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Elicitor induced stevioside

production, in vitro shoot growth in
Stevia rebaudiana
( Bertoni ) plant

Preparation by : Arkan Alsafawi - MSc

Supervisor: Assoc. prof. Günce Şahin
Biology Department
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Abstract
An elicitor can be de ned as a chemical or biochemical compound that is introduced in small
concentrations to a living system to promote the biosynthesis of the target bioactive compound.

Effect of Salicylic Acid (SA) on Growth and Quality of Stevia (Stevia rebaudiana Bert.) Under Salt
Stress.

Effect of Methyl Jasmonate (Me JA) and on In Vitro Growth, Oxidative Defense
System in Stevia rebaudiana.

Effect of Sperimidine (spd) under drought stress in Stevia rebaudiana Bertoni under
in vitro condition

Stevia rebaudiana (Bertoni) as a Multifunctional and Sustainable Crop

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 Salicylic acid introduction
Salicylic acid its derivatives as one of the plant hormones produced by the plant naturally belong to the
group of phenolic acids and consist of a ring linked to the group of hydroxyl and carboxyl group, and
the starting ingredient to form the cinnamic acid. It is mainly manufactured within the plant in
cytoplasmic cell. Symbolized by the symbol SA called chemical ortha hydroxyl benzoic acid chemical
formula is C7H6O3.
The most important effects of salicylic acid are to stimulate the production of antioxidants. Antioxidant
against the effect of free radicals from the group Reactive Oxygen species (ROS) when exposed to heat
stress and stress Drought stress and prevents the oxidation of algebraic and oxytin and cytokinein and
also has a role at the genetic level.

Salicylic acid increases the plant’s response to tolerance and resistance

to various diseases affecting plants as it is found that increasing its
internal concentration activates the protective role of pathogenic
pathogens.

Chemical structure of SA salicylic acid.

 Effect of salicylic acid concentrations and sampling times on stevia

Through this study results showed that, elicitation

with different concentrations of salicylic acid had
a signi cant effect (p<0.05) on stevioside
production (Table 2). Elicitation with salicylic acid
at 30 and 90 mg/L had a positive and signi cant
effect on stevioside production. Addition of 30 and
90 mg/L salicylic acid to the media increased
stevioside synthesis 1.12 (35.65 mg/gDW) and 1.13
(35.78 mg/gDW) fold compared to the control, The
highest stevioside production was 35.05 mg/g DW
at 96 h after elicitation,

The stevioside production increased 1.03, 1.14

and 1.04 fold compared to the 24, 48 and 72 h.

Salicylic acid has an important role in the defense

responses against biotic stresses such a
microbes and herbivores, and abiotic stresses
such as wounding and ozone exposure; therefore,
it is used in plant in vitro cultures as an elicitor
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 Effect of Methyl Jasmonate on In Vitro Growth, and Oxidative Defense

System in Stevia rebaudiana

Methyl jasmonate (abbreviated MeJA) is a volatile organic compound used in plant

defense and many diverse developmental pathways such as seed germination, root
growth, owering, fruit ripening, and senescence. Methyl jasmonate is derived from
jasmonic acid and the reaction is catalyzed by S-adenosyl-L-methionine.

The current research showed that application of potential elicitors, MeJA and SA, to
micro-propagated Stevia rebaudiana plants increased total phenolics, DPPH activ-
ity, and stevioside synthesis when compared to without their application in in vitro
conditions. MeJA and SA treatments stained onto the same gels showed a
substantial increase in the activities of SOD and POX, especially in those of SOD2
and POX1 isoforms, and a decrease in those of CAT antioxidant isozymes. The
stevioside yield increased from 4.1 mg/g dry weight to 18.7 and 19.4 mg/g dry
weight in in vitro plantlets exposed to 50 lM MeJA and 100 lM SA, respectively. The
stevioside production was approximately 3.8-fold higher than that in the control
explants.
Chemical structure of Methyl jasmonate
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 Through this study when MeJA and SA
were not added to the growth medium the
shoots took 8 days to appear and only at
the maximum three shoots appeared.
In contrast, when MeJA and SA were added
to the growth medium the shoots appeared
much earlier and the highest shoot number,
i.e., 3.7 shoots per explant, was recorded at
50 uM SA (Table 1).

Generally, the shoot growth in 50 uM SA

was better than that at MeJA or other SA
concentrations (Table 1).

Moreover, the longest leaf length, i.e., 0.9 cm,

was achieved at 50 uM SA application. As
shown in Table 2, there were significant
differences between MeJA and SA
applications for root formation. A high
percentage of root formation (40–93%) took
place with the supply of the other treatments.
Maximum regeneration percentage (93%) was
obtained with the application of 50 uM SA
(Table 2).

‣ Thus the conclusion from this , exogenous application of MeJA and SA can be used for an extensive in vitro
propagation of Stevia rebaudiana in bioreactors so as to achieve enhanced properties of Stevia rebaudiana.
 Sperimidine introduction and effect on
Stevia rebaudiana
Spermidine is a polyamine compound (C7H19N3) found in ribosomes and living tissues and
having various metabolic functions within organisms. It was originally isolated from semen.

Spermidine maintains cell membrane stability, increases antioxidant enzymes activities,

improving photosystem II (PSII), and relevant gene expression. Spermidine significantly
decreases the H2O2 and O2.- contents

Spermidine Chemical Structure

‣ Through this study the results illustrated that salinity have deleterious impact on the
chlorophyll uorescence parameters of stevia plant due to damaging the photosynthetic
system. On the other hand, application of endophytic fungi associated with spermidine has
bene cial effect on improvement of chlorophyll uorescence parameters and reduction the
negative effect of salinity, nally increased the plant resistance to saline conditions.
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‣ Conclusion In general ,in addition to
this study the results of the present
study indicated a negative effect of
salinity on the growth and
physiological characteristics of the
stevia plants. However, inoculation of
endophytic fungi, particularly co-
inoculation of Pi and Trich, improved
the growth and physiological
parameters and ameliorated adverse
effects of salinity in stevia plants.
‣ Moreover, the spermidine (especially
0.75 mM) induced salt stress tolerance in
stevia plants and showed a synergetic
effects with endophytic fungi in terms of
the mentioned parameters.
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Stevia overview
What is Stevia ?

Stevia is a herbaceous perennial plant, Asteraceae family, Native to

Paraguay (South America) borders of Argentina and Brazil meet.

The scientific name for stevia is Stevia rebaudiana Bertoni

which consists of approximately 230 species of herbaceous,
shrub and sub-shrub plants. Leaves of stevia produce diterpene glycosides (stevioside
and rebaudiosides), non-nutritive, non-toxic, high-potency sweeteners and may
substitute sucrose as well as other synthetic sweetners,
It has medicinal values and uses.

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Extracting the Sweetness

The sweetness found in the stevia plant is Did you know?
released by steeping its dried leaves in The sweet extract of
water, ltering and separating the liquid stevia leaf is up to 300
from the leaves and stems, and further times as sweet as
purifying the plant extract with either water sugar, also without the
or food grade alcohol – all conventional calories.
0% Calorie, 100% Sweet, 100% Nature

plant extraction methods.

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 Is Stevia safe ? 12

Stevia is often touted as a safe and healthy sugar substitute that can sweeten up
foods without the negative health effects linked to re ned sugar.
It’s also associated with several impressive health bene ts, such as reduced calorie
intake, blood sugar levels, and risk of cavities.
However, some concerns exist surrounding stevia’s safety — especially for certain
people who may be more sensitive to its effects.

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How to use stevia as a sugar substitute
Stevia may be used in place of table sugar in your favorite foods and
beverages. A pinch of stevia powder is equal to about one teaspoon of
table sugar.
Tasty ways to use stevia include:

- in coffee or tea
- in homemade lemonade
- sprinkled on hot or cold cereal
- in a smoothie
- sprinkled on unsweetened yogurt
Are stevia plants genetically modi ed?
No, stevia plants are not genetically modi ed. Development of new plant
varieties takes place by selective breeding and not genetic modi cation.
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 Chemical Composition of Stevia
For assessing the chemical constituents the useful part of
the plant is taken which in case of Stevia are its leaves. There
are more than 110 species of stevia present in the world but
amongst all of these only 18 species are known to have sweet
leaves.The major components responsible for the sweet taste
of stevia leaves are the glycosides 1

Steviol glycoside
The active compounds in 2
stevia rebaudiana are steviol
glycosides (mainly stevioside
and rebaudioside).
3

Fig : 1 structure of Steviol glycoside , 2 Stevioside , and 3 Rebaudioside

The sweet property is remarkably bene cial to diabetic patients and people suffering from obesity.
Steviol glycosides are proven to be thermally stable, which makes them more suitable for use in
cooked food and drinks. Due to sweet and therapeutic properties of leaf, S. rebaudiana has
attracted economic and research interests. Toxicological studies based on stevioside have shown
that it does not possess tumor inducing or mutagenic effects. S. rebaudiana is also known to
possess antioxidant activity. Antioxidant delays the oxidation of lipid molecules by inhibiting the
initiation or propagation of scavenging oxidative chain reactions. Therefore, attempts are being
made to enhance its leaf biomass production through cultural techniques. Above and beyond the
realization of various alternative strategies (plant cell and tissue culture), improved extraction
procedures and product improvement (taste and avor improvement) are equally important. The
purpose of this review is to corroborate different scienti c research conducted on Stevia plant to
emphasize its remarkable potential as bene cial agricultural crop.
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 Absorption , Distribution and Metabolism 16

of Stevioside
In the rat, stevioside is converted to steviol by suspensions of rat
intestinal micro ora and the conversion gets completed within two days.
Stevioside appears to be poorly transported across the cell membrane.
No uptake was observed in suspensions of human red blood cells.
Previous work has also shown that little or no stevioside is absorbed in
the blood of humans instead, all kinds of steviol glycosides get
metabolized to steviol and steviol gets absorbed which is also true for
Rebaudioside A and Stevioside. This approach has also been validated
by JECFA thus, concluding the safe use of stevioside
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 Effects on blood pressure and renal function 17

Different research reports based on effects of stevioside have claimed varying

effects on kidney function and blood pressure regulation. It lowers mean arterial
blood pressure alongwith decreasing renal vascular resistance, produces diuresis
and increases fractional excretion of Na+ and K+ . The lack of effect on
glomerular ltration rate implies that stevioside vasodilates both afferent and
efferent arterioles.

Medicinal potential
Stevia extracts have medicinal potential as antihyperglycemic, insulinotropic,
glucagonostic, hypotensive, anti-cancer, antiviral, antimicrobial, antioxidant, anti-
in ammatory, immunostimulatory and chemopreventive agents, as well as for use
as a digestive tonic and for dental and skin care.
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 Antimicrobial activity 18
Stevia has been shown to inhibit the growth and reproduction of bacteria that cause
gum disease and tooth decay, making it an excellent addition to toothpaste and
mouthwash for dental hygiene. Studies indicate that the major cariogenic organism,
Streptococcus mutans, experiences growth suppression and secretes less acid when
grown on media containing stevioside than when grown on sucrose, glucose or fructose
media.
Anti-carcinogenic agent
Stevia leaf extracts and the presense of polyphenolic constituents have shown
inhibitory effect on tumor initiation and promotion. Stevioside, isosteviol, steviol,
leaf aglycones and other metabolites are known to inhibit the tumor formation in
several ways: by blocking Epstein-Barr virus early antigen, induction and also by
reducing production of tumor in two stage mouse skin carcinogenesis model
following exposure to 7,12- dimethylbenz[a]anthracene and 12-O-
tetradecanoylphorbol- 13-acetate.
Stevia under salt stress 19
Through studies on the physiological
and biochemical properties of stevia under
some levels of salinity stress like (0, 50, 100,
150 mM NaCl). Salt stress reduced
chlorophyll a (chlorophyll a), chlorophyll b
(chlorophyll b), total chlorophyll, carotenoid,
and total protein content. The increase in
malondialdehyde (MDA) content was not
signi cant at all NaCl levels, while the
activities of CAT and POX as well as
stevioside and rebaudioside A were
increased under salinity stress. On one side,
chitosan treatments can offset reduced
physiological traits such as photosynthetic
pigments and protein content. On the other
hand, chitosan caused multiple increases in
malondialdi-hyde content, activity of
antioxidant enzymes (catalase and
peroxidase), and steviol glycosides
(stevioside and ribo-dioside A) under salt
stress. We report, for the rst time, about the
potential of chitosan to enhance the salt
tolerance capabilities of stevia by increasing
salt-adapting factors and decreasing harmful
damage from NaCl stress. This Fig. Schematic representation of the physiological, biochemical
and molecular responses to NaCl (1.5 g L−1) of in vitro cultured
Stevia rebaudiana.
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Conclusion 20

Through these studies it can be concluded that sugar is not good for health and excessive and prolonged
consumption of sugar may lead to various health complications like diabetes, liver related issues etc. Many
researches are done to nd an alternative to the white sugar and all the modern science has come up with is the
chemical arti cial sweeteners like aspartame etc which themselves pose threat to health of people when used in
excess. Therefore to nd a healthier and more natural substitute for sugar Stevia rebaudiana is studied. This plant are
leaves have distinct sweet taste. The studies have found that leaves of stevia are 300 times sweeter that the sugar
which is commonly used. Apart from being a natural sweetener Stevia also has many health bene ts like regulating
the blood sugar level and reducing hypertension and in general also it is good for overall health of the body. The
chemical constituents of stevia are discussed in the paper and the most important compounds responsible for the
sweet taste of stevia are stevioside and rebaudioside. Although substantial research is present to prove the existence
of these compounds but still no evident researches have been done on assessing the potential toxicity of the stevia
plant.

Many researches are still going on to assess toxicity of the plant but substantial results were not obtained. There is
still a lot of scope of more research in this area to provide substantial evidence that stevia is safe for consumption and
if there is any toxicity it can be worked upon to be minimized. Hence it can be concluded that though stevia is a very
useful plant but still there is need for more extensive research in this area.

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 The Aim 21

Soil salinity is one of the ecological problems limiting plant growth and
development, especially in semi-arid and arid regions where water availability
is very limited. Stevia rebaudiana Bertoni (Asteraceae) is known for its
sweetness and non-caloric products (steviol glycosides). It can be considered
as a plant for the future sugar industry and human health nutrition. The
present work aims to study the modulation of salt stress effects on Stevia
rebaudiana Bertoni plants by an exogenous application of salicylic acid, Spd
and their combinations.

‣ The aim of this research was to evaluate the effect of controlled elicitation
using salicylic acid (SA), spermidine (Spd), Methyl Jasmonate ( Me Ja) on the
plant performance, SGs contents and the potential role of elicitors in
strengthening tolerance mechanisms in Stevia rebaudiana Bertoni under salt
stress.
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