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International Journal of Pharmacognosy and Phytochemistry, ISSN:2051-7858, Vol.30, Issue.1 1293
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*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*
Production of Flavonoids and Phenolic
Compounds by Elicitation of Iphiona mucronata
(Forssk.) Asch. & Schweinf (Asteraceae) Callus and
Suspension Cultures
*Amal A. Al-Gendy1, 2
1Pharmacognosy Department,
Faculty of Pharmacy,
Zagazig University,
44519, Egypt
Email:amalalgendy@hotmail.com
Riham O. Bakr2
2Pharmacognosy Department,
Faculty of Pharmacy, October
University for Modern Sciences
and Arts (MSA), Egypt
Email:rehambakr@hotmail.com
Omayma D. El-Gindi3
3Pharmacognosy Department,
Faculty of Pharmacy, Egyptian
Russian University (ERU),
Egypt
Email:niron85@hotmail.com
ABSTRACT
Callus and suspension cultures of Iphiona mucronata
grown on Murashige and Skoog medium, supplemented
with 0.1 mg/l kinetin (Kn), 0.1 mg/l naphthalene acetic
acid (NAA) and 5 mg/l ascorbic acid were elicited with
different elicitors. Total flavonoids and phenolics were
determined by aluminum chloride - potassium acetate and
Folin-Ciocalteu colorimetric methods respectively. When
callus and suspension cultures elicited with 10 mg/l yeast
extract (Y), it showed an increase of 2.8 and 3.7 folds for
flavonoids production respectively. When callus cultures
treated with 0.001 mM phenyl alanine (Ph), a slight
increase in flavonoids content reached 1.17 folds, while
treated suspension reached maximum level after 60 hours.
Methyl jasmonate (MJ; 150 µM) increased flavonoids
concentration 4 times for callus and 2.3 times for
suspension. Determination of phenolics for elicited
suspension showed an increase of 2.3 (Y), 2.5 (Ph) and 1.3
(MJ) times. The obtained results favored the use of yeast
extract for high productivity and viability of cell lines.
Keywords- Callus and suspension cultures, elicitors,
flavonoids, Iphiona mucronata, methyl jasmonate,
phenolics, yeast.
1. INTRODUCTION
Flavonoids are of great economic functions as potential
drugs, food nutraceuticals and industrial materials.
Although their importance is manifested by the larger
demands, their low yields in natural plants and the
remarkable complexity for chemical synthesis lead to
shortage of supply. So, an alternative and effective
strategy is focused on elicitation treatment, which
furthermore is considered as a useful experimental system
and a promising production mode for the active
constituents from medicinal plants [1]. A review for in
vitro production of flavonoids is recently published [2].
Application of different strategies in vitro culture as media
manipulation, phytohormone regulation, precursor feeding,
plant cell immobilization, biotransformation and
genetically modified cell can be used to obtain good yield
of secondary metabolites in tissue culture. Elicitation of
tissue culture was found to be more economic [3].
Biotic and abiotic elicitors can enhance secondary
metabolites production. The stimuli are perceived by
receptors activating secondary messengers. These transmit
signals into the cell through signal transduction pathways
leading to gene expression and biochemical changes
resulting in secondary metabolites formation [4, 5]. The
basis for successful elicitation of secondary metabolites is
the choice of suitable elicitor, its concentration, and
optimal time of treatment. Methyl jasmonate activates a
multitude of jasmonate induced proteins which are
associated with accumulation of secondary metabolites [3].
Methyl jasmonate [6], yeast [7] and phenyl alanine [8,9]
have been used for flavonoids and phenolic elicitation in
family Asteraceae, however no records for elicitation have
been performed in genus Iphiona.
Iphiona mucronata (Forssk) Asch. & Schweinf [Astraceae
(Compositae)] is an endangered plant growing in Egyptian
deserts containing polysulphated flavonoids as major
constituents [10]. In a previous study [11], we reported the
best hormonal combination for induction and maintenance
of callus culture of I. mucronata by using Murashige and
Skoog (MS) medium [12], to get the highest growth index
(GI) and flavonoids contents compared with other tested
phytohormones. Moreover, we reported a protocol for
somatic embryogenesis and plant regeneration from callus
and suspension cultures of I. mucronata [13]. In the
current study, yeast, methyl jasmonate and phenyl alanine
were tried in different concentrations to increase the
productivity of total flavonoids and phenolic compounds
and vitality of callus and suspension cultures.
2. EXPERIMENTAL
2.1 Plant material
Aerial parts and seeds of Iphiona mucronata (Forssk.)
Asch. & Schweinf family Asteraceae (Compositae) were
collected from Wadi Hogool area and Al-Sokhna-Katamia
road, Egypt. The plant was identified by Dr. A. Mary,
Faculty of Science, El Azhar University. A voucher
sample (AR-2007) was kept in Pharmacognosy
Department, Faculty of Pharmacy, MSA University,
Egypt.
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2.2 Elicitation of callus culture
Callus was induced using seedling explant grown on MS
medium supplemented with 0.1 mg /l NAA, 0.1 mg /l Kn,
5 mg /l ascorbic acid, 30 g/l sucrose and solidified with 10
g/l agar. Media were adjusted to pH 5.8 using 1 N NaOH
or 1 N HCl, autoclaved at 121 °C for 20 min and incubated
at 25 °C in the dark as previously reported [11]. Known
weight of well grown, friable callus (3 g) was sub-cultured
to fresh media supported with elicitors. Fresh and dry
weights in addition to flavonoids contents were estimated
at zero time, day 5, 21 and 31. The used elicitors were
dried yeast, methyl jasmonate in addition to a feeding
precursor (phenyl alanine) [6], [14], [15]. All results were
recorded in triplicate.
Yeast (Y): Different concentrations of yeast Y1 (10 mg/l),
Y2 (20 mg/l), Y3 (50 mg/l) and Y4 (100 mg/l) were added
at zero time to callus cultures. Control media was prepared
by substituting the yeast extract with distilled water. Fresh
and dry weights of callus elicited with yeast were recorded
to determine the effect of yeast on growth.
Phenyl alanine (Ph): It was used in different
concentrations; Ph1 (1mM), Ph2 (0.01mM), Ph3
(0.05mM) and Ph4 (0.001mM). Control media was
prepared by substituting phenyl alanine with distilled
water.
Methyl jasmonate (MJ): MJ1 (50 µg/l), MJ2 (100 µg/l),
MJ3 (150 µg/l), MJ4 (200 µg/l), MJ5 (300 µg/l) and MJ6
(400 µg/l) in 95% ethanol were used. Control was made
using ethanol 95% instead of methyl jasmonate and also
compared with normal blank with distilled water to
explore ethanol effect.
2.3 Cell suspension culture and growth
parameters
Well grown, friable callus (4-5 g) was planted aseptically
to 50 ml of liquid MS medium (250 ml flask)
supplemented with 0.1 mg/l Kn, 0.1 mg/l NAA, 5 mg/l
ascorbic acid and 3% sucrose. The pH of the media was
adjusted to 5.8±0.1 prior to autoclaving. Cultures were
kept in an incubator shaker at 100 rpm/min, at 25°C in the
dark. The suspension was cultured into fresh medium
every two weeks. Different growth parameters were
estimated. Growth curves for fresh and dry weight were
monitored [16]. Growth index (GI), specific growth rate
(μ) and doubling time (dt) were calculated as follow [17]:
GI = (Ge - Gstart)/ Gstart; where Ge = suspension
mass at the end of each generation (final fresh weight).
Gstart = suspension biomass at zero time (Initial fresh
weight).
μ = (ln x − ln xo)/t; where xo is the initial fresh biomass (or
cell density), x is the biomass (or cell density) at time t,
and μ is the specific growth rate.
Doubling time (dt) = ln (2)/ μ.
2.4 Elicitation of suspension culture
Successful concentrations of elicitors obtained from callus
elicitation were added at 9th day aseptically to suspension
culture. Estimation of flavonoids and phenolic contents
was performed at different intervals and compared with
control. The control samples received equal volumes of
sterile water and ethanol 95% in case of MJ.
2.5 Determination of flavonoids contents
One gram of dried callus or suspension biomass was
refluxed with 25 ml of 95% hot ethanol for 10 min then
left overnight, filtered and adjusted with 80% ethanol (v/v)
to 25 ml. 0.5 ml of each sample was mixed with 1.5 ml
95% ethanol (v/v), 0.1 ml 10% aluminium chloride (w/v),
0.1 ml 1 M potassium acetate and 2.8 ml water.
Aluminium chloride was substituted by the same volume
of distilled water in blank. After incubation at room
temperature for 30 min, the absorbance of the reaction
mixture was measured at 415 nm [18]. For preparation of
calibration curve, standard solutions of 12.5, 25, 50, 80
and 100 µg/ml rutin were dissolved in 80% ethanol (v/v).
0.5 ml of each concentration was separately treated as
mentioned above. Calibration curve was plotted
representing the correlation between absorbance and rutin
concentration.
2.6 Determination of phenolic contents
One gram of dried suspension biomass was extracted with
25 ml methanol. 0.5 ml of each sample was added to 0.5
ml of water, 5 mL of 0.2 N Folin-Ciocalteu reagent (S.d
fine-chem limited), and 4 mL of saturated sodium
carbonate solution (75 g/l).The absorbance was measured
at 765 nm with a Shimadzu UV-visible spectrophotometer
after incubation for 2 h at room temperature [19].
Different concentrations (100, 200, 300, and 400 mg/l) of
gallic acid were prepared for production of calibration
curve. Absorbance was plotted against each concentration
used. Concentration of phenolic content was expressed as
gallic acid equivalent.
3. RESULTS
3.1 Elicitation of callus culture
Yeast: As yeast concentration increased, fresh and dry
weight decreased (Figure 1). Y1 (10 mg /l) showed the
highest fresh and dry weight and kept the vitality even
after 31 days while control became brownish even with
more biomass. Y1 resulted in the maximum increase in the
flavonoids content after 5 days (2825.8 µg/g.DW)
compared with control (1008µg/g.DW). This increase in
the flavonoids content decreased gradually till 31 days
(1266.8µg/g.DW) which was still higher than control
(540.8µg/g.DW) (Figure 2). The other three used
concentrations also showed their peak concentration after
5 days, but with different pattern. Y2 and Y3 flavonoids
concentration decreased till being equal to control, while
Y4 remains higher than the control (1069 µg/g.DW).
Phenyl alanine: Ph3 was the best concentration showing
the highest fresh and dry weight for callus (Figure 3). It
was noticed that phenyl alanine effect was similar to
control through the whole culture but slight increase was
International Journal of Pharmacognosy and Phytochemistry, ISSN:2051-7858, Vol.30, Issue.1 1295
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Figure 1 Effect of yeast elicitation on growth of I.
mucronata callus culture.
A: Fresh weight B: Dry weight
Figure 2 Effect of yeast elicitation on flavonoids contents
of I. mucronata callus culture.
observed in flavonoids content with Ph3 where its
maximum concentration reached 1.17 times control while
Ph1 was 1.05 fold the control. Decreasing phenyl alanine
concentration resulted in a flavonoids content of 0.71 and
0.11 times the control for Ph2 and Ph4, respectively
(Figure 4).
Methyl Jasmonate: MJ3 (150 µM) appeared to be more
effective than other concentrations, beginning after 5 days
(1595.6 µg/g.DW) compared with control (381µg/g. DW).
This increase continued till the 21st day (2193.6µg/g.DW)
compared with control (1559 µg/g.DW) representing 40%
increase although cells are brownish in color and appeared
necrotic but flavonoids production continued (Figure 5).
Through the use of different type of elicitors added at zero
time of subculture and through measuring flavonoidal
Figure 3 Effect of phenyl alanine elicitation on growth of I.
mucronata callus culture.
A: Fresh weight B: Dry weight
concentration through the whole subculture, comparison of
results was represented in figure 6.
3.2 Suspension culture
The obtained results showed that growth index (GI) is
3.59, specific growth rate (μ) is 0.23; while doubling time
(dt) is 3.013. Growth curve is represented in fig. 7. The
graph shows relatively slow growth during the lag phase
(two days) where a small increase in fresh and dry weight
is observed. The exponential phase extends up to 6 days
where the culture growth per time unit increases. By the
9th day stationary phase is reached, while after 15 days
browning of suspension culture occurred indicating the
necessity for subculture every 14 days.
3.2.1 Flavonoids and phenolic contents in suspension
culture
Maximum production of flavonoids and phenolic
accumulation was observed at the 9th day where phenolic
contents reached 6591.7µg/g.DW while flavonoids content
was 1100.7 µg/g.DW, then productivity declined to its
initial value (Figure 8).
3.3 Elicitation of suspension culture
The most successful concentrations of elicitors and
precursor applied for callus culture were used in
suspension culture.
Yeast: The effect of yeast (SY1) began after 3 hours but
the maximum flavonoids content was reached after 12 hr
A
A
B
B
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Figure 4 Effect of phenyl alanine elicitation on flavonoids
contents of I. mucronata callus culture
Figure 5 Effect of methyl jasmonate elicitation on
flavonoids contents of I. mucronata callus culture.
Figure 6 Effect of different elicitors on flavonoids contents
of I. mucronata callus cultures
Figure 7 Growth curves of suspension culture of I.
mucronata
Figure 8 Flavonoids and phenolic contents of I. mucronata
suspension culture.
of elicitation reaching 2036 µg/g.DW comparing with
control reaching 545.4 µg/g.DW (Figure 9A). Yeast
resulted in elicitation of phenolic contents reaching 6132
µg/g.DW compared to 2719 µg/g.DW for control after 12
h and even after its decrease, it will return higher than
control after 48 h (Figure 9B).
Phenyl alanine: Flavonoids content exceeded control
after 48 hours (414.3 µg/g.DW) and reached maximum
level after 60 hours (613.7 µg/g.DW), where flavonoids
were nearly depleted in control cells; then flavonoids
decreased gradually and there was complete depletion
after 6 days at the time the control cells regain their values
(Figure 10A). Phenolic contents were increased to 2.5
times control (3361 µg/g.DW while control 1353
µg/g.DW) after 48 hours but then began to decrease
compared to control till being depleted after 6 days of
exposure which denoted the inhibitory effect of
phenylalanine which may be due to accumulation of
harmful intermediates (Figure 10B).
Methyl Jasmonate: Elicitation of suspension by SMJ3
(150 µM) increased flavonoids and phenolic contents as
shown in figure 11. Flavonoids increased gradually
compared to control suspension (ethanol) and reached
maximum level after 6 hours (3585 µg/g.DW) compared
to control (1610 µg/g.DW) then decreased gradually, but
flavonoids disappeared after 36 hours where cells were
darker in color and necrotic. Similar pattern was observed
for control, which denoted the solvent effect. Upon the use
of SMJ3, slight increase in phenolic contents over control
occurred after 6 hours but declined gradually till complete
depletion after 60 hours.
4. DISCUSSION
4.1 Elicitation of callus culture
The elicited calli showed different responses towards the
applied elicitors and their concentrations. Y1 appeared to
be the best concentration for callus growth and vitality. It
is the lowest concentration resulted in the maximum
increase in the flavonoids content after 5 days, which is
2.8 times the control. Although this increase in the
flavonoids content decreased gradually till 31 days, it is
2.3 times the control in addition to keeping vitality of
cells.
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Fig.9. Flavonoids and phenolic contents of I. mucronata
suspension culture elicited with yeast extract.
A: Flavonoids B: Phenolics
Fig.10. Flavonoids and phenolic contents of I. mucronata
suspension culture elicited with phenyl alanine extract.
A: Flavonoids B: Phenolics
.
Y2 and Y3 highest flavonoid concentration were also
observed after 5 days but decreased till being equal to
control, while Y4 was higher than the control representing
1.9 times.
These results go with the belief that the stronger
stimulation of secondary metabolites by fungal elicitor
often occurs in the late exponential growth stage and early
stationary phase [20]. In a report dealing with silymarin
biosynthesis in milk thistle hairy root culture the results of
yeast elicitation correlating culture time with biosynthesis
reached a maximum by 72 h after culture (2-fold higher
than the control) [7]. However, the use of 16 mg/l yeast
extract inhibited prenylated flavanones in addition to
growth in Sophora flavescens (Fabaceae) callus culture
[21].
The effect of phenyl alanine appeared mainly to cell
viability. When callus cultures treated with 0.001 mM,
they appeared more viable and yellowish in color even
after 31 days. Different reports showed variable effects for
phenylalanine. Although it did not affect flavonoids
production in transformed Scutellaria baicalensis roots
[22], it increased productivity of anthocyanins in callus
culture of Catharanthus roseus which was explained by
the intracellular L-phenylalanine accumulated at high
levels which was used as a biosynthetic precursor material
for anthocyanin and related flavonoids. Moreover, it had a
function as a kind of signal that promoted the transcription
of the genes on the anthocyanin biosynthetic pathway.
Phenylalanine supplementation is expected to increase
metabolic flux through phenyl-propanoid biosynthetic
pathway and elevated level of target compound [23].
Although its effect was weaker when compared with yeast,
but it resembled yeast in its ability to keep the vitality of
cells till the last day of subculture.
Methyl jasmonate: MJ3 (150 µM) appeared to be more
effective than other concentrations which represents 4
times increase in flavonoids concentration after 5 days.
Opposite to yeast effect, flavonoids concentration
increased in presence of MJ till 21 days (1.4 times
ethanolic control) but browning of cells appeared which
may be due to detrimental effect of MJ occurred. In this
case the excretion of metabolites can be explained by
leakage or cell lysis [24]. Ethanol control exhibited a
solvent effect increasing flavonoids concentration when
compared with distilled water so ethanol is considered
partially as elicitor. When MJ was used for 7 days for
flavonoids elicitation in Passiflora quadrangularis callus
cultures, MJ strongly induced orientin production (up to
300 μg/g.DW), and to a lesser extent, isovitexin and
vitexin, but had no effect on isoorientin, relative to
untreated controls [25].
4.2 Suspension culture
The difference in growth rate and doubling time of
suspension (0.23, 3.013) compared to those previously
reported for callus culture of I. mucronata (0.09, 7.17)
represents the main advantage of suspension culture,
which is higher growth rate [11]. The growth of plant cells
A
B
A
B
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Figure 11 Flavonoids and phenolic contents of I.
mucronata suspension culture elicited with methyl
jasmonate.
A: Flavonoids B: Phenolics
is more rapid in suspension than in callus culture and is
also more readily controlled because the culture medium
can be easily amended or changed [26]. Specific growth
rate was similar to that obtained by Euphorbia characias
L. (Euphorbiaceae) suspension culture [27] with (μ) equal
to 0.256 d-1. Hyoscyamus muticus (Solanaceae) also
showed a growth rate of 0.25 d-1 [28]. Upon elicitation of
suspension culture by yeast (SY1), an increase of 3.7 and
2.3 folds of flavonoids and phenolic contents were reached
after 12 hr of elicitation. Phenolics involve beside
flavonoids, lignans, phenolic acids and hydroxycinnamic
acid. So, even in absence of flavonoids the phenolic
contents will remain high. When suspension cultures of
Silybum marianum (L.) treated with 50 µg/ml medium of
yeast extract, an increase in silymarin accumulation was
noticed after 12 h of treatment reaching a maximum
between 24 and 48 h and declining drastically afterwards.
It was found that yeast extract caused 50% and 200%
increase in total silymarin accumulation in suspension
cells and medium respectively. It was suggested that yeast
caused a complex stress response in the cultures in
addition to accumulation of flavonolignans, as for example
lipid peroxidation, and/or activation of other metabolic
pathways [6]. Results of phenyl alanine elicitation were
non significant due to the rapid decrease followed by an
increase in flavonoid and phenolic contents which
appeared unexplained but finally complete cell death
occurred compared with control which is probably due to
accumulation of toxic metabolites.
Although methyl Jasmonate (SMJ3) elicitation for
suspension increased flavonoids level by 2.3 times and
phenolics by 1.3 times after 6 h, depletion occurred after
36h and 60h respectively. This would denote that methyl
jasmonate effect was higher on flavonoids than phenolic
contents. Addition of MJ to a broad range of plant-cell
suspensions resulted in higher accumulation or de novo
synthesis of secondary metabolites. Jasmonates are
expected to be the chemical signal compounds in the
process of elicitation leading to de novo gene transcription,
and finally, the biosynthesis of natural products in cultured
plant cells [29]. In a recent report, MEJA induced
enhancement in phenolic and flavonoid accumulation in
suspension culture of Artemesia absinthium L [30].
Cell cultures of Saussurea medusa (Asteraceae) were
challenged by MJ. The highest jaceosidin and hispidulin
concentrations were achieved being 2.2-fold and 4.2-fold,
respectively, higher than those from controls. L-
phenylalanine ammonia- lyase (PAL) activities were
transiently increased after treatment with MJ, which
suggested that this elicitor modified jaceosidin and
hispidulin production by regulating the phenylpropanoid
pathway [31].
Elicitation of I. mucronata suspension by MJ suppressed
cell growth where cells appeared darker in color and
necrotic. Methyl jasmonate treatment led to a clear
repression of cell growth in suspension culture of Ginkgo
biloba and induced cell browning as well [32]. In view of
the inverse relationship between the production of biomass
and the accumulation of secondary metabolites, the cell
growth inhibition elicited by MJ treatment may favour the
synthesis of secondary metabolites. It was reported that
increased concentrations of MJ (5 - 500 µM) was toxic to
cells and did not induce the phenylpropanoid/flavonoid
pathway genes [33].
5. CONCLUSIONS
Elicitation of Iphiona mucronata (Asteraceae) callus and
suspension cultures using yeast, phenylalanine and methyl
jasmonate increased the flavonoids and phenolic
production. The results favored the use of yeast extract
compared with methyl jasmonate and phenylalanine for
the high productivity and keeping viability of cell lines. To
the best of our knowledge, there is no report for elicitation
in genus iphiona.
ACKNOWLEDGEMENT
The authors are grateful to October University for Modern
Sciences and Arts (MSA) for sponsorship and supplying
the research facilities of this research work.
REFERENCES
[1] Giri, A., and Narasu, L. (2000). Transformed hairy
roots: Recent trends and applications. Biotechnol.
Adv., 18, 1-22.
B
A
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[2] Bharati, A. J., and Bansal, Y. K. (2014). In vitro
production of flavonoids: A review. WJPPS, 3 (6),
508-533.
[3] Sharma, M., et al., (2011). Enhancement of
secondary metabolites in cultured cells through
stress stimulus. American J. Plant Physiol., 6, 50-71.
[4] Sudha, G., and Ravishankar, G. A. (2002).
Involvement and interaction of various signaling
compounds on the plant metabolic events during
defense response, resistance to stress factors,
formation of secondary metabolites and their
molecular aspects. Plant Cell Tiss. Org. Cult., 71,
181-212.
[5] Kákoniová, D., et al., (2009). The possibility to
enhance flavonoids production in Rubia tinctorum l.
callus cultures. Nova Biotechnologica, 9(2), 191-
197.
[6] Sánchez –Sampedro, M. A., et al., (2005). Yeast
extract and methyl jasmonate-induced silymarin
production in cell cultures of Silybum marianum (L.)
Gaertn. J. Biotechnol., 119, 60–69
[7] Hasanloo, H., et al., (2009). Evaluation of the yeast-
extract signaling pathway leading to silymarin
biosynthesis in milk thistle hairy root culture.
World J. Microbiol. Biotechnol. 25 (11), 1901-1909.
[8] Arya, D. (2007). In vitro propagation and
biochemical studies in Puchea lanceolata Oliver &
Hiern: An important medicinal herb. PhD thesis,
University of Rajasthan, Jaipur.
[9] Arya, D., et al., (2008). In vitro propagation and
quercetin quantification in callus cultures of Rasna
(Puchea lanceolata Oliver & Hiern) Indian j.
Biotechnol., 7, 383-387
[10] Ahmed, A. A., et al., (1988). Polysulphated
flavonoids from Iphiona mucronata. Rev.
Latinoamer Quim., 19 (3), 107-109.
[11] Al-Gendy, A. A., et al., (2008). Production of
flavonoids in callus cultures of Iphiona mucronata,
Astraceae. Egypt. J. Biomed. Sci., 28, 142-150
[12] Murashige, T, and Skoog, F. (1962). A revised
medium for rapid growth and bioassay with tobacco
tissue cultures. Physiol. Plant., 15, 473-497.
[13] Al-Gendy, A. A., et al., (2013). Somatic
embryogenesis and plant regeneration from callus
and suspension cultures of Iphiona mucronata
(Forssk). European Scientific Journal, 9 (27), 37-49
[14] Shohael, A. M., et al., (2007). Methyl jasmonate
induced overproduction of eleutherosides in somatic
embryos of Eleutherococcus senticosus cultured in
bioreactors. Electron. J. Biotechnol., 10 (4), 633-
637.
[15] Edahiro, J., et al., (2005). Enhanced accumulation
of anthocyanin in cultured strawberry cells by
repetitive feeding of L-phenylalanine into the
medium. J Biosci. Bioeng., 99 (1), 43–47.
[16] Godoy-Hernández, G. and Vázquez-Flota, F. (2006).
Growth measurements estimation of cell division
and cell expansion. In: Loyola-Vargas VM,
Vázquez-Flota F (ed) “Plant cell culture protocols”.
2nd edn. Humana Press Inc.
[17] Verpoorte, R, et al., (1998). Metabolic engineering
for the improvement of plant secondary metabolite
production. Plant Tiss. Cult. Biotechnol. 4, 3 -19.
[18] Kosalec, I., et al., (2004). Quantitative analysis of
the flavonoids in raw propolis from northern
Croatia. Acta pharma Zagreb., 54, 65-72.
[19] Sellappan, S. and Akoh, C. C. (2002). Flavonoids
and antioxidant capacity of Georgia-Grown Vidalia
Onions. J. Agric. Food Chem., 50, 5338-5342.
[20] Kitamura, Y., et al., (1998). Induction of
furanocoumarin biosynthesis in Glehnia littoralis
cell suspension cultures by elicitor treatment.
Phytochemistry, 48, 113–117.
[21] Yamamoto, H, et al., (1995). Stimulation of
prenylated flavanone production by mannans and
acidic polysaccharides in callus culture of Sophora
flavescens. Phytochemistry, 40 (1), 77-81.
[22] Kuzovkina, I. N., et al., (2001). Flavonoid
production in transformed Scutellaria baicalensis
roots and ways of its regulation. Russ. J. Plant
Physiol., 48 (4), 448-452.
[23] Taha, H. S., et al., (2008). Successful application
for enhancement and production of anthocyanin
pigment from calli cultures of some ornamental
plants. Aust. J. Basic & Appl. Sci., 2 (4), 1148-1156.
[24] Vasconsuelo, A. and Boland, R. (2007). Molecular
aspects of the early stages of elicitation of
secondary metabolites in plants. Plant Sci., 172 (5),
861–875
[25] Antognoni, F., et al., (2007). Induction of flavonoid
production by UV-B radiation in Passiflora
quadrangularis callus cultures. Fitoterapia, 78,
345–352
[26] George, E. F. (2008). Plant tissue culture procedure
– Background. In :( George, E.F, Michael, A.H and
De Klerk, G-J.) plant propagation by tissue culture
3rd Edition. Volume 1. The Background
[27] Fernandes-Ferreira, M., et al., (1989). Calli and
suspension cultures for biomass production of
Euphorbia characias L. subsp. characias.
Biotechnol. Lett., 11, 259-264.
[28] Carvalho, E. B. and Curtis, W. R. (1999). The effect
of inoculum size on the growth of cell and root
cultures of Hyoscyamus muticus: Implications for
reactor inoculation. Biotechnol. Bioprocess Eng., 4,
287-293.
International Journal of Pharmacognosy and Phytochemistry, ISSN:2051-7858, Vol.30, Issue.1 1300
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[29] Sudha, G. and Ravishankar, G. A. (2003).
Elicitation of anthocyanin production in callus
cultures of Daucus carota and the involvement of
methyl jasmonate and salicylic acid. .Acta Physiol.
Plant., 25 (3), 249-256.
[30] Ali, M. et al., (2014). Elicitation of antioxidant
secondary metabolites with jasmonates and
gibberellic acid in cell suspension cultures of
Artemesia absinthium L. Plant Cell Tiss. Organ
Cult. DOI 10.1007/s11240-014-0666-2
[31] Fu, C. X., et al., (2006). Methyl jasmonate
stimulates jaceosidin and hispidulin production in
cell cultures of Saussurea medusa. Appl. Biochem.
Biotechnol., 134 (1), 89-96
[32] Kang, S. M., et al., (2006). Effects of methyl
jasmonate and salicylic acid on the production of
biloalide and ginkgolides in cell cultures of Ginkgo
biloba. In Vitro Cell. Dev. Biol.-plant, 42, 44-49
[33] Suzuki, H., et al., (2005). Methyl jasmonate and
yeast elicitor induce differential transcriptional and
metabolic re-programming in cell suspension
cultures of the model legume Medicago truncatula.
Planta, 220, 696–707.
