Industrial Crops & Products 140 (2019) 111646

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Industrial Crops & Products

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Micronutrients zinc and boron enhance stevioside content in Stevia T

rebaudiana plants while maintaining genetic fidelity
⁎
Pallavi Singh1, Padmanabh Dwivedi
Laboratory of Plant Tissue Culture and Stress Physiology, Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221 005,
India

A R T I C LE I N FO A B S T R A C T

Keywords: Micronutrients such as zinc (Zn) and boron (B) greatly affect plant growth and development. A pot experiment
Boron was conducted in garden soil in the poly house of the Horticulture garden, Banaras Hindu University, Varanasi,
Foliar application India to study the effect of zinc and boron application on stevioside content of Stevia plant. The data from two
Genetic fidelity years (same season) showed that the foliar spraying of zinc sulphate (0.25, 0.50 and 0.75% w/v) and boric acid
ISSR markers
(0.25, 0.50 and 0.75% w/v) alone at all rates significantly increased stevioside content as compared to the
Stevia
Stevioside
control treatment. The most promising results were obtained from plants treated with 0.75% ZnSO4, as there was
Zinc 30 times increase in the peak area noted in HPLC chromatogram. The in vitro raised plants were genetically
stable as demonstrated using ISSR markers.

1. Introduction
Micronutrients in appropriate concentration are essential for plant’s
growth and development; these not only play significant role in most of
the physiological processes like photosynthesis, respiration, various
enzymatic reactions, etc but also form part of cell structure. Their de-
ficiency or inappropriate presence or absence disturbs the metabolic
activities occurring in plants at different stages of development which
finally creates imbalance within, and show symptoms like chlorosis,
necrosis, stunted growth, mottled leaves and many more affected
morphological parameters. As these influence different metabolic
pathways, so affect final product, intermediate products, primary and
secondary metabolites formation. Zinc and Boron are critical micro-
nutrients for healthy plant. Zinc is vital constituent of many enzymes
such as alcoholic dehydrogenase, superoxide dismutase, carbonic an-
hydrase. Besides, synthesis of IAA is also effected by the deficiency of
Zn that is reported to be necessary for optimum tryptophan (precursor
of IAA) levels (Salisbury and Ross, 1992). Zinc greatly affects protein
synthesis and metabolism as again it is an essential part of ribosome and
RNA polymerase. The prime function of boron is linked to the formation
of cell wall and cell membranes, pollination, pollen germination, cell
division, translocation of carbohydrates and metabolism of calcium,
IAA, RNA, etc. (Marschner, 1995). Boron is many times concomitant
with nitrogen, phosphorous, potassium and calcium in plants and hence
required for high yield of crops. Its participation in plant hormone
metabolism is crucial and continuous supply is necessary for plants.
Stevia rebaudiana, a well-known herb belonging to Asteraceae fa-
mily, is used all over the world since ancient times as natural sweetener
with low calories. Stevia usually needs mild temperature and relative
humidity (80%) and regulates blood pressure (Singh and Rao, 2005). It
has got natural antioxidant, sterols and hundreds of phytochemicals.
One of the important glycosides - stevioside highly found in the leaves,
has been recommended for the treatment of metabolic disorder like
diabetes. Stevioside has been reported to counterbalance the hyperse-
cretion of α-cells caused by fatty acids. Further, it increases the action of
fatty acid metabolism genes, proving to have anti-diabetic agent
properties (Hong et al., 2006). Also, it has enhancing effect on the se-
cretion of insulin by directly acting on β-cells without altering the K+-
ATP channel action and cAMP level in the islets, thus authenticating
stevioside and steviol as potent anti-hyperglycemic agents (Jeppesen
et al., 2000). Considering the role of stevioside, attempts have been
made to increase its content through plant tissue culture techniques
(Singh and Dwivedi, 2014). Stevia is safer to be used as sweetening
agents than the synthetic sweeteners like cyclamate and aspartame that
have got carcinogenic properties.
Foliar spray i.e. application of micronutrients in liquid form, to the
leaves especially to stomata and epidermis of the plant is done so that
the deficiency of micronutrients is overcomed and hence yield

⁎
Corresponding author.
E-mail address: pdwivedi25@bhu.ac.in (P. Dwivedi).
1
Current address: Department of Botany, Ramjas College, University of Delhi, Delhi, India.

https://doi.org/10.1016/j.indcrop.2019.111646
Received 11 January 2019; Received in revised form 2 August 2019; Accepted 2 August 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
P. Singh and P. Dwivedi Industrial Crops & Products 140 (2019) 111646

Fig. 1. Foliar spray treated plants (a–f), a Plants before spraying of Boric acid, b Plants after 15 days of spraying of Boric acid, Control - plants sprayed with water, B1
- plants sprayed with 0.25% H3BO3, B2 - plants sprayed with 0.50% H3BO3, B3 - plants sprayed with 0.75% H3BO3, c Plants before spraying of Zinc sulphate, Z1 -
plants sprayed with 0.25% ZnSO4, Z2 - plants sprayed with 0.50% ZnSO4, Z3 - plants sprayed with 0.75% ZnSO4, d Plants after 15 days of spraying of Zinc sulphate, e
plants after 30 days and f 45 days of spraying of Zinc sulphate and Boric acid.

increases. There are surplus reports stating it to be firm and effective
means of addressing nutrient deficiency and increasing yield. Foliar
spraying with Zn as zinc sulphate and B as boric acid significantly
improved growth, yield and yield components along with enhanced
leaves and flower’s nutrients content and plant chemical constituents
i.e. pigments, carbohydrates and flowers oil concentration in Iris plant
(Khalifa et al., 2011). Also, oil percent content was significantly in-
creased by the application of ascorbic acid and zinc application jointly
as paralleled with control plants in Cupressuss empervirens L. (Farahat
et al., 2007). Prior study on Allium cepa concerning foliar application of
Fe, Zn and Mn revealed increase in vital oil, growth and yield of onion
plants (El-Tohamy et al., 2009). Foliar application of minerals like iron,
boron, manganese and copper may be more concrete than application
to soil where these are adsorbed to the soil particles and less available
to the rooting medium (Sarkar et al., 2007; Wissuwa et al., 2008). Foliar
mineral spray ominously affects biomass production of plants
regardless of their growth under non-saline or saline conditions. As
reported, the three micro-elements (B, Zn and Fe) had different effects
on the gentiopicroside content, and proper microelement application
could increase active constituent of gentian (Cai-Lian et al., 2006).
The purpose of the present study was to evaluate the impact of foliar
application of zinc and boron on stevioside content i.e., bioactive
principle of the Stevia, and whether these treatments altered genetic
make-up of plants.
2. Materials and methods
A pot experiment was conducted in garden soil in the poly house of
the Horticulture Garden, BHU, Varanasi to evaluate the response of
foliar spraying of zinc (as zinc sulphate) and boron (as boric acid) on
the stevioside content in the leaves of Stevia rebaudiana. Stevia plants of
25 cm height obtained from nursery were planted in pots (30 cm in

2
P. Singh and P. Dwivedi Industrial Crops & Products 140 (2019) 111646

diameter and 50 cm in depth), each pot filled with garden soil and
organic manure. Plants were sprayed twice with freshly prepared so-
lutions. There were six treatments, comprising of three levels of boron
(0.25, 0.50 and 0.75%, w/v) as boric acid and three levels of Zn (0.25,
0.50 and 0.75%, w/v) as zinc sulphate, while the untreated plants
(control) were sprayed with distilled water. Foliar application of B and
Zn was carried out two times, where the first was after 15 days and the
second applied 30 days after planting. A volume of 50 mL/plant was
sprayed on all the plants in pots with a manual sprayer. Spray was
carried out between 09:00 and 11:00 A.M. The plants were sprayed
with solutions with uniform coverage until the leaves were completely
wet and the solution ran off the leaves. At the time of spray other plants
were covered with plastic sheet to prevent the spreading of the sprayed
nutrients to adjacent pots. Control plants were irrigated with 50 mL of
distilled water. Pest management was carried out during the growth
season according to local practice. The leaves were plucked before and
after 15, 30 and 45 days of spraying for HPLC analysis.

2.1. HPLC quantification of stevioside

Biochemical analysis of the treated plants was performed using

HPLC to confirm the presence of stevioside. Stevioside standard was
obtained from Sigma Ltd., U.S.A. (95% purity).Control and foliar spray-
treated plant leaves were dried at 50 °C for 24 h in dark and pulverized
to uniform size. The HPLC analysis was done using standardized
method (Singh et al., 2014).

2.2. Genetic fidelity

Inter simple sequence repeats (ISSR) marker assays were employed

to evaluate genetic stability of foliar spray-treated plants. Genomic DNA
was isolated from leaves of randomly selected control and treated
plants following the CTAB method (Doyle and Doyle, 1990).

2.3. Polymerase chain reaction (PCR) amplification

Isolated DNA was subjected to polymerase chain reaction (PCR) to

produce fingerprinting patterns using a total of 9 random primers (UBC
series) obtained from OPERON Technologies, USA. PCR experiments
were done following methodology of Singh et al. (2014).

3. Results and discussion

3.1. Stevioside content

Findings of this study indicated that foliar application of zinc sul-

phate and boric acid improved growth of stevia plants as variations was
observed with morphological parameters: size, colour etc. (Fig. 1). It is
to be mentioned that the leaves were larger and greener after the
treatment giving an appearance of increased leaf area (Fig. 1). Plant
leaves act as a source for capturing light and assimilate production.
Foliar spraying of zinc sulphate or boric acid, singly, at all concentra-
tions used significantly increased stevioside content as compared to the
control treatment (Fig. 2A). The second most promising result was
shown by 0.75% H3BO3, as 9 times increase in the peak area was ob-
served (Fig. 2B). Boron is involved in many processes including sugar
transport, cell wall synthesis and maintenance, membrane integrity and
RNA, indole acetic acid (IAA) and phenol metabolism (Dordas and
Brown, 2001). Whereas, the most promising results were obtained from
plants treated with 0.75% ZnSO4, as there was 30 times increase in the
peak area noted (Fig. 2C). Kumar and Arora (2000) on Gladiolus, Fig. 2. HPLC Chromatograms of plants (a–c), a sprayed with water, b sprayed
Munikrishnappa et al. (2002) on Polionthes tuberose found that spraying with0.75% Boric acid, c sprayed with0.75% Zinc sulphate.
zinc sulphate increased plant height, number of leaves as well as fresh
and dry weight of leaves. Iris plant sprayed with Zn at the higher rate
(4.5 g/L) produced the highest values of leaf number/plant (Khalifa
et al., 2011). The high level of Zn (4.5 g/L) results in the highest values

3
P. Singh and P. Dwivedi
of carbohydrate, chl a, carotenoids and flower oil content, while the
highest level of B (20 ppm) produced the highest values of carbohy-
drate, chl b and oil content of iris plant. The middle level of Zn (3.0 g/L)
led to the highest chl b content. On the other hand, the middle level of B
(10 ppm) resulted in the highest content of chla and carotenoids
(Khalifa et al., 2011). The highest percentage of essential oil of Allium
cepawas obtained by adding Zn, especially with the treatment of 0.3 g/L
(El-Tohamy et al., 2009). As reported, Zn (0.3 g/L) treatment increased
the components of Propyl 1-propenyl disulphide, cis; Propyl 1-propenyl
disulphide, trans; Methylpropyltrisulphide; 3,5-Diethyl- 1,2,4-trithio-
nale; 5,6 -Dihydro-6-methyl-2,4-diethyl-4H- 1,3,5-dithiazine; 5,6- b
Dihydro-2,4,6-triethyl-4H- 1,3,5 -dithiazine; 6-Ethyl-4,5,7-trithiade-
cane; 2,4,6-Triethyl- 1,3,5-trithiane and 6-Ethyl-4,5,7,8-tetra-
thiaundecane but 3.4-Dimethylthiophene was decreased. Khalid (1996)
reported that trace elements such as Fe, Zn and Mn increased essential
oil content of different plants such as anise, coriander and sweet fennel.
In our study the mean data of both the seasons suggested that the foliar
spraying of zinc sulphate or boric acid alone increased peak area as
compared to the control treatment, indicating thereby for increased
stevioside content.
3.2. Genetic fidelity
ISSR is a useful marker system which has been successfully em-
ployed to measure genetic stability/instability in tea (Thomas et al.,
2006), Prunus mume (Ning et al., 2007), Swertia chirayita (Joshi and
Dhawan, 2007), Amorphophallus albus (Hu et al., 2008) and Gerbera
(Bhatia et al., 2009). It has several obvious advantages over RAPD, such
as higher reproducibility and in formativeness (Nagaoka and Ogihara,
1997), which makes ISSR more attractive for genotyping and diversity
studies. ISSRs have been used for detecting somaclonal variations in
micropropagated plants (Venkatachalam et al., 2007; Huang et al.,
2009) and for the study of genetic diversity among cultivars and
germplasm in fruit plants, including strawberry, apple, ribes (Korbin
et al., 2002).
ISSR analysis of the treated plants were carried out in comparison to
the control plant to assess whether the foliar spraying of zinc sulphate
and boric acid affected the genetic constitution of the plant, as varia-
tions were observed with morphological parameters: size, colour, etc.
(data not shown, Fig. 1), particularly leaves were larger and greener
4
Industrial Crops & Products 140 (2019) 111646
Fig. 3. ISSR analysis of foliar spray treated plants for the
primer UBC 836, C - plants sprayed with water, B1 - plants
sprayed with 0.25% H3BO3, B2 - plants sprayed with 0.50%
H3BO3, B3 - plants sprayed with 0.75% H3BO3, Z1 - plants
sprayed with 0.25% ZnSO4, Z2 - plants sprayed with 0.50%
ZnSO4, Z3 - plants sprayed with 0.75% ZnSO4.
after the treatment giving an appearance of increased leaf area. The
ISSR profiling with primer UBC-836 for 30 days produced mono-
morphic bright bands; no distinct band was observed among them and
this confirmed that the foliar spraying did not affect genetic integrity of
the plants (Fig. 3).This is the first study, to our knowledge, regarding
positive effect of microelements - Zn and B on growth and increasing
stevioside content in Stevia (which has great commercial implications).
While the treatments led to increased stevioside content, it was realized
to check if Zn and B affect genetic make up of the plants, specially after
short and long term treatments of foliar application. There is a report
available where Zn and B application to strawberry affected fruit de-
velopment and gene expression of related gene expression (Yulia et al.,
2016). Moreover, chances of chromosomal aberration in plants growing
in vivo and subjected to any exogenous treatment are more, hence it was
necessary to validate if Zn and B affect genetic fidelity of plants sub-
jected to Zn and B treatments, both to a short-term and long –term
treatments. We could notice, using ISSR markers which are advanced
molecular markers, that Zn and B treatment maintained genetic fidelity
of the plants as no genetic difference was noticed.
4. Conclusions
To conclude, to our best knowledge, this is the first report of foliar
application of boron and zinc in stevia for increased stevioside content.
Further, ISSR marker assays confirmed the genetic stability of these
micronutrients-treated plants. The results obtained have the bearing in
R&D sector focused on harnessing medicinal properties of stevia plants
in controlling diabetes.
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