Acta agriculturae Slovenica, 117/2, 1–7, Ljubljana 2021
doi:10.14720/aas.2021.117.2.1754 Original research article / izvirni znanstveni članek
An efficient protocol for in vitro regeneration from the nodal explants of Withania 
coagulans (Stocks) Dunal: a valuable medicinal herb
Pari DEHV ARI-NAGAN
1
, Hossein ABBASPOUR
2
*, Mohammad Hasan ASARE
3
, Sara SAADATMAND
1
 
Received July 02, 2020; accepted March 16, 2021.
Delo je prispelo 2. julija 2020, sprejeto 16. marca 2021. 
An efficient protocol for in vitro regeneration from the nodal 
explants of Withania coagulans (Stocks)  Dunal: a valuable 
medicinal herb 
Abstract: In order to develop a protocol for the effective 
micropropagation of the important medicinal plant Withania 
coagulans (Stocks) Dunal, the effects of different concentrations 
and combinations of growth regulators on the nodal explants 
in two independent experiments were investigated. For shoot-
ing, a MS medium fortified with different concentrations and 
combinations of IBA (0.01, 0.1 and 0.5 mg l
-1
), BA (0.5, 1 and 2 
mg l
-1
), Kin (0.5 and 1 mg l
-1
), PG (0.5 mg l
-1
) and GA (0.5 mg 
l
-1
) was used and the highest shooting response, shoot number 
and shoot length were obtained in the MS + IBA (0.01 mg l
-1
) + 
BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) + GA (0.5 mg l
-1
) treatment. In 
the second experiment, the effect of MS supplemented with dif-
ferent combinations and concentrations of IBA (0.1, 0.5, 1 and 
2 mg l
-1
), NAA (0.1 and 1 mg l
-1
) and PG (1 mg l
-1
) on rooting 
of the nodal explants was investigated, which showed that the 
highest rooting response (%) was observed in the MS fortified 
with NAA (0.1 mg l
-1
), NAA (1 mg l
-1
), NAA (0.1 mg l
-1
) + PG 
(1 mg l
-1
), and NAA (1 mg l
-1
) + PG (1 mg l
-1
) treatments, as 
well as the highest number of roots at NAA (0.1 mg l
-1
) and the 
highest root length at IBA (1 mg l
-1
). Our findings highlight a 
complete micropropagation method for W. coagulans from the 
nodal explant that can make a significant contribution to the 
development of W. coagulans material for medical applications.
Key words: Withania coagulans; micropropagation; in vi-
tro; nodal explant; phloroglucinol
1 Islamic Azad University, Faculty of Biological Sciences, Science and Research Branch, Department of Biology, Tehran, Iran
2 Islamic Azad University, Faculty of Biological Sciences, North Tehran Branch, Department of Biology, Tehran, Iran
3 Research Institute of Forests and Rangelands, Tehran, Iran
*Correspondence author: abbaspour75@yahoo.com
Učinkovit protocol za in vitro regeneracijo nodijskih izsečkov 
vrste Withania coagulans (Stocks) Dunal, cenjene zdravilne 
rastline
Izvleček: Z namenom izboljšanja protokola za učinkovito 
mikropropagacijo pomembne zdravilne rastline (Withania 
coagulans (Stocks) Dunal) so bili preučevani učinki različnih 
koncentracij in kombinacij rastnih regulatorjev na izsečkih 
kolenc v dveh neodvisnih poskusih. Za razvoj poganjkov je 
bilo uporabljeno MS gojišče, obogateno z različnimi koncen-
tracijami in kombinacijami IBA (0,01; 0,1 in 0,5 mg l
-1
), BA (0,5; 
1 in 2 mg l
-1
), Kin (0,5 in 1 mg l
-1
), PG (0,5 mg l
-1
) in GA (0,5 
mg l
-1
). Največji odziv v rasti poganjkov, v njihovem številu in 
dolžini je bil dosežen pri obravnavanju MS + IBA (0,01 mg l
-1
) 
+ BA (0,5 mg l
-1
) + PG (0,5 mg l
-1
) + GA (0,5 mg l
-1
). V drugem 
poskusu je bil preučevan učinek MS z dodatkom različnih kon-
centracij in kombinacij IBA (0,1; 0,5; 1 in 2 mg l
-1
), NAA (0,1 in 
1 mg l
-1
) in PG (1 mg l
-1
) na zakoreninjenje nodijskih izsečkov, 
pri čemer je bil dosežen največji odziv zakoreninjenja (%) pri 
obravnavanju MS obogatenim z NAA (0,1 mg l
-1
), NAA (1 mg 
l
-1
), NAA (0,1 mg l
-1
) + PG (1 mg l
-1
), in NAA (1 mg l
-1
) + PG 
(1 mg l
-1
). Največje število korenin je bilo pri obravnavanju s 
NAA (0,1 mg l
-1
), največja dolžina korenin pa pri obravnavanju 
z IBA (1 mg l
-1
). Izsledki raziskave pojasnjujejo celotno metodo 
mikropropagacije vrste W. coagulans iz izsečkov kolenc, kar je 
pomemben prispek k vzgoji sadilnega materiala te vrste za up-
orabo v zdravstvu.
Ključne besede: Withania coagulans; mikropropagacija; 
in vitro; nodijski izsečki; floroglucinol

Acta agriculturae Slovenica, 117/2 – 2021 2
P . DEHV ARI-NAGAN et al.
1 INTRODUCTION
Withania coagulans Dunal is one of the most impor-
tant species in the Solanaceae family, growing mainly in 
the eastern Mediterranean to South Asia, including Iran, 
Afghanistan, Pakistan and India. W. coagulans is widely 
used due to its numerous medicinal properties such as 
hypo-lipidemic, cardiovascular, hepato-protective, anti-
hyperglycemic, anti-diabetic and anti-tumor (Haq et al., 
2013; Maurya & Akanksha, 2010). W. coagulans fruits 
are used in cheese production due to their ability to co-
agulate the milk. The numerous medicinal properties of 
W. coagulans are mainly due to the compounds of witha-
nolides that are naturally synthesized by the plant (Haq 
et al., 2013; Chen et al., 2011). Due to the accumulation 
of the medicinal compound withanolide A in the above-
ground parts of W . coagulans in comparison with the root 
of W . somnifera (L.) Dunal, indicates the economical and 
easy harvesting of withanolides (Rathore et al., 2016). 
Due to the lack of proper cultivation practices, W. c o ag u -
lans plants are harvested from wild, which represents a 
threat to the natural diversity of its germplasm. Reducing 
the chance of seed setting due to self-incompatibility and 
polygamous-dioecious nature of flowers reduces the rate 
of natural regeneration that cannot meet the rate of ex-
ploitation (Rathore et al., 2012; Gilani et al., 2009).
Various factors, such as reproductive failure, habitat 
disturbances, hostile environmental factors and overex-
ploitation, pose a serious threat to valuable medicinal 
plants, which may expose them to complete extinction 
(Gerami et al., 2018; Ghorbani et al. 2018). Therefore, 
collecting plants from nature is not a viable way to meet 
commercial requirements, and it is important to establish 
the appropriate strategies to meet the needs (Ghorbani 
et al. 2019). In vitro culture is one of the biotechnology 
powerful tools that can be effective in the propagation 
of genetically uniform plants from the elite lines in large 
numbers, which can eliminate the need to collect me-
dicinal plants from wild (Ghasemi-Omran et al. 2021). 
Hence the propagation of endangered or rare plants us-
ing in vitro culture can help maintain germplasms and 
prevent extinction (Rathore et al., 2016). Furthermore, 
due to the propagation of genetically uniform plants by 
in vitro culture, it allows the accurate study of stress tol-
erance and the regulation of secondary metabolites be-
tween different treatments, which could have potential 
application in elite breeding lines. A simple and efficient 
method for in vitro propagation of W. coagulans is a ne-
cessity for its sustainable use in order to meet pharma-
ceutical requirements. It can also provide the primary 
needs for genetic improvement through genetic trans-
formation, genetic restoration programs through true-
to-type propagation, and phyto-pharming improvement. 
Therefore, in the current study, the aim was to investigate 
the potential of the nodal explants in order to develop an 
effective protocol for the in vitro propagation of Withania 
coagulans Dunal.
2 MATERIAL AND METHODS
Young, non-lignified stems of W. coagulans were 
collected from Iran (Saravan region, Sistan and Bal-
uchestan Province). The stems were soaked in liquid de-
tergent (10 % (vv
-1
) Teepol) for 5 min after rinsing with 
running tap water for 30 min. After rinsing with running 
tap water (10 min), the stems were cut into 2 cm seg-
ments (explants), each segment containing a node. Then, 
after disinfection with an HgCl
2
 (0.1 %) for 5 min and 
rinsing with sterile distilled water (five times), the nodal 
segments were used for culture.
The explants were implanted vertically on MS me-
dium (Murashige & Skoog, 1962) containing agar (C, 
0.8 %) and sucrose (mg l
-1
, 3 %) at pH 5.8. In order to 
investigate the effect of different hormonal combina-
tions on W. coagulans shooting and rooting, the culture 
media were supplemented with different concentrations 
and combinations of growth regulators. The treatments 
applied for shooting and rooting are represented in Ta-
ble 1. The plant material was kept at 25/18 °C with 14 h 
photoperiod and 60-80 µmol m
−2
 s
−1
 light intensity. After 
6 weeks, the number of differentiated shoots per node, 
shoot length, shoot multiplication percentage, number 
roots per shoot, root length and rooting percentage were 
recorded.
For acclimatization, the rooted plants were trans-
ferred to plastic pots containing an autoclaved mixture of 
cocopeat, soil and sand (1:1:1) after rinsing with tap wa-
ter and removing agar. To retain humidity, the pots were 
covered with clear plastics and kept in the tissue culture 
laboratory. After 10 days, the plastic cover was removed 
from the pots and the pots were kept at 25 °C with 16 
h photoperiod. After 3 weeks, the plantlets were trans-
ferred to normal field conditions.
All experiments were repeated three times and the 
means value were calculated based on four independent 
replicates (Each replication contained 5 explants). Statis-
tical analysis of the results was calculated using SAS v. 
9.1.3 software and the mean comparison was carried out 
with a least significant difference (LSD) test (at the 5 % 
level).

Acta agriculturae Slovenica, 117/2 – 2021 3
An efficient protocol for in vitro regeneration from the nodal explants of Withania coagulans (Stocks) Dunal: a valuable medicinal herb 
3 RESULTS
In the present study, the effect of different concen-
trations and combinations of plant hormones (auxin, 
cytokinin, and gibberellin) and phenolic composition 
(phloroglucinol) on induction of branching in nodal 
explants of W. coagulans under in vitro conditions were 
investigated. The results showed that supplement of MS 
medium with different concentrations and combinations 
of IBA (0.01, 0.1 and 0.5 mgl
-1
), BA (0.5, 1 and 2 mgl
-1
), 
Kin (0.5 and 1 mgl
-1
), GA (0.5 mgl
-1
) and PG (0.5 mgl
-1
) 
increased shooting compared to control treatment (Fig. 
1). As shown in Table 2, the highest increase in the num-
ber of shoots per explant was observed in T9 treatment 
by 170 % compared to T1 (MS medium). Furthermore, a 
Fig. 1: The effect of different treatments on shooting from the nodal explants of W. coagulans in in vitro conditions.
Table 1: Treatments applied in two experiments of the induction of shooting and rooting from the nodal explants of W. 
coagulans
Treatments applied for shooting Treatments applied for rooting
T1 MS (Control) MS (Control)
T2 MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) MS + IBA (0.1 mg l
-1
) 
T3 MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) MS + IBA (0.5 mg l
-1
) 
T4 MS + IBA (0.5 mg l
-1
) + BA (2 mg l
-1
) MS + IBA (1 mg l
-1
) 
T5 MS + Kin (0.5 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) MS + IBA (2 mg l
-1
) 
T6 MS + PG (0.5 mg l
-1
) + BA (1 mg l
-1
) MS + NAA (0.1 mg l
-1
)
T7 MS + Kin (1 mg l
-1
) + IBA (0.5 mg l
-1
) + BA (1 mg l
-1
) MS + NAA (1 mg l
-1
)
T8 MS + IBA (0.1 mg l
-1
) + BA (2 mg l
-1
) MS + IBA (0.1 mg l
-1
) + PG (1 mg l
-1
)
T9 MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) + GA (0.5 mg l
-1
) MS + IBA (1 mg l
-1
) + PG (1 mg l
-1
)
T10 MS + Kin (1 mg l
-1
) + BA (1 mg l
-1
) + PG (0.5 mg l
-1
) MS + NAA (0.1 mg l
-1
) + PG (1 mg l
-1
)
T11 -------------- MS + NAA (1 mg l
-1
) + PG (1 mg l
-1
)
IBA: Indole-3-butyric acid, BA: 6-Benzyladenin, PG: Phloroglucinol, Kin: Kinetin, GA: Gibberellic acid, NAA: 1-Naphthaleneace-
tic acid
high number of shoots per explant was also observed in 
T7, T4 and T3 treatments, respectively (Table 2). The ini-
tiation of bud break and the emergence of buds from ex-
plants were induced within 8 to 10 days in all treatments, 
except for T1 and T10 treatments, which began within 
15 to 20 days. The highest response to nodal segments 
of W. coagulans in terms of shoot multiplication (%) was 
obtained in T9 treatments. Following T9 treatment, the 
highest shoot multiplication was observed in T7, T2 and 
T3 treatments, respectively (Table 2). The results showed 
that adding different concentrations and combinations of 
plant hormones and phenol compound to the MS medi-
um caused a significant increase in shoot length, so that 
the highest shoot length in T9 and T5 treatments was ob-
served by 163 % and 92 %, respectively, compared to the 
MS treatment alone (Table 2).

Acta agriculturae Slovenica, 117/2 – 2021 4
P . DEHV ARI-NAGAN et al.
Table 2: The effect of different concentrations and combinations of growth regulators on shooting from the nodal 
explants of W. coagulans in in vitro conditions.
Treatments
Number of 
shoots/nodes
Shoot 
multiplication (%)
Shoot length 
T1 (MS (Control)) 2.50 ± 0.58 cd 37.75 ± 3.31 f 1.075 ± 0.17 e
T2 (MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) 4.25 ± 0.50 b 72.75 ± 4.57 c 1.810 ± 0.27 bc
T3 (MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) 4.50 ± 0.58 b 72.51 ± 3.70 cd 1.883 ± 0.27 bc
T4 (MS + IBA (0.5 mg l
-1
) + BA (2 mg l
-1
) 4.51 ± 0.48 b 66.50 ± 4.43 d 1.695 ± 0.23 cd
T5 (MS + Kin (0.5 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) 4.25 ± 0.57 b 66.50 ± 4.65 d 2.063 ± 0.25 b
T6 (MS + PG (0.5 mg l
-1
) + BA (1 mg l
-1
) 4.23 ± 0.51 b 67.51 ± 5.51 cd 1.890 ± 0.21 bc
T7 (MS + Kin (1 mg l
-1
) + IBA (0.5 mg l
-1
) + BA (1 mg l
-1
) 4.51 ± 0.56 b 79.75 ± 4.27 b 1.975 ± 0.17 bc
T8 (MS + IBA (0.1 mg l
-1
) + BA (2 mg l
-1
) 3.25 ± 0.51 c 51.00 ± 4.32 e 1.490 ± 0.22 d
T9 (MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) + GA (0.5 
mg l
-1
)
6.75 ± 0.96 a 96.00 ± 3.37 a 2.830 ± 0.17 a
T10 (MS + Kin (1 mg l
-1
) + BA (1 mg l
-1
) + PG (0.5 mg l
-1
) 2.25 ± 0.94 d 52.00 ± 3.37 e 1.680 ± 0.15 cd
Values marked with same letters are not significantly different (LSD, p < 0.05). All the values are means of four replicates ± SD.
IBA: Indole-3-butyric acid, BA: 6-Benzyladenin, PG: Phloroglucinol, Kin: Kinetin, GA: Gibberellic acid, NAA: 1-Naphthaleneace-
tic acid
The results of the present study showed that the MS 
medium fortified with IBA (0.1 mg l
-1
), NAA (0.1 and 1 
mg l
-1
) and combinations of IBA (0.1 mg l
-1
) + PG (1 mg 
l
-1
), NAA (0.1 mg l
-1
) + PG (1 mg l
-1
) and  NAA (1 mg 
l
-1
) + PG (1 mg l
-1
) increased the rooting response of the 
nodal explants relative to the MS medium alone, how-
ever, adding IBA (0.5, 1 and 2 mg l
-1
) and combination of 
IBA (1 mg l
-1
) + PG (1 mg l
-1
) to the MS medium reduced 
the rooting response compared to the MS medium alone. 
The highest percentage of rooting was observed in T6, 
T7, T10 and T11 treatments (T able 3). The results showed 
that different treatments applied, except for T9 treat-
ment, increased the number of roots per explant. The 
highest and lowest number of roots per shoot were re-
corded in T6 (20.5 ± 3.4 per shoot) and T9 (10.5 ± 2.4 per 
shoot) treatments, respectively (Table 3). The results also 
showed that T3, T4, T5, T6, T8, T9 and T10 treatments 
significantly increased the root length and T7 and T11 
treatments reduced the root length compared to the MS 
medium alone, while there was no significant difference 
between control treatment and T2 treatment. The highest 
and lowest root lengths were observed in T4 (4.95 cm) 
and T7 (0.475 cm) treatments, respectively (Table 3).
Table 3: The effect of different concentrations and combinations of growth regulators on rooting from the nodal 
explants of W. coagulans in in vitro conditions
Treatments Rooting response (%) Number of roots Root length
T1 (MS (Control)0 70 ± 8 c 13.0 ± 1.2 de 1.925 ± 0.22 d
T2 (MS + IBA (0.1 mg l
-1
)) 80 ± 8 b 14.0 ± 0.8 d 1.925 ± 0.22 d
T3 (MS + IBA (0.5 mg l
-1
)) 50 ± 7 d 13.8 ± 1.7 d 3.150 ± 0.29 b
T4 (MS + IBA (1 mg l
-1
)) 65 ± 6 c 15.0 ± 2.2 bcd 4.950 ± 0.21 a
T5 (MS + IBA (2 mg l
-1
)) 65 ± 9 c 13.3 ± 1.7 de 2.350 ± 0.13 c
T6 (MS + NAA (0.1 mg l
-1
)) 100 ± 0 a 20.5 ± 3.4 a 3.075 ± 0.10 b
T7 (MS + NAA (1 mg l
-1
)) 100 ± 0 a 17.8 ± 3.1 ab 0.475 ± 0.10 f
T8 (MS + IBA (0.1 mg l
-1
) + PG (1 mg l
-1
)) 85 ± 9 b 14.3 ± 2.5 cd 2.975 ± 0.17 b
T9 (MS + IBA (1 mg l
-1
) + PG (1 mg l
-1
)) 50 ± 7 d 10.5 ± 2.4 e 4.900 ± 0.18 a
T10 (MS + NAA (0.1 mg l
-1
) + PG (1 mg l
-1
)) 100 ± 0 a 17.3 ± 2.6 bc 3.050 ± 0.13 b
T11 (MS + NAA (1 mg l
-1
) + PG (1 mg l
-1
)) 100 ± 0 a 15.5 ± 1.7 bcd 1.050 ± 0.13 e
Values marked with same letters are not significantly different (LSD, p < 0.05). All the values are means of four replicates ± SD.
IBA: Indole-3-butyric acid, BA: 6-Benzyladenin, PG: Phloroglucinol, Kin: Kinetin, GA: Gibberellic acid, NAA: 1-Naphthaleneacetic acid

Acta agriculturae Slovenica, 117/2 – 2021 5
An efficient protocol for in vitro regeneration from the nodal explants of Withania coagulans (Stocks) Dunal: a valuable medicinal herb 
Fig. 2: The rate of hardened plants from well-rooted plantlets obtained from the second experiment (rooting experi-
ment). Values marked with same letters are not significantly different (LSD, p < 0.05).
For acclimatization, the plantlets from the rooting 
experiment were transferred to pots. The results showed 
that the highest acclimatization was observed in T6, T7 
and T10 treatments by 86, 75 and 72 %, respectively. The 
lowest acclimatization was recorded in T9 and T3 treat-
ments by 36 % and 53 %, respectively (Fig. 2).
4 DISCUSSION
The populations of W. coagulans are in their natural 
habitat in Iran in danger of extinction due to the weak 
seed setting and germination created by its poor repro-
ductive system. Irregular and uncontrolled collection of 
W. coagulans for medicinal purposes is another reason 
for the extinction of W . coagulans in Iran. In order to pre-
vent the disappearance of the local W. coagulans popu-
lations, it is therefore necessary to take timely measures 
for their conservation (Gilani et al., 2009). Since there 
are many limitations (low seed viability and self-incom-
patibility) to conventional propagation of W. coagulans 
plants, in vitro culture can be effective in the propagation 
of genetically uniform plants in large numbers (Valiza-
deh & Valizadeh, 2011). Induction of rooting, elongation 
of micro-shoots, differentiation and development of stem 
buds in W. coagulans plants under different concentra-
tions and combinations of plant growth regulators are 
different. The results of the present study showed that the 
highest induction of shooting and shoot length was ob-
tained in the MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + 
PG (0.5 mg l
-1
) + GA (0.5 mg l
-1
) treatment compared to 
other treatments. Saritha & Naidu (2007) indicated that 
MS media fortified with 0.1 mg l
-1
 α-naphthalene acetic 
acid (1.5 mg l
-1
) and 2 mg l
-1
 BA (1.5 mg l
-1
) was the best 
treatment to induce shooting of W. somnifera from ax-
illary buds. In another report, Jain et al. (2011) showed 
that the multiple adventitious shoots of W. coagulans 
plant were differentiated in the MS medium containing 
BA (5 mg l
-1
) and kinetin (0.5 mg l
-1
) from leaf explants. 
Rathore et al. (2016) studied the effect of different con-
centrations of plant hormones (6-Benzylaminopurine 
(BAP), Kin and TDZ) on shoot regeneration from leaf 
explant of W. coagulans and showed that the highest 
shoot regeneration (74 %) was observed under 1 mg l
-
1
BAP treatment. The effect of different concentrations 
and combinations of growth regulators on the callus in-
duction from the nodal explants of W . coagulans was per-
formed by Valizadeh & Valizadeh (2009), who indicated 
that the highest growth of callus was observed on the MS 
medium supplemented with BA (0.25 mg l
-1
) and 2, 4-D 
(4 mg l
-1
). PG is an important phenolic compound that 
effectively induces the growth of root and shoot in stem 
culture. The results of the present study showed that PG 
had a positive effect on the shooting and shoot length, 
and the similar results have been reported on the effect 
of PG on shoot multiplication on Minuartia valentina 

Acta agriculturae Slovenica, 117/2 – 2021 6
P . DEHV ARI-NAGAN et al.
(Pau) Sennen by Ibanez & Amo-Marc (1998). Our find-
ings confirmed that the highest shoot multiplication and 
shooting were achieved on the MS medium containing 
MS + IBA (0.01 mg l
-1
) + BA (0.5 mg l
-1
) + PG (0.5 mg l
-1
) 
+ GA (0.5 mg l
-1
), which can be considered in the propa-
gation of W. coagulans medicinal plant.
Auxin has been reported to induce lateral rooting 
and improve primordium growth (Rathore et al., 2016). 
The results of the present study showed that NAA was 
more effective in rooting and number of roots than IBA. 
Valizadeh & Valizadeh (2011) investigated the effect of 
different concentrations of IBA, auxin and Kin on root-
ing of W. coagulans and showed that the highest percent-
age of rooting and number of roots were obtained under 
the IBA (2 mg l
-1
) treatment. NAA-induced rooting has 
also been reported in other medicinal plants (Ahmed et 
al., 2007b; Sivansean & Murugesan, 2008). In another re-
port, Ahmed et al. (2007a) indicated that auxin and NAA 
treatments induced the highest rate of rooting in stevia 
plant in in vitro condition. In general, various studies 
have shown that various auxin hormones are effective in 
inducing rooting in in vitro conditions. Different effects 
by some compounds can be due to differences in plant 
species, genotype, age and physiological status of the 
mother plants. The results also showed that adding PG to 
the MS medium containing IBA or NAA reduced num-
ber of roots compared to IBA and NAA treatments alone, 
indicating a negative effect of PG on rooting induction.
5 CONCLUSION
In summary, the results of the present study showed 
that the nodal explants of W. coagulans have a high or-
ganogenic potential for rooting and shooting response, 
however, the concentration and combination of growth 
regulators have a significant effect on rooting and shoot-
ing rate. Our findings highlight a complete propagation 
method for W. coagulans plants from the nodal explant 
that can also be used in genetic transformation studies 
to improve the plant and protect plant from extinction.
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