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Micropropagation of Ashwagandha (Withania somnifera)

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Biotechnological Communication Micropropagation of Ashwagandha

(Withania somnifera)

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Biotechnological
Communication
Biosci. Biotech. Res. Comm. 9(1): 88-93 (2016)

Micropropagation of Ashwagandha (Withania somnifera)

Rishikesh H. Autade1*, Sarika A. Fargade2, Amol R. Savant3, Sunil S. Gangurde4,
Rakeshkumar S. Choudhary5 and Suraj S. Dighe6
1,2,3 &6
Department of Plant Biotechnology, College of Agricultural Biotechnology, Loni, ITI Campus,
Chandrapur Road, Tal. - Rahata, Dist. – Ahmednagar, Pin – 413736, Maharashtra, India
4
Biotechnology Center, Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Dist. – Akola, Pin – 444104,
Maharashtra, India.
5
Department of Plant Biotechnology (IABT), University of Agriculture Science Campus, Krishinagar,
Dharwad-580005, Karnataka state, India.

ABSTRACT
In the present work in vitro propagation of a multipurpose medicinal plant, Withania somnifera was done. Direct regen-
eration of nodal explants and their multiplication have been optimized using cytokinin BAP (0.5-4.0 mg/l) and combi-
nation of BAP (0.5 mg/l) + NAA (0.5-3.0 mg/l) respectively. MS media with nodal explants supplemented with BAP (2.0
mg/l) produced maximum average number of shoots (2±0.37) and average shoot length was found to be 2.8±0.15 cm.
Best initiated shoots then sub cultured for shoot multiplication, an improved shoot multiplication in terms of average
number of shoots (5.3±0.41) and average shoot length 6.5±0.12 cm was observed on MS media in combination with
BAP (0.5 mg/l) + NAA (1.5 mg/l). Maximum average number (12± 0.20) and average length 9.8±0.26cm of roots were
observed on MS media supplemented with IBA (2.0 mg/l) out of different IBA (1-5 mg/l) concentrations were taken in
to consideration during the study. Regenerated plantlets were successfully transferred to greenhouse condition.

KEY WORDS: WITHANIA SOMNIFERA, ASHWAGANDHA, MICROPROPAGATION, NODAL EXPLANT, BAP, NAA, IBA.

INTRODUCTION mainstay of about 75-80% of the world population for

primary health care because of the better acceptabil-
Application of biotechnology for conservation of impor- ity with the human body and less side effects (Kamboj,
tant plant species has been given priority under circum- 2000).Many study revealed that cultivation of medicinal
stances, in particular when many valuable plant genetic plants especially high value medicinal plants is creating
resources are getting decimated rapidly from natural new dimension in the field of agriculture. Indian herbal
flora (Kumar et al., 2013). Herbal medicines are still the industry is at blooming stage. However, cultivation

ARTICLE INFORMATION:
*Corresponding Author: [email protected]
Received 10th March, 2016
Accepted after revision 31tst March, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
Thomson Reuters ISI SCI Indexed Journal
NAAS Journal Score : 3.48

88
© A Society of Science and Nature Publication, 2016. All rights
reserved.
Online Contents Available at: http//www.bbrc.in/
 Autade et al.

of medicinal plant is not easy. It is a challenging task The present study was done to determine the effect of
because very little knowledge of seed biology. Efforts growth hormones on shoots initiation, multiplication,
have not been made to search elite specimen and their rooting and hardening of Ashwagandha to standard-
propagation. ize the micro propagation technique in Ashwagandha,
Withania sominifera is a green shrub found through- as very less literature available for plant regeneration
out the drier parts in India, Baluchistan, Pakistan, Afgan- through nodal explant.
istan, Shri Lanka, Congo, South Africa, Egypt, Morocco,
and Jordan. In India, it is widely grown in the prov-
inces of Madhya Pradesh, Uttar Pradesh, plains of Pun- MATERIAL AND METHODS
jab andnorthwestern parts of the India like Gujarat and
Rajasthan Withania sominifera (L.) Dunal, commonly The plant material of Ashwagandha was collected in
called Indian ginseng is a member of the family Solan- plastic bags from Dhanwantari Medicinal and Aro-
aceae, growing up to a height of 30-150 cm. It is con- matic plants project, Mahatma Phule Krishi Vidyapeeth,
sidered as important medicinal plant in the Ayurvedic Rahuri. The plant material was propagated in College of
and indigenous medicinal system of India. It has many Agricultural Biotechnology, Loni, Tal. – Rahata, Dist. –
medicinal properties like anti-inflammatory, anticancer, Ahmednagar, Maharashtra for further use.
antistress, anti-ageing, immune-modular, adaptogenic Young nodal segments were selected as explants and
and shows the free radical scavenging activity It is used were washed under running tap water for 15 minutes.
for treatment of tuberculosis, rheumatism, inflammatory Later immersed in 1% tween-20 for 1 minute and washed
conditions and cardiac diseases. It is also useful as abor- with sterile double distilled water for 2-3 times. The pri-
tificient, amoebicide, anodyne, bactericide, contracep- marily surface sterilized nodal segments then rinsed in
tive and spasmolytic. The roots are also used as sedative 70% ethanol for 1 minute under laminar air flow hood
for senile debility and for the prevention and inhibition and washed with sterile double distilled water for 2-3
of Alzheimer’s disease (Sivanesan, 2007, Rout et. al., times. Finally rinsed with 0.1% mercuric chloride (HgCl2)
2011, Udaykumar et al. 2013 and Darwesh et al., 2014). for 7-8 minutes and washed with sterile double distilled
Recently, Dharajiya et. al., (2014) have analyzed the water for 4-5 times to remove all the surfactants.
antibacterial activity of various solvents viz. aqueous, One by one explants were placed on the filter paper
Hexane, ethyl acetate and methanol extracts of stem of to soak up the extra water. The nodal explants were
Withania somnifera was evaluated against gram nega- then cut from both ends. Finally with the help of forcep
tive bacteria Escherichia coli, Serratia marcescens, the explants were inoculated on the surface of the MS
Pseudomonas aeruginosa and gram positive bacterium media in such a way that ¾ part of the nodal explants
Bacillus cereus by agar well diffusion method. would be in contact with MS media. The culture bottles
Traditionally W. somnifera is propagated from seeds, containing explants inoculated on MS media supple-
but the mature and healthy seeds are not always availa- mented with respective different conc. of BAP and was
ble for germination. The viability period of seeds is very incubated at 25±3°C under white fluorescent light (2000
short and their germination is also poor. The provision lux) for 16/8 hours light and dark conditions. In total 6
of alternative sources of Withania somnifera by encour- weeks of initiation period one subculture was done in
aging its cultivation will go a long way in reducing 3rdweek and data for average shoot number per explants
their heavy dependence on the wild populations. Con- and average shoot length was recorded at the end.
ventional propagation methods have proved to be inad- The best initiated grown shoots were then trans-
equate to meet this challenge. Large scale production planted on MS media supplemented with different con-
through plant in vitro regeneration will provide a means centrations of BAP and NAA. Again the data for average
of putting the plant onto the market at lower prices. shoot number per explant and average shoot length was
Many earlier studies have reported in vitro propaga- recorded after 6 weeks of incubation period.
tion of Ashwagandha by using different explants, such For complete plant development regenerated shoots
as shoot tips (Hadeer et al., 2014; Rani et al., 2014; Baba were excised and transferred to MS medium supple-
et al., 2013; Sivanesan, 2007; Ray and Jha, 2001and Roja mented with different concentrations of IBA and data
and Heble, 1991), axillary bud (Rani and Grover, 1999), of average number of roots and average length of roots
hypocotyl (Kulkarni et al. 2000), cotyledon (Kumar et (cm) was recorded after 6 weeks of inoculation in growth
al. 2013), leaf (Joshi and Padhya, 2010 and Kulkarni chamber. Each treatment was repeated trice and statisti-
et al. 1996), seed (Supe et al., 2006), cotyledonary leaf cal analysis were done by calculating the standard error
segments (Rani et al. 2003), callus of leaf (Arumugam (SE) for the treatments.
and Gopinath 2013), shoot tip and root (Shrivastava and The rooted plants were removed from culture vessels
Dubey, 2007;) and the nodal areas, (Kumar et al., 2011). and washed in running tap water to remove agar. The

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MICROPROPAGATION OF ASHWAGANDHA (WITHANIA SOMNIFERA) 89

Autade et al.

number of roots that developed was counted and the induction with highest shoot number 4.8 shoots/callus
plants were transferred to plastic pots containing sterile and shoot length was 4.3 cm.
soil: sand: vermiculite (1:2:1, v/v/v). Finally initiated elongated shoots were excised and
implanted on MS media augmented with IBA (1-5
mg/l) (Table 3). The optimum root induction i.e. aver-
RESULTS AND DISCUSSION age number of roots (12±0.20) and average root length
(9.8±0.26cm) was obtained on MS media supplemented
During initiation of explants it has been observed that with 2 mg/l IBA (Fig. 4). The rooting results were found
nodal explants cultured on MS media devoid of growth to be consonance with results obtained by Sivanesan
hormone (Cytokinin) failed to induce the initiation of (2007); observed that half strength MS media containing
explants (Table 1). Out of all concentrations of BAP (0.5- 2.0 mg/l IBA was found to be the best. It produced 100%
4.0 mg/l) used for shoot initiation BAP (2 mg/l) pro- rooting with 16 roots/shoot and length was 10.5cm.
moted the shoot initiation i.e. average number of shoot Kumar et al., observed optimum rooting i.e. 72% with
(2±0.37) and average length of shoot (2.8±0.15cm) (Fig. root length 11 cm and number of roots/shoot was 25 on
1-2). Irshad et al., (2013) reported that axillary and api- full strength MS media supplemented with 5.0 mg/l IBA.
cal buds gave maximum response with respective to the At the end, well rooted plants 6.0 - 8.0 cm height
initiation of explant when inoculation on MS media for- obtained from rooting medium were transferred to plas-
tified with 1mg/l BAP. They found 75% response with tic tea cups for hardening (Fig. 5-6). Afterward, individ-
1-3 number of shoot/explant and shoot length was 2 ual cups with single plant were transferred to Polyhouse
cm. Darwesh et al., (2014) also found similar results i.e. and 75% relative humidity was maintained. Overall,
number of shoot 2.57 and shoot length/explant 3.09 but 90% of plants survived in the hardening process (data
with hormonal concentration as 2.0 mg/l BAP and 0.1 not shown) and these plants were established success-
mg/l NAA. fully in the experimental field.When regenerated plant-
Prominent in vitro response (average shoot number lets were transferred to the field, 87% survival rate was
5.3±0.41 and average shoot length 6.5±0.12cm) was obtained by Deshmukh et al. (2012).Arumugam et al.,
observed of best initiated shoots cultured on MS media (2013) observed the regenerated plants were successfully
augmented with BAP (0.5 mg/l) + NAA (1.5 mg/l) (Table hardened and acclimated 85% of plantlets survived well
2).. Increasing concentration of BAP and NAA resulted under natural conditions after transplantation.
in gradually increased in in vitro response of shoots, The production of Ashwagandha roots through con-
while further increased concentration was found to be ventional methods of cultivation (seed) is less than the
directly proportional to poor response of the shoot mul- requirement due to numerous reasons viz. poor yield,
tiplication (Fig. 3). Rani et al., (2014) observed the dif- takes long time, poor viability of seeds, susceptibility of
ferent concentration of NAA and BAP were showing the the seeds and seedlings to fungal infections like seed-
best result for shoot elongation and direct shoot regen- ling mortality and blight, leaf blight, seed rotting (Misra
eration at 0.5 mg/l BAP with 3.0 mg/l and 2.0 mg/l NAA. et al., 1997). This medicinally significant plant species
Similar results were found by Rout et. al., (2011) when has been depleted from its natural habitat and is now
different growth hormone tested in augmentation with included in the list of endangered species (Kanungo
MS media for shoot elongation. 2.0 mg/l BAP with 1.0 and Sahoo, 2011; Patel and Krishnamurthy 2013) by the
mg/l NAA was found to be best eliciting 82.3% shoot International Union for Conservation of Nature and Nat-

Table 1: Effect of BAP on shoot initiation from nodal explant.

Sr. No. MS medium + BAP(mg/l) Avg. shoot number Avg. shoot length (cm)
(mean±SE) (mean±SE)
1. MS medium + 0.0 (control) - -
2. MS medium + 0.5 1 ± 0.28 1 ± 0.26
3. MS medium + 1.0 1.3 ± 0.05 1.6 ± 0.43
4. MS medium + 1.5 1 ± 0.20 1.4 ± 0.25
5. MS medium + 2.0 2 ± 0.37 2.8 ± 0.15
6. MS medium + 2.5 1.3 ± 0.11 1 ± 0.20
7. MS medium + 3.0 1.3 ± 0.41 1.2 ± 0.11
8. MS medium + 3.5 1 ± 0.20 1.6 ± 0.15
9. MS medium + 4.0 1.6 ± 0.41 2.3 ± 0.34

90 MICROPROPAGATION OF ASHWAGANDHA (WITHANIA SOMNIFERA) BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

 Autade et al.

FIGURE 1 TO 6: The successive stages of in vitro propagation of Withania

somnifera. (Fig. 1) Direct regeneration of shoot from nodal segments as a
explants of ashwagandha cultured on Murashige and Skoog media. (Fig. 2)
Initiation of the multiple shoot formation (Fig. 3-4). Development of more
number of multiple shoot. (Fig. 5-6) Root formation from regenerated shoot.

ural Resources (Kavidraet al., 2000; Supeet al., 2006). explant was initiated on MS media supplemented with
The rapid multiplication of Ashwagandhaby tissue cul- different concentration of BAP (0.5 to 4.0 mg/l) resulted
ture techniques can help to solve these problems and the in best response on BAP (2.0mg/l) produced in terms
benefits are extensive in the agricultural world. of average number of shoots 2±0.37 and average shoot
In vitro propagation of Ashwagandha has been length was 2.8±0.15cm. The best initiated shoots then
achieved by using nodal segment as explant. The transferred for multiplication on MS medium supple-

Table 2: Effect of BAP and NAA on shoot multiplication of Ashwagandha.

Sr. No. MS medium +BAP(mg/l) Avg. shoot number Avg. shoot length
+ NAA (mg/l) (mean±SE) (cm) (mean±SE)
1. MS medium + 0.5 + 0.5 2.6 ± 0.25 3.4 ± 0.28
2. MS medium + 0.5 + 1.0 1.6 ± 0.30 2 ± 0.32
3. MS medium + 0.5 + 1.5 5.3 ± 0.41 6.5 ± 0.12
4. MS medium + 0.5 + 2.0 4 ± 0.43 4.7 ± 0.23
5. MS medium + 0.5 + 2.5 2.6 ± 0.36 2.3 ± 0.23
6. MS medium + 0.5 + 3.0 2.5 ± 0.32 2.1 ± 0.11

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MICROPROPAGATION OF ASHWAGANDHA (WITHANIA SOMNIFERA) 91

 Table 3: Effect of IBA on rooting of Ashwagandha.
Sr. no MS medium + IBA Avg. root number Avg. root length
(mg/l) (mean±SE) (cm) (mean±SE)
1. MS medium + 1.0 6.6 ± 0.37 5.3 ± 0.20
2. MS medium + 2.0 12 ± 0.20 9.8 ± 0.26
3. MS medium + 3.0 5.3 ± 0.15 4.7 ± 0.43
4. MS medium + 4.0 3.6 ± 0.25 4 ± 0.32
5. MS medium + 5.0 3.3 ± 0.36 3.7 ± 0.28

mented different concentration of BAP in combination Dharajiya D, Patel P, Patel M. Moitra N (2014). In vitro Anti-
with NNA. After six weeks of incubation BAP and NAA microbial Activity and Qualitative Phytochemical Analysis of
(0.5mg/l+ 1.5mg/l) produced maximum average number Withania somnifera(L.)Dunal Extracts.Int. J. Pharm. Sci. Rev.
of shoot 5.3±0.41 and average shoot length 6.5±0.12cm. Res. 27(2): 349-354.
MS medium supplemented with IBA 2.0mg/l produced Joshi AG, Padhya MA (2010). Shoot regeneration from leaf
maximum average of number roots 12±0.20 and aver- explants of Withania somnifera (L.) Dunal. Nat Sci Biol.
age root length was 9.8±0.26cm. After rooting, rooted 2(1):63-65.
plantlets were successfully established in primary and Kamboj VP (2000). Herbal medicine.Curr. Sci. 78 (1):35-39.
secondary hardening. After 30 days of inoculation on Kanungo S, Sahoo SL (2011). Direct organogenesis of Withania
rooting medium, the rooted plantlets were removed from somnifera L. from apical bud.International Res. J. Biotech. 2(3):
the culture tube and washed with distilled water. These 58-61.
in vitro derived plantlets were transferred to plastic pots Kavidra NT, Neelesh, CS, Vaibhav T, Brahma D (2000).Micro-
containing mixture of sand and FYM in 1:1 ratio for 18 propagation of Centella asiatica (L.) a valuable medicinal herb.
days for hardening. It was concluded from this study Plant Cell Tiss. Org. Cult. 62:175-179.
that plant regeneration from nodal explant of W. som-
Kulkarni AA, Thengane SR, Krishnamurthy KV (1996). Direct
nifera offered a great Potential in agriculture and this in vitro regeneration of leaf explants of Withania somnifera
in genetic transformation of this important species. The (L.) Dunal.Plant Science. 119(1): 163-168.
protocol can be exploited for in vitro generating new
Kulkarni AA, Thengane SR, Krishnamurthy KV (2000). Direct
genetic variability and production of bioactive constitu-
shoot regeneration from node, internode, hypocotyls and
ents from the plant. embryo explants of Withania somnifera. Plant Cell Tissue and
Organ Culture. 62(3): 203-209.
ACKNOWLEDGEMENT Kumar OA, Jyothirmayee G, Tata SS (2011). In vitro plant regen-
eration from leaf explants of Withania somnifera (L) Dunal
The authors are grateful to Principal, College of Agri-
(Ashwagandha) – an important medicinal plant, Research in
cultural Biotechnology, Loni for providing the healthy Biotechnology. 2(5): 34-39.
working environment and facilities during the course of
Kumar OA, Jyothirmayee G, Tata SS (2011).Multiple shoot
the study.
regeneration from nodal explants of Ashwagandha. Asian J of
Exp Biol. 2(4):636-640.
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BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MICROPROPAGATION OF ASHWAGANDHA (WITHANIA SOMNIFERA) 93

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