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African Journal of Agricultural Marketing ISSN 2375-1061 Vol. 8 (9), pp. 001-003, September, 2020. Available online 
at www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 
 

 

Short Communication 

 

An efficient protocol for in vitro clonal propagation of 
natural sweetener plant (Stevia rebaudiana Bertoni) 

 
Ashish Ojha1*, V. N. Sharma1 and Vinay Sharma2

 
 

1
Department of Biotechnology, Plant Tissue Culture and Research Development Laboratory, MIET group of Institutions, 

Meerut- 250005, U. P. India. 
2
Department of Biotechnology and Bioscience, Banasthali University, Banasthali, Rajasthan, India. 

 
Accepted 18 May, 2020 

 
Leaf segments of Stevia were cultured on MS medium supplemented with varying concentrations (0.5, 

1.0, 1.5, 2.0, 2.5 mg/L
-1

) of growth regulators (BAP and 2, 4-D). Ninety one percent aseptic cultures were 

obtained when sterilized with 0.1% HgCl2 for 10 min. The highest amount of callus was obtained in MS 

medium supplemented with1.0 mg/L
-1

 BAP+0.5 mg/L
-1

 2, 4-D, respectively. On the other hand, 2.5 mg/L
-

1
 BAP + 0.5 2,4-D showed lowest performance. Highest shoot was obtained in 2.0 mg/L

-1
 BAP. These 

shoots was transfer into different concentration of IBA, NAA and IAA (0.5. 1.0, 1.5, and 2.0) used. 

Highest rooting percentage was recorded on MS medium with 0.5 mg/L
-1

 IAA. The rooted plantlets were 
transfer into mist chamber with relative humidity for 2 - 4 week after these plants were hardened and 
successfully established in soil. 

 
Key words: Stevia rebaudiana, Sweetener, Callus, leaf. 

 
INTRODUCTION 

 
Stevia (Stevia rebaudiana Bert.) is a small, herbaceous, 
semi-bushy, perennial shrub of Compositae family originated 
from Paraguay. It grows well at the temperature ranging 
between 15 - 30°C. It is one of 154 members of the genus 
Stevia, which produces stevioside, a diterpinoid glycoside 
isolated from plant leaves (Robinson, 1930). Stevioside of 
special intrestto diabetics, persons with hyperglycemia and 
the diet conscious. A sugar-free, no calorie natural 
sweetener is especially helpful for people who are diabetic, 
prone to yeast infections, or trying to lose a few extra 
pounds by controlling calories. The product can be added to 
tea and coffee, cooked or baked goods, processed foods 
and beverages. Dry leaves of this plant are 30 times sweeter 
than sugar with Zero calories. The first report of com-mercial 
cultivation in Paraguay were in 1964 (Sumida, 1968) began 
a large effort aimed at establishment. Stevia as a crop in 
Japan, since then Stevia has been introduced as a crop in a 
number of countries including Brazil, Korea, Mexico, U.S. 
and Canada (Lee et al., 1979). The herbal drug industry is 

considered to be a high growth industry of the late 90s and  
 
 
 
*Corresponding author. E-mail: ashish_ojha1087@rediff 
mail.com. Tel: 09808435371. 
 

 
 
 
seeing the growing demand, it is all set to flourish in the 
next century in ancient Indian traditional Ayurvedic 
system of medicine.  

Stevia has various properties such as antibacterial, 
anticandidal, antifungal, antiviral, cardio tonic (tones, 
balances, strengthens the heart), diuretic, hypoglycemic, 
vasodilator. Seed germination of Stevia is often poor or 
very low (Miyazaki and Watenabe, 1974).  

Therefore, there are basically two options for 

multiplication. The first is tissue culture and second is 

vegetative propogation. 
 
MATERIALS AND METHODS 
 
Plant materials 
 
The explants; leaf segments were used as experimental material. 
The explants were collected from 5 - 6 months old field grown 
plants at FRI (Forest Research Institute, Dehradun) experimental 
field. The explants were treated with Tween 20 (fungicide) 5 - 6 
drops for 5 min with constant shaking for clean fungus content from 
leaf surface then washed thoroughly with distilled water. Then the 
materials were taken under laminar air flow cabinet and surface 
sterilization was done with (0.1% HgCl2) mercuric chloride for 10 
min followed by 4 - 5 times rinse with sterile distilled water to 
remove traces of HgCl2 from material. 

file:///C:\Users\user\Documents\REPUBLICATION\AGRICULTURAL%20SCIENCES\AppData\Local\Temp\www.internationalscholarsjournals.org


 
 
 

 
Table 1. Effects of cytokinin and auxin in MS medium on in vitro shooting and callus formation from leaf segments of Stevia. 

 

Growth regulators (mg/L
-1

) % of shoot Shoots /Cultures Average shoot length (cm) Texture 

BAP (0.5) 45.67 3.13 ± 0.11 2.15 ± 0.28 Green 

BAP (1.0) 56.31 4.33 ± 0.29 2.32 ± 0.27 Green 

BAP (1.5) 60.10 5.40 ± 0.18 2.43 ± 0.11 Green 

BAP (2.0) 76.44 6.78 ± 0.23 3.34 ± 0.55 Green 

BAP (2.5) 58.27 5.12 ± 0.41 2.00 ± 0.42 Green 

 

 Amount of callus Callus status 

BAP + 2-4-D (0.5)+ (0.5) ++++ Good 

BAP + 2-4-D (1.0) + (0.5) +++++++ Best 

BAP + 2-4-D (1.5) + (0.5) ++++ Good 

BAP + 2-4-D (2.0) + (0.5) +++ Average 

BAP + 2-4-D (2.5) + (0.5) + Dead 
 

 
Preparation of culture media 
 
Stock solution of all components were prepared with appropriate 
amount of all the components, Appropriate amount of all stock 
solution were mixed following the standard media preparation 
procedure. The pH of the media was adjusted to 5.6 - 5.8 and 
growth regulators were added at different concentration (0.5, 1.0, 1. 

5, 2.0, 2.5 mg/L
-1

 ). In Each treatment 2 explants were inoculated in 
50 test tubes, 10 test tubes each. 

 

 

medium containing 2 mg/L
-1

 BAP showed the best 
response of multiplication (Uddin et al., 2006). Shoot 
formation with 6.78 ± 0.23 microshoots per explants 

(Table 1). Further more, MS medium containing 0.5 mg/L
-

1
 2,4-D +1.0 mg/L

-1
 BAP also showed the maximum 

callusing in leaf segment explants (Table 1). 

 

 
Inoculation technique 
 

Leaf segments into 1 cm
2
 square pieces and the aseptically 

inoculated onto callus induction media. All inoculation and aseptic 
manipulation were carried out in laminar air flow cabinet. Before 
use, the floor of cabinet was cleaned with 90% ethyl alcohol to 
reduce chance of contamination. The instruments like scalpels, 
forceps, needles etc, were sterilized by an alcoholic dip and flaming 
method inside the laminar air flow chamber. Before the inoculation 
hands were cleaned by spraying 70% ethyl alcohol. After the 
inoculation tubes were shelved in dark condition. The temperature 
of the culture room was maintained at 25 ± 1°C. 

 

Data collection 
 
From the seventh day of inoculation, regular visual observation was 
done up to five weeks to records the days of callus initiation and 
maturation. The explants showing embryogenic callus was recorded 
after 4 - 5 weeks, Histological sections and microscopic observation 
was done under microphotograph. 
 

 

RESULT AND DISCUSSION 

 

In vitro shoot proliferation and multiplication 

 

The auxiliary buds in callus zones proliferated within 6 - 7 
weeks of culture on cytokinin supplemented MS medium. 

When the explants were cultured on 2 mg/L
-1

 BAP 

containing media (76.44%) of them produced shoots. the 
auxiliary shoots proliferated and elongated (3.34 ± 0.55 
cm) within three weeks of culture. In the experiment MS 

 
Rooting induction from microshoots 

 

In vitro raised microshoots (3-4 cm) were excised from 
callus culture and grown on MS medium with growth 
regulators. Initiation of roots from microshoots was very 
slow in NAA. The percentage of shoots that formed roots 
and no of roots/ shoots significantly varied at different 
concentration of IBA, IAA and NAA.  

The highest rooting 98.12% in microshoots was 

obtained as MS medium containing 0.5 mg/L
-1

 IAA with 
3% sucrose with in 2 weeks of microshoot transfer (Table 
2). Further more the no of roots per microshoots was 
optimal (13.7 Roots/ Microshoots) on MS medium 

containing (0.5 mg/L
-1

 IAA) but the average root length 

per shoot was higher (3.65 cm) on 1.5 mg/L
-1

 IBA 
containing MS media (Murashige and Skoog, 1962).  

The effect of IBA, IAA and NAA on root induction was 
positive; however, auxins concentrations had a positive 
effect on the number of roots/ shoots and it was higher in 
the case of IAA than IBA and NAA. Steven et al. (1992) 

reported that 1.0 mg/L
-1

 NAA MS medium showed 
maximum rooting in propagated shoots of Stevia. The 
potential of IBA in root induction has been reported in 
many species (Epstein et al., 1993). In root induction 

experiment, 0.5 mg/L
-1

 IAA was found better as 
maximum roots proliferated that result as better reported 
by Steven et al. (1992). 
 
 
Acclimatization and field establishment 
 

Rooted plantlets were shifted to plastic pots containing 



 
 
 

 
Table 2. Effects of IBA, NAA or IAA on adventitious root formation from micro cuttings in S. rebaudiana.  

 
 Auxins Concentration % of cuttings No. of roots Average root Day of mergence 

 types (mg/l) rooted per shoots length (cm) of roots (days) 

 IAA 0.5 98.12 13.71 2.10 6-12 

  1.0 91.40 9.88 2.35 6-14 

  1.5 74.99 7.31 2.17 6-12 

  2.0 64.31 4.98 3.01 6-17 

 NAA 0.5 81.50 8.65 2.41 6-12 

  1.0 70.20 7.57 2.93 6-12 

  1.5 59.72 5.34 2.42 6-12 

  2.0 54.43 4.45 2.78 6-12 

 IBA 0.5 68.72 7.88 3.07 6-14 

  1.0 60.68 5.95 3.43 6-14 

  1.5 51.85 5.02 3.65 6-14 

  2.0 40.43 3.55 2.98 6-12 
 

 

sterilized soil, sand and peat moss at 1:1:1 ratio covered 
with transparent polythene begs and placed in 
acclimatization room at 28°C with 70 - 90% humidity. 
After the 2 weeks and than completely removed of after 3 
weeks for proper hardening. A total no of 71% plants of 
survived thoroughly this procedure. In many species the 
reduced the survival rate of the explants during 
acclimatization is affiliated to the utilization of high 
concentration of IBA and NAA in rooting medium (Al-
maarrie et al. 1994). 
 

 

ACKNOWLEDGEMENT 

 

We gratefully acknowledge Prof. V. N. Sharma, Director 
Programmer, Biotechnology, Meerut Institute of Engr. and 
Technology, Meerut. For providing his laboratory with 
financial help and giving valuable suggestions from time 
to time during this research work. 
 

 
REFERENCES 
 
Al-Maarrie K, Arnaud Y, Miginiac E (1994). Micropropogation of Pyrus 

communis cultivar, Passe crassane seedlings and cultivar. Williams: 
factors affecting root formation in vitro and ex-vitro. Sci. Hortic. 
Amsterdam. 58:207-214.  

Epstein E, Sagee O, Zahir A (1993). Uptake and metabolism of Indole-3 

acetic acid and Indole-3 butyric acid by Petunia cell suspension 

culture, Plant. Growth Regul., 13:31-40. 

 

 
Lee JI, Kang KK, Lee EU (1979). Studies on new sweetening resource 

plant Stevia (Stevia rebaudiana Bert.) in Koprea. I. Effect of 
transplanting date shifting by cutting and seeding dates on agronomic 
characteristics and dry leaf of Stevia (English abstr.) Res. Rep. ORD., 
21:171-179.  

Miyazaki Y, Watenable H (1974). Studies on the cultivation of Stevia 
rebaudiana Bertoni; on the propogation of the plant (English abst.). 
Jap. J. Trop. Agric., 17:154-157. 

Murashige T, Skoog F (1962). A revised medium for rapid growth and 
bioassays with tobacco tissue cultures, Physiol. Plant, 15:473-497.  

Robinson BL (1930). Contributions from the Grey Herbarium of Harvard 
University. The Grey Herbarium of Harvard University, Cambridge.  

Steven MS, Gail BM, Christopher WWB (1992). Stevioside biosynthesis 
by callus, root, shoot, and riited-shoot cultures in-vitro, Plant. Cell, 
Tiss. Org., 28:151-158. 

Sumida T (1968). Reports on Stevia introduced from Brazil as a new 
sweetness resource in Japan (English summary). J. Cent. Agric. Exp. 
Stn., 31:1-71. 

Uddin MS, Chowdhury MSH, Khan MMH, Belal Uddin M, Ahmed R, 

Baten MA (2006). In vitro propagation of Stevia rebaudiana Bert in 

Bangladesh, Afr. J. Biotechnol., 5(13): 1238-1240. 


