However, in the case of C. orchioides, a completely distinct phenomenon was found. Specifically, calli cultured on the medium put in dark condition were heavier and grew more vigorously than the ones incubated under the light regime of 16 L : 8 D. This finding might be explained by the photodegradation of natural auxins in calli and synthesis of phenolic compounds, which could inhibit enzymes having key roles in cell proliferation (Yeoman, Davidson, 1971).
In addition to factors having positive effects, the present research also figured out inhibiting factors namely YE, AC, and AgNO 3 . Firstly, though Ag 2+ cation (derived from AgNO 3 ) is proven to have the ability to interfere with negative impacts of ethylene by binding to its receptors on the cell membrane (Goren et al., 1984;Yang, Hoffman, 1984;Bleecker et al., 1998), low levels of C. orchioides's callus growth were observed on all media supplemented with AgNO 3 . Secondly, the high adsorption capacity of AC might be the main reason for its inhibitory effect. Specifically, the change of pH and the loss of auxins, cytokinins, and vitamins caused by AC could make calli grow ineffectively in culture medium (Pan, Staden, 1998). Finally, the inverse correlation between YE concentrations and callus growth in our study was also found by Vasil and Hildebrandt (1966). Particularly, when YE quantity increased from 125 to 5000 mg/L, callus proliferation of carrot (Daucus carota), endive (Cichorium endivia) and lettuce (Lactuca sativa) explants fell to the minimum values of 1.320 g, 2.425 g, and 0.478 g respectively.
However, there are also authors including George et al. (2008) who claimed that YE could be used to promote plant growth because of its high amino acid content.

Conclusion
From the results illustrated in Fig. 4, it could be concluded that the following conditions are optimal for callus proliferation of C. orchioides: 1. Half-strength MS medium supplemented with 0.5 mg/L KIN + 30 g/L sucrose + 8 g/L agar.