Int. J. Biosci.

2017

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 10, No. 6, p. 20-28, 2017

RESEARCH PAPER OPEN ACCESS

Evaluation of an important flavonoid silymarin in callus

cultures of Sylibum marianum (L.) Gaertn

Sania1, Safdar Ali Mirza*1, Iram Liaqat2, Nazish Mazhar2

1
Botany Department, GC University, Lahore, Pakistan
2
Zoology Department, GC University, Lahore, Pakistan

Key words: S. marianum, Callus culture, Silymarin, Silybinin

http://dx.doi.org/10.12692/ijb/10.6.20-28 Article published on June 16, 2017

Abstract

Silybum marianum L. Gaertn is an important medicinal herb due to its active secondary metabolite, silymarin
pronounced to be effective in disturbed liver function and ailments. The plant is under harvest pressure for
sylimarin production and is being endangered. With the objective to produce silymarin in vitro, seed
germination were optimized maintaining aseptic conditions. Seedlings raised from sterilized seeds in vitro on
MS medium containing gibberellic acid, were used as explants for callus induction. Among different explants
used, seed and hypocotyls were found substantially suitable explants for callus induction. Significant callus
induction from hypocotyl explant was documented using MS medium added with 2, 4-D whereas seed explant
produced callus under the influence of BAP along with 2, 4-D. Callus cultures from different explants were put to
HPLC analysis to determine silymarin content. Since silymarin is a mixture of different components, all the
callus cultures were same with respect to composition but different in amount of components, seed callus was
observed to contain highest Silybinin content (89%) as compared to the callus cultures of other explants. The in
vitro production of silymarin may prove significant source for the said valuable medicinal compound.
* Corresponding Author: Safdar Ali Mirza  [email protected]

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Introduction Subsequently, plant cell and tissue cultures with

Silybum marianum (L.) Gaertn. is one of the defined production systems which can result in
important medicinal plants of family Asteraceae higher yields and more consistent quality of the
(Balian et al., 2006). Since in Greco-Roman era this products (Neumann et al., 2009), have appeared as
plant has been found to be a herbal remedy for a attractive substitutes for the production of secondary
number of diseases due to its secondary metabolites, metabolites (Ramachandra and Ravishankar 2002).
mostly it was used for treatment of liver, spleen, The source plant is under severe harvest pressure for
kidneys, adverse hepatitis and cirrhosis usually linked extraction of important flavonoid sylimarin from its
with alcohol (Ackerson, 2005). Silybum marianum seeds and is being endangered; the research work was
contains silymarin, a complex of seven flavonolignans initiated with an objective to produce silymarin
and polyphenols (Ball and Kowdley, 2005). Silymarin through in vitro cultures as an alternative source for
is primarily composed of 36.3% silibin, 5.9% sylimarin production and to support conservation of
silidianin, and 5.1% silichristin (Sersen et al., 2006). the plant.
Silymarin, from S. marianum, has undergone
extensive research within the last decades in view of Materials and methods
its medicinal value for treating liver diseases and Seed Collection, Sterilization and Germination
cancer. Pradhan and Girish (2006) examined the Seeds of S. marianum used to conduct the present
silymarin effect on hepatotoxicity and hepatobiliary
research were collected and provided by Muhammad
ailments and proved that silymarin is really useful
Ajaib, Taxonomist and Lecturer, Department of
against liver disorders. In vitro and in vivo studies
Botany. Seeds of S. marianum were sterilized with
have shown that these compounds protect the liver
0.1% HgCl2 under aseptic conditions and put to
from oxidative stress and sustained inflammatory
germination in vitro in Petri plates, the optimized
processes. In view of the protective roles that the
silymarin play, it is now widely used in clinical protocol comprised of seed soaking for 24 h, 5 seeds

applications and established therapies for liver per Petri plate on cotton pad soaked with 20 ml
(Comelli et al., 2007). Silymarin not only protects distilled water, placed in growth room at 26°C ±1
liver from certain toxins and viruses but also temperature.
regenerates damaged tissue of liver (Pradhan and
Girish, 2006). Its polyphenolic flavonoids, have MS Medium and Explant Preparation and Culture
potential for anticancer activities by reducing the Murashige and Skoog (MS) medium (1962) was used
proliferation of tumor cells (Malewicz et al., 2006).
as culture medium prepared and sterilized using
Secondary metabolites in S. marianum, useful to cure
standard protocol. Five days old seedlings were used
many diseases, can be analyzed to separate vitamins
as source of explants. Seed, cotyledonary leaf,
and polyphenols by several methods such as layer
hypocotyl and root were used as explants. The
chromatography (TLC), liquid chromatography
explants were transferred to culture jars containing
(HPLC) and gas spectrometry (Helmja et al., 2007).
Secondary metabolites, produced by plants as defense MS medium supplemented with different

mechanisms (Wink, 1988; Verpoorte et al., 2002) concentrations and combinations of plant growth

show different biological activities, and are used as regulators such as BAP, IBA, TDZ, 2,4-D, NAA etc.,
pharmaceuticals, agrochemicals, flavors, fragrances, under aseptic conditions. The explant containing
colors and food additives. The production of culture jars were transferred to growth room.
secondary metabolites via field cultivation of plants Observations for callus induction were recorded every
has various disadvantages such as low yields, and week and callus cultures were sub-cultured at 15 days
fluctuations in concentrations due to geographical, interval to maintain callus cultures.
seasonal, and environmental variations.

21 Sania et al.
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Standard curve for Silymarin components through by dissolving glacial acetic acid (0.5 ml/100 ml) in
HPLC assay double distilled water (57.5 ml/100 ml), methanol (42
Standard solution was prepared by dissolving ml/100 ml) was added into it by filtration, this mobile
accurately weighed 0.3 g of standard Silymarin phase was double filtered before use. Standard curve
flavonolignins (SO292 Sigma) in 100 ml of methanol plotted was used to compare retention time of the
of HPLC grade. Standard curve for silymarin, assayed peaks of silymarin and its components with explant
through HPLC, was plotted to show retention time of tissues and callus culture samples for appraisal of
the peaks of silymarin main components such as silymarin and its main components. Identification of
silybin, isosilybin, silydianin and silychristin. The specific flavonoids was done by retention times and
analyses of silymarin standard solution was carried by comparing the UV spectra of the peaks with those
out using a Knauer K2600-A Liquid Chromatography of the available standards. The percentage content of
equipped with a Nucleosil C18 (150 × 4.6 mm I.D, 5 sylimarin and its components was calculated by
μm) column. The elution was made in an isocratic comparing peak areas in standard curve.
mode at a flow-rate of 1ml/min and the detection
made at 288 nm under the influence of UV as Results
detector. One analysis required 15 min. the area. Seed germination and callus formation
In vitro seed germination was achieved in 5 day with
Determination of Silymarin in explant tissue and 100% germination. The germination percentage was
callus cultures decreased and germination duration was increased,
Callus cultures obtained from different explants of S. when unsoaked seeds were put to germination. These
marianum were used to analyze silymarin content by 5 day old in vitro grown seedlings were used as source
HPLC analysis. Explant samples and callus cultures of explants for callus induction. Among different
from respective explants such as seed, cotyledonary explants leaf and hypocotyl showed better potential
leaf, root and hypocotyl were oven dried at 50°C for for callus induction than other explants. Among
36 h. Dried callus, 0.2 g of each sample after different concentrations and combinations of plant
overnight soakeing in 5 ml of HNO3 was boiled until growth regulators used, BAP, 2,4-D alone and in
reddish brown fumes appeared, 1.0 ml of H2O2 was combination proved significant for callus induction in
added till the white fumes appeared. The mixture was general (Fig. 1), however in case of hypocotyl, 2,4-D
shaken well during boiling and clear extract was alone and TDZ in combination with 2,4-D both
obtained after filtration. Mobile phase was prepared treatments induced callus effectively (Table 1).

Table 1. Response of explants to plant growth regulators for callus induction.

Explant type PGR (mg/L) Callus characteristics
Index Weight (g) Induction frequency Color Texture
(mean %)
Seed BAP 250 0.21±0.00 79±0.67 Light brown Compact and friable
1.0
2,4-D 165 0.08±0.00 55±0.27 Whitish brown Compact
0.5
2,4-D +BAP 162 0.08±0.00 95±0.27 Whitish brown Compact and friable
0.5 +1.0
Coty- ledonary leaf BAP 225 0.3±0.002 82±0.47 Dark green Granular and friable
2.0
2,4-D 230 0.14±0.001 89±0.45 Variegated brown and green Compact
1.5
2,4-D +BAP 169 0.09±0.00 73±0.31 Dirty green Compact and friable
1.5+2.0
Hypo-cotyl 2,4-D 225 0.2±0.00 90±0.34 Whitish brown Granular and friable
1.0
2,4-D + TDZ 220 0.2±0.00 90±0.34 Whitish brown Granular and friable
1+ 0.05
Root 2,4-D 145 0.06±0.00 80±0.24 Light brown Granular and friable
1.0

The results included in the table are average of three replicates.

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Appraisal of Silymarin in explant tissues and distinct peaks for different components of silymarin.
respective callus cultures through HPLC These components were detected according to their
Analysis of seed tissue and seed callus retention time. The total amount of silymarin was
The analysis of seed tissue and seed callus showed found to be 95.2% and 99.9% respectively, amount of
five distinct peaks for different components of silybinin (88.9%) was observed more in seed tissue
silymarin. These components were detected according and seed callus culture under the combined effect of
to their retention time. The HPLC analysis of seed 2, 4-D and BAP as compared to 2,4-D alone (Table 2),
callus under the influence of 0.5 mg/L 2, 4-D alone the amount of other components is given in the same
and 0.5 mg/L 2, 4-D + 1.0 mg/L BAP showed five Table.

Table 2. Silymarin and silymarin components in seed tissue and callus.

Silymarin components Seed tissue Seed callus Retention Time Comparison
(%age) out of Silymarin (Seed tissue and Seed callus)
2,4-D 2,4-D + BAP Ret. Time Ret. Time (Seed callus)
(0.5 mg/L) (0.5 +1 mg/L) (Seed tissue) 2,4-D 2,4-D + BAP
(0.5 mg/L) (0.5 +1 mg/L)
Taxifolin 5.0 2.5 5.9 2.4 2.093 2.1
Silybinin 89 87.6 88.9 2.70 2.71 2.69
Silydianin 0.3 0.4 1.7 4.89 4.97 4.5
Silychristin 0.3 0.1 0.8 6.34 6.04 5.2
Isosilybinin 4.4 4.6 2.6 8.2 8.4 8.9

Analysis of cotyledonary leaf tissue and callus distinct peaks for different components of silymarin
The chromatograph of cotyledonary leaf tissue (Fig. (detected according to their retention time) under the
2A) and cotyledonary leaf callus (Fig. 2B) showed five influence of 1.5 mg/L 2, 4-D alone and 1.5 mg/L 2, 4-
distinct peaks for different components of silymarin. D with 2.0 mg/L BAP respectively. Amount of
These components were detected according to their silybinin was comparable in cotyledonary leaf tissue
retention time. The total amount of silymarin was and cotyledonary leaf callus induced under the
found to be 97.5% in cotyledonary leaf tissue and exogenous application of 1.5 mg/L 2, 4-D with 2.0
95.5% in cotyledonary leaf callus. The cotyledonary mg/L BAP but quite low in cotyledonary leaf callus
leaf tissue and cotyledonary leaf callus showed five under 2,4-D alone (Table 3).

Table 3. Silymarin and silymarin components in cotyledonary leaf tissue and callus.
Percentage of Silymarin Cotyl leaf tissue Cotyl leaf callus Retention Time Comparison (cotyledonary leaf tissue and callus)
components BAP BAP+2,4-D Ret. Time Ret. Time
(%age) out of Silymarin (2 mg/L) (2+1.5 mg/L) (Cotyl leaf tissue) (Cotyl leaf callus)
BAP BAP + 2,4-D
(2 mg/L) (2+1.5 mg/L)
Taxifolin 1.5 2.1 7.3 2.07 2.024 2.077
Silybinin 70 40.3 69.8 2.71 2.639 2.703
Silydianin 20 42.3 3.3 2.33 2.884 4.558
Silychristin 2.0 1.0 0.1 6.071 6.971 6.04
Isosilybinin 4.1 10.8 1.7 9.01 9.346 9.06

Analysis of hypocotyls tissue and callus The total amount of silymarin was found 98.4% in
The analysis of hypocotyl tissue and hypocotyl callus hypocotyl tissue from in vitro grown seedlings, 90.5%
showed five distinct peaks for different components and 95.4% in hypocotyl callus induced under 2,4-D
of silymarin detected according to their retention alone and 2,4-D with TDZ respectively.
time as was in previously mentioned sample analyses.

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The Table 4 shows the amounts of components of tissue and hypocotyl callus induced under the
silymarin detected according to their retention time. exogenous application of 1 mg/L 2, 4-D alone and 1
Amount of silybinin was comparable in hypocotyl mg/L 2, 4-D with 0.05 TDZ (Table 4).

Table 4. Silymarin and silymarin components in hypocotyl tissue and callus.

Silymarin components Hypocotyl Hypocotyl callus Retention Time Comparison
(%age) out of tissue (Hypocotyl tissue and callus)
Silymarin
2,4-D (1 mg/L) 2,4-D + TDZ Ret. Time Ret. Time (Hypocotyl callus)
(1+0.05 mg/L) (Hypocotyl tissue) 2,4-D (1 mg/L) 2,4-D + TDZ
(1+0.05 mg/L)
Taxifolin 4.0 1.6 4.3 2.32 2.0 2.0
Silybinin 83 82.3 84.3 2.56 2.7 2.7
Silydianin 3.0 0.6 2.9 4.6 4.7 4.5
Silychristin 2.5 0.1 2.3 5.9 6.0 5.4
Isosilybinin 6.9 5.9 1.6 8.1 8.4 8.4

Analysis of root tissue and callus The amounts of components of silymarin (Table 5)
The analysis of root tissue and root callus showed the were detected according to their retention time.
same five distinct peaks for different components of Amount of silybinin was same in root tissue and root
silymarin detected according to their retention time callus but the amount of other components of
but the total amount of silymarin was found quite less silymarin was less in root callus (Table 5).
in root callus induced under 2,4-D.

Table 5. Silymarin and silymarin components in root tissue and callus.

Silymarin Root tissue Root callus Retention Time Comparison
components 2,4-D (1 mg/L) (Root tissue and callus)
(%age) out of
2,4-D (1 mg/L) Ret. Time Ret. Time
Silymarin
(Root tissue) (Root callus)
2,4-D (1 mg/L)
2,4-D (1 mg/L)
Taxifolin 0.9 0.3 2.0 2.0
Silybinin 6.9 6.9 2.8 2.7
Silydianin 5.5 0.8 4.6 4.5
Silychristin 1.6 1.9 8.4 5.4
Isosilybinin 6.9 5.9 - -

Discussion of growth regulators for growth and proliferation of

Prescription drugs and intermediate medicinal biomass. Different researchers through experimental
compounds derived from plants constitute 25 of results have described that the exogenous application
pharmaceuticals. Plant tissue or organ cultures offer of growth regulators influenced growth in in vitro
alternate source for the production of secondary cultures (Vanhala et al., 1998; Weathers et al., 2005;
metabolites. Many investigators have reported Ali et al., 2010). In general, the plant growth
production of useful compounds in both callus and regulator type, combination and concentration are
suspension cultures (Sanchez-Sampedro et al., 2005, crucial factors in cell and organ growth proliferation.
Sánchez-Sampedro et al., 2007, Rady et al., 2014). In The type and concentration of auxin or cytokinin,
vitro cultures generally require an exogenous supply or the auxin to cytokinin ratio, altered biomass

24 Sania et al.
 Int. J. Biosci. 2017

growth in cultured cells in present study (Table 1) al., (1992), Cimino, et al,. (2006), Shilpashree and
as has been described by many researchers, Nair et Ravishankar (2009) and Tanveer et al., (2012).

Fig. 1. Callus induction in different explants from in vitro grown seedlings of Silybum marianum. A) Callus
induction from seeds on MS medium supplemented with 1.0 mg/L BAP (2x) (Culture of 7 day). B) Callus
induction in hypocotyl explant on MS medium supplemented with 1.5 mg/L 2,4-D (1x) (Culture of 14 day).

Plant tissue and shoot cultures have been found monnieri and regenerated shoots resulted in a 3-fold
promising for many medicinal plants to accumulate increase in bacoside A, when compared to field-grown
secondary metabolites to a greater extent than that by plants (Praveen et al., 2009). Callus cultures
natural plants and secondary metabolite production produced significant amount of sylimarin in present
can be improved by different factors Mantell and study as shown by chemical analyses of callus cultures
Smith (1984). For example, ginsengoside from Panax by HPLC and all callus samples were the same in view
ginseng (27% in cell dry weight in culture: 4.4% in of the presence of silymarin components (Table 2 to
whole plant (Linden, 2000), shoot cultures of Bacopa 5) including silybinin (Fig. 2 & 3).

Fig. 2. HPLC chromatographs: A) Cotyledonary leaf tissue B) Cotyledonary leaf callus under the influence of 2.0
mg/L BAP.

25 Sania et al.
 Int. J. Biosci. 2017

The amount of silymarin in callus cultures was except callus samples from root of S. marianum had
comparable to plant tissues as detection and appraisal lower quantities of silybinin. Seed callus showed
of β-Phellandrene has been described by Ali et al., higher quantities of all the components as source of
(2015) in callus cultures of Momordica charantia, silymarin.

Fig. 3. Silybinin content of Silymarin in explant tissues and related callus cultures.

Acknowledgement Ball KR, Kowdley KV. 2005. A review of Silybum

Botany Department, GC University Lahore is marianum (milk thistle) as a treatment for alcoholic
acknowledged for provision of research facilities. liver disease. Journal of Clinical
Gastroenterology 39(6), 520-528.
References
http://dx.doi.org/10.1097/01.mcg.0000165668.7953
Ackerson A. 2005.Milk thistle better nutrition.
http://findarticles/8582801. 0.a0

Ali S, Tariq A, Ajaib M, Khan KM. 2015. Cimino C, Vairo CS, Spina F, Natalucci C,
Appraisal of β-Phellandrene in Callus Cultures of Priolo N. 2006. Callus culture for biomass
Momordica charantia L. Cultivars, Jaunpuri and production of Milk thistle as a potential source of
Jhalri. Journal of Chemical Society of Pakistan 37 milk clotting peptidases. Electronic Journal of
(4), 817-823.
Biotechnology 9(3), Special Issue, 2006.
http://dx.doi.org/10.2225/vol9-issue3-fulltext-14
Ali S, Khan MS, Iqbal J. 2010. Genotype
independent in vitro regeneration system in elite
Comelli F, Giagnoni G, Bettoni I, Colleoni M,
varieties of sugarcane. Pakistan Journal of Botany
42(6), 3783-3790. Costa B. 2007. The inhibition of monoa-cylglycerol
lipase by URB602 showed an anti-inflammatory and
Balian S, Ahmad S, Zafar R. 2006. Anti- anti-nociceptive effect in a murine model of acute
inflammatory activity of leaf and leaf callus of inflammation. British Journal of Pharmacology 152,
Silybum marianum (L.) Gaertn. in albino rats.
787–94.
Research Letters 38(3), 213-214.
http://dx.doi.org/10.1038/sj.bjp.0707425
http://dx.doi.org/10.4103/0253-7613.25815

26 Sania et al.
 Int. J. Biosci. 2017

Helmja K, Vaher M, Puessa T, Kamsol K, Orav Pradhan SC, Girish C. 2006. Hepatoprotective
A, Kaijurand M. 2007. Bioactive components of the herbal drug, silymarinfrom experimental
hop strobilus and Comparison of different extraction pharmacology to clinical medicine. Indian Journal of
methods by capillaryelectrophoretic and Medical Research 124(5), 491-504. PMID:17213517
chromatographic methods. Journal of
Chromatography 2, 222-229. Praveen N, Naik PM, Manohar SH, Nayeem A,

http://dx.doi.org/10.1016/j.chroma.2006.12.067 Murthy HN. 2009. In vitro regeneration of Brahmi

shoots using semisolid and liquid cultures and
Linden JC. 2000. Secondary products from plant quantitative analysis of bacoside A. Acta Physiology
tissue culture. Biotechnology Vol. VI. EOLSS Plant 31, 723–728.
Pulication, Paris France. http://dx.doi.org/10.1016/j.phymed.2007.03.010
http://www.eolss.net/Eolss-sampleAllChapter.aspx
Rady MR, Matter MA, Ghareeb HA, Hanafy
Malewicz B, Wang Z, Jiang C, Guo J, Cleary MS, Saker MM, Eid SA, Hammoda FM,
MP, Grande JP, Lu J. 2006. Enhancement of Imbaby SI, Nazief NH. 2014. In vitro cultures of
mammary carcinogenesis in two rodents models by Silybum marianum and silymarin accumulation,
silymarin dietary supplements. Journal of Journal of Genetic Engineering and Biotechnology
Carcinogenesis 27(9), 1739-1747. 12(1), 75-79.
http://dx.doi.org/10.1093/carcin/bgl032 http://dx.doi.org/10.1016/j.jgeb.2013.11.003

Mantell SH, Smith H. 1984. Culture factors that Ramachandra RS, Ravishankar GA. 2002. Plant
influence secondary metabolite accumulation in plant cell cultures: chemical factories of secondary
cell and tissue cultures. In: Mantell SH, Smith H. metabolites. Biotechnology Advances 20, 101–153.
(Eds.) Plant Biotechnology. Cambridge University http://dx.doi.org/10.1016/S07349750(02)00007-1
Press, Cambridge, 75–108 P.
http://dx.doi.org/10.1080/02648725.1984.10647794 Sánchez-Sampedro A, Kim HK, Choi YH,
Verpoorte R, Corchete P. 2007. Metabolomic
Murashige T, Skoog F. 1962. A revised medium alterations in elicitor treated Silybum marianum
for rapid growth and bioassays with tabacco tissue suspension cultures monitored by nuclear magnetic
cultures. Physiologia Plantarum 115, 493-497. resonance spectroscopy. Journal of Biotechnology
130, 133–42.
Nair AJ, Sudhakaran PR, Madhusudana JR, http://dx.doi.org/10.1016/j.jbiotec.2007.03.007
Ramakrishna SV. 1992. Berberine synthesis
by callus and cell suspension cultures of Coscinium Sanchez-Sampedro MA, Fernandez-Tarago J,
fenestratum, Plant Cell Tissue and Organ Culture 29, Corchete P. 2005. Yeast extract and methyl
7–10. jasmonate induced silymarin production in cell
http://dx.doi.org/10.1007/BF00036139 culture of Silybum marianum L. Gaerth. Journal of
Biotechnology. 119, 60-69.
Neumann J, Zeindl-Eberhart E, Kirchner T, http://dx.doi.org/10.1016/j.jbiotec.2005.06.012
Jung A. 2009. Frequency and type of KRAS
mutations in routine diagnostic analysis of metastatic Sersen F, Vencel T, Annus J. 2006. Silymarin and
colorectal cancer. Pathology Research and Practice its components scavenge phenylglyoxylicketyl
205(12), 858-862. radicals. Fitoterapia 77, 525–529.
http://dx.doi.org/10.1016/j.prp.2009.07.010 http://dx.doi.org/10.1016 /j.fitote. 2006.06.005

27 Sania et al.
 Int. J. Biosci. 2017

Shilpashree HP, Ravishankar R. 2009. In vitro Verpoorte R, Contin A, Memelink J. 2002.

Plant Regeneration and accumulation of flavonoids in Biotechnology for the production of plant secondary
Hypericum mysorense. International Journal of metabolites. Photochemistry Reviews 1, 13–25.
Integrated Biology 8, 43-49. http://dx.doi.org/10.1023/A:1015871916833

Tanveer H, Ali S, Asi MR. 2012. Appraisal of an Weathers PJ, Bunk G, McCoy MC. 2005. The
important flavonoid, quercetin in callus cultures of effect of phytohormones on growth and artemisinin
Citrullus colocynthis. International Journal of production in Artemisia annuahairy roots. In vitro
Agriculture Biology 14, 528–532. Cellular and Developmental Biology Plant 41:47–53.
http://www.fspublishers.org http://dx.doi.org/10.1079/IVP2004604

Vanhala L, Eeva M, Lapinjoki S, Hiltunen R, Wink M. 1988. Plant breeding: importance of plant
Oksman-Caldentey KM. 1998. Effect of growth secondary metabolites for protection against
regulators on transformed root cultures of pathogens and herbivores. Theoretical and Applied
Hyoscyamus muticus. Journal of Plant Physiology Genetics 75, 225–233.
153, 475–481. http://dx.doi.org/10.1007/BF00303957
http://dx.doi.org/10.1016/S0176-1617(98)801776

28 Sania et al.