BioRisk | a 2 | 3-225 (2022) fete ag ar ei aia rts
doi: 10.3897/biorisk.17.77452 RESEARCH ARTICLE & B lO R IS kK

https://biorisk.pensoft.net

Light and auxin treatments affect morphogenesis and
polyphenolics productivity in Artemisia alba
Turra cell aggregates in vitro

Dobrina Pecheva!, Kalina Danova?

| Faculty of Biology, Sofia University St. Kliment Ohridski, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria
2 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. Georgi
Bonchev Str. 61.9, 1113 Sofia, Bulgaria

Corresponding author: Kalina Danova (k_danova@abv.bg)

Academic editor: Stephka Chankova | Received 1 November 2021 | Accepted 15 December 2021 | Published 21 April 2022

Citation: Pecheva D, Danova K (2022) Light and auxin treatments affect morphogenesis and polyphenolics
productivity in Artemisia alba Turra cell aggregates in vitro. In: Chankova S, Peneva V, Metcheva R, Beltcheva M,
Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 213-225. https://doi.org/10.3897/
biorisk.17.77452

Abstract

Artemisia alba Turra is an essential oil-bearing shrub, with a Euro-Mediterranean distribution widespread
in the south-eastern parts of Europe. Phytochemical investigations have evidenced the presence of volatile
mono- and sesquiterpene derivatives, as well as non-volatile sesquiterpenoids, flavonoids and phenolic
acids contributing to the anti-inflammatory, antimicrobial, antioxidant and pro-apoptotic activity of dif-
ferent preparations, obtained from the plant. The current research aims at elucidation of the potential
for biotechnological polyphenolic compounds productivity of non-differentiated cell lines of the plant.
For this purpose, non-differentiated cell aggregates were initiated from either leaf or root explants of the
sterile grown plant. They were cultivated either in the dark or at 16/8 h photoperiod in liquid media,
supplemented with \’-benzyladenine (BA) as auxin. The cytokinin effects of indole-3-butyric acid (IBA)
and 1-naphthalene acetic acid (NAA) were compared. It was established that NAA supplementation was
superior to IBA and light treatment — to dark growth conditions in terms of polyphenolics productivity. In
addition, NAA supplementation led to better expressed compaction and larger size of the cell aggregates
as compared with IBA. The results of the present experiment indicate that secondary metabolites produc-
tivity in vitro is a dynamic process closely related to the plant’s growth and development and is in close

relation to the interactions of the plant with its environmental conditions.

Keywords

Artemisia alba Turra cell suspensions, auxins, cytokinins, polyphenolics productivity in vitro

Copyright Dobrina Pecheva & Kalina Danova. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

214 Dobrina Pecheva & Kalina Danova / BioRisk 17: 213-225 (2022)

Introduction

The production, accumulation and translocation of secondary metabolites within the
integral plant organism are determined by the presence and organisation of highly spe-
cialised anatomical structures. Thus, growth, development and morphogenesis of the
plant individual play crucial roles for the production and accumulation of secondary
metabolites. Since growth and development are dynamically related to the interactions
of the plants with their surrounding environment, the latter plays a crucial role in de-
termining the plants secondary metabolite profile. Plant cell tissue and organ culture
represents the convenience of a controlled environment with the flexibility to alter se-
lected factors related to in vitro cultivation and, thus, modify the production of desired
secondary metabolites in vitro (Danova 2018).

Genus Artemisia, tribe Anthemideae, subtribe Artemisiinae is one of the largest in
the Asteraceae family with more than 500 species and subspecies (Zhen et al. 2010).
Representatives of the genus have been included in traditional medicinal preparations
from centuries for treatment of conditions such as fever, high blood pressure, diabetes,
gastrointestinal disorders, parasites etc. (Tu 2016; Zeb et al. 2018; Danova 2020). Scien-
tific evidence for the pharmacological potential of Artemisia species is the presence of trit-
erpenes, steroids, hydrocarbons, polyacetylenes, flavonoids, coumarins, mono- and ses-
quiterpenoids isolated from representatives of the genus (Maggio et al. 2012). Research
has confirmed the cytotoxic, antihepatotoxic, anti-bacterial, antifungal and antioxidant
properties of preparations of the species (Tan et al. 1998; Bora and Sharma 2011).

A, alba Turra (syn. A. lobelii All, A. camphorata Vill., Artemisia biasolettiana Vis,
A, suavis Jord., A. incanescens Jord.) is an essential oil-bearing shrub natively distrib-
uted in south-eastern Europe (Radulovi¢ and Blagojevi¢ 2010; Biondi and Galdenzi
2012). Its decoction has been traditionally used in the Mediterranean Region as a
tonic and stomach digestive (Rigat et al. 2007). Phytochemical studies on the volatile
(camphor, 1,8—cineole and artemisia ketone dominating components of the essential
oil) and non-volatile (kaempferol, luteolin and apigenin derivatives, rutin, oxygenated
sesquiterpenoids, chlorogenic acid, dicaffeoylquinic acids, scopoletin, umbeliferone
etc.) constituents of the species have been performed, giving the scientific grounds for
the established anti-microbial, anti-inflammatory, antioxidant properties of the species
(Stojanovic et al. 2000; Staliriska et al. 2005; Danova et al. 2020).

In previous research conducted on differentiated shoot cultures of the species, it
was established that the development of the root system as a result of exogenous plant
growth regulators treatment was decisive for the profile of the essential oils, derived
from the aerial parts of A. a/ba Turra. Thus, plantlets with well-developed root system
(in plant growth regulators-free media, as well as in media supplemented with 0.5 mg/l
or 1.0 mg/I indole-3-butyric acid (IBA)) were characterised with over 2.5 times high-
er ratio of the mono/sesquiterpenoids content in the essential oil, as compared with
plantlets with supressed rooting and callus formation at the explant base (in media
where 0.5 mg/l or 1.0 mg/l IBA were combined with 0.2 mg/l benzyl adenine (BA))
(Danova et al. 2018).

Polyphenolics productivity in Artemisia alba Turra cell aggregates 215

Continuing the previous research on Artemisia alba’s biotechnological properties,
we set as an aim to develop a fast-growing non-differentiated in vitro system of Artemi-
sia alba Vurra with high biosynthetic potential regarding the production of polyphe-
nolics. A comparative analysis of the morphogenic and biosynthetic response of liquid
cell-aggregate cultures derived from leaf and root explants with different photoperiod
and different type of auxin (IBA or 1-Naphthylacetic acid, NAA), in the presence of
the same cytokinin (BA) was conducted.

Materials and methods

Plant material

Shoot cultures were initiated through surface sterilisation of stem explants of field
grown A. alba as previously described (Danova et al. 2012). Stock shoot cultures were
kept on plant growth regulators (PGR) — free medium supplemented with the Mu-
rashige and Skoog (Murashige and Skoog 1962) macro- and micro-salts medium.
Stock cultures were kept at a temperature of 25 + 0.1 °C, 16/8 h photoperiod, light
intensity 60 pmol m’s", period of sub-cultivation 4 months.

Tissue culture experiment

For the needs of this research, leaf (Al) and root (A2) explants of the in vitro grown
stock plants were used. Five leaf (A1) and root (A2) explants were inoculated into two
types of liquid media with total volume 50 ml in Erlenmeyer flasks (Table 1). Media
were prepared with macro- and micro-elements and vitamins according to Murashige
and Skoog (1962), 30 g.I' sucrose and the cytokinin benzyladenine. We experimented
with two types of auxin — indole-3-butyric acid and naphthylacetic acid as shown in
Table 1. For each treatment, five separate culture vessels were placed in two types of
light conditions — 16/8 photoperiod and in the dark. All flasks were placed on an or-
bital shaker, 100 rpm. That led to the formation of the eight experimental lines (Table
1). The biological experiment of non-differentiated cell aggregate culture induction
from explants of the sterile grown plan was repeated in triplicate.

Table |. The eight A. a/ba experimental variants.

BA [mg.I"] IBA [mg.I"] NAA [mg.I"] Photoperiod
1 Al ER_3 hv 0.1 1.5 - 16/8
2 A2 ER_3 hv 0.1 i) - 16/8
3 Al ER_3NAA hy 0.1 s 1.5 16/8
4 A2 ER_3NAA hv 0.1 2 1.5 16/8
5 Al ER_3 0.1 1.5 - Dark
6 A2 ER_3 0.1 1.5 - Dark
7 Al ER_3NAA 0.1 - 1.5 Dark
8 A2 ER_3NAA 0.1 - 1.5 Dark

216 Dobrina Pecheva & Kalina Danova / BioRisk 17: 213—225 (2022)

Morphometric observations

The dynamics of the morphometric response of explants was tracked by stereomicros-
copy using the Stereomicroscope Leica M60. Stereomicroscopy was performed 2 and 4
months after the initial induction of the cell lines. For the observations, plant material
from at least two separate culture vessels was sampled.

Biochemical analyses

The quantitative content of malondialdehyde (Dhindsa et al. 1981) and H,O, (Jessup
et al. 1994) were assayed spectrophotometrically.

Phytochemical analyses

Spectrophotometric determination of the total content of phenolic (Singleton et al.
1999) and flavonoid compounds (Zhishen et al. 1999) was performed. For the analy-
ses, plant material from at least four separate culture vessels was sampled. Measure-
ments were performed in triplicate.

Statistical processing

The respective number of samples tested, measurements and biological repetitions
have been mentioned at each method. The SEM values (standard error of the mean)
are reflected in Figures. The means have been compared by t-test of unequal vari-
ances at P < 0.05. Unless otherwise stated, differences are considered statistically
significant at P < 0.05.

Results and discussion

Impact of growth regulators and photoperiod on growth and development

Changes in the fresh/dry weight ratio, two and four months after induction of the
suspension lines are shown in Figure 1. Biomass accumulation was influenced both by
the type of initial explant (leaf or root) and the photoperiod. When comparing this
parameter 2 and 4 months after initiation, the different capacity of dry biomass ac-
cumulation (expressed by a lower FW/DW ratio) in the different treatments is more
noticeable. Two months after the initial induction, the FW/DW ratio of the eight
lines has relatively similar values, the variants grown in the light tending to be more
hydrated (expressed by a higher FW/DW ratio), compared to those grown in the dark
(except A2 ER_3NAA).

In addition, a higher water accumulation tendency was observed in lines obtained
from root (A2) vs. the ones obtained from leaf explant (A1). Two months after the in-

Polyphenolics productivity in Artemisia alba Turra cell aggregates 247)

duction, no cell lines, being strongly productive in terms of dry biomass accumulation,
could be observed, the FW/DW parameter being relatively similar in-between them.

However, after two consecutive passages of the suspension cultures (4 months after
induction), cell lines grown in the light showed a significant drop in the FW/DW ra-
tio, indicating the tendency of higher dry biomass accumulation.

Some of the cell lines grown in the dark (Al ER_3, Al ER_3NAA and A2
ER_3NAA) show a higher result compared to the ones grown in the light, indicating
higher hydration, as the difference in leaf explants treated with IBA (Al ER_3) is the
most significant. This may be due to both reduced abilities to accumulate dry biomass
and differences in the density of cell aggregates in cultures and the change in osmotic
pressure in cells. The largest differences in the FW/DW ratio are observed in leaf ex-
plants grown in the dark Al ER_3 and Al ER_3NAA, which could be explained by
the impaired photosynthetic ability due to lack of light (Fig. 1).

Stereomicroscopy of the eight plant lines 2 months after induction.

When performing the stereomicroscopy 2 months after induction, the presence of
differentiated plant organs and tissues was still observed (Fig. 2). It was more pro-
nounced in root (A2) vs. stem (A1) explants and in NAA treatments as compared with
IBA ones. In the light-grown lines, intense green pigmentation was clearly observed,
as compared with the ones grown in the dark, including both light-grown root lines,
which is a visual indication of the formation of photosynthetic pigments. In the case
of leaf explants with both auxins, callus formation was observed and, in the variant
treated with IBA in light, it was less dense, lighter in colour and presence of antho-

A.alba FW/DW - 2 and 4 months after induction

[FW/DW ratio]

2 months after induction &4 months after induction

Figure |. Changes in the FW/DW ratio 2 and 4 months after initial induction of the cell lines, respec-

tively. Same letters denote statistically non-significant differences.

218 Dobrina Pecheva & Kalina Danova / BioRisk 17: 213—225 (2022)

~—

A1ER 3 hy A2 ER 3 hy Al ER_3NAA hy

ALER 3 A2 ER 3 AL ER 3NAA A2 ER_3NAA

Figure 2. Stereomicroscopic imaging of the eight plant lines 2 months after initial line induction.

cyanin pigments was observed, in contrast to the variant treated with NAA, in which
we observed intense green colouration, denser cell aggregates and relatively stronger
organogenesis. The tendency for stronger organogenesis under the influence of NAA
was maintained in the dark, as the leaf explant clearly showed many etiolated (due to

lack of light) stems.

Stereomicroscopy of the eight cell lines 4 months after induction

When performing the same analysis 4 months after the initial induction (after two
passages of the induced structures described earlier), the degree of differentiation de-
creased significantly, as indirect organogenesis was again observed more clearly in the
lines obtained from root explants (A2) (Fig. 3). There were no significant differences in
terms of indirect organogenesis when comparing the NAA and IBA treatments.

ae oe a

sd em

—
A1 ER 3 hv A2 ER_3 hy Al ER_3NAA hy A2 ER_3NAA hy

ak,

|
A1ER 3 A2 ER 3 A1 ER 3NAA A2 ER 3NAA

Figure 3. Stereomicroscopic imaging of the eight plant lines 4 months after initial line induction.

Polyphenolics productivity in Artemisia alba Turra cell aggregates 219

IBA-treated lines formed larger cell aggregates, which, however, were less dense, more
brittle and looser. The cell aggregates obtained by NAA treatment were smaller in size,
but significantly denser and more compact. In the case of aggregates, obtained from leaf
explants in the light, we can assume the presence of photosynthesis to some extent, due to
the preservation of the intense green colour. After stabilisation, the A2 lines, both the light
and dark, formed aggregates with white to brownish colour, without green colouration.

Photographic characterszation of the eight cell lines 6 months after the initial
induction

Six months after the initial induction and after 3 passages, all cultures were already
completely de-differentiated (Fig. 4). The formation of cell aggregates was also ob-
served in the eight cell lines and they were relatively larger in size in the lines induced
by leaf (Al), as compared with the ones obtained from the root (A2) explant. In the
lines obtained from leaf explant grown in the dark (Al ER_3, Al ER_3NAA), there
was a gradual loss of colour in the de-differentiated cultures. A weak green staining
was only preserved in the lines obtained from light-grown leaf explants (Al ER_3 hy,
Al ER_3NAA hy), being weaker in the NAA treatment. Sporadic morphogenesis was
only observed in the A2 lines, in support of the assumption that they were more dif-
ficult to bring to a fully de-differentiated culture.

MDA content in the eight experimental lines

Changes in malondialdehyde content in the eight plant lines are shown in Figure 5. In
both light and dark, MDA is relatively high in leaf explants compared to root and the
trend is more visible in variants grown in the presence of light.

Again, this may be due to the more active photosynthesis under illumination and
the presence of a larger relative amount of photosynthetic tissue in the original stem

Al ER 3 hy A2 ER 3 hy | Al ER_3NAA hy A2 ER 3NAA hy

id
a 4

Al ER_3 AZ ER 3 Al ER_3NAA A2 ER_3NAA

Figure 4. Photographic characterisation of the eight plant lines 4 months after initial line induction.

220 Dobrina Pecheva & Kalina Danova / BioRisk 17: 213—225 (2022)

og

Gs

:

Figure 5. Changes in malondialdehyde (umol.g’ FW) content in lines, induced from leaf and root of A.
alba under the influence of different growth regulators and at different photoperiods. p < 0.05.Statistically

insignificant differences in the average values of the measured parameters are indicated in identical letters.

explant. The drop of MDA levels in the absence of light may be due to inhibition of
photosynthetic processes in Al lines, respectively generation of less active forms of
oxygen and lower lipid peroxidation. We can note that the trend observed in MDA
was also maintained in the amount of HO, — light-grown variants, expressing signifi-
cantly higher values than in the dark-grown ones, which can again be explained by the
decrease in photosynthetic activity.

Hydrogen peroxide content in the eight experimental lines

Changes in hydrogen peroxide content in the eight plant lines are shown in Figure 6.

There was a slight decrease in the amount of hydrogen peroxide in the root ex-
plants treated with NAA compared to those treated with IBA.

The relations with the type of explant and the auxin applied was also clear — when
using NAA, A2 lines always had lower levels of HO; ; respectively lower levels of oxida-
tive stress, while, when using IBA, there was an inverse dependency — always A2 lines
had higher levels of H,O,, regardless of the light treatment. When comparing the two
parameters, we can note, as a tendency, that both MDA and H,O, were higher in light-
grown lines, probably due to the generation of ROS from the ongoing photosynthetic
processes. However, in light-derived A2 lines explants, the lower MDA as compared to
AJ lines did not correlate with higher H,O, levels, indicating that, in the presence of
light, the root tissue generated high Os levels, which led to activation of the antioxi-
dant protection of the cells and a correspondingly lower degree of lipid oxidation. In the

case of lines obtained from leaf explants grown in the light, both parameters were high

Polyphenolics productivity in Artemisia alba Turra cell aggregates 221

Figure 6. Changes in hydrogen peroxide (nmol.g! FW) content in lines, induced from leaf and root of A.
alba under the influence of different growth regulators and at different photoperiods. p < 0.05. Statistically

insignificant differences in the average values of the measured parameters are indicated in identical letters

and, in the case of lines obtained from leaf explants grown in the dark, they dropped.
Probably the Al lines, grown in the light, retain their photosynthetic ability to some
extent and, accordingly, this leads to the generation of both MDA and H,O,, while, in
the dark, this ability is limited and we see that, both morphologically, they completely
lose their green pigmentation, together with the significant drop of MDA and H,O,,
indicating lower levels of stress, less ROS and a lower degree of lipid peroxidation.

Content of phenols and flavonoids in the eight experimental lines

Phenolic and flavonoid compounds content in the eight plant lines is presented in
Figure 7 and Figure 8, respectively.

Phenolic compounds are part of the plant’s cellular response to various types of
stress. Accordingly, we can assume that their content would increase with a source of
stress, such as light.

The experiments show a dependence with a similar trend — the variants grown in
the 16/8 photoperiod produced higher amounts of phenolic compounds than those
grown in the dark (Fig. 7). There was also a tendency for the amount of phenolic
compounds to be significantly higher in the root explants obtained lines, as compared
to the leaf ones. Their amount was significantly higher (58% and 140%) in the root-
derived lines treated with NAA, as compared to the root-derived explants treated with
IBA. The trend that we observed in the levels of total phenolic compounds was ana-
logical as far as flavonoids were concerned (Fig. 8). Again, we observed higher flavo-
noid levels in the light-grown lines, as compared to the dark-grown ones, which can be

222 Dobrina Pecheva & Kalina Danova / BioRisk 17: 213—225 (2022)

Total phenolics

[mg.g FW"]

Figure 7. Phenolics content (mg. g' FW) in lines induced from leaf and root explants of A. a/ba under
the influence of different growth regulators and at different photoperiods.

Flavonoids

[mg.g FW"!]

Figure 8. Flavonoid content (mg. g' FW) in lines induced from leaf and root explants of A. a/ba under
the influence of different growth regulators and at different photoperiods.

explained by the higher photosynthetic activity. Thus, the observations on the degree
of de-differentiation and its correlation with the levels of MDA, H,O, and phenolic
compounds showed that, in the lines initiated by root explant (A2), de-differentiation
was much slower, a higher degree of tissue hyperhydricity in the early stages of devel-
opment was observed and scarce root morphogenesis still occurred even after several
consecutive passages. Nevertheless, root-derived cell aggregates of A. alba (with the

Polyphenolics productivity in Artemisia alba Turra cell aggregates 225

exception of A2_ER3) were shown to possess the highest biosynthetic capacity of phe-
nolic and flavonoid compounds. Flavonoids have a wide range of applications in phar-
macy, well known pharmacological properties, for example, activation or inhibition
of specific enzymes, incl. cyclo-oxygenase, lipoxygenase, detoxification of carcinogens
and other proven health benefits for humans and animals (Pollastro et al. 2018). They
are thought to be involved in controlling the growth and differentiation of plant cells
and tissues. Accordingly, higher levels of phenolic and flavonoid compounds of those
lines may be related to their impaired ability to de-differentiate. Phenolic compounds
have a high absorption capacity; however, evidence for their direct involvement in
photosynthetic processes is not yet known (Agati et al. 2012).

Root explants treated with NAA and grown in the light (A2_ER3 hv) showed the
best values of all four studied parameters (lipid peroxidation, oxidative stress, phenolic
and flavonoid productivity). They were characterised by a strong hydration after the initial
induction of the culture, which, however, decreased significantly after the second passage.
Accordingly, this line could be distinguished as relatively more productive in this respect.

Artemisia alba Vurra in vitro was shown to respond to the introduction in a de-dif-
ferentiated state by activating both biochemical and physiological mechanisms, which
were characterised by great dynamics.

The aim of this experiment was to establish the most competent cell lines in terms
of biosynthesis of phenolics and flavonoids, experimenting with the presence or absence
of light, the type of plant tissue used for induction of cell line (stem and root explants)
and with the type of auxin added — IBA and NAA. Regarding the formation of cell ag-
gregates and the differentiation of cultures, clear differences were observed according to
the type of auxin used. Lines treated with IBA were easier to de-differentiate, forming
bulky cell aggregates, which were, however, less dense. NAA-treated lines show more
difficult de-differentiation, especially in those derived from root explants (A2), but af-
ter de-differentiation, they formed cell aggregates relatively smaller in size, denser and
with a lower fresh/dry weight ratio, respectively, with better biomass accumulation.

In terms of stress markers, again light-grown cell lines showed elevated values com-
pared to those grown in the dark, with the levels of MDA being significantly higher in
leaf explants (A1), showing high levels of lipid peroxidation.

In terms of the amount of phenolic and flavonoid compounds, the root lines had a sig-
nificantly higher synthetic capacity compared to those obtained from leaf explants. There
was also a clear dependence on the type of auxin used and, again, NAA shows better results.

Conclusion

When we take into consideration everything said above, we can conclude that, de-
spite the slower and more difficult de-differentiation, in terms of the synthesis of
phenolic and flavonoid compounds, the most favourable parameters have been es-
tablished for the lines obtained from root explants, when NAA was applied as auxin.
A2 ER_3NAA_hv and A2 ER_3NAA stand out as the most productive lines for the

224 Dobrina Pecheva & Kalina Danova / BioRisk 17: 213-225 (2022)

production of the target compounds. This may provide future guidance for the devel-
opment of high-yielding root lines for the synthesis of secondary metabolites from the
studied plant Artemisia alba Turra.

Acknowledgements

The authors acknowledge the financial support of grant KI1-06-H39/6, National Sci-
entific Fund, Bulgaria.

References

Agati G, Azzarello E, Pollastri S, Tattini M (2012) Flavonoids as antioxidants in plants: Lo-
cation and functional significance. Plant Science 196: 67-76. https://doi.org/10.1016/j.
plantsci.2012.07.014

Biondi E, Galdenzi D (2012) Phytosociological analysis of the grasslands of Montagna dei Fiori
(central Italy) and syntaxonomic review of the class Festuco-Brometea in the Apennines.
Plant Sociology 49: 91-112. https://doi.org/10.7338/pls201249 1/05

Bora KS, Sharma A (2011) The Genus Artemisia: A Comprehensive Review. Pharmaceutical
Biology 49(1): 101-109. https://doi.org/10.3109/13880209.2010.497815

Danova K (2018) Roles of developmental patterns and morphogenesis in the secondary me-
tabolite production of conventionally and biotechnologically cultivated medicinal and
aromatic plants. Recent Advances in Plant Research, Series: Plant Science Research and
Practices, NOVA Science Publishers, New York, 1—55.

Danova K (2020) Artemisia alba Turra as a flexible model to study interrelations between de-
velopmental patterns and secondary metabolites productivity in plant cell tissue and organ
culture conditions. In: Roe J (Ed.) Artemisia: Classification, Cultivation and Uses, Chapter
3, Nova Science Publishers, New York, 81-118.

Danova K, Todorova M, Trendafilova A, Evstatieva L (2012) Cytokinin and auxin effect on
the terpenoid profile of the essential oil and morphological characteristics of shoot cul-
tures of Artemisia alba. Natural Product Communications 7(8): 1075-1076. https://doi.
org/10.1177/1934578X1200700827

Danova K, Motyka V, Todorova M, Trendafilova A, Krumova S, Dobrev P, Andreeva T, Oreshk-
ova T, Taneva S, Evstatieva L (2018) Effect of cytokinin and auxin treatments on mor-
phogenesis, terpenoid biosynthesis, photosystem structural organization, and endogenous
isoprenoid cytokinin profile in Artemisia alba Turra in vitro. Journal of Plant Growth Regu-
lation 37(2): 403-418. https://doi.org/10.1007/s00344-017-9738-y

Danova K, Trendafilova A, Motyka V, Dobrev P, Ivanova V, Todorova M (2020) Therapeutic
potential and biotechnological utilization of the indigenous biosynthetic capacity of Arte-
misia alba Turra: A Review. Ecologia Balkanica (Special Issue): 257-273.

Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence: Correlated with increased

levels of membrane permeability and lipid peroxidation, and decreased levels of superox-

Polyphenolics productivity in Artemisia alba Turra cell aggregates 225

ide dismutase and catalase. Journal of Experimental Botany 32(1): 93-101. https://doi.
org/10.1093/jxb/32.1.93

Jessup W, Dean RT, Gebicki JM (1984) Iodometric determination of hydroperoxides in lipids
and proteins. Methods in Enzymology 233: 289-303. https://doi.org/10.1016/S0076-
6879(94)33032-8

Maggio A, Rosselli S, Bruno M, Spadaro V, Raimondo FM, Senatore F (2012) Chemical compo-
sition of essential oil from Italian populations of Artemisia alba Turra Asteraceae. Molecules
(Basel, Switzerland) 17(9): 10232-10241. https://doi.org/10.3390/molecules1709 10232

Murashige T, Skoog FA (1962) A revised medium for rapid growth and _ bioassays
with tobacco tissue culture. Physiologia Plantarum 15(3): 473-497. https://doi.
org/10.1111/j.1399-3054.1962.tb08052.x

Pollastro F Minassi A, Fresu LG (2018) Cannabis phenolics and their bioactivities. Current Medici-
nal Chemistry 25(10): 1160-1185. https://doi.org/10.2174/0929867324666170810164636

Rigat M, Bonet MA, Garcia S, Garnatje T, Vallés J (2007) Studies on pharmaceutical eth-
nobotany in the high river Ter valley (Pyrenees, Catalonia, Iberian Peninsula). Journal of
Ethnopharmacology 113(2): 267-277. https://doi.org/10.1016/j.jep.2007.06.004

Radulovi¢ N, Blagojevié P (2010) Volatile profiles of Artemisia alba from contrasting serpentine
and calcareous habitats. Natural Product Communications 5(7): 1117-1122. https://doi.
org/10.1177/1934578X1000500729

Singleton VL, Orthofer R, Lamuela-Raventés RM (1999) Analysis of total phenols and other
oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in
Enzymology 299: 152-178. https://doi.org/10.1016/S0076-6879(99)99017-1

Stalinska K, Guzdek A, Rokicki M, Koj A (2005) Transcription factors as targets of the anti-
inflammatory treatment. A cell culture study with extracts from some Mediterranean diet
plants. Journal of Physiology and Pharmacology 56: 157-169.

Stojanovic G, Palic R, Mitrovic J, Djokovic D (2000) Chemical composition and antimicrobial
activity of the essential oil of Artemisia lobelii All. The Journal of Essential Oil Research
12(5): 621-624. https://doi.org/10.1080/10412905.2000.9712172

Tan RX, Zheng WE, Tang HQ (1998) Biologically active substances from the genus Artemisia.
Planta Medica 64(04): 295-302. https://doi.org/10.1055/s-2006-957438

Tu Y (2016) Artemisinin-a gift from traditional Chinese medicine to the world (Nobel lec-
ture). Angewandte Chemie International Edition 55(35): 10210-10226. https://doi.
org/10.1002/anie.201601967

Zeb S, Ali A, Zaman W, Zeb S, Ali S, Ullah E Shakoor A (2018) Pharmacology, taxonomy and
phytochemistry of the genus Artemisia specifically from Pakistan: A comprehensive review.
Pharmaceutical and Biomedical Research 4: 1-12. https://doi.org/10.18502/pbr.v4i4.543

Zhen L, Chen S, Chen F, Fang W, Li J, Wang H (2010) Karyotype and meiotic analysis of five
species in the genus Artemisia. Caryologia 63(4): 382-390. https://doi.org/10.1080/0008
7114.2010.10589750

Zhishen J, Mengcheng T, Jianming W (1999) The determination of flavonoid contents in mul-
berry and their scavenging effects on superoxide radicals. Food Chemistry 64(4): 555-559.
https://doi.org/10.1016/S0308-8 146(98)00102-2