Enhancement of Growth and Alkaloids Accumulation in Hyoscyamus muticus L . Callus Cultures by High Salt Concentration

a Botany and Microbiology Department, Faculty of Science, Suez University, Suez, Egypt, b Agric. Botany Department, Faculty of Agriculture, Suez Canal University, Ismailia, 41522, Egypt, c Pharmacognosy Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, d Horticulture Department, Faculty of Agriculture, Suez Canal University, Ismailia, 41522, Egypt, e Environmental Sciences Department, Suez University, Suez, Egypt.


Introduction
Salt stress is an important abiotic elicitor affecting plant growth, development, morphogenesis and the accumulation of secondary metabolites such as phenols, terpenes and alkaloids (Naik and Alkhayri, 2016;Selmar, 2008;Haghighi et al., 2012).NaCl stressor had stimulating or inhibiting effect on growth or synthesis of natural products according to its concentration, exposure period and plant species.In this respect, low concentration of NaCl (2000 ppm) enhanced the production of secondary metabolites in Mentha longifolia in vitro cultures but high concentration (6000 ppm) inhibited growth and yellowing cells (El-shennawy et al., 2017).Also, low NaCl level (1000 ppm) increased the total phenolics, total flavonoids and total tannins in callus culture of Fenugreek (Trigonella foenum-graecum ), while the higher concentration (3000 ppm) significantly decreased the secondary metabolite accumulation (Hussein and Aqlan, 2012).However, 25mM of NaCl stressed callus produced about three times of pilocarpine more than the unstressed callus (Abreu et al., 2005).Although root growth was decreased, secondary metabolite accumulation was increased with elevating level of NaCl compared control in madder plants ( Asci et al., 2018).Sitosterol content was increased in Nitraria tangutorum cell suspension at 250 mM NaCl treatment (Ni et al., 2015).Low increment of canavanine content was recorded in Sutherlandia frutescens in vitro shoot culture after exposure to 100 mM NaCl (Colling et al., 2010).Using of NaCl as an elicitor improved the synthesis of both vinblastine and vincristine in Catharanthus roseus embryogenic tissue culture (Fatima et al., 2015).
Tropane alkaloids are plant derived organic compounds that contain a tropane ring in their chemical structure and they are among the oldest medicines known to man.Tropane alkaloids have been found in different plant families, Brassicaceae, Convolvulaceae, Erythroxylaceae, Euphorbiaceae, Olacaceae, Proteaceae, and Rhizophoraceae, but they are best known for their occurrence in the family Solanaceae (Griffin and Lin, 2000).Hyoscyamus muticus, commonly known as the Egyptian henbane, is a shrub belongs to family Solanaceae, is a rich source of tropane alkaloids which have mydriatic, anticholinergic and antispasmodic properties.Hyoscyamine represents 90% of the total alkaloids in addition to small amounts of hyoscine in Hyoscyamus muticus (Batanouny et al., 1999).The present study was carried out to investigate the effect of salt stress by different concentration of NaCl on callus growth, histological structure, biochemical parameters, and alkaloid production from Hyoscyamu muticus L.

Seeds sterilization and germination:
Wild Hyoscyamus muticus L. seeds were collected from Saint Catherine peninsula and identified according to Tackholm, (1974).To break a seed dormancy, it were immersed in 250 mg l -1 of gibberellic acid (GA 3 ) for 24 h at lab. temperature (25±0.5˚C)(Alaghemand et al., 2013).Then washed with tap water and surface-sterilized with 70 % ethanol for 2 min., then in 25 % commercial bleach (containing 5.25 % sodium hypochlorite) with a drops of tween-80 for 20 min and finally rinsed 3 times with double distilled sterilized water.The sterilized seeds were cultured in hormone-free MS medium (Murashige and Skoog), supplemented with 7 g l -1 agar and 15 g l -1 sucrose and maintained at 25 ± 1 °C under light condition (1,500 lux, 16 h/day) for germination.After germination, plantlets have been obtained for explants preparation.

2.3.Elicitation treatments:
After four weeks, callus were removed from vessels under aseptic condition and cut a suitable weight (500 mg) and subculture on the same medium for further proliferation.NaCl added to the medium at different concentrations 6, 8 and 10 dS/m to initiate salt stress.Callus cultures were incubated at 25 ± 1 °C under light condition (1,500 lux, 16 h/day).After 21 days treatment callus were harvested and washed with distilled water to remove remains agar and dried by filter paper.

2.4.Vegetative measurements:
Forty pieces of callus fresh weight (FW) and dry weight (DW) after drying at 70 o C until constant weight was recorded.Water content (WC) of callus was calculated using FW and DW values according to Henson et al. (1981).

2.5.Biochemical determinations:
Photosynthetic pigments (mg 100 g -1 FW) in callus (chl.a, b and carotenoids) were extracted with 85% of acetone and estimated spectrophotometrically at 662, 644 and 440.5 nm (Arnon, 1949).Anthocyanins concentration (mg g -1 FW) was determined after digesting callus with 1% of HCl (v/v) in methanol and kept overnight at 4ºC (Lange et al., 1971).Then centrifuged at 3000 rpm for 10 min at 5°C.The values are calculated as E 535 -0.25 (E 650 ) /g FW.Total carbohydrates (mg g -1 DW) were determined according to Hedge and Hofreiter, (1962), dried samples digested by 5 ml of 2.5 N HCl for 3 hours at 100 o C .Then naturalized with sodium carbonate.Sample was made volume up to 10 ml and then filtrated.50 µl was diluted to 1ml with distilled water.To each tube, 1ml of 5% phenol and 5ml of 96% H2SO4 were added.After 10 min, tubes were shaken at 25-30 0 C for 20 min.Optical density was measured at 490 nm as reported in.Total protein (mg/g FW) was determined by Bradford method (Bradford, 1976) at 595 nm.Proline as mg g -1 FW was estimated with ninhydrin reagent as described by Bates et al. (1973), the red color intensity was measured at 520 nm against the toluene blank.Total glutathione (µmol /mg protein) was determined at 412 nm by 5-5'-Dithiobis (2nitrobenzoic acid) (DTNB) reagent (Griffith, 1980).For determination of total free amino acids and free phenols alcohol extraction of callus was prepared as Abdel-Rahman et al. (1975).Free phenolics (mg g -1 FW) were determined by a modified Folin-Ciocalteu method and measured at 650 nm according to Horwitz et al., (1970).Total free amino acids (mg g -1 FW) was estimated using the method of Rosen colour (1957) with ninhydrin reagent .The blue colored was measured against blank sample at 570 nm.Hydrogen peroxide (mmol/g FW) was determined at 390 nm by the modified method according to Shi et al., (2007) from Sun et al., (2007).Malondialdehyde (µmol.g - FW) determined by the thiobarbituric acid (TBA) reaction as described by Gallego et al., (1996) from Heath and Packer, (1968).All spectrophotometric analyses were done using UV/VIS spectrophotometer, PG instrument Ltd, USA.

SDS-PAGE of soluble proteins
One dimensional SDS-PAGE gel electrophoresis based on the method of Laemmli, (1970) was used to fractionate the soluble proteins in callus.Twenty milligrams of callus were dispersed in 1 ml SDS 10% with 100 μl β-mercaptoethanol for 15 min, then centrifuged at 11000 rpm for 10 min.Twenty μl of extraction were mixed with 20 μl of SDSloading sample buffer (SDS 4%, β-mercaptoethanol 3%, glycerol 20%, Tris HCl 50 mM pH 6.8 and bromophenol blue traces), heated at 96 °C for 3 min and 10 μl aliquot was electrophoresed (10 μl of protein/ lane).The resolving and stacking gels were prepared according to the standard procedure of Davis, (1964).The electrode buffer contained 50 mM TRIS, glycine 0.384 M and SDS 0.1%.The protein bands were developed with Commassie Brilliant Blue R-250 dye (0.2% solution, freshly prepared in 45% methanol, 10% glacial acetic acid and 45% distilled water) at room temperature overnight.The gel was photographed and made by scan apparatus as densitometric (optical density) analysis at 600 nm using standard maker protein (Pharmacia).

Histological investigations
For longitudinal sections (15 μm thick), callus was fixed in formalin acetic acid (FAA), then dehydrated with ethanol series and cleared with ethanol-xylene.Then samples were embedded in paraffin wax at 45-55ºC (Johansen, 1940).Sections were cut with steel blade on rotary microtome.The fixed sections were stained with Safranin O-Fastgreen double stain.After staining, sections mounted in Canada balsam (Sass, 1961).Observation and photomicrographs were achieved using research microscope (LEICA DM500) fitted with digital camera (LEICA ICC50).For staining alkaloid, callus was macerated and stained with Dragendorff's reagent.

2.9.Alkaloids determination
For alkaloids extraction, powdered dry callus was percolated overnight in methanol till complete exhaustion, and then methanol was removed by distillation under For alkaloids extraction.The residue obtained was stirred with 0.1 N HCl and extracted with CH 3 Cl.The chloroform layer was washed with 0.1 N HCl and discarded.The combined acidic layer was then rendered alkaline with NH 4 OH and extracted with CH 3 Cl.The organic layer was collected then dried under vacuum to afford the crude alkaloid extract (Karawya et al., 1975).Total alkaloids concentration was determined using the method reported in Ajanal et al., 2012; which in brief depends on the reaction between Bromocresol green and alkaloids in pH 4.7 to form a yellow colored complex which is extractable in chloroform and the color intensity obey Beer Lambert's law.
Atropine standard solution was made by dissolving 1 mg of Atropine (Sigma) in 10 ml distilled water.The absorbance of the complex in chloroform was measured at spectrum of 470 nm in UV/VIS spectrophotometer, PG instrument Ltd, USA against the blank prepared as above but without atropine.High-Performance Thin-Layer Chromatography (HPTLC) was performed on 20 cm ×10 cm HPTLC silica gel 60 F 254 plates (Merck) with a mobile phase consisting of chloroform : methanol : acetone : aqueous ammonia (25%) 75 : 15 : 10 : 1.6 (v/v/v/v) (Jaremicz et al., 2014).Hyoscyamine standard (Sigma) was diluted by chloroform to final concentration 20, 40, 60, 80, 120, 160 µg/ml.All samples and standards were applied to the plates by means of CAMAG Linomat 5 with dosing syringe 100 μL as 7 mm bands with 10.5 mm distance between tracks, application X 15 mm and 13 mm application Y edges of plate and the application volume was 1-20 µL for samples and 2-8 µL for standard.Loaded HPTLC plates were developed to a distance of 50 mm in Camag Automatic Developing Chamber CADC 2 at room temperature.The development occurring in a two-steps (Preconditioning with 10 ml mobile phase for 5 minutes and development with 25ml mobile phase for 20 minutes).The plate was developed to a distance of 50 mm and dried for 5 min by a stream of warm air, then the plates derivatized with 200 ml Dragendorff's reagent using Chromatogram Immersion Device.Then the plate was scanned and examined densitometry at l = 550 nm by means of CAMAG TLC Scanner 4 with slit dimension of 6 x 0.30 mm.

Statistical analysis
All data were statistically analyzed as randomized complete blocks design (Steel et al., 1997).

Growth, histological description and alkaloid crystals of callus cells:
The stimulating effect of different levels of NaCl on callus growth after 21days showed in

The vegetative growth and total carbohydrates of callus:
The fresh (FW) and dry (DW) weight of henbane callus were gradually increased with increment of NaCl level in MS medium.High level of NaCl (10 dS/m) increased the FW and DW by 4.1 and 2.8 fold more than control, respectively (Figure 2A,B).Slight increment (1%) of water content in salt stressed callus compared to unstressed callus was observed (Figure 2C).Total carbohydrate content significantly reduced under all NaCl levels.The highest reduction of total carbohydrates (24.3%) was determined in 10dS/m of NaCl stressed callus compared to control as showed in (Figure 2D).

pigments:
Photosynthetic pigments (Chl.a, b, and carotenoid) concentrations were gradually decreased in callus under all NaCl levels compared to untreated one.The highest reduction of Chl.a and carotenoid concentration by 31.5 and 26 % was observed at high NaCl level (10 dS/m) lower than control, respectively (Figure 3A,C).Also, the highest reduction of Chl.b (28.1%) was observed in callus treated with moderate level of NaCl (8 dS/m) compared to control (Figure 3B).In contrary, anthocyanin content was increased 3 fold in 10 dS/m of NaCl stressed callus compared to untreated one as shown in Figure (3D).

3.1.4.Nonenzymatic antioxidants and H 2 O 2
Both free phenolics and glutathione concentrations had opposite trend in response to NaCl treatments.So free phenolics were decreased by 21 and 30% (Figure 4B) the glutathione content was increased by 34 and 20 % (Figure 4A) in 6 and 8 dS/m of NaCl stressed callus respectively compared to control.Also, the content of both phenolics and glutathione increased by 8 and 4.6% in 10 dS/m of NaCl streesed callus more than control, respectively but without significant differences.

3.1.5.Enzymatic antioxidants and MDA
The activity of peroxidase (POD) and superoxide dismutase (SOD) were increased with rising of NaCl level in MS medium, while the catalase (CAT) activity was decreased as shown in Figure (5) .Activity of POD and SOD increased by 2.6 and 2.3 fold in 10dS/m of NaCl stressed callus more than untreated callus, respectively.However, malondialdehyde (MDA) content was decreased under salt stress condition compared to normal one.The minimum concentration (1.27 µmol/g FW) of MDA was found in 8 dS/m of NaCl stressed with 56.7% reduction lower than control (Figure 5D).

Alkaloids and nitrogenous compounds
Protein content was gradually decreased by 41.2, 17.1 and 11.7 % in, 6, 8 and 10 dS/m of NaCl stressed callus respectively, compared to control (Figure 6B).The concentration of free amino acids was decreased in callus under both low and moderate level by 48 and 18.4 % compared to control, respectively.Increment of free amino acids content was observed in 10 dS/m of NaCl stressed callus without significant differences with control (Figure 6A).Total alkaloids content was increased 2-fold in callus treated with 10dS/m of NaCl compared to untreated callus or wild leaves.Moreover, low and moderate level of NaCl reduced the total alkaloid by 22.7 and 11.5%, respectively compared to control or wild plants (Figure 6C).In contrary, hyoscyamine content was decreased under all NaCl levels.In this respect, wild leaves synthesized the highest amount (2.8 mg/g DW) of hyoscyamine which was double amount as low NaCl level (Figure 7).

Disscusion:
Creating a case of salt stress in the medium of plant callus cultures is a potential method for secondary metabolites accumulation which may be much more effective than studies performed on intact plant due to the absence of root barrier (Vanishree et al., 2004;Namdeo, 2007).Results reported herein showed that the total alkaloids concentration had doubled under high level of NaCl (8 dS/m) compared to unstressed callus or wild leaves, while the hyoscyamine accumulation was higher in wild leaves compared to all NaCl stressed callus (Figure 7).However, the concentration of hyoscyamine was increased by 40 % in callus treated with low level of NaCl (6dS/m) compared to untreated one.Results were agreed with Mahajan et al., (2005) who reported that high salt stress may be increase or decrease a specific secondary metabolite in plants.
The data obtained from our investigation revealed that callus cells of Egyptian henbane were tolerant to NaCl salt stress due to the enhancement of both fresh and dry biomass as shown in (Figure 2).These findings were found in different plant species such as in vitro Cassava plantlets treated with 10mM NaCl (Cheng et al., 2018).In contrary, Aljibouri et al., (2012) found a reduction of both FW and DW of Hyoscyamus niger callus after application of abiotic elicitors.Results cleared that Photosynthetic pigments such a Chl a, b and carotenoids were susceptible to salt stress due to its reduction under all NaCl levels, while anthocyanin was a stable pigments, so it increased by 3.1 fold compared to unstressed callus (Figure 3).Decreasing the Chls.content in the salt-affected plants could be due to the destruction of the alkaloid protein complex or degradation of Chls.by chlorophyllase (Rao and Rao, 1981).Under salt stress the photosynthetic pigments were decreased in callus of different species such as of Capsicum annuum (Sen and Alikamanoglu, 2011) and wheat (El-Kaaby et al., 2017).
Our results reinforced the hypothesis that anthocyanin participated in salt resistance mechanism in tolerant plant species as tomato and red cabbage (Steyn et al., 2002and Winkel-Shirley, 2002, Eryılmaz, 2006).
High salt concentrations resulted in oxidative stress due to the formation of reactive oxygen species.Effective enzymatic and nonenzymatic antioxidant system is one of the most common protection mechanism that plants evolve to stabilize important macromolecules which deleteriously affected by the ROS.(Shi et al. 2006).Results cleared that henbane callus depend on peroxidase (POD) and superoxide dismutase (SOD) more than catalase (CAT) as enzymatic antioxidant mechanism to quench the ROS under salt stress, as POD and SOD were significantly increased by 2.6 and 2.3 fold in 10 dS/m of NaCl stressed callus compared to control, respectively, while CAT activity was decreased (Figure 5).This findings were agreed with Foyer and Noctor (2000) who reported that, SOD is a key enzyme that plays an important role in cellular defense and catalyze the dismutation of superoxide radicals to H 2 O 2 and superoxide.Also, Yamasaki et al., (1997) reported that POD is oxidoreductase, which efficiently catalyze the oxidation of H 2 O 2 with phenolic compounds or ascorbic acid.Enhancement of POD and SOD activity was detected in different plant species under salt stress as Jatropha curcas callus ( Kumar et al., 2008) and Punica granatum callus (Bonyanpour and Khosh-Khui, 2013) Capsicum annuum callus (El-Kaaby et al., (2017) callus cultures of sugarcane (Munir and Aftab, 2013).Reduction of CAT activity also reported in callus of different species such as Solanum tuberosum (Queirós et al., 2011) and Salsola baryosma (Sharma and Ramawat, 2014).Results also showed that, glutathione content was significantly increased by 34 and 20 % at 6 and 8 dS/m, respectively compared to control.This finding was in line with Queirós et al., (2011) who found the reduction of glutathione reductase activity in saltexposed callus of Solanum tuberosum.Free amino acids were slightly increased by 10.7 % in 10 dS/m of NaCl stressed callus compared to control (Figure 6A).Results were coordinated with Hartzendorf and Rolletschek, 2001 who found a significantly increment of total free amino acids after application of NaCl in Phragmites australis.However, both free phenolics and proline content were decreased in callus under all NaCl levels (Figure 4).This result was in opposite with Ahmed et al., (2012).who found increment of total phenolic in Garden cress cultured under NaCl treatment, in Trianthema triquetra callus cultures (Sharma and Ramawat, 2014).Also, proline increased in callus culture of Salsola baryosma, Trianthema triquetra and Zygophyllum simplex under the salinity stress (Sharma and Ramawat, (2014)./gFW and 1.53 mmol /gFW, respectively) in henbane callus under all NaCl levels may be attributed with effective function of anthocyanin, glutathione, POD and SOD activity in scavenging the ROS under salt stress.This result was in line with Queirós et al., (2011) who found that lipids peroxidation were found in salt sensitive callus in Solanum tuberosum.The H 2 O 2 concentration was increased in four cassava cultivars in vitro shoots and decreased in another one under salt stress (Cheng et al., 2018).
Protein (Figure 6B) and carbohydrate content (Figure 2D) were reduced in all NaCl levels.This reduction may be due to its degradation by active ROS (El-Mashad and Mohamed, 2012).Protein degradation may be essential to provide amino acids for synthesis of new proteins as osmotin suited for growth or survival under stress conditions and also substrates for energy production (Kocisko et al., 1994).Reduction of protein content was occurred in different plants under salt stress as wheat (Sen and Alikamanoglu, 2011), Salsola baryosma, Trianthema triquetra and Zygophyllum simplex (Sharma and Ramawat, 2014).In contrary, soluble sugars content increased in Atriplex halimus NaCl stresses (Martinez et al., 2004).
Results reported herein showed that osmotin protein with molecular weight 32 KDa was highly expressed in 10 dS/m of NaCl stressed callus.Results were in line with Hassanein, (1999) who reported that proteins with MW 127 and 52kDa highly induced in peanut under NaCl stress.Also, Kumar et al., (2008) found that protein bands with MW 98, 69, 66 and 64 kDa were highly expressed in NaCl treated callus of Jatropha curcas while other bands with MW as 83, 58 and 43 kDa proteins were low expressed.

Conclusion:
NaCl at 10 dS/m gave the highest content of total alkaloids in Egyptian henbane callus culture.Hyoscyamine concentration was high in wild leaves than salt stressed callus.High accumulation of alkaloids and antioxidant agents under saline condition improved callus growth.Protein bands can be used as stable indicator for callus tolerance to salt stress.

Figure 1 :
Figure 1: Effect of different concentrations of NaCl on growth (A) , histology (B) and orangestained alkaloid crystals (C) in callus of Hyoscyamus muticus L., after 21 days

Figure ( 1 ).
Shape of callus cells differed according to NaCl levels as shown in Figure (1B).Cells had ovate or spherical shape in 8dS/m of NaCl and unstressed callus while it was amoeboid in low or high levels of salt stressed one.The highest values of cell length and width were found in 6 and 8 dS/m of NaCl stressed callus compared to control or High level of NaCl stressed one.Lysiogenous intercellular space was abundant in high level of NaCl exposed callus compared to schizogenous one in other treatments.Higher abundance of orangestained crystals of alkaloids in callus cells was found in all salt stressed callus cells especially under 10 dS/m of NaCl as shown in Figure (1C).
Figure (4C) indicated the slight reduction in proline content at 6 and 8 dS/m of NaCl stressed callus, but marked reduction occurred at 10 dS/m by 50.6 % in relation to control.The lowest value (1.53 mmol/g FW) of H 2 O 2 was determined in 10 dS/m of NaCl stressed callus with reduction by 16.8 % compared to untreated one (Figure 4D).

Figure 2 :
Figure 2: Effect of different concentrations of NaCl on fresh, dry weight ,water content and total carbohydrate of callus of Hyoscyamus muticus L., after 21 days.

Figure 3 :
Figure 3: Effect of different concentrations of NaCl on pigments (Chl a, Chl b, carotenoid and anthocyanin) of callus of Hyoscyamus muticus L., after 21 days.
Soluble protein of NaCl stressed or unstressed callus were electrophoretic analyzed by one dimensional SDS-PAGE gel as showed in Figure (8).Densitometric analysis revealed that soluble protein was fractioned into 7 varied bands with molecular weights (MW) 240, 109 , 44, 38, 32, 16 and 10 KDa.The unique band (109 KDa) was only expressed in 10 dS/m stressed callus.Bands with MW 240, 16 and 10 had high expression (as high optical density) in 8 dS/m of NaCl stressed callus.While, bands with MW 44, 38 and 32 had high expression in 10 dS/m of NaCl stressed callus.Protein bands were similar by 100% between unstressed callus and 6 or 8 dS/m of NaCl stressed one, then similarity % was low with 10 dS/m of NaCl stressed one.

Figure 6 :
Figure 6: Effect of different concentrations of NaCl on free amino acids (A), protein (B), total alkaloid (C), and hyoscyamine (D) of callus of Hyoscyamus muticus L., after 21 days.

Table 1 :
Description of Hyoscyamus muticus L. callus after 21 days as affected by different concentrations of NaCl.