Horticultural

Communication

Biosci. Biotech. Res. Comm. 11(3): 451-460 (2018)

Standardization of various factors for production of

adventitious roots in selected varieties of

Withania

somnifera

and estimation of total withanolides by High

Performance Liquid Chromatography

Sindhu Rangaraju, A.N. Lokesha and Chenna Reddy Aswath

Division of Floriculture and Medicinal Crops ICAR-Indian Institute of Horticultural Research, Hesaraghatta

Lake Post, Bangalore-89

ABSTRACT

Withania somnifera (Dunal) popularly known as Ashwagandha, “Winter Cherry” and “Indian Ginseng”. Its roots and

leaves are used in a number of preparations for their anti-in ammatory, anticonvulsive, antitumor properties besides

promoting vigour and stamina. Ashwagandha contains very high concentration of metabolites like steroidal lactones

(Withanolides), alkaloids and  avonoids, so it is used in more than 200 commercially ayurvedic formulations. The

annual requirement of Withania sominifera in India is about 9127 MT where as the estimated production in India is

only 5905 MT. This requirement can be met by mass cultivation of adventitious roots using bioreactors. Adventitious

roots induced by this form are considered to be genetically uniform, true to its type that gives rise to mass production

of desired pharmaceutical compound. Seeds of varieties like Jawahar Ashwagandh-20 (JA-20), Arka Ashwagandha

(AA), IIHR WS-48 and IIHR WS-32 have been raised in in-vitro conditions. Adventitious roots were induced from

in-vitro leaves by varying factors. Half strength MS medium yielded more roots than full strength MS medium, com-

bination of IAA and IBA (ranging from 0.025-0.01mg/l) were found to be ideal for adventitious root induction for

each variety. Sucrose concentration (3-4%) in half strength MS media yielded more adventitious roots, with a light

intensity of 16 hours photoperiod than darkness.

KEY WORDS:

WITHANIA SOMNIFERA

(DUNAL), IAA, IBA, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

451

ARTICLE INFORMATION:

*Corresponding Author: sindhurangaraju63@gmail.co.in

Received 1

July, 2018

Accepted after revision 19

Sep, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.3/14

452 STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Sindhu, Lokesha and Aswath

INTRODUCTION

Withania somnifera (Dunal) popularly known as Ash-

wagandha, Indian ginseng and Winter cherry belongs

to the family Solanaceae. The plant’s Latin name liter-

ally means, “sweat of a horse” due to the scent of the

roots. The plant is commonly found in Africa, Mediter-

ranian, India and North America. It is an erect branched

under shrub up to 1.25 m in height, minute, smooth

and shiny hairs throughout the plant. Leaves are ovate,

with hairs and soft to touch. Flowers are greenish, the

roots are  eshy, papery and whitish brown in color. The

stems are around 3 to 4 feet in height. One plant sur-

vives for up to 4 to 5 years. Its stem contains  ber like

texture. The leaves are oval shaped, 2 to 4 inches long

and contain  ber. The  owers are blooming at the base

of the stems are small, somewhat long with chimney

shape and yellowish green in color. The  owers bloom

from the base of the leaves and become red when wipe.

The seeds are small, heart shaped, smooth and  at. The

roots are rough, white from within, strong, transparent,

thick and one to one and half feet long, (Geetha et al.,

2018).

In Ayurveda and Unani medications the herb is

majorly used for its high rejuvenating power. It is called

as “The natural stress buster” due to its ability in mak-

ing the human body copes with different kinds of stress

(Rao, 2012). The roots of Ashwagandha help in boost-

ing immunity power of the body. It is commonly pre-

scribed for hiccup, bronchitis, dropsy, rheumatism and

female disorders, the roots of this plant also prescribed

for general sexual weakness in human beings (Kattimani

et al., 2000). Its roots and leaves are used in a number

of preparations for their anti-in ammatory, anticonvul-

sive, antitumor, immuno-suppressive and antioxidant

properties besides promoting vigor and stamina. Ashwa-

gandha is increasingly becoming a popular adaptogenic

herb and is available throughout the western world as

a dietary supplement. Ashwagandha contains very high

concentration of metabolites like steroidal lactones

(Withanolides), alkaloids and  avonoids, so it is used

in more than 200 commercially ayurvedic formulations.

Adventitious roots are the roots that are induced at

unusual sites such as roots forming on leaves, which

grow and branch rapidly (Dubrovsky and Rost., 2003).

The roots induced by this form are considered to be

genetically uniform, true to its type, that gives rise to

mass production of desired pharmaceutical compound

(Goel et al ., 2009). Adventitious root cultures provide a

preferred platform to produce commercially important

secondary metabolites (Khan et al 2017). Adventitious

roots can harbor medicinally important compounds

through different strategies like elicitation, temperature

stress etc., (Rani et al., 2017).

The technique of micro propagation is applied with the

objective of enhancing the rate of multiplication. Through

the culture over a million of plants can be grown from a

small piece of plant tissue within 12 months. Such prolif-

erative rate of multiplication cannot be expected by any

in-vivo methods. Large scale production through plant

in-vitro regeneration will provide a means of putting

the plant onto the market at lower prices. In addition,

the technique is cost effective, relatively simple and can

be performed by semi-skilled persons. A sustained sup-

ply of the source material often becomes dif cult due to

the factors like environmental changes, cultural practices,

diverse geographical distribution, labour cost, selection of

the superior plant stock, over exploitation by pharmaceu-

tical industry (Kaur et al 2017)

Optimization of various tissue culture techniques

become very important to explore W. somnifera at

different aspects, as plants obtained from  elds ﬁleds

are not enough for all in vitro studies. Therefore, eff-

cient tissue culture techniques like, micropropaogation,

regeneration, organogenesis, hairy root production, etc.

have been established, (Vibha pandey et al. 2017).

The requirement of dried plant material for witha-

nolides drug production in India is estimated to be 9127

tonnes against the annual production of 5905 tonnes

(Sharda et al., 2007). Moreover,  eld cultivation is time

consuming, laborious and not able to meet the Ashwa-

gandha global market requirement (Sivanandan et al.,

2012b; 2013a). To improve the commercial cultivation of

Ashwagandha, biological advances must be made that

should either increase yield or reduce time gap to assure

quality (Banerjee et al., 1994).

The provision of alternative sources of Withania

somnifera by encouraging its cultivation will go a long

way in reducing their heavy dependence on the wild

populations and also major diseases of plant like seed

rot and blight can be overcome. The main objective of

this research is to develop a reproducible protocol for

adventitious root induction from in-vitro leaves, and

comparative analysis of withanolides present in in vitro

adventitious roots roots of different varieties and selec-

tion of best variety for mass propagation in bioreactors.

MATERIALS AND METHODS

Selection and establishment of plant material for micro-

propogation.

Seeds of four high yielding varieties of Withania som-

nifera like JA-20 (released variety from MPKVV, Madhya

Pradesh) used as check in AICRP national trials, Arka Ash-

wagandha (released variety from IIHR, Bangalore), IIHR

WS-32 and IIHR WS-48 were selected for the experiment.

The seeds were soaked in 300 ppm of gibberellic acid

for 12 hours and washed with water. The seeds were

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS 453

Sindhu, Lokesha and Aswath

pre-treated using 100 mg Dithane M-45 fungicide for 15

minutes, washed with sterile water followed by 70%(v/v)

ethanol for 1 minute, washed with sterile water and then

with 0.1% (w/v) sodium hypochlorite(5% (w/v) avail-

able chlorine, NICE Kochi Solution) for 4 minutes. Then

seeds were washed with sterile double distilled water

2-3 times to remove traces of sodium hypochlorite and

dried. The seeds were then inoculated into half strength

MS medium (Murashige and Skoog , 1962).

Procedure for adventitious root induction

Leaves from two month old explants were taken for pro-

duction of adventitious roots, before inoculation, leaves

were cut in middle with sterilized scalpel and placed on

MS culture medium with the adaxial surface down. Dif-

ferent factors were varied–

1. Strength of medium: Half strength and full strength

MS medium with selected combination of auxins

were used to study adventitious root induction.

2. Sucrose concentration: Carbohydrate source plays

an important role in maintaining osmoticum in

plant tissue culture. Root initiation and develop-

ment is a high energy process which requires the

expense of available metabolic substrates such as

sugars. Sucrose at different concentrations (2-7%)

were tried.

3. Auxin treatment: In order to determine the opti-

mal conditions for adventitious root induction, we

tested various concentration of auxins (IAA and

IBA) ranging from 0.025-0.01mg/L in 5 different

combinations and compared it with control (with-

out IAA and IBA).

4. Light intensity: The cultures in half strength MS

medium with selected auxin combination were in-

cubated at 25±2°C with 16 hours photoperiod un-

der cool  uorescent light and for dark treatment

the bottles were placed in shelves without light.

The adventitious roots were observed after 15 days

and parameters like number of roots per explant

and percentage of explants response for root

induction were studied.

The roots were subjected for HPLC analysis to esti-

mate the total withanolide content.

5. Extraction of bioactive principles from W.somnif-

era

The adventitious roots extracted from in-vitro were

washed twice with milli-Q water to remove the traces

of agar, dried and powder dried using pestle and mortar.

They were assessed for different components that contrib-

ute to total withanolides. The analysis was carried out by

HPLC method (Agarwal and Murali, 2010). Two grams of

dry root powder was extracted with 50 mL of methanol

on boiling water bath for about 20 minutes and transfer

the extract to a 250 mL beaker. Repeat the process 3-4

times till the extract was colorless. Then collected all the

extracts and made up the volume to 100mL with metha-

nol, mixed well and  ltered through 0.45 micron mem-

brane  lter and these were subjected to analysis by HPLC

with Photo Diode Array detector. Seven standards such

as Withanoside IV, Withanoside V, Withaferine A, Witha-

nolide A, Withanolide B, 12- deoxy Withanostramolide

and Withanone were used to quantify the amount of vari-

ous withanolides present in the root samples. Chromato-

gram was recorded at 227nm wavelength and later calcu-

late the contents of individual withanolides by the using

the formula and expressed as mg/100g dry weight basis.

Area of the sample 

Standard Wt. (mg) 

Sample dilution 

Purity of standard

-------------------------- 

100

Area of the standard 

Standard dilution 

sample weight (mg)  100

RESULTS AND DISCUSSION

The results from the present study demonstrated that

standardization of different factors like strength of the

medium, effect of photoperiod, sucrose concentration

, combinations of auxins at different concentrations

is essential for effective adventitious root induction in

Withania somnifera.

In this study to determine the effects of media

strength half strength MS medium with a combination

of 0.25mg/l IAA and 0.75 mg/l IBA had higher number

of induction response and also higher number of roots

per explants (Table no 1 and 2). The results of the pre-

sent study is similar to results of the previous studies

on adventitious root induction in Withania somnifera

by Wadegaonkar et al (2006) and Praveen and Murthy

(2010), where half strength MS medium was chosen suit-

able for adventitious root induction. It contradicts with

Yin et al (2013) induced adventitious roots in Pseudos-

tellaria heterophylla in full strength MS medium with

3mg/L IBA using root explants.

Highest root induction response (98.2% in JA 20)

and highest number of roots per explant (16 roots in

WS 32) was observed in bottles placed under 16 hours

photoperiod compared to darkness in all the varieties of

Withania somnifera (Table 3 and 4). Explants incubated

under darkness induced profuse callusing which subse-

quently turned brownish and hindered the induction of

roots. Roots initiated were thick and longer in braches

under 16 hours photoperiod compared to thin and brittle

roots in darkness.

Sindhu, Lokesha and Aswath

454 STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 1. Effect of strength of the medium on the induction response (%)

Treatment Arka Ashwagandha JA 20 IIHR WS 32 IIHR WS 48

Half MS 98.00±0.173a 98.00±0.520a 98.60±0.387a 97.40±0.316a

Full MS 95.4±0.173b 96.20±0.520b 96.40±0.387b 96.40±0.316b

Table 2. Effect of strength of the medium on number of roots per explant

Treatment Arka Ashwagandha JA 20 IIHR WS 32 IIHR WS 48

Half MS 13.80±0.070a 13.60±0.173a 14.20±0.173a 12.60±0.141a

Full MS 13.20±0.070b 13.20±0.173b 13.80±0.173b 12.40±0.141b

Table 3. Effect of photoperiod on adventitious root induction

Treatments

Induction Response (%) No of roots per explant

AA JA 20 WS 32 WS 48 AA JA 20 WS 32 WS 48

16 hr photoperiod 97.8 98.2 97.6 97.6 13.5 15.25 16 16.45

Darkness 91.8 91.6 92 91.8 5.1 5.1 5.05 5.6

S.Em 0.2151 0.245

CD 5% 0.619 1.75

CD 1% 0.81 2.36

CV 0.717% 4.69%

Table 4. Effect of photoperiod on number of days taken for adventitious root induction

Treatment Arka Ashwagandha JA 20 IIHR WS 32 IIHR WS 48

16 hours Photoperiod 24.00±0.316a 26.00±0.346a 26.60±0.141a 26.00±0.346a

Darkness 15.00±0.316b 14.80±0.346b 15.80±0.141b 14.80±0.346b

Table 5. Effect of Sucrose concentration on adventitious root induction response (%)

Treatments (Induction

response %)

Arka Ashwagandha JA 20 WS 32 WS 48

2% Sucrose 88.00±0.397b 82.40±0.465 b 80.40±0.389 b 88.20±0.499b

3% Sucrose 96.60±0.397a 86.80±0.465a 85.80 ±0.389a 98.40±0.499a

4% Sucrose 79.40±0.397c 78.60±0.465c 75.40±0.389c 77.00±0.499c

5% Sucrose 61.40±0.397d 61.60±0.465d 61.20±0.389 d 61.00 ±0.499 d

6% Sucrose 33.60±0.397e 35.60±0.465e 31.60±0.389 e 34.60±0.499 e

7% Sucrose 10.20±0.397f 11.00±0.465f 10.20 ±0.389f 10.60 ±0.499f

Praveen and Murthy (2010) also established adventi-

tious roots from leaf segments of Withania somnifera on

half strength MS medium(0.8%) agar with 0.5mg/L IBA,

30g/L sucrose incubated under 16 hours photoperiod

with 100% of explants response for root induction.

Table 1- Values are mean ± standard error of  ve

replications in three independent experiments, each

with three explants per treatment. Means followed by

the same letter are not signi cantly different at P<0.05

according to Duncan multiple range test in all tables

Data were scored after15 days of culture

Table 2- Values are mean ± standard error of  ve

replications in three independent experiments, each

with three explants per treatment. Means followed by

the same letter are not signi cantly different at P<0.05

according to Duncan Range Multiple Test. Data were

scored after 15 days of culture

Sindhu, Lokesha and Aswath

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS 455

Table 6. Effect of Sucrose concentration on number of adventitious roots per

explant

Treatments Arka Ashwagandha JA 20 WS 32 WS 48

2% Sucrose 11.40±0.319c 11.80±0.331c 10.60±0.294c 11.80±0.261c

3% Sucrose 17.60±0.319a 17.20±0.331a 15.00±0.294a 17.20±0.261a

4% Sucrose 12.80±0.319b 14.00±0.331b 12.40±0.294b 14.20±0.261b

5% Sucrose 8.80±0.319d 10.20±0.331d 9.8 ±0.294c 10.40±0.261d

6% Sucrose 7.40±0.319e 7.60±0.331e 8.60±0.294e 7.40±0.261e

7% Sucrose 5.40±0.319 f 5.60±0.331f 5.20±0.294e 5.40±0.261f

Table 7. Effect of auxins on adventitious root induction response (%)

Treatments Arka Ashwagandha JA 20 WS 32 WS 48

0 IAA+0 IBA 0.000±0.284f 0.000±0.330f 0.000±0.614f 0.000±0.703

0.25 IAA+0.75 IBA 96.60±0.284a 97.20±0.330a 85.80±0.614b 98.40±0.703

0.5 IAA+0.5 IBA 80.80±0.284c 80.60±0.330c 80.40±0.614c 82.20±0.703

0.75 IAA+0.25 IBA 76.80±0.284d 77.40±0.330d 75.60±0.614d 75.00±0.703

0 IAA+1 IBA 83.6±0.284 b 82.40±0.330b 97.6±0.614a 85.60±0.703

1 IAA+0 IBA 72.4±0.284e 68.00±0.330e 67.80±0.614e 65.00±0.703e

Table 8. Effect of auxins on number of adventitious roots per explant

Treatments Arka Ashwagandha JA 20 WS 32 WS 48

0 IAA+0 IBA 0.000±0.257d 0.000±0.245e 0.000±0.238f 0.000±0.371e

0.25 IAA+0.75 IBA 17.60±0.257a 17.20±0.245a 15.00±0.238b 17.20±0.371a

0.5 IAA+0.5 IBA 13.80±0.257b 13.80±0.245b 14.20±0.238c 12.20±0.371c

0.75 IAA+0.25 IBA 11.60±0.257c 11.60±0.245c 11.60±0.238d 13.00±0.371bc

0 IAA+1 IBA 13.60±0.257b 14.40±0.245b 17.40±0.238a 13.60±0.371b

1 IAA+0 IBA 11.40±0.257c 9.20±0.245d 8.80±0.238e 9.80±0.371d

Table 3- Values are mean ± standard error of  ve

replications in three independent experiments, each with

three explants per treatment. Data were scored after 15

days of culture. Growth Conditions- Media- Half MS

supplement with selected auxin combination, photoper-

iod-16 hours, culture period-3 weeks at 25±2°C

Table 4- Values are mean ± standard error of  ve

replications in three independent experiments, each

with three explants per treatment. Means followed by

the same letter are not signi cantly different at P<0.05

according to Duncan Range Multiple Test. Data were

scored after 15 days of culture. Growth Conditions-

Media- Half MS supplement with selected auxin com-

bination, photoperiod-16 hours and complete darkness,

culture period-3 weeks at 25±2°C

Carbohydrate plays an important role in maintain-

ing osmoticum in plant tissue culture. Sucrose is con-

sidered as an unquestionably important carbon and

energy source which is found in abundance in phloem

sac involved in developmental process. From the obser-

vations of the above study, the optimal condition for

adventitious root induction in Withania somnifera was

half strength MS medium with 3% sucrose concentra-

tion, (Table 5 and 6).

It has been documented earlier that a sucrose concen-

tration (3%) was suitable for hairy root growth, whereas

a too low or too higher a concentration of sucrose

was adverse to adventitious root growth inW. somnif-

era(Sivanandhan et al. 2012 a). A lower concentration

cannot provide enough energy and therefore may not be

able to act as building blocks. However, higher sucrose

concentration exhibited negative effect in growing cells.

Nagella and Murthy (2010) recorded that 3 % sucrose

was suitable for biomass accumulation and withanolide

A production in cell suspension culture ofW.somnifera.

Sucrose at higher concentrations in the nutrient medium

normally reduces cell biomass due to the increase of

osmotic potential which subsequently reduces the

uptake of nutrients. A similar result was obtained by

Zhang et al. (2012) inPeriploca sepiumadventitious root

culture and by Sivanandhan et al. (2012 a) inW. somnif-

eraadventitious root and hairy root cultures.

Sindhu, Lokesha and Aswath

456 STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

The lowest induction response was observed in 7%

sucrose concentration, from this result it was noticed

that the number of roots per explant started decreasing

at a higher root concentration of 5% and above. Higher

amount of sucrose can retard the development of cul-

tured cells (Wu et al., 2006) by causing cessation of the

cell cycle when other nutrients are limited (Gould et al.,

1981).

Table 5- Values are mean ± standard error of  ve

replications in three independent experiments, each

with three explants per treatment. Means followed by

the same letter are not signi cantly different at P<0.05

according to Duncan Range Multiple Test. Data were

scored after15 days of culture. Growth Conditions-

Media- Half MS supplement with selected auxin com-

bination, photoperiod-16 hours, culture period-3 weeks

at 25±2°C

Table 6- Values are mean ± standard error of  ve

replications in three independent experiments, each

with three explants per treatment. Means followed by

the same letter are not signi cantly different at P<0.05

according to Duncan Range Multiple Test. Data were

scored after15 days of culture. Growth Conditions-

Media- Half MS supplement with selected auxin com-

bination, photoperiod-16 hours, culture period-3 weeks

at 25±2°C.

Development of roots or shoots from explants involved

in organogenesis depends on morphogenetic potentiality

of the cells. Dedifferentiation, induction of organogenesis

pathway and development of organ are the three distinct

stages during organogenesis (de Kler et al., 1997). Supple-

mentation of exogenous auxin is essential for adventitious

root development (Pop et al., 2011). IAA and IBA induced

adventitious roots from leaf explants after 12 days of cul-

ture. Protuberances developed in leaf explants within a

week from the cut ends and adventitious roots directly

developed from these protuberances in another week. The

percentage of explants response for root induction and

number of roots initiated per explants were recorded after

3 weeks of culture.

Table 9. Analysis of bioactive principles from W. sominifera using HPLC.

Variety names Withanoside IV Withaferin A Withanolide A Withanolide B Total Withanolide

Arka Ashwagandha 0.061±0.001 a 0.023±0.000a 0.000±0.000b 0.000±0.000b 0.084±0.001a

JA 20 0.018±0.001d 0.018±0.000c 0.002±0.000a 0.005±0.000a 0.043±0.001d

IIHR WS 32 0.0034±0.001b 0.022±0.000b 0.000±0.000b 0.000±0.000b 0.057±0.001b

IIHR WS 48 0.030±0.001c 0.017±0.000b 0.000±0.000b 0.000±0.000b 0.047±0.000c

FIGURE 1A. 1) 2 month old explant of Withania somnifera , 2) culturing of

leaves for adventitious root induction 3) best hormone treatment for adven-

titious root induction 4) adventitious root induction observed after 7 days

of culture 5) mass production of adventitious roots in bottles 6) adventi-

tious roots are washed and measured. Growth Conditions- Media- Half MS

supplement with selected auxin combination, photoperiod-16 hours, culture

period-3 weeks at 25±2°C

Sindhu, Lokesha and Aswath

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS 457

FIGURE 1B. Best culture conditions for mass production of adventi-

tious roots in bottles. a, a1- Arka Ashwagandha variety with an auxin

concentration of 0.25mg/L IAA and 0.75mg/L IBA, b,b1- JA 20 variety

with an auxin concentration of 0.25mg/L IAA and 0.75mg/L IBA, c,c1

– IIHR WS 32 variety with an auxin concentration of 0.50mg/L IAA

and 0.50mg/L IBA,d,d1 – IIHR WS 48 variety with an auxin concentra-

tion of 0.25mg/L IAA and 0.75mg/L IBA. Growth Conditions- Media-

Half MS supplement with 3% sucrose concentration, photoperiod-16

hours, culture period-3 weeks at 25±2°C.

FIGURE 1C. Data represents mean ± standard error of three replicates; each experi-

ment was repeated thrice.Comparision of total withanolide, withanolide B, withnolide

A, withaferin A, and Withanoside IV among different varieties of Ashwagandha.

Taiz and Zeiger (2002) reported that roots may

require a less concentration of auxin to grow, but root

growth is strongly inhibited by its higher level because

at this level, auxin induces the production of ethylene,

a root growth inhibitor. The adventitious roots were also

induced in leaf explants of Withania somnifera using

a combination of IAA and IBA by Praveen and Murthy

(2010). Combination of IBA and IAA performed better

than individual treatment of auxin upon adventitious

root induction in Withania somnifera (Sivanandan et al

2012a). Wadegoankar et al (2006) reported that a combi-

nation of IAA and IBA was effective in adventitious rot

induction in leaves of Withania somnifera .

Hence the best auxin concentration for adventitious

root induction with 96.6% root induction with 18.25

roots per explant was observed in Arka Ashwagan-

dha variety with an auxin concentration of 0.25mg/L

IAA and 0.75mg/L where as a combination 0.25mg/L

IAA and 0.75mg/L IBA yielded in 97.2% root induction

response and 15.12 roots per explant in JA 20. Induc-

tion response of 97.2% with 18.5 roots per explant was

observed in an auxin concentration 0.25mg/L IAA and

0.75mg/L IBA of in IIHR WS 48 and induction response

of 97.6% with 18.38 roots per explant was observed

in explants inoculated into half strength MS medium

with 0.50 mg/L IAA and 0.50 mg/L IBA for IIHR WS 32.

Sindhu, Lokesha and Aswath

458 STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

(Table 7 and 8), At the end of 15 day after explant inocu-

lation, the length of the root was between 0.5-1.0 cm and

it was observed that the root length steadily increased

with the increase in growth period with was approxi-

mately 4-5 cm after 30 days. These results indicate fast

growing nature of adventitious roots.

Table 7-Values are mean ± standard error of  ve rep-

lications in three independent experiments, each with

three explants per treatment. Means followed by the same

letter are not signi cantly different at P<0.05 accord-

ing to Duncan Range Multiple Test. Data were scored

after15 days of culture. Growth Conditions- Media- Half

MS supplement with selected auxin combination, photo-

period-16 hours, culture period-3 weeks at 25±2°C.

Table 8- Values are mean ± standard error of  ve

replications in three independent experiments, each

with three explants per treatment. Means followed by

the same letter are not signi cantly different at P<0.05

according to Duncan Range Multiple Test. Data were

scored after15 days of culture. Growth Conditions-

Media- Half MS supplement with selected auxin com-

bination, photoperiod-16 hours, culture period-3 weeks

at 25±2°C.

Variation persist in accumulation of withanolides due

to plant parts, developmental stages (Praveen and Mur-

thy, 2010), plant part obtained from different types of

cultures (Sharada et al., 2007; Singh et al., 2017) of W.

somnifera. These studies establish relationship between

morphology/condition of plant tissue and withanolide

contents. Sivanandhan et al., 2012b, 2013b; Singh et al.,

2017) used in vitro grown plants in different studies to

develop adventitious roots, using different growth con-

ditions. These developed roots were harvested to extract

different combinations of withanolides.

The results for total withanolides analyzed in adven-

titious roots of four genotypes revealed signi cant dif-

ference among them (Table 9).

Among the genotypes, Arka Ashwagandha recorded

high total withanolide content of 0.084% when com-

pared to check JA-20 (0.043%). Among seven witha-

nolides analyzed, withanoside V, 12- deoxy Witha-

nostramolide and Withanone were not detected in all

genotypes and withanoside IV constitutes highest in all

the genotypes. Withanolide A and B were detected only

in JA-20. Arka Ashwagandha and IIHR WS-32 contain

high withaferin A and Withanoside IV when compared

to Check JA-20.

Table 9-Values are mean ± standard error of three

repeated experiments, each experiment was repeated

thrice. Means followed by the same letter are not signif-

icantly different at P<0.05 according to Duncan Range

Multiple Test.

CONCLUSION

From the present research a standard protocol has been

developed for mass production of adventitious roots

from in-vitro leaves in Withania somnifera. A variety

with highest total withanolide content has been identi-

FIGURE 1D. Standard HPLC chromatogram of extracts of W. somnifera roots. A.Withanoside IV (Rt=15.69),

B. Withanoside V (Rt=19.72), C. Withaferin A (Rt=20.06), D. 12-Deoxy Withastramonolide (Rt= 21.35), E.

Withanolide A (Rt=22.37), F. Withanolide B (Rt=25.42)

Sindhu, Lokesha and Aswath

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS STANDARDIZATION OF VARIOUS FACTORS FOR PRODUCTION OF ADVENTITIOUS ROOTS 459

 ed in comparision to JA 20. The requirement of dried

plant material for withanolides drug production in India

is high. Moreover,  eld cultivation is time consuming,

laborious and not able to meet the Ashwagandha global

market requirement. By transferring these roots in to

suspension culture and mass propagating it in bioreac-

tors reduce time gap compared to  eld grown roots and

assure good quality Withania somnifera roots with high

total withanolide content to cater the global demand.

Large scale production through plant in vitro regenera-

tion will provide a means of placing the plant onto the

market at lower prices. In addition, the technique is cost

effective, relatively simple and can be performed by

semi-skilled persons.

ACKNOWLEDGEMENTS

The research was conducted in Indian Institute of Hor-

ticulture Research and was supported by centre for Post

Graduation Studies Jain University. The author also

acknowledges Department of Science and Technology

for providing DST-Inspire fellowship for conducting this

research.

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