Impaginato 255 Adv. Hort. Sci., 2020 34(3): 255­263 DOI: 10.13128/ahsc­7767 Enhancement of Pentacyclic Triterpenoids (Betulinic and Oleanolic acids) production from callus cultures of Lantana camara L. A.M. AL­Ashoush 1, R.A. Shibli 1 (*), R.W. Tahtamouni 2, T.S. Al­Qudah 3, B.B. Abu­Irarmaileh 3 1 Department of Horticulture and Agronomy, Faculty of Agriculture, University of Jordan, Amman, Jordan. 2 Department of Biotechnology, Faculty of Agricultural Technology, Al‐ Balqa Applied University, Amman, Jordan. 3 Hamdi Mango Center for Scientific Research (HMCSR), University of Jordan, Amman, Jordan. Key words: elicitation, growth regulators, heavy metals, in vitro, Lantana, Lantana camara L., sugars. Abstract: Lantana camara L. is an ornamental plant with high medicinal value. This study aimed to investigate the possibility of enhancing the production of betulinic and oleanolic acids in Lantana callus by adding different types and lev­ els of chemical elicitors (NaCl, sugars, growth regulators and heavy metals) to Murashige and Skoog (MS) medium. Data revealed that, adding NaCl to the cul­ ture medium affected callus fresh weight and color negatively, but it increased the extracted amounts of oleanolic and betulinic acids significantly to reach maximum levels of 0.702 and 0.051 mg/g DW at 120 mM NaCl compared to 0.659 mg/g DW and 0.014 mg/g DW obtained in the control. Meanwhile, increasing glucose level to 36.02 g/l in the medium maximized oleanolic acid accumulation to 0.829 mg/g DW, while betulinic acid accumulation reached 0.038 mg/g DW at 54.03 g/l glucose. In growth regulators experiment, highest callus fresh weight was observed in the control medium, while it declined to the minimum at 0.50 mg/l of Thiadiazuron (TDZ). Maximum values of both acids (0.685 and 0.033 mg/g DW) were recorded in MS medium plus 1.0 mg/l TDZ. Callus fresh weight decreased significantly in response to heavy metals addition, while adding chromium at 0.08 mg/l improved production of oleano­ lic acid to reach the maximum of 0.676 mg/g DW. Meanwhile betulinic acid was maximized at 0.057 mg/g DW in callus cultures exposed to 0.08 mg/l cobalt. 1. Introduction Lantana camara L. is a popular flowering ornamental plant (Charitha and Ranwala, 2018), belonging to the family Verbenaceae (Mishra, 2015). It is native to tropical and sub­tropical areas of America (Singh and Saxena, 2016), West Africa (Waoo et al., 2015 a) and tropical Asia (*) Corresponding author: r.shibli@ju.edu.jo Citation: AL­ASHOUSH A.M., SHIBLI R.A., TAHTAMOUNI R.W., AL­QUDAH T.S., ABU­IRARMAILEH B.B., 2020 ­ Enhancement of Pentacyclic Triterpenoids (Betulinic and Oleanolic acids) production from callus cultures of Lantana camara L. ­ Adv. Hort. Sci., 34(3): 255­263 Copyright: © 2020 AL­Ashoush A.M., Shibli R.A., Tahtamouni R.W., Al­Qudah T.S., Abu­Irarmaileh B.B. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 30 december 2019 Accepted for publication 26 May 2020 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(3): 255­263 256 (Ghisalberti, 2000). This plant is routinely used as an evergreen aromatic ornamental or hedge shrub (Bhakta and Ganjewala , 2009; Zoubiri and Baaliouamer, 2012). It’s commonly named as Lantana, red sage, Surinam tea plant, Spanish flag and West Indian lantana (Kalita et al., 2012). Lantana camara L. is also listed as one of the most important medicinal plants, as it possesses many dis­ tinguished medicinal properties (Waoo et al., 2014). The interest in Lantana camara L. has recently, increased because it is an excellent source of many chemical compounds of medicinal potential (Srivastava et al., 2011; Saxena et al., 2012; Waoo et al., 2014) like pentacyclic triterpenoids which includes many active compounds such as betulinic and oleanolic acids (Kensa, 2011; Venkatachalam et al., 2011; Kazmi et al., 2012; Mariajancyrani et al., 2014). Betulinic acid has recently gained increased atten­ tion as it possesses a remarkable variety of biological and medicinal properties (Moghaddam et al., 2012; Pandey et al., 2015). Oleanolic acid was also reported to possess many important biological activities (Ghosh et al., 2010; Xia et al., 2011; Singh et al., 2012). So producing such valuable compounds in commercial amounts is of great importance. In vitro culture was utilized as an efficient approach for production of secondary metabolites. Currently, in vitro culture techniques are used to facilitate the pos­ sibility of making quantitative and qualitative elicita­ tion of the production of plant secondary metabolites by changing the culturing media composition (Affonso et al., 2007; Alenizi et al., 2020). According to Taiz and Zeiger (2002), adding elevated levels of growth regula­ tors, sugars and heavy metals was found to drive the plant cell machinery to produce more secondary metabolites rather than cell division. For example, Pasqua et al. (2005) reported successful enhancement of anthocyanin production in Camptotheca acuminata cell cultures by adding different types and levels of growth regulators and sugars to the growth media. In addition, Lee et al. (2011) were able to produce more rutin from callus and adventitious roots of white mul­ berry tree by adding auxins, cytokinins, and nitrogen to the growth medium. Heavy metals were also found to increase the pro­ duction of bioactive compounds in many plants (Verpoorte et al., 2002). So, this study was carried out to enhance the possibility of production of betulinic and oleanolic acids in Lantana callus cul­ tures by modifying the culture medium using differ­ ent types and levels of growth regulators, sugars, NaCl, and heavy metals in callus culture media. 2. Materials and Methods Mother stock culture establishment and maintenance In vitro grown callus cultures of Lantana camara L. were implemented by the Plant Tissue Culture and Microbiology Laboratories at Hamdi Mango Center for Scientific Research (HMCSR). The cultures were subcultured on callus maintenance semi solid medi­ um consisting of 4.4 g/l of Murashige and Skoog (Murashige and Skoog, 1962) MS premix (Duchefa Biochemie) plus 34.2 g/l sucrose in addition to 1.0 mg/l Kinetin and 2.0 mg/l 2,4­D. Cultures were main­ tained in the growth room under dark conditions at 24± 1°C. Subculturing of callus was performed every 3­4 weeks by subdividing callus under sterile condi­ tions. Enhancement of betulinic and oleanolic acids produc‐ tion To study the possibility of enhancing production of betulinic and oleanolic acids in Lantana camara L. callus cultures, different types and levels of chemical elicitors (NaCl, sugars, growth regulators, heavy met­ als) were added to the culture medium. In NaCl experiment, callus clumps of 1.0 g were subcultured onto fresh MS callus maintenance medi­ um described earlier and supplemented with NaCl at different concentrations (0, 40, 80, 120 and 160 mM). Meanwhile, the effect of sugars on betulinic and oleanolic acids was experimented by transferring 1.0 g of callus clumps onto maintenance medium supple­ mented with different types and levels of sugars: sucrose (34.2, 68.4 and 102.6 g/l); glucose or fructose (18.01, 36.02 and 54.03 g/l). In heavy metals experiment, lead (Pb), cobalt (Co) or chromium (Cr) were prepared from their salts; lead nitrate Pb (NO3)2, potassium chromate (K2CrO4), and cobalt (II) chloride (CoCl2.6H2O) respectively. A stock solution of 100 mg/l was prepared from each salt. After that the requested amount of each heavy metal (0.08, 0.16 and 0.24 mg/l) was added to the callus medium prepared as described earlier. To test the effect of plant growth regulators thidi­ azuron (TDZ) and kinetin on betulinic and oleanolic acids production, the explants were transferred onto hormone free MS medium for one week to remove any carry over effect of callus maintenance media. Next, callus clumps of 1.0 g were subcultured onto full strength MS solid medium supplemented with different levels (0.5, 1.0 and 1.5 mg/l) of either TDZ Al‐Ashoush et al. ‐ Enhancement of Pentacyclic Triterpenoids production in Lantana camara L. 257 or Kinetin in combination with 2.0 mg/l 2,4­D. All cultures were maintained in the growth room under dark conditions at 24±1°C, and data were col­ lected after 6 weeks for callus fresh weight and color before oven drying. Extraction Calluses from each treatment were oven dried at 50oC for 2 days. Then, the dried callus was ground using liquid nitrogen. Next, 100 mg were taken from the dried matter of each treatment and soaked in 10 ml of methanol and water in a ratio of 1:1. The sam­ ples were then placed on a shaker at slow speed for at least 72 hours at room temperature (Hussain et al., 2012). The plant extract was filtered and cen­ trifuged at 5000 g for 10 min. The resulting super­ natant was dried in a rotary evaporator at 40oC. Next, the dried extract was resuspended in the mobile phase for HPLC analysis. Preparation of betulinic and oleanolic acids stock solutions and working standards Betulinic and oleanolic acid stock solutions at con­ centration of 1000 mg/l were prepared by weighing 5 mg of each in 5 ml volumetric flask, dissolved and brought to volume by methanol HPLC grade. Both stock solutions were stored at 4oC in dark. Working solutions were prepared by serially diluting stock solutions using the mobile phase at concentrations of 800, 400, 200, 100, 50, 25 and 12.5 mg/l. Fresh work­ ing standards were prepared daily. Betulinic and oleanolic acids were eluted at 15.8 and 19 min, respectively. Calibration curves were constructed for betulinic and oleanolic acids before starting chemical analysis (r²= 0.9999). HPLC instrumentation and conditions HPLC analysis was performed using a Shimadzu­LC system (Shimadzu Corporation, Kyoto, Japan) equipped with a CBM­20A controller, LC­20AT pump, and SPD­20AV UV/VIS detector Chromatographic separations were achieved using an Agilent Eclipse Plus C18 column and were carried out using an iso­ cratic flow rate of 1 ml/min, a column temperature of 25o C, a mobile phase of acetonitrile: methanol: acidi­ fied water (70:20:10 v/v), with pH adjusted to 2.8 using 85% phosphoric acid. The ultraviolet (UV) detection was set at 210 nm. The injection volume was 20 μl of sample solution. Total run time was 19 min for each injection. Data were acquired and processed with LC­Solution software (Shimadzu Corporation, Kyoto, Japan). Statistical analysis Each treatment was arranged in a complete ran­ domized design (CRD) and replicated ten times with two callus clumps/replicate. HPLC analysis was con­ ducted on three replicates for each treatment with two samples/replicate. The collected data were sta­ tistically analyzed using SPSS analysis system. The analysis of variance (ANOVA) was used and mean separation was done according to the Tukey’s HSD at probability level of 0.05. 3. Results Effect of NaCl After 6 weeks of incubation under different levels of NaCl, the obtained results indicated that, callus fresh weight and color (callus quality) declined in response to NaCl and the effects became more severe as NaCl level increased (Table 1). The maxi­ mum fresh weight 8.6 g was recorded in the control, while brownish yellow callus with minimal fresh weight value of (2.6 g) was obtained on the medium supplemented with 160 mM NaCl (Table 1). On the other hand, adding NaCl to the growth medium enhanced the production of oleanolic acid in the callus cultures in response to NaCl level reaching the maximum level of 0.702 mg/g DW at 120 mM NaCl (mM) Weight (g) Color Oleanolic acid (mg/g DW) Betulinic acid (mg/g DW) Control 8.60 ± 0.36 a * White 0.659 ± 0.011 d 0.014 ± 0.0003 e 40 5.90 ± 0.35 b White 0.668± 0.0028 c 0.021± 0.0531 d 80 4.70 ± 0.59 bc Yellow 0.674± 0.0124 b 0.043 ± 0.0081 b 120 4.11 ± 0.32 c Yellow 0.702 ± 0.0088 a 0.051 ± 0.0083 a 160 2.62 ± 0.53 d Yellow to brown 0.656± 0.0096 d 0.035± 0.0057 c Table 1 ­ Effect of NaCl on callus fresh weight, color and levels of oleanolic and betulinic acids in callus culture of Lantana camara * Values represent means ± standard error. Control: represents callus maintenance medium consisting of solid MS medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­D + 1.0 mg/l kinetin. Means with different letters are significantly different according to Tukey HSD range test at P≤0.05. Adv. Hort. Sci., 2020 34(3): 255­263 258 NaCl compared to 0.659 mg/g DW extracted from the control treatment (Table 1). Also, the production of betulinic acid was enhanced by adding NaCl. The maximum value 0.051 mg/g DW was obtained at 120 mM NaCl compared to 0.014 mg/g DW extracted from the control (Table 1). Effect of sugars Sucrose. Our results revealed that, increasing sucrose concentration in the MS media led to a dra­ matic decline in fresh weight and quality of Lantana camara L. callus cultures (Table 2). Fresh weight of the white callus decreased significantly from 8.6 g in con­ trol treatment to reach the minimum (3.1 g) at 102.6 g/l sucrose where it turned dark brown (Table 2). Meanwhile, in response to level of sucrose, we observed significant increases in oleanolic and betulin­ ic acids production. The maximum amounts of oleano­ lic acid (0.97 mg/g DW) and betulinic acid (0.035 mg/g DW) were recorded in media with 102.6 g/l of sucrose. This level of sucrose enhanced the production of betulinic and oleanolic acids in callus cultures to levels that exceeded values of those extracted from naturally growing plants (Table 2). On the other hand, callus was not healthy and the growth was very limited in all treatments compared to the control (Table 2). Glucose. Adding glucose resulted in a significant decline in callus growth and quality as shown in Table 2. Instead increasing glucose level in the medium resulted in increasing level of oleanolic acid to reach the maximum at 36.02 g/l glucose (0.829 mg/g DW) compared to control (0.659 mg/g DW). Meanwhile, betulinic acid accumulation increased to 2.5 times when 54.03 g/l glucose was added to the media com­ pared to the control (0.014 mg/g DW) as shown in Table 2. Fructose. The obtained data indicated that, fruc­ tose was not a good choice for Lantana camara L. callus cultures in terms of growth, color and sec­ ondary metabolite production. Callus fresh weight decreased gradually with increasing fructose levels in the medium and minimum fresh weight of the brown callus (1.55 g) was obtained on 54.03 g/l fructose (Table 2). Meanwhile, adding fructose to the culture medium at levels higher than 18.01 g/l negatively affected oleanolic accumulation compared to the control, and the same for betulinic acid (Table 2). Effect of plant growth regulators Effect of Kinetin. Data obtained revealed a signifi­ cant increase in fresh weight (8.6 g) at Kinetin con­ centration of 1.0 mg/l (Table 3). Meanwhile maxi­ mum amounts of oleanolic acid and betulinic acids (0.67 and 0.021 mg/g DW) were extracted from cal­ lus cultures grown on media supplemented with low rate of kinetin (0.5 mg/l), while the amount of fresh weight and both acids declined significantly as kinetin level increased in the media (Table 3). * Values represent means ± standard error. Control: represents callus maintenance medium consisting of solid MS medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­D + 1.0 mg/l kinetin. Means with different letters are significantly different according to Tukey HSD range test at P≤0.05. Table 2 ­ Effect of sucrose; glucose and fructose on callus fresh weight, color and production of betulinic acid and oleanolic acid in callus culture of Lantana camara Carbohydrate (g/l) Weight (g) Color Oleanolic acid (mg/g DW) Betulinic acid (mg/g DW) Sucrose Control (34.2) 8.60 ± 0.36 a* White 0.659± 0.011 c 0. 014 ± 0.0003 c 68.4 3.21 ± 0.16 b Yellow 0.824 ± 0.0082 b 0.027± 0.013 b 102.6 3.11 ± 0.32 b dark brown 0.970± 0.0081 a 0.035± 0.0291 a Glucose Control 8.60 ± 0.36 a White 0.659± 0.011 c 0.014 ± 0.0003 c 18.01 4.49 ± 0.197 b Yellow 0.765± 0.0093 b 0.025 ± 0.0084 b 36.02 3.80 ± 0.56 bc Yellow start to be brown 0.829± 0.0089 a 0.036± 0.0189 a 54.03 3.00 ± 0.46 c Yellow start to be brown 0.764± 0.0077 b 0. 038 ± 0.0095 a Fructose Control 8.60± 0.36 a White 0.659 ± 0.011 a 0.014 ± 0.0003 a 18.01 4.40 ± 0.22 b Yellow 0.661 ± 0.0035 a 0.012 ± 0.0066 ab 36.02 2.34 ± 0.47 c Yellow 0.514 ± 0.0092 b 0.010 ± 0.0090 b 54.03 1.55 ± 0.13 d Brown 0.380 ± 0.0060 c 0.010± 0.0089 b Al‐Ashoush et al. ‐ Enhancement of Pentacyclic Triterpenoids production in Lantana camara L. 259 Effect of TDZ. The highest fresh weight of callus (8.6 g) was obtained in the control treatment fol­ lowed by that obtained on 1.5 mg/l TDZ in combina­ tion with 2.0 mg/l 2,4­D (Table 3). However, the resulted callus was of good quality as it remained white in all treatments levels (Table 3). Moreover, a significant increase in oleanolic acid and betulinic acids was obtained in response to TDZ, and maxi­ mum values of both acids 0.685 and 0.033 mg/g DW were recorded in callus cultures grown in MS medi­ um plus 1.0 mg/l TDZ, while adding higher levels of TDZ negatively impacted on oleanolic and betulinic acids (Table 3). * Values represent means ± standard error. Control: represents callus maintenance medium consisting of solid MS medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­D + 1.0 mg/l kinetin. Means with different letters are significantly different according to Tukey HSD range test at P≤0.05. Table 3 ­ Effect of different growth regulators in combination with 2.0 mg/l of 2, 4­D on callus fresh weight, color and production of betulinic and oleanolic acids in callus culture of Lantana camara Growth regulator (mg/l) Weight (g) Color Oleanolic acid (mg/g DW) Betulinic acid (mg/g DW) Kinetin 0.5 4.63 ± 0.55 b* White 0.672 ± 0.0091 a 0.021 ± 0.0067 a 1.0 (control) 8.60 ± 0.36 a White 0. 659 ± 0.011 b 0.014 ± 0.0003 b 1.5 4.72 ± 0.44 b Yellow 0. 621 ± 0.0001 c 0.014 ± 0.0010 b TDZ Control 8.60 ± 0.36 a White 0.659 ± 0.011 c 0.014 ± 0.0003 c 0.5 1.40 ± 0.14 c Yellow 0. 651 ± 0.0088 d 0.025±0.023 b 1.0 3.44 ± 0.63 b White 0.685 ± 0.0082 a 0.033±0.009 a 1.5 3.68 ± 0.23 b White 0.668±0.0045 b 0.012± 0.0072 c Effect of heavy metals Effect of cobalt. Callus fresh weight and color were negatively affected by adding cobalt to the media at levels higher than 0.08 mg/l. Minimum weight of 5.32 g was recorded at Co level of 0.24 mg/l (Table 4). On the other hand, callus remained healthy white despite of Co addition at all levels (Table 4, Fig. 1). Meanwhile, amounts of oleanolic acid were negatively affected by adding Co to the media at all levels compared to the quantity extract­ ed from the control (Table 4). On the other hand, production of betulinic acid was significantly improved by adding Co to the medium. At 0.24 mg/l * Values represent means ± standard error. Control: represents callus maintenance medium consisting of solid MS medium + 34.2 g/l sucrose+ 2.0 mg/l 2, 4­D + 1.0 mg/l kinetin. Means with different letters are significantly different according to Tukey HSD range test at P≤0.05. Table 4 ­ Effect of heavy metals on callus fresh weight, color and production of betulinic and oleanolic acids in callus culture of Lantana camara Heavy metal (mg/l) Weight (g) Color Oleanolic acid (mg/g DW) Betulinic acid (mg/g DW) Cobalt Control 8.60 ±0.36 a * White 0.659 ± 0.011 a 0.014 ± 0.0003 d 0.08 6.87 ± 0.59 ab White 0.501 ± 0.0066 b 0.020 ± 0.0003 c 0.16 5.41 ± 0.71 b White 0.472 ± 0.0046 c 0.033± 0.0072 b 0.24 5.32 ± 0.75 b White 0.215 ± 0.0082 d 0.057± 0.0102 a Lead Control 8.60± 0.36 a White 0.659 ±0.011 a 0.014 ± 0.0003 b 0.08 3.80 ± 0.76 b White with red spots 0.344 ± 0.001b 0.020 ± 0.009 a 0.16 3.42 ± 0.90 b Yellow with red spots 0.343 ± 0.006 b 0.021 ± .0001 a 0.24 1.57 ± 0.10 b Yellow with red spots 0.117± 0.0085 c 0. 016 ± 0.008 b Chromium Control 8.6 ±0.36 a White 0.659 ± 0.011 c 0.014 ± 0.0003 a 0.08 3.60 ± 0.54 b Yellow with brown spots 0.676 ± 0.001 a 0.013 ± 0.0003 a 0.16 2.81 ± 0.66 b Yellow with brown spots 0.665 ± 0.0083 b 0.010 ± 0.0085 b 0.24 0.99 ± 0.13 c Yellow with brown spots 0.435 ± 0.0071 d 0.010± .0069 b 260 Adv. Hort. Sci., 2020 34(3): 255­263 it was four times higher than the control as shown in Table 4. Effect of lead. Data revealed that, callus growth, color and oleanolic acid production decreased dra­ matically with increasing Pb level in the media (Table 4, Fig. 2). The minimal value for fresh weight (1.57 g) was recorded in cultures grown in media supple­ mented with 0.24 mg/l Pb. Furthermore, adding Pb to the medium determined the appearance of red spots on the white callus at all Pb levels (Table 4, Fig. 2). Meanwhile, production of betulinic acid was improved significantly by adding either 0.08 or 0.16 mg/l Pb while it tended to decline at 0.24 mg/l Pb (Table 4). Effect of chromium. The results obtained showed that callus growth was negatively affected by adding Cr in the medium (Table 4). Furthermore, adding Cr to the medium resulted in development of brown spots on the white callus at all levels (Table 4). Moreover, chromium had improved the production of oleanolic acid to reach the maximum (0.676 mg/g DW) at 0.08 mg/l, while it tended to decline at higher Cr levels (Table 4). Meanwhile, amounts of betulinic acid decreased in response to Cr to reach minimum level of (0.010 mg/g DW) at either 0.16 or 0.24 mg/l, respectively (Table 4). 4. Discussion and Conclusions Effect of NaCl Adding NaCl to the culture medium resulted in reduction in most growth parameters in callus cul­ tures of Lantana camara which agrees with Nikman, et al. (2004) and Nikman et al. (2006) studies on Nicotiana seedlings and on calli and seedlings of two Trigonella species on culture media supplemented with elevated levels of NaCl, where growth and quali­ ty of the cultures declined sharply at levels high lev­ els. Generally, quality parameters including color, declined in tissue cultured plant material of many plant species when exposed to osmotic stress due to addition of salts or sugars to the media (Taiz and Zeiger, 2002). This could be due to the continuous accumulation of osmotica, dehydration and phenolic compounds accumulation that results in a shift in color toward brown which might determine, with time, cell death. Meanwhile, enhanced accumulation of oleanolic and betulinic acids in Lantana camara callus cultures was obtained at high NaCl levels agrees with Parida and Das (2005) who reported that salt stress might induce the production of secondary metabolites to maintain cell turgidity and to reduce dehydration. Also, Wang et al. (2015) confirmed in their study on cotton that salinity stress resulted from adding NaCl to the growth medium induced the production of some secondary metabolites such as gossypol, flavonoids and tannin. Effect of sugars It was clear from our data, that increasing sugar concentration in the medium inversely affected callus fresh weight and color. Elevated levels of sugars would make them act as osmotic agents instead of energy sources, and this would hinder growth rate of the explants (Tahtamouni et al., 2016). Consequently cell volume would decrease as a result of low turgor pressure which would be translated in a form of growth reduction (Taiz and Zeiger, 2002). Meanwhile, according to our results increasing sucrose level in the medium led to significant increase in production of oleolinic acid and betulinic acid. Generally, high levels of sucrose were reported to increase the secondary metabolite production in plant tissue cultures (Bandhakavi and Kamarapu, 2016). Similar results were also recorded in different species such as, grape berries (Dai et al., 2014), Ginkgo biloba (Park et al., 2004), and Clematis pitcher (Kawa­Miszczak et al., 2009). Fig. 2 ­ Effect of lead on callus fresh weight and color in callus culture of Lantana camara L. after 6 weeks of incubation in A medium containing 0.08; B medium containing 0.16 and C medium containing 0.24 mg/l lead. Fig. 1 ­ Effect of cobalt on callus fresh weight and color in callus culture of Lantana camara L. after 6 weeks of incubation in A medium containing 0.08; B medium containing 0.16 and C medium containing 0.24 mg/l cobalt. Al‐Ashoush et al. ‐ Enhancement of Pentacyclic Triterpenoids production in Lantana camara L. 261 Enhancement of secondary metabolites production in response to glucose was also obtained by Verma et al. (2012) in their study about improving alkaloid con­ tent in callus culture of Catharanthus roseus. Effect of plant growth regulators Callus fresh weight was influenced by the level of Kinetin in the medium. This was in full agreement with Singh and Saxena findings (2016) where maxi­ mum callus growth was obtained when 2, 4­D was used in combination with Kinetin to induce callusing in yellow variety of Lantana camara L. Effect of heavy metals Our results showed that, increasing levels of the experimented heavy metals in the culture medium led to a gradual decline in callus fresh weight and color. A decline in cultures growth in response to addition of chromium to the medium was reported by Waoo et al. (2015 a) who observed that increasing chromium in the medium decreased shoot length and percentage of survival in Lantana camara L. Similar response was also reported in Waoo et al. (2015 b) study about the toxic effects of different lead concentrations on in‐ vitro grown shoot cultures of Lantana camara. Our results had also indicated that production of oleanolic and betulinic acids varied with type and level of the added heavy metal. Chemical elicitation was commonly combined with osmotic stress that usually reduces growth (Taiz and Zeiger, 2002). In many studies, increasing biosynthesis of secondary metabolites by stressing the plant using chemical and/ or physical elicitation agents was possible. But adding such elicitors usually resulted in decreasing biomass of the plant, and consequently the levels of secondary metabolites was unchanged or declined, although the concentration is strongly enhanced at tissue or cell level (Gershenzon, 1984; Selmar and Kleinwächter, 2013; Paulsen and Selmar, 2016). Therefore, although biosynthesis of these com­ pounds increased significantly in plant material like callus cultures or microshoots (Paulsen and Selmar, 2016) due to chemical elicitation, the total biomass sharply decline because of the negative effects of the elicitors. So, the total biosynthesis of secondary metabolites is dramatically decreased. It can be concluded from our study that enhance­ ment of oleanolic and betulinic acids production in Lantana camara callus cultures is possible by adding some types of chemical elicitors to the culture medi­ um. Meanwhile, obtaining enough plant material is crucial to guarantee sustainable production of sec­ ondary metabolites in commercial amounts. This would open the gate for conducting research to improve our methodology to maintains proper amounts of plant material with enhanced levels of both acids. Acknowledgements Authors would like to thank the University of Jordan, The Deanship of Academic Research, for grant­ ing this research fund. Further thanks are extended to Hamdi Mango Center for Scientific Research, the Faculty of Agriculture: the Department of Horticulture and Crop Sciences at the University of Jordan. 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