ORIGINAL PAPER

Rapid plant regeneration and analysis of genetic fidelityin micropropagated plants of Vitex trifolia: an importantmedicinal plant

Naseem Ahmad • Saad Bin Javed • Md Imran Khan •

Mohammad Anis

Received: 12 October 2012 / Revised: 29 March 2013 / Accepted: 3 April 2013 / Published online: 1 May 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract An efficient in vitro regeneration protocol of a

valuable medicinal plant, Vitex trifolia has been success-

fully established using nodal segments as explants. Three

different cytokinins (BA, Kn, 2iP) and auxins (NAA, IAA,

IBA) in different concentrations and combinations, evalu-

ated as supplements to Murashige and Skoog’s medium

showed to have a marked influence on the regeneration

output. Among all the single cytokinin treatments MS

medium supplemented with 5.0 lM BA produced the

maximum number of shoots yielding 8.20 ± 0.37 shoots

per explant with 4.8 ± 0.43 cm shoot length after 8 weeks

of culture. Combined with low auxin concentrations, all the

three cytokinins at their optimal concentrations synergis-

tically enhanced the regeneration credentials. However,

MS medium enriched with 5.0 lM BA and 0.5 lM NAA

yielded the best possible regeneration in the species with a

regeneration percentage of 97.33 ± 2.67 % and amounting

to 16.80 ± 0.58 shoots per explant with 6.20 ± 0.25 cm

mean shoot length at the end of 8 weeks in culture. Ex vitro

rooting of in vitro derived microshoots was achieved by

20 min 500 lM IBA treatment followed by transfer to

thermocol cups containing sterile soilrite. A 95 % plantlets

survived acclimatization procedure to the field. Genetic

conformity of the regenerated plants was established

through RAPD. All the bands visualized on agarose gels

were monomorphic with that of the donor plant indicating

the clonal nature of the regenerants.

Keywords Genetic integrity � Clonal propagation � Nodal

segments � Plant growth regulators � Chaste tree

Abbreviations

BA 6-Benzyladenine

2iP 2-Isopentenyladenine

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

Kn 6-Furfurylaminopurine

PGRs Plant growth regulators

MS Murashige and Skoog (1962)

NAA a naphthalene acetic acid

Introduction

In vitro culture of plant cells, tissues, and organs for mass

propagation is a common practice nowadays. The success

of this technique has been phenomenal and rendered sup-

port systems for a variety of recalcitrant plants. The main

beneficiary of this technique has been the medicinal plants

whose wild and natural stocks are fast depleting. Moreover,

the additional benefit for biomass production, yield of

secondary metabolites and bioactive molecules has created

a sensation to explore the regeneration potential of many

medicinal plants in recent years. Such a technique can

facilitate sufficient yield of secondary metabolites, while at

the same time the natural stocks are conserved. Vitex tri-

folia is an important medicinal shrub finding prominent

position in many widely practiced pharmacopeias. The

plant and its extracts are used to improve memory, relieve

pain, cure fever (Bhattacharjee and De 2005), and also

regarded as anti-bacterial, anti-inflammatory, anti-diabetic,

Communicated by B. Borkowska.

N. Ahmad (&) � S. B. Javed � M. I. Khan � M. Anis

Plant Biotechnology Laboratory, Department of Botany, Aligarh

Muslim University, Aligarh 202 002, India

e-mail: naseemptc1@rediffmail.com; naseembot@gmail.com

123

Acta Physiol Plant (2013) 35:2493–2500

DOI 10.1007/s11738-013-1285-y

and anti-cancerous (Pullaiah and Naidu 2003). Habitat

destruction and over harvesting for medicinal uses pose

threats to many medicinal plants including V. trifolia.

Being shrubby in nature and restricted distribution along

beaches and sandy soils make this particular plant highly

vulnerable to human exploitation. Although it can be

propagated through cuttings and suckers, no one ever cared

to take trouble and, thus, heavily relied on its collection

and gathering rather than cultivation. Such practices ham-

per the natural regeneration process, thus affecting the

ecological balance.

With such natural/ecological constraints, overcoming

the shortcomings of supply and demand is a serious issue.

Some alternative procedure which can maintain the bal-

ance, while at the same time the conservation aspects are

not affected will be necessary if the demand is to be met in

future. This, thus necessitates the intervention of biotech-

nological tools like plant tissue culture which may facilitate

mass multiplication for use by conversationalist in general

and pharmaceuticals in particular. Some very good reports

on in vitro propagation of the Vitex species like Vitex

negundo are available (Sahoo and Chand 1998; Thiru-

vengadam and Jayabalan 2000; Chandramu et al. 2003;

Vadawale et al. 2006; Ahmad and Anis 2007a; Usha et al.

2007; Afroz et al. 2008; Ahmad et al. 2008; Ahmad and

Anis 2011; Ahmad et al. 2013). However, no report on the

successful in vitro mass multiplication of V. trifolia is

available except a single preliminary report (Hiregoudar

et al. 2006) with limited insight about the process. Some

very intriguing and important aspects of in vitro regener-

ation like genetic fidelity analysis are also missing. We for

the first time report an efficient reliable reproducible pro-

tocol keeping all these aspects through the present

communication.

Materials and methods

Establishment of aseptic culture

Plants of V. trifolia were collected from Calangute beach,

Goa and successfully maintained at the Botanical garden,

Aligarh Muslim University, Aligarh. Axillary shoots from

a single plant designated as mother plant were collected for

use in the present experiments. These were washed under

running tap water for 20 min, treated with a laboratory

detergent (Labolene, Qualigens, India) 5 % (v/v) for 5 min

followed by 3–4 washing with sterile distilled water. The

plant material was surface sterilized with 0.1 % (w/v)

HgCl2 for 4 min followed by repeated washes with sterile

distilled water. Nodal segments bearing two axillary buds

were excised aseptically and cultured on sterile shoot

induction medium.

Media and culture conditions

The nutrient medium used in all the experiments consisted

of Murashige and Skoog (MS) (Murashige and Skoog

1962) with 3 % (w/v) sucrose (Qualigens, India) supple-

mented with various concentrations of different PGRs. All

the salts used were of analytical grade. The pH of the

medium was adjusted to 5.8 using 1 N NaOH and the

medium was solidified with 0.8 % (w/v) bacteriological

grade agar (Qualigens, India) before autoclaving at 121 �C

for 15 min. All the culture vials were incubated in culture

room at 25 ± 2 �C under 16/8 h (light/dark) cycle with a

light intensity of 50 lmol-2 s-1 supplied by cool white

fluorescent lamps (2 tubes 9 40 Watt, Philips, India) and

with 60–65 % relative humidity.

Shoot initiation and multiplication

Murashige and Skoog’s medium supplemented with dif-

ferent plant growth regulators viz. BA, Kn and 2iP at

various concentrations (0.0, 0.1, 0.5, 1.0, 2.5, 5.0, and

10 lM) either singly or in combination with IAA, IBA or

NAA (0.1, 0.5, 1.0, and 2.0 lM) were used for morpho-

genic response. Cultures were subcultured onto fresh media

after every 3 weeks up to a maximum of five subculturings.

The frequency of explants producing shoots, number of

shoots per explant and shoot length were recorded after

every 2 weeks of culture.

Ex vitro rooting and acclimatization

Individual in vitro raised microshoots (4–5 cm) were

excised from culture and their basal cut end were dipped in

different auxins viz. IBA, NAA and IAA of various

strengths (100, 200, 300, 500, and 1,000 lM) for 20 min,

followed by transfer to thermocol cups containing sterile

soilrite. Data on percentage of rooting, mean number of

roots and root length per shoot were recorded after 4 weeks

of transfer. Potted plantlets were covered with transparent

polythene bags to ensure high humidity, maintained in a

growth room and watered every alternate day with half-

strength MS salts lacking organic supplements. Polythene

bags were opened after 2 weeks to acclimatize the plants to

field conditions. After 4 weeks, the acclimatized plants

were transferred to pots containing normal garden soil and

maintained in greenhouse.

Molecular screening and genetic fidelity analysis

Molecular screening of the micropropagated plants was

performed by RAPD technique. Genomic DNA from the

mother plant and ten randomly selected acclimatized plants

were extracted using young leaves following the

2494 Acta Physiol Plant (2013) 35:2493–2500

123

hexadecyltrimethylammonium bromide (CTAB) method

described by Doyle and Doyle (1990). The extracted DNA

was tested for purity and concentrations derived using

Nanodrop spectrophotometer (Implen, Germany). RAPD

primers kits (OPA and OPB) were tested for random

amplification using PCR on a thermocycler (Biometra, T

Gradient Thermoblock, Germany). The PCR amplification

mixture (20 ll) consisted of 10 9 buffer (2 ll), 25 mM

MgCl2 (1.2 ll), 10 mM dNTPs (0.4 ll), 2 lM primers, 3

Unit Taq polymerase (0.2 ll), and 20 ng Template DNA.

PCR amplification program consisted of 45 cycles, each

cycle having a 94 �C denaturation step for 5 min, a 35 �C

annealing for 1 min, a 72 �C elongation for 1 min followed

by a final extension step at 72 �C for 10 min. DNA

amplification products were fractioned by electrophoresis

in 0.8 % (w/v) agarose gels with 3 ll ethidium bromide in

TAE buffer (pH 8.0) run at 50 V for 2 h and visualized on

a UV transilluminator (Bio Rad, Hercules, CA, USA). Only

well-defined and reproducible bands were scored. Bands

with the same migration were considered to be homologous

fragments, regardless of intensity. To assess the consis-

tency of band profiles PCR amplification was carried out

three times.

Statistical analysis

Each treatment consisted of 20 replicates and all the

experiments were repeated thrice. The data on various

parameters were subjected to one-way Analysis of Vari-

ance (ANOVA) using SPSS Ver 16 (SPSS Inc., Chicago,

USA). The significance of differences among means was

carried out using Duncan’s multiple range test at P = 0.05.

Fig. 1 a Bud break in nodal

segment of Vitex trifolia.

b Multiple shoot induction on

MS ? BA (5.0 lM) after

4 weeks of culture. c, d Cultures

showing multiple shoot

formation from nodal segments

of V. trifolia on MS ? BA

(5.0 lM) after 8 weeks of

culture

Concentration (µM)

Res

po

nd

ing

ep

lan

ts (

%)

0

20

40

60

80

100BAKN2iP

Control 0.1 0.5 1.0 2.5 5.0 7.5 10.0

a

b bcc

d

e

f

a

c

b

dc

e

f

a

c

b

c c

d

e

f

a

Concentration (µM)

Mea

n n

o o

f sh

oo

ts/ e

xpla

nt

(Bar

s)

0

2

4

6

8

10

Mea

n s

ho

ot

len

gth

/ exp

lan

t in

cm

(lin

es)

-2

0

2

4

BAKN2iPBAKN2iP

Control 0.1 0.5 1.0 2.5 5.0 7.5 10.0

h

g gg

e

a

b

c

g g

fg

ef

ccd

de

gg

g

e

c

de

e

h

cg

be

ad ac

a

ad bf

eg

cgbg

egdg

abac

g fg

bg

be

ac

acae

b

Fig. 2 a and b Effect of various cytokinins on multiple shoot

regeneration from nodal segments of V. trifolia in MS medium after

8 weeks of incubation. The bars/lines represent mean ± SE. Bars/

lines denoted by the same letter within response variables are not

significantly different (P = 0.05) using DMRT

Acta Physiol Plant (2013) 35:2493–2500 2495

123

The results were expressed as the mean ± SE of three

repeated experiments.

Results and discussions

Shoot initiation and multiplication

Effect of cytokinins

All the three cytokinins (BA, Kn, and 2iP) used for pro-

voking bud break (Fig. 1a) in the present study produced

the desired results, though, with different frequencies

(Fig. 2a). Apart from the cytokinin type, concentrations

also had a marked influence on shoot development. Of the

different concentrations tested, 5.0 lM was found to be

most effective with all the three cytokinins (Fig. 1b).

Increasing and decreasing the concentration proved to be

less productive for the species and resulted in low regen-

eration frequencies and shoot length, leaf size, leaf area,

etc. Such promotive effect of a particular concentration and

cytokinin type has been reported with other medicinal

plants like Vitex negundo (Ahmad and Anis 2011) and

Salix tetrasperma (Khan et al. 2011). Thus, BA at 5.0 lM

concentration yielded the optimum result forming

8.20 ± 0.37 shoots with average shoot length

4.8 ± 0.43 cm (Fig. 2b) after 8 weeks of culture (Fig. 1c

and d); Kn and 2iP at the same concentration gave lesser

Fig. 3 a Shoot proliferation on

MS ? BA (5.0 lM) ?NAA

(0.5 lM) after 8 weeks of

culture. b Ex vitro rooted

plantlets using 500 lM IBA.

c 4-week-old acclimatized

plantlets of V. trifolia in Soilrite

2496 Acta Physiol Plant (2013) 35:2493–2500

123

number of shoots with less pronounced shoot growth

(Fig. 2b). In many studies involving in vitro culture (Ah-

mad et al. 2008; Khan et al. 2011), BA has earned the

distinction of being the most effective cytokinin. The

structural stability and the ability of the plant cells to easily

assimilate the compound make this particular cytokinin

stand out among others in most of the cases. Thus, the

order of effectiveness of cytokinins on nodal segments in

shoot multiplication revealed as BA [ Kn and 2iP.

Effect of combination of cytokinins and auxins

Auxins though invariably used for indirect organogenesis or

for induction of rooting, has been proved to be quite effective

when used in conjunction with cytokinins for promoting

in vitro shoot growth as in Mucuna pruriens (Faisal et al.

2006). Similarly, the effectiveness of auxins for promoting

shoot differentiation in combination with cytokinins was

found prominent in this species. Three auxins (NAA, IAA,

and IBA) in different concentrations were tested against the

optimal cytokinin concentration of three cytokinins (BA, Kn,

and 2iP). This strategy proved highly beneficial as all com-

binations resulted in high shoot yield over the single cytoki-

nin treatment. However, the best result was obtained in MS

medium containing 5.0 lM BA in combination with 0.5 lM

NAA (Fig. 3a) which produced 16.80 ± 0.58 shoots per

explant with 6.20 ± 0.25 cm shoot length in 97.33 ± 2.67 %

cultures after 8 weeks of culture (Table 1). Other combina-

tions of BA with IAA or IBA also gave good results though

with lesser effectiveness (Table 1). Combinations of auxins

(NAA, IAA and IBA) along with Kn (5.0 lM) or 2iP

(5.0 lM) also yielded favorable results, multiplication and

elongation of shoots were enhanced with an optimum

response at 0.5 lM of NAA supplementation (Tables 2, 3).

Hence, the effectiveness of auxins for affecting multiple

shoot regeneration synergistically with cytokinins (BA, Kn,

and 2iP) followed the order of effectiveness NAA [ IAA or

IBA. Such order of effectiveness has been reported in a

number of recalcitrant woody plants like Pterocarpus,

Balanites aegyptiaca (Siddique and Anis 2009), Salix (Khan

et al. 2011).

Ex vitro Rooting

Rooting of in vitro raised microshoots was achieved by ex

vitro method. Among the tested auxins, IBA proved to be the

most effective. Maximum number of roots per shootlet

(10.40 ± 0.68) with maximum rooting frequency (97.67 ±

1.45 %) and mean individual root length (3.5 ± 0.57 cm)

was observed with 500 lM IBA treatment, after 4 weeks of

transfer (Figs. 3b, 4a, b). NAA and IAA at the same con-

centration produced 3.4 ± 1.02 and 4.0 ± 0.70 roots,

respectively. Effectiveness of the three may be listed as

IBA [ NAA or IAA (Fig. 4a, b). These findings on the

superiority of IBA over other auxins (NAA and IAA) for ex

vitro rooting have also been reported in Vitex negundo

Table 1 Effect of various auxins with the optimal concentration

of BA (5.0 lM) on direct shoot regeneration from nodal segments of

V. trifolia in MS medium after 8 weeks of culture

Growth regulators

(lM)

% Response Mean no. of

shoots

Mean shoot

length (cm)

IAA IBA NAA

0.1 69.0 ± 2.08e–g 8.20 ± 0.80ef 4.20 ± 0.30b–g

0.5 77.6 ± 2.18cd 13.0 ± 0.54bc 5.40 ± 0.29a–c

1.0 82.3 ± 1.45c 9.80 ± 0.91de 5.60 ± 0.38ab

1.5 67.6 ± 2.18fg 7.40 ± 0.81e–g 3.70 ± 0.46e–g

2.0 64.0 ± 2.00g 6.20 ± 0.58fg 3.44 ± 0.38fg

0.1 66.6 ± 1.66fg 7.40 ± 0.74e–g 3.08 ± 0.44g

0.5 75.6 ± 2.33c–e 10.8 ± 0.58cd 4.90 ± 0.33b–e

1.0 81.6 ± 4.40cd 7.40 ± 0.87e–g 5.14 ± 0.32a–d

1.5 68.3 ± 3.75e–g 6.40 ± 0.67 fg 2.90 ± 0.44g

2.0 61.6 ± 2.02g 5.00 ± 0.63g 3.10 ± 0.42g

0.1 90.0 ± 1.15b 11.8 ± 0.86b–d 4.40 ± 0.29c–f

0.5 97.3 ± 2.66a 16.8 ± 0.58a 5.26 ± 0.27a–d

1.0 89.3 ± 1.76b 14.0 ± 0.70b 6.20 ± 0.25a

1.5 81.0 ± 2.08c 9.60 ± 0.97de 4.06 ± 0.53d–g

2.0 74.4 ± 1.00d–f 7.00 ± 0.94fg 3.84 ± 0.46e–g

Values represent mean ± standard error of 20 replicates per treatment in

three repeated experiments. Means followed by the same letter are not

significantly different (P = 0.05) using Duncan’s multiple range test

Table 2 Effect of various auxins with the optimal concentration of

Kn (5.0 lM) on direct shoot regeneration from nodal segments of

Vitex negundo in MS medium after 8 weeks of culture

Growth regulators

(lM)

% Response Mean no.of

shoots

Mean shoot

length (cm)

IAA IBA NAA

0.1 64.0 ± 3.05d–f 6.20 ± 1.06ce 4.20 ± 0.30c–g

0.5 74.3 ± 2.33bc 9.00 ± 1.00bc 5.40 ± 0.29a–c

1.0 71.0 ± 2.08cd 6.80 ± 0.96ce 5.60 ± 0.38ab

1.5 68.3 ± 4.40c–e 6.20 ± 1.11c 3.70 ± 0.46e–g

2.0 58.3 ± 2.02f 4.80 ± 0.86e 3.44 ± 0.38fg

0.1 62.3 ± 1.45e 5.60 ± 0.81e 3.08 ± 0.42g

0.5 71.6 ± 2.88cd 8.40 ± 0.74bc 4.90 ± 0.33b–e

1.0 69.0 ± 2.08c–e 6.00 ± 0.83ce 5.14 ± 0.32a–d

1.5 67.3 ± 3.92c–e 5.60 ± 1.02e 2.96 ± 0.44g

2.0 59.3 ± 2.96f 4.20 ± 0.73e 3.10 ± 0.42g

0.1 85.0 ± 1.73a 8.80 ± 1.20bc 4.40 ± 0.29b–f

0.5 87.3 ± 2.66a 13.4 ± 1.53a 5.26 ± 0.27a–d

1.0 85.0 ± 1.73a 11.0 ± 0.89a–c 6.20 ± 0.25a

1.5 79.6 ± 2.33ab 6.80 ± 0.96ce 4.06 ± 0.53d–g

2.0 67.6 ± 1.45cd 5.00 ± 0.54e 3.84 ± 0.46e–g

Values represent mean ± standard error of 20 replicates per treatment in three

repeated experiments. Means followed by the same letter are not significantly

different (P = 0.05) using Duncan’s multiple range test

Acta Physiol Plant (2013) 35:2493–2500 2497

123

(Ahmad and Anis 2007a) and Cyamopsis tetragonoloba

(Ahmad and Anis 2007b). This technique of ex vitro rooting

is technically advantageous as this eliminates the additional

in vitro rooting step and combines the steps of rooting and

acclimatization of regenerants. Thus, the cost and time

required for clonal multiplication is greatly reduced.

Hardening and acclimatization

This step was combined with the ex vitro rooting procedure

and achieved using soilrite (Fig. 3c). Watering with half-

strength MS salt solutions every alternate day was done to

nourish the plants. The plantlets after 2 weeks were subjected

to slow hardening as described in ‘‘Materials and methods’’

before fully acclimatizing to soilrite. Thereafter, the plantlets

were transferred to pots filled with normal garden soil and

maintained in a greenhouse. Finally, they were shifted to field

conditions with a final survival rate of 90 % where they grew

well and exhibited normal morphology. Such a technique

involving a series of slow hardening in a particular environ-

ment before transfer to another environment has found suc-

cess with many micropropagation protocols (Husain and Anis

2009; Siddique and Anis 2009).

Molecular screening and genetic stability

Micropropagated plants though originally destined to be

clones being vegetative in nature and products of the same

mother plant have been found to vary with regard to their

genetic composition (Larkin and Scowcroft 1981). Such

variations called ‘somaclonal variations’ are a potential

drawback during micropropagation of an elite plant, where

clonal fidelity is required to maintain the advantages of the

desired genotype (Rahman and Rajora 2001). These vari-

ations may be avoided by keeping a stringent quality check

using various molecular markers. Recently, DNA-based

molecular markers have been a preferred method for

establishing the genetic fidelity (Martin et al. 2004). RAPD

or Randomly Amplified Polymorphic DNAs technique is

the most common and cost effective way of analyzing the

genetic fidelity of micropropagated plants (Bindya and

Kanwar 2003; Fatima et al. 2012). Out of 40 primers tested

(Table 4) for random amplification, 33 primers generated

good amplification visible by resolution of bands on aga-

rose gel electrophoresis (Table 4). Of these two primers

(OPB 10 and OPB 17) were selected based on the number

of bands produced and resolution for testing the genetic

fidelity among the regenerants. All the bands generated by

these primers were monomorphic with the mother plant

Table 3 Effect of various auxins with the optimal concentration of

2iP (5.0 lM) on direct shoot regeneration from nodal segments of

Vitex negundo in MS medium after 8 weeks of culture

Growth regulators

(lM)

% Response Mean no. of

shoots

Mean shoot

length (cm)

IAA IBA NAA

0.1 62.0 ± 3.05de 5.80 ± 0.73de 4.30 ± 0.33c–h

0.5 75.3 ± 2.60bc 9.40 ± 1.16bc 5.56 ± 0.28ab

1.0 72.0 ± 3.05c 7.20 ± 0.96c–e 5.68 ± 0.37a–c

1.5 70.0 ± 2.88 cd 6.60 ± 0.87c–e 3.84 ± 0.46gh

2.0 59.0 ± 1.33e 5.20 ± 0.86e 3.52 ± 0.37gh

0.1 61.0 ± 1.6de 6.20 ± 0.91de 3.04 ± 0.40 h

0.5 73.3 ± 2.40bc 8.80 ± 0.80cd 4.94 ± 0.30b–e

1.0 68.3 ± 2.02cd 6.40 ± 0.67c–e 5.20 ± 0.29a–e

1.5 69.0 ± 3.70c 5.80 ± 1.01de 3.04 ± 0.45 h

2.0 58.3 ± 3.33e 4.60 ± 0.81e 3.14 ± 0.42 h

0.1 85.6 ± 2.33a 9.40 ± 1.24bc 4.50 ± 0.35b–g

0.5 88.3 ± 1.66a 14.0 ± 1.37a 5.42 ± 0.28a–d

1.0 86.0 ± 2.00a 11.8 ± 0.86ab 6.28 ± 0.25a

1.5 80.6 ± 2.96ab 7.60 ± 1.12c–e 4.22 ± 0.62d–h

2.0 69.0 ± 2.06cd 5.60 ± 0.67e 3.98 ± 0.53e–h

Values represent mean ± standard error of 20 replicates per treatment in three

repeated experiments. Means followed by the same letter are not significantly dif-

ferent (P = 0.05) using Duncan’s multiple range test

Concentration (µM)

Mea

n n

o o

f ro

ots

/sh

oo

t (B

ars)

0

2

4

6

8

10

12

14

16

Mea

n r

oo

t le

ng

th in

cm

(lin

es)

-2

-1

0

1

2

3

4IAAIBANAAIAAIBANAA

Control 100 200 300 500 1000

f

efefc-f c-e

cd cd

f f

c

b

ab

f

c

c-e

g g g

d-f

aba-d

f

c-f

a-f

a

ab

ef

b-f

a-ca-e

b

Concentration (µM)

% R

oo

tin

g r

esp

on

se

0

20

40

60

80

100 IAAIBA

NAA

Control 100 200 300 500 1000

e e

f

g g

aa

b

e

c

g

cdd

e

g

f

a

Fig. 4 a and b Effect of various auxins for ex vitro root induction in

regenerated shootlets of V. trifolia after 4 weeks of transplantation.

The bars/lines represent mean ± SE. Bars/lines denoted by the same

letter within response variables are not significantly different

(P = 0.05) using DMRT

2498 Acta Physiol Plant (2013) 35:2493–2500

123

Table 4 Randomly amplified

polymorphic DNA (RAPD)

primers used for screening the

Banding pattern in Vitex species

S. no. Kit A Kit B

Primers Sequence (50-30) No. of bands Primers Sequence (50-30) No. of bands

1 OPA-01 CAGGCCCTTC 3 OPB 01 GTTTCGCTCC 7

2 OPA-02 TGCCGAGCTG 4 OPB 02 TGATCCCTGG 5

3 OPA-03 AGTCAGCCAC 2 OPB 03 CATCCCCCTG 4

4 OPA-04 AATCGGGCTG 2 OPB 04 GGACTGGAGT 3

5 OPA-05 AGGGGTCTTG 0 OPB 05 TGCGCCCTTC 4

6 OPA-06 GGTCCCTGAC 1 OPB 06 TGCTCTGCCC 5

7 OPA-07 GAAACGGGTG 3 OPB 07 GGTGACGCAG 3

8 OPA-08 GTGACGTAGG 0 OPB 08 GTCCACACGG 2

9 OPA-09 GGGTAACGCC 3 OPB 09 TGGGGGACTC 6

10 OPA-10 GTGATCGCAG 5 OPB 10 CTGCTGGGAC 9

11 OPA-11 CAATCGCCGT 0 OPB 11 GTAGACCCGT 4

12 OPA-12 TCGGCGATAG 5 OPB 12 CCTTGACGCA 2

13 OPA-13 CAGCACCCAC 3 OPB 13 TTCCCCCGCT 2

14 OPA-14 TCTGTGCTGG 0 OPB 14 TCCGCTCTGG 1

15 OPA-15 TTCCGAACCC 0 OPB 15 GGAGGGTGTT 3

16 OPA-16 AGCCAGCGAA 5 OPB 16 TTTGCCCGGA 1

17 OPA-17 GACCGCTTGT 6 OPB 17 AGGGAACGAG 6

18 OPA-18 AGGTGACCGT 7 OPB 18 CCACAGCAGT 4

19 OPA-19 CAAACGTCGG 0 OPB 19 ACCCCCGAAG 1

20 OPA-20 GTTGCGATCC 0 OPB 20 GGACCCTTAC 2

Total no. of bands 123

Fig. 5 a and b Randomly

amplified polymorphic DNA

(RAPD) amplification pattern

generated with OPB 10 (a) and

OPB 18 (b) among regenerated

plants of V. trifolia with mother

plant. M, marker (kDNA/

EcoR1 ? HindIII indicated in

bp); P, mother plant; Lane 1–10,

regenerated plants

Acta Physiol Plant (2013) 35:2493–2500 2499

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(Fig. 5a, b) and, thus, genetically uniform. This suggested

that the genetic integrity of the mother plant was main-

tained in the regenerants and, thus, they are clones of the

mother plant. No genetic changes have, thus, emerged due

to culture stress or the technique adopted in the present

study. Such strategy adopting RAPD technique for genetic

fidelity analysis has been well documented in literature

(Ahmad and Anis 2011; Faisal et al. 2012).

Conclusions and future prospects

Medicinal plant resources have been the most affected and

neglected part of the phytodiversity. Plant tissue culture serves

as a savior of such plants and makes it possible to generate a

large number of propagules for rehabilitation and use by

pharmaceutical companies. The present communication is a

successful attempt at this and would facilitate successful

conservation of V. trifolia, an important medicinal undershrub.

The protocol developed encompasses the various factors and

considerations of any micropropagation system to emerge as a

reliable method for mass propagation of the species. Extending

further support of the claim is the genetic fidelity analysis that

ensures a stringent quality check on the products. Thus, the

elite nature of the mother plant is maintained in the regener-

ants. The protocol will also help future researchers to follow

suit and develop efficient protocols for other medicinal plants.

Author Contribution N. Ahmad designed the whole

experiment and performed genetic integrity experiments.

M.I. Khan is responsible for carrying out the experiments

and data collection. S�B. Javed has done the statistical

analysis of data. The whole experiment was guided by the

Group Leader (M. Anis) who also edited the manuscript.

Acknowledgments The award of DST, Young Scientist (SR/FT/LS-

014/2009) scheme to Naseem Ahmad by the Department of Science and

Technology (DST), Government of India, New Delhi, is greatly

acknowledged. Research support from the Department of Science and

Technology (Govt. of India), New Delhi under the DST-FIST (2011) and

UGC-SAP (2009) Programme, is also acknowledged.

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